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Article 1.The Annexes to Directive 93/85/EEC are hereby replaced by the...
Article 2.(1) Member States shall adopt and publish, by 31 March 2007 at...
Article 3.This Directive shall enter into force on the third day...
3.1.1. Remove with a clean and disinfected scalpel or vegetable knife the...
3.1.2. Collect the heel end cores in unused disposable containers which...
3.1.3. Process the heel end cores by one of the following...
3.1.4. Decant the supernatant. If excessively cloudy, clarify either by slow...
3.1.5. Concentrate the bacterial fraction by centrifugation at 7 000 g for...
3.1.6. Resuspend the pellet in 1,5 ml pellet buffer (Appendix 3). Use 500 µl...
3.1.7. It is imperative that all C. m. subsp. sepedonicus positive...
3.2.1. With a clean disinfected knife or pruning shears, remove a...
3.2.2. Process the stem segments by one of the following procedures:...
3.2.3. Decant the supernatant after settling for 15 minutes.
3.2.4. Further clarification of the extract or concentration of the bacterial fraction...
3.2.5. Divide the neat or concentrated sample extract into 2 equal parts....
4.1. Prepare the test slides by one of the following procedures:...
4.2. Dry the droplets at ambient temperatures or by warming at...
4.3. IF procedure: According to test slide preparation in 4.1(i): Prepare a...
4.3.1. Arrange the slides on moist paper. Cover each test window...
4.3.2. Incubate the slides on moist paper under a cover for...
4.3.3. Shake the droplets off each slide and rinse carefully with...
4.3.4. Arrange the slides on moist paper. Cover the test windows...
4.3.5. Incubate the slides on moist paper under a cover for...
4.3.6. Shake the droplets of conjugate off the slide. Rinse and...
4.3.7. Pipette 5 to 10 µl of 0,1M phosphate-buffered glycerol (Appendix 3) or a commercially...
4.4.1. Examine test slides on an epifluorescence microscope with filters suitable...
4.4.2. Observe for bright fluorescing cells with characteristic morphology of C....
4.4.3. There are several problems inherent to the specificity of the...
4.4.4. Consider only fluorescing cells with typical size and morphology at...
5.1.2. Pipette 100 µl of each sample extract into an Eppendorf tube...
5.1.3. Remove the supernatant and dissolve the pellet in 500 µl of...
5.1.4. Centrifuge for 8 min. at 7 000 g, remove the supernatant and...
5.1.5. Spot 16 µl of the fixed suspensions onto a clean multitest...
5.1.6. Air-dry the slides (or on slide dryer at 37 °C) and...
5.2. Pre-hybridisation and hybridisation
5.2.1. Prepare a lysozyme solution containing 10 mg lysozyme (Sigma L–6876) in...
5.2.2. Dehydrate the cells in a graded ethanol series of 50 %,...
5.2.3. Prepare a moist incubation chamber by covering the bottom of...
5.2.4. Prepare the hybridisation solution (Appendix 7) allowing 45 µl per slide, and...
5.2.6. Apply two coverslips (24 × 24 mm) to each slide without...
5.2.7. Prepare three beakers containing 1 l of Ultra pure water...
5.2.8. Remove the coverslips from the slides and place the slides...
5.2.9. Wash away excess probe by incubation for 15 mins. in...
5.2.10. Transfer the slide holder to 1/2 hybmix washing solution and incubate for a...
5.3.1. Observe the slides immediately with a microscope fitted for epifluorescence...
5.3.2. Observe for bright fluorescing cells with characteristic morphology of C....
5.3.3. If any contamination is suspected the test must be repeated....
5.3.4. There are several problems inherent to the specificity of the...
5.3.5. Consider only fluorescing cells with typical size and morphology, see...
6.2.1. Prepare test and control templates for PCR according to the validated...
6.2.2. Prepare the appropriate PCR reaction mix in a contamination-free environment...
6.2.4. Incorporate a negative control sample containing only PCR reaction mix...
6.2.5. Place tubes in the same thermal cycler which was used...
7.1. Distribute the whole of the remaining test aliquot of the...
7.5. As the positive controls, inoculate 5 plants with an aqueous...
7.6. As the negative control, inoculate 5 plants with sterile pellet...
7.7. Incubate plants in quarantine facilities for up to four weeks...
7.8. Examine regularly for symptoms starting after a week. Count the...
7.9. As soon as symptoms in eggplants are observed reisolation should...
7.10. Under certain circumstances, in particular where growing conditions are not...
8. ISOLATION OF C. M. SUBSP. SEPEDONICUS
Laboratories involved in optimisation and validation of protocols
Preparation of positive and negative controls for the core screening tests PCR/IF and FISH
Prepare 10 sterile 1,5 ml microvials with 900 µl of the resuspended...
Transfer 100 µl of the suspension of C. m. subsp. sepedonicus...
Establish decimal levels of contamination by further diluting in the...
The six contaminated microvials will be used as positive controls....
Prepare aliquots of 100 µl in sterile 1,5 ml microvials thus obtaining...
The presence and quantification of C. m. subsp. sepedonicus in...
For the PCR test perform DNA extraction from positive and...
Gram stain procedure (Hucker's modification) (Doetsch, 1981)
1. Anonymous, 1987. Scheme of the detection and diagnosis of the...
2. Bradbury, J. F., 1970. Isolation and preliminary study of bacteria...
3. Dinesen, I. G., 1984. The extraction and diagnosis of Corynebacterium...
4. Doetsch, R. N., 1981. Determinative methods of light microscopy. In:...
5. Hugh, R. and Leifson, F., 1953. The taxonomic significance of...
6. Janse, J. D., 1991. Infra- and intra-specific classification of Pseudomonas solanacearum...
7. Janse, J. D. and J. Van Vaerenbergh. The interpretation of...
8. Jansing, H. and K. Rudolph, 1998. Physiological capabilities of Clavibacter...
9. Kovacs, N., 1956. Identification of Pseudomonas pyocyanea by the oxidase...
10. Klement Z.; Rudolph, K and D. C. Sands, 1990. Methods...
11. Lelliott, R. A., 1966. The plant pathogenic coryneform bacteria. J....
13. Lelliott, R. A. and P. W., Sellar, 1976. The detection...
14. Li, X. and S.H. de Boer, 1995. Selection of Polymerase...
16. Pastrik, K.-H. and R.A. Rainey. 1999. Identification and differentiation of...
17. Pastrik, K.-H., 2000. Detection of Clavibacter michiganensis ssp. sepedonicus in...
18. Ramamurthi, C. S., 1959. Comparative studies on some Gram-positive phytopathogenic...
19. Schaad, W., Berthier-Schaad, Y., Sechler, A. and Knorr, D. (1999)...
20. Schaad, W. 2001. Laboratory guide for identification of plant pathogenic...
21. Skerman, V. B. D., 1967. A guide to the identification...
22. Smith, N. C.; Hennesy, J; Stead, D.E., 2001. Repetetive sequence-derived...
24. Stead, D.E. 1992. Grouping of plant pathogenic and some other...