Decree No. 331 / 2004 Coll.
Decree on measures to ensure protection against introduction and spread of potato ring agent and bacterial brown rot agent
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331
DECLARATION
of 4 May 2004
on measures to ensure protection against the introduction and spread of potato ring agent and bacterial brown rot agent
According to Section 88 (2) of Act No. 326 / 2004 Coll., on Phytosanitary Care and on the Amendment of Certain Related Acts (hereinafter "the Act '), the Ministry of Agriculture provides for the implementation of § 71 (1) (i) of the Act:
Subject matter
This decree implements the relevant provisions of the European Union1) and provides for measures to be applied in the territory of the Czech Republic against the spread of Clavibacter michiganensis (Smith) Davis et al. subsp. sepedonicus (Spieckermann et Kotthoff) Davis et al., agents of bacterial potato ring rot, and Ralstonia solanacearum (Smith) Yabuuchi et al., originators of bacterial brown rot to be
(a) prevent the occurrence and spread of:
(b) identify the occurrence and extent of the enlargement; and
(c) in the event of an outbreak, they have been prevented from spreading and protective measures taken to eradicate them.
Terms
For the purposes of this decree:
(a) the agent of ring rot - Clavibacter michiganensis (Smith) Davis et al. sepedonicus (Spieckermann et Kotthoff) Davis et al.,
(b) the agent of brown rot - Ralstonia solanacearum (Smith) Yabuuchi et al.,
(c) host plants -
1. potato ring agent (Solanum tuberosum L.), with the exception of potato seed, and other plants of the family Solanaceae, for which the possibility of infection by ring agent in natural conditions has been confirmed,
2. the agent of brown rot - potatoes (Solanum tuberosum L.) with the exception of potato seeds, tomato (Solanum lycopersicum L.) other than seeds and fruits, aubergines of the aftertaste (Solanum dulcamara L.) and other plants of the eggplant family (Solanaceae) in which the possibility of infection by the agent of brown rot in natural conditions has been confirmed,
(d) detection survey - survey to detect the occurrence of a ring agent or a brown rot agent in a specific territory or part;
(e) the demarcation survey - the survey to determine the extent and origin of the infestation by the agent of ring rot or by the agent of brown rot, to determine the method of spreading the infection and to determine the boundaries of the territory to which the exceptional plant health measures under Section 76 of the Act apply;
(f) likely infestation by the agent of the ring rot or the agent of the brown rot - suspected of the presence of the agent of the ring rot or the agent of the brown rot in the plant, consignment, lot, plant, machine, means of transport, storage or parts thereof and any other objects, including packaging material, pursuant to Article 11 (1) of the Act;
(g) possible extension of the ring rot agent or the brown rot agent - suspected occurrence of the ring rot agent or the brown rot agent on the property, in the premises or in the territory, pursuant to Article 11 (1) of the Act,
(h) by infestation by the agent of the ring rot or the agent of the brown rot - the occurrence of the agent of the ring rot or the agent of the brown rot pursuant to Article 11 (1) of the Act;
(i) quarantine territory - the territory defined by the Constitution, which is subject to exceptional plant health measures pursuant to Article 76 (1) of the Act on the grounds of confirmation of the occurrence of a ring agent or a brown rot agent;
(j) the safety zone - the territory defined by the Constitution, which is subject to exceptional plant health measures under Section 76 of the Act on grounds of suspicion of the presence of a ring agent or a brown rot agent;
(k) clones related to lots - lots of host plants which have been produced by vegetative propagation from one original lot; for:
1. sister clones refer to clones related lots which were produced in the same year by vegetative propagation from a single parent lot, in the case of tomato plants which grew from the same seed lot,
2. The parent of a clone relative of a lot is considered by a cloned relative of a sister of a clone relative of a lot that was immediately formed from it.
Exploration of the incidence of ring agent and brown rot agent
(1) The Institute shall each year carry out a systematic survey, in accordance with Section 10 (1) of the Act, of the occurrence of the circling agent and of the brown rot agent within the scope of paragraphs 2 to 7.
(2) The detection survey for the occurrence of ring rot agent and brown rot agent are subject to:
(a) potato propagating material (2) originating in the Czech Republic within the scope of § 8 (2);
(b) randomly selected lots of potato propagating material (2) originating in the Member States of the European Union (hereinafter referred to as the Member State);
(c) randomly selected lots of seed potatoes originating in the Czech Republic and other Member States;
(d) all potato lots grown in a safety zone as defined in Article 5 (1) (c).
(3) A demarcation survey on the occurrence of ring agent (s) or brown rot agent (s) shall be subject to all host plant batches identified as suspected of occurrence of ring agent (s) or brown rot agent (s) and all host plant batches produced in the quarantine area as defined in Article 5 (1) (c).
(4) The detection of ring rot agent and brown rot agent in plant health checks on consignments imported from third countries and exported to third countries shall be subject to:
(a) all imported potato propagating material batches (2) and randomly selected batches of other potatoes;
(b) all potato lots intended for export to third countries.
(5) In addition, a detection survey for the occurrence of brown rot agent is subject to:
(a) tomato plants intended for further cultivation;
(b) water resources used for the irrigation of host plants by the producer of brown rot including accompanying host plants and waste water from processing plants which process potato tubers and, where appropriate, solid waste from such plants.
(6) In addition, a demarcation survey of the presence of the agent of brown rot is subject to the presence or confirmation of the presence of a water source used for the irrigation of parts suspected of the occurrence and of the weeds host plants, or to the growing substrate at the places of production where those lots originate.
(7) The incidence of ring rot agent and brown rot agent is detected
(a) laboratory testing of samples of potato tubers taken from the crops or landfills in the cases referred to in paragraph 2 (a) and (b), paragraph 3 and paragraph 4 (b), in the case referred to in paragraph 4 (a), from imported lots;
(b) by examining whole potato plants and cutting flesh of tubers, in crops or in landfills, and, where appropriate, by laboratory testing samples of potato tubers, in the cases referred to in paragraph 2 (c) and (d);
(c) inspection of tomato crops at the appropriate time, followed by laboratory testing of plant samples with symptoms of infestation by the agent of brown rot in the cases referred to in paragraph 5 (a);
(d) laboratory testing of samples of water, growing medium, solid waste or host plants of coastal vegetation in the case referred to in paragraph 5 (b) and paragraph 6.
(8) The method of inspection of the lot and the number, origin, composition and duration of sampling carried out in the survey carried out in accordance with paragraphs 2 to 7, the scope of the survey and the transmission of information on the samples collected and tested shall be determined by the Institute in accordance with Article 10 (1) of the Act on the basis of recognised scientific and statistical principles and the biology of the ring agent and of the brown rot agent, taking into account the systems of potato production and other host plants and depending on the current situation in the occurrence or the level of risk of introduction of the ring agent and the brown rot agent and shall be published in the Official Journal of the Central Control and Testing Institute of the Agricultural Organisation ("Bulletin '). Testing of the samples taken for the detection and determination of the ring agent and the brown rot agent shall be carried out in accordance with the method set out in Annex 1 or established by the Institute in accordance with Section 10 (1) of the Act and published in the Bulletin.
(9) Samples officially collected or tested by a person responsible for this activity by the Institute may also be used for the survey carried out in accordance with paragraphs 2 and 4, provided that it is ensured, on the basis of the regular supervision of that administration, that the samples are taken and tested in a manner determined in accordance with paragraph 8.
(10) The results of the survey carried out in accordance with paragraphs 2 to 6 shall be notified in writing by the Institute to the legal or natural person who owns or is authorised to place the lot on the market or cultivate it or grow it without undue delay.
(11) The details of the survey referred to in paragraphs 2 to 8 shall be submitted by the Institute once a year in accordance with Article 10 (5) of the Act to the other Member States and to the Commission in order to ensure a comparable level of confirmation of the absence of the circling agent and of the brown rot agent between Member States. The results of official surveys carried out pursuant to paragraphs 2 to 6 shall be notified once a year by the Institute to the other Member States and to the Commission, in the case of research in potato propagating material, (2) propagated only for planting on the parcels of the propagator, by 1 September, in other cases by 1 June. Data on the details and results of the survey concerning host plant crops shall always relate to the production of the previous year. The details of this notification shall be confidential.
Measures to detect suspected occurrence of ring agent or brown rot agent
(1) In accordance with § 11 (1) of the Act, the presence of a ring rot agent or a brown rot agent is considered to be suspected
(a) by the constitution of a verified detection of visual signs of infestation by these harmful organisms and, in the case of the agent of brown rot, a positive result of the rapid screening test referred to in Annex 1, Section I, number 1 and Section II; or
(b) the positive result of the screening test carried out by or under the supervision of the Institute as set out in Annex 1.
(2) In the event of a suspected occurrence of a ring agent or of a brown rot agent, the Institute shall, in the context of a professional investigation pursuant to Section 76 (7) of the Act and the Specific Legislation (3), ensure that official laboratory testing is carried out in accordance with the procedure laid down in Section 3 (8) and subject to the conditions laid down in Annex 2 (B), in order to confirm or deny such suspicion.
(3) The Institute shall immediately initiate an investigation in accordance with Section 76 (2) of the Act in order to establish the origin of the suspected occurrence and in accordance with Section 11 (1) of the Act, following a suspicion of the occurrence of ring rot agent or brown rot agent
(a) prohibit the movement of all lots or consignments from which samples have been taken, except those subject to measures under the supervision of the Institute, which excludes the identifiable risk of spreading the ring agent or the brown rot agent; and
(b) order appropriate measures based on the estimated risk of further possible spread of the ring rot agent or brown rot agent to prevent such spread, including the prohibition of movement or the provision of official supervision of the movement of all other host plant batches on land and objects associated with the suspected occurrence and, where appropriate, outside such parcels and objects.
(4) Where there is a suspected risk of infestation of lots of host plants or, in the case of an agent of brown rot, surface water originating in another Member State or in another Member State, the Institute shall immediately communicate to the official phytosanitary service of the Member State concerned the details of that suspicion and shall cooperate with that State in an appropriate manner.
(5) If the Czech Republic is informed of a suspected occurrence of a ring rot agent or a brown rot agent which may originate in the Czech Republic or which may threaten the Czech Republic, the Institute shall order appropriate measures in accordance with paragraphs 2 and 3.
Measures to confirm the occurrence of ring agent or brown rot agent
(1) If the official laboratory testing of the samples taken in accordance with § 3 (8) confirms the presence of the ring agent or of the brown rot agent, the Institute in accordance with § 76 (2) of the Act and taking into account the current scientific results, the biology of the ring agent and the brown rot agent and the way in which the host plants are grown, placed on the market and industrial processing
(a) identify as infested plants, consignments or lots, and equipment, machinery, means of transport, warehouses or parts thereof and any other objects including the packaging material from which the sample was taken and which have come into contact with the specified plant material from which the sample was taken, and the place or places of production, including those where the complete replacement of the growing medium is possible and the land on which the infested plants were grown and from which the sample was taken;
(b) carry out expert investigations to determine the extent of the probable contamination and the primary source of the disease in accordance with point 1 of Annex No 3, including testing of cloned host plant batches as referred to in Article 6, and determine, in accordance with point 2 of Annex No 3, the extent of the likely contamination through pre-harvest or post-harvest contact, and on the basis of cloning, cultivation, irrigation and watering or other cultivation links with the infected object or object identified under point (a);
(c) define the quarantine area on the basis of the designation of the contaminated objects or objects referred to in (a) and the safety zone on the basis of the determination of the extent of the likely contamination referred to in (b) and the possible extension of the circling agent or brown rot agent in accordance with the provisions of point 3 of Annex 3.
(2) In case of confirmation of the presence of the agent of brown rot with regard to surface water, waste water from the processing and packaging plant and with regard to host plants of the eggplant family from accompanying vegetation, through which the cultivation of potatoes, the Institute, through irrigation, watering or flooding of surface water, could be at risk
(a) ensure official laboratory testing of samples of surface water and, where appropriate, host plants from the aubergine family in the manner set out in Annex No 1, at appropriate times to determine the extent of contamination;
(b) indicate, in accordance with the positive results of the official laboratory testing referred to in (a), the surface water from which the samples were taken as contaminated;
(c) define the quarantine area on the basis of the designation of surface water as contaminated in accordance with (b) and the safety zone on the basis of the determination of the extent of likely contamination by the agent of brown rot and its possible spread in accordance with point 3 of Annex 3.
(3) The Institute shall immediately notify the other Member States and the Commission, in accordance with the provisions of point 4 of Annex No 3, of the confirmed presence of the ring agent or of the brown rot agent referred to in paragraph 1 (a) or paragraph 2 (b), as applicable, and communicate details of the establishment of the quarantine area and the safety zone referred to in paragraph 1 (c) or paragraph 2 (c), as appropriate. The details of this notification shall be confidential.
(4) If another Member State confirms the presence of a ring agent or a brown rot agent in a plant, consignment or lot originating in the Czech Republic in a notification made by analogy in accordance with paragraph 3, the Institute shall, as appropriate and in accordance with the provisions of paragraphs 1 and 2, identify the contaminated objects and objects, determine the extent of the likely contamination and define the quarantine area and, where appropriate, the safety zone.
(5) By means of an official communication in the Bulletin, the Institute sets out the principles for conducting the expert investigation referred to in paragraph 1 (b) and for determining the extent of the quarantine territory and the safety zone as defined in paragraph 1 (c), paragraph 2 (c) and paragraph 4.
Testing of clones related parts of host plants
(1) The Institute shall provide laboratory testing in accordance with § 4 (2) of all supervised lots of host plants of clones related to lots that have been identified as infected under § 5 (1) (a), but in any case all clones related to the propagating material2) to the extent necessary to determine the initial source of the disease and the extent of possible contamination in order to determine the level of risk of further spread of the ring agent or of the brown rot agent.
(2) According to the results of the testing referred to in paragraph 1, the Institute shall, if necessary, identify other objects and objects as contaminated, extend the extent of possible contamination and re-establish the quarantine area and safety zone in accordance with points (a), (b) and (c) of Article 5 (1).
Measures to prevent the spread of ring rot agent and brown rot agent
(1) The plant health administration shall, in accordance with Article 11 (1) of the Act, order the following exceptional plant health measures under Article 76 (2) of the Act:
(a) a lot of host plants designated as contaminated in accordance with Article 5 (1) (a) shall not be planted and shall be handled under the supervision of the Institute in the manner set out in point 1 of Annex No 4, provided that the Institute has previously verified that there is no identifiable risk of spreading the ring agent or the brown rot agent and that the disposal requirements set out in Annex 5 are met in the case of industrial processing of that lot;
(b) a lot of host plants identified as likely to be contaminated in accordance with Article 5 (1) (b) may be used under the supervision of the Institute in the manner set out in points 2 and 3 of Annex No 4, where appropriate, unless the Institute has verified in advance that there is no identifiable risk of spreading the ring agent or the brown rot agent, and that the disposal requirements laid down in Annex 5, in the case of industrial processing of that lot, are met, and that, on the basis of the results of the testing of cloned related host plant parts referred to in Article 6, the batch is not identified as contaminated in accordance with Article 5 (1) (a);
(c) equipment, machinery, means of transport, warehouses or parts thereof, and any other objects, including packaging material, identified as infested in accordance with Article 5 (1) (a) or identified as likely to be infested in accordance with Article 5 (1) (b), shall either be destroyed or subjected to cleansing and disinfection using the appropriate methods set out in Annex 6, provided that, after cleaning and disinfection, such objects and objects are not considered to be contaminated or likely to be contaminated;
(d) in the quarantine territory and safety zone defined in accordance with § 5 (1) (c) or, where applicable, § 5 (2) (c), the measures provided for in the event of occurrence of ring agent in point 4 of Annex 4 and in the case of the occurrence of brown rot agent in point 5 of Annex 4 shall be implemented.
(2) The Institute may, in accordance with Article 11 (1) of the Act, impose more stringent exceptional plant health measures against the dissemination of ring agent or brown rot agent than those referred to in paragraph 1, provided that such measures are scientifically justified, satisfactory and effective. Details of these measures and the measures ordered under paragraph 1 (d) within the quarantine territory and in the security zone shall be communicated by the Institute to the other Member States and to the Commission together with the registration number of the obliged persons concerned, registered under Article 12 (1) of the Act.
(3) Pursuant to Article 76 (4) (b) of the Act, the Institute shall abolish the exceptional plant health measures ordered pursuant to paragraph 1 and paragraph 2 after their fulfilment and after the expiry of the period laid down to comply with them.
(4) In accordance with Section 7 (1) of the Act, the possession of a ring rot agent and a brown rot agent shall be prohibited, except for the retention of the ring rot agent for scientific, breeding or diagnostic purposes authorised by the Institute pursuant to Section 8 (1) of the Act, provided that the protective measures do not thereby interfere with their occurrence or risk of spreading them.
Testing of propagating material2) potato
(1) Propagating material2) Potato grown on the territory of the Czech Republic and marketed must be subjected to laboratory testing carried out in the framework of a screening survey pursuant to § 3 (2), to the extent specified in paragraph 2 and in accordance with § 3 (8) and (9), and found free from ring agent and brown rot agent.
(2) The testing referred to in paragraph 1 shall be carried out in the relevant harvest year:
(a) plants of breeding propagating material (4) of potato intended for the production of pre-stages and representative samples of all lots of potato propagating material, provided that, during the last harvest year, in a lot of propagating material2) of the potato originating in the Czech Republic, the presence of the ring variety agent has been confirmed, or
(b) if the presence of the agent of brown rot has been confirmed in a lot of propagating material2) of potato originating in the Czech Republic during the last harvest year, either:
1. plants of breeding propagating material (4) of potato and pre-stages and representative samples of all lots of potato basic propagating material in cases of a confirmed clonic link with a focus on that link; or
2. representative samples of all lots of potato basic propagating material or pre-stages and potato breeding material4) in cases where there is no evidence of a clone link; or
(c) plants of potato breeding material (4) or representative samples of all lots of potato basic propagating material or pre-stages, or of all lots of potato propagating material intended for propagation in the following year, provided that the presence of ring agent or brown rot agent has not been confirmed in the last harvest year in the potato reproductive material2) of origin of the Czech Republic.
Efficacy
This decree shall take effect on the day of its publication.
Minister:
Ing. Palas v. r.
Příloha č. 1
Annex No 1 to Decree No 331 / 2004 Coll.
A. METHODS OF DIAGNOSIS, DETECTION AND IDENTIFICATION OF THE ORIGIN OF COLUMN
The presented progress diagrams describe the different procedures that are included in:
(i) the diagnosis of ring size in potato tubers and potato plants;
(ii) detection of Clavibacter michiganensis subsp. sepedonicus in samples of potato tubers and potato plants;
(iii) identification of Clavibacter michiganensis subsp. sepedonicus.
GENERAL PRINCIPLES
Optimised protocols for different methods, validated reagents and details for the preparation of test and control materials are given in the Appendices. The list of laboratories involved in the optimisation and validation of protocols is contained in Appendix 1.
Since the protocols contain the detection of the quarantine organism and include the use of viable cultures of Clavibacter michiganensis subsp. sepedonicus as control materials, it is necessary to operate under appropriate quarantine conditions with appropriate waste disposal facilities and under the conditions of relevant authorisations issued by the Institute.
The test parameters shall ensure a permanent and reproducible detection of Clavibacter michiganensis subsp. sepedonicus as established thresholds for the selected methods.
The preparation of positive controls is absolutely essential.
Testing according to required thresholds also includes correct setting, maintenance and calibration of equipment, careful handling and storage of reagents and any measures to avoid contamination between samples, e.g. separation of positive controls from tested samples. Standard quality management must be applied in order to avoid administrative and other errors, particularly in labelling and documentation.
The suspected occurrence as referred to in § 4 (1) indicates a positive result from the diagnostic or screening tests carried out on the sample as shown in the following flow diagrams.
If the first screening test (IF or PCR / FISH) is positive, there is a suspected ring agent infection and a second screening test must be performed. If the second screening test is positive, then the suspected occurrence is confirmed and testing according to the scheme must be continued. If the second screening test is negative, then the sample is not considered to be infected by the ring agent.
Therefore, a positive IF test under § 4 (1) is defined as a positive IF test result confirmed by a second screening test (PCR / FISH).
The confirmed presence requires the isolation and identification of a pure culture of Clavibacter michiganensis subsp. sepedonicus with a pathogenicity confirmation.
1. Use of progress diagrams
1.1. Progress diagram for the diagnosis of bacterial circling in potato tubers and potato plants showing signs of bacterial circling
The test procedure is intended for potato tubers and plants with suspected occurrence or typical signs of circling. Includes a rapid screening test, isolation of the pathogen from the infected conductive tissue on the diagnostic medium and, in the case of a positive result, identification of the clavibacter michiganensis subsp. sepedonicus culture.
(1) The description of the symptoms is in section 2.
(2) The appropriate tests are: - IF test (section 4), - PCR test (section 6), - FISH test (section 5).
(3) Although the isolation of pathogen from plant material with typical symptoms of spreading suspensions to media is not complicated, cultivation in advanced stages of infection may not be successful. Saprophytic bacteria that grow on the infected tissue may overgrow or suppress the pathogen on the isolation medium. It is therefore recommended to use non-selective and selective media, preferably MTNA (section 8) or biotest (section 7).
(4) A description of the colony's typical morphology is in Section 8.
(5) If the isolation test is negative but the symptoms of the disease are typical, the isolation must be performed again.
(6) Reliable identification of the pure culture Clavibacter michiganensis subsp. sepedonicus is achieved using the tests referred to in Section 9.
(7) The pathogenicity test is described in Section 10.
1.2. Progressive diagram for the diagnosis of bacterial ring count in samples of unsignified potato tubers
The testing procedure is designed to detect latent infections in potato tubers. A positive result from at least two screening tests, each based on a different biological principle, shall be supplemented by the isolation of the pathogen and subsequently, in case of isolation of typical colonies, by confirming that the pure culture is Clavibacter michiganensis subsp. sepedonicus. A positive result of only one screening test is not sufficient to make the sample considered suspicious.
Screening and isolation tests shall allow detection threshold 103 to 104 cells / ml of resuspended pellet included as positive controls in each series of tests.
(1) The standard sample size is 200 tubers, although the procedure can be applied to a smaller number if 200 tubers are not available.
(2) Methods of extraction and concentration of pathogen are described in section 3.1.
(3) If at least two tests based on different biological principles are positive, isolation and confirmation must be carried out. At least one screening test shall be carried out. If this test is negative, the sample is considered negative. If this test is positive, one or more screening tests based on different biological principles are necessary to verify the first positive result. If the second or other test is negative, the sample is considered negative. No further tests necessary.
(4) Immunofluorescence (IF) test. For IF testing, polyclonal antibody is always used, and other monoclonal antibodies allow greater accuracy (see section 4).
(5) PCR test. Appropriate validated PCR reagents and protocols shall be used (see Section 6).
(6) FISH test. Validated reagents and protocols shall be used (see Section 5).
(7) Selective isolation. This may in many cases be an appropriate method for direct isolation of Clavibacter michiganensis subsp. sepedonicus using MTNA medium or NCP-88 medium and dilution of resuspended pellet 1 / 100. Typical colonies can be obtained within 3-10 days after spreading to medium. The pathogen culture can then be cleaned and identified. In order to fully exploit the potential of the test, careful preparation of the cuticle tissue requires the reduction of secondary bacteria that are competing with Clavibacter michiganensis subsp. sepedonicus on the medium and which may overgrow the pathogen. If the culture method fails so, the isolation must be made from plants used for biotest (see Section 8).
(8) Biotest is used to isolate Clavibacter michiganensis subsp. sepedonicus from potato extract pellets by selective enrichment in eggplant plants (Solanum melongena). The test requires optimal incubation conditions for this method. Bacterial inhibitors of Clavibacter michiganensis subsp. sepedonicus on MTNA or NCP-88 are unlikely to interfere with this test (see section 7).
(9) Typical colony morphology is described in Section 8.
(10) Cultivation or biotests may fail because of competition or inhibition of saprophytic bacteria. If the results of the screening tests are positive but the isolation tests are negative, the isolation tests shall be repeated from the same pellet or by additional removal of the conductive mesh near the navel end of the tubers of the same sample and, if necessary, further samples shall be tested.
(11) Reliable identification of clean cultures suspected of Clavibacter michiganensis subsp. sepedonicus shall be achieved by using the tests described in Section 9.
(12) The pathogenicity test is described in section 10.
1.3. Progressive diagram for the diagnosis of bacterial ring rot in samples of unsignified potato plants
(1) Recommended sample sizes - see Section 3.2.
(2) Methods of extraction and concentration of pathogen are described in section 3.2.
(3) If at least two tests based on different biological principles are positive, isolation and confirmation must be carried out. At least one screening test shall be carried out. If this test is negative, the sample is considered negative. If this test is positive, a second or more screening tests based on different biological principles are necessary to verify the first positive result. If the second or further test is negative, the sample is considered negative. No further tests necessary.
(4) The selective isolation test and typical colony morphology are described in Section 8.
(5) The IF test is described in Section 4.
(6) The PCR test is described in Section 6.
(7) The FISH test is described in Section 5.
(8) The biotest is described in Section 7.
(9) Cultivation or bioassay may fail due to competition or inhibition of saprophytic bacteria. If the results of the screening tests are positive but the isolation tests are negative, the isolation tests shall be repeated and additional samples shall be tested if necessary.
(10) Reliable identification of pure cultures suspected to be Clavibacter michiganensis subsp. sepedonicus shall be achieved using the tests described in Section 9.
(11) The pathogenicity test is described in section 10.
2. Visual examination for the presence of circling symptoms
2.1. Potato plants
In European climate conditions, the signs in the field are rarely found and often at the end of the season. In addition, symptoms are hidden or confused with symptoms of other diseases, age or mechanical damage. Therefore, the symptoms may be easily overlooked during field inspections. The symptoms of fainting are very different from those of brown rot; fainting is usually slow and initially limited to the edges of the leaves. Young infected leaves often continue to grow despite infection, although growth in infected areas is limited. This creates unusually shaped leaves. Leaves affected by clogging conductive tissues on the lower part of the stem often have chlorotic, yellow to orange intercostal parts. Infected uterine leaves, leaves and even stems may eventually die. Often leaves and tubers are only smaller. Occasionally, plants are stunted. The colour images of a number of symptoms are available at http: / / forumeuropa.eu.int / Public / irc / sanco / Home / main.
2.2. Potato tubers
The earliest symptoms are weak glass or translucency of the skin without softening around the vascular system, especially near the navel. The ring of vascular bundles at the navel end of the tuber may have slightly darker coloring than usual. The first well-identified symptom is the yellowish colour of the ring of the vascular bundles and a condition where, when the tuber is pressed gently, they emerge from the blood vessels of the cheese column. This exudate contains millions of bacteria. The conductive tissue may turn brown and the symptoms on the tubers are similar at this stage to the signs of brown rot caused by Ralstonia solanacearum. Initially, these symptoms may be limited to one part of the ring, may not be just near the navel part and may gradually spread to the entire ring. With the procedure of infection, the conductive tissues are destroyed: the external cortical part can be separated from the internal cortical part. In advanced stages of infection, cracks appear on the surface of the tubers, often with red-brown edges. Recently, there have been several cases in Europe where the middle crust is rotting with the vascular ring, causing secondary damage to the formation of internal cavities and necrosis. Secondary fungal or bacterial infection may mask the symptoms and may be difficult, if not impossible, to distinguish the advanced signs of circling from other potato rot. Atypical symptoms are possible. The colour images of a number of symptoms are available at http: / / forumeuropa.eu.int / Public / irc / sanco / Home / main.
3. Preparation of samples
3.1. Potato tubers
Note:
- Standard sample size is 200 tubers per test. More intensive sampling requires more tests on samples of this size. The larger number of tubers in the sample leads to a slower or more complex interpretation of the results. However, the procedure may also be used appropriately for samples with less than 200 tubers, provided that fewer tubers are available.
- Validation of all the following detection methods is based on testing of samples of 200 tubers.
- The potato extract described below can also be used to detect the causative of brown rot, Ralstonia solanacearum.
Optional treatment before sample preparation:
The tubers are washed. Appropriate disinfectants (containing chlorine if PCR test is to be carried out to remove possible pathogenic DNA) and detergents between each sample shall be used. The tubers let the air dry. This washing procedure is particularly useful if there is too much soil in the sample and PCR test or direct insulation is to be carried out.
3.1.1. With a clean and disinfected scalpel or knife or potato scraper, the skin at the navel end of each tuber is removed. Care shall be taken to cut the conical incisions of the conductive mesh from the navel ends of the potatoes. The excess tissue not including vascular bundles shall be minimised. After removal, the flaps must be processed within 24 hours or preserved at -20 ° C for a maximum period of two weeks.
All tubers with suspected signs of bacterial circling shall be set aside and tested separately.
If signs of ring ulceration are detected when the stem is cut from the navel end, a visual examination of this tuber shall be carried out after the tuber has been cut at the navel end. All incised tubers with suspected symptoms are allowed to cork for 2 days at room temperature and are kept in quarantine at 4 - 10 ° C until all tests are completed. All tubers in the sample shall be kept in accordance with Annex 2.
3.1.2. The flaps of the navel shall be collected in unused disposable containers which are closed and / or sealed (in case the containers are reused, thoroughly cleaned and disinfected with chlorine-containing products). It is best to process the flaps from the navel end immediately, if this is not possible, stored in a container without the addition of a buffer, refrigerated for a maximum of 72 hours or at room temperature for a maximum of 24 hours. The drying and suberalisation of the props and the growth of saprophytes during storage may prevent the detection of the presence of a ring-ring bacterium.
3.1.3. Trimmings from the navel end shall be processed using one of the following procedures:
(a) the effluents are poured in sufficient quantities (approximately 40 ml) of the extraction buffer (Appendix 3) and shaken in a rotary shaker (50-100 rpm) for 4 hours at below 24 ° C or for 16-24 hours chilled;
or
(b) the pieces are homogenised with a sufficient quantity (approximately 40 ml) of the extraction buffer (Appendix 3), either in a blender or crushed in a sealed disposable maceration bag using a rubber fat or a suitable grinding device.
Note:
There is a high risk of cross-contamination of samples when samples are homogenised using a mixer. Safety precautions must be taken to avoid aerosol formation or spillage during extraction. Freshly sterilised blade (s) and container (s) must be used for each sample. If a PCR test is to be used, the transfer of DNA to containers or grinding equipment is to be avoided, crushing in disposable bags and using a disposable tube is recommended.
3.1.4. The supernatant decanting. If it is excessively cloudy, be cleaned either by slow centrifugation (at a maximum of 180 g for 10 minutes at 4- 10 ° C) or by vacuum filtration (40-100µm), wash the filter with an addition (10 ml) of the extraction buffer (Appendix 3).
3.1.5. The bacterial fraction is concentrated by centrifugation at 7000 g for 15 minutes (or 10,000 g for 10 minutes) at a temperature of 4- 10 ° C and the supernatant is removed without agitation of the pellet.
3.1.6. Resuspend the pellet in 1,5 ml pellet buffer (Appendix 3). 500 µl for Clavibacter michiganensis subsp. sepedonicus, 500 µl for Ralstonia solanacearum and 500 µl for reference purposes shall be used. Add sterile glycerol to a final concentration of 10- 25% (v / v) to 500 µl of the reference ratio and to the remaining part of the sample, mix by swirling and store at -16 to -24 ° C (weeks) or at -68 to -86 ° C (months). Extracts shall be stored at 4- 10 ° C during testing.
Repeated freezing and defrosting is not recommended.
Where transport of the extract is required, transport in the cooling box shall be ensured within 24 to 48 hours.
3.1.7. It is essential that with all positive controls and samples Clavibacter michiganensis subsp. sepedonicus was treated separately to prevent contamination. This also applies to the sliders for IF tests and all tests.
3.2. Potato plants
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Regulation Information
| Citation | Decree No 331 / 2004 Coll., on measures to ensure protection against the introduction and spread of potato ring agent and bacterial brown rot agent |
|---|---|
| Regulation Type | Order |
| Author | - |
| Collection | Code of Laws |
| Date of Promulgation | 31.05.2004 |
|---|---|
| Effective from | 31.05.2004 |
| Effective until | - |
| Status | Valid |
The regulation text is for informational purposes only.
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