1998L0057 — EN — 21.08.1998 — 000.001 — 1
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)
Corrected by:
►C1Corrigendum, OJ L 218, 18.8.1999, p. 30 (98/57)
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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 Commission(1),
Having regard to the Opinion of the European Parliament(2),
Having regard to the Opinion of the Economic and Social
Committee(3),
Whereas the harmful organism Ralstonia solanacearum (Smith)
Yabuuchi et al. was previously known as Pseudomonas solanacearum
(Smith) Smith; whereasRalstonia 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 scientificdevelopment;
Whereas potato and tomato production occupies an important place in
Community agriculture; whereas the potato and tomato yield isconstantly threatened by harmful organisms;
Whereas, through the protection of potato and tomato cultivation against
such harmful organisms, not only should productive capacity be main-tained 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 alimited effect if such organisms were not controlled simultaneously andmethodically throughout the Community and were not prevented fromspreading;
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 andtomatoes; whereas disease outbreaks caused by this pathogen haveoccurred in some parts of the Community and some limited sources ofinfection still exist;
Whereas there is a considerable risk to potato and tomato cultivation
throughout the Community if effective measures are not taken, withrespect to these crops, to locate this organism and determine its distribu-tion, to prevent it from occurring and spreading, and, if found, toprevent 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 totake additional or stricter measures where necessary, provided that thereis no hindrance to the movement of potatoes or tomatoes within theCommunity, except insofar as laid down in Council Directive 77/93/EEC of 21 December 1976 on protective measures against the intro-duction into the Community of organisms harmful to plants or plantproducts and against their spread within the Community (
4); whereas
such measures must be notified to the other Member States and to theCommission;
Whereas the measures have to take into account that systematic official
surveys are necessary to locate the pathogen; whereas such surveysshould include inspection procedures and, where appropriate, given that
(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).
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under certain environmental circumstances the disease can remain latent
and unobserved both in the growing crop of potatoes and in storedpotato tubers, should include sampling and testing procedures; whereasspread of the pathogen within the growing crop is not the most impor-tant factor, but whereas the pathogen can spread by surface water andcertain associated wild solanaceous plants, and therefore the irrigationof potato and tomato crops using contaminated water appears to pose arisk for infection of such crops; whereas also the pathogen can existthrough the winter in self-sown (volunteer) potato and tomato plantsand these may be a source of infection being carried from one season tothe next; whereas the pathogen is spread also by the contamination ofpotatoes through contact with infected potatoes and through contactwith planting, harvesting and handling equipment or transport andstorage containers which have become contaminated with the organismby previous contact with infected potatoes;
Whereas spread of the pathogen can be reduced or prevented by decon-
tamination of such objects; wheras any such contamination of seedpotatoes poses a major risk for the spread of the pathogen; similarly thelatent infection of seed potatoes poses a major risk for the spread of thepathogen and this can be prevented by the use of seed potatoesproduced in an officially approved programme whereby seed potatoeshave 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 condi-
tions is incomplete and it is anticipated that a review of the measuresproposed will be necessary within several seasons; similarly improve-ments to the test procedure are anticipated in the light of furtherresearch especially on the sensitivity and specificity of test methods inorder 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 MemberStates to prevent the introduction of the pathogen into their territory, itis desirable for Member States to cooperate closely with the Commis-sion within the Standing Committee of Plant Health (hereinafterreferred to as ‘the Committee'),
HAS ADOPTED THIS DIRECTIVE:
Article 1
This Directive concerns the measures to be taken within the Member
States against Ralstonia solanacearum (Smith) Yabuuchi et al., previ-
ously known as Pseudomonas solanacearum (Smith) Smith, (hereinafter
referred to as ‘the organism'),in order to, with respect to the host plantsof the organism listed in Annex I Section 1, (hereinafter referred to as‘the listed plant material'):
(a) locate it and determine its distribution;
(b) prevent its occurrence and spread; and
(c) if found, to prevent its spread and to control it with the aim of
eradication.
Article 2
1. Member States shall conduct annual systematic official surveys for
the organism on the listed plant material originating in their territory. Inorder to identify other possible sources of contamination threatening theproduction of the listed plant material, Member States shall carry out arisk assessment and, unless no risk of spread of the organism has beenidentified during that assessment, they shall, in production areas of thelisted plant material, conduct targeted official surveys for the organismon plants other than the listed plant material, including wild solanaceoushost plants, as well as on surface water which is used for irrigation orspraying of listed plant material and on liquid waste discharged fromindustrial processing or packaging premises handling listed plantmaterial and used for irrigation or spraying of listed plant material. Theextent of these targeted surveys shall be determined according to the
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risk identified. Member States may also conduct official surveys for theorganism on the material, such as growing medium, soil and solid wastefrom industrial processing or packaging premises.
2. The official surveys provided for in paragraph 1 shall be carried
out:
(a) for the listed plant material, according to the details set down in
point 1 of Section II, Annex I; and,
(b) for host plants other than the listed plant material, and for water
including liquid waste, in accordance with appropriate methods and,where appropriate, samples shall be taken and subjected to officialor officially supervised laboratory testing;
(c) where appropriate for other material, in accordance with appropriate
methods.
For these surveys, further details of the inspection procedures and the
number, origin, stratification and timing of collection of samples shallbe decided by the responsible official bodies within the meaning ofDirective 77/93/EEC based on sound scientific and statistical principlesand the biology of the organism and taking into account in the MemberState concerned, the particular production systems of the listed plantmaterial 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 andto the Commission in accordance with the provisions of point 2 ofSection II, Annex I. These notifications shall be submitted by 1 Juneexcept for potatoes use as farm-saved seed for which the notificationshall be submitted by 1 September. The details and results for cropsshall be related to the previous year’s production. The details of thesenotifications 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:
— the appropriate methods for the surveys and the laboratory testing
provided for in paragraph 2, first subparagraph, (b).
5. The following provisions may be adopted in accordance with the
procedure laid down in Article 16a of Directive 77/93/EEC:
— the appropriate methods for the surveys provided for in paragraph 2,
first subparagraph, (c),
— further details of the surveys provided for in paragraph 2, second
subparagraph, with a view to ensuring comparable levels of assur-ance between Member States.
Article 3
Member States shall ensure that the suspected occurrence or confirmed
presence of the organism in their territory shall be reported to their ownresponsible official bodies.
Article 4
1. In each case of suspected occurrence, the responsible official
bodies of the Member State(s) concerned shall ensure completion ofofficial or officially supervised laboratory testing using, for the listedplant material, the relevant method set out in Annex II and in accord-ance with the conditions specified in point 1 of Annex III, or, in allother cases, any other officially approved method, in order to confirm orrefute the suspected occurrence. In the case of confirmation, the require-ments 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:
(i) diagnostic symptoms of the disease caused by the organism have
been seen and a positive result in the rapid screening test(s), asspecified in Annex II section I, point 1 and section II has beenobtained; or,
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(ii) a positive result in the screening test(s) as specified in Annex II
section I, point 2 and section III has been obtained,
the responsible official bodies of the Member States shall, in relation totheir own production:
(a) prohibit the movement of plants and tubers from all crops, lots or
consignments from which the samples have been taken, exceptunder their control and provided that it has been established thatthere is no identifiable risk of the organism spreading;
(b) take steps to trace the origin of the suspected occurrence;
(c) introduce appropriate additional precautionary measures based on
the level of estimated risk, particularly in relation to production ofthe listed plant material and the movement of seed potato lots otherthan those referred to in point (a), produced on the place of produc-tion from which the samples referred to in point (a) were taken, inorder to prevent any spread of the organism.
3. In those cases of suspected occurrence where there is a risk of
contamination of the listed plant material or surface water from or intoanother Member State(s), the Member State in which the suspectedoccurrence has been reported shall immediately notify, according to therisk identified, the details of the said suspected occurrence to the otherMember State(s) concerned, and there whall be appropriate cooperationbetween the said Member States. The Member State(s) so notified shallintroduce precautionary measures in accordance with paragraph 2(c)and take any further action, as appropriate, in accordance with para-graphs 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:
— the measures referred to in paragraph 2(c).
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 allother cases, any other officially approved method, confirms the pres-ence of the organism in a sample taken pursuant to this Directive, theresponsible official bodies of a Member State, having regard to soundscientific principles, the biology of the organism and the particularproduction, marketing and processing systems of the host plants of theorganism in that Member State, shall:
(a) for the listed plant material;
(i) establish an investigation to determine the extent and primary
source(s) of the contamination in accordance with the provi-sions of Annex IV, with further testing in accordance withArticle 4(1), on, at least, all clonally related seed potato stocks,and,
(ii) designate as contaminated the listed plant material, consign-
ment and/or lot from which the sample was taken, and themachinery, vehicle, vessel, store, or units thereof, and anyother objects including packaging material which have been incontact with the listed plant material from which the samplewas taken; also designate as contaminated, where appropriate,the field(s), unit(s) of protected crop production and place(s) ofproduction from which the listed plant material was harvestedand from which the sample was taken; and for those samplestaken in the growing season, designate as contaminated thefield(s), place(s) of production, and, where appropriate, unit(s)of protected crop production from which the sample was taken,and,
(iii) determine, in accordance with the provisions of point 1 of
Annex V, the extent of probable contamination through pre- orpost-harvest contact, through production, irrigation or sprayinglinks or through clonal relationship with the designatedcontamination, and,
(iv) demarcate a zone on the basis of the designation of contamina-
tion under point (ii), the determination of the extent of prob-able contamination under point (iii), and the possible spread of
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the organism, in accordance with the provisions of point 2(i) ofAnnex V;
b) for crops of host plants other than those mentioned under point (a)
where production of the listed plant material is identified at risk,
(i) establish an investigation in accordance with point (a)(i); and
(ii) designate as contaminated the host plants of the organism from
which the sample was taken; and
(iii) determine the probable contamination and demarcate a zone in
accordance with points (a)(iii) and (iv), respectively, in relationto production of the listed plant material;
c) for surface water (including liquid waste discharges from industrial
processing or packaging premises handling listed plant material)and associated wild solanaceous host plants, where production ofthe listed plant material is identiefied at risk through irrigation,spraying, or flooding of the surface water,
(i) establish an investigation including an official survey at appro-
priate times on samples of surface water and if present wildsolanaceous host plants to establish the extent of the contami-nation; and
(ii) designate as contaminated the surface water from which the
sample(s) was taken, to the extent appropriate and on the basisof the investigation under point (i); and
(iii) determine the probable contamination and demarcate a zone on
the basis of the designation of contamination under point (ii),and the possible spread of the organism taking into account theprovisions of points 1 and 2(ii) of Annex V.
2. Member States shall immediately notify the other Member States
and the Commission, in accordance with the provisions of point 3 ofAnnex V, of any contamination designated under paragraphs 1(a)(ii)and 1(c)(ii) and the details of the zone demarcation under paragraph1(a)(iv) and, where applicable, under paragraph 1(c)(iii). The details ofthe notification under this paragraph may be submitted to theCommittee.
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 thenotification under this subparagraph shall immediately be submitted tothe 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 shallestablish an investigation in accordance with paragraph 1(a)(i) and,where applicable, paragraph 1(c)(i) and take further action, as appro-priate, in accordance with paragraphs 1 and 2.
Article 6
1. Member States shall prescribe that the listed plant material desig-
nated to be contaminated under Article 5(1)(a)(ii) may not be plantedand that, under the control and approval of their responsible officialbodies, it shall be subjected to one of the provisions of point 1 of AnnexVI, such that it is established that there is no identifiable risk of theorganism spreading.
2. Member States shall prescribe that the listed plant material deter-
mined 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 contaminatedunder Article 5(1)(a)(iii) may not be planted and shall, under the controlof their responsible official bodies, be put to appropriate use or disposalas specified in point 2 of Annex VI, such that it is established that thereis 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 packagingmaterial, designated as contaminated under Article 5(1)(a)(ii) or deter-mined as probably contaminated under Article 5(1)(a)(iii) and (c)(iii),shall either be destroyed or decontaminated using appropriate methods
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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 demarcatedunder Article 5(1)(a)(iv) and (c)(iii), a series of measures, as specifiedin points 4.1 and 4.2 of Annex VI, shall be implemented. Details ofthese measures shall be notified annually to the other Member Statesand to the Commission. The details of this notification may besubmitted 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 linefrom potato material obtained under an officially approved programmewhich has been found free of the organism in official or officiallysupervised testing using the relevant method set out in Annex II.
The aforesaid testing shall be carried out by a Member State:
(a) in cases where there have been confirmed finding(s) of the
organism in their own seed potato production,
(i) by testing of the earlier propagations, including the initial clonal
selection and systematic testing of basic seed potato clones; or
(ii) in cases where it has been established that there is no clonal
relationship, by testing of all basic seed potato clones or earlierpropagations including the initial clonal selection, and
b) in other cases, either on each plant of the initial clonal selection or
on representative samples of the basic seed potatoes or earlierpropagations.
2. The following provisions may be adopted in accordance with the
procedure laid down in Article 16a of Directive 77/93/EEC:
— the detailed rules of application of paragraph 1, second subpara-
graph, point (a),
— the rules concerning the representative samples provided for in
paragraph 1, second subparagraph, point (b).
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 inArticles 6 and 8 of this Directive in accordance with the provisions laiddown in Directive 95/44/EC (
1) 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 theorganism or to prevent it from spreading, insofar as they are in compli-ance 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 besubmitted to the Committee.
(1) 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).
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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 inaccordance with the procedure laid down in Article 16a of Directive77/93/EEC. In the case of the methods laid down in Annex II and themeasures in paragraphs 4.1 and 4.2 of Annex VI to this Directive areport shall be prepared by the Commission reviewing these methodsand measures in the light of experience gained and the report shall besubmitted 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 witheffect from 21 August 1999. They shall forthwith inform the Commis-sion thereof.
When Member States adopt these measures, they shall contain a refer-
ence to this Directive or shall be accompanied by such reference on theoccasion of their official publication. The methods of making suchreference 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 inthe field governed by this Directive. The Commission shall inform theother 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 Ralstoniasolanacearum(Smith)Yabuuchietal. referred
to in Article 1
Plants (including tubers), other than true seed, of Sonanum
tuberosum L.Potato
Plants, other than fruits and seeds, of Lycopersicon lycopersicum
(L.) Karsten ex Farw.Tomato
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 MemberState 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 instore. These samples shall be subjected to official or officially super-vised 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 methodset 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 indus-
trial processing or packaging premises,
— number and timing of samples,
— area/river/location of premises sampled, as appropriate,— method of analysis.
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ANNEX II
TEST SCHEME FOR DIAGNOSIS, DETECTION AND IDENTIFICA-
TION OFRALSTONIA SOLANACEARUM (SMITH) YABUUCHI ET AL.
SCOPE OF THE TEST SCHEME
The presented scheme describes the various procedures involved in:
(i) diagnosis of brown rot in potato tubers and of bacterial wilt in potato and
tomato plants;
(ii) detection of Ralstonia solanacearum in samples of potato tubers;
(iii)identification of Ralstonia solanacearum .
In the Appendices, details are provided for the preparation of the test materials,
i.e. growth media, buffers, solutions, reagents.
CONTENTS:
SECTION I: Application of the test scheme … 9
1. Diagnosis of brown rot in potato tubers and of bacterial wilt in potato
and tomato plants …9
2. Detection and identification of Ralstonia solanacearum in samples of
potato tubers …11
SECTION II: Diagnosis of brown rot in potato tubers and of bacterial wilt in potato
and tomato plants …13
1. Symptoms … 13
2. Rapid screening test(s) … 133. Isolation procedure … 144. Confirmation test(s) … 14
SECTION III: Detection and identification of Ralstonia solanacearum in samples of
potato tubers …17
1. Preparation of the sample for testing … 17
2. Immunofluorescence (IF) test … 183. Enzyme Linked Immuno-Sorbent Assay test (ELISA) … 204. Polymerase-Chain Reaction test (PCR )… 2 0
5. Selektive plating test … 226. Bioassay test … 237. Enrichment tests … 238. Pathogenicity test … 23
Appendix 1 Nutrient media for isolation and culture of Ralstonia solanacearum …2 4
Appendix 2 Materials for sample preparation … 25
Appendix 3 Materials for the IF test … 26
Appendix 4 Determination of the level of contamination in the IF test … 27
Appendix 5 Materials for the ELISA test … 28
Appendix 6 Materials for the PCR test … 29
Appendix 7 Materials for the selective plating and enrichment test(s) … 29
References … 30
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SECTION I
APPLICATION OF THE TEST SCHEME
1.Diagnosis of brown rot in potato tubers and of bacterial wilt in
potato and tomato plants
The testing procedure is intended for potato tubers with symptoms typical
or suspect of brown rot and for potato and tomato plants with symptomstypical or suspect of bacterial wilt. It involves a rapid screening test,isolation of the pathogen from infected vascular tissue on diagnosticmedia and, in case of a positive result, identification of the culture asRalstonia solanacearum .
Flow chart diagram presentation
Flowchart references:
(1) Description of symptoms is provided in section II.1.
(2) Rapid screening tests facilitate presumptive diagnosis.
Appropriate tests are:
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— Streaming test of vascular stem tissue (section II.2),
— Test for poly- β-hydroxybutyrate granules (section II.2),
— IF test (section III.2),— ELISA test (section III.3),— PCR test (section III.4).
(
3) Although isolation of the pathogen from plant material with typical
symptoms by dilution plating is straightforward, culturing may failfrom advanced stages of infection. Saprophytic bacteria which growon diseased tissue may outgrow or inhibit the pathogen on the isola-tion medium. If the isolation test is negative, but disease symptomsare typical, then isolation must be repeated, preferably by a selectiveplating test.
(
4) Reliable identification of a pure culture of Ralstonia solanacearum is
achieved by at least one of the tests listed in section II.4.1 in combi-nation with a pathogenicity test (section II.4.3). Strain characterisationis optional but recommended for each new case.
2.Detection and identification of Ralstonia solanacearum in samples of
potato tubers
The procedure is intended for detection of latent infections in potato
tubers by one or, preferably, more screening test(s) which, if positive, arecomplemented by the isolation of the pathogen; followed by, in case ofisolation of typical colonies, identification of a pure culture as Ralstonia
solanacearum .
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Flow chart diagram presentation
Flowchart references:
(1)Sample size
The standard size is 200 tubers. However, the procedure can beapplied conveniently for samples with less than 200 tubers.
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(2)Screening test(s)
A single test may not be sufficiently sensitive or reliable to detectRalstonia solanacearum in a sample. Therefore, more than one test is
recommended and these tests should preferably be based on differentbiological principles.
(
3)Immunofluorescence (IF) test
The IF test is a well established screening test. This is an advantageover other tests which are not yet fully developed or validated. Thetest is used for many other statutory bacteria, e.g. Clavibacter michi-
ganensis subsp.sepedonicus . With the reading parameters specified in
this method it is a sensitive test (sensitivity threshold of 10
3—104cells
per ml of potato extract pellet).The critical factor for the reliability of the test result is the quality ofthe antiserum. Only antiserum with a high titre (minimum 2 000 forthe crude antiserum) is acceptable and all tests must be carried out atthe antiserum titre or one dilution below the titre. The indirect methodis preferred. The direct method can be applied if the test has a level ofsensitivity and specificity equivalent to that of the indirect method.The IF test has the advantage of subjective interpretation of cellstaining morphology and fluorescence intensity which provide infor-mation on reaction specificity. Cross reactions by serologically relatedbacteria from soil or associated with potato tissues with cellmorphology of Ralstonia solanacearum are common. The IF test can
be used as a sole screening test although in cases where cross-reac-tions are suspected an additional screening test based on a differentbiological principle should be done. In such cases selective plating isthe most appropriate test.
(
4)Selective plating
With the modified SMSA medium and testing methodology specifiedin this method, this is a sensitive and selective test for Ralstonia
solanacearum . The result is available 3—6 days after sample prepara-
tion. The pathogen is obtained directly in culture and can be readilyidentified. For full exploitation of its potential, the test requires carefulpreparation of heel ends to avoid secondary bacteria associated withthe potato tuber which are competitors with Ralstonia solanacearum
on the medium and may affect the development of the pathogen. Somestrains may grow poorly as the components of the medium may affectthe target organism. Care is also required to differentiate Ralstonia
solanacearum from other bacteria which may develop on the medium.
Selective plating can be used as a sole screening test provided that incases where inhibition of Ralstonia solanacearum by other bacteria on
the plates is suspected and a negative test result is obtained the sampleis retested using a different test to confirm or refute the diagnosis. Insuch cases, the IF test is the most appropriate test.
(
5)ELISA test
The ELISA test is generally less sensitive than the IF test (sensitivitythreshold of 10
4—105cells per ml of potato extract pellet). The test is
cheap and fast but generally more vulnerable to false positive (crossreactions) and false negative results (inhibition by phenolic moleculesin the potato extract). The requirements for antiserum specificity areextremely high. The ELISA test cannot be used as a single screeningtest.
(
6)PCR test
PCR has the potential for very sensitive detection although the test isreadily inhibited by plant or tuber extract components resulting infalse negatives. Some potato cultivars contain more inhibitors thanothers. It is therefore necessary to remove these inhibitors. The inhibi-tion can be reduced by dilution but the populations of Ralstonia
solanacearum are also diluted. Great care has to be taken in all steps
of sample and test preparation to prevent contamination which would
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result in false positive tests. False positives could also arise from
sequence homology of other organisms. For these reasons the directPCR cannot be used as a sole screening test.
(
7)Enrichment test
Incubating potato extract pellet samples in a semi-selective broth, suchas modified SMSA broth, allows multiplication of Ralstonia solana-
cearum. More important perhaps, it also dilutes potential inhibitors of
the ELISA or PCR test. Ralstonia solanacearum in enrichment broth
can thus be detected by IF, ELISA and PCR. We do not recommendthat direct plating is made from the enriched broths. These enrichmentmethods have not been thoroughly tried and tested. They are includedhere because they have good potential. However, because of therelative lack of experience with them, they cannot be used as soledetection methods.
(
8)Bioassay test
The bioassay test is used for isolation of Ralstonia solanacearum from
potato extract pellets by selective enrichment in a host plant and canbe done on tomato plants or eggplants. The test requires optimalincubation conditions as specified in this method. Bacteria inhibitorytoRalstonia solanacearum on the SMSA medium will most likely not
interfere in this test.
(
9)Confirmation test(s)
Reliable identification of a pure culture of Ralstonia solanacearum is
achieved by at least one of the tests listed in section II.4.1. in combi-nation with a pathogenicity test (section II.4.3.). Strain characterisationis optional but recommended for each new case.
SECTION II
Diagnosis of brown rot in potato tubers and of bacterial wilt in potato and
tomato plants
1.Symptoms
1.1 Symptoms in potato
The potato plant. The early stage of infection is wilting of the leaves
towards the top of the plant at high temperatures during the day withrecovery at night. Wilting becomes rapidly irreversible and results in thedeath of the plant. The vascular tissue in transversely cut stems fromwilted plants may become brown and a milky ooze exudes from the cutsurface or can be easily expressed by squeezing. When a cut stem isplaced vertically in water, threads of slime will stream from the vascularbundles.
The potato tuber. Potato tubers must be cut transversely close to the heel
(= stolon) end. The early stage of infection is a glassy yellow to lightbrown discoloration of the vascular ring from which a pale cream oozeemerges spontaneously after a few minutes or when gentle pressure isapplied with the thumbs on the skin near the cut surface. Later, thevascular discoloration becomes more distinct brown and the necrosis canextend into the parenchymatous tissue. In advanced stages, the infectionbreaks outwards from the heel end and the eyes which may result inreddish-brown, slightly sunken lesions on the skin from which bacteriamay ooze, causing soil particles to adhere.
1.2 Symptoms in tomato
The tomato plant. The first visible symptom is the flaccid appearance of
the youngest leaves. Under favourable environmental conditions for thepathogen (soil temperature about 25 °C saturated humidity), epinasty and
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wilting of one side or of the whole plant follows within a few days
which leads to total plant collapse. Under less favourable conditions (soiltemperature below 21 °C) large numbers of adventitous roots may
develop on the stem. It is possible to observe a greasy cord along thestem which is evidence of the necrosis in the vascular system. When thestem is cut crosswise discoloured brown vascular tissues of the stemexude drops of white or yellowish bacterial ooze.
2.Rapid screening tests
Rapid screening tests facilitate presumptive diagnosis. Use one or more of
the following tests:
Stem streaming test
The presence of Ralstonia solanacearum in wilting potato or tomato
stems can be assessed by the following simple presumptive test:
Cut the stem just above the soil level. Place the cut surface in a beaker
with water. Shortly after, threads of bacterial slime will stream spontane-ously out of the vascular bundles. Any other bacteria causing vascularinfection in potato or tomato plants will not show this phenomenon.
Detection of poly- β-hydroxybutyrate (PHB) granules
The PHB granules in the cells of Ralstonia solanacearum are visualised
by staining with Nile blue A or with Sudan black B.
Either prepare a smear of the ooze or the suspended tissue on a micro-
scope slide or prepare a smear of a 48 hour culture on YPGA or SPA(Appendix 1). Prepare positive control smears of a biovar2/race 3 strainand, if considered useful, a negative control smear of a heterologeousstrain. Allow to dry. Pass the lower surface of the slide several timesrapidly through the flame until the smear is fixed.
Nile blue test
(1) Flood the fixed smear with 1 % aqueous solution of Nile blue A.
Incubate for 10 minutes at 55 °C.
(2) Drain off the staining solution. Wash briefly in gently running tap
water. Remove excess water with tissue paper.
(3) Flood the smear with 8 % aqueous acetic acid.
Incubate for one minute at ambient temperature.
(4) Wash in gently running tap water. Blot dry on tissue paper.(5) Remoisten with a drop of water. Apply a coverslip.(6) Examine the stained smear with an epifluorescence microscope at
450 nm under oil immersion at a magnification of 1 000.
Observe for bright orange fluorescence of PHB granules. Also observe
under normal light to ensure that the granules are intracellular and thatcell morphology is typical of Ralstonia solanacearum .
Sudan black test
(1) Flood the fixed smear with 0,3 % Sudan black B solution in 70 %
ethanol. Incubate for 10 minutes at ambient temperature.
(2) Drain off the staining solution. Wash briefly in tap water. Remove
excess water. Remove excess water with tissue paper.
(3) Dip the smear briefly in xylol. Blot dry on tissue paper.
Caution! Xylol is a harmful product. Work in a fume cabinet.
(4) Flood the smear with 0,5 % (w/v) aqueous safranin and leave for 10
seconds at ambient temperature.
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Caution! Safranin is a harmful product. Work in a fume cabinet.
(5) Wash in gently running tap water. Blot dry on tissue paper. Apply a
coverslip.
(6) Examine the stained smear with a light microscope using transmitted
light under oil immersion at a magnification of 1 000.
PHB granules in cells of Ralstonia solanacearum stain blue-black.
The cell wall stains pink.
Other tests
Other appropriate screening tests are the IF test (section III.2), the ELISA
test (section III.3) and the PCR test (section III.4).
3.Isolation procedure
3.1 Remove ooze or sections of discoloured tissue from the vascular ring in
the potato tuber or from the vascular strands in the stem of potato ortomato plants. Suspend in a small volume of sterile distilled water or50 mM phosphate buffer. Leave for 5—10 minutes on the bench.
3.2 Prepare a series of decimal dilutions of the suspension, e.g. 1/10 and
1/100 or more as considered appropriate.
3.3 Transfer a standard volume of the suspension and the dilutions on to a
general nutrient medium (NA, YPGA and SPA, Appendix 1) and/or on toKelman’s tetrazolium medium (Appendix 1) and/or on to SMSA selectivemedium (Appendix 7). Spread or streak with an appropriate dilutionplating technique. If considered useful, prepare separate plates of eachmedium used with a diluted cell suspension culture of a virulent biovar2/race 3 strain of Ralstonia solanacearum as a positive control.
3.4 Incubate the plates for three days at 28 °C. Incubation may be prolonged
to six days if growth is slow, but colonies on SMSA plates often becomeatypical and die off.
On the general nutrient media, virulent isolates of Ralstonia solana-
cearumdevelop pearly-white, flat, irregular and fluidal colonies often
with characteristic whorls.
On Kelman’s tetrazolium medium, typical colonies of virulent isolates of
Ralstonia solanacearum are cream, flat, irregular and fluidal with blood
red coloured whorls in the centre. Avirulent forms of Ralstonia solana-
cearumdevelop butyrous, deep-red colonies.
On SMSA medium, typical colonies of virulent isolates of Ralstonia
solanacearum are milky-white, flat, irregular and fluidal with blood red
colouration in the centre.
Avirulent forms of Ralstonia solanacearum develop less fluidal colonies
which are completely pink to red on the SMSA medium.
3.5 Purify colonies with characteristic morphology by subculture on a general
nutrient medium. Avoid regular subculturing which may induce loss ofvirulence.
4.Confirmation test(s)
4.1 Identification of Ralstonia solanacearum
Identify pure cultures of Ralstonia solanacearum by at least one of the
following procedures:
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Nutritional and enzymatic tests
Note:Include appropriate control strains in each test used.
The following phenotypic properties of Ralstonia solanacearum are
universally present or absent:
Fluorescent pigment −
PHB inclusions +Oxidation/Fermentation (O/F) test O +/F −Catalase +Kovac’s oxidase +
Reduction of nitrate +
Utilisation of citrate +
Growth at 40 °C −
Growth in 1 % NaCl +
Growth in 2 % NaCl −Arginine dihydrolase −Gelatin liquefaction −Starch hydrolysis −Aesulin hydrolysis −Levan production −
Media and methods are provided in Lelliott & Stead (1987)
IF-test
Prepare a suspension of 10
6cells per ml from the culture and the control
strain(s). Prepare a series of twofold dilutions of the antiserum. Applythe IF procedure (section III.2). The IF titre of the culture must beequivalent to that of the positive control.
ELISA test
Prepare a suspension of >10
6cells per ml from the culture and the
control strain(s). Apply the ELISA procedure (section III.3). The ELISAvalue of the culture must be equivalent to that of the positive control.
PCR test
Prepare a suspension of 10
6cells per ml from the culture and the control
strain(s). Apply the PCR procedure (section III.4). The PCR product ofthe culture must have the same size and restrictive enzyme analysis(REA) pattern as that of the positive control.
Fluorescent in-situ hybridisation (FISH)
Prepare a suspension of 10
6cells per ml from the culture and the control
strain(s). Apply the FISH procedure (van Beuningen et al, 1995) with the
OLI-1 PCR primer (Seal et al, 1993) The culture must show the same
reaction as the positive control.
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Biovar
1 2 3 4 5
biovar 2 biovar 2-A biovar 2-TProtein profiling
Denatured whole cell proteins are separated by polyacrylamide gel elec-
trophoresis — PAGE (Stead, 1992a).
Fatty acid profiling (FAP)
Grow the culture and a positive control strain for 48 hours at 28 °Co n
trypticase soy agar and apply the FAP procedure (Janse, 1991; Stead,1992a; Stead, 1992b). The profile of the culture must be identical to thatof the positive control. Under the specified conditions, characteristicfatty acids are 14:0 3OH, 16:0 2OH, 16:1 2OH und 18:1 2OH.
Strain characterisation
Strain characterisation is optional but is recommended for each new case
using at least one of the following: —
Biovar determination
Ralstonia solanacearum is separated into biovars on the basis of the
ability to produce acid from three hexose alcohols and three sugars(Hayward, 1964 & 1994):
Utilisation of:
— maltose
− + + − +
— lactose − + + − +
— cellobiose − + + − +
— mannitol − − + + +
— sorbitol − − + + −
— dulcitol − − + + −
Additional tests differentiate biovar 2 in subphenotypes (Hayward,
1994):
utilisation of trehalose − + +
utilisation of inositol + − +
utilisation of D-ribose − − +
pectolytic activity low low high
Race determination
The race (Buddenhagen et al., 1962) can be determined on the basis of
a pathogenicity test in tomato plants or eggplants and in tobacco plantsand by a hypersensitivity reaction (HR) test in tobacco leaves (Lozanoand Sequeira, 1970):
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Race (*)
1 2 3
Reaction in: — tomato plants/eggplants wilting no reaction wilting
— tobacco plants wilting no reaction no reaction
— tobacco leaves necrosis
(48 hrs)
and
wilting
(7—8
days)HR (12—24
hrs)chlorosis
(2—8 days)
(*) Race 4 (pathogenic on ginger and a few other hosts) and race 5 (pathogenic on mulberry only) are not
included.
Race characterisation by the pathogenicity test or tobacco hypersensi-
tivity test may not be highly reliable and instead can be deduced fromthe biovar and the natural host of origin.
The culture can be further characterized by:
Genomic fingerprinting
Molecular differentiation of strains in the Ralstoniasolanacearum
complex can be done by:
RFLP analysis (Cook et al, 1989)
Repetitive sequence PCR [REP-, ERIC- & BOX-PCR (Louws et al,
1995; Smith et al, 1995)]
4.3.Pathogenicity test
This test is for confirmation of the diagnosis of Ralstonia solanacearum
and for the assessment of the virulence of the cultures identified asRalstonia solanacearum .
Prepare an inoculum of 10
6cells per ml from the culture and a positive
control strain. Inoculate 5—10 tomato plants or eggplants at, preferably,the third true leaf stage or older (section III, 6.). Incubate for up to twoweeks at 22 °C—28 °C and high relative humidity with daily watering.
Observe for wilting and/or epinasty, chlorosis, stunting.
Isolate from symptomatic plants as follows:
— Remove a section of tissue from the stem two cm above the inocula-
tion point.
— Comminute and suspend in a small volume of sterile distilled water
or 50 mM phosphate buffer. Then plate, incubate, and check fortypical colonies of Ralstonia solanacearum .
SECTION III
DETECTION AND IDENTIFICATION OF RALSTONIA SOLANA-
CEARUM IN SAMPLES OF POTATO TUBERS
Note: The standard sample size is 200 tubers. However, the procedure can be
applied conveniently for samples with less than 200 tubers.
1.Preparation of the sample for testing
Note:The potato extract pellet obtained in this procedure can also be
used for detection of Clavibacter michiganensis subsp.sepedo-
nicus.
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Pre-testing options if considered useful:
(i) Incubate the sample at 25—30 °C for up to two weeks to encourage
multiplication of low Ralstoniasolanacearum populations.
(ii) Wash the tubers under running water with appropriate disinfectants
and detergents. Air dry the tubers.
1.1 Remove with a clean and disinfected scalpel or vegetable knife the skin at
the heel end of the tuber so that the vascular tissues first become visible.Carefully cut out a small conical core (3—5 mm diameter) of vasculartissue at the heel end. Keep the amount of non-vascular tissue to aminimum. Process each of the tubers in the sample.
Note:Visual examination of the tubers (Section II.1) can be done at this
stage. Set aside any tuber with symptoms or severe rotting andtest separately (section II).
1.2 Collect the heel ends in a closed container. Preferably, the heel ends
should be processed immediately. If this is not possible, store them fornot more than 24 hours or, at 4 °C, for not longer than 72 hours.
1.3 Process the heel ends by one of the following procedures:
(i) Transfer the heel ends into an appropriate container.
Add a sufficient volume of maceration buffer (Appendix 2) to cover
the heel ends.
Comminute the heel ends in a Waring Blender or by Ultra Thurrax
until complete homogenisation has just been achieved. Avoid exces-sive homogenisation.
Allow the macerate to soak for 15—30 minutes.
(ii) Transfer the heel ends into an appropriate container.
Add a sufficient volume of maceration buffer to cover the heel ends.Place the container on a rotary shaker.Incubate at 50—100 rpm for 4 hours at 20 °C—22 °C or for 16—24
hours at 4 °C.
(iii) Transfer the heel ends into a strong disposable maceration bag (e.g.
Stomacher bag with dimensions 105 mm × 150 mm, radiationsterile).
Crush the heel ends carefully with an appropriate tool, e.g. a
hammer, until complete homogenisation has been achieved.
Add a sufficient volume of maceration buffer to cover the heel ends.Allow the macerate to settle for 15—30 minutes.
1.4 Extract the bacteria from the processed heel ends by one of the following
procedures:
(i) Decant the macerate gently in a centrifuge tube while leaving the
debris in the container or bag. If the decanted macerate is cloudy,centrifuge at not more than 180 g for 10 minutes at a temperaturebelow 10 °C.
Centrifuge the decanted macerate, or the supernatant from the first
centrifugation step, at 7000 g for 15 minutes or at 10 000 g for 10minutes at a temperature below 10 °C.
Discard the supernatant without disturbing the pellet.
(ii) Filter the macerate through a filtration system with pore size of
40—100 µm. Enhance filtration using a vacuum pump.
Collect the filtrate in a centrifuge tube.Wash the filter with maceration buffer.Centrifuge filtrate at 7000 g for 15 minutes or at 10 000 g for 10
minutes at a temperature below 10 °C.
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Discard the supernatant without distrubing the pellet.
1.5 Resuspend the pellet in 1 ml pelletbuffer (Appendix 2).
Divide in two equal parts and transfer each part to a microvial.Use one microvial for testing. Conserve the remainder of this extract at
4°C during testing.
Add 10—25 % (v/v) of sterile glycerol to the other microvial. Vortex.
Store at −18 °C (weeks) or at −70 °C (months).
2.IF-test
Use antiserum for Ralstonia solanacearum , preferably to race 3/biovar 2.
Determine the titre on a suspension of 10
6cells per ml from the homolo-
geous strain of Ralstonia solanacearum with an appropriate dilution of
the fluorescein isothiocyanate (FITC) conjugate, according to the manu-facturer’s recommendations. The crude antiserum should have an IF titreof at least 1:2 000.
Use multiwell microscope slides with preferably 10 windows of at least
6 mm diameter.
Include a FITC conjugate control on each test slide. The test should be
repeated with a PBS control included if any positive cell is observed inthe FITC control.
Prepare separate positive control slides with a suspension of 10
6cells per
ml from a strain of the appropriate race/biovar of Ralstonia solana-
cearum. Use one slide in each set of tests.
2.1 Prepare the test slides by one of the following procedures:
(i) For pellets with relative little starch:
Pipette a measured standard volume (15 µl is appropriate for 6 mm
window diameter — scale up volume for larger windows) of theresuspended pellet on a row of windows. The remaining row can beused as duplicate or for a second sample as presented in Figure 1.
(ii) For other pellets:
Prepare decimal dilutions, viz. 1/10, 1/100 and 1/1 000 of the resus-
pended pellet in pellet buffer. Pipette a measured standard volume(15 µl is appropriate for 6 mm window diameter — scale up volumefor larger windows) of the resuspended pellet and each dilution on arow of windows. The remaining row can be used as duplicate or for asecond sample as presented in Figure 2.
2.2 Let the droplets dry. Fix the bacterial cells to the slide either by heating,
flaming or with 95 % ethanol.
2.3 IF procedure
(i) According to test slide preparation in 2.1 (i):
Prepare a set of twofold dilutions of the antiserum in IF buffer
(Appendix 3):
of the titre (T/4 ), of the titre (T/2 ), the titre (T) and twice the
titre (2T).
(ii) According to test slide preparation in 2.1 (ii):
Prepare the working dilution (WD) of the antiserum in IF buffer. The
working dilution is the dilution of the antiserum with optimumspecificity and is usually half of the titre.
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FITC T/4 T/2 T 2T = >Twofold dilutions
of antiserum
FITC Standard dilution of antiserum
Neat Neat 1/10 1/100 1/1 000 = >Decimal dilution
of resuspendedpelletFigure 1
Preparation of the test slide according to 2.1 (i) and 2.3 (i)
One standard dilution of resuspended pellet
T = titre
Sample 1 • 1 • 2 • 3 • 4 • 5
Duplicate of sample 1 or sample 2 •6•7•8•9•1 0
Figure 2
Preparation of the test slide according to 2.1 (ii) and 2.3 (ii)
Sample 1 • 1 • 2 • 3 • 4 • 5
Duplicate of sample 1 or sample 2 •6•7•8•9•1 0
2.3.1 Arrange the slides on damp tissue paper.
Cover the test windows with the antiserum dilution(s). Apply PBS on the
FITC windows. The volume of antiserum applied on the windows mustbe equivalent to the volume of extract applied.
2.3.2 Incubate under a cover for 30 minutes at ambient temperature.2.3.3 Shake the droplets of antiserum off the slide and rinse the slides carefully
with IF buffer. Wash for five minutes in IF buffer-Tween and subse-quently for five minutes in IF buffer (Appendix 3). Carefully removeexcess moisture.
2.3.4 Arrange the slides on damp tissue paper. Cover the test windows and the
FITC window with the dilution of FITC conjugate used to determine thetitre. The volume of conjugate applied on the windows must be identicalto the volume of antiserum applied.
2.3.5 Incubate under a cover for 30 minutes at ambient temperature.2.3.6 Shake the droplets of conjugate off the slide. Rinse and wash as before
(2.3.3). Carefully remove excess moisture.
2.3.7Pipette 5—10 µl 0,1 M phosphate-buffered glycerol (Appendix 3) or a
similar mountant on each window and apply a coverslip.
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2.4 Reading the IF test
Examine test slides on an epifluorescence microscope with filters suitable
for excitation of FITC, under oil immersion at a magnification of 500—1 000. Scan windows across two diameters at right angles and around theperimeter.
Check the positive control slide first. Cells must be bright fluorescent and
completely stained.
Note:The test must be repeated if the staining is aberrant.Read the test
slides. Observe first for absence of fluorescing cells in the FITC controlwindows. Fluorescing cells in the FITC control indicate non-specificbinding of the conjugate, autofluorescence or contamination:
Note:Repeat the test if such is observed.Observe for bright fluorescing
cells with characteristic morphology of Ralstonia solanacearum in the
test windows. The fluorescence intensity must be equivalent to the posi-tive control strain at the same antiserum dilution. Cells with incompletestaining or with weak fluorescence must be disregarded, unless there aremany such cells (see interpretation of the IF test result).
Interpretation of the IF test result:
(i) If bright fluorescing cells with characteristic morphology are not
found, then the IF test is negative.
(ii) If bright fluorescing cells with characteristic morphology are found,
then determine the mean number of cells per microscope field andcalculate the number of cells (N) per ml of resuspended pellet(Appendix 4).
A population of approximately 10
3cells per ml of resuspended pellet
is considered to be the limit of detection for the IF test,
— for samples with N >103cells per ml of resuspended pellet, the
IF test is considered positive,
— for samples with ►C1N<103cells◄per ml of resuspended
pellet, the IF test may be considered positive.
(iii) If large numbers ( >105cells per ml) of incomplete or weakly
fluorescing cells are seen at the titre of the antiserum, a second testshould be carried out:
— either a test based on a different biological principle, or
— a repeat IF test, with either a second antiserum or a ten-fold
dilution of the pellet.
3.ELISA test
Based upon Robinson-Smith et al., 1995
Use antiserum for Ralstonia solanacearum , preferably to race 3/biovar 2.
Determine the titre on a suspension of 10
6cells per ml from the homolo-
geous strain of Ralstonia solanacearum .
The use of NUNC-Polysorb microtitre plates is recommended.
Include a negative potato extract control and a phosphate buffered saline
(PBS) control.
Use a suspension of >106cells per ml from a strain of the appropriate
race/biovar of Ralstonia solanacearum as the positive control. Test in an
identical manner as the sample(s) but well-separated from the samples onthe microtitre plate.
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3.1 Pipette 100—200 µl of the resuspended pellet in a microvial.
Heat for 4 minutes at 100 °C. Remove the microvial on ice.
3.2 Add an equal volume of double strength carbonate coating buffer
(Appendix 5). Vortex.
3.3 Apply 100 µl aliquots to each of at least two wells of the microtitre plate.
Incubate for one hour at 37 °C or overnight at 4 °C.
3.4 Flick out the extracts from the wells. Wash the wells three times with
PBS-Tween (Appendix 5), leaving the last washing solution in the wellsfor at least five minutes.
3.5 Prepare the appropriate dilution of Ralstonia solanacearum antiserum in
blocking buffer (Appendix 5). Apply 100 µl of antiserum dilution to thewells.
Incubate for one hour at 37 °C.
3.6 Flick out the antiserum from the wells. Wash the wells as before (3.4).3.7Prepare the appropriate dilution of alkaline phosphatase conjugate in
blocking buffer. Apply 100 µl of conjugate dilution to the wells.
Incubate for one hour at 37 °C.
3.8 Flick out the conjugate from the wells. Wash the wells as before (3.4 and
3.6).
3.9 Prepare the alkaline phosphatase substrate solution (Appendix 5). Apply
100 µl to the wells. Incubate for 30 minutes to one hour in the dark atambient temperature.
3.10 Read absorbance at 409 nm.
Interpretation of the ELISA test:
The ELISA test is negative if the optical density (OD) of the sample is
>2 × OD of the negative control.
The ELISA test is positive if the optical density (OD) of the sample is
>2 × OD of the negative control.
4.PCR test
Based on Seal et al., 1993
Note:Filterplugged pipette tips must be used during all stages of
sample preparation and other manipulations involvingPCR.Prepare a suspension of 10
6cells per ml from a race 3/
biovar 2 strain of Ralstonia solanacearum as the positive control.
Test in an identical manner as the sample(s).
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Component Quantity Final concentration4.1 Pipette 100 µl of the resuspended pellet into a microvial.
Alternatively, transfer 90 µl of the resuspended pellet to a microvial
containing 10 µl of 0,5M NaOH. Mix by repeatedly inverting the micro-vial.
4.2 Heat for four minutes at 100 °C. Remove the microvial immediately on
ice.
4.3 Prepare at least two decimal dilutions, e.g. 1/10 and 1/100 or more if
considered useful, in sterile distilled or ultra pure water (UPW).
4.4 Prepare the PCR reaction mix (Appendix 6) in a sterile vial by adding the
following components in the following order:
For a 50 µl reaction volume:
Sterile distilled or UPW 30,8 µl—33,8 µl
10 × PCR Buffer 5,0 µl 1 ×d-ATP 1,0 µl 0,2 mMd-CTP 1,0 µl 0,2 mMd-GTP 1,0 µl 0,2 mMd-TTP 1,0 µl 0,2 mMPrimer OLI-1 (20 µM) 2,5 µl 1 µmPrimer Y-2 (20 µM) 2,5 µl 1 µmTaq polymerase (5 U/µl)
0,2 µl 1,0 U
Total volume 45 µl—48 µl
For more reactions:
Calculate the quantity of each component for the required number of reactions.Mix the components and transfer 45 µl—48 µl of the mix into sterile PCR vials.Keep the vials with the PCR reaction mix on ice.
For 25 µl reaction volumes:
Scale down components accordingly.
4.5 PCR amplification
4.5.1 Optional: Pulse centrifuge the vials with the boiled sample and positive
control.
Add, in the specified order, 2—5 µl of the sample(s), water control and
positive control to the vials with the PCR reaction mix. Place the vials inthe heating block of the DNA thermal cycler.
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4.5.2 Run the following programme:
1 cycle of:(i) 2 minutes at 96 °C:denaturation of template
50 cycles of
(ii) 20 seconds at 94 °C:denaturation
(iii) 20 seconds at 68 °C:annealing of primers
(iv) 30 seconds at 72 °C:extension of copy1 cycle of:
(v) 10 minutes at 72 °C:further extension1 cycle of:
(vi) hold at 4 °C
Note: These parameters are for a Perkin Elmer 9600. Other thermal cycles
may require mineral oil overlay in the PCR reaction vials and/or modi-fication of the duration of step (ii), (iii) and (iv) in the amplificationprofile.
4.5.3 Remove the vials from the thermal cycler. Analyse the PCR product. If
not done immediately, store the vials at 4 °C for use in the same day or at
−18°C for longer.
4.6 Analysis of the PCR product
The PCR fragments are detected by agarose gelelectrophoresis and
staining with ethidium bromide.
4.6.1 Prepare an appropriate agarose gel by gently bringing to the boil agarose
in tris-acetate-electrophoresis (TAE) buffer.
4.6.2 Cool the molten agarose to 50—60 °C, pour into the mould of the
electrophoresis unit and insert the comb. Let the solution solidify.
4.6.3 Remove the comb. Submerge the gel in TAE so that it is just covered
(2—3 mm) with the buffer.
4.6.4 Place 3 µl droplets of loading buffer on parafilm. Add 12 µl of the PCR
product from either the samples, the positive control or the water controland mix by gentle aspiration in the pipette tip before loading. The givenvolumes may be modified to fit with the capacity of the wells in theagarose gel.
4.6.5 Carefully load the wells of the gel. Include for reference an appropriate
DNA marker in at least one well.
4.6.6 Connect the wires on the power supply and the electrophoresis equip-
ment. Run the gel at 5—8 V/cm until the front of the tracking indicator iswithin 1 cm of the end of the gel.
4.6.7Switch off the power supply. Disconnect the wires from the electrophor-
esis unit.
Carefully remove the gel. Soak it in ethidium bromide solution for
30—45 minutes.
Note:Wear disposable gloves at all times when handling ethidium
bromide which is a powerful mutagen!
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4.6.8 Destain in distilled water for 10—15 minutes.
4.6.9 Visualise the amplified DNA fragment(s) by UV transillumination. The
PCR product of Ralstonia solanacearum with primer set OLI-1 and Y-2
is 288 bp in length. Check against the DNA marker and against thepositive control.
Note:The water control must be negative in each case. If positive,
repeat the test.
4.6.10 Photograph the gel if a permanent record is required.
4.6.11 Confirm the authenticity of the amplified fragment by restriction enzyme
analysis (REA).
4.7Restriction Enzyme Analysis (REA)
4.7.1 Transfer 8,5 µl from the PCR product (4.5.3) to a new microvial. Add
1 µl of 10 × enzyme buffer and 0,5 µl of restriction enzyme AvaII.
4.7.2 Mix by gentle aspiration in the pipette tip. If drops remain on the walls of
the vial, pulse spin in a microcentrifuge. Incubate for one hour at 37 °C.
4.7.3 Analyse the digested PCR fragment by agarose gelelectrophoresis as
before (4.6).
Interpretation of the PCR test result:
The PCR test is negative if the characteristic 288 bp fragment is not
detected and the fragment is detected for the positive control strain ofRalstonia solanacearum .
The PCR test is positive if the 288 bp fragment is detected and REA-
analysis of the amplified fragment is identical with the positive controlstrain of Ralstonia solanacearum .
5.Selective plating test
Based upon Elphinstone et al., 1996
5.1 Perform the test by an appropriate dilution plating technique, e.g.:
(i) Prepare at least two decimal dilutions, viz. 1/10 and 1/100 or more if
considered useful, of the resuspended pellet in pelletbuffer. Pipette ameasured standard volume (50—100 µl) of the resuspended pelletand each dilution onto modified SMSA selective medium (Appendix7) and spread with a glass rod over the whole surface of the medium.
If considered useful, also perform a dilution streak of a 10 µl loopful
of the resuspended pellet. Flame the loop between streaks.
(ii) Transfer a measured standard volume (50—100 µl) of the resus-
pended pellet onto modified SMSA selective medium and spreadwith a glass rod over the whole surface of the medium. Streak therod without flaming on at least two other modified SMSA plates.
5.2 Apply, by the same dilution plating technique, a suspension of 10
6cells
per ml from a virulent race 3/biovar 2 strain of Ralstonia solanacearum
as the positive control on a set of separate modified SMSA plates.
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5.3 Incubate the plates at 28 °C. Start reading the plates after three days. If
negative, incubate further up to six days. Colonies of virulent isolates ofRalstonia solanacearum are milky-white, flat, irregular and fluidal with
blood red coloration in the centre and showing internal streaking orwhorling.
5.4 Purify colonies with characteristic morphology by subculturing onto a
general nutrient medium (Appendix 1).
5.5 Identify pure cultures (section II, 4.1) and confirm Ralstonia solana-
cearumcultures by a pathogenicity test (section II, 4.3).
Interpretation of the selective plating test result:
The selective plating test is negative if no bacterial colonies are isolated
after six days or if no colonies characteristic of Ralstonia solanacearum
are isolated, provided that no inhibition is suspected by colonies of otherbacteria and that colonies characteristic of Ralstonia solanacearum are
found in the positive controls.
The selective plating test is positive if colonies characteristic of Ralstonia
solanacearum are isolated.
6.Bioassay test
Based upon Janse, 1988.
6.1 Use 10 test plants of susceptible tomato or eggplant seedlings at the third
true leaf stage for each sample. Do not water the testplants for 24 hoursbefore inoculation.
6.2 Distribute 100 µl of resuspended pellet between the testplants. Inoculate
in the stem between the cotyledons and at one or more other places.
6.3 Inoculate, by the same technique, 10 seedlings with a suspension of 10
6
cells per ml from a virulent biovar 2/race 3 strain of Ralstonia solana-
cearumas the positive control and with pelletbuffer as the negative
control. Separate the positive control plants from the other plants to avoidcross-contamination.
6.4 Grow the test plants further for up to four weeks at 22 °C—28 °C and
high relative humidity with daily watering. Observe for wilting, epinasty,chlorosis and/or stunting.
6.5 Isolate from infected plants (section II). Identify pure cultures with char-
acteristic morphology (section II.4.1) and confirm Ralstonia solana-
cearumcultures by a pathogenicity test (section II.4.3).
6.6 If considered useful, check absence of infection in batches of test plants
not showing any indication of infection. Remove from each test plant aone cm section of stem from two cm above the inoculation point. Homog-enise the tissues in maceration buffer. Perform dilution plating (sectionIII.5.1). If positive, identify pure cultures with characteristic morphology(section II.4.1) and confirm Ralstonia solanacearum cultures by a patho-
genicity test (section II.4.3).
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Interpretation of the bioassay test result:
The bioassay test is negative if test plants are not infected by Ralstonia
solanacearum , and provided that Ralstonia solanacearum is detected in
the positive controls.
The bioassay test is positive if test plants are infected by Ralstonia
solanacearum .
7.Enrichment tests
Based upon Elphinstone et al., 1996
7.1 Transfer 100 µl of the resuspended pellet in three ml of modified SMSA
broth (Appendix 7).
7.2 Incubate for 48 hours, and in any case not longer than 72 hours, at 28 °C
with the cap of the tube loosely fitted for aeration.
7.3 Tighten cap and vortex. Aliquot for the IF test (this section, 2.), the
ELISA test (this section, 3.) and/or the PCR test (this section, 4.).
8.Pathogenicity test
Refer to Section II.4.3.
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Appendix 1
Nutrient media for isolation and culture of Ralstonia solanacearum
Nutrient Agar (NA)
Nutrient Agar (Difco) 23 g
Distilled water 1 litre
Prepare litre volumes of medium in one litre flasks.
Dissolve the ingredients.Sterilise by autoclaving at 121 °C for 15 minutes.
Cool to 50 °C. Pour plates.
Yeast-Peptone-Glucose-Agar (YPGA)
Yeast Extract (Difco) 5 g
Bacto Peptone (Difco) 5 gD(+) Glucose (monohydrate) 10 gBacto Agar (Difco) 15 gDistilled water 1 litre
Prepare litre volumes of medium in one litre flasks.
Dissolve the ingredients.Sterilise by autoclaving at 121 °C for 15 minutes.
Cool to 50 °C. Pour plates.
Sucrose Peptone Agar (SPA)
Sucrose 20 g
Peptone 5 gK
2HPO4 0,5 g
MgSO4·7H2O 0,25 g
Bacto-Agar (Difco) 15 g
Distilled water 1 litre
Prepare litre volumes of medium in one litre flasks.
Dissolve the ingredients. Adjust to pH 7,2—7,4 if necessary.Sterilise by autoclaving at 121 °C for 15 minutes.
Cool to 50 °C. Pour plates.
Kelman’s tetrazolium medium
Casamino acids (Difco) 1 g
Bacto-Peptone (Difco) 10 gDextrose 5 gBacto-Agar (Difco) 15 gDistilled water 1 litre
Prepare litre volumes of medium in one litre flasks.
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Dissolve the ingredients. Sterilise by autoclaving at 121 °C for 15 minutes.
Cool to 50 °C.
Add a filter-sterilised aqueous solution of triphenyl tetrazolium chloride (Sigma)
to obtain a final concentration of 50 mg per litre.
Pour plates.
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Appendix 2
Materials for sample preparation
Maceration Buffer: 50 mM phophate buffer, pH 7,0
This buffer is used for tissue maceration.
Na2HPO4 4,26 g
KH2PO4 2,72 g
Distilled water 1 litre
Dissolve the ingredients and check pH. Aliquot as considered appropriate.
Sterilise by autoclaving at 121 °C for 15 minutes.
The addition of 5 % polyvinylpyrrolidone-40000 MWT (PVP-40) is recom-
mended when performing the direct PCR test to reduce the incidence of amplifi-cation inhibition by aromatic molecules in the extract.
The addition of a deflocculant, an antifoam agent or an anti-oxidant is recom-
mended using the Waring Blender or Ultra Turrax homogenisation procedure formaceration of the potato tissue cores.
Lubrol flakes 0,5 g per litre
DC Silicone antifoam 1,0 ml per litreTetrasodiumpyrophosphate 1,0 g per litre
Autoclave separately. Add to the desired concentration.
Pelletbuffer: 10 mM phosphate buffer, pH 7,2This buffer is used for resuspension and dilution of potato heel and pellets.
Na
2HPO4·12 H2O 2,7g
Na H2PO4·2H2O 0,4 g
Distilled water 1 litre
Dissolve the ingredients and check pH. Aliquot as considered appropriate.
Sterilise by autoclaving at 121 °C for 15 minutes.
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Appendix 3
Materials for the IF test
IF-buffer: 10 mM phosphate buffered saline (PBS) pH 7,2
This buffer is used for dilution of antisera.
Na2HPO4·12 H2O 2,7g
NaH2PO4·2H2O 0,4 g
NaCl 8,0 g
Distilled water 1 litre
Dissolve the ingredients and check pH. Aliquot as considered appropriate.
Sterilise by autoclaving at 121 °C for 15 minutes.
IF-buffer-TweenThis buffer is used for washing the slides. Add 0,1 % Tween 20 to the IF buffer.0,1 M Phosphate buffered glycerol pH 7,6This buffer is used as a mountant fluid on the windows of the IF slide to enhance
fluorescence.
Na
2HPO4·12 H2O 3,2 g
NaH2PO4·2H2O 0,15 g
Glycerol 50 ml
Distilled water 100 ml
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Appendix 4
Determination of contamination level in the IF test
Surface area (S) of window of multispot slide
=πD2
4
Where D = diameter of window (1)
Surface area (s) of objective field
=πd2
4
Where d = diameter of field (2)
Calculate d either by direct measurement or from the following formulae:
s=πi2
G2K2×4
where
i = field coefficient (depends upon ocular type and varies from 8 to 24),K = tube coefficient (1 or 1,25),G = magnification of 100x, 40x etc. objective. (3)
from (2)
d=4s
π√(4)
from (3)
d=4×πi2
G2K2×4
π=i
GK√
Count the number of typical fluorescent cells per field (c).
Calculate the number of typical fluorescent cells per window (C).
C=cS
s
Calculate the number of typical fluorescent cells per ml pellet (N)
N=C×1 000
y×F
wherey= volume of pellet on window,F= pellet dilution factor.
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Appendix 5
Materials for the ELISA test
Double strength Carbonate Coating Buffer, pH 9,6
Na2CO3 6,36 g
NaHCO3 11,72 g
Distilled water 1 litre
Dissolve the ingredients and check pH. Aliquot as considered appropriate.
Sterilise by autoclaving at 121 °C for 15 minutes.
Sodium sulphite at a final concentration of 0,2 % may be added as anti-oxidant if
the extract contains a high fraction of aromatic molecules.
10 × Phosphate Buffered Saline (PBS), pH 7,4
NaCl 80 g
KH2PO4 2g
Na2HPO4× 12H2O2 9 g
KCl 2 g
Distilled water 1 litre
Dissolve the ingredients and check pH. Aliquot as considered appropriate.
Sterilise by autoclaving at 121 °C for 15 minutes.
Phosphate Buffered Saline-Tween (PBS-T)
10 × PBS 100 ml
10 % Tween 20 5 mlDistilled water 895 ml
Blocking (antibody) Buffer (must be freshly prepared)
10 × PBS 10 ml
Polyvinylpyrrolidone-44000 MWT (PVP-44) 2 g10 % Tween 20 0,5 gMilk powder 0,5 gDistilled water to 100 ml
Alkaline phosphatase substrate solution pH 9,8
Diethanolamine 97ml
Distilled water 800 ml
Mix and adjust to pH 9,8 with concentrated HCI.
Make up to one litre with distilled water.Add 0,2 g MgCl
2.
Dissolve two phosphatase substrate five mg tablets (Sigma) per 15 ml of solu-
tion.
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Appendix 6
Materials for the PCR test
Oligonucleotide primer sequence
Primer OLI-1 5 /prime1-GGGGGTAGCTTGCTACCTGCC-3 /prime1
Primer Y-2 5 /prime1CCCACTGCTGCCTCCCGTAGGAGT-3 /prime1
For materials see Seal et al(1993).
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Appendix 7
Materials for selective plating and enrichment tests
SMSA Selective medium (Engelbrecht, 1994 as modified by Elphinstone et
al, 1996) but delete the agar and the tetrazolium salts.
Basal medium
Casamino acids (Difco) 1 gBacto peptone (Difco) 10 gGlycerol 5 mlAgar (Difco) 15 gDistilled water 1 litre
Prepare litre volumes of medium in one litre flasks.
Dissolve the ingredients and check pH. Adjust pH, if necessary, to 6,5 before
autoclaving. Ralstonia solanacearum will not grow well on the medium at pH
>7,0.
Sterilise by autoclaving at 121 °C for 15 minutes.
Cool to 50 °C.
Add the following ingredients (all from Sigma) to obtain the specified final
concentrations:
Crystal violet 5 mg per litre
Polymixin B sulphate 100 mg per litre (approximately 600 000
units)Sigma P-1004
Bacitracin (*) 25 mg per litre (approximately 1 250
units)Sigma B-0125
Chloramphenicol 5 mg per litre Sigma C-3175
Penicillin-G 0,5 mg per litre (approximately 825 units) Sigma P-3032Tetrazolium salts 50 mg per litre
(*) If considered necessary, increasing bacitracin concentration to 300 ppm may reduce contaminating saprophytic
bacteria without reducing the recovery of Ralstonia solanacearum .
Dissolve the ingredients in 70 % ethanol to the given concentrations for the
volume of medium prepared. Some ingredients, viz.polymixin B and chloram-
phenicol require slight warming and shaking.
SMSA broth (Elphinstone et al., 1996)
Prepare as for the SMSA selective medium but delete the agar.Dispense in three ml aliquots into 30 ml disposable Universal tubes.
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References
Buddenhagen, I.W.; Sequeira, L. and Kelman, A., 1962. Description of races in
Pseudomonas solanacearum .Phytopathology 52, 726.
Cook, D.; Elizabeth B. and Sequeira L., 1989. Genetic diversity of Pseudomonas
solanacearum : detection of restriction fragment length polymorphism with DNA
probes that specify virulence and hypersensitive respons. Molecular Plant-
Microbe Interactions 2, 113—121.
Dinesen I.G. and DeBoer, S.H., 1995. Extraction of Clavibacter michiganensis
subsp.sepedonicus from composite samples of potato tubers. American Potato
Journal72, 133—142.
Elphinstone, J.G.; Hennessy, J.; Wilson, J. and Stead, D.E., 1996. Sensitivity of
different methods for the detection of Pseudomonas solanacearum (Smith) Smith
in potato tuber extracts. EPPO Bulletin 26.
Engelbrecht, M.C., 1994. Modification of a semi-selective medium for the isola-
tion and quantification of Pseudomonas solanacearum. ACIAR Bacterial Wilt
Newsletter 10, 3—5.
Hayward, A.C., 1964. Characteristics of Pseudomonas solanacearum. Journal of
Applied Bacteriology 27, 265—277.
Hayward, A.C., 1994. Systematic and phylogeny of Pseudomonas solanacearum
and related bacteria. In: Bacterial Wilt: the disease and its causative agent,
Pseudomonas solanacearum (eds. A.C. Hayward and G.L. Hartman) CAB Inter-
national Oxford, 127—135.
Janse, J.D., 1988. A detection method for Pseudomonas solanacearum in symp-
tomless potato tubers and some data on its sensitivity and specificity. EPPO
Bulletin18, 343—351.
Janse, J.D., 1991. Infra- and intraspecific classification of Pseudomonas solana-
cearumstrains using whole cell fatty acid analysis. Systematic and Applied
Microbiology 14, 335—345.
Kelman, A., 1954. The relationship of pathogenicity in Pseudomonas solana-
cearumto colony appearance on a tetrazolium medium. Phytopathology 64,
293—695.
Lelliot, R.A. and Stead, D.E., 1987. Methods for the diagnosis of bacterial
diseases of plants. (T.F. Preece ed.) Blackwell Scientific Publications, Oxford.
216 pp.
Louws, F.J.; Fulbright, D.W.; Stephens, C.T. and De Bruijn, F.J., 1995. Differen-
tiation of genomic structure by rep-PCR fingerprinting to rapidly classifyXanthomonas campestris pv.vesicatoria. Phytopathology 85, 528—536.
Lozano, J.C. and Sequeira, L., 1970. Differentiation of races of Pseudomonas
solanacearum by a leaf infiltration technique. Phytopathology 60, 838.
Mirza, M.S.; Rademaker, J.W.L.; Janse, J.D. and Akkermans, A.D.L., 1993.
Specific 16S ribosomal RNA targeted oligonucleotide probe against Clavibacter
michiganensis subsp.sepedonicus. Canadian Journal of Microbiology 39,
1029—1034.
Robinson-Smith, A.; Jones, P.; Elphinstone, J.G. and Forde, S.M.D., 1995.
Production of antibodies to Pseudomonas solanacearum , the causative agent of
bacterial wilt. Food and Agricultural Immunology 7, 67—79.
Seal, S.E.; Jackson, L.A.; Young, J.P.W. and Daniels, M.J., 1993. Differentiation
ofPseudomonas solanacearum, P. syzygii, P. picketti and the blood disease
bacterium by partial 16S rRNA sequencing: construction of oligonucleotideprimers for sensitive detection by polymerase chain reaction. Journal of General
Microbiology 139, 1587—1594.
Smith, J.J.; Offord, L.C.; Holderness, M. and Saddler, G.S., 1995. Genetic
diversity of Burkholderia solanacearum (synonym Pseudomonas solanacearum )
race 3 in Kenya. Applied and Environmental Microbiology 61, 4262—4268.
Stead, D.E., 1992a. Techniques for detecting and identifying plant pathogenic
bacteria. In: Techniques for rapid detection of plant pathogens (eds. J.M. Duncan
and L. Torrance). Blackwell Scientific Publications, Oxford, 76—111.
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Stead, D.E., 1992b. Grouping of plant pathogenic and some other Pseudomonas
spp. using cellular fatty acid profiles. International Journal of Systematic Bacter-
iology42, 281—295.
Van Beuningen, A.; Derks, H. and Janse J.D., 1995. Detection and identification
ofClavibacter michiganensis subsp.sepedonicus with special attention to fluor-
escentin-situhybridisation (FISH) using a 16S rRNA targeted oligonucleotide
probe.Züchtungsforschung 2, 266—269.
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ANNEX III
1. For each suspected occurrence for which a positive result in the screening
test(s) has been identified according to, for the listed plant material, therelevant method set out in Annex II or, in all other cases, any other officiallyapproved method, and confirmation or refutation by completion of the saidmethod is awaited, there should be retention and appropriate conservation of:
— wherever possible, the lot, or part thereof (from which the sample has
been taken) in its original packaging with label,
— wherever possible, the remaining part of the samples,— any remaining extract and additional prepared material for the screening
test(s) e.g. immunofluorescence slides, and,
— all relevant documentation,
until the completion of the said method.
2. In the case of confirmation of the organism, there should be retention and
appropriate conservation of:
— the material specified in paragraph 1, and
— a sample of the infected tomato or eggplant material inoculated with the
tuber or plant extract, where appropriate, and
— the isolated culture of the organism,
until at least one month after the notification procedure under Article 5(2).
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ANNEX IV
The elements in the investigation referred to in Article 5(1)(a)(i) shall include
where relevant:
(i) places of production,
— growing or having grown, potatoes which are clonally related to potatoes
found to be infected with the organism,
— growing or having grown tomatoes which are from the same source as
tomatoes found to be infected with the organism,
— growing or having grown, potatoes or tomatoes which have been placed
under official control because of the suspected occurrence of theorganism,
— growing or having grown, potatoes which are clonally related to potatoes
that have been grown on places of production found to be infested withthe organism,
— growing potatoes or tomatoes and located in the neighbourhood of
infested places of production, including such places of productionsharing production equipment and facilities directly or through acommon contractor,
— using surface water for irrigation or spraying from any source confirmed
or suspected to be infested with the organism,
— using surface water for irrigation or spraying from a source used in
common with places of production confirmed or suspected to be infestedwith the organism,
— flooded or have been flooded with surface water confirmed or suspected
to be infested with the organism; and,
(ii) surface water used for irrigation or spraying of, or which has flooded field(s)
or place(s) of production confirmed to be infested with the organism.
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ANNEX V
1. The elements in the determination of the extent of probable contamination
under Article 5(1)(a)(iii) and 5(1)(c)(iii), shall include where relevant:
— the listed plant material grown at a place of production designated as
contaminated under Article 5(1)(a)(ii),
— place(s) of production with a production link to the listed plant material
designated as contaminated under Article 5(1)(a)(ii), including thosesharing production equipment and facilities directly or through a commoncontractor,
— the listed plant material produced in the place(s) of production referred to
in the previous indent, or present in such place(s) of production during theperiod when the listed plant material designated as contaminated underArticle 5(1)(a)(ii), was present on the places of production referred to inthe first indent,
— stores handling the listed plant material from the above places of produc-
tion,
— any machinery, vehicle, vessel, store, or units thereof, and any other
objects including packaging material, that may have come into contactwith the listed plant material designated as contaminated under Article5(1)(a)(ii),
— any of the listed plant material stored in, or in contact with, any of the
structures or objects listed in the previous indent, prior to the cleansingand disinfection of such structures and objects,
— as a result of the investigation and testing under Article 5(1)(a)(i), in the
case of potato, those tubers or plants with a sister or parental clonalrelationship to, and in the case of tomato, those plants with the samesource as, the listed plant material designated to be contaminated underArticle 5(1)(a)(ii) and for which, although they may have tested negativefor the organism, it appears that contamination is probable through aclonal link,
— place(s) of production of the listed plant material referred to in the
previous indent,
— place(s) of production of the listed plant material using water for irriga-
tion or spraying which has been designated as contaminated under Article5(1)(c)(ii),
— listed plant material produced on fields flooded with surface water
confirmed to be infested with the organism.
2. The determination of the possible spread under Article 5(1)(a)(iv) and
5(1)(c)(iii) shall include:
(i) in cases under Article 5(1)(a)(iv), a consideration of the following
elements,
— the proximity of other places of production growing the listed plant
material,
— the common production and use of seed potato stocks,— places of production using surface water for irrigation or spraying of
listed plant material in cases where there is or has been risk of surfacewater run-off from, or flooding of, place(s) of production designatedto be contaminated under Article 5(1)(a)(ii);
(ii) in cases where surface water has been designated as contaminated under
Article 5(1)(c)(ii):
— place(s) of production producing listed plant material adjacent to, or
at risk from flooding by, the surface water designated as contami-nated,
— any discrete irrigation basin associated with the surface water desig-
nated as contaminated.
3. The details of the notification referred to in the first subparagraph of Article
5(2) shall include:
— date of reporting of suspected occurrence under Article 4 and dates of
sampling and confirmation of the presence of the organism under Article5, as appropriate,
— a description of the elements of the designated contamination and zone
demarcation.
4. The details of the additional notification referred to in the second subpara-
graph of Article 5(2) shall include:
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— for any potato consignment or lot designated as contaminated, the certi-
ficates prescribed in Articles 7 and 8 of Directive 77/93/EEC, the passportnumber or registration number of potato producers, collective warehousesand dispatching centres, as appropriate,
— for any tomato plant consignment or lot designated as contaminated the
crtificates prescribed in Articles 7 or 8 of Directive 77/93/EEC andpassport number, in accordance with the listing in Annex V, Part A,Section I.2.2 to Directive 77/93/EEC,
— the variety name and category for seed potato stocks, and where possible
in all other cases,
— such other information relating to the confirmed outbreak as the Commis-
sion may require.
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ANNEX VI
1. With refrence to Article 6(1) the provisions shall be:
— incineration, or
— use as animal feed after heat treatment such that there is no risk of the
organism surviving, or
— deep burial, at a disposal site at which there is no risk of seepage to
agricultural land or contact with water sources which could be used forirrigation of agricultral land, or
— industrial processing through direct and immediate delivery to a
processing plant with officially approved waste disposal facilities whichconform to the provisions laid down in Annex VII to this Directive, or
— other measures, provided that it has been established that there is no
identifiable risk of the organism spreading; such measures to be imme-diately notified to the Commission and to the other Member States.
2. The appropriate use or disposal of the listed plant material referred to in
Article 6(2), under the control of the responsible official bodies of theMember State(s) concerned, with appropriate communication betweenresponsible official bodies to ensure such control at all times and approvalby the responsible official body of the Member State where the potatoes areto be packed or processed in respect of the waste disposal facilities referredto in the first and second indents, shall be:
(i) for potato tubers,
— use as ware potatoes intended for consumption and packed at sites
with appropriate waste disposal facilities, ready for direct deliveryand use without repacking, and intended for such direct deliveryand use, or
— use as ware potatoes intended for industrial processing, and
intended for direct and immediate delivery to a processing plantwith appropriate waste disposal facilities, or
— some other use or disposal, provided that it is established that there
is no identifiable risk of the organism spreading, and subject toapproval by the said responsible official bodies. Such measures tobe immediately notified to the Commission and to the otherMember States.
(ii) for other plant parts including stem and foliage debris,
— destruction, or
— some other use or disposal, provided that it is established that there
is no identifiable risk of the organism spreading; such measuresshall be notified to the Commission and to other Member States.
3. The appropriate methods for decontamination of the objects referred to in
Article 6(3) shall be cleansing and, where appropriate, disinfection, suchthat there is no identifiable risk of the organism spreading and shall beemployed under the supervision of the responsible official bodies of theMember States.
4. The series of measures to be implemented by Member States within the
demarcated zone(s) established under Article 5(1)(a)(iv) and (c)(iii) andreferred to in Article 6(4) shall include:
4.1. In cases where places of production have been designated as contaminated
under Article 5(1)(a)(ii):
(a) in a field or unit of protected crop production designated to be contami-
nated under Article 5(1)(a)(ii), either
(i) during at least the four growing years following the designated
contamination,
— measures shall be taken to eliminate volunteer potato and
tomato plants as well as other host plants of the organismincluding solanaceous weeds, and
— the following shall not be planted:
— potato tubers or plants,
— tomato plants and seeds,— taking into account the biology of the organism,
— other host plants,
— plants of species of Brassica, for which there is an
identified risk of the organism surviving,
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— crops for which there is an identified risk of the organism
spreading;
— in the first potato or tomato cropping season following the
period specified in the preceding indent, and on the conditionthat the field has been found free from volunteer potato andtomato plants and other host plants including solanaceousweeds for at least the two consecutive growing years prior toplanting,
— in the case of potatoes, officially certified seed potatoes
shall be planted for ware production only and,
— an official survey including testing, as detailed in Article
2(1), shall be conducted,
— in the potato or tomato cropping season succeeding that
referred to in the previous indent and following an appropriaterotation cycle, in the case of potatoes officially certified seedpotatoes shall be planted for either seed or ware production andin the case of potatoes and tomatoes an official survey asdetailed in Article 2(1), shall be conducted; or
(ii) during the five growing years following that of the designated
contamination,
— measures shall be taken to eliminate volunteer potato and
tomato plants as well as other host plants of the organismincluding solanaceous weeds, and
— the field shall be established and maintained during the first
three years either, in bare fallow or, in cereals according to therisk identified, or, in permanent pasture with frequent closecutting or intensive grazing or, as grass for seed production,followed by planting in the succeeding two years with non-hostplants of the organism for which there is no identified risk ofthe organism surviving or spreading,
— in the first potato or tomato cropping season following the
period specified in the preceding indent,
— in the case of potatoes, officially certified seed potatoes
shall be planted for either seed or ware production,
and an official survey including testing, as detailed in Article
2(1), shall be conducted;
(b) in other fields:
— in the growing year following the designated contamination:
— either no potato tubers or plants, or other host plants of the
organism shall be planted, and measures shall be taken to elimi-nate volunteer potato and tomato plants and other host plantsincluding solanaceous weeds as appropriate, or
— in the case of potato tubers, officially certified seed potatoes
may be planted for ware production only, on the condition thatthe responsible official bodies are satisfied that the risks ofvolunteer potato and tomato plants and other host plants of theorganism including solanaceous weeds, have been eliminated.The growing crop shall be inspected at appropriate times andvolunteer potato plants shall be tested for the organism; inaddition, for potatoes, the harvested tubers shall be inspected;
— for the first growing year following that specified in the first indent,
— in the case of potatoes, only officially certified seed potatoes
shall be planted for either seed or ware production,
— for at least the second growing year following that in the first
indent,
— in the case of potatoes, only officially certified seed potatoes or
seed potatoes grown under official control from officially certi-fied seed potatoes shall be planted for either seed or wareproduction,
— in each of the growing years referred to in the previous indents
measures shall be taken to eliminate volunteer potato and tomatoplants and other host plants of the organism including solanaceousweeds, and an official survey as detailed in Article 2(1), shall beconducted, and in the cases where seed potatoes are planted for seedproduction testing of tubers shall be carried out;
(c) immediately following the designation of contamination under Article
5(1)(a)(ii) and in each of the subsequent growing years up to and
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including the first permissible potato or tomato cropping season on the
field(s) designated as contaminated, as detailed in paragraph (a):
— all machinery and storage facilities on the place of production and
involved in potato or tomato production shall be cleansed and,where appropriate, disinfected using appropriate methods, as speci-fied in point 3,
— official controls on irrigation and spraying programmes, including a
ban thereof, shall be introduced as appropriate in order to preventthe spread of the organism;
(d) in a unit of protected crop production designated as contaminated under
Article 5(1)(a)(ii) where complete replacement of the growing mediumis possible,
— no potato tubers or plants, or other host plants of the organism
including tomato plants and seeds shall be planted unless the saidunit has been subjected to officially supervised measures to elimi-nate the organism and to remove all host plant material, including,at least, a complete change in growing medium and cleansing and,where appropriate, disinfection of the said unit and all equipment,and subsequently has been granted approval for potato or tomatoproduction by the responsible official bodies, and
— for potato production, this production shall be from officially certi-
fied seed potatoes, or from mini-tubers or micro-plants derived fromtested sources,
— official controls on irrigation and spraying programmes, including a
ban thereof, shall be introduced as appropriate, in order to preventthe spread of the organism.
4.2 Within the demarcated zone, without prejudice to the measures detailed
under 4.1, the Member States shall:
(a) immediately, and for at least three growing years, after the designated
contamination:
(aa) in cases where the demarcated zone has been determined under
Article 5(1)(a)(iv),
— ensure supervision by their responsible official bodies of prem-
ises growing, storing or handling potato tubers or tomatoes,together with premises which operate machinery for potato ortomato production under contract,
— require cleansing and, where appropriate, disinfection of
machinery and stores on such premises, using appropriatemethods, as specified under point 3,
— require the planting of only certified seed or seed grown under
official control for all potato crops within that zone, andtesting after harvest of seed potato crops grown in places ofproduction determined as probably contaminated under Article5(1)(a)(iii),
— require the separate handling of harvested potato seed stocks to
those of ware on all premises within the zone,
— conduct an official survey as detailed in Article 2(1),
(ab) in cases where surface water has been designated as contaminated
under Article 5(1)(c)(ii) or included in the elements for thepossible spread of the organism in accordance with Annex V point2,
— conduct an annual survey at appropriate times including
sampling of surface water and appropriate solanaceous hostplants in the relevant water sources and testing in accordancewith
— for listed plant material, the relevant method set out in
Annex II, or
— in other cases, any other officially approved method,
— introduce official controls on irrigation and spraying
programmes, including a ban on the use of the water desig-nated as contaminated for the irrigation and spraying of listedplant material, and, where appropriate, other host plants inorder to prevent the spread of the organism. This ban may bereviewed on the basis of the results obtained in the said annualsurvey,
— in cases where liquid waste discharges are contaminated, intro-
duce official controls on the disposal of waste from industrialprocessing or packaging premises handling listed plantmaterial;
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(b) establish a programme, where appropriate, for the replacement of all
seed potato stocks over an appropriate period of time.
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ANNEX VII
The officially approved waste disposal facilities referred to in Annex VI para-
graph 1, fourth indent shall conform to the following provisions such that the riskof spreading the organism is obviated:
(i) potato and tomato processing waste (including rejected potatoes and peelings
and tomatoes) and any other solid waste associated with the potatoes andtomatoes shall be disposed by either,
— deep burial at disposal site at which there is no risk of seepage to
agricultural land or contact with water sources which could be used forirrigation of agricultural land. The waste shall be conveyed directly tothe site under containment conditions such that there is no risk of loss ofthe waste, or
— incineration;
(ii) liquid processing waste: prior to disposal, liquid waste containing suspended
solids shall be subjected to filtration or settlement processes to remove suchsolids. These solids shall be disposed of as set out in subparagraph (i).
The liquid waste shall then be either:
— heated to a minimum of 70 °C during at least 30 minutes, prior to
disposal, or
— otherwise disposed of subject to official approval and under official
control such that there is no risk that the waste could come into contactwith agricultural land or water sources which could be used for irrigationof agricultural land. The details thereof shall be notified to the otherMember States and to the Commission.