MT
CONTROL OF RALSTONIA SOLANACEARUM (SMITH) YABUUCHI ET AL REGULATIONS * (smith)
Table of Provisions (23)
Statutory Text (23 Articles)
CONTROL OF RALSTONIS SOLANACEARUM
(SMITH) YABUUCI ET AL
SUBSIDIARY LEGISLATION 433.07
CONTROL OF RALSTONIA SOLANACEARUM
(SMITH) YABUUCHI ET AL REGULATIONS *
15th May, 2004
LEGAL NOTICE 270 of 2004, as amended by Legal Notices 33 and 42 of
2007.
The title of these regulations is the Control of Ralstonia
solanacearum (Smith) Yabuuchi et al Regulations.
SECTION I
List of host plants of Ralstonia solanacearum (Smith) Yabuuchi et al.
referred to in regulation 2
Plants (including tubers), other than true seed,
of Solanum tuberosum L.
Potato
Plants, other than fruits and seeds, of Lycopersicon
lycopersicum (L.) Karsten ex Farw.
Tomato
SECTION II
1. The official surveys referred to in regulation 4(a) shall be based on the
biology of the organism and the particular production systems in Malta and shall
(i)
in the case of potato,
at appropriate times, visual inspection of the growing crop, and/or
sampling of both seed and other potatoes in the growing season or
in store. These samples shall be subjected to official or officially
supervised visual inspection by cutting of tubers, and
in the case of seed potatoes, and where appropriate for other
potatoes, official or officially supervised laboratory testing using
the method set out in Schedule II,
(ii) in the case of tomato,
visual inspection, at appropriate times, of at least the growing
crop of plants intended for replanting for professional use.
2. The notification of the official surveys referred to in regulation 5 shall
(i)
in the case of surveys on potatoes,
estimated total area grown, in hectares, of seed and other
potatoes,
stratification by seed category and ware, and where appropriate,
by region,
number and timing of samples taken for testing,
number of visual inspections in the field,
number (and size of sample) of visual inspections on tubers;
(ii) in the case of surveys on, at least, the growing crop of plants of tomato
intended for replanting for professional use,
estimated total number of plants,
number of visual inspections;
(iii) in the case of surveys on host plants other than potatoes and tomatoes,
including wild solanaceous host plants,
species,
number and timing of samples taken,
area sampled, as appropriate,
method of analysis;
CONTROL OF RALSTONIS SOLANACEARUM
(SMITH) YABUUCI ET AL
(iv) in the case of surveys on water and liquid waste discharges from
industrial processing or packaging premises,
-
number and timing of samples,
-
area/ location of premises sampled, as appropriate,
-
method of analysis.
The scope of these regulations is to lay down the measures
to be taken against Ralstonia solanacearum (Smith) Yabuuchi et
al., previously known as Pseudomonas solanacearum (Smith)
Smith, (hereinafter referred to as "the organism"), in order to, with
respect to the host plants of the organism listed in Schedule I
Section I, to these regulations (hereinafter referred to as "the listed
plant material")
(a) locate it and determine its distribution;
(b) prevent its occurrence and spread; and
(c) if found, to prevent its spread and to control it with the
aim of eradication.
Substituted by
L.N. 33 of 2007;
TEST SCHEME FOR DIAGNOSIS, DETECTION
L.N. 42 of 2007.
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), 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, then the suspicion is confirmed (suspected occurrence) and the testing
according to the scheme must be continued. If the second screening test is negative,
then the sample is considered not contaminated with R. solanacearum.
Confirmed presence as referred to in regulation 7(1) implies the isolation and
identification of a pure culture of R. solanacearum with confirmation of
pathogenicity.
CONTROL OF RALSTONIS SOLANACEARUM
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SECTION I
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.
Streaming test for bacterial ooze from vascular stem tissue is described in Section
VI.A.1.
Test for poly--hydroxybutyrate granules in bacterial cells is described in Section
VI.A.2.
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.
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 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.
CONTROL OF RALSTONIS SOLANACEARUM
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The standard sample size is 200 tubers, although the procedure can be used with
smaller samples if 200 tubers are not available.
Pathogen extraction and concentration methods are described in Section III.1.1.
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 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.
( )
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.
CONTROL OF RALSTONIS SOLANACEARUM
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3. Scheme for detection and identification of Ralstonia solanacearum in
samples of asymptomatic potato, tomato or other host plants
( 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.
( ) 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. 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.
CONTROL OF RALSTONIS SOLANACEARUM
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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--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.
4. 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. 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.
CONTROL OF RALSTONIS SOLANACEARUM
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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 1cm 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 1cm
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.
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 m ay be achieved by filtra tion and/or
centrifugation as described in Section III.1.1.3 - 1.1.5.
CONTROL OF RALSTONIS SOLANACEARUM
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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
See Section IV.2.1. for recommended sampling procedures.
Pathogen concentration methods are described in Section IV.2.1. Concentration
increases population of both pathogen and competing sapr ophytic bac teri a a nd is
recommended only if it does not result in inhibition of the isolation test.
The selective isolation test is described in Section VI.A.4.
The bioassay test is described in Section VI.A.9.
Enrichment PCR methods are described in Section VI.A.4.2. and Section VI.A.6.
Enrichment ELISA methods are described in Section VI.A.4.2. and Section VI.A.8.
Typical colony morphology is described in Section II.3.d.
Culturing can fail due to competition of inhibition by saprophytic bacteria. If high
CONTROL OF RALSTONIS SOLANACEARUM
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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 30cm and within 2m 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.
CONTROL OF RALSTONIS SOLANACEARUM
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SECTION V
1. Scheme for detection and identification of R. solanacearum in soil
See Section V.2.1. for recommended sampling procedures.
The selective isolation test is described in Section VI.A.4.
Enrichment PCR methods are described in Section VI.A.4.2. and Section VI.A.6.
The bioassay is described in Section VI.A.9.
Typical colony morphology is described in Section II.3.d.
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.
Reliable identification of pure presumptive R. solanacearum cultures is achieved
using the tests described in Section VI.B.
( 8 )
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.
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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% aqueous 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
CONTROL OF RALSTONIS SOLANACEARUM
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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 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.
5. 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 website
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
CONTROL OF RALSTONIS SOLANACEARUM
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(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)
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.
6. 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.
CONTROL OF RALSTONIS SOLANACEARUM
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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.
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.
Add 100 µl sample extract and place in a heating block or water bath at
95°C for 10 min.
Put tube on ice for 5 min.
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.
Add 220 µl of Easy DNA® solution A (Invitrogen), mix well by
vortexing and incubate at 65°C for 30 min.
Add 100 µl of Easy DNA® solution B (Invitrogen), vortex vigorously
until the precipitate runs freely in the tube and the sample is uniformly
viscous.
Add 500 µl of chloroform and vortex until the viscosity decreases and
the mixture is homogeneous.
Centrifuge at 15 000 g for 20 min at 4°C to separate phases and form the
interphase.
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.
Place tubes in the same thermal cycler which was used in preliminary
CONTROL OF RALSTONIS SOLANACEARUM
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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 ìl 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 ( = 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
b u ff e r ( s e e A p p e n d i x 6 ) . R e s o l v e t h e d i g e s t e d f r a g m e n t s b y a g a r o s e g e l
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 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.
7. 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
CONTROL OF RALSTONIS SOLANACEARUM
(SMITH) YABUUCI ET AL
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 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 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.
8. 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 website 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 website
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
CONTROL OF RALSTONIS SOLANACEARUM
(SMITH) YABUUCI ET AL
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)
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).
Add an equal volume of double strength coating buffer (Appendix 4)
and vortex.
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).
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.
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).
Add 100 µl to each well and incubate for one hour at 37°C.
Flick out the antibody solution from the wells and wash as before (4).
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.
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
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.
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.
Wash the wells three times with PBS-Tween (Appendix 4).
Prepare an appropriate dilution of R. solanacearum-specific monoclonal
antibodies in PBS (Appendix 4) also containing 0,5% bovine serum
albumin (BSA) and add 190 µl to each well. Incubate at 37°C for two
hours.
Wash the wells three times with PBS-Tween (Appendix 4).
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.
9. 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 shown 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
CONTROL OF RALSTONIS SOLANACEARUM
(SMITH) YABUUCI ET AL
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
+
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
The Plant Health Department shall conduct annual
systematic official surveys for the organism on the listed plant
material originating in Malta. In order to identify other possible
sources of contamination threatening the production of the listed
plant material, the Plant Health Department shall carry out a risk
assessment and, unless no risk of spread of the organism has been
identified during that assessment, it shall, in production areas of the
listed plant material, conduct targeted official surveys for the
organism on plants other than the listed plant material, including
wild solanaceous host plants, as well as on surface water which is
used for irrigation or spraying of listed plant material and on liquid
waste discharged from industrial processing or packaging premises
handling listed plant material and used for irrigation or spraying of
listed plant material. The extent of these targeted surveys shall be
determined according to the risk identified. The Plant Health
Department may also conduct official surveys for the organism on
the material, such as growing medium, soil and solid waste from
industrial processing or packaging premises.
Substituted by
L.N. 33 of 2007;
L.N. 42 of 2007.
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 regulation 7(2).
CONTROL OF RALSTONIS SOLANACEARUM
(SMITH) YABUUCI ET AL
The official surveys provided for in regulation 3 shall be
carried out
(a) for the listed plant material, according to the details set
down in point 1 of Section II, Schedule I; and,
(b) for host plants other than the listed plant material, and
*These regulations were repealed by Legal Notice 84 of 2023.
CONTROL OF RALSTONIS SOLANACEARUM
(SMITH) YABUUCI ET AL
for water including liquid waste, in accordance with
appropriate methods and, where appropriate, samples
shall be taken and subjected to official or officially
supervised laboratory testing;
(c) where appropriate for other material, in accordance
with appropriate methods. For these surveys, further
details of the inspection procedures and the number,
origin, stratification and timing of collection of
samples shall be decided by the Plant Health
Department based on sound scientific and statistical
principles and the biology of the organism and taking
into account the particular production systems of the
listed plant material and, as appropriate, of other host
plants of the organism.
Substituted by
L.N. 33 of 2007;
L.N. 42 of 2007.
The elements in the investigation referred to in regulation 7(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;
(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.
The details and results of the official surveys provided for
in regulation 3 shall be notified each year to the other Member
States and to the Commission in accordance with the provisions of
point 2 of Section II, Schedule I. These notifications shall be
submitted by the 1st June except for potatoes use as farm-saved
seed for which the notification shall be submitted by the 1st
September. The details and results for crops shall be related to the
previous year’s production.
Substituted by
L.N. 33 of 2007;
L.N. 42 of 2007.
1. The elements to be considered in the determination of the extent of probable
contamination under regulation 7(1)(a)(iii) and 5(1)(c)(iii), shall include
the listed plant material grown at a place of production designated as
contaminated under regulation 7(1)(a)(ii),
place(s) of production with some production link to the listed plant
material designated as contaminated under regulation 7(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 regulation 7(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 as contaminated under
regulation 7(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 regulation 7(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
regulation 7(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
regulation 7(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 regulations 7(1)(a)(iv) and 7(1)(c)(iii) shall include
(i)
in cases under regulation 7(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
CONTROL OF RALSTONIS SOLANACEARUM
(SMITH) YABUUCI ET AL
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 regulation 7(1)(a)(ii);
(ii) in cases where surface water has been designated as contaminated under
regulation 7(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 regulation 7(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 regulation 6(3), the Plant Health Department in thr area 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 regulation 7(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
4. The details of the additional notification referred to in regulation 7(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.
CONTROL OF RALSTONIS SOLANACEARUM
(SMITH) YABUUCI ET AL
(1) The suspected occurrence or confirmed presence of the
organism in Malta shall be reported to the Plant Health Department.
(2) In each case of suspected occurrence, the Plant Health
Department shall ensure completion of official or officially
supervised laboratory testing using, for the listed plant material, the
relevant method set out in Schedule II and in accordance with the
conditions specified in point 1 of Schedule III, or, in all other cases,
any other officially approved method, in order to confirm or refute
t h e s u s p e c t e d o c c u r r e n c e . I n t h e c a s e o f c o n f i r m a t i o n, t he
requirements laid down in point 2 of Schedule III shall apply.
(3) Pending the confirmation or refutation of a suspected
occurrence under subregulation (2), in each case of suspected
occurrence where, either
(i)
diagnostic symptoms of the disease caused by the
organism have been seen and a positive result in the
rapid screening test(s), as specified in Schedule II
section I, point 1 and section II has been obtained; or
(ii) a positive result in the screening test(s) as specified in
Schedule II section I, point 2 and section III has been
obtained,
the Plant Health Department shall
(a) prohibit the movement of plants and tubers from all
crops, lots or consignments from which the samples
have been taken, except under their control and
provided that it has been established that there is no
identifiable risk of the organism spreading;
(b) take steps to trace the origin of the suspected
occurrence;
CONTROL OF RALSTONIS SOLANACEARUM
(SMITH) YABUUCI ET AL
(c) introduce
appropriate
additional
precautionary
measures based on the level of estimated risk,
particularly in relation to production of the listed plant
material and the movement of seed potato lots other
than those referred to in point (a), produced on the
place of production from which the samples referred to
in point (a) were taken, in order to prevent any spread
of the organism.
(4) In those cases of suspected occurrence where there is a risk
of contamination of the listed plant material or surface water from
or into another Member State(s), where the suspected occurrence
has been reported in Malta the Plant Health Department shall
immediately notify, according to the risk identified, the details of
the said suspected occurrence to any other Member Stateconcerned,
and shall cooperate with the said Member States. Where any other
Member State so notifies the Plant Health Department, the Plant
Health Department shall introduce precautionary measures in
accordance with subregulation (3)(c) and take any further action, as
appropriate.
Substituted by
L.N. 33 of 2007;
L.N. 42 of 2007.
1. The provisions referred to in regulation 8(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 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.
2. The appropriate use or disposal of the listed plant material referred to in
regulation 8(2), under the control of the responsible official bodies of the Plant
Health Department concerned, with appropriate communication between responsible
official bodies to ensure such control at all times and approval by the responsible
official body of the area 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.
3. The appropriate methods for decontamination of the objects referred to in
regulation 8(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 Plant Health Department.
4. The series of measures to be implemented by the Plant Health Department
within the demarcated zone(s) established under regulation 7(1)(a)(iv) and (c)(iii)
and referred to in regulation 8(4) shall include
4.1. In cases where places of production have been designated as contaminated
under regulation 7(1)(a)(ii)
(a) in a field or unit of protected crop production designated to be
contaminated under regulation 7(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 regulation 3 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
CONTROL OF RALSTONIS SOLANACEARUM
(SMITH) YABUUCI ET AL
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;
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 paragraph 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 paragraph 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
regulation 7(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 regulation 7(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 paragraph 4.1, the Plant Health Department 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 paragraph 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
regulation 7(1)(a)(iv),
- ensure supervision by their responsible official bodies of
CONTROL OF RALSTONIS SOLANACEARUM
(SMITH) YABUUCI ET AL
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 regulation 7(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 regulation 3;
(bb) in cases where surface water has been designated as
contaminated under regulation 7(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.
(1) If official or officially supervised laboratory testing,
using, for the listed plant material, the relevant method set out in
Schedule II or, in all other cases, any other officially approved
method, confirms the presence of the organism in a sample taken
pursuant to these regulations, the Plant Health Department, having
regard to sound scientific principles, the biology of the organism
and the particular production, marketing and processing systems of
the host plants of the organism in Malta, shall
(a) for the listed plant material
(i)
establish an investigation to determine the extent
and primary source(s) of the contamination in
accordance with the provisions of Schedule IV,
with further testing in accordance with
regulation 6(2), on, at least, all clonally related
seed potato stocks, and
(ii) designate as contaminated the listed plant
material, consignment and/or lot from which the
sample was taken, and the machinery, vehicle,
vessel, store, or units thereof, and any other
objects including packaging material which have
been in contact with the listed plant material
from which the sample was taken; also designate
as contaminated, where appropriate, any field,
unit of protected crop production and place of
production from which the listed plant material
was harvested and from which the sample was
taken; and for those samples taken in the
growing season, designate as contaminated any
field, place of production, and, where
appropriate, unit of protected crop production
from which the sample was taken, and
(iii) determine, in accordance with the provisions of
point 1 of Schedule V, the extent of probable
CONTROL OF RALSTONIS SOLANACEARUM
(SMITH) YABUUCI ET AL
contamination through pre- or post-harvest
contact, through production, irrigation or
spraying links or through clonal relationship
with the designated contamination, and
(iv)
demarcate a zone on the basis of the designation
of contamination under paragraph (a)(ii), the
determination of the extent of probable
contamination under paragraph (a)(iii), and the
possible spread of the organism, in accordance
with the provisions of point 2(i) of Schedule V;
(b) for crops of host plants other than those mentioned
under paragraph (a) where production of the listed
plant material is identified at risk,
(i)
establish an investigation in accordance with
paragraph (a)(i); and
(ii) designate as contaminated the host plants of the
organism from which the sample was taken; and
(iii) determine the probable contamination and
demarcate a zone in accordance with paragraphs
(a)(iii) and (a)(iv), respectively, in relation to
production of the listed plant material;
(c) for surface water (including liquid waste discharges
from industrial processing or packaging premises
handling listed plant material) and associated wild
solanaceous host plants, where production of the listed
plant material is identified at risk through irrigation,
spraying, or flooding of the surface water,
(i)
establish an investigation including an official
survey at appropriate times on samples of
surface water and if present wild solanaceous
host plants to establish the extent of the
contamination; and
(ii) designate as contaminated the surface water
from which any sample was taken, to the extent
appropriate and on the basis of the investigation
under subparagraph (i); and
(iii) determine the probable contamination and
demarcate a zone on the basis of the designation
of contamination under subparagraph (ii), and
the possible spread of the organism taking into
account the provisions of points 1 and 2(ii) of
Schedule V.
(2) The Plant Health Department shall immediately notify the
other Member States and the Commission, in accordance with the
p r ov i s i on s of p oi nt 3 o f S c he du l e V, o f a n y c o nt a m i n a t io n
designated under subregulation (1)(a)(ii) and (c)(ii) and the details
of the zone demarcation under subregulation (1)(a)(iv) and, where
applicable, under subregulation (1)(c)(iii). The Plant Health
Department shall at the same time submit to the Commission the
additional notification set out at point 4 of Schedule V.
CONTROL OF RALSTONIS SOLANACEARUM
(SMITH) YABUUCI ET AL
(3) Where the Plant Health Department receives a notification
from other Member States in terms of paragraph 2 of Article 5 of
th e E u r op e a n Co u n c i l D i r e c t i v e 9 8 /5 7 , i t w i l l e s t a bl i s h a n
investigation in accordance with subregulation (1)(a)(i) and, where
applicable, subregulation (1)(c)(i) and take further action, as
appropriate, in accordance with subregulations (1) and (2).
Substituted by
L.N. 33 of 2007;
L.N. 42 of 2007.
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 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.
(1) The listed plant material designated to be contaminated
under regulation 7(1)(a)(ii) may not be planted and, under the
control and approval of the Plant Health Department, it shall be
subjected to one of the provisions of point 1 of Schedule VI, such
that it is established that there is no identifiable risk of the
organism spreading.
(2) The listed plant material determined as probably
contaminated under regulation 7(1)(a)(iii) and (c)(iii) including
listed plant material for which a risk has been identified, produced
on places of production determined as probably contaminated
under regualtion 7(1)(a)(iii) may not be planted and shall, under the
control of the Plant Health Department, be put to appropriate use or
disposal as specified in point 2 of Schedule VI, such that it is
established that there is no identifiable risk of the organism
spreading.
(3) Any machinery, vehicle, vessel, store, or units thereof, and
any other objects including packaging material, designated as
contaminated under regulation 7(1)(a)(ii) or determined as
probably contaminated under regulations 7(1)(a)(iii) and (c)(iii),
shall either be destroyed or decontaminated using appropriate
methods as specified in point 3 of Schedule VI. After
decontamination, any such objects shall no longer be considered
contaminated.
(4) Without prejudice to the measures implemented under
subregulations (1), (2) and (3), in the zone demarcated under
regulation 7(1)(a)(iv) and (c)(iii), a series of measures, as specified
in points 4.1 and 4.2 of Schedule VI, shall be implemented. Details
of these measures shall be notified annually to the other Member
States and to the Commission.
Seed potatoes shall meet the requirements of the Plant
Quarantine Act, and shall derive in direct line from potato material
obtained under an officially approved programme which has been
found free of the organism in official or officially supervised
testing using the relevant method set out in Schedule II. The testing
shall be carried out by the Plant Health Department
(a) in cases where there has been confirmed any finding of
the organism in local seed potato production,
(i)
by testing of the earlier propagations, including
the initial clonal selection and systematic testing
of basic seed potato clones; or
(ii) in cases where it has been established that there
is no clonal relationship, by testing of all basic
seed potato clones or earlier propagations
including the initial clonal selection, and
CONTROL OF RALSTONIS SOLANACEARUM
(SMITH) YABUUCI ET AL
(b) in other cases, either on each plant of the initial clonal
selection or on representative samples of the basic
seed potatoes or earlier propagations.
The holding and handling of the organism is hereby
banned.
Without prejudice to the provisions of the Plant Quarantine
Act, the Plant Health Department may authorise derogations from
the measures referred to in regulations 8 and 10 in accordance with
th e prov isions laid down in th e Plan t Quaran tine (Harm ful
Organisms) Regulations, for trial or scientific purposes, and for
work on varietal selections.
(1) The Plant Health may adopt such additional or stricter
measures as may be required to combat the organism or to prevent
it from spreading, insofar as they are in compliance with the
provisions of the Plant Quarantine Act.
(2) The details of these measures shall be notified to the other
Member States and to the Commission.
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).
CONTROL OF RALSTONIS SOLANACEARUM
(SMITH) YABUUCI ET AL
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
Biovar 2T
(Worldwide
(Found in Chile
(Found in
distribution)
and Colombia) tropical areas)
Utilisation of trehalose
-
+
+
Utilisation of meso-inositol
+
-
+
Utilisation of D ribose
-
-
+
Pectolytic activity ( 1 )
low
low
high
( 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.
CONTROL OF RALSTONIS SOLANACEARUM
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Appendix 1
Laboratories involved in optimisation and validation of protocols
Laboratory (1)
Location
Country
Agentur
für
Gesundheit
u n d Vienna and Linz
Austria
Ernährungssicherheit
Departement Gewasbescherming
Merelbeke
Belgium
Plantedirektoratet
Lyngby
Denmark
Central Science Laboratory
York
England
Scottish Agricultural Science Agency
Edinburgh
Scotland
Laboratoire National de la Protection des Angers
France
Végétaux, Unité de Bactériologie
Laboratoire National de la Protection des Le Rheu
France
V é g é t a u x , St a t i o n d e Q u a r a n t a i n e d e l a
Pomme de Terre
Biologische Bundesanstalt
Kleinmachnow
Germany
Pflanzenschutzamt Hannover
Hannover
Germany
State Laboratory
Dublin
Ireland
D i p a r t i m e n t o d i S c i e n z e e Te c n o l o g i e Bologna
Italy
Agroambientali
Regione Veneto Unità Periferica per i Servizi Verona
Italy
Fitosanitari
Nederlandse Algemene Keuringsdienst
Emmeloord
Netherlands
Plantenziektenkundige Dienst
Wageningen
Netherlands
Direcção-Geral de Protecção das Culturas
Lisbon
Portugal
Centro Diagnostico de Aldearrubia
Salamanca
Spain
I n s t i t u t o Va l e n c i a n o d e I n v e s t i g a c i o n e s Valencia
Spain
Agrarias
Swedish University of Agricultural Sciences
Uppsala
Sweden
(1) Contact scientists: see web site http://forum.europa.eu.int/Public/irc/sanco/
Home/main.
CONTROL OF RALSTONIS SOLANACEARUM
(SMITH) YABUUCI ET AL
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
CONTROL OF RALSTONIS SOLANACEARUM
(SMITH) YABUUCI ET AL
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 wi ll be slower, althoug h 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.
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 HPO 4
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
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
Country of origin Biovar
#
NCPPB 4153
CFBP 4582, Pr 3020, EURS11
Egypt
NCPPB 4154
CFBP 4585, 550, EURS21
Turkey
NCPPB 3857
CFBP 4587, Pr 1140, EURS26
England
NCPPB 1584
CFBP 4598, EURS49
Cyprus
NCPPB 2505
CFBP 4599, EURS50
Sweden
NCPPB 4155
CFBP 4601, 502, EURS55
Belgium
NCPPB 4156 (*)
71(*)
PD 2762, CFBP 3857
Netherlands
NCPPB 4157
LNPV 15.59
France
NCPPB 4158
CFBP 4608, Port 448, EURS80
Portugal
NCPPB 4160
IVIA-1632-2
Spain
NCPPB 4161
B3B
Germany
NCPPB 325
CFBP 2047, KEL60-1, R842
USA
NCPPB 3967
CFBP 4610, R285, GONg7
Costa Rica
NCPPB 4028
CFBP 4611, R303/571, CIP310,
Colombia
SEQ205
NCPPB 3985
CFBP 4612, R578, CIP312
Peru
2T
NCPPB 3989
CFBP 4613, R568, CIP226
Brazil
2T
NCPPB 3996
CFBP 3928, R276/355, CIP72,
Peru
SEQ225
NCPPB 3997
CFBP 4614, R280/363, CIP49,
Australia
HAY0131a
NCPPB 4029
CFBP 4615, R297/349, CIP121,
Sri Lanka
CMIb2861
NCPPB 4005
CFBP 4616, R470
Philippines
NCPPB 4011
CFBP 4617, R288, HEmps2
China
CONTROL OF RALSTONIS SOLANACEARUM
(SMITH) YABUUCI ET AL
(*)
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)
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.
CONTROL OF RALSTONIS SOLANACEARUM
(SMITH) YABUUCI ET AL
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
Quantity (per l)
Lubrol flakes
Deflocculant (*)
0,5 g
DC silicone antifoam
Anti-foam agent (*)
1,0 ml
Tetrasodium pyrophosphate
Anti-oxidant
1,0 g
Polyvinylpyrrolidone-40000
(PVP-40)
Binding of PCR inhibitors
50 g
(*)
For use with homogenisation extraction method
1.2. Pellet buffer (10 mM phosphate buffer, pH 7,2)
This buffer is used for resuspension and dilution of potato tuber heel-end core
extracts following concentration to a pellet by centrifugation.
Na 2 HPO 4 .12H 2 O
2,7 g
NaH 2 PO 4 .2H 2 O
0,4 g
Distilled water
1,0 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 HPO 4 .12H 2 O
2,7 g
NaH 2 PO 4 .2H 2 O
0,4 g
NaCL
8,0 g
Distilled water
1,0 l
Dissolve ingredients, check pH and sterilise by autoclaving at 121°C for 15
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).
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
Na2HPO 4 .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.
CONTROL OF RALSTONIS SOLANACEARUM
(SMITH) YABUUCI ET AL
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 = i 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
F = dilution factor of re-suspended pellet.
CONTROL OF RALSTONIS SOLANACEARUM
(SMITH) YABUUCI ET AL
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
Final concentration
reaction
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 (5Uµl) ( 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
Final concentration
reaction
Sterile UPW
16,025 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 (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. solanacearum-
specific 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'
CONTROL OF RALSTONIS SOLANACEARUM
(SMITH) YABUUCI ET AL
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
Final concentration
reaction
Sterile UPW
12,625 µl
10X PCR buffer ( 1 ) (15 mM MgCl 2 )
2,5 µl
1X (1,5 mM MgCl 2 )
BSA (fraction V) (10%)
0,25 µl
0,1%
d-nTP mix (20 mM)
0,125 µl
0,1 mM
Primer 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
Final concentration
reaction
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
Final concentration
reaction
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)
CONTROL OF RALSTONIS SOLANACEARUM
(SMITH) YABUUCI ET AL
(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.
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
184,8
231,0
3x hybmix
30,0
120,0
180,0
240,0
300,0
1% SDS
0,9
3,6
5,4
7,2
9,0
Formamide
27,0
108,0
162,0
216,0
270,0
Probe EUB 338 (100 ng/µl)
4,5
18,0
27,0
36,0
45,0
Probe OLI-1 or OLI-2 (100 ng/µl)
4,5
18,0
27,0
36,0
45,0
Total volume (µl)
90,0
360,0
540,0
720,0
900,0
CONTROL OF RALSTONIS SOLANACEARUM
(SMITH) YABUUCI ET AL
Store all solutions containing light sensitive oligo-probes in the dark at -20°C.
NB
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,
night 14 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
CONTROL OF RALSTONIS SOLANACEARUM
(SMITH) YABUUCI ET AL
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