Regulations, 2007.

Suppliment tal-Gazzetta tal-Gvern ta’ Malta, Nru. 18,040, 23 ta’ Frar, 2007

Taqsima B

L.N. 33 of 2007

PLANT QUARANTINE ACT

(ACT NO. XVIII OF 2001)

Control of Ralstonia solanacearum (Smith) Yabuuchi et al (Amendments)

Regulations, 2007.

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:-

Title and scope.

(1) The title of these regulations is Control of Ralstonia solanacearum (Smith)

Yabuuchi et al (Amendments) Regulations, 2007, and they shall be read and construed as

one with the Control of Ralstonia solanacearum (Smith) Yabuuchi et al Regulations,

2004, hereinafter referred to as ‘ the principal regulations’. These regulations shall come

in force on 1 st April 2007.

(2) The scope of these regulations is to transpose Council Directive 2006/63/EC

amending the Annex II and VII to Council Directive 98/57/EEC on the control of

Ralstonia solanacearum (Smith) Yabuuchi et al.

Amends Schedules II to VII of the principal regulations.

Schedules II to VII of the principal regulations shall be replaced by the text in Annex to

this regulation.

B

ANNEX

‘SCHEDULE II

TEST SCHEME FOR DIAGNOSIS, DETECTION AND IDENTIFICATION OF

RALSTONIA SOLANACEARUM (SMITH) YABUUCHI ET AL.

SCOPE OF THE TEST SCHEME

The presented scheme describes the various procedures involved in

(i) Diagnosis of brown rot in potato tubers and of bacterial wilt in potato, tomato and

some other host plants;

(ii) Detection of Ralstonia solanacearum in samples of potato tubers, potato-, tomato-

and other host plants, water and soil;

(iii) Identification of Ralstonia solanacearum (R. solanacearum).

GENERAL PRINCIPLES

Optimised protocols for the various methods, validated reagents and details for the

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

laboratories that were included in optimization and validation of protocols is provided in

Appendix 1.

Since the protocols involve detection of a quarantine organism and will include the use of

viable cultures of R. solanacearum as control materials, it will be necessary to perform

the procedures under suitable quarantined conditions with adequate waste disposal

facilities and under the conditions of appropriate licences as issued by the official plant

quarantine authorities.

Testing parameters must assure consistent and reproducible detection of levels of R.

solanacearum at the set thresholds of the selected methods.

Precise preparation of positive controls is imperative.

Testing according to the required thresholds also implies correct settings, maintenance

and calibration of equipment, careful handling and preservation of reagents and all

measures to prevent contamination between samples, e.g. separation of positive controls

from test samples. Quality control standards must be applied to avoid administrative and

other errors, especially concerning labelling and documentation.

A suspected occurrence, as referred to in Regulation 6(3) of the principal regulations

implies a positive result in diagnostic or screening tests performed on a sample as

specified in flow charts below. A positive first screening test (IF test, PCR/FISH,

selective isolation) must be confirmed by a second screening test based on a different

biological principle.

If the first screening test is positive, then contamination with R. solanacearum is

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

positive, than the suspicion is confirmed (suspected occurrence) and the testing according

to the scheme must be continued. If the second screening test is negative, then the sample

is considered not contaminated with R. solanacearum.

Confirmed presence as referred to in regulation 7(1) in the principal regulations implies

the isolation and identification of a pure culture of R. solanacearum with confirmation of

pathogenicity.

SECTION I

B

APPLICATION OF THE TEST SCHEME

1. Detection scheme for the diagnosis of brown rot and bacterial wilt (Ralstonia

solanacearum) in potato tubers and potato, tomato or other host plants with symptoms

of brown rot or bacterial wilt.

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

suspect of brown rot or vascular wilt. It involves a rapid screening test, isolation of the

pathogen from infected vascular tissue on (selective) medium and, in case of a positive result,

identification of the culture as Ralstonia solanacearum.

( 1 ) For description of symptoms see Section II.1.

( 2 ) Rapid diagnostic tests facilitate presumptive diagnosis but are not essential. A negative

result does not always guarantee absence of the pathogen

( 3 ) Streaming test for bacterial ooze from vascular stem tissue is described in Section VI.A.1.

( 4 ) Test for poly-β-hydroxybutyrate granules in bacterial cells is described in Section VI.A.2.

( 5 ) Serological agglutination tests on bacterial ooze or extracts from symptomatic tissue are

described in Section VI.A.3.

( ) If test on bacterial ooze suspended in water or symptomatic tissue extracts is described in

Section VI.A.5.

( ) FISH test on bacterial ooze suspended in water or symptomatic tissue extracts is described

in Section VI.A.7.

( ) ELISA test on bacterial ooze suspended in water or symptomatic tissue extracts is

described in Section VI.A.8.

( ) PCR test on bacterial ooze suspended in water or symptomatic tissue extracts is described

in Section VI.A.6.

( 10 ) The pathogen is usually easily isolated from symptomatic plant material by dilution

plating (Section II.3).

( 11 ) Typical colony morphology is described in Section II.3.d.

( 12 ) Culturing may fail from advanced stages of infection due to competition or overgrowth by

saprophytic bacteria. If disease symptoms are typical, but the isolation test is negative,

then the isolation must be repeated, preferably using a selective plate test.

( 13 ) Reliable identification of pure cultures of presumptive R. solanacearum isolates is

achieved using the tests described in Section VI.B. Sub-specific characterization is

optional but recommended for each new case.

( 14 ) The pathogenicity test is described in Section VI.C.

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 and for all other cases, the relevant

methods set out in Schedule II, and confirmation or refutation by completion of the said

methods is awaited, there should be 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,

— a sample of the infected tomato or eggplant material inoculated with the tuber or plant

extract, where appropriate,

And

— the isolated culture of the organism,

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

Scheme for detection and identification of Ralstonia solanacearum in samples of

asymptomatic potato tubers

Principle

The testing procedure is intended for detection of latent infections in potato tubers. A positive

result from at least two screening tests (3), based on different biological principles, must be

B

complemented by the isolation of the pathogen; followed by, in case of isolation of typical

colonies, confirmation of a pure culture as R. solanacearum A positive result from only one of

the screening tests is not sufficient to consider the sample suspect.

Screening tests and isolation tests must permit detection of 103 to 104 cells/ml of resuspended

pellet, included as positive controls in each series of tests.

( 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 III.1.1.

( 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 or other tests are negative the sample is considered

negative. Further tests are not necessary.

( ) The IF test is described in Section VI.A.4.

( 5 ) The selective isolation test is described in Section VI.A.4.

( 6 ) PCR tests are described in Section VI.A.6.

( 7 ) The FISH test is described in Section VI.A.7.

( 8 ) ELISA tests are described in Section VI.A.8.

( 9 ) The bioassay is described in Section VI.A.9.

( 10 ) Typical colony morphology is described in Section II.3.d.

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

clear positive results are obtained in screening test, 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.

( 12 ) Reliable identification of pure cultures of presumptive R. solanacearum isolates is

achieved using the tests described in Section VI.B.

( 13 ) The pathogenicity test is described in Section VI.C.

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

(i) places of production,

— growing or having grown, potatoes which are clonally related to potatoes found to be

infected with the organism,

— growing or having grown, tomatoes which are from the same source as tomatoes found to

be infected with the organism,

— growing or having grown, potatoes or tomatoes which have been placed under official

control because of the suspected occurrence of the organism,

— growing or having grown, potatoes which are clonally related to potatoes that have been

grown on places of production found to be infested with the organism,

— growing potatoes or tomatoes and located in the neighbourhood of infested places of

production, including such places of production sharing production equipment and facilities

directly or through a common contractor,

— using surface water for irrigation or spraying from any source confirmed or suspected to be

contaminated with the organism,

— using surface water for irrigation or spraying from a source used in common with places of

production confirmed or suspected to be infested with the organism,

— flooded or have been flooded with surface water confirmed or suspected to be

contaminated with the organism;

And

(ii) surface water used for irrigation or spraying of, or which has flooded field(s) or place(s) of

production confirmed to be infested with the organism.

Scheme for detection and identification of Ralstonia solanacearum in samples of

asymptomatic potato, tomato or other host plants

B

( 1 ) See section III.2.1. for recommended sample sizes.

( 2 ) Pathogen extraction and concentration methods are described in Section III.2.1.

( 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 or other tests are negative the sample is considered

negative. Further tests are not necessary.

( 4 ) The selective isolation test is described in Section VI.A.4.

( 5 ) The IF test is described in Section VI.A.5.

( 6 ) PCR tests are described in Section VI.A.6.

( 7 ) The FISH test is described in Section VI.A.7.

( 8 ) ELISA tests are described in Section VI.A.8.

( 9 ) The bioassay is described in Section VI.A.9.

( 10 ) Typical colony morphology is described in Section II.3.d

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

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

repeat the isolation tests.

( 12 ) Reliable identification of pure presumptive R. solanacearum cultures is achieved using

the tests described in Section VI.B.

( 13 ) The pathogenicity test is described in Section VI.C.

SECTION II

DETAILED METHODS FOR DETECTION OF RALSTONIA SOLANACEARUM IN

POTATO TUBERS AND POTATO, TOMATO OR OTHER HOST PLANTS WITH

SYMPTOMS OF BROWN ROT OR BACTERIAL WILT

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

1.1. Symptoms on potato

The potato plant. The early stage of infection in the field is recognised by wilting of the

leaves towards the top of the plant at high temperatures during the day with recovery at night.

In early stages of wilting leaves remain green, but later yellowing and brown necrosis

develops. Epinasty also occurs. Wilting of one shoot or whole plants becomes rapidly

irreversible and results in the collapse and death of the plant. The vascular tissue of

transversely cut stems from wilted plants usually appears brown and a milky bacterial ooze

exudes from the cut surface or can be expressed by squeezing. When a cut stem is placed

vertically in water, threads of slime will stream from the vascular bundles.

The potato tuber. Potato tubers must be cut transversely close to the heel (stolon end) or

longitudinally over the stolon end. The early stage of infection is recognised by a glassy

yellow to light brown discolouration of the vascular ring from which a pale cream bacterial

ooze emerges spontaneously after some minutes. Later, the vascular discolouration becomes a

more distinct brown and necrosis can extend into the parenchymatous tissue. In advanced

stages, infection breaks outwards from the heel end and the eyes from which bacterial slime

may ooze causing soil particles to adhere. Reddish-brown slightly sunken lesions may appear

on the skin due to collapse of vascular tissues internally. Secondary development of fungal

and bacterial soft rots is common in the advanced stages of the disease.

1.2. Symptoms on tomato

The tomato plant. The first visible symptom is the flaccid appearance of the youngest leaves.

B

Under favourable environmental conditions for the pathogen (soil temperatures of

approximately 25 °C; saturated humidity), epinasty and wilting of one side or of the whole

plant follows within a few days leading to total plant collapse. Under less favourable

conditions (soil temperature below 21 °C), less wilting occurs, but large numbers of

adventitious roots may develop on the stem. It is possible to observe watersoaked streaks from

the base of the stem which is evidence of necrosis in the vascular system. When the stem is

cut crosswise, discoloured brown vascular tissues exude white or yellowish bacterial ooze.

1.3. Symptoms on other hosts

Solanum dulcamara and S. nigrum plants. Under natural conditions, wilting symptoms are

rarely observed in these weed hosts unless soil temperatures exceed 25 °C or inoculum levels

are extremely high (e.g. as for S. nigrum growing adjacent to diseased potato or tomato

plants). When wilting does occur, the symptoms are as described for tomato. Non-wilting S.

dulcamara plants growing with stems and roots in water may show internal light brown

discolouration of vascular tissues on transverse section of the stem base or underwater stem

parts. Bacteria may ooze from cut vascular tissues or form threads of slime if the cut stem is

placed vertically in water, even in the absence of wilting symptoms.

2. Rapid screening tests

Rapid screening tests may facilitate presumptive diagnosis but are not essential. Use one or

more of the following validated tests

2.1. Stem streaming test

(See Section VI.A.1.)

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

Characteristic PHB granules in the cells of R. solanacearum are visualised by staining heat-

fixed smears of bacterial ooze from infected tissue on a microscope slide with Nile Blue A or

Sudan Black (See Section VI.A.2.).

2.3. Serological agglutination tests

(See Section VI.A.3.)

2.4. Other tests

Further appropriate rapid screening tests include the IF test (see Section VI.A.5.), FISH test

(see Section VI.A.7.), ELISA tests (see Section VI.A.8.) and PCR tests (see Section VI.A.6).

3. Isolation procedure

(a) Remove ooze or sections of discoloured tissue from the vascular ring in the potato tuber or

from the vascular strands in stems of potato, tomato or other wilting host plants. Suspend in a

small volume of sterile distilled water or 50mM phosphate buffer (Appendix 4) and leave for

5 to 10 minutes.

(b) Prepare a series of decimal dilutions of the suspension.

(c) Transfer 50-100 µl of the suspension and dilutions to a general nutrient medium (NA,

YPGA or SPA; see Appendix 2) and/or to Kelman’s tetrazolium medium (Appendix 2) and/or

a validated selective medium (e.g. SMSA; see Appendix 2). Spread or streak with an

appropriate dilution plating technique. If useful, prepare separate plates with a diluted cell

suspension of R. solanacearum biovar 2 as a positive control.

(d) Incubate the plates for two to six days at 28 °C.

— On the general nutrient media, virulent isolates of R. solanacearum develop pearly cream-

white, flat, irregular and fluidal colonies often with characteristic whorls in the centre.

Avirulent forms of R. solanacearum form small round non-fluidal, butyrous colonies which

are entirely cream-white.

— On Kelman’s tetrazolium and SMSA media, the whorls are blood red in colour. Avirulent

forms of Ralstonia solanacearum form small round non-fluidal, butyrous colonies which are

entirely deep red.

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

under Article 5(1)(a)(iii) and 5(1)(c)(iii), shall include

— the listed plant material grown at a place of production designated as contaminated under

Article 5(1)(a)(ii),

— place(s) of production with some production link to the listed plant material designated as

contaminated under Article 5(1)(a)(ii), including those sharing production equipment and

facilities directly or through a common contractor,

— the listed plant material produced in the place(s) of production referred to in the previous

indent, or present in such place(s) of production during the period when the listed plant

material designated as contaminated under Article 5(1)(a)(ii), was present on the place of

production referred to in the first indent,

— premises handling the listed plant material 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 listed plant material designated

B

as contaminated under Article 5(1)(a)(ii),

— any of the listed plant material stored in, or in contact with, any of the structures or objects

listed in the previous indent, prior to the cleansing and disinfection of such structures and

objects,

— as a result of the investigation and testing under Article 5(1)(a)(i), in the case of potato,

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

those plants with the same source as, the listed plant material designated to be contaminated

under Article 5(1)(a)(ii) 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,

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

— place(s) of production of the listed plant material using water for irrigation or spraying

which has been designated as contaminated under Article 5(1)(c)(ii),

— listed plant material produced on fields flooded with surface water confirmed to be

contaminated.

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

5(1)(a)(iv) and 5(1)(c)(iii) shall include

(i) in cases under Article 5(1)(a)(iv),

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

— the common production and use of seed potato stocks,

— places of production using surface water for irrigation or spraying of listed plant material

in cases where there is or has been risk of surface water run-off from, or flooding of, place(s)

of production designated to be contaminated under Article 5(1)(a)(ii).

(ii) in cases where surface water has been designated as contaminated under Article

5(1)(c)(ii)

— place(s) of production producing listed plant material adjacent to, or at risk from flooding

by, the surface water designated as contaminated,

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

— water bodies connected with the surface water designated as contaminated, taking into

account

— the direction and rate of flow of the water designated as contaminated,

— the presence of wild solanaceous host plants.

3. The notification referred to in the first subparagraph of Article 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 II, at least

— for potatoes,

(a) the variety name of the lot,

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

— for tomato plants, the variety name of the lot and, where applicable, the category.

— without prejudice to the requirements for notification of a suspected occurrence in Article

4 (3), the Member State in which the occurrence has been confirmed shall, when there is a

risk of contamination of listed plant material from or into another Member States(s),

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

Article 5(3), such as

(a) the variety name of the potato or tomato lot,

(b) the name and address of the consignor and the consignee,

(c) the date of delivery of the potato or tomato lot,

(d) the size of the potato or tomato lot delivered,

(e) 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.

4. The details of the additional notification referred to in the second subparagraph of Article

5(2) shall be provided as follows

After all investigations have been finalised, for each case

(a) the date that contamination was confirmed,

(b) a brief description of the investigation carried out to identify the source and possible

spread of the contamination, including the level of sampling undertaken,

(c) information on the identified or presumed sources(s) of contamination,

(d) details of the extent of the designated contamination, including the number of places of

production and for potatoes, the number of lots with an indication of the variety and, if seed

potatoes, category,

(e) details of the zone demarcation, including the number of places of production, not

designated as contaminated, but included in the zone,

(f) details of water designation, including the name and location of the water body and the

extent of the designation/irrigation ban,

(g) for any tomato plant consignment or lot designated as contaminated, the certificates

prescribed in Article 13(1)(ii) of Directive 2000/29/EC and passport number, in accordance

with the listing in Annex V, Part A, Section I.2.2 to Directive 2000/29/EC,

(h) such other information relating to the confirmed outbreak(s) as the Commission may

require.

Identification tests for R. solanacearum

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

VI.B.

SECTION III

1. Detailed methods for detection and identification of Ralstonia solanacearum in

samples of asymptomatic potato tubers

1.1. Sample preparation

Note

— 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 ring rot

bacterium, Clavibacter michiganensis subsp. sepedonicus.

Optional pre-treatment in advance to sample preparation

(a) Incubation of samples at 25 to 30 °C, for up to two weeks before testing, to encourage

multiplication of any R. solanacearum populations.

(b) Wash the tubers. Use appropriate disinfectants (chlorine compounds when PCR-test is to

be used in order to remove 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.

1.1.1. Remove with a clean and disinfected scalpel or vegetable knife the skin at the heel

(stolon) end of each tuber so that the vascular tissues become visible. Carefully cut out a small

core of vascular tissue at the heel end and keep the amount of non-vascular tissue to a

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

Note: Set aside any (rotting) tubers with suspected brown rot symptoms and test separately.

If during removal of the heel end core suspect symptoms of brown rot are observed, a visual

inspection of this tuber should be done and the tuber cut near the heel end. Any cut tuber with

suspected symptoms should be kept for at least two days at room temperature in order to

allow suberisation and stored refrigerated (at 4 to 10 °C) under proper quarantine conditions.

B

All tubers including those with suspicious symptoms should be kept according to Schedule

III.

1.1.2. Collect the heel end cores in unused disposable containers which can be closed and/or

sealed (in case containers are reused they should be thoroughly cleaned and disinfected using

chlorine compounds). Preferably, the heel end cores should be processed immediately. If this

is not possible, store them in the container, without addition of buffer, refrigerated for not

longer than 72 hours or for not longer than 24 hours at room temperature.

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

(a) Cover the cores with sufficient volume (approximately 40 ml) of extraction buffer

(Appendix 4) and agitate on a rotary shaker (50-100 rpm) for 4 hours below 24 °C or for 16 to

24 hours refrigerated,

or

(b) Homogenise the cores with sufficient volume (approximately 40 ml) of extraction buffer

(Appendix 4), either in a blender (e.g. Waring or Ultra Thurax) or by crushing in a sealed

disposable maceration bag (e.g. Stomacher or Bioreba strong guage polythene, 150 mm × 250

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

Note: The risk of cross-contamination of samples is high when samples are homogenized

using a blender. Take precautions to avoid aerosol generation or spillage during the extraction

process. Ensure that freshly sterilised blender blades and vessels are used for each sample. If

the PCR test is to be used, avoid carry-over of DNA on containers or grinding apparatus.

Crushing in disposable bags and use of disposable tubes is recommended where PCR is to be

used.

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

centrifugation (at not more than 180 g for 10 minutes at a temperature between 4 to 10 °C) or

by vacuum filtration (40 to 100 µm), washing the filter with additional (approximately 10 ml)

extraction buffer.

1.1.4. Concentrate the bacterial fraction by centrifugation at 7 000 g for 15 minutes (or 10 000

g for 10 minutes) at a temperature between 4 to 10 °C and discard the supernatant without

disturbing the pellet.

1.1.5. Resuspend the pellet in 1.5 ml pellet buffer (Appendix 4). Use 500 µl to test for R.

solanacearum, 500 µl for Clavibacter michiganensis subsp. sepedonicus and 500 µl for

reference purposes. Add sterile glycerol to final concentration of 10 to 25 % (v/v) to the 500

µl of the reference aliquot and to the remaining test aliquot, vortex and store at – 16 to – 24 °C

(weeks) or at – 68 to – 86 °C (months). Preserve the test aliquots at 4 to 10 °C during testing.

Repeated freezing and thawing is not advisable.

If transport of the extract is required, ensure delivery in a cool box within 24 to 48 hours.

1.1.6. It is imperative that all R. solanacearum positive controls and samples are treated

separately to avoid contamination. This applies to IF slides and to all tests.

1.2. Testing

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

Selective isolation (see Section VI.A.4.)

IF test (see Section VI.A.5.)

PCR tests (see Section VI.A.6.)

FISH test (see Section VI.A.7.)

ELISA tests (see Section VI.A.8.)

Bioassay (see Section VI.A.9.)

2. Detailed methods for detection and identification of R. solanacearum in samples of

asymptomatic potato, tomato or other host plants

2.1. Sample preparation

Note: For detection of latent R. solanacearum populations it is advised to test composite

samples. The procedure can be conveniently applied for composite samples of up to 200 stem

parts. Where surveys are performed they should be based on a statistically representative

sample of the plant population under investigation.

2.1.1. Collect 1 to 2 cm stem segments in a closed sterile container according to the following

sampling procedures

Nursery tomato seedlings: With a clean disinfected knife, remove a 1 cm segment from the

base of each stem, just above the soil level.

Field or glasshouse grown tomato plants: With a clean disinfected knife, remove the

lowermost side shoot from each plant by cutting just above the joint with the main stem.

Remove the lowermost 1cm segment from each side shoot.

Other hosts: With a clean disinfected knife or pruning shears, remove a 1 cm segment from

the base of each stem, just above the soil level. In the case of S. dulcamara or other host

plants growing in water, remove 1-2 cm sections from underwater stems or stolons with

aquatic roots.

When sampling a particular location it is recommended to test a statistically representative

sample of at least 10 plants per sampling point of each potential weed host. Pathogen

detection will be most reliable during late spring, summer and autumn seasons, although

natural infections can be detected all year round in the perennial Solanum dulcamara growing

in watercourses. Known hosts include volunteer potato plants (groundkeepers), Solanum

dulcamara, S. nigrum, Datura stramonium and other members of the family Solanaceae.

Further hosts are Pelargonium spp. and Portulaca oleracea. Some European weed spp. which

may potentially harbour R. solanacearum biovar 2/Race 3 populations in roots and/or

rhizospheres under specific environmental conditions include Atriplex hastata, Bidens pilosa,

Cerastium glomeratum, Chenopodium album, Eupatorium cannabinum, Galinsoga

parviflora, Ranunculus scleratus, Rorippa spp, Rumex spp., Silene alba, S. nutans., Tussilago

farfarra and Urtica dioica.

Note: Visual examination for internal symptoms (vascular staining or bacterial ooze) can be

done at this stage. Set aside any stem segments with symptoms and test separately (See

Section II).

2.1.2. Disinfect stem segments briefly with ethanol 70 % and immediately blot dry on tissue

paper. Then process the stem segments by one of the following procedures: either,

(a) Cover the segments with sufficient volume (approximately 40 ml) of extraction buffer

(Appendix 4) and agitate on a rotary shaker (50 to 100 rpm) for four hours below 24 °C or for

16 to 24 hours refrigerated, or

(b) Process immediately by crushing the segments in a strong maceration bag (e.g. Stomacher

or Bioreba) with an appropriate volume of extraction buffer (Appendix 4) using a rubber

mallet or appropriate grinding apparatus (e.g. Homex). If this is not possible, store the stem

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

temperature.

B

2.1.3. Decant the supernatant after settling for 15 minutes.

2.1.4. Further clarification of the extract or concentration of the bacterial fraction are not

usually required but may be achieved by filtration and/or centrifugation as described in

Section III.1.1.3 – 1.1.5.

2.1.5. Divide the neat or concentrated sample extract into two equal parts. Maintain one half

at 4 to 10 °C during testing and store the other half with 10 to 25 % (v/v) sterile glycerol at –

16 to – 24 °C (weeks) or at – 68 to – 86 °C (month) in case further testing is required.

2.2. Testing

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

Selective isolation (see Section VI.A.4.)

IF test (see Section VI.A.5.)

PCR tests (see Section VI.A.6.)

FISH test (see Section VI.A.7.)

ELISA tests (see Section VI.A.8.)

Bioassay (see Section VI.A.9.)

SECTION IV

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

( 1 ) See section IV.2.1. for recommended sampling procedures.

( 2 ) Pathogen concentration methods are described in Section IV.2.1. Concentration increases

population of both pathogen and competing saprophytic bacteria and is recommended

only if it does not result in inhibition of the isolation test.

( 3 ) The selective isolation test is described in Section VI.A.4.

( 4 ) The bioassay test is described in Section VI.A.9.

( 5 ) Enrichment PCR methods are described in Section VI.A.4.2. and section VI.A.6.

( 6 ) Enrichment ELISA methods are described in Section VI.A.4.2. and Section VI.A.8.

B

( 7 ) Typical colony morphology is described in Section II.3.d.

( 8 ) Culturing can fail due to competition of inhibition by saprophytic bacteria. If high

saprophyte population are suspected to affect the reliability of the isolation, then repeat

the isolation tests after dilution of the sample in sterile water.

( 9 ) Reliable identification of pure presumptive R. solanacearum cultures is achieved using the

tests described in Section VI.B.

( 10 ) The pathogenicity test is described in Section VI.C.

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

Principle

The validated detection scheme, described in this section, is applicable for pathogen detection

in samples of surface water and can also be applied for testing samples of potato processing or

sewage effluents. However, it is important to note that the expected sensitivity of detection

will vary with the substrate. Sensitivity of the isolation test is affected by populations of

competing saprophytic bacteria which are generally much higher in potato processing and

sewage effluents than in surface water. Whereas the scheme below is expected to detect as

few as 103 cells per litre in surface water the sensitivity of detection in potato processing or

sewage effluents is likely to be significantly lower. For this reason, it is recommended to test

effluents after any purification treatments (e.g. sedimentation or filtration) during which

saprophytic bacterial populations are reduced. The limitations in sensitivity of the test scheme

should be considered when assessing the reliability of any negative results obtained. Whereas

this scheme has been successfully used in survey work to determine presence or absence of

the pathogen in surface water, its limitations should be realised when used in similar surveys

of potato processing or sewage effluents.

2.1. Sample preparation

Note

— Detection of R. solanacearum in surface water is most reliable during late spring, summer

and autumn seasons when water temperatures exceed 15 °C.

— Repeated sampling at different times in the above mentioned period at designated sampling

points will increase the reliability of detection by reducing the effects of climatic variation.

— Take into account the effects of heavy rainfall and the geography of the watercourse to

avoid extensive dilution effects that may obscure presence of the pathogen.

— Take surface water samples in the vicinity of host plants if these hosts are present.

2.1.1. At selected sampling points, collect water samples by filling disposable sterile tubes or

bottles at a depth if possible below 30 cm and within 2 m from the bank. For processing and

sewage effluents, collect samples from the point of effluent discharge. Sample sizes up to 500

ml per sampling point are recommended. If smaller samples are preferred, it is advisable to

take samples on at least three occasions per sampling point, each sample consisting of two

replicated sub-samples of at least 30 ml. For intensive survey work, select at least three

sampling points per 3 km of watercourse and ensure that tributaries entering the watercourse

are also sampled.

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

2.1.3. If required, the bacterial fraction may be concentrated using one of the following

methods

(a) Centrifuge 30 to 50 ml sub-samples at 10 000 g for 10 minutes (or 7 000 g for 15 minutes)

preferably at 4 to 10 °C, discard the supernatant and resuspend the pellet in 1 ml pellet buffer

(Appendix 4).

(b) Membrane filtration (minimum pore size 0,45 µm) followed by washing the filter in 5 to

10 ml pellet buffer and retention of the washings. This method is suitable for larger volumes

of water containing low numbers of saprophytes.

Concentration is usually not advisable for samples of potato processing or sewage effluent

since increased populations of competing saprophytic bacteria will inhibit detection of

Ralstonia solanacearum.

2.2. Testing

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

SECTION V

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

B

( 1 ) See Section V.2.1. for recommended sampling procedures.

( 2 ) The selective isolation test is described in Section VI.A.4.

( 3 ) Enrichment PCR methods are described in Section VI.A.4.2. and Section VI.A.6.

( 4 ) The bioassay is described in Section VI.A.9.

( 5 ) Typical colony morphology is described in Section II.3.d.

( 6 ) Culturing can fail due to competition or inhibition by saprophytic bacteria. If high

saprophyte populations are suspected to affect the reliability of the isolation, then repeat

the isolation tests after further dilution of the sample.

( 7 ) Reliable identification of pure presumptive R. solanacearum cultures is achieved using the

tests described in Section VI.B.

( ) The pathogenicity test is described in Section VI.C.

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

Principles

The validated detection scheme, described in this section, is applicable for pathogen detection

in soil samples but can also be used to test samples of solid potato processing waste or sewage

sludge. However, it should be noted that these methods are insufficiently sensitive to

guarantee detection of low and/or irregularly dispersed populations of Ralstonia

solanacearum that may occur in naturally infested samples of these substrates.

The limitations in sensitivity of this test scheme should be considered when assessing the

reliability of any negative results obtained and also when used in surveys to determine

presence or absence of the pathogen in soils or sludges. The most reliable test for presence of

the pathogen in a field soil is to plant a susceptible host and monitor it for infection, but even

with this method low levels of contamination will escape detection.

2.1. Sample preparation

2.1.1. Sampling of field soil should follow standard principals used for nematode sampling.

Collect 0,5 to 1 kg of soil per sample from 60 sites per 0,3 ha from a depth of 10 to 20 cm (or

in a grid of 7 x 7 metres) If the pathogen is suspected to be present, increase the number of

collection points to 120 per 0,3 ha. Maintain samples at 12 to 15 °C prior to testing. Sample

potato processing and sewage sludges by collecting a total of 1 kg from sites representing the

total volume of sludge to be tested. Mix each sample well before testing.

2.1.2. Disperse sub-samples of 10 to 25 g of soil or sludge by rotary shaking (250 rpm) in 60

to 150 ml extraction buffer (Appendix 4) for up to two hours. If required, addition of 0,02 %

sterile Tween-20 and 10 to 20 g sterile gravel may assist dispersion.

2.1.3. Maintain the suspension at 4 °C during testing.

2.2. Testing

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

SECTION VI

OPTIMISED PROTOCOLS FOR DETECTION AND IDENTIFICATION OF R.

SOLANACEARUM

A. DIAGNOSTIC AND DETECTION TESTS

1. Stem streaming test

The presence of R. solanacearum in stems of wilting potato, tomato or other host plants can

be indicated by the following simple presumptive test: Cut the stem just above the soil level.

Suspend the cut surface in a tube of clean water. Observe for characteristic spontaneous

streaming of threads of bacterial slime from the cut vascular bundles after a few minutes.

2. Detection of poly-ß-hydroxybutyrate granules

1. Prepare a smear of bacterial ooze from infected tissue or from a 48-hour culture on YPGA

or SPA medium (Appendix 2) on a microscope slide.

B

2. Prepare positive control smears of a biovar 2 strain of R. solanacearum and, if considered

useful, a negative control smear of a known PHB negative sp.

3. Allow to air dry and pass the lower surface of each slide rapidly above a flame to fix the

smears.

4. Stain preparation with either Nile Blue or Sudan Black and observe microscopically as

described below

Nile blue test

(a) Flood each slide with 1 % aquous solution of Nile Blue A and incubate for 10 minutes at

55 °C.

(b) Drain off the staining solution. Wash briefly in gently running tap water. Remove excess

water with tissue paper.

(c) Flood the smear with 8 % aqueous acetic acid and incubate for one minute at ambient

temperature.

(d) Wash briefly in gently running tap water. Remove excess water with tissue paper.

(e) Re-moisten with a drop of water and apply a coverslip.

(f) Examine the stained smear with an epifluorescence microscope at 450 nm under oil

immersion at a magnification of 600 to 1 000 using an oil- or water-immersion objective.

(g) Observe for bright orange fluorescence of PHB granules. Also observe under transmitted

normal light to ensure that the granules are intracellular and that cell morphology is typical of

R. solanacearum.

Sudan Black test

(a) Flood each slide with 0,3 % Sudan Black B solution in 70 % ethanol and incubate for 10

minutes at ambient temperature.

(b) Drain off the staining solution and wash briefly in tap water, removing excess water with

tissue paper.

(c) Dip the slides briefly in xylol and blot dry on tissue paper. Caution: Xylol is harmful, take

necessary safety precautions and work in a fume cupboard.

(d) Flood the slides with 0,5 % (w/v) aqueous safranin and leave for 10 seconds at ambient

temperature. Caution: Safranin is harmful, take necessary safety precautions and work in a

fume cupboard.

(e) Wash in gently running tap water, blot dry on tissue paper and apply a coverslip.

(f) Examine stained smears with a light microscope using transmitted light under oil

immersion at a magnification of 1 000 using an oil-immersion objective.

(g) Observe for blue-black staining of PHB granules in cells of R. solanacearum with pink-

stained cell walls.

3. Serological agglutination tests

Agglutination of R. solanacearum cells in bacterial ooze or symptomatic tissue extracts is best

observed using validated antibodies (see Appendix 3) labelled with appropriate coloured

markers such as red Staphylococcus aureus cells or coloured latex particles. If using a

commercially available kit (see Appendix 3), follow the manufacturers instructions.

Otherwise perform the following procedure

(a) Mix drops of a suspension of labelled antibody and bacterial ooze (approximately 5 µl

each) on windows of multiwell test slides.

(b) Prepare positive and negative controls using suspensions of R. solanacearum biovar 2 and

a heterologous strain.

(c) Observe for agglutination in positive samples after gentle mixing for 15 seconds.

4. Selective isolation

4.1. Selective plating

Note: Before using this method for the first time, perform preliminary tests to ensure

reproducible detection of 103 to 104 colony forming units of R. solanacearum per ml added

to extracts from samples which previously tested negative.

Use an appropriately validated selective medium such as SMSA (as modified by Elphinstone

et al., 1996; see Appendix 2).

Care is required to differentiate R. solanacearum from other bacteria able to develop colonies

on the medium. Furthermore, colonies of R. solanacearum may show atypical morphology if

plates are overcrowded or antagonistic bacteria are also present. Where effects of competition

or antagonism are suspected, the sample should be re-tested using a different test.

Highest sensitivity of detection by this method can be expected when using freshly prepared

sample extracts. However, the method is also applicable for use with extracts which have

been stored under glycerol at – 68 to – 86 °C.

As positive controls, prepare decimal dilutions from a suspension of 106 cfu per ml of a

virulent biovar 2 strain of R. solanacearum (e.g. NCPPB 4156 = PD 2762 = CFBP 3857). To

avoid any possibility of contamination, prepare positive controls totally separately from

samples to be tested.

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

should be tested before it is used to test routine samples.

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

4.1.1. Perform an appropriate dilution plating technique aiming to ensure that any background

saprophytic colony forming populations are diluted out. Spread 50 - 100 µl per plate of

sample extract and each dilution.

4.1.2. Incubate plates at 28 °C. Read plates after 48 hours and daily thereafter up to six days.

Typical R. solanacearum colonies on SMSA medium are milky white, flat, irregular and

fluidal and after three days incubation develop pink to blood-red coloration in the centre with

internal streaking or whorling. (see website

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

Note: Atypical colonies of R. solanacearum sometimes form on this medium. These may be

small, round, entirely red in colour

and non-fluidal or only partially fluidal and therefore difficult to distinguish from saprophytic

colony-forming bacteria.

4.1.3. Purify presumptive R. solanacearum colonies after streaking or dilution plating onto a

general nutrient medium to obtain isolated colonies (see Appendix 2).

4.1.4. Store cultures short-term in sterile water (pH 6 to 8, chlorine free) at room temperature

in the dark, or long term in a suitable cryoprotectant medium at – 68 to – 86 °C or lyophilised.

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

Section VI. C).

Interpretation of selective plating test results

The selective plating test is negative if no bacterial colonies are observed after six days or if

no presumptive colonies typical of R. solanacearum are found, provided that no inhibition is

B

suspected due to competition or antagonism by other bacteria and that typical R.

solanacearum colonies are found in the positive controls.

The selective plating test is positive if presumptive R. solanacearum colonies are isolated.

4.2. Enrichment procedure

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

This procedure can be used to selectively increase R. solanacearum populations in sample

extracts and increase sensitivity of detection. The procedure also effectively dilutes inhibitors

of the PCR reaction (1:100). It should be noted, however, that enrichment of R. solanacearum

can fail due to competition or antagonism by saprophytic organisms which are often

simultaneously enriched. For this reason, isolation of R.solanacearum from enriched broth

cultures may be difficult. In addition, since populations of serologically related saprophytes

can be increased, the use of specific monoclonal antibodies rather than polyclonal antibodies

is recommended where the ELISA test is to be used.

4.2.1. For enrichment-PCR, transfer 100 µl of sample extract into 10 ml of enrichment broth

(Appendix 2) previously aliquoted into DNA-free tubes or flasks. For enrichment-ELISA,

higher proportions of sample extract to broth can be used (e.g. 100 µl in 1,0 ml of enrichment

broth).

4.2.2. Incubate for 72 hours at 27 to 30 °C in shaking culture or static culture with caps

loosely- fitted to permit aeration.

4.2.3. Mix well before using in ELISA or PCR tests.

4.2.4. Treat enriched broth in an identical manner as the sample(s) in the above tests.

Note: If inhibition of enrichment of R. solanacearum is anticipated, due to high populations of

certain competing saprophytic bacteria, enrichment of sample extracts before any

centrifugation or other concentration steps may give better results.

1. The provisions referred to in Article 6(1) 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 organism into the environment e.g. through seepage to

agricultural land or contact with water sources which could be used for irrigation of

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

B

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 options shall be disposed of

by officially approved methods in accordance with Schedule VII to these principal

regulations.

2. The appropriate use or disposal of the listed plant material referred to in Article 6(2), under

the control of the responsible official bodies of the Member State(s) concerned, with

appropriate communication between responsible official bodies to ensure such control at all

times and approval by the responsible official 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 indents, shall be

(i) for potato tubers,

— 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.

(ii) for other plant parts including stem and foliage debris,

— destruction,

or

— some other use or disposal, provided that it is established that there is no identifiable risk

of the organism spreading; and subject to approval by the said responsible official bodies.

or

3. The appropriate methods for decontamination of the objects referred to in Article 6(3) shall

be cleansing and, where appropriate, disinfection, such 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 by Member States within the demarcated zone(s)

established under Article 5(1)(a)(iv) and (c)(iii) and referred to in Article 6(4) shall include

4.1. In cases where places of production have been designated as contaminated under Article

5(1)(a)(ii)

(a) in a field or unit of protected crop production designated to be contaminated under Article

5(1)(a)(ii), either

(i) during at least the four growing years following the designated contamination,

— measures shall be taken to eliminate volunteer potato and tomato plants as well as other

host plants of the organism including solanaceous weeds,

and

— the following shall not be planted

— potato tubers, plants or true seeds,

— tomato plants and seeds,

— taking into account the biology of the organism,

— other host plants,

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

surviving,

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

— in the first potato or tomato cropping season following the period specified in the

preceding indent, and on the condition that the field has been found free from volunteer potato

and tomato plants and other host plants including solanaceous weeds during official

inspections for at least the two consecutive growing years prior to planting,

— in the case of potatoes, only ware potato production shall be allowed,

— in the case of potatoes and tomatoes, the harvested potato tubers, or the tomato plants, as

appropriate, shall be tested according to the procedure detailed in Schedule II;

— in the potato or tomato 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, an official survey as detailed in Article 2(1), shall be conducted;

or

(ii) during the five growing years following that of the designated contamination,

— measures shall be taken to eliminate volunteer potato and tomato plants as well as other

naturally found host plants of the organism including solanaceous weeds,

and

— the field shall be established and maintained during the first three years either, in bare

fallow or, in cereals according to the risk identified, or, in permanent pasture with frequent

close cutting or intensive grazing or, as grass for seed production, followed by planting in the

succeeding two years with non-host plants of the organism for which there is no identified

risk of the organism surviving or spreading,

— in the first potato or tomato cropping season following the period specified in the

preceding indent, and on the condition that the field has been found free from volunteer potato

and tomato plants and other host plants including solanaceous weeds during official

inspections for at least the two consecutive growing years prior to planting,

— in the case of potatoes, seed or ware potato production shall be allowed,

— the harvested potato tubers, or the tomato plants, as appropriate, shall be tested according

to the procedure detailed in Schedule II;

(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 tomato

plants and other naturally found host plants of the organism including solanaceous weeds as

appropriate have been eliminated

— in the growing year following that of the designated contamination,

— either no potato tubers or plants or true seeds, or other host plants of the organism shall be

planted,

or

— in the case of potato tubers, certified seed potatoes may be planted for ware production

only,

— in the case of tomato plants, tomato plants grown from seed which meets the requirements

of Directive 2000/29/EC may be planted for fruit production only;

B

— in the second growing year following that of the designated contamination,

— in the case of potatoes, only certified seed potatoes or seed potatoes officially tested for the

absence of brown rot and grown under official control on places of production other than

those referred to in 4.1 shall be planted for either seed or ware production,

— in the case of tomatoes, only tomato plants grown from seed which meets the requirements

of Directive 2000/29/EC or, if vegetatively propagated, from tomato plants produced from

such seed and grown under official control on places of production other than those referred

to in 4.1, shall be planted for either plant or fruit production;

— for at least in the third growing year following that of the designated contamination,

— in the case of potatoes, 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 the case of tomatoes, only tomato plants grown from seed which meets the requirements

of Directive 2000/29/EC or tomato plants grown under official control from such plants shall

be planted for either plant or fruit 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, an official inspection of the growing crop at appropriate times shall be conducted

and in each potato field, official testing of the harvested potatoes shall be conducted according

to the procedure detailed in Schedule II;

(c) immediately following the designation of contamination under Article 5(1)(a)(ii) and after

the first subsequent growing year

— all machinery and storage facilities on the place of production and involved in potato or

tomato production shall be cleansed and, where appropriate, disinfected using appropriate

methods, as specified in point 3,

— official controls on irrigation and spraying programmes, including a ban thereof, shall be

introduced as appropriate in order to prevent the spread of the organism;

(d) in an unit of protected crop production designated as contaminated under Article

5(1)(a)(ii) where complete replacement of the growing medium is possible,

— no potato tubers, plants or true seeds, or other host plants of the organism including tomato

plants and 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, where

appropriate, disinfection of the said unit and all equipment, and subsequently has been granted

approval for potato or tomato production by the responsible official bodies,

and

— for potato production, this production shall be from certified seed potatoes, or from mini-

tubers or microplants derived from tested sources,

— for tomato production, this production shall be from seed which meets the requirements of

Directive 2000/29/EC or, if vegetatively propagated, from tomato plants produced from such

seed and grown under official control,

— official controls on irrigation and spraying programmes, including a ban thereof, shall be

introduced as appropriate, in order to prevent the spread of the organism;

4.2. Within the demarcated zone, without prejudice to the measures detailed under 4.1, the

Member States shall

(a) immediately following the designated contamination ensure that all machinery and storage

facilities on such premises and involved with potato or tomato production be cleansed and

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

(b) immediately, and for at least three growing years following the designated contamination

(ba) in cases where the demarcated zone has been determined under Article 5(1)(a)(iv),

— ensure supervision by their responsible official bodies of premises growing, storing or

handling potato tubers or tomatoes, together with premises which operate machinery for

potato or tomato production 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 determined as probably contaminated under Article 5(1)(a)(iii),

— 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, where appropriate, disinfection to be

carried out between the handling of seed and ware stocks,

— require the planting of only tomato plants grown from seed which meets the requirements

of Directive 2000/29/EC or, if vegetatively propagated, from tomato plants produced from

such seed and grown under official control, for all tomato crops within that zone,

— conduct an official survey as detailed in Article 2(1);

(bb) in cases where surface water has been designated as contaminated under Article

5(1)(c)(ii) or included in the elements for the possible spread of the organism in accordance

with Schedule V point 2,

— conduct an annual survey at appropriate times including sampling of surface water and

where appropriate solanaceous host plants in the relevant water sources and testing in

accordance with the relevant methods set out in Schedule II for the listed plant material and

for other cases,

— introduce official controls on irrigation and spraying programmes, including a ban on the

use of the water designated as contaminated for the irrigation and spraying of listed plant

material, and, where appropriate, other host plants in order to prevent the spread of the

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

survey, and designations revoked where the responsible official bodies are satisfied that the

surface water is no longer contaminated. The use of water subject to a ban may be permitted,

under official control, for irrigation and spraying of host plants, where officially approved

techniques are employed which eliminate the organism and prevent its spread,

— in cases where liquid waste discharges are contaminated, introduce official controls on the

disposal of solid or liquid waste discharges from industrial processing or packaging premises

handling listed plant material;

(c) establish a programme, where appropriate, for the replacement of all seed potato stocks

over an appropriate period of time.

IF Test

Principle

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

robustness to achieve the required thresholds.

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

Isolation, PCR or FISH test must be performed as a second screening test. When the IF test is

used as the second screening test and the IF reading is positive, further testing according to

the flow scheme is required to complete the analysis.

Note: Use a validated source of antibodies to R. solanacearum (see web site

http://forum.europa.eu.int/Public/irc/sanco/Home/main). It is recommended that the titre is

determined for each new batch of antibodies. The titre is defined as the highest dilution at

which optimum reaction occurs when testing a suspension containing 105 to 106 cells per ml

of the homologous strain of R. solanacearum and using an appropriate fluorescein

isothiocyanate (FITC) conjugate according to the manufacturer’s recommendations. Validated

polyclonal antisera all had an IF titre of at least 1:2 000. During testing, the antibodies should

be used at a working dilution(s) close to or at the titre.

The test should be performed on freshly-prepared sample extracts. If necessary, it can be

successfully performed on extracts stored at – 68 to – 86 °C under glycerol. Glycerol can be

removed from the sample by addition of 1 ml pellet buffer (Appendix 4), re-centrifugation for

15 minutes at 7 000 g and re-suspension in an equal volume of pellet buffer. This is often not

necessary, especially if samples are fixed to the slides by flaming.

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

of R. solanacearum, suspended in potato extract, as specified in Appendix 3 B, and optionally

in buffer.

Naturally infected tissue (maintained by lyophilisation or freezing at – 16 to – 24 °C) should

be used where possible as a similar control on the same slide.

As negative controls, aliquots of sample extract which previously tested negative for R.

solanacearum can be used.

Standardised positive and negative control materials available for use with this test are listed

in Appendix 3.

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

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

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

(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, 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.

5.2. Dry the droplets at ambient temperature or by warming to temperatures of 40 to 45 °C.

Fix the bacterial cells to the slide either by heating (15 minutes at 60 °C), flaming, with 95 %

ethanol or according to specific instructions from the suppliers of the antibodies.

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

necessary (up to a maximum of three months) prior to further testing.

5.3. IF procedure

(i) According to test slide preparation in 5.1(i)

Prepare a set of twofold dilutions The first well should have 1/2 of the titre (T/2), the others

1/4 of the titre (T/4), 1/2 of the titre (T/2), the titre (T) and twice the titre (2T).

(ii) According to test slide preparation in 5.1(ii)

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

the specificity.

Figure 1. Preparation of the test slide according to 5.1(i) and 5.3(i)

B

Figure 2. Preparation of the test slide according to 5.1(ii) and 5.3(ii).

5.3.1. Arrange the slides on moist tissue paper. Cover each test window completely with the

antibody dilution(s). The volume of antibody applied on each window must be at least the

volume of extract applied.

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

suppliers of the antibodies

5.3.2. Incubate the slides on moist paper under a cover for 30 minutes at ambient temperature

(18 to 25 °C).

5.3.3. Shake the droplets off each slide and rinse carefully with IF buffer. Wash by

submerging for five minutes in IF buffer-Tween (Appendix 4) and subsequently in IF buffer.

Avoid causing aerosols or droplet transfer that could result in cross-contamination. Carefully

remove excess moisture by blotting gently.

5.3.4. Arrange the slides on moist paper. Cover the test windows with the dilution of FITC

conjugate used to determine the titre. The volume of conjugate applied on the windows must

be identical to the volume of antibody applied.

5.3.5. Incubate the slides on moist paper under a cover for 30 minutes at ambient temperature

(18 to 25 °C).

5.3.6. Shake the droplets of conjugate off the slide. Rinse and wash as before (5.3.3).

Carefully remove excess moisture.

5.3.7. Pipette 5 - 10 µl of 0,1M phosphate-buffered glycerol (Appendix 4) or a commercially

antifading mountant on each window and apply a coverslip.

5.4. Reading the IF test

5.4.1 Examine test slides on an epifluorescence microscope with filters suitable for excitation

of FITC, under oil or water immersion at a magnification of 500-1 000. Scan windows across

two diameters at right angles and around the perimeter. For samples showing no or low

number of cells observe at least 40 microscope fields.

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

at the determined antibody titre or working dilution. The IF test (Section VI.A.5.) must be

repeated if the staining is aberrant.

5.4.2. Observe for bright fluorescing cells with characteristic morphology of R. solanacearum

in the test windows of the test slides (see website

http://forum.europa.eu.int/Public/irc/sanco/Home/main). The fluorescence intensity must be

equivalent to the positive control strain at the same antibody dilution. Cells with incomplete

staining or with weak fluorescence must be disregarded.

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

slides in a batch show positive cells due to the contamination of buffer or if positive cells are

found (outside of the slide windows) on the slide coating.

5.4.3. There are several problems inherent to the specificity of the immunofluorescence test.

Background populations of fluorescing cells with atypical morphology and cross reacting

saprophytic bacteria with size and morphology similar to R. solanacearum are likely to occur

in potato heel end core and stem segment pellets.

5.4.4. Consider only fluorescing cells with typical size and morphology at the titre or working

dilution of the antibodies as in 5.3.

5.4.5. Interpretation of the IF reading

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

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

of resuspended pellet (Appendix 5).

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

pellet. The sample is considered potentially contaminated and further testing is required.

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

pellet and the sample is considered negative. Further testing is not required.

The officially approved waste disposal methods referred to in Schedule VI paragraph 1, shall

conform to the following provisions such that any identifiable risk of spreading the organism

is obviated

(i) potato and tomato waste (including rejected potatoes and peelings and tomatoes) and any

other solid waste associated with the potatoes and tomatoes (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

B

identifiable risk of escape of the organism into the environment e.g. through seepage to

agricultural land or contact with water sources which could be used for irrigation of

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 subparagraph (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 or water

sources which could be used for irrigation of 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 tests

Principles

When the PCR test is used as the principal screening test and found to be positive, isolation or

IF must be performed as a second compulsory screening test. When PCR is used as the second

screening test and found to be positive, further testing according to the flow scheme is

required to complete the diagnosis.

Full exploitation of this method as principal screening test is only recommended when

specialised expertise has been acquired.

Note: Preliminary testing with this method should permit reproducible detection of 103 to 104

cells of R. solanacearum per ml added to sample extracts which previously tested negative.

Optimisation experiments may be required to achieve maximum levels of sensitivity and

specificity in all laboratories.

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

an internal control.

Use appropriate precautions to avoid contamination of sample with target DNA. The PCR test

should be performed by experienced technicians, in dedicated molecular biology laboratories,

in order to minimise the possibility of contamination with target DNA.

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

final samples in the procedure, to make evident whether any carry over of DNA has occurred.

B

The following negative controls should be included in the PCR test

— Sample extract that previously tested negative for R. solanacearum,

— 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 R. solanacearum has been added (preparation see

Appendix 3 B).

— A suspension of 106 cells per ml of R. solanacearum in water from a virulent isolate (e.g.

NCPPB 4156 = PD 2762 = CFBP 3857; see Appendix 3 B).

— If possible use also 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, be

advisible to prepare extracts from washed potatoes if PCR protocols are to be used.

Standardized positive and negative control material available for use with this test are listed in

Appendix 3).

6.1. DNA purification methods

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

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

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

substrates, thus removing inhibitors of PCR and other enzymatic reactions and concentrating

target DNA in the sample extract. The following method has been optimised for use with the

validated PCR methods shown in Appendix 6.

(a) Method according to Pastrik (2000)

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 water bath at 95 °C for 10 min.

3) Put tube on ice for 5 min.

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 min.

5) Add 220 µl of Easy DNA® solution A (Invitrogen), mix well by vortexing and incubate at

65 °C for 30 min.

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.

7) Add 500 µl of chloroform and vortex until the viscosity decreases and the mixture is

homogeneous.

8) Centrifuge at 15 000 g for 20 min at 4 °C to separate phases and form the interphase.

9) Transfer the upper phase into a fresh Eppendorf tube.

10) Add 1 ml of 100 % ethanol (– 20 °C) vortex briefly and incubate on ice for 10 min.

11) Centrifuge at 15 000 g for 20 min at 4 °C and remove ethanol from pellet.

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

13) Centrifuge at 15 000 g for 10 min at 4 °C, save the pellet and remove ethanol.

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

15) Resuspend the pellet in 100 µl sterile UPW and leave at room temperature for at least 20

minutes.

16) Store at – 20 °C until required for PCR.

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

containing DNA for the PCR.

(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. P C R

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

VI.A.6.). Prepare one decimal dilution of sample DNA extract (1:10 in UPW).

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

according to the published protocols (Appendix 6). Where possible, it is recommended to use

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

6.2.3. Add 2-5 µl of DNA extract per 25 µl PCR reaction in sterile PCR tubes according to the

PCR protocols, (see Appendix 6).

6.2.4. Incorporate a negative control sample containing only PCR reaction mix and add the

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

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

the appropriately optimised PCR programme (Appendix 6).

6.3. Analysis of the PCR product

6.3.1. Resolve PCR amplicons by agarose gel electrophoresis. Run at least 12 µl of amplified

DNA reaction mixture from each sample mixed with 3 µl loading buffer (Appendix 6) in 2,0

% (w/v) agarose gels in tris-acetate-EDTA (TAE) buffer (Appendix 6) at 5 to 8 V per cm. Use

an appropriate DNA marker, e.g. 100 bp ladder.

6.3.2. Reveal DNA bands by staining in ethidium bromide (0,5 mg per L) for 30 to 60 minutes

taking appropriate precautions for handling this mutagen.

6.3.3. Observe stained gel under short wave UV transillumination (λ = 302 nm) for amplified

PCR products of the expected size (Appendix 6) and document.

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

restriction enzyme analysis on a sample of the remaining amplified DNA by incubating at the

optimum temperature and time with an appropriate enzyme and buffer (see Appendix 6).

Resolve the digested fragments by agarose gel electrophoresis as before and observe

characteristic restriction fragment pattern under UV transillumination after ethidium bromide

staining and compare with the undigested and digested positive control.

Interpretation of the PCR test result

The PCR test is negative if the R. solanacearum-specific PCR amplicon of expected size is

not detected for the sample in question but is detected for all positive control samples (in case

of multiplex PCR with plant specific internal control primers: a second PCR-product of

expected size must be amplified with the sample in question).

The PCR test is positive if the R. solanacearum-specific PCR amplicon of expected size and

restriction pattern (when required) is detected, providing that it is not amplified from any of

the negative control samples. Reliable confirmation of a positive result can also be obtained

by repeating the test with a second set of PCR primers (Appendix 6).

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

B

positive control sample containing R. solanacearum in water but negative results are obtained

from positive controls with R. solanacearum in potato extract. In multiplex PCR protocols

with internal PCR controls, inhibition of the reaction is indicated when neither of the two

amplicons are obtained.

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

negative controls.

FISH test

Principle

When the FISH test is used as the first screening test and found to be positive, Isolation or the

IF test must be performed as a second compulsory screening test. When the FISH test is used

as the second screening test and found to be positive, further testing according to the flow

scheme is required to complete the diagnosis.

Note: Use validated R. solanacearum-specific oligo-probes (see Appendix 7). Preliminary

testing with this method should permit reproducible detection of at least 103 to 104 cells of R.

solanacearum per ml added to sample extracts which previously tested negative.

The following procedure should preferably be performed on freshly prepared sample extract

but can also be successfully performed on sample extract that has been stored under glycerol

at – 16 to – 24 or – 68 to – 86 °C.

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

solanacearum.

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

solanacearum biovar 2 (e.g. strain NCPPB 4156 = PD 2762 = CFBP 3857, see Appendix 3)

in 0,01M phosphate buffer (PB) from a 3 to 5 day culture). Prepare separate positive control

slides of the homologous strain or any other reference strain of R. solanacearum, suspended

in potato extract, as specified in Appendix 3 B.

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

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

Standardized positive and negative control material available for use with this test are listed in

Appendix 3A).

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

7.1. Potato extract fixation

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

7.1.1. Prepare fixative solution (see Appendix 7).

7.1.2. Pipette 100 µl of each sample extract into an Eppendorf tube and centrifuge for 7

minutes at 7 000 g.

7.1.3. Remove the supernatant and dissolve the pellet in 200 µl of fixative prepared < 24

hours previously. Vortex and incubate for one hour in the refrigerator.

7.1.4. Centrifuge for 7 minutes at 7 000 g, remove the supernatant and resuspend the pellet in

75 µl 0,01M PB (see Appendix 7).

7.1.5. Spot 16 µl of the fixed suspensions onto a clean multitest slide as shown in Fig. 7.1.

Applying two different samples per slide, undiluted and use 10 µl to make a 1:100 dilution (in

0,01 M PB). The remaining sample solution (49 µl) can be stored at – 20 °C after addition of

one volume of 96 % ethanol. In case the FISH assay requires repeating, remove the ethanol by

centrifugation and add an equal volume of 0,01 PB (mix by vortexing).

Fig. 7.1 Layout for FISH slide

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

At this stage the procedure may be interrupted and the hybridisation continued the following

day. Slides should be stored dust-free and dry at room temperature.

7.2. Hybridisation

7.2.1. Dehydrate the cells in a graded ethanol series of 50 %, 80 % and 96 % for one minute

each. Air dry the slides in a slide-holder.

7.2.2. Prepare a moist incubation chamber by covering the bottom of an air-tight box with

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

hybridisation oven at 45 °C for at least 10 minutes.

7.2.3. Apply 10 µl of hybridisation solution (Appendix 7) to eight windows (windows 1, 2, 4,

5, 6, 7, 9 and 10; see Fig 7.1) of each slide leaving the two centre windows (3 and 8) empty.

7.2.4. Apply coverslips (24 × 24 mm) to the first and last four windows without trapping air.

Place the slides in the pre-warmed moist chamber and hybridise for five hours in the oven at

45 °C in the dark.

7.2.5. Prepare three beakers containing 1 l of Milli Q (molecular grade) water, 1 l of 1x

hybmix (334 ml 3x hybmix and 666 ml Milli Q water) and 1 l of 1/8x hybmix (42 ml 3x

hybmix and 958 ml Milli Q water). Pre-incubate each in a waterbath at 45 °C.

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

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

45 °C.

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

minutes.

7.2.9. Dip the slides briefly in Milli Q water and place them on filter paper. Remove excess

moisture by covering the surface gently with filter paper. Pipette 5 to 10 µl of anti-fading

mountant solution (e.g. Vectashield, Vecta Laboratories, CA, USA or equivalent) on each

window and apply a large coverslip (24 × 60 mm) over the whole slide.

7.3. Reading the FISH test

7.3.1. Observe the slides immediately with a microscope fitted for epifluorescence

microscopy at 630 or 1 000 × magnification under immersion oil. With a filter suitable for

B

fluorescein isothiocyanate (FITC) eubacterial cells (including most gram negative cells) in the

sample are stained fluorescent green. Using a filter for tetramethylrhodamine-5-

isothiocyanate, Cy3-stained cells of R. solanacearum appear fluorescent red. Compare cell

morphology with that of the positive controls. Cells must be bright fluorescent and completely

stained The FISH test (Section VI.A.7.) must be repeated if the staining is aberrant. Scan

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

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

7.3.2. Observe for bright fluorescing cells with characteristic morphology of R. solanacearum

in the test windows of the test slides (see web site

http://forum.europa.eu.int/Public/irc/sanco/Home/main). The fluorescence intensity must be

equivalent or better than that of the positive control strain. Cells with incomplete staining or

with weak fluorescence must be disregarded.

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

all slides in a batch show positive cells due to the contamination of buffer or if positive cells

are found (outside of the slide windows) on the the slide coating.

7.3.4. There are several problems inherent to the specificity of the FISH test. Background

populations of fluorescing cells with atypical morphology and cross reacting saprophytic

bacteria with size and morphology similar to R. solanacearum may occur, although much less

frequent as in the IF test, in potato heel end core and stem segment pellets.

7.3.5. Consider only fluorescing cells with typical size and morphology.

7.3.6. Interpretation of the FISH test result

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

morphology typical of R. solanacearum are observed using the FITC filter and bright red

fluorescent cells using the rhodamine filter in all positive controls and not in any of the

negative controls. If bright fluorescing cells with characteristic morphology are found,

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

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

cells per ml of resuspended pellet are considered potentially contaminated. Further testing is

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

considered negative.

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

of R. solanacearum are not observed using the rhodamine filter, provided that typical bright

red fluorescent cells are observed in the positive control preparations when using the

rhodamine filter.

ELISA tests

Principle

ELISA can only be used as an optional test in addition to IF, PCR or FISH due to a relatively

low sensitivity of this test. When DAS ELISA is used enrichment and the use of monoclonal

antibodies are compulsory (see web site

http://forum.europa.eu.int/Public/irc/sanco/Home/main). Enrichment of the samples before

using ELISA may be useful in order to increase the sensitivity of the test, but it can fail due to

competition by other organisms in the sample.

Note: Use an validated source of antibodies to R. solanacearum (see web site

http://forum.europa.eu.int/Public/irc/sanco/Home/main) It is recommended that the titre is

determined for each new batch of antibodies. The titre is defined as the highest dilution at

which optimum reaction occurs when testing a suspension containing 105 to 106 cells per ml

of the homologous strain of R. solanacearum and using appropriate secondary antibody

conjugates according to the manufacturer’s recommendations. During testing, the antibodies

should be used at a working dilution close to or at the titre of the commercial formulation.

Determine the titre of the antibodies on a suspension of 105 to 106 cells per ml of the

homologous strain of R. solanacearum.

Include a sample extract that previously tested negative for R. solanacearum and a suspension

of a non-cross reacting bacterium in phosphate buffered saline (PBS) as negative controls.

As positive control use aliquots of sample extract, that previously tested negative, mixed with

103 to 104 cells per ml of R. solanacearum biovar 2 (e.g. strain NCPPB 4156 = PD 2762 =

CFBP 3857, see Appendix 2 A and B). For comparison of results on each plate use a standard

suspension of 105 to 106 cells per ml in PBS of R. solanacearum. Ensure positive controls are

well separated on the microtitre plate from the sample(s) under test.

Standardised positive and negative control materials available for use with this test are listed

in Appendix 3 A.

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

Two ELISA protocols have been validated.

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

1) Use 100 to 200 µl aliquots of sample extract. (Heating at 100 °C for four minutes in a

waterbath or heating block may reduce non-specific results in some cases).

2) Add an equal volume of double strength coating buffer (Appendix 4) and vortex.

3) Apply 100 µl aliquots to each of at least two wells of a microtitre plate (e.g. Nunc-Polysorp

or equivalent) and incubate for one hour at 37 °C or overnight at 4 °C. 27.7.2006 EN Official

Journal of the European Union L 206/69

4) Flick out the extracts from the wells. Wash the wells three times with PBS-Tween

(Appendix 4), leaving the last washing solution in the wells for at least five minutes.

5) Prepare the appropriate dilution of antibodies against-R. solanacearum in blocking buffer

(Appendix 4). For validated commercial antibodies, use the recommended dilutions (usually

twice as concentrated as the titre).

6) Add 100 µl to each well and incubate for one hour at 37 °C.

7) Flick out the antibody solution from the wells and wash as before (4).

8) Prepare the appropriate dilution of secondary antibody-alkaline phosphatase conjugate in

blocking buffer. Add 100 µl to each well and incubate for one hour at 37 °C.

9) Flick out conjugated antibody from wells and wash as before (4).

10) Add 100 µl alkaline phosphatase substrate solution (Appendix 4) to each well. Incubate in

the dark at ambient temperature and read absorbance at 405 nm at regular intervals within 90

minutes.

(b) DASI ELISA

1) Prepare the appropriate dilution of anti-R. solanacearum polyclonal immunoglobulins in

coating buffer pH 9.6 (Appendix 4). Add 200 µl to each well. Incubate at 37 °C for four to

five hours or at 4 °C for 16 hours.

2) Wash the wells three times with PBS-Tween (Appendix 4).

Add 190 µl of sample extract to at least two wells. Also add positive and negative controls in

two wells each per plate. Incubate for 16 hr at 4 °C.

3) Wash the wells three times with PBS-Tween (Appendix 4).

4) Prepare an appropriate dilution of R. solanacearum-specific monoclonal antibodies in PBS

B

(Appendix 4) also containing 0,5 % bovine serum albumin (BSA) and add 190 µl to each

well. Incubate at 37 °C for two hours.

5) Wash the wells three times with PBS-Tween (Appendix 4).

6) Prepare an appropriate dilution of anti-mouse immunoglobulins conjugated with alkaline

phosphatase in PBS. Add 190 µl to each well. Incubate at 37 °C for two hours.

7) Wash the wells three times with PBS-Tween (Appendix 4).

8) Prepare an alkaline phosphatase substrate solution containing 1 mg p-nitrophenyl

phosphate per ml of substrate buffer (Appendix 4). Add 200 µl to each well. Incubate in the

dark at ambient temperature and read absorbance at 40 nm at regular intervals within 90

minutes.

Interpretation of ELISA test results

The ELISA test is negative if the average optical density (OD) reading from duplicate sample

wells is < 2x OD of that in the negative sample extract control well, providing the OD for the

positive controls are all above 1,0 (after 90 minutes incubation with the substrate) and are

greater than twice the OD obtained for negative sample extracts.

The ELISA test is positive if the average OD readings from duplicate sample wells is > 2x

OD in the negative sample extract well provided that OD readings in all negative control

wells are < 2x those in the positive control wells.

Negative ELISA readings in positive control wells indicate that the test has not been

performed correctly or that it has been inhibited. Positive ELISA readings in negative control

wells indicate that cross-contamination or nonspecific antibody binding has occurred.

Bioassay test

Note: Preliminary testing with this method should permit reproducible detection of 103 to 104

colony-forming units of R. solanacearum per ml added to sample extracts that previously

tested negative (preparation see Appendix 3).

Highest sensitivity of detection can be expected when using freshly prepared sample extract

and optimal growth conditions. However, the method can be successfully applied to extracts

that have been stored under glycerol at – 68 to – 86 °C.

The following protocol is based upon Janse (1988)

9.1. Use 10 test plants of a susceptible tomato cultivar (e.g. Moneymaker or cultivar with

equivalent susceptibility as determined by the testing laboratory) at the third true leaf stage for

each sample. For cultural details, see Appendix 8. Alternatively, use eggplants (e.g. cultivar

Black Beauty or cultivars with equivalent susceptibility), use only plants at leaf stage two to

three up to full expansion of the third true leaf. Symptoms have been show to be less severe

and to develop more slowly in eggplant. Where possible, it is therefore recommended to use

tomato seedlings.

9.2. Distribute 100 µl of sample extract between the test plants.

9.2.1. Syringe inoculation

Inoculate the plant stems just above the cotyledons using a syringe fitted with a hypodermic

needle (not less than 23G). Distribute the sample between the test plants.

9.2.2. Slit inoculation

Holding the plant between two fingers, pipette a drop (approximately 5 - 10 µl) of the

suspended pellet on the stem between the cotyledons and the first leaf.

Using a sterile scalpel, make a diagonal slit, about 1.0 cm long and approximately 2/3 of the

stem thickness deep, starting the cut from the pellet drop.

Seal the cut with sterile vaseline from a syringe.

9.3. Inoculate by the same technique, five seedlings with an aqueous suspension of 105 to 106

cells per ml prepared from a 48 hr culture of a virulent biovar 2 strain of R. solanacearum as a

positive control and with pellet buffer (Appendix 4) as negative control. Separate positive and

negative control plants from the other plants to avoid cross-contamination.

9.4. Grow the test plants in quarantine facilities for up to four weeks at 25 to 30 °C and high

relative humidity with appropriate watering to prevent waterlogging or wilting through water

deficiency. To avoid contamination incubate positive control and negative control plants on

clearly separated benches in a glasshouse or growth chamber or, in case space is limited,

ensure strict separation between treatments. If plants for different samples must be incubated

close together, separate them with appropriate screens. When fertilising, watering, inspecting

and any other manipulations take great care to avoid cross-contamination. It is essential to

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

bacterium from sample to sample.

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

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

R. solanacearum (Section VI.B.).

9.6. If no symptoms are observed after three weeks perform IF/PCR/Isolation on a composite

sample of 1 cm stem sections of each test plant taken above the inoculation site. If the test is

positive perform dilution plating (section 4.1).

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

Interpretation of the bioassay test results

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

symptoms, the bacteria can be reisolated from these plants and no symptoms are found on the

negative controls.

The bioassay test is negative if test plants are not infected by R. solanacearum, and provided

that R. solanacearum is detected in positive controls.

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

B. IDENTIFICATION TESTS

Identify pure cultures of presumptive R. solanacearum isolates using at least two of the

following tests based on different biological principles.

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

1. Nutritional and enzymatic identification tests

Determine the following phenotypic properties, which are universally present or absent in R.

solanacearum, according to the methods of Lelliott and Stead (1987), Klement et al. (1990),

Schaad (2001).

Test

Expected result

Fluorescent pigment production

–

Poly-ß-hydroxybutyrate inclusions

+

Oxidation/fermentation (O/F) test

O+/F–

Catalase activity

+

Kovac’s oxidase test

+

B

Reduction of nitrate

+

Utilisation of citrate

+

Growth at 40 °C

-

Growth in 1 % NaCl

+

Growth in 2 % NaCl

-

Arginine dihydrolase activity

-

Gelatine liquefaction

-

Starch hydrolysis

-

Aesculin hydrolysis

-

Levan production

-

2. IF test

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

2.2. Prepare a twofold dilution series of an appropriate antiserum (see website

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

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

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

positive control. L 206/72 EN Official Journal of the European Union 27.7.2006

3. ELISA test

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

to this method.

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

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

solanacearum.

3.3. A positive ELISA test is achieved if the ELISA reading obtained from the culture is at

least half that obtained for the positive control.

4. PCR tests

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

4.2. Heat 100 µl of the cell suspension in closed tubes in a heating block or boiling waterbath

at 100 °C for four minutes. The samples may then be stored at – 16 to – 24 °C until required.

4.3. Apply appropriate PCR procedures to amplify R. solanacearum-specific amplicons (e.g.

Seal et al. (1993); Pastrik and Maiss (2000); Pastrik et al. (2002); Boudazin et al. (1999);

Opina et al. (1997), Weller et al. (1999).

4.4. A positive identification of R. solanacearum is achieved if the PCR amplicons are the

same size and have the same restriction fragment length polymorphisms as for the positive

control strain.

5. FISH test

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

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

oligo-probes (Appendix 7).

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

the positive control.

6. Fatty acid profiling (FAP)

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

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

6.3. A positive FAP test is achieved if the profile of the presumptive culture is identical to that

of the positive control. The presence of characteristic fatty acids are 14:0 3OH, 16:0 2OH,

16:1 2OH and 18:1 2OH and absence of 16:0 3OH is highly indicative of a Ralstonia sp.

7. Strain characterisation methods

Strain characterisation using one of the following methods is recommended for each new case

of isolation of R. solanacearum.

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

7.1. Biovardetermination

R. solanacearum is separated into biovars on the basis of the ability to utilise and/or oxidise

three disaccharides and three hexose alcohols (Hayward, 1964 and Hayward et al., 1990).

Growth media for the biovar test is described in Appendix 2. The test can be successfully

performed by stab inoculating the media with pure cultures of R. solanacearum isolates and

incubating at 28 °C. If the media are dispensed into sterile 96 well cell culture plates (200 µl

per well) colour change from olive green to yellow can be observed within 72 hours,

indicating a positive test result.

Biovar

Utilisation of

Maltose

-

+

+

Lactose

-

+

+

D (+) Cellobiose

-

+

+

Mannitol

-

-

+

Sorbitol

-

-

+

Dulcitol

-

-

+

Additional tests differentiate biovar 2 sub-phenotypes

Biovar 2A

Biovar 2A

(Worldwide distribution)

(Found in Chile and

Colombia)

B

Utilisation of trehalose

-

+

Utilisation o fmeso-inositol

+

-

Utilisation of D ribose

-

-

Pectolytic activity ( 1 )

low

low

( 1 ) See Lelliott and Stead (1987)

7.2. Genomic fingerprinting

Molecular differentiation of strains in the R. solanacearum complex can be achieved using

several techniques, including

7.2.1. Restriction fragment length polymorphism (RFLP) analysis (Cook et al., 1989).

7.2.2. Repetitive sequence PCR using REP, BOX and ERIC primers (Louws et al., 1995;

Smith et al., 1995).

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

7.3. PCR methods

Specific PCR primers (Pastrik et al, 2002; see Appendix 6) can be used to differentiate strains

belonging to division 1 (biovars 3, 4 and 5) and division 2 (biovars 1, 2A and 2T) of R.

solanacearum, as originally defined by RFLP analysis (Cook et al., 1989) and 16S rDNA

sequencing (Taghavi et al., 1996).

C. CONFIRMATION TEST

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

solanacearum and for assessment of virulence of cultures identified as R. solanacearum.

1) Prepare an inoculum of approximately 106 cells per ml from a 24 to 48 hour culture of the

isolate to be tested and an appropriate positive control strain of R. solanacearum (e.g. NCPPB

4156 = PD 2762 = CFBP 3857; see Appendix 3).

2) Inoculate 5 to 10 susceptible tomato or eggplant seedlings at the third true leaf stage (see

Section VI.A.9).

3) Incubate for up to two weeks at 25 to 28 °C and high relative humidity with appropriate

watering to avoid waterlogging or drought stress. With pure cultures typical wilting should be

obtained within 14 days. If after this period symptoms are not present, the culture cannot be

confirmed as being a pathogenic form of R. solanacearum.

4) Observe for symptoms of wilting and/or epinasty, chlorosis and stunting.

5) Isolate from symptomatic plants by removing a section of stem about 2 cm above the

inoculation point. Comminute and suspend in a small volume of sterile distilled water or 50

mM phosphate buffer (Appendix 4). Isolate from the suspension by dilution spreading or

streaking on a suitable medium, preferably onto a selective medium (Appendix 2), incubate

for 48 to 72 hours at 28 °C and observe the formation of colonies typical of R. solanacearum.

Appendix 1

Laboratories involved in optimisation and validation of protocols

Laboratory (1)

Location

Agentur für Gesundheit und Ernährungssicherheit

Vienna and Linz

Departement Gewasbescherming

Merelbeke

Plantedirektoratet

Lyngby

B

Central Science Laboratory

Scottish Agricultural Science Agency

Laboratoire National de la Protection des Végétaux, Unité de Bactériologie

Laboratoire National de la Protection des Végétaux, Station de Quarantaine de la

Pomme de Terre

Biologische Bundesanstalt

Pflanzenschutzamt Hannover

State Laboratory

Dipartimento di Scienze e Tecnologie Agroambientali

Regione Veneto Unità Periferica per i Servizi Fitosanitari

Nederlandse Algemene Keuringsdienst

Plantenziektenkundige Dienst

Direcção-Geral de Protecção das Culturas

B

Centro Diagnostico de Aldearrubia

Salamanca

Instituto Valenciano de Investigaciones Agrarias

Valencia

Swedish University of Agricultural Sciences

Uppsala

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

Appendix 2

Media for isolation and culture of R. solanacearum

(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.

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 minutes.

Sucrose Peptone Agar (SPA)

Sucrose

20,0 g

Bacto-Peptone (Difco)

5,0 g

K 2 HPO 4

0,5 g

MgSO 4 .7H 2 O

0,25 g

Bacto-Agar (Difco)

15,0 g

Distilled water

1,0 l

pH 7,2 – 7,4

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

Kelman’s Tetrazolium Medium

Casamino acids (Difco)

1,0 g

Bacto-Peptone (Difco)

10,0 g

Dextrose

5,0 g

Bacto-Agar (Difco)

15,0 g

Distilled water

1,0 l

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

Cool to 50 °C and add a filter-sterilised solution of 2,3,5-triphenyl tetrazolium chloride

(Sigma) to obtain a final concentration

of 50 mg per l.

(b) Validated selective growth media

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

Basal medium

Casamino acids (Difco)

1,0 g

Bacto-Peptone (Difco)

10,0 g

Glycerol

5,0 ml

Bacto-Agar (Difco); see Note 2.

15,0 g

Distilled water

1,0 L

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

Cool to 50 °C and add filter-sterilised aqueous stock solutions of the following ingredients to

obtain the specified final concentrations

Crystal Violet (Sigma)

5 mg per l

Polymixin-B-Sulphate (Sigma P-1004)

600 000 U (approximately 100 mg) per

l

Bacitracin (Sigma B-0125)

1 250 U (approximately 25 mg) per l

Chloramphenicol (Sigma C-3175)

5 mg per l

Penicillin-G (Sigma P-3032)

825 U (approximately 0,5 mg) per l

2,3,5-triphenyl tetrazolium chloride (Sigma)

50 mg per l

Note

1. Use of reagents other than those specified above may affect growth of R. solanacearum.

2. Oxoid Agar #1 can be used in place of Bacto-Agar (Difco). In this case growth of R.

solanacearum will be slower, although growth of competing saprophytes may also be

reduced. Typical colonies of R. solanacearum may take 1 to 2 days longer to form and the red

colouration may be lighter and more diffuse than on Bacto-Agar.

3. Increasing bacitracin concentration to 2 500 U per l may reduce populations of competing

bacteria without affecting growth of Ralstonia solanacearum.

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

Plates should be free from surface condensation before use.

Avoid excess drying of plates.

B

Quality control should be performed after preparation of each new batch of medium by

plating a suspension of a reference culture of R. solanacearum (see Appendix 3) and

observing formation of typical colonies after incubation at 28 °C for two to five days.

(c) Validated enrichment media

SMSA Broth (Elphinstone et al., 1996)

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

chloride.

Sucrose

10 g

Proteose peptone

5 g

K 2 HPO4

0,5 g

MgSO 4

0,25 g

NaNO 3

0,25 g

Distilled water

1 l

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

Add antibiotic stock solutions as for SMSA broth.

Appendix 3

A. Commercially available standardised control material

(a) Bacterial isolates

The following bacterial isolates are recommended for use as standard reference material either

as positive controls (Table 1) or during optimisation of tests to avoid cross-reactions (Table

2). All strains are commercially available from

1. National Collection of Plant Pathogenic Bacteria (NCPPB), Central Science Laboratory,

York, UK

2. Culture Collection of the Plant Protection Service (PD), Wageningen, the Netherlands.

3. Collection Française de Bactéries Phytopathogènes (CFBP), INRA Station

Phytobactériologie, Angers, France.

Table 1 SMT reference panel of isolates of R. solanacearum

NCPPB code

SMT

Other codes

#

NCPPB 4153

CFBP 4582, Pr 3020, EURS11

NCPPB 4154

CFBP 4585, 550, EURS21

NCPPB 3857

CFBP 4587, Pr 1140, EURS26

NCPPB 1584

CFBP 4598, EURS49

NCPPB 2505

CFBP 4599, EURS50

NCPPB 4155

CFBP 4601, 502, EURS55

NCPPB 4156

71(*)

PD 2762, CFBP 3857

(*)

NCPPB 4157

LNPV 15.59

NCPPB 4158

CFBP 4608, Port 448, EURS80

NCPPB 4160

IVIA-1632-2

NCPPB 4161

B3B

NCPPB 325

CFBP 2047, KEL60-1, R842

NCPPB 3967

CFBP 4610, R285, GONg7

NCPPB 4028

CFBP 4611, R303/571, CIP310, SEQ205

NCPPB 3985

CFBP 4612, R578, CIP312

NCPPB 3989

CFBP 4613, R568, CIP226

NCPPB 3996

CFBP 3928, R276/355, CIP72, SEQ225

NCPPB 3997

CFBP 4614, R280/363, CIP49, HAY0131a

NCPPB 4029

CFBP 4615, R297/349, CIP121, CMIb2861

NCPPB 4005

CFBP 4616, R470

NCPPB 4011

CFBP 4617, R288, HEmps2

(*) Use as standard reference strain of R. solanacearum biovar 2 (race 3).

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

culture collection.

Table 2: SMT reference panel of serologically- or genetically-related bacteria for use in

optimisation of detection tests

NCPPB code

SMT

Other code

Identification

#

NCPPB 4162

CFBP 1954

Bacillus polymyxa (1)

NCPPB 4163

CFBP 1538

Pseudomonas marginalis pv. marginalis

(1)

NCPPB 4164

—

CFBP 2227

Burkholderia cepacia (2)

NCPPB 4165

—

CFBP 2459

Ralstonia pickettii (2)

NCPPB 4166

CFBP 3567

Ralstonia pickettii (1)

CSL Pr1150

NCPPB 4167

CFBP 4618

Ralstonia sp. (1)

PD 2778

NCPPB 1127

CFBP 3575

Burkholderia andropogonis (1)

NCPPB 353

CFBP 3572

Burkholderia caryophylli (1)

NCPPB 945

CFBP 3569

Burkholderia cepacia (1)

NCPPB 3708

CFBP 3574

Burkholderia glumae (1)

NCPPB 3590

CFBP 3573

Burkholderia plantarii (1)

NCPPB 3726

CFBP 3568

Banana Blood Disease Bacterium (1) (2)

(3)

NCPPB 4168

CFBP 4619

Enterobacter sp. (1)

IPO S339

NCPPB 4169

IPO 1695

Enterobacter sp. (1)

NCPPB 4170

CFBP 4621

Ochrobactrum anthropi (1) (2)

IPO S306

NCPPB 4171

CFBP 4622

Curtobacterium sp. (1) (2)

IPO 1693

NCPPB 4172

IPO 1696a

Pseudomonas sp. (1)

B

NCPPB 4173

—

PD 2318

Aureobacterium sp. (2)

NCPPB 4174

IVIA 1844.06

Flavobacterium sp. (1) (2)

(1) Potential cross-reacting strain in serological tests (IF and/or ELISA) with polyclonal

antisera.

(2) Strain from which PCR product can be amplified in some laboratories of a similar size to

that expected using specific primers OLI-1 and Y-2 (see Appendix 6).

(3) Likely to cross-react in most tests but known to occur only on banana in Indonesia.

(b) Commercially available standardised control material

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

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

tests.

Freeze dried pellet of potato extract from 200 healthy potato tubers containing 103 to 104 and

104 to 106 cells R. solanacearum biovar 2 (strain NCPPB 4156 = PD 2762 = CFBP 3857) as

positive controls for serological and PCR tests. Since cell viability is affected during freeze-

drying, these are not suitable as standard controls for isolation or bioassay tests.

Formalin-fixed suspensions of R. solanacearum biovar 2 (strain NCPPB 4156 = PD 2762 =

CFBP 3857) at 106 cells per ml as positive controls for serological tests.

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

FISH

Produce a 48 hour culture of a virulent strain of R. solanacearum race3/biovar2 (e.g. strain

NCPPB 4156 = PD 2762 = CFBP 3857) on basal SMSA medium and suspend in 10 mM

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

obtained by a faintly turbid suspension equivalent to an optical density of 0,15 at 600 nm.

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

be free from R. solanacearum.

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

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

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

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

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

microvials will be used as negative controls. Label the microvials accordingly.

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

control sample. Store at – 16 to – 24 °C until use.

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

confirmed by IF.

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

each series of test samples.

For IF and FISH tests perform assays on positive and negative control samples with each

series of test samples.

For IF, FISH and PCR assays R. solanacearum must be detected in at least the 106 and 104

cells/ml of the positive controls and not in any of the negative controls.

Appendix 4

Buffers for test procedures

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

1. Buffers for extraction procedure

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

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

shaking.

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

Lubrol flakes

Deflocculant (*)

DC silicone antifoam

Anti-foam agent (*)

Tetrasodium pyrophosphate

Anti-oxidant

Polyvinylpyrrolidone-40000 (PVP-40)

Binding of PCR inhibitors

(*) 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

1,0 l

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

2. Buffers for the IF test

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

This buffer is used for dilution of antibodies

Na 2 HPO4.12H 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 minutes.

2.2. IF-buffer-Tween

This buffer is used to wash slides.

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

2.3. Phosphate buffered glycerol, pH 7,6

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

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).

B

3. Buffers for the Indirect ELISA test

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

Na 2 CO 3

6,36 g

NaHCO 3

11,72 g

Distilled water

1,00 l

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

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

oxidised aromatic compounds.

3.2. 10X Phosphate buffered saline (PBS), pH 7,4

NaCL

80,0 g

KH 2 PO 4

2,0 g

Na2HPO4.12H 2 O

29,0 g

KCl

2,0 g

Distilled water

1,0 L

3.3. PBS-Tween

10X PBS

100 ml

10 % Tween

20 5 ml

Distilled water

895 ml

3.4. Blocking (antibody) buffer (must be freshly prepared).

10X PBS

10,0 ml

Polyvinylpyrrolidone-44000 (PVP-44)

2,0 g

10 % Tween

20 0,5 ml

Milk powder

0,5 g

Distilled water

make up to 100 ml

3.5. Alkaline phosphatase substrate solution, pH 9,8

Diethanolamine

97 ml

Distilled water

800 ml

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

Make up to 1 L with distilled water.

Add 0,2 g MgCl 2 .

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

4. Buffers for DASI ELISA test

4.1. Coating buffer, pH 9,6

Na 2 CO 3

1,59 g

NaHCO 3

2,93 g

Distilled water

1 000 ml

Dissolve ingredients and check pH 9,6

4.2. 10X Phosphate saline buffer(PBS) pH 7,2 to 7,4

NaCl

80,0 g

NaH 2 PO 4 .2 H 2 O

4,0 g

Na 2 HPO 4 .12H 2 O

27,0 g

Distilled water

1 000 ml

4.3. PBS-Tween

10X PBS

50 ml

10 % Tween

20 5 ml

Distilled water

950 ml

4.4. Substrate buffer, pH 9,8

Diethanolamine

100 ml

Distilled water

900 ml

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

Appendix 5

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 (100 ×, 40 × etc.).

3. Calculate the number of typical fluorescent cells per ml of re-suspended pellet (N).

N = C × 1 000/y × F

where y = volume of re-suspended pellet on each window

and F = dilution factor of re-suspended pellet.

Appendix 6

Validated PCR protocols and reagents

NB: Preliminary testing should permit reproducible detection of 103 to 104 cells of R.

solanacearum per ml of sample extract.

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

bacterial strains (see Appendix 3).

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

1.1. Oligonucleotide primers

Forward primer OLI-1 5′-GGG GGT AGC TTG CTA CCT GCC-3′

Reverse primer Y-2

5′-CCC ACT GCT GCC TCC CGT AGG AGT-3′

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

1.2. PCR reaction mix

Reagent

Quantity per reaction

Final

concentr

B

ation

Sterile UPW

17,65 µl

10X PCR buffer (1) (15 mM MgCl 2 )

2,5 µl

1X (1,5

mM

MgCl 2 )

dNTP mix (20 mM)

0,25 µl

0,2 mM

Primer OLI-1 (20 µM)

1,25 µl

1µM

Primer Y-2 (20 µM)

1,25 µl

1µM

Taq polymerase (5U/µl) (1)

0,1 µl

0,5 U

Sample volume

2,0 µl

Total volume

25 µl

(1) Method was validated using Taq polymerase from Perkin Elmer (AmpliTaq) and Gibco

BRL.

1.3. PCR reaction conditions

Run the following programme

1 cycle of

(i)

2 minutes at 96 °C (denaturation of template DNA)

35 cycles of

(ii)

20 seconds at 94 °C (denaturation of template DNA)

(iii)

20 seconds at 68 °C (annealing of primers)

(iv)

30 seconds at 72 °C (extension of copy)

1 cycle of

(v)

10 minutes at 72 °C (final extension)

(vi)

hold at 4 °C.

NB: This programme was optimised for use with a Perkin Elmer 9600 thermal cycler.

Modification of the duration steps of cycles (ii), (iii) and (iv) may be required for use with

other models.

1.4. Restriction enzyme analysis of amplicon.

PCR products amplified from R. solanacearum DNA produce a distinctive restriction

fragment length polymorphism with enzyme Ava II after incubation at 37 °C.

2. PCR protocol of Pastrik and Maiss (2000)

2.1. Oligonucleotide primers

Forward primer Ps-1

5′- agt cga acg gca gcg ggg g -3′

Reverse primer Ps-2

5′- ggg gat ttc aca tcg gtc ttg ca -3′

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

2.2. PCR reaction mix

Reagent

Quantity per reaction

Final

concentr

ation

Sterile UPW

16,025 µl

10X PCR buffer (1)

2,5 µl

1X (1,5

mM

MgCl2)

BSA (fraction V) (10 %)

0,25 µl

0,1 %

d-nTP mix (20 mM)

0,125 µl

0,1 mM

Primer Ps-1 (10 µM)

0,5 µl

0,2 µM

Primer Ps-2 (10 µM)

0,5 µl

0,2 µM

Taq polymerase (5U/µl) (1)

0,1 µl

0,5 U

Sample volume

5,0 µl

Total volume

25,0 µl

(1) Methods were validated using Taq polymerase from Perkin Elmer (AmpliTaq) and Gibco

BRL.

N.B. Originally optimised for MJ Research PTC 200 thermocycler with Gibco Taq

Polymerase.

Perkin Elmer AmpliTaq and buffer can also be used at the same concentrations.

2.3. PCR reaction conditions

Run the following programme

1 cycle of

(i)

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

35 cycles of

(ii)

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

(iii)

30 seconds at 68 °C (annealing of primers)

(iv)

45 seconds at 72 °C (extension of copy)

1 cycle of

(v)

5 minutes at 72 °C (final extension)

(vi)

hold at 4 °C.

NB: This programme is optimised for use with an MJ Research PTC 200 thermal cycler.

Modification of the duration steps of cycles (ii), (iii) and (iv) may be required for use with

other models.

2.4. Restriction enzyme analysis of amplicon.

PCR products amplified from R. solanacearum DNA produce a distinctive restriction

fragment length polymorphism with enzyme Taq I after incubation at 65 °C for 30 minutes.

The restriction fragments obtained from R. solanacearumspecific fragment are 457 bp and 96

bp in size.

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

3.1. Oligonucleotide primers

Forward primer RS-1-F 5′- ACT AAC GAA GCA GAG ATG CAT TA -3′

Reverse primer RS-1-R 5′- CCC AGT CAC GGC AGA GAC T -3′

Forward primer NS-5-F 5′- AAC TTA AAG GAA TTG ACG GAA G -3′

Reverse primer NS-6-R 5′- GCA TCA CAG ACC TGT TAT TGC CTC -3′

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

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

3.2. PCR reaction mix

Reagent

Quantity per reaction

Final

concentr

ation

Sterile UPW

12,625 µl

10X PCR buffer (1) (15 mM MgCl 2 )

2,5 µl

1X (1,5

mM

MgCl 2 )

B

BSA (fraction V) (10 %)

0,25 µl

0,1 %

d-nTP mix (20 mM)

0,125 µl

0,1 mM

Primer RS-1-F (10 µM)

2,0 µl

0,8 µM

Primer RS-1-R (10 µM)

2,0 µl

0,8 µM

Primer NS-5-F (10 µM) (2)

0,15 µl

0,06 µM

Primer NS-6-R (10 µM) (2)

0,15 µl

0,06 µM

Taq polymerase (5 U/µl) (1)

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) and Gibco

BRL.

(2) Concentration of primers NS-5-F and NS-6-R were optimised for potato heel end core

extraction using the homogenization method and DNA purification according to Pastrik

(2000) (see Section VI.A.6.1.a.). Re-optimisation of reagent concentrations will be required if

extraction by shaking or other DNA isolation methods are used.

3.3. PCR reaction conditions

Run the following programme

1 cycle of

(i)

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

35 cycles of

(ii)

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

(iii)

30 seconds at 58 °C (annealing of primers)

(iv)

45 seconds at 72 °C (extension of copy)

1 cycle of

(v)

5 minutes at 72 °C (final extension)

(vi)

hold at 4 °C.

NB: This programme is optimised for use with an MJ Research PTC 200 thermal cycler.

Modification of the duration steps of cycles (ii), (iii) and (iv) may be required for use with

other models.

3.4. Restriction enzyme analysis of amplicon.

PCR products amplified from R. solanacearum DNA produce a distinctive restriction

fragment length polymorphism with enzyme Bsm I or an Isoschizomere (e.g. Mva 1269 I)

after incubation at 65 °C for 30 minutes.

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

4.1. Oligonucleotide primers

Forward primer Rs-1-F 5′- ACT AAC GAA GCA GAG ATG CAT TA -3′

Reverse primer Rs-1-R 5′- CCC AGT CAC GGC AGA GAC T -3′

Reverse primer Rs-3-R 5′- TTC ACG GCA AGA TCG CTC -3′

Expected amplicon size from R. solanacearum template DNA

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

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

4.2. PCR reaction mix

(a) Biovar 1/2-specific PCR

Reagent

Quantity per reaction

Final

concentr

ation

Sterile UPW

12,925 µl

10X PCR Buffer (1)

2,5 µl

1X (1,5

mM

MgCl 2 )

BSA (fraction V) (10 %)

0,25 µl

0,1 %

d-NTP mix (20mM)

0,125 µl

0,1 mM

Primer Rs-1-F (10 µM)

2 µl

0,8 µM

Primer Rs-1-R (10 µM)

2 µl

0,8 µM

Taq polymerase (5U/µl) (1)

0,2 µl

1 U

Sample volume

5,0 µl

Total volume

25,0 µl

(1) Methods have been validated using Taq polymerase from Perkin Elmer (AmpliTaq) and

Gibco BRL.

(b) Biovar 3/4/5-specific PCR

Reagent

Quantity per reaction

Final

concentr

ation

Sterile UPW

14,925 µl

10X PCR Buffer (1)

2,5 µl

1X (1,5

mM

MgCl 2 )

BSA (fraction V) (10 %)

0,25 µl

0,1 %

dNTP mix (20 mM)

0,125 µl

0,1 mM

Primer Rs-1-F (10 µM)

1 µl

0,4 µM

Primer Rs-3-R (10 µM)

1 µl

0,4 µM

Taq polymerase (5 U/µl) (1)

0,2 µl

1 U

Sample volume

5,0 µl

Total volume

25,0 µl

(1) Methods have been validated using Taq polymerase from Perkin Elmer (AmpliTaq) and

Gibco BRL.

4.3. PCR reaction conditions

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

1 cycle of

(i)

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

35 cycles of

(ii)

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

(iii)

30 seconds at 58 °C (annealing of primers)

(iv)

45 seconds at 72 °C (extension of copy)

1 cycle of

(v)

5 minutes at 72 °C (final extension)

(vi) hold at 4 °C.

NB: This programme was optimised for use with an MJ Research PTC 200 thermal cycler.

Modification of the duration steps of cycles (ii), (iii) and (iv) may be required for use with

other models.

B

4.4. Restriction enzyme analysis of amplicon.

PCR products amplified from R. solanacearum DNA using primers Rs-1-F and Rs-1-R

produce a distinctive restriction fragment length polymorphism with enzyme Bsm I or an

Isoschizomere (e.g. Mva 1269 I) after incubation at 65 °C for 30 minutes. PCR products

amplified from R. solanacearum DNA using primers Rs-1-F and Rs-3-R have no restriction

sites.

5. Preparation of the loading buffer

5.1. Bromphenol blue (10 %-stock solution)

Bromphenol blue

5 g

Distilled water (bidest)

50 ml

5.2. Loading buffer

Glycerol (86 %)

3,5 ml

Bromphenol blue (5,1)

300 µl

Distilled Water (bidest)

6,2 ml

6. 10X Tris Acetate EDTA (TAE) buffer, pH 8.0

Tris buffer

48,40 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

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

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

2. Fixative solution

(WARNING! THE FIXATIVE CONTAINS PARAFORMALDEHYDE WHICH IS TOXIC.

WEAR GLOVES AND DO NOT INHALE. IT IS ADVISABLE TO WORK IN A FUME

CUPBOARD.)

(i) Heat 9 ml molecular grade water (e.g. Ultra pure water (UPW)) to about 60 °C and add 0,4

g paraformaldehyde. Paraformaldehyde dissolves after adding 5 drops of 1N NaOH and

stirring with a magnetic stirrer.

(ii) Adjust pH to 7.0 by addition of 1ml of 0,1M phosphate buffer (PB; pH 7,0) and 5 drops of

1N 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.

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 %

Formamide

30 %,

probe EUB 338

5 ng/µl

probe OLI-1 or OLI-2

5 ng/µl

Prepare quantities of hybridisation solution according to the calculations in Table 1. For each

slide (containing 2 different samples in duplicate) 90 µl hybridisation solution is required.

IMPORTANT: FORMAMIDE IS VERY TOXIC SO WEAR GLOVES AND TAKE NECESSARY

SAFETY PRECAUTIONS!

Table 1 Suggested quantities for preparation of hybridisation mix

Number of slides

Sterile UPW

23,1

92,4

138,6

3x hybmix

30,0

120,0

180,0

1 % SDS

0,9

3,6

5,4

Formamide

27,0

108,0

162,0

Probe EUB 338 (100 ng/µl)

4,5

18,0

27,0

Probe OLI-1 or OLI-2 (100 ng/µl)

4,5

18,0

27,0

Total volume (µl) 90,0

360,0

540,0

B

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 minutes.

Appendix 8

Eggplant and tomato culture

Sow seeds of tomato (Lycopersicon esculentum) or eggplant (Solanum melongena) in

pasteurised seed compost. Transplant seedlings with fully expanded cotyledons (10 to 14

days) into pasteurised potting compost.

Eggplants or tomatoes should be grown in a glasshouse with the following environmental

conditions prior to inoculation

Day length

14 hours or natural day length if greater;

Temperature

day 21 to 24 °C,

night14 to 18 °C.

Susceptible variety of tomato

“‘Moneymaker’”

Susceptible variety of eggplant

“‘Black Beauty’”

Suppliers

see website

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

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