Commission Directive 2006/56/EC of 12 June 2006 amending the Annexes to Council Directive 93/85/EEC on the control of potato ring rot
| Laboratory | Location | Country |
|---|---|---|
| Agentur für Gesundheit und Ernährungssicherheit | Vienna and Linz | Austria |
| Departement Gewasbescherming | Merelbeke | Belgium |
| Plantedirektoratet | Lyngby | Denmark |
| Central Science Laboratory | York | England |
| Scottish Agricultural Science Agency | Edinburgh | Scotland |
| Laboratoire National de la Protection des Végétaux, Unité de Bactériologie | Angers | France |
| Laboratoire National de la Protection des Végétaux, Station de Quarantaine de la Pomme de Terre | Le Rheu | France |
| Biologische Bundesanstalt | Kleinmachnow | Germany |
| Pflanzenschutzamt Hannover | Hannover | Germany |
| State Laboratory | Dublin | Ireland |
| Plantenziektenkundige Dienst | Wageningen | Netherlands |
| Norwegian Crop Research Institute, Plant Protection Centre | Aas | Norway |
| Direcção-Geral de Protecção das Culturas | Lisbon | Portugal |
| Nacionalni institut za biologijo | Ljubljana | Slovenia |
| Centro de Diagnostico de Aldearrubia | Salamanca | Spain |
(i) Diagnosis of ring rot in potato tubers and plants; (ii) Detection of Clavibacter michiganensis ssp.sepedonicus in samples of potato tubers and plants;(iii) Identification of Clavibacter michiganensis ssp.sepedonicus (C. m. subsp.sepedonicus ).
The 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. Validation of all detection methods described below is based on testing of samples of 200 tubers. The potato extract described below can also be used for detection of the potato brown rot bacterium, Ralstonia solanacearum.
(a) cover the cores with sufficient volume (approximately 40 ml) of extraction buffer (Appendix 3) and agitate on a rotary shaker (50 to 100 rpm) for four hours below 24 °C or for 16 to 24 hours refrigerated, or (b) homogenise the cores with sufficient volume (approximately 40 ml) of extraction buffer (Appendix 3), 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).
(a) cover the segments with sufficient volume (approximately 40 ml) of extraction buffer (Appendix 3) and agitate on a rotary shaker (50 to 100 rpm) for four hours below 24 °C or for 16 to 24 hours refrigerated, or (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 3) 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.
(i) For pellets with relatively little starch sediment: Pipette 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. (ii) For other pellets: Prepare decimal dilutions (1/10 and 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.
(i) According to test slide preparation in 4.1(i): Prepare a set of twofold dilutions of the antibody in IF buffer. 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). (ii) According to test slide preparation in 4.1(ii): Prepare the working dilution (WD) of the antibody in IF buffer. The working dilution affects the specificity.
| Dilutions of resuspended pellet | |||||||
| 1/100 | 1/1 | 1/1 | 1/1 | 1/1 | | Dilution of resuspended pellet | |
| (T = titre) | T/2 | T/4 | T/2 | T | 2T | | Twofold dilutions of antiserum/antibody |
| Sample 1 |  |  | | | | ||
| 1 | 2 | 3 | 4 | 5 | |||
| Duplicate of sample 1 or sample 2 | | | | | | ||
| 6 | 7 | 8 | 9 | 10 | |||
| Working dilution of antiserum/antibody | |||||||
| 1/1 | 1/10 | 1/100 | empty | empty | | Decimal dilution of resuspended pellet | |
| Sample 1 | | | | | | ||
| 1 | 2 | 3 | 4 | 5 | |||
| Duplicate of sample 1 or sample 2 | | | | | | ||
| 6 | 7 | 8 | 9 | 10 | |||
(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 4). The IF reading is positive for samples with at least 5 × 10 3 typical cells per ml of resuspended pellet. The sample is considered potentially contaminated. and further testing is required.(ii) The IF reading is negative for samples with less than 5 × 10 3 cells per ml resuspended pellet and the sample is considered negative. Further testing is not required.
| Sample 1 | Blank | Blank | Blank | Sample 2 |
 |  | | | |
| window 1 | window 2 | window 3 | window 4 | window 5 |
| Sample 1 | Blank | Blank | Blank | Sample 2 |
| | | | |
| window 6 | window 7 | window 8 | window 9 | window 10 |
| Coverslip 1 | Coverslip 2 | |||
(i) Valid FISH test results are obtained if bright green fluorescent cells of size and morphology typical of C. m. subsp.sepedonicus are observed using the FITC filter and if 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.(ii) The FISH test is negative if bright red fluorescent cells with size and morphology typical of C. m. subsp.sepedonicus 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.
sample extract that previously tested negative for C. m. subsp.Sepedonicus ,buffer controls used for extracting the bacterium and the DNA from the sample, PCR-reaction mix.
aliquots of resuspended pellets to which C. m. subsp.sepedonicus has been added (preparation see Appendix 2),a suspension of 10 6 cells per ml ofC. m. subsp.sepedonicus in water from a virulent isolate (e.g. NCPPB 2140 or NCPPB 4053),if possible also use DNA extracted from positive control samples in the PCR test.
1. 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. 2. Add 100 µl sample extract and place in a heating block or waterbath at 95 °C for 10 minutes. 3. Put tube on ice for five minutes. 4. 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 minutes. 5. Add 220 µl of Easy DNA ® solution A (Invitrogen), mix well by vortexing and incubate at 65 °C for 30 minutes.6. 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.7. Add 500 µl of chloroform and vortex until the viscosity decreases and the mixture is homogeneous. 8. Centrifuge at 15000 g for 20 minutes at 4 °C to separate phases and form the interphase.9. Transfer the upper phase into a fresh Eppendorf tube. 10. Add 1 ml of 100 % ethanol ( –20 °C) vortex briefly and incubate on ice for 10 minutes.11. Centrifuge at 15000 g for 20 minutes at 4 °C and remove ethanol from pellet.12. Add 500 µl 80 % ethanol ( –20 °C) and mix by inverting the tube.13. Centrifuge at 15000 g for 10 minutes at 4 °C, save the pellet and remove ethanol.14. Allow the pellet to dry in air or in a DNA speed vac. 15. Resuspend the pellet in 100 µl sterile UPW and leave at room temperature for at least 20 minutes. 16. Store at –20 °C until required for PCR.17. Spin down any white precipitate by centrifugation and use 5 µl of the supernatant containing DNA for the PCR.
| Oxidation/Fermentation (O/F) test | Inert or weakly oxidative |
| Oxidase activity | – |
| Growth at 37 °C | – |
| Urease activity | – |
| Aesculin hydrolysis | + |
| Starch hydrolysis | – or weak |
| Tolerance of 7 % NaCl | – |
| Indole production | – |
| Catalase activity | + |
| H | – |
| Citrate utilization | – |
| Gelatin liquefaction | – |
| Acid glycerol | – |
| Acid from lactose | – or weak |
| Acid from rhamnose | – |
| Acid from salicin | – |
| Gram stain (Appendix 9) | + |
(a) Prepare a suspension of approximately 10 6 cells per ml in IF buffer (Appendix 3).(b) Prepare a 2-fold dilution series of an appropriate antiserum. (c) Apply the IF procedure (section 4). (d) A positive IF test is achieved if the IF titre of the culture is equivalent to that of the positive control.
(a) Prepare a suspension of approximately 10 6 cells per ml in ultra pure water (UPW).(b) Heat 100 µl of the cell suspension in closed tubes in a heating block or boiling waterbath at 100 °C for four minutes. If required, addition of freshly-prepared NaOH to a final concentration of 0,05M may assist cell lysis. The samples may then be stored at –16 to–24 °C until required.(c) Apply appropriate PCR procedures to amplify C. m. subsp.sepedonicus specific amplicons (e. g. Pastrik, 2000; see Appendix 4; Li and de Boer, 1995; Millset al. , 1997; Pastrik and Rainey, 1999; Schaadet al. , 1999.(d) A positive identification of C. m. subsp.sepedonicus is achieved if the PCR amplicons are the same size and have the same restriction fragment length polymorphisms as for the positive control strain.
(a) Prepare a suspension of approximately 10 6 cells per ml in UPW.(b) Apply the FISH procedure (section 5). (c) A positive FISH test is achieved if the same reactions are achieved from the culture and the positive control.
(a) Grow the culture on trypticase soy agar (Oxoid) for 72 hours at 21 °C (+/– 1°). (b) Apply an appropriate FAP procedure (Janse, 1991; Stead, 1992). (c) A 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 15:1 Anteiso A, 15:0 Iso, 15:0 Anteiso, 16:0 Iso, 16:0 and 17:0 Anteiso is highly indicative of C. m. sepedonicus . Other genera such asCurtobacterium, Arthrobacter andMicrococcus also have some of these acids but 15:1 Anteiso A is a rare acid in these bacteria but occurs in allClavibacter spp. at between 1 to 5 %. InC. m. sepedonicus the value is usually around 5 %.
(a) Prepare a suspension of approximately 10 6 cells per ml in UPW.(b) Apply the test according the procedure (Smith et al. , 2001).
| Na | 4,26 g |
| KH | 2,72 g |
| Distilled water | 1.00 L |
| Purpose | Quantity (per L) | |
|---|---|---|
| Lubrol flakes | Deflocculant | 0,5 g |
| DC silicone antifoam | Anti-foam agent | 1,0 ml |
| Tetrasodium pyrophosphate | Anti-oxidant | 1,0 g |
| Polyvinylpyrrolidone- | Binding of PCR inhibitors | 50 g |
| Na | 2,7 g |
| NaH | 0,4 g |
| Distilled water | 1,00 L |
| Na | 2,7 g |
| NaH | 0,4 g |
| NaCl | 8,0 g |
| Distilled water | 1.0 L |
| Na | 3,2 g |
| NaH | 0,15 g |
| Glycerol | 50 ml |
| Distilled water | 100 ml |
1. Count the mean number of typical fluorescent cells per field of view (c) 2. Calculate the number of typical fluorescent cells per microscope slide window (C) C = c × S/s where S = surface area of window of multispot slide, and s = surface area of objective field. s = πi 2 /4G2 K2 where i = field coefficient (varies from 8 to 24 depending upon ocular type) K = tube coefficient (1 or 1,25) G = magnification of objective (100x, 40x etc.). 3. Calculate the number of typical fluorescent cells per ml of resuspended pellet (N) N = C × 1000 /y × Fwhere y = volume of resuspended pellet on each window, and F = dilution factor of resuspended pellet.
| Nutrient Agar (Difco) | 23,0 g |
| Distilled water | 1,0 L |
| Yeast Extract (Difco) | 5,0 g |
| Bacto-Peptone (Difco) | 5,0 g |
| D(+) Glucose (monohydrate) | 10,0 g |
| Bacto-Agar (Difco) | 15,0 g |
| Distilled water | 1,0 L |
| Bacto-Yeast-Extract (Difco) | 2,0 g |
| D(+) Glucose (monohydrate) | 2,5 g |
| K | 0,25 g |
| KH | 0,25 g |
| MgSO | 0,1 g |
| MnSO | 0,015 g |
| NaCl | 0,05 g |
| FeSO | 0,005 g |
| Bacto-Agar (Difco) | 18 g |
| Distilled water | 1,0 L |
| Yeast extract (Difco) | 2,0 g |
| Mannitol | 2,5 g |
| K | 0,25 g |
| KH | 0,25 g |
| NaCl | 0,05 g |
| MgSO | 0,1 g |
| MnSO | 0,015 g |
| FeSO | 0,005 g |
| Agar (Oxoid no. 1) | 16,0 g |
| Distilled water | 1,0 L |
| Nutrient agar (Difco) | 23 g |
| Yeast extract (Difco) | 2 g |
| D-mannitol | 5 g |
| K | 2 g |
| KH | 0,5 g |
| MgSO | 0,25 g |
| Distilled water | 1,0 L |
| Forward primer PSA-1 | 5′- ctc ctt gtg ggg tgg gaa aa -3′ |
| Reverse primer PSA-R | 5′- tac tga gat gtt tca ctt ccc c -3′ |
| Forward primer NS-7-F | 5′- gag gca ata aca ggt ctg tga tgc -3′ |
| Reverse Primer NS-8-R | 5’- tcc gca ggt tca cct acg ga -3’ |
| Reagent | Quantity per reaction | Final concentration |
|---|---|---|
| Sterile UPW | 15,725 µl | |
| 10x PCR buffer | 2,5 µl | 1x (1,5 mM MgCl |
| BSA (fraction V) (10 %) | 0,25 µl | 0,1 % |
| d-nTP mix (20 mM) | 0,125 µl | 0,1 mM |
| Primer PSA-1 (10 µM) | 0,5 µl | 0,2 µM |
| Primer PSA-R (10 µM) | 0,5 µl | 0,2 µM |
| Primer NS-7-F (10 µM) | 0,1 µl | 0,04 µM |
| Primer NS-8-R (10 µM) | 0,1 µl | 0,04 µM |
| Taq polymerase (5 U/µl) | 0,2 µl | 1,0 U |
| Sample volume | 5,0 µl | |
| Total volume | 25,0 µl |
| 1 cycle of: | (i) | 3 minutes at 95 °C (denaturation of template DNA) |
| 10 cycles of: | (ii) | 1 minute at 95 °C (denaturation of template DNA) |
| (iii) | 1 minute at 64 °C (annealing of primers) | |
| (iv) | 1 minute at 72 °C (extension of copy) | |
| 25 cycles of: | (v) | 30 seconds at 95 °C (denaturation of template DNA) |
| (vi) | 30 seconds at 62 °C (annealing of primers) | |
| (vii) | 1 minute at 72 °C (extension of copy) | |
| 1 cycle of: | (viii) | 5 minutes at 72 °C (final extension) |
| (ix) | hold at 4 °C. |
| Bromphenol blue | 5 g |
| Distilled Water (bidest) | 50 ml |
| Glycerol (86 %) | 3,5 ml |
| Bromphenol blue (5.1) | 300 µl |
| Distilled Water (bidest) | 6,2 ml |
| Tris buffer | 48,4 g |
| Glacial acetic acid | 11,42 ml |
| EDTA (disodium salt) | 3,72 g |
| Distilled water | 1,00 L |
| 5′- ttg cgg ggc gca cat ctc tgc acg -3′ | |
| Non-specific eubacterial probe EUB-338-FITC: | 5′- gct gcc tcc cgt agg agt -3′ |
(i) Heat nine ml molecular grade water (e.g. Ultra pure water (UPW)) to about 60 °C and add 0,4 g paraformaldehyde. Paraformaldehyde dissolves after adding five drops of 1N NaOH and stirring with a magnetic stirrer. (ii) Adjust pH to 7,0 by addition of 1ml of 0,1 M phosphate buffer (PB; pH 7,0) and five drops of 1 N 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] (iii) Filter the solution through a 0,22 µm membrane filter and keep dust-free at 4 °C until further use. (iv) Note: Alternative fixative solution: 96 % ethanol.
| NaCl | 2,7 M |
| Tris-HCl | 60 mM (pH 7,4) |
| EDTA (filter sterilised and autoclaved) | 15 mM |
| Sodium dodecyl sulphate (SDS) | 0,01 % |
| probe EUB 338 | 5 ng/μl |
| probe CMSCY301 | 5 ng/μl |
| 2 slides | 8 slides | |
|---|---|---|
| Sterile UPW | 50,1 | 200,4 |
| 3x hybmix | 30,0 | 120,0 |
| 1 % SDS | 0,9 | 3,6 |
| Probe EUB 338 (100 ng/μl) | 4,5 | 18,0 |
| Probe CMSCY301 (100 ng/μl) | 4,5 | 18,0 |
| Total volume (μl) | 90,0 | 360,0 |
| Na | 8,52 g |
| KH | 5,44 g |
| Distilled water | 1,00 L |
| Day length: | 14 hours or natural day length if greater; | |
| Temperature: | day: | 21 to 24 °C, |
| night: | 15 °C. |
| Susceptible varieties of eggplant: | "Black Beauty", |
| "Long Tom", | |
| "Rima", | |
| "Balsas" |
1. The elements to be considered in the determination of the extent of probable contamination under Article 5(1)(b) shall include: tubers or plants grown at a place of production designated as contaminated under Article 5(1)(a), place(s) of production with some production link to the tubers or plants, designated as contaminated under Article 5(1)(a), including those sharing production equipment and facilities directly or through a common contractor, tubers or plants 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 tubers or plants designated as contaminated under Article 5(1)(a) were present on the place of production referred to in the first indent, premises handling potatoes from the places of production referred to in the above indents, any machinery, vehicle, vessel, store, or units thereof, and any other objects including packaging material, that may have come into contact with the tubers or plants designated as contaminated under Article 5(1)(a), any tubers or plants 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, as a result of the testing under Article 6, those tubers or plants with a sister or parental clonal relationship to the tubers or plants designated to be contaminated under Article 5(1)(a) 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, and place(s) of production of the tubers or plants referred to in the previous indent.
2. The elements to be considered in the determination of the possible spread under Article 5(1)(c) shall include: the proximity of other places of production growing potatoes or other host plants, the common production and use of seed potato stocks.
3. The notification referred to in the first subparagraph of Article 5(2) shall be provided as follows: immediately after the presence of the organism has been confirmed by laboratory testing, using the methods set out in Annex I, at least: the variety name of the potato lot, the type (ware, seed, etc.) and where applicable the seed category of potatoes,
when there is a risk of contamination of potatoes from or into another Member States(s), the Member State in which the occurrence has been confirmed shall immediately notify the Member State(s) concerned of information needed to comply with Article 5(3), such as: the variety name of the potato lot, the name and address of the consignor and the consignee, the date of delivery of the potato lot, the size of the potato lot delivered, 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.
The Commission shall be notified immediately when such information has been provided. After all investigations have been finalised, for each case: the date that contamination was confirmed, a brief description of the investigation carried out to identify the source and possible spread of the contamination, including the level of sampling undertaken, information on the identified or presumed sources(s) of contamination, details of the extent of the designated contamination, including the number of places of production and the number of lots with an indication of the variety and, if seed potatoes, category, details of the zone demarcation, including the number of places of production, not designated as contaminated, but included in the zone, such other information relating to the confirmed outbreak(s) as the Commission may require.
1. The officially supervised measures referred to in Article 7(1) shall be: use as animal feed after heat treatment such that there is no risk of the organism surviving, or disposal at an officially approved dedicated waste disposal site at which there is no identifiable risk of escape of the pathogen into the environment e.g. through seepage to agricultural land, or incineration, or industrial 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, or other 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.
Any remaining waste associated with and arising from the above shall be disposed of by officially approved methods in accordance with Annex V to this Directive. 2. The appropriate use or disposal of tubers or plants determined as probably contaminated under Article 5(1)(b) and referred to in Article 7(2), under the control of the responsible official bodies of the Member States concerned, with appropriate communication between responsible official bodies to ensure such control at all times and approval by the responsible official bodies 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 indent, shall be: use 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, or use 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, or some 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.
3. The appropriate methods for cleansing and disinfecting of the objects referred to in Article 7(3) shall be those for which it has been established 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. 4. The series of measures to be implemented by Member States within the demarcated zone established under Article 5(1)(c) and referred to in Article 7(4) shall include: 4.1. in places of production designated as contaminated under Article 5(1)(a): (a) in a field designated to be contaminated under Article 5 (1)(a), either (i) during at least the three growing years following the year of the designated contamination, measures shall be taken to eliminate volunteer potato plants and other naturally found host plants of the organism, and no potato tubers, plants or true seeds, or other naturadlly found host plants of the organism, or crops for which there is an identified risk of the organism spreaing, shall be planted,
in the first potato cropping season following the period specified in the preceding indent, and on the condition that the field has been found free from volunteer potato plants and other naturally found host plants of the organism during official inspections for at least the two consecutive growing years prior to planting, only ware potato production shall be allowed and the harvested tubers shall be tested according to the procedure detailed in Annex I; in the potato 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, potatoes may be planted for either seed or ware production, and an official survey as detailed in Article 2(1), shall be conducted; or
(ii) during the four growing years following that of the designated contamination, measures shall be taken to eliminate volunteer potato plants and other naturally found host plants of the organism, and the field shall be laid to, and maintained either, in bare fallow or in permanent pasture with frequent close cutting or intensive grazing,
in the first potato cropping season following the period specified in the preceding indent, and on the condition that the field has been found free from volunteer potato plants and other naturally found host plants of the organism during official inspections for at least the two consecutive growing years prior to planting, seed or ware potato production shall be allowed and the harvested tubers shall be tested according to the procedure detailed in Annex I;
(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 other naturally found host plants of the organism have been eliminated: in the growing year following that of the designated contamination either no potato tubers, plants or true seeds or other naturally found host plants of the organism shall be planted, or certified seed potatoes may be planted for ware production only,
in the second growing year following that of the designated contamination, only certified seed or seed potatoes officially tested for the absence of ring 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, for at least the third growing year following the designated contamination, only certified seed potatoes or seed potatoes grown under official control from certified seed potatoes shall be planted for either seed or ware production, in 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 in each potato field official testing of the harvested potatoes shall be conducted according to the procedure detailed in Annex I,
(c) immediately following the designation of contamination under Article 5 (1)(a) and after the first subsequent growing year, all machinery and storage facilities on the place of production and involved in potato production shall be cleansed and disinfected as appropriate using appropriate methods, as specified in 3; (d) in an unit of protected crop production where complete replacement of the growing medium is possible, no tubers, plants or true 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 disinfection of the production unit, and all equipment, and, subsequently has been granted approval for potato production by the responsible official bodies, and potato production shall be from certified seed potatoes, or from mini-tubers or micro-plants derived from tested sources;
4.2. within the demarcated zone, without prejudice to the measures detailed under 4.1, the Member States shall: (a) immediately following the designated contamination, ensure that all machinery and storage facilities on such premises and involved with potato production be cleansed and disinfected, as appropriate, and using appropriate methods, as specified in 3; (b) immediately, and for at least three growing seasons, after the designated contamination: ensure supervision by their responsible official bodies of premises growing, storing or handling potato tubers, together with premises which operate potato machinery under contract, require 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 designated as probably contaminated under Article 5(1)(b), require the separate handling of harvested seed potato stocks to those of ware on all premises within the zone, or a system of cleansing and disinfection to be carried out between the handling of seed and ware stocks, conduct an official survey as detailed in Article 2(1);
(c) establish a programme, where appropriate, for the replacement of all seed potato stocks over an appropriate period of time.
| Iodine | 1 g |
| Potassium iodide | 2 g |
| Distilled water | 300 ml |
| Safranin O | 2,5 g |
| 95 % ethanol | 100 ml |
1. Prepare smears, air dry and heat fix. 2. Flood slide with crystal violet solution for one minute. 3. Wash briefly with tap water. 4. Flood with Lugol's iodine for one minute. 5. Wash with tap water and blot dry. 6. Decolourise with 95 % ethanol, added dropwise, until no further colour is removed or immerse with gentle agitation for 30 seconds. 7. Wash in tap water and blot dry. 8. Flood with safranin solution for 10 seconds. 9. Wash with tap water and blot dry.
1. For each suspected occurrence for which a positive result in the screening test (s) has been identified according to the methods set out in Annex I, and confirmation or refutation by completion of the said methods is awaited, there should be the retention and appropriate conservation of: all tubers sampled and, wherever possible, all plants sampled, any remaining extract and additional prepared material for the screening test(s), e.g. immunofluorescence slides, and all relevant documentation,
until the completion of the said methods. Retention of the tubers will enable variety testing to be undertaken where appropriate. 2. In the case of positive confirmation of the organism, there should be retention and appropriate conservation of: the material specified in paragraph 1, and a sample of the infected eggplant material inoculated with the tuber or plant extract, and the isolated culture of the organism,
until at least one month after the notification procedure under Article 5(2).
(i) potato waste (including rejected potatoes and peelings) and any other solid waste associated with the potatoes (including soil, stones and other debris) shall be disposed by either, disposal 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. The waste shall be conveyed directly to the site under containment conditions such that there is no risk of loss of the waste, or incineration, or other 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.
(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). The liquid waste shall then be either: heated to a minimum of 60 °C throughout the entire volume during at least 30 minutes prior to disposal, or otherwise 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. The details thereof shall be notified to the other Member States and to the Commission.