http://www.legislation.gov.uk/id/eudr/1998/57/contents

SECTION IList of host plants of Ralstonia solanacearum (Smith) Yabuuchi et al. referred to in Article 1SECTION IISurveys1.The official surveys referred to in Article 2(2)(a) shall be...2.The notification of the official surveys referred to in Article...

ANNEX II TEST SCHEME FOR DIAGNOSIS, DETECTION AND IDENTIFICATION OF RALSTONIA SOLANACEARUM (SMITH) YABUUCHI ET AL.

SCOPE OF THE TEST SCHEME CONTENTS GENERAL PRINCIPLES SECTION I APPLICATION OF THE TEST SCHEME 1.Detection scheme for the diagnosis of brown rot and bacterial...2.Scheme for detection and identification of Ralstonia solanacearum in samples of...Principle: 3.Scheme for detection and identification of Ralstonia solanacearum in samples of...SECTION II DETAILED METHODS FOR DETECTION OF RALSTONIA SOLANACEARUM IN POTATO TUBERS AND POTATO, TOMATO OR OTHER HOST PLANTS WITH SYMPTOMS OF BROWN ROT OR BACTERIAL WILT 1.Symptoms (see website http://forum.europa.eu.int/Public/irc/sanco/Home/main) 1.1.Symptoms on potato 1.2.Symptoms on tomato 1.3.Symptoms on other hosts 2.Rapid screening tests 2.1.Stem streaming test 2.2.Detection of poly-ß-hydroxybutyrate (PHB) granules 2.3.Serological agglutination tests 2.4.Other tests 3.Isolation procedure 4.Identification tests for R. solanacearum SECTION III1.Detailed methods for detection and identification of Ralstonia solanacearum in samples...1.1.Sample preparation Note: 1.1.1.Remove with a clean and disinfected scalpel or vegetable knife the...1.1.2.Collect the heel end cores in unused disposable containers which...1.1.3.Decant the supernatant. If excessively cloudy, clarify either by slow...1.1.4.Concentrate the bacterial fraction by centrifugation at 7 000 g...1.1.5.Resuspend the pellet in 1.5 ml pellet buffer (Appendix 4). Use 500 µl...1.1.6.It is imperative that all R. solanacearum positive controls and...1.2.Testing 2.Detailed methods for detection and identification of R. solanacearum in samples...2.1.Sample preparation 2.1.1.Collect 1 to 2 cm stem segments in a closed sterile container...2.1.2.Disinfect stem segments briefly with ethanol 70 % and immediately blot dry...2.1.3.Decant the supernatant after settling for 15 minutes. 2.1.4.Further clarification of the extract or concentration of the bacterial fraction...2.1.5.Divide the neat or concentrated sample extract into two equal parts....2.2.Testing SECTION IV1.Scheme for detection and identification of R. solanacearum in water 2.Methods for detection and identification of R. solanacearum in water Principle 2.1.Sample preparation Note: 2.1.1.At selected sampling points, collect water samples by filling disposable...2.1.2.Transport samples in cool dark conditions (4 to 10 °C) and test...2.1.3.If required, the bacterial fraction may be concentrated using one...2.2.Testing SECTION V1.Scheme for detection and identification of R. solanacearum in soil 2.Methods for detection and identification of R. solanacearum in soil Principles 2.1.Sample preparation 2.1.1.Sampling of field soil should follow standard principals used for...2.1.2.Disperse sub-samples of 10 to 25 g of soil or sludge by...2.1.3.Maintain the suspension at 4 °C during testing. 2.2.Testing SECTION VI OPTIMISED PROTOCOLS FOR DETECTION AND IDENTIFICATION OF R. SOLANACEARUM A.DIAGNOSTIC AND DETECTION TESTS 1.Stem streaming test 2.Detection of poly-ß-hydroxybutyrate granules Nile blue test: Sudan Black test: 3.Serological agglutination tests 4.Selective isolation 4.1.Selective plating 4.1.1.Perform an appropriate dilution plating technique aiming to ensure that...4.1.2.Incubate plates at 28 °C. Read plates after 48 hours and daily...4.1.3.Purify presumptive R. solanacearum colonies after streaking or dilution plating...4.1.4.Store cultures short-term in sterile water (pH 6 to 8, chlorine...4.1.5.Identify presumptive cultures (see Section VI.B.) and perform a pathogenicity test...Interpretation of selective plating test results 4.2.Enrichment procedure 4.2.1.For enrichment-PCR, transfer 100 µl of sample extract into 10 ml of...4.2.2.Incubate for 72 hours at 27 to 30 °C in shaking culture or...4.2.3.Mix well before using in ELISA or PCR tests. 4.2.4.Treat enriched broth in an identical manner as the sample(s)...5.IF Test Principle 5.1.Prepare the test slides by one of the following procedures:...5.2.Dry the droplets at ambient temperature or by warming to...5.3.IF procedure 5.3.1.Arrange the slides on moist tissue paper. Cover each test...5.3.2.Incubate the slides on moist paper under a cover for...5.3.3.Shake the droplets off each slide and rinse carefully with...5.3.4.Arrange the slides on moist paper. Cover the test windows...5.3.5.Incubate the slides on moist paper under a cover for...5.3.6.Shake the droplets of conjugate off the slide. Rinse and...5.3.7.Pipette 5 - 10 µl of 0,1M phosphate-buffered glycerol (Appendix 4) or a...5.4.Reading the IF test: 5.4.1Examine test slides on an epifluorescence microscope with filters suitable...5.4.2.Observe for bright fluorescing cells with characteristic morphology of R. solanacearum...5.4.3.There are several problems inherent to the specificity of the...5.4.4.Consider only fluorescing cells with typical size and morphology at...5.4.5.Interpretation of the IF reading: 6.PCR tests Principles 6.1.DNA purification methods (a)Method according to Pastrik (2000) (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 2-5 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 L) for...6.3.3.Observe stained gel under short wave UV transillumination (λ = 302 nm) for...6.3.4.For all new findings/cases verify authenticity of the PCR amplicon...Interpretation of the PCR test result: 7.FISH test Principle 7.1.Potato extract fixation 7.1.1.Prepare fixative solution (see Appendix 7). 7.1.2.Pipette 100 µl of each sample extract into an Eppendorf tube...7.1.3.Remove the supernatant and dissolve the pellet in 200 µl of...7.1.4.Centrifuge for 7 minutes at 7 000 g, remove the supernatant...7.1.5.Spot 16 µl of the fixed suspensions onto a clean multitest...7.1.6.Air-dry the slides (or on slide dryer at 37 °C) and...7.2.Hybridisation 7.2.1.Dehydrate the cells in a graded ethanol series of 50 %,...7.2.2.Prepare a moist incubation chamber by covering the bottom of...7.2.3.Apply 10 μl of hybridisation solution (Appendix 7) to eight windows...7.2.4.Apply coverslips (24 × 24 mm) to the first and last...7.2.5.Prepare three beakers containing 1 l of Milli Q (molecular grade)...7.2.6.Remove the coverslips from the slides and place the slides...7.2.7.Wash away excess probe by incubation for 15 minutes in the...7.2.8.Transfer the slide holder to 1/8 hybmix washing solution and incubate for...7.2.9.Dip the slides briefly in Milli Q water and place...7.3.Reading the FISH test 7.3.1.Observe the slides immediately with a microscope fitted for epifluorescence...7.3.2.Observe for bright fluorescing cells with characteristic morphology of R. solanacearum...7.3.3.If any contamination is suspected the test must be repeated....7.3.4.There are several problems inherent to the specificity of the...7.3.5.Consider only fluorescing cells with typical size and morphology. 7.3.6.Interpretation of the FISH test result: 8.ELISA tests Principle (a)Indirect ELISA (Robinson Smith et al. , 1995) (b)DASI ELISA Interpretation of ELISA test results: 9.Bioassay test 9.1.Use 10 test plants of a susceptible tomato cultivar (e.g....9.2.Distribute 100 µl of sample extract between the test plants. 9.2.1.Syringe inoculation 9.2.2.Slit inoculation 9.3.Inoculate by the same technique, five seedlings with an aqueous...9.4.Grow the test plants in quarantine facilities for up to...9.5.Isolate from infected plants (Section II.3.) and identify purified cultures of presumptive...9.6.If no symptoms are observed after three weeks perform IF/PCR/Isolation...9.7.Identify any purified cultures of presumptive R. solanacearum (Section VI.B.). Interpretation of the bioassay test results B.IDENTIFICATION TESTS 1.Nutritional and enzymatic identification tests 2.IF test 2.1.Prepare a suspension of approximately 10 6 cells per ml...2.2.Prepare a twofold dilution series of an appropriate antiserum (see...2.3.Apply the IF procedure (Section VI.A.5.). 2.4.A positive IF test is achieved if the IF titre...3.ELISA test 3.1.Prepare a suspension of approximately 10 8 cells per ml...3.2.Perform an appropriate ELISA procedure with a specific monoclonal antibody...3.3.A positive ELISA test is achieved if the ELISA reading...4.PCR tests 4.1.Prepare a suspension of approximately 10 6 cells per ml...4.2.Heat 100 µl of the cell suspension in closed tubes in...4.3.Apply appropriate PCR procedures to amplify R. solanacearum -specific amplicons...4.4.A positive identification of R. solanacearum is achieved if the...5.FISH test 5.1.Prepare a suspension of approximately 10 6 cells per ml...5.2.Apply the FISH procedure (Section VI.A.7.) with at least 2 R....5.3.A positive FISH test is achieved if the same reactions...6.Fatty acid profiling (FAP) 6.1.Grow the culture on trypticase soy agar (Oxoid) for 48 hours...6.2.Apply an appropriate FAP procedure (Janse, 1991; Stead, 1992). 6.3.A positive FAP test is achieved if the profile of...7.Strain characterisation methods 7.1.Biovar determination 7.2.Genomic fingerprinting 7.2.1.Restriction fragment length polymorphism (RFLP) analysis (Cook et al. , 1989)....7.2.2.Repetitive sequence PCR using REP, BOX and ERIC primers (Louws...7.2.3.Amplified fragment length polymorphism (AFLP) analysis (Van der Wolf et al....7.3.PCR methods C.CONFIRMATION TEST

Appendix 1 Laboratories involved in optimisation and validation of protocols

Appendix 2 Media for isolation and culture of R. solanacearum

(a)General growth media Nutrient Agar (NA) Yeast Peptone Glucose Agar (YPGA) Sucrose Peptone Agar (SPA) Kelman’s Tetrazolium Medium (b)Validated selective growth media SMSA medium (Englebrecht, 1994 as modified by Elphinstone et al. ,...Note: 1.Use of reagents other than those specified above may affect...2.Oxoid Agar #1 can be used in place of Bacto-Agar...3.Increasing bacitracin concentration to 2 500 U per l may...(c)Validated enrichment media SMSA Broth (Elphinstone et al. , 1996) Modified Wilbrink broth (Caruso et al. , 2002)

Appendix 3

A.Commercially available standardised control material (a)Bacterial isolates (b)Commercially available standardised control material B.Preparation of positive and negative controls for the core screening...

Appendix 4 Buffers for test procedures

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 3.Buffers for the Indirect ELISA test 3.1.Double strength coating buffer, pH 9,6. 3.2.10X Phosphate buffered saline (PBS), pH 7,4 3.3.PBS-Tween 3.4.Blocking (antibody) buffer (must be freshly prepared). 3.5.Alkaline phosphatase substrate solution, pH 9,8 4.Buffers for DASI ELISA test 4.1.Coating buffer, pH 9,6 4.2.10X Phosphate saline buffer(PBS) pH 7,2 to 7,4 4.3.PBS-Tween 4.4.Substrate buffer, pH 9,8

Appendix 5 Determination of contamination level in IF and FISH tests

Count the mean number of typical fluorescent cells per field...

Appendix 6 Validated PCR protocols and reagents

1.PCR protocol of Seal et al. (1993) 1.1.Oligonucleotide primers 1.2.PCR reaction mix 1.3.PCR reaction conditions 1.4.Restriction enzyme analysis of amplicon. 2.PCR protocol of Pastrik and Maiss (2000) 2.1.Oligonucleotide primers 2.2.PCR reaction mix 2.3.PCR reaction conditions 2.4.Restriction enzyme analysis of amplicon. 3.Multiplex PCR protocol with internal PCR control (Pastrik et al. ,...3.1.Oligonucleotide primers 3.2.PCR reaction mix 3.3.PCR reaction conditions 3.4.Restriction enzyme analysis of amplicon. 4.R. solanacearum biovar-specific PCR protocol (Pastrik et al. , 2001) 4.1.Oligonucleotide primers 4.2.PCR reaction mix 4.3.PCR reaction conditions 4.4.Restriction enzyme analysis of amplicon. 5.Preparation of the loading buffer 5.1.Bromphenol blue (10 %- stock solution ) 5.2.Loading buffer 6.10X Tris Acetate EDTA (TAE) buffer, pH 8.0

Appendix 7 Validated reagents for FISH test

1.Oligo-probes 2.Fixative solution 3.3X Hybmix 4.Hybridisation solution 5.0,1M Phosphate buffer, pH 7,0

Appendix 8 Eggplant and tomato culture

Sow seeds of tomato ( Lycopersicon esculentum ) or eggplant...

Eggplants or tomatoes should be grown in a glasshouse with...

REFERENCES 1.Amann, R.I., L. Krumholz and D.A. Stahl. 1990. Fluorescent-oligonucleotide probing...2.Anon. 1998. Council Directive 98/57/EC of 20 July 1998 on the control...3.Boudazin, G., A.C. Le Roux, K. Josi, P. Labarre and...4.Caruso, P., Gorris, M.T., Cambra, M., Palomo, J.L., Collar, J...5.Cook, D., Barlow, E. and Sequeira, L. 1989. Genetic diversity...6.Elphinstone, J.G., Hennessy, J., Wilson, J.K. and Stead, D.E. 1996....7.Englebrecht, M.C. (1994) Modification of a semi-selective medium for the...8.Hayward, A.C. 1964. Characteristics of Pseudomonas solanacearum . Journal of...9.Hayward, A.C., El-Nashaar, H.M., Nydegger, U. and De Lindo, L....10.Ito, S., Y. Ushijima, T. Fujii, S. Tanaka, M. Kameya-Iwaki, S....11.Janse, J.D. (1988) A detection method for Pseudomonas solanacearum in...12.Janse, J.D. 1991. Infra- and intra-specific classification of Pseudomonas solanacaerum...13.Kelman, A. 1954. The relationship of pathogenicity of Pseudomonas solanacearum...14.Klement Z.; Rudolph, K and D.C. Sands, 1990. Methods in...15.Lelliott, R.A. and Stead, D.E. 1987. Methods for the diagnosis...16.Lopez, M.M., Gorris, M.T., Llop, P., Cubero, J., Vicedo, B.,...17.Louws, F.J., Fulbright, D.W., Stephens, C.T. and De Bruijn, F.J.,...18.Louws, F.J., Fulbright, D.W., Stephens, C.T. and De Bruijn, F.J....19.Opina, N., F. Tavner, G. Holloway, J.-F Wang, T.-H Li,...20.Pastrik, K.H. and Maiss, E. 2000. Detection of R. solanacearum in...21.Pastrik, K.H., Elphinstone, J.G. and Pukall, R. 2002. Sequence analysis and...22.Robinson-Smith, A., Jones, P., Elphinstone, J.G. and Forde, S.M.D. (1995)...23.Schaad, W. 2001. Laboratory guide for identification of plant pathogenic...24.Seal, S.E., L.A. Jackson, J.P.W. Young, and M.J. Daniels. 1993....25.Smith, J.J., Offord, L.C., Holderness, M. and Saddler, G.S. 1995. Genetic...26.Stead, D.E. 1992. Grouping of plant pathogenic and some other...27.Taghavi, M., Hayward, A.C., Sly, L.I., Fegan, M. 1996. Analysiss of...28.Van Der Wolf, J.M., Bonants, P.J.M., Smith, J.J., Hagenaar, M.,...29.Weller, S.A., Elphinstone, J.G., Smith, N., Stead, D.E. and Boonham,...30.Wullings, B.A., A.R. van Beuningen, J.D. Janse and A.D.L. Akkermans....

ANNEX III

For each suspected occurrence for which a positive result in...

ANNEX IV

The elements in the investigation referred to in Article 5(1)(a)(i) shall include...

places of production, growing or having grown, potatoes which are...

ANNEX V

The elements to be considered in the determination of the...

ANNEX VI

The provisions referred to in Article 6(1) shall be: use as animal...

ANNEX VII

The officially approved waste disposal methods referred to in Annex VI paragraph 1,...

potato and tomato waste (including rejected potatoes and peelings and tomatoes)...

The options described in this Annex also apply to the...

Council Directive 98/57/EC

of 20 July 1998

on the control of Ralstonia solanacearum (Smith) Yabuuchi et al.

THE COUNCIL OF THE EUROPEAN UNION,

Having regard to the Treaty establishing the European 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 the harmful organism Ralstonia solanacearum (Smith) Yabuuchi et al. was previously known as Pseudomonas solanacearum (Smith) Smith; whereas Ralstonia solanacearum (Smith) Yabuuchi et al. is likely to become the generally accepted name for the organism; whereas the present Directive should take account of this scientific development;

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

Whereas, through the protection of potato and tomato 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 if such organisms were not controlled simultaneously and methodically throughout the Community and were not prevented from spreading;

Whereas one of the harmful organisms on potatoes and tomatoes is Ralstonia solanacearum (Smith) Yabuuchi et al., the pathogenic agent of the potato brown rot disease and of bacterial wilt in potatoes and tomatoes; whereas disease outbreaks caused by this pathogen have occurred in some parts of the Community and some limited sources of infection still exist;

Whereas there is a considerable risk to potato and tomato cultivation throughout the Community if effective measures are not taken, with respect to these crops, to locate this organism 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 or tomatoes within the Community, except insofar as laid down in Council Directive 77/93/EEC of 21 December 1976 on protective measures against the introduction into the Community of organisms harmful to plants or plant products and against their spread within the Community4; whereas such measures must be notified to the other Member States and to the Commission;

Whereas the measures have to take into account that systematic official surveys are necessary to locate the pathogen; whereas such surveys should include inspection procedures and, where appropriate, given that under certain environmental circumstances the disease can remain latent and unobserved both in the growing crop of potatoes and in stored potato tubers, should include sampling and testing procedures; whereas spread of the pathogen within the growing crop is not the most important factor, but whereas the pathogen can spread by surface water and certain associated wild solanaceous plants, and therefore the irrigation of potato and tomato crops using contaminated water appears to pose a risk for infection of such crops; whereas also the pathogen can exist through the winter in self-sown (volunteer) potato and tomato plants and these may be a source of infection being carried from one season to the next; whereas the pathogen is spread also 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 spread of the pathogen can be reduced or prevented by decontamination of such objects; wheras any such contamination of seed potatoes poses a major risk for the spread of the pathogen; similarly the latent infection of seed potatoes poses a major risk for the spread of the pathogen and this can be prevented by the use of seed potatoes produced in an officially approved programme whereby seed potatoes have been tested and found free from infection;

Whereas the current knowledge of the biology and epidemiology of Ralstonia solanacearum (Smith) Yabuuchi et al. under European conditions is incomplete and it is anticipated that a review of the measures proposed will be necessary within several seasons; similarly improvements to the test procedure are anticipated in the light of further research especially on the sensitivity and specificity of test methods in order to select and standardise the optimum test methods available;

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: