{
  "url": "https://openlegislation.uk/eu-directive-2006-63/annex",
  "json_url": "https://openlegislation.uk/eu-directive-2006-63/annex.json",
  "legislation": "eudr/2006/63",
  "legislation_title": "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.",
  "legislation_url": "https://openlegislation.uk/eu-directive-2006-63",
  "path": "annex",
  "kind": "annex",
  "number": null,
  "heading": null,
  "text": "ANNEX II\nTEST SCHEME FOR DIAGNOSIS, DETECTION AND IDENTIFICATION OF RALSTONIA SOLANACEARUM (SMITH) YABUUCHI ET AL.\nSCOPE OF THE TEST SCHEME\nThe presented scheme describes the various procedures involved in:\n(i) Diagnosis of brown rot in potato tubers and of bacterial wilt in potato, tomato and some other host plants;\n(ii) Detection of Ralstonia solanacearum in samples of potato tubers, potato-, tomato- and other host plants, water and soil;\n(iii) Identification of Ralstonia solanacearum (R. solanacearum).\nCONTENTS\nPage\nGeneral principles\n40\nSECTION I:\nApplication of the test scheme\n40\n1.\nDetection scheme for the diagnosis of brown rot and bacterial wilt (R. solanacearum) in potato tubers and potato, tomato or other host plants with symptoms of brown rot or bacterial wilt\n40\n2.\nScheme for detection and identification of R. solanacearum in samples of asymptomatic potato tubers\n43\n3.\nScheme for detection and identification of R. solanacearum in samples of asymptomatic, potato, tomato or other host plants\n46\nSECTION II:\nDetailed methods for detection of R. solanacearum in potato tubers and potato, tomato or other host plants with symptoms of brown rot or bacterial wilt\n48\n1.\nSymptoms\n48\n2.\nRapid screening tests\n48\n3.\nIsolation procedure\n49\n4.\nIdentification tests for R. solanacearum\n49\nSECTION III:\n1.\nDetailed methods for detection and identification of R. solanacearum in samples of asymptomatic potato tubers\n49\n1.1.\nSample preparation\n49\n1.2.\nTesting\n51\n2.\nDetailed methods for detection and identification of R. solanacearum in samples of asymptomatic potato, tomato or other plants\n51\n2.1.\nSample preparation\n51\n2.2.\nTesting\n52\nSECTION IV:\n1.\nScheme for detection and identification of R. solanacearum in water\n53\n2.\nMethods for detection and identification of R. solanacearum in water\n55\n2.1.\nSample preparation\n55\n2.2.\nTesting\n55\nSECTION V:\n1.\nScheme for detection and identification of R. solanacearum in soil\n56\n2.\nMethods for detection and identification of R. solanacearum in soil\n58\n2.1.\nSamples preparation\n58\n2.2.\nTesting\n58\nSECTION VI:\nOptimised protocols for detection and identification of R. solanacearum\n58\nA\nDiagnostic and detection tests\n58\n1.\nStem streaming test\n58\n2.\nDetection of poly-ß-hydroxybutyrate granules\n58\n3.\nSerological agglutination tests\n59\n4.\nSelective isolation\n60\n4.1.\nSelective plating\n60\n4.2.\nEnrichment procedure\n60\n5.\nImmunofluorescence test (IF test)\n61\n6.\nPolymerase chain reaction test (PCR test)\n64\n6.1.\nDNA purification methods\n65\n(a)\nMethod according to Pastrik (2000)\n65\n(b)\nOther methods\n65\n6.2.\nPCR\n66\n6.3.\nAnalysis of the PCR product\n66\n7.\nFluorescent in-situ hybridization test (FISH test)\n67\n8.\nEnzyme Linked ImmunoSorbent Assay (ELISA) tests\n69\n(a)\nIndirect ELISA\n69\n(b)\nDASI (Double-Antibody Sandwich Indirect) ELISA\n70\n9.\nBioassay test\n71\nB\nIdentification tests\n72\n1.\nNutritional and enzymatic identification tests\n72\n2.\nIF test\n72\n3.\nELISA test\n73\n4.\nPCR test\n73\n5.\nFISH test\n73\n6.\nFatty acid profiling (FAP)\n73\n7.\nStrain characterisation methods\n73\n7.1.\nBiovar determination\n73\n7.2.\nGenomic fingerprinting\n74\n7.3.\nPCR methods\n74\nC\nConfirmation test\n74\nAppendix 1\nLaboratories involved in optimisation and validation of protocols\n76\nAppendix 2\nMedia for isolation and culture of R. solanacearum\n77\nAppendix 3\n(A)\nCommercially available standardised control material\n79\n(B)\nPreparation of controls\n80\nAppendix 4\nBuffers for test procedures\n82\nAppendix 5\nDetermination of contamination level in IF and FISH tests\n85\nAppendix 6\nValidated PCR protocols and reagents\n86\nAppendix 7\nValidated reagents for FISH test\n91\nAppendix 8\nCulture conditions for tomato and eggplant\n93\nReferences\n94\nGENERAL PRINCIPLES\nOptimiseOptimised protocols for the various methods, validated reagents and details for the preparation of test and control materials are provided in the Appendices. A list of the laboratories that were included in optimization and validation of protocols is provided in Appendix 1.\nSince the protocols involve detection of a quarantine organism and will include the use of viable cultures of R. solanacearum as control materials, it will be necessary to perform the procedures under suitable quarantined conditions with adequate waste disposal facilities and under the conditions of appropriate licences as issued by the official plant quarantine authorities.\nTesting parameters must assure consistent and reproducible detection of levels of R. solanacearum at the set thresholds of the selected methods.\nPrecise preparation of positive controls is imperative.\nTesting according to the required thresholds also implies correct settings, maintenance and calibration of equipment, careful handling and preservation of reagents and all measures to prevent contamination between samples, e.g. separation of positive controls from test samples. Quality control standards must be applied to avoid administrative and other errors, especially concerning labelling and documentation.\nA suspected occurrence, as referred to in Article 4(2) of Directive 98/57/EC implies a positive result in diagnostic or screening tests performed on a sample as specified in flow charts below. A positive first screening test (IF test, PCR/FISH, selective isolation) must be confirmed by a second screening test based on a different biological principle.\nIf the first screening test is positive, then contamination with R. solanacearum is suspected and a second screening test must be done. If the second screening test is positive, than the suspicion is confirmed (suspected occurrence) and the testing according to the scheme must be continued. If the second screening test is negative, then the sample is considered not contaminated with R. solanacearum.\nConfirmed presence as referred to in Article 5(1) in Directive 98/57/EC implies the isolation and identification of a pure culture of R. solanacearum with confirmation of pathogenicity.\nSECTION I\nAPPLICATION OF THE TEST SCHEME\n1.\nDetection scheme for the diagnosis of brown rot and bacterial wilt (Ralstonia solanacearum) in potato tubers and potato, tomato or other host plants with symptoms of brown rot or bacterial wilt.\nThe testing procedure is intended for potato tubers and plants with symptoms typical or suspect of brown rot or vascular wilt. It involves a rapid screening test, isolation of the pathogen from infected vascular tissue on (selective) medium and, in case of a positive result, identification of the culture as Ralstonia solanacearum.\n2.\nScheme for detection and identification of Ralstonia solanacearum in samples of asymptomatic potato tubers\nPrinciple:\nThe testing procedure is intended for detection of latent infections in potato tubers. A positive result from at least two screening tests (3), based on different biological principles, must be complemented by the isolation of the pathogen; followed by, in case of isolation of typical colonies, confirmation of a pure culture as R. solanacearum A positive result from only one of the screening tests is not sufficient to consider the sample suspect.\nScreening tests and isolation tests must permit detection of 103 to 104 cells/ml of resuspended pellet, included as positive controls in each series of tests.\n3.\nScheme for detection and identification of Ralstonia solanacearum in samples of asymptomatic potato, tomato or other host plants\nSECTION II\nDETAILED METHODS FOR DETECTION OF RALSTONIA SOLANACEARUM IN POTATO TUBERS AND POTATO, TOMATO OR OTHER HOST PLANTS WITH SYMPTOMS OF BROWN ROT OR BACTERIAL WILT\n1.\nSymptoms (see website http://forum.europa.eu.int/Public/irc/sanco/Home/main)\n1.1.\nSymptoms on potato\nThe potato plant. The early stage of infection in the field is recognised by wilting of the leaves towards the top of the plant at high temperatures during the day with recovery at night. In early stages of wilting leaves remain green, but later yellowing and brown necrosis develops. Epinasty also occurs. Wilting of one shoot or whole plants becomes rapidly irreversible and results in the collapse and death of the plant. The vascular tissue of transversely cut stems from wilted plants usually appears brown and a milky bacterial ooze exudes from the cut surface or can be expressed by squeezing. When a cut stem is placed vertically in water, threads of slime will stream from the vascular bundles.\nThe potato tuber. Potato tubers must be cut transversely close to the heel (stolon end) or longitudinally over the stolon end. The early stage of infection is recognised by a glassy yellow to light brown discolouration of the vascular ring from which a pale cream bacterial ooze emerges spontaneously after some minutes. Later, the vascular discolouration becomes a more distinct brown and necrosis can extend into the parenchymatous tissue. In advanced stages, infection breaks outwards from the heel end and the eyes from which bacterial slime may ooze causing soil particles to adhere. Reddish-brown slightly sunken lesions may appear on the skin due to collapse of vascular tissues internally. Secondary development of fungal and bacterial soft rots is common in the advanced stages of the disease.\n1.2.\nSymptoms on tomato\nThe tomato plant. The first visible symptom is the flaccid appearance of the youngest leaves. Under favourable environmental conditions for the pathogen (soil temperatures of approximately 25 °C; saturated humidity), epinasty and wilting of one side or of the whole plant follows within a few days leading to total plant collapse. Under less favourable conditions (soil temperature below 21 °C), less wilting occurs, but large numbers of adventitious roots may develop on the stem. It is possible to observe watersoaked streaks from the base of the stem which is evidence of necrosis in the vascular system. When the stem is cut crosswise, discoloured brown vascular tissues exude white or yellowish bacterial ooze.\n1.3.\nSymptoms on other hosts\nSolanum dulcamara and S. nigrum plants. Under natural conditions, wilting symptoms are rarely observed in these weed hosts unless soil temperatures exceed 25 °C or inoculum levels are extremely high (e.g. as for S. nigrum growing adjacent to diseased potato or tomato plants). When wilting does occur, the symptoms are as described for tomato. Non-wilting S. dulcamara plants growing with stems and roots in water may show internal light brown discolouration of vascular tissues on transverse section of the stem base or underwater stem parts. Bacteria may ooze from cut vascular tissues or form threads of slime if the cut stem is placed vertically in water, even in the absence of wilting symptoms.\n2.\nRapid screening tests\nRapid screening tests may facilitate presumptive diagnosis but are not essential. Use one or more of the following validated tests:\n2.1.\nStem streaming test\n(See Section VI.A.1.)\n2.2.\nDetection of poly-ß-hydroxybutyrate (PHB) granules\nCharacteristic PHB granules in the cells of R. solanacearum are visualised by staining heat-fixed smears of bacterial ooze from infected tissue on a microscope slide with Nile Blue A or Sudan Black (See Section VI.A.2.).\n2.3.\nSerological agglutination tests\n(See Section VI.A.3.)\n2.4.\nOther tests\nFurther appropriate rapid screening tests include the IF test (see Section VI.A.5.), FISH test (see Section VI.A.7.), ELISA tests (see Section VI.A.8.) and PCR tests (see Section VI.A.6).\n3.\nIsolation procedure\n(a) Remove ooze or sections of discoloured tissue from the vascular ring in the potato tuber or from the vascular strands in stems of potato, tomato or other wilting host plants. Suspend in a small volume of sterile distilled water or 50mM phosphate buffer (Appendix 4) and leave for 5 to 10 minutes.\n(b) Prepare a series of decimal dilutions of the suspension.\n(c) Transfer 50-100 µl of the suspension and dilutions to a general nutrient medium (NA, YPGA or SPA; see Appendix 2) and/or to Kelman’s tetrazolium medium (Appendix 2) and/or a validated selective medium (e.g. SMSA; see Appendix 2). Spread or streak with an appropriate dilution plating technique. If useful, prepare separate plates with a diluted cell suspension of R. solanacearum biovar 2 as a positive control.\n(d) Incubate the plates for two to six days at 28 °C.\nOn the general nutrient media, virulent isolates of R. solanacearum develop pearly cream-white, flat, irregular and fluidal colonies often with characteristic whorls in the centre. Avirulent forms of R. solanacearum form small round non-fluidal, butyrous colonies which are entirely cream-white.\nOn Kelman’s tetrazolium and SMSA media, the whorls are blood red in colour. Avirulent forms of Ralstonia solanacearum form small round non-fluidal, butyrous colonies which are entirely deep red.\n4.\nIdentification tests for R. solanacearum\nTests to confirm identity of presumptive isolates of R. solanacearum are shown in Section VI.B.\nSECTION III\n1.\nDetailed methods for detection and identification of Ralstonia solanacearum in samples of asymptomatic potato tubers\n1.1.\nSample preparation\nNote:\nThe standard sample size is 200 tubers per test. More intensive sampling requires more tests on samples of this size. Larger numbers of tubers in the sample will lead to inhibition or difficult interpretation of the results. However, the procedure can be conveniently applied for samples with less than 200 tubers where fewer tubers are available.\nValidation of all detection methods described below is based on testing of samples of 200 tubers.\nThe potato extract described below can also be used for detection of the potato ring rot bacterium, Clavibacter michiganensis subsp. sepedonicus.\nOptional pre-treatment in advance to sample preparation:\n(a) Incubation of samples at 25 to 30 °C, for up to two weeks before testing, to encourage multiplication of any R. solanacearum populations.\n(b) Wash the tubers. Use appropriate disinfectants (chlorine compounds when PCR-test is to be used in order to remove pathogen DNA) and detergents between each sample. Air dry the tubers. This washing procedure is particularly useful (but not required) for samples with excess soil and if a PCR-test or direct isolation procedure is to be performed.\n1.1.1.\nRemove with a clean and disinfected scalpel or vegetable knife the skin at the heel (stolon) end of each tuber so that the vascular tissues become visible. Carefully cut out a small core of vascular tissue at the heel end and keep the amount of non-vascular tissue to a minimum. (see web site http://forum.europa.eu.int/Public/irc/sanco/Home/main).\nNote: Set aside any (rotting) tubers with suspected brown rot symptoms and test separately.\nIf during removal of the heel end core suspect symptoms of brown rot are observed, a visual inspection of this tuber should be done and the tuber cut near the heel end. Any cut tuber with suspected symptoms should be kept for at least two days at room temperature in order to allow suberisation and stored refrigerated (at 4 to 10 °C) under proper quarantine conditions. All tubers including those with suspicioussymptoms should be kept according to Annex III.\n1.1.2.\nCollect the heel end cores in unused disposable containers which can be closed and/or sealed (in case containers are reused they should be thoroughly cleaned and disinfected using chlorine compounds). Preferably, the heel end cores should be processed immediately. If this is not possible, store them in the container, without addition of buffer, refrigerated for not longer than 72 hours or for not longer than 24 hours at room temperature.\nProcess the heel end cores by one of the following procedures: either,\n(a) Cover the cores with sufficient volume (approximately 40 ml) of extraction buffer (Appendix 4) and agitate on a rotary shaker (50-100 rpm) for 4 hours below 24 °C or for 16 to 24 hours refrigerated,\nor\n(b) Homogenise the cores with sufficient volume (approximately 40 ml) of extraction buffer (Appendix 4), either in a blender (e.g. Waring or Ultra Thurax) or by crushing in a sealed disposable maceration bag (e.g. Stomacher or Bioreba strong guage polythene, 150 mm × 250 mm; radiation sterilised) using a rubber mallet or suitable grinding apparatus (e.g. Homex).\nNote: The risk of cross-contamination of samples is high when samples are homogenized using a blender. Take precautions to avoid aerosol generation or spillage during the extraction process. Ensure that freshly sterilised blender blades and vessels are used for each sample. If the PCR test is to be used, avoid carry-over of DNA on containers or grinding apparatus. Crushing in disposable bags and use of disposable tubes is recommended where PCR is to be used.\n1.1.3.\nDecant the supernatant. If excessively cloudy, clarify either by slow speed centrifugation (at not more than 180 g for 10 minutes at a temperature between 4 to 10 °C) or by vacuum filtration (40 to 100 µm), washing the filter with additional (approximately 10 ml) extraction buffer.\n1.1.4.\nConcentrate the bacterial fraction by centrifugation at 7 000 g for 15 minutes (or 10 000 g for 10 minutes) at a temperature between 4 to 10 °C and discard the supernatant without disturbing the pellet.\n1.1.5.\nResuspend the pellet in 1.5 ml pellet buffer (Appendix 4). Use 500 µl to test for R. solanacearum, 500 µl for Clavibacter michiganensis subsp. sepedonicus and 500 µl for reference purposes. Add sterile glycerol to final concentration of 10 to 25 % (v/v) to the 500 µl of the reference aliquot and to the remaining test aliquot, vortex and store at -16 to -24 °C (weeks) or at -68 to -86 °C (months). Preserve the test aliquots at 4 to 10 °C during testing.\nRepeated freezing and thawing is not advisable.\nIf transport of the extract is required, ensure delivery in a cool box within 24 to 48 hours.\n1.1.6.\nIt is imperative that all R. solanacearum positive controls and samples are treated separately to avoid contamination. This applies to IF slides and to all tests.\n1.2.\nTesting\nSee Flow chart and description of the tests and optimised protocols in the relevant appendices:\nSelective isolation (see Section VI.A.4.)\nIF test (see Section VI.A.5.)\nPCR tests (see Section VI.A.6.)\nFISH test (see Section VI.A.7.)\nELISA tests (see Section VI.A.8.)\nBioassay (see Section VI.A.9.)\n2.\nDetailed methods for detection and identification of R. solanacearum in samples of asymptomatic potato, tomato or other host plants\n2.1.\nSample preparation\nNote: For detection of latent R. solanacearum populations it is advised to test composite samples. The procedure can be conveniently applied for composite samples of up to 200 stem parts. Where surveys are performed they should be based on a statistically representative sample of the plant population under investigation.\n2.1.1.\nCollect 1 to 2 cm stem segments in a closed sterile container according to the following sampling procedures:\nNursery tomato seedlings: With a clean disinfected knife, remove a 1 cm segment from the base of each stem, just above the soil level.\nField or glasshouse grown tomato plants: With a clean disinfected knife, remove the lowermost side shoot from each plant by cutting just above the joint with the main stem. Remove the lowermost 1cm segment from each side shoot.\nOther hosts: With a clean disinfected knife or pruning shears, remove a 1 cm segment from the base of each stem, just above the soil level. In the case of S. dulcamara or other host plants growing in water, remove 1-2 cm sections from underwater stems or stolons with aquatic roots.\nWhen sampling a particular location it is recommended to test a statistically representative sample of at least 10 plants per sampling point of each potential weed host. Pathogen detection will be most reliable during late spring, summer and autumn seasons, although natural infections can be detected all year round in the perennial Solanum dulcamara growing in watercourses. Known hosts include volunteer potato plants (groundkeepers), Solanum dulcamara, S. nigrum, Datura stramonium and other members of the family Solanaceae. Further hosts are Pelargonium spp. and Portulaca oleracea. Some European weed spp. which may potentially harbour R. solanacearum biovar 2/Race 3 populations in roots and/or rhizospheres under specific environmental conditions include Atriplex hastata, Bidens pilosa, Cerastium glomeratum, Chenopodium album, Eupatorium cannabinum, Galinsoga parviflora, Ranunculus scleratus, Rorippa spp, Rumex spp., Silene alba, S. nutans., Tussilago farfarra and Urtica dioica.\nNote: Visual examination for internal symptoms (vascular staining or bacterial ooze) can be done at this stage. Set aside any stem segments with symptoms and test separately (See Section II).\n2.1.2.\nDisinfect stem segments briefly with ethanol 70 % and immediately blot dry on tissue paper. Then process the stem segments by one of the following procedures: either,\n(a) Cover the segments with sufficient volume (approximately 40 ml) of extraction buffer (Appendix 4) and agitate on a rotary shaker (50 to 100 rpm) for four hours below 24 °C or for 16 to 24 hours refrigerated, or\n(b) Process immediately by crushing the segments in a strong maceration bag (e.g. Stomacher or Bioreba) with an appropriate volume of extraction buffer (Appendix 4) using a rubber mallet or appropriate grinding apparatus (e.g. Homex). If this is not possible, store the stem segments refrigerated for not longer than 72 hours or for not longer than 24 hours at room temperature.\n2.1.3.\nDecant the supernatant after settling for 15 minutes.\n2.1.4.\nFurther clarification of the extract or concentration of the bacterial fraction are not usually required but may be achieved by filtration and/or centrifugation as described in Section III.1.1.3 – 1.1.5.\n2.1.5.\nDivide the neat or concentrated sample extract into two equal parts. Maintain one half at 4 to 10 °C during testing and store the other half with 10 to 25 % (v/v) sterile glycerol at -16 to -24 °C (weeks) or at -68 to -86 °C (month) in case further testing is required.\n2.2.\nTesting\nSee Flow chart and description of the tests and optimised protocols in the relevant appendices:\nSelective isolation (see Section VI.A.4.)\nIF test (see Section VI.A.5.)\nPCR tests (see Section VI.A.6.)\nFISH test (see Section VI.A.7.)\nELISA tests (see Section VI.A.8.)\nBioassay (see Section VI.A.9.)\nSECTION IV\n1.\nScheme for detection and identification of R. solanacearum in water\n2.\nMethods for detection and identification of R. solanacearum in water\nPrinciple\nThe validated detection scheme, described in this section, is applicable for pathogen detection in samples of surface water and can also be applied for testing samples of potato processing or sewage effluents. However, it is important to note that the expected sensitivity of detection will vary with the substrate. Sensitivity of the isolation test is affected by populations of competing saprophytic bacteria which are generally much higher in potato processing and sewage effluents than in surface water. Whereas the scheme below is expected to detect as few as 103 cells per litre in surface water the sensitivity of detection in potato processing or sewage effluents is likely to be significantly lower. For this reason, it is recommended to test effluents after any purification treatments (e.g. sedimentation or filtration) during which saprophytic bacterial populations are reduced. The limitations in sensitivity of the test scheme should be considered when assessing the reliability of any negative results obtained. Whereas this scheme has been successfully used in survey work to determine presence or absence of the pathogen in surface water, its limitations should be realised when used in similar surveys of potato processing or sewage effluents.\n2.1.\nSample preparation\nNote:\nDetection of R. solanacearum in surface water is most reliable during late spring, summer and autumn seasons when water temperatures exceed 15 °C.\nRepeated sampling at different times in the above mentioned period at designated sampling points will increase the reliability of detection by reducing the effects of climatic variation.\nTake into account the effects of heavy rainfall and the geography of the watercourse to avoid extensive dilution effects that may obscure presence of the pathogen.\nTake surface water samples in the vicinity of host plants if these hosts are present.\n2.1.1.\nAt selected sampling points, collect water samples by filling disposable sterile tubes or bottles at a depth if possible below 30 cm and within 2 m from the bank. For processing and sewage effluents, collect samples from the point of effluent discharge. Sample sizes up to 500 ml per sampling point are recommended. If smaller samples are preferred, it is advisable to take samples on at least three occasions per sampling point, each sample consisting of two replicated sub-samples of at least 30 ml. For intensive survey work, select at least three sampling points per 3 km of watercourse and ensure that tributaries entering the watercourse are also sampled.\n2.1.2.\nTransport samples in cool dark conditions (4 to 10 °C) and test within 24 hours.\n2.1.3.\nIf required, the bacterial fraction may be concentrated using one of the following methods:\n(a) Centrifuge 30 to 50 ml sub-samples at 10 000 g for 10 minutes (or 7 000 g for 15 minutes) preferably at 4 to 10 °C, discard the supernatant and resuspend the pellet in 1 ml pellet buffer (Appendix 4).\n(b) Membrane filtration (minimum pore size 0,45 µm) followed by washing the filter in 5 to 10 ml pellet buffer and retention of the washings. This method is suitable for larger volumes of water containing low numbers of saprophytes.\nConcentration is usually not advisable for samples of potato processing or sewage effluent since increased populations of competing saprophytic bacteria will inhibit detection of Ralstonia solanacearum.\n2.2.\nTesting\nSee flow chart and description of the tests in the relevant appendices.\nSECTION V\n1.\nScheme for detection and identification of R. solanacearum in soil\n2.\nMethods for detection and identification of R. solanacearum in soil\nPrinciples\nThe validated detection scheme, described in this section, is applicable for pathogen detection in soil samples but can also be used to test samples of solid potato processing waste or sewage sludge. However, it should be noted that these methods are insufficiently sensitive to guarantee detection of low and/or irregularly dispersed populations of Ralstonia solanacearum that may occur in naturally infested samples of these substrates.\nThe limitations in sensitivity of this test scheme should be considered when assessing the reliability of any negative results obtained and also when used in surveys to determine presence or absence of the pathogen in soils or sludges. The most reliable test for presence of the pathogen in a field soil is to plant a susceptible host and monitor it for infection, but even with this method low levels of contamination will escape detection.\n2.1.\nSample preparation\n2.1.1.\nSampling of field soil should follow standard principals used for nematode sampling. Collect 0,5 to 1 kg of soil per sample from 60 sites per 0,3 ha from a depth of 10 to 20 cm (or in a grid of 7 x 7 metres) If the pathogen is suspected to be present, increase the number of collection points to 120 per 0,3 ha. Maintain samples at 12 to 15 °C prior to testing. Sample potato processing and sewage sludges by collecting a total of 1 kg from sites representing the total volume of sludge to be tested. Mix each sample well before testing.\n2.1.2.\nDisperse sub-samples of 10 to 25 g of soil or sludge by rotary shaking (250 rpm) in 60 to 150 ml extraction buffer (Appendix 4) for up to two hours. If required, addition of 0,02 % sterile Tween-20 and 10 to 20 g sterile gravel may assist dispersion.\n2.1.3.\nMaintain the suspension at 4 °C during testing.\n2.2.\nTesting\nSee flow chart and description of the tests in the relevant appendices.\nSECTION VI\nOPTIMISED PROTOCOLS FOR DETECTION AND IDENTIFICATION OF R. SOLANACEARUM\nA.\nDIAGNOSTIC AND DETECTION TESTS\n1.\nStem streaming test\nThe presence of R. solanacearum in stems of wilting potato, tomato or other host plants can be indicated by the following simple presumptive test: Cut the stem just above the soil level. Suspend the cut surface in a tube of clean water. Observe for characteristic spontaneous streaming of threads of bacterial slime from the cut vascular bundles after a few minutes.\n2.\nDetection of poly-ß-hydroxybutyrate granules\n1.. Prepare a smear of bacterial ooze from infected tissue or from a 48-hour culture on YPGA or SPA medium (Appendix 2) on a microscope slide.\n2.. Prepare positive control smears of a biovar 2 strain of R. solanacearum and, if considered useful, a negative control smear of a known PHB negative sp.\n3.. Allow to air dry and pass the lower surface of each slide rapidly above a flame to fix the smears.\n4.. Stain preparation with either Nile Blue or Sudan Black and observe microscopically as described below:\nNile blue test:\n(a) Flood each slide with 1 % aquous solution of Nile Blue A and incubate for 10 minutes at 55 °C.\n(b) Drain off the staining solution. Wash briefly in gently running tap water. Remove excess water with tissue paper.\n(c) Flood the smear with 8 % aqueous acetic acid and incubate for one minute at ambient temperature.\n(d) Wash briefly in gently running tap water. Remove excess water with tissue paper.\n(e) Re-moisten with a drop of water and apply a coverslip.\n(f) Examine the stained smear with an epifluorescence microscope at 450 nm under oil immersion at a magnification of 600 to 1 000 using an oil- or water-immersion objective.\n(g) Observe for bright orange fluorescence of PHB granules. Also observe under transmitted normal light to ensure that the granules are intracellular and that cell morphology is typical of R. solanacearum.\nSudan Black test:\n(a) Flood each slide with 0,3 % Sudan Black B solution in 70 % ethanol and incubate for 10 minutes at ambient temperature.\n(b) Drain off the staining solution and wash briefly in tap water, removing excess water with tissue paper.\n(c) Dip the slides briefly in xylol and blot dry on tissue paper. Caution: Xylol is harmful, take necessary safety precautions and work in a fume cupboard.\n(d) Flood the slides with 0,5 % (w/v) aqueous safranin and leave for 10 seconds at ambient temperature. Caution: Safranin is harmful, take necessary safety precautions and work in a fume cupboard.\n(e) Wash in gently running tap water, blot dry on tissue paper and apply a coverslip.\n(f) Examine stained smears with a light microscope using transmitted light under oil immersion at a magnification of 1 000 using an oil-immersion objective.\n(g) Observe for blue-black staining of PHB granules in cells of R. solanacearum with pink-stained cell walls.\n3.\nSerological agglutination tests\nAgglutination of R. solanacearum cells in bacterial ooze or symptomatic tissue extracts is best observed using validated antibodies (see Appendix 3) labelled with appropriate coloured markers such as red Staphylococcus aureus cells or coloured latex particles. If using a commercially available kit (see Appendix 3), follow the manufacturers instructions. Otherwise perform the following procedure:\n(a) Mix drops of a suspension of labelled antibody and bacterial ooze (approximately 5 µl each) on windows of multiwell test slides.\n(b) Prepare positive and negative controls using suspensions of R. solanacearum biovar 2 and a heterologous strain.\n(c) Observe for agglutination in positive samples after gentle mixing for 15 seconds.\n4.\nSelective isolation\n4.1.\nSelective plating\nNote: Before using this method for the first time, perform preliminary tests to ensure reproducible detection of 103 to 104 colony-forming units of R. solanacearum per ml added to extracts from samples which previously tested negative.\nUse an appropriately validated selective medium such as SMSA (as modified by Elphinstone et al., 1996; see Appendix 2).\nCare is required to differentiate R. solanacearum from other bacteria able to develop colonies on the medium. Furthermore, colonies of R. solanacearum may show atypical morphology if plates are overcrowded or antagonistic bacteria are also present. Where effects of competition or antagonism are suspected, the sample should be re-tested using a different test.\nHighest sensitivity of detection by this method can be expected when using freshly prepared sample extracts. However, the method is also applicable for use with extracts which have been stored under glycerol at -68 to -86 °C.\nAs positive controls, prepare decimal dilutions from a suspension of 106 cfu per ml of a virulent biovar 2 strain of R. solanacearum (e.g. NCPPB 4156 = PD 2762 = CFBP 3857). To avoid any possibility of contamination, prepare positive controls totally separately from samples to be tested.\nFor each newly prepared batch of a selective medium its suitability for growth of the pathogen should be tested before it is used to test routine samples.\nTest control material in an identical manner as the sample(s).\n4.1.1.\nPerform an appropriate dilution plating technique aiming to ensure that any background saprophytic colony-forming populations are diluted out. Spread 50 - 100 µl per plate of sample extract and each dilution.\n4.1.2.\nIncubate plates at 28 °C. Read plates after 48 hours and daily thereafter up to six days. Typical R. solanacearum colonies on SMSA medium are milky white, flat, irregular and fluidal and after three days incubation develop pink to blood-red coloration in the centre with internal streaking or whorling. (see website http://forum.europa.eu.int/Public/irc/sanco/Home/main).\nNote: Atypical colonies of R. solanacearum sometimes form on this medium. These may be small, round, entirely red in colour and non-fluidal or only partially fluidal and therefore difficult to distinguish from saprophytic colony-forming bacteria.\n4.1.3.\nPurify presumptive R. solanacearum colonies after streaking or dilution plating onto a general nutrient medium to obtain isolated colonies (see Appendix 2).\n4.1.4.\nStore cultures short-term in sterile water (pH 6 to 8, chlorine free) at room temperature in the dark, or long term in a suitable cryoprotectant medium at -68 to -86 °C or lyophilised.\n4.1.5.\nIdentify presumptive cultures (see Section VI.B.) and perform a pathogenicity test (see Section VI. C).\nInterpretation of selective plating test results\nThe selective plating test is negative if no bacterial colonies are observed after six days or if no presumptive colonies typical of R. solanacearum are found, provided that no inhibition is suspected due to competition or antagonism by other bacteria and that typical R. solanacearum colonies are found in the positive controls.\nThe selective plating test is positive if presumptive R. solanacearum colonies are isolated.\n4.2.\nEnrichment procedure\nUse a validated enrichment medium such as modified Wilbrink broth (see Appendix 2).\nThis procedure can be used to selectively increase R. solanacearum populations in sample extracts and increase sensitivity of detection. The procedure also effectively dilutes inhibitors of the PCR reaction (1:100). It should be noted, however, that enrichment of R. solanacearum can fail due to competition or antagonism by saprophytic organisms which are often simultaneously enriched. For this reason, isolation of R.solanacearum from enriched broth cultures may be difficult. In addition, since populations of serologically related saprophytes can be increased, the use of specific monoclonal antibodies rather than polyclonal antibodies is recommended where the ELISA test is to be used.\n4.2.1.\nFor enrichment-PCR, transfer 100 µl of sample extract into 10 ml of enrichment broth (Appendix 2) previously aliquoted into DNA-free tubes or flasks. For enrichment-ELISA, higher proportions of sample extract to broth can be used (e.g. 100 µl in 1,0 ml of enrichment broth).\n4.2.2.\nIncubate for 72 hours at 27 to 30 °C in shaking culture or static culture with caps loosely- fitted to permit aeration.\n4.2.3.\nMix well before using in ELISA or PCR tests.\n4.2.4.\nTreat enriched broth in an identical manner as the sample(s) in the above tests.\nNote: If inhibition of enrichment of R. solanacearum is anticipated, due to high populations of certain competing saprophytic bacteria, enrichment of sample extracts before any centrifugation or other concentration steps may give better results.\n5.\nIF Test\nPrinciple\nThe use of the IF test as the principal screening test is recommended because of its proven robustness to achieve the required thresholds.\nWhen the IF test is used as the principal screening test and the IF reading is positive, the Isolation, PCR or FISH test must be performed as a second screening test. When the IF test is used as the second screening test and the IF reading is positive, further testing according to the flow scheme is required to complete the analysis.\nNote: Use a validated source of antibodies to R. solanacearum (see web site http://forum.europa.eu.int/Public/irc/sanco/Home/main). It is recommended that the titre is determined for each new batch of antibodies. The titre is defined as the highest dilution at which optimum reaction occurs when testing a suspension containing 105 to 106 cells per ml of the homologous strain of R. solanacearum and using an appropriate fluorescein isothiocyanate (FITC) conjugate according to the manufacturer’s recommendations. Validated polyclonal antisera all had an IF titre of at least 1:2 000. During testing, the antibodies should be used at a working dilution(s) close to or at the titre.\nThe test should be performed on freshly-prepared sample extracts. If necessary, it can be successfully performed on extracts stored at -68 to -86 °C under glycerol. Glycerol can be removed from the sample by addition of 1 ml pellet buffer (Appendix 4), re-centrifugation for 15 minutes at 7 000 g and re-suspension in an equal volume of pellet buffer. This is often not necessary, especially if samples are fixed to the slides by flaming.\nPrepare separate positive control slides of the homologous strain or any other reference strain of R. solanacearum, suspended in potato extract, as specified in Appendix 3 B, and optionally in buffer.\nNaturally infected tissue (maintained by lyophilisation or freezing at -16 to -24 °C) should be used where possible as a similar control on the same slide.\nAs negative controls, aliquots of sample extract which previously tested negative for R. solanacearum can be used.\nStandardised positive and negative control materials available for use with this test are listed in Appendix 3.\nUse multiwell microscope slides with preferably 10 windows of at least 6 mm diameter.\nTest control material in an identical manner as the sample(s).\n5.1.\nPrepare the test slides by one of the following procedures:\n(i) For pellets with relatively little starch sediment:\nPipette a measured standard volume (15 µl is appropriate for 6 mm window diameter – scale up volume for larger windows) of a 1/100 dilution of the resuspended potato pellet onto the first window. Subsequently pipette a similar volume of undiluted pellet (1/1) onto the remaining windows on the row. The second row can be used as duplicate or for a second sample as presented in Figure 1.\n(ii) For other pellets:\nPrepare decimal dilutions (1/10, 1/100) of the resuspended pellet in pellet buffer. Pipette a measured standard volume (15 µl is appropriate for 6 mm window diameter – scale up volume for larger windows) of the resuspended pellet and each dilution on a row of windows. The second row can be used as duplicate or for a second sample as presented in Figure 2.\n5.2.\nDry the droplets at ambient temperature or by warming to temperatures of 40 to 45 °C. Fix the bacterial cells to the slide either by heating (15 minutes at 60 °C), flaming, with 95 % ethanol or according to specific instructions from the suppliers of the antibodies.\nIf necessary, fixed slides may then be stored frozen in a desiccated box for as little time as necessary (up to a maximum of three months) prior to further testing.\n5.3.\nIF procedure\n(i) According to test slide preparation in 5.1(i):\nPrepare a set of twofold dilutions The first well should have 1/2 of the titre (T/2), the others 1/4 of the titre (T/4), 1/2 of the titre (T/2), the titre (T) and twice the titre (2T).\n(ii) According to test slide preparation in 5.1(ii):\nPrepare the working dilution (WD) of the antibody in IF buffer. The working dilution affects the specificity.\nFigure 1.\nPreparation of the test slide according to 5.1(i) and 5.3(i)\nDilutions of resuspended pellet\n1/100\n1/1\n1/1\n1/1\n1/1\n\nDilution of resuspended pellet\n(T = titre)\nT/2\nT/4\nT/2\nT\n2T\n\nTwofold dilutions of antiserum/antibody\nSample 1\n1\n2\n3\n4\n5\nDuplicate of sample1 or sample 2\n6\n7\n8\n9\n10\nFigure 2.\nPreparation of the test slide according to 5.1(ii) and 5.3(ii).\nWorking dilution of antiserum/antibody\n1/1\n1/10\n1/100\nempty\nempty\n\nDecimal dilution of resuspended pellet\nSample 1\n1\n2\n3\n4\n5\nDuplicate of sample 1 or sample 2\n6\n7\n8\n9\n10\n5.3.1.\nArrange the slides on moist tissue paper. Cover each test window completely with the antibody dilution(s). The volume of antibody applied on each window must be at least the volume of extract applied.\nThe following procedure should be carried out in the absence of specific instructions from the suppliers of the antibodies:\n5.3.2.\nIncubate the slides on moist paper under a cover for 30 minutes at ambient temperature (18 to 25 °C).\n5.3.3.\nShake the droplets off each slide and rinse carefully with IF buffer. Wash by submerging for five minutes in IF buffer-Tween (Appendix 4) and subsequently in IF buffer. Avoid causing aerosols or droplet transfer that could result in cross-contamination. Carefully remove excess moisture by blotting gently.\n5.3.4.\nArrange the slides on moist paper. Cover the test windows with the dilution of FITC conjugate used to determine the titre. The volume of conjugate applied on the windows must be identical to the volume of antibody applied.\n5.3.5.\nIncubate the slides on moist paper under a cover for 30 minutes at ambient temperature (18 to 25 °C).\n5.3.6.\nShake the droplets of conjugate off the slide. Rinse and wash as before (5.3.3).\nCarefully remove excess moisture.\n5.3.7.\nPipette 5 - 10 µl of 0,1M phosphate-buffered glycerol (Appendix 4) or a commercially antifading mountant on each window and apply a coverslip.\n5.4.\nReading the IF test:\n5.4.1\nExamine test slides on an epifluorescence microscope with filters suitable for excitation of FITC, under oil or water immersion at a magnification of 500-1 000. Scan windows across two diameters at right angles and around the perimeter. For samples showing no or low number of cells observe at least 40 microscope fields.\nCheck the positive control slide first. Cells must be bright fluorescent and completely stained at the determined antibody titre or working dilution. The IF test (Section VI.A.5.) must be repeated if the staining is aberrant.\n5.4.2.\nObserve for bright fluorescing cells with characteristic morphology of R. solanacearum in the test windows of the test slides (see website http://forum.europa.eu.int/Public/irc/sanco/Home/main). The fluorescence intensity must be equivalent to the positive control strain at the same antibody dilution. Cells with incomplete staining or with weak fluorescence must be disregarded.\nIf any contamination is suspected the test must be repeated. This may be the case when all slides in a batch show positive cells due to the contamination of buffer or if positive cells are found (outside of the slide windows) on the slide coating.\n5.4.3.\nThere are several problems inherent to the specificity of the immunofluorescence test. Background populations of fluorescing cells with atypical morphology and cross reacting saprophytic bacteria with size and morphology similar to R. solanacearum are likely to occur in potato heel end core and stem segment pellets.\n5.4.4.\nConsider only fluorescing cells with typical size and morphology at the titre or working dilution of the antibodies as in 5.3.\n5.4.5.\nInterpretation of the IF reading:\n(i) If bright fluorescing cells with characteristic morphology are found, estimate the average number of typical cells per microscope field and calculate the number of typical cells per ml of resuspended pellet (Appendix 5).\nThe IF reading is positive for samples with at least 5 × 103 typical cells per ml of resuspended pellet. The sample is considered potentially contaminated and further testing is required.\n(ii) The IF reading is negative for samples with less than 5 × 103 cells per ml of resuspended pellet and the sample is considered negative. Further testing is not required.\n6.\nPCR tests\nPrinciples\nWhen the PCR test is used as the principal screening test and found to be positive, isolation or IF must be performed as a second compulsory screening test. When PCR is used as the second screening test and found to be positive, further testing according to the flow scheme is required to complete the diagnosis.\nFull exploitation of this method as principal screening test is only recommended when specialised expertise has been acquired.\nNote: Preliminary testing with this method should permit reproducible detection of 103 to 104 cells of R. solanacearum per ml added to sample extracts which previously tested negative. Optimisation experiments may be required to achieve maximum levels of sensitivity and specificity in all laboratories.\nUse validated PCR reagents and protocols (see Appendix 6). Preferably select a method with an internal control.\nUse appropriate precautions to avoid contamination of sample with target DNA. The PCR test should be performed by experienced technicians, in dedicated molecular biology laboratories, in order to minimise the possibility of contamination with target DNA.\nNegative controls (for DNA extraction and PCR procedures) should always be handled as final samples in the procedure, to make evident whether any carry over of DNA has occurred.\nThe following negative controls should be included in the PCR test:\nSample extract that previously tested negative for R. solanacearum,\nBuffer controls used for extracting the bacterium and the DNA from the sample,\nPCR-reaction mix.\nThe following positive controls should be included:\nAliquots of resuspended pellets to which R. solanacearum has been added (preparation see Appendix 3 B).\nA suspension of 106 cells per ml of R. solanacearum in water from a virulent isolate (e.g. NCPPB 4156 = PD 2762 = CFBP 3857; see Appendix 3 B).\nIf possible use also DNA extracted from positive control samples in the PCR test.\nTo avoid potential contamination prepare positive controls in a separate environment from samples to be tested.\nSample extracts should be as free as possible from soil. It could therefore, in certain cases, be advisible to prepare extracts from washed potatoes if PCR protocols are to be used.\nStandardized positive and negative control material available for use with this test are listed in Appendix 3).\n6.1.\nDNA purification methods\nUse positive and negative control samples as described above (see Appendix 3).\nTest control material in an identical manner as the sample(s).\nA variety of methods are available for purification of target DNA from complex sample substrates, thus removing inhibitors of PCR and other enzymatic reactions and concentrating target DNA in the sample extract. The following method has been optimised for use with the validated PCR methods shown in Appendix 6.\n(a)\nMethod according to Pastrik (2000)\n1)) Pipette 220 µl of lysis buffer (100 mM NaCl, 10 mM Tris-HCl [pH 8,0], 1 mM EDTA [pH 8,0]) into a 1,5 ml Eppendorf tube.\n2)) Add 100 µl sample extract and place in a heating block or water bath at 95 °C for 10 min.\n3)) Put tube on ice for 5 min.\n4)) Add 80 µl Lysozyme stock solution (50 mg Lysozyme per ml in 10 mM Tris HCl, pH 8,0) and incubate at 37 °C for 30 min.\n5)) Add 220 µl of Easy DNA® solution A (Invitrogen), mix well by vortexing and incubate at 65 °C for 30 min.\n6)) Add 100 µl of Easy DNA® solution B (Invitrogen), vortex vigorously until the precipitate runs freely in the tube and the sample is uniformly viscous.\n7)) Add 500 µl of chloroform and vortex until the viscosity decreases and the mixture is homogeneous.\n8)) Centrifuge at 15 000 g for 20 min at 4 °C to separate phases and form the interphase.\n9)) Transfer the upper phase into a fresh Eppendorf tube.\n10)) Add 1 ml of 100 % ethanol (-20 °C) vortex briefly and incubate on ice for 10 min.\n11)) Centrifuge at 15 000 g for 20 min at 4 °C and remove ethanol from pellet.\n12)) Add 500 µl 80 % ethanol (-20 °C) and mix by inverting the tube.\n13)) Centrifuge at 15 000 g for 10 min at 4 °C, save the pellet and remove ethanol.\n14)) Allow the pellet to dry in air or in a DNA speed vac.\n15)) Resuspend the pellet in 100 µl sterile UPW and leave at room temperature for at least 20 minutes.\n16)) Store at -20 °C until required for PCR.\n17)) Spin down any white precipitate by centrifugation and use 5 µl of the supernatant containing DNA for the PCR.\n(b)\nOther methods\nOther DNA extraction methods, e.g. Qiagen DNeasy Plant Kit, could be applied providing that they are proven to be equally as effective in purifying DNA from control samples containing 103 to 104 pathogen cells per ml.\n6.2.\nPCR\n6.2.1.\nPrepare test and control templates for PCR according to the validated protocols (Section VI.A.6.). Prepare one decimal dilution of sample DNA extract (1:10 in UPW).\n6.2.2.\nPrepare the appropriate PCR reaction mix in a contamination-free environment according to the published protocols (Appendix 6). Where possible, it is recommended to use a multiplex PCR protocol that also incorporates an internal PCR control.\n6.2.3.\nAdd 2-5 µl of DNA extract per 25 µl PCR reaction in sterile PCR tubes according to the PCR protocols, (see Appendix 6).\n6.2.4.\nIncorporate a negative control sample containing only PCR reaction mix and add the same source of UPW as used in the PCR mix in place of sample.\n6.2.5.\nPlace tubes in the same thermal cycler which was used in preliminary testing and run the appropriately optimised PCR programme (Appendix 6).\n6.3.\nAnalysis of the PCR product\n6.3.1.\nResolve PCR amplicons by agarose gel electrophoresis. Run at least 12 µl of amplified DNA reaction mixture from each sample mixed with 3 µl loading buffer (Appendix 6) in 2,0 % (w/v) agarose gels in tris-acetate-EDTA (TAE) buffer (Appendix 6) at 5 to 8 V per cm. Use an appropriate DNA marker, e.g. 100 bp ladder.\n6.3.2.\nReveal DNA bands by staining in ethidium bromide (0,5 mg per L) for 30 to 60 minutes taking appropriate precautions for handling this mutagen.\n6.3.3.\nObserve stained gel under short wave UV transillumination (λ = 302 nm) for amplified PCR products of the expected size (Appendix 6) and document.\n6.3.4.\nFor all new findings/cases verify authenticity of the PCR amplicon by performing restriction enzyme analysis on a sample of the remaining amplified DNA by incubating at the optimum temperature and time with an appropriate enzyme and buffer (see Appendix 6). Resolve the digested fragments by agarose gel electrophoresis as before and observe characteristic restriction fragment pattern under UV transillumination after ethidium bromide staining and compare with the undigested and digested positive control.\nInterpretation of the PCR test result:\nThe PCR test is negative if the R. solanacearum-specific PCR amplicon of expected size is not detected for the sample in question but is detected for all positive control samples (in case of multiplex PCR with plant specific internal control primers: a second PCR-product of expected size must be amplified with the sample in question).\nThe PCR test is positive if the R. solanacearum-specific PCR amplicon of expected size and restriction pattern (when required) is detected, providing that it is not amplified from any of the negative control samples. Reliable confirmation of a positive result can also be obtained by repeating the test with a second set of PCR primers (Appendix 6).\nNote: Inhibition of the PCR may be suspected if the expected amplicon is obtained from the positive control sample containing R. solanacearum in water but negative results are obtained from positive controls with R. solanacearum in potato extract. In multiplex PCR protocols with internal PCR controls, inhibition of the reaction is indicated when neither of the two amplicons are obtained.\nContamination may be suspected if the expected amplicon is obtained from one or more of the negative controls.\n7.\nFISH test\nPrinciple\nWhen the FISH test is used as the first screening test and found to be positive, Isolation or the IF test must be performed as a second compulsory screening test. When the FISH test is used as the second screening test and found to be positive, further testing according to the flow scheme is required to complete the diagnosis.\nNote: Use validated R. solanacearum-specific oligo-probes (see Appendix 7). Preliminary testing with this method should permit reproducible detection of at least 103 to 104 cells of R. solanacearum per ml added to sample extracts which previously tested negative.\nThe following procedure should preferably be performed on freshly prepared sample extract but can also be successfully performed on sample extract that has been stored under glycerol at -16 to -24 or -68 to -86 °C.\nAs negative controls, use aliquots of sample extract that previously tested negative for R. solanacearum.\nAs positive controls prepare suspensions containing 105 to 106 cells per ml of R. solanacearum biovar 2 (e.g. strain NCPPB 4156 = PD 2762 = CFBP 3857, see Appendix 3) in 0,01M phosphate buffer (PB) from a 3 to 5 day culture). Prepare separate positive control slides of the homologous strain or any other reference strain of R. solanacearum, suspended in potato extract, as specified in Appendix 3 B.\nThe use of the FITC-labelled eubacterial oligo-probe offers a control for the hybridisation process, since it will stain all eubacteria that are present in the sample.\nStandardized positive and negative control material available for use with this test are listed in Appendix 3A).\nTest control material in an identical manner as the sample(s).\n7.1.\nPotato extract fixation\nThe following protocol is based upon Wullings et al. (1998):\n7.1.1.\nPrepare fixative solution (see Appendix 7).\n7.1.2.\nPipette 100 µl of each sample extract into an Eppendorf tube and centrifuge for 7 minutes at 7 000 g.\n7.1.3.\nRemove the supernatant and dissolve the pellet in 200 µl of fixative prepared < 24 hours previously. Vortex and incubate for one hour in the refrigerator.\n7.1.4.\nCentrifuge for 7 minutes at 7 000 g, remove the supernatant and resuspend the pellet in 75 µl 0,01M PB (see Appendix 7).\n7.1.5.\nSpot 16 µl of the fixed suspensions onto a clean multitest slide as shown in Fig. 7.1. Applying two different samples per slide, undiluted and use 10 µl to make a 1:100 dilution (in 0,01 M PB). The remaining sample solution (49 µl) can be stored at -20 °C after addition of one volume of 96 % ethanol. In case the FISH assay requires repeating, remove the ethanol by centrifugation and add an equal volume of 0,01 PB (mix by vortexing).\nFig. 7.1\nLayout for FISH slide\nSample 1\nBlank\nBlank\nBlank\nSample 2\nwindow 1\nwindow 2\nwindow 3\nwindow 4\nwindow 5\nSample 1\nBlank\nBlank\nBlank\nSample 2\nwindow 6\nwindow 7\nwindow 8\nwindow 9\nwindow 10\nCoverslip 1\nCoverslip 2\n7.1.6.\nAir-dry the slides (or on slide dryer at 37 °C) and fix them by flaming.\nAt this stage the procedure may be interrupted and the hybridisation continued the following day. Slides should be stored dust-free and dry at room temperature.\n7.2.\nHybridisation\n7.2.1.\nDehydrate the cells in a graded ethanol series of 50 %, 80 % and 96 % for one minute each. Air dry the slides in a slide-holder.\n7.2.2.\nPrepare a moist incubation chamber by covering the bottom of an air-tight box with tissue or filter paper soaked in 1x hybmix (Appendix 7). Pre-incubate the box in the hybridisation oven at 45 °C for at least 10 minutes.\n7.2.3.\nApply 10 μl of hybridisation solution (Appendix 7) to eight windows (windows 1, 2, 4, 5, 6, 7, 9 and 10; see Fig 7.1) of each slide leaving the two centre windows (3 and 8) empty.\n7.2.4.\nApply coverslips (24 × 24 mm) to the first and last four windows without trapping air. Place the slides in the pre-warmed moist chamber and hybridise for five hours in the oven at 45 °C in the dark.\n7.2.5.\nPrepare three beakers containing 1 l of Milli Q (molecular grade) water, 1 l of 1x hybmix (334 ml 3x hybmix and 666 ml Milli Q water) and 1 l of 1/8x hybmix (42 ml 3x hybmix and 958 ml Milli Q water). Pre-incubate each in a waterbath at 45 °C.\n7.2.6.\nRemove the coverslips from the slides and place the slides in a slide holder.\n7.2.7.\nWash away excess probe by incubation for 15 minutes in the beaker with 1x hybmix at 45 °C.\n7.2.8.\nTransfer the slide holder to 1/8 hybmix washing solution and incubate for a further 15 minutes.\n7.2.9.\nDip the slides briefly in Milli Q water and place them on filter paper. Remove excess moisture by covering the surface gently with filter paper. Pipette 5 to 10 μl of anti-fading mountant solution (e.g. Vectashield, Vecta Laboratories, CA, USA or equivalent) on each window and apply a large coverslip (24 × 60 mm) over the whole slide.\n7.3.\nReading the FISH test\n7.3.1.\nObserve the slides immediately with a microscope fitted for epifluorescence microscopy at 630 or 1 000 × magnification under immersion oil. With a filter suitable for fluorescein isothiocyanate (FITC) eubacterial cells (including most gram negative cells) in the sample are stained fluorescent green. Using a filter for tetramethylrhodamine-5-isothiocyanate, Cy3-stained cells of R. solanacearum appear fluorescent red. Compare cell morphology with that of the positive controls. Cells must be bright fluorescent and completely stained The FISH test (Section VI.A.7.) must be repeated if the staining is aberrant. Scan windows across two diameters at right angles and around the perimeter. For samples showing no or low number of cells observe at least 40 microscope fields.\n7.3.2.\nObserve for bright fluorescing cells with characteristic morphology of R. solanacearum in the test windows of the test slides (see web site http://forum.europa.eu.int/Public/irc/sanco/Home/main). The fluorescence intensity must be equivalent or better than that of the positive control strain. Cells with incomplete staining or with weak fluorescence must be disregarded.\n7.3.3.\nIf any contamination is suspected the test must be repeated. This may be the case when all slides in a batch show positive cells due to the contamination of buffer or if positive cells are found (outside of the slide windows) on the the slide coating.\n7.3.4.\nThere are several problems inherent to the specificity of the FISH test. Background populations of fluorescing cells with atypical morphology and cross reacting saprophytic bacteria with size and morphology similar to R. solanacearum may occur, although much less frequent as in the IF test, in potato heel end core and stem segment pellets.\n7.3.5.\nConsider only fluorescing cells with typical size and morphology.\n7.3.6.\nInterpretation of the FISH test result:\n(i) Valid FISH test results are obtained if bright green fluorescent cells of size and morphology typical of R. solanacearum are observed using the FITC filter and bright red fluorescent cells using the rhodamine filter in all positive controls and not in any of the negative controls. If bright fluorescing cells with characteristic morphology are found, estimate the average number of typical cells per microscope field and calculate the number of typical cells per ml of resuspended pellet (Appendix 4). Samples with at least 5 × 103 typical cells per ml of resuspended pellet are considered potentially contaminated. Further testing is required. Samples with less than 5 × 103 typical cells per ml of resuspended pellet are considered negative.\n(ii) The FISH test is negative if bright red fluorescent cells with size and morphology typical of R. solanacearum are not observed using the rhodamine filter, provided that typical bright red fluorescent cells are observed in the positive control preparations when using the rhodamine filter.\n8.\nELISA tests\nPrinciple\nELISA can only be used as an optional test in addition to IF, PCR or FISH due to a relatively low sensitivity of this test. When DAS ELISA is used enrichment and the use of monoclonal antibodies are compulsory (see web site http://forum.europa.eu.int/Public/irc/sanco/Home/main). Enrichment of the samples before using ELISA may be useful in order to increase the sensitivity of the test, but it can fail due to competition by other organisms in the sample.\nNote: Use an validated source of antibodies to R. solanacearum (see web site http://forum.europa.eu.int/Public/irc/sanco/Home/main) It is recommended that the titre is determined for each new batch of antibodies. The titre is defined as the highest dilution at which optimum reaction occurs when testing a suspension containing 105 to 106 cells per ml of the homologous strain of R. solanacearum and using appropriate secondary antibody conjugates according to the manufacturer’s recommendations. During testing, the antibodies should be used at a working dilution close to or at the titre of the commercial formulation.\nDetermine the titre of the antibodies on a suspension of 105 to 106 cells per ml of the homologous strain of R. solanacearum.\nInclude a sample extract that previously tested negative for R. solanacearum and a suspension of a non-cross reacting bacterium in phosphate buffered saline (PBS) as negative controls.\nAs positive control use aliquots of sample extract, that previously tested negative, mixed with 103 to 104 cells per ml of R. solanacearum biovar 2 (e.g. strain NCPPB 4156 = PD 2762 = CFBP 3857, see Appendix 2 A and B). For comparison of results on each plate use a standard suspension of 105 to 106 cells per ml in PBS of R. solanacearum. Ensure positive controls are well separated on the microtitre plate from the sample(s) under test.\nStandardised positive and negative control materials available for use with this test are listed in Appendix 3 A.\nTest control material in an identical manner as the sample(s).\nTwo ELISA protocols have been validated.\n(a)\nIndirect ELISA (Robinson Smith et al., 1995)\n1)) Use 100 to 200 µl aliquots of sample extract. (Heating at 100 °C for four minutes in a waterbath or heating block may reduce non-specific results in some cases).\n2)) Add an equal volume of double strength coating buffer (Appendix 4) and vortex.\n3)) Apply 100 µl aliquots to each of at least two wells of a microtitre plate (e.g. Nunc-Polysorp or equivalent) and incubate for one hour at 37 °C or overnight at 4 °C.\n4)) Flick out the extracts from the wells. Wash the wells three times with PBS-Tween (Appendix 4), leaving the last washing solution in the wells for at least five minutes.\n5)) Prepare the appropriate dilution of antibodies against-R. solanacearum in blocking buffer (Appendix 4). For validated commercial antibodies, use the recommended dilutions (usually twice as concentrated as the titre).\n6)) Add 100 µl to each well and incubate for one hour at 37 °C.\n7)) Flick out the antibody solution from the wells and wash as before (4).\n8)) Prepare the appropriate dilution of secondary antibody-alkaline phosphatase conjugate in blocking buffer. Add 100 µl to each well and incubate for one hour at 37 °C.\n9)) Flick out conjugated antibody from wells and wash as before (4).\n10)) Add 100 µl alkaline phosphatase substrate solution (Appendix 4) to each well. Incubate in the dark at ambient temperature and read absorbance at 405 nm at regular intervals within 90 minutes.\n(b)\nDASI ELISA\n1)) Prepare the appropriate dilution of anti-R. solanacearum polyclonal immunoglobulins in coating buffer pH 9.6 (Appendix 4). Add 200 µl to each well. Incubate at 37 °C for four to five hours or at 4 °C for 16 hours.\n2)) Wash the wells three times with PBS-Tween (Appendix 4).\nAdd 190 µl of sample extract to at least two wells. Also add positive and negative controls in two wells each per plate. Incubate for 16 hr at 4 °C.\n3)) Wash the wells three times with PBS-Tween (Appendix 4).\n4)) Prepare an appropriate dilution of R. solanacearum-specific monoclonal antibodies in PBS (Appendix 4) also containing 0,5 % bovine serum albumin (BSA) and add 190 µl to each well. Incubate at 37 °C for two hours.\n5)) Wash the wells three times with PBS-Tween (Appendix 4).\n6)) Prepare an appropriate dilution of anti-mouse immunoglobulins conjugated with alkaline phosphatase in PBS. Add 190 µl to each well. Incubate at 37 °C for two hours.\n7)) Wash the wells three times with PBS-Tween (Appendix 4).\n8)) Prepare an alkaline phosphatase substrate solution containing 1 mg p-nitrophenyl phosphate per ml of substrate buffer (Appendix 4). Add 200 µl to each well. Incubate in the dark at ambient temperature and read absorbance at 40 nm at regular intervals within 90 minutes.\nInterpretation of ELISA test results:\nThe ELISA test is negative if the average optical density (OD) reading from duplicate sample wells is < 2x OD of that in the negative sample extract control well, providing the OD for the positive controls are all above 1,0 (after 90 minutes incubation with the substrate) and are greater than twice the OD obtained for negative sample extracts.\nThe ELISA test is positive if the average OD readings from duplicate sample wells is > 2x OD in the negative sample extract well provided that OD readings in all negative control wells are < 2x those in the positive control wells.\nNegative ELISA readings in positive control wells indicate that the test has not been performed correctly or that it has been inhibited. Positive ELISA readings in negative control wells indicate that cross-contamination or non-specific antibody binding has occurred.\n9.\nBioassay test\nNote: Preliminary testing with this method should permit reproducible detection of 103 to 104 colony-forming units of R. solanacearum per ml added to sample extracts that previously tested negative (preparation see Appendix 3).\nHighest sensitivity of detection can be expected when using freshly prepared sample extract and optimal growth conditions. However, the method can be successfully applied to extracts that have been stored under glycerol at -68 to -86 °C.\nThe following protocol is based upon Janse (1988):\n9.1.\nUse 10 test plants of a susceptible tomato cultivar (e.g. Moneymaker or cultivar with equivalent susceptibility as determined by the testing laboratory) at the third true leaf stage for each sample. For cultural details, see Appendix 8. Alternatively, use eggplants (e.g. cultivar Black Beauty or cultivars with equivalent susceptibility), use only plants at leaf stage two to three up to full expansion of the third true leaf. Symptoms have been show to be less severe and to develop more slowly in eggplant. Where possible, it is therefore recommended to use tomato seedlings.\n9.2.\nDistribute 100 µl of sample extract between the test plants.\n9.2.1.\nSyringe inoculation\nInoculate the plant stems just above the cotyledons using a syringe fitted with a hypodermic needle (not less than 23G). Distribute the sample between the test plants.\n9.2.2.\nSlit inoculation\nHolding the plant between two fingers, pipette a drop (approximately 5 - 10 µl) of the suspended pellet on the stem between the cotyledons and the first leaf.\nUsing a sterile scalpel, make a diagonal slit, about 1.0 cm long and approximately 2/3 of the stem thickness deep, starting the cut from the pellet drop.\nSeal the cut with sterile vaseline from a syringe.\n9.3.\nInoculate by the same technique, five seedlings with an aqueous suspension of 105 to 106 cells per ml prepared from a 48 hr culture of a virulent biovar 2 strain of R. solanacearum as a positive control and with pellet buffer (Appendix 4) as negative control. Separate positive and negative control plants from the other plants to avoid cross-contamination.\n9.4.\nGrow the test plants in quarantine facilities for up to four weeks at 25 to 30 °C and high relative humidity with appropriate watering to prevent waterlogging or wilting through water deficiency. To avoid contamination incubate positive control and negative control plants on clearly separated benches in a glasshouse or growth chamber or, in case space is limited, ensure strict separation between treatments. If plants for different samples must be incubated close together, separate them with appropriate screens. When fertilising, watering, inspecting and any other manipulations take great care to avoid cross-contamination. It is essential to keep glasshouses and growth chambers free of all insect pests since they may transmit the bacterium from sample to sample.\nObserve for symptoms of wilting, epinasty, chlorosis and/or stunting.\n9.5.\nIsolate from infected plants (Section II.3.) and identify purified cultures of presumptive R. solanacearum (Section VI.B.).\n9.6.\nIf no symptoms are observed after three weeks perform IF/PCR/Isolation on a composite sample of 1 cm stem sections of each test plant taken above the inoculation site. If the test is positive perform dilution plating (section 4.1).\n9.7.\nIdentify any purified cultures of presumptive R. solanacearum (Section VI.B.).\nInterpretation of the bioassay test results\nValid Bioassay test results are obtained when plants of the positive control show typical symptoms, the bacteria can be reisolated from these plants and no symptoms are found on the negative controls.\nThe bioassay test is negative if test plants are not infected by R. solanacearum, and provided that R. solanacearum is detected in positive controls.\nThe bioassay test is positive if the test plants are infected by R. solanacearum.\nB.\nIDENTIFICATION TESTS\nIdentify pure cultures of presumptive R. solanacearum isolates using at least two of the following tests based on different biological principles.\nInclude known reference strains where appropriate for each test performed (see Appendix 3).\n1.\nNutritional and enzymatic identification tests\nDetermine the following phenotypic properties, which are universally present or absent in R. solanacearum, according to the methods of Lelliott and Stead (1987), Klement et al. (1990), Schaad (2001).\nTest\nExpected result\nFluorescent pigment production\n–\nPoly-ß-hydroxybutyrate inclusions\n+\nOxidation/fermentation (O/F) test\nO+/F–\nCatalase activity\n+\nKovac’s oxidase test\n+\nReduction of nitrate\n+\nUtilisation of citrate\n+\nGrowth at 40 °C\n–\nGrowth in 1 % NaCl\n+\nGrowth in 2 % NaCl\n–\nArginine dihydrolase activity\n–\nGelatine liquefaction\n–\nStarch hydrolysis\n–\nAesculin hydrolysis\n–\nLevan production\n–\n2.\nIF test\n2.1.\nPrepare a suspension of approximately 106 cells per ml in IF buffer (Appendix 4).\n2.2.\nPrepare a twofold dilution series of an appropriate antiserum (see website http://forum.europa.eu.int/Public/irc/sanco/Home/main).\n2.3.\nApply the IF procedure (Section VI.A.5.).\n2.4.\nA positive IF test is achieved if the IF titre of the culture is equivalent to that of the positive control.\n3.\nELISA test\nNote: If performing only 2 identification tests, do not use another serological test in addition to this method.\n3.1.\nPrepare a suspension of approximately 108 cells per ml in 1X PBS (Appendix 4).\n3.2.\nPerform an appropriate ELISA procedure with a specific monoclonal antibody to R. solanacearum.\n3.3.\nA positive ELISA test is achieved if the ELISA reading obtained from the culture is at least half that obtained for the positive control.\n4.\nPCR tests\n4.1.\nPrepare a suspension of approximately 106 cells per ml in molecular grade sterile water.\n4.2.\nHeat 100 µl of the cell suspension in closed tubes in a heating block or boiling waterbath at 100 °C for four minutes. The samples may then be stored at -16 to -24 °C until required.\n4.3.\nApply appropriate PCR procedures to amplify R. solanacearum-specific amplicons (e.g. Seal et al. (1993); Pastrik and Maiss (2000); Pastrik et al. (2002); Boudazin et al. (1999); Opina et al. (1997), Weller et al. (1999).\n4.4.\nA positive identification of R. solanacearum is achieved if the PCR amplicons are the same size and have the same restriction fragment length polymorphisms as for the positive control strain.\n5.\nFISH test\n5.1.\nPrepare a suspension of approximately 106 cells per ml in UPW.\n5.2.\nApply the FISH procedure (Section VI.A.7.) with at least 2 R. solanacearum-specific oligo-probes (Appendix 7).\n5.3.\nA positive FISH test is achieved if the same reactions are achieved from the culture and the positive control.\n6.\nFatty acid profiling (FAP)\n6.1.\nGrow the culture on trypticase soy agar (Oxoid) for 48 hours at 28 °C.\n6.2.\nApply an appropriate FAP procedure (Janse, 1991; Stead, 1992).\n6.3.\nA positive FAP test is achieved if the profile of the presumptive culture is identical to that of the positive control. The presence of characteristic fatty acids are 14:0 3OH, 16:0 2OH, 16:1 2OH and 18:1 2OH and absence of 16:0 3OH is highly indicative of a Ralstonia sp.\n7.\nStrain characterisation methods\nStrain characterisation using one of the following methods is recommended for each new case of isolation of R. solanacearum.\nInclude known reference strains where appropriate for each test performed (see Appendix 3).\n7.1.\nBiovar determination\nR. solanacearum is separated into biovars on the basis of the ability to utilise and/or oxidise three disaccharides and three hexose alcohols (Hayward, 1964 and Hayward et al., 1990). Growth media for the biovar test is described in Appendix 2. The test can be successfully performed by stab inoculating the media with pure cultures of R. solanacearum isolates and incubating at 28 °C. If the media are dispensed into sterile 96 well cell culture plates (200 µl per well) colour change from olive green to yellow can be observed within 72 hours, indicating a positive test result.\nBiovar\n1\n2\n3\n4\n5\nUtilisation of:\nMaltose\n–\n+\n+\n–\n+\nLactose\n–\n+\n+\n–\n+\nD (+) Cellobiose\n–\n+\n+\n–\n+\nMannitol\n–\n–\n+\n+\n+\nSorbitol\n–\n–\n+\n+\n–\nDulcitol\n–\n–\n+\n+\n–\nAdditional tests differentiate biovar 2 sub-phenotypes\nSee Lelliott and Stead (1987)\nBiovar 2A(Worldwide distribution)\nBiovar 2A(Found in Chile and Colombia)\nBiovar 2T(Found in tropical areas)\nUtilisation of trehalose\n–\n+\n+\nUtilisation of meso-inositol\n+\n–\n+\nUtilisation of D ribose\n–\n–\n+\nPectolytic activity\nlow\nlow\nhigh\n7.2.\nGenomic fingerprinting\nMolecular differentiation of strains in the R. solanacearum complex can be achieved using several techniques, including:\n7.2.1.\nRestriction fragment length polymorphism (RFLP) analysis (Cook et al., 1989).\n7.2.2.\nRepetitive sequence PCR using REP, BOX and ERIC primers (Louws et al., 1995; Smith et al., 1995).\n7.2.3.\nAmplified fragment length polymorphism (AFLP) analysis (Van der Wolf et al., 1998).\n7.3.\nPCR methods\nSpecific PCR primers (Pastrik et al, 2002; see Appendix 6) can be used to differentiate strains belonging to division 1 (biovars 3, 4 and 5) and division 2 (biovars 1, 2A and 2T) of R. solanacearum, as originally defined by RFLP analysis (Cook et al., 1989) and 16S rDNA sequencing (Taghavi et al., 1996).\nC.\nCONFIRMATION TEST\nThe pathogenicity test must be performed as final confirmation of a diagnosis of R. solanacearum and for assessment of virulence of cultures identified as R. solanacearum.\n1)) Prepare an inoculum of approximately 106 cells per ml from a 24 to 48 hour culture of the isolate to be tested and an appropriate positive control strain of R. solanacearum (e.g. NCPPB 4156 = PD 2762 = CFBP 3857; see Appendix 3).\n2)) Inoculate 5 to 10 susceptible tomato or eggplant seedlings at the third true leaf stage (see Section VI.A.9).\n3)) Incubate for up to two weeks at 25 to 28 °C and high relative humidity with appropriate watering to avoid waterlogging or drought stress. With pure cultures typical wilting should be obtained within 14 days. If after this period symptoms are not present, the culture cannot be confirmed as being a pathogenic form of R. solanacearum.\n4)) Observe for symptoms of wilting and/or epinasty, chlorosis and stunting.\n5)) Isolate from symptomatic plants by removing a section of stem about 2 cm above the inoculation point. Comminute and suspend in a small volume of sterile distilled water or 50 mM phosphate buffer (Appendix 4). Isolate from the suspension by dilution spreading or streaking on a suitable medium, preferably onto a selective medium (Appendix 2), incubate for 48 to 72 hours at 28 °C and observe the formation of colonies typical of R. solanacearum.\nAppendix 1\nLaboratories involved in optimisation and validation of protocols\nContact scientists: see web site http://forum.europa.eu.int/Public/irc/sanco/Home/main.\nLaboratory\nLocation\nCountry\nAgentur für Gesundheit und Ernährungssicherheit\nVienna and Linz\nAustria\nDepartement Gewasbescherming\nMerelbeke\nBelgium\nPlantedirektoratet\nLyngby\nDenmark\nCentral Science Laboratory\nYork\nEngland\nScottish Agricultural Science Agency\nEdinburgh\nScotland\nLaboratoire National de la Protection des Végétaux, Unité de Bactériologie\nAngers\nFrance\nLaboratoire National de la Protection des Végétaux, Station de Quarantaine de la Pomme de Terre\nLe Rheu\nFrance\nBiologische Bundesanstalt\nKleinmachnow\nGermany\nPflanzenschutzamt Hannover\nHannover\nGermany\nState Laboratory\nDublin\nIreland\nDipartimento di Scienze e Tecnologie Agroambientali\nBologna\nItaly\nRegione Veneto Unità Periferica per i Servizi Fitosanitari\nVerona\nItaly\nNederlandse Algemene Keuringsdienst\nEmmeloord\nNetherlands\nPlantenziektenkundige Dienst\nWageningen\nNetherlands\nDirecção-Geral de Protecção das Culturas\nLisbon\nPortugal\nCentro Diagnostico de Aldearrubia\nSalamanca\nSpain\nInstituto Valenciano de Investigaciones Agrarias\nValencia\nSpain\nSwedish University of Agricultural Sciences\nUppsala\nSweden\nAppendix 2\nMedia for isolation and culture of R. solanacearum\n(a)\nGeneral growth media\nNutrient Agar (NA)\nNutrient Agar (Difco)\n23,0 g\nDistilled water\n1,0 L\nDissolve ingredients and sterilise by autoclaving at 121 °C for 15 min.\nYeast Peptone Glucose Agar (YPGA)\nYeast extract (Difco)\n5,0 g\nBacto-Peptone (Difco)\n5,0 g\nD(+) Glucose (monohydrate)\n10,0 g\nBacto-Agar (Difco)\n15,0 g\nDistilled water\n1,0 L\nDissolve ingredients and sterilise by autoclaving at 121 °C for 15 minutes.\nSucrose Peptone Agar (SPA)\nSucrose\n20,0 g\nBacto-Peptone (Difco)\n5,0 g\nK2HPO4\n0,5 g\nMgSO4.7H2O\n0,25 g\nBacto-Agar (Difco)\n15,0 g\nDistilled water\n1,0 l\npH 7,2 – 7,4\nDissolve ingredients and sterilise by autoclaving at 121 °C for 15 minutes.\nKelman’s Tetrazolium Medium\nCasamino acids (Difco)\n1,0 g\nBacto-Peptone (Difco)\n10,0 g\nDextrose\n5,0 g\nBacto-Agar (Difco)\n15,0 g\nDistilled water\n1,0 l\nDissolve ingredients and sterilise by autoclaving at 121 °C for 15 minutes.\nCool to 50 °C and add a filter-sterilised solution of 2,3,5-triphenyl tetrazolium chloride (Sigma) to obtain a final concentration of 50 mg per l.\n(b)\nValidated selective growth media\nSMSA medium (Englebrecht, 1994 as modified by Elphinstone et al., 1996)\nBasal medium\nCasamino acids (Difco)\n1,0 g\nBacto-Peptone (Difco)\n10,0 g\nGlycerol\n5,0 ml\nBacto-Agar (Difco); see Note 2.\n15,0 g\nDistilled water\n1,0 L\nDissolve ingredients and sterilise by autoclaving at 121 °C for 15 minutes.\nCool to 50 °C and add filter-sterilised aqueous stock solutions of the following ingredients to obtain the specified final concentrations:\nCrystal Violet (Sigma)\n5 mg per l\nPolymixin-B-Sulphate (Sigma P-1004)\n600 000 U (approximately 100 mg) per l\nBacitracin (Sigma B-0125)\n1 250 U (approximately 25 mg) per l\nChloramphenicol (Sigma C-3175)\n5 mg per l\nPenicillin-G (Sigma P-3032)\n825 U (approximately 0,5 mg) per l\n2,3,5-triphenyl tetrazolium chloride (Sigma)\n50 mg per l\nNote:\n1.\nUse of reagents other than those specified above may affect growth of R. solanacearum.\n2.\nOxoid Agar #1 can be used in place of Bacto-Agar (Difco). In this case growth of R. solanacearum will be slower, although growth of competing saprophytes may also be reduced. Typical colonies of R. solanacearum may take 1 to 2 days longer to form and the red colouration may be lighter and more diffuse than on Bacto-Agar.\n3.\nIncreasing bacitracin concentration to 2 500 U per l may reduce populations of competing bacteria without affecting growth of Ralstonia solanacearum.\nStore media and stock solutions of antibiotics at 4 °C in the dark and use within one month.\nPlates should be free from surface condensation before use.\nAvoid excess drying of plates.\nQuality control should be performed after preparation of each new batch of medium by plating a suspension of a reference culture of R. solanacearum (see Appendix 3) and observing formation of typical colonies after incubation at 28 °C for two to five days.\n(c)\nValidated enrichment media\nSMSA Broth (Elphinstone et al., 1996)\nPrepare as for SMSA selective agar medium but omit Bacto-Agar and 2,3,5-tetrazolium chloride.\nModified Wilbrink broth (Caruso et al., 2002)\nSucrose\n10 g\nProteose peptone\n5 g\nK2HPO4\n0,5 g\nMgSO4\n0,25 g\nNaNO3\n0,25 g\nDistilled water\n1 l\nSterilise by autoclaving at 121 °C for 15 minutes and cool to 50 °C\nAdd antibiotic stock solutions as for SMSA broth.\nAppendix 3\nA.\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 103 to 104 and 104 to 106 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 106 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 × 108 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 106 and 104 cells/ml of the positive controls and not in any of the negative controls.\nAppendix 4\nBuffers for test procedures\nGeneral: Unopened sterilised buffers can be stored for up to one year\n1.\nBuffers for extraction procedure\n1.1.\nExtraction buffer (50 mM phosphate buffer, pH 7,0)\nThis buffer is used for extraction of the bacterium from plant tissues by homogenisation or shaking.\nNa2HPO4 (anhydrous)\n4,26 g\nKH2PO4\n2,72 g\nDistilled water\n1.00 l\nDissolve ingredients, check pH and sterilise by autoclaving at 121 °C for 15 min.\nAdditional components may be useful as follows:\nFor use with homogenisation extraction method\nPurpose\nQuantity (per l)\nLubrol flakes\nDeflocculant\n0,5 g\nDC silicone antifoam\nAnti-foam agent\n1,0 ml\nTetrasodium pyrophosphate\nAnti-oxidant\n1,0 g\nPolyvinylpyrrolidone-40000 (PVP-40)\nBinding of PCR inhibitors\n50 g\n1.2.\nPellet buffer (10 mM phosphate buffer, pH 7,2)\nThis buffer is used for resuspension and dilution of potato tuber heel-end core extracts following concentration to a pellet by centrifugation.\nNa2HPO4.12H2O\n2,7 g\nNaH2PO4.2H2O\n0,4 g\nDistilled water\n1,0 l\nDissolve ingredients, check pH and sterilise by autoclaving at 121 °C for 15 minutes.\n2.\nBuffers for the IF test\n2.1.\nIF-Buffer (10 mM phosphate buffered saline (PBS), pH 7.2)\nThis buffer is used for dilution of antibodies\nNa2HPO4.12H2O\n2,7 g\nNaH2PO4.2H2O\n0,4 g\nNaCL\n8,0 g\nDistilled water\n1,0 l\nDissolve ingredients, check pH and sterilise by autoclaving at 121 °C for 15 minutes.\n2.2.\nIF-buffer-Tween\nThis buffer is used to wash slides.\nAdd 0,1 % Tween 20 to the IF buffer.\n2.3.\nPhosphate buffered glycerol, pH 7,6\nThis buffer is used as a mountant fluid on the windows of IF slides to enhance fluorescence.\nNa2HPO4.12H2O\n3,2 g\nNaH2PO4.2H2O\n0,15 g\nGlycerol\n50 ml\nDistilled water\n100 ml\nAnti-fading mountant solutions are commercially available e.g. Vectashield® (Vector Laboratories) or Citifluor® (Leica).\n3.\nBuffers for the Indirect ELISA test\n3.1.\nDouble strength coating buffer, pH 9,6.\nNa2CO3\n6,36 g\nNaHCO3\n11,72 g\nDistilled water\n1,00 l\nDissolve ingredients, check pH and sterilise by autoclaving at 121 °C for 15 minutes.\nSodium sulphite (0.2 %) may be added as antioxidant if required to prevent build up of oxidised aromatic compounds.\n3.2.\n10X Phosphate buffered saline (PBS), pH 7,4\nNaCL\n80,0 g\nKH2PO4\n2,0 g\nNa2HPO4.12H2O\n29,0 g\nKCl\n2,0 g\nDistilled water\n1,0 L\n3.3.\nPBS-Tween\n10X PBS\n100 ml\n10 % Tween 20\n5 ml\nDistilled water\n895 ml\n3.4.\nBlocking (antibody) buffer (must be freshly prepared).\n10X PBS\n10,0 ml\nPolyvinylpyrrolidone-44000 (PVP-44)\n2,0 g\n10 % Tween 20\n0,5 ml\nMilk powder\n0,5 g\nDistilled water\nmake up to 100 ml\n3.5.\nAlkaline phosphatase substrate solution, pH 9,8\nDiethanolamine\n97 ml\nDistilled water\n800 ml\nMix and adjust to pH 9,8 with concentrated HCl.\nMake up to 1 L with distilled water.\nAdd 0,2 g MgCl2.\nDissolve 2 phosphatase substrate 5 mg tablets (Sigma) per 15 ml of solution.\n4.\nBuffers for DASI ELISA test\n4.1.\nCoating buffer, pH 9,6\nNa2CO3\n1,59 g\nNaHCO3\n2,93 g\nDistilled water\n1 000 ml\nDissolve ingredients and check pH 9,6\n4.2.\n10X Phosphate saline buffer(PBS) pH 7,2 to 7,4\nNaCl\n80,0 g\nNaH2PO4.2 H2O\n4,0 g\nNa2HPO4.12H2O\n27,0 g\nDistilled water\n1 000 ml\n4.3.\nPBS-Tween\n10X PBS\n50 ml\n10 % Tween 20\n5 ml\nDistilled water\n950 ml\n4.4.\nSubstrate buffer, pH 9,8\nDiethanolamine\n100 ml\nDistilled water\n900 ml\nMix and adjust to pH 9,8 with concentrated HCl.\nAppendix 5\nDetermination of contamination level in IF and FISH tests\n1.. Count the mean number of typical fluorescent cells per field of view (c).\n2.. Calculate the number of typical fluorescent cells per microscope slide window (C).\nC = c × S/s\nwhere\nS\n=\nsurface area of window of multispot slide\nand\ns\n=\nsurface area of objective field\ns = πi2/4G2K2\nwhere\ni\n=\nfield coefficient (varies from 8 to 24 depending upon ocular type)\nK\n=\ntube coefficient (1 or 1,25)\nG\n=\nmagnification of objective (100 ×, 40 × etc.).\n3.. Calculate the number of typical fluorescent cells per ml of re-suspended pellet (N).\nN = C × 1 000/y × F\nwhere\ny\n=\nvolume of re-suspended pellet on each window\nand\nF\n=\ndilution factor of re-suspended pellet.\nAppendix 6\nValidated PCR protocols and reagents\nNB: Preliminary testing should permit reproducible detection of 103 to 104 cells of R. solanacearum per ml of sample extract.\nPreliminary testing should also show no false positive results with a panel of selected bacterial strains (see Appendix 3).\n1.\nPCR protocol of Seal et al. (1993)\n1.1.\nOligonucleotide primers\nForward primer OLI-1\n5′-GGG GGT AGC TTG CTA CCT GCC-3′\nReverse primer Y-2\n5′-CCC ACT GCT GCC TCC CGT AGG AGT-3′\nExpected amplicon size from R. solanacearum template DNA = 288 bp\n1.2.\nPCR reaction mix\nMethod was validated using Taq polymerase from Perkin Elmer (AmpliTaq) and Gibco BRL.\nReagent\nQuantity per reaction\nFinal concentration\nSterile UPW\n17,65 µl\n10X PCR buffer (15 mM MgCl2)\n2,5 µl\n1X (1,5 mM MgCl2)\ndNTP mix (20 mM)\n0,25 µl\n0,2 mM\nPrimer OLI-1 (20 µM)\n1,25 µl\n1µM\nPrimer Y-2 (20 µM)\n1,25 µl\n1µM\nTaq polymerase (5U/µl)\n0,1 µl\n0,5 U\nSample volume\n2,0 µl\nTotal volume\n25 µl\n1.3.\nPCR reaction conditions\nRun the following programme:\n1 cycle of:\n(i)\n2 minutes at 96 °C (denaturation of template DNA)\n35 cycles of:\n(ii)\n20 seconds at 94 °C (denaturation of template DNA)\n(iii)\n20 seconds at 68 °C (annealing of primers)\n(iv)\n30 seconds at 72 °C (extension of copy)\n1 cycle of:\n(v)\n10 minutes at 72 °C (final extension)\n(vi)\nhold at 4 °C.\nNB: This programme was optimised for use with a Perkin Elmer 9600 thermal cycler. Modification of the duration steps of cycles (ii), (iii) and (iv) may be required for use with other models.\n1.4.\nRestriction enzyme analysis of amplicon.\nPCR products amplified from R. solanacearum DNA produce a distinctive restriction fragment length polymorphism with enzyme Ava II after incubation at 37 °C.\n2.\nPCR protocol of Pastrik and Maiss (2000)\n2.1.\nOligonucleotide primers\nForward primer Ps-1\n5′- agt cga acg gca gcg ggg g -3′\nReverse primer Ps-2\n5′- ggg gat ttc aca tcg gtc ttg ca -3′\nExpected amplicon size from R. solanacearum template DNA = 553 bp.\n2.2.\nPCR reaction mix\nMethods were validated using Taq polymerase from Perkin Elmer (AmpliTaq) and Gibco BRL.\nN.B. Originally optimised for MJ Research PTC 200 thermocycler with Gibco Taq Polymerase.\nPerkin Elmer AmpliTaq and buffer can also be used at the same concentrations.\nReagent\nQuantity per reaction\nFinal concentration\nSterile UPW\n16,025 µl\n10X PCR buffer\n2,5 µl\n1X (1,5 mM MgCl2)\nBSA (fraction V) (10 %)\n0,25 µl\n0,1 %\nd-nTP mix (20 mM)\n0,125 µl\n0,1 mM\nPrimer Ps-1 (10 µM)\n0,5 µl\n0,2 µM\nPrimer Ps-2 (10 µM)\n0,5 µl\n0,2 µM\nTaq polymerase (5U/µl)\n0,1 µl\n0,5 U\nSample volume\n5,0 µl\nTotal volume:\n25,0 µl\n2.3.\nPCR reaction conditions\nRun the following programme:\n1 cycle of:\n(i)\n5 minutes at 95 °C (denaturation of template DNA)\n35 cycles of:\n(ii)\n30 seconds at 95 °C (denaturation of template DNA)\n(iii)\n30 seconds at 68 °C (annealing of primers)\n(iv)\n45 seconds at 72 °C (extension of copy)\n1 cycle of:\n(v)\n5 minutes at 72 °C (final extension)\n(vi)\nhold at 4 °C.\nNB: This programme is optimised for use with an MJ Research PTC 200 thermal cycler. Modification of the duration steps of cycles (ii), (iii) and (iv) may be required for use with other models.\n2.4.\nRestriction enzyme analysis of amplicon.\nPCR products amplified from R. solanacearum DNA produce a distinctive restriction fragment length polymorphism with enzyme Taq I after incubation at 65 °C for 30 minutes. The restriction fragments obtained from R. solanacearum-specific fragment are 457 bp and 96 bp in size.\n3.\nMultiplex PCR protocol with internal PCR control (Pastrik et al., 2002)\n3.1.\nOligonucleotide primers\nForward primer RS-1-F\n5′- ACT AAC GAA GCA GAG ATG CAT TA -3′\nReverse primer RS-1-R\n5′- CCC AGT CAC GGC AGA GAC T -3′\nForward primer NS-5-F\n5′- AAC TTA AAG GAA TTG ACG GAA G -3′\nReverse primer NS-6-R\n5′- GCA TCA CAG ACC TGT TAT TGC CTC -3′\nExpected amplicon size from R. solanacearum template DNA = 718 bp (RS-primer set)\nExpected amplicon size from the 18S rRNA internal PCR control = 310 bp (NS-primer set).\n3.2.\nPCR reaction mix\nMethods were validated using Taq polymerase from Perkin Elmer (AmpliTaq) and Gibco BRL.\nConcentration of primers NS-5-F and NS-6-R were optimised for potato heel end core extraction using the homogenisation method and DNA purification according to Pastrik (2000) (see Section VI.A.6.1.a.). Re-optimisation of reagent concentrations will be required if extraction by shaking or other DNA isolation methods are used.\nReagent\nQuantity per reaction\nFinal concentration\nSterile UPW\n12,625 µl\n10X PCR buffer (15 mM MgCl2)\n2,5 µl\n1X (1,5 mM MgCl2)\nBSA (fraction V) (10 %)\n0,25 µl\n0,1 %\nd-nTP mix (20 mM)\n0,125 µl\n0,1 mM\nPrimer RS-1-F (10 µM)\n2,0 µl\n0,8 µM\nPrimer RS-1-R (10 µM)\n2,0 µl\n0,8 µM\nPrimer NS-5-F (10 µM)\n0,15 µl\n0,06 µM\nPrimer NS-6-R (10 µM)\n0,15 µl\n0,06 µM\nTaq polymerase (5 U/µl)\n0,2 µl\n1,0 U\nSample volume\n5,0 µl\nTotal volume:\n25,0 µl\n3.3.\nPCR reaction conditions\nRun the following programme:\n1 cycle of:\n(i)\n5 minutes at 95 °C (denaturation of template DNA)\n35 cycles of:\n(ii)\n30 seconds at 95 °C (denaturation of template DNA)\n(iii)\n30 seconds at 58 °C (annealing of primers)\n(iv)\n45 seconds at 72 °C (extension of copy)\n1 cycle of:\n(v)\n5 minutes at 72 °C (final extension)\n(vi)\nhold at 4 °C.\nNB: This programme is optimised for use with an MJ Research PTC 200 thermal cycler. Modification of the duration steps of cycles (ii), (iii) and (iv) may be required for use with other models.\n3.4.\nRestriction enzyme analysis of amplicon.\nPCR products amplified from R. solanacearum DNA produce a distinctive restriction fragment length polymorphism with enzyme Bsm I or an Isoschizomere (e.g. Mva 1269 I) after incubation at 65 °C for 30 minutes.\n4.\nR. solanacearum biovar-specific PCR protocol (Pastrik et al., 2001)\n4.1.\nOligonucleotide primers\nForward primer Rs-1-F\n5′- ACT AAC GAA GCA GAG ATG CAT TA -3′\nReverse primer Rs-1-R\n5′- CCC AGT CAC GGC AGA GAC T -3′\nReverse primer Rs-3-R\n5′- TTC ACG GCA AGA TCG CTC -3′\nExpected amplicon size from R. solanacearum template DNA:\nwith Rs-1-F/Rs-1-R = 718 bp\nwith Rs-1-F/Rs-3-R = 716 bp.\n4.2.\nPCR reaction mix\n(a) Biovar 1/2-specific PCR\nMethods have been validated using Taq polymerase from Perkin Elmer (AmpliTaq) and Gibco BRL.\nReagent\nQuantity per reaction\nFinal concentration\nSterile UPW\n12,925 µl\n10X PCR Buffer\n2,5 µl\n1X (1,5 mM MgCl2)\nBSA (fraction V) (10 %)\n0,25 µl\n0,1 %\nd-NTP mix (20mM)\n0,125 µl\n0,1 mM\nPrimer Rs-1-F (10 µM)\n2 µl\n0,8 µM\nPrimer Rs-1-R (10 µM)\n2 µl\n0,8 µM\nTaq polymerase (5U/µl)\n0,2 µl\n1 U\nSample volume\n5,0 µl\nTotal volume\n25,0 µl\n(b) Biovar 3/4/5-specific PCR\nMethods have been validated using Taq polymerase from Perkin Elmer (AmpliTaq) and Gibco BRL.\nReagent\nQuantity per reaction\nFinal concentration\nSterile UPW\n14,925 µl\n10X PCR Buffer\n2,5 µl\n1X (1,5 mM MgCl2)\nBSA (fraction V) (10 %)\n0,25 µl\n0,1 %\ndNTP mix (20 mM)\n0,125 µl\n0,1 mM\nPrimer Rs-1-F (10 µM)\n1 µl\n0,4 µM\nPrimer Rs-3-R (10 µM)\n1 µl\n0,4 µM\nTaq polymerase (5 U/µl)\n0,2 µl\n1 U\nSample volume\n5,0 µl\nTotal volume\n25,0 µl\n4.3.\nPCR reaction conditions\nRun the following programme for both biovar 1/2- and biovar 3/4/5-specific reactions:\n1 cycle of:\n(i)\n5 minutes at 95 °C (denaturation of template DNA)\n35 cycles of:\n(ii)\n30 seconds at 95 °C (denaturation of template DNA)\n(iii)\n30 seconds at 58 °C (annealing of primers)\n(iv)\n45 seconds at 72 °C (extension of copy)\n1 cycle of:\n(v)\n5 minutes at 72 °C (final extension)\n(vi)\nhold at 4 °C.\nNB: This programme was optimised for use with an MJ Research PTC 200 thermal cycler. Modification of the duration steps of cycles (ii), (iii) and (iv) may be required for use with other models.\n4.4.\nRestriction enzyme analysis of amplicon.\nPCR products amplified from R. solanacearum DNA using primers Rs-1-F and Rs-1-R produce a distinctive restriction fragment length polymorphism with enzyme Bsm I or an Isoschizomere (e.g. Mva 1269 I) after incubation at 65 °C for 30 minutes. PCR products amplified from R. solanacearum DNA using primers Rs-1-F and Rs-3-R have no restriction sites.\n5.\nPreparation of the loading buffer\n5.1.\nBromphenol blue (10 %-stock solution)\nBromphenol blue\n5 g\nDistilled water (bidest)\n50 ml\n5.2.\nLoading buffer\nGlycerol (86 %)\n3,5 ml\nBromphenol blue (5,1)\n300 µl\nDistilled Water (bidest)\n6,2 ml\n6.\n10X Tris Acetate EDTA (TAE) buffer, pH 8.0\nTris buffer\n48,40 g\nGlacial acetic acid\n11,42 ml\nEDTA (disodium salt)\n3,72 g\nDistilled water\n1,00 L\nDilute to 1X before use.\nAlso commercially available (e.g. Invitrogen or equivalent).\nAppendix 7\nValidated reagents for FISH test\n1.\nOligo-probes\nR. solanacearum-specific probe OLI-1-CY3: 5′-ggc agg tag caa gct acc ccc-3′\nNon-specific eubacterial probe EUB-338-FITC: 5′-gct gcc tcc cgt agg agt-3′\n2.\nFixative solution\n(WARNING! THE FIXATIVE CONTAINS PARAFORMALDEHYDE WHICH IS TOXIC. WEAR GLOVES AND DO NOT INHALE. IT IS ADVISABLE TO WORK IN A FUME CUPBOARD.)\n(i) Heat 9 ml molecular grade water (e.g. Ultra pure water (UPW)) to about 60 °C and add 0,4 g paraformaldehyde. Paraformaldehyde dissolves after adding 5 drops of 1N NaOH and stirring with a magnetic stirrer.\n(ii) Adjust pH to 7.0 by addition of 1ml of 0,1M phosphate buffer (PB; pH 7,0) and 5 drops of 1N HCl. Check pH with indicator strips and adjust if necessary with HCl or NaOH. (WARNING! DO NOT USE A PH METER IN SOLUTIONS WITH PARAFORMALEDHYDE.)\n(iii) Filter the solution through a 0,22 µm membrane filter and keep dust-free at 4 °C until further use.\n3.\n3X Hybmix\nNaCl\n2,7 M\nTris-HCl\n60 mM (pH 7,4)\nEDTA (filter sterilised and autoclaved)\n15 mM\nDilute to 1X as required.\n4.\nHybridisation solution\n1X Hybmix\nSodium dodecyl sulphate (SDS)\n0,01 %\nFormamide\n30 %,\nprobe EUB 338\n5 ng/μl\nprobe OLI-1 or OLI-2\n5 ng/μl\nPrepare quantities of hybridisation solution according to the calculations in Table 1. For each slide (containing 2 different samples in duplicate) 90 μl hybridisation solution is required. IMPORTANT: FORMAMIDE IS VERY TOXIC SO WEAR GLOVES AND TAKE NECESSARY SAFETY PRECAUTIONS!\nTable 1\nSuggested quantities for preparation of hybridisation mix\nNB: Store all solutions containing light sensitive oligo-probes in the dark at -20 °C.\nProtect from direct sunlight or electric light during use.\nNumber of slides:\n1\n4\n6\n8\n10\nSterile UPW\n23,1\n92,4\n138,6\n184,8\n231,0\n3x hybmix\n30,0\n120,0\n180,0\n240,0\n300,0\n1 % SDS\n0,9\n3,6\n5,4\n7,2\n9,0\nFormamide\n27,0\n108,0\n162,0\n216,0\n270,0\nProbe EUB 338 (100 ng/μl)\n4,5\n18,0\n27,0\n36,0\n45,0\nProbe OLI-1 or OLI-2 (100 ng/μl)\n4,5\n18,0\n27,0\n36,0\n45,0\nTotal volume (μl)\n90,0\n360,0\n540,0\n720,0\n900,0\n5.\n0,1M Phosphate buffer, pH 7,0\nNa2HPO4\n8,52 g\nKH2PO4\n5,44 g\nDistilled water\n1,00 L\nDissolve ingredients, check pH and sterilise by autoclaving at 121 °C for 15 minutes.\nAppendix 8\nEggplant and tomato culture\nSow seeds of tomato (Lycopersicon esculentum) or eggplant (Solanum melongena) in pasteurised seed compost. Transplant seedlings with fully expanded cotyledons (10 to 14 days) into pasteurised potting compost.\nEggplants or tomatoes should be grown in a glasshouse with the following environmental conditions prior to inoculation:\nDay length\n14 hours or natural day length if greater;\nTemperature\nday 21 to 24 °C,\nnight14 to 18 °C.\nSusceptible variety of tomato\n“‘Moneymaker’”\nSusceptible variety of eggplant\n“‘Black Beauty’”\nSuppliers\nsee website http://forum.europa.eu.int/Public/irc/sanco/Home/main\nREFERENCES\n1.\nAmann, R.I., L. Krumholz and D.A. Stahl. 1990. Fluorescent-oligonucleotide probing of whole cells for determinative, phylogenetic and environmental studies in microbiology. J. Bacteriol. 172: 762-770.\n2.\nAnon. 1998. Council Directive 98/57/EC of 20 July 1998 on the control of Ralstonia solanacearum (Smith) Yabuuchi et al. Official Journal of the European Communities L235, 1-39.\n3.\nBoudazin, G., A.C. Le Roux, K. Josi, P. Labarre and B. Jouan. 1999. Design of division specific primers of Ralstonia solanacearum and application to the identification of European isolates. European Journal of Plant Pathology 105; 373-380.\n4.\nCaruso, P., Gorris, M.T., Cambra, M., Palomo, J.L., Collar, J and Lopez, M.M. 2002. Enrichment Double-Antibody Sandwich Indirect Enzyme-Linked Immunosorbent Assay That Uses a Specific Monoclonal Antibody for sensitive Detection of Ralstonia solanacearum in Asymptomatic Potato Tubers. Applied and Environmental Microbiology, 68, 3634-3638.\n5.\nCook, D., Barlow, E. and Sequeira, L. 1989. Genetic diversity of Pseudomonas solanacearum: detection of restriction fragment length polymorphisms with DNA probes that specify virulence and the hypersensitive response. Molecular Plant-Microbe Interactions 1:113-121.\n6.\nElphinstone, J.G., Hennessy, J., Wilson, J.K. and Stead, D.E. 1996. Sensitivity of detection of Ralstonia solanacearum in potato tuber extracts. EPPO Bulletin 26; 663-678.\n7.\nEnglebrecht, M.C. (1994) Modification of a semi-selective medium for the isolation and quantification of Pseudomonas solanacearum. In: A.C. Hayward (ed.) Bacterial Wilt Newsletter 10, 3-5. Australian Centre for International Agricultural Research, Canberra, Australia.\n8.\nHayward, A.C. 1964. Characteristics of Pseudomonas solanacearum. Journal of Applied Bacteriology 27; 265-277.\n9.\nHayward, A.C., El-Nashaar, H.M., Nydegger, U. and De Lindo, L. 1990. Variation in nitrate metabolism in biovars of Pseudomonas solanacearum. Journal of Applied Bacteriology 69; 269-280.\n10.\nIto, S., Y. Ushijima, T. Fujii, S. Tanaka, M. Kameya-Iwaki, S. Yoshiwara and F. Kishi. 1998. Detection of viable cells of Ralstonia solanacearum in soil using a semi-selective medium and a PCR technique. J. Phytopathology 146; 379-384.\n11.\nJanse, J.D. (1988) A detection method for Pseudomonas solanacearum in symptomless potato tubers and some data on its sensitivity and specificity. Bulletin OEPP/EPPO Bulletin 18, 343-351.\n12.\nJanse, J.D. 1991. Infra- and intra-specific classification of Pseudomonas solanacaerum strains using whole cell fatty-acid analysis. Systematic and Applied Microbiology 14; 335-345.\n13.\nKelman, A. 1954. The relationship of pathogenicity of Pseudomonas solanacearum to colony appearance on a tetrazolium medium. Phytopathology 44; 693-695.\n14.\nKlement Z.; Rudolph, K and D.C. Sands, 1990. Methods in Phytobacteriology. Akadémiai Kiadó, Budapest, 568 pp.\n15.\nLelliott, R.A. and Stead, D.E. 1987. Methods for the diagnosis of bacterial diseases of plants. Blackwell scientific Publications Ltd., Oxford. 216 pp.\n16.\nLopez, M.M., Gorris, M.T., Llop, P., Cubero, J., Vicedo, B., Cambra, M., 1997. Selective enrichment improves selective isolation, serological and molecular detection of plant pathogenic bacteria. In: H.W. Dehne et al., (eds). Klewer Academic Publishers. pp. 117-121.\n17.\nLouws, F.J., Fulbright, D.W., Stephens, C.T. and De Bruijn, F.J., 1994. Specific genomic fingerprints of phytopathogenic Xanthomonas and Pseudomonas pathovars and strains generated with repetitive sequences and PCR. Applied and Environmental Microbiology, 60, 2286-2295.\n18.\nLouws, F.J., Fulbright, D.W., Stephens, C.T. and De Bruijn, F.J. 1995. Differentiation of genomic structure by rep-PCR fingerprinting to rapidly classify Xanthomonas campestris pv. vesicatoria. Phytopathology 85; 528-536.\n19.\nOpina, N., F. Tavner, G. Holloway, J.-F Wang, T.-H Li, R. Maghirang, M. Fegan, A.C. Hayward, V. Krishnapillai, W.F. Hong, B.W. Holloway, J.N. Timmis. 1997. A novel method for development of species and strain-specific DNA probes and PCR primers for identifying Burkholderia solanacearum (formerly Pseudomonas solanacearum). As Pac. J. Mol. Biol. Biotechnol. 5; 19-33.\n20.\nPastrik, K.H. and Maiss, E. 2000. Detection of R. solanacearum in potato tubers by polymerase chain reaction. J. Phytopathology 148; 619-626.\n21.\nPastrik, K.H., Elphinstone, J.G. and Pukall, R. 2002. Sequence analysis and detection of Ralstonia solanacearum by multiplex PCR amplification of 16S-23S ribosomal intergenic spacer region with internal positive control. European Journal of Plant Pathology 108, 831-842.\n22.\nRobinson-Smith, A., Jones, P., Elphinstone, J.G. and Forde, S.M.D. (1995) Production of antibodies to Pseudomonas solanacearum, the causative agent of bacterial wilt. Food and Agricultural Immunology 7, 67-79.\n23.\nSchaad, W. 2001. Laboratory guide for identification of plant pathogenic bacteria. Schaad [Hrsg.]. - 3. ed.; St. Paul, Minnesota: 373 pp.\n24.\nSeal, S.E., L.A. Jackson, J.P.W. Young, and M.J. Daniels. 1993. Detection of Pseudomonas solanacearum, Pseudomonas syzygii, Pseudomonas pickettii and Blood Disease Bacterium by partial 16S rRNA sequencing: construction of oligonucleotide primers for sensitive detection by polymerase chain reaction. J. Gen. Microbiol. 139: 1587-1594.\n25.\nSmith, J.J., Offord, L.C., Holderness, M. and Saddler, G.S. 1995. Genetic diversity of Burkholderia solanacearum (synonym Pseudomonas solanacearum) race 3 in Kenya. Applied and Environmental Microbiology 61; 4262-4268.\n26.\nStead, D.E. 1992. Grouping of plant pathogenic and some other Pseudomonas spp. using cellular fatty-acid profiles. International Journal of Systematic Bacteriology 42; 281-295.\n27.\nTaghavi, M., Hayward, A.C., Sly, L.I., Fegan, M. 1996. Analysiss of the phylogenetic relationships of strains of Burkholderia solanacearum, Pseudomonas syzygii, and the blood disease bacterium of banana based on 16S rRNA gene sequences. International Journal of Systematic Bacteriology 46; 10-15.\n28.\nVan Der Wolf, J.M., Bonants, P.J.M., Smith, J.J., Hagenaar, M., Nijhuis, E., Van Beckhoven, J.R.C., Saddler, G.S., Trigalet, A., Feuillade, R. 1998. Genetic diversity of Ralstonia solanacearum Race 3 in Western Europe as determined by AFLP, RC-PFGE and rep-PCR. In: Prior, P., Allen, C. and Elphinstone, J. (eds.) Bacterial wilt disease: Molecular and Ecological Aspects. Springer (Berlin) pp. 44-49.\n29.\nWeller, S.A., Elphinstone, J.G., Smith, N., Stead, D.E. and Boonham, N. 1999. Detection of Ralstonia solanacearum strains using an automated and quantitative flourogenic 5’ nuclease TaqMan assay. Applied and Environmental Microbiology 66; 2853-2858.\n30.\nWullings, B.A., A.R. van Beuningen, J.D. Janse and A.D.L. Akkermans. 1998. Detection of R. solanacearum, which causes brown rot of potato, by fluorescent in situ hybridization with 23s rRNA-targeted probes. Appl. Environ. Microbiol. 64: 4546-4554.\nANNEX III\n1.. For each suspected occurrence for which a positive result in the screening test(s) has been identified according to, for the listed plant material and for all other cases, the relevant methods set out in Annex II, and confirmation or refutation by completion of the said methods is awaited, there should be retention and appropriate conservation of:\nall tubers sampled and, wherever possible, all plants sampled,\nany remaining extract and additional prepared material for the screening test(s) e.g. immunofluorescence slides,\nand\nall relevant documentation,\nuntil the completion of the said methods.\nRetention of the tubers will enable variety testing to be undertaken where appropriate.\n2.. In the case of positive confirmation of the organism, there should be retention and appropriate conservation of:\nthe material specified in paragraph 1,\na sample of the infected tomato or eggplant material inoculated with the tuber or plant extract, where appropriate,\nand\nthe isolated culture of the organism,\nuntil at least one month after the notification procedure under Article 5(2).\nANNEX IV\nThe elements in the investigation referred to in Article 5(1)(a)(i) shall include where relevant:\n(i) places of production,\ngrowing or having grown, potatoes which are clonally related to potatoes found to be infected with the organism,\ngrowing or having grown, tomatoes which are from the same source as tomatoes found to be infected with the organism,\ngrowing or having grown, potatoes or tomatoes which have been placed under official control because of the suspected occurrence of the organism,\ngrowing or having grown, potatoes which are clonally related to potatoes that have been grown on places of production found to be infested with the organism,\ngrowing potatoes or tomatoes and located in the neighbourhood of infested places of production, including such places of production sharing production equipment and facilities directly or through a common contractor,\nusing surface water for irrigation or spraying from any source confirmed or suspected to be contaminated with the organism,\nusing surface water for irrigation or spraying from a source used in common with places of production confirmed or suspected to be infested with the organism,\nflooded or have been flooded with surface water confirmed or suspected to be contaminated with the organism;\nand\n(ii) surface water used for irrigation or spraying of, or which has flooded field(s) or place(s) of production confirmed to be infested with the organism.\nANNEX V\n1.. The elements to be considered in the determination of the extent of probable contamination under Article 5(1)(a)(iii) and 5(1)(c)(iii), shall include:\nthe listed plant material grown at a place of production designated as contaminated under Article 5(1)(a)(ii),\nplace(s) of production with some production link to the listed plant material designated as contaminated under Article 5(1)(a)(ii), including those sharing production equipment and facilities directly or through a common contractor,\nthe listed plant material produced in the place(s) of production referred to in the previous indent, or present in such place(s) of production during the period when the listed plant material designated as contaminated under Article 5(1)(a)(ii), was present on the place of production referred to in the first indent,\npremises handling the listed plant material from the places of production referred to in the above indents,\nany machinery, vehicle, vessel, store, or units thereof, and any other objects including packaging material, that may have come into contact with the listed plant material designated as contaminated under Article 5(1)(a)(ii),\nany of the listed plant material stored in, or in contact with, any of the structures or objects listed in the previous indent, prior to the cleansing and disinfection of such structures and objects,\nas a result of the investigation and testing under Article 5(1)(a)(i), in the case of potato, those tubers or plants with a sister or parental clonal relationship to, and in the case of tomato, those plants with the same source as, the listed plant material designated to be contaminated under Article 5(1)(a)(ii) and for which, although they may have tested negative for the organism, it appears that contamination is probable through a clonal link. Variety testing may be undertaken to verify the identity of the contaminated and clonally related tubers or plants,\nplace(s) of production of the listed plant material referred to in the previous indent,\nplace(s) of production of the listed plant material using water for irrigation or spraying which has been designated as contaminated under Article 5(1)(c)(ii),\nlisted plant material produced on fields flooded with surface water confirmed to be contaminated.\n2.. The elements to be considered in the determination of the possible spread under Article 5(1)(a)(iv) and 5(1)(c)(iii) shall include:\n(i) in cases under Article 5(1)(a)(iv),\nthe proximity of other places of production growing the listed plant material,\nthe common production and use of seed potato stocks,\nplaces of production using surface water for irrigation or spraying of listed plant material in cases where there is or has been risk of surface water run-off from, or flooding of, place(s) of production designated to be contaminated under Article 5(1)(a)(ii).\n(ii) in cases where surface water has been designated as contaminated under Article 5(1)(c)(ii):\nplace(s) of production producing listed plant material adjacent to, or at risk from flooding by, the surface water designated as contaminated,\nany discrete irrigation basin associated with the surface water designated as contaminated,\nwater bodies connected with the surface water designated as contaminated, taking into account:\nthe direction and rate of flow of the water designated as contaminated,\nthe presence of wild solanaceous host plants.\n3.. The notification referred to in the first subparagraph of Article 5(2) shall be provided as follows:\nimmediately after the presence of the organism has been confirmed by laboratory testing, using the methods set out in Annex II, at least:\nfor potatoes,\n(a) the variety name of the lot,\n(b) the type (ware, seed, etc.) and where applicable the seed category,\nfor tomato plants, the variety name of the lot and, where applicable, the category.\nwithout prejudice to the requirements for notification of a suspected occurrence in Article 4 (3), the Member State in which the occurrence has been confirmed shall, when there is a risk of contamination of listed plant material from or into another Member States(s), immediately notify the Member State(s) concerned of information needed to comply with Article 5(3), such as:\n(a) the variety name of the potato or tomato lot,\n(b) the name and address of the consignor and the consignee,\n(c) the date of delivery of the potato or tomato lot,\n(d) the size of the potato or tomato lot delivered,\n(e) a copy of the plant passport or at least the plant passport number where appropriate, or where appropriate the registration number of the grower or merchant, and a copy of the delivery notice.\nThe Commission shall be notified immediately when such information has been provided.\n4.. The details of the additional notification referred to in the second subparagraph of Article 5(2) shall be provided as follows:\nAfter all investigations have been finalised, for each case:\n(a) the date that contamination was confirmed,\n(b) a brief description of the investigation carried out to identify the source and possible spread of the contamination, including the level of sampling undertaken,\n(c) information on the identified or presumed sources(s) of contamination,\n(d) details of the extent of the designated contamination, including the number of places of production and for potatoes, the number of lots with an indication of the variety and, if seed potatoes, category,\n(e) details of the zone demarcation, including the number of places of production, not designated as contaminated, but included in the zone,\n(f) details of water designation, including the name and location of the water body and the extent of the designation/irrigation ban,\n(g) for any tomato plant consignment or lot designated as contaminated, the certificates prescribed in Article 13(1)(ii) of Directive 2000/29/EC and passport number, in accordance with the listing in Annex V, Part A, Section I.2.2 to Directive 2000/29/EC,\n(h) such other information relating to the confirmed outbreak(s) as the Commission may require.\nANNEX VI\n1. The provisions referred to in Article 6(1) shall be:\nuse as animal feed after heat treatment such that there is no risk of the organism surviving,\nor\ndisposal at an officially approved dedicated waste disposal site atwhich there is no identifiable risk of escape of the organism into the environment e.g. through seepage to agricultural land or contact with water sources which could be used for irrigation of agricultural land,\nor\nincineration,\nor\nindustrial processing through direct and immediate delivery to a processing plant with officially approved waste disposal facilities for which it has been established that there is no identifiable risk of the organism spreading, and with a system of cleansing and disinfection of at least the departing vehicles,\nor\nother measures, provided that it has been established that there is no identifiable risk of the organism spreading; such measures and their justification to be notified to the Commission and to the other Member States.\nAny remaining waste associated with and arising from the above options shall be disposed of by officially approved methods in accordance with Annex VII to this Directive.\n2. The appropriate use or disposal of the listed plant material referred to in Article 6(2), under the control of the responsible official bodies of the Member State(s) concerned, with appropriate communication between responsible official bodies to ensure such control at all times and approval by the responsible official body of the Member State where the potatoes are to be packed or processed in respect of the waste disposal facilities referred to in the first and second indents, shall be:\n(i) for potato tubers,\nuse as ware potatoes intended for consumption, packed ready for direct delivery and use without repacking, on a site with appropriate waste disposal facilities. Potatoes intended for planting may only be handled at the same site, if this is done separately or after cleansing and disinfection,\nor\nuse as ware potatoes intended for industrial processing, and intended for direct and immediate delivery to a processing plant with appropriate waste disposal facilities and a system of cleansing and disinfection of at least the departing vehicles,\nor\nsome other use or disposal, provided that it is established that there is no identifiable risk of the organism spreading and subject to approval by the said responsible official bodies.\n(ii) for other plant parts including stem and foliage debris,\ndestruction,\nor\nsome other use or disposal, provided that it is established that there is no identifiable risk of the organism spreading; and subject to approval by the said responsible official bodies.\n3. The appropriate methods for decontamination of the objects referred to in Article 6(3) shall be cleansing and, where appropriate, disinfection, such that there is no identifiable risk of the organism spreading and shall be employed under the supervision of the responsible official bodies of the Member States.\n4. The series of measures to be implemented by Member States within the demarcated zone(s) established under Article 5(1)(a)(iv) and (c)(iii) and referred to in Article 6(4) shall include:\n4.1. In cases where places of production have been designated as contaminated under Article 5(1)(a)(ii):\n(a) in a field or unit of protected crop production designated to be contaminated under Article 5(1)(a)(ii), either\n(i) during at least the four growing years following the designated contamination,\nmeasures shall be taken to eliminate volunteer potato and tomato plants as well as other host plants of the organism including solanaceous weeds,\nand\nthe following shall not be planted:\npotato tubers, plants or true seeds,\ntomato plants and seeds,\ntaking into account the biology of the organism,\nother host plants,\nplants of species of Brassica, for which there is an identified risk of the organism surviving,\ncrops for which there is an identified risk of the organism spreading;\nin the first potato or tomato cropping season following the period specified in the preceding indent, and on the condition that the field has been found free from volunteer potato and tomato plants and other host plants including solanaceous weeds during official inspections for at least the two consecutive growing years prior to planting,\nin the case of potatoes, only ware potato production shall be allowed,\nin the case of potatoes and tomatoes, the harvested potato tubers, or the tomato plants, as appropriate, shall be tested according to the procedure detailed in Annex II;\nin the potato or tomato cropping season succeeding that referred to in the previous indent and following an appropriate rotation cycle, which shall be at least two years if seed potatoes are to be grown, an official survey as detailed in Article 2(1), shall be conducted;\nor\n(ii) during the five growing years following that of the designated contamination,\nmeasures shall be taken to eliminate volunteer potato and tomato plants as well as other naturally found host plants of the organism including solanaceous weeds,\nand\nthe field shall be established and maintained during the first three years either, in bare fallow or, in cereals according to the risk identified, or, in permanent pasture with frequent close cutting or intensive grazing or, as grass for seed production, followed by planting in the succeeding two years with non-host plants of the organism for which there is no identified risk of the organism surviving or spreading,\nin the first potato or tomato cropping season following the period specified in the preceding indent, and on the condition that the field has been found free from volunteer potato and tomato plants and other host plants including solanaceous weeds during official inspections for at least the two consecutive growing years prior to planting,\nin the case of potatoes, seed or ware potato production shall be allowed,\nthe harvested potato tubers, or the tomato plants, as appropriate, shall be tested according to the procedure detailed in Annex II;\n(b) in all other fields of the contaminated place of production and on the condition that the responsible official bodies are satisfied that the risk of volunteer potato plants and tomato plants and other naturally found host plants of the organism including solanaceous weeds as appropriate have been eliminated:\nin the growing year following that of the designated contamination,\neither no potato tubers or plants or true seeds, or other host plants of the organism shall be planted,\nor\nin the case of potato tubers, certified seed potatoes may be planted for ware production only,\nin the case of tomato plants, tomato plants grown from seed which meets the requirements of Directive 2000/29/EC may be planted for fruit production only;\nin the second growing year following that of the designated contamination,\nin the case of potatoes, only certified seed potatoes or seed potatoes officially tested for the absence of brown rot and grown under official control on places of production other than those referred to in 4.1 shall be planted for either seed or ware production,\nin the case of tomatoes, only tomato plants grown from seed which meets the requirements of Directive 2000/29/EC or, if vegetatively propagated, from tomato plants produced from such seed and grown under official control on places of production other than those referred to in 4.1, shall be planted for either plant or fruit production;\nfor at least in the third growing year following that of the designated contamination,\nin the case of potatoes, only certified seed potatoes or seed potatoes grown under official control from certified seed potatoes shall be planted for either seed or ware production,\nin the case of tomatoes, only tomato plants grown from seed which meets the requirements of Directive 2000/29/EC or tomato plants grown under official control from such plants shall be planted for either plant or fruit production;\nin each of the growing years referred to in the previous indents, measures shall be taken to eliminate volunteer potato plants and other naturally found host plants of the organism if present and, an official inspection of the growing crop at appropriate times shall be conducted and in each potato field, official testing of the harvested potatoes shall be conducted according to the procedure detailed in Annex II;\n(c) immediately following the designation of contamination under Article 5(1)(a)(ii) and after the first subsequent growing year:\nall machinery and storage facilities on the place of production and involved in potato or tomato production shall be cleansed and, where appropriate, disinfected using appropriate methods, as specified in point 3,\nofficial controls on irrigation and spraying programmes, including a ban thereof, shall be introduced as appropriate in order to prevent the spread of the organism;\n(d) in an unit of protected crop production designated as contaminated under Article 5(1)(a)(ii) where complete replacement of the growing medium is possible,\nno potato tubers, plants or true seeds, or other host plants of the organism including tomato plants and seeds shall be planted unless the production unit has been subjected to officially supervised measures to eliminate the organism and to remove all host plant material, including, at least, a complete change in growing medium and cleansing and, where appropriate, disinfection of the said unit and all equipment, and subsequently has been granted approval for potato or tomato production by the responsible official bodies,\nand\nfor potato production, this production shall be from certified seed potatoes, or from mini-tubers or micro-plants derived from tested sources,\nfor tomato production, this production shall be from seed which meets the requirements of Directive 2000/29/EC or, if vegetatively propagated, from tomato plants produced from such seed and grown under official control,\nofficial controls on irrigation and spraying programmes, including a ban thereof, shall be introduced as appropriate, in order to prevent the spread of the organism;\n4.2. Within the demarcated zone, without prejudice to the measures detailed under 4.1, the Member States shall:\n(a) immediately following the designated contamination ensure that all machinery and storage facilities on such premises and involved with potato or tomato production be cleansed and disinfected, as appropriate, and using appropriate methods, as specified in 3,\n(b) immediately, and for at least three growing years following the designated contamination:\n(ba) in cases where the demarcated zone has been determined under Article 5(1)(a)(iv),\nensure supervision by their responsible official bodies of premises growing, storing or handling potato tubers or tomatoes, together with premises which operate machinery for potato or tomato production under contract,\nrequire the planting of only certified seed or seed grown under official control for all potato crops within that zone, and testing after harvest of seed potato crops grown in places of production determined as probably contaminated under Article 5(1)(a)(iii),\nrequire the separate handling of harvested seed potato stocks to those of ware on all premises within the zone, or a system of cleansing and, where appropriate, disinfection to be carried out between the handling of seed and ware stocks,\nrequire the planting of only tomato plants grown from seed which meets the requirements of Directive 2000/29/EC or, if vegetatively propagated, from tomato plants produced from such seed and grown under official control, for all tomato crops within that zone,\nconduct an official survey as detailed in Article 2(1);\n(bb) in cases where surface water has been designated as contaminated under Article 5(1)(c)(ii) or included in the elements for the possible spread of the organism in accordance with Annex V point 2,\nconduct an annual survey at appropriate times including sampling of surface water and where appropriate solanaceous host plants in the relevant water sources and testing in accordance with the relevant methods set out in Annex II for the listed plant material and for other cases,\nintroduce official controls on irrigation and spraying programmes, including a ban on the use of the water designated as contaminated for the irrigation and spraying of listed plant material, and, where appropriate, other host plants in order to prevent the spread of the organism. This ban may be reviewed on the basis of the results obtained in the said annual survey, and designations revoked where the responsible official bodies are satisfied that the surface water is no longer contaminated. The use of water subject to a ban may be permitted, under official control, for irrigation and spraying of host plants, where officially approved techniques are employed which eliminate the organism and prevent its spread,\nin cases where liquid waste discharges are contaminated, introduce official controls on the disposal of solid or liquid waste discharges from industrial processing or packaging premises handling listed plant material;\n(c) establish a programme, where appropriate, for the replacement of all seed potato stocks over an appropriate period of time.\nANNEX VII\nThe officially approved waste disposal methods referred to in Annex VI paragraph 1, shall conform to the following provisions such that any identifiable risk of spreading the organism is obviated:\n(i) potato and tomato waste (including rejected potatoes and peelings and tomatoes) and any other solid waste associated with the potatoes and tomatoes (including soil, stones and other debris) shall be disposed by either,\ndisposal at an officially approved dedicated waste disposal site at which there is no identifiable risk of escape of the organism into the environment e.g. through seepage to agricultural land or contact with water sources which could be used for irrigation of agricultural land. The waste shall be conveyed directly to the site under containment conditions such that there is no risk of loss of the waste,\nor\nincineration,\nor\nother measures, provided that it has been established that there is no identifiable risk of the organism spreading; such measures to be notified to the Commission and to the other Member States.\n(ii) liquid waste: prior to disposal, liquid waste containing suspended solids shall be subjected to filtration or settlement processes to remove such solids. These solids shall be disposed of as set out in subparagraph (i).\nThe liquid waste shall then be either:\nheated to a minimum of 60 °C throughout the entire volume during at least 30 minutes prior to disposal,\nor\notherwise disposed of subject to official approval and under official control such that there is no identifiable risk that the waste could come into contact with agricultural land or water sources which could be used for irrigation of agricultural land. The details thereof shall be notified to the other Member States and to the Commission.\nThe options described in this Annex also apply to the waste associated with handling, disposal and processing of contaminated lots.",
  "extent": "E+W+S+N.I.",
  "status": null,
  "version": "current",
  "annotations": [
    {
      "type": "footnote",
      "label": "f1",
      "text": "See Lelliott and Stead (1987)"
    },
    {
      "type": "footnote",
      "label": "f2",
      "text": "Contact scientists: see web site http://forum.europa.eu.int/Public/irc/sanco/Home/main."
    },
    {
      "type": "footnote",
      "label": "f3",
      "text": "Use as standard reference strain of R. solanacearum biovar 2 (race 3)."
    },
    {
      "type": "footnote",
      "label": "f4",
      "text": "Potential cross-reacting strain in serological tests (IF and/or ELISA) with polyclonal antisera."
    },
    {
      "type": "footnote",
      "label": "f5",
      "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": "f6",
      "text": "Likely to cross-react in most tests but known to occur only on banana in Indonesia."
    },
    {
      "type": "footnote",
      "label": "f7",
      "text": "For use with homogenisation extraction method"
    },
    {
      "type": "footnote",
      "label": "f8",
      "text": "Method was validated using Taq polymerase from Perkin Elmer (AmpliTaq) and Gibco BRL."
    },
    {
      "type": "footnote",
      "label": "f9",
      "text": "Methods were validated using Taq polymerase from Perkin Elmer (AmpliTaq) and Gibco BRL."
    },
    {
      "type": "footnote",
      "label": "f10",
      "text": "Methods were validated using Taq polymerase from Perkin Elmer (AmpliTaq) and Gibco BRL."
    },
    {
      "type": "footnote",
      "label": "f11",
      "text": "Concentration of primers NS-5-F and NS-6-R were optimised for potato heel end core extraction using the homogenisation method and DNA purification according to Pastrik (2000) (see Section VI.A.6.1.a.). Re-optimisation of reagent concentrations will be required if extraction by shaking or other DNA isolation methods are used."
    },
    {
      "type": "footnote",
      "label": "f12",
      "text": "Methods have been validated using Taq polymerase from Perkin Elmer (AmpliTaq) and Gibco BRL."
    },
    {
      "type": "footnote",
      "label": "f13",
      "text": "Methods have been validated using Taq polymerase from Perkin Elmer (AmpliTaq) and Gibco BRL."
    }
  ],
  "outstanding_effects": [],
  "provenance": {
    "source": "legislation.gov.uk",
    "source_url": "https://www.legislation.gov.uk/eudr/2006/63/annex",
    "retrieved_at": "2026-11-01T15:22:34.779162+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."
}