331/2004 Sb.
The DECREE
from day 4. May 2004
on measures to ensure the protection against the introduction and spread of the agent
the originator of the potato ring rot disease and brown rot
Change: 328/2008 Sb.
The Ministry of agriculture provides under section 88, paragraph. 2 of the Act No. 326/2004
Coll., on phytosanitary care and amendments to some related laws,
(hereinafter referred to as "the Act") for the implementation of section 71, paragraph. 1 (a). I) of the Act:
§ 1
The subject of the edit
This Decree incorporates the relevant provisions of European Community ^ 1)
and regulates the measures, which shall apply to the territory of the Czech Republic against the
spreading Clavibacter michiganensis (Smith) Davis et al. SSP..
sepedonicus (Spieckermann et Kotthoff) Davis et al., agents
potato ring rot, Ralstonia solanacearum (Smith)
Yabuuchi et al., the originators of bacterial brown rot in order to
and prevent its occurrence and spread),
(b) determine the location of and) range extension and
(c)) when found, to prevent their spread and to control it with
safeguard measures with a view to their eradication.
§ 2
The concepts of
For the purposes of this Decree:
and the originator of the ring rot bacterium) Clavibacter michiganensis (Smith)
Davis et al SSP. sepedonicus (Spieckermann et Kotthoff) Davis et al.,
(b)) the originator of the brown rot bacterium Ralstonia solanacearum (Smith)
Yabuuchi et al.,
(c)) host plants-
1. the originator-potatoes (Solanum tuberosum l.), with the exception of the
the seeds of potato, and other plants of the Solanaceae family (Solanaceae),
which was confirmed by the possibility of infection by the originator in the ring
natural conditions,
2. the agent of brown rot-potatoes (Solanum tuberosum l.), with the exception of the
the seeds of the potato, the tomato (Lycopersicon lycopersicum l.) seeds
and fruits, Eggplant nights (Solanum dulcamara l.) and other plants of the
Solanaceae (Solanaceae), which was confirmed by the possibility of infection
the originator of the brown rot in natural conditions,
d) detection survey-a survey for the presence of the agent
ring rot or the agent of brown rot in a particular territory or in certain
lot,
e-survey vymezovacím survey) to determine the extent and origin of contamination
the originator of the ring or the originator of the brown rot, to determine how to
the spread of infection and to determine the boundaries of the territory covered by the Special
plant-health measures under section 76 of the Act,
(f) the probable originator of ring rot) infestation or the originator of the Brown
rot-suspected presence of ring rot agent or agent Brown
rot in the plant, consignment, consignment, devices, machines, transport
resource inventory or parts thereof, and any other objects
including packaging material, under section 11 (2). 1 of the Act,
(g) the possible extension of ring rot agent) or the agent of brown rot
-suspected presence of ring rot agent or agent of brown rot
on land, in the object or in the territory, under section 11 (2). 1 of the Act,
(h)) infestation of ring rot or the originator of the originator of the brown rot-
the presence of ring rot agent or agent of brown rot under section 11
paragraph. 1 of the Act,
I) quarantine the territory-territory defined State phytosanitary administration
(hereinafter referred to as "phytosanitary administration"), which are subject to special
plant-health measures under section 76, paragraph. 1 of the law because of the confirmation
the presence of ring rot agent or agent of brown rot,
j) safety zone-the area designated on the phytosanitary administration
relating emergency phytosanitary measures under section 76 of the Act
because of the suspected presence of ring rot agent or agent Brown
rot,
clonally related to) the lots-batches of host plants, which
was established by vegetative propagation from one of the original batch; While for:
1. sister clonally related lot lot, consider clonally related
incurred in the same year in a vegetative way from single parent
of the lot, in the case of tomato plants that grew from the same lot
seed,
2. parental clonally related game considers the batches of clonally
related sister clonally related topics, that it immediately
arose.
§ 3
A survey of the occurrence of ring rot agent and the agent of brown rot
(1) the phytosanitary administration carried out annually in accordance with section 10, paragraph 1. 1
a systematic survey of the occurrence of the Act, the originator of the ring and the originator of the
brown rot in the range referred to in paragraphs 2 to 7.
(2) Detekčnímu survey of ring rot agent and agent Brown
rot shall be subject to
and) propagating material of potato ^ 2) originating in the Czech Republic
the scope according to § 8, paragraph. 2,
(b) selected at random) propagating material of potato ^ 2)
originating in the Member States of the European Union (hereinafter referred to as "Member State"),
c) randomly selected batches of potatoes originating in the Czech nesadbových
Republic and the other Member States,
(d)) all the potato cultivated in the safety zone defined
under section 5 (3). 1 (a). (c)).
(3) the survey on the occurrence of the Vymezovacímu ring rot agent or agent
brown rot are subject to all host plants labeled
as the originator of occurrence is suspected ring rot or the originator of the Brown
rot and all host plants produced in
quarantine the territory defined pursuant to section 5 (3). 1 (a). (c)).
(4) the detection of ring rot agent and the agent of brown rot in
plant-health checks of consignments imported from third countries and
exported to third countries shall be subject to
and all imported batches) propagating material of potato ^ 2) and
randomly selected topics and other potatoes,
(b)) all the potato lot intended for export to third countries.
(5) Detekčnímu survey on the occurrence of the cause of brown rot, moreover, are subject to
and) growths tomato plants intended for further cultivation,
(b) water resources) used to host plants, the cause of Brown závlaze
rot including accompanying the host plants and the waste water from
processing undertakings, which are industrially processed potato tubers,
where solid wastes from these enterprises.
(6) Vymezovacímu survey of the prevalence of the agent of brown rot, moreover, are subject to
in the case of suspected occurrence or confirmation of its occurrence of water resources
used to závlaze lots of suspected occurrence of weed and the host
plants, growing medium, where appropriate, in places of production where
These sessions.
(7) the presence of ring rot agent and the agent of brown rot is determined
and laboratory testing of samples) in Potato tubers samples taken in cases of
referred to in paragraph 2 (a). and (b))), paragraph 3 and in paragraph 4 (b).
(b)) of the stands or from landfills, in the case referred to in paragraph 4 (b). and)
from imported parts,
(b) the inspection of the whole potato) and cut the pulp of tubers in the stands
or in landfills, where appropriate, laboratory testing of samples of tubers
the potato, in the cases referred to in paragraph 2 (a). (c)), and (d)),
(c) inspection of the tomato crops) at an appropriate time with a subsequent laboratory
the testing of samples of the plants with symptoms of brown rot in the originator of the
the cases referred to in paragraph 5 (a). and)
(d) laboratory testing of samples of the water), the growing medium, hard
waste or host plants of the coastal vegetation in the case referred
in paragraph 5 (a). (b)) and in paragraph 6.
(8) the method of inspection lot and the number, origin, composition and duration of the subscription
the samples in the survey, carried out in accordance with paragraphs 2 to 7, the scope of this
survey and method of conveying information about the samples taken and tested
samples determined in accordance with section 10, paragraph 1. the plant health Act
management on the basis of sound scientific and statistical principles and biology
the originator of ring rot and brown rot agent, taking into account the
systems of production of potatoes and other host plants and in
Depending on the current situation of the occurrence of or on the level of risk
the introduction of ring rot agent and the agent of brown rot and be published in the
Journal of the State plant health management (hereinafter referred to as "the Gazette"). Testing
samples for the detection and diagnosis of ring rot agent and
the causative agent of brown rot shall be carried out in the manner specified in annex No. 1.
or laid down by the phytosanitary administration in accordance with section 10, paragraph 1. 1
the law and published in the Gazette.
(9) to the survey carried out in accordance with paragraphs 2 and 4, you can also use
the samples tested officially collected or the natural or legal person,
responsible for this activity, the Ministry of agriculture, pursuant to section 71, paragraph. 1
(a). (b) the plant protection act or the Administration) under section 72, paragraph. 5 (a).
even if the law) will ensure regular monitoring on the basis of this
the Administration, that the samples will be collected and tested in the manner prescribed by the
of paragraph 8.
(10) the results of the survey carried out in accordance with paragraphs 2 to 6 Announces
phytosanitary administration in writing without undue delay of the legal or
a natural person who owns the lot of potato or tomato plants and
or is entitled to this batch of placing on the market or grown or
as in the case of propagating material of a ^ 2) potato and tomato plants
also the central control and testing Institute of agriculture.
(11) the details of the implementation of the survey in accordance with paragraphs 2 to 8 shall submit to the
phytosanitary administration, in accordance with section 10, paragraph 1. 5 of the Act, once a year in
the range specified in annex No. 2 section, and the other Member States and the
The Commission, in order to ensure a comparable level of confirmation of the absence of
ring rot agent and the agent of brown rot as between Member States.
The results of the official surveys carried out in accordance with paragraphs 2 to 6 Announces
phytosanitary Administration once a year to the other Member States and the Commission,
in the case of a survey in the propagating material of the potato, ^ 2) množeném
only for planting on the grounds of the producer, to 1. September, in other
cases up to 1. June. Information notice about the details and results of the
a survey concerning the crops of host plants, always relate to the
the production of the previous year. The details of this notification shall be confidential.
§ 4
Measures for the detection of suspected occurrence, the originator of the diagnosis or
the causative agent of brown rot
(1) for the suspected presence of ring rot agent or agent Brown
rot in accordance with § 11 (1). 1 of the Act shall be deemed to
and phytosanitary Administration) verified the findings of the Visual symptoms
the infestation of these harmful organisms and, in the case of the agent of brown rot
a positive result in the rapid screening test according to annex No 1
section I and section II, number 1, or
(b) positive screening test) carried out by the
phytosanitary administration or under its supervision in the manner prescribed in
Annex No 1.
(2) in the case of suspicion of the presence of ring rot agent or agent
brown rot phytosanitary administration in vocational investigation under section
paragraph 76. 7 of the Act and special legislation ^ 3) shall ensure implementation of the
the official laboratory testing, and in the manner prescribed by section 3 of the
paragraph. 8 and in compliance with the conditions laid down in annex 2, part B,
order to confirm or deny this suspicion.
(3) the phytosanitary Administration begins immediately after the discovery of a suspected
the presence of ring rot agent or agent of brown rot of professional
the investigation under section 76, paragraph. 2 of the Act in order to determine the origin of the suspect
the occurrence of and in accordance with § 11 (1). 1 of the law
and the move) disables all lots or consignments from which they were removed
samples, with the exception of those which are subject to measures under the supervision of the
the phytosanitary administration, which eliminates the risk of discoverable extension
ring rot agent or agent of brown rot, and
(b) shall order appropriate measures) based on the level of estimated risk
possible further spread of ring rot agent or agent Brown
rot in order to prevent the spread of the movement, including the prohibition or
ensure official supervision of the movement of all other lots
host plants in the grounds and in the buildings connected with the suspect
from the presence of, or outside of these land and buildings.
(4) When a suspected risk of contamination of the hands of host plants
or, in the case of the agent of brown rot, surface waters originating from another
Member State or in another Member State, it shall notify the
phytosanitary administration official phytosanitary service immediately
the Member State concerned for details concerning this suspicion and
It will be with this State to cooperate in an appropriate manner.
(5) if the suspected presence of ring rot agent or
the causative agent of brown rot, which may have originated in the Czech Republic, or
that may threaten the Czech Republic, the Czech Republic, informed
phytosanitary administration orders the appropriate measures referred to in paragraphs 2 and 3.
§ 5
Measures in case of confirmation of ring rot agent or agent
brown rot
(1) if the official laboratory testing of samples taken in a manner
laid down pursuant to article 3, paragraph 3. 8 confirm the presence of ring rot agent or
the causative agent of brown rot, phytosanitary administration in accordance with section 76, paragraph. 2
the law and taking into account the latest scientific results, biology
ring rot agent and the agent of brown rot and the manner of cultivation,
the placing on the market and the industrial processing of host plants
and as contaminated plants) marks, the consignment or lot, and equipment,
machinery, transport equipment, warehouses, or parts thereof, and any other
objects including packaging material, from which the sample was taken, and that
come into contact with the listed plant material from which the sample was
removed, and the place or places of production, including those where the full
replacement of the growing medium, and the land on which the infected plants
produced and from which the sample was taken,
(b)) performs a professional investigation aimed to determine the extent of the likely
contamination and the initial source of infection in accordance with the provisions of point 1
Annex 3, including the testing of clonally related lots of host
According to § 6 of the plant and shall, in accordance with the provisions of point 2 of the annex No 3
the extent of probable contamination through předsklizňového or
post-harvest contact, and on the basis of clonal relatedness, cultivation,
irrigation and watering or other growing ties with an infected
the subject or the object, designated in accordance with subparagraph (a)),
(c)) shall establish a quarantine on the basis of the designation of contaminated items or
objects under (a)), and the safety zone on the basis of the determination of the
the extent of probable contamination under point (a) (b)), and the possible extension of the
ring rot agent or agent of brown rot in accordance with
the provisions of section 3 of annex 3.
(2) in case of confirmation of the occurrence of the cause of brown rot with regard to
surface water, waste water from processing and packaging equipment
potatoes and with regard to the host plants of the Solanaceae from
the accompanying vegetation through which irrigation,
watering or flooding of the surface water could be jeopardised by growing
potato phytosanitary administration
and) will ensure the official laboratory testing of samples of the surface
water treatment and, where appropriate solanaceous host plants in the manner
laid down in annex No. 1 at appropriate times in order to determine the scope of
contamination,
(b)) marks in accordance with the positive results of the official laboratory
testing in accordance with point (a)) the surface water from which they have been collected
samples as contaminated,
(c)) shall establish a quarantine on the basis of the designation of surface water for
contaminated according to point (b)), and the safety zone on the basis of the determination of the
the extent of probable contamination, the originator of the brown rot and its possible
the extension referred to in point 3 of annex 3.
(3) the phytosanitary administration shall immediately notify the other Member States
and the Commission, in accordance with the provisions of point 4 of the annex No 3 confirmed presence of
ring rot agent or agent of brown rot in accordance with paragraph 1
(a). and, if applicable, paragraph 2) (a). (b)), and shall communicate the details of the definition of
the quarantine of the territory and the safety zone referred to in paragraph 1 (b). (c)),
paragraph 2 (b), if applicable. (c)). the details of this notification is
confidential.
(4) if other Member States will confirm the presence of ring rot agent or
the causative agent of brown rot in the plant, the consignment or lot originating in the Czech
Republic in the notification by the mutatis mutandis in accordance with paragraph 3,
phytosanitary administration adequately and in accordance with the
the provisions of paragraphs 1 and 2 shall identify the infected objects and objects provides
the extent of probable contamination and demarcate a quarantine, where applicable,
safety zone.
(5) the phytosanitary administration lays down the official communication in Journal of policy
for the conduct of professional investigation referred to in paragraph 1 (b). (b)) and for the determination
the range of the quarantine territory and security zone, by vymezovaných
paragraph 1 (b). (c) paragraph 2 (b)). (c)) and paragraph 4.
§ 6
Testing the clonally related lots of host plants
(1) the phytosanitary administration shall ensure that laboratory testing in accordance with section 4 of the
paragraph. 2 all dohledaných parts of host plants of clonally
related to topics that have been designated as contaminated under article 5 (3). 1
(a). and), in each case, however, all clonally related sessions
propagating material ^ 2), and to the extent necessary to determine the
the primary source of the disease and the extent of possible contamination with the aim to establish
risk of further spread of ring rot agent or agent Brown
rot.
(2) in accordance with the outcome of the testing referred to in paragraph 1, the phytosanitary administration in
If necessary, additional courses and marks the objects as contaminated,
the range of possible contamination and newly define quarantine territory and security
the zone pursuant to section 5 (3). 1 (a). and (b))) and (c)).
section 7 of the
Measures against the spread of the agent of ring rot and brown rot agent
(1) the phytosanitary administration in accordance with § 11 (1). 1 of the law shall prescribe the
emergency phytosanitary measures under section 76, paragraph. 2 of the Act:
and a lot of host plants) designated as contaminated under article 5 (3). 1
(a). and) must not be under the supervision of sázena and phytosanitary management with
It shall be disposed of in the manner referred to in point 1 of the annex No 4, if
phytosanitary administration in advance validates that there is no recognizable
the risk of extension of ring rot agent or agent of brown rot and that
compliance with the requirements on the disposal of waste, laid down in the annex.
5, in the case of industrial processing of this batch,
b) lot of host plants identified as probably contaminated
under section 5 (3). 1 (a). (b)) may be used under the supervision of the
plant health management in the manner referred to in section 2 or 3 of annex
No 4, if phytosanitary administration in advance to verify that there is no
recognizable risk or the originator of the extension ring rot agent
brown rot, and that compliance with the requirements on the disposal of waste,
laid down in annex 5, in the case of industrial processing of this
of the lot, and if it is not this lot on the basis of the result of testing clonally
relatives of lots of host plants under section 6 is marked as
contaminated according to § 5 (3). 1 (a). and)
(c)), machines, means of transport, warehouses, or their parts and
any other objects including packaging material, designated as contaminated
under section 5 (3). 1 (a). and probably) or marked as infected
under section 5 (3). 1 (a). (b)), will either be destroyed or subjected to cleaning and
disinfection using the appropriate methods referred to in annex No 6 with the fact that after the
implementation of the cleansing and disinfection of the infected objects and objects for
not likely to be contaminated are not considered,
(d)) in the quarantine territory and safety zone, as defined in § 5 (3).
1 (a). (c)), where appropriate, under section 5 (3). 2 (a). (c)), must be made
the measures provided for in the case of ring rot agent in point 4
Annex No. 4, and in the case of the occurrence of the cause of brown rot in point 5 of the annex to
4.
(2) the phytosanitary Administration may order under section 11 (2). 1 of the law
more stringent emergency phytosanitary measures against the spread of the agent
ring rot or the agent of brown rot than the measures referred to in
paragraph 1, if such measures are technically justified, acceptable and
effective. Details of these measures and on the measures imposed by
paragraph 1 (b). (d)) inside the quarantine territory and in the safety zone
tells the phytosanitary administration of the other Member States and the Commission together
with the registration number of the concerned persons, registered under the mandatory section
12 paragraph. 1 of the Act.
(3) the phytosanitary administration cancels pursuant to section 76, paragraph. 4 (b). (b)) of the Act
emergency phytosanitary measures ordered in accordance with paragraph 1 and the
paragraph 2 after their meeting and the expiry of the period laid down for their
meet the.
(4) possession of ring rot agent and the agent of brown rot is in
accordance with section 7 (2). 1 of the Act shall be prohibited, with the exception of their possession to
scientific or diagnostic purposes, in order to allow
phytosanitary administration pursuant to section 8 (2). 1 of the Act, unless this would
impaired by protective measures even when they occur does not risk
their spread.
§ 8
Testing of propagating material ^ 2) potato
(1) the propagating material ^ 2) potato, produced on the territory of the Czech
Republic and placed on the market, shall be subjected to laboratory testing,
undertaken in the framework of the detection of the survey pursuant to section 3 (3). 2, to the extent
in accordance with paragraph 2 and in accordance with § 3 (1). 8 and 9, and found free of
ring rot agent and the agent of brown rot.
(2) the Test referred to in paragraph 1, shall be subjected to in the harvest year
and breeding) plants propagating material ^ 4) potatoes intended
for the production of předstupňů and representative samples of all lots
propagating material of potato, if it was in the last past
harvest year in lot of propagating material ^ 2) potato originating in
The Czech Republic confirmed the presence of ring rot agent, and or
(b)) was in the last, if the previous harvest year in pot
propagating material ^ 2) potato originating in the Czech Republic confirmed
the presence of the causative agent of brown rot, either
1. propagating material of plants breeding ^ 4) potato and
předstupňů and representative samples of all lots of the basic
propagating material of potato in cases of confirmed clone
context, focusing on the link, or
2. representative samples of all lots of basic propagating
material předstupňů and the breeding of potato or propagating
potato material ^ 4) in cases in which the demonstrably does not exist
No cloning of a link, or
(c) breeding) plants propagating material of potato ^ 4) or
representative samples of all lots of basic propagating material
potato or předstupňů, or all lots of propagating material
potato intended for reproduction in the following year, if the occurrence of the originator
diagnosis of the cause of brown rot was not even in the last past
harvest year has been confirmed in a consignment of propagating material ^ 2) potato
originating in the Czech Republic.
§ 9
The effectiveness of the
This Decree shall take effect on the date of its publication.
Minister:
Ing. Palas in r.
Annex 1
AND METHODS OF DIAGNOSIS, DETECTION AND IDENTIFICATION OF THE CAUSATIVE AGENT OF RING ROT
Submitted by progress diagrams describe the various procedures that are
part of:
I) diagnosis of ring rot in Potato tubers and potato plants,
II) detection
Clavibacter michiganensis
subsp. sepedonicus in samples of potato tubers and potato,
III) identification
Clavibacter michiganensis
subsp.
sepedonicus
.
THE GENERAL PRINCIPLES OF
Optimized protocols for the various methods, validated reagents and
details for the preparation of test and control materials are
listed in the appendices. The list of laboratories that participated in the
optimization and validation of protocols is in Appendix 1.
Because the protocols contain the detection of a quarantine organism and include
the use of viable cultures
Clavibacter michiganensis
subsp.
sepedonicus
as control materials, it is necessary to work for appropriate quarantine
conditions with adequate waste disposal facilities and under the conditions
the relevant authorisations issued by the phytosanitary administration. Test
the parameters must ensure stable and reproducible detection of levels
Clavibacter michiganensis subsp. sepedonicus as thresholds
selected methods.
Completely necessary is to prepare positive controls. Testing in accordance with
the required thresholds also involves the correct Setup, maintenance and calibration
equipment, careful handling and storage of reagents and all
measures to prevent contamination between samples, for example. Department
positive controls from test samples. Must be applied
the standard of quality management in order to avoid administrative and other
errors, especially in the labelling and documentation.
A suspected occurrence, as referred to in section 4, paragraph 4. 1 indicates a positive
the result of the diagnostic or screening tests carried out on the
of the sample, as shown in the below route diagrams.
If the first screening test (IF or PCR/FISH) is positive, there is a
the suspected infection, the originator of the ring and the other must be made
screening test. If the second screening test is positive, then it is
suspicion of the occurrence of confirmed and must continue testing according to
the given schema. If the second screening test is negative, then the sample
It is not considered to be infected by the originator. For this reason, it is
a positive IF test referred to in section 4, paragraph 4. 1 is defined as the positive outcome of the
If test confirmed a second screening test (PCR/FISH).
Confirmed presence requires the isolation and identification of a pure culture
Clavibacter michiganensis
subsp.
sepedonicus
with confirmation of pathogenicity.
1. Use progressive charts
1.1. Flowchart for the diagnosis of ring rot in
Potato tubers and potato plants with symptoms of
bacterial ring rot
The testing procedure is intended for Potato tubers and plants with suspicion of
typical or ring rot symptoms. Includes quick
screening test, isolation of the pathogen from infected vascular tissue on
diagnostic media and, in case of a positive result, identification of the
culture
Clavibacter michiganensis
subsp.
sepedonicus
.
(1) a description of the symptoms is in section 2.
(2) the Appropriate tests are:-the IF test (section 4),
-PCR test (section 6),
-FISH test (section 5).
(3) Although isolation of the pathogen from plant material with typical
the symptoms with the suspensions on the media is not complicated, the cultivation in the
advanced stages of infection may fail. Saprophytic bacteria
which grow on the infected pattern may outgrow or inhibit
the pathogen on the isolation medium. Therefore, it is recommended to use a non-selective
even a selective media, preferably the MTNA (section 8) or bioassay (section 7).
(4) a description of the typical morphology of the colony is in section 8.
(5) If the insulation test is negative, but the symptoms of the disease are
typical insulation must be performed again.
(6) Reliable identification of a pure culture of Clavibacter michiganensis
subsp. sepedonicus is achieved using the tests referred to in section 9.
(7) the pathogenicity Test is described in section 10.
1.2. Flowchart for the diagnosis of ring rot in
samples of asymptomatic Potato tubers the testing procedure is intended for
detection of latent infections in Potato tubers. A positive result from
at least two screening tests, each of which is based on another
biological principle, must be complemented by the isolation of the pathogen, and subsequently, in
the case of isolation of typical colonies, confirmation that a pure culture is
Clavibacter michiganensis
subsp.
sepedonicus
. A positive result only one screening test is not
sufficient to ensure that the sample was considered a suspect.
Screening and isolation tests must allow for the detection of 103 to 104
cells per ml of resuspended Pellet, included as positive controls in each
a series of tests.
(1) the standard sample size is 200 tubers, although the procedure can be used
the smaller the number, if 200 tubers is not available.
(2) the methods of extraction and concentration of the pathogen are described in section 3.1.
(3) if at least two tests based on different biological
the principles of positive, must be carried out in isolation and confirmation. Performs
at least one screening test. If this test is negative, it is
a sample is considered negative. If this test is positive,
It is for the verification of the first positive result in one or more essential
more screening tests based on different biological
principles. If it is a second or additional test is negative, the sample is
considered negative. Further tests are necessary.
(4) Test immunofluorescence (IF).
For the examination IF they always use a Polyclonal Antibody, more
monoclonal antibodies will allow more precision (see section 4).
(5) the PCR test.
Apply appropriate validated PCR reagents and protocols (see section 6).
(6) the FISH test.
Apply validated reagents and protocols (see section 5).
(7) the Selective isolation.
This can be in many cases a suitable method for the direct isolation
Clavibacter michiganensis subsp. sepedonicus using MTNA medium or
NCP-88 medium and dilution of resuspended pellet 1/100. Typical colonies can be
get within 3-10 days after crushing to the media. The culture of the pathogen can be
then clean and identify. For full use of the potential test
requires careful preparation of the tissue from the heel to reduce
secondary bacteria which are in competition with Clavibacter michiganensis
subsp. sepedonicus to the media and that the pathogen can become. If
cultivation method as follows, must be made from the isolation of plants
used for the bioassay test (see section 8).
(8) bioassay test is used for the isolation of Clavibacter michiganensis subsp.
sepedonicus from pellets by selective enrichment of potato extracts
in plants the Eggplant (Solanum melongena). The test requires
the optimal incubation conditions laid down for that method. Bacterial
inhibitors of Clavibacter michiganensis subsp. sepedonicus to MTNA or
NCP-88 medium may interfere with this test are not (see section 7).
(9) the typical colony morphology is described in section 8.
(10) the Cultivation or bioassays may fail for reasons of competition or
inhibition of saprophytic bacteria. If the results of the screening
tests positive, but isolation tests negative, repeat the insulation
tests of the same or additional pellets by removing the vascular tissue of the
near the end of the tubers of the same sample and, if necessary,
tests of other samples.
(11) the Reliable identification of pure cultures suspected to Clavibacter
michiganensis subsp. sepedonicus is achieved using the tests that are described in the
section 9.
(12) the pathogenicity Test is described in section 10.
1.3. Flowchart for the diagnosis of ring rot in
samples of asymptomatic potato
(1) the recommended sample sizes-see section 3.2.
(2) the methods of extraction and concentration of the pathogen are described in section 3.2.
(3) if at least two tests based on different biological
the principles of positive, must be carried out in isolation and confirmation. Performs
at least one screening test. If this test is negative, it is
a sample is considered negative. If this test is positive,
It is the first positive result for authentication necessary to perform the second
or more screening tests based on different biological
principles. If it is a second or additional test is negative, the sample is
considered negative. Further tests are necessary.
(4) Selective insulation test and the typical morphology of the colony are described
in section 8.
(5) IF test is described in section 4.
(6) the PCR test is described in section 6.
(7) the FISH test is described in section 5.
(8) Bioassay is described in section 7.
(9) the Cultivation or the bioassay test can fail for reasons of competition or
inhibition of saprophytic bacteria. If the results of the screening
the positive tests, but the tests are negative, the insulation is insulation
tests and, if necessary, carry out a further test of the samples.
(10) the Reliable identification of pure cultures, where it is suspected that
These are the Clavibacter michiganensis subsp. sepedonicus, reaches
using tests described in section 9.
(11) the pathogenicity Test is described in section 10.
2. Visual examination for the presence of symptoms of c. sepedonicum
(I)
2.1. potato Plants
In European climatic conditions with symptoms in the field found just
rarely and often until the end of the season. In addition, the symptoms are
hidden or changes with symptoms of other diseases, old age or mechanical
damage. Therefore, the symptoms can be when checking on the easy
overlooked. Symptoms of wilting symptoms are very different from the Brown
rot; wilting is usually slow and initially limited to the edges of the leaves.
Young infected leaves often despite the infection continues to grow, even if growth
in infected places is limited. This generated an unusually shaped
the leaves. Leaves affected by blocking vascular tissue on the bottom of the stem
they often have chlorotic, yellow to Orange, intercostal areas.
The infected leaflets, leaves and even stems may eventually
die. The leaves and tubers are often only smaller. Occasionally they are
the plants were stunted. Color slides of a series of symptoms are on the Internet
page http://forum.europa.eu.int/Public/irc/sanco/Home/main.
2.2. Potato tubers
The earliest symptoms are a slight glassiness or translucence of the tissue
without softening around the vascular system, particularly near the navel. Ring
the vascular on the heel end may be slightly darker
coloring than usual. The first readily identifiable symptom is yellowish
discoloration of the vascular ring and status when a gentle pressing
roots emerge from the vessels posts cheesy. This exudation contains
millions of bacteria. Conductive tissue may become Brown on standing and symptoms on tubers
at this stage are similar to the symptoms of brown rot caused by Ralstonia
solanacearum. Initially, these symptoms may be limited to one part of the
the ring may not be just near the heel and can
gradually spread to the whole ring. With the procedure, infection leads to destruction
Vascular tissues: the outer part of the korová may be separated from the inner cortical
part. In advanced stages of infection appear on the surface of the tuber
the cracks, often with russet edge. The last time in Europe
There were several cases where the Central cortex rots with the ring volume
Vascular, which occurs secondary to the creation of internal cavities
and necrosis. Secondary fungal or bacterial infection symptoms
mask and can be difficult, if not impossible, to distinguish advanced symptoms
potato ring rot from other hnilob. Can atypical
the symptoms. Color slides of a series of symptoms are on the website
http://forum.europa.eu.int/Public/irc/sanco/Home/main.
3. Preparation of samples
3.1. Potato tubers
Note:
-The standard sample size is 200 tubers on 1 test. More intense
sampling requires more tests on samples of this size. Greater
the amount of tubers in the sample leads to inhibition or difficult interpretation
the results. However, the procedure can be conveniently used for samples with less than 200
tubers, if available, a smaller amount of tubers.
-Validation of all detection methods listed below is based on the
testing of samples of the size of 200 tubers.
-The potato extract described below can also be used to determine the originator of the
brown rot,
Ralstonia solanacearum
.
Optional treatment prior to sample preparation:
The tubers are washed. Appropriate means of disinfection are used (with the content
chlorine, when the PCR test is to be performed to remove the possible
pathogen DNA) and detergents between each sample. Tubers
to dry in the air. This washing procedure is especially useful when
It is in the sample too much soil and if you want to perform the PCR test or
direct isolation.
3.1.1. With a clean and disinfected scalpel or a knife or scraper
the potatoes will remove the peel on the heel end of each tuber. Carefully
to cut taper vascular tissue cores from the heel ends
the potatoes. To a minimum will reduce the excess part of the wire netting not involving vascular
volumes. After removing the cores must be processed heel ends into
24 hours or conserved at-20 ° c for no longer than two weeks.
All tubers with suspected symptoms of ring rot can be
party and test separately. If you are in the excision of výkrojku
the heel end detected symptoms of ring rot, the Visual
examination of the tubers after cutting the tuber at the heel end. All
incised tubers with suspected symptoms will leave korkovatět for 2
days at room temperature and kept in quarantine at a temperature of 4-10
° C until the completion of all tests. All tubers in the sample shall be kept
in accordance with Annex 2.
3.1.2. The cores from the heel is collected in the unused containers on
one use that are closing and/or sealable (in the case of
the containers are reused, it must be thoroughly cleaned and
disinfected with chlorine compounds). The best way is to handle
the cores from the heel end immediately if this is not possible, keep the
in the container without adding buffer, refrigerated for up to 72 hours, or
at room temperature for up to 24 hours. Drying and suberizace potato
and the growth of Saprophytes can be increased during storage may prevent the detection of the
the bacteria causing the cause.
3.1.3. the heel end cores shall be processed by one of the following
procedures:
and once the zalijí) sufficient quantity (approximately 40 ml)
extraction buffer (Appendix 3), and třepají in the Rotary shaker (50-100
Rev/min) for 4 hours at a temperature lower than 24 ° c or for 16-24
hours refrigerated;
or
b) cores with a sufficient quantity of tissues (approximately 40 ml)
extraction buffer (Appendix 3), either in a blender or crushing in a sealed
disposable maceration bag using a rubber or tloučku
the milling device.
Note:
Samples are homogenized using a blender there is a high risk of
cross-contamination of samples. It is necessary to make the security measures for the
avoid aerosol generation or spillage during extraction. For each sample
must be used freshly sterilized Blade (knife) and containers. If it has a
be used in the PCR test, it is necessary to prevent the transmission of DNA on containers or grinding
the device, it is recommended that crushing in disposable bags and use
the tube on one use.
3.1.4. Decant the supernatant. If excessively cloudy, clear the
either a slow centrifugation (at a maximum of 180 g for 10 minutes at
a temperature of 4-10 ° c), or vacuum filtration (40-100 µm), the filter is washed out
the addition of (10 ml) extraction buffer (Appendix 3).
3.1.5. The bacterial fraction by centrifugation at 7000 g until after a period of
15 minutes (or 10000 g for 10 minutes) at a temperature of 4-10 ° c, and deletes
the supernatant without disturbing the Pellet.
3.1.6. Resuspenduje with pellets in 1.5 ml pellet buffer (Appendix 3).
500 µ l is used for test on Clavibacter michiganensis subsp.
sepedonicus, 500 µ l of Ralstonia solanacearum and 500 µ l of the reference
purposes. Add sterile glycerol to final concentration of 10-25% (v/v)
to 500 µ l of the reference aliquot and to the remaining portion of the sample, mix
with the swirl and stores at-16 to-24 ° c (weeks) or at
-68 to-86 ° c (months). Extracts during testing must be kept at
a temperature of 4-10 ° c.
Repeated freezing and thawing is not recommended.
If the transport is required to extract, shall ensure that the transport in a cool box
within 24 to 48 hours.
3.1.7. It is imperative that all positive controls and
samples of Clavibacter michiganensis subsp. sepedonicus are treated separately,
in order to avoid contamination. This also applies to IF slides on tests and
all of the tests.
3.2. potato Plants
Note:
For the detection of latent stocks Clavibacter michiganensis subsp.
sepedonicus are recommended checking the combined samples. The procedure is the
suitable for combined samples up to 200 parts of the stems. (If done
detailed surveys, they should be based on a statistically representative
sample plant population, which is the subject of the investigation.)
3.2.1. Using a clean disinfected knife or pruning shears,
Deletes the part about the size of 1-2 cm from the bottom side of each stem directly
above the surface of the Earth. Parts of the stems are disinfected with ethanol 70% briefly,
immediately dry with tissue paper and collected in a closed sterile container.
3.2.2. Part of the stalks are processed by one of the following procedures:
and section zalijí) of sufficient quantity (approximately 40 ml) of extraction
buffer (Appendix 3), and třepají in the Rotary shaker (50 to 100 rpm) for
4 hours at a temperature lower than 24 ° c or for 16 to 24 hours refrigerated,
or
(b)) section immediately crushed hard maceration bag with an appropriate
the amount of extraction buffer (Appendix 3) using a rubber tloučku
or suitable grinding equipment. If this is not possible, store the parts
stems in a cool place for a maximum of 72 hours or up to 24 hours at room
temperature.
3.2.3. After the establishment, which is to last 15 minutes, decant supernatant.
3.2.4 Further purification of the extract or concentration of the bacterial fraction
It is usually not necessary, but it can be achieved by filtration and/or centrifugation
as described in section 3.1.3.-3.1.6.
3.2.5 Clean or concentrated sample extract will be divided into 2 of the same
parts, one half is maintained during the test at a temperature of 4-10 ° c and
the other half is stored with 10-25% (v/v) sterile glycerol at
a temperature of-16 to-24 ° c (weeks) or at-68 to-86 ° c (months)
If you need further testing.
4. IF test
The PRINCIPLE of the
It is recommended to use the IF test as the principal screening test, because
was demonstrated its ability to achieve the required thresholds.
Used as the principal screening test and its result is
positive, it must be performed as a second screening test or PCR test
The FISH. If the test IF used as the second screening test and the
the result is positive, it is necessary to complete the analysis of the additional testing
According to the flow scheme.
Note:
When the IF test is used as the principal screening test is used, always
Polyclonal Antibody. In the case of a positive result IF test with
Polyclonal Antibody can be another examination of the sample with the monoclonal
more accurate, but the antibody less sensitive.
Use antibodies against a known strain of the agent-
ATCC 33113 (NCPPB 2137), or NCPPB 2140. It is recommended to determine the titre for the
each new series against substances. 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 the agent of ring rot and
When using an appropriate fluorescein isothiocyanate (FITC)
According to the manufacturer's recommendations. Unprocessed Polyclonal or monoclonal
the antibodies should have an IF titre of at least 1:2000. During the test should
the antibodies used in the working dilution (WD) close to or at the titre
the titre. Use validated antibodies (see website
http://forum.europa.eu.int/Public/irc/sanco/Home/main).
The test shall be performed on freshly prepared sample extracts. In
If necessary, can be successfully performed on extracts stored
at-68 to-86 ° c under glycerol. Glycerol can be removed from the sample
by adding 1 ml pellet buffer (Appendix 4), again by centrifugation after
for 15 minutes at 7000 g and re-suspension in the same amount of pellet
buffer. It is often not necessary, especially if slides samples
fixed flame (see 4.2).
Prepare separate positive control slides of the homologous strain
or with another tribe, the originator of comparative diagnosis of suspended
in the extract from the potatoes referred to in Appendix 2, and optionally in buffer.
Naturally infected tissue (maintained by lyophilization or
freezing at-16 to-24 ° c) should be in accordance with the options to use
as a parallel control on the same slide slide.
As negative controls shall apply an aliquot of sample extracts
which previously tested negative.
Apply to microscopic slides with more cutouts, best with 10 Windows on
diameter of at least 6 mm.
Test control material shall be carried out in the same way as the test
of the samples.
4.1. prepare the test Slides by one of the following procedures:
and) for pellets with relatively low amounts of starch sediment:
Pour a measured standard volume (for 6 mm window diameter is
appropriate 15 µ l-higher volume for larger Windows) of the resuspended
potato pellet 1/100 to the first window. Subsequently pipette
the same amount of undiluted Pellet (1/1) into the remaining wells in the series.
The second row can be used as duplicate or for a second sample as presented in
Figure 1.
(b)) For other pellets:
Prepare decimal dilutions (1/10 and 1/100) of the resuspended pellet in
Pellet buffer. Pipette a measured standard volume (with a window of
diameter 6 mm is recommended 15 µ l-higher volume for larger Windows)
1/100 of the resuspended pellet and each solution to the wells family. The second
the series can be used as duplicate or for a second sample as presented in Figure 2.
4.2. Dry the droplets at ambient temperature or heating to a temperature of 40
up to 45 ° c. The bacterial cells to the slide either by heating for (15 minutes
at a temperature of 60 ° c), over the flame or by using 95% ethanol, or by
Special instructions to suppliers of the antibodies.
If necessary, you can then fixated slides before using
store frozen in a dry box after the shortest possible period of time (up to a maximum
3 months).
4.3. IF Test Procedure:
and the preparation of slides on) under the test in section 4.1 (a). and): prepare
the set of solutions of double antibody in IF buffer. The first hole should
be the titre (T/2), in the other quarter of the titre (T/4), the titre (T/2), the titre (T) and
twice the titre (2T).
(b) training slides on) under the test in section 4.1 (a). (b)): prepare
the working dilution (WD) antibody in IF buffer. The working dilution affects
accuracy.
Figure 1: the preparation of slides on the test according to section 4.1. (a). ) and section
4.3. (b). and)
Figure 2: the preparation of the test slide according to section 4.1. (a). (b)) and section
4.3 b)
4.3.1. The slides will be arranged on the moistened paper. Each
the test window completely cover the dilution of antibodies. The quantity of
antibodies to each window must be at least the same as the quantity
used the extract.
If specific instructions are not available from the supplier of antibodies,
proceed as follows:
4.3.2. incubate the slides on moist paper under a cover for
30 minutes at room temperature (18-25 ° c).
4.3.3. Shaken off drops from all the slides and this carefully
Rinse the buffer IF. Washed by submerging for 5 minutes in IF buffer
Tween (Appendix 3) and subsequently in IF buffer. It is necessary to prevent
aerosol or droplet transfer that could cause mutual
contamination, and carefully remove excess moisture with a soft blow dry.
4.3.4. The slides will be placed on moist paper. Cover the test Windows with the
the dilution of FITC conjugate, fixing the titre. The amount of conjugate
applied to the wells must be the same as the amount used antibodies.
4.3.5. incubate the slides covered with damp paper for 30 minutes
at room temperature (18-25 ° c).
4.3.6. Drops of conjugate shaken off of slides and this will rinse and
washed as before (4.3.3).
Carefully remove excess moisture.
4.3.7. Pipette 5-10 µ l 0, 1 m phosphate buffer solution with glycerol
(Appendix 3) or a commercial cover of fluids and attach to each
coverslip.
4.4. evaluation of the IF test
4.4.1. Test slides on an epifluorescence microscope with the viewers
filters suitable for excitation of FITC, under oil or water immersion at
magnification up to 1000 x. Examine the window in two orthogonal
diameters and around the perimeter. For samples with no or a small number of cells
examines at least 40 microscope fields. First, check the positive
a control sample. The cells must be bright fluorescent and completely stained in
the specified titre or working dilution of antibodies. If the colours
derogations must be repeated IF test (section 4).
4.4.2. Observe the bright Fluorescing cells with characteristic
morphology of Clavibacter michiganensis subsp. sepedonicus in the test
the Windows of the slides (see Web site
http://forum.europa.eu.int/Public/irc/sanco/Home/main). The intensity of the
the fluorescence must be compared to the positive control strain in
the same dilution of antibodies is the same or better. Cells with incomplete staining
or a weak fluorescence must be taken into account. When the suspicion of any
contamination of the test must be repeated. This can happen when all
the slides in the group show positive cells due to the contamination of buffer or
When the finding of positive cells (outside of the slide Windows) on the surface
slides.
4.4.3. There are several problems inherent to the accuracy
the immunofluorescence test. In the background of potato heel and parts
the stem may occur populations of Fluorescing cells with
atypical morphology and cross reacting saprophytic bacteria with
size and morphology similar to Clavibacter michiganensis subsp.
sepedonicus.
4.4.4. Take into account only Fluorescing cells with typical size
and morphology at the titre or working dilution of the antibodies as in section 4.3.
4.4.5. Interpretation of the IF test result:
and) when bright Fluorescing cells with characteristic morphology are found,
estimates of the average number of typical cells per microscope field and
calculate the number of typical cells per ml of resuspended Pellet (Appendix
4. IF Result is) positive for samples, where is the number of typical cells
1 ml of resuspended pellet at least 5x103. The sample is considered to
potentially infected and required further testing.
(b)) the result of the IF test is negative for samples that contain less than
5x103 cells per ml of resuspended pellet and the sample is considered to
negative. Further testing is not necessary.
5. FISH Test
The PRINCIPLE of the
When used as the first screening test and the FISH test is positive, the
It must be as a second compulsory screening test done IF test. When
The fish test performs as the second screening test is positive, it is to
the completion of the diagnosis must be further testing according to the flow scheme.
Note:
Use the validated oligosondy specific for Clavibacter
michiganensis subsp. sepedonicus (Appendix 7). The initial testing of this
method should permit reproducible detection of at least 103-104 cells
Clavibacter michiganensis subsp. sepedonicus per ml added to extracts
from the sample that were previously tested with negative result.
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 control applies an aliquot part of the extract of the
the sample, which was previously tested on Clavibacter michiganensis subsp.
sepedonicus with negative result.
As a positive control, prepare suspensions containing 105 to 106
the cells of Clavibacter michiganensis subsp. sepedonicus per ml (e.g. strain
NCPPB 4053 or PD 406) in 0.01 M phosphate buffer (PB) from 3-5 days
culture (preparation see Appendix 2). Prepare a separate slide with
homologous strain or any other
the reference strain of Clavibacter michiganensis subsp. sepedonicus
the suspended in potato extract, as specified in Appendix 2.
The use of FITC the FITC-labelled probe provides control
the process of hybridization, because it turns all eubakterie present in the
sample.
Test control material is done the same way as samples.
5.1. potato extract Fixation
5.1.1. Prepare fixative solution (see Appendix 7)
5.1.2. Pipette 100 µ l of the each sample extract into Eppendorfovy
Eppendorf tube and centrifuge for 8 minutes at 7000 g.
5.1.3. Remove the supernatant and dissolve pellets in 500 µ l fixation
the solution prepared max. 24 hours in advance. Mix and incubate for
overnight at 4 ° c.
An alternative fixative is 96% ethanol. For its use is
dissolve the pellets from step 5.1.2. in 50 µ l 0.01 M PB and 50 µ l of 96% ethanol.
Mix by vortexing and incubate at 4 ° c for 30-60
minutes.
5.1.4. Centrifuge for 8 minutes at 7000 g, remove the
the supernatant and resuspenduje with pellets in the 75 µ l 0.01 M PB (see Appendix 3).
5.1.5. Action 16 µ l fixed suspension on a clean glass slide, window-type 10
as shown in Figure 3, with 2 different samples on one
slide, undiluted and diluted 1:100 with the use of 10 µ l (in 0.01 M PB).
The remaining sample solution (50 µ l) can be stored at-20 ° c
Add 1 volume of 96% ethanol. In the event that the FISH
the method again, remove the ethanol by centrifugation, add the same
the quantity of 0.01 M PB and mix by vortexing.
Figure 3. Layout for FISH slide
5.1.6. The slides left to dry in the air (or dryer slides
at a temperature of 37 ° c) and fixed above the flame.
In this phase is the possibility to interrupt the procedure and continue further hybridization
the day. The slides should be stored protected from dust and dry at
room temperature.
5.2. Předhybridizace and hybridization
5.2.1. Prepare a lysozyme solution containing 10 mg lysozyme (Sigma
L-6876) in 10 ml of buffer (100 mM Tris-HCl, 50 mM EDTA, pH 8.0). This
the solution can be stored, but only one should freeze and leave
thawed. Each sample well cover approximately 50 µ l of lysozyme
and incubate for 10 minutes at room temperature. Then immerse the
slides in demineralised water, only once, and the dried filter
the paper.
Alternatively, add 50 µ l instead of lysozyme proteases to 40-400 µ g ml-1
in buffer (20 mM Tris-HCl, 2 mM CaCl2, pH 7.4) to each well and incubate for
at a temperature of 37 ° c for 30 minutes.
5.2.2. dry Cells in a graded series of 50%, 80% and 96% ethanol
each time for 1 minute. Slides to air-dry in the
the bracket slides.
5.2.3. Prepare a moist incubation Chamber by covering the bottom of an airtight
box with tissue or filter paper soaked in 1 x hybmix
(Appendix 7). Přeinkubuje box in the hybridisation oven at 55
° C for at least 10 minutes.
5.2.4. Prepare the hybridisation solution (Appendix 7) with 45 µ l per 1 slide
and předinkubuje for 5 minutes at a temperature of 55 ° c.
5.2.5. The slides will be placed on a hot plate at a temperature of 45 ° c and to
each of the 4 Windows on a slide/sklíčcích add 10 µ l
hybridisation solution.
5.2.6. Each slide cover 2 canopy slides (24 x 24 mm)
to them without trapping air. The slides are placed in a preheated moist Chamber
and hybridizuje 14-18 h in an oven at a temperature of 55 ° c in the dark.
5.2.7. Prepare three beakers containing 1 l of sterile water (Ultra pure
water = UPW), 1 l of 1 x hybmix (334 ml 3 x hybmix and 666 ml UPW) and 1 l of 1/2
x hybmix (167 ml 3 x hybmix and 833 ml UPW). Each of them is přeinkubuje
in a water bath at 55 ° c.
5.2.8. Removed the cover slides and slides are placed in the holder
slides.
5.2.9. Flushes the excess sample incubation for 15 minutes in the beaker
with 1 x hybmix at a temperature of 55 ° c.
5.2.10. The bracket slides moves into wash solution 1/2 hybmix and
leave to incubate for a further 15 minutes.
5.2.11. Slides are dipped briefly in UPW and puts on the filter paper.
Remove excess moisture by covering the surface with light filter
the paper. Pipette 5-10 µ l of the solution (e.g. covering. Vectashield,
Vecta Laboratories, CA, USA or similar) to each well and the entire glass slide
to cover a large a glass coverslip (24 x 60 mm).
5.3. Assessment of the FISH test
5.3.1. viewing slides immediately with a microscope fitted for
epifluorescence microscopy magnification 630x or 1 000x.us under oil
immersion. 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 cells Clavibacter michiganensis
subsp. sepedonicus Appear fluorescent dyed red. Compares the
Cell morphology with the morphology of the positive control samples.
The cells must be bright fluorescent and completely stained. Test FISH (section
9.4) must be repeated if the coloring of the derogation. Viewing window
across two diameters at right angles and around the perimeter. For samples, where
are not observed, no or little cells, observed at least 40 fields
the microscope.
5.3.2. is looking for bright Fluorescing cells with morphology
characteristic for Clavibacter michiganensis subsp. sepedonicus in
the Windows of the test slides (see Web site
http://forum.europa.eu.int/Public/irc/sanco/Home/main). The intensity of the
the fluorescence must match or be better than positive
the control strain. Cells that are not fully colored or exhibit
weak fluorescence, shall not be taken into account.
5.3.3. When any contamination is suspected the test must be repeated.
From any contamination is suspected the test must be repeated. It is
can happen when all slides in a group show positive cells
due to the contamination of buffer or in finding positive cells (outside the window
slides) on the surface of the slides.
5.3.4. There are several problems inherent to the accuracy of the FISH test. In
pellets from potato heel end core and stem may occur
populations of Fluorescing cells with atypical morphology and
cross reacting saprophytic bacteria with size and morphology
similar to Clavibacter michiganensis subsp. sepedonicus, although much
less often than the IF test.
5.3.5. Account shall be taken only Fluorescing cells with typical size
and morphology, see 5.3.2.
5.3.6. Interpretation of the FISH test result:
and the FISH test) results are valid if they are using a FITC filter
bright green fluorescent cells with size and morphology typical of
Clavibacter michiganensis subsp. sepedonicus and using
the rhodamine filter bright red fluorescent cells are observed in the
all of the positive controls and not in any negative
checks. If bright Fluorescing cells are present with the typical
morphology, estimates the average number of typical cells 1
a microscope field and calculate the number of typical cells per ml
the resuspended Pellet (Appendix 4). Samples that contain at least 5 x
103 typical cells per ml of resuspended Pellet is considered to be
probably infected with Clavibacter michiganensis subsp. sepedonicus.
Further testing is required. Samples that contain less than 5 x 103
typical cells per ml of resuspended Pellet is considered to be
negative.
(b)) the FISH test is negative if using the rhodamine
the filter is not observed bright red fluorescent cells with size
and morphology typical of Clavibacter michiganensis subsp. sepedonicus,
If these are typical bright red fluorescent cells in
observed using the rhodamine filter in the positive controls.
6. PCR test
The PRINCIPLE of the
If the PCR test as the principal screening test and it is positive,
It must be as a second compulsory screening test done IF test. If
The PCR test is used as the second screening test is positive, it is for the
the completion of the diagnosis must be further testing according to the flow scheme.
The use of this method in its entirety as the principal screening test
It is recommended only if the required specialized expertise.
Note:
Preliminary testing with this method should permit reproducible
detection of 103 to 104 cells of Clavibacter michiganensis subsp. sepedonicus
to 1 ml added to sample extracts which previously tested
a negative result. In order to achieve maximum sensitivity and accuracy in
all laboratories optimization attempts may be required.
Use validated PCR reagents and protocols. Priority is used
method with internal control.
It is necessary to use appropriate security measures to prevent the
sample contamination with target DNA. The PCR test should be performed by experienced
technicians in laboratories specializing in molecular biology, to
to minimize the possibility of contamination with target DNA.
With negative controls (for DNA extraction and PCR progresses) should always
treated as final samples to make it clear, if there was a
transfer of DNA.
The PCR test should include the following negative controls:
-sample extract that previously tested negative for Clavibacter
michiganensis subsp. sepedonicus with a negative result,
-buffer controls used for extracting the bacterium and the DNA from the sample,
-The PCR reaction mix.
The following should be included in the positive control:
-an aliquot of resuspended pellets to which was added the Clavibacter
michiganensis subsp. sepedonicus (preparation see Appendix 2),
-a suspension of 106 cells per ml of Clavibacter michiganensis subsp.
sepedonicus in water from a virulent isolate (e.g.. NCPPB 2140 or NCPPB
4053),
-If possible, use when performing PCR DNA extracted from also
positive control samples.
To avoid possible contamination, prepare positive controls in the
a separate environment from samples to be tested.
Extracts of samples should be free from soil. In the case of
the use of PCR to prepare extracts of umytých potatoes.
6.1. DNA purification Methods
Shall the above-described the positive and negative control samples.
Prepare control material in the same way as samples.
The purification of target DNA from complex sample substrates are available
different methods for 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
The PCR method, shown in Appendix 6.
6.1. Method according to Pastrik) (2000)
1. Pour 220 µ l lýzového buffer (100 mM NaCl, 10 mM Tris-HCl [pH
8,0], 1 mM EDTA [pH 8,0]) to a volume of 1.5 ml Eppendorf tube.
2. Add 100 µ l sample extract and place in a heating block or
water bath at 95 ° c for 10 minutes.
3. Insert the Microtest tube on ice for 5 minutes.
4. Add 80 µ l lysozyme stock solution (50 mg lysozyme per ml in
10 mM Tris HCl, pH 8.0) and incubate at 37 ° c for 30
minutes.
5. Add 220 µ l Easy DNA (R) and the solution (Invitrogen), mix well
shaking and incubated at 65 ° c for 30 minutes.
6. Add 100 µ l Easy DNA (R) solution B (Invitrogen) and
mix thoroughly by shaking up the sediment itself will flow into the tubes and sample
begins to be uniformly viscous.
7. Add 500 µ l of chloroform and stir by shaking, until the viscosity
reduced and the mixture becomes homogeneous.
8. Centrifuge at 15000 g for 20 minutes at 4 ° c for the Department
phases and form the interphase.
9. Transfer the upper phase into a clean microfuge tube.
10. Add 1 ml of 100% ethanol (-20 ° c), mix thoroughly by shaking and
incubate on ice for 10 minutes.
11. Centrifuge at 15000 g for 20 minutes at 4 ° c and is removed from the
pellets of ethanol.
12. Add 500 µ l 80% ethanol (-20 ° c) and mix by inversion
Eppendorf tube.
13. Centrifuge at 15000 g for 10 minutes at 4 ° c, the pellets are
preserves and remove ethanol.
14. Pellets are allowed to dry in air or in a DNA speed vac.
15. resuspend the Pellets are in 100 µ l of sterile water (UPW) and allow to stand
at least 20 minutes at room temperature.
16. Store at-20 ° c until use in PCR.
17. any white precipitate by centrifugation and are removed for PCR
uses 5 µ l of supernatant containing DNA.
6.1. (b)) other methods
Other DNA extraction methods could be used, if it is proved that the
purification of DNA are from control samples containing 103 to 104
pathogenic cells per ml as efficient.
6.2 the PCR
6.2.1. Prepare test samples and controls for PCR according to the
a validated Protocol (Appendix 6). Prepare one decimal solution
DNA sample (1:10 in sterile water).
6.2.2. Prepare the appropriate PCR reaction mix in an environment where
There is no risk of contamination, according to a published Protocol (Appendix 6).
Validated PCR Protocol is a multiplex reaction that also includes
internal PCR control.
6.2.3. Into sterile PCR vials add 5 µ l of the extract DNA at 25
µ l of the PCR reaction.
6.2.4. Includes negative control sample containing only to the reaction
The PCR mixture and add the same sterile water UPW, which was used to
The PCR mixture instead of the sample.
6.2.5. The PCR tubes are placed into the same thermal cycler which was
used during initial testing and shall be suitably optimized
The PCR programme (Appendix 6)
6.3. analysis of the PCR product
6.3.1. Distributed amplikony PCR in agarose gel electrophoresis.
Spotted at least 12 µ l of the amplified DNA reaction mixture from each
sample mixed with 3 µ l loading buffer (Appendix 6) in 2.0% (w/v)
agarose gel in Trisacetát-EDTA (TAE) buffer (Appendix 6) in 5-8 in the
cm. Apply appropriate DNA marker, for example. 100 bp ladder.
6.3.2. Detect the strips of DNA staining in ethidium bromide (0.5 mg per
l) for 30-45 minutes, using appropriate security measures for the
handling this mutagen.
6.3.3. In obarveném and UV (short wavelength, e.g. 302 nm) observe
the gel is looking for amplified PCR products of the expected size and
the result is documented.
6.3.4. For all new findings, check the authenticity of the PCR Amplicon
by performing restriction enzyme analysis in the remaining sample
The Amplified DNA by incubating at the optimum temperature and time with an appropriate
the cleavage enzyme and buffer (see Appendix 6). Shall resolve
fragments in agarose gel electrophoresis, and observe the
the characteristic restriction fragment pattern under UV transillumination after
ethidium bromide staining and compares with the undigested and digested
the positive control.
Interpretation of the result of the PCR test: the PCR test is negative if the Amplicon
typical for
Clavibacter michiganensis subsp. sepedonicus was not the expected size
detected in a given sample, but was detected in all positive
the control samples (in the case of multiple PCR with plant internal
control primers: a second PCR-product of expected size must be
amplified with the sample).
The PCR test is positive if the detected Amplicon was typical for
Clavibacter michiganensis subsp. sepedonicus expected size and
the model (if required), provided that it was not amplified in any
from the negative control samples. Reliable confirmation of a positive
result can be achieved also by repeating the test with a second set of PCR primers
(section 9.3).
Note:
You can have the suspicion on the inhibition of the PCR, if the expected Amplicon is obtained
from the positive control sample containing Clavibacter michiganensis
subsp. sepedonicus in water, but from a positive control with Clavibacter
michiganensis subsp. sepedonicus in potato extract were negative.
In the multiple PCR Protocols with internal PCR controls, and inhibition of the
reaction, if not obtained any of the two amplicons are obtained.
Contamination may be suspected if the expected Amplicon is obtained from
one or more of the negative tests.
7. Test on Eggplant
Note:
Preliminary testing with this method should permit reproducible
detection of 103 to 104 units of Clavibacter michiganensis subsp.
sepedonicus colony-forming per ml added to sample extracts
that have been tested negative (preparation see Appendix 2)
The highest sensitivity of detection can be expected when using freshly
the prepared sample extract and optimal growth conditions.
However, the method can be successfully applied to extracts that have been stored in the
glycerol at-68 to-86 ° c.
Some varieties of Eggplant provide an excellent selective
enriching the medium for the growth of Clavibacter michiganensis subsp.
sepedonicus even in the absence of symptoms, and they also provide
excellent basic confirmation test.
To reduce the risk of false negative results should be
optimal growth conditions.
Details of cultivation are listed in Appendix 8.
7.1. the Pellets according to 3.1.5. is distributed among the eggplants one of the below
the above methods (7.2, 7.3, 7.4). Use only plants at the stage of
2-3 sheets to full deployment of the third true leaf. In order to ensure
full use of the resuspended pellet and the efficient inoculation will be for
the following procedures need 15-25 of Eggplant on the sample.
7.2. the notch and Inoculation
7.2.1. each pot horizontally (to the point of pots with a diameter of
10 cm foam polystyrene block is appropriate with the hollow parts of the
dimensions: 5 cm, depth 10 cm in width and 15 cm length). For each test
the sample is placed between the stem and block strip of sterile aluminium foil.
The plant is able to fix it on the spot rubber strap around the block.
7.2.2. Using a scalpel is carried out between the cotyledons and the first
sheet of longitudinal or slightly diagonal cut 0.5 to 1.0 cm long and deep
approximately three-fourths the diameter of the stem.
7.2.3. The notch to hold open the tip of the blade by using a scalpel and spotted
the seed with a brush on the eyeliner or fine artist's
with a brush dipped into pellets. The rest of the pellets are distributed among all the
The Eggplant test plants.
7.2.4. The cut with sterile Vaseline applied to coincide the injection
syringe with a volume of 2 ml.
7.3 slotted Inoculation II
7.3.1. On the stem of the plant held between two fingers, pipette uterine
the first true leaves and leaf drop (approximately 5 to 10 µ l) suspended
the pellets.
7.3.2. Using a sterile scalpel, make a notch (at an angle of sešikmený
approximately 5 °), 1.0 cm long and approximately 2/3 of the thickness of
the stem, while the cut will start in place of the suspended pellet drops.
7.3.3. The cut is covers sterile Vaseline from a syringe.
7.4. Syringe inoculation
7.4.1 For reduction of the internal tension of the cells (turgoru) with eggplants
the day before the inoculation of nezalévají.
7.4.2 the Eggplant Stems just above the cotyledons inokulují
using a syringe with a hypodermic needle (not less than 23 G). Pellets
shall be divided between the eggplants.
7.5. As a positive control, 5 plants the same inoculation
method (7.2, 7.3 or 7.4) 105 water up to 106 cells per ml
the known culture the causative agent of ring rot and where possible even grids of
naturally infected Potato tubers.
4.7. the negative control should 5 plants with sterile 0.05 M
PBS the same inoculation method (7.3 or 7.4 7.2).
4.8. After inoculation the plants let incubate in quarantine in appropriate
terms and conditions (Appendix 8) for up to 4 weeks at a temperature of 18-24 ° c. Plants
incubate for sufficient illumination and high humidity (70-80%) and
bathe, so as to avoid intake of water or wilting due to
the lack of water. Cells of Clavibacter michiganensis subsp. sepedonicus
die at temperatures above 30 ° c and an optimum temperature is 21 ° c. In order to
avoid contamination incubate plants for positive and negative
control in the greenhouse or growth Chamber to clearly separate the shelves,
or when lack of space will ensure strict separation between treatment with
them. If the plants must be for different samples incubated close together,
Detaches the appropriate partitions. When fertilizing, watering, and other
the handling is committed to helping to prevent
contamination. It is vital that greenhouses and growth Chambers were protected
before all the insects because they could transfer bacteria from one
the sample on the second.
4.8. Regularly after eight days of counting plants showing
the symptoms. The originator of the ring with wilt in Eggplant
the leaves, which can start as marginal or mezižilková weakness
(loss of turgoru). May be withered mesh at first, dark green or
spotted, but before the znekrotizováním lighter. Her place among the
rot often does it feel greasy watery appearance. Nekrotická mesh
are often bright yellow edge. Plants always neodumírají; the longer
period to the appearance of the symptoms, the greater the chance of survival.
Plants may outgrow the infection. Young eggplants are
much more sensitive to low concentrations of ring rot agent than
the older plants, therefore it is necessary to use plants in the phase three worksheets
and or shortly before her. Wilt can also cause the population of the other
bacteria or fungi present in the tuber tissue pellet of tubers. They include
Erwinia carotovora subsp. carotovora and e. carotovora subsp.
atroseptica, Phoma exigua var. foveata, as well as a large concentration of
saprophytic bacteria. Such Wilts can be distinguished from the cases
caused by the originator of the ring, since whole leaves quickly wilt
or whole plants. You may also prepare a Gram stain: this test
distinguish Clavibacter michiganensis subsp. sepedonicus from Erwinia spp.
4.9. When the Eggplant develops symptoms, should be
made using parts of the tissue isolation wilted leaves or stems
plants. The surface of the leaves and stems of the eggplants with the vydezinfikuje
by wiping with 70% ethanol. Performs a test or PCR on the Eggplant and sauce
isolates are suitable (selective) media (see section 8). It can also
prepare a Gram stain (Appendix 9). Net culture suspicious of
the presence of Clavibacter michiganensis subsp. sepedonicus,
identify and confirm pathogenicity (see section 9 and 10).
7.10. In certain circumstances, in particular where growing conditions are not
the optimum, it may happen that the Clavibacter michiganensis subsp.
sepedonicus remains in eggplants as latent
infection even after the end of the incubation period of up to 4 weeks. If you are not
After 4 weeks, observed no signs, the test shall be carried out IF/PCR on
composite sample parts of the stems with a length of 1 cm from each of the tested plants
taken above the inoculation site. If the test is positive, it should be
insulation made on suitable (selective) media procedure referred to in
section 8. Net culture suspicious of the presence of Clavibacter
michiganensis subsp. sepedonicus identify and confirm with the
pathogenicity (see section 9 and 10).
8. Isolation of Clavibacter michiganensis subsp. sepedonicus
The diagnosis can be confirmed only if the originator of the ring
isolated and identified. Although it is the originator of ring rot
demanding organism, it is possible to isolate him from the tissues shortfall
symptoms of infection.
However, it may be outgrown by rapidly growing saprophytic bacteria and, therefore,
It is not recommended to carry out the insulation directly from the tuber tissue Pellet (3.1.5)
or stem (3.2). Direct isolation of Clavibacter michiganensis subsp.
sepedonicus is maybe the use of selective media and an appropriate dilution of
the resuspended pellet from the heel bulbs or from the stems of the plant.
The insulation is made from all symptomatic Potato tubers or stem and from plants
Eggplant, which do not show symptoms, but which was
a positive result in the test IF/PCR composite samples (see section 4.9).
If you need to perform the maceration of the stems of the eggplants should be performed
in accordance with section 3.1.2.
As positive controls, prepare decimal dilutions of the suspension of 106 cfu
per ml of Clavibacter michiganensis subsp. sepedonicus (e.g.. NCPPB 4053
or PD 406). To avoid any risk of contamination, prepare
the positive control separately from samples to be tested.
For each newly prepared batch of selective media should be before
using the usual test samples to test its suitability for growth
the pathogen.
Control material shall be tested in the same way as samples.
8.1. the Smear on the selective medium
8.1.1. From 100 µ l aliquots of resuspended potato sample
pellets or Eggplant juice prepare ten-fold dilutions
in pellet buffer (Appendix 3).
8.1.2. Isolation from undiluted potato pellet usually fails due to
demanding growth conditions of Clavibacter michiganensis subsp.
sepedonicus and competition Saprophytes can be increased. Whereas, the bacteria is the
in infected tissues usually present in high concentrations,
You can saprofyty usually rinse dilution, while the pathogen remains.
Therefore, it is recommended to be applied 100 µ l of each sample, in ředění1/100 to
1/10 000 to the MTNA medium or NCP-88 medium (Appendix 5), using the
AIDS for roztěrům (burning) and roztěrů techniques.
8.1.3. incubate Plates in the dark place at a temperature of 21-23 ° c.
8.1.4. the initial check shall include a comparison with the control plates
dishes and counting colonies similar to Clavibacter michiganensis
subsp. sepedonicus are carried out after three days, with another count after
5.7, and 10 days.
8.2. Purification of suspicious colonies
Note:
The subculture of colonies similar to Clavibacter michiganensis subsp.
sepedonicus for Eggplant inoculation and/or subsequent
the identification should be grown on media YGM; inoculation and identification
should be done before the media are too overgrown i.e. best
After 3-5 days.
8.2.1. The colony similar to Clavibacter michiganensis subsp. sepedonicus,
spread on one of the following media (composition given in Appendix 5):
-nutrient dextrose agar (for subculture only),
yeast peptone glucose agar-,
-yeast extract agar with mineral salts.
Incubation takes place at a temperature of 21-24 ° c for a maximum of 10 days.
The originator of the ring grows slowly and usually creates the colony size
pinheads, domed, creamy-colored.
8.2.2. Again, carry out a smear to guarantee purity. The subcultures are
the growth rate improves. Typical colonies are creamy white or color
ivory, sometimes yellow, round, smooth, raised, convex-domed,
mucoid-fluidal, with entire edges and usually have an average of 1-3 mm.
A simple Gram stain (Appendix 9) may help to select colonies for
For more testing.
8.2.3. identify Presumptive cultures (see section 9) and
pathogenicity test (see section 10).
9. The identification of the
Net culture likely isolated culture of Clavibacter michiganensis
subsp. sepedonicus are identified for the use of at least two
the following tests based on different biological principles. In
If necessary, include for each test a known reference
tribe.
9.1. Nutritional and enzymatic identification tests
Determine the following phenotypic properties. All media should
be incubated at 21 ° c and after six days should be evaluated.
If there is no growth, incubate for a maximum of 20 days.
All tests must include a known strain of Clavibacter
michiganensis subsp. sepedonicus. Nutritional and physiological tests must
by using seed from nutrient agar subcultures. Morphological
the comparison must be made from nutrient dextrose agar cultures from.
The expected test result
Oxidation-fermentation (O/F) inert or weakly
oxidation
-Oxidase activity
The activity of catalase +
Reduction of nitrate-
Urease-activity
The Formation Of H2S-
Indole production-
The use of the citrate-
Hydrolysis of starch-or weak
Growth at 37 ° c
Growth in 7% NaCl-
Hydrolysis of gelatin-
Hydrolysis of eskulinu +
Acid from:
-glycerol-
-lactose-or weak
-rhamnose-
-salicin-
Gram stain (Appendix 9)
9.2. IF test
and) prepare a suspension of approximately 106 cells per ml in IF buffer (Appendix on
3).
(b)) prepare a series of twofold dilutions of the antiserum suitable.
(c)) shall be carried out IF procedure (section 4).
(d)) of a positive result IF test is achieved if the IF titre of the culture
corresponds to a titre of the positive control.
9.3. PCR test
and) prepare a suspension of approximately 106 cells per ml in upw
(UPW)
b) heat 100 µ l of cell suspension in closed tubes in
heating block or boiling waterbath at 100 ° c for 4
minutes. If necessary, you may add fresh cells promote Lysis
the prepared NaOH to a final concentration of 0.05 M. the samples can then be
store at a temperature of-16 to-24 ° c until use.
(c)) for the amplification of Clavibacter michiganensis subsp. sepedonicus
specific amplicons shall apply appropriate PCR procedures (eg. Pastrik,
2000; see Appendix 4; Li and de Boer, 1995; Mills et al., 1997; Pastrik and
Rainey, 1999; Schaad et al., 1999).
(d)) identification of Clavibacter michiganensis subsp. sepedonicus is
a positive, if they are the same size and amplikony of the PCR are the same
the variety of the length of the fragment as the positive control strain.
9.4. FISH test
and) prepare a suspension of approximately 106 cells per ml in UPW.
(b)), the procedure Executes FISH (section 5)
(c) the FISH Test is positive), if the same reactions are achieved from the culture and
the positive control.
9.5. Fatty acid profiling (FAP)
and the culture is grown) trypticase soy agar (Oxoid) for 72
hours at 21 ° c (+/-1 ° c).
(b)) shall be used appropriate FAP procedure (Janse, 1991; Stead, 1992).
(c) a positive FAP Test) if it is identical with the suspected profile of culture
profile of the positive control. The presence of characteristic fatty
acids 15:1 Anteiso A, 15:0 Iso, 15:0 Anteiso, 16:0 Iso, 16:0 and 18:0
Anteiso clearly demonstrates the presence of Clavibacter michiganensis subsp.
sepedonicus. Other genera such as Curtobacterium, Arthrobacter and Micrococcus
They also contain some of these acids but 15:1 Anteiso A is for these
unusual bacteria, which is acid, however, occurs in all
Clavibacter spp. in the range of 1-5%. For Clavibacter michiganensis subsp.
sepedonicus the value is usually around 5%.
9.6. BOX-PCR
and) prepare a suspension of approximately 106 cells per ml in UPW.
(b) the test procedure shall be carried out) by Smith et al., 2001.
10. pathogenicity Test
For the final confirmation of ring rot agent and for the determination of virulence
cultures identified as Clavibacter michiganensis subsp.
sepedonicus pathogenicity test must be performed.
10.1. Prepare the test lasts approximately 106 cells per ml from a 3-day
cultures of isolated substances tested and the appropriate positive strain
Clavibacter michiganensis subsp. sepedonicus.
10.2. Inoculate 5-10 stems of young seedlings in Eggplant
3 true leaf stage.
10.3. Incubate at 18-24 ° c with sufficient light and high
relative humidity with appropriate watering to avoid waterlogging
or desiccation. With pure cultures should occur within 2 weeks typical
Wilt, but plants that after this time, do not show any
symptoms of infection should be incubated for up to 3 weeks at temperatures
favourable for the growth of Eggplant, but not exceeding 25 ° c.
If after 3 weeks, symptoms of infection, cannot be
culture considered pathogenic form of Clavibacter michiganensis subsp.
sepedonicus.
10.4. The insulation is carried out from the plants with symptoms of infection by removing the part of the
the stem two cm above the inoculation point. Tissue is crushed and suspended in
a small volume of sterile distilled water or 50 mM phosphate buffer.
Isolates from the suspension or deposition of rozetřením to the MTNA and YPGA,
incubate for 3 to 5 days at a temperature of 21-23 ° c, and track the emergence of
colonies typical for Clavibacter michiganensis subsp. sepedonicus.
Appendix 1
Laboratories involved in optimisation and validation of protocols
------------------------------------------------------ -------------------------------------------
Laboratory (1) instead of the country
------------------------------------------------------ -------------------------------------------
Agencies für Gesundheit und Vienna and Linz Austria
Ernährungssicherheit
Department Of Gewasbescherming Merelbeke Belgium
Plantedirektoratet Lyngby Denmark
The Central Science Laboratory, York, England
The Scottish Agricultural Science Agency, Edinburgh, Scotland
Laboratoire National de la Protection Angers France
Végétaux, unit de Bactériologie
Laboratoire National de la Protection Le Rheu France
Végétaux, Station de Quarantaine de la Pomme
de Terre
Biologische Bundesanstalt Kleinmachnow Germany
Pflanzenschutzamt Hannover Hannover Germany
State Laboratory, Dublin Ireland
Plantenziektenkundige Dienst Wageningen University Netherlands
The Norwegian Crop Research Institute, Plant Aas Norway
Protection Centre
Direcçăo-General de Protecçăo das Culturas Lisbon Portugal
Nacionalni institut za biologijo Ljubljana Slovenia
Centro de Diagnóstico de Aldearrubia Salamanca Spain
------------------------------------------------------ -------------------------------------------
(1) contact person: see Web site
http://forum.europa.eu.int/Public/irc/sanco/Home/main
Appendix 2
Preparation of positive and negative controls for the examination of the potato tests
PCR/IF and FISH
Produced with the 72 hour culture of a virulent strain of c. m. subsp.
sepedonicus (NCPPB 4053 or PD 406) on MTNA and the basic medium
suspended in 10 mMfosfátového buffer to obtain the density of approximately 1
up to 2 x 108 cells capable of making colonies/ml. It usually reaches
through the slightly turbid suspension equivalent optical density
0.20-600 nm. Tissue cores shall be taken from the heel ends 200 tubers
taken from the production of the variety, with white skin, that is certain, that is
free from c. m. subsp. sepedonicus.
The heel end cores shall be processed in the usual manner and resuspenduje with pellets
in 10 ml.
Prepare 10 sterile 1.5 ml microvials with a volume of 900 mikrol
the resuspended Pellet.
Transfer the suspension had 100 c. m. subsp. sepedonicus in the first
Eppendorf tube. Lets shake.
In the next five microvials decimal dilutions are made.
Six contaminated microvials will be used as the positive control.
Four non-contaminated microvials will be used as the negative control.
Microtest tube shall bear a label.
Prepare aliquots of 100 mikrol in sterile tubes
about the volume of 1.5 ml, which gets 9 copies of each sample.
Storage takes place at a temperature of-16 to-24 ° c until use.
The presence and quantity of c. m. subsp. sepedonicus in the control samples
should first be confirmed by immunofluorescence.
For the PCR test DNA extraction from positive and negative
control samples with each series of test samples.
For IF and FISH tests shall be carried out quantitative analyses of positive and
negative control samples with each series of test samples.
In the quantitative analysis of IF, FISH and PCR c. m. subsp.
sepedonicus is detected in at least the 106 and 104 cells/ml of the positive controls
and may not be detected in any of the negative controls.
Appendix 3
Buffers for test procedures
General: Unopened sterilized buffers can be stored for up to one
of the year.
1. buffers for extraction
1.1. The extraction buffer (50 mM phosphate buffer, pH 7.0)
This buffer is used to extract the bacteria from plant tissues
by homogenisation or shaking.
Na2 HPO4 (anhydrous) 4.26 g
Kh2po4 2.72 g
Distilled water 1.00 l
Dissolve ingredients, check pH and sterilization
autoclave at 121 ° c for 15 min.
May be useful in the following folder:
The purpose of the quantity (on the
litre)
Lubrol flakes deflocculant (*) 0.5 g
DC silicone Antifoam agent odpěňovač (*) 1.0 ml
Tetrasodiumpyrofosfát Antioxidant agent 1.0 g
Polyvinylpyrrolidone-40000 (PVP-40) the binding of PCR inhibitors, 50 g
-------------------------------------------
(*) for use in the extraction by homogenisation
1.2. Peletový buffer (10 mM phosphate buffer, pH 7.2)
This buffer is used for resuspension and dilution of the extracts of the potato from
the potato tuber heel after centrifugation
concentrated into pellets.
Na2hpo4 12H2O 2.7 g
NaH2PO4. 2H2O 0.4 g
Distilled water 1.00 l
Dissolve ingredients, check pH and sterilization
autoclave at 121 ° c for 15 min.
2. buffers for the IF test
2.1. buffers for the IF (10 mM phosphate buffered saline (PBS),
pH 7.2)
This buffer is used for dilution of antibodies.
Na2hpo4 12H2O 2.7 g
NaH2PO4. 2H2O 0.4 g
NaCl 8.0 g
Distilled water 1.00 l
Dissolve ingredients, check pH and sterilization
autoclave at 121 ° c for 15 min
2.2. IF-buffer-Tween
This buffer is used for the washing of glasses. Add 0.1% Tween 20 to the buffer
for IF.
2.3. Phosphate buffered glycerol, pH 7.6
This buffer is used as a top solution for the window slides on IF the tests to
the increase in fluorescence.
Na2hpo4 12H2O 3.2 g
NaH2PO4. 2H2O 0.15 g
Glycerol 50 ml
Distilled water 100 ml
Cover solutions are commercially available, such as. Vectashield (Vector (R)
Laboratories) or Citifluor ® (Leica).
Appendix 4
Determination of the concentration of positive cells IF and FISH
1. Calculate the average number of typical fluorescent cells per
field of view (c).
2. Calculate the number of typical fluorescent cells per window
the microscopic slides (C).
(C) = c x S/s,
where S = the area of one field on a slide with more wells and
s = surface area of objective field.
s = đi2/2, 4G2K
where i = field coefficient (ranging from 8-24 depending on type of eyepiece),
K = tube coefficient (1 or 1,25),
G = magnification of the lens (100 x, 40 x, etc.).
3. Calculate the number of characteristic of Fluorescing cells per 1 ml
the resuspended Pellet (N).
N = C × 1 000/y x F,
where y = volume of re-suspended pellet on each window, and
F = dilution factor of re-suspended pellet
Appendix 5
Media for isolation and culture of c. m. subsp. sepedonicus
1. General growth media
Nutrient agar (Nutrient agar = on)
Nutrient agar (Difco) 23 g
Distilled water 1.00 l
Dissolve ingredients and autoclave at 121 ° c
for 15 min.
Nutrient dextrose agar (Nutrient:-dextrose agar = NDA)
Bakto difco nutrient agar containing 1% of D (+) glucose (monohydrate).
Autoclave sterilization is performed at 121 C for 20 min.
Kvasnično-peptone-glucose agar (Yeast peptone glucose agar = YPGA)
Yeast extract (Difco) 5.0 g
Baktopepton (Difco) 5.0 g
D (+) glucose (monohydrate) 10.0 g
Baktoagar (Difco) 15.0 g
Distilled water 1.00 l
Dissolve ingredients and autoclave at 121 ° c
for 15 min.
Medium with kvasnicovým extract and mineral salts (Yeast extract
mineral salts medium = YGM)
Yeast extract 2.0 g
(Difco)
D (+) glucose 2.5 g
(monohydrate)
K2hpo4 0.25 g
Kh2po4 0.25 g
MgSO4. 7 H2O 0.1 g
MnSO4. H2O 0.015 g
NaCl 0.05 g
Feso4. 7 H2O 0.005 g
Baktoagar (Difco) 18.0 g
Distilled water 1.00 l
Dissolve ingredients and sterilization in an autoclave 0.5 l media at
115 ° c for 20 min.
(b) Validated selective growth media)
MTNA Medium
Unless otherwise stated, all the ingredients come from the media, BDH.
Yeast extract 2.0 g
(Difco)
Mannitol 2.5 g
K2hpo4 0.25 g
Kh2po4 0.25 g
MgSO4. 7 H2O 0.1 g
MnSO4. H2O 0.015 g
NaCl 0.05 g
Feso4. 7 H2O 0.005 g
Agar (Oxoid No. 1) 16.0 g
Distilled water 1.00 l
Dissolve ingredients, pH to 7.2. After autoclaving (at 121 ° c
After a period of 15 min.) and cooling down to 50 ° C, add the antibiotics:
trimethoprim 0.06 g, nalidixic acid 0.002 g, amphotericin B 0.01 g.
In stock are the solutions of the antibiotics: trimethoprim (Sigma), and
nalidixic acid (Sigma) (both 5 mg/ml) in 96% methanol, amphotericin B
(Sigma) (1 mg/ml) in dimethyl sulfoxide. Stock solutions are
sterilized by filtration.
Note:
The durability of the basic media is 3 months. After the addition of the antibiotics is
shelf life 1 month when stored in a cool place up to 8 +/-2 ° C.
NCP-88 medium
Nutrient agar (Difco) 23.0 g
Yeast extract 2.0 g
(Difco)
D-mannitol 5.0 g
K2hpo4 2.0 g
0.5 g of kh2po4
MgSO4. 7 H2O 0.25 g
Distilled water 1.00 l
Dissolve ingredients, adjust the pH to 7.2. After autoclaving and cooling
50 ° c, add the following antibiotics: polymyxin B sulphate (Sigma)
0, 003g, nalidixic acid (Sigma) 0.008 g, acid (Sigma) 0.2 g.
Dissolve antibiotics for preparation of stock solutions as follows:
nalidixic acid in 0.01 M NaOH, cycloheximide in 50% ethanol, polymyxin B
sulphate in distilled water. Stock solutions to sterilize by filtration.
Note:
The durability of the basic media is 3 months. After the addition of the antibiotics is
shelf life 1 month when stored in a cool place up to 8 +/-2 ° C.
Appendix 6
Validated PCR Protocols and reagents
Note:
The initial testing should permit reproducible detection of at least 103
up to 104 cells of c. m. subsp. sepedonicus per ml of sample extract.
Initial testing would not also show no false positive
the results of the Group of the selected strains of bacteria.
1. PCR Protocol with internal PCR control (Pastrik, 2000)
1.1. Oligonukleotidní primers
Primer PSA-1 5 '-ctc ctt gtg ggg tgg gaa aa-3 '
Primer PSA-R 5 '-tac tga gat gtt tca ctt ccc c-3 '
Primer NS-7-F 5 '-gag gca ata aca ggt ctg tga tgc-3 '
Primer NS-8-R 5 '-tcc gca ggt tca cct acg ga-3 '
Expected Amplicon size from the DNA of c. m. subsp. sepedonicus template
= 502 bp (PSA-primer set).
Expected Amplicon size from the 18S rRNA internal PCR control = 377
BP (NS-primer set).
1.2. PCR reaction mix
------------------------------------- ------------------------- ------------------------
The quantity of reagent on the reaction of the final concentration
------------------------------------- ------------------------- ------------------------
Sterile water (UPW) 15.725 µ l
10 x PCR buffer (1) (15 mM MgCl2) 2.5 µ l of 1 x (1.5 mM MgCl2)
BSA (fraction V) (10%) 0.25 µ l 0.1%
D-nTP mix (20 mM) l 0.1 µ 0.125 mM
Primer PSA-1 (10 µ M) 0.5 0.2 µ µ l M
Primer PSA-R (10 µ M) 0.5 0.2 µ µ l M
Primer NS-7-F (10 µ M) (2) 0.1 µ µ l 0.04 M
Primer NS-8-R (10 µ M) (2) 0.1 µ µ l 0.04 M
Taq polymerase (5 U/µ l) (1) 0.2 µ l 1.0 for
Sample quantity 5.0 µ l
------------------------------------- ------------------------- ------------------------
The total volume of 25.0 µ l
------------------------------------- ------------------------- ------------------------
(1) Methods were validated using Taq polymerase Perkin Elmer
(AmpliTaq or Gold) and Gibco BRL.
(2) concentration of primers NS-7 F and NS-8-R has been optimized for
potato heel extraction using the homogenisation method and
DNA purification according to Pastrik (2000) (see section 6.1) and 6.2).
When you use the extraction by shaking or other DNA isolation methods is needed
new optimization the concentration of the reagent.
1.3. PCR Reaction conditions
Proceed according to the following programme:
1 cycle of: i) 3 minutes at a temperature of 95 ° c (denaturation of the DNA of the matrix)
10 cycles ii) 1 minute at 95 ° c (denaturation of the DNA of the matrix)
III) 1 minute at 64 ° c (Primer)
IV) 1 minute at a temperature of 72 ° c (extension of copy)
25 cycles) for 30 seconds at a temperature of 95 ° c (denaturation of the DNA of the matrix)
vi) for 30 seconds at a temperature of 62 ° c (Primer)
VII) 1 minute at a temperature of 72 ° c (extension of copy)
1 cycle viii) 5 minutes at 72 ° c (final extension)
IX) maintained at 4 ° c
Note:
This program is optimized for use with heating cyklerem MJ
Research PTC 200. When using other models may be needed modification
steps of cycles (ii), (iii) (iv))))), vi) and (vii)).
1.4. Analysis of ampliconu restrictive enzyme
PCR products amplified from the DNA of c. m. subsp. sepedonicus produce
the typical variety of the length of the fragment with enzyme Bgl II after
incubation at 37 ° c for 30 minutes. Fragments obtained from the
fragment specific for c. m. subsp. sepedonicus are 282 bp
and 220 bp.
2. preparation of the loading buffer
2.1. Bromphenol blue (10%-stock solution)
Bromphenol blue 5 g
Distilled water 50 ml
Coating buffer
Glycerol (86%) 3.5 ml
Bromphenol blue 300 µ l
6.2 ml distilled water
3. Buffer 10 x TRIS-acetate-EDTA (TAE) buffer, pH 8.0
TRIS 48.4 g
11.42 ml glacial acetic acid
EDTA (sodium salt) 3.72 (g)
Distilled water 1.00 l
Before use, dilute to 1 x.
Also, commercially available (eg. Invitrogen or equivalent).
Appendix 7
Validated reagents for FISH test
1. Oligosondy
Cms-specific probe CMS-Appear-01:5 '-ttg cgg ggc gca cat ctc tgc acg-3 '
Non-specific eubacterial probe EUB-338-FITC: 5′-gct gcc tcc cgt agg agt-3′
2. Fixative solution
[NOTE: FIXATIVE SOLUTION CONTAINS PARAFORMALDEHYDE WHICH IS TOXIC!
THE USE OF GLOVES AND DO NOT BREATHE. IT IS RECOMMENDED TO WORK IN A FUME CUPBOARD.]
I) heat 9 ml molecular grade water (e.g. clean. Ultra pure water =
(UPW)) at a temperature of around 60 ° c and add 0.4 g paraformaldehyde.
Paraformaldehyde dissolves after adding 5 drops of 1N NaOH and stirring
magnetic stirrer.
(ii) adjust pH to) 7.0 by adding 1 ml 0.1 M phosphate buffer (PB; pH
7.0) and 5 drops of 1N HCl. The indicator Strip, check the pH and
If necessary, adjust with HCl or NaOH.
[Warning: DO NOT USE in SOLUTIONS with PARAFORMALEDHYDEM pH METER!]
III) filter the solution through a membrane filter, 0.22 µm and stored
protected against dust at 4 ° c until further use.
(iv)) Note:
Alternative fixative solution: 96% ethanol
.
3.3 x Hybmix
NaCl 2.7 M
60 mM TRIS-HCl (pH 7.4)
EDTA (filter sterilised through and 15 mM
autoclaved)
Dilute to 1 x as required.
4. Hybridisation solution
1 x Hybmix
Sodium dodecyl sulfate (SDS) 0.01%
Probe EUB 338 5 ng/mikrol
Probe CMSCY301 5 ng/mikrol
Prepare quantities of hybridisation solution according to the calculations in the table.
For each slide (containing 2 different samples in duplicate) must be 90 mikrol
hybridisation solution.
Table: recommended quantities for the preparation of hybridisation mix
------------------------------------- -------------------------
2 slides 8 slides
------------------------------------- -------------------------
Sterile ultra clean water 50.1 200.4
3 x hybmix 30.0 120.0
1% SDS 0.9 3.6
probe EUB 338 (100 ng/mikrol) 18.0 4.5
probe CMSCY301 (100 ng/mikrol) 18.0 4.5
------------------------------------- -------------------------
Total volume (mikrol) 90.0 360.0
------------------------------------- -------------------------
Note.:
All solutions containing light-sensitive oligosondy shall be kept in the
dark place at a temperature of-20 ° c. During use, it is necessary to protect against
direct sunlight or electric light.
5.0, 1 m phosphate buffer, pH 7.0
Na2hpo4 8.52 g
Kh2 PO4 5.44 g
Distilled water 1.001
Dissolve ingredients, check pH and Sterilise autoclaving
at a temperature of 121 ° c for 15 minutes.
Appendix 8
Growing Eggplant
The seeds of Eggplant (Solanum melongena) in vysejí
pasteurized výsevního medium. Seedlings with fully expanded
the cotyledons (10 to 14 days) is přepichují in pasteurized
the growing medium.
Eggplants should be grown in a greenhouse under the following conditions:
Duration of day: 14 hours or natural day length if greater;
Temperature: day: 21 to 24 ° c,
night: 15 ° c.
Suitable varieties of eggplants
"Black Beauty",
"Long Tom",
"Rima"
"Balsas".
Supplier: see Web site
http://forum.europa.eu.int/Public/irc/sanco/Home/main
Appendix 9
Gram stain in Huckerově modification (Doetsch, 1981) ^ (1)
Crystal Violet solution
Dissolve 2 g Crystal Violet in 20 ml 95% ethanol.
Dissolve 0.8 g ammonium oxalate in 80 ml of distilled water. Both
the solutions are mixed.
Lugol's iodine solution
Iodine 1 g
Potassium iodide 2 g
Distilled water 300 ml
Solids spread together with the tloučku in the bowl. Nasypou to
water and blended in a closed container to the dissolution.
Safraninový solution of contrast dye
Stock solution:
Safranin O 2.5 g
95% ethanol 100 ml
Mix and store.
Dilution: 1:10 for the preparation of a working solution.
How to dye
1. Prepare smears, air dry and fix the heating.
2. Slide with Crystal Violet solution and allow to Act 1
minute.
3. wash briefly under running water.
4. Embedding with Lugolovým jódovým and allow the solution to act for
one minute.
5. Rinse under running water and dry with tissue paper.
6. Odbarvuje using a drop of 95% ethanol,
If the dye is flushed out, or dipping in gentle agitation
ethanol for 30 seconds.
7. Rinse under running water and dry with tissue paper.
8. Embedding with the safraninovým solution and leave to act for 10 s.
9. Rinse under running water and dry with tissue paper.
Gram-positive bacteria, Gram-negative bacteria, Violet Blue
turns růžovočerveně.
(1) can also be used commercially available solutions or colouring
kits.
LITERATURE
1. Anonymous, 1987. The scheme of the detection and diagnosis of the ring
rot bacterium Corynebacterium sepedonicum in batches of potato tubers.
Commission of the European Communities, Luxembourg. Publ EUR 11 288 EN
21 pp.
2. Bradbury, j. f., 1970. Isolation and preliminary study of bacteria
from plants. Rev. pl. Path., 49, 213-218.
3. the Dinesen, I. G., 1984. The extraction and diagnosis of
Corynebacterium sepedonicum from diseased potato tubers. EPPO Bull. 14
(2), 147-152.
4. Doetsch, r. n., 1981. Determinative methods of light microscopy. In:
Manual of methods for general bacteriology, American Society for
Microbiology, Washington, 21-23.
5. Hugh, r. and Leifson, f., 1953. The taxonomic significance of
fermentative versus oxidative metabolism of carbohydrates by various
Gram-negative bacteria. J. 7., 66, 24-26.
6. Janse, J. D., 1991. Infra-and intra-specific classification of
Pseudomonas solanacearum strains using whole cell fattyacid analysis.
Systematic and Applied Microbiology 14; 335-345.
7. Janse, j. d. and j. Van Vaerenbergh. The interpretation of the EC
method for the detection of latent ring rot infections (Corynebacterium
sepedonicum) in potato. EPPO Bull., no. 17, 1987, pp. 1-10.
8. Jansing, h. and k. Rudolph, 1998. Physiological capabilities of
Clavibacter michiganensis subsp. sepedonicus and development of and
semi-selective medium. Journal of Plant Diseases and Protection, 105,
590-601.
9. Kovacs, n., 1956. Identification of Pseudomonas pyocyanea by the
oxidase reaction. Nature, Lond., 178, 703.
10. Clement Z.; Rudolph, and d. c. Sands, 1990. Methods in
Phytobacteriology. Akadémiai Kiadó, Budapest, 568 pp.
11. Lelliott, r. a., 1966. The plant pathogenic coryneform bacteria. (J).
Appl. 7.29, 114-118.
12. Lelliott, r. a., e. Billing and a. c. Hayward, 1966. And the determinative
scheme for the fluorescent plant pathogenic pseudomonads j. Appl.
7., 29, 470-489.
13. Lelliott, r. a., and p. w., Sellar, 1976. The detection of latent ring
rot (Corynebacterium sepedonicum (Spiek. et Kotth.) Skapt. et Burkh.)
in potato stocks. EPPO Bull., 6 (2), 101-106.
14. If X and de Boer, S.H. 1995. Selection of Polymerase Chain Reaction
primers from RNA intergenic spacer region for specific detection of
Clavibacter michiganensis subsp. sepedonicus. Phytopathology, 85,
837-842.
15. Mills, d., Russell, b., w. and j., w. H, 1997. Specific detection
of Clavibacter michiganensis subsp. sepedonicus by amplification of
three unique DNA sequences isolated by subtraction hybridization.
Phytopathology, 87, 8, 853-861.
16. Pastrik. -H and an R.a.. Rainey. 1999. Identification and
differentiation of Clavibacter michiganensis subspecies would be followed
chain reaction-based techniques. J. Phytopathology 147; 687-693.
17. Pastrik, k.-h., 2000. Detection of Clavibacter michiganensis subsp.
sepedonicus in potato tubers by multiplex PCR with coamplification of
host DNA. European Journal of Plant Pathology, 106, 155-165.
18. Ramamurthi, c. s., 1959. Comparative studies on some Gram-positive
phytopathogenic bacteria and their relationship to the Corynebacteria.
Mem. Cornell agric. Exp. Sta., 366, 52 pp.
19. Schaad, w., Berthier-Schaad, y., Sechler, a. and Knorr, d. (1999)
Detection of Clavibacter michiganensis subsp. sepedonicus in potato
tubers by BIO-PCR and an automated real-time fluorescence detection
System. Plant Disease 83; 1095-1100.
20. Schaad, w. 2001. Laboratory guide for identification of plant
pathogenic bacteria. Schaad [Hrsg.]. – 3. Ed.; St. Paul, Minnesota:
373 pp.
21. Skerman, v. B. D., 1967. A guide to the identification of the
Genera of bacteria. 2nd ed., William and Wilkins Company, Baltimore.
22. Smith, N. (C); Hennesy, J; Stead, D.E., 2001. Repetetive
sequence-derived PCR profiling using the BOX-A1 of Ralstonia solanacearum
a primer for rapid identification of plant pathogen Clavibacter
michiganensis subsp. sepedonicus. European Journal of Plant Pathology,
107 (7), 739-748.
23. Sneath, p. h. a. and v. g. Collins, 1974. And a study in test
reproductibility between laboratories: report of Pseudomonas working
party. Antonie van Leeuwenhoek, 40, 481-527.
24. Stead, D.E., 1992. The grouping of plant pathogenic and some other
Pseudomonas spp. using cellular fatty-acid profiles. International
Journal of Systematic Bacteriology 42; 281-295.
25. Wullings, b. a.; van Beuningen, a. r.; Janse, J. D., and A. D. L.
Akkermans, 1998. Detection of Ralstonia solanacearum, which causes
brown rot of potato, by fluorescent in situ hybridization with 23s
rRNA-targeted probes. Appl. Environ. Microbiol. 64, 4546-4554.
(B) Methods Of Diagnosis, Detection And Identification Of The Causative Agent Of Brown Rot
Submitted by progress diagrams describe the various procedures that are
part of:
I) diagnosis of brown rot in Potato tubers and of bacterial wilt
potato, tomato and some other host plants,
II) detection of Ralstonia solanacearum in samples of potato tubers, plants
potato, tomato plants and other host plants, and in samples
water and soil,
(iii)) identification of Ralstonia solanacearum (r. solanacearum)
THE GENERAL PRINCIPLES OF
Optimized protocols for the various methods, reagents and approved
details for the preparation of test and control materials are
listed in the appendices. The list of laboratories that participated in the
optimization and validation of protocols is in Appendix 1.
Because the protocols contain the detection of a quarantine organism and include
the use of viable cultures of r. solanacearum as control
materials, it is necessary to work for appropriate quarantine conditions with
the appropriate device for the disposal of waste and the conditions
laid down in the authorization of plant health management.
Testing parameters must assure the permanent and reproducible detection of
levels of r. solanacearum as the thresholds of the selected methods.
Essential is the exact preparation of positive controls.
Testing according to the required thresholds also involves the correct settings,
maintenance and calibration of equipment, careful handling agents and their
retention and all measures to prevent contamination between samples,
for example. separation of the positive controls from test samples. Must be
applied quality management standard, in order to avoid administrative
and other errors, especially in the labelling and documentation.
A suspected occurrence, as referred to in section 4, paragraph 4. 1 indicates a positive
the result of the diagnostic or screening tests carried out on the
of the sample, as shown in the below route diagrams.
The first positive 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, there is a suspicion of
infection of r. solanacearum and must be made second screening test.
If the second screening test is positive, then the suspicion is
confirmed, and must continue to test according to the scheme.
If the second screening test is negative, then the sample is
considered as infected with r. solanacearum.
Confirmed presence under section. 5 (3). 1 requires the isolation and identification of
pure cultures of r. solanacearum with confirmation of pathogenicity.
1. Use progressive charts
1.1. Flowchart for the diagnosis of brown rot and bacterial
Wilt (Ralstonia solanacearum) in Potato tubers and potato,
tomato plants and other host plants with symptoms of Brown
rot or bacterial wilt
The testing procedure is intended for Potato tubers and plants with suspicion of
typical or symptoms of brown rot or bacterial
wilt. It involves a rapid screening test, isolation of the pathogen from
infected vascular tissue on the selective medium and in the case of
a positive identification of the culture of Ralstonia solanacearum.
-----
1) description of the symptoms is located in section 2.1.
2) rapid diagnostic tests facilitate presumptive diagnosis but are not
the perfect. A negative result indicates the absence of the pathogen always.
3) Test on the discharge of mucus from the vascular stem is described in section
6.1.1.
4) Test for the presence of poly-beta-hydroxybutyrate granules is described in
section 6.1.2.
5) serological agglutination tests of bacterial ooze or extracts from
mesh reporting symptoms are described in section 6.1.3.
6) IF Test of bacterial slime slurried in water or extracts
mesh reporting symptoms is described in section 6.1.5.
7) Test of FISH bacterial ooze or the suspended in water extracts
mesh reporting symptoms is described in section 6.1.7.
8) ELISA Test of bacterial ooze or the suspended in water
extracts of tissue exhibiting symptoms is described in section 6.1.8.
9) the PCR Test of bacterial slime slurried in water or extracts
mesh reporting symptoms is described in section 6.1.6.
10) the pathogen is usually easily izolovatelný from the plant material
reporting symptoms of the dilution method roztěrů (gradual dilution)
(see section 2.3).
11) Typical colony morphology is described in section 2.3. d.
12) Cultivation may fail when the advanced stages of infection for reasons
competition or a lush growth of saprophytic bacteria. If you are
typical symptoms of infection, but the isolation test is negative, the
Repeat the isolation, preferably by selective substrates.
13) reliable identification of pure cultures of r. solanacearum is carried out
using the tests described in point 6.2. Partial specific characteristics is the
optional, but recommended for each new case.
14) pathogenicity Test is described in section 6.3.
1.2. Flowchart for the detection and identification of Ralstonia
solanacearum in samples of asymptomatic Potato tubers
Testing is intended for detection of latent infections in Potato tubers.
A positive result from at least two screening tests (see note in
Diagram 3), each of which is based on a different biological principle,
must be complemented by the isolation of the pathogen and, in case of isolation of typical
the colonies, confirmation that a pure culture of r. solanacearum. Positive
the result of only one screening test is not sufficient to
that sample was considered a suspect. Screening tests and isolation
tests must allow for the detection of 103 to 104 cells/ml of resuspended
Pellet, included as positive controls in each series of tests.
-----
1) Standard sample size is 200 tubers, although the procedure can be used
the smaller the number, if 200 tubers is not available.
2) extraction methods and the concentration of the pathogen are described in section 3.1.1.
3) if they are at least two tests based on different biological
the principles of positive, must be carried out in isolation and confirmation. Performs
at least one screening test. If the test is negative, the sample is
considered negative. In the event that the test is positive, it is for the
the validation of the first positive result in one or more of the essential
screening tests based on different biological principles.
If the second or subsequent test is negative, the sample is considered to
negative. Further tests are necessary.
4) IF Test is described in section 6.1.5.
5) selective isolation Test is described in section 6.1.4.
6) PCR Tests are described in section 6.1.6.
7) the FISH Test is described in section 6.1.7.
8) ELISA tests are reviewed in section 6.1.8.
9) Bioassay is described in section 6.1.9.
10) Typical colony morphology is described in section 2.3. d.
11) Cultivation or the bioassay test can fail for reasons of competition or
inhibition of saprophytic bacteria. If the screening tests
obtained positive results, but the insulation tests are negative, you must
Repeat isolation tests from the same or additional pellets by removing
vascular tissue near the heel end of the tubers of the same sample
Alternatively, perform the test with the other samples.
12) Reliable identification of pure cultures of suspects on the R.
solanacearum is achieved by the tests described in section 6.2.
13) pathogenicity Test is described in section 6.3.
Flow chart for the detection and identification of Ralstonia solanacearum in
samples of asymptomatic potato, tomato plants
asymptomatic, or other host plants
asymptomatic
-----
1) see section 3.2.1. where sample sizes are recommended.
2) extraction methods and the concentration of the pathogen are described in section 3.2.1.
3) if they are at least two tests based on different biological
the principles of positive, must be carried out in isolation and confirmation. Performs
at least one screening test. If the test is negative, the sample is
considered negative. In the event that the test is positive,
for the validation of the first positive result in the second or more
screening tests based on different biological principles.
If the second or subsequent negative tests, the sample for the
negative. Further tests are necessary.
4) Selective isolation test is described in section 6.1.4.
5) IF Test is described in section 6.1.5.
6) PCR Tests are described in section 6.1.6.
7) the FISH Test is described in section 6.1.7.
8) ELISA tests are described in section 6.1.8.
9) Bioassay is described in section 6.1.9.
10) Typical colony morphology is described in section 2.3. d.
11) Cultivation or the bioassay test can fail for reasons of competition or
inhibition of saprophytic bacteria. If the screening tests
obtained positive results, but the insulation tests are negative, repeat
the insulation tests.
12) Reliable identification of pure cultures, where it is suspected that
This is the r. solanacearum is achieved by the tests described in section
6.2.
13) pathogenicity Test is described in section 6.3.
2. The diagnosis of the cause of brown rot
2.1. Symptoms
2.1.1. The symptoms in potato
Potato plant.
The early stage of infection in the field conditions, wilting of leaves recognizes
towards the top of the plant at high temperatures during the day, while in the
night recovery occurs. In the early stages of wilting leaves remain
Green, but later turn yellow and Brown necrosis develops. Also occurs
to bending the leaves down. Wilting of one side shoot or whole plants
quickly becoming irreversible and results in the collapse and plants. From the vascular
volumes across the stumps of dead plants usually flows out of the stems Brown and
Milky bacterial ooze or the ooze out. During immersion
uříznutého stem vertically in water the fibers from the vascular bundles
slime.
The tuber of the potato. Potato tubers must be cut transversely close across the heel
over and over the stolon end lengthwise. In the early stage of infection by
láhvově yellow to light brown discoloration of the vascular ring from
After a few minutes which emerges a pale cream bacterial
slime. Later, he becomes a more significant vascular discoloration Brown and necrosis
may extend into the parenchymatous tissue. In more advanced stages, the
the infection spreads outwards from the heel end of the tubers and buds may escape
bacterial slime that hot particles of soil. On skin
may appear reddish-brown, slightly sunken spots as a result of
the internal collapse of the vascular tissue. In more advanced stages of infection is
usually the secondary development of soft hnilob bacterial disease
origin.
2.1.2. The symptoms in tomato
Tomato plant. The first visible symptom is the flaccid appearance
the youngest leaves. Under favourable conditions for the pathogen (soil temperature
around 25 ° C, saturated humidity), within a few
days unfold twisting the sheets down and wilting on one side
plants or whole plants, which ends its full odumřením. For
less favourable conditions for the pathogen (soil temperature less than 21şC)
the plant so much nevadne, but on the stem can consist of a greater number of
the side shoots. It is possible to observe watersoaked streaks from the bottom
the stem, which are evidence of necrosis in the vascular system. When the cross section
stem brown coloured conductive tissue secrete a white or yellowish
bacterial slime.
2.1.3. The symptoms for other hosts
Solanum dulcamara and s. nigrum. Under normal conditions, are
These wild host plants rarely observed symptoms
Wilt, if soil temperatures exceed 25 ° C or is extremely
the high concentration of inoculum (e.g. for plants growing in s. nigrum
diseased potato or tomato plants). When wilt symptoms are
the same as for tomato plants. Timeless plant., dulcamara
It has stems and roots in water, may show internal light brown
coloring vascular tissue on the bottom of the cross section of the stem or parts
the stem under water. From the cut vascular bundles can bacteria or
can form the threads of slime, if the stem is cut vertically into the submerged
water, even in the absence of signs of wilting.
2.2. Rapid screening tests
Rapid screening tests may help to advance the diagnosis, but are not
sufficient. Apply one or more of the following tests:
2.2.1. Test on the discharge of mucus from the stem (see 6.1.1.)
2.2.2. Test for the presence of poly-beta-hydroxybutyrate (PHB) granules
Characteristic PHB granules in the cells of r. solanacearum are
visualised by staining heat-fixed spots of bacterial ooze from
infected tissue on a microscope slide with Nile blue A or
Sudan black (see section 6.1.2).
2.2.3. Serological agglutination tests
(See section 6.1.3).
2.2.4. Other tests
Other appropriate rapid screening tests are the IF test (see section
6.1.5.), FISH test (see section 6.1.7.), ELISA tests (see section 6.1.8.) and
The PCR tests (see section 6.1.6).
2.3. Procedure for the isolation of the
and Removes the mucus or) layer of coloured tissue from the vascular ring
the potato tuber or from the vascular fibre stem plants potato, tomato
or other wilting host plants. Suspended in a small amount of
of sterile distilled water or 50 mm phosphate buffer (Appendix 4) and
leave for 5-10 minutes.
(b) prepare a series of decimal) the dilution of the suspension.
(c)) transfer 50-100 of the suspension and had a solution to universal breeding ground
(NA, YPGA neboSPA; see Appendix 2) and/or to Kelman's tetrazolové media
(Appendix 2) and/or validated selective medium (eg. SMSA, see.
Appendix 2). Grind the dilution method roztěrů. Prepare a
Alternatively, a separate dish with a diluted cell suspension of R.
solanacearum biovar 2 for positive control.
d) incubate for 2-6 days at a temperature of 28 ° C.
-To create a universal culture of virulent isolates of R.
solanacearum Pearly cream-white, flat, irregular and fluidal
colonies often with characteristic whorls in the Centre. Aviruletní
forms of r. solanacearum form small round non-fluidal, butyrous colonies
that are completely creamy white.
-For the Kelman and SMSA media tetrazolového are blood-
the red-coloured. Aviruletní forms of r. solanacearum form small round
non-fluidal, butyrous colonies which are entirely deep red.
2.4 identification tests of Ralstonia solanacearum
Tests confirming the presence of r. solanacearum in the suspect isolates are
described in section 6.2.
3. Detection and identification of the causative agent of brown rot in samples of tubers
potato
3.1. Detailed methods for detection and identification of Ralstonia solanacearum
in samples of asymptomatic Potato tubers
3.1.1. Preparation of samples
Note:
-The standard sample size is 200 tubers per test. More intense
sampling requires more tests on samples of this size. Greater
the amount of tubers in the sample leads to inhibition or difficult interpretation
the results. The procedure can be conveniently used for samples with less than 200 tubers,
If the available smaller quantities of tubers.
-Validation of all detection methods listed below is based on the
testing of samples of the size of 200 tubers.
-The potato Extract described below can also be used to determine the
the originator of the potato ring rot disease. Clavibacter michiganensis
subsp. sepedonicus.
Optional treatment before the preparation of the sample
Incubation of samples) at 2-30 ° C for up to 2 weeks before
testing to support the propagation of all r. solanacearum populations.
(b)), the tubers are washed. Appropriate means of disinfection are used (with the content
chlorine, when the PCR test is to be performed, for the Elimination of pathogenic
DNA) and detergents between each sample. Tubers to dry
on the air. This washing procedure is useful especially in the case when it is in
samples of soil or too if the PCR test is to be performed, or
direct isolation.
3.1.1.1. With a clean and disinfected scalpel or knives
vegetables removes the skin on the heel end of each tuber so that the
visible conductive mesh. Carefully cut a small conductive cores
strands on the heel end. The amount of tissue not involving the vascular bundles are
reduced to a minimum.
Note:
All the (rotting) tubers with suspected symptoms of brown rot can be
party and test separately.
If they are detected when symptoms of výkrojku excision of suspected Brown
rot, a Visual examination of the tubers of tuber of such cut on
the heel end. All incised tubers with suspected symptoms
shall be kept for at least 2 days at room temperature, so that it may be
to zkorkovatění (suberizaci), and then store in a cool place (4-10th.
(C)) for proper quarantine conditions. All tubers including those with
suspicious symptoms should be kept according to annex III.
3.1.1.2. The cores from the heel is placed in unused containers
one use that are closing and/or sealable (in the case of
containers are reused, it must be thoroughly cleaned and
disinfected with chlorine compounds). The best way is to handle
the cores from the heel end immediately. If this is not possible, store in
the container without adding buffer; in a cool place for a maximum of 72 hours or at room
temperature (18-25 ° C) for a maximum of 24 hours.
Process the heel ends shall be processed by one of the following procedures:
and with a sufficient amount of Zalijí) (approximately 40 ml) of extraction buffer
(Appendix 4) and swirling in the Rotary shaker (50 to 100 rpm) for four hours
at a temperature lower than 24 ° C or for 16-24 hours refrigerated, or
(b) the quantity of the tissues with sufficient) (approximately 40 ml) of extraction
buffer (Appendix 4), either in a blender (e.g.. Waring or Ultra Thurax) or
crushing in a sealed disposable maceration bag (e.g. strong
Stomacher or Bioreba polyethylene bag, 150 mm x 250 mm, pickled
radiation) using a rubber tloučku or suitable grinding equipment (eg.
Homex).
Note:
Samples are homogenized using a blender there is a high risk of cross-
contamination of the samples. It is necessary to avoid formation of aerosol or spillage during the
the extraction process. For each sample, apply freshly sterilized
the knife (blade) and containers. When the PCR test is necessary to prevent the transmission of DNA at
containers or grinding apparatus. The PCR test is recommended to use
crushing in disposable bags and use disposable tubes.
3.1.1.3. decant the Supernatant. If it is too cloudy, clear the
either a slow centrifugation (maximum 180 g for 10 minutes at a temperature of
4-10 ° C), or vacuum filtration (40-100 µm), the filter is washed out by the addition of
(10 ml) extraction buffer.
3.1.1.4. The bacterial fraction is concentrated by centrifugation, 7 g for 15
minutes (or 10000 g for 10 minutes) at a temperature of 4-10 ° C and
remove the supernatant without disturbing the Pellet.
3.1.1.5. Pellets are resuspenduje in 1.5 ml pellet buffer (Appendix 4).
Apply 500 µ l for r. solanacearum, 500 µ l for Clavibacter
michiganensis subsp. sepedonicus and 500 µ l for reference purposes. Adds
sterile glycerol to final concentration of 10-25% (v/v) to 500 µ l
the reference aliquot and to the remaining portion of the sample, mix
swirl and stored at a temperature of-16 to-24 ° C (weeks) or at-68 to
-86 ° C (months). Extracts shall be kept during the test at a temperature of
4-10 ° C.
Repeated freezing and thawing is not recommended.
If you need to transport the extract, shall ensure that the transport in a cool box with
delivery within 24 to 48 hours.
3.1.1.6. It is imperative that all positive controls and samples of R.
solanacearum were stored and processed separately to avoid
cross-contamination. It applies to the slides IF and all tests.
3.1.2. Test
Follow the diagrams and descriptions of the tests and optimised protocols in the
the relevant appendices:
Selective isolation (see section 6.1.4).
The if test (see section 6.1.5.)
The PCR tests (see section 6.1.6).
The FISH test (see section 6.1.7.)
ELISA tests (see section 6.1.8.)
The bioassay test (see section 6.1.9.)
3.2. Detailed methods for detection and identification of r. solanacearum in
samples of asymptomatic potato, tomato or other
host plants
3.2.1. Preparation of samples
Note:
For the purposes of detection of latent r. solanacearum populations it is recommended that
test composite samples. The procedure can be used for mixed samples of
the number of up to 200 stem parts. If the survey is carried out, it should be
based on a statistically representative sample of the investigated plant
of the population.
3.2.1.1. In a closed sterile container is placed 1-2 cm long pieces
the stems according to the following procedure for sampling:
Tomato seedlings: with a clean disinfected knife
Removes a 1 cm from the base of each stem, just above the soil level.
Plants from the field or glasshouse: with a clean disinfected knife removes the
the lowermost side shoot from each plant just above the uříznutý
by joining with the main stem. Remove the lowermost 1 cm segment from
each stem. Other host plants: a clean disinfected knife
or pruning shears, remove a 1 cm segment from the base of each
the stem just above the soil level. In the case of the Eggplant (s. dulcamara) or
other host plants growing in the water are taken 1-2 cm parts
of the stem under water or rhizomes with roots.
When sampling a particular area it is recommended to test statistically
a representative sample of at least 10 plants per sampling point 1
plevelnou for each potential host plant. Detection
the pathogen will be most reliable during late spring, summer and fall,
Although natural infections can be detected throughout the year for multiannual
Solanum dulcamara growing in watercourses. Among the known host
the plants include weed plants potato, Solanum dulcamara, S.
nigrum, Datura stramonium and other members of the family Solanaceae. Other
host plants are plants of the genus Pelargonium spp. and
Portulaca oleracea. Some European weed, which can host
the population of r. solanacearum biovar 2/Race 3 in the roots and/or oddencích for
specific 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:
At this stage it is possible to perform a Visual investigation of internal symptoms
(coloring of the blood vessels or bacterial ooze). Any part of the stem with the symptoms
aside and tested separately (see section 2).
3.2.1.2. The part of the stem is disinfected with ethanol shortly 70% and immediately Blot dry
with tissue paper. Then the process part of the stem of one of the following
procedures:
zalijí is part of) a sufficient quantity (approximately 40 ml) of extraction
buffer (Appendix 4) and shake on the Rotary shaker (50 to 100 rpm) 4
hours at a temperature of 24oC or 16 to 24 hours refrigerated,
or
(b)) in the process immediately by crushing hard maceration bag (e.g.. Stomacher lab-Blender
or Bioreba) with an appropriate amount of extraction buffer (Appendix 4)
using a rubber tloučku or suitable grinding equipment (eg. Homex).
If this is not possible, a part of the stems in a cool place for up to 72 hours
or at room temperature (18-25 ° c) for 24 hours.
3.2.1.3. once established, to take a 15-minute, decant the supernatant.
3.2.1.4. the further purification of the extract or concentration of bacterial
the fraction is usually not necessary, but can be performed by filtration and/or
centrifugation as described in section 3.1.1.3. to be applied.
3.2.1.5. Pure or concentrated sample extract will be divided into two
the same parts. One part shall be tested at a temperature of 4-10 ° C and the second part is
stored in the 10-25% (v/v) sterile glycerol at-16 to-24th.
C (weeks) or at-68 to-86oC (month) for any further testing.
3.2.2. Testing
Follow the diagrams and descriptions of the tests and optimised protocols in the
the relevant appendices:
Selective isolation (see section 6.1.4).
The IF test (see section 6.1.5.)
The PCR tests (see section 6.1.6).
The FISH test (see section 6.1.7.)
ELISA tests (see section 6.1.8.)
The bioassay test (see section 6.1.9.)
4. Detection and identification of the causative agent of brown rot in the water
4.1 flowchart for the detection and identification of r. solanacearum in
water
-----
(1) see section 4.2.1., which lists the recommended sampling procedures.
(2) method of the concentration of the pathogen are described in section 4.2.1.
Increases the concentration of the population as a pathogen, so competitive
saprophytic bacteria and is recommended only if does not prevent
izolačnímu test.
(3) the Selective isolation test is described in section 6.1.4.
(4) Bioassay is described in section 6.1.9.
(5) the enrichment-PCR Methods are described in the additional section. and
6.1.6.
(6) the enrichment-ELISA test Methods are described in the additional section. and
6.1.8.
(7) the typical colony morphology is described in section 2.3. d.
(8) the Cultivation can fail due to competition or inhibition
saprophytic bacteria. If it is suspected that the United saprofytická
the population will affect the reliability of the insulation, the insulation tests are repeated after
diluting the sample with sterile water.
(9) reliable identification of suspected pure cultures of r. solanacearum
is achieved by the tests described in section 6.2.
(10) the pathogenicity Test is described in section 6.3.
4.2. Methods for detection and identification of r. solanacearum in water
The principle of the
The validated detection scheme described in this section is applicable to the
pathogen detection in samples of surface water and can also be used to
testing of samples of potato processing waste water and waste water.
The expected sensitivity of detection, however, vary depending on the substrate.
Sensitivity of the test is affected by the selective isolation of populations
certain competing saprophytic bacteria, which is generally much more in
waste water from the potato processing and sewage effluents than in surface water.
While the below diagram assumes the detection of about 103 cells/liter
the surface of the water, the sensitivity of detection in the waste water of processing potato
and waste water is likely to be substantially lower. For this reason,
It is recommended to test the waste water after performing any cleaning
procedure (e.g. sedimentation or filtration), which will reduce
saprophytic bacterial populations. Limitations of sensitivity test
the schema should be taken into account when assessing the reliability of the
the case of getting negative results. Whereas this scheme
successfully used to map the presence or absence of the pathogen in the
surface water, it is necessary to realize its limitations when used for
similar mapping in the waste water from the potato processing and sewage
the water.
4.2.1. Preparation of samples
Note:
-Detection of r. solanacearum in surface water is most reliable in
during late spring, summer and fall, when water temperature exceeds 15
St. (C).
-Repeated sampling at different times during the above period on
designated sampling points will increase the reliability of the findings by reducing
the effects of fluctuations in weather conditions.
-Due to heavy rainfall and geography of water flow may be significant
dilution and thus disguising the presence of the pathogen.
-Water samples shall be collected in the vicinity of host plants, if
are present.
4.2.1.1. On the selected sampling locations samples of water into the
sterile tubes or bottles on one use, preferably 30 cm
beneath the surface and 2 m from the Bank. When the sampling of waste water processing
potatoes and waste water samples from the site of waste water outlet.
Recommended sample size is 500 ml. smaller is preferred
size, it is recommended to remove samples of at least 3 times for each
sample site, each of which will contain 2 recurring incremental samples
at least 30 ml. Intensive mapping shall be at least
3 sampling sites on each of 3 km of the water flow and sample also
tributaries.
4.2.1.2. the samples shall be transported in a cool and dark place (4-10 ° C) and test
within 24 hours.
4.2.1.3. If needed, can be made of the concentration of bacterial
the fractions using the following methods:
and) centrifuge 30-50 ml sub-samples at 10 000 g for 10 minutes
(or 7000 g for 15 minutes) preferably at 4-10 C, st.
the supernatant liquid above the sediment and resuspenduje with pellets in 1 ml of buffer
(Appendix 4).
(b)) shall be carried out through the membrane filtration (0.45 µm pore size minimum)
followed by washing the filter in a 5-10 ml pellet buffer and interception
the filtrate. This method is suitable for large volumes of water, which contain
a small number of Saprophytes can be increased.
Concentration is usually not recommended for samples of potato processing
or waste water, because of increased populations of competing
saprophytic bacteria inhibit the detection of Ralstonia solanacearum.
4.2.2. Test
See the flow chart and description of the tests in the relevant appendices.
5. Detection and identification of the causative agent of brown rot in the soil
5.1. Flowchart for the detection and identification of r. solanacearum in
the soil
-----
(1) see section 5.2.1. where are the recommended sampling procedures
(2) Selective isolation test is described in section 6.1.4.
(3) the Enrichment of the PCR tests are described in the additional section. and 6.1.6.
(4) Bioassay is described in section 6.1.9.
(5) the typical colony morphology is described in section 2.3. d.
(6) the Cultivation may fail for reasons of competition or inhibition
saprophytic bacteria. It is suspected that the influence of the saprophytic
the population on the reliability of isolation, repeated tests of the selective isolation
After a further dilution of the sample.
(7) reliable identification of suspected pure cultures of r. solanacearum
is achieved by the tests described in section 6.2.
(8) the pathogenicity Test is described in section 6.3.
5.2. Methods for detection and identification of r. solanacearum in soil
The principles of the
Valid detection scheme described in this section is applicable to the detection of
the pathogen in the soil samples, but can also be used to test
samples of the solid waste in the potato processing or sewage sludge from water.
However, it is to be noted that these methods are not sensitive enough to
guarantee detection of low or irregularly dispersed populations
Ralstonia solanacearum that may occur in naturally
infected samples of these substrates.
When evaluating the reliability of all obtained negative results and also
When compiling the reports that determine the presence of the pathogen in the soil or
sludge is the need to take into account the limited sensitivity of this test
the schema. The most reliable test for the presence of the pathogen in the soil of the field is
plant a susceptible host plants and its monitor, if it is not
infected, but also when using this method escapes the low degree of contamination
attention.
5.2.1. Preparation of samples
5.2.1.1. A sampling of field soil should follow the standard principles
used for nematode sampling. One sample shall be collected up to 0.5
1 kg of soil from the 60 seats on the surface from a depth of 0.3 ha 10-20 cm., if
the suspected presence of the pathogen, zvyší is the number of collection points to 120 from
Desktop 0.3 ha. Before testing the samples shall be kept at a temperature of 12-15
St. C. Kal potato processing and sewage samples colors with
Assembly of a total of 1 kg of the places representing the total volume
the sludge to be tested. Before testing each sample, mix well.
5.2.1.2. The incremental samples 10-25 g of soil or sludge scatters Rotary
shaking (250 RPM) in 60-150 ml of extraction buffer (Appendix 4) after
for 2 hours. If necessary, add 0.02% sterile Tween-20 and 10-20 g
sterile gravel may assist dispersion.
5.2.1.3. During testing, it keeps the suspension in a temperature of 4 ° C.
5.2.2. Test
Flow chart and description of the tests are in the Appendix.
6. Optimized protocols for the detection and identification of r. solanacearum
6.1. Diagnostic and detection tests
6.1.1. Test on the discharge of mucus from the stem
The presence of r. solanacearum in wilting potato stalks,
tomato or other host plants may show the following
a simple test of probable occurrence of: cutting off the stem just above the
the level of the country. Cut the stem delves into the tube with clean water.
Monitors, that after a few minutes the characteristic
spontaneously flowing threads of bacterial slime from the přeříznutých vascular
volumes.
6.1.2. Test for the presence of poly-beta-hydroxybutyrate granules
1. Prepare a smear of bacterial ooze from infected tissue or
of the 48-hour culture on YPGA or SPA (Appendix 2)
a microscope slide.
2. Prepare a smear of the positive control strain of biovar 2 R.
solanacearum and, where applicable, a negative control smear of a known PHB
negative sp.
3. allow to dry Smears and the bottom surface of each slide
quickly stretched over the flame to the smears they.
4. Sections stained with either Nile blue or Sudan black and performs
microscopic observation according to the following description:
Nile blue test:
and Both slides) zakápnou 1% aqueous Nile blue and
incubate for 10 minutes at a temperature of 55 ° C.
b) removes dye solution. Briefly Rinse gently running water from the
the tap. Excess water with tissue paper supernatant.
(c) Prepare the zakápne) 8% aqueous acetic acid solution and allow to
incubate for 1 minute at room temperature.
d) Briefly Rinse gently running water from the tap.
Excess water with tissue paper supernatant.
e) moistened with a drop of water again and coverslip.
(f) Examine the stained smear with) on an epifluorescence microscope at 450 nm
under oil or water immersion up to 1000 x 600 x magnification with the use of
lens for oil or water imersi.
g) Watches that the PHB granules bright orange
fluorescence. Also observed in transmitted normal light, whether
the granules are intracellular and that cell morphology is typical of R.
solanacearum.
Sudan black test:
and all of the slides) Zakápnou 0.3% Sudan black B solution in 70% of the
ethanol and incubate for 10 minutes at room temperature.
b) removes dye solution and rinse briefly in gently running tap water
from the tap and apply suction to excess water with tissue paper.
(c)) the slides briefly in xylol delves and SAP, with tissue paper.
Caution: Xylol is harmful, it is necessary to comply with the necessary
the safety measures and work in a fume cupboard.
d) slides zakápnou 0.5% (w/v) aqueous safraninu and leave
10 seconds at room temperature.
Caution: safranin is harmful, it is necessary to comply with the necessary
the safety measures and work in a fume cupboard.
e) Slides with gently running water from the tap, then the
excess water with tissue paper and a coverslip.
(f) Examine the stained smear with) microscope with transmitted light under the
oil immersion at a magnification of 1 000x.us.
(g)) is looking for the blue-black coloring PHB granules in the cells of r. solanacearum with
pink-coloured walls of the cells.
6.1.3. Serological agglutination tests
Agglutination of r. solanacearum cells in bacterial ooze or in extracts
of the tissues with the symptoms is best observable using validated
antibodies (see. Appendix 3) labelled relevant dyed
značkovači, for example. the red cells of Staphylococcus aureus or
dyed latex particles. When using commercially available
equipment (see Appendix 3), it shall proceed according to the instructions of the manufacturer. Otherwise,
the procedure is the following:
and Mixed with drops of suspension) labeled antibody and bacterial slime
(approximately 5 µ l of each substance) on the window of the test víceokénkového
the slides.
b) prepare positive and negative control samples of the use of suspensions
r. solanacearum biovar 2 and a heterologous strain.
(c)), whether Watching in positive samples after gentle mixing for
15 seconds of agglutination.
6.1.4. Selective isolation
6.1.4.1. The vaccination on fertile ground
Note:
Before this method is applied for the first time, carry out a preliminary test to
to ensure reproducible detection of 103 to 104 units that make up
colonies of r. solanacearum per ml added to sample extracts which
previously tested with negative result.
Apply duly validated selective growth medium, for example. SMSA (as amended by the
Elphinstone et al., 1996; see Appendix 2).
Resolution of r. solanacearum from other bacteria capable of the soil
growth requires a lot of attention. Furthermore, colonies of r. solanacearum may have
atypical morphology if plates are overcrowded or are
also present antagonistic bacteria. It is suspected that the competition
or inhibition, the sample should be re-tested using a different test.
When using freshly prepared extracts from samples can expect
the sensitivity of detection of this method will be very high. The method is, however,
also applicable for the extracts which have been stored under glycerol at
from-68 to-86 ° C.
Positive controls, prepare decimal dilutions of the suspension as 106
cfu/ml of a virulent biovar 2 strain of r. solanacearum (e.g.. NCPPB 4156 =
PD 2762 = CFBP 3857). To avoid possible contamination, prepare
positive control samples completely separately from the samples for testing. U
each newly prepared batch of selective medium should be
its use for routine testing of the samples tested her
suitability for culture of the pathogen. Control material is tested the same
manner as the sample (s).
1. Perform a dilution in order to ensure that all population
saprophytic bacteria were excluded. Apply 50-100 µ l of the extract
sample plate for each dilution.
2. The dishes are incubated at a temperature of 28 ° C After 48 hours.
check their condition and then inspections are carried out daily for 6 days.
Typical colonies of r. solanacearum on the SMSA medium are milky white,
flat, irregular and fluidal and after 3 days incubation develop pink
When the blood-red coloring in the middle with the internal strips or spirals.
Note:
Sometimes form on this medium atypical colonies of r. solanacearum. Can
be small, round, entirely red and non-fluidal or only partially fluidal
and, therefore, difficult to distinguish from saprophytic bacteria forming
the colony.
3. Projected r. solanacearum colonies are spread or vaccinate
method of dilution on fertile ground to obtain isolated
the colony (see Appendix 2).
4. Culture is kept in the short-term in sterile water (pH 6-8, without
chlorine) at room temperature in the dark or in the long term in an appropriate
protective medium at-68 to-86oC or freeze-dried.
5. Identify suspicious cultures (see section 6.2), and test
pathogenicity (see section 6.3).
Interpretation of test results, vaccination on selective media. Test
vaccination on selective media is negative, if not after 6 days
selective bacteria or are not found any suspicious colonies
typical of r. solanacearum, and provided that no inhibition
other bacteria and are found in control samples of typical
colonies of r. solanacearum.
Test on selective media is positive if they are isolated
suspicious colonies of r. solanacearum.
additional. Enrichment tests
Apply validated enrichment media, eg. modified broth
Wilbrink (see Appendix 2).
This procedure can be used for selective stocks zoom R.
solanacearum in extracts from samples and increase sensitivity of detection. This
will and the dilution potential inhibitors of PCR (1:100).
Enrichment of r. solanacearum may, however, be unsuccessful for reasons of competition
or antagonism by saprophytic organisms which are often at the same time
also monetized. For this reason, it may be the isolation of r. solanacearum from
enriched culture difficult. In addition, because it can lead to the development of
populations of serologically related Saprophytes can be increased, for the ELISA test is recommended
used instead of the specific monoclonal Polyclonal Antibodies
the antibodies.
1. For enrichment-PCR is transferred 100 ul of sample extract into 10
ml enriched broth (Appendix 2), which is to prepare in advance
sterile test tubes or flasks. For enrichment-ELISA test may be
used a larger share of sample extract to broth (e.g. 100 ul in 1.0 ml
enriched broth).
2. Incubate for 72 hours at a temperature of 27 to 30 ° c in shaking culture or static
culture with caps, which will allow the aeration.
3. Before starting the ELISA or PCR content, mix well.
4. With enriched bujónem is treated in the same way as the samples in the above
those tests.
Note:
If inhibition of enrichment of r. solanacearum due to high
the concentration of competing saprophytic bacteria, can be achieved through better
the results of the enrichment of sample extracts before any centrifugation
or other procedures increase the concentration.
6.1.5. IF Test
Procedure
Use the IF test as the principal screening test is recommended because
proven reliability in achieving the required thresholds.
If the IF test as the main, and the result is positive, the IF test
must be used as the second screening test, PCR or isolation test
The FISH. When the IF test is used as the second screening test and the
positive, it is necessary to perform additional testing according to the flow scheme
that analysis was complete.
Note:
Apply validated source of antibodies to r. solanacearum. It is recommended that
determine the titre of antibodies for every new batch. The titre is defined as the
the highest dilution at which optimum reaction occurs when testing a
a suspension containing 105 to 106 cells/ml of the homologous strain of r.
solanacearum and using an appropriate fluorescein
isothiocyanate (FITC) in accordance with the manufacturer's recommendations. The validated
Polyclonal Antisera have all the titre of at least 1:2 000. During
testing, the antibodies should be used at a working dilution (s) close
or the same as the titre.
The test should be performed on freshly prepared sample extracts of samples.
If necessary, it can be successfully performed on extracts
at-68 to-86 ° c under glycerol. Glycerol can be from the sample
removed by addition of 1 ml of buffer (Appendix 4), re-centrifugation
at 7000 g and re-suspension in the same amount of pellet buffer. Often it
It is not necessary, especially if samples are fixed to the slides.
Prepare separate positive control slides of the homologous strain
or any other reference strain of r. solanacearum is suspended in the
potato extract, as specified in Appendix 3 (B), and, optionally, in the
buffer.
Naturally infected tissue (maintained by lyophilization or
freezing at a temperature of-16 to 24 ° c) should be used
as a parallel control on the same slide slide.
As negative controls are applied aliquot parts of sample extracts,
which previously tested negative.
Standardized materials for the positive and the negative control, which can be
use for this test are listed in Appendix 3. Shall apply
microscopic slides with more cutouts, if possible with 10 Windows with a diameter
at least 6 mm.
Test control material shall be carried out in the same way as the assay samples.
6.1.5.1. Prepare slides to test one of the following
procedures:
I) for pellets with relatively little starch sediment:
Pour a measured standard volume (15 µ l is suitable for the diameter of the
6 mm window-for larger Windows is greater in proportion)
the dilution of the resuspended potato pellet in 1/100 to the first window.
Subsequently, the same amount of undiluted Pellet (1/1) to the
the remaining wells in the series. The second row can be used as double
or for a second sample, as shown in the figure. 1.
II) for other pellets:
Prepare decimal dilutions (1/10, 1/100) of the resuspended pellet in
Pellet buffer. Pour a measured standard volume (15 l is µ
appropriate for 6 mm window diameter-for larger Windows volume in
the ratio) of the resuspended pellet and each dilution to the series
Wells. The second row can be used as a reserve or for a second
the pattern, as shown in Fig. 2.
6.1.5.2. Dry the droplets at ambient temperature or heating to a temperature
40 to 45 ° c. The bacterial cells to the slide either by heating for (15
minutes at a temperature of 60 ° c), flame, 95% ethanol or according to the
specific instructions from the supplier of antibodies.
If necessary, fixed slides may be stored in the frozen
in the dry state of boxing for as short a period (maximum 3 months) before the
additional testing.
6.1.5.3. IF Procedure
I) according to test slide preparation in the paragraph. 5.1. i): prepare a set of
twofold dilution series. The first hole should be 1/2 of the titre (T/2), the other
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 the paragraph. 5.1. ii): prepare
the working dilution (BC) of the antibody in IF buffer. The working dilution affects
accuracy.
Figure 1. Preparation of the test slide according to paragraph 2(a). 6.1.5.1. i) and even 6.1.5.3.)
Figure 2. Preparation of the test slide according to paragraph 2(a). 6.1.5.1. ii), and
6.1.5.3. ii)
1. The slides will be arranged on moist tissue paper. Each
the test window completely cover the dilution of antibodies. The quantity of
antibodies to each window must be at least the same as the quantity
used the extract.
If specific instructions are not available from the supplier of antibodies,
proceed as follows:
2. incubate the slides on moist paper under a cover for 30
minutes at room temperature (18-25 ° C).
3. Shaken off drops from all the slides and this carefully
Rinse the buffer IF. Washed by submerging for 5 minutes in IF buffer
Tween (Appendix 4) and subsequently in IF buffer. It is necessary to prevent
aerosol or droplet transfer that could cause mutual
contamination. Carefully remove excess moisture with a soft blow dry.
4. place the Slides on moist paper. Cover the test Windows with the
the dilution of FITC conjugate, fixing the titre. The amount of conjugate
applied to the wells must be the same as the amount used antibodies.
5. incubate the Slides on moist paper covered for 30 minutes at
room temperature (18-25 ° C).
6. Shaken off drops of conjugate from the slides. Crystal rinse and wash
as before (3). Carefully remove excess moisture.
7. Pipette 5-10 ul of 0, 1 m phosphate buffer solution with glycerol (Appendix
4) or commercial cover of fluids and attach to each cover
slide.
6.1.5.4. The evaluation of the IF test
1. Viewing the slides on an epifluorescence microscope with a test with
filters suitable for excitation of FITC, under oil or water immersion at
magnification up to 1 000x.us. Examine the window in two orthogonal
diameters and around the perimeter. For samples with no or a small number of cells
examines at least 40 microscope fields.
First, check the positive control sample. The cells must be clearly
fluorescent and completely stained in the specified the titre of the antibodies or
the working dilution. If the colours of the derogation, the IF test must be
repeated (paragraph 6.1.5).
2. Observe the bright Fluorescing cells with characteristic morphology
R. solanacearum in the test Windows of the test slides. The intensity of fluorescence
must be when compared to the positive control strain at the same dilution
the antibodies the same or better. Cells with incomplete staining or weak
fluorescence cannot be taken into account.
From any contamination is suspected the test must be repeated. It is
can happen when all slides in a group show positive cells
due to the contamination of buffer or in finding positive cells (outside the window
slides) on the surface of the slides.
3. There are several problems inherent to the accuracy
the immunofluorescence test. In the background of potato heel and parts
the stem may occur populations of Fluorescing cells with
atypical morphology and cross reacting saprophytic bacteria with
size and morphology similar to r. solanacearum.
4. Take into account only Fluorescing cells with typical size and
morphology at the titre or working dilution of the antibodies as in section
6.1.5.3.
5. Interpretation of test results IF:
even) when bright Fluorescing cells with characteristic morphology are found,
estimates of the average number of typical cells per microscope field and
calculate the number of typical cells per ml of resuspended Pellet (Appendix
5).
IF the result is positive for samples, where is the number of typical cells per 1 ml
the resuspended pellet at least 5 x 103. The sample is considered to
potentially infected and required further testing.
(ii) IF test) result is negative for samples that contain less than
5 x 103 cells per ml of resuspended pellet and the sample is considered to
negative. Further testing is not necessary.
6.1.6. PCR Tests
The principles of the
If the PCR test as the principal screening test and the result is
positive, must be compulsorily made second screening test
isolation or IF. If the PCR is used as the second screening test and the
is positive, it is necessary to perform additional testing according to the flow scheme
the diagram to the analysis was complete.
The use of this method in its entirety as the principal screening test
It is recommended only if the required specialized expertise.
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. The achievement of
maximum sensitivity and accuracy in all laboratories may require
the optimization attempts. Apply approved reagents and PCR Protocols (see
Appendix 6). If possible, select a method, with the internal control.
It is necessary to use appropriate security measures to prevent contamination of the
a sample of the target DNA. The PCR test should be carried out by experienced technicians, in
dedicated molecular biology laboratories, in order to
minimize the possibility of contamination with target DNA.
With negative controls (during the extraction of DNA and PCR) should always
treated as final samples to make it clear, if there was a
transfer of DNA.
The PCR test should include the following negative controls:
-sample extract that previously tested negative for r. solanacearum with
a negative result,
-buffer controls used for extracting the bacterium and the DNA from the sample,
-The PCR reaction mix.
The following should be included in the positive control:
-an aliquot of resuspended pellets to which has been added to R.
solanacearum (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 the DNA in the PCR also extracted from the positive
control samples.
To prevent possible contamination, prepare positive controls
in an environment that is separate from the samples that will be tested.
Extracts of samples should be free from soil. In the case of
the use of PCR to prepare extracts of umytých potatoes.
Standardized materials for the positive and the negative control, which can be
use for this test are listed in Appendix 3.
6.1.6.1. the methods of purification of DNA
Use the above described positive and negative control samples (see.
Appendix 3).
Test control material shall be carried out in the same way as the assay samples.
The purification of target DNA from complex sample substrates are available
different methods for inhibitors of PCR and other enzymatic reactions and
concentrating target DNA in the sample. The following method has been
optimized for use with approved PCR methods referred to in
Appendix 6.
and according to Patrick's Method) (2000)
1. Pour 220 µ l lyzátového buffer (100 mm NaCl, 10 mM Tris-HCl
(pH 8.0), 1 mM EDTA (pH 8.0)) in 1.5 ml Eppendorf tube Eppendorfovy.
2. Add 100 µ l sample extract and Microtest tube is placed in the
a heating block or water bath with a temperature of 95 ° c for 10 min.
3. Insert the Microtest tube on 5 min.
4. Add 80 µ l stock solution 50 mg lysozyme lysozyme (1 ml/10
mm TRIS HCL, pH 8.0) and incubate at 37 ° c for 30 minutes.
5. Add 220 µ l of the solution Easy DNA (R) and (Invitrogen), mix well
shaking and incubated at 65 ° c for 30 minutes.
6. Add 100 µ l of the solution Easy DNA (R) (B) (Invitrogen), thoroughly
mix thoroughly by shaking, if the sample begins to be uniformly viscous.
7. Add 500 µ l of chloroform and mix until the viscosity decreases and the mixture
it becomes homogeneous.
8. To separate phases and form the interphase with the mixture in the centrifuge for 15 000
g for 20 min at 4 ° c.
9. the upper phase into a fresh Eppendorf tube.
10. Add 1 ml of 100% ethanol (-20 ° c), mix thoroughly by shaking and briefly
incubate on ice for 10 min.
11. Centrifuge at 15000 g for 20 min at 4 ° c and
Remove ethanol from Pellet.
12. Add 500 µ l 80% ethanol (-20 ° c) and mix překlápěním
Eppendorf tube.
13. Centrifuge at 15000 g for 10 minutes at 4 ° c,
preserves the pellets and remove ethanol.
14. Pellets are allowed to dry in air or in BOTTOM of evaporator.
15. the resuspenduje 100 Pellets in µ l of sterile UPW and leave to stand in the
room temperature for at least 20 minutes.
16. Store at-20 ° c until use in PCR.
17. any white precipitate is removed by centrifugation. 5 µ l of supernatant
containing DNA for the PCR test is used.
(b)) other methods
Other methods of DNA extraction, for example. Qiagen DNeasy Plant Kit, could
use, if proved, that for the purification of DNA from control
samples containing 103 to 104 pathogen cells per ml as well
effective.
6.1.6.2. The PCR.
1. Prepare test samples and controls for PCR according to the approved
protocols (6.1.6). Prepare ten-fold dilutions of the sample, extract the DNA
(1:10 in sterile water).
2. Prepare the appropriate PCR reaction mix in the environment in which
There is no contamination, according to the published protocols (Appendix 6). If
possible, it is recommended to use a multiplex PCR protocol that also
includes internal PCR Protocol.
3. Into sterile PCR vials add 5 µ l of the extract DNA at 25 µ l
The PCR reaction by the PCR Protocols (see Appendix 6).
4. Add negative control samples containing only to the reaction mixture
PCR and adds the same source of UPW as was the one that was used in the mixture
PCR instead of the sample.
5. The PCR tubes are placed in the same thermal cycler which was used
during the initial testing, and runs the appropriately optimised PCR programme
(Appendix 6).
6.1.6.3. the analysis of the PCR product
1. the Amplikony shall be allocated in agarose gel electrophoresis. Spotted with
at least 12 µ l mixture of amplified DNA from each sample with 3 µ l
loading buffer (Appendix 6) in 2.0% (w/v) agarose gel in
TRIS-acetate-EDTA (TAE) buffer (Appendix 6) in 5-8 V/cm shall apply.
the appropriate DNA marker, for example. (ladder) 100 bp.
2. Detect DNA bands by staining in ethidium bromide (0.5 mg/l) after
for 30-60 minutes for the use of appropriate security measures for the
handling this mutagen.
3. In obarveném and UV (short wavelength e.g. 302 nm) observe gel
looking for amplified PCR products of the expected size and the result
shall be documented.
4. For all new findings/cases, checks the authenticity of the PCR Amplicon
by performing restriction enzyme analysis in the remaining sample
The Amplified DNA by incubating at the optimum temperature and time the same
the cleavage enzyme and buffer (see Appendix 6). The digested fragments is
splits the electrophoresis in agarose gel and observe the characteristic
polymorphism pattern under UV light after ethidium bromide staining
and compares with the undigested and digested positive control.
Interpretation of results of PCR
The PCR test is negative if specific PCR Amplicon for R.
solanacearum expected size is not detected in the sample, but
is detected for all positive control samples (in the case of
multiple PCR with primers specific for internal control
plant: a second PCR product of expected size must be amplified
with the sample). The PCR test is positive if it is detected
for the specific PCR Amplicon of expected size of r. solanacearum and
(or) the model, provided that it is not amplified in any sample
the negative control. Reliable confirmation of a positive result can also be
get a repeat test with a second set of PCR primers Appendix 6).
Note:
May be suspected if the inhibition of the PCR obtained the expected
Amplicon from the positive control sample containing r. solanacearum in
the water, while a sample of r. solanacearum positive controls in the
potato extract negative results are obtained. In multiplex
PCR Protocols with internal PCR controls inhibition reaction
If there is no from two amplicons are obtained.
If the expected Amplicon is obtained from one or more samples
negative controls, contamination is suspected.
6.1.7. FISH Test
The principle of the
When used as the first screening test and the FISH test is positive, the
It must be as a second compulsory screening test performed or IF isolation
test. When the FISH test performs as the second screening test and the
a positive diagnosis is necessary to complete more testing in the
flow diagram.
Note:
Use an approved oligosondy r. solanacearum-specific
(Appendix 7). Initial testing with this method should permit
reproducible detection of at least 103-104 cells of r. solanacearum per ml
added to extracts from the sample that were previously tested with
a negative result. The following procedure should preferably be
made with freshly prepared sample extract but can also be successfully
performed on sample extract that has been stored under glycerol at-16 to
-24 ° c or-68 to-86 ° c.
As negative control applies an aliquot part of the extract of the sample,
that previously tested negative for r. solanacearum with negative result.
As a positive control, prepare suspensions containing 105 to 106
cells per ml of r. solanacearum biovar 2 (strain NCPPB 4156 for example = PD 2762
= CFBP 3857, see Appendix 3) in 0, 01 m phosphate buffer (PB) of 3-5 days
culture. Prepare separate positive control slides
samples of the homologous strain or any other reference strain of R.
solanacearum, suspended in potato extract, as specified in the
Appendix 3 (B). the use of FITC the FITC-labelled probe provides
control the process of hybridization, because turns all eubakterie
present in the sample.
Standardized positive and negative control material
used in this test is given in Appendix 3, point and Test control.
the material shall be made in the same manner as the sample (s).
6.1.7.1. potato extract Fixation
The following protocol is based on the Wullingse et al. (1998).
1 prepare fixative solution (see Appendix 7).
2. Pour 100 µ l of each sample extract into Eppendorfovy
Eppendorf tube and centrifuge for 7 minutes at 7000 g.
3. Remove the supernatant and dissolve the pellets in the 200 µ l fixation
the solution prepared max. 24 hours in advance. Shake and incubate for 1
an hour in the refrigerator.
4. Centrifuge for 7 minutes at 7000 g, remove the supernatant and
resuspenduje with pellets in the 75 µ l 0, 01 m PB (see Appendix 7).
5. Action 16 µ l fixed suspension on net 10 window-type glass, as
shown in Figure 7.1, with 2 different samples on one
slide, undiluted and diluted 1:100 with the use of 10 µ l (in 0.01 M PB).
The remaining sample solution (49 µ l) can be stored at-20 ° c
Add 1 volume of 96% ethanol. In the event that the FISH
the method again, remove the ethanol by centrifugation and add the same
the quantity of 0.01 PB (mix by vortexing).
6. allow Slides to dry in the air (or in the dryer when the slides
a temperature of 37 ° c) and fixed above the flame.
At this stage it is possible to interrupt the procedure and continue further hybridization
the day. The slides should be stored protected from dust and dry at
room temperature.
6.1.7.2. Hybridization
1. Dehydratují the cells in a gradual series of ethanol 50%, 80% and 96%,
each time for 1 minute. Dry air in the holder.
2. Prepare a moist incubation Chamber by covering the bottom of an airtight box
cloth or filter paper sodden hybridisation mixture (1 x
hybmix, Appendix 7). Before incubation heats up box in the hybridisation oven at
a temperature of 45 ° c for at least 10 minutes.
3. Apply 10 µ l of hybridisation solution (Appendix 7) to 8 wells
(window 1, 2, 4, 5, 6, 7, 9 and 10; see fig. 7.1) on each slide,
While the two central Windows (3 and 8) is empty.
4. Attached the cover slides (24 x 24 mm) on the first and last 4 Windows
and so, under them without trapping air. The slides are placed in advance
heated moist Chamber and allow it to run through the process of hybridization for 5
hours in the oven at 45 ° c in the dark.
5. Prepare three beakers containing 1 l of water with molecular quality
(Milli Q), 1 l of 1 x hybmix (334 ml 3 x hybmix and 666 ml of water with molecular
quality) and 1 l of 1/8 x hybmix (42 ml 3 x hybmix and 958 ml of water with
Molecular quality). Let incubate in a water bath at a temperature of 45
° C.
6. Removed the cover slides and slides are placed in the holder
slides.
7. Wash the excess sample incubation for 15 minutes in the beaker with 1
x hybmix at 45 ° C.
8. slide-Holder moves into wash solution 1/8 hybmix and allow
They incubate for a further 15 minutes.
9. The slides briefly immersed in UPW (for example. Milli Q water) and put on the
filter paper. Remove excess moisture by covering the surface with light
filter paper. Pipette 5-10 µ l of the solution (e.g. covering.
Vectashield, Vecta Laboratories, CA, USA or similar) to each
the window and the whole slide covers a large a glass coverslip (24 x 60 mm).
6.1.7.3. FISH test
1. Slides immediately viewing under a microscope suitable for
epifluorescence microscopy, magnification x 630 or 1000 x under
oil immersion. With a filter suitable for fluorescein isothiocyanate (FITC)
eubacterial cells (including most gram-negative cells) in the
the sample are stained fluorescent green. Using a filter for
tetramethylrhodamine-5-isothiocyanate stained cells of r. solanacearum
Appear fluorescent red. Compares the cell morphology with
the morphology of the positive control samples. The cells must be clearly
fluorescent and completely stained. The FISH test (paragraph 6.1.7) must be
repeated, if the coloring of the derogation. Viewing window across
two diameters at right angles and around the perimeter. In the case of samples, where there are
observed no or little cells, observed at least 40 fields
the microscope.
2. Looking for the bright Fluorescing cells with characteristic morphology are found,
for R. solanacearum in the test Windows of the test slides. The intensity of the
the fluorescence must match or be better than positive
the control strain. Cells that are not fully colored or exhibit
weak fluorescence, shall not be taken into account.
3. any contamination is suspected the test must be repeated. It
may occur in the case when all slides in a batch show positive
the cells due to the contamination of buffer or if positive cells are
found (outside of the slide Windows) on the cover slides.
4. the specificity of the test FISH carries with it a number of problems. Is
likely that the tissues of potato tubers and stem
potato occurs when cells on the populations of fluorescent
background with atypical morphology and cross reacting saprophytic to
bacteria size and morphology similar to r. solanacearum, and when
much less frequently than the IF test.
5. Account shall be taken only Fluorescing cells with typical size and
morphology.
6. Interpretation of the results of the test FISH
even the FISH test) results are valid if they are using a FITC filter
bright green fluorescent cells with size and morphology typical of
R. solanacearum and using the rhodamine filter bright red
fluorescent cells are observed in all positive controls and are not
observed in any of the negative controls. If present it clearly
Fluorescing cells with characteristic morphology are found, estimate the average number of
typical cells per microscope field and calculate the number of typical cells
in 1 ml of resuspended Pellet (Appendix 4). Samples that contain
at least 5 × 103 typical cells per ml of resuspended Pellet,
considered probably infected. Further testing is required. Samples,
that contain less than 5 × 103 typical cells per ml of resuspended
the pellets are considered as negative.
(ii)) the FISH test is negative if using the rhodamine
the filter is not observed bright red fluorescent cells with size
and morphology typical of r. solanacearum are these typical
bright red fluorescent cells using the rhodamine filter
observed in the positive controls.
6.1.8. ELISA tests
The principle of the
ELISA tests can only be used as an optional test in addition to the tests IF, PCR
or FISH for its relatively low sensitivity. When you use the DAS ELISA is
enrichment and use of monoclonal antibodies required. Enrichment of samples
before using the ELISA test may increase the sensitivity of the test, but may
also meet with failure because of competition from other organisms in the sample.
Note:
Apply validated source of antibodies to r. solanacearum. It is recommended that
determine the titre of antibodies for every new batch. The titre is defined as the
the highest dilution at which optimum reaction occurs when testing a
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
the manufacturer's recommendations. During testing, the antibodies should be used in
Working dilution, which is close to or the same as at the titre of the commercial
formulation.
Determine the titre of antibodies for the suspension of 105 to 106 cells per ml
the homologous strain of r. solanacearum.
The sample that was previously tested as r. solanacearum negative and
a suspension of bacteria without interaction in saline phosphate
buffer (PBS) as negative control samples.
As a positive control samples are aliquots of sample shall
the extracts which previously tested negative, with 103
up to 104 cells per ml of r. solanacearum biovar 2 (strain NCPPB 4156 eg. =
PD 2762 = CFBP 3857, see Appendix 2 A and B). For a comparison of the results of the
each plate uses the standard suspension of 105 to 106 cells per ml
in the PBS of r. solanacearum. Positive control samples must be on
microtitre plate well separated from the samples for testing.
Standardized positive and negative materials, which are used for
This test are listed in Appendix 3, point and Test the control material.
shall be carried out in the same manner as the sample (s).
Two ELISA protocols are validated.
and Indirect ELISA test) (Robinson Smith et al., 1995)
1) use 100-200 µ l sample extract. (Heated to 100 ° c on 4
minutes in a water bath or heating box may in some cases
reduce non-specific results).
2) adds the same amount twice as strong uhličitanového cover
buffer (Appendix 4) and mix.
3) is 100 µ l Dispense into each well of the microtitre plate (eg.
Nunc-Polysorp or equivalent) and incubate 1 hour at
temperature 37 ° c or 14-18 h at 4 ° c.
4) extracts by pouring from holes. The wells three times with PBS-Tween vymyji
(Appendix 4) and the last washing solution in the hole for at least 5
minutes.
5) prepare the appropriate dilution of antibodies to r. solanacearum in
blocking buffer (Appendix 4). For validated commercial antibodies,
apply the recommended dilution (usually double the concentration than is
titre).
6) 100 µ l is added to each well and incubate 1 hour at
a temperature of 37 ° c.
7) antibody Solution is poured from the wells and the wells are washed as before
(point 4).
8) prepare the appropriate dilution of alkaline phosphatase in blocking
buffer. Add 100 µ l to each well and incubate 1 hour at
a temperature of 37 ° c.
9) Conjugate with spills from wells and the wells are washed as before (point 4).
10. Add 100 µ) l of substrate solution of alkaline phosphatase
(Appendix 4) into each well. Incubated in the dark at ambient
temperature and read absorbance at 405 nm in a regular 90minutových
intervals.
(b)) the DAS-ELISA
1) prepare the appropriate dilution of immunoglobulin in polyklonálního
uhličitanovém buffer pH 9.6 (Appendix 4). Add 200 µ l into each well.
Incubated at a temperature of 37 ° c for 4 to 5 hours or 4 ° C for 16
hours.
2) Wells three times good rinse using the PBS-Tween (Appendix
4). Add 190 µ l sample extract into at least two wells. Add
also, the positive and negative control samples of the two wells on each
plate. Leave to incubate for 16 hours at a temperature of 4 ° c.
3) Wells three times well rinsed with the use of PBS-Tween (Appendix 4).
4) prepare the appropriate dilution of specific monoclonal antibodies to R.
solanacearum in PBS (Appendix 4) containing 0.5% bovine serum
albumin (BSA), add 190 µ l to each well and leave to stand for 2 hours
at a temperature of 37 ° c.
5) Wells three times well rinsed with the use of PBS-Tween (Appendix 4).
6) prepare a dilution of protimyšího immunoglobulin conjugated
alkaline phosphatase in PBS. Add 190 µ l to each well and leave to
incubate for 2 hours at a temperature of 37 ° c.
7) Wells three times well rinsed with the use of PBS-Tween (Appendix 4).
8) prepare a solution of 1 mg p-NPP/ml alkaline phosphatase substrátovém
buffer (Appendix 4). Add 200 µ l to each well and incubate in the dark
at room temperature. Absorbance at 405 nm is read at regular
intervals for 90 minutes.
Interpretation of the results of the ELISA test
The ELISA test is negative if the average value of the optical density (OD)
from the wells with the same samples is less than twice the OH with the negative
the control sample, if all the values of the OH positive control
the samples are greater than 1.0 (after 90 minutes of incubation with the substrate) and
they are more than twice the OH is obtained from the negative pattern
extracts. The ELISA test is positive if the average value of the OH wells
the same sample is greater than twice the OH in the negative of the extract
the sample to be tested, if the value of the OH in all negative control
the samples are less than twice the values in the positive control
formulas.
Negative ELISA readings in positive control wells indicate that
the test has not been performed correctly or that was inhibited. Positive values
ELISA in the negative control wells indicate that there has been a mutual
contamination or non-specific binding of the antibodies.
6.1.9. Bioassay test
Note.:
Preliminary testing with this method should permit reproducible
detection of 103 to 104 colony-forming units of r. solanacearum per ml
added to the sample extract that previously tested negative
the result of the (preparation see Appendix 3).
The highest sensitivity of detection can be expected when using freshly
the prepared sample extract and optimal growth conditions.
The method can be successfully applied to extracts of which are preserved in the
glycerol in temperature-68 to-86 ° c. The following protocol is based on the
Janseho (1988):
6.1.9.1. the 10 test plants of susceptible tomato varieties
(e.g. Moneymaker or cultivar with equivalent sensitivity
by the test lab) in the third true leaf stage for each
sample. Cultivation details see Appendix 8. Or use the Eggplant
(e.g. cultivar Black Beauty or cultivars with equivalent susceptibility),
but the only plants in phase 2. up to 3. the leaves unfold in the third until the full
the right of the sheet. Symptoms in eggplants are less pronounced and develop
more slowly. If possible, we recommend that you use tomato seedlings.
6.1.9.2. Distributed 100 µ l sample extract between the test plants.
1. Syringe inoculation
Inokulují with plant stems just above the cotyledons using a syringe with
a hypodermic needle (not less than 23 g). The sample shall be divided between the test
plants.
2. Inoculation notch
The plant is holding between two fingers, pipette, and for about 5-10
µ l of the suspended pellet on the stem between the cotyledons and the first leaf.
With a sterile scalpel make the cross section of about 1 cm long and deep
approximately 2/3 of the stem thickness, while the cut will begin at the site drops
the resuspended Pellet.
The cut is then sealed sterile Vaseline from a syringe.
6.1.9.3. The same technique with nainokulujte 5 seedlings water 105
up to 106 cells per ml, prepared from the 48-hour culture of a virulent
biovar 2 strain of r. solanacearum as a positive control sample, and
with pellet buffer (Appendix 4) as negative control sample. Separates the
positive and negative control plants from other plants, in order to
prevent cross-contamination.
6.1.9.4. grow the test plants in the quarantine facility 4
weeks at a temperature of 25-30 ° c and high relative humidity with appropriate
watering to avoid waterlogging as wilting due to
the lack of water. To prevent contamination, must be held
positive and negative control plants in completely separate places
the greenhouse or in the growing Chamber or otherwise strictly isolated. They have to
plants from different samples incubated close together,
Detaches the appropriate screens. When fertilizing, watering, and viewing all
other activities, special attention to avoid
cross-contamination. Necessary is to keep the greenhouse or growing space
without insect pests, because they could transfer bacteria between samples.
Looking for symptoms of wilting, epinasty, chloróz or stunting of the plant.
6.1.9.5. From infected plants to carry out insulation (paragraph 2.3) and
identify pure cultures are suspected (paragraph 6.2.).
6.1.9.6. If during 3 weeks no symptoms are observed,
the test shall be carried out IF/PCR/isolation on a composite sample of segments of straw
long 1 cm of each test plant taken above the place
inoculation. If the test is positive, the dilution plating (section
4.1).
6.1.9.7. Identify all the pure culture with suspicion on the R.
solanacearum (section 6.2).
Interpretation of the bioassay test results
Valid bioassay test results are obtained, if the positive control
the test plants show typical symptoms, bacteria can be from
These plants again isolated and negative control plants
do not show any symptoms. The bioassay test is negative if the plants
they are not infected by r. solanacearum, if the organism was
detected in the positive control test plants. The bioassay test is
positive if the test plants are infected by R.
solanacearum.
6.2. Identification tests
Identify pure cultures isolated suspect organism R.
solanacearum using at least two of the following tests based on
different biological principles. Include, where appropriate, of the known reference
for each of the test strains (see Appendix 3).
6.2.1. Nutritional and enzymatic identification tests Determine the following
phenotypic properties, which are universally present or absent in
R. solanacearum, Lelliott and Stead (1987), Klement et al.
(1990), Schaad (2001).
The text of the expected result
Fluorescent pigment production-
Inclusion Of PHB +
Oxidation fermentation test +/F-
Catalase activity +
Oxidázový test according to Kovacse +
Reduction of nitrates +
The use of the citrate industry conducted +
Growth at 40 ° C-
Growth in 1% NaCl +
Growth in 2% NaCl-
Arginine dihydrolase activity-
Ztekucení gelatin-
Hydrolysis of starch-
Hydrolysis of eskulinu-
Production of levanu-
Media and methods see Lelliot & Stead (1987).
6.2.2. IF Test
6.2.2.1. Prepare a suspension of approximately 106 cells per ml in IF buffer
(Appendix 4).
6.2.2.2. Prepare a twofold dilution series of an appropriate antiserum series.
6.2.2.3. the method shall be applied IF (6.1.5).
6.2.2.4. IF Test is positive, the if titre of the culture is equivalent to the titer of
the positive control.
6.2.3. The ELISA Test
Note:
When performing only 2 identification tests are not used in addition to this
Another serological test methods.
6.2.3.1. Prepare a suspension of approximately 108 cells per 1 ml in 1 x PBS
(Appendix 4).
6.2.3.2. the ELISA test shall be carried out with a specific monoclonal antibody
to r. solanacearum.
6.2.3.3. The ELISA Test is positive if the value of the outcome
obtained from this culture is at least half in comparison with the value of the
the positive control.
6.2.4. PCR Tests
6.2.4.1. Prepare a suspension of approximately 106 cells per ml of sterile water
(UPW = ultra pure water).
transfer. Heat 100 µ l of cell suspension in closed tubes in
heating block or boiling waterbath at 100 ° c for four minutes. Samples
they can be kept at a temperature of-16 to-24 ° c to the next.
6.2.4.3. the procedures shall be used for the amplification of the PCR amplicons are obtained
r. solanacearum-specific (eg. Seal et al. (1993); Pastrik and
Maiss (2000); Pastrik et al. (2002); Boudazin et al. (1999); Opina et
Al., (1997), Weller et al. (1999).
6.2.4.4. identification of r. solanacearum is positive if
amplikony PCR are the same size and have the same variety the length
the fragments as the positive control strain.
6.2.5. Test FISH
6.2.5.1. Prepare a suspension of approximately 106 cells per ml in UPW.
6.2.5.2. the method of FISH (6.1.7) with at least 2 oligosondami
specific for r. solanacearum (Appendix 7).
6.2.5.3. FISH Test is positive if the culture and a positive
control reaches the same reactions.
6.2.6. The analysis of the fatty acid profiling (FAP)
6.2.6.1. Growing culture on trypticase soy agar (Oxoid) for 48
hours at a temperature of 28 ° c.
6.2.6.2. use the method of FAP procedure (Janse, 1991; Stead, 1992).
6.2.6.3. FAP Test is positive if the profile is suspicious of culture
the same as the profile of the positive control. The presence of the characteristic
fatty acids are 14:0 3OH, 16:0 2OH, 16:1 2OH and 18:1 2OH and absence
16:0 3 OH highlights of Ralstonia sp.
6.2.7. the characteristics of the Methods of the tribes
For each new case of isolation of r. solanacearum is recommended
characteristics of strain by using one of the following methods. For each
the test is carried out, where appropriate, shall include known reference strains (see.
Appendix 3).
6.2.7.1. biovar Determination
R. solanacearum biovar differing usage and/or
the oxidation of the three disaccharides and three hexose alcohols (Hayward, 1964 and
Hayward et al., 1990). Solid for biovar test is described in Appendix
2. Test can be successfully performed by vaccination in the broth with the net culture
R. solanacearum and incubating at 28 ° c. If it is solid
divided into sterile plates with 96 wells (200 µ l per 1 hole),
coloring is amended during the 72 hours from olive green to yellow and means
a positive test result.
6.2.7.2. Genomic variability
Molecular identification of strains of r. solanacearum complex can be achieved
using several techniques, which include:
1. analysis of the polymorphism of length polymorphism RFLP ANALYSIS (Cook et
Al., 1989).
2. Repetitive sequence PCR using REP, BOX of primers and ERIC (Louws
et al., 1995; Smith et al., 1995).
3. analysis of length polymorphism AFLP fragments (Van amplifikovaných
der Wolf et al., 1998).
6.2.7.3. PCR Methods
Specific PCR primers (Pastrik et al, 2002; see Appendix 6) can be used
to differentiate strains belonging to group 1 (biovars 3, 4 and 5), and
Group 2 (biovars 1, 2A and 2T) of r. solanacearum, as originally
defined by analysis of RFLP ANALYSIS (Cook et al., 1989) and 16S rDNA sequencing
(Taghavi et al., 1996).
6.3. the confirmatory test (assays)
As final confirmation of a diagnosis of r. solanacearum and for
the virulence of cultures identified as r. solanacearum must be
performed assays.
1) prepare a seed density of approximately 106 cells per ml from 24 to
48 hour cultures to test, and the positive control strain of R.
solanacearum (e.g.. NCPPB 4156 = PD 2762 = CFBP 3857; see Appendix 3).
2) Amounts to 5 to 10 susceptible tomato or Eggplant seedlings at the stage 3
true leaf (see paragraph 6.1.9).
3) Seedlings with let incubate for up to 2 weeks at a temperature of 25-28 ° c and
high relative humidity with appropriate watering to, without issue
or getting more land. With pure cultures typical wilting
should occur within 14 days. If, after this time the signs, culture
cannot be considered as a pathogenic form of r. solanacearum.
4) looking for symptoms of wilting and/or epinasty, chlorosis and/or stunting.
5) isolate from symptomatic plants by removing
the segment of the stem about 2 cm above the inoculation point. Segment, and
suspended in a small volume of sterile distilled water or 50 mM
phosphate buffer (Appendix 4). Isolation of suspension shall be
After the dilution, if possible on a selective medium (Appendix 2).
After incubation of 48 to 72 hours at a temperature of 28 ° c to evaluate the occurrence of
typical colonies of r. solanacearum.
Appendix 1
Laboratories involved in optimisation and validation of protocols
------------------------------------------------ ----------------------------------
Laboratory (1) instead of the country
------------------------------------------------ ----------------------------------
Für Gesundheit und Ernährungssicherheit agencies in Vienna and Linz Austria
Department Of Gewasbescherming Merelbeke Belgium
Plantedirektoratet Lyngby Denmark
The Central Science Laboratory, York, England
The Scottish Agricultural Science Agency, Edinburgh, Scotland
Laboratoire National de la Protection Végétaux,
Unit de Bactériologie Angers France
Laboratoire National de la Protection Végétaux,
Station de Quarantaine de la Pomme de Terre, Le Rheu France
Biologische Bundesanstalt Kleinmachnow Germany
Pflanzenschutzamt Hannover Hannover Germany
State Laboratory, Dublin Ireland
Dipartimento di Scienze e Tecnologie
Agroambientali Bologna Italy
Regione Veneto Unita Periferica per i Servizi
Fitosanitari Verona Italy
Nederlandse Algemene Keuringsdienst Emmeloord Netherlands
Plantenziektenkundige Dienst Wageningen University Netherlands
Direcçao-General de Protecçao das Culturas Lisbon Portugal
Centro de Diagnóstico de Aldearrubia Salamanca Spain
The Instituto Valenciano de Estudios Agrarias Valencia Spain
Swedish University of Agricultural Sciences Uppsala Sweden
------------------------------------------------ ----------------------------------
(1) Contacts: see Web site
http://forum.europa.eu.int/Public/irc/sanco/Home/main
Appendix 2
Media for isolation and culture of r. solanacearum
and) medium for the isolation and culture
Nutrient agar (NA)
Nutrient agar (Difco) 23.0 g
Distilled water 1.00 l
Dissolve ingredients and sterilují pressure at a temperature of 121 ° c for 15 minutes.
Kvasnično-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 1.00 l
Dissolve ingredients and sterilují pressure at a temperature of 121 ° c for 15 minutes.
Sacharózopeptonový agar (SPA)
Sucrose 20.0 g
Bacto-Peptone (Difco) 5.0 g
K2 HPO4 0.5 g
MgSO4. 7 H2O 0.25 g
Bacto-Agar (Difco) 15.0 g
Distilled water 1.00 l
pH 7,2-7,4
Dissolve ingredients and sterilují in an autoclave at 121 ° c for 15 minutes.
To Kelman's tetrazolium medium
casein hydrolysate (Difco) 1.0 g
Bacto-Peptone (Difco) 10.0 g
5.0 g dextrose
Bacto-Agar (Difco) 15.0 g
Distilled water 1.00 l
Dissolve ingredients and sterilují in an autoclave at 121 ° c for 15 minutes.
Cool to 50 ° c and add a filter-sterilised
2, 3, 5-triphenyl-triphenyl tetrazolium chloride (Sigma), until the
achieve a final concentration of 50 mg per litre.
b) Validated selective medium
Solid SMSA MEDIUM (Englebrecht, 1994, as amended by Elphinstone et al., 1996)
Basic solid
Casamino acids (Difco) 1.0 g
Bacto-Peptone (Difco) 10.0 g
5.0 ml of glycerol
Bacto-Agar (Difco) (see note 2) 15.0 g
Distilled water 1.00 l
Dissolve ingredients and sterilují in an autoclave at 121 ° c for 15 minutes.
Cool to 50 ° c and add a filter-sterilised
in aqueous solution the following folders to a specified
the final concentration of:
Crystal Violet (Sigma) 5 mg/l
Polymyxin-B-sulphate (Sigma P1004) 600 000 (approximately 100 mg)/l
Bacitracin (Sigma B-0125) 1 250 U (approximately 25 mg)/l
Chloramphenicol (Sigma C-3175) 5 mg/l
Penicillin-G (Sigma P-3032) 825 U (approximately 0.5 mg)/l
2, 3, 5-tetrazolium chloride (Sigma) 50
mg/l
Notes:
1. The use of other than the above reagents may affect growth of r.
solanacearum.
2. The place of Bacto-Agar (Difco) can be used Oxoid Agar # 1. In such a
case growth of r. solanacearum will be slower, but it may also limit the
the growth of competing Saprophytes can be increased. Typical colonies of r. solanacearum may
take 1-2 days longer and the red coloring may be lighter and
more widely distributed than when using Bacto-Agar.
3. Increasing bacitracin concentration to 2 500 U/l may reduce population
competing bacteria without affecting the growth of Ralstonia solanacearum.
Solutions of antibiotics is kept and stored at 4 ° c in the dark and
must be consumed within 1 month.
Plates should be free from surface condensation before use.
Necessary to avoid excessive dryness of the media. Each new batch
broth should be under the control of the quality of the broth after
suspension of a reference culture of r. solanacearum (see Appendix 3), and
the observation that after 2 to 5 days at 28 ° c appear typical
the colony.
(c) Validated the enriched broth)
Solid SMSA (Elphinstone et al., 1996) shall be carried out as preparation for the
SMSA selective agarovou breeding ground, but omits the Bacto-Agar and
2, 3, 5-tetrazolium chloride.
Modified Wilbrink solid (Caruso et al., 2002)
10 g sucrose.
Proteázový peptone 5 g
K2 HPO4 0.5 g
0.25 g MgSO4
Nano3 0.25 g
Distilled water 1.00 l
Steriluje pressure at a temperature of 121 ° c for 15 minutes and cool na50 ° c.
Add antibiotic stock solutions as a breeding ground for the SMSA.
Appendix 3
A. Commercial standardized control material
and Isolated the bacterial culture) Following the isolated bacterial
culture are recommended as a standard reference material either to the
positive control (table 1) or during optimisation of tests, in order to
avoid cross-reactions (table 2). All strains are commercially
available and can be obtained from the following collections:
1. the national collection of plant pathogenic bacteria (NCPPB), Central
Science Laboratory, York, UK.
2. the collection of cultures section of the plant protection service (PD), Wageningen, the Netherlands.
3. the French collection of plant pathogenic bacteria (CFBP), Institute of
fytobakteriologie INRA, Angers, France.
Table 1: Reference panel isolated bacterial cultures of r. solanacearum SMT
-------------------------------------------------------- --------------------------
NCPPB Code SMT
# Other codes, country of origin, Biovar
-------------------------------------------------------- --------------------------
CFBP 4582 NCPPB 4153 6, Pr 3020, EURS11 Egypt 2
CFBP 4585 NCPPB 4154 10, 550, EURS21 Turkey 2
CFBP 4587 NCPPB 3857 12, Pr 1140, EURS26 England 2
CFBP 4598 NCPPB 1584 23, EURS49 Cyprus 2
CFBP 4599, NCPPB 2505 24 EURS50 Sweden 2
CFBP 4601, NCPPB 4155 26 502, EURS55 Belgium 2
NCPPB 4156 (*) (*) 71 PD 2762, CFBP 3857 Netherlands 2
NCPPB 4157 66 LNPV 15.59 France 2
CFBP 4608 NCPPB 4158 39, Port 448, EURS80 Portugal 2
NCPPB 4160 69 IVIA-1632-2 Spain 2
NCPPB B3B 4161 76 Germany 2
NCPPB 325 41 CFBP 2047, KEL60-1, R842 USA 1
CFBP 4610 NCPPB 3967 42, R285, GONg7 Costa Rica 1
CFBP 4611 NCPPB 4028 43, R303/571, CIP310,
SEQ205 Colombia 2
CFBP 4612 NCPPB 3985 44, R578, CIP312 Peru 2T
CFBP 4613 NCPPB 3989 45, R568, CIP226 Brazil 2T
CFBP 3928, NCPPB 3996 46 R276/355, CIP72,
SEQ225 Peru 3
CFBP 4614, NCPPB 3997 47 R280/363, CIP49,
HAY0131 Australia 3
CFBP 4615 NCPPB 4029 48, R297/349, CIP121,
CMIb2861 Sri Lanka 4
CFBP 4616, NCPPB 4005 49 R470 Philippines 4
CFBP 4617, NCPPB 4011 50 R288, HEmps2 China 5
-------------------------------------------------------- --------------------------
(*) As standard reference strain of r. solanacearum is used
biovar 2 (genus 3).
Note:
The reliability of the above strains can be guaranteed only in case that the
they come from the original collections of cultures.
Table 2: SMT Reference panel of serologically-or genetically
relatives of bacteria to optimize the detection tests
-------------------------------------- --------------------------------------
NCPPB Code SMT
# Other Identification codes
-------------------------------------- --------------------------------------
NCPPB 4162 51 CFBP 1954 Bacillus polymyxa (1)
NCPPB 4163 52 CFBP 1538 Pseudomonas marginalis
PV. marginalis (1)
NCPPB 4164-CFBP 2227 Burkholderia cepacia complex (2)
NCPPB 4165-2459 Ralstonia pickettii CFBP (2)
NCPPB 4166 58 CFBP 3567,
Ralstonia pickettii Pr1150 CSL (1)
NCPPB 4167 60 CFBP 4618,
PD 2778 Ralstonia SP. (1)
NCPPB 1127 53 CFBP 3575 Burkholderia andropogonis (1)
NCPPB Burkholderia caryophylli 353 54 CFBP 3572 (1)
NCPPB 945 55 CFBP 3569 Burkholderia cepacia complex (1)
NCPPB Burkholderia glumae 3708 56 CFBP 3574 (1)
NCPPB 3590 57 CFBP 3573 Burkholderia plantarii (1)
NCPPB 3726 59 CFBP 3568 Banana Blood Disease Bacterium (1) (2) (3)
NCPPB 4168 61 CFBP 4619,
IPO S339 Enterobacter SP. (1)
NCPPB 4169 62 IPO 1695 Enterobacter SP. (1)
NCPPB 4170 63 CFBP 4621,
IPO S306 Ochrobactrum anthropi (1) (2)
NCPPB 4171 64 CFBP 4622,
IPO 1693 Curtobacterium SP. (1) (2)
NCPPB 4172 65 IPOS 1696a Pseudomonas SP. (1)
NCPPB 4173-PD 2318 Aureobacterium SP. (2)
NCPPB 4174 81 IVIA Flavobacterium 1844.06 SP. (1) (2)
-------------------------------------- --------------------------------------
(1) Potential cross-reacting strain in serological tests (IF
and/or ELISA) with Polyclonal antiserum.
(2) the tribe, from which can be amplified in certain laboratories product
PCR on a similar size, such as the expected size when using
specific primers OLI-1 and Y-2 (see Appendix 6).
(3) the likelihood of cross reactions in most tests, but the occurrence of known
only for bananas in Indonesia.
(b)) a commercial standardized control material
The following standard control material can be obtained from the collection
the cultures of the NCPPB.
Frozen dried pellet of potato extract from 200 healthy tubers
the potato to the negative control of all tests. Frozen dried pellets
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.
Because the viability of the cell is affected during freeze-drying, these are not
appropriate as standards in tests of isolation and pathogenicity tests. In
formaldehyde fixed suspension of r. solanacearum biovar 2 (strain NCPPB
4156 = PD 2762 = CFBP 3857) with 106 cells per 1 ml positive control
serological tests.
(B) preparation of positive and negative controls for screening tests
PCR/IF and FISH
Cultivating the 48-hour culture a virulent strain of r. solanacearum race
3/biovar 2 (e.g. strain NCPPB 4156 = PD 2762 = CFBP 3857) on the SMSA medium
soil and suspended in 10 mM phosphate buffer to obtain the cell
the density of approximately 2 × 108 colony-forming cells per ml. Usually
She gently reaches a muddy suspension equivalent to an optical density of 0.15
at 600 nm.
Remove the pulp from the heel ends 200 tubers from the production of varieties with white
skin that is free from r. solanacearum. Process is
the heel end of the above procedure, and resuspenduje with pellets in 10 ml.
Prepare 10 sterile 1.5 ml microvials with a volume of 900 µ l
the resuspended Pellet.
Transfer 100 µ l of the suspension of r. solanacearum in the first place and
shake.
Ten-fold dilutions shall be carried out in the next five microvials.
These six contaminated microvials will be used as a control
samples. Four non-contaminated microvials will be used as a control
negative samples. Microtest tube must be properly marked.
Prepare aliquots of 100 µ l in sterile tubes on the volume
1.5 ml, which gets 9 copies of each sample. Storage is
at a temperature of-16 to-24 ° c until use.
The presence and quantity of r. solanacearum in the control samples should
be the first to test IF confirmed.
For the PCR test DNA extraction from positive and negative
control samples with each series of test samples.
For IF and FISH tests perform assays positive
negative control samples with each series of test samples.
In the quantitative analysis of IF, FISH and PCR of r. solanacearum
detected in at least 106 and 10 4 cells/ml positive controls and not
be detected in any of the negative controls.
Appendix 4
Buffers for test procedures
General: Unopened sterilized buffers can be stored for up to one
of the year.
1. buffers for extraction
1.1. The extraction buffer (50 mM phosphate buffer, pH 7.0)
This buffer is used to extract the bacteria from plant tissues
by homogenisation or shaking.
Na2 HPO4 (anhydrous) 4.26 g
Kh2po4 2.72 g
Distilled water 1.00 l
Dissolve ingredients, check pH and Sterilise by autoclaving at 121 ° c
for 15 min.
May be useful in the following folder:
The purpose of the quantity (per litre)
Lubrol flakes deflocculant (*) 0.5 g
DC silicone Antifoam agent odpěňovač (*) 1.0 ml
Tetrasodiumpyrofosfát Antioxidant agent 1.0 g
Polyvinylpyrrolidone-40000 (PVP-40) the binding of PCR inhibitors, 50 g
---------------------------------------
(*) for use in the extraction by homogenisation
1.2. Peletový buffer (10 mM phosphate buffer, pH 7.2)
This buffer is used for resuspension and dilution of the extracts of the potato from
the potato tuber heel after centrifugation
concentrated into pellets.
Na2hpo4 12H2O 2.7 g
NaH2PO4. 2H2O 0.4 g
Distilled water 1.00 l
Dissolve ingredients, check pH and Sterilise by autoclaving at 121 ° c
for 15 min.
2. buffers for the IF test
IF the buffer (10 mM phosphate buffered saline (PBS), pH
7.2)
This buffer is used for dilution of antibodies.
Na2hpo4 12H2O 2.7 g
NaH2PO4. 2H2O 0.4 g
NaCl 8.0 g
Distilled water 1.00 l
Dissolve ingredients, check pH and Sterilise by autoclaving at 121 ° c
for 15 min.
If-buffer-Tween
This buffer is used for rinsing the slides. Add 0.1%
Tween 20 to the IF buffer.
Phosphate buffered glycerol, pH 7.6
This buffer is used as a top solution for the window slides on IF the tests to
the increase in fluorescence.
Na2hpo4 12H2O 3.2 g
NaH2PO4. 2H2O 0.15 g
Glycerol 50 ml
Distilled water 100 ml
Cover solutions are commercially available, such as. Vectashield ® (Vector
Laboratories) or Citifluor (R) (Leica).
3. Buffers for the indirect ELISA
3.1. Double strength coating buffer, pH 9.6.
Na2CO3 6.36 g
Nah CO3 11.72 g
Distilled water 1.00 l
Dissolve ingredients, check pH and autoclaving at steriluje
a temperature of 121 ° c for 15 minutes.
If the extract contains a high proportion of aromatic molecules is possible as
antioxidant add sodium sulfite (0.2%).
3.2.10 X phosphate buffered solution of sodium chloride (10 x
The PBS), pH 7.4
NaCl 80.0 g
2.0 g of kh2po4
Na2hpo4. 12H2O 29.0 g
KCL 2.0 g
Distilled water 1.00 l
3.3. PBS-Tween
10 x 100 ml PBS
10% Tween 20 5 ml
895 ml distilled water
3.4. Blocking buffer (must be freshly prepared).
10 x 10.0 ml of PBS
Polyvinylpyrrolidone-44000
(PVP-44) 2.0 g
10% Tween 20 0.5 ml
Milk powder 0.5 g
Distilled water to make up to 100 ml
3.5. alkaline phosphatase substrate Solution, pH 9.8
Diethanolamine 97 ml
Distilled water 800 ml
Dissolve and adjust the pH with concentrated HCl to 9.8.
Make up to 1 l with distilled water.
Add 0.2 g MgCl2.
Dissolve two 5 mg tablets 2 phosphatase substrate (Sigma) in a 15 ml
the solution.
4. buffers for DASI ELISA
4.1. Coating buffer, pH 9.6.
Na2CO3 1.59 g
Nah CO3 2.93 g
Distilled water to 1 000 ml
Dissolve ingredients and check pH 9.6.
4.2.10 x fosfátosolný buffer (PBS) pH 7.2-7.4
NaCl 80.0 g
12H2O 27 g na2hpo4.
NaH2PO4. 2H2O 4 g
Distilled water to 1 000 ml
4.3. PBS-Tween
10 x PBS 50 ml
10% Tween 20 5 ml
Distilled water 950 ml
4.4. Substrate buffer solution, pH 9.8
Diethanolamine 100 ml
Distilled water 900 ml
Mix and sets the value of the pH 9.8 with concentrated HCl.
Appendix 5
Determination of the concentration of positive cells IF and FISH
1. Calculate the average number of typical fluorescent cells per
field of view (c).
2. Calculate the number of typical fluorescent cells per window
the microscopic slides (C).
(C) = c x S/s,
where S = the area of one field on a slide with porthole Windows and more
s = surface area of objective field.
s = píi2/2, 4G2K
where i = field coefficient (in the range of 8-24 depending on type of eyepiece),
K = tube coefficient (1 or 1,25),
G = magnification of the lens (100 x, 40 x, etc.).
3. Calculate the number of characteristic of Fluorescing cells per 1 ml
the resuspended Pellet (N).
N = C × 1 000/y x 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
Note:
The initial testing should permit reproducible detection of at least 103
up to 104 cells of r. solanacearum per ml of sample extract. Welcome
testing should also not show no false positive results
a group of selected strains of bacteria (see Appendix 3).
1. PCR Protocol of Seal et al. (1993)
1.1. A mismatched primers
Direct 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 template r. solanacearum DNA = 288 bp
1.2. PCR reaction mix
The quantities of reagents in the reaction, the final concentration
Sterile UPW 17.65 µ l
10 x PCR buffer (1) (15 mM MgCl2) 2.5 µ l of 1 x (1.5 mM MgCl2)
A mixture of dntps are licensed (20 mm) 0.2 0.25 µ l mM
Primer OLI-1 (20 µ M) µ 1.25 l 1 µ M
Primer Y-2 (20 µ M) µ 1.25 l 1 µ M
Taq polymerasa (5U/µ l) (1) 0.5 0.1 µ l U
Quantity of sample of 2.0 l µ
The total quantity of 25 µ l
(1) the method has been validated using Taq polymerase Perkin Elmer
(AmpliTaq) and Gibco BRL.
1.3. PCR Reaction conditions
Carry out the following process:
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 (primer DNA hybridization with templátovou)
IV) 30 seconds at 72 ° c (synthesis)
1 cycle of: v) 10 minutes at 72 ° c (final fusion)
vi) leaves at a temperature of 4 ° c.
Note:
This program has been optimized for use with a Perkin Elmer
9600. The use of other instruments may require adjustment of the individual steps
cycle ii), (iii)) and (iv)).
1.4. Restriction enzyme analysis of Amplicon.
PCR products amplified from r. solanacearum DNA produce
length polymorphism with enzyme Ava II after incubation
at a temperature of 37 ° c.
2. PCR Protocol of Pastrik and Maisse (2000)
2.1. A mismatched primers
Direct 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 '
The expected size of the Amplicon DNA of r. solanacearum template DNA = 553
BP.
2.2. PCR reaction mix
The quantities of reagents in the reaction, the final concentration
Sterile UPW 16.025 µ l
10 x PCR buffer (1) 2.5 µ l of 1 x (1.5 mM MgCl2)
BSA (fraction V) (10%) 0.25 µ l 0.1%
A mixture of dntps are licensed (20 mm) l 0.1 µ 0.125 mM
Primer Ps-1 (10 µ M) 0.5 0.2 µ µ l M
Primer Ps-2 (10 µ M) 0.5 0.2 µ µ l M
Taq polymerasa (5U/µ l) (1) 0.5 0.1 µ l U
The amount of 5.0 µ l sample
The total quantity of 25.0 µ l
(1) the method has been validated using Taq polymerase Perkin Elmer
(AmpliTaq) and Gibco BRL.
Note:
Originally optimised for MJ Research PTC 200 cycler with
polymerasou Gibco Taq Polymerization. Perkin Elmer AmpliTaq and buffer may
also be used in the same concentrations.
2.3. PCR Reaction conditions
Carry out the following process:
1 cycle 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 (primer DNA hybridization with templátovou)
IV) 45 seconds at 72 ° c (synthesis)
1 cycle of: v) 5 minutes at 72 ° c (extension synthesis)
vi) leaves at a temperature of 4 ° c.
Note:
This programme was optimised for use with MJ Research
PTC 200. The use of other instruments may require adjustment
the individual steps of the cycle ii), (iii)) and (iv)).
2.4. Restriction enzyme analysis of Amplicon
PCR products amplified from r. solanacearum DNA produce
a clear length polymorphism with enzyme Taq I after
the incubation time of 30 minutes at a temperature of 65 ° c. Restriction fragments
r. solanacearum-specific size 457 bp and 96 bp.
3. multiplex PCR Protocol with internal control (Pastrik et al.,
2002)
3.1. A mismatched primers
Direct 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 '
Direct 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 template r. solanacearum DNA = 718 bp
(RS-primer set).
Expected Amplicon size 18S rRNA internal PCR control = 310 bp
(NS-primer set).
3.2. PCR reaction mix
The quantities of reagents in the reaction, the final concentration
Sterile UPW 12.625 µ l
10 x PCR buffer (1) (15 mM MgCl2) 2.5 µ l of 1 x (1.5 mM MgCl2)
BSA (fraction V) (10%) 0.25 µ l 0.1%
A mixture of dntps are licensed (20 mm) l 0.1 µ 0.125 mM
Primer RS-1-F (10 µ M) µ µ 2.0 l 0.8 M
Primer RS-1-R (10 µ M) µ µ 2.0 l 0.8 M
Primer NS-S-F (10 µ M) (2) 0.15 µ l 0.06 µ M
Primer NS-6-R (10 µ M) (2) 0.15 µ l 0.06 µ M
Taq polymerase (5U/µ l) (1) 0.2 µ l 1.0 for
The amount of 5.0 µ l sample
The total quantity of 25.0 µ l
-----
(1) the method has been validated using Taq polymerase Perkin Elmer
(AmpliTaq) and Gibco BRL.
(2) concentration of primers NS-5-F and NS-6-R were optimised for
potato tuber heel parts using the homogenisation
method and DNA purification according to Pastrik (2000) "(see 6.1.6.1)). The use of the
extraction by shaking or other DNA isolation methods may require
a new implementation of the optimization of the concentrations of the reagents.
3.3. PCR Reaction conditions
Carry out the following process:
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 (primer DNA hybridization with templátovou)
IV) 45 seconds at 72 ° c (synthesis)
1 cycle of: v) 5 minutes at 72 ° c (final fusion)
vi) leave at 4 ° c.
Note:
This programme was optimised for use with MJ Research
PTC 200. The use of other instruments may require adjustment of the individual
steps of the cycle ii), (iii)) and (iv)).
3.4. Restriction enzyme analysis of Amplicon
PCR products amplified from r. solanacearum DNA produce
a clear length polymorphism with enzyme Bsm I
or an Isoschizomere (eg. Mva 1269 I) after incubation period of 30 minutes
a temperature of 65 ° c.
4. PCR protocol specific for r. solanacearum biovar (Pastrik et al,
2001)
4.1. A mismatched primers
Direct 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 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
and Protocol-specific PCR) biovar 1
The quantities of reagents in the reaction, the final concentration
0Sterilní UPW 12.925 µ l
10 x PCR buffer (1) 2.5 µ l of 1 x (1.5 mM MgCl2)
BSA (fraction V) (10%) 0.25 µ l 0.1%
A mixture of dntps are licensed (20 mm) l 0.1 µ 0.125 mM
Primer Rs-1-F (10 µ M) µ µ 2.0 l 0.8 M
Primer Rs-1-R (10 µ M) µ µ 2.0 l 0.8 M
Taq polymerase (5U/µ l) (1) 0.2 µ l 1.0 for
The amount of 5.0 µ l sample
The total quantity of 25.0 µ l
(1) the method has been validated using Taq polymerase Perkin Elmer
(AmpliTaq) and Gibco BRL.
(b) the specific PCR Protocol) Biovar 3/4/5
The quantities of reagents in the reaction, the final concentration
Sterile UPW 14.925 µ l
10 x PCR buffer (1) 2.5 µ l of 1 x (1.5 mM MgCl2)
BSA (fraction V) (10%) 0.25 µ l 0.1%
A mixture of dntps are licensed (20 mm) l 0.1 µ 0.125 mM
Primer Rs-1-F (10 µ M) µ l 1.0 0.4 µ M
Primer Rs-3-R (10 µ M) µ l 1.0 0.4 µ M
Taq polymerase (5U/µ l) (1) 0,2 µ l 1.0 for
The amount of 5.0 µ l sample
The total quantity of 25.0 µ l
----
(1) the method has been validated using Taq polymerase Perkin Elmer
(AmpliTaq) and Gibco BRL.
4.3. PCR Reaction conditions
The following shall be a circular process for both responses specific to the
biovar 1/2-and biovar 3/4/5:
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 (primer DNA hybridization with templátovou)
IV) 45 seconds at 72 ° c (synthesis)
1 cycle of: v) 5 minutes at 72 ° c (final fusion)
vi) leaves at a temperature of 4 ° c.
Note:
This programme was optimised for use with MJ Research
PTC 200. The use of other instruments may require adjustment of the individual
steps of the cycle ii), (iii)) and (iv)).
4.4. Restriction enzyme analysis of Amplicon.
PCR products amplified r. solanacearum DNA using primers
RS-1-F and Rs-1-R create distinct polymorphism length restriction
fragments with enzyme Bsm I or an Isoschizomere (eg. Mva 1269 I) after
the incubation time of 30 minutes at a temperature of 65 ° c. PCR products resulting from
amplification from r. solanacearum DNA using primers Rs-1-F and Rs-3-R do not have
no restriction sites.
5. preparation of the loading buffer
5.1. Bromphenol blue (10%-stock solution)
Bromphenol blue 5 g
Distilled water (double-distilled) water 50 ml
5.2. Coating buffer
Glycerol (86%) 3.5 ml
Bromphenol blue (5.1) 300 µ l
Distilled water (double-distilled) water (bidestilát) 6.2 ml
6. Buffer 10 x TRIS-acetate-EDTA (TAE) buffer, pH 8.0
TRIS 48.4 g
11.42 ml glacial acetic acid
EDTA (sodium salt) 3.72 (g)
Distilled water 1.00 l
Before use, dilute to 1 x.
Also, commercially available (eg. Invitrogen or equivalent).
Appendix 7
Validated reagents for FISH test
1. Oligosondy
Probe for r. solanacearum-specific OLI-1-Appear: 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
[NOTE: FIXATIVE SOLUTION CONTAINS PARAFORMALDEHYDE WHICH IS TOXIC.
USE GLOVES AND NEVDECHNOUT. IT IS RECOMMENDED TO WORK IN A FUME CUPBOARD.]
I) heat 9 ml molecular grade water (e.g. clean. Ultra pure water =
(UPW)) at a temperature of around 60 ° c and add 0.4 g paraformaldehyde.
Paraformaldehyde dissolves after adding 5 drops of 1N NaOH and stirring
magnetic stirrer.
(ii) adjust pH to) 7.0 by adding 1 ml 0.1 M phosphate buffer (PB; pH
7.0) and 5 drops of 1N HCl. Check the pH with indicator Strip and in
If necessary, adjust with HCl or NaOH. [WARNING:
SOLUTIONS WITH PH METER PARAFORMALEDHYDEM BYPASS!]
III) Filter the solution through a membrane filter, 0.22 µm and stored
protected against dust at 4 ° c until further use.
3.3 x Hybmix
NaCl 2.7 M
60 mM TRIS-HCl (pH 7.4)
EDTA (filter sterilised and autoclaved) 15 mM dilute to 1 x,
According to the needs.
4. Hybridisation solution
1 x Hybmix
Sodium dodecyl sulfate (SDS) 0.01%
30% formamide
Probe EUB 338 5 ng/l µ
Probe OLI-1 or OLI2 5 ng/l µ
Prepare quantities of hybridisation solution according to the calculations in table 1.
For each slide (containing 2 different samples in duplicate) must be 90 µ l
hybridisation solution. WARNING: FORMAMIDE IS VERY TOXIC,
THE USE OF GLOVES AND TAKE NECESSARY SAFETY PRECAUTIONS!
Table 1. The recommended quantities for the preparation of hybridisation mix
------------------------------ ----------------------------------------
The number of slides 1 4 6 8 10
------------------------------ ----------------------------------------
Sterile ultra clean water 23.1 92.4 138.6 184.8 231.0
3 x Hybmix 30.0 120.0 180.0 240.0 300.0
1% SDS 0.9 3.6 5.4 7.2 9.0
Formamide 27.0 108.0 162.0 270.0 216.0
Probe EUB 338 (100 ng/µ l) 4.5 18.0 27.0 36.0 45.0
Probe OLI-1 or OLI2 (100 ng/µ l) 4.5 18.0 27.0 36.0 45.0
------------------------------ ----------------------------------------
The total amount of 540.0 720.0 900.0 360.0 90.0
------------------------------ ----------------------------------------
Note:
All solutions containing light-sensitive oligosondy shall be kept in the
dark place at a temperature of-20 ° c. During use, protects against direct
sunlight or electric light.
5.0, 1 m phosphate buffer, pH 7.0
Na2HPO 48.52 g
Kh2 PO4 5.44 g
Distilled water 1.00 l
Dissolve ingredients, check pH and Sterilise autoclaving at
a temperature of 121 ° c for 15 minutes.
Appendix 8
The cultivation of Eggplant and tomato
Vysejí, seeds of tomato (Lycopersicon esculentum) or Eggplant (Solanum
melongena) in pasteurised výsevního substrate. Seedlings with fully
expanded cotyledons (10 to 14 days) to navel
pasteurized potting compost. Eggplants and tomato plants should
should grow in the greenhouse under the following conditions:
Duration of day: 14 hours or natural day length if greater;
Temperature: day: 21 to 24 ° c,
night: 14 to 18 ° c.
Good variety of eggplants: "Black Beauty"
Good variety of tomato "Moneymaker"
Vendors: see Web site
http://forum.europa.eu.int/Public/irc/sanco/Home/main.
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Bacteriology 46; 10-15.
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Annex 2
And the range of information. a survey of the occurrence of the originator
ring rot and brown rot agent pursuant to § 3 (2). 11
Information about the originator of the survey carried out by the ring and
the causative agent of brown rot under section 3 (3). 11 includes:
and for the presence of the agent) in the diagnosis and the cause of Brown
rot in the stands, in the landfills and in consignments of potatoes:
-the distribution of the propagating material of ^ 1) (breakdown by categories) and
other potatoes (potato distribution for consumer and industrial
the purposes of the)
-the total area of cultivation of seed and other potatoes in hectares,
-the number and acreage of previewed and sampled stands in hectares,
-the number of watched and sampled lots in the Czech Republic or the CZECH REPUBLIC harvested
imported tubers,
-sampling time, and the number of samples taken from the crops of potatoes, and
consignments harvested tubers and results of testing;
(b) in the determination of the occurrence of the originator) of brown rot in tomato plants:
-the number of controlled undertakings,
-the number of checks and watched the plant (i guess),
-the number of samples taken and results of testing;
(c)) in determining the occurrence of the cause of brown rot in the stands of other
host plants than potato and tomato, including host
solanaceous plants in coastal vegetation:
-the number of controlled companies, the kind and the number of (i guess) previewed
plants,
-the number of samples of the plants, the type of tests and results
testing,
-number and designation of the water bodies, where samples of plants;
(d)) in determining the occurrence of the cause of brown rot in water sources
used to host plants and závlaze in waste waters from the
undertakings which have processed industrially potatoes:
-number and designation of the water bodies and the number of enterprises, from where they were removed
water samples,
-the number of samples of water, sampling time, the type of tests and
the results of the test;
(B). Preservation of the tested samples, and lots, and the substantiation of the testing
1. If a suspicion of the presence of ring rot agent or brown
rot on the basis of a positive result the testing carried out the procedures
According to annex No 1, must be up to the confirmation or refutation of this
the result of the next procedure according to annex No 1 and in an appropriate manner
preserved:
-the whole lot of the plants from which the sample was taken, or part thereof
in its original packaging with label, if that is possible,
-all tubers and, where possible, plants of the sample taken,
-any remaining extract and additional prepared material for the
the screening test (s) (s), for example. slides for immunofluorescence test, and
-all relevant documentation
The preservation of the tubers will enable testing of varietal identity or use
Another method of testing.
2. In the case of confirmation of the occurrence of the originator of ring rot or brown
rot must be for a minimum period of one month from the official announcement
the result of the test in the manner prescribed in section 5 (3). 3 retained and suitable
conservation of:
-the material specified in paragraph 1,
-a sample of the infected tomato or Eggplant
inoculated with the tuber or plant (in the case of brown rot,
where appropriate), and
-the isolated culture of the originator of ring rot or brown rot.
----------------
1) § 2 (2). 1 (a). (b)) Act No. 219/2003 Coll., on the circulation of
the seed and the seed of cultivated plants and amending certain laws, as amended by
amended.
Annex 3
The implementation of professional investigation and determination of the extent of probable
contamination of the originator of the kroužkovitostií or the originator of the brown rot and range
the possible extension of the notification and confirmed the occurrence of the
ring rot agent or agent of brown rot
1. Professional investigation under section 5 (3). 1 (a). (b)) is focused on:
-the place of production,
in which they have been or are grown, potatoes which are clonally
related with the plants of the lot in which the potato was confirmed infestations
the originator of the ring or the originator of the brown rot,
in which they have been or are grown tomato plants originating from the same lot
seed or planting tomato plants as plants, in which the contamination was confirmed
the originator of the brown rot,
in which they have been or are cultivated plants, and in the case of the agent
brown rot of tomato, which were also due to the presence of
the originator of the diagnosis of the cause of brown rot or undergo vocational
the investigation referred to in section 4, paragraph 4. 3,
in which they have been or are cultivated plants, and in the case of the agent
brown rot of tomato, and which also lies in the vicinity of the infected place
production, including the place of production, where they were jointly or through
common contractual partner used agricultural machinery or equipment,
which was used to závlaze or spraying of surface water resources in
which it was confirmed that contaminated the originator of the brown rot, or that
is the originator of the infestation of brown rot suspicious,
-on which was used to závlaze or spraying of surface water
the source, which confirmed that it is infested with the originator of the brown rot
or who is of contamination by the originator of the suspect, brown rot
that was or is flooded with surface water contaminated by the originator Brown
rot or surface water from this contamination suspected,
and
-surface water that was used to závlaze or spraying of land
or place of production, in which it was confirmed that they are contaminated by the originator of the
brown rot, or that this field or place of production has flooded.
2. In the determination of the extent of probable contamination, the originator of the
diagnosis of the cause of brown rot or under section 5 (3). 1 (a). (b))
to be assessed, in particular:
-host plants grown at a place of production designated as
infected under section 5 (3). 1 (a). and)
-the place of production or premises with some type of connection to the
the host plants designated as contaminated under article 5(4). 1 (a). and)
including those that have joint production plants and equipment, and in the
which have been jointly used agricultural machines, and it directly or
through a common contractor,
-host plants produced in the place (s) of production referred to (-s)
in the previous indent, or present in such place (s) (s)
during the period when the host plants designated as contaminated under article
5 (3). 1 (a). and there were in such areas) or places
production referred to in the first indent,
-Joint warehouses storing the host plants of the above places
production,
-equipment, machines, means of transport, warehouse or its part and
any other objects including packaging material, that may have come into
contact with host plants designated as contaminated under article 5 (3).
1 (a). and) during the previous 12 months or a shorter period, if the
It is technically justified,
-host plants, which are stored or are in contact with the
structures or objects listed in the previous indent, to
implementation of the cleansing and disinfection of such structures and objects in a way
laid down in annex 6,
– host plants, which are based on testing in accordance with § 6
the same clonal origin as host plants designated as contaminated
under section 5 (3). 1 (a). and), and for which the plant health management
the basis of the professional investigation under section 76, paragraph. 2 the Act marks due to the
clone connection likely contamination, including plant
tested with a negative result; in case of doubt about the identity of
contaminated and clonally related host plants of phytosanitary
the administration shall determine the testing of the variety,
-the place of production, in which the host plants listed in
the previous indent grown or stored,
-the place of production, in which the surface water used for spraying or
for irrigation of the origin of the resources identified under section 5 (3). 2 (a). (b))
as the originator of the brown rot infested,
-host plants of the agent of brown rot on the grounds
the flooded water, designated under section 5 (3). 2 (a). (b)) for the contaminated
the originator of the brown rot.
3. In determining the extent of the possible extension of ring rot agent or
the causative agent of brown rot under section 5 (3). 1 (a). (c)) must be assessed
in particular:
-the proximity of other places of production growing potatoes or other
the host plants,
-the place of planting or use stored seed potatoes together
-the place of production, on which are the host plants irrigated or
postřikovány surface water, if it exists, or existed
the danger that this water has flooded the place of production designated under section 5 of
paragraph. 1 (a). and as contaminated by the originator), brown rot,
-the place of production, on which are the host plants grown
immediately adjacent to the surface water designated under section 5 (3). 2 (a). (b))
as contaminated by the originator of brown rot, or that may be affected by this
surface water flooded,
-each individual irrigation reservoir, which is associated with the surface
water identified under section 5 (3). 2 (a). (b)) for the contaminated causing the Brown
rot,
-water objects that are associated with the surface water designated in accordance with
§ 5 (3). 2 (a). (b)) in the originator of the brown rot infested, with
taking account of the direction of flow and amount of flow of the water and to volunteer
increasing host plants of the agent of brown rot of the family
Solanaceae.
4. the formalities for the notification of the occurrence of ring rot agent confirmed or
the causative agent of brown rot under section 5 (3). 3.
4.1. immediately after the confirmation of the presence of ring rot agent or
brown rot laboratory tests using the methods set out in the
Annex No 1 shall notify the Commission and the Member States at least:
-the variety name of the lot concerned potato, kind of supply (drinking,
seedlings, etc.), in the case of seed of the category of the propagating material,
-the variety name of the lot concerned, tomato and, where appropriate, of the category
propagating material.
Without prejudice to the requirements for the reporting of suspected occurrence, the originator of the
diagnosis of brown rot or pursuant to section 4, paragraph 4. 4, if it is confirmed by the
occurrence in the game of potato or tomato plants originating from another Member
State (of other Member States) or there is a risk of contamination by potato or
tomato in another Member State (the other Member States) must be
shall immediately be notified to the State concerned (the States concerned) the information necessary to
to meet the measures according to section 5 (3). 4, in particular:
-the variety name of the lot concerned potato or tomato plants,
-the name and address of the consignor and the consignee of a batch,
-the date of delivery and the quantity of the delivered batches,
-the identification of the appropriate phytosanitary certificate under section 21
paragraph. 1 (a). (d) where applicable, a copy of the law) or number of the plant
the passport or registration number of the grower or merchant and a copy of the packing slip
the worksheet.
4.2. After all, the investigation shall be notified:
-the date of the confirmation of the designated contamination,
-a brief description of the investigation carried out to determine the source and possible
spread of the contamination, including the extent of the sampling carried out,
-information about the identified or presumed sources (sources) contamination,
-details of the extent of the contamination, including the number of places of production and the number of
lots with an indication of the variety and for seed potatoes of a category of propagating
material,
-details of the established quarantine territory and security zone, including the
the number of places of production, not designated as contaminated, but are
in the quarantine the territory or in the safety zone,
-in the case of the agent of brown rot for details on determination of water resources,
including the name and location of the water object and scope of the marking area
the prohibition of irrigation;
-for any tomato plant consignment or lot designated for
contaminated, appropriate phytosanitary certificate pursuant to § 21. 1
(a). (d)) of the Act and the number of the said plant Passport;
-For more information related to a confirmed (i) source (source) contamination,
where appropriate, the Commission shall request.
Annex 4
Measures following confirmation of the presence of ring rot agent or agent
brown rot
1.
The way of loading with the lots designated as contaminated the host plants
With host plants, designated as contaminated under article 5 (3). 1
(a). and) must be loaded on the basis of the regulation of plant health management
According to § 7 (2). 1 (a). and some of the below) manners:
and transportation to the controlled landfill) and immediate převrstvení suitable material
(soil, debris, etc.) so, to prevent theft and to further
the spread of ring rot agent or agent of brown rot; may be
While used just such a dump, for which there is no danger
leakage on the agricultural land arable land or, in the case of tubers
the originator of the brown rot infected into surface waters used for
irrigation of agricultural land;
(b)) in the case of potato tubers in the pařících heat treatment facilities,
where are the tubers exposed to a temperature of 75 ° C for at least
ten minutes, and then be fed to farm animals;
(c)) in the case of potato tubers, through direct and immediate delivery
batch in a processing plant for heat treatment of tubers
industrial or food purposes, which are the tubers exposed
temperature of at least 75 ° C for at least 10 minutes or, in the case of
loading with lots on the territory of the Czech Republic, processed in a
technological procedure, approved by the phytosanitary administration
excludes the distribution of ring rot agent and the agent of brown rot;
the movement of contaminated lots enables the phytosanitary administration only to enterprises
This administration in writing for this purpose approved, on the basis of
Verify that in the enterprise system of cleansing and disinfection of storage
space and departing vehicles and that the firm meets the requirements for the
disposal or destruction of waste, laid down in annex No 5,
(d) in the case of ring rot agent) handling of tubers with silážováním
silage corn in terms of cut tubers and their uniform
layering in the entire volume of the preserved corn; plant health
the Administration will examine the specific conditions of the silage before using this
measures and sets out specific procedures to eliminate the possibility of the spread of
the originator of the ring,
(e) other action) has been approved in writing in advance to plant
the Administration, if it has been demonstrated that there is no identifiable risk of the
extension of ring rot or agent of brown rot, such measures
phytosanitary Administration must be ordered by a certain person, including
the determination of the conditions for risk-free loading of the waste,
f) combustion taking account of the special law ^ 1).
When disposing of any waste associated with the above
options, or arising from the above options must be satisfied
waste disposal requirements set out in annex 5.
The measures under (a)) can be used to load with the lots marked
as contaminated just in case, when it was not possible to completely use the procedure in the
(a) (b)) to (e)). The measures imposed in accordance with subparagraphs (d) and (e)))
phytosanitary administration shall immediately communicate to the Commission and other Member
States. If the measures in subparagraphs) to (e)) carried out on the territory of the
another Member State, the person shall inform the povinnná
phytosanitary administration, which shall notify this information to the official organization
plant health care, the Member State concerned.
2.
The way of loading with host plants marked for
likely to be contaminated
With host plants, designated as probably contaminated
under section 5 (3). 1 (a). (b)), it must be loaded on the basis of regulation
plant health management according to § 7 (2). 1 (a). (b)) by one of the below
the following:
consumption of potato tubers) packaging adapted for direct delivery to the place of
the consumption and use without repacking as ware potatoes, at the place with the
appropriate waste disposal according to the requirements laid down in
Annex No 5; moving parts suspected of contamination by the originator of the Brown
rot enables the phytosanitary administration only to jobs by the Administration
approved for that purpose in writing, and on the basis of the verification that, in undertaking
System disinfection of storage areas and departing vehicles and the
that the firm meets the requirements for the disposal of waste, laid down in the
Annex No 5,
(b)) in the case of potato tubers in the pařících heat treatment facilities,
where are the tubers exposed to a temperature of 75 ° C for at least
ten minutes, and then be fed to farm animals;
(c)) in the case of potato tubers, through direct and immediate delivery
batch in a processing plant for heat treatment of tubers
industrial or food purposes, which are the tubers exposed
temperature of at least 75 ° C for at least 10 minutes or, in the case of
loading with lots on the territory of the Czech Republic, processed in a
technological procedure, approved by the phytosanitary administration
excludes the distribution of ring rot agent and the agent of brown rot;
moving parts suspected of contamination, authorises phytosanitary administration
only to undertakings by the Administration in writing for that purpose on the territory of the Czech
Republic approved, on the basis of the verification that, in undertaking works
a system of disinfection of storage areas and departing vehicles and the company
meets the requirements for the disposal of waste, laid down in annex No 5,
(d) in the case of ring rot agent) handling of tubers with silážováním
silage corn in terms of cut tubers and their uniform
layering in the entire volume of the preserved corn; plant health
the Administration will examine the specific conditions of the silage before using this
measures and sets out specific procedures to eliminate the possibility of the spread of
the originator of the ring,
(e) in the case of ring rot agent) and propagating material in potato
planting tubers in the quarantine the territory defined pursuant to section 5 (3). 1 (a). (c))
under the conditions that:
-tubers were produced in this quarantine territory, and it has been demonstrated that the
not to their immediate contact with the tubers of a batch marked for
contaminated,
– quarantine territory not in closed growing area for the production of
propagating material of potato under a special law ^ 2)
– the stand will be controlled by phytosanitary administration and tested according
Annex No 1 on the occurrence of the originator of the ring,
-tubers harvested from the crop will be used only in the manner
referred to in subparagraph (b)). (d)), unless the tubers from the lot designated as the
contaminated,
(f) other action) has been approved in writing in advance to plant
Administration and in which there is no identifiable risk of spreading of the originator
ring rot nor the agent of brown rot, such measures must be
phytosanitary Administration ordered a mandatory person including
the determination of the conditions for risk-free loading of the waste.
When disposing of any waste associated with the above
options, or arising from the above options must be satisfied
waste disposal requirements set out in annex 5.
The measures ordered in accordance with points (d)), e) and (f))
phytosanitary Administration immediately to the Commission and the other Member States.
If the measures in subparagraphs) to (d)) carried out on the territory of another
the Member State required the person about it in advance, inform the
phytosanitary administration, which shall notify this information to the official organization
plant health care, the Member State concerned so as to make it possible
ensure the official control over the fulfilment of the measures ordered.
3.
The treatment of the whole plant and post-harvest residues to plant All plants
a batch of suspected of contamination or infested is removed from the land, or
the object and stored in controlled landfills in accordance with the wording in paragraph 1.
(a). and) or burned in a way that does not breach statutory
the provisions on the protection of the atmosphere. This way is recommended especially for
tomato plants.
Cultivated plants of a batch of suspected of contamination or infested it is possible
in the case of ring rot agent instead of the measures referred to in points 1.
and 2. or incineration to dispose of a herbicide to the time when you can
guarantee and liquidation of underground plant parts, and consequently the vegetation God.
The measures ordered under this section, inform the phytosanitary administration
immediately notify the Commission and the other Member States.
4.
The quarantine measures in the territory after the confirmation of the causative agent
ring rot
4.1. In the quarantine area that includes a place of production designated as
the originator of ring rot infested, pursuant to section 5 (3). 1 (a). and)
phytosanitary administration after confirmation of ring rot agent
orders under section 7 (2). 1 (a). (d)), that the user of the land in this area:
4.1.1. the land designated as infested with the originator of the kroužkovitostií according to the
§ 5 (3). 1 (a). and):
and) for three cycles of the period following the year of confirmation
the presence of the agent of the potato ring rot grow including seed, and must
systematically search for and dispose of wild potato and
other wild host plants of ring rot agent (§
76 (1) (a). point 2). the Act)
or
This plot of land after four growing seasons to maintain as black fallow
or after the first year of the black úhoru for three cycles
zatravnit or sow clover, alfalfa or jetelotrávou
vojtěškotrávou and stand on it to keep mowing as the lowest
stubble, or graze intensively, and it must search for and delete
wild potato plants and other naturally found host
plants of ring rot agent (§ 76 para 1), point 2. of the Act);
(b)) may grow potatoes in the first year following the fulfilment of the
the provisions referred to under (a). and), but only with the proviso that
-during the previous two years was based on the written request of the
the user of the land to plant the Administration verified that the plot was
found free from volunteer potato plants and other naturally
found host plants of the originator of the ring,
-will be used for planting, propagating materiál3) or farm sadba4)
produced under the control of plant health management and tested according
Annex No 1 on the occurrence of the originator of the ring with a negative result, and
-harvest will be intended only for drinking purposes or industrial
processing (§ 76 para 1) point 1. the Act)
(c)) after completion of the other emergency phytosanitary measures
ordered in the quarantine the territory referred to in point 4 may in the next harvest
the period following the appropriate rotation cycle, which shall be
at least 2 years, grow potatoes or seed on consumerism with the condition,
the phytosanitary Administration will carry out a survey of the occurrence of the originator of the enclosing
ring rot under section 3 (3). 3, will be used for planting, propagating
material ^ 3) or farm seed ^ 4) cultured under control
the phytosanitary administration and tested according to annex No 1 on the occurrence of the originator
ring rot with the result of the negative, and the harvested tubers shall be tested
According to annex No 1 on the occurrence of the originator.
4.1.2. the land other than designated as infested with the originator of the
ring rot under section 5 (3). 1 (a). and), which was in
confirmation of the causative agent of the potato ring rot grown, not after
for the two following cycles grow potatoes; /§ 76
(1) (a). and point 1). law/;
4.1.3. the land other than designated as infested with the originator of the
ring rot under section 5 (3). 1 (a). and), which was not in
confirmation of the causative agent of the potato ring rot grown, may only be in
growing season, which is followed by the year of confirmation of the causative agent
ring rot, grow potatoes, but only on condition that
-phytosanitary Administration finds no risk of the spread of protokolárně
ring rot agent volunteer potato plants or other
naturally found host plants of the originator of the ring, and
-harvest will be intended only for drinking purposes or industrial
processing
/section 76, paragraph 1, point (a). and point 1). law/;
4.1.4. for four complete cycles of the confirmation
the presence of ring rot agent to find and dispose of the wild
potato plants and other naturally found host plants of the originator
ring/section 76, paragraph 1, point (a). point 2). law/and harvested potatoes
must be tested in accordance with Annex No 1 [section 76, paragraph 1 (b), (d))
the law];
4.1.5. must secure separate handling and separate storage
^ 3) propagating material and the seed potato farmers ' ^ 4) produced
under the control of plant health management and tested according to annex No 1 on the
the presence of ring rot agent with the result of the negative from the other
potato/§ 76 (1) (a). and point 1). law/;
4.1.6. shall in the manner set out in annex No 6:
-once to perform the cleaning and disinfecting of the objects and items that
they came into contact with the contaminated lots or lots may
contaminated, and no later than that time before they come into contact with other
host plants/parts of section 76, paragraph 1, point (a). (c)))/Act;
– carry out the cleaning of machinery, transport equipment, storage space,
sorting lines, containers, pallets and other items after each use when
production of potato tubers for the time specified phytosanitary Administration [section 76
paragraph. 1 (a). point 2). the law]; cleansing machines is carried out after their
their work on one piece of land with vegetation, at least one potato
the cleansing of the storage spaces, containers, pallets, třídiček, transport
resources and other objects always pick one lot or
their work with one of the lots;
-perform for three full cycles of the confirmation of the occurrence of the
the originator of the ring final cleaning after harvesting techniques
the harvest and the final cleansing and disinfection of all equipment, transport
resources, sorting lines, containers, pallets, and other objects used
When post-harvest, loading and removal, which must be
terminated before using/§ 76 (1) (a). point 2). law/;
4.1.7. may be for growing potato after a period of three years following the year
the diagnosis of ring rot agent only use propagating
material ^ 3) or farm seed ^ 4) cultured under control
the phytosanitary administration and tested according to annex No 1 on the occurrence of the originator
with the result, negative ring/section 76, paragraph 1, point (a). and point 1).
law/;
4.1.8. may not dropping off a sliced potato tuber or dropping off the tubers
potato using impale on the exposition/section 76, paragraph. 1 (a). and point 1).
law/;
4.1.9. may not reproduce ^ 1) propagating material or for custom
the need for the vegetation period immediately following the growing
the period during which, or from which the harvest was the presence of ring rot agent
detected/section 76, paragraph. 1 (a). and point 1). law/;
4.1.10. may move outside this territory only a batch of potato, which
they were the official laboratory testing found free from the originator
ring rot and lot marked phytosanitary administration as
contaminated or potentially contaminated in the fulfilment of all the conditions in the
nařízeném measures/section 76, paragraph. 1 (a). and point 1). law/.
The harvested Potato tubers packaging adapted for direct delivery to the place of
consumption on the territory of the Czech Republic and intended for use without repacking
as ware potatoes can move outside the quarantine territory immediately after the
Subscribe to the official sample if the lot has been inspected at the time of the
harvest to plant the Administration on the occurrence of the symptoms of the originator
ring rot, including tours of the cut flesh of the tuber, with negative
as a result.
4.1.11. may export plants propagated in this territory to the
of third countries only if they complied with official import quarantine
the conditions laid down by the authority of the importing country phytosanitary care/§ 76
paragraph. 1 (a). and point 1). law/;
4.1.12. must not use agricultural tools and machinery,
used in the cultivation, harvesting, post-harvest, transport and
storage potatoes that came into contact with the infected parts, after
period of one year from the time of confirmation of the occurrence of the originator of the outside of the ring
This territory without proper cleaning and disinfection operations carried out in the manner specified in the
Annex No 6/section 76, paragraph. 1 (a). point 2). law/;
4.2. in confirmation of the causative agent of ring rot in production systems, where
It is possible the complete replacement of the growing medium (e.g. in glasshouses
breeder's or in the cultivation of minihlízek, meristémů, etc.) user
land:
and grow potatoes) must not, including seed, if you are not under the control of
the phytosanitary Administration to eradicate the originator of the ring, including
remove all plants or other plants of the family
Solanaceae, the complete replacement of the growing medium and cleansing and disinfection
all objects, machinery and equipment referred to in
Annex No 6/section 76, paragraph 1, point (a). and the law/);
(b)) must establish more potato cultivation of propagating material
derived from plants to test for the presence of ring rot agent with
negative result/§ 76 (1) (a). and point 1). law/.
4.3. Determine volunteer potato plants in quarantine territory
in accordance with section 4.1.4. the user must land in this area report
plant health management, and prior to disposal, these plants must allow
their official testing for the presence of the agent. When
the diagnosis of ring rot agent when this testing is
the land it considers land infested/§ 76 (1) (a). and)
point 2. law/.
4.4. the phytosanitary Administration conducts random surveillance of land and
objects, which are grown or stored, potatoes or with
These plants are treated, in particular the implementation of cleansing and disinfection
machinery that is used in the stands and in the harvested potato
Potato tubers also in other places of production.
4.5. In the safety zone, as defined in the place of production, on the basis of the
probable contamination and possible extension or the originator
ring rot under section 5 (3). 1 (a). (c)), the phytosanitary administration
orders under section 7 (2). 1 (a). (d)), that the user of the land in this area
must for three full cycles of the confirmation of the occurrence of the
the originator of the ring:
and final cleaning) harvesting techniques after their harvest and
final cleansing and disinfection of all machines, means of transport,
sorting lines, containers, pallets, and other objects used in the
post-harvest, loading and removal, which shall be completed
before starting the new production of the season/§ 76 (1) (a). point 2).
law/;
(b)) must secure separate handling and separate storage
propagating material of potato ^ 3) ^ 4) farm seed propagated
under the control of plant health management and tested according to annex No 1 on the
the presence of ring rot agent with the result of the negative from the other
potato [section 76, paragraph 1 (b), point 1). the law];
(c)) must only be used for the cultivation of potato planting materiál3) or
farm seed ^ 4) grown under the control of plant health management
and tested according to annex No 1 on the presence of ring rot agent with
the result of the negative/§ 76 (1) (a). and point 1). law/.
5.
The quarantine measures in the territory after the confirmation of the causative agent of brown rot
5.1. In the quarantine area that includes a place of production designated as
the originator of the brown rot infested, pursuant to section 5 (3). 1 (a). and) or
the surface water designated as contaminated under article 5 (3). 2 (a). (b)),
phytosanitary administration after the confirmation of the causative agent of brown rot
orders under section 7 (2). 1 (a). (d)), that the user of the land in this area:
5.2.1. on land designated as infested with the originator of the brown rot under section
5 (3). 1 (a). and):
and) shall not for a period of four cycles grow host plants
the causative agent of brown rot including seed potato and tomato plants and other plants
proven to allow his transfer, i.e.. the plants, which are harvested
the parts that are in direct contact with the soil and to which the following
the harvest of the Earth stays (e.g. plants intended for cultivation with the roots,
some species of the genus Brassica, bulevnaté, onion flowers and tubers)/section
paragraph 76. 1 (a). and point 1). law/, and must find and dispose of
Wild host plants including weed/section 76, paragraph. 1 (a).
point 2). law/or
(b)) must land after the first three growing seasons to maintain as a black
fallow or cultivate cereals, often chopped or intensely
forage or grazed by the permanent grass seed/section 76, paragraph. 1 (a). point 2).
law/and in the next two years on it may grow only nehostitelské
plants that do not allow survival or transfer agent of brown rot, and
It must be in the course of these five years to find and dispose of volunteer
host plants including weed/section 76, paragraph. 1 (a). and)
law/;
(c) in the case of mode), may in accordance with subparagraph (a)) to grow the potato or tomato in
the first growing season following the fulfilment of the provisions of the
referred to under (a)), but only with the proviso that
– during the previous two years was based on the written request of the
the user of the land to plant the Administration verified that the plot was
found free of flora, potato, tomato and other
host plants including solanaceous weeds, and
– in the case of the cultivation of potatoes will be used for planting, propagating
material ^ 3), and
– the harvest will be intended only for drinking purposes or industrial
processing,
– in the case of potato and tomato plants will be harvested Potato tubers, or
tomato plants tested in accordance with Annex No 1;
then may grow a potato or a tomato until after the appropriate rotation
crops provided for plant protection in the case of the cultivation and administration
potatoes for planting propagating material used ^ 3), and the harvest must
be intended only for drinking purposes or for industrial processing,
for cultivation of seed potatoes must be made in advance
take-up survey of the occurrence of the cause of brown rot under section 3 (3). 3 [section
paragraph 76. 1 (a). and point 1). the law];
(d) in the case of mode), may in accordance with subparagraph (b)) to grow the potato or tomato in
the first growing season following the fulfilment of the provisions of the
referred to under point (b)), but only with the proviso that
– during the previous two years was based on the written request of the
the user of the land to plant the Administration verified that the plot was
found free of flora, potato, tomato and other
the causative agent of brown rot of host plants, including weed from family
Solanaceae, and
– in the case of the cultivation of potatoes will be used for planting, propagating
materiál3), and
-potatoes may be grown for seed or consumerist purposes or to
the processing industry, and in the case of potato and tomato plants will be harvested
Potato tubers or plants of tomato, tested the procedure laid down in
Annex No 1 [section 76, paragraph 1 (b), point 1). the law];
(e)) may, after fulfilment of the other emergency phytosanitary measures
ordered in the quarantine the territory referred to in section 5 for the first time on this land
grow potatoes or other host plant of the agent Brown
rot, only applies when propagating material ^ 3), which was officially
recognised by a special Act ^ 5), and of the intended first cultivation
host plants on this land to inform in writing
phytosanitary administration in that time, to be able to perform
take-up survey of the occurrence of the cause of brown rot under section 3 (3). 3.
(f) shall not be carried out) artificial irrigation for five cycles
following confirmation of the causative agent of brown rot/§ 76
paragraph. 1 (a). point 2). law/.
5.2.2. on the land other than designated as infested with the originator of the Brown
rot under section 5 (3). 1 (a). and):
and in growing period), the following growing season, in which the
or of whose harvest has been detected the presence of the causative agent of brown rot, including
seed potato and tomato plants, grow potatoes or other host
plants of the agent of brown rot and must find and dispose of the targeted
Flora of tomato, potato and other host
plants, including host species weed/§ 76 (1) (a). and) point
2. law/, or
– to grow potatoes, provided that the plant will be used only
propagating materiál3), which was officially recognized by the Special
zákona5), and production is intended exclusively for potable purposes or to
processing [section 76, paragraph 1 (b), point 2). the law],
– may grow tomato only from seed which meets the requirements referred to in
special legal předpisu6), and only for fruit production [section 76
paragraph. 1 (a). point 2). the law];
(b)) may be for a period of two years following the year of implementation of the measures referred to in
(a))
– use only to the cultivation of potato planting materiál3) or
farm seed ^ 4) grown under the control of plant health management
and tested according to annex No 1 on the occurrence of the cause of brown rot with
the result, negative
– for growing tomato plants use only seed which meets the requirements of
under special legislation ^ 6), or tomato plants
produced from the seed of the plant under the supervision of the management of [section
paragraph 76. 1 (a). point 2). the law];
(c)) shall, immediately after the indication of the place of production of the contaminated and subsequently to the
the time of the first permissible potato or tomato plants growing every year
perform the cleaning and disinfecting of all machinery and storage facilities
used in the cultivation of the potato or tomato plants in the manner specified in the
Annex No 6 [section 76, paragraph 1 (b), point 2). the law];
(d)) may be carried out only with the consent and the moisture under the supervision
the phytosanitary administration, provided that there is no possibility of any
identifiable risk of spreading the cause of brown rot/section 76, paragraph. 1 (a).
point 2). law/,
(e)) should for three full cycles of the confirmation of the occurrence of the
search for cause of brown rot and dispose of flora
potato and other naturally found host plants of the originator of the Brown
rot [section 76, paragraph 1 (b), point 2). the law].
5.3. not dropping off a sliced potato tuber or dropping off the tubers
potato using impale on the exposition/section 76, paragraph. 1 (a). and point 1).
law/;
5.4. allowed to move outside this territory only a batch of potato that were
the official laboratory testing found free from the causative agent of brown rot
and the lot designated as contaminated the phytosanitary administration or
probably infected if you meet all the conditions in nařízeném measures
/section 76, paragraph 1, point (a). and point 1). law/. The harvested Potato tubers packaging
adapted for direct delivery to the place of consumption on the territory of the Czech Republic and
designed for use without repacking as ware potatoes can move
outside quarantine territory immediately after collection of the official sample, if the
the relevant batch inspection of the plant at the time of harvest management
on the occurrence of the cause of brown rot symptoms, including the cutting of the flesh tours
tubers, with negative results.
5.5. shall not export plants propagated in this territory to third
countries only if they complied with official import quarantine conditions,
laid down by the authority of the importing country phytosanitary care/section 76, paragraph. 1
(a). and point 1). law/;
3.5. shall not use agricultural tools and machinery,
used in the cultivation, harvesting, post-harvest, transport and
storage potatoes that came into contact with the infected parts, after
period of one year from the time of confirmation of the causative agent of brown rot out
This territory without proper cleaning and disinfection operations carried out in the manner specified in the
Annex No 6/section 76, paragraph 1, point (a). point 2). law/;
5.7. in confirmation of the causative agent of brown rot in production systems, where
It is possible the complete replacement of the growing medium (e.g. in glasshouses
breeder's or in the cultivation of minihlízek, meristémů, etc.) user
land:
and may not grow potatoes) or other host plants of the agent Brown
including seed rot of potato and tomato plants, unless under control
the phytosanitary Administration to eradicate the cause of brown rot, including
delete all of the potato and the tomato, the complete replacement of the growing
medium and cleansing and disinfection of all the buildings, machinery and equipment
made in the manner indicated in the annex No 6/section 76, paragraph. 1 (a). and),/;
(b)) must establish more potato cultivation after the consent of the plant
administration of planting materiálu3) officially recognised under the Special
zákona5) or obtained from the plants to test for the presence of the agent Brown
rot with negative result/section 76, paragraph. 1 (a). and point 1). law/;
(c)) must be used only for the cultivation of tomato seed, which corresponds to the
the requirements under the special rule of předpisu6), or tomato plants
produced from the seed of the plant under the supervision of the management of [section
paragraph 76. 1 (a). point 2). the law];
(d)) may be artificially irrigated cropping of potato or tomato plants only with the consent of
plant health and plant health management will allow the Administration to carry out
official control of irrigation systems in the quarantine territory/§ 76 (1)
(a). point 2). law/.
3.6. the findings of the flora in the potato or tomato plants
quarantine territory according to paragraph 5.2.2. (a). and the user must land in)
This report and the administration of the territory of the plant health before disposing of these
plants must make their official testing for the presence of the agent
brown rot. When the confirmation of the causative agent of brown rot in this
testing the competent land considered land infested/§ 76
(1) (a). point 2). law/.
3.7. the phytosanitary Administration conducts random surveillance of land and
objects in which plants are grown or stored potato and tomato plants
or will these plants be treated confidentially, in particular the implementation of cleansing and
disinfection of machinery, which is used in the stands and potato
tomato and Potato tubers harvested in other places
the production.
5.10. In the safety zone, as defined in the place of production, on the basis of the
probable contamination or possible spread of the agent of brown rot
under section 5 (3). 1 (a). (c)), the phytosanitary administration orders pursuant to section 7 of the
paragraph. 1 (a). (d)), that the user of the land in this area must, for a period of three
complete growing period of the confirmation of the occurrence of the cause of brown rot:
and final cleaning) harvesting techniques after their harvest and
final cleansing and disinfection of all machines, means of transport,
sorting lines, containers, pallets, and other objects used in the
post-harvest, loading and removal, which shall be completed
before starting the new production of the season/§ 76 (1) (a). point 2).
law/;
(b) secure the separate handling and) separate storage of propagating
material ^ 3) potato and farm sadby4) grown under control
the phytosanitary administration and tested according to annex No 1 on the occurrence of the originator
brown rot with negative result from other potato [§
paragraph 76. 1 (a). and point 1). the law];
(c)) for the cultivation of potato to use just propagating materiál3) or
farm seed ^ 4) produced under the control of plant health management and
tested according to annex No 1 on the occurrence of the cause of brown rot, with the result
negative [section 76, paragraph 1 (b), point 1). the law];
(d)) for the cultivation of tomato, use only seed which meets the requirements of
under special legislation ^ 6), or tomato plants
produced from the seed of the plant under the supervision of the management of [section
paragraph 76. 1 (a). point 2). the law].
5.11. In the quarantine the territory defined pursuant to section 5 (3). 2 (a). (c)) on the
the basis of the designation of contaminated surface water for the originator of the brown rot
or in the safety zone designated under section 5 (3). 2 (a). (c))
phytosanitary Administration:
and a survey of the occurrence of each year) is the causative agent of brown rot, and it
sampling of surface water and possible host plants of the family
the water resources Solanaceae as laid down by
phytosanitary administration and then by testing in accordance with the relevant
the methods set out in annex 1;
(b)) control irrigation and spraying systems and, where appropriate,
lays down conditions of artificial irrigation of host plants
the causative agent of brown rot, to prevent its spread, including the prohibition of
use of the water designated as contaminated for the irrigation and spraying
host plants of the causative agent of brown rot. This ban may be
revoked in the case, which is based on the results of the survey carried out by the
evidence that surface water is no longer infected, or that are used by the
for the irrigation and spraying technology approved plant protection
the Administration, which exclude the presence of the causative agent of brown rot and its dissemination;
(c)) in the case of contamination by sewage disposal control
contaminated waste water from industrial processing and packaging
plants which handle with the potatoes.
-----------------
1) Act No. 86/2002 Coll., on the protection of the atmosphere and some of the other
laws (the law on the protection of the atmosphere), as amended.
2) section 7 (1). 4 of Act No. 219/2003 Coll., on marketing of seeds and plants
cultivated plants and amending certain laws, as amended
regulations.
3) § 2 (2). 1 (a). (b)) Act No. 219/2003 Coll., on the circulation of
the seed and the seed of cultivated plants and amending certain laws, as amended by
amended.
4) section 19a of Act No. 408/2000 Coll., on the protection of rights to plant varieties and
Amendment of the Act No. 92/1996 Coll., on plant varieties, seeds and seed potatoes grown
the plant, in the wording of later regulations, (law on the protection of rights to
varieties), as amended.
5) section 6 of Act No. 219/2003 Coll., on marketing of seeds and plants
cultivated plants and on amendment to certain laws.
6) Annex No 4 of Decree No 215/2008 Coll., on measures against the introduction into the
and the dissemination of harmful organisms of plants or plant products.
Annex 5
The requirements on the disposal of waste in the industrial processing of the lot
the host plants, designated as contaminated under article 5 (3). 1 (a). and)
or likely to be contaminated under article 5 (3). 1 (a). (b))
The method and place of disposal of the waste in the industrial processing of the lot
the host plants must be in accordance with the particular legal
Regulation ^ 1) Phytosanitary administration allowed the firm to handle
contaminated or potentially contaminated batches of the host plants for the
industrial or food purposes in accordance with annex 4 paragraph 1 (c).
(c) and paragraph 2 (a)). (c)) must satisfy the following requirements on the
disposal of waste, in order to avoid any risk of the spread of the agent
ring rot or the agent of brown rot:
1. Solid waste from processing tubers of the potato and the tomato plant Waste
Potato tubers, potato peels or fruits of tomato and other solid waste
resulting from the processing of potato tubers (e.g. soil, stones, etc.)
tomato plant must be:
and removed to a controlled landfill) waste and immediately převrstveny the appropriate
material (soil, debris, etc.). Waste must be brought directly to the
landfill and must be packed in such a way that during transport is excluded
the danger of falling out of the waste from means of transport.
Can be used for just such a landfill, which is out of the question
the risk of leakage of ring rot agent or brown rot in the environment,
for example. percolation into the agricultural arable land or, in the case of
tubers infected by the originator of brown rot, into surface waters used
for irrigation of agricultural land, or
(b)) burned, taking into account special law ^ 2), or
(c) disposed of by other measures), which has been approved in writing in advance
phytosanitary administration and if it has been demonstrated that there is no
recognizable risk or the originator of the ring extension Brown
rot. These measures shall be communicated to the Commission and the phytosanitary administration
the other Member States.
2. Waste water processing tubers of potato and tomato plants Before
disposal of waste water must be, that contain on the surface of the hard
floating material, filter or divert to the settling tanks to
These materials have been deprived of water drifting. Deposits must be
disposed of according to the procedure referred to under point 1.
Waste water is then:
– before being introduced to warm up for at least 30 minutes at a temperature of
a minimum of 60 ° c inside the volume,
or
-otherwise disable way beforehand by the phytosanitary administration
so, to avoid any risk that the waste comes into contact with agricultural
land or water sources which could be used for irrigation
agricultural land; the details of this method of disposal shall communicate to the
the phytosanitary administration of the other Member States and the Commission.
-----------------
1) Law No 185/2001 Coll., on waste, as amended by later regulations.
2) Act No. 86/2002 Coll., on the protection of the atmosphere and some of the other
laws (the law on the protection of the atmosphere), as amended.
Annex 6
Cleansing and disinfection of objects and items contaminated or suspected of
contamination of the originator of the ring or the originator of the brown rot
1. Mechanical cleansing of objects and items and disposal of debris and residues
All solid impurities, the remains of plants and tubers are collected (smetením,
industrial vacuum-cleaning it with a vacuum cleaner or vyfoukáním inaccessible places
machinery and equipment with compressed air) and take to the landfill odpadu1).
For safer disposal is appropriate material in advance by further disinfected
that procedure or převrstvením Earth prosypat chlorine
lime or burnt lime in a quantity of 5 kg on 1 m3.
The method and place of disposal must be in accordance with the particular legal
předpisem1) and must be approved by the phytosanitary administration.
2. Washing and disinfection of objects and subjects
Any application of disinfectant or resource must
be preceded by a thorough mechanical cleansing of disinfected objects and
articles referred to in point 1 of this annex. Subsequently, machines and objects
equipment wash water pressure with detergent, rinse with clean water and
leave to dry. Then disinfect and available for use against
ring rot agents or agent of brown rot plant
the Administration approved products or disinfectants used
in accordance with the label or the phytosanitary administration of the approved
technological procedure.
3. Clearance items in hot water and steam
This is done by soaking for at least 30 minutes at a water temperature of 65 ° C
or 15 minutes at a water temperature of 80 ° C to the urban planning of hot steam is
stable or portable steam generators, pressure steam temperature
must be at least 80 ° C at the time of exposure of at least 30 minutes. When
a temperature of over 100 ° C exposure time can be reduced to 15 minutes.
4. Disinfection of soil and organic wastes
Propařením shall be carried out at a temperature of 120 ° C or repeated propařením
at a temperature of 70-80 ° C, or by boiling in liquid phase with water.
-----------------
1) Law No 185/2001 Coll., on waste, as amended
regulations.
1) Council Directive 93/85/EEC of 4 March. October 1985 for the protection against
bacterial ring rot of potato.
Council Directive 98/57/EC of 20 July 1998. July 1998 on protection against
Ralstonia solanacearum (Smith) Yabuuchi et al.
Commission Directive 2006/56/EC of 12 July 2005. June 2006 amending
the annex to Council Directive 93/85/EEC.
Commission Directive 2006/63/EC of 14 July 1999. July 2006 amending
annexes II to VII to Council Directive 98/57/EC.
2) § 2 (2). 1 (a). (b)) Act No. 219/2003 Coll., on the circulation of
the seed and the seed of cultivated plants and amending certain laws, as amended by
amended.
section 31, paragraph 3). 2 Decree No 215/2008 Coll., on measures against the introduction into the
and the dissemination of harmful organisms of plants or plant products.
4) § 2 (2). 1 (a). o) Act No. 219/2003 Coll., on the circulation of
the seed and the seed of cultivated plants and amending certain laws, as amended by
amended.
