Decree No. 328 / 2008 Coll.
Decree amending Decree No. 331 / 2004 Coll., on measures to ensure protection against the introduction and spread of potato ring agent and bacterial brown rot agent
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328
DECLARATION
of 19 August 2008
amending Decree No 331 / 2004 Coll., on measures to ensure protection against the introduction and spread of potato ring agent and bacterial brown rot agent
According to § 88 (2) of Act No. 326 / 2004 Coll., on Phytosanitary Care and on the Amendment of Certain Related Acts, as amended by Act No. 131 / 2006 Coll. and Act No. 249 / 2008 Coll., (hereinafter "the Act '), the Ministry of Agriculture provides for the implementation of § 71 (1) (h) of the Act:
Decree No 331 / 2004 Coll., on measures to ensure protection against the introduction and spread of potato ring agent and bacterial brown rot agent, is amended as follows:
1. Paragraph 1, including footnote 1, reads as follows:
Subject matter
This decree implements the relevant regulations of the European Communities (1) and regulates 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., originators 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.
1) Council Directive 93 / 85 / EEC of 4 October 1985 on the protection against potato ring rot. Council Directive 98 / 57 / EC of 20 July 1998 on protection against Ralstonia solanacearum (Smith) Yabuuchi et al. Commission Directive 2006 / 63 / EC of 14 July 2006 amending Annexes II to VII to Council Directive 98 / 57 / EC. ';
2. In Article 2 (k) (1), the words "in the case of tomato plants which have grown from the same seed lot," shall be added at the end of the text of point 1.
3. in Article 3 (2) (a), footnote 2 reads:
"(2) Paragraph 2 (1) (b) of Act No. 219 / 2003 Coll., on the circulation of seeds and propagating plants and amending certain laws, as amended."
4. in Article 3 (4) (a), the word "potato" is replaced by "potato" (2);
5. in Article 4 (2), footnote 3 reads:
"3) Paragraph 31 (2) of Decree No 215 / 2008 Coll., on measures to prevent the introduction and spread of harmful organisms of plants and plant products."
6. In Article 6 (1), the word "material 'is replaced by" material2),';
7. in Article 7 (1) (b):
"(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 plant health administration in the manner set out in points 2 and 3 of Annex No 4, where appropriate, provided that the plant health authority has verified in advance that there is no identifiable risk of spreading the ring agent or the brown rot agent and that the requirements for disposal laid down in Annex 5, in the case of industrial processing of that lot, are met, and that, on the basis of the testing of cloned host plant parts referred to in Article 6, this lot is not identified as contaminated in accordance with Article 5 (1) (a),";
8. in Article 8 (2) (a), footnote 4 reads:
"4) Paragraph 2 (1) (o) of Act No. 219 / 2003 Coll., on the circulation of seeds and propagating plants and amending certain laws, as amended."
9. In Article 8 (2) (c), the words "or all lots of potato propagating material intended for propagation in the following year 'shall be inserted after the words" or pre-stages';
10. Annex No 1 shall read as follows:
"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 appropriate authorisations issued by the plant health authority.
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 the final concentration of 10- 25% (v / v) to the 500 μl 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
Note:
For the detection of latent populations of Clavibacter michiganensis subsp. sepedonicus, it is recommended to check the combined samples. The procedure is suitable for combined samples of up to 200 parts of stems. (Where detailed surveys are carried out, they should be based on a statistically representative sample of the plant population under investigation.)
3.2.1. A clean disinfected knife or secateurs shall be removed from the bottom of each stem directly above the surface of the ground.
Part of the stems are briefly disinfected with ethanol 70%, immediately dried with absorbent paper and collected in a closed sterile container.
3.2.2. Parts of stems shall be processed by one of the following procedures:
(a) the parts are watered with a sufficient quantity (approximately 40 ml) of the extraction buffer (Appendix 3) and shaken in a rotary shaker (50- 100 rpm) for a period of 4 hours at below 24 ° C or for a period of 16-24 hours chilled,
or
(b) the parts are immediately crushed in a solid maceration bag with an adequate amount of extraction buffer (Appendix 3) using a rubber bat or a suitable grinding device. If this is not possible, parts of the stalks shall be stored refrigerated for a maximum of 72 hours or a maximum of 24 hours at room temperature.
3.2.3. The supernatant is decanted after settling for 15 minutes.
3.2.4 Further purification of the extract or concentration of the bacterial fraction is usually not necessary, but can be achieved by filtration and / or centrifugation as described in section 3.1.3.-3.1.6.
3.2.5 Clean or concentrated sample extract is divided into 2 equal parts, one half is maintained during testing at 4- 10 ° C and the other half is stored at 10- 25% (v / v) sterile glycerol at -16 to -24 ° C (weeks) or at -68 to -86 ° C (months) if further testing is necessary.
4. IF test
PRINCIP
It is recommended to use the IF test as the main screening test as it has been shown to reach the required thresholds.
If the main screening test is used and the result is positive, a PCR or FISH test shall be performed as the second screening test. If the IF test is used as a second screening test and the result is positive, further testing according to the flow diagram is necessary to complete the analysis.
Note:
If the IF test is used as the main investigation test, polyclonal antibody is always used. In case of a positive IF test result with polyclonal antibody, further examination of the monoclonal antibody sample may be more accurate but less sensitive.
Antibodies against known strain of ring agent - ATCC33113 (NCPPB 2137) or NCPPB 2140 are used. It is recommended to determine the titre for each new series of antibodies. The titre is determined as the highest dilution at which an optimal reaction occurs in the test of a suspension containing 105 to 106 cells per ml of the homologous strain of the ring agent and using a suitable fluorescence isothiocyanate conjugate (FITC) as recommended by the manufacturer. Unprocessed polyclonal or monoclonal antibodies should have IF titre at least 1: 2000. During the test, antibodies should be used in working dilution (WD) near the titre or on the titre. Validated antibodies are used (see website http: / / forum.eu.int / Public / irc / sanco / Home / main.
The test is performed on freshly prepared sample extracts. If necessary, it can be successfully performed on extracts stored at - 68 to - 86 ° C in glycerol. Glycerol can be removed from the sample by adding 1 ml of pellet buffer (Appendix 4), centrifugation for 15 minutes at 7,000 g and re-suspension at the same amount of pellet buffer. This is often not necessary, especially if the slides with the samples are fixed by flame (see 4.2).
A separate positive control slide shall be prepared with a homologous strain or other comparator strain of ring agent suspended in a potato leaching according to Appendix 2 and optional in the buffer.
The skin infected by natural means (stored by lyophilisation or freezing at -16 to -24 ° C) should be used as a parallel control on the same slide as possible.
The aliquot parts of sample extracts previously tested with negative results shall be used as negative controls.
Microscopic slides with multiple windows shall be used, preferably with 10 windows with a minimum diameter of 6 mm.
The control material shall be tested in the same way as the sample test.
4.1. The test slip is prepared by one of the following procedures:
(a) For pellets with a relatively low quantity of starch sediment:
Pipette the measured standard quantity (15 μl is suitable for windows of 6 mm diameter - for larger windows of volume higher) of the resuspended 1 / 100 potato pellet solution into the first window. Subsequently, the same amount of undiluted pellet (1 / 1) is piped into the remaining windows in the row. The second row may be used as a duplicate or for the second sample as shown in Figure 1.
(b) For other pellets:
Decimal dilution (1 / 10 and 1 / 100) of the resuspended pellet in pellet buffer shall be prepared. Pipette the measured standard quantity (15 μl is suitable for 6 mm diameter windows - higher volume windows) of the resuspended 1 / 100 pellet and each solution into a series of windows. The second row may be used as a duplicate or for the second sample as shown in Figure 2.
4.2. Dry the drops at room temperature or warm to 40 to 45 ° C. Bacterial cells are fixed to the slide either by heating (15 minutes at 60 ° C), above the flame or by using 95% ethanol or according to specific antibody suppliers' instructions.
If necessary, fixed slides may then be stored frozen in a dry box for as short a period as possible (maximum 3 months) before further use.
4.3. IF test procedure:
(a) According to the preparation of the test slide in section 4.1 (a):
Prepare a set of double antibody solutions in IF buffer. In the first hole, the titre is ½ (T / 2), the other titre (T / 4), the titre ½ (T / 2), the titre (T) and the double titre (2T).
(b) According to the preparation of the test slide in section 4.1 (b):
Prepare working dilution (WD) of antibodies in IF buffer. Working dilution affects accuracy.
Figure 1: Preparation of the test slide according to Section 4.1 (a) and Section 4.3 (a)
Figure 2: Preparation of the test slide according to sections 4.1 (b) and 4.3 (b)
4.3.1. The slides are arranged on wet paper. Each test window is covered by complete dilution of antibodies. The amount of antibody on each window shall be at least equal to the quantity of extract used.
If specific instructions from the antibody supplier are not available, the following shall be followed:
4.3.2. The slides are incubated on wet paper covered for 30 minutes at room temperature (18- 25 ° C).
4.3.3. Shake off drops from all slides and rinse them thoroughly with IF buffer. Wash by immersion for 5 minutes in IF Tween buffer (Appendix 3) and then in IF buffer. The formation of aerosol or transfer of droplets which could cause mutual contamination should be avoided and the excess moisture removed carefully by gently drying.
4.3.4. Place it on wet paper. The test windows shall be covered by dilution of the FITC conjugate determining the titre. The amount of conjugate placed in the windows must be the same as the amount of antibody used.
4.3.5. The covered slides are incubated on wet paper for 30 minutes at room temperature (18- 25 ° C).
4.3.6. The conjugate drops are shaken from the slides and rinsed and washed as before (4.3.3.).
Carefully remove excess moisture.
4.3.7. Pipette 5-10 μl of 0,1M phosphate buffer with glycerol (Appendix 3) or commercial covering fluid into each window and insert a cover slide.
4.4. Evaluation of IF test
4.4.1. Test slides are examined with an epifluorescence microscope with filters suitable for excitation of FITC under oil or water imersi at an magnification of 500 to 1000 times. The windows shall be examined in two perpendicular diameters and around the perimeter. For samples with no or small number of cells, at least 40 microscope fields shall be examined.
A positive control sample shall be checked first. The cells must be clearly fluorescent and completely dyed in a specified antibody titre or working dilution. If the colour is different, the IF test shall be repeated (section 4).
4.4.2. Clear fluorescent cells with the characteristic morphology of Clavibacter michiganensis subsp. sepedonicus are observed in slide testing windows (see website http: / / forum.europa.eu.int / Public / irc / sanco / Home / main. Fluorescence intensity must be the same or better when compared to a positive control strain in the same antibody dilution. Cells with incomplete discolouration or weak fluorescence cannot be taken into account.
If any contamination is suspected, the test shall be repeated. This can happen when all the slides in the group show positive cells due to the contamination of the buffer or when positive cells (outside the slide windows) are detected on the slide surface.
4.4.3. There are several problems relevant to the accuracy of the immunofluorescence test. An accompanying population of fluorescent cells with atypical morphology and cross-reacting saprophytic bacteria with size and morphology similar to Clavibacter michiganensis subsp. sepedonicus may occur in the navel parts of the potato and stem.
4.4.4. Only fluorescent cells with typical size and morphology in titre or working dilution of antibodies according to section 4.3 are taken into account.
4.4.5. Interpretation of IF test result:
(a) When clearly fluorescent cells with typical morphology are detected, the average number of typical cells in 1 microscopic field is estimated and the number of typical cells per ml of resuspended pellet (Appendix 4) is calculated.
The IF result is positive for samples where the number of typical cells per ml of resuspended pellet is at least 5x103. The sample is considered potentially infected and further testing is mandatory.
(b) The IF test result is negative for samples containing less than 5x103 cells per ml of resuspended pellet and the sample is considered negative. Further testing is not necessary.
5. FISH test
PRINCIP
When a FISH test is used as the first screening test and is positive, the IF test must be performed as the second mandatory screening test. When the FISH test is performed as a second screening test and is positive, further testing according to the progress diagram is necessary to complete the diagnosis.
Note:
Validated oligoprobes specific to Clavibacter michiganensis subsp. sepedonicus (Appendix 7) are used. Initial testing by this method should allow reproducible detection of at least 103- 104 Clavibacter michiganensis subsp cells. sepedonicus per ml added to sample extracts previously tested with negative results.
The following procedure should preferably be carried out with freshly prepared extracts, but may be successfully performed with an extract that has been stored in glycerol at -16 to -24 or -68 to -86 ° C.
The aliquot part of the sample extract previously tested for Clavibacter michiganensis subsp. sepedonicus shall be used as a negative control.
A suspension containing 105 to 106 cells of Clavibacter michiganensis subsp. sepedonicus per ml (e.g. NCPPB strain 4053 or PD 406) in 0,01 M phosphate buffer (PB) from 3-5 day culture is prepared (see Appendix 2 for preparation). Separate slides with positive control samples of the homological strain or other reference strain Clavibacter michiganensis subsp. sepedonicus suspended in potato extract according to Appendix 2 shall be prepared.
The use of FITC-labelled eubacterial oligoprobes provides a control of the hybridization process, as it will colour all eubacteria present in the sample.
The test of the control material shall be carried out in the same way as for the samples.
5.1. Fixing of potato extract
5.1.1. Prepare the fixation solution (see Appendix 7)
5.1.2. Pipette 100 μl of each sample extract into the Eppendorf microcentrifuge and centrifuge for 8 minutes to 7,000 g.
5.1.3. Remove the supernatant and dissolve the pellet in 500 μl of the fixation solution of the prepared max. 24 hours in advance. Shake and incubate overnight at 4 ° C.
The alternative fixative agent is 96% ethanol. For its use, the pellet is dissolved from step 5.1.2 in 50 μl 0,01 M PB and 50 μl 96% ethanol. Mix by shaking and incubate at 4 ° C for 30-60 minutes.
5.1.4. Centrifuge for 8 minutes at 7,000 g, remove the supernatant and re-suspend the pellet in 75 μl 0,01 M PB (see Appendix 3).
5.1.5. The 16 μl fixed suspension shall be dripped to a clean 10-window slide, as shown in Figure 3, using 2 different samples per slide, undiluted and diluted with 1: 100 using 10 μl (in 0,01 M PB). The remaining sample solution (49μl) may be stored at -20 ° C after addition of 1 volume of 96% ethanol. If the FISH method has to be repeated, remove ethanol by centrifugation, add the same amount of 0,01 M PB and mix by shaking.
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Regulation Information
| Citation | Decree No. 328 / 2008 Coll., amending 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 | 02.09.2008 |
|---|---|
| Effective from | 02.09.2008 |
| Effective until | - |
| Status | Valid |
The regulation text is for informational purposes only.
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