Regulations, 2004

Suppliment tal-Gazzetta tal-Gvern ta’ Malta, Nru. 17,584, 14 ta’ Mejju, 2004

L.N. 270 of 2004

PLANT QUARANTINE ACT, 2001

(ACT NO. XVIII OF 2001)

Control of Ralstonia solanacearum (Smith) Yabuuchi et al

Regulations, 2004

IN exercise of the powers conferred by article 32 of the Plant

Quarantine Act 2001, the Minister for Rural Affairs and the Environment

has made the following regulations:-

The title of these regulations is the Control of Ralstonia Title and

commencement

solanacearum (Smith) Yabuuchi et al Regulations, 2004 and they shall

come into force on 15th May, 2004.

detection of Ralstonia solanacearum in samples of potato tubers;

positive result, identification of the culture as Ralstonia solanacearum.

The presented scheme describes the various procedures involved in

identification of Ralstonia solanacearum.

APPLICATION OF THE TEST SCHEME

SCOPE OF THE TEST SCHEME

SECTION I

(iii)

(ii)

(i)

(3) Although isolation of the pathogen from plant material with typical symptoms by dilution plating is straightforward, culturing may fail

isolation medium. If the isolation test is negative, but disease symptoms are typical, then isolation must be repeated, preferably by a

from advanced stages of infection. Saprophytic bacteria which grow on diseased tissue may outgrow or inhibit the pathogen on the

- Test for poly-ß-hydroxybutyrate granules (section II.2),

Streaming test of vascular stem tissue (section II.2),

(2) Rapid screening tests facilitate presumptive diagnosis.

(1) Description of symptoms is provided in section II.1.

- ELISA test (section III.3),

- PCR test (section III.4).

- IF test (section III.2),

selective plating test.

-

Flowchart references

Appropriate tests are

(4) Reliable identification of a pure culture of Ralstonia solanacearum is achieved by at least one of the tests listed in section II.4.1 in

complemented by the isolation of the pathogen; followed by, in case of isolation of typical colonies, identification of a pure culture as Ralstonia

combination with a pathogenicity test (section II.4.3). Strain characterisation is optional but recommended for each new case.

The procedure is intended for detection of latent infections in potato tubers by one or, preferably, more screening test(s) which, if positive, are

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

solanacearum.

antiserum) is acceptable and all tests must be carried out at the antiserum titre or one dilution below the titre. The indirect method is preferred. The direct

solanacearum. The result is available 3—6 days after sample preparation. The pathogen is obtained directly in culture and can be readily identified. For full

(5) ELISA test: The ELISA test is generally less sensitive than the IF test (sensitivity threshold of 10 4 —10 5 cells per ml of potato extract pellet). The test is

validated. The test is used for many other statutory bacteria, e.g. Clavibacter michiganensis subsp. sepedonicus. With the reading parameters specified in

(4) Selective plating: With the modified SMSA medium and testing methodology specified in this method, this is a sensitive and selective test for Ralstonia

(3) Immunofluorescence (IF) test: The IF test is a well established screening test. This is an advantage over other tests which are not yet fully developed or

contamination which would result in false positive tests. False positives could also arise from sequence homology of other organisms. For these reasons the

The critical factor for the reliability of the test result is the quality of the antiserum. Only antiserum with a high titre (minimum 2 000 for the crude

specificity. Cross reactions by serologically related bacteria from soil or associated with potato tissues with cell morphology of Ralstonia solanacearum are

(2) Screening test(s): A single test may not be sufficiently sensitive or reliable to detect Ralstonia solanacearum in a sample. Therefore, more than one test

(6) PCR test: PCR has the potential for very sensitive detection although the test is readily inhibited by plant or tuber extract components resulting in false

negatives. Some potato cultivars contain more inhibitors than others. It is therefore necessary to remove these inhibitors. The inhibition can be reduced by

(7) Enrichment test: Incubating potato extract pellet samples in a semi-selective broth, such as modified SMSA broth, allows multiplication of Ralstonia

develop on the medium. Selective plating can be used as a sole screening test provided that in cases where inhibition of Ralstonia solanacearum by other

components of the medium may affect the target organism. Care is also required to differentiate Ralstonia solanacearum from other bacteria which may

cheap and fast but generally more vulnerable to false positive (cross reactions) and false negative results (inhibition by phenolic molecules in the potato

bacteria on the plates is suspected and a negative test result is obtained the sample is retested using a different test to confirm or refute the diagnosis. In

dilution but the populations of Ralstonia solanacearum are also diluted. Great care has to be taken in all steps of sample and test preparation to prevent

The IF test has the advantage of subjective interpretation of cell staining morphology and fluorescence intensity which provide information on reaction

common. The IF test can be used as a sole screening test although in cases where cross-reactions are suspected an additional screening test based on a

exploitation of its potential, the test requires careful preparation of heel ends to avoid secondary bacteria associated with the potato tuber which are

competitors with Ralstonia solanacearum on the medium and may affect the development of the pathogen. Some strains may grow poorly as the

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

extract). The requirements for antiserum specificity are extremely high. The ELISA test cannot be used as a single screening test.

method can be applied if the test has a level of sensitivity and specificity equivalent to that of the indirect method.

different biological principle should be done. In such cases selective plating is the most appropriate test.

this method it is a sensitive test (sensitivity threshold of 10 3 —10 4 cells per ml of potato extract pellet).

is recommended and these tests should preferably be based on different biological principles.

direct PCR cannot be used as a sole screening test.

such cases, the IF test is the most appropriate test.

Flowchart references

thumbs on the skin near the cut surface. Later, the vascular discoloration becomes more distinct brown and the necrosis can extend into the parenchymatous

(9) Confirmation test(s): Reliable identification of a pure culture of Ralstonia solanacearum is achieved by at least one of the tests listed in section II.4.1. in

The potato plant. The early stage of infection is wilting of the leaves towards the top of the plant at high temperatures during the day with recovery at night.

solanacearum. More important perhaps, it also dilutes potential inhibitors of the ELISA or PCR test. Ralstonia solanacearum in enrichment broth can thus

plant collapse. Under less favourable conditions (soil temperature below 21 °C) large numbers of adventitous roots may develop on the stem. It is possible

tissue. In advanced stages, the infection breaks outwards from the heel end and the eyes which may result in reddish-brown, slightly sunken lesions on the

The tomato plant. The first visible symptom is the flaccid appearance of the youngest leaves. Under favourable environmental conditions for the pathogen

be detected by IF, ELISA and PCR. We do not recommend that direct plating is made from the enriched broths. These enrichment methods have not been

thoroughly tried and tested. They are included here because they have good potential. However, because of the relative lack of experience with them, they

(8) Bioassay test: The bioassay test is used for isolation of Ralstonia solanacearum from potato extract pellets by selective enrichment in a host plant and

discoloration of the vascular ring from which a pale cream ooze emerges spontaneously after a few minutes or when gentle pressure is applied with the

(soil temperature about 25 °C saturated humidity), epinasty and wilting of one side or of the whole plant follows within a few days which leads to total

can be done on tomato plants or eggplants. The test requires optimal incubation conditions as specified in this method. Bacteria inhibitory to Ralstonia

Wilting becomes rapidly irreversible and results in the death of the plant. The vascular tissue in transversely cut stems from wilted plants may become

brown and a milky ooze exudes from the cut surface or can be easily expressed by squeezing. When a cut stem is placed vertically in water, threads of

The potato tuber. Potato tubers must be cut transversely close to the heel (= stolon) end. The early stage of infection is a glassy yellow to light brown

to observe a greasy cord along the stem which is evidence of the necrosis in the vascular system. When the stem is cut crosswise discoloured brown

combination with a pathogenicity test (section II.4.3.). Strain characterisation is optional but recommended for each new case.

Diagnosis of brown rot in potato tubers and of bacterial wilt in potato and tomato plants

solanacearum on the SMSA medium will most likely not interfere in this test.

vascular tissues of the stem exude drops of white or yellowish bacterial ooze.

skin from which bacteria may ooze, causing soil particles to adhere.

slime will stream from the vascular bundles.

cannot be used as sole detection methods.

1.2 Symptoms in tomato

1.1 Symptoms in potato

SECTION II

1. Symptoms

Cut the stem just above the soil level. Place the cut surface in a beaker with water. Shortly after, threads of bacterial slime will stream spontaneously out of

(4) Flood the smear with 0,5 % (w/v) aqueous safranin and leave for 10 seconds at ambient temperature. Caution! Safranin is a harmful product. Work in a

Prepare positive control smears of a biovar2/race 3 strain and, if considered useful, a negative control smear of a heterologeous strain. Allow to dry. Pass

Either prepare a smear of the ooze or the suspended tissue on a microscope slide or prepare a smear of a 48 hour culture on YPGA or SPA (Appendix 1).

Observe for bright orange fluorescence of PHB granules. Also observe under normal light to ensure that the granules are intracellular and that cell

The presence of Ralstonia solanacearum in wilting potato or tomato stems can be assessed by the following simple presumptive test

(6) Examine the stained smear with a light microscope using transmitted light under oil immersion at a magnification of 1 000.

(6) Examine the stained smear with an epifluorescence microscope at 450 nm under oil immersion at a magnification of 1 000.

(1) Flood the fixed smear with 0,3 % Sudan black B solution in 70 % ethanol. Incubate for 10 minutes at ambient temperature.

the vascular bundles. Any other bacteria causing vascular infection in potato or tomato plants will not show this phenomenon.

(2) Drain off the staining solution. Wash briefly in tap water. Remove excess water. Remove excess water with tissue paper.

The PHB granules in the cells of Ralstonia solanacearum are visualised by staining with Nile blue A or with Sudan black B.

Other appropriate screening tests are the IF test (section III.2), the ELISA test (section III.3) and the PCR test (section III.4).

(3) Dip the smear briefly in xylol. Blot dry on tissue paper. Caution! Xylol is a harmful product. Work in a fume cabinet.

(2) Drain off the staining solution. Wash briefly in gently running tap water. Remove excess water with tissue paper.

Flood the fixed smear with 1 % aqueous solution of Nile blue A. Incubate for 10 minutes at 55 °C.

(3) Flood the smear with 8 % aqueous acetic acid. Incubate for one minute at ambient temperature.

Rapid screening tests facilitate presumptive diagnosis. Use one or more of the following tests

the lower surface of the slide several times rapidly through the flame until the smear is fixed.

PHB granules in cells of Ralstonia solanacearum stain blue-black. The cell wall stains pink.

(5) Wash in gently running tap water. Blot dry on tissue paper. Apply a coverslip.

(4) Wash in gently running tap water. Blot dry on tissue paper.

(5) Remoisten with a drop of water. Apply a coverslip.

Detection of poly-ß-hydroxybutyrate (PHB) granules

morphology is typical of Ralstonia solanacearum.

2. Rapid screening tests

Stem streaming test

Sudan black test

fume cabinet.

Nile blue test

Other tests

(1)

On SMSA medium, typical colonies of virulent isolates of Ralstonia solanacearum are milky-white, flat, irregular and fluidal with blood red colouration

3.1 Remove ooze or sections of discoloured tissue from the vascular ring in the potato tuber or from the vascular strands in the stem of potato or tomato

technique. If considered useful, prepare separate plates of each medium used with a diluted cell suspension culture of a virulent biovar 2/race 3 strain of

On Kelman's tetrazolium medium, typical colonies of virulent isolates of Ralstonia solanacearum are cream, flat, irregular and fluidal with blood red

Kelman's tetrazolium medium (Appendix 1) and/or on to SMSA selective medium (Appendix 7). Spread or streak with an appropriate dilution plating

3.5 Purify colonies with characteristic morphology by subculture on a general nutrient medium. Avoid regular subculturing which may induce loss of

3.4 Incubate the plates for three days at 28 °C. Incubation may be prolonged to six days if growth is slow, but colonies on SMSA plates often become

3.3 Transfer a standard volume of the suspension and the dilutions on to a general nutrient medium (NA, YPGA and SPA, Appendix 1) and/or on to

On the general nutrient media, virulent isolates of Ralstonia solanacearum develop pearly-white, flat, irregular and fluidal colonies often with

Avirulent forms of Ralstonia solanacearum develop less fluidal colonies which are completely pink to red on the SMSA medium.

plants. Suspend in a small volume of sterile distilled water or 50 mM phosphate buffer. Leave for 5—10 minutes on the bench.

3.2 Prepare a series of decimal dilutions of the suspension, e.g. 1/10 and 1/100 or more as considered appropriate.

coloured whorls in the centre. Avirulent forms of Ralstonia solanacearum develop butyrous, deep-red colonies.

The following phenotypic properties of Ralstonia solanacearum are universally present or absent

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

Note: Include appropriate control strains in each test used.

Ralstonia solanacearum as a positive control.

4.1 Identification of Ralstonia solanacearum

Nutritional and enzymatic tests

4. Confirmation test(s)

3. Isolation procedure

characteristic whorls.

Fluorescent pigment

atypical and die off.

in the centre.

virulence.

-

Oxidation/Fermentation (O/F) test

Arginine dihydrolase

Growth in 1 % NaCl

Growth in 2 % NaCl

Utilisation of citrate

Reduction of nitrate

Gelatin liquefaction

Starch hydrolysis

Kovac's oxidase

Growth at 40 °C

PHB inclusions

Catalase

O +/F -

+

+

+

+

+

+

-

-

-

-

-

Prepare a suspension of 10 6 cells per ml from the culture and the control strain(s). Apply the PCR procedure (section III.4). The PCR product of the culture

Grow the culture and a positive control strain for 48 hours at 28 °C on trypticase soy agar and apply the FAP procedure (Janse, 1991; Stead, 1992a; Stead,

1992b). The profile of the culture must be identical to that of the positive control. Under the specified conditions, characteristic fatty acids are 14:0 3OH,

Prepare a suspension of 10 6 cells per ml from the culture and the control strain(s). Apply the FISH procedure (van Beuningen et al, 1995) with the OLI-1

Prepare a suspension of > 10 6 cells per ml from the culture and the control strain(s). Apply the ELISA procedure (section III.3). The ELISA value of the

Prepare a suspension of 10 6 cells per ml from the culture and the control strain(s). Prepare a series of twofold dilutions of the antiserum. Apply the IF

Strain characterisation is optional but is recommended for each new case using at least one of the following: —

Denatured whole cell proteins are separated by polyacrylamide gel electrophoresis — PAGE (Stead, 1992a).

must have the same size and restrictive enzyme analysis (REA) pattern as that of the positive control.

procedure (section III.2). The IF titre of the culture must be equivalent to that of the positive control.

PCR primer (Seal et al, 1993) The culture must show the same reaction as the positive control.

Media and methods are provided in Lelliott & Stead (1987)

culture must be equivalent to that of the positive control.

Fluorescent in-situ hybridisation (FISH)

16:0 2OH, 16:1 2OH und 18:1 2OH.

Fatty acid profiling (FAP)

Strain characterisation

Biovar determination

Aesulin hydrolysis

Levan production

Protein profiling

ELISA test

PCR test

IF-test

-

-

Ralstonia solanacearum is separated into biovars on the basis of the ability to produce acid from three hexose alcohols and three sugars (Hayward, 1964 &

Biovar

— cellobiose

— maltose

— lactose

Utilisation of

1994)

— mannitol

— sorbitol

— dulcitol

+

+

+

+

+

+

+

+

-

-

-

-

-

-

+

+

+

+

+

+

+

+

-

-

-

-

-

-

-

-

biovar 2-A

biovar 2-T

biovar 2

+

+

+

+

+

-

-

-

-

Additional tests differentiate biovar 2 in subphenotypes (Hayward, 1994)

utilisation of trehalose

utilisation of D-ribose

utilisation of inositol

The race (Buddenhagen et al., 1962) can be determined on the basis of a pathogenicity test in tomato plants or eggplants and in tobacco plants and by a

Race 4 (pathogenic on ginger and a few other hosts) and race 5 (pathogenic on mulberry only) are not included.

hypersensitivity reaction (HR) test in tobacco leaves (Lozano and Sequeira, 1970)

high

low

low

— tomato plants/eggplants

Race determination

pectolytic activity

— tobacco plants

no reaction

no reaction

Reaction in

wilting

wilting

wilting

Race 1

preferably, the third true leaf stage or older (section III, 6.). Incubate for up to two weeks at 22 °C—28 °C and high relative humidity with daily watering.

Race characterisation by the pathogenicity test or tobacco hypersensitivity test may not be highly reliable and instead can be deduced from the biovar and

- Comminute and suspend in a small volume of sterile distilled water or 50 mM phosphate buffer. Then plate, incubate, and

This test is for confirmation of the diagnosis of Ralstonia solanacearum and for the assessment of the virulence of the cultures identified as Ralstonia

solanacearum. Prepare an inoculum of 10 6 cells per ml from the culture and a positive control strain. Inoculate 5—10 tomato plants or eggplants at,

The potato extract pellet obtained in this procedure can also be used for detection of Clavibacter michiganensis subsp. sepedonicus.

Incubate the sample at 25—30 °C for up to two weeks to encourage multiplication of low Ralstoniasolanacearum populations.

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

DETECTION AND IDENTIFICATION OF RALSTONIA SOLANACEARUM IN SAMPLES OF POTATO TUBERS

Remove a section of tissue from the stem two cm above the inoculation point.

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

Molecular differentiation of strains in the Ralstonia solanacearum complex can be done by

check for typical colonies of Ralstonia solanacearum.

Observe for wilting and/or epinasty, chlorosis, stunting.

Preparation of the sample for testing

Isolate from symptomatic plants as follows

The culture can be further characterized by

necrosis (48 hrs) and wilting (7—8 days)

Pre-testing options if considered useful

RFLP analysis (Cook et al, 1989)

-

the natural host of origin.

Genomic fingerprinting

4.3 Pathogenicity test

chlorosis (2—8 days)

— tobacco leaves

HR (12—24 hrs)

SECTION III

no reaction

(i)

1. Note

Carefully cut out a small conical core (3—5 mm diameter) of vascular tissue at the heel end. Keep the amount of non-vascular tissue to a minimum. Process

Decant the macerate gently in a centrifuge tube while leaving the debris in the container or bag. If the decanted macerate is cloudy, centrifuge

Filter the macerate through a filtration system with pore size of 40—100 µm. Enhance filtration using a vacuum pump. Collect the filtrate in a

Transfer the heel ends into an appropriate container. Add a sufficient volume of maceration buffer to cover the heel ends. Place the container

Note: Visual examination of the tubers (Section II.1) can be done at this stage. Set aside any tuber with symptoms or severe rotting and test separately

Comminute the heel ends in a Waring Blender or by Ultra Thurrax until complete homogenisation has just been achieved. Avoid excessive

1.5 Resuspend the pellet in 1 ml pelletbuffer (Appendix 2). Divide in two equal parts and transfer each part to a microvial. Use one microvial for testing.

1.2 Collect the heel ends in a closed container. Preferably, the heel ends should be processed immediately. If this is not possible, store them for not more

Use antiserum for Ralstonia solanacearum, preferably to race 3/biovar 2. Determine the titre on a suspension of 10 6 cells per ml from the homologeous

Transfer the heel ends into a strong disposable maceration bag (e.g. Stomacher bag with dimensions 105 mm × 150 mm, radiation sterile).

centrifugation step, at 7 000 g for 15 minutes or at 10 000 g for 10 minutes at a temperature below 10 °C. Discard the supernatant without

Crush the heel ends carefully with an appropriate tool, e.g. a hammer, until complete homogenisation has been achieved. Add a sufficient

1.1 Remove with a clean and disinfected scalpel or vegetable knife the skin at the heel end of the tuber so that the vascular tissues first become visible.

at not more than 180 g for 10 minutes at a temperature below 10 °C. Centrifuge the decanted macerate, or the supernatant from the first

Transfer the heel ends into an appropriate container. Add a sufficient volume of maceration buffer (Appendix 2) to cover the heel ends.

Conserve the remainder of this extract at 4 °C during testing. Add 10—25 % (v/v) of sterile glycerol to the other microvial. Vortex. Store at - 18 °C

centrifuge tube. Wash the filter with maceration buffer. Centrifuge filtrate at 7 000 g for 15 minutes or at 10 000 g for 10 minutes at a

Wash the tubers under running water with appropriate disinfectants and detergents. Air dry the tubers.

on a rotary shaker. Incubate at 50—100 rpm for 4 hours at 20 °C—22 °C or for 16—24 hours at 4 °C.

volume of maceration buffer to cover the heel ends. Allow the macerate to settle for 15—30 minutes.

temperature below 10 °C. Discard the supernatant without distrubing the pellet.

1.4 Extract the bacteria from the processed heel ends by one of the following procedures

homogenisation. Allow the macerate to soak for 15—30 minutes.

1.3 Process the heel ends by one of the following procedures

than 24 hours or, at 4 °C, for not longer than 72 hours.

disturbing the pellet.

each of the tubers in the sample.

(weeks) or at - 70 °C (months).

2. IF-test

(section II).

(iii)

(ii)

(ii)

(ii)

(i)

(i)

According to test slide preparation in 2.1 (i): Prepare a set of twofold dilutions of the antiserum in IF buffer (Appendix 3): ¼ of the titre (T/4 ),

For pellets with relative little starch: Pipette a measured standard volume (15 µl is appropriate for 6 mm window diameter — scale up volume

standard volume (15 µl is appropriate for 6 mm window diameter — scale up volume for larger windows) of the resuspended pellet and each

Prepare separate positive control slides with a suspension of 10 6 cells per ml from a strain of the appropriate race/biovar of Ralstonia solanacearum. Use

According to test slide preparation in 2.1 (ii): Prepare the working dilution (WD) of the antiserum in IF buffer. The working dilution is the

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

for larger windows) of the resuspended pellet on a row of windows. The remaining row can be used as duplicate or for a second sample as

For other pellets: Prepare decimal dilutions, viz. 1/10, 1/100 and 1/1 000 of the resuspended pellet in pellet buffer. Pipette a measured

strain of Ralstonia solanacearum with an appropriate dilution of the fluorescein isothiocyanate (FITC) conjugate, according to the manufacturer's

dilution on a row of windows. The remaining row can be used as duplicate or for a second sample as presented in Figure 2.

2.2 Let the droplets dry. Fix the bacterial cells to the slide either by heating, flaming or with 95 % ethanol.

dilution of the antiserum with optimum specificity and is usually half of the titre.

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

recommendations. The crude antiserum should have an IF titre of at least 1:2 000.

½ of the titre (T/2 ), the titre (T) and twice the titre (2T).

2.1 Prepare the test slides by one of the following procedures

Preparation of the test slide according to 2.1 (i) and 2.3 (i)

One standard dilution of resuspended pellet

presented in Figure 1.

one slide in each set of tests.

2.3 IF procedure

Figure 1

(ii)

(ii)

control.

(i)

(i)

2.3.3 Shake the droplets of antiserum off the slide and rinse the slides carefully with IF buffer. Wash for five minutes in IF buffer-Tween and subsequently

2.3.1 Arrange the slides on damp tissue paper. Cover the test windows with the antiserum dilution(s). Apply PBS on the FITC windows. The volume of

2.3.4 Arrange the slides on damp tissue paper. Cover the test windows and the FITC window with the dilution of FITC conjugate used to determine the

magnification of 500—1 000. Scan windows across two diameters at right angles and around the perimeter. Check the positive control slide first. Cells

2.4 Reading the IF test : Examine test slides on an epifluorescence microscope with filters suitable for excitation of FITC, under oil immersion at a

2.3.7 Pipette 5—10 µl 0,1 M phosphate-buffered glycerol (Appendix 3) or a similar mountant on each window and apply a coverslip.

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

The test must be repeated if the staining is aberrant.

titre. The volume of conjugate applied on the windows must be identical to the volume of antiserum applied.

antiserum applied on the windows must be equivalent to the volume of extract applied.

for five minutes in IF buffer (Appendix 3). Carefully remove excess moisture.

2.3.2 Incubate under a cover for 30 minutes at ambient temperature.

2.3.5 Incubate under a cover for 30 minutes at ambient temperature.

Preparation of the test slide according to 2.1 (ii) and 2.3 (ii)

must be bright fluorescent and completely stained. Note

Figure 2

(iii) If large numbers (> 10 5 cells per ml) of incomplete or weakly fluorescing cells are seen at the titre of the antiserum, a second test should be carried out

3.5 Prepare the appropriate dilution of Ralstonia solanacearum antiserum in blocking buffer (Appendix 5). Apply 100 µl of antiserum dilution to the wells.

buffered saline (PBS) control. Use a suspension of > 10 6 cells per ml from a strain of the appropriate race/biovar of Ralstonia solanacearum as the positive

strain of Ralstonia solanacearum. The use of NUNC-Polysorb microtitre plates is recommended. Include a negative potato extract control and a phosphate

Read the test slides. Observe first for absence of fluorescing cells in the FITC control windows. Fluorescing cells in the FITC control indicate non-specific

3.4 Flick out the extracts from the wells. Wash the wells three times with PBS-Tween (Appendix 5), leaving the last washing solution in the wells for at

Use antiserum for Ralstonia solanacearum, preferably to race 3/biovar 2. Determine the titre on a suspension of 10 6 cells per ml from the homologeous

Observe for bright fluorescing cells with characteristic morphology of Ralstonia solanacearum in the test windows. The fluorescence intensity must be

equivalent to the positive control strain at the same antiserum dilution. Cells with incomplete staining or with weak fluorescence must be disregarded,

If bright fluorescing cells with characteristic morphology are found, then determine the mean number of cells per microscope field and

A population of approximately 10 3 cells per ml of resuspended pellet is considered to be the limit of detection for the IF test,

3.3 Apply 100 µl aliquots to each of at least two wells of the microtitre plate. Incubate for one hour at 37 °C or overnight at 4 °C.

- for samples with N > 10 3 cells per ml of resuspended pellet, the IF test may be considered positive.

3.1 Pipette 100—200 µl of the resuspended pellet in a microvial. Heat for 4 minutes at 100 °C. Remove the microvial on ice.

for samples with N > 10 3 cells per ml of resuspended pellet, the IF test is considered positive,

If bright fluorescing cells with characteristic morphology are not found, then the IF test is negative.

control. Test in an identical manner as the sample(s) but well-separated from the samples on the microtitre plate.

- a repeat IF test, with either a second antiserum or a ten-fold dilution of the pellet.

Repeat the test if such is observed.

3.2 Add an equal volume of double strength carbonate coating buffer (Appendix 5). Vortex.

calculate the number of cells (N) per ml of resuspended pellet (Appendix 4).

either a test based on a different biological principle, or

unless there are many such cells (see interpretation of the IF test result).

binding of the conjugate, autofluorescence or contamination: Note

Based upon Robinson-Smith et al., 1995

Interpretation of the IF test result

Incubate for one hour at 37 °C.

-

-

3. ELISA Test

least five minutes.

(ii)

(i)

3.9 Prepare the alkaline phosphatase substrate solution (Appendix 5). Apply 100 µl to the wells. Incubate for 30 minutes to one hour in the dark at ambient

4.1 Pipette 100 µl of the resuspended pellet into a microvial. Alternatively, transfer 90 µl of the resuspended pellet to a microvial containing 10 µl of 0,5M

3.7 Prepare the appropriate dilution of alkaline phosphatase conjugate in blocking buffer. Apply 100 µl of conjugate dilution to the wells. Incubate for one

Prepare a suspension of 10 6 cells per ml from a race 3/biovar 2 strain of Ralstonia solanacearum as the positive control. Test in an identical manner as the

4.3 Prepare at least two decimal dilutions, e.g. 1/10 and 1/100 or more if considered useful, in sterile distilled or ultra pure water (UPW).

Filterplugged pipette tips must be used during all stages of sample preparation and other manipulations involving PCR.

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

The ELISA test is negative if the optical density (OD) of the sample is < 2 × OD of the negative control.

The ELISA test is positive if the optical density (OD) of the sample is > 2 × OD of the negative control.

3.8 Flick out the conjugate from the wells. Wash the wells as before (3.4 and 3.6).

4.2 Heat for four minutes at 100 °C. Remove the microvial immediately on ice.

3.6 Flick out the antiserum from the wells. Wash the wells as before (3.4).

NaOH. Mix by repeatedly inverting the microvial.

3.10 Read absorbance at 409 nm.

Interpretation of the ELISA test

For a 50 µl reaction volume

Based on Seal et al., 1993

Final concentration

4. PCR test

hour at 37 °C.

temperature.

Component

sample(s).

Quantity

Note

Taq polymerase (5 U/µl)

Sterile distilled or UPW

Primer OLI-1 (20 µM)

Primer Y-2 (20 µM)

10 × PCR Buffer

30,8 µl—33,8 µl

0,2 mM

0,2 mM

0,2 mM

0,2 mM

d-ATP

d-CTP

d-GTP

d-TTP

5,0 µl

1,0 µl

1,0 µl

1,0 µl

1,0 µl

2,5 µl

2,5 µl

0,2 µl

1,0 U

1 µm

1 µm

1 ×

45 µl—48 µl

4.5.1 Optional: Pulse centrifuge the vials with the boiled samp le and positive control. Add, in the specified order, 2—5 µl of the sample(s), water control

For more reactions: Calculate the quantity of each component for the required number of reactions. Mix the components and transfer 45 µl—48 µl of the

mix into sterile PCR vials. Keep the vials with the PCR reaction mix on ice. For 25 µl reaction volumes: Scale down components accordingly.

and positive control to the vials with the PCR reaction mix. Place the vials in the heating block of the DNA thermal cycler.

4.5.2 Run the following programme

denaturation of template

4.5 PCR amplification

20 seconds at 94 °C

2 minutes at 96 °C

Total volume

denaturation

50 cycles of

1 cycle of

(ii)

(ii)

(i)

Note: These parameters are for a Perkin Elmer 9600. Other thermal cycles may require mineral oil overlay in the PCR reaction vials and/or modification

of the duration of step (ii), (iii) and (iv) in the amplification profile.

hold at 4 °C

annealing of primers

20 seconds at 94 °C

30 seconds at 72 °C

10 minutes at 72 °C

extension of copy

further extension

1 cycle of

1 cycle of

(iv)

(vi)

(v)

4.6.4 Place 3 µl droplets of loading buffer on parafilm. Add 12 µl of the PCR product from either the samples, the positive control or the water control and

4.6.6 Connect the wires on the power supply and the electrophoresis equipment. Run the gel at 5—8 V/cm until the front of the tracking indicator is within

4.6.7 Switch off the power supply. Disconnect the wires from the electrophoresis unit. Carefully remove the gel. Soak it in ethidium bromide solution for

4.6.9 Visualise the amplified DNA fragment(s) by UV transillumination. The PCR product of Ralstonia solanacearum with primer set OLI -1 and Y-2 is

4.5.3 Remove the vials from the thermal cycler. Analyse the PCR product. If not done immediately, store the vials at 4 °C for use in the same day or at

mix by gentle aspiration in the pipette tip before loading. The given volumes may be modified to fit with the capacity of the wells in the agarose gel.

4.6.2 Cool the molten agarose to 50—60 °C, pour into the mould of the electrophoresis unit and insert the comb. Let the solution solidify.

4.6 Analysis of the PCR product. The PCR fragments are detected by agarose gelelectrophoresis and staining with ethidium bromide.

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

4.6.5 Carefully load the wells of the gel. Include for reference an appropriate DNA marker in at least one well.

Note:Wear disposable gloves at all times when handling ethidium bromide which is a powerful mutagen!

4.6.3 Remove the comb. Submerge the gel in TAE so that it is just covered (2—3 mm) with the buffer.

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

288 bp in length. Check against the DNA marker and against the positive control.

Note:The water control must be negative in each case. If positive, repeat the test.

4.6.10 Photograph the gel if a permanent record is required.

4.6.8 Destain in distilled water for 10—15 minutes.

4.7 Restriction Enzyme Analysis (REA)

1 cm of the end of the gel.

- 18 °C for longer.

30—45 minutes.

measured standard volume (50—100 µl) of the resuspended pellet and each dilution onto modified SMSA selective medium (Appendix 7) and

Transfer a measured standard volume (50—100 µl) of the resuspended pellet onto modified SMSA selective medium and spread with a glass

5.2 Apply, by the same dilution plating technique, a suspension of 10 6 cells per ml from a virulent race 3/biovar 2 strain of Ralstonia solanacearum as the

isolated, provided that no inhibition is suspected by colonies of other bacteria and that colonies characteristic of Ralstonia solanacearum are found in the

4.7.2 Mix by gentle aspiration in the pipette tip. If drops remain on the walls of the vial, pulse spin in a microcentrifuge. Incubate for one hour at 37 °C.

Prepare at least two decimal dilutions, viz. 1/10 and 1/100 or more if considered useful, of the resuspended pellet in pelletbuffer. Pipette a

spread with a glass rod over the whole surface of the medium. If considered useful, also perform a d ilution streak of a 10 µl loopful of the

Ralstonia solanacearum are milky -white, flat, irregular and fluidal with blood red coloration in the centre and showing internal streaking or whorling.

The PCR test is positive if the 288 bp fragment is detected and REA -analysis of the amplified fragment is identical with the positive control strain of

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

The selective plating test is negative if no bacterial colonies are isolated after six days or if no colonies characteristic of Ralstonia solanacearum are

5.3 Incubate the plates at 28 °C. Start reading the plates after three days. If negative, incubate further up to six days. Colonies of virulent isolates of

4.7.1 Transfer 8,5 µl from the PCR product (4.5.3) to a new microvial. Add 1 µl of 10 × enzyme buffer and 0,5 µl of restriction enzyme Ava II.

rod over the whole surface of the medium. Streak the rod without flaming on at least two other modified SMSA plates.

5.5 Identify pure cultures (section II, 4.1) and confirm Ralstonia solanacearum cultures by a pathogenicity test (section II, 4.3).

5.4 Purify colonies with characteristic morphology by subculturing onto a general nutrient medium (Appendix 1).

4.7.3 Analyse the digested PCR fragment by agarose gelelectrophoresis as before (4.6).

5.1 Perform the test by an appropriate dilution plating technique, e.g.

resuspended pellet. Flame the loop between streaks.

positive control on a set of separate modified SMSA plates.

Interpretation of the selective plating test result

Interpretation of the PCR test result

Based upon Elphinstone et al., 1996

5. Selective plating test

Ralstonia solanacearum.

solanacearum.

(ii)

(i)

section of stem from two cm above the inoculation point. Homogenise the tissues in maceration buffer. Perform dilution plating (section III.5.1). If positive,

6.1 Use 10 test plants of susceptible tomato or eggplant seedlings at the third true leaf stage for each sample. Do not water the testplants for 24 hours before

6.3 Inoculate, by the same technique, 10 seedlings with a suspension of 10 6 cells per ml from a virulent biovar 2/race 3 strain of Ralstonia solanacearum as

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

6.6 If considered useful, check absence of infection in batches of test plants not showing any indication of infection. Remove from each test plant a one cm

identify pure cultures with characteristic morphology (section II.4.1) and confirm Ralstonia solanacearum cultures by a pathogenicity test (section II.4.3).

the positive control and with pelletbuffer as the negative control. Separate the positive control plants from the other plants to avoid cross-contamination.

6.5 Isolate from infected plants (section II). Identify pure cultures with characteristic morphology (section II.4.1) and confirm Ralstonia solanacearum

6.4 Grow the test plants further for up to four weeks at 22 °C—28 °C and high relative humidity with daily watering. Observe for wilting, epinasty,

6.2 Distribute 100 µl of resuspended pellet between the testplants. Inoculate in the stem between the cotyledons and at one or more other places.

7.2 Incubate for 48 hours, and in any case not longer than 72 hours, at 28 °C with the cap of the tube loosely fitted for aeration.

The selective plating test is positive if colonies characteristic of Ralstonia solanacearum are isolated.

7.1 Transfer 100 µl of the resuspended pellet in three ml of modified SMSA broth (Appendix 7).

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

cultures by a pathogenicity test (section II.4.3).

Interpretation of the bioassay test result

Based upon Elphinstone et al., 1996

7. Enrichment tests

chlorosis and/or stunting.

Based upon Janse, 1988.

6. Bioassay test

positive controls.

inoculation.

controls.

7.3 Tighten cap and vortex. Aliquot for the IF test (this section, 2.), the ELISA test (this section, 3.) and/or the PCR test (this section, 4.).

Nutrient media for isolation and culture of Ralstonia solanacearum

Bacto Peptone (Difco)

Nutrient Agar (Difco)

Yeast Extract (Difco)

Appendix 1

Distilled water

Prepare ½ litre volumes of medium in one litre flasks.

Sterilise by autoclaving at 121 °C for 15 minutes.

Yeast-Peptone-Glucose-Agar (YPGA)

8. Pathogenicity test

Cool to 50 °C. Pour plates.

Dissolve the ingredients.

Refer to Section II.4.3.

Nutrient Agar (NA)

1 litre

23 g

5 g

5 g

D(+) Glucose (mo nohydrate)

Bacto Agar (Difco)

Bacto-Agar (Difco)

Distilled water

Distilled water

MgSO 4 ·7H 2 O

Sucrose

K 2 HPO 4

Peptone

Dissolve the ingredients. Adjust to pH 7,2—7,4 if necessary.

Prepare ½ litre volumes of medium in one litre flasks.

Prepare ½ litre volumes of medium in one litre flasks.

Sterilise by autoclaving at 121 °C for 15 minutes.

Sterilise by autoclaving at 121 °C for 15 minutes.

Sucrose Peptone Agar (SPA)

Cool to 50 °C. Pour plates.

Cool to 50 °C. Pour plates.

Dissolve the ingredients.

0,25 g

1 litre

1 litre

0,5 g

10 g

15 g

20 g

15 g

5 g

Add a filter-sterilised aqueous solution of triphenyl tetrazolium chloride (Sigma) to obtain a final concentration of 50 mg per litre.

Materials for sample preparation

Casamino acids (Difco)

Bacto-Peptone (Difco)

Bacto-Agar (Difco)

Distilled water

Appendix 2

Na 2 HPO 4

Dextrose

KH 2 PO 4

Dissolve the ingredients. Sterilise by autoclaving at 121 °C for 15 minutes.

Prepare ½ litre volumes of medium in one litre flasks.

Maceration Buffer: 50 mM phophate buffer, pH 7,0

This buffer is used for tissue maceration.

Kelman's tetrazolium medium

Cool to 50 °C.

Pour plates.

4,26 g

2,72 g

1 litre

10 g

15 g

1 g

5 g

The addition of a deflocculant, an antifoam agent or an anti-oxidant is recommended using the Waring Blender or Ultra Turrax homogenisation procedure

The addition of 5 % polyvinylpyrrolidone-40000 MWT (PVP-40) is recommended when performing the direct PCR test to reduce the incidence of

Tetrasodiumpyrophosphate

DC Silicone antifoam

Na 2 HPO 4 ·12 H 2 O

Na H 2 PO 4 ·2 H 2 O

Distilled water

Distilled water

Lubrol flakes

This buffer is used for resuspension and dilution of potato heel and pellets.

Dissolve the ingredients and check pH. Aliquot as considered appropriate.

Dissolve the ingredients and check pH. Aliquot as considered appropriate.

amplification inhibition by aromatic molecules in the extract.

Autoclave separately. Add to the desired concentration.

Sterilise by autoclaving at 121 °C for 15 minutes.

Sterilise by autoclaving at 121 °C for 15 minutes.

Pelletbuffer: 10 mM phosphate buffer, pH 7,2

for maceration of the potato tissue cores.

1,0 ml per litre

0,5 g per litre

1,0 g per litre

1 litre

1 litre

2,7 g

0,4 g

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

Materials for the IF test

Na 2 HPO 4 ·12 H 2 O

Na 2 HPO 4 ·12 H 2 O

NaH 2 PO 4 ·2 H 2 O

NaH 2 PO 4 ·2 H 2 O

Distilled water

Appendix 3

Glycerol

NaCl

This buffer is used for washing the slides. Add 0,1 % Tween 20 to the IF buffer.

Dissolve the ingredients and check pH. Aliquot as considered appropriate.

IF-buffer: 10 mM phosphate buffered saline (PBS) pH 7,2

Sterilise by autoclaving at 121 °C for 15 minutes.

0,1 M Phosphate buffered glycerol p H 7,6

This buffer is used for dilution of antisera.

IF-buffer-Tween

0,15 g

1 litre

2,7 g

0,4 g

8,0 g

3,2 g

Determination of contamination level in the IF test

field coefficient (depends upon ocular type and varies from 8 to 24),

Calculate d either by direct measurement or from the following formulae: s = (p i 2 )/(G 2 K 2 × 4)

Materials for the ELISA test

Calculate the number of typical fluorescent cells per ml pellet (N) N = C × 1 000/y × F

Distilled water

Appendix 4

Appendix 5

Calculate the number of typical fluorescent cells per window (C). C = c S/s

magnification of 100x, 40x etc. objective.

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

Surface area (S) of window of multispot slide = (p D 2 )/4

volume of pellet on window,

tube coefficient (1 or 1,25),

from (3) d = v((4 × (p i 2 )/(G 2 K 2 × 4))/p) = i/(GK)

pellet dilution factor.

Surface area (s) of objective field = (p d 2 )/4

Where D = diameter of window

Where d = diameter of field

from (2) d = v(4s)/p

=

=

=

=

=

where

where

K

G

y

i

F

100 ml

50 ml

(1)

(2)

(3)

(4)

Sodium sulphite at a final concentration of 0,2 % may be added as anti-oxidant if the extract contains a high fraction of aromatic molecules.

Na 2 HPO 4 × 12H 2 O

Distilled water

Distilled water

NaHCO 3

KH 2 PO 4

Na 2 CO 3

NaCl

KCl

Dissolve the ingredients and check pH. Aliquot as considered appropriate.

Dissolve the ingredients and check pH. Aliquot as considered appropriate.

Double strength Carbonate Coating Buffer, pH 9,6

Sterilise by autoclaving at 121 °C for 15 minutes.

Sterilise by autoclaving at 121 °C for 15 minutes.

10 × Phosphate Buffered Saline (PBS), pH 7,4

11,72 g

6,36 g

1 litre

1 litre

80 g

29 g

2 g

2 g

Polyvinylpyrrolidone-44000 MWT (PVP-44)

Diethanolamine

10 % Tween 20

10 % Tween 20

Distilled water

Distilled water

Milk powder

10 × PBS

10 × PBS

Blocking (antibody) Buffer (must be freshly prepared)

Alkaline phosphatase substrate solution pH 9,8

Phosphate Buffered Saline-Tween (PBS-T)

to 100 ml

100 ml

895 ml

10 ml

97 ml

0,5 g

0,5 g

5 ml

2 g

SMSA Selective medium (Engelbrecht, 1994 as modified by Elphinstone et al, 1996) but delete the agar and the tetrazolium salts.

Materials for selective plating and enrichment tests

Casamino acids (Difco)

Bacto peptone (Difco)

Distilled water

Appendix 7

Glycerol

Dissolve two phosphatase substrate five mg tablets (Sigma) per 15 ml of solution.

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

5'CCCACTGCTGCCTCCCGTAGGAGT-3'

Make up to one litre with distilled water.

For materials see Seal et al (1993).

Add 0,2 g MgCl 2 .

Basal medium

Primer Y-2

800 ml

10 g

1 g

Materials for

Oligonucleotid

CTTGCTACC

the PCR test

GGGGGTAG

Appendix 6

Primer OLI-1

sequence

TGCC-3'

e primer

5'-

If considered necessary, increasing bacitracin concentration to 300 ppm may reduce contaminating saprophytic bacteria without reducing the recovery of Ralstonia

Dissolve the ingredients and check pH. Adjust pH, if necessary, to 6,5 before autoclaving. Ralstonia solanacearum will not grow well on the medium at pH

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

Distilled water

Agar (Difco)

Sterilise by autoclaving at 121 °C for 15 minutes. Cool to 50 °C.

Prepare ½ litre volumes of medium in one litre flasks.

(approximately 600 000 units)

Polymixin B sulphate

100 mg per litre

Sigma P-1004

Crystal violet

5 mg per litre

Bacitracin 2

1 litre

> 7,0.

5 ml

15 g

solanacearum.

Dissolve the ingredients in 70 % ethanol to the given concentrations for the volume of medium prepared. Some ingredients, viz. polymixin B and

Prepare as for the SMSA selective medium but delete the agar.

chloramphenicol require slight warming and shaking.

SMSA broth (Elphinstone et al., 1996)

(approximately 1 250 units)

(approximately 825 units)

Chloramphenicol

Tetrazolium salts

0,5 mg per litre

25 mg per litre

50 mg per litre

Sigma B-0125

Sigma C-3175

Sigma P-3032

5 mg per litre

Penicillin -G

Dispense in three ml aliquots into 30 ml disposable Universal tubes.

relevant method set out in Schedule II or, in all other cases, any other officially approved method, and confirmation or refutation by completion of the said

1. For each suspected occurrence for which a positive result in the screening test(s) has been identified according to, for the listed plant material, the

wherever possible, the lot, or part thereof (from which the sample has been taken) in its original packaging with label,

a sample of the infected tomato or eggplant material inoculated with the tuber or plant extract, where appropriate, and

- any remaining extract and additional prepared material for the screening test(s) e.g. immunofluorescence slides, and,

2. In the case of confirmation of the organism, there should be retention and appropriate conservation of

until at least one month after the notification procedure under regulation 7(2) of these regulations.

The scope of these regulations is to lay down the measures to Scope.

be taken against Ralstonia solanacearum (Smith) Yabuuchi et al.,

previously known as Pseudomonas solanacearum (Smith) Smith,

(hereinafter referred to as ‘the organism’), in order to, with respect to

the host plants of the organism listed in Schedule I Section I, to these

regulations (hereinafter referred to as ‘the listed plant material’):(a)

locate it and determine its distribution;(b) prevent its occurrence and

spread; and(c) if found, to prevent its spread and to control it with the

aim of eradication.

- wherever possible, the remaining part of the samples,

method is awaited, there should be retention and appropriate conservation of

the material specified in paragraph 1, and

the isolated culture of the organism,

- all relevant documentation,

until the completion of the said method.

-

-

-

-

- using surface water for irrigation or spraying from a source used in common with places of production confirmed or suspected

surface water used for irrigation or spraying of, or which has flooded field(s) or place(s) of production confirmed to be infested with the organism.

- growing or having grown, potatoes which are clonally related to potatoes that have been grown on places of production found

- growing potatoes or tomatoes and located in the neighbourhood of infested places of production, including such places of

- growing or having grown tomatoes which are fro m the same source as tomatoes found to be infected with the organism,

- growing or having grown, potatoes or tomatoes which have been placed under official control because of the suspected

- using surface water for irrigation or spraying from any source confirmed or suspected to be infested with the organism,

growing or having grown, potatoes which are clonally related to potatoes found to be infected with the organism,

- flooded or have been flooded with surface water confirmed or suspected to be infested with the organism; and,

production sharing production equipment and facilities directly or through a common contractor,

The elements in the investigation referred to in sub-regulation 7(1)(a)(i) of these regulations shall include where relevant

The Plant Health Department shall conduct annual systematic Official surveys.

official surveys for the organism on the listed plant material originating

in Malta. In order to identify other possible sources of contamination

threatening the production of the listed plant material, the Plant Health

Department shall carry out a risk assessment and, unless no risk of spread

of the organism has been identified during that assessment, it shall, in

production areas of the listed plant material, conduct targeted official

surveys for the organism on plants other than the listed plant material,

including wild solanaceous host plants, as well as on surface water which

is used for irrigation or spraying of listed plant material and on liquid

waste discharged from industrial processing or packaging premises

handling listed plant material and used for irrigation or spraying of listed

plant material. The extent of these targeted surveys shall be determined

according to the risk identified. The Plant Health Department may also

conduct official surveys for the organism on the material, such as

growing medium, soil and solid waste from industrial processing or

packaging premises.

to be infested with the organism,

to be infested with the organism,

occurrence of the organism,

places of production,

-

(ii)

(i)

1. The elements in the determination of the extent of probable contamination under sub-regulations 7(1)(a)(iii) and 7(1)(c)(iii) of these regulations, shall

- any machinery, vehicle, vessel, store, or units thereof, and any other objects including packaging material, that may have come

- the listed plant material produced in the place(s) of production referred to in the previous indent, or present in such place(s) of

tubers or plants with a sister or parental clonal relationship to, and in the case of tomato, those plants with the same source as,

- any of the listed plant material stored in, or in contact with, any of the structures or objects listed in the previous indent, prior

risk of surface water run-off from, or flooding of, place(s) of production designated to be contaminated under sub-regulation

- places of production using surface water for irrigation or spraying of listed plant material in cases where there is or has been

the listed plant material grown at a place of production designated as contaminated under sub-regulation 7(1)(a)(ii) of these

into contact with the listed plant material designated as contaminated under sub-regulation 7(1)(a)(ii) of these regulations,

production during the period when the listed plant material designated as contaminated under sub-regulation 7(1)(a)(ii) of

- place(s) of production with a production link to the listed plant material designated as contaminated under sub-regulation

7(1)(a)(ii) to these regulations, including those sharing production equipment and facilities directly or through a common

- as a result of the investigation and testing under sub-regulation 7(1)(a)(i) of these regulations, in the case of potato, those

the listed plant material designated to be contaminated under sub-regulation (1)(a)(ii) of these regulations and for which,

although they may have tested negative for the organism, it appears that contamination is probable through a clonal link,

- place(s) of production of the listed plant material using water for irrigation or spraying which has been designated as

- listed plant material produced on fields flooded with surface water confirmed to be infested with the organism.

in cases where surface water has been designated as contaminated under sub-reglation 7(1)(c)(ii) of these regulations

2. The determination of the possible spread under sub-regulations 7(1)(a)(iv) and 7(1)(c)(iii) of these regulations shall include

these regulations, was present on the places of production referred to in the first indent,

- place(s) of production of the listed plant material referred to in the previous indent,

- stores handling the listed plant material from the above places of production,

the proximity of other places of production growing the listed plant material,

contaminated under sub-regulation 7(1)(c)(ii) of these regulations,

in cases under sub-regulation 7(1)(a)(iv), a consideration of the following elements,

to the cleansing and disinfection of such structures and objects,

The official surveys provided for in regulation 3 of these

regulations shall be carried out

(a) for the listed plant material, according to the details set

down in point 1 of Section II, Schedule I to these regulations; and,

(b) for host plants other than the listed plant material, and

for water including liquid waste, in accordance with appropriate

methods and, where appropriate, samples shall be taken and

subjected to official or officially supervised laboratory testing;

(c) where appropriate for other material, in accordance with

appropriate methods.For these surveys, further details of the

inspection procedures and the number, origin, stratification and

timing of collection of samples shall be decided by the Plant Health

Department based on sound scientific and statistical principles and

the biology of the organism and taking into account the particular

production systems of the listed plant material and, as appropriate,

of other host plants of the organism.

- the common production and use of seed potato stocks,

(1)(a)(ii) of these regulations;

regulations,

contractor,

-

-

include where relevant

(ii)

(i)

date of reporting of suspected occurrence under regulation 6 to these regulations and dates of sampling and confirmation of the

place(s) of production producing listed plant material adjacent to, or at risk from flooding by, the surface water designated as

Organisms) Regulations 2004 (L.N. 97 of 2004), the passport number or registration number of potato producers, collective

for any potato consignment or lot designated as contaminated, the certificates prescribed in The Plant Quarantine (Harmful

- for any tomato plant consignment or lot designated as contaminated the crtificates prescribed in The Plant Quarantine

- the variety name and category for seed potato stocks, and where possible in all other cases,

- such other information relating to the confirmed outbreak as the Commission may require.

any discrete irrigation basin associated with the surface water designated as contaminated.

- a description of the elements of the designated contamination and zone demarcation.

(Harmful Organisms) Regulations 2004 (L.N. 97 of 2004), and passport number

presence of the organism under regulation 7 to these regulations, as appropriate,

3. The details of the notification referred to in sub-regulation 7(2) to these regulations shall include

4. The details of the additional notification referred to sub-regulation 7(2) shall include

warehouses and dispatching centres, as appropriate,

contaminated,

-

-

-

-

- other measures, provided that it has been established that there is no identifiable risk of the organism spreading; such measures

Department, with appropriate communication between responsible official bodies to ensure such control at all times and approval by the responsible official

subject to approval by the said responsible official bodies. Such measures to be immediately notified to the Commission and

use as ware potatoes intended for industrial processing, and intended for direct and immediate delivery to a processing plant

use as ware potatoes intended for consumption and packed at sites with appropriate waste disposal facilities, ready for direct

appropriate, disinfection, such that there is no identifiable risk of the organism spreading and shall be employed under the supervision of the Plant Health

4. The series of measures to be implemented within the demarcated zone(s) established under sub-regulations 7 (1)(a)(iv) and (c)(iii) of these regulations

2. The appropriate use or disposal of the listed plant material referred to in sub-regulation 8 (2) of these regulations, under the control of the Plant Health

some other use or disposal, provided that it is established that there is no identifiable risk of the organism spreading; such

body of the Member State where the potatoes are to be packed or processed in respect of the waste disposal facilities referred to in the first and second

deep burial, at a disposal site at which there is no risk of seepage to agricultural land or contact with water sources which

industrial processing through direct and immediate delivery to a processing plant with officially approved waste disposal

some other use or disposal, provided that it is established that there is no identifiable risk of the organism spreading, and

3. The appropriate methods for decontamination of the objects referred to in sub-regulation 8 (3) of these regulations shall be cleansing and, where

use as animal feed after heat treatment such that there is no risk of the organism surviving, or

facilities which conform to the provisions laid down in Schedule VII to these regulations, or

delivery and use without repacking, and intended for such direct delivery and use, or

4.1. In cases where places of production have been designated as contaminated under sub-regulation 7 (1)(a)(ii)

to be immediately notified to the Commission and to the other Member States.

measures shall be notified to the Commission and to other Member States.

The details and results of the official surveys provided for in

regulation 3 of these regulations shall be notified each year to the other

Member States and to the Commission in accordance with the provisions

of point 2 of Section II, Schedule I to these regulations. These

notifications shall be submitted by the 1st June except for potatoes use

as farm-saved seed for which the notification shall be submitted by the

1st September. The details and results for crops shall be related to the

previous year’s production.

1. With refrence to sub-regulation 8(1) of these regulations the provisions shall be

could be used for irrigation of agricultral land, or

with appropriate waste disposal facilities, or

and referred to in sub-regulation 8 (4) of these regulations shall include

for other plant parts including stem and foliage debris,

to the other Member States.

incineration, or

destruction, or

for potato tubers,

-

-

-

-

-

-

-

-

-

indents, shall be

Department.

(ii)

(i)

either no potato tubers or plants, or other host plants of the organism shall be planted, and measures shall be taken to eliminate

the field has been found free from volunteer potato and tomato plants and other host plants including solanaceous weeds for at

measures shall be taken to eliminate volunteer potato and tomato plants as well as other host plants of the organism including

measures shall be taken to eliminate volunteer potato and tomato plants as well as other host plants of the organism including

rotation cycle, in the case of potatoes officially certified seed potatoes shall be planted for either seed or ware production and

followed by planting in the succeeding two years with non-host plants of the organism for which there is no identified risk of

in the first potato or tomato cropping season following the period specified in the preceding indent, and on the condition that

in the case of potatoes and tomatoes an official survey as detailed in regulation 3 of these regulations, shall be conducted; or

in the case of potato tubers, officially certified seed potatoes may be planted for ware production only, on the condition that

the field shall be established and maintained during the first three years either, in bare fallow or, in cereals according to the

the responsible official bodies are satisfied that the risks of volunteer potato and tomato plants and other host plants of the

risk identified, or, in permanent pasture with frequent close cutting or intensive grazing or, as grass for seed production,

in the potato or tomato cropping season succeeding that referred to in the previous indent and following an appropriate

in the case of potatoes, officially certified seed potatoes shall be planted for either seed or ware production,

volunteer potato and tomato plants and other host plants including solanaceous weeds as appropriate, or

an official survey including testing, as detailed in regulation 3 of these regulations, shall be conducted,

in the case of potatoes, officially certified seed potatoes shall be planted for ware production only and,

in the first potato or tomato cropping season following the period specified in the preceding indent,

in a field or unit of protected crop production designated to be contaminated under sub-regulation 7 (1)(a)(ii), either

plants of species of Brassica, for which there is an identified risk of the organism surviving,

and an official survey including testing, as detailed in regulation 3 of these regulations, shall be conducted;

crops for which there is an identified risk of the organism spreading;

in the growing year following the designated contamination

during at least the four growing years following the designated contamination,

least the two consecutive growing years prior to planting,

during the five growing years following that of the designated contamination,

taking into account the biology of the organis m,

the organism surviving or spreading,

the following shall not be planted

tomato plants and seeds,

solanaceous weeds, and

solanaceous weeds, and

potato tubers or plants,

other host plants,

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in other fields

(b)

(ii)

(a)

(i)

in a unit of protected crop production designated as contaminated under sub-regulation 7(1)(a)(ii) of these regulations where complete replacement

official controls on irrigation and spraying programmes, including a ban thereof, shall be introduced as appropriate, in order to

official controls on irrigation and spraying programmes, including a ban thereof, shall be introduced as appropriate in order to

said unit has been subjected to officially supervised measures to eliminate the organism and to remove all host plant material,

including, at least, a complete change in growing medium and cleansing and, where appropriate, disinfection of the said unit

all machinery and storage facilities on the place of production and involved in potato or tomato production shall be cleansed

organism including solanaceous weeds, have been eliminated. The growing crop shall be inspected at appropriate times and

no potato tubers or plants, or other host plants of the organism including tomato plants and seeds shall be planted unless the

ensure supervision by their responsible official bodies of premises growing, storing or handling potato tubers or tomatoes,

and all equipment, and subsequently has been granted approval for potato or tomato production by the responsible official

regulation 3 of these regulations, shall be conducted, and in the cases where seed potatoes are planted for seed production

in the case of potatoes, only officially certified seed potatoes or seed potatoes grown under official control from officially

in each of the growing years referred to in the previous indents measures shall be taken to eliminate volunteer potato and

for potato production, this production shall be from officially certified seed potatoes, or from mini-tubers or micro-plants

volunteer potato plants shall be tested for the organism; in addition, for potatoes, the harvested tubers shall be inspected;

immediately following the designation of contamination under sub-regulation 7(1)(a)(ii) of these regulations and in each of the subsequent

tomato plants and other host plants of the organism including solanaceous weeds, and an official survey as detailed in

growing years up to and including the first permissible potato or tomato cropping season on the field(s) designated as contaminated, as detailed in

in the case of potatoes, only officially certified seed potatoes shall be planted for either seed or ware production,

together with premises which operate machinery for potato or tomato production under contract,

4.2 Within the demarcated zone, without prejudice to the measures detailed under 4.1, the Plant Health Department shall

in cases where the demarcated zone has been determined under sub-regulation 7(1)(a)(iv) of these regulations,

and, where appropriate, disinfected using appropriate methods, as specified in point 3,

certified seed potatoes shall be planted for either seed or ware production,

for at least the second growing year following that in the first indent,

for the first growing year following that specified in the first indent,

immediately, and for at least three growing years, after the designated contamination

testing of tubers shall be carried out;

prevent the spread of the organism;

prevent the spread of the organism.

derived from tested sources,

bodies, and

of the growing medium is possible,

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paragraph (a)

(aa)

(d)

(c)

(a)

require the planting of only certified seed or seed grown under official control for all potato crops within that zone, and testing

in cases where surface water has been designated as contaminated under sub-regulation 7(1)(c)(ii) of these regulations or included in the elements

conduct an annual survey at appropriate times including sampling of surface water and appropriate solanaceous host plants in

require cleansing and, where appropriate, disinfection of machinery and stores on such premises, using appropriate methods,

prevent the spread of the organism. This ban may be reviewed on the basis of the results obtained in the said annual survey,

in cases where liquid waste discharges are contaminated, introduce official controls on the disposal of waste from industrial

after harvest of seed potato crops grown in places of production determined as probably contaminated under sub-regulation

contaminated for the irrigation and spraying of listed plant material, and, where appropriate, other host plants in order to

introduce official controls on irrigation and spraying programmes, including a ban on the use of the water designated as

require the separate handling of harvested potato seed stocks to those of ware on all premises within the zone,

establish a programme, where appropriate, for the replacement of all seed potato stocks over an appropriate period of time.

for listed plant material, the relevant method set out in Schedule II, or

in other cases, any other officially approved method,

conduct an official survey as detailed in regulation 3 of these regulations,

processing or packaging premises handling listed plant material;

the relevant water sources and testing in accordance with

for the possible spread of the organism in accordance with Schedule V point 2,

as specified under point 3,

ii.

i.

7(1)(a)(iii),

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(ab)

(b)

otherwise disposed of subject to official approval and under official control such that there is no risk that the waste could come

potato and tomato processing waste (including rejected potatoes and peelings and tomatoes) and any other solid waste associated with the potatoes

be used for irrigation of agricultural land. The waste shall be conveyed directly to the site under containment conditions such

into contact with agricultural land or water sources which could be used for irrigation of agricultural land. The details thereof

deep burial at disposal site at which there is no risk of seepage to agricultural land or contact with water sources which could

The officially approved waste disposal facilities referred to in Schedule VI paragraph 1, fourth indent shall conform to the following provisions such that

liquid processing waste: prior to disposal, liquid waste containing suspended solids shall be subjected to filtration or settlement processes to

heated to a minimum of 70 °C during at least 30 minutes, prior to disposal, or

shall be notified to the other Member States and to the Commission.

(1) The suspected occurrence or confirmed presence of the

organism in Malta shall be reported to the Plant Health Department.

(2) In each case of suspected occurrence, the Plant Health

Department shall ensure completion of official or officially supervised

laboratory testing using, for the listed plant material, the relevant method

set out in Schedule II to these regulations and in accordance with the

conditions specified in point 1 of Schedule III, or, in all other cases, any

other officially approved method, in order to confirm or refute the

suspected occurrence. In the case of confirmation, the requirements laid

down in point 2 of ScheduleIII shall apply.

(3) Pending the confirmation or refutation of a suspected

occurrence under regulation 6(2) of these regulations, in each case of

suspected occurrence where, either

(i) diagnostic symptoms of the disease caused by the

organism have been seen and a positive result in the rapid screening

test(s), as specified in Schedule II section I, point 1 and section II

has been obtained; or,

(ii) a positive result in the screening test(s) as specified in

Schedule II section I, point 2 and section III has been obtained,the

Plant Health Department shall

(a) prohibit the movement of plants and tubers from

all crops, lots or consignments from which the samples have

been taken, except under their control and provided that it

has been established that there is no identifiable risk of the

organism spreading;

(b) take steps to trace the origin of the suspected

occurrence;

(c) introduce appropriate additional precautionary

measures based on the level of estimated risk, particularly in

relation to production of the listed plant material and the

movement of seed potato lots other than those referred to in

point (a), produced on the place of production from which

the samples referred to in point (a) were taken, in order to

prevent any spread of the organism.

(4) In those cases of suspected occurrence where there is a

risk of contamination of the listed plant material or surface water from

or into another Member State(s), where the suspected occurrence has

been reported in Malta the Plant Health Department shall immediately

notify, according to the risk identified, the details of the said suspected

occurrence to any other Member Stateconcerned, and shall cooperate

with the said Member States. Where any other Member State so notifies

the Plant Health Department, the Plant Health Department shall

introduce precautionary measures in accordance with regulation 3(ii)(c)

of these regulations and take any further action, as appropriate.

remove such solids. These solids shall be disposed of as set out in subparagraph (i).

that there is no risk of loss of the waste, or

the risk of spreading the organism is obviated

incineration;

and tomatoes shall be disposed by either,

The liquid waste shall then be either

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(ii)

(i)

Mitbug[ fl-Istamperija tal-Gvern — Printed at the Government Printing Press

(1) lf official or officially supervised laboratory testing, Where the presence

of organism is

using, for the listed plant material, the relevant method set out in confirmed

Schedule II to these regulations or, in all other cases, any other officially

approved method, confirms the presence of the organism in a sample

taken pursuant to these regulations, the Plant Health Department, having

regard to sound scientific principles, the biology of the organism and

the particular production, marketing and processing systems of the host

plants of the organism in Malta, shall

(a)

for the listed plant material;

(i) establish an investigation to determine the extent

and primary source(s) of the contamination in accordance with

the provisions of Schedule IV to these regulations, with further

testing in accordance with regulation 6(2) of these regulations,

on, at least, all clonally related seed potato stocks, and,

(ii) designate as contaminated the listed plant material,

consignment and/or lot from which the sample was taken,

and the machinery, vehicle, vessel, store, or units thereof, and

any other objects including packaging material which have

been in contact with the listed plant material from which the

sample was taken; also designate as contaminated, where

appropriate, any field, unit of protected crop production and

place of production from which the listed plant material was

harvested and from which the sample was taken; and for those

samples taken in the growing season, designate as

contaminated any field, place of production, and, where

appropriate, unit of protected crop production from which

the sample was taken, and,

(iii) determine, in accordance with the provisions of

point 1 of Schedule V to these regulations, the extent of

probable contamination through pre- or post-harvest contact,

through production, irrigation or spraying links or through

clonal relationship with the designated contamination, and,

(iv) demarcate a zone on the basis of the designation

of contamination under point (a) (ii) of this sub-regulation,

the determination of the extent of probable contamination

under point (a) (iii) of this sub-regulation, and the possible

spread of the organism, in accordance with the provisions of

point 2(i) of Schedule V to these regulations;

(b) for crops of host plants other than those mentioned under

point (a) where production of the listed plant material is identified

at risk,

(i) establish an investigation in accordance with point

(a)(i) of this sub-regulation;and

(ii) designate as contaminated the host plants of the

organism from which the sample was taken; and

(iii) determine the probable contamination and

demarcate a zone in accordance with points (a)(iii) and (a)

(iv) of this sub-regulation, respectively, in relation to

production of the listed plant material;

(c) for surface water (including liquid waste discharges from

industrial processing or packaging premises handling listed plant

material) and associated wild solanaceous host plants, where

production of the listed plant material is identified at risk through

irrigation, spraying, or flooding of the surface water,

(i) establish an investigation including an official

survey at appropriate times on samples of surface water and

if present wild solanaceous host plants to establish the extent

of the contamination; and

(ii) designate as contaminated the surface water from

which any sample was taken, to the extent appropriate and

on the basis of the investigation under point (c)(i) of this sub-

regulation; and

(iii) determine the probable contamination and

demarcate a zone on the basis of the designation of

contamination under point (c )(ii) of this sub-regulation, and

the possible spread of the organism taking into account the

provisions of points 1 and 2 (ii) of Schedule V to these

regulations.

(2) The Plant Health Department shall immediately notify

the other Member States and the Commission, in accordance with the

provisions of point 3 of Schedule V, of any contamination designated

under paragraphs l(a)(ii) and l(c)(ii) of this regulation and the details of

the zone demarcation under paragraph 1 (a) (iv) of this regulation and,

where applicable, under paragraph l(c)(iii) of this regulation. The Plant

Health Department shall at the same time submit to the Commission

the additional notification set out at point 4 of Schedule V to these

regulations.

(3) Where the Plant Health Department receives a

notification from other Member States in terms of paragraph 2 of Article

5 of the European Council Directive 98/57, it establish an investigation

in accordance with paragraph l(a)(i) of this regulation and, where

applicable, paragraph l(c)(i) of this regulation and take further action,

as appropriate, in accordance with regulations (7)1 and (7)2 of these

regulations.

(1) The listed plant material designated to be contaminated

under sub- regulation 7(1)(a)(ii) of these regulations may not be planted

and, under the control and approval of the Plant Health Department, it

shall be subjected to one of the provisions of point 1 of Schedule VI to

these regulations, such that it is established that there is no identifiable

risk of the organism spreading.

(2) The listed plant material determined as probably

contaminated under sub-regulation 7(1)(a) (iii) and (c)(iii) of these

regulations including listed plant material for which a risk has been

identified, produced on places of production determined as probably

contaminated under sub-regualtion 7(1)(a)(iii) of these regulations may

not be planted and shall, under the control of the Plant Health

Department, be put to appropriate use or disposal as specified in point 2

of Schedule VI to these regulations, such that it is established that there

is no identifiable risk of the organism spreading.

(3) Any machinery, vehicle, vessel, store, or units thereof,

and any other objects including packaging material, designated as

contaminated under sub regulation 7(1)(a)(ii) of these regulations or

determined as probably contaminated under sub-regulations 7(1)(a)(iii)

and (c)(iii) of these regulations, shall either be destroyed or

decontaminated using appropriate methods as specified in point 3 of

Schedule Vl to these regulations. After decontamination, any such

objects shall no longer be considered contaminated.

(4) Without prejudice to the measures implemented under

sub-regulations 8(1), 8(2) and 8(3) above, in the zone demarcated under

regulation 7(1)(a)(iv) and (c)(iii) of these regualtions, a series of

measures, as specified in points 4.1 and 4.2 of Schedule VI to these

regulations, shall be implemented. Details of these measures shall be

notified annually to the other Member States and to the Commission.

Seed potatoes shall meet the requirements of the Plant

Quarantine Act, 2001 and shall derive in direct line from potato material

obtained under an officially approved programme which has been found

free of the organism in official or officially supervised testing using the

relevant method set out in Schedule II to these regulations. The testing

shall be carried out by the Plant Health Department

(a) in cases where there has been confirmed any finding of

the organism in local seed potato production,

(i) by testing of the earlier propagations, including the

initial clonal selection and systematic testing of basic seed

potato clones; or

(ii) in cases where it has been established that there is

no clonal relationship, by testing of all basic seed potato clones

or earlier propagations including the initial clonal selection,

and

(b) in other cases, either on each plant of the initial clonal

selection or on representative samples of the basic seed potatoes

or earlier propagations.

The holding and handling of the organism is hereby Prohibition of

holding and

banned.

handling of

organism.

Without prejudice to the provisions of the Plant Quarantine Derogations for

Act, 2001, the Plant Health Department may authorise derogations from experimental

scientific

the measures referred to in regulations 8 and 10 of these regulations in purposesAct XVIII

of 2001L.N. 97 of

accordance with the provisions laid down in the Plant Quarantine 2004.

(Harmful Organisms) Regulations, 2004 for trial or scientific purposes,

and for work on varietal selections.

(1) The Plant Health may adopt such additional or stricter Additional

measuresAct XVIII

measures as may be required to combat the organism or to prevent it of 2001.

from spreading, insofar as they are in compliance with the provisions

of the Plant Quarantine Act, 2001.(2) The details of these measures

shall be notified to the other Member States and to the Commission.

- in the case of seed potatoes, and where appropriate for other potatoes, official or officially supervised laboratory testing using the method

1. The official surveys referred to in regulation 4(a) shall be based on the biology of the organism and the particular production systems in Malta and shall

at appropriate times, visual inspection of the growing crop, and/or sampling of both seed and other potatoes in the growing season or in

store. These samples shall be subjected to official or officially supervised visual inspection by cutting of tubers, and

List of host plants of Ralstonia solanacearum (Smith) Yabuuchi et al. referred to in regulation 2 of these regulations

- visual inspection, at appropriate times, of at least the growing crop of plants intended for replanting for professional use.

(ii) in the case of surveys on, at least, the growing crop of plants of tomato intended for replanting for professional use,

(iii) in the case of surveys on host plants other than potatoes and tomatoes, including wild solanaceous host plants,

stratification by seed category and ware, and where appropriate, by region,

estimated total area grown, in hectares, of seed and other potatoes,

number (and size of sample) of visual inspections on tubers;

2. The notification of the official surveys referred to in regulation 5 shall include

SCHED ULE I

SECTION II

Tomato

SECTION I

number and timing of samples taken for testing,

number of visual inspections in the field,

Potato

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

estimated total number of plants,

number of visual inspections;

Plants (including tubers), other than true seed, of Solanum tuberosum L.

in the case of surveys on potatoes,

set out in Schedule II,

in the case of tomato,

in the case of potato,

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Surveys

comprise

(ii)

(i)

(i)

(iv) in the case of surveys on water and liquid waste discharges from industrial processing or packaging premises,

area/ location of premises sampled, as appropriate,

number and timing of samples taken,

number and timing of samples,

area sampled, as appropriate,

method of analysis;

method of analysis.

species,

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The testing procedure is intended for potato tubers with symptoms typical or suspect of brown rot and for potato and tomato plants with symptoms typical

or suspect of bacterial wilt. It involves a rapid screening test, isolation of the pathogen from infected vascular tissue on diagnostic media and, in case of a

Test scheme for diagnosis, detection and identification of Ralstonia solanacearum (Smith) Yabuuchi et al.

diagnosis of brown rot in potato tubers and of bacterial wilt in potato and tomato plants;

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