MT
Control of Ralstonia solanacearum (Smith) Yabuuchi et al
Table of Provisions (19)
Statutory Text (19 Articles)
Suppliment tal-Gazzetta tal-Gvern ta’ Malta, Nru. 18,046, 9 ta’ Marzu, 2007
L.N. 42 of 2007
PLANT QUARANTINE ACT
(CAP. 433)
Control of Ralstonia solanacearum (Smith) Yabuuchi et al
(Amendment) (No. 2) Regulations, 2007
IN exercise of the powers conferred by article 32 of the Plant
Quarantine Act, the Minister for Rural Affairs and the Environment
has made the following regulations:-
(1) The title of these regulations is Control of Ralstonia Title,
solanacearum (Smith) Yabuuchi et al (Amendment) (No. 2) commencement
and scope.
Regulations, 2007, and they shall be read and construed as one with L.N. 270 of
the Control of Ralstonia solanacearum (Smith) Yabuuchi et al 2004.
Regulations,
2004, hereinafter referred to as "the principal
regulations".
(2) These regulations shall come into force on the 1st April,
2007. (3) The scope of these regulations is to transpose Council
Directive 2006/63/EC amending Annexes II to VII of Council
Directive 98/57/EEC on the control of Ralstonia solanacearum
(Smith) Yabuuchi et al.
"SCHEDULE II
TEST SCHEME FOR DIAGNOSIS, DETECTION AND IDENTIFICATION
OF RALSTONIA SOLANACEARUM (SMITH) YABUUCHI ET AL.
SCOPE OF THE TEST SCHEME
The presented scheme describes the various procedures involved in
(i)
Diagnosis of brown rot in potato tubers and of bacterial wilt in
potato, tomato and some other host plants;
(ii) Detection of Ralstonia solanacearum in samples of potato tubers,
potato-, tomato- and other host plants, water and soil;
(iii)
Identification of Ralstonia solanacearum (R. solanacearum).
GENERAL PRINCIPLES
Optimised protocols for the various methods, validated reagents and details
for the preparation of test and control materials are provided in the Appendices. A
list of the laboratories that were included in optimization and validation of
protocols is provided in Appendix 1.
Since the protocols involve detection of a quarantine organism and will
include the use of viable cultures of R. solanacearum as control materials, it will
be necessary to perform the procedures under suitable quarantined conditions with
adequate waste disposal facilities and under the conditions of appropriate licences
as issued by the official plant quarantine authorities.
Testing parameters must assure consistent and reproducible detection of
levels of R. solanacearum at the set thresholds of the selected methods.
Precise preparation of positive controls is imperative.
Testing according to the required thresholds also implies correct settings,
maintenance and calibration of equipment, careful handling and preservation of
reagents and all measures to prevent contamination between samples, e.g.
separation of positive controls from test samples. Quality control standards must
be applied to avoid administrative and other errors, especially concerning labelling
and documentation.
A suspected occurrence, as referred to in regulation 6(3), 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.
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.
For description of symptoms see Section II.1.
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-β-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.
( 9 )
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.
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.
The IF test is described in Section VI.A.4.
( 5 )
The selective isolation test is described in Section VI.A.4.
( 6 )
PCR tests are described in Section VI.A.6.
( 7 )
The FISH test is described in Section VI.A.7.
( 8 )
ELISA tests are described in Section VI.A.8.
( 9 )
The bioassay is described in Section VI.A.9.
( 10 )
Typical colony morphology is described in Section II.3.d.
( 11 ) Culturing of bioassays can fail due to competition or inhibition by saprophytic bacteria. If clear
positive results are obtained in screening test, but the isolation tests are negative, then repeat the isolation tests
from the same pellet or by taking additional vascular tissue near the heel end from cut tubers of the same
sample and, if necessary, test additional samples.
( 12 ) Reliable identification of pure cultures of presumptive R. solanacearum isolates is achieved
using the tests described in Section VI.B.
( 13 )
The pathogenicity test is described in Section VI.C.
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.
( 10 )
Typical colony morphology is described in Section II.3.d
( 11 ) Culturing or bioassays can fail due to competition or inhibition by saprophytic bacteria. If
positive results are obtained in screening test, but the isolation tests are negative, then repeat the isolation tests.
( 12 ) Reliable identification of pure presumptive R. solanacearum cultures is achieved using the tests
described in Section VI.B.
( 13 )
The pathogenicity test is described in Section VI.C.
SECTION II
DETAILED METHODS FOR DETECTION OF RALSTONIA SOLANACEARUM
IN POTATO TUBERS AND POTATO, TOMATO OR OTHER HOST PLANTS
WITH SYMPTOMS OF BROWN ROT OR BACTERIAL WILT
1. Symptoms (see website http://forum.europa.eu.int/Public/irc/sanco/
Home/main)
1.1. Symptoms on potato
The potato plant. The early stage of infection in the field is recognised by
wilting of the leaves towards the top of the plant at high temperatures during the
day with recovery at night. In early stages of wilting leaves remain green, but later
yellowing and brown necrosis develops. Epinasty also occurs. Wilting of one shoot
or whole plants becomes rapidly irreversible and results in the collapse and death
of the plant. The vascular tissue of transversely cut stems from wilted plants
usually appears brown and a milky bacterial ooze exudes from the cut surface or
can be expressed by squeezing. When a cut stem is placed vertically in water,
threads of slime will stream from the vascular bundles.
The potato tuber. Potato tubers must be cut transversely close to the heel
(stolon end) or longitudinally over the stolon end. The early stage of infection is
recognised by a glassy yellow to light brown discolouration of the vascular ring
from which a pale cream bacterial ooze emerges spontaneously after some
minutes. Later, the vascular discolouration becomes a more distinct brown and
necrosis can extend into the parenchymatous tissue. In advanced stages, infection
breaks outwards from the heel end and the eyes from which bacterial slime may
ooze causing soil particles to adhere. Reddish-brown slightly sunken lesions may
appear on the skin due to collapse of vascular tissues internally. Secondary
development of fungal and bacterial soft rots is common in the advanced stages of
the disease.
1.2. Symptoms on tomato
The tomato plant. The first visible symptom is the flaccid appearance of the
youngest leaves. Under favourable environmental conditions for the pathogen (soil
temperatures of approximately 25°C; saturated humidity), epinasty and wilting of
one side or of the whole plant follows within a few days leading to total plant
collapse. Under less favourable conditions (soil temperature below 21°C), less
wilting occurs, but large numbers of adventitious roots may develop on the stem. It
is possible to observe watersoaked streaks from the base of the stem which is
evidence of necrosis in the vascular system. When the stem is cut crosswise,
discoloured brown vascular tissues exude white or yellowish bacterial ooze.
1.3. Symptoms on other hosts
Solanum dulcamara and S. nigrum plants. Under natural conditions, wilting
symptoms are rarely observed in these weed hosts unless soil temperatures exceed
25°C or inoculum levels are extremely high (e.g. as for S. nigrum growing adjacent
to diseased potato or tomato plants). When wilting does occur, the symptoms are as
described for tomato. Non-wilting S. dulcamara plants growing with stems and
roots in water may show internal light brown discolouration of vascular tissues on
transverse section of the stem base or underwater stem parts. Bacteria may ooze
from cut vascular tissues or form threads of slime if the cut stem is placed
vertically in water, even in the absence of wilting symptoms.
2. Rapid screening tests
Rapid screening tests may facilitate presumptive diagnosis but are not
essential. Use one or more of the following validated tests
2.1. Stem streaming test
(See Section VI.A.1.)
2.2. Detection of poly-β-hydroxybutyrate (PHB) granules
Characteristic PHB granules in the cells of R. solanacearum are visualised by
staining heat-fixed smears of bacterial ooze from infected tissue on a microscope
slide with Nile Blue A or Sudan Black (See Section VI.A.2.).
2.3. Serological agglutination tests
(See Section VI.A.3.)
2.4. Other tests
Further appropriate rapid screening tests include the IF test (see Section
VI.A.5.), FISH test (see Section VI.A.7.), ELISA tests (see Section VI.A.8.) and
PCR tests (see Section VI.A.6).
3. Isolation procedure
(a)
Remove ooze or sections of discoloured tissue from the vascular ring in
the potato tuber or from the vascular strands in stems of potato, tomato or other
wilting host plants. Suspend in a small volume of sterile distilled water or 50mM
phosphate buffer (Appendix 4) and leave for 5 to 10 minutes.
(b) Prepare a series of decimal dilutions of the suspension.
(c) Transfer 50-100 µl of the suspension and dilutions to a general nutrient
medium (NA, YPGA or SPA; see Appendix 2) and/or to Kelman’s tetrazolium
medium (Appendix 2) and/or a validated selective medium (e.g. SMSA; see
Appendix 2). Spread or streak with an appropriate dilution plating technique. If
useful, prepare separate plates with a diluted cell suspension of R. solanacearum
biovar 2 as a positive control.
(d) Incubate the plates for two to six days at 28 °C.
-
On the general nutrient media, virulent isolates of R.
solanacearum develop pearly cream-white, flat, irregular and fluidal colonies
often with characteristic whorls in the centre. Avirulent forms of R.
solanacearum form small round non-fluidal, butyrous colonies which are
entirely cream-white.
-
On Kelman’s tetrazolium and SMSA media, the whorls are blood
red in colour. Avirulent forms of Ralstonia solanacearum form small round
non-fluidal, butyrous colonies which are entirely deep red.
Schedules II to VII of the principal regulations shall be Substitutes
substituted by Schedules II to VII respectively contained in the Schedules
VII of the
principal
Schedule to these regulations.
regulations.
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).
The Control of Ralstonia solanacearum (Smith) Yabuuchi Revocation of
et al (Amendments) Regulations, 2007, published by Legal Notice 33 L.N. 33 of 2007.
of 2007 are hereby revoked.
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
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.
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.
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 may be achieved by filtration and/or
centrifugation as described in Section III.1.1.3 - 1.1.5.
2.1.5. Divide the neat or concentrated sample extract into two equal parts.
Maintain one half at 4 to 10°C during testing and store the other half with 10 to
25% (v/v) sterile glycerol at -16 to -24°C (weeks) or at -68 to -86°C (month) in
case further testing is required.
2.2. Testing
See Flow chart and description of the tests and optimised protocols in the
relevant appendices
Selective isolation (see Section VI.A.4.)
IF test (see Section VI.A.5.)
PCR tests (see Section VI.A.6.)
FISH test (see Section VI.A.7.)
ELISA tests (see Section VI.A.8.)
Bioassay (see Section VI.A.9.)
SECTION IV
1. Scheme for detection and identification of R. solanacearum in water
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 saprophytic bacteria and 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.
( 8 )
Culturing can fail due to competition of inhibition by saprophytic bacteria. If high saprophyte
population are suspected to affect the reliability of the isolation, then repeat the isolation tests after dilution of
the sample in sterile water.
( 9 )
Reliable identification of pure presumptive R. solanacearum cultures is achieved using the tests
described in Section VI.B.
( 10 )
The pathogenicity test is described in Section VI.C.
2. Methods for detection and identification of R. solanacearum in
water
Principle
The validated detection scheme, described in this section, is applicable for
pathogen detection in samples of surface water and can also be applied for testing
samples of potato processing or sewage effluents. However, it is important to note
that the expected sensitivity of detection will vary with the substrate. Sensitivity of
the isolation test is affected by populations of competing saprophytic bacteria
which are generally much higher in potato processing and sewage effluents than in
surface water. Whereas the scheme below is expected to detect as few as 103 cells
per litre in surface water the sensitivity of detection in potato processing or sewage
effluents is likely to be significantly lower. For this reason, it is recommended to
test effluents after any purification treatments (e.g. sedimentation or filtration)
during which saprophytic bacterial populations are reduced. The limitations in
sensitivity of the test scheme should be considered when assessing the reliability
of any negative results obtained. Whereas this scheme has been successfully used
in survey work to determine presence or absence of the pathogen in surface water,
its limitations should be realised when used in similar surveys of potato processing
or sewage effluents.
2.1. Sample preparation
Note
-
Detection of R. solanacearum in surface water is most reliable
during late spring, summer and autumn seasons when water temperatures
exceed 15°C.
-
Repeated sampling at different times in the above mentioned
period at designated sampling points will increase the reliability of detection
by reducing the effects of climatic variation.
-
Take into account the effects of heavy rainfall and the geography
of the watercourse to avoid extensive dilution effects that may obscure
presence of the pathogen.
-
Take surface water samples in the vicinity of host plants if these
hosts are present.
2.1.1. At selected sampling points, collect water samples by filling
disposable sterile tubes or bottles at a depth if possible below 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.
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.
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
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.
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 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
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 provided.
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.
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 (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.
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 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
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 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,
-
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.
PCR tests
Principles
When the PCR test is used as the principal screening test and found to be
positive, isolation or IF must be performed as a second compulsory screening test.
When PCR is used as the second screening test and found to be positive, further
testing according to the flow scheme is required to complete the diagnosis.
Full exploitation of this method as principal screening test is only
recommended when specialised expertise has been acquired.
Note: Preliminary testing with this method should permit reproducible
detection of 103 to 104 cells of R. solanacearum per ml added to sample extracts
which previously tested negative. Optimisation experiments may be required to
achieve maximum levels of sensitivity and specificity in all laboratories.
Use validated PCR reagents and protocols (see Appendix 6). Preferably
select a method with an internal control.
Use appropriate precautions to avoid contamination of sample with target
DNA. The PCR test should be performed by experienced technicians, in dedicated
molecular biology laboratories, in order to minimise the possibility of
contamination with target DNA.
Negative controls (for DNA extraction and PCR procedures) should always
be handled as final samples in the procedure, to make evident whether any carry
over of DNA has occurred.
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
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
(a)
Method according to Pastrik (2000)
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.
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.
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.
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
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
Add 2-5 µl of DNA extract per 25 µl PCR reaction in sterile PCR
tubes according to the PCR protocols, (see Appendix 6).
6.2.4. Incorporate a negative control sample containing only PCR reaction
mix and add the same source of UPW as used in the PCR mix in place of sample.
6.2.5. Place tubes in the same thermal cycler which was used in preliminary
testing and run the appropriately optimised PCR programme (Appendix 6).
6.3. Analysis of the PCR product
6.3.1. Resolve PCR amplicons by agarose gel electrophoresis. Run at least
12 µl of amplified DNA reaction mixture from each sample mixed with 3 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 (λ = 302
nm) for amplified PCR products of the expected size (Appendix 6) and document.
6.3.4. For all new findings/cases verify authenticity of the PCR amplicon
by performing restriction enzyme analysis on a sample of the remaining amplified
DNA by incubating at the optimum temperature and time with an appropriate
enzyme and buffer (see Appendix 6). Resolve the digested fragments by agarose
gel electrophoresis as before and observe characteristic restriction fragment pattern
under UV transillumination after ethidium bromide staining and compare with the
undigested and digested positive control.
Interpretation of the PCR test result
The PCR test is negative if the R. solanacearum-specific PCR amplicon of
expected size is not detected for the sample in question but is detected for all
positive control samples (in case of multiplex PCR with plant specific internal
control primers: a second PCR-product of expected size must be amplified with the
sample in question).
The PCR test is positive if the R. solanacearum-specific PCR amplicon of
expected size and restriction pattern (when required) is detected, providing that it
is not amplified from any of the negative control samples. Reliable confirmation of
a positive result can also be obtained by repeating the test with a second set of PCR
primers (Appendix 6).
Note: Inhibition of the PCR may be suspected if the expected amplicon is
obtained from the 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.
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.".
FISH test
Principle
When the FISH test is used as the first screening test and found to be
positive, Isolation or the IF test must be performed as a second compulsory
screening test. When the FISH test is used as the second screening test and found
to be positive, further testing according to the flow scheme is required to complete
the diagnosis.
Note: Use validated R. solanacearum-specific oligo-probes (see Appendix
7). Preliminary testing with this method should permit reproducible detection of at
least 103 to 104 cells of R. solanacearum per ml added to sample extracts which
previously tested negative.
The following procedure should preferably be performed on freshly prepared
sample extract but can also be successfully performed on sample extract that has
been stored under glycerol at -16 to -24 or -68 to -86°C.
As negative controls, use aliquots of sample extract that previously tested
negative for R. solanacearum.
As positive controls prepare suspensions containing 105 to 106 cells per ml
of R. solanacearum biovar 2 (e.g. strain NCPPB 4156 = PD 2762 = CFBP 3857,
see Appendix 3) in 0,01M phosphate buffer (PB) from a 3 to 5 day culture).
Prepare separate positive control slides of the homologous strain or any other
reference strain of R. solanacearum, suspended in potato extract, as specified in
Appendix 3 B.
The use of the FITC-labelled eubacterial oligo-probe offers a control for the
hybridisation process, since it will stain all eubacteria that are present in the
sample.
Standardized positive and negative control material available for use with
this test are listed in Appendix 3A).
Test control material in an identical manner as the sample(s).
7.1. Potato extract fixation
The following protocol is based upon Wullings et al. (1998)
7.1.1.
Prepare fixative solution (see Appendix 7).
7.1.2. Pipette 100 µl of each sample extract into an Eppendorf tube and
centrifuge for 7 minutes at 7 000 g.
7.1.3. Remove the supernatant and dissolve the pellet in 200 µl of fixative
prepared < 24 hours previously. Vortex and incubate for one hour in the
refrigerator.
7.1.4. Centrifuge for 7 minutes at 7 000 g, remove the supernatant and
resuspend the pellet in 75 µl 0,01M PB (see Appendix 7).
7.1.5. Spot 16 µl of the fixed suspensions onto a clean multitest slide as
shown in Fig. 7.1. Applying two different samples per slide, undiluted and use 10
µl to make a 1:100 dilution (in 0,01 M PB). The remaining sample solution (49 µl)
can be stored at -20°C after addition of one volume of 96% ethanol. In case the
FISH assay requires repeating, remove the ethanol by centrifugation and add an
equal volume of 0,01 PB (mix by vortexing).
Fig. 7.1 Layout for FISH slide
7.1.6.
Air-dry the slides (or on slide dryer at 37°C) and fix them by
flaming.
At this stage the procedure may be interrupted and the hybridisation
continued the following day. Slides should be stored dust-free and dry at room
temperature.
7.2. Hybridisation
7.2.1. Dehydrate the cells in a graded ethanol series of 50%, 80% and 96%
for one minute each. Air dry the slides in a slide-holder.
7.2.2. Prepare a moist incubation chamber by covering the bottom of an air-
tight box with tissue or filter paper soaked in 1x hybmix (Appendix 7). Pre-
incubate the box in the hybridisation oven at 45°C for at least 10 minutes.
7.2.3. Apply 10 µl of hybridisation solution (Appendix 7) to eight windows
(windows 1, 2, 4, 5, 6, 7, 9 and 10; see Fig 7.1) of each slide leaving the two centre
windows (3 and 8) empty.
7.2.4. Apply coverslips (24 × 24 mm) to the first and last four windows
without trapping air. Place the slides in the pre-warmed moist chamber and
hybridise for five hours in the oven at 45°C in the dark.
7.2.5. Prepare three beakers containing 1 l of Milli Q (molecular grade)
water, 1 l of 1x hybmix (334 ml 3x hybmix and 666 ml Milli Q water) and 1 l of 1/
8x hybmix (42 ml 3x hybmix and 958 ml Milli Q water). Pre-incubate each in a
waterbath at 45°C.
7.2.6.
Remove the coverslips from the slides and place the slides in a slide
holder.
7.2.7. Wash away excess probe by incubation for 15 minutes in the beaker
with 1x hybmix at 45°C.
7.2.8. Transfer the slide holder to 1/8 hybmix washing solution and
incubate for a further 15 minutes.
7.2.9. Dip the slides briefly in Milli Q water and place them on filter paper.
Remove excess moisture by covering the surface gently with filter paper. Pipette 5
to 10 µl of anti-fading mountant solution (e.g. Vectashield, Vecta Laboratories,
CA, USA or equivalent) on each window and apply a large coverslip (24 × 60 mm)
over the whole slide.
7.3. Reading the FISH test
7.3.1. Observe the slides immediately with a microscope fitted for
epifluorescence microscopy at 630 or 1 000 × magnification under immersion oil.
With a filter suitable for 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.
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 test are listed in Appendix 3 A.
Test control material in an identical manner as the sample(s).
Two ELISA protocols have been validated.
(a)
Indirect ELISA (Robinson Smith et al., 1995)
1)
Use 100 to 200 µl aliquots of sample extract. (Heating at 100°C
for four minutes in a waterbath or heating block may reduce non-specific
results in some cases).
2)
Add an equal volume of double strength coating buffer
(Appendix 4) and vortex.
3)
Apply 100 µl aliquots to each of at least two wells of a microtitre
plate (e.g. Nunc-Polysorp or equivalent) and incubate for one hour at 37°C or
overnight at 4°C. (27.7.2006 EN Official Journal of the European Union L
206/69).
4)
Flick out the extracts from the wells. Wash the wells three times
with PBS-Tween (Appendix 4), leaving the last washing solution in the wells
for at least five minutes.
5)
Prepare the appropriate dilution of antibodies against-R.
solanacearum in blocking buffer (Appendix 4). For validated commercial
antibodies, use the recommended dilutions (usually twice as concentrated as
the titre).
6)
Add 100 µl to each well and incubate for one hour at 37°C.
7)
Flick out the antibody solution from the wells and wash as before
(4).
8)
Prepare the appropriate dilution of secondary antibody-alkaline
phosphatase conjugate in blocking buffer. Add 100 µl to each well and
incubate for one hour at 37°C.
9)
Flick out conjugated antibody from wells and wash as before (4).
10) Add 100 µl alkaline phosphatase substrate solution (Appendix 4)
to each well. Incubate in the dark at ambient temperature and read
absorbance at 405 nm at regular intervals within 90 minutes.
(b) DASI ELISA
1)
Prepare the appropriate dilution of anti-R. solanacearum
polyclonal immunoglobulins in coating buffer pH 9.6 (Appendix 4). Add 200
µl to each well. Incubate at 37°C for four to five hours or at 4°C for 16 hours.
2)
Wash the wells three times with PBS-Tween (Appendix 4).
Add 190 µl of sample extract to at least two wells. Also add
positive and negative controls in two wells each per plate. Incubate for 16 hr
at 4°C.
3)
Wash the wells three times with PBS-Tween (Appendix 4).
4)
Prepare an appropriate dilution of R. solanacearum-specific
monoclonal antibodies in PBS (Appendix 4) also containing 0,5% bovine
serum albumin (BSA) and add 190 µl to each well. Incubate at 37°C for two
hours.
5)
Wash the wells three times with PBS-Tween (Appendix 4).
6)
Prepare an appropriate dilution of anti-mouse immunoglobulins
conjugated with alkaline phosphatase in PBS. Add 190 µl to each well.
Incubate at 37°C for two hours.
7)
Wash the wells three times with PBS-Tween (Appendix 4).
8)
Prepare an alkaline phosphatase substrate solution containing 1
mg p-nitrophenyl phosphate per ml of substrate buffer (Appendix 4). Add
200 µl to each well. Incubate in the dark at ambient temperature and read
absorbance at 40 nm at regular intervals within 90 minutes.
Interpretation of ELISA test results
The ELISA test is negative if the average optical density (OD) reading from
duplicate sample wells is < 2x OD of that in the negative sample extract control
well, providing the OD for the positive controls are all above 1,0 (after 90 minutes
incubation with the substrate) and are greater than twice the OD obtained for
negative sample extracts.
The ELISA test is positive if the average OD readings from duplicate sample
wells is > 2x OD in the negative sample extract well provided that OD readings in
all negative control wells are < 2x those in the positive control wells.
Negative ELISA readings in positive control wells indicate that the test has
not been performed correctly or that it has been inhibited. Positive ELISA readings
in negative control wells indicate that cross-contamination or nonspecific antibody
binding has occurred.
Bioassay test
Note: Preliminary testing with this method should permit reproducible
detection of 103 to 104 colony-forming units of R. solanacearum per ml added to
sample extracts that previously tested negative (preparation see Appendix 3).
Highest sensitivity of detection can be expected when using freshly prepared
sample extract and optimal growth conditions. However, the method can be
successfully applied to extracts that have been stored under glycerol at -68 to -
86°C.
The following protocol is based upon Janse (1988)
9.1. Use 10 test plants of a susceptible tomato cultivar (e.g. Moneymaker or
cultivar with equivalent susceptibility as determined by the testing laboratory) at
the third true leaf stage for each sample. For cultural details, see Appendix 8.
Alternatively, use eggplants (e.g. cultivar Black Beauty or cultivars with
equivalent susceptibility), use only plants at leaf stage two to three up to full
expansion of the third true leaf. Symptoms have been 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 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
7.2006).
3. ELISA test
Note: If performing only 2 identification tests, do not use another
serological test in addition to this method.
3.1. Prepare a suspension of approximately 108 cells per ml in 1X PBS
(Appendix 4).
3.2. Perform an appropriate ELISA procedure with a specific monoclonal
antibody to R. solanacearum.
3.3. A positive ELISA test is achieved if the ELISA reading obtained from
the culture is at least half that obtained for the positive control.
4. PCR tests
4.1. Prepare a suspension of approximately 106 cells per ml in molecular
grade sterile water.
4.2. Heat 100 µl of the cell suspension in closed tubes in a heating block or
boiling waterbath at 100°C for four minutes. The samples may then be stored at -
16 to -24°C until required.
4.3. Apply appropriate PCR procedures to amplify R. solanacearum-specific
amplicons (e.g. Seal et al. (1993); Pastrik and Maiss (2000); Pastrik et al. (2002);
Boudazin et al. (1999); Opina et al. (1997), Weller et al. (1999).
4.4. A positive identification of R. solanacearum is achieved if the PCR
amplicons are the same size and have the same restriction fragment length
polymorphisms as for the positive control strain.
5. FISH test
5.1. Prepare a suspension of approximately 106 cells per ml in UPW.
5.2. Apply the FISH procedure (Section VI.A.7.) with at least 2 R.
solanacearum-specific oligo-probes (Appendix 7).
5.3. A positive FISH test is achieved if the same reactions are achieved from
the culture and the positive control.
6. Fatty acid profiling (FAP)
6.1. Grow the culture on trypticase soy agar (Oxoid) for 48 hours at 28°C.
6.2. Apply an appropriate FAP procedure (Janse, 1991; Stead, 1992).
6.3. A positive FAP test is achieved if the profile of the presumptive culture
is identical to that of the positive control. The presence of characteristic fatty acids
are 14:0 3OH, 16:0 2OH, 16:1 2OH and 18:1 2OH and absence of 16:0 3OH is
highly indicative of a Ralstonia sp.
7. Strain characterisation methods
Strain characterisation using one of the following methods is recommended
for each new case of isolation of R. solanacearum.
Include known reference strains where appropriate for each test performed
(see Appendix 3).
7.1. Biovardetermination
R. solanacearum is separated into biovars on the basis of the ability to utilise
and/or oxidise three disaccharides and three hexose alcohols (Hayward, 1964 and
Hayward et al., 1990). Growth media for the biovar test is described in Appendix
2. The test can be successfully performed by stab inoculating the media with pure
cultures of R. solanacearum isolates and incubating at 28°C. If the media are
dispensed into sterile 96 well cell culture plates (200 µl per well) colour change
from olive green to yellow can be observed within 72 hours, indicating a positive
test result.
Biovar
Utilisation of
Maltose
-
+
+
-
+
Lactose
-
+
+
-
+
D (+) Cellobiose
-
+
+
-
+
Mannitol
-
-
+
+
+
Sorbitol
-
-
+
+
-
Dulcitol
-
-
+
+
-
Additional tests differentiate biovar 2 sub-phenotypes
Biovar 2A
Biovar 2A
Biovar 2T
(Worldwide
(Found in
(Found in
distribution)
Chile and
tropical
Colombia)
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.
Appendix 1
Laboratories involved in optimisation and validation of protocols
Laboratory (1)
Location
Country
Agentur
für
Gesundheit
und
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
Végétaux, Station de Quarantaine de la
Pomme de Terre
Biologische Bundesanstalt
Kleinmachnow
Germany
Pflanzenschutzamt Hannover
Hannover
Germany
State Laboratory
Dublin
Ireland
Dipartimento di Scienze e Tecnologie
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
Instituto Valenciano de Investigaciones
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.
Appendix 2
Media for isolation and culture of R. solanacearum
(a)
General growth media
Nutrient Agar (NA)
Nutrient Agar (Difco)
23,0 g
Distilled water
1,0 L
Dissolve ingredients and sterilise by autoclaving at 121°C for 15 min.
Yeast Peptone Glucose Agar (YPGA)
Yeast extract (Difco)
5,0 g
Bacto-Peptone (Difco)
5,0 g
D(+) Glucose (monohydrate)
10,0 g
Bacto-Agar (Difco)
15,0 g
Distilled water
1,0 L
Dissolve ingredients and sterilise by autoclaving at 121°C for 15 minutes.
Sucrose Peptone Agar (SPA)
Sucrose
20,0 g
Bacto-Peptone (Difco)
5,0 g
K 2 HPO 4
0,5 g
MgSO 4 .7H 2 O
0,25 g
Bacto-Agar (Difco)
15,0 g
Distilled water
1,0 l
pH 7,2 - 7,4
Dissolve ingredients and sterilise by autoclaving at 121°C for 15 minutes.
Kelman’s Tetrazolium Medium
Casamino acids (Difco)
1,0 g
Bacto-Peptone (Difco)
10,0 g
Dextrose
5,0 g
Bacto-Agar (Difco)
15,0 g
Distilled water
1,0 l
Dissolve ingredients and sterilise by autoclaving at 121°C for 15 minutes.
Cool to 50°C and add a filter-sterilised solution of 2,3,5-triphenyl tetrazolium
chloride (Sigma) to obtain a final concentration of 50 mg per l.
(b) Validated selective growth media
SMSA medium (Englebrecht, 1994 as modified by Elphinstone et al., 1996)
Basal medium
Casamino acids (Difco)
1,0 g
Bacto-Peptone (Difco)
10,0 g
Glycerol
5,0 ml
Bacto-Agar (Difco); see Note 2.
15,0 g
Distilled water
1,0 L
Dissolve ingredients and sterilise by autoclaving at 121°C for 15 minutes.
Cool to 50°C and add filter-sterilised aqueous stock solutions of the
following ingredients to obtain the specified final concentrations
Crystal Violet (Sigma)
5 mg per l
Polymixin-B-Sulphate (Sigma P-1004)
600 000 U (approximately 100
mg) per l
Bacitracin (Sigma B-0125)
1 250 U (approximately 25
mg) per l
Chloramphenicol (Sigma C-3175)
5 mg per l
Penicillin-G (Sigma P-3032)
825 U (approximately 0,5 mg)
per l
2,3,5-triphenyl tetrazolium chloride (Sigma) 50 mg per l
Note
1. Use of reagents other than those specified above may affect growth of
R. solanacearum.
2. Oxoid Agar #1 can be used in place of Bacto-Agar (Difco). In this case
growth of R. solanacearum will be slower, although growth of competing
saprophytes may also be reduced. Typical colonies of R. solanacearum may take 1
to 2 days longer to form and the red colouration may be lighter and more diffuse
than on Bacto-Agar.
3. Increasing bacitracin concentration to 2 500 U per l may reduce
populations of competing bacteria without affecting growth of Ralstonia
solanacearum.
Store media and stock solutions of antibiotics at 4°C in the dark and use
within one month.
Plates should be free from surface condensation before use.
Avoid excess drying of plates.
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
A.
Commercially available standardised control material
(a)
Bacterial isolates
The following bacterial isolates are recommended for use as standard
reference material either as positive controls (Table 1) or during optimisation of
tests to avoid cross-reactions (Table 2). All strains are commercially available
from
1. National Collection of Plant Pathogenic Bacteria (NCPPB), Central
Science Laboratory, York, UK.
2. Culture Collection of the Plant Protection Service (PD), Wageningen,
the Netherlands.
3. Collection Française de Bactéries Phytopathogènes (CFBP), INRA
Station Phytobactériologie, Angers, France.
Table 1 SMT reference panel of isolates of R. solanacearum
NCPPB code
Other codes
Country of
SMT
Biovar
origin
#
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
71(*)
NCPPB 4156 (*)
PD 2762, CFBP 3857
Netherlands
NCPPB 4157
LNPV 15.59
France
NCPPB 4158
C F B P 4 6 0 8 , P o r t 4 4 8 , Portugal
EURS80
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
C F B P 4 6 11 , R 3 0 3 / 5 7 1 , Colombia
CIP310, SEQ205
44 CFBP 4612, R578, CIP312
2T
NCPPB 3985
Peru
45 CFBP 4613, R568, CIP226
2T
NCPPB 3989
Brazil
46 CFBP 3928, R276/355, CIP72, Peru
NCPPB 3996
SEQ225
CFBP 4614, R280/363, CIP49, Australia
HAY0131a
C F B P 4 6 1 5 , R 2 9 7 / 3 4 9 , Sri Lanka
CIP121, CMIb2861
CFBP 4616, R470
Philippines
CFBP 4617, R288, HEmps2 China
(*) 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
SMT Other code
Identification
#
51 CFBP 1954 Bacillus polymyxa (1)
52 CFBP 1538 Pseudomonas marginalis pv. marginalis (1)
CFBP 2227 Burkholderia cepacia (2)
-
-
CFBP 2459 Ralstonia pickettii (2)
58 CFBP 3567 Ralstonia pickettii (1)
CSL Pr1150
60 CFBP 4618 Ralstonia sp. (1)
PD 2778
53 CFBP 3575 Burkholderia andropogonis (1)
54 CFBP 3572 Burkholderia caryophylli (1)
55 CFBP 3569 Burkholderia cepacia (1)
56 CFBP 3574 Burkholderia glumae (1)
57 CFBP 3573 Burkholderia plantarii (1)
59 CFBP 3568 Banana Blood Disease Bacterium (1) (2) (3)
61 CFBP 4619 Enterobacter sp. (1)
IPO S339
62 IPO 1695
Enterobacter sp. (1)
63 CFBP 4621 Ochrobactrum anthropi (1) (2)
IPO S306
64 CFBP 4622 Curtobacterium sp. (1) (2)
IPO 1693
65 IPO 1696a
Pseudomonas sp. (1)
PD 2318
Aureobacterium sp. (2)
-
81 IVIA 1844.06 Flavobacterium sp. (1) (2)
(1) Potential cross-reacting strain in serological tests (IF and/or ELISA) with polyclonal antisera.
(2) Strain from which PCR product can be amplified in some laboratories of a similar size to that expected
using specific primers OLI-1 and Y-2 (see Appendix 6).
(3) Likely to cross-react in most tests but known to occur only on banana in Indonesia.
(b) Commercially available standardised control material
The following standard control material is available from the NCPPB culture
collection.
Freeze dried pellet of potato extract from 200 healthy potato tubers as
negative control for all tests.
Freeze dried pellet of potato extract from 200 healthy potato tubers
containing 103 to 104 and 104 to 106 cells R. solanacearum biovar 2 (strain
NCPPB 4156 = PD 2762 = CFBP 3857) as positive controls for serological and
PCR tests. Since cell viability is affected during freeze-drying, these are not
suitable as standard controls for isolation or bioassay tests.
Formalin-fixed suspensions of R. solanacearum biovar 2 (strain NCPPB
4156 = PD 2762 = CFBP 3857) at 106 cells per ml as positive controls for
serological tests.
B. Preparation of positive and negative controls for the core screening
tests PCR/IF and FISH
Produce a 48 hour culture of a virulent strain of R. solanacearum race3/
biovar2 (e.g. strain NCPPB 4156 = PD 2762 = CFBP 3857) on basal SMSA
medium and suspend in 10 mM phosphate buffer to obtain a cell density of
approximately 2 × 108 cfu per ml. This is usually obtained by a faintly turbid
suspension equivalent to an optical density of 0,15 at 600 nm.
Remove the heel end cores of 200 tubers taken from a white skin variety
production known to be free from R. solanacearum.
Process the heel ends as usual and resuspend the pellet in 10 ml.
Prepare 10 sterile 1,5 ml microvials with 900 µl of the resuspended pellet.
Transfer 100 µl of the suspension of R. solanacearum to the first microvial.
Vortex.
Establish decimal levels of contamination by further diluting in the next five
microvials.
The six contaminated microvials will be used as positive controls. The four
non-contaminated microvials will be used as negative controls. Label the
microvials accordingly.
Prepare aliquots of 100 µl in sterile 1,5 ml microvials thus obtaining nine
replicas of each control sample. Store at -16 to -24°C until use.
The presence and quantification of R. solanacearum in the control samples
should be first confirmed by IF.
For the PCR test perform DNA extraction from positive and negative control
samples with each series of test samples.
For IF and FISH tests perform assays on positive and negative control
samples with each series of test samples.
For IF, FISH and PCR assays R. solanacearum must be detected in at least
the 106 and 104 cells/ml of the positive controls and not in any of the negative
controls.
Appendix 4
Buffers for test procedures
General: Unopened sterilised buffers can be stored for up to one year
1. Buffers for extraction procedure
1.1. Extraction buffer (50 mM phosphate buffer, pH 7,0)
This buffer is used for extraction of the bacterium from plant tissues by
homogenisation or shaking.
Na 2 HPO 4 (anhydrous)
4,26 g
KH 2 PO 4
2,72 g
Distilled water
1.00 l
Dissolve ingredients, check pH and sterilise by autoclaving at 121°C for 15
min.
Additional components may be useful as follows
Purpose
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.
Make up to 1 L with distilled water.
Add 0,2 g MgCl 2 .
Dissolve 2 phosphatase substrate 5 mg tablets (Sigma) per 15 ml of solution.
4. Buffers for DASI ELISA test
4.1. Coating buffer, pH 9,6
Na 2 CO 3
1,59 g
NaHCO 3
2,93 g
Distilled water
1 000 ml
Dissolve ingredients and check pH 9,6
4.2. 10X Phosphate saline buffer(PBS) pH 7,2 to 7,4
NaCl
80,0 g
NaH 2 PO 4 .2 H 2 O
4,0 g
Na 2 HPO 4 .12H 2 O
27,0 g
Distilled water
1 000 ml
4.3. PBS-Tween
10X PBS
50 ml
10% Tween 20
5 ml
Distilled water
950 ml
4.4. Substrate buffer, pH 9,8
Diethanolamine
100 ml
Distilled water
900 ml
Mix and adjust to pH 9,8 with concentrated HCl.
Appendix 5
Determination of contamination level in IF and FISH tests
1. Count the mean number of typical fluorescent cells per field of view (c).
2. Calculate the number of typical fluorescent cells per microscope slide
window (C).
C = c × S/s
where S = surface area of window of multispot slide
and s = surface area of objective field
s = πi 2 /4G 2 K 2
where i = field coefficient (varies from 8 to 24 depending upon ocular type)
K = tube coefficient (1 or 1,25)
G = magnification of objective (100 ×, 40 × etc.).
3. Calculate the number of typical fluorescent cells per ml of re-suspended
pellet (N).
N = C × 1 000/y × F
where y = volume of re-suspended pellet on each window
and
F = dilution factor of re-suspended pellet.
Appendix 6
Validated PCR protocols and reagents
NB: Preliminary testing should permit reproducible detection of 103 to 104
cells of R. solanacearum per ml of sample extract.
Preliminary testing should also show no false positive results with a panel of
selected bacterial strains (see Appendix 3).
1. PCR protocol of Seal et al. (1993)
1.1. Oligonucleotide primers
Forward primer OLI-1 5'-GGG GGT AGC TTG CTA CCT GCC-3'
Reverse primer Y-2
5'-CCC ACT GCT GCC TCC CGT AGG AGT-3'
Expected amplicon size from R. solanacearum template DNA = 288 bp
1.2. PCR reaction mix
Reagent
Quantity per
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'
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)
(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
NB: Store all solutions containing light sensitive oligo-probes in the dark at -20°C.
Protect from direct sunlight or electric light during use.
5. 0,1M Phosphate buffer, pH 7,0
Na 2 HPO 4
8,52 g
KH 2 PO 4
5,44 g
Distilled water
1,00 L
Dissolve ingredients, check pH and sterilise by autoclaving at 121 °C for 15
minutes.
Appendix 8
Eggplant and tomato culture
Sow seeds of tomato (Lycopersicon esculentum) or eggplant (Solanum
melongena) in pasteurised seed compost. Transplant seedlings with fully expanded
cotyledons (10 to 14 days) into pasteurised potting compost.
Eggplants or tomatoes should be grown in a glasshouse with the following
environmental conditions prior to inoculation
Day length
14 hours or natural day length if greater;
Temperature
day 21 to 24°C,
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
REFERENCES
1. Amann, R.I., L. Krumholz and D.A. Stahl. 1990. Fluorescent-
oligonucleotide probing of whole cells for determinative, phylogenetic and
environmental studies in microbiology. J. Bacteriol. 172: 762-770.
2. Anon. 1998. Council Directive 98/57/EC of 20 July 1998 on the control
of Ralstonia solanacearum (Smith) Yabuuchi et al. Official Journal of the
European Communities L235, 1-39.
3. Boudazin, G., A.C. Le Roux, K. Josi, P. Labarre and B. Jouan. 1999.
Design of division specific primers of Ralstonia solanacearum and application to
the identification of European isolates. European Journal of Plant Pathology 105;
373-380.
4. Caruso, P., Gorris, M.T., Cambra, M., Palomo, J.L., Collar, J and Lopez,
M.M. 2002. Enrichment Double-Antibody Sandwich Indirect Enzyme-Linked
Immunosorbent Assay That Uses a Specific Monoclonal Antibody for sensitive
Detection of Ralstonia solanacearum in Asymptomatic Potato Tubers. Applied
and Environmental Microbiology, 68, 3634-3638.
5. Cook, D., Barlow, E. and Sequeira, L. 1989. Genetic diversity of
Pseudomonas solanacearum: detection of restriction fragment length
polymorphisms with DNA probes that specify virulence and the hypersensitive
response. Molecular Plant-Microbe Interactions 1:113-121.
6. Elphinstone, J.G., Hennessy, J., Wilson, J.K. and Stead, D.E. 1996.
Sensitivity of detection of Ralstonia solanacearum in potato tuber extracts. EPPO
Bulletin 26; 663-678.
7. Englebrecht, M.C. (1994) Modification of a semi-selective medium for
the isolation and quantification of Pseudomonas solanacearum. In: A.C. Hayward
(ed.) Bacterial Wilt Newsletter 10, 3-5. Australian Centre for International
Agricultural Research, Canberra, Australia.
8. Hayward, A.C. 1964. Characteristics of Pseudomonas solanacearum.
Journal of Applied Bacteriology 27; 265-277.
9. Hayward, A.C., El-Nashaar, H.M., Nydegger, U. and De Lindo, L.
1990. Variation in nitrate metabolism in biovars of Pseudomonas solanacearum.
Journal of Applied Bacteriology 69; 269-280.
10. Ito, S., Y. Ushijima, T. Fujii, S. Tanaka, M. Kameya-Iwaki, S.
Yoshiwara and F. Kishi. 1998. Detection of viable cells of Ralstonia solanacearum
in soil using a semi-selective medium and a PCR technique. J. Phytopathology
146; 379-384.
11. Janse, J.D. (1988) A detection method for Pseudomonas solanacearum
in symptomless potato tubers and some data on its sensitivity and specificity.
Bulletin OEPP/EPPO Bulletin 18, 343-351.
12. Janse, J.D. 1991. Infra- and intra-specific classification of Pseudomonas
solanacaerum strains using whole cell fatty-acid analysis. Systematic and Applied
Microbiology 14; 335-345.
13. Kelman, A. 1954. The relationship of pathogenicity of Pseudomonas
solanacearum to colony appearance on a tetrazolium medium. Phytopathology 44;
693-695.
14. Klement Z.; Rudolph, K and D.C. Sands, 1990. Methods in
Phytobacteriology. Akadémiai Kiadó, Budapest, 568 pp.
15. Lelliott, R.A. and Stead, D.E. 1987. Methods for the diagnosis of
bacterial diseases of plants. Blackwell scientific Publications Ltd., Oxford. 216 pp.
16. Lopez, M.M., Gorris, M.T., Llop, P., Cubero, J., Vicedo, B., Cambra,
M., 1997. Selective enrichment improves selective isolation, serological and
molecular detection of plant pathogenic bacteria. In: H.W. Dehne et al., (eds).
Klewer Academic Publishers. pp. 117-121.
17. Louws, F.J., Fulbright, D.W., Stephens, C.T. and De Bruijn, F.J., 1994.
Specific genomic fingerprints of phytopathogenic Xanthomonas and Pseudomonas
pathovars and strains generated with repetitive sequences and PCR. Applied and
Environmental Microbiology, 60, 2286-2295.
18. Louws, F.J., Fulbright, D.W., Stephens, C.T. and De Bruijn, F.J. 1995.
Differentiation of genomic structure by rep-PCR fingerprinting to rapidly classify
Xanthomonas campestris pv. vesicatoria. Phytopathology 85; 528-536.
19. Opina, N., F. Tavner, G. Holloway, J.-F Wang, T.-H Li, R. Maghirang,
M. Fegan, A.C. Hayward, V. Krishnapillai, W.F. Hong, B.W. Holloway, J.N.
Timmis. 1997. A novel method for development of species and strain-specific
DNA probes and PCR primers for identifying Burkholderia solanacearum
(formerly Pseudomonas solanacearum). As Pac. J. Mol. Biol. Biotechnol. 5; 19-
20. Pastrik, K.H. and Maiss, E. 2000. Detection of R. solanacearum in
potato tubers by polymerase chain reaction. J. Phytopathology 148; 619-626.
21. Pastrik, K.H., Elphinstone, J.G. and Pukall, R. 2002. Sequence analysis
and detection of Ralstonia solanacearum by multiplex PCR amplification of 16S-
23S ribosomal intergenic spacer region with internal positive control. European
Journal of Plant Pathology 108, 831-842.
22. Robinson-Smith, A., Jones, P., Elphinstone, J.G. and Forde, S.M.D.
(1995) Production of antibodies to Pseudomonas solanacearum, the causative
agent of bacterial wilt. Food and Agricultural Immunology 7, 67-79.
23. Schaad, W. 2001. Laboratory guide for identification of plant
pathogenic bacteria. Schaad [Hrsg.]. - 3. ed.; St. Paul, Minnesota: 373 pp.
24. Seal, S.E., L.A. Jackson, J.P.W. Young, and M.J. Daniels. 1993.
Detection of Pseudomonas solanacearum, Pseudomonas syzygii, Pseudomonas
pickettii and Blood Disease Bacterium by partial 16S rRNA sequencing
construction of oligonucleotide primers for sensitive detection by polymerase
chain reaction. J. Gen. Microbiol. 139: 1587-1594.
25. Smith, J.J., Offord, L.C., Holderness, M. and Saddler, G.S. 1995.
Genetic diversity of Burkholderia solanacearum (synonym Pseudomonas
solanacearum) race 3 in Kenya. Applied and Environmental Microbiology 61;
4262-4268.
26. Stead, D.E. 1992. Grouping of plant pathogenic and some other
Pseudomonas spp. using cellular fatty-acid profiles. International Journal of
Systematic Bacteriology 42; 281-295.
27. Taghavi, M., Hayward, A.C., Sly, L.I., Fegan, M. 1996. Analysiss of the
phylogenetic relationships of strains of Burkholderia solanacearum, Pseudomonas
syzygii, and the blood disease bacterium of banana based on 16S rRNA gene
sequences. International Journal of Systematic Bacteriology 46; 10-15.
28. Van Der Wolf, J.M., Bonants, P.J.M., Smith, J.J., Hagenaar, M., Nijhuis,
E., Van Beckhoven, J.R.C., Saddler, G.S., Trigalet, A., Feuillade, R. 1998. Genetic
diversity of Ralstonia solanacearum Race 3 in Western Europe as determined by
AFLP, RC-PFGE and rep-PCR. In: Prior, P., Allen, C. and Elphinstone, J. (eds.)
Bacterial wilt disease: Molecular and Ecological Aspects. Springer (Berlin) pp. 44-
29. Weller, S.A., Elphinstone, J.G., Smith, N., Stead, D.E. and Boonham, N.
1999. Detection of Ralstonia solanacearum strains using an automated and
quantitative flourogenic 5’ nuclease TaqMan assay. Applied and Environmental
Microbiology 66; 2853-2858.
30. Wullings, B.A., A.R. van Beuningen, J.D. Janse and A.D.L.
Akkermans. 1998. Detection of R. solanacearum, which causes brown rot of
potato, by fluorescent in situ hybridization with 23s rRNA-targeted probes. Appl.
Environ. Microbiol. 64: 4546-4554.