1998L0057 — EN — 30.07.2006 — 001.001

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This document is meant purely as a documentation tool and the institutions do not assume any liability for its contents

►B	COUNCIL DIRECTIVE 98/57/EC of 20 July 1998 on the control of Ralstonia solanacearum (Smith) Yabuuchi et al. (OJ L 235, 21.8.1998, p.1)

Amended by:

		Official Journal
		No	page	date
►M1	COMMISSION DIRECTIVE 2006/63/CE of 14 July 2006	L 206	36	27.7.2006

Corrected by:

C1	Corrigendum, OJ L 218, 18.8.1999, p. 30  (98/57)

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▼B

COUNCIL DIRECTIVE 98/57/EC

of 20 July 1998

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

Article 1

This Directive concerns the measures to be taken within the Member States against Ralstonia solanacearum (Smith) Yabuuchi et al., previously known as Pseudomonas solanacearum (Smith) Smith, (hereinafter referred to as ‘the organism’), in order to, with respect to the host plants of the organism listed in Annex I Section 1, (hereinafter referred to as ‘the listed plant material’):

Article 2

1.  Member States shall conduct annual systematic official surveys for the organism on the listed plant material originating in their territory. In order to identify other possible sources of contamination threatening the production of the listed plant material, Member States shall carry out a risk assessment and, unless no risk of spread of the organism has been identified during that assessment, they shall, in production areas of the listed plant material, conduct targeted official surveys for the organism on plants other than the listed plant material, including wild solanaceous host plants, as well as on surface water which is used for irrigation or spraying of listed plant material and on liquid waste discharged from industrial processing or packaging premises handling listed plant material and used for irrigation or spraying of listed plant material. The extent of these targeted surveys shall be determined according to the risk identified. Member States may also conduct official surveys for the organism on the material, such as growing medium, soil and solid waste from industrial processing or packaging premises.

2.  The official surveys provided for in paragraph 1 shall be carried out:

For these surveys, further details of the inspection procedures and the number, origin, stratification and timing of collection of samples shall be decided by the responsible official bodies within the meaning of Directive 77/93/EEC based on sound scientific and statistical principles and the biology of the organism and taking into account in the Member State concerned, the particular production systems of the listed plant material and, as appropriate, of other host plants of the organism.

3.  The details and results of the official surveys provided for in paragraph 1 shall be notiefied each year to the other Member States and to the Commission in accordance with the provisions of point 2 of Section II, Annex I. These notifications shall be submitted by 1 June except for potatoes use as farm-saved seed for which the notification shall be submitted by 1 September. The details and results for crops shall be related to the previous year's production. The details of these notifications may be submitted to the Committee.

4.  The following provision shall be adopted in accordance with the procedure laid down in Article 16a of Directive 77/93/EEC:

5.  The following provisions may be adopted in accordance with the procedure laid down in Article 16a of Directive 77/93/EEC:

Article 3

Member States shall ensure that the suspected occurrence or confirmed presence of the organism in their territory shall be reported to their own responsible official bodies.

Article 4

1.  In each case of suspected occurrence, the responsible official bodies of the Member State(s) concerned shall ensure completion of official or officially supervised laboratory testing using, for the listed plant material, the relevant method set out in Annex II and in accordance with the conditions specified in point 1 of Annex III, or, in all other cases, any other officially approved method, in order to confirm or refute the suspected occurrence. In the case of confirmation, the requirements laid down in point 2 of Annex III shall apply.

2.  Pending the confirmation or refutation of a suspected occurrence under paragraph 1, in each case of suspected occurrence where, either:

the responsible official bodies of the Member States shall, in relation to their own production:

3.  In those cases of suspected occurrence where there is a risk of contamination of the listed plant material or surface water from or into another Member State(s), the Member State in which the suspected occurrence has been reported shall immediately notify, according to the risk identified, the details of the said suspected occurrence to the other Member State(s) concerned, and there whall be appropriate cooperation between the said Member States. The Member State(s) so notified shall introduce precautionary measures in accordance with paragraph 2(c) and take any further action, as appropriate, in accordance with paragraphs 1 and 2.

4.  The following provision may be adopted in accordance with the procedure laid down in Article 16a of Directive 77/93/EEC:

Article 5

1.  If official or officially supervised laboratory testing, using, for the listed plant material, the relevant method set out in Annex II or, in all other cases, any other officially approved method, confirms the presence of the organism in a sample taken pursuant to this Directive, the responsible official bodies of a Member State, having regard to sound scientific principles, the biology of the organism and the particular production, marketing and processing systems of the host plants of the organism in that Member State, shall:

2.  Member States shall immediately notify the other Member States and the Commission, in accordance with the provisions of point 3 of Annex V, of any contamination designated under paragraphs 1(a)(ii) and 1(c)(ii) and the details of the zone demarcation under paragraph 1(a)(iv) and, where applicable, under paragraph 1(c)(iii). The details of the notification under this paragraph may be submitted to the Committee.

Member States shall at the same time submit to the Commission the additional notification set out at point 4 of Annex V. The details of the notification under this subparagraph shall immediately be submitted to the members of the Committee.

3.  As a result of the notification under paragraph 2 and the elements mentioned therein, other Member States detailed in the notification shall establish an investigation in accordance with paragraph 1(a)(i) and, where applicable, paragraph 1(c)(i) and take further action, as appropriate, in accordance with paragraphs 1 and 2.

Article 6

1.  Member States shall prescribe that the listed plant material designated to be contaminated under Article 5(1)(a)(ii) may not be planted and that, under the control and approval of their responsible official bodies, it shall be subjected to one of the provisions of point 1 of Annex VI, such that it is established that there is no identifiable risk of the organism spreading.

2.  Member States shall prescribe that the listed plant material determined as probably contaminated under Article 5(1)(a) (iii) and (c)(iii) including listed plant material for which a risk has been identified, produced on places of production determined as probably contaminated under Article 5(1)(a)(iii) may not be planted and shall, under the control of their responsible official bodies, be put to appropriate use or disposal as specified in point 2 of Annex VI, such that it is established that there is no identifiable risk of the organism spreading.

3.  Member States shall prescribe that any machinery, vehicle, vessel, store, or units thereof, and any other objects including packaging material, designated as contaminated under Article 5(1)(a)(ii) or determined as probably contaminated under Article 5(1)(a)(iii) and (c)(iii), shall either be destroyed or decontaminated using appropriate methods as specified in point 3 of Annex VI. After decontamination, any such objects shall no longer be considered contaminated.

4.  Without prejudice to the measures implemented under paragraphs 1, 2 and 3, Member States shall prescribe that, in the zone demarcated under Article 5(1)(a)(iv) and (c)(iii), a series of measures, as specified in points 4.1 and 4.2 of Annex VI, shall be implemented. Details of these measures shall be notified annually to the other Member States and to the Commission. The details of this notification may be submitted to the Committee.

Article 7

1.  Member States shall prescribe that seed potatoes shall meet the requirements of Directive 77/93/EEC and shall derive in direct line from potato material obtained under an officially approved programme which has been found free of the organism in official or officially supervised testing using the relevant method set out in Annex II.

The aforesaid testing shall be carried out by a Member State:

2.  The following provisions may be adopted in accordance with the procedure laid down in Article 16a of Directive 77/93/EEC:

Article 8

Member States shall ban the holding and handling of the organism.

Article 9

Without prejudice to the provisions of Directive 77/93/EEC, Member States may authorise derogations from the measures referred to in Articles 6 and 8 of this Directive in accordance with the provisions laid down in Directive 95/44/EC ( 5 ) for trial or scientific purposes, and for work on varietal selections.

Article 10

Member States may adopt in relation to their own production such additional or stricter measures as may be required to combat the organism or to prevent it from spreading, insofar as they are in compliance with the provisions of Directive 77/93/EEC.

The details of these measures shall be notified to the other Member States and to the Commission. The details of this notification may be submitted to the Committee.

Article 11

Amendments to the Annexes to this Directive, to be made in the light of developments in scientific or technical knowledge, shall be adopted in accordance with the procedure laid down in Article 16a of Directive 77/93/EEC. In the case of the methods laid down in Annex II and the measures in paragraphs 4.1 and 4.2 of Annex VI to this Directive a report shall be prepared by the Commission reviewing these methods and measures in the light of experience gained and the report shall be submitted to the Committee before 1 January 2002.

Article 12

1.  Member States shall bring into force the laws, regulations and administrative provisions necessary to comply with this Directive with effect from 21 August 1999. They shall forthwith inform the Commission thereof.

When Member States adopt these measures, they shall contain a reference to this Directive or shall be accompanied by such reference on the occasion of their official publication. The methods of making such reference shall be adopted by Member States.

2.  The Member States shall immediately communicate to the Commission the main provisions of domestic law which they adopt in the field governed by this Directive. The Commission shall inform the other Member States thereof.

Article 13

This Directive shall enter into force on the day of its publication in the Official Journal of the European Communities.

Article 14

This Direcitve is addressed to the Member States.

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ANNEX I

SECTION I

List of host plants of Ralstonia solanacearum (Smith) Yabuuchi et al. referred to in Article 1

SECTION II

Surveys

1.

The official surveys referred to in Article 2(2)(a) shall be based on the biology of the organism and the particular production systems in the Member State concerned and shall comprise: (i) in the case of potato, — at appropriate times, visual inspection of the growing crop, and/or sampling of both seed and other potatoes in the growing season or in store. These samples shall be subjected to official or officially supervised visual inspection by cutting of tubers, and — in the case of seed potatoes, and where appropriate for other potatoes, official or officially supervised laboratory testing using the method set out in Annex II, (ii) in the case of tomato, — visual inspection, at appropriate times, of at least the growing crop of plants intended for replanting for professional use.

2.

The notification of the official surveys referred to in Article 2(3) shall include: (i) in the case of surveys on potatoes, — estimated total area grown, in hectares, of seed and other potatoes, — stratification by seed category and ware, and where appropriate, by region, — number and timing of samples taken for testing, — number of visual inspections in the field, — number (and size of sample) of visual inspections on tubers; (ii) in the case of surveys on, at least, the growing crop of plants of tomato intended for replanting for professional use, — estimated total number of plants, — number of visual inspections; (iii) in the case of surveys on host plants other than potatoes and tomatoes, including wild solanaceous host plants, — species, — number and timing of samples taken, — area/river sampled, as appropriate, — method of analysis; (iv) in the case of surveys on water and liquid waste discharges from industrial processing or packaging premises, — number and timing of samples, — area/river/location of premises sampled, as appropriate, — method of analysis.

▼M1

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ANNEX II

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

SCOPE OF THE TEST SCHEME

The presented scheme describes the various procedures involved in:

CONTENTS

GENERAL PRINCIPLES

OptimiseOptimised protocols for the various methods, validated reagents and details for the preparation of test and control materials are provided in the Appendices. A list of the laboratories that were included in optimization and validation of protocols is provided in Appendix 1.

Since the protocols involve detection of a quarantine organism and will include the use of viable cultures of R. solanacearum as control materials, it will be necessary to perform the procedures under suitable quarantined conditions with adequate waste disposal facilities and under the conditions of appropriate licences as issued by the official plant quarantine authorities.

Testing parameters must assure consistent and reproducible detection of levels of R. solanacearum at the set thresholds of the selected methods.

Precise preparation of positive controls is imperative.

Testing according to the required thresholds also implies correct settings, maintenance and calibration of equipment, careful handling and preservation of reagents and all measures to prevent contamination between samples, e.g. separation of positive controls from test samples. Quality control standards must be applied to avoid administrative and other errors, especially concerning labelling and documentation.

A suspected occurrence, as referred to in Article 4(2) of Directive 98/57/EC implies a positive result in diagnostic or screening tests performed on a sample as specified in flow charts below. A positive first screening test (IF test, PCR/FISH, selective isolation) must be confirmed by a second screening test based on a different biological principle.

If the first screening test is positive, then contamination with R. solanacearum is suspected and a second screening test must be done. If the second screening test is positive, than the suspicion is confirmed (suspected occurrence) and the testing according to the scheme must be continued. If the second screening test is negative, then the sample is considered not contaminated with R. solanacearum.

Confirmed presence as referred to in Article 5(1) in Directive 98/57/EC implies the isolation and identification of a pure culture of R. solanacearum with confirmation of pathogenicity.

SECTION I

APPLICATION OF THE TEST SCHEME

1.   Detection scheme for the diagnosis of brown rot and bacterial wilt (Ralstonia solanacearum) in potato tubers and potato, tomato or other host plants with symptoms of brown rot or bacterial wilt.

The testing procedure is intended for potato tubers and plants with symptoms typical or suspect of brown rot or vascular wilt. It involves a rapid screening test, isolation of the pathogen from infected vascular tissue on (selective) medium and, in case of a positive result, identification of the culture as Ralstonia solanacearum.

2.   Scheme for detection and identification of Ralstonia solanacearum in samples of asymptomatic potato tubers

Principle:

The testing procedure is intended for detection of latent infections in potato tubers. A positive result from at least two screening tests (3), based on different biological principles, must be complemented by the isolation of the pathogen; followed by, in case of isolation of typical colonies, confirmation of a pure culture as R. solanacearum A positive result from only one of the screening tests is not sufficient to consider the sample suspect.

Screening tests and isolation tests must permit detection of 103 to 104 cells/ml of resuspended pellet, included as positive controls in each series of tests.

3.   Scheme for detection and identification of Ralstonia solanacearum in samples of asymptomatic potato, tomato or other host plants

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

The potato plant. The early stage of infection in the field is recognised by wilting of the leaves towards the top of the plant at high temperatures during the day with recovery at night. In early stages of wilting leaves remain green, but later yellowing and brown necrosis develops. Epinasty also occurs. Wilting of one shoot or whole plants becomes rapidly irreversible and results in the collapse and death of the plant. The vascular tissue of transversely cut stems from wilted plants usually appears brown and a milky bacterial ooze exudes from the cut surface or can be expressed by squeezing. When a cut stem is placed vertically in water, threads of slime will stream from the vascular bundles.

The potato tuber. Potato tubers must be cut transversely close to the heel (stolon end) or longitudinally over the stolon end. The early stage of infection is recognised by a glassy yellow to light brown discolouration of the vascular ring from which a pale cream bacterial ooze emerges spontaneously after some minutes. Later, the vascular discolouration becomes a more distinct brown and necrosis can extend into the parenchymatous tissue. In advanced stages, infection breaks outwards from the heel end and the eyes from which bacterial slime may ooze causing soil particles to adhere. Reddish-brown slightly sunken lesions may appear on the skin due to collapse of vascular tissues internally. Secondary development of fungal and bacterial soft rots is common in the advanced stages of the disease.

1.2.   Symptoms on tomato

The tomato plant. The first visible symptom is the flaccid appearance of the youngest leaves. Under favourable environmental conditions for the pathogen (soil temperatures of approximately 25 °C; saturated humidity), epinasty and wilting of one side or of the whole plant follows within a few days leading to total plant collapse. Under less favourable conditions (soil temperature below 21 °C), less wilting occurs, but large numbers of adventitious roots may develop on the stem. It is possible to observe watersoaked streaks from the base of the stem which is evidence of necrosis in the vascular system. When the stem is cut crosswise, discoloured brown vascular tissues exude white or yellowish bacterial ooze.

1.3.   Symptoms on other hosts

Solanum dulcamara and S. nigrum plants. Under natural conditions, wilting symptoms are rarely observed in these weed hosts unless soil temperatures exceed 25 °C or inoculum levels are extremely high (e.g. as for S. nigrum growing adjacent to diseased potato or tomato plants). When wilting does occur, the symptoms are as described for tomato. Non-wilting S. dulcamara plants growing with stems and roots in water may show internal light brown discolouration of vascular tissues on transverse section of the stem base or underwater stem parts. Bacteria may ooze from cut vascular tissues or form threads of slime if the cut stem is placed vertically in water, even in the absence of wilting symptoms.

2.   Rapid screening tests

Rapid screening tests may facilitate presumptive diagnosis but are not essential. Use one or more of the following validated tests:

2.1.   Stem streaming test

(See Section VI.A.1.)

2.2.   Detection of poly-ß-hydroxybutyrate (PHB) granules

Characteristic PHB granules in the cells of R. solanacearum are visualised by staining heat-fixed smears of bacterial ooze from infected tissue on a microscope slide with Nile Blue A or Sudan Black (See Section VI.A.2.).

2.3.   Serological agglutination tests

(See Section VI.A.3.)

2.4.   Other tests

Further appropriate rapid screening tests include the IF test (see Section VI.A.5.), FISH test (see Section VI.A.7.), ELISA tests (see Section VI.A.8.) and PCR tests (see Section VI.A.6).

3.   Isolation procedure

4.   Identification tests for R. solanacearum

Tests to confirm identity of presumptive isolates of R. solanacearum are shown in Section VI.B.

SECTION III

1.   Detailed methods for detection and identification of Ralstonia solanacearum in samples of asymptomatic potato tubers

1.1.   Sample preparation

Note:

Optional pre-treatment in advance to sample preparation:

1.1.1.

Remove with a clean and disinfected scalpel or vegetable knife the skin at the heel (stolon) end of each tuber so that the vascular tissues become visible. Carefully cut out a small core of vascular tissue at the heel end and keep the amount of non-vascular tissue to a minimum. (see web site http://forum.europa.eu.int/Public/irc/sanco/Home/main). Note: Set aside any (rotting) tubers with suspected brown rot symptoms and test separately. If during removal of the heel end core suspect symptoms of brown rot are observed, a visual inspection of this tuber should be done and the tuber cut near the heel end. Any cut tuber with suspected symptoms should be kept for at least two days at room temperature in order to allow suberisation and stored refrigerated (at 4 to 10 °C) under proper quarantine conditions. All tubers including those with suspicioussymptoms should be kept according to Annex III.

1.1.2.

Collect the heel end cores in unused disposable containers which can be closed and/or sealed (in case containers are reused they should be thoroughly cleaned and disinfected using chlorine compounds). Preferably, the heel end cores should be processed immediately. If this is not possible, store them in the container, without addition of buffer, refrigerated for not longer than 72 hours or for not longer than 24 hours at room temperature. Process the heel end cores by one of the following procedures: either, (a) Cover the cores with sufficient volume (approximately 40 ml) of extraction buffer (Appendix 4) and agitate on a rotary shaker (50-100 rpm) for 4 hours below 24 °C or for 16 to 24 hours refrigerated, or (b) Homogenise the cores with sufficient volume (approximately 40 ml) of extraction buffer (Appendix 4), either in a blender (e.g. Waring or Ultra Thurax) or by crushing in a sealed disposable maceration bag (e.g. Stomacher or Bioreba strong guage polythene, 150 mm × 250 mm; radiation sterilised) using a rubber mallet or suitable grinding apparatus (e.g. Homex). Note: The risk of cross-contamination of samples is high when samples are homogenized using a blender. Take precautions to avoid aerosol generation or spillage during the extraction process. Ensure that freshly sterilised blender blades and vessels are used for each sample. If the PCR test is to be used, avoid carry-over of DNA on containers or grinding apparatus. Crushing in disposable bags and use of disposable tubes is recommended where PCR is to be used.

1.1.3.

Decant the supernatant. If excessively cloudy, clarify either by slow speed centrifugation (at not more than 180 g for 10 minutes at a temperature between 4 to 10 °C) or by vacuum filtration (40 to 100 µm), washing the filter with additional (approximately 10 ml) extraction buffer.

1.1.4.

Concentrate the bacterial fraction by centrifugation at 7 000 g for 15 minutes (or 10 000 g for 10 minutes) at a temperature between 4 to 10 °C and discard the supernatant without disturbing the pellet.

1.1.5.

Resuspend the pellet in 1.5 ml pellet buffer (Appendix 4). Use 500 µl to test for R. solanacearum, 500 µl for Clavibacter michiganensis subsp. sepedonicus and 500 µl for reference purposes. Add sterile glycerol to final concentration of 10 to 25 % (v/v) to the 500 µl of the reference aliquot and to the remaining test aliquot, vortex and store at 16 to 24 °C (weeks) or at 68 to 86 °C (months). Preserve the test aliquots at 4 to 10 °C during testing. Repeated freezing and thawing is not advisable. If transport of the extract is required, ensure delivery in a cool box within 24 to 48 hours.

1.1.6.

It is imperative that all R. solanacearum positive controls and samples are treated separately to avoid contamination. This applies to IF slides and to all tests.

1.2.   Testing

See Flow chart and description of the tests and optimised protocols in the relevant appendices:

2.   Detailed methods for detection and identification of R. solanacearum in samples of asymptomatic potato, tomato or other host plants

2.1.   Sample preparation

Note: For detection of latent R. solanacearum populations it is advised to test composite samples. The procedure can be conveniently applied for composite samples of up to 200 stem parts. Where surveys are performed they should be based on a statistically representative sample of the plant population under investigation.

2.1.1.

Collect 1 to 2 cm stem segments in a closed sterile container according to the following sampling procedures: Nursery tomato seedlings: With a clean disinfected knife, remove a 1 cm segment from the base of each stem, just above the soil level. Field or glasshouse grown tomato plants: With a clean disinfected knife, remove the lowermost side shoot from each plant by cutting just above the joint with the main stem. Remove the lowermost 1cm segment from each side shoot. Other hosts: With a clean disinfected knife or pruning shears, remove a 1 cm segment from the base of each stem, just above the soil level. In the case of S. dulcamara or other host plants growing in water, remove 1-2 cm sections from underwater stems or stolons with aquatic roots. When sampling a particular location it is recommended to test a statistically representative sample of at least 10 plants per sampling point of each potential weed host. Pathogen detection will be most reliable during late spring, summer and autumn seasons, although natural infections can be detected all year round in the perennial Solanum dulcamara growing in watercourses. Known hosts include volunteer potato plants (groundkeepers), Solanum dulcamara, S. nigrum, Datura stramonium and other members of the family Solanaceae. Further hosts are Pelargonium spp. and Portulaca oleracea. Some European weed spp. which may potentially harbour R. solanacearum biovar 2/Race 3 populations in roots and/or rhizospheres under specific environmental conditions include Atriplex hastata, Bidens pilosa, Cerastium glomeratum, Chenopodium album, Eupatorium cannabinum, Galinsoga parviflora, Ranunculus scleratus, Rorippa spp, Rumex spp., Silene alba, S. nutans., Tussilago farfarra and Urtica dioica. Note: Visual examination for internal symptoms (vascular staining or bacterial ooze) can be done at this stage. Set aside any stem segments with symptoms and test separately (See Section II).

2.1.2.

Disinfect stem segments briefly with ethanol 70 % and immediately blot dry on tissue paper. Then process the stem segments by one of the following procedures: either, (a) Cover the segments with sufficient volume (approximately 40 ml) of extraction buffer (Appendix 4) and agitate on a rotary shaker (50 to 100 rpm) for four hours below 24 °C or for 16 to 24 hours refrigerated, or (b) Process immediately by crushing the segments in a strong maceration bag (e.g. Stomacher or Bioreba) with an appropriate volume of extraction buffer (Appendix 4) using a rubber mallet or appropriate grinding apparatus (e.g. Homex). If this is not possible, store the stem segments refrigerated for not longer than 72 hours or for not longer than 24 hours at room temperature.

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 are not usually required but may be achieved by filtration and/or centrifugation as described in Section III.1.1.3 – 1.1.5.

2.1.5.

Divide the neat or concentrated sample extract into two equal parts. Maintain one half at 4 to 10 °C during testing and store the other half with 10 to 25 % (v/v) sterile glycerol at 16 to 24 °C (weeks) or at 68 to 86 °C (month) in case further testing is required.

2.2.   Testing

See Flow chart and description of the tests and optimised protocols in the relevant appendices:

SECTION IV

1.   Scheme for detection and identification of R. solanacearum in water

2.   Methods for detection and identification of R. solanacearum in water

Principle

The validated detection scheme, described in this section, is applicable for pathogen detection in samples of surface water and can also be applied for testing samples of potato processing or sewage effluents. However, it is important to note that the expected sensitivity of detection will vary with the substrate. Sensitivity of the isolation test is affected by populations of competing saprophytic bacteria which are generally much higher in potato processing and sewage effluents than in surface water. Whereas the scheme below is expected to detect as few as 103 cells per litre in surface water the sensitivity of detection in potato processing or sewage effluents is likely to be significantly lower. For this reason, it is recommended to test effluents after any purification treatments (e.g. sedimentation or filtration) during which saprophytic bacterial populations are reduced. The limitations in sensitivity of the test scheme should be considered when assessing the reliability of any negative results obtained. Whereas this scheme has been successfully used in survey work to determine presence or absence of the pathogen in surface water, its limitations should be realised when used in similar surveys of potato processing or sewage effluents.

2.1.   Sample preparation

Note:

2.1.1.

At selected sampling points, collect water samples by filling disposable sterile tubes or bottles at a depth if possible below 30 cm and within 2 m from the bank. For processing and sewage effluents, collect samples from the point of effluent discharge. Sample sizes up to 500 ml per sampling point are recommended. If smaller samples are preferred, it is advisable to take samples on at least three occasions per sampling point, each sample consisting of two replicated sub-samples of at least 30 ml. For intensive survey work, select at least three sampling points per 3 km of watercourse and ensure that tributaries entering the watercourse are also sampled.

2.1.2.

Transport samples in cool dark conditions (4 to 10 °C) and test within 24 hours.

2.1.3.

If required, the bacterial fraction may be concentrated using one of the following methods: (a) Centrifuge 30 to 50 ml sub-samples at 10 000 g for 10 minutes (or 7 000 g for 15 minutes) preferably at 4 to 10 °C, discard the supernatant and resuspend the pellet in 1 ml pellet buffer (Appendix 4). (b) Membrane filtration (minimum pore size 0,45 µm) followed by washing the filter in 5 to 10 ml pellet buffer and retention of the washings. This method is suitable for larger volumes of water containing low numbers of saprophytes. Concentration is usually not advisable for samples of potato processing or sewage effluent since increased populations of competing saprophytic bacteria will inhibit detection of Ralstonia solanacearum.

2.2.   Testing

See flow chart and description of the tests in the relevant appendices.

SECTION V

1.   Scheme for detection and identification of R. solanacearum in soil

2.   Methods for detection and identification of R. solanacearum in soil

Principles

The validated detection scheme, described in this section, is applicable for pathogen detection in soil samples but can also be used to test samples of solid potato processing waste or sewage sludge. However, it should be noted that these methods are insufficiently sensitive to guarantee detection of low and/or irregularly dispersed populations of Ralstonia solanacearum that may occur in naturally infested samples of these substrates.

The limitations in sensitivity of this test scheme should be considered when assessing the reliability of any negative results obtained and also when used in surveys to determine presence or absence of the pathogen in soils or sludges. The most reliable test for presence of the pathogen in a field soil is to plant a susceptible host and monitor it for infection, but even with this method low levels of contamination will escape detection.

2.1.   Sample preparation

2.1.1.

Sampling of field soil should follow standard principals used for nematode sampling. Collect 0,5 to 1 kg of soil per sample from 60 sites per 0,3 ha from a depth of 10 to 20 cm (or in a grid of 7 x 7 metres) If the pathogen is suspected to be present, increase the number of collection points to 120 per 0,3 ha. Maintain samples at 12 to 15 °C prior to testing. Sample potato processing and sewage sludges by collecting a total of 1 kg from sites representing the total volume of sludge to be tested. Mix each sample well before testing.

2.1.2.

Disperse sub-samples of 10 to 25 g of soil or sludge by rotary shaking (250 rpm) in 60 to 150 ml extraction buffer (Appendix 4) for up to two hours. If required, addition of 0,02 % sterile Tween-20 and 10 to 20 g sterile gravel may assist dispersion.

2.1.3.

Maintain the suspension at 4 °C during testing.

2.2.   Testing

See flow chart and description of the tests in the relevant appendices.

SECTION VI

OPTIMISED PROTOCOLS FOR DETECTION AND IDENTIFICATION OF R. SOLANACEARUM

A.   DIAGNOSTIC AND DETECTION TESTS

1.   Stem streaming test

The presence of R. solanacearum in stems of wilting potato, tomato or other host plants can be indicated by the following simple presumptive test: Cut the stem just above the soil level. Suspend the cut surface in a tube of clean water. Observe for characteristic spontaneous streaming of threads of bacterial slime from the cut vascular bundles after a few minutes.

2.   Detection of poly-ß-hydroxybutyrate granules

Nile blue test:

Sudan Black test:

3.   Serological agglutination tests

Agglutination of R. solanacearum cells in bacterial ooze or symptomatic tissue extracts is best observed using validated antibodies (see Appendix 3) labelled with appropriate coloured markers such as red Staphylococcus aureus cells or coloured latex particles. If using a commercially available kit (see Appendix 3), follow the manufacturers instructions. Otherwise perform the following procedure:

4.   Selective isolation

4.1.   Selective plating

Note: Before using this method for the first time, perform preliminary tests to ensure reproducible detection of 103 to 104 colony-forming units of R. solanacearum per ml added to extracts from samples which previously tested negative.

Use an appropriately validated selective medium such as SMSA (as modified by Elphinstone et al., 1996; see Appendix 2).

Care is required to differentiate R. solanacearum from other bacteria able to develop colonies on the medium. Furthermore, colonies of R. solanacearum may show atypical morphology if plates are overcrowded or antagonistic bacteria are also present. Where effects of competition or antagonism are suspected, the sample should be re-tested using a different test.

Highest sensitivity of detection by this method can be expected when using freshly prepared sample extracts. However, the method is also applicable for use with extracts which have been stored under glycerol at 68 to 86 °C.

As positive controls, prepare decimal dilutions from a suspension of 106 cfu per ml of a virulent biovar 2 strain of R. solanacearum (e.g. NCPPB 4156 = PD 2762 = CFBP 3857). To avoid any possibility of contamination, prepare positive controls totally separately from samples to be tested.

For each newly prepared batch of a selective medium its suitability for growth of the pathogen should be tested before it is used to test routine samples.

Test control material in an identical manner as the sample(s).

4.1.1.

Perform an appropriate dilution plating technique aiming to ensure that any background saprophytic colony-forming populations are diluted out. Spread 50 - 100 µl per plate of sample extract and each dilution.

4.1.2.

Incubate plates at 28 °C. Read plates after 48 hours and daily thereafter up to six days. Typical R. solanacearum colonies on SMSA medium are milky white, flat, irregular and fluidal and after three days incubation develop pink to blood-red coloration in the centre with internal streaking or whorling. (see website http://forum.europa.eu.int/Public/irc/sanco/Home/main). Note: Atypical colonies of R. solanacearum sometimes form on this medium. These may be small, round, entirely red in colour and non-fluidal or only partially fluidal and therefore difficult to distinguish from saprophytic colony-forming bacteria.

4.1.3.

Purify presumptive R. solanacearum colonies after streaking or dilution plating onto a general nutrient medium to obtain isolated colonies (see Appendix 2).

4.1.4.

Store cultures short-term in sterile water (pH 6 to 8, chlorine free) at room temperature in the dark, or long term in a suitable cryoprotectant medium at 68 to 86 °C or lyophilised.

4.1.5.

Identify presumptive cultures (see Section VI.B.) and perform a pathogenicity test (see Section VI. C).

The selective plating test is negative if no bacterial colonies are observed after six days or if no presumptive colonies typical of R. solanacearum are found, provided that no inhibition is suspected due to competition or antagonism by other bacteria and that typical R. solanacearum colonies are found in the positive controls. The selective plating test is positive if presumptive R. solanacearum colonies are isolated.

4.2.   Enrichment procedure

Use a validated enrichment medium such as modified Wilbrink broth (see Appendix 2).

This procedure can be used to selectively increase R. solanacearum populations in sample extracts and increase sensitivity of detection. The procedure also effectively dilutes inhibitors of the PCR reaction (1:100). It should be noted, however, that enrichment of R. solanacearum can fail due to competition or antagonism by saprophytic organisms which are often simultaneously enriched. For this reason, isolation of R.solanacearum from enriched broth cultures may be difficult. In addition, since populations of serologically related saprophytes can be increased, the use of specific monoclonal antibodies rather than polyclonal antibodies is recommended where the ELISA test is to be used.

4.2.1.

For enrichment-PCR, transfer 100 µl of sample extract into 10 ml of enrichment broth (Appendix 2) previously aliquoted into DNA-free tubes or flasks. For enrichment-ELISA, higher proportions of sample extract to broth can be used (e.g. 100 µl in 1,0 ml of enrichment broth).

4.2.2.

Incubate for 72 hours at 27 to 30 °C in shaking culture or static culture with caps loosely- fitted to permit aeration.

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) in the above tests. Note: If inhibition of enrichment of R. solanacearum is anticipated, due to high populations of certain competing saprophytic bacteria, enrichment of sample extracts before any centrifugation or other concentration steps may give better results.

5.   IF Test

Principle

The use of the IF test as the principal screening test is recommended because of its proven robustness to achieve the required thresholds.

When the IF test is used as the principal screening test and the IF reading is positive, the Isolation, PCR or FISH test must be performed as a second screening test. When the IF test is used as the second screening test and the IF reading is positive, further testing according to the flow scheme is required to complete the analysis.

Note: Use a validated source of antibodies to R. solanacearum (see web site http://forum.europa.eu.int/Public/irc/sanco/Home/main). It is recommended that the titre is determined for each new batch of antibodies. The titre is defined as the highest dilution at which optimum reaction occurs when testing a suspension containing 105 to 106 cells per ml of the homologous strain of R. solanacearum and using an appropriate fluorescein isothiocyanate (FITC) conjugate according to the manufacturer’s recommendations. Validated polyclonal antisera all had an IF titre of at least 1:2 000. During testing, the antibodies should be used at a working dilution(s) close to or at the titre.

The test should be performed on freshly-prepared sample extracts. If necessary, it can be successfully performed on extracts stored at 68 to 86 °C under glycerol. Glycerol can be removed from the sample by addition of 1 ml pellet buffer (Appendix 4), re-centrifugation for 15 minutes at 7 000 g and re-suspension in an equal volume of pellet buffer. This is often not necessary, especially if samples are fixed to the slides by flaming.

Prepare separate positive control slides of the homologous strain or any other reference strain of R. solanacearum, suspended in potato extract, as specified in Appendix 3 B, and optionally in buffer.

Naturally infected tissue (maintained by lyophilisation or freezing at 16 to 24 °C) should be used where possible as a similar control on the same slide.

As negative controls, aliquots of sample extract which previously tested negative for R. solanacearum can be used.

Standardised positive and negative control materials available for use with this test are listed in Appendix 3.

Use multiwell microscope slides with preferably 10 windows of at least 6 mm diameter.

Test control material in an identical manner as the sample(s).

5.1.   Prepare the test slides by one of the following procedures:

5.2.

Dry the droplets at ambient temperature or by warming to temperatures of 40 to 45 °C. Fix the bacterial cells to the slide either by heating (15 minutes at 60 °C), flaming, with 95 % ethanol or according to specific instructions from the suppliers of the antibodies. If necessary, fixed slides may then be stored frozen in a desiccated box for as little time as necessary (up to a maximum of three months) prior to further testing.

5.3.

IF procedure (i) According to test slide preparation in 5.1(i): Prepare a set of twofold dilutions The first well should have 1/2 of the titre (T/2), the others 1/4 of the titre (T/4), 1/2 of the titre (T/2), the titre (T) and twice the titre (2T). (ii) According to test slide preparation in 5.1(ii): Prepare the working dilution (WD) of the antibody in IF buffer. The working dilution affects the specificity. Figure 1.  Preparation of the test slide according to 5.1(i) and 5.3(i)   Dilutions of resuspended pellet 1/100 1/1 1/1 1/1 1/1  Dilution of resuspended pellet (T = titre) T/2 T/4 T/2 T 2T  Twofold dilutions of antiserum/antibody Sample 1   1 2 3 4 5 Duplicate of sample1 or sample 2 6 7 8 9 10 Figure 2.  Preparation of the test slide according to 5.1(ii) and 5.3(ii).   Working dilution of antiserum/antibody 1/1 1/10 1/100 empty empty  Decimal dilution of resuspended pellet Sample 1     1 2 3 4 5 Duplicate of sample 1 or sample 2 6 7 8 9 10 5.3.1. Arrange the slides on moist tissue paper. Cover each test window completely with the antibody dilution(s). The volume of antibody applied on each window must be at least the volume of extract applied. The following procedure should be carried out in the absence of specific instructions from the suppliers of the antibodies: 5.3.2. Incubate the slides on moist paper under a cover for 30 minutes at ambient temperature (18 to 25 °C). 5.3.3. Shake the droplets off each slide and rinse carefully with IF buffer. Wash by submerging for five minutes in IF buffer-Tween (Appendix 4) and subsequently in IF buffer. Avoid causing aerosols or droplet transfer that could result in cross-contamination. Carefully remove excess moisture by blotting gently. 5.3.4. Arrange the slides on moist paper. Cover the test windows with the dilution of FITC conjugate used to determine the titre. The volume of conjugate applied on the windows must be identical to the volume of antibody applied. 5.3.5. Incubate the slides on moist paper under a cover for 30 minutes at ambient temperature (18 to 25 °C). 5.3.6. Shake the droplets of conjugate off the slide. Rinse and wash as before (5.3.3). Carefully remove excess moisture. 5.3.7. Pipette 5 - 10 µl of 0,1M phosphate-buffered glycerol (Appendix 4) or a commercially antifading mountant on each window and apply a coverslip.

5.4.

Reading the IF test: 5.4.1 Examine test slides on an epifluorescence microscope with filters suitable for excitation of FITC, under oil or water immersion at a magnification of 500-1 000. Scan windows across two diameters at right angles and around the perimeter. For samples showing no or low number of cells observe at least 40 microscope fields. Check the positive control slide first. Cells must be bright fluorescent and completely stained at the determined antibody titre or working dilution. The IF test (Section VI.A.5.) must be repeated if the staining is aberrant. 5.4.2. Observe for bright fluorescing cells with characteristic morphology of R. solanacearum in the test windows of the test slides (see website http://forum.europa.eu.int/Public/irc/sanco/Home/main). The fluorescence intensity must be equivalent to the positive control strain at the same antibody dilution. Cells with incomplete staining or with weak fluorescence must be disregarded. If any contamination is suspected the test must be repeated. This may be the case when all slides in a batch show positive cells due to the contamination of buffer or if positive cells are found (outside of the slide windows) on the slide coating. 5.4.3. There are several problems inherent to the specificity of the immunofluorescence test. Background populations of fluorescing cells with atypical morphology and cross reacting saprophytic bacteria with size and morphology similar to R. solanacearum are likely to occur in potato heel end core and stem segment pellets. 5.4.4. Consider only fluorescing cells with typical size and morphology at the titre or working dilution of the antibodies as in 5.3. 5.4.5. Interpretation of the IF reading: (i) If bright fluorescing cells with characteristic morphology are found, estimate the average number of typical cells per microscope field and calculate the number of typical cells per ml of resuspended pellet (Appendix 5). The IF reading is positive for samples with at least 5 × 103 typical cells per ml of resuspended pellet. The sample is considered potentially contaminated and further testing is required. (ii) The IF reading is negative for samples with less than 5 × 103 cells per ml of resuspended pellet and the sample is considered negative. Further testing is not required.

6.   PCR tests

Principles

When the PCR test is used as the principal screening test and found to be positive, isolation or IF must be performed as a second compulsory screening test. When PCR is used as the second screening test and found to be positive, further testing according to the flow scheme is required to complete the diagnosis.

Full exploitation of this method as principal screening test is only recommended when specialised expertise has been acquired.

Note: Preliminary testing with this method should permit reproducible detection of 103 to 104 cells of R. solanacearum per ml added to sample extracts which previously tested negative. Optimisation experiments may be required to achieve maximum levels of sensitivity and specificity in all laboratories.

Use validated PCR reagents and protocols (see Appendix 6). Preferably select a method with an internal control.

Use appropriate precautions to avoid contamination of sample with target DNA. The PCR test should be performed by experienced technicians, in dedicated molecular biology laboratories, in order to minimise the possibility of contamination with target DNA.

Negative controls (for DNA extraction and PCR procedures) should always be handled as final samples in the procedure, to make evident whether any carry over of DNA has occurred.

The following negative controls should be included in the PCR test:

The following positive controls should be included:

To avoid potential contamination prepare positive controls in a separate environment from samples to be tested.

Sample extracts should be as free as possible from soil. It could therefore, in certain cases, be advisible to prepare extracts from washed potatoes if PCR protocols are to be used.

Standardized positive and negative control material available for use with this test are listed in Appendix 3).

6.1.   DNA purification methods

Use positive and negative control samples as described above (see Appendix 3).

Test control material in an identical manner as the sample(s).

A variety of methods are available for purification of target DNA from complex sample substrates, thus removing inhibitors of PCR and other enzymatic reactions and concentrating target DNA in the sample extract. The following method has been optimised for use with the validated PCR methods shown in Appendix 6.

(a)   Method according to Pastrik (2000)

(b)   Other methods

Other DNA extraction methods, e.g. Qiagen DNeasy Plant Kit, could be applied providing that they are proven to be equally as effective in purifying DNA from control samples containing 103 to 104 pathogen cells per ml.

6.2.   PCR

6.2.1.

Prepare test and control templates for PCR according to the validated protocols (Section VI.A.6.). Prepare one decimal dilution of sample DNA extract (1:10 in UPW).

6.2.2.

Prepare the appropriate PCR reaction mix in a contamination-free environment according to the published protocols (Appendix 6). Where possible, it is recommended to use a multiplex PCR protocol that also incorporates an internal PCR control.

6.2.3.

Add 2-5 µl of DNA extract per 25 µl PCR reaction in sterile PCR tubes according to the PCR protocols, (see Appendix 6).

6.2.4.

Incorporate a negative control sample containing only PCR reaction mix and add the same source of UPW as used in the PCR mix in place of sample.

6.2.5.

Place tubes in the same thermal cycler which was used in preliminary testing and run the appropriately optimised PCR programme (Appendix 6).

6.3.   Analysis of the PCR product

6.3.1.

Resolve PCR amplicons by agarose gel electrophoresis. Run at least 12 µl of amplified DNA reaction mixture from each sample mixed with 3 µl loading buffer (Appendix 6) in 2,0 % (w/v) agarose gels in tris-acetate-EDTA (TAE) buffer (Appendix 6) at 5 to 8 V per cm. Use an appropriate DNA marker, e.g. 100 bp ladder.

6.3.2.

Reveal DNA bands by staining in ethidium bromide (0,5 mg per L) for 30 to 60 minutes taking appropriate precautions for handling this mutagen.

6.3.3.

Observe stained gel under short wave UV transillumination (λ = 302 nm) for amplified PCR products of the expected size (Appendix 6) and document.

6.3.4.

For all new findings/cases verify authenticity of the PCR amplicon by performing restriction enzyme analysis on a sample of the remaining amplified DNA by incubating at the optimum temperature and time with an appropriate enzyme and buffer (see Appendix 6). Resolve the digested fragments by agarose gel electrophoresis as before and observe characteristic restriction fragment pattern under UV transillumination after ethidium bromide staining and compare with the undigested and digested positive control.

Interpretation of the PCR test result:

The PCR test is negative if the R. solanacearum-specific PCR amplicon of expected size is not detected for the sample in question but is detected for all positive control samples (in case of multiplex PCR with plant specific internal control primers: a second PCR-product of expected size must be amplified with the sample in question).

The PCR test is positive if the R. solanacearum-specific PCR amplicon of expected size and restriction pattern (when required) is detected, providing that it is not amplified from any of the negative control samples. Reliable confirmation of a positive result can also be obtained by repeating the test with a second set of PCR primers (Appendix 6).

Note: Inhibition of the PCR may be suspected if the expected amplicon is obtained from the positive control sample containing R. solanacearum in water but negative results are obtained from positive controls with R. solanacearum in potato extract. In multiplex PCR protocols with internal PCR controls, inhibition of the reaction is indicated when neither of the two amplicons are obtained.

Contamination may be suspected if the expected amplicon is obtained from one or more of the negative controls.

7.   FISH test

Principle

When the FISH test is used as the first screening test and found to be positive, Isolation or the IF test must be performed as a second compulsory screening test. When the FISH test is used as the second screening test and found to be positive, further testing according to the flow scheme is required to complete the diagnosis.

Note: Use validated R. solanacearum-specific oligo-probes (see Appendix 7). Preliminary testing with this method should permit reproducible detection of at least 103 to 104 cells of R. solanacearum per ml added to sample extracts which previously tested negative.

The following procedure should preferably be performed on freshly prepared sample extract but can also be successfully performed on sample extract that has been stored under glycerol at 16 to 24 or 68 to 86 °C.

As negative controls, use aliquots of sample extract that previously tested negative for R. solanacearum.

As positive controls prepare suspensions containing 105 to 106 cells per ml of R. solanacearum biovar 2 (e.g. strain NCPPB 4156 = PD 2762 = CFBP 3857, see Appendix 3) in 0,01M phosphate buffer (PB) from a 3 to 5 day culture). Prepare separate positive control slides of the homologous strain or any other reference strain of R. solanacearum, suspended in potato extract, as specified in Appendix 3 B.

The use of the FITC-labelled eubacterial oligo-probe offers a control for the hybridisation process, since it will stain all eubacteria that are present in the sample.

Standardized positive and negative control material available for use with this test are listed in Appendix 3A).

Test control material in an identical manner as the sample(s).

7.1.   Potato extract fixation

The following protocol is based upon Wullings et al. (1998):

7.1.1.

Prepare fixative solution (see Appendix 7).

7.1.2.

Pipette 100 µl of each sample extract into an Eppendorf tube and centrifuge for 7 minutes at 7 000 g.

7.1.3.

Remove the supernatant and dissolve the pellet in 200 µl of fixative prepared < 24 hours previously. Vortex and incubate for one hour in the refrigerator.

7.1.4.

Centrifuge for 7 minutes at 7 000 g, remove the supernatant and resuspend the pellet in 75 µl 0,01M PB (see Appendix 7).

7.1.5.

Spot 16 µl of the fixed suspensions onto a clean multitest slide as shown in Fig. 7.1. Applying two different samples per slide, undiluted and use 10 µl to make a 1:100 dilution (in 0,01 M PB). The remaining sample solution (49 µl) can be stored at 20 °C after addition of one volume of 96 % ethanol. In case the FISH assay requires repeating, remove the ethanol by centrifugation and add an equal volume of 0,01 PB (mix by vortexing). Fig. 7.1  Layout for FISH slide Sample 1 Blank Blank Blank Sample 2 window 1 window 2 window 3 window 4 window 5 Sample 1 Blank Blank Blank Sample 2 window 6 window 7 window 8 window 9 window 10 Coverslip 1   Coverslip 2

7.1.6.

Air-dry the slides (or on slide dryer at 37 °C) and fix them by flaming. At this stage the procedure may be interrupted and the hybridisation continued the following day. Slides should be stored dust-free and dry at room temperature.

7.2.   Hybridisation

7.2.1.

Dehydrate the cells in a graded ethanol series of 50 %, 80 % and 96 % for one minute each. Air dry the slides in a slide-holder.

7.2.2.

Prepare a moist incubation chamber by covering the bottom of an air-tight box with tissue or filter paper soaked in 1x hybmix (Appendix 7). Pre-incubate the box in the hybridisation oven at 45 °C for at least 10 minutes.

7.2.3.

Apply 10 μl of hybridisation solution (Appendix 7) to eight windows (windows 1, 2, 4, 5, 6, 7, 9 and 10; see Fig 7.1) of each slide leaving the two centre windows (3 and 8) empty.

7.2.4.

Apply coverslips (24 × 24 mm) to the first and last four windows without trapping air. Place the slides in the pre-warmed moist chamber and hybridise for five hours in the oven at 45 °C in the dark.

7.2.5.

Prepare three beakers containing 1 l of Milli Q (molecular grade) water, 1 l of 1x hybmix (334 ml 3x hybmix and 666 ml Milli Q water) and 1 l of 1/8x hybmix (42 ml 3x hybmix and 958 ml Milli Q water). Pre-incubate each in a waterbath at 45 °C.

7.2.6.

Remove the coverslips from the slides and place the slides in a slide holder.

7.2.7.

Wash away excess probe by incubation for 15 minutes in the beaker with 1x hybmix at 45 °C.

7.2.8.

Transfer the slide holder to 1/8 hybmix washing solution and incubate for a further 15 minutes.

7.2.9.

Dip the slides briefly in Milli Q water and place them on filter paper. Remove excess moisture by covering the surface gently with filter paper. Pipette 5 to 10 μl of anti-fading mountant solution (e.g. Vectashield, Vecta Laboratories, CA, USA or equivalent) on each window and apply a large coverslip (24 × 60 mm) over the whole slide.

7.3.   Reading the FISH test

7.3.1.

Observe the slides immediately with a microscope fitted for epifluorescence microscopy at 630 or 1 000 × magnification under immersion oil. With a filter suitable for fluorescein isothiocyanate (FITC) eubacterial cells (including most gram negative cells) in the sample are stained fluorescent green. Using a filter for tetramethylrhodamine-5-isothiocyanate, Cy3-stained cells of R. solanacearum appear fluorescent red. Compare cell morphology with that of the positive controls. Cells must be bright fluorescent and completely stained The FISH test (Section VI.A.7.) must be repeated if the staining is aberrant. Scan windows across two diameters at right angles and around the perimeter. For samples showing no or low number of cells observe at least 40 microscope fields.

7.3.2.

Observe for bright fluorescing cells with characteristic morphology of R. solanacearum in the test windows of the test slides (see web site http://forum.europa.eu.int/Public/irc/sanco/Home/main). The fluorescence intensity must be equivalent or better than that of the positive control strain. Cells with incomplete staining or with weak fluorescence must be disregarded.

7.3.3.

If any contamination is suspected the test must be repeated. This may be the case when all slides in a batch show positive cells due to the contamination of buffer or if positive cells are found (outside of the slide windows) on the the slide coating.

7.3.4.

There are several problems inherent to the specificity of the FISH test. Background populations of fluorescing cells with atypical morphology and cross reacting saprophytic bacteria with size and morphology similar to R. solanacearum may occur, although much less frequent as in the IF test, in potato heel end core and stem segment pellets.

7.3.5.

Consider only fluorescing cells with typical size and morphology.

7.3.6.

Interpretation of the FISH test result: (i) Valid FISH test results are obtained if bright green fluorescent cells of size and morphology typical of R. solanacearum are observed using the FITC filter and bright red fluorescent cells using the rhodamine filter in all positive controls and not in any of the negative controls. If bright fluorescing cells with characteristic morphology are found, estimate the average number of typical cells per microscope field and calculate the number of typical cells per ml of resuspended pellet (Appendix 4). Samples with at least 5 × 103 typical cells per ml of resuspended pellet are considered potentially contaminated. Further testing is required. Samples with less than 5 × 103 typical cells per ml of resuspended pellet are considered negative. (ii) The FISH test is negative if bright red fluorescent cells with size and morphology typical of R. solanacearum are not observed using the rhodamine filter, provided that typical bright red fluorescent cells are observed in the positive control preparations when using the rhodamine filter.

8.   ELISA tests

Principle

ELISA can only be used as an optional test in addition to IF, PCR or FISH due to a relatively low sensitivity of this test. When DAS ELISA is used enrichment and the use of monoclonal antibodies are compulsory (see web site http://forum.europa.eu.int/Public/irc/sanco/Home/main). Enrichment of the samples before using ELISA may be useful in order to increase the sensitivity of the test, but it can fail due to competition by other organisms in the sample.

Note: Use an validated source of antibodies to R. solanacearum (see web site http://forum.europa.eu.int/Public/irc/sanco/Home/main) It is recommended that the titre is determined for each new batch of antibodies. The titre is defined as the highest dilution at which optimum reaction occurs when testing a suspension containing 105 to 106 cells per ml of the homologous strain of R. solanacearum and using appropriate secondary antibody conjugates according to the manufacturer’s recommendations. During testing, the antibodies should be used at a working dilution close to or at the titre of the commercial formulation.

Determine the titre of the antibodies on a suspension of 105 to 106 cells per ml of the homologous strain of R. solanacearum.

Include a sample extract that previously tested negative for R. solanacearum and a suspension of a non-cross reacting bacterium in phosphate buffered saline (PBS) as negative controls.

As positive control use aliquots of sample extract, that previously tested negative, mixed with 103 to 104 cells per ml of R. solanacearum biovar 2 (e.g. strain NCPPB 4156 = PD 2762 = CFBP 3857, see Appendix 2 A and B). For comparison of results on each plate use a standard suspension of 105 to 106 cells per ml in PBS of R. solanacearum. Ensure positive controls are well separated on the microtitre plate from the sample(s) under test.

Standardised positive and negative control materials available for use with this test are listed in Appendix 3 A.

Test control material in an identical manner as the sample(s).

Two ELISA protocols have been validated.

(a)   Indirect ELISA (Robinson Smith et al., 1995)

(b)   DASI ELISA

Interpretation of ELISA test results:

The ELISA test is negative if the average optical density (OD) reading from duplicate sample wells is < 2x OD of that in the negative sample extract control well, providing the OD for the positive controls are all above 1,0 (after 90 minutes incubation with the substrate) and are greater than twice the OD obtained for negative sample extracts.

The ELISA test is positive if the average OD readings from duplicate sample wells is > 2x OD in the negative sample extract well provided that OD readings in all negative control wells are < 2x those in the positive control wells.

Negative ELISA readings in positive control wells indicate that the test has not been performed correctly or that it has been inhibited. Positive ELISA readings in negative control wells indicate that cross-contamination or non-specific antibody binding has occurred.

9.   Bioassay test

Note: Preliminary testing with this method should permit reproducible detection of 103 to 104 colony-forming units of R. solanacearum per ml added to sample extracts that previously tested negative (preparation see Appendix 3).

Highest sensitivity of detection can be expected when using freshly prepared sample extract and optimal growth conditions. However, the method can be successfully applied to extracts that have been stored under glycerol at 68 to 86 °C.

The following protocol is based upon Janse (1988):

9.1.

Use 10 test plants of a susceptible tomato cultivar (e.g. Moneymaker or cultivar with equivalent susceptibility as determined by the testing laboratory) at the third true leaf stage for each sample. For cultural details, see Appendix 8. Alternatively, use eggplants (e.g. cultivar Black Beauty or cultivars with equivalent susceptibility), use only plants at leaf stage two to three up to full expansion of the third true leaf. Symptoms have been show to be less severe and to develop more slowly in eggplant. Where possible, it is therefore recommended to use tomato seedlings.

9.2.

Distribute 100 µl of sample extract between the test plants. 9.2.1.   Syringe inoculation Inoculate the plant stems just above the cotyledons using a syringe fitted with a hypodermic needle (not less than 23G). Distribute the sample between the test plants. 9.2.2.   Slit inoculation Holding the plant between two fingers, pipette a drop (approximately 5 - 10 µl) of the suspended pellet on the stem between the cotyledons and the first leaf. Using a sterile scalpel, make a diagonal slit, about 1.0 cm long and approximately 2/3 of the stem thickness deep, starting the cut from the pellet drop. Seal the cut with sterile vaseline from a syringe.

9.3.

Inoculate by the same technique, five seedlings with an aqueous suspension of 105 to 106 cells per ml prepared from a 48 hr culture of a virulent biovar 2 strain of R. solanacearum as a positive control and with pellet buffer (Appendix 4) as negative control. Separate positive and negative control plants from the other plants to avoid cross-contamination.

9.4.

Grow the test plants in quarantine facilities for up to four weeks at 25 to 30 °C and high relative humidity with appropriate watering to prevent waterlogging or wilting through water deficiency. To avoid contamination incubate positive control and negative control plants on clearly separated benches in a glasshouse or growth chamber or, in case space is limited, ensure strict separation between treatments. If plants for different samples must be incubated close together, separate them with appropriate screens. When fertilising, watering, inspecting and any other manipulations take great care to avoid cross-contamination. It is essential to keep glasshouses and growth chambers free of all insect pests since they may transmit the bacterium from sample to sample. Observe for symptoms of wilting, epinasty, chlorosis and/or stunting.

9.5.	Isolate from infected plants (Section II.3.) and identify purified cultures of presumptive R. solanacearum (Section VI.B.).

9.6.	If no symptoms are observed after three weeks perform IF/PCR/Isolation on a composite sample of 1 cm stem sections of each test plant taken above the inoculation site. If the test is positive perform dilution plating (section 4.1).

9.7.	Identify any purified cultures of presumptive R. solanacearum (Section VI.B.).

Valid Bioassay test results are obtained when plants of the positive control show typical symptoms, the bacteria can be reisolated from these plants and no symptoms are found on the negative controls. The bioassay test is negative if test plants are not infected by R. solanacearum, and provided that R. solanacearum is detected in positive controls. The bioassay test is positive if the test plants are infected by R. solanacearum.

B.   IDENTIFICATION TESTS

Identify pure cultures of presumptive R. solanacearum isolates using at least two of the following tests based on different biological principles.

Include known reference strains where appropriate for each test performed (see Appendix 3).

1.   Nutritional and enzymatic identification tests

Determine the following phenotypic properties, which are universally present or absent in R. solanacearum, according to the methods of Lelliott and Stead (1987), Klement et al. (1990), Schaad (2001).

2.   IF test

2.1.	Prepare a suspension of approximately 106 cells per ml in IF buffer (Appendix 4).

2.2.	Prepare a twofold dilution series of an appropriate antiserum (see website http://forum.europa.eu.int/Public/irc/sanco/Home/main).

2.3.	Apply the IF procedure (Section VI.A.5.).

2.4.	A positive IF test is achieved if the IF titre of the culture is equivalent to that of the positive control.

3.   ELISA test

Note: If performing only 2 identification tests, do not use another serological test in addition to this method.

3.1.	Prepare a suspension of approximately 108 cells per ml in 1X PBS (Appendix 4).

3.2.	Perform an appropriate ELISA procedure with a specific monoclonal antibody to R. solanacearum.

3.3.	A positive ELISA test is achieved if the ELISA reading obtained from the culture is at least half that obtained for the positive control.

4.   PCR tests

4.1.	Prepare a suspension of approximately 106 cells per ml in molecular grade sterile water.

4.2.	Heat 100 µl of the cell suspension in closed tubes in a heating block or boiling waterbath at 100 °C for four minutes. The samples may then be stored at 16 to 24 °C until required.

4.3.	Apply appropriate PCR procedures to amplify R. solanacearum-specific amplicons (e.g. Seal et al. (1993); Pastrik and Maiss (2000); Pastrik et al. (2002); Boudazin et al. (1999); Opina et al. (1997), Weller et al. (1999).

4.4.	A positive identification of R. solanacearum is achieved if the PCR amplicons are the same size and have the same restriction fragment length polymorphisms as for the positive control strain.

5.   FISH test

5.1.	Prepare a suspension of approximately 106 cells per ml in UPW.

5.2.	Apply the FISH procedure (Section VI.A.7.) with at least 2 R. solanacearum-specific oligo-probes (Appendix 7).

5.3.	A positive FISH test is achieved if the same reactions are achieved from the culture and the positive control.

6.   Fatty acid profiling (FAP)

6.1.	Grow the culture on trypticase soy agar (Oxoid) for 48 hours at 28 °C.

6.2.	Apply an appropriate FAP procedure (Janse, 1991; Stead, 1992).

6.3.	A positive FAP test is achieved if the profile of the presumptive culture is identical to that of the positive control. The presence of characteristic fatty acids are 14:0 3OH, 16:0 2OH, 16:1 2OH and 18:1 2OH and absence of 16:0 3OH is highly indicative of a Ralstonia sp.

7.   Strain characterisation methods

Strain characterisation using one of the following methods is recommended for each new case of isolation of R. solanacearum.

Include known reference strains where appropriate for each test performed (see Appendix 3).

7.1.   Biovar determination

R. solanacearum is separated into biovars on the basis of the ability to utilise and/or oxidise three disaccharides and three hexose alcohols (Hayward, 1964 and Hayward et al., 1990). Growth media for the biovar test is described in Appendix 2. The test can be successfully performed by stab inoculating the media with pure cultures of R. solanacearum isolates and incubating at 28 °C. If the media are dispensed into sterile 96 well cell culture plates (200 µl per well) colour change from olive green to yellow can be observed within 72 hours, indicating a positive test result.

Additional tests differentiate biovar 2 sub-phenotypes

7.2.   Genomic fingerprinting

Molecular differentiation of strains in the R. solanacearum complex can be achieved using several techniques, including:

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 et al., 1995; Smith et al., 1995).

7.2.3.

Amplified fragment length polymorphism (AFLP) analysis (Van der Wolf et al., 1998).

7.3.   PCR methods

Specific PCR primers (Pastrik et al, 2002; see Appendix 6) can be used to differentiate strains belonging to division 1 (biovars 3, 4 and 5) and division 2 (biovars 1, 2A and 2T) of R. solanacearum, as originally defined by RFLP analysis (Cook et al., 1989) and 16S rDNA sequencing (Taghavi et al., 1996).

C.   CONFIRMATION TEST

The pathogenicity test must be performed as final confirmation of a diagnosis of R. solanacearum and for assessment of virulence of cultures identified as R. solanacearum.

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Appendix 1

Laboratories involved in optimisation and validation of protocols

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Appendix 2

Media for isolation and culture of R. solanacearum

(a)   General growth media

Nutrient Agar (NA)

Dissolve ingredients and sterilise by autoclaving at 121 °C for 15 min.

Yeast Peptone Glucose Agar (YPGA)

Dissolve ingredients and sterilise by autoclaving at 121 °C for 15 minutes.

Sucrose Peptone Agar (SPA)

Dissolve ingredients and sterilise by autoclaving at 121 °C for 15 minutes.

Kelman’s Tetrazolium Medium

Dissolve ingredients and sterilise by autoclaving at 121 °C for 15 minutes.

Cool to 50 °C and add a filter-sterilised solution of 2,3,5-triphenyl tetrazolium chloride (Sigma) to obtain a final concentration of 50 mg per l.

(b)   Validated selective growth media

SMSA medium (Englebrecht, 1994 as modified by Elphinstone et al., 1996)

Dissolve ingredients and sterilise by autoclaving at 121 °C for 15 minutes.

Cool to 50 °C and add filter-sterilised aqueous stock solutions of the following ingredients to obtain the specified final concentrations:

Note:

Store media and stock solutions of antibiotics at 4 °C in the dark and use within one month.

Plates should be free from surface condensation before use.

Avoid excess drying of plates.

Quality control should be performed after preparation of each new batch of medium by plating a suspension of a reference culture of R. solanacearum (see Appendix 3) and observing formation of typical colonies after incubation at 28 °C for two to five days.

(c)   Validated enrichment media

SMSA Broth (Elphinstone et al., 1996)

Prepare as for SMSA selective agar medium but omit Bacto-Agar and 2,3,5-tetrazolium chloride.

Modified Wilbrink broth (Caruso et al., 2002)

Sterilise by autoclaving at 121 °C for 15 minutes and cool to 50 °C

Add antibiotic stock solutions as for SMSA broth.

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Appendix 3

A.   Commercially available standardised control material

(a)   Bacterial isolates

The following bacterial isolates are recommended for use as standard reference material either as positive controls (Table 1) or during optimisation of tests to avoid cross-reactions (Table 2). All strains are commercially available from:

Note: Authenticity of the above strains can be guaranteed only if obtained from an authentic culture collection.

(b)   Commercially available standardised control material

The following standard control material is available from the NCPPB culture collection.

Freeze dried pellet of potato extract from 200 healthy potato tubers as negative control for all tests.

Freeze dried pellet of potato extract from 200 healthy potato tubers containing 103 to 104 and 104 to 106 cells R. solanacearum biovar 2 (strain NCPPB 4156 = PD 2762 = CFBP 3857) as positive controls for serological and PCR tests. Since cell viability is affected during freeze-drying, these are not suitable as standard controls for isolation or bioassay tests.

Formalin-fixed suspensions of R. solanacearum biovar 2 (strain NCPPB 4156 = PD 2762 = CFBP 3857) at 106 cells per ml as positive controls for serological tests.

B.   Preparation of positive and negative controls for the core screening tests PCR/IF and FISH

Produce a 48 hour culture of a virulent strain of R. solanacearum race3/biovar2 (e.g. strain NCPPB 4156 = PD 2762 = CFBP 3857) on basal SMSA medium and suspend in 10 mM phosphate buffer to obtain a cell density of approximately 2 × 108 cfu per ml. This is usually obtained by a faintly turbid suspension equivalent to an optical density of 0,15 at 600 nm.

Remove the heel end cores of 200 tubers taken from a white skin variety production known to be free from R. solanacearum.

Process the heel ends as usual and resuspend the pellet in 10 ml.

Prepare 10 sterile 1,5 ml microvials with 900 µl of the resuspended pellet.

Transfer 100 µl of the suspension of R. solanacearum to the first microvial. Vortex.

Establish decimal levels of contamination by further diluting in the next five microvials.

The six contaminated microvials will be used as positive controls. The four non-contaminated microvials will be used as negative controls. Label the microvials accordingly.

Prepare aliquots of 100 µl in sterile 1,5 ml microvials thus obtaining nine replicas of each control sample. Store at 16 to 24 °C until use.

The presence and quantification of R. solanacearum in the control samples should be first confirmed by IF.

For the PCR test perform DNA extraction from positive and negative control samples with each series of test samples.

For IF and FISH tests perform assays on positive and negative control samples with each series of test samples.

For IF, FISH and PCR assays R. solanacearum must be detected in at least the 106 and 104 cells/ml of the positive controls and not in any of the negative controls.

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Appendix 4

Buffers for test procedures

General: Unopened sterilised buffers can be stored for up to one year

1.   Buffers for extraction procedure

1.1.   Extraction buffer (50 mM phosphate buffer, pH 7,0)

This buffer is used for extraction of the bacterium from plant tissues by homogenisation or shaking.

Dissolve ingredients, check pH and sterilise by autoclaving at 121 °C for 15 min.

Additional components may be useful as follows:

1.2.   Pellet buffer (10 mM phosphate buffer, pH 7,2)

This buffer is used for resuspension and dilution of potato tuber heel-end core extracts following concentration to a pellet by centrifugation.

Dissolve ingredients, check pH and sterilise by autoclaving at 121 °C for 15 minutes.

2.   Buffers for the IF test

2.1.   IF-Buffer (10 mM phosphate buffered saline (PBS), pH 7.2)

This buffer is used for dilution of antibodies

Dissolve ingredients, check pH and sterilise by autoclaving at 121 °C for 15 minutes.

2.2.   IF-buffer-Tween

This buffer is used to wash slides.

Add 0,1 % Tween 20 to the IF buffer.

2.3.   Phosphate buffered glycerol, pH 7,6

This buffer is used as a mountant fluid on the windows of IF slides to enhance fluorescence.

Anti-fading mountant solutions are commercially available e.g. Vectashield® (Vector Laboratories) or Citifluor® (Leica).

3.   Buffers for the Indirect ELISA test

3.1.   Double strength coating buffer, pH 9,6.

Dissolve ingredients, check pH and sterilise by autoclaving at 121 °C for 15 minutes.

Sodium sulphite (0.2 %) may be added as antioxidant if required to prevent build up of oxidised aromatic compounds.

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

Mix and adjust to pH 9,8 with concentrated HCl.

Make up to 1 L with distilled water.

Add 0,2 g MgCl2.

Dissolve 2 phosphatase substrate 5 mg tablets (Sigma) per 15 ml of solution.

4.   Buffers for DASI ELISA test

4.1.   Coating buffer, pH 9,6

Dissolve ingredients and check 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

Mix and adjust to pH 9,8 with concentrated HCl.

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Appendix 5

Determination of contamination level in IF and FISH tests

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Appendix 6

Validated PCR protocols and reagents

NB: Preliminary testing should permit reproducible detection of 103 to 104 cells of R. solanacearum per ml of sample extract.

Preliminary testing should also show no false positive results with a panel of selected bacterial strains (see Appendix 3).

1.   PCR protocol of Seal et al. (1993)

1.1.   Oligonucleotide primers

Expected amplicon size from R. solanacearum template DNA = 288 bp

1.2.   PCR reaction mix

1.3.   PCR reaction conditions

Run the following programme:

NB: This programme was optimised for use with a Perkin Elmer 9600 thermal cycler. Modification of the duration steps of cycles (ii), (iii) and (iv) may be required for use with other models.

1.4.   Restriction enzyme analysis of amplicon.

PCR products amplified from R. solanacearum DNA produce a distinctive restriction fragment length polymorphism with enzyme Ava II after incubation at 37 °C.

2.   PCR protocol of Pastrik and Maiss (2000)

2.1.   Oligonucleotide primers

Expected amplicon size from R. solanacearum template DNA = 553 bp.

2.2.   PCR reaction mix

2.3.   PCR reaction conditions

Run the following programme:

NB: This programme is optimised for use with an MJ Research PTC 200 thermal cycler. Modification of the duration steps of cycles (ii), (iii) and (iv) may be required for use with other models.

2.4.   Restriction enzyme analysis of amplicon.

PCR products amplified from R. solanacearum DNA produce a distinctive restriction fragment length polymorphism with enzyme Taq I after incubation at 65 °C for 30 minutes. The restriction fragments obtained from R. solanacearum-specific fragment are 457 bp and 96 bp in size.

3.   Multiplex PCR protocol with internal PCR control (Pastrik et al., 2002)

3.1.   Oligonucleotide primers

Expected amplicon size from R. solanacearum template DNA = 718 bp (RS-primer set)

Expected amplicon size from the 18S rRNA internal PCR control = 310 bp (NS-primer set).

3.2.   PCR reaction mix

3.3.   PCR reaction conditions

Run the following programme:

NB: This programme is optimised for use with an MJ Research PTC 200 thermal cycler. Modification of the duration steps of cycles (ii), (iii) and (iv) may be required for use with other models.

3.4.   Restriction enzyme analysis of amplicon.

PCR products amplified from R. solanacearum DNA produce a distinctive restriction fragment length polymorphism with enzyme Bsm I or an Isoschizomere (e.g. Mva 1269 I) after incubation at 65 °C for 30 minutes.

4.   R. solanacearum biovar-specific PCR protocol (Pastrik et al., 2001)

4.1.   Oligonucleotide primers

Expected amplicon size from R. solanacearum template DNA:

with Rs-1-F/Rs-1-R = 718 bp

with Rs-1-F/Rs-3-R = 716 bp.

4.2.   PCR reaction mix

4.3.   PCR reaction conditions

Run the following programme for both biovar 1/2- and biovar 3/4/5-specific reactions:

NB: This programme was optimised for use with an MJ Research PTC 200 thermal cycler. Modification of the duration steps of cycles (ii), (iii) and (iv) may be required for use with other models.

4.4.   Restriction enzyme analysis of amplicon.

PCR products amplified from R. solanacearum DNA using primers Rs-1-F and Rs-1-R produce a distinctive restriction fragment length polymorphism with enzyme Bsm I or an Isoschizomere (e.g. Mva 1269 I) after incubation at 65 °C for 30 minutes. PCR products amplified from R. solanacearum DNA using primers Rs-1-F and Rs-3-R have no restriction sites.

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

Dilute to 1X before use.

Also commercially available (e.g. Invitrogen or equivalent).

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Appendix 7

Validated reagents for FISH test

1.   Oligo-probes

R. solanacearum-specific probe OLI-1-CY3: 5′-ggc agg tag caa gct acc ccc-3′

Non-specific eubacterial probe EUB-338-FITC: 5′-gct gcc tcc cgt agg agt-3′

2.   Fixative solution

(WARNING! THE FIXATIVE CONTAINS PARAFORMALDEHYDE WHICH IS TOXIC. WEAR GLOVES AND DO NOT INHALE. IT IS ADVISABLE TO WORK IN A FUME CUPBOARD.)

3.   3X Hybmix

Dilute to 1X as required.

4.   Hybridisation solution

Prepare quantities of hybridisation solution according to the calculations in Table 1. For each slide (containing 2 different samples in duplicate) 90 μl hybridisation solution is required. IMPORTANT: FORMAMIDE IS VERY TOXIC SO WEAR GLOVES AND TAKE NECESSARY SAFETY PRECAUTIONS!

5.   0,1M Phosphate buffer, pH 7,0

Dissolve ingredients, check pH and sterilise by autoclaving at 121 °C for 15 minutes.

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Appendix 8

Eggplant and tomato culture

Sow seeds of tomato (Lycopersicon esculentum) or eggplant (Solanum melongena) in pasteurised seed compost. Transplant seedlings with fully expanded cotyledons (10 to 14 days) into pasteurised potting compost.

Eggplants or tomatoes should be grown in a glasshouse with the following environmental conditions prior to inoculation:

REFERENCES

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ANNEX III

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ANNEX IV

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

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ANNEX V

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ANNEX VI

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ANNEX VII

The officially approved waste disposal methods referred to in Annex VI paragraph 1, shall conform to the following provisions such that any identifiable risk of spreading the organism is obviated:

The options described in this Annex also apply to the waste associated with handling, disposal and processing of contaminated lots.

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( 1 ) OJ C 124, 21.4.1997, p. 12 and OJ C 108, 7.4.1998, p. 85.

( 2 ) OJ C 14, 19.1.1998, p. 34.

( 3 ) OJ C 206, 7.7.1997, p. 57.

( 4 ) OJ L 26, 31.1.1977, p. 20. Directive as last amended by Commission Directive 98/2/EC (OJ L 15, 21.1.1998, p. 34).

( 5 ) OJ L 184, 3.8.1995, p. 34. Directive as last amended by Commission Directive 97/46/EC (OJ L 204, 31.7.1997, p. 43).