Control of Potato Ring Rot (Amendment) Regulations, 2007

Suppliment tal-Gazzetta tal-Gvern ta’ Malta, Nru. 18,061, 5 ta’ April, 2007

L.N. 90 of 2007

PLANT QUARANTINE ACT

(CAP. 433)

Control of Potato Ring Rot (Amendment) Regulations, 2007

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

Quarantine Act, the Minister for Rural Affairs and the Environment

has made the following regulations:-

(1) The title of these regulations is the Control of Potato Title,

Ring Rot (Amendment) Regulations, 2007 and they shall be read and commencement

and scope.

construed as one with the Control of Potato Ring Rot Regulations, L.N. 290 of

2004, hereinafter in these regulations referred to as "the principal 2004.

regulations".

(2) These regulations shall come into force on the 1st April,

2007. (3) The scope of these regulations is to transpose Council

Directive 2006/56/EC amending the Annexes to Council Directive

93/85/EEC on the control of potato ring rot.

(Regulation 2)

"SCHEDULE I

TEST SCHEME FOR DIAGNOSIS, DETECTION AND IDENTIFICATION OF

THE RING ROT BACTERIUM, CLAVIBACTER MICHIGANENSIS (Smith)

Davis et al. ssp. SEPEDONICUS (Spieckermann et Kotthoff) Davis et al.

SCOPE OF THE TEST SCHEME

The presented scheme describes the various procedures involved in

(i)

Diagnosis of ring rot in potato tubers and plants;

(ii) Detection of Clavibacter michiganensis ssp. sepedonicus in samples of

potato tubers and plants;

(iii) Identification of Clavibacter michiganensis ssp. sepedonicus (C. m.

subsp. sepedonicus).

GENERAL PRINCIPLES

Optimized protocols for the various methods, validated reagents and details

for the preparation of test and control materials are provided in the Appendices. A

list of the laboratories that were included in optimization and validation of

protocols is provided in Appendix 1.

Since the protocols involve detection of a quarantine organism and will

include the use of viable cultures of C. m. subsp. sepedonicus as control materials,

it will be necessary to perform the procedures under suitable quarantined

conditions with adequate waste disposal facilities and under the conditions of

appropriate licences as issued by the official plant quarantine authorities.

Testing parameters must assure consistent and reproducible detection of

levels of C. m. subsp. sepedonicus at the set thresholds of the selected methods.

Precise preparation of positive controls is imperative.

Testing according to the required thresholds also implies correct settings,

maintenance and calibration of equipment, careful handling and preservation of

reagents and all measures to prevent contamination between samples, e.g.

separation of positive controls from test samples. Quality control standards must

be applied to avoid administrative and other errors, especially concerning labelling

and documentation.

A suspected occurrence, as referred to in regulation 4(3) implies a positive

result in diagnostic or screening tests performed on a sample as specified in flow

charts.

If the first screening test (IF or PCR/FISH) is positive, then contamination

with Cms is suspected and a second screening test must be done. If the second

screening test is positive, then the suspicion is confirmed (suspected occurrence)

and the testing according to the scheme must be continued. If the second screening

test is negative, then the sample is considered not contaminated with Cms.

Therefore a positive IF test as referred to in regulation 4(3) is defined by a

positive IF reading confirmed by a second screening test (PCR/FISH).

Confirmed presence as referred to in regulation 5(1) implies the isolation and

identification of a pure culture of C. m. subsp. sepedonicus with confirmation of

pathogenicity.

1. FLOW CHART DIAGRAM PRESENTATION

1.1. Detection scheme for the diagnosis of Ring Rot in potato tubers and

potato plants with symptoms of ring rot

The testing procedure is intended for potato tubers and plants with symptoms

typical or suspect of ring rot. It involves a rapid screening test, isolation of the

pathogen from infected vascular tissue on diagnostic media and, in case of a

positive result, identification of the culture as C. m. subsp. sepedonicus.

( 1 )

Description of symptoms is provided in section 2.

( 2 )

Appropriate tests are

–

IF-test (section 4).

–

PCR test (section 6).

–

FISH test (section 5).

( 3 ) Although isolation of the pathogen from plant material with typical symptoms by dilution plating

is straightforward, culturing may fail from advance stages of infection. Saprophytic bacteria which grow on

diseased tissue may outgrow or inhibit the pathogen on the isolation medium. Therefore it is recommended to

use both non selective and selective media, preferably MTNA (section 8) or Bioassay test (section 7).

( 4 )

Description of typical colony morphology is provided in section 8.

( 5 )

If the isolation test is negative, but diseases symptoms are typical, then isolation must be repeated.

( 6 ) Reliable identification of a pure culture of C. m. subsp. Sepedonicus is achieved by using the tests

listed in section 9.

( 7 )

The pathogenicity is described in section 10.

1.2. Scheme for detection and identification of Clavibacter michiganensis

ssp. sepedonicus in samples of asymptomatic potato tubers

Principle

The testing procedure is intended for detection of latent infections in potato

tubers. A positive result from at least two screening tests, based on different

biological principles, must be complemented by the isolation of the pathogen;

followed by, in case of isolation of typical colonies, confirmation of a pure culture

as C. m. subsp. sepedonicus. A positive result from only one of the screening tests

is not sufficient to consider the sample suspect.

Screening tests and isolation tests must permit a detection threshold of 103 to

104 cells/ml resuspended pellet, included as positive controls in each series of

tests.

(1) The standard sample size is 200 tubers although the procedure can be used with smaller samples if

200 tubers are not available.

(2) Pathogen extraction and concentration methods are described in section 3.1.

(3) If at least two tests based on different biological principles are positive, isolation and

confirmation have to be done. Perform at least one screening test. When this test is negative, the sample is

considered to be negative in case this test is positive a second or more screening tests based on different

biological principles are required to verify the first positive result. If the second ore other tests are negative the

sample is considered negative. Further tests are not necessary.

(4) Immunofluorescence (IF) test.

Always use a polyclonal antibody for IF screening, additional monoclonal antibodies may provide

more specifity (see section 4).

(5)

PCR test.

Use appropriately validated PCR reagents and protocols (see section 6).

(6)

Fish test.

Use validated reagents and protocols (see section 5).

(7)

Selective isolation.

With the MTNA medium of NCP-88 medium and a 1/100 dilution of the resuspended pellet, this

is in many cases a suitable method for direct isolation of C. m. subsp sepedonicus. Typical colonies can be

obtained 3 to 10 days after planting. The pathogen then can be purified and identified. For full exploitation of

its potential, the test requires careful preparation of heel end cores to avoid secondary bacteria associated with

the potato tuber which are competitors with C. m. subsp sepedonicus on the medium and may overgrow the

pathogen. If the plate test fails isolation must be done from plants used for the bio assay (see section 8).

(8) The bioassay test is used for isolation of C. m. subsp sepedonicus from potato extract pellets by

selective enrichment in eggplants (Solanum melongena). The test requires optimal incubation conditions as

specified in this method. Bacteria inhibitory to C. m. subsp sepedonicus on the MTNA or NCP-88 medium will

most likely not interfere in this test (see section 7).

(9)

Typical colony morphology is described in section 8.

(10) Culturing or bioassays can fail due to competition or inhibition by saprophytic bacteria. If

positive results are obtained in screening tests, but the isolation tests are negative, then repeat the isolation

tests from the same pellet or by taking additional vascular tissue near the heel end from cut tubers of the same

sample and, if necessary, test additional samples.

(11) Reliable identification of pure presumptive C. m. subsp sepedonicus cultures is achieved using the

tests described in section 9.

(12) The pathogenicity test is described in section 10.

1.3. Scheme for detection and identification of Clavibacter michiganensis

ssp. sepedonicus in samples of asymptomatic potato plants

(1) See section 3.2 for recommended sample sizes.

(2) Pathogen extraction and concentration methods are described in section 3.2.

(3) If at least two tests based on different biological principles are positive, isolation and

confirmation have to be done.

Perform at least one screening test. When this test is negative the sample is considered to be

negative. In case this test is positive a second or more screening tests based on different biological principles

are required to verify the first positive result. If the second or other tests are negative the sample is considered

negative. Further tests are not necessary.

(4) The selective isolation test and typical colony morphology are described in section 8.

(5) The IF test is described in section 4.

(6) PCR tests are described in section 6.

(7) The FISH test is described in section 5.

(8)

The bioassay is described in section 7.

(9) Culturing or bioassays can fail due to competition of inhibition by saprophytic bacteria. If

positive results are obtained in screening tests but the isolation tests are negative, then repeat the isolation

tests, and if necessary, test additional samples.

(10) Reliable identification of pure presumptive C. m. subsp sepedonicus cultures is achieved using the

tests described in section 9.

(11) The pathogenicity test is described in section 10.

2. VISUAL EXAMINATION FOR RING ROT SYMPTOMS

2.1. Potato plants

Under European climatic conditions symptoms are rarely found in the field

and often only at the end of the season. Moreover the symptoms are frequently

masked or confused by/with other diseases, senescence or mechanical damages.

Therefore it may be easy to miss symptoms in field inspections. Wilting symptoms

are very different from those of brown rot; wilting is usually slow and initially

limited to the leaf margins. Young infected leaves often continue to expand

although, less so in the infected zones. This creates odd shaped leaves. Leaves

affected by blocking of the vascular tissues further down the stem often develop

chlorotic, yellow to orange, intercostal areas. Infected leaflets, leaves and even

stems may eventually die. Often leaves and tubers are simply reduced in size.

Occasionally plants are stunted. Coloured pictures of a range of symptoms can be

found on the web site http://forum.europa.eu.int/Public/irc/sanco/Home/main

2.2. Potato tubers

The earliest symptoms are a slight glassiness or translucence of the tissue

without softening around the vascular system, particularly near the heel end. The

vascular ring at the heel end may be slightly darker in colour than normal. The first

readily identifiable symptom is one whereby the vascular ring has a yellowish

coloration and when the tuber is gently squeezed, pillars of cheese-like material

emerge from the vessels. This exudation contains millions of bacteria. Browning

of the vascular tissue may develop and tuber symptoms at this stage are similar to

those of brown rot caused by Ralstonia solanacearum. At first, these symptoms

may be restricted to one part of the ring, not necessarily close to the heel end and

may gradually extend to the whole ring. As the infection progresses, destruction of

the vascular tissue occurs; the outer cortex may become separated from the inner

cortex. In advanced stages of infection, cracks appear on the surface of the tuber,

which are often reddish-brown at their margins. Recently in Europe several cases

have occurred where the central cortex rots at the same time as the vascular ring

resulting in secondary invasion with internal hollowing and necrosis. Secondary

fungal or bacterial invasion may mask the symptoms and it may be difficult, if not

impossible, to distinguish advanced ring rot symptoms from other tuber rots.

Atypical symptoms may be possible. Coloured pictures of a range of symptoms

can be found on the web site http://forum.europa.eu.int/Public/irc/sanco/Home/

main

3. SAMPLE PREPARATION

3.1. Potato tubers

Note

-

The standard sample size is 200 tubers per test. More intensive

sampling requires more tests on samples of this size. Larger numbers of

tubers in the sample will lead to inhibition or difficult interpretation of the

results. However, the procedure can be conveniently applied for samples with

less than 200 tubers where fewer tubers are available.

-

Validation of all detection methods described below is based on

testing of samples of 200 tubers.

-

The potato extract described below can also be used for detection

of the potato brown rot bacterium, Ralstonia solanacearum.

Optional pre-treatment in advance to sample preparation

Wash the tubers. Use appropriate disinfectants (chlorine compounds when

PCR-test is to be used in order to remove eventual pathogen DNA) and detergents

between each sample. Air dry the tubers. This washing procedure is particularly

useful (but not required) for samples with excess soil and if a PCR-test or direct

isolation procedure is to be performed.

3.1.1. Remove with a clean and disinfected scalpel or vegetable knife the

skin at the heel end of each tuber so that the vascular tissue becomes visible.

Carefully cut out a small core of vascular tissue at the heel end and keep the

amount of non-vascular tissue to a minimum (see web site: http://

forum.europa.eu.int/Public/irc/sanco/Home/main)

Note

Set aside any tubers with suspected ring rot symptoms and test separately.

If during removal of the heel end core suspect symptoms of ring rot are

observed, the tuber should be visually inspected after cutting near the heel end.

Any cut tuber with suspected symptoms should be suberised at room temperature

for two days and stored under quarantine (at 4 to 10°C) until all tests have been

completed. All tubers in the sample (including those with suspicious symptoms)

should be kept according to Schedule II.

3.1.2. Collect the heel end cores in unused disposable containers which can

be closed and/or sealed (in case containers are reused they should be thoroughly

cleaned and disinfected using chlorine compounds). Preferably, the heel end cores

should be processed immediately. If this is not possible, store them in the

container, without addition of buffer, refrigerated for not longer than 72 hours or

for not longer than 24 hours at room temperature. Drying and suberisation of cores,

and growth of saprophytes during storage may hinder detection of the ring rot

bacterium.

3.1.3. Process the heel end cores by one of the following procedures: either,

(a)

cover the cores with sufficient volume (approximately 40 ml) of

extraction buffer (Appendix 3) and agitate on a rotary shaker (50 to 100 rpm)

for four hours below 24°C or for 16 to 24 hours refrigerated, or

(b) homogenise the cores with sufficient volume (approximately 40

ml) of extraction buffer (Appendix 3), either in a blender (e.g. Waring or

Ultra Thurax) or by crushing in a sealed disposable maceration bag (e.g.

Stomacher or Bioreba strong guage polythene, 150 mm × 250 mm; radiation

sterilised) using a rubber mallet or suitable grinding apparatus (e.g. Homex).

Note

The risk of cross-contamination of samples is high when samples are

homogenized using a blender. Take precautions to avoid aerosol generation or

spillage during the extraction process. Ensure that freshly sterilised blender blades

and vessels are used for each sample. If the PCR test is to be used, avoid carry-

over of DNA on containers or grinding apparatus. Crushing in disposable bags and

use of disposable tubes is recommended where PCR is to be used.

3.1.4. Decant the supernatant. If excessively cloudy, clarify either by slow

speed centrifugation (at not more than 180 g for 10 minutes at a temperature

between 4 to 10°C) or by vacuum filtration (40 to 100 µm), washing the filter with

additional (10 ml) extraction buffer (Appendix 3).

3.1.5. Concentrate the bacterial fraction by centrifugation at 7 000 g for 15

minutes (or 10 000 g for 10 minutes) at a temperature between 4 to 10°C and

discard the supernatant without disturbing the pellet.

3.1.6. Resuspend the pellet in 1,5 ml pellet buffer (Appendix 3). Use 500 µl

to test for C. m. subsp. sepedonicus, 500 µl for Ralstonia solanacearum and 500 µl

for reference purposes. Add sterile glycerol to final concentration of 10 to 25% (v/

v) to the 500 µl of the reference aliquot and to the remaining test aliquot, vortex

and store at -16 to -24°C (weeks) or at -68 to -86°C (months). Preserve the test

aliquots at 4 to 10°C during testing.

Repeated freezing and thawing is not advisable.

If transport of the extract is required, ensure delivery in a cool box within 24

to 48 hours.

3.1.7. It is imperative that all C. m. subsp. sepedonicus positive controls

and samples are treated separately to avoid contamination. This applies to IF slides

and to all tests.

3.2. Potato plants

Note

For detection of latent C. m. subsp. sepedonicus populations it is advised to

test composite samples. The procedure can be conveniently applied for composite

samples of up to 200 stem parts. (Where surveys are performed they should be

based on a statistically representative sample of the plant population under

investigation.)

3.2.1. With a clean disinfected knife or pruning shears, remove a 1 to 2 cm

segment from the base of each stem, just above the soil level.

Disinfect stem segments briefly with ethanol 70% and immediately blot dry

on tissue paper.

Collect stem segments in a closed sterile container according to the following

sampling procedures

3.2.2. Process the stem segments by one of the following procedures

either,

(a)

cover the segments with sufficient volume (approximately 40 ml)

of extraction buffer (Appendix 3) and agitate on a rotary shaker (50 to 100

rpm) for four hours below 24°C or for 16 to 24 hours refrigerated, or

(b) process immediately. By crushing the segments in a strong

maceration bag (e.g. Stomacher or Bioreba) with an appropriate volume of

extraction buffer (Appendix 3) using a rubber mallet or appropriate grinding

apparatus (e.g. Homex). If this is not possible, store the stem segments

refrigerated for not longer than 72 hours or for not longer than 24 hours at

room temperature.

3.2.3. Decant the supernatant after settling for 15 minutes.

3.2.4. Further clarification of the extract or concentration of the bacterial

fraction are not usually required but may be achieved by filtration and/or

centrifugation as described in section 3.1.4 to 3.1.6.

3.2.5. Divide the neat or concentrated sample extract into 2 equal parts.

Maintain one half at 4 to 10°C during testing and store the other half with 10 to

25% (v/v) sterile glycerol at -16 to -24°C (weeks) or at -68 to -86°C (months) in

case further testing is required.

Schedules I, II, III and IV of the principal regulations shall Substitutes

be substituted by Schedules I, II, III and IV respectively, set out in Schedules

III and IV of the

principal

Schedule I to these regulations.

regulations.

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

screening test (s) has been identified according to the methods set out in Schedule

I, and confirmation or refutation by completion of the said methods is awaited,

there should be the retention and appropriate conservation of

-

all tubers sampled and, wherever possible, all plants sampled,

-

any remaining extract and additional prepared material for the

screening test(s), e.g. immunofluorescence slides, and

-

all relevant documentation,

until the completion of the said methods.

Retention of the tubers will enable variety testing to be undertaken where

appropriate.

2. In the case of positive confirmation of the organism, there should be

retention and appropriate conservation of

-

the material specified in paragraph 1, and

-

a sample of the infected eggplant material inoculated with the

tuber or plant extract, and

-

the isolated culture of the organism,

until at least one month after the notification procedure under regulation 5(2).

Schedule V, as set out in Schedule II to these regulations, Adds Schedule

shall be added immediately after Schedule IV of the principal V

principal

regulations.

regulations.

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

probable contamination under regulation 5(1)(b) shall include

-

tubers or plants grown at a place of production designated as

contaminated under regulation 5(1)(a),

-

place(s) of production with some production link to the tubers or

plants, designated as contaminated under regulation 5(1)(a), including those

sharing production equipment and facilities directly or through a common

contractor,

-

tubers or plants produced in the place(s) of production referred to

in the previous indent, or present in such place(s) of production during the

period when the tubers or plants designated as contaminated under regulation

5(1)(a) were present on the place of production referred to in the first indent,

-

premises handling potatoes from the places of production

referred to in the above indents,

-

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

other objects including packaging material, that may have come into contact

with the tubers or plants designated as contaminated under regulation 5(1)(a),

-

any tubers or plants stored in, or in contact with, any of the

structures or objects listed in the previous indent, prior to the cleansing and

disinfection of such structures and objects,

-

as a result of the testing under regulation 5(3), those tubers or

plants with a sister or parental clonal relationship to the tubers or plants

designated to be contaminated under regulation 5(1)(a) and for which,

although they may have tested negative for the organism, it appears that

contamination is probable through a clonal link. Variety testing may be

undertaken to verify the identity of the contaminated and clonally related

tubers or plants, and

-

place(s) of production of the tubers or plants referred to in the

previous indent.

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

spread under regulation 5(1)(c) shall include

-

the proximity of other places of production growing potatoes or

other host plants,

-

the common production and use of seed potato stocks.

3. The notification referred to in the first subparagraph of regulation 5(2)

shall be provided as follows

-

immediately after the presence of the organism has been

confirmed by laboratory testing, using the methods set out in Schedule I, at

least

-

the variety name of the potato lot,

-

the type (ware, seed, etc.) and where applicable the seed

category of potatoes,

-

when there is a risk of contamination of potatoes from or into

another Member States(s), the competent authority shall immediately notify

the Member State(s) concerned of information needed to comply with

regulation 5(3), such as

-

the variety name of the potato lot,

-

the name and address of the consignor and the consignee,

-

the date of delivery of the potato lot,

-

the size of the potato lot delivered,

-

a copy of the plant passport or at least the plant passport

number where appropriate, or where appropriate the registration number

of the grower or merchant and a copy of the delivery notice.

The Commission shall be notified immediately when such information has

been provided.

-

After all investigations have been finalised, for each case

-

the date that contamination was confirmed,

-

a brief description of the investigation carried out to

identify the source and possible spread of the contamination, including

the level of sampling undertaken,

-

information on the identified or presumed sources(s) of

contamination,

-

details of the extent of the designated contamination,

including the number of places of production and the number of lots

with an indication of the variety and, if seed potatoes, category,

-

details of the zone demarcation, including the number of

places of production, not designated as contaminated, but included in

the zone,

-

such other information relating to the confirmed

outbreak(s) as the Commission may require

IF TEST

Principle

The use of the IF test as the principal screening test is recommended because

of its proven robustness to achieve the required thresholds.

When the IF test is used as the principal screening test and the IF reading is

positive, the PCR or FISH test must be performed as a second screening test. When

the IF test is used as the second screening test and the IF reading is positive,

further testing according to the flow scheme is required to complete the analysis.

Note

Always use a polyclonal antibody, when the IF test is used as the principal

screening test. In case of a positive IF reading with a polyclonal antibody further

screening of the sample with a monoclonal antibody may provide more specificity

but can be less sensitive.

Use antibodies to a reference strain of C. m. subsp. sepedonicus. It is

recommended that the titre is determined for each new batch of antibodies. The

titre is defined as the highest dilution at which optimum reaction occurs when

testing a suspension containing 105 to 106 cells per ml of the homologous strain of

C. m. subsp. sepedonicus and using an appropriate fluorescein isothiocyanate

(FITC) conjugate according to the manufacturer’s recommendations. The crude

polyclonal or monoclonal antibodies should have an IF titre of at least 1:2000.

During testing, the antibodies should be used at a working dilution(s) (WD) close

to or at the titre. Use validated antibodies (see web site http://forum.europa.eu.int/

Public/irc/sanco/Home/main).

The test should be performed on freshly-prepared sample extracts. If

necessary, it can be successfully performed on extracts stored at -68 to -86°C under

glycerol. Glycerol can be removed from the sample by addition of 1 ml pellet

buffer (Appendix 4), re-centrifugation for 15 minutes at 7 000 g and resuspension

in an equal volume of pellet buffer. This is often not necessary, especially if slides

samples are fixed to the slides by flaming (see 2.2).

Prepare separate positive control slides of the homologous strain or any other

reference strain of C. m. subsp. sepedonicus, suspended in potato extract, as

specified in Appendix 2, and optionally in buffer.

Naturally infected tissue (maintained by lyophilization or freezing at -16 to

-24°C) should be used where possible as a similar control on the same slide.

As negative controls, use aliquots of sample extract which previously tested

negative.

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

mm diameter.

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

4.1. Prepare the test slides by one of the following procedures

(i)

For pellets with relatively little starch sediment

Pipette a measured standard volume (15 µl is appropriate for 6 mm window

diameter-scale up volume for larger windows) of a 1/100 dilution of the

resuspended potato pellet onto the first window. Subsequently pipette a similar

volume of undiluted pellet (1/1) onto the remaining windows on the row. The

second row can be used as duplicate or for a second sample as presented in Figure

1. (ii) For other pellets

Prepare decimal dilutions (1/10 and 1/100) of the resuspended pellet in pellet

buffer. Pipette a measured standard volume (15 µl is appropriate for 6 mm window

diameter-scale up volume for larger windows) of the resuspended pellet and each

dilution on a row of windows. The second row can be used as duplicate or for a

second sample as presented in Figure 2.

4.2. Dry the droplets at ambient temperatures or by warming at temperature

of 40 to 45°C. Fix the bacterial cells to the slide either by heating (15 minutes at

60°C), flaming, with 95% ethanol or according to specific instructions from the

suppliers of the antibodies.

If necessary, fixed slides may then be stored frozen in a desiccated box for as

little time as necessary (up to a maximum of 3 months) prior to further testing.

4.3. IF procedure

(i)

According to test slide preparation in 4.1(i)

Prepare a set of twofold dilutions of the antibody in IF buffer. The first well

should have 1/2 of the titre (T/2), the others 1/4 of the titre (T/4), 1/2 of the titre (T/

2), the titre (T) and twice the titre (2T).

(ii) According to test slide preparation in 4.1(ii)

Prepare the working dilution (WD) of the antibody in IF buffer. The working

dilution affects the specificity.

Figure 1. Preparation of the test slide according to 4.1(i) and 4.3(i)

Figure 2. Preparation of the test slide according to 4.1(ii) and 4.3(ii)

4.3.1. Arrange the slides on moist paper. Cover each test window

completely with the antibody dilution(s). The volume of antibody applied on each

window must be at least the volume of extract applied.

The following procedure should be carried out in the absence of specific

instructions from the suppliers of the antibodies

4.3.2. Incubate the slides on moist paper under a cover for 30 minutes at

ambient temperature (18 to 25°C).

4.3.3. Shake the droplets off each slide and rinse carefully with IF buffer.

Wash by submerging for 5 minutes in IF buffer-Tween (Appendix 3) and

subsequently for 5 minutes in IF buffer. Avoid causing aerosols or droplet transfer

that could result in cross-contamination. Carefully remove excess moisture by

blotting gently.

4.3.4. Arrange the slides on moist paper. Cover the test windows with the

dilution of FITC conjugate used to determine the titre. The volume of conjugate

applied on the windows must be identical to the volume of antibody applied.

4.3.5. Incubate the slides on moist paper under a cover for 30 minutes at

ambient temperature (18 to 25°C).

4.3.6. Shake the droplets of conjugate off the slide. Rinse and wash as

before (4.3.3).

Carefully remove excess moisture.

4.3.7. Pipette 5 to 10 µl of 0,1M phosphate-buffered glycerol (Appendix 3)

or a commercially antifading mountant on each window and apply a coverslip.

4.4. Reading the IF test

4.4.1. Examine test slides on an epifluorescence microscope with filters

suitable for excitation of FITC, under oil or water immersion at a magnification of

500 to 1 000. Scan windows across two diameters at right angles and around the

perimeter. For samples showing no or low number of cells observe at least 40

microscope fields.

Check the positive control slide first. Cells must be bright fluorescent and

completely stained at the determined antibody titre or working dilution. The IF test

(section 4) must be repeated if the staining is aberrant.

4.4.2. Observe for bright fluorescing cells with characteristic morphology

of C. m. subsp. sepedonicus in the test windows of the test slides (see web site

http://forum.europa.eu.int/Public/irc/sanco/Home/main).

The

fluorescence

intensity must be equivalent or better to the positive control strain at the same

antibody dilution. Cells with incomplete staining or with weak fluorescence must

be disregarded.

If any contamination is suspected the test must be repeated. This may be the

case when all slides in a batch show positive cells due to the contamination of

buffer or if positive cells are found (outside of the slide windows) on the slide

coating.

4.4.3. There are several problems inherent to the specificity of the

immunofluorescence test. Background populations of fluorescing cells with

atypical morphology and cross reacting saprophytic bacteria with size and

morphology similar to C. m. sepedonicus are likely to occur in potato heel end core

and stem segment pellets.

4.4.4. Consider only fluorescing cells with typical size and morphology at

the titre or working dilution of the antibodies as in 4.3.

4.4.5. Interpretation of the IF reading

(i) If bright fluorescing cells with characteristic morphology are found,

estimate the average number of typical cells per microscope field and calculate the

number of typical cells per ml of resuspended pellet (Appendix 4).

The IF reading is positive for samples with at least 5 × 103 typical cells per

ml of resuspended pellet. The sample is considered potentially contaminated. and

further testing is required.

(ii) The IF reading is negative for samples with less than 5 × 103 cells per

ml resuspended pellet and the sample is considered negative. Further testing is not

required.

1. The officially supervised measures referred to in regulation 5(4) of the

principal regulations shall be

-

use as animal feed after heat treatment such that there is no risk

of the organism surviving, or

-

disposal at an officially approved dedicated waste disposal site at

which there is no identifiable risk of escape of the pathogen into the

environment e.g. through seepage to agricultural land, or

-

incineration, or

-

industrial processing through direct and immediate delivery to a

processing plant with officially approved waste disposal facilities for which

it has been established that there is no identifiable risk of the organism

spreading, and with a system of cleansing and disinfection of at least the

departing vehicles, or

-

other measures, provided that it has been established that there is

no identifiable risk of the organism spreading; such measures and their

justification to be notified to the Commission and to the other Member

States.

Any remaining waste associated with and arising from the above shall be

disposed of by officially approved methods in accordance with Schedule V.

2. The appropriate use or disposal of tubers or plants determined as

probably contaminated under regulation 5(1)(b) and referred to in regulation 5(5),

under the control of the responsible official bodies of the Member States

concerned, with appropriate communication between responsible official bodies to

ensure such control at all times and approval by the responsible official bodies 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 indent, shall be

-

use as ware potatoes intended for consumption, packed ready for

direct delivery and use without repacking, on a site with appropriate waste

disposal facilities. Potatoes intended for planting may only be handled at the

same site, if this is done separately or after cleansing and disinfection, or

-

use as ware potatoes intended for industrial processing, and

intended for direct and immediate delivery to a processing plant with

appropriate waste disposal facilities and a system of cleansing and

disinfection of at least the departing vehicles, or

-

some other use or disposal, provided that it is established that

there is no identifiable risk of the organism spreading and subject to approval

by the said responsible official bodies.

3. The appropriate methods for cleansing and disinfecting of the objects

referred to in regulation 5(6) shall be those for which it has been established that

there is no identifiable risk of the organism spreading and shall be employed under

the supervision of the responsible official bodies of the Member States.

4. The series of measures to be implemented within the demarcated zone

established under regulation 5(1)(c) and referred to in regulation 5(7) shall

include

4.1. in places of production designated as contaminated under regulation

5(1)(a)

(a)

in a field designated to be contaminated under regulation 5(1)(a),

either

(i) - during at least the three growing years following the year

of the designated contamination,

-

measures shall be taken to eliminate volunteer

potato plants and other naturally found host plants of the

organism, and

-

no potato tubers, plants or true seeds, or other

naturally found host plants of the organism, or crops for

which there is an identified risk of the organism spreading,

shall be planted,

- in the first potato cropping season following the period

specified in the preceding indent, and on the condition that the field has

been found free from volunteer potato plants and other naturally found

host plants of the organism during official inspections for at least the

two consecutive growing years prior to planting, only ware potato

production shall be allowed and the harvested tubers shall be tested

according to the procedure detailed in Schedule I;

- in the potato cropping season succeeding that referred to

in the previous indent and following an appropriate rotation cycle,

which shall be at least two years if seed potatoes are to be grown,

potatoes may be planted for either seed or ware production, and an

official survey as detailed in regulation 3(1) shall be conducted; or

(ii) - during the four growing years following that of the

designated contamination,

-

measures shall be taken to eliminate volunteer

potato plants and other naturally found host plants of the

organism, and

-

the field shall be laid to, and maintained either, in

bare fallow or in permanent pasture with frequent close

cutting or intensive grazing,

- in the first potato cropping season following the period

specified in the preceding indent, and on the condition that the field has

been found free from volunteer potato plants and other naturally found

host plants of the organism during official inspections for at least the

two consecutive growing years prior to planting, seed or ware potato

production shall be allowed and the harvested tubers shall be tested

according to the procedure detailed in Schedule I;

(b) in all other fields of the contaminated place of production, and on

the condition that the responsible official bodies are satisfied that the risk of

volunteer potato plants and other naturally found host plants of the organism

have been eliminated

-

in the growing year following that of the designated

contamination either no potato tubers, plants or true seeds or other

naturally found host plants of the organism shall be planted, or

-

certified seed potatoes may be planted for ware

production only,

-

in the second growing year following that of the

designated contamination, only certified seed or seed potatoes officially

tested for the absence of ring rot and grown under official control on

places of production other than those referred to in paragraph 4.1 shall

be planted for either seed or ware production,

-

for at least the third growing year following the

designated contamination, only certified seed potatoes or seed potatoes

grown under official control from certified seed potatoes shall be

planted for either seed or ware production,

-

in each of the growing years referred to in the previous

indents, measures shall be taken to eliminate volunteer potato plants and

other naturally found host plants of the organism if present and in each

potato field official testing of the harvested potatoes shall be conducted

according to the procedure detailed in Schedule I,

(c)

immediately following the designation of contamination under

regulation 5(1)(a) and after the first subsequent growing year, all machinery

and storage facilities on the place of production and involved in potato

production shall be cleansed and disinfected as appropriate using appropriate

methods, as specified in paragraph 3;

(d) in an unit of protected crop production where complete

replacement of the growing medium is possible,

-

no tubers, plants or true seeds shall be planted unless the

production 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

disinfection of the production unit, and all equipment, and,

subsequently has been granted approval for potato production by the

responsible official bodies, and

-

potato production shall be from certified seed potatoes, or

from mini-tubers or micro-plants derived from tested sources;

4.2. within the demarcated zone, without prejudice to the measures detailed

under paragraph 4.1, the competent authority shall

(a)

immediately following the designated contamination, ensure that

all machinery and storage facilities on such premises and involved with

potato production be cleansed and disinfected, as appropriate, and using

appropriate methods, as specified in paragraph 3;

(b) immediately, and for at least three growing seasons, after the

designated contamination

-

ensure supervision by their responsible official bodies of

premises growing, storing or handling potato tubers, together with

premises which operate potato machinery under contract,

-

require the planting of only certified seed or seed grown

under official control for all potato crops within that zone, and testing

after harvest of seed potato crops grown in places of production

designated as probably contaminated under regulation 5(1)(b),

-

require the separate handling of harvested seed potato

stocks to those of ware on all premises within the zone, or a system of

cleansing and disinfection to be carried out between the handling of

seed and ware stocks,

-

conduct an official survey as detailed in regulation 3(1)(a);

(c)

establish a programme, where appropriate, for the replacement of

all seed potato stocks over an appropriate period of time.".

FISH TEST

Principle

When the FISH test is used as the first screening test and found to be

positive, the IF test must be performed as a second compulsory screening test.

When the FISH test is used as the second screening test and found to be positive,

further testing according to the flow scheme is required to complete the diagnosis.

Note

Use validated C. m. subsp. sepedonicus-specific oligo-probes (Appendix 7).

Preliminary testing with this method should permit reproducible detection of at

least 103 to 104 cells of C. m. subsp. sepedonicus per ml added to sample extracts

which previously tested negative.

The following procedure should preferably be performed on freshly prepared

sample extract but can also be successfully performed on sample extract that has

been stored under glycerol at -16 to -24°C or -68 to -86°C.

As negative controls, use aliquots of sample extract that previously tested

negative for C. m. subsp. sepedonicus.

As positive controls prepare suspensions containing 105 to 106 cells per ml

of C. m. subsp. sepedonicus (e.g. strain NCPPB 4053, or PD 406) in 0,01M

phosphate buffer (PB) from a three to five day culture (preparation see Appendix

2). Prepare separate positive control slides of the homologous strain or any other

reference strain of C. m. subsp. sepedonicus, suspended in potato extract, as

specified in Appendix 2.

The use of the FITC-labelled eubacterial oligo-probe offers a control for the

hybridisation process, since it will stain all eubacteria that are present in the

sample.

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

5.1. Potato extract fixation

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

5.1.1. Prepare fixative solution (see Appendix 7).

5.1.2. Pipette 100 µl of each sample extract into an Eppendorf tube and

centrifuge for eight min at 7 000 g.

5.1.3. Remove the supernatant and dissolve the pellet in 500 µl of fixative

prepared < 24 hours previously. Vortex and incubate overnight at 4°C.

An alternative fixative is 96% ethanol. To use this dissolve the pellet from

step 5.1.2 in 50 µl 0,01M PB and 50 µl 96% ethanol. Vortex mix and incubate at

4°C for 30 to 60 minutes.

5.1.4. Centrifuge for 8 min. at 7 000 g, remove the supernatant and

resuspend the pellet in 75 µl 0,01M PB (see Appendix 3).

5.1.5. Spot 16 µl of the fixed suspensions onto a clean multitest slide as

shown in Fig. 3. Apply two different samples per slide, undiluted and use 10 µl to

make a 1:100 dilution (in 0,01M PB). The remaining sample solution (49 µl) can

be stored at -20°C after addition of 1 volume of 96% ethanol. In case the FISH

assay requires repeating, remove the ethanol by centrifugation and add an equal

volume of 0,01M PB (mix by vortexing).

Figure 3. Layout for FISH slide

5.1.6. Air-dry the slides (or on slide dryer at 37°C) and fix them by flaming.

At this stage the procedure may be interrupted and the hybridisation

continued the following day. Slides should be stored dust-free and dry at room

temperature.

5.2. Pre-hybridisation and hybridization

5.2.1. Prepare a lysozyme solution containing 10 mg lysozyme (Sigma L-

6876) in 10 ml buffer (100 mM Tris-HCl, 50 mM EDTA, pH8.0). This solution can

be stored but it should only be freeze-thawed once. Cover each sample well with

approximately 50 µl of lysozyme solution and incubate for 10 minutes at room

temperature. Then dip the slides in demineralised water, once only and dry with

filter paper.

Alternatively, instead of lysozyme add 50 µl of 40 to 400 µg ml -1 proteinase

K in buffer (20 mM Tris-HCl, 2 mM CaCl 2 , pH 7,4) to each well and incubate at

37°C for 30 minutes.

5.2.2. Dehydrate the cells in a graded ethanol series of 50%, 80% and 96%

for one minute each. Air dry the slides in a slide-holder.

5.2.3. Prepare a moist incubation chamber by covering the bottom of an air-

tight box with tissue or filter paper soaked in 1x hybmix (Appendix 7). Pre-

incubate the box in the hybridisation oven at 55°C for at least 10 minutes.

5.2.4. Prepare the hybridisation solution (Appendix 7) allowing 45 µl per

slide, and preincubate for five minutes at 55°C.

5.2.5. Place slides on a hot plate at 45°C and apply 10 µl of hybridization

solution to each of the four wells on the slide(s).

5.2.6. Apply two coverslips (24 × 24 mm) to each slide without trapping

air. Place the slides in the pre-warmed moist chamber and hybridise overnight in

the oven at 55°C in the dark.

5.2.7. Prepare three beakers containing 1 l of Ultra pure water (UPW), 1 l

of 1x hybmix (334 ml 3x hybmix and 666 ml UPW) and 1 l of 1/2x hybmix (167

ml 3x hybmix and 833 ml UPW). Pre-incubate each in a waterbath at 55°C.

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

holder.

5.2.9. Wash away excess probe by incubation for 15 mins. in the beaker

with 1x hybmix at 55°C.

5.2.10. Transfer the slide holder to 1/2 hybmix washing solution and

incubate for a further 15 mins.

5.2.11. Dip the slides briefly in UPW and place them on filter paper. Remove

excess moisture by covering the surface gently with filter paper. Pipette 5 to 10 µl

of anti-fading mountant solution (e.g. Vectashield, Vecta Laboratories, CA, USA

or equivalent) on each window and apply a large coverslip (24 × 60 mm) over the

whole slide.

5.3. Reading the FISH test

5.3.1. Observe the slides immediately with a microscope fitted for

epifluorescence microscopy at 630 or 1000x magnification under immersions oil.

With a filter suitable for fluorescein isothiocyanate (FITC) eubacterial cells

(including most gram negative cells) in the sample are stained fluorescent green.

Using a filter for tetramethylrhodamine-5-isothiocyanate, Cy3-stained cells of C.

m. subsp. sepedonicus appear fluorescent red. Compare cell morphology with that

of the positive controls. Cells must be bright fluorescent and completely stained

The FISH test (section 9.4) must be repeated if the staining is aberrant. Scan

windows across two diameters at right angles and around the perimeter. For

samples showing no or low number of cells observe at least 40 microscope fields.

5.3.2. Observe for bright fluorescing cells with characteristic morphology

of C. m. subsp. sepedonicus in the test windows of the test slides (see web site

http://forum.europa.eu.int/Public/irc/sanco/Home/main).

The

fluorescence

intensity must be equivalent or better than that of the positive control strain. Cells

with incomplete staining or with weak fluorescence must be disregarded.

5.3.3. If any contamination is suspected the test must be repeated. This may

be the case when all slides in a batch show positive cells due to the contamination

of buffer or if positive cells are found (outside of the slide windows) on the slide

coating.

5.3.4. There are several problems inherent to the specificity of the FISH

test. Background populations of fluorescing cells with atypical morphology and

cross reacting saprophytic bacteria with size and morphology similar to C. m.

subsp. sepedonicus may occur, although much less frequent than in the IF test, in

potato heel end core and stem segment pellets.

5.3.5. Consider only fluorescing cells with typical size and morphology, see

in 5.3.2.

5.3.6. Interpretation of the FISH test result

(i) Valid FISH test results are obtained if bright green fluorescent cells of

size and morphology typical of C. m. subsp. sepedonicus are observed using the

FITC filter and if bright red fluorescent cells using the rhodamine filter in all

positive controls and not in any of the negative controls. If bright fluorescing cells

with characteristic morphology are found, estimate the average number of typical

cells per microscope field and calculate the number of typical cells per ml of

resuspended pellet (Appendix 4). Samples with at least 5 × 103 typical cells per ml

of resuspended pellet are considered potentially contaminated. Further testing is

required. Samples with less than 5 × 103 typical cells per ml of resuspended pellet

are considered negative.

(ii) The FISH test is negative if bright red fluorescent cells with size and

morphology typical of C. m. subsp. sepedonicus are not observed using the

rhodamine filter, provided that typical bright red fluorescent cells are observed in

the positive control preparations when using the rhodamine filter.

(Regulation 3)

"SCHEDULE V

The officially approved waste disposal methods referred to in Schedule IV

paragraph 1, shall conform to the following provisions such that any identifiable

risk of spreading the organism is obviated

(i) potato waste (including rejected potatoes and peelings) and any other

solid waste associated with the potatoes (including soil, stones and other debris)

shall be disposed by either,

-

disposal at an officially approved dedicated waste disposal site at

which there is no identifiable risk of escape of the organism into the

environment e.g. through seepage to agricultural land. The waste shall be

conveyed directly to the site under containment conditions such that there is

no risk of loss of the waste, or

-

incineration, or

-

other measures, provided that it has been established that there is

no identifiable risk of the organism spreading; such measures to be notified

to the Commission and to the other Member States.

(ii) liquid waste: prior to disposal, liquid waste containing suspended solids

shall be subjected to filtration or settlement processes to remove such solids. These

solids shall be disposed of as set out in paragraph (i).

The liquid waste shall then be either

-

heated to a minimum of 60°C throughout the entire volume

during at least 30 minutes prior to disposal, or

-

otherwise disposed of subject to official approval and under

official control such that there is no identifiable risk that the waste could

come into contact with agricultural land. The details thereof shall be notified

to the other Member States and to the Commission.

The options described in this Schedule also apply to the waste associated

with handling, disposal and processing of contaminated lots.".

PCR TEST

Principles

When the PCR test is used as the principal screening test and found to be

positive, the IF test must be performed as a second compulsory screening test.

When the PCR test is used as the second screening test and found to be positive,

further testing according to the flow scheme is required to complete the diagnosis.

Full exploitation of this method as principal screening test is only

recommended when specialised expertise has been acquired.

Note

Preliminary testing with this method should permit reproducible detection of

103 to 104 cells of C. m. subsp. sepedonicus per ml added to sample extracts which

previously tested negative. Optimisation experiments may be required to achieve

maximum levels of sensitivity and specificity in all laboratories.

Use validated PCR reagents and protocols. Preferably select a method with

an internal control.

Use appropriate precautions to avoid contamination of sample with target

DNA. The PCR test should be performed by experienced technicians, in dedicated

molecular biology laboratories, in order to minimise the possibility of

contamination with target DNA.

Negative controls (for DNA extraction and PCR procedures) should always

be handled as final samples in the procedure, to make evident whether any carry

over of DNA has occurred.

The following negative controls should be included in the PCR test

-

sample extract that previously tested negative for C. m. subsp.

Sepedonicus,

-

buffer controls used for extracting the bacterium and the DNA

from the sample,

-

PCR-reaction mix.

The following positive controls should be included

-

aliquots of resuspended pellets to which C. m. subsp. sepedonicus

has been added (preparation see Appendix 2),

-

a suspension of 106 cells per ml of C. m. subsp. sepedonicus in

water from a virulent isolate (e.g. NCPPB 2140 or NCPPB 4053),

-

if possible also use DNA extracted from positive control samples

in the PCR test.

To avoid potential contamination prepare positive controls in a separate

environment from samples to be tested.

Sample extracts should be as free as possible from soil. It could therefore in

certain cases advisible to prepare extractions from washed potatoes if PCR

protocols are to be used.

6.1. DNA purification methods

Use positive and negative control samples as described above.

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

A variety of methods are available for purification of target DNA from

complex sample substrates, thus removing inhibitors of PCR and other enzymatic

reactions and concentrating target DNA in the sample extract.

The following method has been optimised for use with the validated PCR

method shown in Appendix 6.

6.1.(a) Method according to Pastrik (2000)

1. Pipette 220 µl of lysis buffer (100 mM NaCl, 10 mM Tris-HCl [pH 8,0],

1 mM EDTA [pH 8,0]) into a 1,5 ml Eppendorf tube.

2. Add 100 µl sample extract and place in a heating block or waterbath at

95°C for 10 minutes.

3. Put tube on ice for five minutes.

4. Add 80 µl Lysozyme stock solution (50 mg lysozyme per ml in 10 mM

Tris HCl, pH 8,0) and incubate at 37°C for 30 minutes.

5. Add 220 µl of Easy DNA® solution A (Invitrogen), mix well by

vortexing and incubate at 65°C for 30 minutes.

6. Add 100 µl of Easy DNA® solution B (Invitrogen), vortex vigorously

until the precipitate runs freely in the tube and the sample is uniformly viscous.

Add 500 µl of chloroform and vortex until the viscosity decreases and

the mixture is homogeneous.

Centrifuge at 15 000 g for 20 minutes at 4°C to separate phases and

form the interphase.

Transfer the upper phase into a fresh Eppendorf tube.

Add 1 ml of 100% ethanol (-20°C) vortex briefly and incubate on ice for

10 minutes.

Centrifuge at 15 000 g for 20 minutes at 4°C and remove ethanol from

pellet.

Add 500 µl 80% ethanol (-20°C) and mix by inverting the tube.

Centrifuge at 15 000 g for 10 minutes at 4°C, save the pellet and remove

ethanol.

Allow the pellet to dry in air or in a DNA speed vac.

Resuspend the pellet in 100 µl sterile UPW and leave at room

temperature for at least 20 minutes.

Store at -20°C until required for PCR.

Spin down any white precipitate by centrifugation and use 5 µl of the

supernatant containing DNA for the PCR.

6.1.(b) Other methods

Other DNA extraction methods (e.g. Qiagen DNeasy Plant Kit) could be

applied providing that they are proven to be equally as effective in purifying DNA

from control samples containing 103 to 104 pathogen cells per ml.

6.2. PCR

6.2.1. Prepare test and control templates for PCR according to the validated

protocol (Appendix 6). Prepare one decimal dilution of sample DNA extract (1:10

in UPW).

6.2.2. Prepare the appropriate PCR reaction mix in a contamination-free

environment according to the published protocol (Appendix 6). The validated PCR

protocol is a multiplex reaction that also incorporates an internal PCR control.

6.2.3. Add 5 µl of DNA extract per 25 µl PCR reaction in sterile PCR

tubes.

6.2.4. Incorporate a negative control sample containing only PCR reaction

mix and add the same source of UPW as used in the PCR mix in place of sample.

6.2.5. Place tubes in the same thermal cycler which was used in preliminary

testing and run the appropriately optimized PCR programme (Appendix 6).

6.3. Analysis of the PCR product

6.3.1. Resolve PCR amplicons by agarose gel electrophoresis. Run at least

12 µl of amplified DNA reaction mixture from each sample mixed with 3 µl

loading buffer (Appendix 6) in 2,0% (w/v) agarose gels in tris-acetate-EDTA

(TAE) buffer (Appendix 6) at 5 to 8 V per cm. Use an appropriate DNA marker,

e.g. 100 bp ladder.

6.3.2. Reveal DNA bands by staining in ethidium bromide (0,5 mg per L)

for 30 to 45 min taking appropriate precautions for handling this mutagen.

6.3.3. Observe stained gel under short wave UV transillumination (e.g. λ =

302 nm) for amplified PCR products of the expected size (Appendix 6) and

document.

6.3.4. For all new findings/cases verify authenticity of the PCR amplicon

by performing restriction enzyme analysis on a sample of the remaining amplified

DNA by incubating at the optimum temperature and time with an appropriate

enzyme and buffer (see Appendix 6). Resolve the digested fragments by agarose

gel electrophoresis as before and observe characteristic restriction fragment pattern

under UV transillumination after ethidium bromide staining and compare with the

undigested and digested positive control.

Interpretation of the PCR test result

The PCR test is negative if the C. m. subsp. sepedonicus-specific PCR

amplicon of expected size is not detected for the sample in question but is detected

for all positive control samples (in case of multiplex PCR with plant specific

internal control primers: a second PCR-product of expected size must be amplified

with the sample in question).

The PCR test is positive if the C. m. subsp. sepedonicus-specific PCR

amplicon of expected size and restriction pattern (when required) is detected,

providing that it is not amplified from any of the negative control samples.

Reliable confirmation of a positive result can also be obtained by repeating the test

with a second set of PCR primers (section 9.3).

Note

Inhibition of the PCR may be suspected if the expected amplicon is obtained

from the positive control sample containing C. m. subsp. sepedonicus in water but

negative results are obtained from positive controls with C. m. subsp. sepedonicus

in potato extract. In multiplex PCR protocols with internal PCR controls,

inhibition of the reaction is indicated when neither of the two amplicons are

obtained.

Contamination may be suspected if the expected amplicon is obtained from

one or more of the negative controls.

7. BIOASSAY TEST

Note

Preliminary testing with this method should permit reproducible detection of

103 to 104 colony-forming units of C. m. subsp. sepedonicus per ml added to

sample extracts that previously tested negative (preparation see Appendix 2).

Highest sensitivity of detection can be expected when using freshly prepared

sample extract and optimal growth conditions. However, the method can be

successfully applied to extracts that have been stored under glycerol at -68 to -

86°C.

Some varieties of eggplant provide an excellent selective enrichment medium

for the growth of C. m. subsp. Sepedonicus even in the absence of symptoms and

also provide an excellent confirmatory host test.

Growth conditions should be optimal to reduce the risk of false negative test

results.

For cultural details, see Appendix 8.

7.1. Distribute the whole of the remaining test aliquot of the resuspended

pellet from section 3.1.6 or 3.2.5 between eggplants by one of the methods given

below (7.3 or 7.4). Use only plants at leaf stage two to three up to full expansion of

the third true leaf. In order to ensure complete utilisation of the resuspended pellet

as well as effective inoculation the procedures outlined below will require 15 to 25

eggplants per sample.

7.2. Do not water eggplants for one to two days prior to inoculation to

reduce turgor pressure.

7.3. Slit inoculation

7.3.1. Holding the plant between two fingers, pipette a drop (approximately

5 to 10 µl) of the suspended pellet on the stem between the cotyledons and the first

leaf.

7.3.2. Using a sterile scalpel, make a diagonal slit, about 1,0 cm long and

approximately 2/3 of the stem thickness deep, starting the cut from the pellet drop.

7.3.3. Seal the cut with sterile vaseline from a syringe.

7.4. Syringe inoculation

Inoculate the eggplant stems just above the cotyledons using a syringe fitted

with a hypodermic needle (not less than 23 G). Distribute the sample between the

eggplants.

7.5. As the positive controls, inoculate 5 plants with an aqueous suspension

of 105 to 106 cells per ml of a known culture of C. m. subsp. sepedonicus and,

where possible, with naturally infected tuber tissue (see section 4) by the same

inoculation method (7.3 or 7.4).

7.6. As the negative control, inoculate 5 plants with sterile pellet buffer by

the same inoculation method (7.3 or 7.4).

7.7. Incubate plants in quarantine facilities for up to four weeks at 18 to

24°C. Incubate plants with sufficient light and high humidity (70 to 80%) and

water to prevent water logging or wilting through water deficiency. C. m.

sepedonicus cells are killed at temperatures above 30°C and the optimum

temperature is 21°C. To avoid contamination incubate positive control and

negative control plants on clearly separated benches in a glasshouse or growth

chamber or, in case space is limited, ensure strict separation between treatments. If

plants for different samples must be incubated close together, separate them with

appropriate screens. When fertilising, watering, inspecting and any other

manipulations take great care to avoid cross-contamination. It is essential to keep

glasshouses and growth chambers free of all insect pests since they may transmit

the bacterium from sample to sample.

7.8. Examine regularly for symptoms starting after a week. Count the

number of plants showing symptoms. C. m. subsp. sepedonicus causes leaf wilting

in eggplants which may commence as a marginal or interveinal flaccidity. Wilted

tissue may initially appear dark green or mottled but turns paler before becoming

necrotic. Interveinal wilts often have a greasy water-soaked appearance. Necrotic

tissue often has a bright yellow margin. Plants are not necessarily killed; the longer

the period before symptoms develop, the greater the chance of survival. Plants may

outgrow the infection. Young eggplants are much more susceptible to low

populations of C. m. subsp. Sepedonicus than are older plants, hence the necessity

to use plants at or just before leaf stage 3.

Wilts may also be induced by populations of other bacteria or fungi present in

the tuber tissue pellet. These include Ralstonia solanacearum, Erwinia carotovora

subsp. carotovora and E. carotovora subsp. atroseptica, Erwinia chrysanthemi,

Phoma exigua var. foveata, as well as large populations of saprophytic bacteria. In

particular Erwinia chrysanthemi can cause leaf symptoms and wilt that is very

similar to symptoms of C. m. sepedonicus. The only difference is blackening of the

stems in case in Erwinia chrysanthemi infections. Other wilts can be distinguished

from those caused by C. m. subsp. sepedonicus since whole leaves or whole plants

wilt rapidly. Also a Gram stain can be prepared: this test will differentiate C. m.

subsp. sepedonicus from Erwinia spp.

7.9. As soon as symptoms in eggplants are observed reisolation should be

performed, using sections of wilted leaf tissue or stem tissue from plants (see 3.1.3

for tissue maceration). Surface disinfect the eggplant leaves and stems by wiping

with 70% ethanol. Perform an IF test or PCR on the eggplant sap and isolate on to

suitable (selective) media (see section 8). A Gram stain (Appendix 9) may also be

prepared. Identify purified cultures of presumptive C. m. subsp. sepedonicus and

confirm pathogenicity (see section 9 and 10).

7.10. Under certain circumstances, in particular where growing conditions

are not optimal, it may be possible for C. m. subsp. sepedonicus to exist as a latent

infection within eggplants even after incubation periods up to 4 weeks. If no

symptoms are observed after 4 weeks perform IF/PCR on a composite sample of 1

cm stem sections of each test plant taken above the inoculation site. If the test is

positive reisolation on suitable (selective) media should be performed following

the procedure under section 8. Identify purified cultures of presumptive C. m.

subsp. sepedonicus and confirm pathogenicity (section 9 and 10).

Interpretation of the bioassay test result.

Valid Bioassay test results are obtained when plants of the positive control

show typical symptoms, the bacteria can be reisolated from these plants and no

symptoms are found on the negative controls.

The bioassay test is negative if test plants are not infected by C. m. subsp.

sepedonicus, and provided that C. m. subsp. sepedonicus is detected in positive

controls.

The bioassay test is positive if the test plants are infected by C. m. subsp.

sepedonicus.

8. ISOLATION OF C. M. SUBSP. SEPEDONICUS

Note

Diagnosis is only completed if C. m. subsp. sepedonicus is isolated,

subsequently identified (see section 9). and confirmed by a pathogenicity test

(section 10). Although C. m. subsp. sepedonicus is a fastidious organism, it can be

isolated from symptomatic tissue.

However, it may be outgrown by rapidly growing saprophytic bacteria and,

therefore, isolations directly from the tuber or stem tissue pellet (section 3.1.6 or

3.2.5) is difficult. With selective medium and appropriate dilution of the

resuspended pellet from the heel end cores or stems of potatoes direct isolation of

C. m. subsp. sepedonicus may be possible.

Isolations shall be made from all symptomatic potato tubers or stem

segments and from eggplants were no symptoms are observed but IF/PCR test

from the composite sample was positive (see section 7.10). Maceration of

eggplants stems when necessary should be carried out as described in section

3.1.3.

As positive controls, prepare decimal dilutions from a suspension of 10 6 cfu

per ml of C. m. subsp. sepedonicus (e.g. NCPPB 4053 or PD 406). To avoid any

possibility of contamination, prepare positive controls totally separated from

samples to be tested.

For each newly prepared batch of a selective medium its suitability for

growth of the pathogen should be tested before it is used to test routine samples.

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

8.1. Selective plating

8.1.1. From a 100 µl aliquot from a resuspended potato pellet sample or

eggplant sap make 10-fold dilutions in pellet buffer (Appendix 3).

8.1.2. Isolation from undiluted potato pellet usually fails due to the

fastidious growth habit of Cms and competition by saprophytes. Since the

bacterium is usually present in high populations in infected tissues, the

saprophytes can usually be diluted out, whilst the pathogen remains. It is therefore

recommended to spread 100 µl from each of the samples, 1/100 up to 1/10 000

dilutions onto MTNA medium or NCP-88 medium (Appendix 5) (if using 90 mm

diameter petri dishes- adjust volume for alternative dish sizes), using spreaders

(hockey sticks) and the spread plate technique.

Note

An alternative strategy is to spread out the initial 100 µl potato pellet aliquot

onto a first agar plate with a spreader and then remove the spreader to a second

agar plate, streaking out any residue left on the spreader; finally repeat this with a

third plate, thus giving a dilution plating effect via the spreader.

8.1.3. Incubate plates in the dark at 21 to 23°C.

8.1.4. Initial examinations of the plates including, by reference to the

control plates, counts of any C. m. subsp. sepedonicus like colonies are made after

3 days, with further counts after 5, 7, eventually 10 days.

8.2. Purification of suspicious colonies

Note

Subculturing of C. m. subsp. sepedonicus-like colonies should be carried out

onto YGM media for eggplant inoculation and/or subsequent identification; this

should be done before the plates become too overgrown i.e. preferably after three

to five days.

8.2.1. Streak C. m. subsp. sepedonicus-like colonies on to one of the

following media: (formulae are given in Appendix 5)

nutrient dextrose agar (for use in subculturing only),

yeast peptone glucose agar,

yeast extract mineral salts agar.

Incubate at 21°C to 24°C for up to 10 days.

C. m. subsp. sepedonicus is slow-growing, usually producing pin-point,

cream, domed colonies within 10 days. Photos of typical colonies of C. m. subsp.

sepedonicus (see web site http://forum.europa.eu.int/Public/irc/sanco/Home/

main).

8.2.2. Re-streak to establish purity.

Growth rates are improved with subculture. Typical colonies are creamy-

white or ivory, occasionally yellow, rounded, smooth, raised, convex-domed,

mucoid-fluidal, with entire edges and usually 1 to 3 mm in diameter.

A simple Gram stain (Appendix 9) may help to select colonies for further

testing.

8.2.3. Identify presumptive cultures (see section 9) and perform a

pathogenicity test (see section 10).

9. IDENTIFICATION

Identify pure cultures of presumptive C. m. subsp. sepedonicus isolates using

at least two of the following tests based on different biological principles.

Include known reference strains where appropriate for each test performed.

9.1. Nutritional and enzymatic identification tests

Determine the following phenotypic properties which are universally present

or absent in C. m. subsp. sepedonicus, according to the methods of Lelliott and

Stead (1987), Klement et al. (1990), Schaad (2001), Anonymous (1987).

All media should be incubated at 21°C and examined after six days. If no

growth has occurred, incubate for up to 20 days.

All tests must include a known C. m. subsp. sepedonicus control. Nutritional

and physiological tests must be made using inocula from nutrient agar subcultures.

Morphological comparisons must be made from nutrient dextrose agar cultures.

Tests

Expected result

Oxidation/Fermentation (O/F) test

Inert or weakly oxidative

Oxidase activity

-

Growth at 37°C

-

Urease activity

-

Aesculin hydrolysis

+

Starch hydrolysis

- or weak

Tolerance of 7% NaCl

-

Indole production

-

Catalase activity

+

H 2 S production

-

Citrate utilization

-

Gelatin liquefaction

-

Acid glycerol

-

Acid from lactose

- or weak

Acid from rhamnose

-

Acid from salicin

-

Gram stain (Appendix 9)

+

9.2. IF-test

(a)

Prepare a suspension of approximately 106 cells per ml in IF

buffer (Appendix 3).

(b)

Prepare a 2-fold dilution series of an appropriate antiserum.

(c)

Apply the IF procedure (section 4).

(d) A positive IF test is achieved if the IF titre of the culture is

equivalent to that of the positive control.

9.3. PCR test

(a)

Prepare a suspension of approximately 106 cells per ml in ultra

pure water (UPW).

(b) Heat 100 µl of the cell suspension in closed tubes in a heating

block or boiling waterbath at 100°C for four minutes. If required, addition of

freshly-prepared NaOH to a final concentration of 0,05M may assist cell

lysis. The samples may then be stored at -16 to -24°C until required.

(c)

Apply appropriate PCR procedures to amplify C. m. subsp.

sepedonicus specific amplicons (e. g. Pastrik, 2000; see Appendix 4; Li and

de Boer, 1995; Mills et al., 1997; Pastrik and Rainey, 1999; Schaad et al.,

1999. (d) A positive identification of C. m. subsp. sepedonicus is achieved

if the PCR amplicons are the same size and have the same restriction

fragment length polymorphisms as for the positive control strain.

9.4. FISH test

(a)

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

(b)

Apply the FISH procedure (section 5).

(c)

A positive FISH test is achieved if the same reactions are

achieved from the culture and the positive control.

9.5. Fatty acid profiling (FAP)

(a)

Grow the culture on trypticase soy agar (Oxoid) for 72 hours at

21°C (+/– 1°).

(b)

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

(c)

A positive FAP test is achieved if the profile of the presumptive

culture is identical to that of the positive control. The presence of

characteristic fatty acids are 15:1 Anteiso A, 15:0 Iso, 15:0 Anteiso, 16:0 Iso,

16:0 and 17:0 Anteiso is highly indicative of C. m. sepedonicus. Other genera

such as Curtobacterium, Arthrobacter and Micrococcus also have some of

these acids but 15:1 Anteiso A is a rare acid in these bacteria but occurs in all

Clavibacter spp. at between 1 to 5%. In C. m. sepedonicus the value is

usually around 5%.

9.6. BOX-PCR

(a)

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

(b)

Apply the test according the procedure (Smith et al., 2001).

10. CONFIRMATION TEST

The pathogenicity test must be performed as final confirmation of a diagnosis

of C. m. subsp. sepedonicus and for assessment of virulence of cultures identified

as C. m. subsp. sepedonicus

10.1. Prepare an inoculum of approximately 106 cells per ml from three day

cultures of the isolate to be tested and an appropriate positive control strain of C.

m. subsp. sepedonicus.

10.2. Inoculate 5 to 10 eggplant stems of young seedlings at leaf stage 3

(section 7.3 or 7.4).

10.3. Incubate at 18 to 24°C with sufficient light and high relative humidity

with appropriate watering to avoid waterlogging or drought stress (section 7.7).

With pure cultures, typical wilting should be obtained within two weeks but plants

not showing symptoms (see section 7.8) after this time should be incubated up to

three weeks at temperatures conducive to eggplant growth but not exceeding 25°C

(Appendix 8). If after three weeks symptoms are not present, the culture cannot be

confirmed as being a pathogenic form of C. m. subsp. sepedonicus.

10.4. Isolate from symptomatic plants by removing a section of stem 2 cm

above the inoculation point. Comminute and suspend in a small volume of sterile

distilled water or 50 mM phosphate buffer (Appendix 3). Isolate from the

suspension by dilution spreading or streaking onto MTNA and YPGA (Appendix

5), incubate for three to five days at 21 to 23°C and observe the formation of

colonies typical of C. m. subsp. sepedonicus.

Appendix 1

Laboratories involved in optimisation and validation of protocols

Laboratory ( 1 )

Location

Country

Agentur für Gesundheit und Ernährungssicherheit

Vienna and Linz

Austria

Departement Gewasbescherming

Merelbeke

Belgium

Plantedirektoratet

Lyngby

Denmark

Central Science Laboratory

York

England

Scottish Agricultural Science Agency

Edinburgh

Scotland

Laboratoire National de la Protection des Végétaux,

Angers

France

Unité de Bactériologie

Laboratoire National de la Protection des Végétaux,

Le Rheu

France

Station de Quarantaine de la Pomme de Terre

Biologische Bundesanstalt

Kleinmachnow

Germany

Pflanzenschutzamt Hannover

Hannover

Germany

State Laboratory

Dublin

Ireland

Plantenziektenkundige Dienst

Wageningen

Netherlands

Norwegian Crop Research Institute, Plant Protection

Aas

Norway

Centre

Direcção-Geral de Protecção das Culturas

Lisbon

Portugal

Nacionalni institut za biologijo

Ljubljana

Slovenia

Centro de Diagnostico de Aldearrubia

Salamanca

Spain

( 1 ) Contact scientists: see website http://forum.europa.eu.int/Public/irc/sanco/Home/main

Appendix 2

Preparation of positive and negative controls for the core screening tests

PCR/IF and FISH

Produce a 72 hour culture of a virulent strain of C. m. subsp. sepedonicus

(NCPPB 4053 or PD 406) on MTNA basal medium and suspend in 10 mM

phosphate buffer to obtain a cell density of approximately 1 to 2 × 108 cfu per ml.

This is usually obtained by a faintly turbid suspension equivalent to an optical

density of 0,20 at 600 nm.

Remove the heel end cores of 200 tubers taken from a white skin variety

production known to be free from C. m. subsp. sepedonicus.

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

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

Transfer 100 µl of the suspension of C. m. subsp. sepedonicus to the first

microvial. Vortex.

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

microvials.

The six contaminated microvials will be used as positive controls. The four

non-contaminated microvials will be used as negative controls. Label the

microvials accordingly.

Prepare aliquots of 100 µl in sterile 1,5 ml microvials thus obtaining nine

replicas of each control sample. Store at -16 to -24°C until use.

The presence and quantification of C. m. subsp. sepedonicus in the control

samples should be first confirmed by IF.

For the PCR test perform DNA extraction from positive and negative control

samples with each series of test samples.

For IF and FISH tests perform assays on positive and negative control

samples with each series of test samples.

For IF, FISH and PCR assays C. m. subsp. sepedonicus must be detected in at

least the 106 and 104 cells/ml of the positive controls and not in any of the

negative controls.

Appendix 3

Buffers for test procedures

GENERAL: Unopened sterilized buffers can be stored for up to one year.

1. Buffers for extraction procedure

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

This buffer is used for extraction of the bacterium from plant tissues by

homogenisation or shaking.

Na 2 HPO 4 (anhydrous)

4,26 g

KH 2 PO 4

2,72 g

Distilled water

1.00 L

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

min.

Additional components may be useful as follows

Purpose

Quantity (per L)

Lubrol flakes

Deflocculant (*)

0,5 g

DC silicone antifoam

Anti-foam agent (*)

1,0 ml

Tetrasodium pyrophosphate

Anti-oxidant

1,0 g

Polyvinylpyrrolidone-40 000 (PVP-40)

Binding of PCR inhibitors

50 g

(*) For use with homogenisation extraction method.

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

This buffer is used for resuspension and dilution of potato tuber heel-end

core extracts following concentration to a pellet by centrifugation.

Na 2 HPO 4 .12H 2 O

2,7 g

NaH 2 PO 4 .2H 2 O

0,4 g

Distilled water

1,00 L

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

min.

2. Buffers for the IF test

2.1. IF-Buffer (10 mM phosphate buffered saline (PBS), pH 7,2)

This buffer is used for dilution of antibodies.

Na 2 HPO 4 .12H 2 O

2,7 g

NaH 2 PO 4 .2H 2 O

0,4 g

NaCl

8,0 g

Distilled water

1.0 L

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

min.

2.2. IF-buffer-Tween

This buffer is used to wash slides.

Add 0,1% Tween 20 to the IF buffer.

2.3. Phosphate buffered glycerol, pH 7,6

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

enhance fluorescence.

Na 2 HPO 4 .12H 2 O

3,2 g

NaH 2 PO 4 .2H 2 O

0,15 g

Glycerol

50 ml

Distilled water

100 ml

Anti-fading mountant solutions are commercially available e.g. Vectashield®

(Vector Laboratories) or Citifluor® (Leica).

Appendix 4

Determination of contamination level in IF and FISH tests

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

2. Calculate the number of typical fluorescent cells per microscope slide

window (C)

C = c × S/s

Where

S = surface area of window of multispot slide, and

s = surface area of objective field.

s = πi 2 /4G 2 K 2 where

i =

field coefficient (varies from 8 to 24 depending

upon ocular type)

K = tube coefficient (1 or 1,25)

G = magnification of objective (100x, 40x etc.).

3. Calculate the number of typical fluorescent cells per ml of resuspended

pellet (N)

N = C × 1 000/y × F

where

y = volume of resuspended pellet on each window, and

F = dilution factor of resuspended pellet.

Appendix 5

Media for isolation and culture of C. m. subsp. sepedonicus

(a)

General growth media

Nutrient agar (NA)

Nutrient Agar (Difco)

23,0 g

Distilled water

1,0 L

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

Nutrient dextrose agar (NDA)

Difco bacto nutrient agar containing 1% D(+) glucose (monohydrate).

Sterilize by autoclaving at 115°C for 20 minutes.

Yeast peptone glucose agar (YPGA)

Yeast Extract (Difco)

5,0 g

Bacto-Peptone (Difco)

5,0 g

D(+) Glucose (monohydrate)

10,0 g

Bacto-Agar (Difco)

15,0 g

Distilled water

1,0 L

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

Yeast extract mineral salts medium (YGM)

Bacto-Yeast-Extract (Difco)

2,0 g

D(+) Glucose (monohydrate)

2,5 g

K 2 HPO 4

0,25 g

KH 2 PO 4

0,25 g

MgSO 4 .7H 2 O

0,1 g

MnSO 4 .H 2 O

0,015 g

NaCl

0,05 g

FeSO 4 .7H 2 O

0,005 g

Bacto-Agar (Difco)

18 g

Distilled water

1,0 L

Dissolve ingredients and sterilize 0,5 litre volumes of medium by autoclaving

at 115°C for 20 minutes.

(b) Validated selective growth media

MTNA medium

Unless otherwise stated all media components are from BDH.

Yeast extract (Difco)

2,0 g

Mannitol

2,5 g

K 2 HPO 4

0,25 g

KH 2 PO 4

0,25 g

NaCl

0,05 g

MgSO 4 .7H 2 O

0,1 g

MnSO 4 .H 2 O

0,015 g

FeSO 4 .7H 2 O

0,005 g

Agar (Oxoid no. 1)

16,0 g

Distilled water

1,0 L

Dissolve ingredients, adjust pH to 7,2. After autoclaving (at 121°C for 15

minutes) and cooling down to 50°C, add the antibiotics: trimethoprim 0,06 g,

nalidixic acid 0,002 g, amphotericin B 0,01 g.

Stock antibiotic solutions: trimethoprim (Sigma) and nalidixic acid (Sigma)

(both at 5 mg/ml), in 96 % methanol, amphotericin B (Sigma) (1 mg/ml) in

dimethyl sulfoxide. Stock solutions are filter-sterilized.

Note

Durability of basal medium is three months. After antibiotics are added

durability is one month when stored refrigerated.

NCP-88 medium

Nutrient agar (Difco)

23 g

Yeast extract (Difco)

2 g

D-mannitol

5 g

K 2 HPO 4

2 g

KH 2 PO 4

0,5 g

MgSO 4 .7H 2 O

0,25 g

Distilled water

1,0 L

Dissolve ingredients, adjust pH to 7,2. After autoclaving and cooling down to

50°C, add the following antibiotics

Polymyxin B sulphate (Sigma) 0,003 g, nalidixic acid (Sigma) 0,008 g,

Cycloheximide (Sigma) 0,2 g.

Dissolve antibiotics in stock solutions as follows: nalidixic acid in 0,01 M

NaOH, cycloheximide in 50% ethanol, polymyxin B sulphate in distilled water.

Stock solutions are filter-sterilized.

Note

Durability of basal medium is three months. After antibiotics are added

durability is one month when stored refrigerated.

Appendix 6

Validated PCR protocol and reagents

Note

Preliminary testing should permit reproducible detection of at least 103 to

104 cells of C. m. sepedonicus per ml of sample extract.

Preliminary testing should also show no false positive results with a panel of

selected bacterial strains.

1. Multiplex PCR protocol with internal PCR control (Pastrik, 2000)

1.1. Oligonucleotide primers

Forward primer PSA-1

5'- ctc ctt gtg ggg tgg gaa aa -3'

Reverse primer PSA-R

5'- tac tga gat gtt tca ctt ccc c -3'

Forward primer NS-7-F

5'- gag gca ata aca ggt ctg tga tgc -3'

Reverse Primer NS-8-R

5’- tcc gca ggt tca cct acg ga -3’

Expected amplicon size from C. m. subsp. sepedonicus template DNA =

502 bp (PSA-primer set).

Expected amplicon size from the 18S rRNA internal PCR control = 377

bp (NS-primer set).

1.2. PCR reaction mix

Reagent

Quantity per reaction Final concentration

Sterile UPW

15,725 µl

10x PCR buffer ( ) (15 mM MgCl 2 )

2,5 µl

1x (1,5 mM MgCl 2 )

BSA (fraction V) (10%)

0,25 µl

0,1%

d-nTP mix (20 mM)

0,125 µl

0,1 mM

Primer PSA-1 (10 µM)

0,5 µl

0,2 µM

Primer PSA-R (10 µM)

0,5 µl

0,2 µM

Primer NS-7-F (10 µM) ( )

0,1 µl

0,04 µM

Primer NS-8-R (10 µM) ( )

0,1 µl

0,04 µM

Taq polymerase (5 U/µl) ( )

0,2 µl

1,0 U

Sample volume

5,0 µl

Total volume

25,0 µl

(1)

Methods were validated using Taq polymerase from Perkin Elmer (AmpliTaq or Gold) and Gibco BRL.

(2) Concentration of primers NS-7 F and NS-8-R were optimised for potato heel end core extraction using

the homogenization method and DNA purification according to Pastrik (2000) (see section 6.1.a) and 6.2). Re-

optimisation of reagent concentrations will be required if extraction by shaking or other DNA isolation

methods are used.

1.3. PCR reaction conditions

Run the following programme

1 cycle of

(i)

3 minutes at 95°C (denaturation of template DNA)

10 cycles of: (ii)

1 minute at 95°C (denaturation of template DNA)

(iii)

1 minute at 64°C (annealing of primers)

(iv)

1 minute at 72°C (extension of copy)

25 cycles of: (v)

30 seconds at 95°C (denaturation of template DNA)

(vi)

30 seconds at 62°C (annealing of primers)

(vii) 1 minute at 72°C (extension of copy)

1 cycle of

(viii) 5 minutes at 72°C (final extension)

(ix)

hold at 4°C.

Note

This programme is optimised for use with an MJ Research PTC 200 thermal

cycler. Modification of the duration steps of cycles (ii), (iii) (iv), (v), (vi) and (vii)

may be required for use with other models.

1.4. Restriction enzyme analysis of amplicon.

PCR products amplified from C. m. subsp. sepedonicus DNA produce a

distinctive restriction fragment length polymorphism with enzyme Bgl II after

incubation at 37°C for 30 minutes. The restriction fragments obtained from C. m.

subsp. sepedonicus-specific fragment are 282 bp and 220 bp in size.

2. Preparation of the Loading buffer

2.1. Bromphenol blue (10%-stock solution)

Bromphenol blue

5 g

Distilled Water (bidest)

50 ml

2.2. Loading buffer

Glycerol (86%)

3,5 ml

Bromphenol blue (5.1)

300 µl

Distilled Water (bidest)

6,2 ml

3. 10x Tris Acetate EDTA (TAE) buffer, pH 8,0

Tris buffer

48,4 g

Glacial acetic acid

11,42 ml

EDTA (disodium salt)

3,72 g

Distilled water

1,00 L

Dilute to 1x before use.

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

Appendix 7

Validated reagents for FISH test

1. Oligo-probes

Cms-specific probe CMS-CY3-01

5'- ttg cgg ggc gca cat ctc tgc acg -3'

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

2. Fixative solution

[WARNING! THE FIXATIVE CONTAINS PARAFORMALDEHYDE WHICH

IS TOXIC. WEAR GLOVES AND DO NOT INHALE. IT IS ADVISABLE TO WORK

IN A FUME CUPBOARD]

(i) Heat nine ml molecular grade water (e.g. Ultra pure water (UPW)) to

about 60°C and add 0,4 g paraformaldehyde. Paraformaldehyde dissolves after

adding five drops of 1N NaOH and stirring with a magnetic stirrer.

(ii) Adjust pH to 7,0 by addition of 1ml of 0,1 M phosphate buffer (PB; pH

7,0) and five drops of 1 N HCl. Check pH with indicator strips and adjust if

necessary with HCl or NaOH.

[WARNING! DO NOT USE A PH METER IN SOLUTIONS WITH

PARAFORMALEDHYDE]

(iii) Filter the solution through a 0,22 µm membrane filter and keep dust-

free at 4°C until further use.

(iv) Note

Alternative fixative solution: 96% ethanol.

3. 3x Hybmix

NaCl

2,7 M

Tris-HCl

60 mM (pH 7,4)

EDTA (filter sterilised and autoclaved)

15 mM

Dilute to 1x as required.

4. Hybridisation solution

1x Hybmix

Sodium dodecyl sulphate (SDS)

0,01%

probe EUB 338

5 ng/µl

probe CMSCY301

5 ng/µl

Prepare quantities of hybridisation solution according to the calculations in

Table. For each slide (containing two different samples in duplicate) 90 µl

hybridisation solution is required.

Table: Suggested quantities for preparation of hybridisation mix

2 slides

8 slides

Sterile UPW

50,1

200,4

3x hybmix

30,0

120,0

1% SDS

0,9

3,6

Probe EUB 338 (100 ng/µl)

4,5

18,0

Probe CMSCY301 (100 ng/µl)

4,5

18,0

Total volume (µl)

90,0

360,0

NB. Store all solutions containing light sensitive oligo-probes in the dark at -

20°C. Protect from direct sunlight or electric light during use.

5. 0,1M Phosphate buffer, pH 7,0

Na 2 HPO 4

8,52 g

KH 2 PO 4

5,44 g

Distilled water

1,00 L

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

min.

Appendix 8

Eggplant culture

Sow seeds of eggplant (Solanum melongena) in pasteurized seed compost.

Transplant seedlings with fully expanded cotyledons (10 to 14 days) into

pasteurized potting compost.

Eggplants should be grown in a glasshouse with the following environmental

conditions

Day length

14 hours or natural day length if greater;

Temperature

day

21 to 24°C,

night

15°C.

Susceptible varieties of eggplant

‘Black Beauty’,

‘Long Tom’,

‘Rima’,

‘Balsas’

Supplier: see website http://forum.europa.eu.int/Public/irc/sanco/Home/main

Appendix 9

Gram stain procedure (Hucker’s modification) (Doetsch, 1981) ( * )

Crystal violet solution

Dissolve 2 g crystal violet in 20 ml 95% ethanol.

*Commercially available solutions and staining kits can also be used.

Dissolve 0,8 g ammonium oxalate in 80 ml distilled water.

Mix the two solutions.

Lugol’s iodine

Iodine

1 g

Potassium iodide

2 g

Distilled water

300 ml

Grind the solids together in a pestle and mortar. Add to the water and stir to

dissolve in a closed container.

Safranin counterstain solution

Stock solution

Safranin O

2,5 g

95% ethanol

100 ml

Mix and store.

Dilute: 1:10 to obtain a working solution.

Staining procedure

1. Prepare smears, air dry and heat fix.

2. Flood slide with crystal violet solution for one minute.

3. Wash briefly with tap water.

4. Flood with Lugol’s iodine for one minute.

5. Wash with tap water and blot dry.

6. Decolourise with 95% ethanol, added dropwise, until no further colour

is removed or immerse with gentle agitation for 30 seconds.

7. Wash in tap water and blot dry.

8. Flood with safranin solution for 10 seconds.

9. Wash with tap water and blot dry.

Gram positive bacteria stain violet-blue; Gram negative bacteria stain pink-

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