The harmful organism Ralstonia solanacearum (Smith) Yabuuchi et al., formerly known by Pseudomonas solanacearum (Smith) Smith, is a cause of the "brown rot" diseases in potato and potato tubers. "bacterial wilt" in potato, tomato and other species of solanaceae, and poses a serious threat to the production of these crops.

Because outbreaks of the diseases mentioned in some parts of the European Community have occurred in recent years and some outbreaks of isolated infection still exist, and in particular with regard to Spain, certain intercepts in Castilla y León and Galicia and, this bacterium is present with a distribution located on the island of La Palma in the Canary Islands; considering the economic importance of these plant products; and taking into account that the current knowledge of the biology and epidemiology of Ralstonia solanacearum under the conditions It is incomplete and that the need to revise in future the measures and the procedures for analysis established here, it is necessary to adopt the necessary minimum measures provided for in Council Directive 98 /57/EC, of July 20, 1998, on the control of Ralstonia solanacearum (Smith) Yabuuchi et al. by the appropriate eradication and control programme and without prejudice to the adoption of the most stringent additional measures necessary.

Consequently, by means of this Royal Decree, Directive 98 /57/EC of the Council of 20 July is incorporated into national law, in accordance with the powers conferred on the State by the Article 149.1.13.a of the Spanish Constitution in terms of bases and coordination of the general planning of economic activity.

In the preparation of this provision, the Autonomous Communities and the sectors affected have been consulted.

In its virtue, on the proposal of the Minister of Agriculture, Fisheries and Food, prior to the report of the Minister of Public Administrations, in agreement with the State Council, and after deliberation in the Council of Ministers of the October 1999,

D I S P O N G O:

Article 1. Object.

This provision establishes the eradication and control programme of the organism Ralstonia solanacearum (Smith) Yabuuchi et al., formerly called Pseudomonas solanacearum (Smith) Smith (hereinafter referred to as ' the Article 12 of Royal Decree 1190/1998 of 12 June 1998 regulating the national programmes for the eradication or control of organisms harmful to plants not yet established in the national territory, for the purpose of which, in respect of the host plants of the organism referred to in Section I of the Annex I of this Royal Decree (hereinafter referred to as "the indicated plant material"), the necessary measures are taken to:

(a) locate the organism and determine its distribution; (b) prevent its occurrence and spread; and (c) if it appears, prevent its spread and control it with a view to its eradication.

2. The measures referred to in this Royal Decree are declared to be of public use in accordance with the provisions of Royal Decree 1190/1998.

Article 2. Scope and validity of the program.

1. The programme to be approved and the measures taken by the dimanants shall apply throughout the national territory.

2. As long as the Commission of the European Communities does not rule on this, the duration of the programme is limited. At all times and as a result of the disease situation, the Ministry of Agriculture, Fisheries and Food may make any amendments deemed necessary or to determine its conclusion.

Article 3. Official examinations.

1. The official bodies responsible for each Autonomous Community, referred to in Article 11.2 (b) of Royal Decree 2071/1993 of 26 November 1993 on protective measures against the introduction and dissemination in the territory of the Member States of the National and European Community harmful organisms for plants or plant products, as well as for the export and transit to third countries and the competent authority of the Autonomous Community of the Canary Islands shall carry out annually systematic official examinations to detect the possible presence of the organism in the material indicated plant originating in its territory.

In order to determine other possible sources of contamination for the cultivation of the indicated plant material, the official bodies responsible for each Autonomous Community shall carry out a risk assessment and, unless there has been no risk of spreading during that assessment, they shall carry out, in the areas producing such material, official examinations specifically geared to the detection of the organism in plants other than that material, including wild host solanaceae as well as in surface waters which are used to water such material and in the liquid waste discharged by industrial processing or packaging plants in which the material is handled and used to water the plant material indicated.

The extent of these targeted examinations will be determined based on the risk observed.

The official bodies responsible for each Autonomous Community may also carry out official examinations for the detection of the organism in other materials, such as the culture medium, soil and solid waste. from the industrial processing or packaging plant.

2. The official examinations provided for in Article 3 (1) shall be carried out:

(a) in the case of the plant material indicated, in accordance with the rules laid down in point 1 of Section II of Annex I to this Royal Decree; and (b) in the case of the host plants other than the material plant and water, including liquid waste, using appropriate methods and, where appropriate, taking samples and submitting them for official laboratory analysis or under official supervision; (c) where appropriate, in the case of other materials, using appropriate methods.

For the conduct of these examinations, details of the inspection procedures as well as the number, origin and classification of the samples and the calendar

of their collection will be decided by the official bodies responsible for each Autonomous Community based on sound scientific and statistical principles and in the biology of the organism and taking into account, according to the region the specific systems used for the production of the indicated plant material and, where appropriate, other host plants of the organism.

3. The details and results of the official examinations provided for in Article 3 (1) shall be notified to the Ministry of Agriculture, Fisheries and Food and, in turn, through the appropriate course, to the other Member States and to the European Commission each year, following, for that purpose, the provisions of point 2 of Section II of Annex I to this Royal Decree. These notifications shall be submitted by 15 May at the latest, except in the case of potatoes used for sowing on their own holding, which shall be submitted by 15 August at the latest. The details and results relating to the crops will relate to the production of the previous year.

Article 4. Obligation to notify.

The producers, the collective warehouses and the centres of dispatch of the materials referred to in Section I of Annex I to this Royal Decree shall immediately notify the official bodies responsible for each Autonomous community, in which their parcels or establishments are situated, the suspected occurrence or the confirmed presence of the organism in the material produced or marketed.

Article 5. Precautionary measures.

1. In all cases of suspected outbreak, the official bodies responsible for each Autonomous Community concerned shall ensure that the official laboratory analysis or official supervision in which it is applied is carried out for plant material. indicated, the relevant method set out in Annex II and under the conditions set out in point 1 of Annex III to this Royal Decree or, in all other cases, any other officially approved method for confirming or clearing the suspect in question. In the event of confirmation, the requirements set out in point 2 of Annex III to this Royal Decree shall apply.

2. Pending the confirmation or dissipation of the suspicion referred to in Article 5 (1), in all cases where:

(i) symptoms of diseases caused by the organism have been observed and positive results have been obtained in the rapid test or screening referred to in point 1 of Section I and point 1 of the Section II of Annex II to this Royal Decree; or (ii) a positive result has been obtained in the test or screening referred to in point 2 of Section I and Section III of Annex II to this Royal Decree, the bodies officials responsible for each Autonomous Community in respect of their own production:

(a) prohibit the movement of plants and tubers from all crops, lots or consignments from which the samples have been taken, unless such movement takes place under its control and provided that it has been established that the there is no identifiable risk of the organism spreading; (b) they shall take the necessary measures to discover the origin of the suspected outbreak; (c) establish, in particular, the production of the indicated plant material and the movement of seed potatoes other than those referred to in point (a), produced at the place of production from which the samples referred to in point (a) have been obtained, additional preventive measures which, on the basis of the estimated level of risk, are appropriate to prevent any spread of the organism.

3. In cases of suspected outbreak where there is a risk of contamination of the indicated plant material or of the surface waters coming from or directed to another or other Autonomous Communities or, where appropriate, another Member State, the Community Autonomous in which the suspected outbreak has been observed, shall immediately inform the Ministry of Agriculture, Fisheries and Food of the details of such outbreak and of the level of risk identified for it, through the channel. report to the Autonomous Communities or to the Member States concerned, and the Autonomous Communities cooperate accordingly. The Autonomous Communities to which this report shall apply shall apply preventive measures in accordance with Article 5 (2) (c) and shall take any other measure appropriate in accordance with the provisions of paragraph 1. and 2 of the same.

Article 6. Confirmation of the presence of the organism.

1. If the presence of the organism in a sample, taken in compliance with this Royal Decree, is confirmed by the analysis of official laboratories or under official supervision in which the method used for the plant material is used set out in Annex II to this Royal Decree or, for all other cases, any other officially approved method, the official bodies responsible for each Autonomous Community, on the basis of that effect in sound scientific principles, the biology of the organism and the specific systems of production, marketing and processing to be used in their territory for the host plants of the body, shall take the following measures:

(a) in the case of the following plant material:

i) undertake an investigation to determine the extent and source or primary sources of the contamination, in accordance with the provisions of Annex IV to this Royal Decree and by making further analyses, in accordance with the Article 4 (1), at least, in all stocks of clonically related seed potatoes;

(ii) declare contaminated the indicated plant material, the consignment or the batch of which the sample has been taken, as well as the machinery, vehicle, ship, warehouse, or units thereof, and any other objects, including the packing material, which has been in contact with the plant material indicated to which the sample is taken; likewise, the field (s), the unit (s) of production of protected crops and the site (s) shall be contaminated, where appropriate; or places of production where the indicated plant material has been harvested and from which the In addition, in the case of samples taken during the cultivation phase, the field (s), the place (s) of production (s), the place (s) of production and, where appropriate, the unit (s) of production of protected crops from which the product has been produced shall be contaminated. taken the sample;

(iii) shall determine, in accordance with the provisions of point 1 of Annex V to this Royal Decree, the extent of the contamination likely to have occurred from prior or post-harvest contacts, production, irrigation or by cloning with the declared pollution; and

iv) will delimit an area based on three factors:

the pollution declaration referred to in point (ii) above, the extent of the likely contamination to be determined under point (iii) above and the possible spread of the organism, according to the provisions of point 2 (i) of Annex V to this Royal Decree.

(b) in the case of crops of host plants other than the material referred to in point (a) where a risk has been identified for the production of the indicated plant material:

(i) undertake an investigation in accordance with point (a) (i);

(ii) shall declare contaminated the host plants of the body from which the sample has been taken; and (iii) with respect to the production of the indicated plant material, determine the extent of the likely contamination and delimit an area in accordance with points (iii) and (iv) of point (a) respectively.

(c) in the case of surface waters (including discharges of liquid waste from industrial plants for processing or packaging in which the indicated plant material is handled) and in the case of solanaceae wild host of the associated organism in which, due to irrigation, spraying or flooding with those waters, a risk has been identified for the production of the indicated plant material:

(i) undertake an investigation to determine the extent of the contamination, by conducting an official examination of the surface waters and, where appropriate, the wild host solanaceae at the appropriate time. body;

(ii) shall, in so far as it is relevant and on the basis of the investigation referred to in (i), declare to be contaminated the surface waters from which the sample or samples have been taken; and,

(iii) determine the extent of the likely contamination and delimit an area according to the pollution declaration referred to in point (ii) above and the possible spread of the organism, taking into account provisions of point 1 and point (ii) of point 2 of Annex V to this Royal Decree.

2. In accordance with the provisions of points 3 and 4 of Annex V to this Royal Decree, the Autonomous Communities shall immediately notify the Ministry of Agriculture, Fisheries and Food and, in turn, the Ministry of Agriculture, to the other Member States and to the European Commission, any case of contamination declared under point (a) (ii) and point (ii) (ii) of Article 6 (1) (c), as well as the data relating to the area which has been defined in accordance with Article 6 (1) (a) (iv) and, where applicable, point (iii) of the point (c) of that paragraph.

3. As a result of the notification provided for in paragraph 2 and of the data contained therein, the Autonomous Communities concerned shall undertake an investigation in accordance with Article 6 (1) (a) (i) and, where appropriate, with point (i) of point (c) of the same paragraph, and shall take the appropriate additional measures in accordance with Article 6 (1) and (2).

4. The Ministry of Agriculture, Fisheries and Food shall establish a register of the holdings, the collective warehouses and the holding centres concerned.

Article 7. Plant health measures to be adopted on contaminated material.

1. The planting of the indicated plant material which has been declared contaminated by virtue of Article 6 (1) (a) (ii) and under the control and approval of the official bodies responsible for each Community shall be prohibited. Autonomous, such material shall be subject to one of the provisions of point 1 of Annex VI to this Royal Decree so that it can be established that there is no identifiable risk of the organism spreading.

2. The planting of the plant material indicated which, pursuant to Article 6 (1) (c) (iii) (iii) and (iii), has been deemed to have been likely to be contaminated, including the plant material indicated, is prohibited. in respect of which the existence of a risk has been detected, produced in places of production within the meaning of Article 6 (1) (a) (iii) have been deemed likely to be contaminated and, under the control of the official bodies responsible for each Autonomous Community, such material shall be used for appropriate or shall be disposed of in accordance with point 2 of Annex VI to this Royal Decree so that it can be established that there is no identifiable risk of the organism spreading.

3. Any machinery, vehicle, craft, warehouse or units of these and any other objects, including packaging material, which have been declared to be contaminated by virtue of point (a) (ii) and (iii) of paragraph 1 (c) Article 6 or which have been deemed likely to be contaminated by virtue of paragraph 1 (a) (iii) of the same Article shall be destroyed or decontaminated by appropriate methods in accordance with point 3 of Annex VI to the Royal Decree.

After decontamination, all these objects will no longer be considered contaminated.

4. Without prejudice to the measures to be carried out pursuant to Article 7 (1), (2) and (3), in the area defined in accordance with Article 6 (1) (a) and (iii) (iv), a number of measures shall be applied in respect of the measures referred to in Article 7 (1) (a) and (iii). in accordance with points 4.1 and 4.2 of Annex VI to this Royal Decree. Details of these measures shall be notified to the Ministry of Agriculture, Fisheries and Food each year and shall, in turn, through the appropriate channel, to the other Member States and to the European Commission.

5. Plant health measures ordered by the official bodies responsible for each Autonomous Community referred to in Article 7 (1), (2), (3) and (4) shall be carried out by the owner of the material concerned.

In the event that the persons concerned do not execute in due time and form the measures referred to in the preceding paragraph, the Autonomous Community shall execute them, with their own means or by using other services, expenditure relating to the persons concerned, the amount of which may be required by way of award, irrespective of the penalties to be imposed.

Article 8. Measures applicable to prevent contamination by seed potatoes.

1. Seed potatoes shall meet the requirements of Royal Decree 2071/1993 and proceed, in direct line, of potatoes which, having been obtained in the framework of an officially approved programme, have yielded negative results in respect of the presence of potatoes. of the body in official or officially supervised analysis where the relevant method set out in Annex II to this Royal Decree has been used.

2. Such analyses shall be carried out:

(a) in cases where the presence of the organism has been confirmed in the production of seed potatoes, i) analysing the material previously propagated, including the initial clonal selection and analysing systematically the clones of basic seed potatoes, or

(ii) in cases where it has been demonstrated that there is no clonal relationship, analysing all clones of basic seed potatoes or previously propagated material, including initial clonal selection, and

(b) in other cases, by analysing in each of the plants of the initial clonal selection or representative samples of the basic seed potatoes or of the material previously propagated.

Article 9. Prohibition.

The conservation and manipulation of the body referred to in this Royal Decree is prohibited.

Article 10. Exceptions for scientific purposes.

Without prejudice to the provisions of Royal Decree, 2071/1993, exceptions to the provisions of Articles 7 and 9 of this Royal Decree may be authorized, in accordance with the rules laid down for the conduct of tests. scientific research or studies in the field of varietal selections in Royal Decree 401/1996 of 1 March establishing the conditions for the introduction into the national territory of certain harmful organisms, plants, plant products and other objects for the purpose of testing, scientific and for the activity of selection of varieties and in Royal Decree 39/1998 of 16 January amending Royal Decree 401/1996 of 1 March 1996 laying down the conditions for the introduction into the national territory of certain Plant harmful organisms, plant products and other objects, for testing purposes, for scientific purposes and for the activity of variety selection.

Article 11. Additional measures.

1. The competent bodies of the Autonomous Communities may take the additional or stricter measures necessary to combat the organism or prevent its spread, provided that such measures are in accordance with the provisions of the Royal Decree 2071/1993.

2. The details of these measures shall be notified to the Ministry of Agriculture, Fisheries and Food and shall, in turn, notify the other Member States and the Commission, through the appropriate channel.

Article 12. Compensation.

1. The system of compensation provided for in Article 18 of Royal Decree 1190/1998 of 12 June 1998 governing the national programmes for the eradication or control of organisms harmful to plants not yet established in the national territory.

2. The costs incurred and the plant material destroyed in the application of an official measure are not to be compensated where the owner of the plants or plant products concerned has failed to comply with the rules in force and, in particular, Royal Decree 2071/1993 of 26 November 1993 on protective measures against the introduction and dissemination in the national territory and the European Economic Community of organisms harmful to plants or plant products; and for export and transit to third countries.

3. In the event that the person affected by the contamination of a lot supplied by a producer, storekeeper or operator who had failed to comply with the legislation in force, he would receive compensation from any of these subjects, and Part of the Administration shall reimburse the Administration for the compensation received.

4. The Ministry of Agriculture, Fisheries and Food, within the limits established by the appropriations available for these purposes, shall participate, in accordance with its budgets, in the amount of 50 per 100 of the expenditure incurred in the implementation of the programme.

Single transient arrangement. Provisional measures.

As long as the European Commission does not decide and to ensure comparable levels of safety between the different Autonomous Communities, it may be adopted, after a favourable report by the National Phytosanitary Committee:

The appropriate methods for the examinations and analyses provided for in points (b) and (c) of the first subparagraph of Article 3 (2) and any accuracy of the procedures provided for in the second subparagraph of paragraph 2 of this Article.

The additional measures provided for in Article 5 (2) (c).

The detailed rules for the development of Article 8 (2) (a) and the provisions applicable to the representative samples referred to in point (b) of the same paragraph and Article.

The Ministry of Agriculture, Fisheries and Food shall request its adoption to the European Commission by means of the channel provided for in Article 16a of Council Directive 77 /93/EEC of 21 December 1976 on the measures to protect against the introduction into the Community of organisms harmful to plants or plant products and against their spread within the Community.

Additional disposition first. Communication to the Ministry of Agriculture, Fisheries and Food.

Without prejudice to the notifications provided for in Articles 3 and 5 (3), Article 6 (2) and Article 11, the competent bodies of the Autonomous Communities shall communicate to the Ministry of Agriculture, Fisheries and Food:

(a) The results of the investigations referred to in Article 6 (3) of this Royal Decree.

(b) Data on the register of affected holdings, collective warehouses and dispatch centres in each Autonomous Community.

c) The phytosanitary measures carried out by the Autonomous Communities referred to in Article 7 of this Royal Decree.

d) The analyses established in Article 8 of this Royal Decree, carried out by the competent bodies of the Autonomous Communities.

Additional provision second. Applicable general rules.

Royal Decree 2071/1993 of 26 November 1993 on protective measures against the introduction into the national territory and the European Economic Community of organisms harmful to plants or plant products Royal Decree 1190/1998 of 12 June 1998 regulating the national programmes for the eradication or control of organisms harmful to plants not yet established in the national territory shall apply without prejudice to the rules specific to this Royal Decree.

Additional provision third. Basic character.

The provisions of this Royal Decree will have the character of basic regulations, in accordance with the provisions of Article 149.1.13.a of the Constitution, which reserves the State the competence in the field of bases and coordination of the general planning of economic activity.

Final disposition first. Faculty of development.

The Minister of Agriculture, Fisheries and Food is empowered to make, within the scope of his powers, the necessary provisions for the development and implementation of this Royal Decree and, in particular, prior report of the (a) the national plant health committee, the provisional rules for the application of Article 8 (2) (a) and (b) and, in the light of developments in scientific or technical knowledge, the amendments to be made to the Annexes to this Royal Decree, as long as the European Commission does not resolve this matter.

Final disposition second. Entry into force.

This Royal Decree shall enter into force on the day following that of its publication in the "Official Gazette of the State".

Given in Madrid to October 22, 1999.

JOHN CARLOS R.

The Minister of Agriculture, Fisheries and Food,

JESUS POSADA MORENO

ANNEX I

SECTION I

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

1)

Plants (including tubers), other than true seeds, of Solanum tuberosum L.: Patata.

Plants, other than seeds and fruits, of Lycopersicum lycopersicum (L.) Karsten ex Farw.: Tomato.

SECTION II

Exams

1. The official examinations referred to in Article 3 (2) (a) shall be based on the biology of the body and on the specific production systems used in the Autonomous Community concerned and shall include the following:

i) in the case of potatoes:

at appropriate times, a visual inspection of the growing crop, and/or samples of seed potatoes and other potatoes during the growth phase or stored; these samples shall be subjected to a visual inspection official or under official supervision, for which the tubers shall be dried, and in the case of seed potatoes and, where appropriate, other potatoes, official laboratory analysis or under official supervision in which the method is used set out in Annex II to this Royal Decree;

ii) in the case of tomatoes:

at the appropriate time, a visual inspection of at least the growing growth of plants intended for replanting for professional use.

2. The notification of the official examinations provided for in Article 3 (3) shall include the following

:

i) in the case of potato tests:

the estimated total area, in hectares, of the crops of seed potatoes and other potatoes,

the classification by categories (of sowing and consumption) and, where appropriate, by regions,

the amount of samples taken for analysis and the time of collection,

the number of visual inspections performed in the field,

the number of visual inspections performed on tubers (and the quantity of samples);

(ii) in the case of examinations, at least,

growing growth of tomato plants intended for replanting for professional use:

the estimated total amount of plants,

the number of visual inspections;

(iii) in the case of examinations of host plants other than potato and tomato, including the host wild solanaceae:

the species,

the amount of samples taken and the time of their collection,

the area or river to be sampled, as appropriate,

the method of analysis;

(iv) in the case of water tests and discharges of liquid waste from industrial processing or packaging plants:

the quantity of samples taken and the time of collection, the area, river or location of the installation which is the subject of sampling, as appropriate, the method of analysis.

ANNEX II

Method of diagnosis, detection and identification of Ralstonia solanacearum (Smith) Yabuuchi et al.

This method describes the various procedures involved in:

i) the diagnosis of rottenness for potato tubers and bacterial wilt in potato and tomato plants;

(ii) the detection of Ralstonia solanacearum in samples of potato tubers;

iii) the identification of Ralstonia solanacearum.

Details are provided in the Appendices for the preparation of materials for testing, among others, nutritional means, tampons, solutions and reagents.

SECTION I

Application of the method

1. Diagnosis of parda rot in potato tubers and bacterial wilt in potato and tomato plants:

The analysis procedure refers to potato tubers with typical symptoms of brown rot and potato and tomato plants which have typical symptoms or which allow for the suspicion of wilting. bacterial and includes a rapid selection test, the isolation of the pathogen from the infected vascular tissue in an appropriate culture medium, and, in the event of a positive result, the identification of the culture as Ralstonia solanacearum.

Organization chart

(VIEW PAGE 38743).

Notes:

(1) The description of the symptoms is presented in section II, point 1.

(2) Rapid selection tests facilitate a presumptive diagnosis.

These are appropriate tests:

the test for the exudation of the stem vascular tissue (section II point 2), the polybutyrate granules detection test (section II point 2), the IF test (section III point 2), the ELISA test (point 3 of the Section III), the PCR test (Section III point 4).

(3) Although the isolation of the pathogen from plant material with typical symptoms is simple by sowing dilutions, the crop may fail in advanced states of infection. The saprophyte bacteria that grow in the diseased tissue can mask or inhibit the pathogen in the isolation medium. If the isolation is negative, but the symptoms of the disease are typical, the isolation should be repeated, preferably using a selective medium.

(4) Reliable identification of a pure culture of Ralstonia solanacearum is achieved using at least one of the tests listed in section 4.1 of Section II in combination with a pathogenicity test (point 4.3 of Section II). The characterization of the strain is optional, although it is recommended each time a new case is presented.

2. Detection and identification of Ralstonia solanacearum in samples of potato tubers:

The procedure is intended for the detection of latent infections in potato tubers by one or, preferably, several selection tests which, if positive, are complemented by the isolation of the pathogen; Isolation of typical colonies, followed by the identification of a pure culture such as Ralstonia solanacearum.

Organization chart

(VIEW PAGE 38743).

Notes:

(1) Sample size: The normal sample size is 200 tubers. However, the procedure can be conveniently adapted for samples with less than 200 tubers.

(2) Selection Tests: A single test may not be sensitive or reliable enough to detect Ralstonia solanacearum in a sample. It is therefore recommended that more than one test be carried out and based on different biological principles.

(3) Immunofluorescence Test (IF): The IF test is a fully recognized selection test, which is an advantage to other tests that have not yet been fully validated or completely validated. The test is used for many other bacteria, for example Clavibacter michiganensis ssp.

sepedonicus. The reading parameters specified in this method make it a sensitive test (with detection limits of 103-104 cells per ml of resuspended precipitate). The indirect method is preferred. The direct method can be applied if the test has a degree of sensitivity and specificity equivalent to that of the indirect method.

The critical factor in the reliability of the results is the quality of the antiserum. Only an anti-serum with a high titre (at least 2000 for crude anti-serum) is acceptable and all tests must be performed at the dilution of the antiserum titre or with a dilution below it.

The IF test presents the advantage of the subjective interpretation of the morphology of cell staining and fluorescence intensity, which provide information on the specificity of the reaction. Cross-reactions produced by serologically related bacteria from the soil or associated with potato tissues, with cellular morphology of Ralstonia solanacearum, are common. Although the IF test can be used as the only screening test, when it is suspected that there have been cross-reactions, an alternative selection test based on different biological principles will be agreed. In such cases sowing in selective means is the most appropriate test.

(4) Seed testing in selective medium: The modified SMSA medium and the procedure specified in this method make this a sensitive and selective test for the detection of Ralstonia solanacearum, the results of which are can be known three to six days after sowing. The pathogen is obtained directly in culture and can be easily identified.

In order to take full advantage of the potential of the test, it is necessary to carefully prepare the cots to avoid other bacteria associated with the potato tubers that compete with Ralstonia solanacearum in the medium and can affect the development of the pathogen. Some strains may grow insufficiently, as the components of the medium may affect the body sought. Care should also be taken to differentiate Ralstonia solanacearum from other bacteria that may develop in the medium. Although sowing in selective medium can be used as a single test for selection, when negative results are obtained and there is a suspicion that there has been an inhibition of the growth of Ralstonia solanacearum, due to the presence of other bacteria in the middle, it will be convenient to perform a second selection test.

In such a case, the IF test will be the most appropriate.

(5) ELISA test: In general, ELISA test is less sensitive than IF test (detection limits:

104-105 cells per ml of potato extract precipitate). This is a cheap and rapid test, which, however, in terms of results, is generally more exposed to false positive results (cross-reactions) and false negatives (inhibition caused by phenolic molecules present in the the potato extract). The antiserum must have a very high specificity. The ELISA test cannot be used as the only test for selection.

(6) PCR test: This test has the potential for a high detection sensitivity, but it can be easily inhibited by components of the plant extract or tuber, resulting in a false negative result. Some potato cultivars contain more inhibitors than others, which need to be removed. Inhibition can be reduced by dilution, although the populations of Ralstonia solanacearum are also diluted. A lot of care must be taken at all stages of sample preparation and testing to avoid contamination that would result in false positive results. False-positive results can also be given due to homologous sequences from other organisms. Hence, the direct PCR test cannot be used as the only selection test.

(7) Enrichment Test: Incubation of potato extract samples in a semi-elective broth, such as modified SMSA broth, allows for the multiplication of Ralstonia solanacearum and, more importantly perhaps, also dilutes the potential inhibitors of the ELISA or PCR tests. In this way, Ralstonia solanacearum can be detected by the IF, ELISA and PCR tests in an enrichment broth.

We do not recommend the production of direct crops from enriched caldos, as these enrichment methods have not been tested and thoroughly tested.

If they are included here it is because they have considerable potential. However, due to the relative lack of experience they have, they cannot be used as unique detection methods.

(8) Bioassay test: The bioassay test is used for the isolation of Ralstonia solanacearum from potato extract by selective enrichment of the bacteria in a host plant, and may be performed in plants. of tomato or in aubergines. It requires the incubation conditions to be optimal, as specified in this method. It is highly likely that the inhibitory bacteria of Ralstonia solanacearum in SMSA medium do not interfere in this test.

(9) confirmatory tests: The reliable identification of a pure crop of Ralstonia solanacearum is carried out by one of the tests listed in section 4.1 of Section II, in combination with a test of pathogenicity (section 4.3 of Section II).

Although the characterization of the strain is optional, it is recommended in each new case.

SECTION II

Diagnostic of rot in potato tubers and bacterial wilt in potato and tomato plants

1. Symptoms:

1.1 Potato symptoms:

On the floor. The initial phase of the infection is characterized by a wilting of the leaves in ascending progression towards the upper end of the plant, under the effect of the high daytime temperatures, with a recovery during the night. The wilting is quickly irreversible and causes the plant to die. The vascular tissue of the stems of the marchted plants cut transversely may become brown, and on the surface of the cut a white-off mucosal exudate is usually observed or can be extracted by squeezing the stem. If a cut stem is placed vertically in water, the vascular beams are made out of viscous threads of said exudate.

In the tuber. Potato tubers should be cut transversely close to the basal part (stolon).

In the initial phase of the infection a discoloration occurs between vitreous yellow and clear pardo of the vascular line, from which a pale cream mucosal exudate usually flows spontaneously after several minutes or when it is done. a slight pressure with the thumbs on the skin near the surface of the cut.

Subsequently, vascular discoloration takes on a more marked brown tone and the necrosis can extend to the parenchymal tissue. In the advanced stages, the infection progresses from the basal and the eyes, which can result in slightly sunken reddish-brown lesions in the skin, causing bacteria to flow, which causes the skin to become particles of the skin. soil.

1.2 Tomato symptoms:

On the floor. The first visible symptom is the flaccid aspect of the younger leaves. In environmental conditions favourable to the pathogen (soil temperature of about 25 oC, saturated humidity), epinastia and wilting occurs in part or all of the plant in a few days, resulting in the total collapse of the plant. itself. In less favourable conditions (soil temperature below 21 oC) numerous adventitious stem roots may arise. Sometimes a grating cord is observed along the stem that shows the existence of necrosis in the vascular system. By transversely cutting the stem, the vascular tissues thereof, which present a brown discoloration, exude drops of white or yellowish bacterial liquid.

2. Quick Selection Tests: Quick selection tests facilitate a presumptive diagnosis.

Realse one or more of the following tests:

Stem Exudation Test: The presence of Ralstonia solanacearum in marchite potato or tomato stems can be evaluated using the simple test shown below.

Cut the stem just above the ground level.

Place the cut surface in a beaker containing water. Soon after, the vascular beams will spontaneously emerge threads of bacterial exudate. This phenomenon will not occur with any other bacteria causing vascular infections in potato or tomato plants.

Test for the detection of polybutyrate granules (PHB): The granules of PHB of the Ralstonia solanacearum cells are displayed by dyeing with blue Nile A or black Sudan B.

Prepare a smear of the exudate or suspicious tissue on a slide, or a smear of a 48-hour culture on the YPGA or SPA (Appendix 1). Prepare positive control smear of a strain of biovar 2 and race 3 and, if considered useful, a smear of a negative control. Let dry.

Quickly pass through the flame the lower face of the slide until the smear is fixed.

Nile Blue Test:

1. Bathe the frotis fixed with aqueous solution at 1 per 100 blue Nile A. Incubar for 10 minutes at 55 ° C.

2. Drain the staining solution. Gently wash the tap for a few moments. Remove leftover water with a paper handkerchief.

3. Bathe the frotis with aqueous acetic acid at 8 per 100. Incubate for 1 minute at room temperature.

4. Gently wash the tap for a few moments.

Dry with a paper handkerchief.

5. Moisten again with a drop of water.

Tape with a bucket.

6. Examine the tinted smear with a microscope equipped with epifluence at 450 nm with immersion oil, at 1,000 increases.

Observe whether the bright orange fluorescence of the PHB granules occurs. Also observe with normal light to make sure that the granules are intracellular and that the cell morphology is typical of Ralstonia solanacearum.

Sudan Black Test:

1. Bathe the frotis fixed with a solution of black Sudan B to 0.3 per 100 in ethanol of 70 per 100. Leave in incubation for 10 minutes at room temperature.

2. Drain the staining solution. Wash gently with tap water for a few moments. Remove leftover water with a paper handkerchief.

3. Briefly immerse the smear in xylol. Dry with a paper handkerchief. Caution when using xylol which is a harmful product. Work on the smoke campaign.

4. Bathe the frotis with aqueous safranin at 0.5 per 100 (w/v) and leave for 10 minutes at room temperature. Caution when using safranin which is a harmful product. Work on the smoke campaign.

5. Gently wash the tap for a few moments.

Dry with a paper handkerchief. Cover with a cutback.

6. Examine the staining with an optical microscope with light transmitted with immersion oil, at 1,000 increases.

The PHB granules of the Ralstonia solanacearum cells are dyed in bluish black, while the walls of the cells make it pink.

Other tests: Other suitable tests for a quick selection are the IF test (Section III point 2), the ELISA test (section III point 3) and the PCR test (section III point 4).

3. Isolation process:

3.1 Coger exudate or small sections of discoloured tissue from the tuber vascular ring or from the stem vascular beams of the potato or tomato plant. Put into suspension in a reduced volume of sterile distilled water or 50mM phosphate buffer.

Let rest from 5 to 10 minutes.

3.2 Prepare decimal dilutions of the suspension, for example 1/10 and 1/100, or more if deemed necessary.

3.3 Add a standard volume of suspension and dilutions to a general nutritional medium (NA, YPGA and SPA; Appendix 1) and/or to a Kelman tetrazolium medium (Appendix 1) and/or to a selective medium SMSA (Appendix 7). Extend or make stretch marks with an appropriate plate dilution technique. If considered useful, prepare a set of distinct plates with a culture of a diluted cell suspension of a virulent strain of Ralstonia solanacearum of biovar 2 and race 3 to be used as positive control.

3.4 Leave the plates in incubation for three days at 28oC.

Incubation can last up to six days if growth is slow, but colonies in SMSA often become atypical and die.

In a general nutritional medium, the virulent isolates of Ralstonia solanacearum develop pearly, flat, irregular and fluid white colonies, which frequently present the characteristic verticides.

In the middle of Kelman's tetrazolium, the typical colonies of virulent isolates of Ralstonia solanacearum are cream-colored, flat, irregular, with blood-red rings in the center. The avian colonies of Ralstonia solanacearum are butirous and strong red.

In the SMSA medium, the typical colonies of the virulent isolates of Ralstonia solanacearum are milky white, flat, irregular and fluid, and have centers of a red blood color.

The avian forms of Ralstonia solanacearum develop less fluid colonies, the color of which oscillates between completely pink and red in the SMSA medium.

3.5 Purify the colonies of characteristic morphology by subculture in a general nutritional medium.

Avoid continuous subcultures that can lead to a loss of virulence.

4. Confirmation tests:

4.1 Identification of Ralstonia solanacearum:

Identify the pure crops of Ralstonia solanacearum by at least one of the following procedures.

Note: Include the appropriate controls in each test.

Nutritional and enzymatic tests: The following phenotypic properties of Ralstonia solanacearum are universally present or absent:

Fluorescent pigment .................................-

PHB inclusions ...................................... +

O/F Test ............................................... O + /F

Catalasa .................................................... +

Kovacs oxidase ...................................... +

Reduction of nitrates .................................. +

Use of citrate .................................... +

Growth at 40 oC ....................................-

Growth in NaCl to 1 per 100 ................... +

Growth in NaCl to 2 per 100 ...................-

Arginine Dihydrolase .................................-

Liquefaction of gelatin ...................................-

Hydrolysis of starch ...................................-

Hydrolysis of the Sculpin ................................-

Levano production ...................................-

Media and methods are found in Lelliott and Stead (1987)

IF test: Prepare a suspension of 106 cells per ml of the culture and positive control strains.

Prepare a series of dilutions to 1/2 of the antiserum.

Apply the IF procedure (section III point 2).

The crop IF title must be equivalent to that of positive control.

ELISA test: Prepare a suspension of more than 106 cells per ml of culture and positive control strains. Apply the ELISA procedure (Section III point 3). The ELISA value of the crop must be equivalent to that of positive control.

PCR test: Prepare a suspension of 106 cells per ml of culture and positive control strains.

Apply the PCR procedure (section III point 4).

The PCR product of the crop must be the same size and subsequently the same bands in the analysis with the restriction enzyme (REA), which the positive control.

Fluorescent in situ hybridization (Fluorescent in situ hybridisation or FISH): Preparing a suspension of 106 cells per ml of the culture and positive control strains. Apply the FISH procedure (Van Beuningen et al., 1995) with the initiator OLI-1 PCR (Seal et al., 1993).

The crop must show the same reaction as the positive control.

Protein profiles: Denatured whole cell proteins are separated by polyacrylamide gel electrophoresis (PAGE) according to Stead (1992a).

Fatty acid profiles (Fatty acid profiling or FAP):

Maintain the culture and the positive control strain on soy tripticase agar for 48 hours at 28 oC and apply the FAP procedure (Janse, 1991; Stead, 1992a; Stead 1992b). The crop profile should be identical to that of positive control. In the specified conditions, the characteristic fatty acids are 14: 0 3OH, 16: 0 2OH, 16: 1 2OH and 18: 1 2OH.

4.2 Characterization of the strains: The characterization of the strains is optional, but it is recommended in each new case, using at least one of the following tests:

Determination of biovars: Ralstonia solanacearum is divided into biovars based on the ability to produce acid from three alcoholic hexosas and three sugars (Hayward, 1964 and 1994).

(VIEW PAGE 38746)

Biovar 1 2 3 4 5

Using:

Maltose ......-+ +-+

Lactose ......-+ +-+

Celobious ....-+ +-+

Mannitol .....--+ + +

Sorbitol ......--+ +

Dulcitol ......--+ +

Biovar 2 differs in subphenotypes by further testing (Hayward, 1994):

Biovar 2 Biovar 2-A Biovar 2-T

Use of trehalose. -+ +

Using inositol .. +-+

Using D-ribose. --+

High Low Low ... Activity

Determination of breeds: Race (Buddenhagen et al., 1962) can be determined by a pathogenicity test in tomato plants or in aubergines and in tobacco plants, and by a hypersensitivity reaction (HR) Tobacco (Lozano and Sequeira, 1970):

Race (*) 1 2 3

Reaction to:

Tomato plants/eggplants. Marching No reaction Marching

Tobacco plants. Withering No reaction No reaction

Tobacco sheets. Necrosis (48 h) wilting (7-8 days) HR (12-24 h) Chlorosis (2-8 days)

(*) Race 4 (pathogenic in ginger and some other host) and race 5 (pathogenic only in the morera) are not included.

The characterization of the breed through pathogenicity or hypersensitivity tests in tobacco leaves may not be very reliable, but may be deduced from the biovar and the original host.

The culture can be further characterized by the following:

Obtaining the genomic fingerprint.

The molecular differentiation of the strains in the Ralstonia solanacearum complex can be performed as follows:

RFLP analysis (Cook et al., 1989).

repetitive sequence PCR [REP-, ERIC-& BOX-PCR (Louws et al., Smith et al., 1995)].

4.3 Pathogenicity test: This test is intended to confirm the diagnosis of Ralstonia solanacearum and check the virulence of the cultures identified as Ralstonia solanacearum.

Prepare an inoculum of 106 cells per ml of culture and a positive control strain. Inoculate from 5 to 10 tomato or eggplant plants, preferably in the phase of the third true or later leaf (Section III point 6). Leave in incubation for up to two weeks between 22 and 28 oC with high relative humidity and daily irrigation.

Observe whether wilting and/or epinastia, chlorosis, or dwarfism occur.

Isolate the bacteria from symptomatic plants as follows:

separate sections of the stem 2 cm per oak from the inoculation point, dilate and suspend in a small volume of sterile water or in 50mM phosphate buffer. Sow, leave in incubation and search for colonies with typical morphology of Ralstonia solanacearum.

SECTION III

Detection and identification of Ralstonia solanacearum in potato tubers samples

Note: The normal sample size is 200 tubers. However, the procedure can be conveniently adapted for samples with less than 200 tubers.

1. Preparing the sample for analysis:

Observation: The potato extract obtained with this procedure can also be used for the detection of Clavibacter michiganensis ssp. sepedonicus.

Pre-analysis options, if you consider them useful:

i) incubate the sample at 25-30 oC for a period of up to two weeks to promote the multiplication of the latent populations of Ralstonia solanacearum;

ii) wash the tubers with running water using appropriate disinfectants and detergents. Air dry the tubers.

1.1 Remove the epidermis from the basal (navel) part of the tuber with a scalpel or a clean, disinfected knife, so that the vascular tissues remain in sight. Carefully remove a small conical wedge (3 to 5 mm in diameter) of vascular tissue from the basal portion of each tuber. Remove the minimum possible volume of non-vascular tissue. Process each of the sample tubers.

Observation: The visual examination of the tubers (section II, point 1) can be performed at this stage.

Remove tubers that have symptoms or are very rotten and analyze them individually (section II).

1.2 Place the wedges in a closed container. They should be processed immediately. If not possible, they may be stored for a period of up to 24 hours or, if maintained at 4 oC, not exceeding 72 hours.

1.3 Process the wedges by one of the following methods:

i) Place the wedges in a suitable container.

Add maceration buffer solution (Appendix 2) in sufficient quantity to cover them.

Crumble them into a Waring Blender or Ultra Thurrax shredder until you achieve a complete but not excessive homogenization.

Leave the macerate to soak from 15 to 30 minutes.

ii) Place the wedges in a suitable container.

Add maceration buffer in sufficient quantity to cover them.

Place the container in a rotating stirrer.

Incubate at 50-100 rpm for 4 hours at 20-22 oC or for 16-24 hours at 4 oC.

iii) Place the wedges within a disposable maceration bag that is resistant (e.g., a Stomacher bag, 105 x 150 mm, sterile by radiation).

Carefully manage wedges using a suitable instrument, such as a hammer, until you achieve complete homogenization.

Add maceration buffer in sufficient quantity to cover the wedges.

Let the macerate rest for 15-30 minutes.

1.4 Extract the bacteria from the wedges processed by one of the following procedures:

i) gently decant the macerate into a centrifuge tube and leave the waste in the container or bag. If the decantado is cloudy, it shall be centrifuged at a maximum of 180 g for 10 minutes at a temperature of less than 10 oC.

Centrifuge the decanted or supernatant obtained in the first centrifugation, at 7,000 g for 15 minutes or at 10,000 g for 10 minutes at a temperature of less than 10 oC.

Remove the supernatant without disturbing the precipitate.

ii) Filter the macerate by means of a filtration system whose pores are 40-100 mm.

Accelerate filtration using a vacuum pump.

Collect filtering in a centrifuge tube.

Wash the filter with a maceration buffer.

Centrifuge the filtrate to 7,000 g for 15 minutes or 10,000 g for 10 minutes at a temperature below 10 oC.

Discard the supernatant without disturbing the precipitate.

1.5 Resuspend the precipitate in 1 ml of precipitate buffer (Appendix 2).

Split into two equal parts and put each part in a microvial.

Use a single microvial for testing. Keep the excess extract at 4 oC during the analysis.

Add sterile glycerol to 10-25 per 100 (v/v) to the other microvial. Homogenize by agitation. Save to -18 oC (weeks) or -70 oC (months).

2. Immunofluorescence test (IF): Use an antiserum for Ralstonia solanacearum, preferably for race 3 and biovar 2. Determine the title in a suspension of 106 cells per ml of the homologous strain of Ralstonia solanacearum with a dilution The invention is suitable for the conjugate of fluorescein isothiocyanate (FITC), according to the recommendations of the manufacturer. The crude anti-serum should present an IF titre of at least 1:2,000.

Use multiple-well slides, preferably with 10 wells of at least 6 mm in diameter.

Include in each slide a FITC conjugate control. The test with the included PBS control should be repeated if any positive cell is observed in the FITC control.

Prepare positive controls on another slide with a suspension of 106 cells per ml of a suitable breed/biovar strain of Ralstonia solanacearum.

Include a slide in each test series.

2.1 Prepare the slides according to one of the following procedures:

i) For precipitates with a relatively small amount of starch:

Set with a pipette a given volume (15 ml is sufficient for 6 mm diameter wells; increase

tar the volume if the diameter is greater) than the resuspended precipitate in a row of wells. The other row may be used as a duplicate or for a second sample, as shown in Figure 1.

ii) For other precipitates:

Prepare decimal dilutions, for example 1/10, 1/100 and 1/1,000, of the resuspended precipitate in precipitate buffer. A measured normalized volume (15 ml is sufficient for 6 mm diameter wells; increase the volume if the diameter is greater) of the resuspended precipitate and of each dilution in a row of wells with a pipette. The other row may be used as a duplicate or for a second sample, as shown in Figure 2.

2.2 Let the droplets dry. Fix the bacterial cells to the slide by heating it, flaming it or by ethanol of 95 per 100.

2.3 IF Procedure:

i) If the slide has been prepared according to point 2.1 (i): Prepare a set of dilutions to 1/2 of the antiserum in the IF buffer (Appendix 3): 1/4 of the title (T/4), 1/2 of the title (T/2), the title (T) and twice the title (2T).

(ii) If the slide has been prepared in accordance with point 2.1 (ii): Prepare the working dilution (DT) of the antiserum in the IF buffer. The dilution of work is the dilution of the antiserum with optimal specificity, and usually is at the middle of the title.

(VIEW PAGE 38748)

FIGURE 1

Preparing the slides according to points 2.1 and 2.3 (i)

NORMALIZED DILUTION OF RESUSPENDED PRECIPITATE

(T = title)

FITC T/4 T/2 T 2T Dilutions to 1/2 of Antiserum

Sample 1 J1 J2 J3 J4 J5 Duplicate of sample 1 or sample 2 J6 J7 J8 J9 J10

FIGURE 2

Preparing the slides according to points 2.1 and 2.3 (ii)

FITC NORMALIZED ANTISERUM DILUTION

Nothing 1/10 1/100 1/1,000 Decimal Precipitated Suspended

Sample 1 J1 J2 J3 J4 J5 Duplicate of sample 1 or sample 2 J6 J7 J8 J9 J10

2.3.1 Place the slides in a wetted paper handkerchief.

Add the antiserum dilution or dilutions to the appropriate wells. Apply PBS to FITC wells.

The volume of the antiserum applied in the wells should be equivalent to the volume of extract applied.

2.3.2 Tape and leave incubate for 30 minutes at room temperature.

2.3.3 Turn off the antiserum droplets from the slide holder and carefully rinse the slides with IF buffer. Wash for 5 minutes with IF-Tween tampon. Repeat the operation with the IF buffer (Appendix 3). Carefully remove excess moisture.

2.3.4 Place the slides in a wetted paper handkerchief.

Cover the wells in question and the FITC pot with the dilution of the FITC conjugate used to determine the title. The volume of conjugate applied in the wells should be identical to the applied antiserum volume.

2.3.5 Tape and leave in incubation for 30 minutes at room temperature.

2.3.6 Turn off the conjugate droplets from the slide. Rinse and wash as before (2.3.3). Carefully remove excess moisture.

2.3.7 Echar with a pipette of 5 to 10 ml of 0,1 M glycerol phosphate buffer (Appendix 3) or a similar volume in each well and place a bucket.

2.4 IF test reading:

Browse the slides in an epifluorescent microscope with filters suitable for the FITC excitation, with immersion oil and 500-1,000 increases. Walk the wells along two perpendicular diameters to each other and around the perimeter.

First, check positive control. The cells must be bright fluorescent and be completely dyed.

Note: The test must be repeated if the staining is not good at positive control.

Read the slides. First observe the absence of fluorescent cells in the FITC wells. If they exist, it would indicate a non-specific binding of the conjugate, autofluorescence or contamination.

Note: In this case, repeat the test.

Check for bright fluorescent cells with characteristic morphology of Ralstonia solanacearum in the problem wells. The intensity of the fluorescence must be equivalent to that of the positive control strain at the same dilution of the antiserum. Cells that have incomplete staining or whose fluorescence is scarce should be discarded unless there are many cells of this type (see the interpretation of the IF test results).

Interpretation of IF test results:

i) The test will be negative if bright fluorescent cells with characteristic morphology are not found in the sample.

ii) If bright fluorescent cells with characteristic morphology are found, determine the average number of cells per microscopic field and calculate the number of cells (N) per ml of resuspended precipitate (Appendix 4).

The detection limit for the IF test is considered to be around 103 cells per ml of resuspended precipitate:

in the samples with NT103 cells per ml, the IF test is considered positive, in the samples with N " 103 cells per ml, the IF test can be considered positive.

iii) If there is a large number of cells (T105 cells per ml) completely dyed or with weak fluorescence in the title, a second test must be performed:

either a test based on a different biological principle, either repeat the IF test, either with a second antiserum or with a decimal dilution of the precipitate.

3. Enzyme immunoabsorption test (Enzyme Linked Immuno Sorbent Assay or ELISA) (based on Robinson-Smith et al., 1995).

Use antiserum for Ralstonia solanacearum, preferably of race 3 and biovar 2. Determine the title in a suspension of 106 cells per ml of the homologous strain of Ralstonia solanacearum.

It is recommended to use NUNC Polysorp microtitling plates.

Include a negative potato extract control and a salt phosphate buffer (PBS) control.

Use a suspension of more than 106 cells per ml of a strain of Ralstonia solanacearum biovar 2, race 3, as positive control. Process it in the same way as the sample or samples, but well separated from them in the microtitling plate.

3.1 Echar with a pipette of 100 to 200 ml of the resuspended precipitate in a microvial.

Heat for 4 minutes at 100 oC. Place the microvial on ice.

3.2 Add an equal volume of double-concentration carbonate upholstery buffer (Appendix 5).

Homogenize by agitation.

3.3 Add 100 ml aliquots to each of at least two wells of the microtitling plate.

Leave in incubation for one hour at 37 oC or for one night at 4 oC.

3.4 Remove the extracts from the wells. Wash these three times with PBS-Tween (Appendix 5), leaving in them the last washing solution for at least 5 minutes.

3.5 Prepare for proper antiserum dilution of

Ralstonia solanacearum in blocking buffer (Appendix 5). Pour 100 ml of antiserum dilution into the wells.

Leave in incubation for an hour at 37 oC.

3.6 Remove the antiserum from the wells. Wash these as in point 3.4.

3.7 Prepare for adequate dilution of alkaline phosphatase conjugate in blocking buffer. Pour 100 ml of conjugate dilution into the wells.

Leave in incubation for an hour at 37 oC.

3.8 Remove the conjugate from the wells. Wash these as in points 3.4 and 3.6.

3.9 Prepare the alkaline phosphatase substrate solution (Appendix 5). Pour 100 ml into the wells. Leave in incubation for 30 minutes to an hour in the dark at room temperature.

3.10 Read the absorbance at 405 nm.

ELISA test interpretation: The ELISA test will be negative if the optical density (DO) of the sample is R 2 x DO of the negative control.

The ELISA test will be positive if the DO of the sample is T 2 x DO of the negative control.

4. Chain polymerization test (Polymerase Chain Reaction or PCR) (Based on Seal et al., 1993).

Observation: Filter-adjusted pipette tips should be used at all stages of sample preparation and whenever PCR is to be handled.

Prepare a suspension of 106 cells per ml of a strain of Ralstonia solanacearum of race 3 and biovar 2 as positive control. Act in the same way as with the sample or samples.

4.1 Echar with a pipette 100 ml of the resuspended precipitate in a microvial.

Another alternative is to take 90 ml of the resuspended precipitate in a microvial containing 10 ml of NaOH 0.5 M. Mix by repeatedly reversing the vial.

4.2 Heat for 4 minutes at 100 oC. Place the microvial on ice immediately.

4.3 Prepare at least two decimal dilutions, for example 1/10 and 1/100, or more if considered useful, in sterile distilled water or ultrapure (UPW).

4.4 Prepare the PCR reaction mixture (Appendix 6) in a sterile vial by adding the following components in the order listed:

50 µ l reaction volume

Component/Quantity µ l/Final Concentration

Sterile or ultra-pure distilled water. 30.8-33.8

10 x PCR buffer ..................... 5.0 1 x

d-ATP ..................................... 1.0 0.2 mM

d-CTP ..................................... 1.0 0.2mM

d-GTP ..................................... 1.0 0.2 mM

d-TTP ..................................... 1.0 0.2 mM

Initiator OLI-1 (20 µ M) .............. 2.5 1 mM

Initiator Y-2 (20 µ M) ................ 2.5 1 mM

Taq Polymerase (5 U/ µ l) ............. 0.2 1.0 U

Total Volume .................... 45-48

For more reactions:

Calculate the amount of each component for the number of reactions required.

Mix the components together and put 45-48 ml of the mixture into sterile vials.

Place the vials with the mixture of the PCR reaction on ice.

For 25 ml reaction volumes

Reduce the components proportionally.

4.5 Amplification of the PCR.

4.5.1 Optional: Centrifuge with impulses the vials containing the boiled sample and the positive control.

Add, in the order indicated, 2-5 ml of the sample or samples, water control and positive control to the vials containing the PCR reaction mixture.

Place the vials in the DNA thermocycler heater.

4.5.2 Apply the following program:

A cycle of:

i) Two minutes at 96 oC: denaturation of the mold chain;

50 cycles of:

ii) Twenty seconds at 94 oC: denaturation, iii) Twenty seconds at 68 oC: initiator hybridization, iv) Thirty seconds at 72 oC: copy extension;

A cycle of:

v) Ten minutes at 72 oC: new extension;

A cycle of:

vi) Temperature maintenance at 4 oC.

Observation: These parameters are for a Perkin Elmer 9600 apparatus. For other thermocyclers, a layer of mineral oil may be required in the vials of the PCR reaction and/or modify the duration of phases (ii), (iii) and (iv) of amplification.

4.5.3 Remove the vials from the thermocycler. Analyze the product of the PCR. If this operation is not done immediately, store the vials at 4 oC if they are to be used on the same day or -18 oC, if the waiting period is to be greater.

4.6 PCR product analysis:

The PCR fragments are detected by agarose gel electrophoresis and ethidium bromide staining.

4.6.1 Prepare an appropriate agarose gel by gently bringing the agarose to a boil in a tris-acetate buffer for electrophoresis (SAD).

4.6.2 Cool the molten agarose to 50-60 oC, pour it into the mold of the electrophoresis unit and insert the comb. Let the gel solidify.

4.6.3 Retire the comb. Dip the gel into SAD so that it is covered (2-3 mm) with the tampon.

4.6.4 Put 3 ml droplets of load buffer into parafilm. Add 12 ml of the PCR product from the samples, from the positive control or from the water control, and mix by gently vacuuming at the tip of the pipette before loading. The given volumes can be modified to fit the ability of the wells in the agarose gel.

4.6.5 Carefully load the gel wells. Include an appropriate DNA marker at least in a well to serve as a reference.

4.6.6 Connect the cables to the electricity generator and the electrophoresis equipment. Leave the gel to 5-8 V/cm until the front of the tracking indicator is 1 cm from the end of the gel.

4.6.7 Cut off electricity supply.

Disconnect the cables from the electrophoresis unit.

Carefully remove the gel. Immerse it in an etidium bromide solution for a period of between thirty-forty-five minutes.

Observation: Disposable gloves should be worn each time etidium bromide is handled, as it is a very strong mutagen.

4.6.8 Destinage in distilled water for 10-15 minutes.

4.6.9 Display the fragment or the DNA fragments amplified by UV transience. The PCR product of Ralstonia solanacearum with the set of initiators OLI-1 and Y-2 has a length of 288 bp. Collate with the DNA marker and with positive control.

Observation: Water control should be negative in any case. Otherwise, repeat the test.

4.6.10 Make a picture of the gel, if a permanent test is needed.

4.6.11 Confirm the authenticity of the amplified fragment using a restriction enzyme (REA) analysis.

4.7 Restriction Enzyme Analysis (REA):

4.7.1 Echar 8.5 ml of the PCR product (4.5.3) in a new microvial. Add 1 ml of enzyme 10 ^ and 0.5 ml of enzyme restriction AvaII.

4.7.2 Mix by gently vacuuming at the tip of the pipette. If there are drops left in the vial walls, give a pulse in a microflow. Leave to incubate for one hour at 37 oC.

4.7.3 Analyze the fragment of the digested PCR by means of agarose gel electrophoresis, as indicated in section 4.6.

Interpretation of PCR test results:

The PCR test is negative if the characteristic 288 bp fragment is not detected and if it is detected for the positive control strain of Ralstonia solanacearum.

The PCR test is positive if the 288 bp fragment is detected and the REA analysis of the amplified fragment is identical to that of the positive control strain of Ralstonia solanacearum.

5. Seed testing in selective medium (based on Elphinstone et al., 1996):

5.1 Perform the test using an appropriate plate dilution technique, such as:

i) Prepare at least two decimal dilutions, for example 1/10 and 1/100, or more, if necessary, of the resuspended sediment in precipitate buffer.

Take with a pipette a measured normalized volume (50-100 ml) of the resuspended sediment and of each dilution in the modified SMSA selective medium (Appendix 7) and extend with a glass rod all over the surface of the medium.

If it is considered useful, also perform a dilution test with a handle loaded with 10 ml of the resuspended sediment. Flout the handle between each seeding.

ii) Put a measured standard volume (50-100 ml) of the resuspended sediment into the modified SMSA selective medium and extend with a glass rod all over the middle surface. Pass the rod without flaming it at least for two other modified SMSA plates.

5.2 Add, by the same dilution technique in plates, a suspension of 106 cells per ml of a strain of Ralstonia solanacearum of race 3 and biovar 2 as positive control to a different set of modified SMSA plates.

5.3 Let the plates be incubated at 28 oC. Start your reading after three days. If the result is negative, wait up to six days at most. The virulent isolates of Ralstonia solanacearum develop milky, flat, irregular and fluid white colonies, with blood red centers and which may present internal streaks or spirals.

5.4 Purify the colonies of characteristic morphology by subculture in a general nutritional medium (Appendix 1).

5.5 Identify pure crops (section 4.1 of Section II) and confirm the crops of Ralstonia solanacearum by means of a pathogenicity test (section 4.3 of Section II).

Interpretation of seeding results in selective media:

The seeding in selective medium is negative if bacterial colonies are not isolated after six days or isolated colonies of Ralstonia solanacearum are isolated, provided there is no suspicion of inhibition produced by colonies of other bacteria and that in the positive controls are found typical colonies of Ralstonia solanacearum.

Seeding in selective medium is positive if typical colonies of Ralstonia solanacearum are isolated.

6. Bioassay tests (based on Janse, 1988):

6.1 Use in each sample 10 tomato or eggplant seedlings of varieties that are sensitive to Ralstonia solanacearum, in the phase of the third true leaf. Plants should not be irrigated during the 24 hours prior to inoculation.

6.2 Distribute between the plants 100 ml of resuspended sediment, inoculating it on the stem, between the cotyledons, and on one or several other points.

6.3 Using this same technique, inoculate 10 seedlings with a suspension of 106 cells per ml of a virulent strain of Ralstonia solanacearum of race 3 and biovar 2 as positive control and with precipitate buffer as control negative. Physically separate the inoculated plants with the positive control of the others to avoid contamination.

6.4 Let plants continue to grow for up to four weeks at 22-28 oC with high relative humidity and daily irrigation. Check for symptoms of wilting, epinasty, chlorosis and/or reduced growth.

6.5 Isolate of infected plants (section II).

Identify the characteristic morphology pure cultures (section 4.1 of section II) and confirm the crops of Ralstonia solanacearum by a pathogenicity test (section 4.3 of section II).

6.6 If it is considered useful, check for infection in lots that do not show symptoms.

Separate from the stem of each plant a section of 1 cm that is 2 cm above the point of inoculation.

Homogenize the tissues in maceration buffer.

Carry out the sowing by dilution in plates (section 5.1 of Section III). If a positive result is to be obtained, identify the characteristic morphology pure cultures (section 4.1 of Section II) and confirm the presence of Ralstonia solanacearum crops by means of a pathogenicity test (section 4.3 of Section II).

Interpretation of the results of the bioassay test:

The bioassay test is negative if the plants are not infected by Ralstonia solanacearum, provided that Ralstonia solanacearum is detected in the positive controls.

The bioassay test is positive if the plants are infected by Ralstonia solanacearum.

7. Enrichment tests (based on Elphinstone et al., 1996):

7.1 Echar 100 ml of resuspended sediment in 3 ml modified SMSA broth (Appendix 7).

7.2 Let be incubated at 28 oC for 48 hours and, in any case, for a maximum of 72 hours, keeping the tube stopper without squeezing for aeration.

7.3 Adjust the stopper and mix. Make aliquot parts for the IF test (paragraph 2 of this section), the ELISA test (paragraph 3 of this section) and/or the PCR test (paragraph 4 of this section).

8. Pathogenicity tests:

See section 4.3 of Section II.

APPENDIX 1

Nutritive means for the isolation and cultivation of Ralstonia solanacearum

Nutritional Agar (NA):

Nutrient Agar (Difco): 23 g.

Distilled water: 1 l.

Prepare volumes of 0.5 litres of the medium in 1 l flasks.

Dissolve the ingredients.

Sterilize in a autoclave at 121 oC for 15 minutes.

Cool up to 50 oC. Distribute on plates.

Agar peptone glucose (YPGA) agar:

yeast extract (Difco): 5 g.

Peptone Bacto (Difco): 5 g.

D (+) glucose (monohydrate): 10 g.

Bacto agar (Difco): 15 g.

Distilled water: 1 l.

Prepare 0.5 l volumes of the media in 1 l flasks.

Dissolve the ingredients.

Sterilize in a autoclave at 121 oC for 15 minutes.

Cool up to 50 oC. Distribute on plates.

Sucrose and peptone agar (SPA):

Sucrose: 20 g.

Peptone: 5 g.

K2HPO4: 0.5 g.

MgSO4 7H2O: 0.25 g.

Bacto agar (Difco): 15 g.

Distilled water: 1 l.

Prepare 0.5 l volumes of the media in 1 l flasks.

Dissolve the ingredients. If necessary, adjust the pH to 7.2-7.4.

Sterilize in a autoclave at 121 oC for 15 minutes.

Cool up to 50 oC. Distribute on plates.

Kelman's tetrazolium medium:

Casamino acids (Difco): 1 g.

Peptone Bacto (Difco): 10 g.

Dextrose: 5 g.

Bacto agar (Difco): 15 g.

Distilled water: 1 l.

Prepare volumes of 0.5 litres of the medium in 1 l flasks.

Dissolve the ingredients.

Sterilize in a autoclave at 121 oC for 15 minutes.

Cool up to 50 oC.

Add an aqueous solution of triphenyl tetrazolium chloride (Sigma), sterilized by filtration, to achieve a final concentration of 50 mg/l.

Distribute on plates.

APPENDIX 2

Materials for the preparation of samples

Maceration buffer: 50 mM phosphate buffer, pH 7.0:

This buffer is used to macerate tissues.

Na2HPO4: 4.26 g.

KH2PO4: 2.72 g.

Distilled water: 1 l.

Dissolve the ingredients and check the pH. Prepare the necessary aliquots.

Sterilize in a autoclave at 121 oC for 15 minutes.

For direct PCR testing, it is recommended to add polyvinylpyrrolidone-40,000 MWT (PVP-40) to 5 per 100, to reduce the incidence of amplification inhibition by aromatic molecules present in the extract.

In the case of the use of the homogenization procedures with Waring Blender or with Ultra Turrax for maceration of the potato tissues, it is recommended to use a deflocculant, an antifoaming agent or a Antioxidant.

Lubricating flakes: 0.5 g/l.

Antifoaming DC silicone: 1.0 ml/l.

Tetrasodiophosphate: 1.0 g/l.

Sterilize separately in a autoclave. Add until the desired concentration is obtained.

Precipitate buffer: 10 mM phosphate buffer, pH 7.2:

This buffer is used for resuspension and dilution of the precipitates and potato wedges.

Na2HPO4 12H2O: 2.7 g.

NaH2PO4 2H2O: 0,4 g.

Distilled water: 1 l.

Dissolve the ingredients and check the pH. Prepare the necessary aliquots.

Sterilize in a autoclave at 121 oC for 15 minutes.

APPENDIX 3

Materials for the IF test

IF buffer: saline phosphate buffer (PBS) 10 mM, pH 7.2:

This buffer is used for the dilution of antisera.

Na2HPO4 12H2O: 2.7 g.

NaH2PO4 2H2O: 0,4 g.

NaCl: 8.0 g.

Distilled water: 1 l.

Dissolve the ingredients and check the pH. Prepare the necessary aliquots.

Sterilize in a autoclave at 121 oC for 15 minutes.

IF-Tween tampon:

This buffer is used to wash the slides.

Add 0.1 per 100 from Tween 20 to the IF buffer.

Glycerol phosphate 0.1 M, pH 7.6:

This buffer is used as a mounting fluid in the wells of the IF slides to increase fluorescence.

Na2HPO4 12H2O: 3,2 g.

NaH2PO4 2H2O: 0.15 g.

Glycerol: 50 ml.

distilled water: 100 ml.

APPENDIX 4

(VIEW PAGE 38752)

Determining the level of contamination in the IF test

Multiple-object slide-box surface (S)

= pD2/4

in which D = diameter of the pot. [1]

Target field surface (s)

= pd2/4

on which d = diameter of the field. [2]

Calculate d well by direct measurement, either with the following formulas:

(VIEW PAGE 38752)

p i 2 s = G2K2 ^ 4 [3]

in that

i = field coeffecy (depends on the type of the eye and varies from 8 to 24),

K = tube coefficient (1 or 1.25),

G = increase (100x, 40x, etc.) of the target.

From [2]:

4 s d = " p [4] From [3]:

p i 2 4 ^

G 2K 2 ^ 4 i d = = " p GK

Count the number of typical fluorescent cells per field (c).

Calculate the number of typical fluorescent cells per well (C):

S C = c s

Calculate the number of typical fluorescent cells per ml of precipitate (N):

1,000

N = C ^ ^ F and

What = volume of precipitate in the well,

F = dilution factor of the precipitate.

APPENDIX 5

Materials for ELISA Test

Double carbonate buffer for upholstery, pH 9.6:

Na2CO3: 6.36 g.

NaHCO3: 11.72 g.

Distilled water: 1 l.

Dissolve the ingredients and check the pH. Prepare the necessary aliquots.

Sterilize in a autoclave at 121 oC for 15 minutes.

If the extract contains a high proportion of aromatic molecules, it can be added as a sodium sulphite antioxidant with a final concentration of 0.2 per 100.

Saline phosphate buffer (PBS) 10 x, pH 7.4:

NaCl: 80 g.

KH2PO4: 2 g.

Na2HPO4 12H2O: 29 g.

KCl: 2 g.

Distilled water: 1 l.

Dissolve the ingredients and check the pH. Prepare the necessary aliquots.

Sterilize in a autoclave at 121 oC for 15 minutes.

Salino phosphate buffer-Tween (PBS-T):

PBS 10 x: 100 ml.

10 per 100 Tween solution 20: 5 ml.

distilled water: 895 ml.

Lock buffer (antibodies) (should be prepared at the time of use):

PBS 10 x: 10 ml.

Polyvinylpyrrolidona-44000 MWT (PVP-44): 2.0 g.

Solution to 10 per 100 of Tween 20: 0.5 g.

Milk powder: 0.5 g.

Distilled Water: Up to 100 ml.

alkaline phosphatase substrate solution, pH 9.8:

Diethanolamine: 97 ml.

distilled water: 800 ml.

Mix and adjust the pH to 9.8 with concentrated HCl.

Complete up to 1 l with distilled water.

Add 0.2 g of MgCl2.

Dissolve two 5 mg lozenges of phosphatase substrate (Sigma) for every 15 ml of solution.

APPENDIX 6

Materials for PCR Test

Initiator oligonucleotide sequence:

Initiator OLI-1: 5 '-GGGGGTAGCTTGCTACCTGCC-3' Initiator Y-2: 5 'CCCACTGCTGCCTCCCGTAGGAGT-3' For materials see Seal et al., 1993.

APPENDIX 7

Materials for seed testing in selective and enrichment media

Selective medium SMSA (Engelbrecht, 1994, as amended by Elphinstone et al., 1996):

Casamino acid base medium (Difco): 1 g.

Peptone Bacto (Difco): 10 g.

Glycerol: 5 ml.

agar (Diffo): 15 g.

Distilled water: 1 l.

Prepare 0.5 ls volumes from the media into 1 l flasks.

Dissolve the ingredients and check the pH. If necessary, adjust the pH to 6.5 before sterilizing.

Ralstonia solanacearum does not develop well at pH 7.0.

Sterilize in a autoclave at 121 oC for 15 minutes.

Cool up to 50 oC.

Add the following ingredients (all Sigma) to obtain the specified final concentrations:

Glass violet: 5 mg/l.

Polymyxin B sulfate: 100 mg/l (approximately 600,000 units) Sigma P-1004.

Bacitracin (*): 25 mg/l (approximately 1,250 units) Sigma B-0125.

Chloranphenicol: 5 mg/l Sigma C-3175.

Penicillin G: 0.5 mg/l (approximately 825 unides9 Sigma P-3032.

tetrazolium salts: 50 mg/l.

Dissolve the ingredients in 70 per 100 ethanol to obtain the indicated concentrations for the prepared diluent volume. Some ingredients, such as polymyxin B and chloramphenicol, require slight warming and agitation.

Broth SMSA (Elphinstone et al., 1996).

Prepare identically to the selective medium SMSA, but suppress the agar and the tetrazolium salts.

Distribute in 3 ml aliquots, in 30 ml Universal disposable tubes.

(*) If deemed necessary, the increased concentration of bacitracinahasta300ppmcan be educatedby bacteriassaprophytes without reducing the recovery of Ralstonia solanacearum.

REFERENCES

Buddenhagen, I. W., Sequeira, L., and Kelman, A., 1962:

"Description of races in Pseudomonas solanacearum", Phytopathology, number 52, page 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 responds," Molecular Plant-Microbe Interactions, number 2, pages 113-121.

Dinesen, I. G., and DeBoer, S. H., 1995: " Extraction of Clavibacter michiganensis subsp. sepedonicus from composite samples of potato tubers, " American Potato Journal, number 72, pages 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, number 26.

Engelbrecht, M. C., 1994: "Modification of a semilective medium for the isolation and quantification of Pseudomonas solanacearum," ACIAR Bacterial Wilt Newsletter, number 10, pages 3-5.

Hayward A. C., 1964: "Characteristics of Pseudomonas solanacearum," Journal of Applied Bacteriology, number 27, pages 265-277.

Hayward A. C., 1994: "Systematic and phylogeny of Pseudomonas solanacearum and related bacteria,"

Bacterial Wilt: The disease and its causative agent, Pseudomonas solanacearum, A. C. Hayward and G. L.

Hartman, eds., Cab International Oxford, pages 127-135.

Janse, J. D., 1988: "A detection method for Pseudomonas solanacearum in symptomless potato tubers and some data on its sensitivity and specification", EPPO Bulletin, number 18, pages 343-351.

Janse, J. D., 1991: "Infra-and intraspecific classification of Pseudomonas solanacearum strains using whole cell fatty acid analysis," Systematic and Applied Microbiology, number 14, pages 335-345.

Kelman, A., 1954: "The relationship of pathogen in Pseudomonas solanacearum to colony appearance on a tetrazolium medium," Phytopathology, number 64, pages 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 pages Louws, F. J., Fulbright, D. W., Stephens, C. T., and Brujin, F. J., 1995: "Differentiation of genomic structure by rep-PCR fingerprinting to rapidly classify Xanthomonas campestris pv. vesicatoria," Phytopathology, number 85, pages 528-536.

Lozano, J. C., and Sequeira, L., 1970: "Differentiation of races of Pseudomonas solanacearum by a leaf infiltration technique," Phytopathology, number 60, page 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, number 39, pages 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, number 7, pages 67-79.

Seal, S. E., Jackson, L. A., Young, J. P. W., and Daniels, M.J., 1993: " Differentiation of Pseudomonas solanacearum, P. syzygii, P. picketti and the blood disease bacterial by partial 16S rRNA sequencing:

construction of oligonucleotide primers for sensitive detection by polymerase chain reaction, " Journal of General Microbiology, number 139, pages 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, number 61, pages 4262-4268.

Stead, D. E., 1992a: "Techniques for detecting and identifying plant pathogenic bacteria," Techniques for rapid detection of plant pathogens, J. M. Duncan and L. Torrance, eds., Blackwell Scientific Publications, Oxford, pages 76-111.

Stead, D. E., 1992b: " Grouping of plant pathogenic and some other Pseudomonas spp. using cellular fatty acid profiles ", International Journal of Systematic Bacteriology, number 42, pages 281-295.

Van Beuningen, A., Derks, H., and Janse J. D., 1995:

" Detection and identification of Clavibacter michiganensis subsp. sepedonicus with special attention to fluorescent in-situ hybridisation (FISH) using a 16S rRNA targeted oligonucleotide probe, " Zuchtungsforschung, number 2, pages 266-269.

ANNEX III

1. In all cases where the presence of the organism is suspected and a positive result has been obtained in detection tests carried out in accordance with the relevant method set out in Annex II to this Royal Decree for the material For all other cases, indicated or any other officially approved method, the conclusion of such a method for confirming or dispelling the suspicion, shall be expected to be retained and maintained conveniently until that time:

-whenever possible, the lot, or the part of it (from which the sample was taken) in its original packaging with a label,-wherever possible, the remaining part of the samples,-any extract that is over and any additional material that has been prepared for testing or screening (e.g., the immunofluorescence plates),-all relevant documentation.

2. In cases where the presence of the organism is finally confirmed, they shall be retained and maintained conveniently for at least the month following the notification procedure referred to in Article 6 (2):

-the material referred to in point 1, and

-a sample of the infected tomato or eggplant material that has been inoculated with the tuberculo extract or plant where appropriate, and

-the isolated culture of the organism.

ANNEX IV

The elements of the investigation provided for in Article 6 (1) (a) (i) shall, where appropriate, be as follows:

i) the places of production:

-in which potatoes are grown or have been grown which are clonically related to -those where the infection has been found by the organism,

-in which tomatoes are grown or have been grown which come from the same sources as those where the infection has been proven by the organism,

-in which potatoes or tomatoes have been grown or have been grown under official control because the presence of the organism is suspected,

-where potatoes are grown or have been grown which are clonically related to those that have been grown in places of production of which the infection is suspected by the organism,

-in which potatoes or tomatoes are grown and which are located in the vicinity of the infected places of production, including those where equipment and production facilities are shared directly or by intervention by a common contractor,

-in which surface water is used for irrigation or surface water of any source in which it has been confirmed or suspected of infection by the organism,

-where surface waters of a source operated in common with places of production in which it has been confirmed or suspected of infection by the organism are used for irrigation or spraying work,

-which are or have been flooded with surface waters in which the infection is confirmed or suspected by the organism; and (ii) the surface waters used for irrigation or spraying, or for the flood, of a field or fields or of a place or places of production where the infection has been confirmed by the organism.

ANNEX V

1. In determining, in accordance with point (a) (iii) and Article 6 (1) (c) (iii), the extent of the likely contamination, the following elements shall be included, where appropriate:

-the indicated plant material obtained at a place of production which has been declared contaminated by virtue of Article 6 (1) (a) (ii),

-the place or places of production having a production relationship with the indicated plant material which has been declared contaminated by virtue of Article 6 (1) (a) (ii), including those places that share production equipment and facilities directly or through the intervention of a common contractor,

-the indicated plant material which has been produced in the place or places of production referred to in the previous indent or which was present in such places during the time when the indicated plant material was declared contaminated by virtue of Article 6 (1) (a) (ii), shall be present in the places of production referred to in the first indent,

-warehouses which have handled the indicated plant material from the production sites referred to in the previous indents,

-any machinery, vehicle, ship, warehouse or units of these and any other objects, including packaging material, which may have been in contact with the indicated plant material contaminated by virtue Article 6 (1) (a) (ii) of Article 6 (1) (a

,

-any indicated plant material which has been stored or has been in contact with any of the structures and objects referred to in the previous indent prior to the cleaning and disinfection of these structures,

-as a result of the research and analysis referred to in Article 6 (1) (a) (i): in the case of potatoes, those tubers or plants having a fraternal or fraternal relationship (a) parental leave with the indicated plant material contaminated by virtue of Article 6 (1) (a) (ii) and which, according to the results of the investigation, are likely to be contaminated; and, in the case of tomatoes, those plants which come from the same sources as said material and with respect to which, although the body's detection tests have been negative, the contamination appears likely via a clonal link,

-the place or places of production of the plant material referred to in the previous indent,

-the place or places of production of the plant material indicated in those used for irrigation or spraying of water which has been declared contaminated by virtue of paragraph 1 (c) (ii). Article 6,

-the indicated plant material produced in fields waterlogged with surface waters in which the infection has been confirmed by the organism.

2. For the purposes of determining the possible spread referred to in point (a) (iv) and point (iii) of Article 6 (1) (c), the following elements shall be taken into account:

(i) in the cases referred to in Article 6 (1) (a) (iv):

-the proximity of other production sites where the indicated plant material is grown,

-the common production and use of stocks of seed potatoes,-the places of production where surface water is used for the irrigation or spraying of the plant material indicated where the existing or existing plant material has been risk of runoff, or of flooding, of surface water from a place or places of production that have been declared contaminated by virtue of paragraph

(ii) in Article 6 (1) (a);

(ii) in cases where contaminated surface waters have been declared under point (c) (ii) of Article 6 (1) (c):

-the place (s) producing the plant material adjacent to the surface waters declared contaminated, or which risk being flooded with these waters,-any separate source of irrigation that is communicated in some form with the surface waters declared contaminated.

3. The notification referred to in the first subparagraph of Article 6 (2) shall include the following

:

-the date on which the alleged presence of the organism has been reported pursuant to Article 5 and the dates of sampling and confirmation of the presence of the organism in accordance with Article 6, the cases,-a description of the elements of the pollution declaration and the delimitation of areas.

4. The notification referred to in the first subparagraph of Article 6 (2) shall include the following

:

-in the case of any consignment or batch of contaminated potatoes, the accompanying documents prescribed in Article 7 of Royal Decree 2071/1993, the passport number or registration number of the producers of potato, collective warehouses and distribution centres, as appropriate,

-in the case of any consignment or batch of tomatoes declared as contaminated, the accompanying documents prescribed in Article 7 of Royal Decree 2071/1993 and the passport number in accordance with the list set out in point 2.2 of Section I of Part A of Annex V to Royal Decree 2071/1993.

-in the case of stocks of seed potatoes and, if possible, in all other cases, the name and category of the variety,

-any other data required by the Ministry of Agriculture, Fisheries and Food relating to the confirmed outbreak.

ANNEX VI

1. With regard to Article 7 (1), the following shall be

:

-incineration,

-the use as feed, after appropriate heat treatment, so that there is no risk of survival of the organism, or

-deep burial in a landfill where there is no risk of filtration to agricultural land or contact with water sources that can be used for irrigation of such lands,

-industrial processing, after direct and immediate delivery of the material to a processing plant with facilities which are officially approved for the disposal of waste and comply with the provisions of the Annex VII to this Royal Decree, or,

-other measures, provided that it has been established that there is no identifiable risk of spread of the organism and provided that such measures are immediately notified to the Ministry of Agriculture, Fisheries and The Commission and the other Member States are to be fed and in turn, through the appropriate channel.

2. The appropriate use or disposal of the plant material referred to in Article 7 (2) shall be carried out under the control of the responsible official bodies concerned, as well as the appropriate communication between them. (a) to ensure at all times that control, and with the approval of the official body of each Autonomous Community where the potatoes are to be packaged or processed, as regards the landfills referred to in the first and second indents second, and consist of the following:

i) in the case of potato tubers:

-the use as consumption potatoes that are packed in places with appropriate waste disposal facilities, ready for delivery and direct use without need for new packaging, and intended for such delivery and use direct, or

-use as consumption potatoes intended for industrial processing and direct and immediate delivery to a processing plant with appropriate waste disposal facilities, or

-some other type of use or disposal, provided that it is established that there is no identifiable risk of the organism spreading, and after approval of those responsible official bodies. Those measures shall be notified immediately to the Ministry of Agriculture, Fisheries and Food and in turn, through the appropriate channel, to the Commission and to the other Member States;

(ii) in the case of other parts of plants, including stem and leaf detritos:

-the destruction, or

-some other type of use or disposal, provided it is established that there is no identifiable risk of the organism's spread and provided that such use or disposal is notified to the Ministry of Agriculture, Fisheries and Food and feed, in turn, through the appropriate channel, to the Commission and to the other Member States.

3. The appropriate methods for the decontamination of the objects referred to in Article 7 (3) shall consist of a cleaning and, where appropriate, a disinfection which will allow the identification of any identifiable risk of the organism's spread to be excluded; these operations shall be carried out under the supervision of the official bodies responsible for the Autonomous Communities.

4. The series of measures referred to in Article 7 (4) to be applied within the area or demarcated areas established by virtue of point (a) (iv) and (iii) of Article 6 (1) (c) will be as follows:

4.1. In cases where, pursuant to Article 6 (1) (a) (ii), contaminated sites of production have been declared, the measures shall consist of the following:

(a) in the fields or units of production of protected crops which have been declared contaminated by virtue of that same provision:

i) for at least four years of cultivation following the declaration of contamination:

-measures shall be taken to remove the potatoes and spontaneous tomateras as well as other host plants of the organism, including the weeds solanaceae;-they shall not be planted:

potatoes and potato tubers, tomato potatoes and tomato seeds,

-taking into account the biology of the organism:

other host plants, plants of Brassica species for which there is an identified risk of survival or spread of the organism, other crops for which there is an identified risk of survival or spread of the body;

-in the first season of growing potatoes or tomatoes following the period indicated in the previous indent and, provided that it has been established that during at least the two years of vegetation immediately preceding the planting, the field was free of potatoes and spontaneous tomateras and other host plants of the organism, including the weeds solanaceae:

in the case of potatoes, officially certified seed potatoes shall be planted for the production of potatoes for consumption only, and an official examination shall be carried out, accompanied by appropriate analyses, as provided for in Article 3 (1);

-in the season of growing potatoes or tomatoes as indicated in the preceding indent and after an appropriate rotation cycle, the planting of certified seed potatoes shall be carried out in the case of potatoes. officially for the production of seed potatoes or potatoes and, in the case of potatoes and tomatoes, for the official examination provided for in Article 3 (1);

(ii) during the five years of cultivation following that of the pollution declaration:

-measures shall be taken to remove spontaneous potatoes and tomateras as well as other plants which may be a host of the organism, including solanaceous weeds, and

-for the first three years, the field shall be left and maintained, either in full fallow or for the cultivation of cereals, in accordance with the risk determined either as permanent pasture, with intense and frequent mowing or intensive grazing, or as a grassland for seed production, and then in the subsequent two years, plants which are not the host of the organism for which there is no identified risk of survival, or propagation of this;

-in the first season of growing potatoes or tomatoes following the period indicated in the previous indent:

in the case of potatoes, only officially certified seed potatoes shall be planted for the production of seed potatoes or for consumption, and the official examination shall be carried out, accompanied by the appropriate analyses, provides for Article 3 (1).

b) in other fields, the measures shall consist of the following:

-during the crop year following the pollution declaration:

no potatoes or potato tubers or other plants that can be a host of the organism will be planted, and measures will be taken to remove the potatoes and spontaneous tomateras as well as other plants that may be a host of the potato. (a) a body, including any maleal weed, or in the case of potato tubers, may be planted exclusively officially certified seed potatoes for the production of potatoes for consumption, provided that the organisms responsible officers have been fully convinced of the elimination of risks through spontaneous plants of potatoes and tomateras and other hosts of the organism, including the weeds solanaceae; during the growth phase, the crops shall be inspected at the appropriate time, the spontaneous potatoes shall be subjected to analysis for the possible detection of the organism and, in the end, the harvested tubers shall be inspected; in the first year of cultivation following the one indicated in the preceding indent:

in the case of potatoes, only seed potatoes officially certified for the production of seed potatoes or for consumption shall be planted; for at least the second year of cultivation following the one indicated in the first hyphen:

in the case of potatoes, they shall be planted for the production of seed potatoes or for consumption only officially certified seed potatoes or seed potatoes which have been grown under official control from officially certified seed potatoes; in each of the years of cultivation indicated in the previous indents, measures shall be taken to remove the potatoes and spontaneous tomateras as well as other plants which may be the host of the organism, In addition, the official examination referred to in paragraph 1 shall be carried out. Article 3 and, in cases where seed potatoes are planted for the production of the same type of potatoes, the tubers shall be subjected to analysis.

(c) Immediately after the contamination has been declared pursuant to Article 6 (1) (a) (ii), and in each subsequent growing year up to, including, the first growing season permissible of potatoes or tomatoes in the field or fields declared contaminated as referred to in point (a) above:

-all machinery and storage facilities in the place of production used in the production of potatoes or tomatoes shall be cleaned and, where appropriate, disinfected using appropriate methods in the manner in which they are available, point 3, and

-in order to prevent the spread of the organism, official controls shall be carried out on irrigation and spraying plans and, if necessary, the same shall be prohibited.

(d) In the case of units of production of protected crops which have been declared contaminated by virtue of Article 6 (1) (a) (ii), and where a total substitution of the products is possible, culture media:

-potatoes and potato tubers or other plants which may be a host of the organism, including tomatoes and tomato seeds, shall not be planted unless, on the one hand, those units have been officially submitted to the supervised that, having as their object the removal of the organism and the removal of all the host plant material, include at least a complete change of the means of cultivation and a cleaning and, where appropriate, a disinfection of such units and the entire team and that, on the other hand, the responsible official bodies have given subsequently their authorisation for the production of potatoes or tomatoes, and in addition the production shall, in the case of potato, officially certified seed potatoes or mini-tubers or microplants obtained from analysed sources, Furthermore, in order to prevent the spread of the organism, official controls shall be carried out on irrigation and sprinkling plans and, where appropriate, shall be prohibited.

4.2. Within the area defined and without prejudice to the measures provided for in point 4.1, the Autonomous Communities shall:

(a) immediately after the declaration of contamination and for a period of at least three years of cultivation:

aa) in cases where the delimitation of the area has been effected pursuant to Article 6 (1) (a) (iv):

-shall ensure that their responsible official bodies supervise the facilities they cultivate, store or handle potato or tomato tubers, as well as other facilities they use in the framework of a contract machinery for the production of potatoes or tomatoes,

-require that the machinery and warehouses of such installations be cleaned and, where appropriate, disinfected using appropriate methods as set out in point 3,

-will require that for all potato crops within the demarcated area, only certified seed or seed grown under official control is planted, and an analysis is carried out after harvesting the crops from seed potatoes in places of production determined as likely to be contaminated under Article 6 (1) (a) (iii),

-shall provide that in all facilities located in the area the stocks harvested from seed potatoes and potatoes for consumption are to be handled separately,

-shall carry out the official examination provided for in Article 3 (1

;

ab) in cases where, pursuant to Article 6 (1) (c) (ii), contaminated surface waters have been declared or where, at the disposal of point 2 of Annex V to this Royal Decree, they have been including those waters between the factors of the possible spread of the organism:

-shall carry out an annual examination, at appropriate times, with a sampling of the surface waters and of the solanaceous host plants present in the relevant water sources, as well as analysis according to:

in the case of the plant material indicated, the corresponding method set out in Annex II to this Royal Decree, or in other cases, any other officially approved method;

-in order to prevent the spread of the organism, will establish official controls of irrigation and spraying plans as well as a prohibition (revisable in the light of the results of the above mentioned annual examination) of the use of the waters declared contaminated for the irrigation and spraying of the plant material indicated and, where appropriate, other host plants;-in cases where they have been contaminated by discharges of liquid waste, they shall establish official controls on the disposal of waste from industrial plants for processing or packaging handling the indicated plant material;

(b) shall, where appropriate, establish a programme for the replacement of all stocks of seed potatoes within an appropriate period.

ANNEX VII

The officially approved waste disposal facilities referred to in the fourth indent of point 1 of Annex VI to this Royal Decree shall comply with the provisions set out below for the purpose of avoid any risk of spreading the organism:

(i) residues resulting from the processing of potatoes and tomatoes (including rejected potatoes and tomatoes and peelings), as well as any other solid waste accompanying potatoes and tomatoes, shall be disposed of:

-by means of its deep burial in a landfill where there is no risk of filtration to agricultural land or contact with water sources that can be used for the irrigation of such lands; the waste will be directly to the landfill under the conditions necessary to avoid any risk of loss of the site, or

-by incineration;

(ii) liquid waste from processing containing suspended solid elements shall be filtered or subjected to sedimentation processes for the separation of those elements. These shall be disposed of in the manner set out in point (i).

For your part, liquid waste will be subject to one of the following measures:

-heating to a minimum temperature of 70 0C for at least 30 minutes prior to disposal, or removal by another system which, officially approved and applied under official control, disposes any risk of contact waste with agricultural land or with water sources that can be used for the irrigation of agricultural land. The characteristics of such a system shall be notified to the Ministry of Agriculture, Fisheries and Food and in turn, through the appropriate channel, to the other Member States and to the Commission.