GENERAL PRINCIPLES 1.FLOW CHART DIAGRAM PRESENTATION 1.1.Detection scheme for the diagnosis of Ring Rot in potato...1.2.Scheme for detection and identification of Clavibacter michiganensis ssp. sepedonicus in...Principle 1.3.Scheme for detection and identification of Clavibacter michiganensis ssp. sepedonicus in...2.VISUAL EXAMINATION FOR RING ROT SYMPTOMS 2.1.Potato plants 2.2.Potato tubers 3.SAMPLE PREPARATION 3.1.Potato tubers Note: 3.1.1.Remove with a clean and disinfected scalpel or vegetable knife the...Note: 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...Note: 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...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.Potato plants Note: 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.IF TEST Principle Note: 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.Reading the IF test: 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...4.4.5.Interpretation of the IF reading: 5.FISH TEST Principle Note: 5.1.Potato extract fixation 5.1.1.Prepare fixative solution (see Appendix 7). 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...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...5.2.5.Place slides on a hot plate at 45 °C and apply...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...5.2.11.Dip the slides briefly in UPW and place them on...5.3.Reading the FISH test 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...5.3.6.Interpretation of the FISH test result: 6.PCR TEST Principles Note: 6.1.DNA purification methods 6.1.(a)Method according to Pastrik (2000) 6.1.(b)Other methods 6.2.PCR 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...6.2.3.Add 5 µl of DNA extract per 25 µl PCR reaction in...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...6.3.Analysis of the PCR product 6.3.1.Resolve PCR amplicons by agarose gel electrophoresis. Run at least...6.3.2.Reveal DNA bands by staining in ethidium bromide (0,5 mg per...6.3.3.Observe stained gel under short wave UV transillumination (e.g. λ...6.3.4.For all new findings/cases verify authenticity of the PCR amplicon...Note: 7.BIOASSAY TEST Note: 7.1.Distribute the whole of the remaining test aliquot of the...7.2.Do not water eggplants for one to two days prior...7.3.Slit inoculation 7.3.1.Holding the plant between two fingers, pipette a drop (approximately...7.3.2.Using a sterile scalpel, make a diagonal slit, about 1,0 cm...7.3.3.Seal the cut with sterile vaseline from a syringe. 7.4.Syringe inoculation 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 Note: 8.1.Selective plating 8.1.1.From a 100 µl aliquot from a resuspended potato pellet sample...8.1.2.Isolation from undiluted potato pellet usually fails due to the...Note: 8.1.3.Incubate plates in the dark at 21 to 23 °C. 8.1.4.Initial examinations of the plates including, by reference to the...8.2.Purification of suspicious colonies Note: 8.2.1.Streak C. m. subsp. sepedonicus –like colonies on to one...8.2.2.Re-streak to establish purity. 8.2.3.Identify presumptive cultures (see section 9) and perform a pathogenicity test...9.IDENTIFICATION 9.1.Nutritional and enzymatic identification tests 9.2.IF-test 9.3.PCR test 9.4.FISH test 9.5.Fatty acid profiling (FAP) 9.6.BOX-PCR 10.CONFIRMATION TEST 10.1.Prepare an inoculum of approximately 10 6 cells per ml...10.2.Inoculate 5 to 10 eggplant stems of young seedlings at leaf...10.3.Incubate at 18 to 24 °C with sufficient light and high relative...10.4.Isolate from symptomatic plants by removing a section of stem...1.Buffers for extraction procedure 1.1.Extraction buffer (50 mM phosphate buffer, pH 7,0) 1.2.Pellet buffer (10 mM phosphate buffer, pH 7,2) 2.Buffers for the IF test 2.1.IF-Buffer (10 mM phosphate buffered saline (PBS), pH 7,2) 2.2.IF-buffer-Tween 2.3.Phosphate buffered glycerol, pH 7,6 (a)General growth media Nutrient agar (NA) Nutrient dextrose agar (NDA) Yeast peptone glucose agar (YPGA) Yeast extract mineral salts medium (YGM) (b)Validated selective growth media MTNA medium Note: NCP-88 medium Note: Note: 1.Multiplex PCR protocol with internal PCR control (Pastrik, 2000) 1.1.Oligonucleotide primers 1.2.PCR reaction mix 1.3.PCR reaction conditions Note: 1.4.Restriction enzyme analysis of amplicon. 2.Preparation of the Loading buffer 2.1.Bromphenol blue (10 %-stock solution) 2.2.Loading buffer 3.10x Tris Acetate EDTA (TAE) buffer, pH 8,0 1.Oligo-probes 2.Fixative solution 3.3x Hybmix 4.Hybridisation solution 5.0,1M Phosphate buffer, pH 7,0 Crystal violet solution Lugol's iodine Safranin counterstain solution Staining procedure REFERENCES 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....12.Lelliott, R. A., E. Billing and A. C. Hayward, 1966....13.Lelliott, R. A. and P. W., Sellar, 1976. The detection...14.Li, X. and S.H. de Boer, 1995. Selection of Polymerase...15.Mills, D., Russell, B., W. and J., W. Hanus, 1997....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...23.Sneath, P. H. A. and V. G. Collins, 1974. A study...24.Stead, D.E. 1992. Grouping of plant pathogenic and some other...25.Wullings, B. A.; van Beuningen, A. R.; Janse, J. D....

Council Directive 93/85/EEC

of 4 October 1993

on the control of potato ring rot

THE COUNCIL OF THE EUROPEAN COMMUNITIES,

Having regard to the Treaty establishing the European Economic Community, and in particular Article 43 thereof,

Having regard to the proposal from the Commission1,

Having regard to the opinion of the European Parliament2,

Having regard to the opinion of the Economic and Social Committee3,

Whereas potato production occupies an important place in Community agriculture; whereas the potato yield is constantly threatened by harmful organisms;

Whereas, through the protection of potato cultivation against such harmful organisms, not only should productive capacity be maintained but agricultural productivity should also be increased;

Whereas protective measures to prevent the introduction of harmful organisms into the territory of a Member State would have only a limited effect were such organisms not controlled simultaneously and methodically throughout the Community and not prevented from spreading;

Whereas one of the harmful organisms on potatoes is Clavibacter michiganensis (Smith) Davis et al. ssp. sepedonicus (Spieckermann et Kotthoff) Davis et al., the pathogenic agent of the potato ring rot disease; whereas this disease has occurred in some parts of the Community and some limited sources of infection still exist;

Whereas there is a considerable risk to potato cultivation throughout the Community if effective measures are not taken to locate this disease and determine its distribution, to prevent it from occurring and spreading, and, if found, to prevent its spread and to control it with the aim of eradication;

Whereas, in order to ensure this, certain measures must be taken within the Community; whereas Member States must, in addition, be able to take additional or stricter measures where necessary, provided that there is no hindrance to the movement of potatoes within the Community, except in so far as laid down in Council Directive 77/93/EEC of 21 December 1976 on protective measures against the introduction into the Member States of organisms harmful to plants or plant products4; whereas such measures must be notified to the other Member States and to the Commission;

Whereas Council Directive 80/665/EEC of 24 June 1980 on the control of potato ring rot5, laid down minimum measures to be taken by the Member States against potato ring rot;

Whereas, since then, there have been significant developments in the understanding of potato ring rot disease and the detection of the potato ring rot pathogen;

Whereas the application of the Community plant health regime to the Community as an area without internal frontiers has called for the re-examination and revision of some provisions of Directive 80/665/EEC;

Whereas, as a result of such re-examination, the provisions of Directive 80/665/EEC have been found insufficient, and further specification of measures is necessary;

Whereas, in that situation, Directive 80/665/EEC should be repealed and the necessary measures adopted;

Whereas the measures have to take into account, first, that the disease can remain latend and unobserved both in the growing crop and in stored tubers, and so can be effectively prevented only by production and use of seed potatoes free from infection and, secondly, that systematic official surveys are necessary to locate it; whereas spread of the pathogen within the growing crop is not the most important factor, but whereas the pathogen can exist through the winter in self-sown (volunteer) potato plants and these are the major source of infection being carried from one season to the next; whereas the pathogen is spread mainly by the contamination of potatoes through contact with infected potatoes and through contact with planting, harvesting and handling equipment or transport and storage containers which have become contaminated with the organism by previous contact with infected potatoes; whereas such contaminated objects can remain infectious for some time after such contamination; whereas spread of the pathogen can be reduced or prevented by disinfection of such objects; whereas any such contamination of seed potatoes poses a major risk for the spread of the pathogen;

Whereas, for the determination of the details of such general measures, as well as for those stricter or additional measures taken by Member States to prevent the introduction of the pathogen into their territory, it is desirable for Member States to cooperate closely with the Commission within the Standing Committee of Plant Health (hereinafter referred to as ‘the Committee’),

HAS ADOPTED THIS DIRECTIVE: