Council Directive 98/57/EC of 20 July 1998 on the control of Ralstonia solanacearum (Smith) Yabuuchi et al.
Date: 1998-07-20
Institutions: Council of the European Union
Subjects: food inspection, fruit vegetable, plant disease, plant health control, potato, protection of plant life

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I

(Acts  whose  publication  is  obligatory)

COUNCIL  DIRECTIVE  98/57/EC

of  20  July  1998

on  the  control  of  Ralstonia  solanacearum  (Smith)  Yabuuchi  et  al.

THE  COUNCIL  OF  THE  EUROPEAN  UNION,

Having  regard  to  the  Treaty  establishing  the  European
Community,  and  in  particular  Article  43  thereof,

Having  regard  to  the  proposal  from  the  Commission (1),

Having  regard 
Parliament (2),

to 

the  Opinion  of 

the  European

Having regard to the Opinion of the Economic and Social
Committee (3),

Whereas  the  harmful  organism  Ralstonia  solanacearum
(Smith)  Yabuuchi  et  al.  was  previously  known  as
Pseudomonas  solanacearum 
(Smith)  Smith;  whereas
Ralstonia  solanacearum  (Smith)  Yabuuchi  et  al.  is  likely
to become the generally accepted name for the organism;
whereas the present Directive should take account of this
scientific development;

Whereas  potato  and  tomato  production  occupies  an
important  place  in  Community  agriculture;  whereas  the
potato  and  tomato  yield  is  constantly  threatened  by
harmful  organisms;

Whereas,  through  the  protection  of  potato  and  tomato
cultivation  against  such  harmful  organisms,  not  only
should productive capacity be maintained but agricultural
productivity  should  also  be  increased;

Whereas  protective  measures  to  prevent  the  introduction
of  harmful  organisms  into  the  territory  of  a  Member
State  would  have  only  a  limited  effect  if  such  organisms
were  not  controlled  simultaneously  and  methodically
throughout the Community and were not prevented from
spreading;

(1) OJ  C  124,  21.4.1997,  p.  12  and
OJ  C  108,  7.4.1998,  p.  85.
(2) OJ  C  14,  19.1.1998,  p.  34.
(3) OJ  C  206,  7.7.1997,  p.  57.

Whereas  one  of  the  harmful  organisms  on  potatoes  and
tomatoes  is  Ralstonia  solanacearum  (Smith)  Yabuuchi  et
al., the pathogenic agent of the potato brown rot disease
and  of  bacterial  wilt  in  potatoes  and  tomatoes;  whereas
disease  outbreaks  caused  by  this  pathogen  have  occurred
in  some  parts  of  the  Community  and  some  limited
sources  of  infection  still  exist;

Whereas  there  is  a  considerable  risk  to  potato  and
tomato cultivation throughout the Community if effective
measures  are  not  taken,  with  respect  to  these  crops,
to  locate  this  organism  and  determine  its  distribution,
to prevent it from occurring and spreading, and, if found,
to  prevent  its  spread  and  to  control  it  with  the  aim
of  eradication;

Whereas,  in  order  to  ensure  this,  certain  measures  must
be  taken  within  the  Community;  whereas  Member  States
must,  in  addition,  be  able  to  take  additional  or  stricter
measures  where  necessary,  provided  that  there  is  no
hindrance  to  the  movement  of  potatoes  or  tomatoes
within  the  Community,  except  insofar  as  laid  down  in
Council  Directive  77/93/EEC  of  21  December  1976  on
protective  measures  against  the  introduction  into  the
Community  of  organisms  harmful  to  plants  or  plant
products 
the
Community (4);  whereas  such  measures  must  be  notified
to  the  other  Member  States  and  to  the  Commission;

spread  within 

against 

their 

and 

Whereas  the  measures  have  to  take  into  account  that
systematic  official  surveys  are  necessary  to  locate  the
pathogen; whereas such surveys should include inspection
procedures  and,  where  appropriate,  given  that  under
certain  environmental  circumstances  the  disease  can
remain  latent  and  unobserved  both  in  the  growing  crop
of  potatoes  and  in  stored  potato  tubers,  should  include
sampling  and  testing  procedures;  whereas  spread  of  the
pathogen  within  the  growing  crop  is  not  the  most
important  factor,  but  whereas  the  pathogen  can  spread
by  surface  water  and  certain  associated  wild  solanaceous
irrigation  of  potato  and
plants,  and  therefore  the 

(4) OJ  L  26,  31.1.1977,  p.  20.  Directive  as  last  amended  by
Commission  Directive  98/2/EC  (OJ  L  15,  21.1.1998,  p.  34).

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tomato crops using contaminated water appears to pose a
risk  for  infection  of  such  crops;  whereas  also  the
pathogen  can  exist  through  the  winter  in  self-sown
(volunteer) potato and tomato plants and these may be a
source  of  infection  being  carried  from  one  season  to  the
next;  whereas  the  pathogen  is  spread  also  by  the
contamination  of  potatoes  through  contact  with  infected
potatoes  and  through  contact  with  planting,  harvesting
and  handling  equipment  or  transport  and  storage
containers  which  have  become  contaminated  with  the
organism  by  previous  contact  with  infected  potatoes;

Whereas  spread  of  the  pathogen  can  be  reduced  or
prevented  by  decontamination  of  such  objects;  wheras
any  such  contamination  of  seed  potatoes  poses  a  major
risk  for  the  spread  of  the  pathogen;  similarly  the  latent
infection  of  seed  potatoes  poses  a  major  risk  for  the
spread  of  the  pathogen  and  this  can  be  prevented  by  the
use  of  seed  potatoes  produced  in  an  officially  approved
programme  whereby  seed  potatoes  have  been  tested  and
found  free  from  infection;

solanacearum 

Whereas  the  current  knowledge  of  the  biology  and
(Smith)
epidemiology  of  Ralstonia 
Yabuuchi  et  al.  under  European  conditions  is  incomplete
and  it  is  anticipated  that  a  review  of  the  measures
proposed  will  be  necessary  within  several  seasons;
test  procedure  are
to 
similarly 
anticipated  in  the  light  of  further  research  especially  on
the  sensitivity  and  specificity  of  test  methods  in  order  to
select  and  standardise 
test  methods
available;

the  optimum 

improvements 

the 

Whereas,  for  the  determination  of  the  details  of  such
general  measures,  as  well  as  for  those  stricter  or
additional  measures  taken  by  Member  States  to  prevent
the  introduction  of  the  pathogen  into  their  territory,  it  is
desirable for Member States to cooperate closely with the
Commission  within  the  Standing  Committee  of  Plant
Health  (hereinafter  referred  to  as  ‘the  Committee’),

HAS  ADOPTED  THIS  DIRECTIVE:

Article  1

This  Directive  concerns  the  measures  to  be  taken  within
the  Member  States  against  Ralstonia  solanacearum
(Smith)  Yabuuchi 
et  al.,  previously  known  as
Pseudomonas  solanacearum  (Smith)  Smith,  (hereinafter
referred to as ‘the organism’), in order to, with respect to
the  host  plants  of  the  organism  listed  in  Annex  I
Section  1,  (hereinafter  referred  to  as  ‘the  listed  plant
material’):

(a)

locate  it  and  determine  its  distribution;

(b) prevent  its  occurrence  and  spread;  and

(c)

if found, to prevent its spread and to control it with
the  aim  of  eradication.

Article  2

1. Member  States  shall  conduct  annual  systematic
official  surveys  for  the  organism  on  the  listed  plant
material originating in their territory. In order to identify
other  possible  sources  of  contamination  threatening  the
production  of  the  listed  plant  material,  Member  States
shall  carry  out  a  risk  assessment  and,  unless  no  risk  of
spread  of  the  organism  has  been  identified  during  that
assessment,  they  shall,  in  production  areas  of  the  listed
plant  material,  conduct  targeted  official  surveys  for  the
organism  on  plants  other  than  the  listed  plant  material,
including  wild  solanaceous  host  plants,  as  well  as  on
surface  water  which  is  used  for  irrigation  or  spraying  of
listed plant material and on liquid waste discharged from
industrial  processing  or  packaging  premises  handling
listed plant material and used for irrigation or spraying of
listed plant material. The extent of these targeted surveys
shall  be  determined  according  to  the  risk  identified.
Member  States  may  also  conduct  official  surveys  for  the
organism  on  the  material,  such  as  growing  medium,  soil
and  solid  waste  from  industrial  processing  or  packaging
premises.

The  official  surveys  provided  for  in  paragraph  1

2.
shall  be  carried  out:

(a)

for  the  listed  plant  material,  according  to  the  details
set  down  in  point  1  of  Section  II,  Annex  I;  and,

(b)

for  host  plants  other  than  the  listed  plant  material,
and  for  water  including  liquid  waste,  in  accordance
with  appropriate  methods  and,  where  appropriate,
samples  shall  be  taken  and  subjected  to  official  or
officially  supervised  laboratory  testing;

(c) where  appropriate  for  other  material,  in  accordance

with  appropriate  methods.

For  these  surveys,  further  details  of  the  inspection
procedures  and  the  number,  origin,  stratification  and
timing  of  collection  of  samples  shall  be  decided  by  the
responsible  official  bodies  within 
the  meaning  of
Directive  77/93/EEC  based  on  sound  scientific  and
statistical  principles  and  the  biology  of  the  organism  and
taking  into  account  in  the  Member  State  concerned,  the
particular  production  systems  of  the  listed  plant  material
and,  as  appropriate,  of  other  host  plants  of  the
organism.

3.
The  details  and  results  of  the  official  surveys
provided  for  in  paragraph  1  shall  be  notiefied  each  year

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to  the  other  Member  States  and  to  the  Commission  in
accordance  with  the  provisions  of  point  2  of  Section  II,
Annex I. These notifications shall be submitted by 1 June
except  for  potatoes  use  as  farm-saved  seed  for  which  the
notification  shall  be  submitted  by  1  September.  The
to
details  and  results 
the  previous  year’s  production.  The  details  of  these
notifications  may  be  submitted  to  the  Committee.

for  crops  shall  be  related 

the responsible official bodies of the Member States shall,
in  relation  to  their  own  production:

(a) prohibit  the  movement  of  plants  and  tubers  from  all
crops,  lots  or  consignments  from  which  the  samples
have  been  taken,  except  under  their  control  and
provided that it has been established that there is no
identifiable  risk  of  the  organism  spreading;

The 

following  provision  shall  be  adopted 

4.
in
accordance  with  the  procedure  laid  down  in  Article  16a
of  Directive  77/93/EEC:

— the  appropriate  methods  for  the  surveys  and  the
laboratory  testing  provided  for  in  paragraph  2,  first
subparagraph,  (b).

5.
in
The  following  provisions  may  be  adopted 
accordance  with  the  procedure  laid  down  in  Article  16a
of  Directive  77/93/EEC:

— the  appropriate  methods  for  the  surveys  provided  for

in  paragraph  2,  first  subparagraph,  (c),

— further  details  of 

the  surveys  provided 

in
paragraph  2,  second  subparagraph,  with  a  view  to
ensuring  comparable  levels  of  assurance  between
Member  States.

for 

Article  3

Member States shall ensure that the suspected occurrence
or  confirmed  presence  of  the  organism  in  their  territory
shall  be  reported  to  their  own  responsible  official
bodies.

Article  4

In  each  case  of 

suspected  occurrence, 

1.
the
responsible  official  bodies  of  the  Member  State(s)
concerned  shall  ensure  completion  of  official  or  officially
supervised  laboratory  testing  using,  for  the  listed  plant
material,  the  relevant  method  set  out  in  Annex  II  and  in
accordance  with  the  conditions  specified  in  point  1  of
Annex  III,  or,  in  all  other  cases,  any  other  officially
approved  method,  in  order  to  confirm  or  refute  the
suspected  occurrence.  In  the  case  of  confirmation,  the
requirements  laid  down  in  point  2  of  Annex  III  shall
apply.

Pending 

the  confirmation  or  refutation  of  a
2.
suspected  occurrence  under  paragraph  1,  in  each  case  of
suspected  occurrence  where,  either:

(i) diagnostic  symptoms  of  the  disease  caused  by  the
organism  have  been  seen  and  a  positive  result  in  the
rapid  screening  test(s),  as  specified  in  Annex  II
section  I,  point  1  and  section  II  has  been  obtained;
or,

(ii) a positive result in the screening test(s) as specified in
Annex  II  section  I,  point  2  and  section  III  has  been
obtained,

(b)

take  steps  to  trace  the  origin  of  the  suspected
occurrence;

(c)

introduce  appropriate  additional  precautionary
measures  based  on  the  level  of  estimated  risk,
particularly  in  relation  to  production  of  the  listed
plant material and the movement of seed potato lots
other  than  those  referred  to  in  point  (a),  produced
on  the  place  of  production  from  which  the  samples
referred  to  in  point  (a)  were  taken,  in  order  to
prevent  any  spread  of  the  organism.

identified, 

3.
In  those  cases  of  suspected  occurrence  where  there
is  a  risk  of  contamination  of  the  listed  plant  material  or
surface  water  from  or  into  another  Member  State(s),  the
Member  State  in  which  the  suspected  occurrence  has
been  reported  shall  immediately  notify,  according  to  the
risk 
the  said  suspected
occurrence  to  the  other  Member  State(s)  concerned,  and
there  whall  be  appropriate  cooperation  between  the  said
Member  States.  The  Member  State(s)  so  notified  shall
introduce  precautionary  measures  in  accordance  with
paragraph  2(c)  and 
further  action,  as
appropriate,  in  accordance  with  paragraphs  1  and  2.

the  details  of 

take  any 

The 

following  provision  may  be  adopted 

4.
in
accordance  with  the  procedure  laid  down  in  Article  16a
of  Directive  77/93/EEC:

— the  measures  referred  to  in  paragraph  2(c).

Article  5

1.
If  official  or  officially  supervised  laboratory  testing,
using,  for  the  listed  plant  material,  the  relevant  method
set  out  in  Annex  II  or,  in  all  other  cases,  any  other
officially  approved  method,  confirms  the  presence  of  the
organism  in  a  sample  taken  pursuant  to  this  Directive,
the responsible official bodies of a Member State, having
regard  to  sound  scientific  principles,  the  biology  of  the
organism  and  the  particular  production,  marketing  and
processing  systems  of  the  host  plants  of  the  organism  in
that  Member  State,  shall:

(a)

for  the  listed  plant  material;

primary 

(i) establish  an 
and 

investigation  to  determine  the
the
of 
source(s) 
extent 
the
accordance  with 
contamination 
provisions  of  Annex  IV,  with  further  testing  in
accordance  with  Article  4(1),  on,  at  least,  all
clonally  related  seed  potato  stocks,  and,

in 

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(ii) designate  as  contaminated  the 

listed  plant
material,  consignment  and/or  lot  from  which
the  sample  was  taken,  and  the  machinery,
vehicle,  vessel,  store,  or  units  thereof,  and  any
other  objects 
including  packaging  material
which have been in contact with the listed plant
material from which the sample was taken; also
designate  as  contaminated,  where  appropriate,
the field(s), unit(s) of protected crop production
and  place(s)  of  production  from  which  the
listed  plant  material  was  harvested  and  from
which  the  sample  was  taken;  and  for  those
samples  taken  in  the  growing  season,  designate
as  contaminated 
field(s),  place(s)  of
production,  and,  where  appropriate,  unit(s)  of
protected  crop  production  from  which  the
sample  was  taken,  and,

the 

(iii) determine  the  probable  contamination  and
demarcate  a  zone  on 
the
designation  of  contamination  under  point  (ii),
and  the  possible  spread  of  the  organism  taking
into account the provisions of points 1 and 2(ii)
of  Annex  V.

the  basis  of 

2. Member  States  shall  immediately  notify  the  other
Member  States  and  the  Commission,  in  accordance
with  the  provisions  of  point  3  of  Annex  V,  of  any
contamination  designated  under  paragraphs  1(a)(ii)  and
1(c)(ii)  and  the  details  of  the  zone  demarcation  under
paragraph  1(a)(iv)  and,  where  applicable,  under
paragraph  1(c)(iii).  The  details  of  the  notification  under
this  paragraph  may  be  submitted  to  the  Committee.

(iii) determine, in accordance with the provisions of
point  1  of  Annex  V,  the  extent  of  probable
contamination  through  pre-  or  post-harvest
contact, 
irrigation  or
spraying  links  or  through  clonal  relationship
with  the  designated  contamination,  and,

through  production, 

Member  States  shall  at  the  same  time  submit  to  the
Commission the additional notification set out at point 4
of  Annex  V.  The  details  of  the  notification  under  this
subparagraph  shall  immediately  be  submitted  to  the
members  of  the  Committee.

(iv) demarcate  a  zone  on 

the  basis  of 

the
designation  of  contamination  under  point  (ii),
the  determination  of  the  extent  of  probable
contamination  under  point 
the
possible  spread  of  the  organism,  in  accordance
with  the  provisions  of  point  2(i)  of  Annex  V;

(iii),  and 

(b)

for  crops  of  host  plants  other  than  those  mentioned
under  point  (a)  where  production  of  the  listed  plant
material  is  identified  at  risk,

(i) establish  an  investigation  in  accordance  with

point  (a)(i);  and

(ii) designate as contaminated the host plants of the
organism  from  which  the  sample  was  taken;
and

(iii) determine  the  probable  contamination  and
demarcate  a  zone  in  accordance  with  points
(a)(iii)  and  (iv),  respectively,  in  relation  to
production  of  the  listed  plant  material;

(c)

for  surface  water  (including  liquid  waste  discharges
from  industrial  processing  or  packaging  premises
handling  listed  plant  material)  and  associated  wild
solanaceous  host  plants,  where  production  of  the
listed  plant  material  is  identiefied  at  risk  through
irrigation,  spraying,  or  flooding  of  the  surface
water,

(i) establish  an  investigation  including  an  official
survey  at  appropriate  times  on  samples  of
surface  water  and  if  present  wild  solanaceous
host  plants  to  establish  the  extent  of  the
contamination;  and

(ii) designate  as  contaminated  the  surface  water
from  which  the  sample(s)  was  taken,  to  the
extent  appropriate  and  on  the  basis  of  the
investigation  under  point  (i);  and

3.
As  a  result  of  the  notification  under  paragraph  2
and the elements mentioned therein, other Member States
detailed in the notification shall establish an investigation
in  accordance  with  paragraph  1(a)(i)  and,  where
applicable,  paragraph  1(c)(i)  and  take  further  action,  as
appropriate,  in  accordance  with  paragraphs  1  and  2.

Article  6

1. Member  States  shall  prescribe  that  the  listed  plant
material  designated 
contaminated  under
to  be 
Article 5(1)(a)(ii) may not be planted and that, under the
control  and  approval  of  their  responsible  official  bodies,
it shall be subjected to one of the provisions of point 1 of
Annex  VI,  such  that  it  is  established  that  there  is  no
identifiable  risk  of  the  organism  spreading.

2. Member  States  shall  prescribe  that  the  listed  plant
material  determined  as  probably  contaminated  under
Article  5(1)(a)  (iii)  and  (c)(iii)  including  listed  plant
material  for  which  a  risk  has  been  identified,  produced
on  places  of  production  determined  as  probably
contaminated  under  Article  5(1)(a)(iii)  may  not  be
planted  and  shall,  under  the  control  of  their  responsible
official  bodies,  be  put  to  appropriate  use  or  disposal  as
specified  in  point  2  of  Annex  VI,  such  that  it  is
established  that  there  is  no  identifiable  risk  of  the
organism spreading.

3. Member  States  shall  prescribe  that  any  machinery,
vehicle,  vessel,  store,  or  units  thereof,  and  any  other
objects 
including  packaging  material,  designated  as
contaminated  under  Article  5(1)(a)(ii)  or  determined
as  probably  contaminated  under  Article  5(1)(a)(iii)  and

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(c)(iii),  shall  either  be  destroyed  or  decontaminated  using
appropriate methods as specified in point 3 of Annex VI.
After  decontamination,  any  such  objects  shall  no  longer
be  considered  contaminated.

in 

the 

that, 

4. Without  prejudice  to  the  measures  implemented
under  paragraphs  1,  2  and  3,  Member  States  shall
prescribe 
zone  demarcated  under
Article  5(1)(a)(iv)  and  (c)(iii),  a  series  of  measures,  as
specified  in  points  4.1  and  4.2  of  Annex  VI,  shall  be
implemented.  Details  of  these  measures  shall  be  notified
annually  to  the  other  Member  States  and  to  the
Commission.  The  details  of  this  notification  may  be
submitted  to  the  Committee.

Article  7

1. Member  States  shall  prescribe  that  seed  potatoes
shall  meet  the  requirements  of  Directive  77/93/EEC  and
shall  derive  in  direct  line  from  potato  material  obtained
under  an  officially  approved  programme  which  has  been
found  free  of  the  organism  in  official  or  officially
supervised  testing  using  the  relevant  method  set  out  in
Annex  II.

The  aforesaid  testing  shall  be  carried  out  by  a  Member
State:

(a)

in  cases  where  there  have  been  confirmed  finding(s)
of 
seed  potato
production,

the  organism 

their  own 

in 

(i) by  testing  of  the  earlier  propagations,  including
the initial clonal selection and systematic testing
of  basic  seed  potato  clones;  or

(ii)

in  cases  where  it  has  been  established  that  there
is  no  clonal  relationship,  by  testing  of  all  basic
seed  potato  clones  or  earlier  propagations
including  the  initial  clonal  selection,  and

(b)

in  other  cases,  either  on  each  plant  of  the  initial
clonal  selection  or  on  representative  samples  of  the
basic  seed  potatoes  or  earlier  propagations.

in
The  following  provisions  may  be  adopted 
2.
accordance  with  the  procedure  laid  down  in  Article  16a
of  Directive  77/93/EEC:

— the  detailed  rules  of  application  of  paragraph  1,

second  subparagraph,  point  (a),

— the  rules  concerning 

the  representative  samples
provided  for  in  paragraph  1,  second  subparagraph,
point  (b).

Article  8

Article  9

to 

Without  prejudice 
the  provisions  of  Directive
77/93/EEC,  Member  States  may  authorise  derogations
from  the  measures  referred  to  in  Articles  6  and  8  of  this
Directive  in  accordance  with  the  provisions  laid  down  in
Directive 95/44/EC (1) for trial or scientific purposes, and
for  work  on  varietal  selections.

Article  10

Member  States  may  adopt  in  relation  to  their  own
production such additional or stricter measures as may be
required  to  combat  the  organism  or  to  prevent  it  from
spreading,  insofar  as  they  are  in  compliance  with  the
provisions  of  Directive  77/93/EEC.

The  details  of  these  measures  shall  be  notified  to  the
other Member States and to the Commission. The details
of  this  notification  may  be  submitted  to  the  Committee.

Article  11

Amendments to the Annexes to this Directive, to be made
in  the  light  of  developments  in  scientific  or  technical
knowledge,  shall  be  adopted 
in  accordance  with
the  procedure  laid  down  in  Article  16a  of  Directive
77/93/EEC.  In  the  case  of  the  methods  laid  down  in
Annex  II  and  the  measures  in  paragraphs  4.1  and  4.2  of
Annex  VI  to  this  Directive  a  report  shall  be  prepared  by
the  Commission  reviewing  these  methods  and  measures
in  the  light  of  experience  gained  and  the  report  shall  be
submitted  to  the  Committee  before  1  January  2002.

Article  12

1. Member  States  shall  bring  into  force  the  laws,
regulations  and  administrative  provisions  necessary  to
comply  with  this  Directive  with  effect  from  21  August
1999.  They  shall  forthwith  inform  the  Commission
thereof.

When  Member  States  adopt  these  measures,  they  shall
contain  a  reference  to  this  Directive  or  shall  be
accompanied  by  such  reference  on  the  occasion  of  their
official  publication.  The  methods  of  making  such
reference  shall  be  adopted  by  Member  States.

2.
The  Member  States  shall  immediately  communicate
to  the  Commission  the  main  provisions  of  domestic  law
which  they  adopt  in  the  field  governed  by  this  Directive.
The  Commission  shall  inform  the  other  Member  States
thereof.

Member States shall ban the holding and handling of the
organism.

(1) OJ  L  184,  3.8.1995,  p.  34.  Directive  as  last  amended  by
Commission  Directive  97/46/EC  (OJ  L  204,  31.7.1997,
p.  43).

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Article  13

Done  at  Brussels,  20  July  1998.

This  Directive  shall  enter  into  force  on  the  day  of  its
publication  in  the  Official  Journal  of  the  European
Communities.

Article  14

This  Direcitve  is  addressed  to  the  Member  States.

For  the  Council

The  President

W.  MOLTERER

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L 235/7

ANNEX  I

SECTION  I

List  of  host  plants  of  Ralstonia  solanacearum  (Smith)  Yabuuchi  et  al.  referred  to  in  Article  1

Plants  (including  tubers),  other  than  true  seed,  of  Sonanum  tuberosum  L.

Potato

Plants,  other  than  fruits  and  seeds,  of  Lycopersicon  lycopersicum  (L.)  Karsten  ex  Farw.

Tomato

SECTION  II

Surveys

1. The official surveys referred to in Article 2(2)(a) shall be based on the biology of the organism and the

particular  production  systems  in  the  Member  State  concerned  and  shall  comprise:

(i)

in  the  case  of  potato,

— at  appropriate  times,  visual  inspection  of  the  growing  crop,  and/or  sampling  of  both  seed  and
other potatoes in the growing season or in store. These samples shall be subjected to official or
officially  supervised  visual  inspection  by  cutting  of  tubers,  and

— in  the  case  of  seed  potatoes,  and  where  appropriate  for  other  potatoes,  official  or  officially

supervised  laboratory  testing  using  the  method  set  out  in  Annex  II,

(ii)

in  the  case  of  tomato,

— visual  inspection,  at  appropriate  times,  of  at  least  the  growing  crop  of  plants  intended  for

replanting  for  professional  use.

2. The  notification  of  the  official  surveys  referred  to  in  Article  2(3)  shall  include:

(i)

in  the  case  of  surveys  on  potatoes,

— estimated  total  area  grown,  in  hectares,  of  seed  and  other  potatoes,

— stratification  by  seed  category  and  ware,  and  where  appropriate,  by  region,

— number  and  timing  of  samples  taken  for  testing,

— number  of  visual  inspections  in  the  field,

— number  (and  size  of  sample)  of  visual  inspections  on  tubers;

(ii)

in the case of surveys on, at least, the growing crop of plants of tomato intended for replanting for
professional  use,

— estimated  total  number  of  plants,

— number  of  visual  inspections;

(iii)

in the case of surveys on host plants other than potatoes and tomatoes, including wild solanaceous
host  plants,

— species,

— number  and  timing  of  samples  taken,

— area/river  sampled,  as  appropriate,

— method  of  analysis;

(iv)

in the case of surveys on water and liquid waste discharges from industrial processing or packaging
premises,

— number  and  timing  of  samples,

— area/river/location  of  premises  sampled,  as  appropriate,

— method  of  analysis.

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21.8.98

ANNEX  II

TEST  SCHEME  FOR  DIAGNOSIS,  DETECTION  AND  IDENTIFICATION  OF
RALSTONIA  SOLANACEARUM  (SMITH)  YABUUCHI  ET  AL.

SCOPE  OF  THE  TEST  SCHEME

The  presented  scheme  describes  the  various  procedures  involved  in:

(i) diagnosis  of  brown  rot  in  potato  tubers  and  of  bacterial  wilt  in  potato  and  tomato  plants;

(ii) detection  of  Ralstonia  solanacearum  in  samples  of  potato  tubers;

(iii)

identification  of  Ralstonia solanacearum.

In the Appendices, details are provided for the preparation of the test materials, i.e. growth media, buffers,
solutions,  reagents.

CONTENTS:

SECTION  I:

Application  of  the  test  scheme 

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Diagnosis of brown rot in potato tubers and of bacterial wilt in potato and tomato
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

plants 

9

9

2. Detection and identification of Ralstonia solanacearum in samples of potato tubers

11

SECTION  II: Diagnosis  of  brown  rot  in  potato  tubers  and  bacterial  wilt  in  potato  tomato  plants

13

1. Symptoms  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

2. Rapid  screening  test(s)  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

3.

Isolation  procedure  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

4. Confirmation  test(s)  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

13

13

14

14

SECTION  III: Detection  and  identification  of  Ralstonia  solanacearum  in  samples  of  potato  tubers

17

1. Preparation  of  the  sample  for  testing 

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

2.

Immunofluorescence  (IF)  test  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

3. Enzyme  Linked  Immuno-Sorbent  Assay  test  (ELISA) 

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

4. Polymerase  Chain  Reaction  test  (PCRTM)  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

5. Selective  plating  test  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

6. Bioassay  test  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

7. Enrichment  tests  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

8. Pathogenicity  test  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Appendix  1

Nutrient  media  for  isolation  and  culture  of  Ralstonia  solanacearum 

. . . . . . . . . . . . . . . . . .

Appendix  2 Materials  for  sample  preparation  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Appendix  3 Materials  for  the  IF  test 

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Appendix  4

Determination  of  the  level  of  contamination  in  the  IF  test  . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Appendix  5 Materials  for  the  ELISA  test  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Appendix  6 Materials  for  the  PCR  test 

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Appendix  7 Materials  for  the  selective  plating  and  enrichment  test(s)  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

17

18

20

20

22

23

23

23

24

25

26

27

28

29

29

References  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

30

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L 235/9

SECTION  I

APPLICATION  OF  THE  TEST  SCHEME

1.

Diagnosis  of  brown  rot  in  potato  tubers  and  of  bacterial  wilt  in  potato  and  tomato  plants

The  testing  procedure  is  intended  for  potato  tubers  with  symptoms  typical  or  suspect  of  brown  rot  and  for  potato  and
tomato  plants  with  symptoms  typical  or  suspect  of  bacterial  wilt.  It  involves  a  rapid  screening  test,  isolation  of  the
pathogen from infected vascular tissue on diagnostic media and, in case of a positive result, identification of the culture as
Ralstonia solanacearum.

Flow  chart  diagram  presentation

potato or tomato plants or potato tubers with symptoms (1)

rapid screening-tests (2)

ISOLATION

colonies with typical morphology

YES

NO (3)

CONFIRMATION TEST(S)

identification (4) of a pure 
culture as
Ralstonia solanacearum 
plus pathogenicity test (4)

YES

NO

plants and/or tubers
not infected by
Ralstonia solanacearum

plants and/or tubers infected by
Ralstonia solanacearum

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21.8.98

Flowchart  references:

(1) Description  of  symptoms  is  provided  in  section  II.1.

(2) Rapid  screening  tests  facilitate  presumptive  diagnosis.

Appropriate  tests  are:
— Streaming  test  of  vascular  stem  tissue  (section  II.2),
— Test  for  poly-ß-hydroxybutyrate  granules  (section  II.2),
— IF  test  (section  III.2),
— ELISA  test  (section  III.3),
— PCR  test  (section  III.4).

(3) Although  isolation  of  the  pathogen  from  plant  material  with  typical  symptoms  by  dilution  plating  is  straightforward,
culturing may fail from advanced stages of infection. Saprophytic bacteria which grow on diseased tissue may outgrow
or inhibit the pathogen on the isolation medium. If the isolation test is negative, but disease symptoms are typical, then
isolation  must  be  repeated,  preferably  by  a  selective  plating  test.

(4) Reliable  identification  of  a  pure  culture  of  Ralstonia  solanacearum  is  achieved  by  at  least  one  of  the  tests  listed  in
section  II.4.1  in  combination  with  a  pathogenicity  test  (section  II.4.3).  Strain  characterisation  is  optional  but
recommended  for  each  new  case.

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L 235/11

2.

Detection  and  identification  of  Ralstonia  solanacearum  in  samples  of  potato  tubers

The  procedure  is  intended  for  detection  of  latent  infections  in  potato  tubers  by  one  or,  preferably,  more  screening  test(s)
which, if positive, are complemented by the isolation of the pathogen; followed by, in case of isolation of typical colonies,
identification  of  a  pure  culture  as  Ralstonia solanacearum.

Flow  chart  diagram  presentation

sample of potato tubers (1)

SCREENING TEST(S) (2)

IF-test (3) and/or 
selective plating (4) with optional 
additional tests:

ELISA-test (5)
PCR-test (6)
Enrichment test(s) (7)

at least one
test positive

all tests 
done, negative

sample not
infected by
Ralstonia
solanacearum

ISOLATION TEST(S)

selective
plating

and/or

bio
assay test (8)

Colonies with typical morphology

YES

NO

CONFIRMATION TEST(S)

identification (9) of the culture as
Ralstonia solanacearum plus
pathogenicity test (9)

YES

NO

sample infected by
Ralstonia solanacearum

sample not
infected by
Ralstonia
solanacearum

sample not
infected by
Ralstonia
solanacearum

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21.8.98

Flowchart  references:

(1) S a m p l e   s i z e

The  standard  size  is  200  tubers.  However,  the  procedure  can  be  applied  conveniently  for  samples  with  less  than  200
tubers.

(2) S c r e e n i n g   t e s t ( s )

A single test may not be sufficiently sensitive or reliable to detect Ralstonia solanacearum in a sample. Therefore, more
than  one  test  is  recommended  and  these  tests  should  preferably  be  based  on  different  biological  principles.

(3) I m m u n o f l u o r e s c e n c e   ( I F )   t e s t

The IF test is a well established screening test. This is an advantage over other tests which are not yet fully developed
or  validated.  The  test  is  used  for  many  other  statutory  bacteria,  e.g.  Clavibacter  michiganensis  subsp.  sepedonicus.
With the reading parameters specified in this method it is a sensitive test (sensitivity threshold of 103—104  cells per ml
of  potato  extract  pellet).

The critical factor for the reliability of the test result is the quality of the antiserum. Only antiserum with a high titre
(minimum  2000  for  the  crude  antiserum)  is  acceptable  and  all  tests  must  be  carried  out  at  the  antiserum  titre  or  one
dilution  below  the  titre.  The  indirect  method  is  preferred.  The  direct  method  can  be  applied  if  the  test  has  a  level  of
sensitivity  and  specificity  equivalent  to  that  of  the  indirect  method.

The IF test has the advantage of subjective interpretation of cell staining morphology and fluorescence intensity which
provide  information  on  reaction  specificity.  Cross  reactions  by  serologically  related  bacteria  from  soil  or  associated
with  potato  tissues  with  cell  morphology  of  Ralstonia  solanacearum  are  common.  The  IF  test  can  be  used  as  a  sole
screening  test  although  in  cases  where  cross-reactions  are  suspected  an  additional  screening  test  based  on  a  different
biological  principle  should  be  done.  In  such  cases  selective  plating  is  the  most  appropriate  test.

(4) S e l e c t i v e   p l a t i n g

With the modified SMSA medium and testing methodology specified in this method, this is a sensitive and selective test
for  Ralstonia  solanacearum.  The  result  is  available  3—6  days  after  sample  preparation.  The  pathogen  is  obtained
directly  in  culture  and  can  be  readily  identified.  For  full  exploitation  of  its  potential,  the  test  requires  careful
preparation  of  heel  ends  to  avoid  secondary  bacteria  associated  with  the  potato  tuber  which  are  competitors  with
Ralstonia  solanacearum  on  the  medium  and  may  affect  the  development  of  the  pathogen.  Some  strains  may  grow
poorly  as  the  components  of  the  medium  may  affect  the  target  organism.  Care  is  also  required  to  differentiate
Ralstonia solanacearum from other bacteria which may develop on the medium. Selective plating can be used as a sole
screening  test  provided  that  in  cases  where  inhibition  of  Ralstonia  solanacearum  by  other  bacteria  on  the  plates  is
suspected  and  a  negative  test  result  is  obtained  the  sample  is  retested  using  a  different  test  to  confirm  or  refute  the
diagnosis.  In  such  cases,  the  IF  test  is  the  most  appropriate  test.

(5) E L I S A   t e s t

The ELISA test is generally less sensitive than the IF test (sensitivity threshold of 104—105  cells per ml of potato extract
pellet).  The  test  is  cheap  and  fast  but  generally  more  vulnerable  to  false  positive  (cross  reactions)  and  false  negative
results  (inhibition  by  phenolic  molecules  in  the  potato  extract).  The  requirements  for  antiserum  specificity  are
extremely  high.  The  ELISA  test  cannot  be  used  as  a  single  screening  test.

(6) P C R   t e s t

PCR  has  the  potential  for  very  sensitive  detection  although  the  test  is  readily  inhibited  by  plant  or  tuber  extract
components  resulting  in  false  negatives.  Some  potato  cultivars  contain  more  inhibitors  than  others.  It  is  therefore
necessary  to  remove  these  inhibitors.  The  inhibition  can  be  reduced  by  dilution  but  the  populations  of  Ralstonia
solanacearum  are  also  diluted.  Great  care  has  to  be  taken  in  all  steps  of  sample  and  test  preparation  to  prevent
contamination  which  would  result  in  false  positive  tests.  False  positives  could  also  arise  from  sequence  homology  of
other  organisms.  For  these  reasons  the  direct  PCR  cannot  be  used  as  a  sole  screening  test.

(7) E n r i c h m e n t   t e s t

Incubating potato extract pellet samples in a semi-selective broth, such as modified SMSA broth, allows multiplication
of  Ralstonia  solanacearum.  More  important  perhaps,  it  also  dilutes  potential  inhibitors  of  the  ELISA  or  PCR  test.
Ralstonia solanacearum in enrichment broth can thus be detected by IF, ELISA and PCR. We do not recommend that
direct plating is made from the enriched broths. These enrichment methods have not been thoroughly tried and tested.
They  are  included  here  because  they  have  good  potential.  However,  because  of  the  relative  lack  of  experience  with
them,  they  cannot  be  used  as  sole  detection  methods.

(8) B i o a s s a y   t e s t

The bioassay test is used for isolation of Ralstonia solanacearum from potato extract pellets by selective enrichment in
a  host  plant  and  can  be  done  on  tomato  plants  or  eggplants.  The  test  requires  optimal  incubation  conditions  as
specified  in  this  method.  Bacteria  inhibitory  to  Ralstonia  solanacearum  on  the  SMSA  medium  will  most  likely  not
interfere  in  this  test.

(9) C o n f i r m a t i o n   t e s t ( s )

Reliable  identification  of  a  pure  culture  of  Ralstonia  solanacearum  is  achieved  by  at  least  one  of  the  tests  listed  in
section  II.4.1.  in  combination  with  a  pathogenicity  test  (section  II.4.3.).  Strain  characterisation  is  optional  but
recommended  for  each  new  case.

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L 235/13

Diagnosis  of  brown  rot  in  potato  tubers  and  of  bacterial  wilt  in  potato  and  tomato  plants

SECTION  II

1.

1.1

Symptoms

S y m p t o m s   i n   p o t a t o

The  potato  plant.  The  early  stage  of  infection  is  wilting  of  the  leaves  towards  the  top  of  the  plant  at  high  temperatures
during  the  day  with  recovery  at  night.  Wilting  becomes  rapidly  irreversible  and  results  in  the  death  of  the  plant.  The
vascular  tissue  in  transversely  cut  stems  from  wilted  plants  may  become  brown  and  a  milky  ooze  exudes  from  the  cut
surface or can be easily expressed by squeezing. When a cut stem is placed vertically in water, threads of slime will stream
from  the  vascular  bundles.

The potato tuber. Potato tubers must be cut transversely close to the heel (= stolon) end. The early stage of infection is a
glassy yellow to light brown discoloration of the vascular ring from which a pale cream ooze emerges spontaneously after
a  few  minutes  or  when  gentle  pressure  is  applied  with  the  thumbs  on  the  skin  near  the  cut  surface.  Later,  the  vascular
discoloration  becomes  more  distinct  brown  and  the  necrosis  can  extend  into  the  parenchymatous  tissue.  In  advanced
stages,  the  infection  breaks  outwards  from  the  heel  end  and  the  eyes  which  may  result  in  reddish-brown,  slightly  sunken
lesions  on  the  skin  from  which  bacteria  may  ooze,  causing  soil  particles  to  adhere.

1.2

S y m p t o m s   i n   t o m a t o

The  tomato  plant.  The  first  visible  symptom  is  the  flaccid  appearance  of  the  youngest  leaves.  Under  favourable
environmental conditions for the pathogen (soil temperature about 25 °C saturated humidity), epinasty and wilting of one
side or of the whole plant follows within a few days which leads to total plant collapse. Under less favourable conditions
(soil  temperature  below  21 °C)  large  numbers  of  adventitous  roots  may  develop  on  the  stem.  It  is  possible  to  observe  a
greasy  cord  along  the  stem  which  is  evidence  of  the  necrosis  in  the  vascular  system.  When  the  stem  is  cut  crosswise
discoloured  brown  vascular  tissues  of  the  stem  exude  drops  of  white  or  yellowish  bacterial  ooze.

2.

Rapid  screening  tests

Rapid  screening  tests  facilitate  presumptive  diagnosis.  Use  one  or  more  of  the  following  tests:

Stem  streaming  test

The  presence  of  Ralstonia  solanacearum  in  wilting  potato  or  tomato  stems  can  be  assessed  by  the  following  simple
presumptive  test:

Cut the stem just above the soil level. Place the cut surface in a beaker with water. Shortly after, threads of bacterial slime
will stream spontaneously out of the vascular bundles. Any other bacteria causing vascular infection in potato or tomato
plants  will  not  show  this  phenomenon.

Detection  of  poly-ß-hydroxybutyrate  (PHB)  granules

The  PHB  granules  in  the  cells  of  Ralstonia  solanacearum  are  visualised  by  staining  with  Nile  blue  A  or  with  Sudan
black  B.

Either prepare a smear of the ooze or the suspended tissue on a microscope slide or prepare a smear of a 48 hour culture
on  YPGA  or  SPA  (Appendix  1).  Prepare  positive  control  smears  of  a  biovar2/race  3  strain  and,  if  considered  useful,  a
negative  control  smear  of  a  heterologeous  strain.  Allow  to  dry.  Pass  the  lower  surface  of  the  slide  several  times  rapidly
through  the  flame  until  the  smear  is  fixed.

N i l e   b l u e   t e s t

(1) Flood  the  fixed  smear  with  1 %  aqueous  solution  of  Nile  blue  A.

Incubate  for  10  minutes  at  55 °C.

(2) Drain  off  the  staining  solution.  Wash  briefly  in  gently  running  tap  water.  Remove  excess  water  with  tissue  paper.

(3) Flood  the  smear  with  8 %  aqueous  acetic  acid.

Incubate  for  one  minute  at  ambient  temperature.

(4) Wash  in  gently  running  tap  water.  Blot  dry  on  tissue  paper.

(5) Remoisten  with  a  drop  of  water.  Apply  a  coverslip.

(6) Examine the stained smear with an epifluorescence microscope at 450 nm under oil immersion at a magnification of

1 000.

Observe for bright orange fluorescence of PHB granules. Also observe under normal light to ensure that the granules are
intracellular  and  that  cell  morphology  is  typical  of  Ralstonia solanacearum.

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S u d a n   b l a c k   t e s t

(1) Flood  the  fixed  smear  with  0,3 %  Sudan  black  B  solution  in  70 %  ethanol.  Incubate  for  10  minutes  at  ambient

temperature.

(2) Drain  off  the  staining  solution.  Wash  briefly  in  tap  water.  Remove  excess  water.  Remove  excess  water  with  tissue

paper.

(3) Dip  the  smear  briefly  in  xylol.  Blot  dry  on  tissue  paper.

Caution!  Xylol  is  a  harmful  product.  Work  in  a  fume  cabinet.

(4) Flood  the  smear  with  0,5 %  (w/v)  aqueous  safranin  and  leave  for  10  seconds  at  ambient  temperature.

Caution!  Safranin  is  a  harmful  product.  Work  in  a  fume  cabinet.

(5) Wash  in  gently  running  tap  water.  Blot  dry  on  tissue  paper.  Apply  a  coverslip.

(6) Examine the stained smear with a light microscope using transmitted light under oil immersion at a magnification of

1 000.

PHB  granules  in  cells  of  Ralstonia  solanacearum  stain  blue-black.  The  cell  wall  stains  pink.

O t h e r   t e s t s

Other  appropriate  screening  tests  are  the  IF  test  (section  III.2),  the  ELISA  test  (section  III.3)  and  the  PCR  test
(section  III.4).

Isolation  procedure

Remove  ooze  or  sections  of  discoloured  tissue  from  the  vascular  ring  in  the  potato  tuber  or  from  the  vascular  strands  in
the  stem  of  potato  or  tomato  plants.  Suspend  in  a  small  volume  of  sterile  distilled  water  or  50  mM  phosphate  buffer.
Leave  for  5—10  minutes  on  the  bench.

Prepare  a  series  of  decimal  dilutions  of  the  suspension,  e.g.  1/10  and  1/100  or  more  as  considered  appropriate.

Transfer  a  standard  volume  of  the  suspension  and  the  dilutions  on  to  a  general  nutrient  medium  (NA,  YPGA  and  SPA,
Appendix 1) and/or on to Kelman’s tetrazolium medium (Appendix 1) and/or on to SMSA selective medium (Appendix 7).
Spread  or  streak  with  an  appropriate  dilution  plating  technique.  If  considered  useful,  prepare  separate  plates  of  each
medium  used  with  a  diluted  cell  suspension  culture  of  a  virulent  biovar  2/race  3  strain  of  Ralstonia  solanacearum  as  a
positive  control.

Incubate  the  plates  for  three  days  at  28 °C.  Incubation  may  be  prolonged  to  six  days  if  growth  is  slow,  but  colonies  on
SMSA  plates  often  become  atypical  and  die  off.

On the general nutrient media, virulent isolates of Ralstonia solanacearum develop pearly-white, flat, irregular and fluidal
colonies  often  with  characteristic  whorls.

On Kelman’s tetrazolium medium, typical colonies of virulent isolates of Ralstonia solanacearum are cream, flat, irregular
and  fluidal  with  blood  red  coloured  whorls  in  the  centre.  Avirulent  forms  of  Ralstonia  solanacearum  develop  butyrous,
deep-red  colonies.

On  SMSA  medium,  typical  colonies  of  virulent  isolates  of  Ralstonia  solanacearum  are  milky-white,  flat,  irregular  and
fluidal  with  blood  red  colouration  in  the  centre.

Avirulent  forms  of  Ralstonia  solanacearum  develop  less  fluidal  colonies  which  are  completely  pink  to  red  on  the  SMSA
medium.

Purify  colonies  with  characteristic  morphology  by  subculture  on  a  general  nutrient  medium.  Avoid  regular  subculturing
which  may  induce  loss  of  virulence.

Confirmation  test(s)

Identification  of  Ralstonia solanacearum

Identify  pure  cultures  of  Ralstonia  solanacearum  by  at  least  one  of  the  following  procedures:

N u t r i t i o n a l   a n d   e n z y m a t i c   t e s t s

Note:  Include  appropriate  control  strains  in  each  test  used.

3.

3.1

3.2

3.3

3.4

3.5

4.

4.1

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L 235/15

The  following  phenotypic  properties  of  Ralstonia  solanacearum  are  universally  present  or  absent:

Fluorescent  pigment

PHB  inclusions

−

+

Oxidation/Fermentation  (O/F)  test

O +/F −

Catalase

Kovac’s  oxidase

Reduction  of  nitrate

Utilisation  of  citrate

Growth  at  40 °C

Growth  in  1 %  NaCl

Growth  in  2 %  NaCl

Arginine dihydrolase

Gelatin  liquefaction

Starch hydrolysis

Aesulin hydrolysis

Levan  production

+

+

+

+

−

+

−

−

−

−

−

−

Media  and  methods  are  provided  in  Lelliott  &  Stead  (1987)

I F - t e s t

Prepare a suspension of 106  cells per ml from the culture and the control strain(s). Prepare a series of twofold dilutions of
the antiserum. Apply the IF procedure (section III.2). The IF titre of the culture must be equivalent to that of the positive
control.

E L I S A   t e s t

Prepare  a  suspension  of  .106  cells  per  ml  from  the  culture  and  the  control  strain(s).  Apply  the  ELISA  procedure
(section  III.3).  The  ELISA  value  of  the  culture  must  be  equivalent  to  that  of  the  positive  control.

P C R   t e s t

Prepare  a  suspension  of  106  cells  per  ml  from  the  culture  and  the  control  strain(s).  Apply  the  PCR  procedure
(section  III.4).  The  PCR  product  of  the  culture  must  have  the  same  size  and  restrictive  enzyme  analysis  (REA)  pattern  as
that  of  the  positive  control.

F l u o r e s c e n t   i n - s i t u   h y b r i d i s a t i o n   ( F I S H )

Prepare  a  suspension  of  106  cells  per  ml  from  the  culture  and  the  control  strain(s).  Apply  the  FISH  procedure  (van
Beuningen  et  al,  1995)  with  the  OLI-1  PCR  primer  (Seal  et  al,  1993)  The  culture  must  show  the  same  reaction  as  the
positive  control.

P r o t e i n   p r o f i l i n g

Denatured  whole  cell  proteins  are  separated  by  polyacrylamide  gel  electrophoresis  —  PAGE  (Stead,  1992a).

F a t t y   a c i d   p r o f i l i n g   ( F A P )

Grow the culture and a positive control strain for 48 hours at 28 °C on trypticase soy agar and apply the FAP procedure
(Janse,  1991;  Stead,  1992a;  Stead,  1992b).  The  profile  of  the  culture  must  be  identical  to  that  of  the  positive  control.
Under  the  specified  conditions,  characteristic  fatty  acids  are  14:0  3OH,  16:0  2OH,  16:1  2OH  und  18:1  2OH.

4.2.

Strain characterisation

Strain  characterisation  is  optional  but  is  recommended  for  each  new  case  using  at  least  one  of  the  following:  —

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B i o v a r   d e t e r m i n a t i o n

Ralstonia  solanacearum  is  separated  into  biovars  on  the  basis  of  the  ability  to  produce  acid  from  three  hexose  alcohols
and  three  sugars  (Hayward,  1964  &  1994):

Utilisation  of:

— maltose

— lactose

— cellobiose

— mannitol

— sorbitol

— dulcitol

1

−

−

−

−

−

−

2

+

+

+

−

−

−

Biovar

3

+

+

+

+

+

+

4

−

−

−

+

+

+

5

+

+

+

+

−

−

Additional  tests  differentiate  biovar  2  in  subphenotypes  (Hayward,  1994):

utilisation  of  trehalose

utilisation  of  inositol

utilisation  of  D-ribose

pectolytic activity

R a c e   d e t e r m i n a t i o n

biovar 2

biovar 2-A

biovar  2-T

−

+

−

low

+

−

−

low

+

+

+

high

The race (Buddenhagen et al., 1962) can be determined on the basis of a pathogenicity test in tomato plants or eggplants
and  in  tobacco  plants  and  by  a  hypersensitivity  reaction  (HR)  test  in  tobacco  leaves  (Lozano  and  Sequeira,  1970):

Reaction  in:

— tomato  plants/eggplants

— tobacco  plants

— tobacco leaves

1

wilting

wilting

necrosis
(48  hrs)  and
wilting  (7—8  days)

Race (*)

2

no  reaction

no  reaction

HR  (12—24  hrs)

3

wilting

no  reaction

chlorosis
(2—8  days)

(*) Race  4  (pathogenic  on  ginger  and  a  few  other  hosts)  and  race  5  (pathogenic  on  mulberry  only)  are  not  included.

Race characterisation by the pathogenicity test or tobacco hypersensitivity test may not be highly reliable and instead can
be  deduced  from  the  biovar  and  the  natural  host  of  origin.

The  culture  can  be  further  characterized  by:

G e n o m i c   f i n g e r p r i n t i n g

Molecular  differentiation  of  strains  in  the  Ralstoniasolanacearum complex  can  be  done  by:

RFLP  analysis  (Cook  et  al,  1989)

Repetitive  sequence  PCR  [REP-,  ERIC-  &  BOX-PCR  (Louws  et  al,  1995;  Smith  et  al,  1995)]

4.3.

Pathogenicity  test

This  test  is  for  confirmation  of  the  diagnosis  of  Ralstonia  solanacearum  and  for  the  assessment  of  the  virulence  of  the
cultures  identified  as  Ralstonia solanacearum.

Prepare an inoculum of 106  cells per ml from the culture and a positive control strain. Inoculate 5—10 tomato plants or
eggplants  at,  preferably,  the  third  true  leaf  stage  or  older  (section  III,  6.).  Incubate  for  up  to  two  weeks  at  22 °C—28 °C
and  high  relative  humidity  with  daily  watering.  Observe  for  wilting  and/or  epinasty,  chlorosis,  stunting.

Isolate  from  symptomatic  plants  as  follows:

— Remove  a  section  of  tissue  from  the  stem  two  cm  above  the  inoculation  point.

— Comminute and suspend in a small volume of sterile distilled water or 50 mM phosphate buffer. Then plate, incubate,

and  check  for  typical  colonies  of  Ralstonia solanacearum.

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DETECTION  AND  IDENTIFICATION  OF RALSTONIA SOLANACEARUM IN  SAMPLES  OF  POTATO  TUBERS

Note: The  standard  sample  size  is  200  tubers.  However,  the  procedure  can  be  applied  conveniently  for  samples  with  less  than  200

SECTION  III

tubers.

1.

Preparation  of  the  sample  for  testing

1.1

1.2

1.3

Note: The  potato  extract  pellet  obtained  in  this  procedure  can  also  be  used  for  detection  of  Clavibacter  michiganensis

subsp.  sepedonicus.

Pre-testing  options  if  considered  useful:

(i)

Incubate  the  sample  at  25—30 °C  for  up  to  two  weeks  to  encourage  multiplication  of  low  Ralstoniasolanacearum
populations.

(ii) Wash  the  tubers  under  running  water  with  appropriate  disinfectants  and  detergents.  Air  dry  the  tubers.

Remove  with  a  clean  and  disinfected  scalpel  or  vegetable  knife  the  skin  at  the  heel  end  of  the  tuber  so  that  the  vascular
tissues  first  become  visible.  Carefully  cut  out  a  small  conical  core  (3—5  mm  diameter)  of  vascular  tissue  at  the  heel  end.
Keep  the  amount  of  non-vascular  tissue  to  a  minimum.  Process  each  of  the  tubers  in  the  sample.

Note:  Visual  examination  of  the  tubers  (Section  II.1)  can  be  done  at  this  stage.  Set  aside  any  tuber  with  symptoms  or

severe  rotting  and  test  separately  (section  II).

Collect  the  heel  ends  in  a  closed  container.  Preferably,  the  heel  ends  should  be  processed  immediately.  If  this  is  not
possible,  store  them  for  not  more  than  24  hours  or,  at  4 °C,  for  not  longer  than  72  hours.

Process  the  heel  ends  by  one  of  the  following  procedures:

(i) Transfer  the  heel  ends  into  an  appropriate  container.

Add  a  sufficient  volume  of  maceration  buffer  (Appendix  2)  to  cover  the  heel  ends. 

Comminute  the  heel  ends  in  a  Waring  Blender  or  by  Ultra  Thurrax  until  complete  homogenisation  has  just  been
achieved.  Avoid  excessive  homogenisation.

Allow  the  macerate  to  soak  for  15—30  minutes.

(ii) Transfer  the  heel  ends  into  an  appropriate  container.

Add  a  sufficient  volume  of  maceration  buffer  to  cover  the  heel  ends.

Place  the  container  on  a  rotary  shaker.

Incubate  at  50—100  rpm  for  4  hours  at  20 °C—22 °C  or  for  16—24  hours  at  4 °C.

(iii) Transfer  the  heel  ends  into  a  strong  disposable  maceration  bag  (e.g.  Stomacher  bag  with  dimensions  105  mm  ×

150  mm,  radiation  sterile).

Crush  the  heel  ends  carefully  with  an  appropriate  tool,  e.g.  a  hammer,  until  complete  homogenisation  has  been
achieved.

Add  a  sufficient  volume  of  maceration  buffer  to  cover  the  heel  ends.

Allow  the  macerate  to  settle  for  15—30  minutes.

1.4

Extract  the  bacteria  from  the  processed  heel  ends  by  one  of  the  following  procedures:

(i) Decant  the  macerate  gently  in  a  centrifuge  tube  while  leaving  the  debris  in  the  container  or  bag.  If  the  decanted

macerate  is  cloudy,  centrifuge  at  not  more  than  180  g  for  10  minutes  at  a  temperature  below  10 °C.

Centrifuge the decanted macerate, or the supernatant from the first centrifugation step, at 7 000 g for 15 minutes or
at  10 000  g  for  10  minutes  at  a  temperature  below  10 °C.

Discard  the  supernatant  without  disturbing  the  pellet.

(ii) Filter  the  macerate  through  a  filtration  system  with  pore  size  of  40—100  em.  Enhance  filtration  using  a  vacuum

pump.

Collect  the  filtrate  in  a  centrifuge  tube.

Wash  the  filter  with  maceration  buffer.

Centrifuge  filtrate  at  7 000  g  for  15  minutes  or  at  10 000  g  for  10  minutes  at  a  temperature  below  10 °C.

Discard  the  supernatant  without  distrubing  the  pellet.

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1.5

Resuspend  the  pellet  in  1  ml  pelletbuffer  (Appendix  2).

Divide  in  two  equal  parts  and  transfer  each  part  to  a  microvial.

Use  one  microvial  for  testing.  Conserve  the  remainder  of  this  extract  at  4 °C  during  testing.

Add  10—25 %  (v/v)  of  sterile  glycerol  to  the  other  microvial.  Vortex.  Store  at  − 18 °C  (weeks)  or  at  − 70 °C  (months).

2.

IF-test

Use antiserum for Ralstonia solanacearum, preferably to race 3/biovar 2. Determine the titre on a suspension of 106  cells
per  ml  from  the  homologeous  strain  of  Ralstonia  solanacearum  with  an  appropriate  dilution  of  the  fluorescein
isothiocyanate (FITC) conjugate, according to the manufacturer’s recommendations. The crude antiserum should have an
IF  titre  of  at  least  1:2000.

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

Include  a  FITC  conjugate  control  on  each  test  slide.  The  test  should  be  repeated  with  a  PBS  control  included  if  any
positive  cell  is  observed  in  the  FITC  control.

Prepare separate positive control slides with a suspension of 106  cells per ml from a strain of the appropriate race/biovar
of  Ralstonia solanacearum.  Use  one  slide  in  each  set  of  tests.

2.1

Prepare  the  test  slides  by  one  of  the  following  procedures:

(i) For  pellets  with  relative  little  starch:

2.2

2.3

Pipette a measured standard volume (15 el is appropriate for 6 mm window diameter — scale up volume for larger
windows)  of  the  resuspended  pellet  on  a  row  of  windows.  The  remaining  row  can  be  used  as  duplicate  or  for  a
second  sample  as  presented  in  Figure  1.

(ii) For  other  pellets:

Prepare  decimal  dilutions,  viz.  1/10,  1/100  and  1/1000  of  the  resuspended  pellet  in  pellet  buffer.  Pipette  a  measured
standard  volume  (15  el  is  appropriate  for  6  mm  window  diameter  —  scale  up  volume  for  larger  windows)  of  the
resuspended  pellet  and  each  dilution  on  a  row  of  windows.  The  remaining  row  can  be  used  as  duplicate  or  for  a
second  sample  as  presented  in  Figure  2.

Let  the  droplets  dry.  Fix  the  bacterial  cells  to  the  slide  either  by  heating,  flaming  or  with  95 %  ethanol.

IF  procedure

(i) According  to  test  slide  preparation  in  2.1  (i):

Prepare  a  set  of  twofold  dilutions  of  the  antiserum  in  IF  buffer  (Appendix  3):

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  2.1  (ii):

Prepare  the  working  dilution  (WD)  of  the  antiserum  in  IF  buffer.  The  working  dilution  is  the  dilution  of  the
antiserum  with  optimum  specificity  and  is  usually  half  of  the  titre.

Figure  1

Preparation  of  the  test  slide  according  to  2.1  (i)  and  2.3  (i)

One  standard  dilution  of  resuspended  pellet

FITC

T/4

Sample  1

Duplicate  of
sample  1  or
sample  2

Q

1

Q

6

Q

2

Q

7

T  =  titre

T/2

Q

3

Q

8

T

2T

Twofold  dilutions  of
antiserum

Q

4

Q

9

Q

5

Q

10

(cid:222)
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Figure  2

Preparation  of  the  test  slide  according  to  2.1  (ii)  and  2.3  (ii)

FITC

Standard  dilution  of  antiserum

Neat

Q

1

Q

6

Neat

Q

2

Q

7

Sample  1

Duplicate  of
sample  1
or  sample  2

1/10

1/100

1/1000

Decimal  dilution  of
resuspended  pellet

Q

3

Q

8

Q

4

Q

9

Q

5

Q

10

2.3.1

Arrange  the  slides  on  damp  tissue  paper.

Cover the test windows with the antiserum dilution(s). Apply PBS on the FITC windows. The volume of antiserum applied
on  the  windows  must  be  equivalent  to  the  volume  of  extract  applied.

Incubate  under  a  cover  for  30  minutes  at  ambient  temperature.

Shake  the  droplets  of  antiserum  off  the  slide  and  rinse  the  slides  carefully  with  IF  buffer.  Wash  for  five  minutes  in  IF
buffer-Tween  and  subsequently  for  five  minutes  in  IF  buffer  (Appendix  3).  Carefully  remove  excess  moisture.

Arrange  the  slides  on  damp  tissue  paper.  Cover  the  test  windows  and  the  FITC  window  with  the  dilution  of  FITC
conjugate used to determine the titre. The volume of conjugate applied on the windows must be identical to the volume of
antiserum  applied.

Incubate  under  a  cover  for  30  minutes  at  ambient  temperature.

Shake  the  droplets  of  conjugate  off  the  slide.  Rinse  and  wash  as  before  (2.3.3).  Carefully  remove  excess  moisture.

Pipette  5—10  el  0,1  M  phosphate-buffered  glycerol  (Appendix  3)  or  a  similar  mountant  on  each  window  and  apply  a
coverslip.

2.3.2

2.3.3

2.3.4

2.3.5

2.3.6

2.3.7

2.4

Reading  the  IF  test

Examine test slides on an epifluorescence microscope with filters suitable for excitation of FITC, under oil immersion at a
magnification  of  500—1000.  Scan  windows  across  two  diameters  at  right  angles  and  around  the  perimeter.

Check  the  positive  control  slide  first.  Cells  must  be  bright  fluorescent  and  completely  stained.  Note:  The  test  must  be
repeated  if  the  staining  is  aberrant.

Read  the  test  slides.  Observe  first  for  absence  of  fluorescing  cells  in  the  FITC  control  windows.  Fluorescing  cells  in  the
FITC  control  indicate  non-specific  binding  of  the  conjugate,  autofluorescence  or  contamination:  Note:  Repeat  the  test  if
such  is  observed.

Observe  for  bright  fluorescing  cells  with  characteristic  morphology  of  Ralstonia  solanacearum  in  the  test  windows.  The
fluorescence  intensity  must  be  equivalent  to  the  positive  control  strain  at  the  same  antiserum  dilution.  Cells  with
incomplete staining or with weak fluorescence must be disregarded, unless there are many such cells (see interpretation of
the  IF  test  result).

Interpretation  of  the  IF  test  result:

(i)

If  bright  fluorescing  cells  with  characteristic  morphology  are  not  found,  then  the  IF  test  is  negative.

(ii)

If  bright  fluorescing  cells  with  characteristic  morphology  are  found,  then  determine  the  mean  number  of  cells  per
microscope  field  and  calculate  the  number  of  cells  (N)  per  ml  of  resuspended  pellet  (Appendix  4).

A population of approximately 103  cells per ml of resuspended pellet is considered to be the limit of detection for the
IF  test,

— for  samples  with  N  .  103  cells  per  ml  of  resuspended  pellet,  the  IF  test  is  considered  positive,

— for  samples  with  N  .  103  cells  per  ml  of  resuspended  pellet,  the  IF  test  may  be  considered  positive.

(cid:222)
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(iii)

If large numbers (. 105  cells per ml) of incomplete or weakly fluorescing cells are seen at the titre of the antiserum, a
second  test  should  be  carried  out:

— either  a  test  based  on  a  different  biological  principle,  or

— a  repeat  IF  test,  with  either  a  second  antiserum  or  a  ten-fold  dilution  of  the  pellet.

3.

ELISA  test

Based  upon  Robinson-Smith  et  al.,  1995

Use antiserum for Ralstonia solanacearum, preferably to race 3/biovar 2. Determine the titre on a suspension of 106  cells
per  ml  from  the  homologeous  strain  of  Ralstonia solanacearum.

The  use  of  NUNC-Polysorb  microtitre  plates  is  recommended.

Include  a  negative  potato  extract  control  and  a  phosphate  buffered  saline  (PBS)  control.

Use  a  suspension  of  .  106  cells  per  ml  from  a  strain  of  the  appropriate  race/biovar  of  Ralstonia  solanacearum  as  the
positive  control.  Test  in  an  identical  manner  as  the  sample(s)  but  well-separated  from  the  samples  on  the  microtitre
plate.

Pipette  100—200  el  of  the  resuspended  pellet  in  a  microvial.

Heat  for  4  minutes  at  100 °C.  Remove  the  microvial  on  ice.

Add  an  equal  volume  of  double  strength  carbonate  coating  buffer  (Appendix  5).  Vortex.

Apply 100 el aliquots to each of at least two wells of the microtitre plate. Incubate for one hour at 37 °C or overnight at
4 °C.

Flick out the extracts from the wells. Wash the wells three times with PBS-Tween (Appendix 5), leaving the last washing
solution  in  the  wells  for  at  least  five  minutes.

Prepare  the  appropriate  dilution  of  Ralstonia  solanacearum  antiserum  in  blocking  buffer  (Appendix  5).  Apply  100  el  of
antiserum  dilution  to  the  wells.

Incubate  for  one  hour  at  37 °C.

Flick  out  the  antiserum  from  the  wells.  Wash  the  wells  as  before  (3.4).

Prepare the appropriate dilution of alkaline phosphatase conjugate in blocking buffer. Apply 100 el of conjugate dilution
to  the  wells.

Incubate  for  one  hour  at  37 °C.

Flick  out  the  conjugate  from  the  wells.  Wash  the  wells  as  before  (3.4  and  3.6).

Prepare  the  alkaline  phosphatase  substrate  solution  (Appendix  5).  Apply  100 el  to  the  wells.  Incubate  for  30  minutes  to
one  hour  in  the  dark  at  ambient  temperature.

3.1

3.2

3.3

3.4

3.5

3.6

3.7

3.8

3.9

3.10

Read  absorbance  at  409  nm.

Interpretation  of  the  ELISA  test:
The  ELISA  test  is  negative  if  the  optical  density  (OD)  of  the  sample  is  ,  2 × OD  of  the  negative  control.
The  ELISA  test  is  positive  if  the  optical  density  (OD)  of  the  sample  is  .  2 × OD  of  the  negative  control.

4.

PCR  test

Based  on  Seal  et  al.,  1993

Note: Filterplugged  pipette  tips  must  be  used  during  all  stages  of  sample  preparation  and  other  manipulations  involving

PCR.

Prepare  a  suspension  of  106  cells  per  ml  from  a  race  3/biovar  2  strain  of  Ralstonia solanacearum as  the  positive  control.
Test  in  an  identical  manner  as  the  sample(s).

4.1

Pipette  100  el  of  the  resuspended  pellet  into  a  microvial.

Alternatively, transfer 90 el of the resuspended pellet to a microvial containing 10 el of 0,5M NaOH. Mix by repeatedly
inverting  the  microvial.

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4.2

4.3

4.4

4.5

4.5.1

Heat  for  four  minutes  at  100 °C.  Remove  the  microvial  immediately  on  ice.

Prepare  at  least  two  decimal  dilutions,  e.g.  1/10  and  1/100  or  more  if  considered  useful,  in  sterile  distilled  or  ultra  pure
water  (UPW).

Prepare  the  PCR  reaction  mix  (Appendix  6)  in  a  sterile  vial  by  adding  the  following  components  in  the  following
order:

For  a  50  el  reaction  volume:

Component

Quantity

Final
concentration

Sterile  distilled  or  UPW

10 × PCR  Buffer

d-ATP

d-CTP

d-GTP

d-TTP
Primer  OLI-1  (20  eM)
Primer  Y-2  (20  eM)
Taq  polymerase  (5U/el)

30,8  el—33,8  el
5,0  el
1,0  el
1,0  el
1,0  el
1,0  el
2,5  el
2,5  el
0,2  el

1 ×

0,2  mM

0,2  mM

0,2  mM

0,2  mM
1  eM
1  eM

1,0  U

Total  volume

45  el—48  el

For  more  reactions:

Calculate  the  quantity  of  each  component  for  the  required  number  of  reactions.

Mix  the  components  and  transfer  45  el—48  el  of  the  mix  into  sterile  PCR  vials.

Keep  the  vials  with  the  PCR  reaction  mix  on  ice.

For  25  el  reaction  volumes:

Scale  down  components  accordingly.

PCR  amplification

Optional:  Pulse  centrifuge  the  vials  with  the  boiled  sample  and  positive  control.

Add, in the specified order, 2—5 el of the sample(s), water control and positive control to the vials with the PCR reaction
mix.  Place  the  vials  in  the  heating  block  of  the  DNA  thermal  cycler.

4.5.2

Run  the  following  programme:

1  cycle  of:

(i) 2  minutes  at  96 °C:  denaturation  of  template

50  cycles  of
(ii) 20  seconds  at  94 °C:  denaturation
(iii) 20  seconds  at  68 °C:  annealing  of  primers
(iv) 30  seconds  at  72 °C:  extension  of  copy

1  cycle  of:
(v) 10  minutes  at  72 °C:  further  extension

1  cycle  of:
(vi) hold  at  4 °C

Note: These  parameters  are  for  a  Perkin  Elmer  9600.  Other  thermal  cycles  may  require  mineral  oil  overlay  in  the  PCR

reaction  vials  and/or  modification  of  the  duration  of  step  (ii),  (iii)  and  (iv)  in  the  amplification  profile.

4.5.3

Remove  the  vials  from  the  thermal  cycler.  Analyse  the  PCR  product.  If  not  done  immediately,  store  the  vials  at  4 °C  for
use  in  the  same  day  or  at  −18 °C  for  longer.

4.6

Analysis  of  the  PCR  product

The  PCR  fragments  are  detected  by  agarose  gelelectrophoresis  and  staining  with  ethidium  bromide.

4.6.1

Prepare  an  appropriate  agarose  gel  by  gently  bringing  to  the  boil  agarose  in  tris-acetate-electrophoresis  (TAE)  buffer.

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4.6.2

4.6.3

4.6.4

4.6.5

4.6.6

Cool  the  molten  agarose  to  50—60 °C,  pour  into  the  mould  of  the  electrophoresis  unit  and  insert  the  comb.  Let  the
solution  solidify.

Remove  the  comb.  Submerge  the  gel  in  TAE  so  that  it  is  just  covered  (2—3  mm)  with  the  buffer.

Place  3  el  droplets  of  loading  buffer  on  parafilm.  Add  12  el  of  the  PCR  product  from  either  the  samples,  the  positive
control  or  the  water  control  and  mix  by  gentle  aspiration  in  the  pipette  tip  before  loading.  The  given  volumes  may  be
modified  to  fit  with  the  capacity  of  the  wells  in  the  agarose  gel.

Carefully  load  the  wells  of  the  gel.  Include  for  reference  an  appropriate  DNA  marker  in  at  least  one  well.

Connect the wires on the power supply and the electrophoresis equipment. Run the gel at 5—8 V/cm until the front of the
tracking  indicator  is  within  1  cm  of  the  end  of  the  gel.

4.6.7

Switch  off  the  power  supply.  Disconnect  the  wires  from  the  electrophoresis  unit.

Carefully  remove  the  gel.  Soak  it  in  ethidium  bromide  solution  for  30—45  minutes.

Note: Wear  disposable  gloves  at  all  times  when  handling  ethidium  bromide  which  is  a  powerful  mutagen!

4.6.8

4.6.9

4.6.10

4.6.11

4.7

4.7.1

4.7.2

Destain  in  distilled  water  for  10—15  minutes.

Visualise  the  amplified  DNA  fragment(s)  by  UV  transillumination.  The  PCR  product  of  Ralstonia  solanacearum  with
primer  set  OLI-1  and  Y-2  is  288  bp  in  length.  Check  against  the  DNA  marker  and  against  the  positive  control.

Note: The  water  control  must  be  negative  in  each  case.  If  positive,  repeat  the  test.

Photograph  the  gel  if  a  permanent  record  is  required.

Confirm  the  authenticity  of  the  amplified  fragment  by  restriction  enzyme  analysis  (REA).

Restriction  Enzyme  Analysis  (REA)

Transfer 8,5 el from the PCR product (4.5.3) to a new microvial. Add 1 el of 10 × enzyme buffer and 0,5 el of restriction
enzyme  Ava II.

Mix  by  gentle  aspiration  in  the  pipette  tip.  If  drops  remain  on  the  walls  of  the  vial,  pulse  spin  in  a  microcentrifuge.
Incubate  for  one  hour  at  37 °C.

4.7.3

Analyse  the  digested  PCR  fragment  by  agarose  gelelectrophoresis  as  before  (4.6).

Interpretation  of  the  PCR  test  result:

The PCR test is negative if the characteristic 288 bp fragment is not detected and the fragment is detected for the positive
control  strain  of  Ralstonia solanacearum.

The  PCR  test  is  positive  if  the  288  bp  fragment  is  detected  and  REA-analysis  of  the  amplified  fragment  is  identical  with
the  positive  control  strain  of  Ralstonia solanacearum.

5.

Selective  plating  test

Based  upon  Elphinstone  et  al.,  1996

5.1

Perform  the  test  by  an  appropriate  dilution  plating  technique,  e.g.:

(i) Prepare at least two decimal dilutions, viz. 1/10 and 1/100 or more if considered useful, of the resuspended pellet in
pelletbuffer.  Pipette  a  measured  standard  volume  (50—100  el)  of  the  resuspended  pellet  and  each  dilution  onto
modified  SMSA  selective  medium  (Appendix  7)  and  spread  with  a  glass  rod  over  the  whole  surface  of  the  medium.
If  considered  useful,  also  perform  a  dilution  streak  of  a  10  el  loopful  of  the  resuspended  pellet.  Flame  the  loop
between  streaks.

(ii) Transfer a measured standard volume (50—100 el) of the resuspended pellet onto modified SMSA selective medium
and  spread  with  a  glass  rod  over  the  whole  surface  of  the  medium.  Streak  the  rod  without  flaming  on  at  least  two
other  modified  SMSA  plates.

Apply,  by  the  same  dilution  plating  technique,  a  suspension  of  106  cells  per  ml  from  a  virulent  race  3/biovar  2  strain  of
Ralstonia  solanacearum  as  the  positive  control  on  a  set  of  separate  modified  SMSA  plates.

Incubate the plates at 28 °C. Start reading the plates after three days. If negative, incubate further up to six days. Colonies
of virulent isolates of Ralstonia solanacearum are milky-white, flat, irregular and fluidal with blood red coloration in the
centre  and  showing  internal  streaking  or  whorling.

5.2

5.3

5.4

Purify  colonies  with  characteristic  morphology  by  subculturing  onto  a  general  nutrient  medium  (Appendix  1).

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5.5

Identify  pure  cultures  (section  II,  4.1)  and  confirm  Ralstonia  solanacearum  cultures  by  a  pathogenicity  test  (section  II,
4.3).

Interpretation  of  the  selective  plating  test  result:

The  selective  plating  test  is  negative  if  no  bacterial  colonies  are  isolated  after  six  days  or  if  no  colonies  characteristic  of
Ralstonia  solanacearum  are  isolated,  provided  that  no  inhibition  is  suspected  by  colonies  of  other  bacteria  and  that
colonies  characteristic  of  Ralstonia  solanacearum  are  found  in  the  positive  controls.

The  selective  plating  test  is  positive  if  colonies  characteristic  of  Ralstonia  solanacearum  are  isolated.

6.

Bioassay  test

Based  upon  Janse,  1988.

6.1

6.2

6.3

6.4

6.5

6.6

Use 10 test plants of susceptible tomato or eggplant seedlings at the third true leaf stage for each sample. Do not water the
testplants  for  24  hours  before  inoculation.

Distribute 100 el of resuspended pellet between the testplants. Inoculate in the stem between the cotyledons and at one or
more  other  places.

Inoculate, by the same technique, 10 seedlings with a suspension of 106  cells per ml from a virulent biovar 2/race 3 strain
of  Ralstonia  solanacearum  as  the  positive  control  and  with  pelletbuffer  as  the  negative  control.  Separate  the  positive
control  plants  from  the  other  plants  to  avoid  cross-contamination.

Grow the test plants further for up to four weeks at 22 °C—28 °C and high relative humidity with daily watering. Observe
for  wilting,  epinasty,  chlorosis  and/or  stunting.

Isolate from infected plants (section II). Identify pure cultures with characteristic morphology (section II.4.1) and confirm
Ralstonia  solanacearum  cultures  by  a  pathogenicity  test  (section  II.4.3).

If considered useful, check absence of infection in batches of test plants not showing any indication of infection. Remove
from  each  test  plant  a  one  cm  section  of  stem  from  two  cm  above  the  inoculation  point.  Homogenise  the  tissues  in
maceration  buffer.  Perform  dilution  plating  (section  III.5.1).  If  positive,  identify  pure  cultures  with  characteristic
morphology  (section  II.4.1)  and  confirm  Ralstonia  solanacearum  cultures  by  a  pathogenicity  test  (section  II.4.3).

Interpretation  of  the  bioassay  test  result:

The  bioassay  test  is  negative  if  test  plants  are  not  infected  by  Ralstonia  solanacearum,  and  provided  that  Ralstonia
solanacearum is  detected  in  the  positive  controls.

The  bioassay  test  is  positive  if  test  plants  are  infected  by  Ralstonia solanacearum.

7.

Enrichment  tests

Based  upon  Elphinstone  et  al.,  1996

7.1

7.2

7.3

Transfer  100  el  of  the  resuspended  pellet  in  three  ml  of  modified  SMSA  broth  (Appendix  7).

Incubate  for  48  hours,  and  in  any  case  not  longer  than  72  hours,  at  28 °C  with  the  cap  of  the  tube  loosely  fitted  for
aeration.

Tighten cap and vortex. Aliquot for the IF test (this section, 2.), the ELISA test (this section, 3.) and/or the PCR test (this
section,  4.).

8.

Pathogenicity  test

Refer  to  Section  II.4.3.

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Appendix  1

Nutrient  media  for  isolation  and  culture  of  Ralstonia  solanacearum

N u t r i e n t   A g a r   ( N A )

Nutrient  Agar  (Difco)

Distilled  water

23  g

1  litre

Prepare  1⁄2  litre  volumes  of  medium  in  one  litre  flasks.

Dissolve  the  ingredients.

Sterilise  by  autoclaving  at  121 °C  for  15  minutes.

Cool  to  50 °C.  Pour  plates.

Y e a s t - P e p t o n e - G l u c o s e - A g a r   ( Y P G A )

Yeast  Extract  (Difco)

Bacto  Peptone  (Difco)

D(+)  Glucose  (monohydrate)

Bacto  Agar  (Difco)

Distilled  water

5  g

5  g

10  g

15  g

1  litre

Prepare  1⁄2  litre  volumes  of  medium  in  one  litre  flasks.

Dissolve  the  ingredients.

Sterilise  by  autoclaving  at  121 °C  for  15  minutes.

Cool  to  50 °C.  Pour  plates.

S u c r o s e   P e p t o n e   A g a r   ( S P A )

Sucrose

Peptone

K2HPO4

MgSO4·7H2O

Bacto  Agar  (Difco)

Distilled  water

20  g

5  g

0,5  g

0,25  g

15  g

1  litre

Prepare  1⁄2  litre  volumes  of  medium  in  one  litre  flasks.

Dissolve  the  ingredients.  Adjust  to  pH  7,2—7,4  if  necessary.

Sterilise  by  autoclaving  at  121 °C  for  15  minutes.

Cool  to  50 °C.  Pour  plates.

K e l m a n ’ s   t e t r a z o l i u m   m e d i u m

Casamino  acids  (Difco)

Bacto  Peptone  (Difco)

Dextrose

Bacto  Agar  (Difco)

Distilled  water

1  g

10  g

5  g

15  g

1  litre

Prepare  1⁄2  litre  volumes  of  medium  in  one  litre  flasks.

Dissolve  the  ingredients.  Sterilise  by  autoclaving  at  121 °C  for  15  minutes.

Cool  to  50 °C.

Add  a  filter-sterilised  aqueous  solution  of  triphenyl  tetrazolium  chloride  (Sigma)  to  obtain  a  final
concentration  of  50  mg  per  litre.

Pour  plates.

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Appendix  2

Materials  for  sample  preparation

M a c e r a t i o n   B u f f e r :   5 0   m M   p h o p h a t e   b u f f e r ,   p H   7 , 0

This  buffer  is  used  for  tissue  maceration.

Na2HPO4

KH2PO4

Distilled  water

4,26  g

2,72  g

1  litre

Dissolve  the  ingredients  and  check  pH.  Aliquot  as  considered  appropriate.

Sterilise  by  autoclaving  at  121 °C  for  15  minutes.

The  addition  of  5 %  polyvinylpyrrolidone-40000  MWT  (PVP-40)  is  recommended  when  performing  the
direct  PCR  test  to  reduce  the  incidence  of  amplification  inhibition  by  aromatic  molecules  in  the  extract.

The  addition  of  a  deflocculant,  an  antifoam  agent  or  an  anti-oxidant  is  recommended  using  the  Waring
Blender  or  Ultra  Turrax  homogenisation  procedure  for  maceration  of  the  potato  tissue  cores.

Lubrol  flakes

0,5  g  per  litre

DC  Silicone  antifoam

1,0  ml  per  litre

Tetrasodiumpyrophosphate

1,0  g  per  litre

Autoclave  separately.  Add  to  the  desired  concentration.

P e l l e t b u f f e r :   1 0   m M   p h o s p h a t e   b u f f e r ,   p H   7 , 2

This  buffer  is  used  for  resuspension  and  dilution  of  potato  heel  and  pellets.

Na2HPO4·12H2O

NaH2PO4·2H2O

Distilled  water

2,7  g

0,4  g

1  litre

Dissolve  the  ingredients  and  check  pH.  Aliquot  as  considered  appropriate.

Sterilise  by  autoclaving  at  121 °C  for  15  minutes.

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Appendix  3

Materials  for  the  IF  test

I F - b u f f e r :   1 0   m M   p h o s p h a t e   b u f f e r e d   s a l i n e   ( P B S )   p H   7 , 2

This  buffer  is  used  for  dilution  of  antisera.

Na2 HPO4·12  H2O

NaH2 PO4·2  H2O

NaCl

Distilled  water

2,7  g

0,4  g

8,0  g

1  litre

Dissolve  the  ingredients  and  check  pH.  Aliquot  as  considered  appropriate.

Sterilise  by  autoclaving  at  121 °C  for  15  minutes.

I F - b u f f e r - T w e e n

This  buffer  is  used  for  washing  the  slides.  Add  0,1 %  Tween  20  to  the  IF  buffer.

0 , 1   M   P h o s p h a t e   b u f f e r e d   g l y c e r o l   p H   7 , 6

This  buffer  is  used  as  a  mountant  fluid  on  the  windows  of  the  IF  slide  to  enhance  fluorescence.

Na2 HPO4·12  H2O

NaH2 PO4·2  H2O

Glycerol

Distilled  water

3,2  g

0,15  g

50  ml

100  ml

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Appendix  4

Determination  of  contamination  level  in  the  IF  test

Surface  area  (S)  of  window  of  multispot  slide

=

(cid:240) D2
4

Where  D  =  diameter  of  window

Surface  area  (s)  of  objective  field

=

(cid:240) d2
4

Where  d  =  diameter  of  field

Calculate  d  either  by  direct  measurement  or  from  the  following  formulae:

s =

(cid:240) i2
G2 K2 × 4

where i = field  coefficient  (depends  upon  ocular  type  and  varies  from  8  to  24),

K = tube  coefficient  (1  or  1,25),

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

from  (2)

d = ˛W4 s

(cid:240)

(1)

(2)

(3)

(4)

from  (3)

d = ˛WW4 ×

(cid:240) i2
G2 K2 × 4

(cid:240)

=

i
GK

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

Calculate  the  number  of  typical  fluorescent  cells  per  window  (C).

C = c

S
s

Calculate  the  number  of  typical  fluorescent  cells  per  ml  pellet  (N)

N = C ×

1 000
y

× F

where y = volume  of  pellet  on  window,

F = pellet  dilution  factor.

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Appendix  5

Materials  for  the  ELISA  test

D o u b l e   s t r e n g t h   C a r b o n a t e   C o a t i n g   B u f f e r ,   p H   9 , 6

Na2CO3

NaHCO3

Distilled  water

6,36  g

11,72  g

1  litre

Dissolve  the  ingredients  and  check  pH.  Aliquot  as  considered  appropriate.

Sterilise  by  autoclaving  at  121 °C  for  15  minutes.

Sodium  sulphite  at  a  final  concentration  of  0,2 %  may  be  added  as  anti-oxidant  if  the  extract  contains  a
high  fraction  of  aromatic  molecules.

1 0 × P h o s p h a t e   B u f f e r e d   S a l i n e   ( P B S ) ,   p H   7 , 4

NaCl

KH2PO4

Na2HPO4 · 12H2O

KCl

Distilled  water

80  g

2  g

29  g

2  g

1  litre

Dissolve  the  ingredients  and  check  pH.  Aliquot  as  considered  appropriate.

Sterilise  by  autoclaving  at  121 °C  for  15  minutes.

P h o s p h a t e   B u f f e r e d   S a l i n e - T w e e n   ( P B S - T )

10 × PBS

10 %  Tween  20

Distilled  water

100  ml

5  ml

895  ml

B l o c k i n g   ( a n t i b o d y )   B u f f e r   ( m u s t   b e   f r e s h l y   p r e p a r e d )

10 × PBS

10  ml

Polyvinylpyrrolidone-44000  MWT  (PVP-44)

2,0  g

10 %  Tween  20

Milk  powder

Distilled  water

0,5  g

0,5  g

to  100  ml

A l k a l i n e   p h o s p h a t a s e   s u b s t r a t e   s o l u t i o n   p H   9 , 8

Diethanolamine

Distilled  water

97  ml

800  ml

Mix  and  adjust  to  pH  9,8  with  concentrated  HCI.

Make  up  to  one  litre  with  distilled  water.

Add  0,2  g  MgCl2.

Dissolve  two  phosphatase  substrate  five  mg  tablets  (Sigma)  per  15  ml  of  solution.

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Appendix  6

Materials  for  the  PCR  test

O l i g o n u c l e o t i d e   p r i m e r   s e q u e n c e

Primer  OLI-1

59-GGGGGTAGCTTGCTACCTGCC-39

Primer  Y-2

59-CCCACTGCTGCCTCCCGTAGGAGT-39

For  materials  see  Seal  et  al  (1993).

Appendix  7

Materials  for  selective  plating  and  enrichment  tests

S M S A   S e l e c t i v e   m e d i u m   (Engelbrecht,  1994  as  modified  by  Elphinstone  et  al,  1996)  but  delete  the
agar  and  the  tetrazolium  salts.

Basal  medium

Casamino  acids  (Difco)

Bacto  peptone  (Difco)

Glycerol

Agar  (Difco)

Distilled  water

1  g

10  g

5  ml

15  g

1  litre

Prepare  1/2  litre  volumes  of  medium  in  one  litre  flasks.

Dissolve  the  ingredients  and  check  pH.  Adjust  pH,  if  necessary,  to  6,5  before  autoclaving.  Ralstonia
solanacearum will  not  grow  well  on  the  medium  at  pH  . 7,0.

Sterilise  by  autoclaving  at  121 °C  for  15  minutes.

Cool  to  50 °C.

Add  the  following  ingredients  (all  from  Sigma)  to  obtain  the  specified  final  concentrations:

Crystal  violet

5  mg  per  litre

Polymixin  B  sulphate

100  mg  per  litre

(approximately  600 000  units)

Sigma  P-1004

Bacitracin (*)

25  mg  per  litre

(approximately  1 250  units)

Sigma  B-0125

Chloramphenicol

5  mg  per  litre

Sigma  C-3175

Penicillin-G

0,5  mg  per  litre

(approximately  825  units)

Sigma  P-3032

Tetrazolium salts

50  mg  per  litre

Dissolve  the  ingredients  in  70 %  ethanol  to  the  given  concentrations  for  the  volume  of  medium  prepared.
Some  ingredients,  viz.  polymixin  B  and  chloramphenicol  require  slight  warming  and  shaking.

S M S A   b r o t h   (Elphinstone  et  al.,  1996)

Prepare  as  for  the  SMSA  selective  medium  but  delete  the  agar.

Dispense  in  three  ml  aliquots  into  30  ml  disposable  Universal  tubes.

(*) If considered necessary, increasing bacitracin concentration to 300 ppm may reduce contaminating saprophytic bacteria

without  reducing  the  recovery  of  Ralstonia solanacearum.

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References

Buddenhagen, I.W.; Sequeira, L. and Kelman, A. 1962. Description of races in Pseudomonas solanacearum.
Phytopathology  52,  726.

Cook, D.; Elizabeth B. and Sequeira L., 1989. Genetic diversity of Pseudomonas solanacearum: detection of
restriction  fragment  length  polymorphism  with  DNA  probes  that  specify  virulence  and  hypersensitive
respons.  Molecular  Plant-Microbe  Interactions  2,  113—121.

Dinesen  I.G.  and  DeBoer,  S.H.  1995.  Extraction  of  Clavibacter  michiganensis  subsp.  sepedonicus  from
composite  samples  of  potato  tubers.  American  Potato  Journal  72,  133—142.

Elphinstone,  J.G.;  Hennessy,  J.;  Wilson,  J.  and  Stead,  D.E.  1996.  Sensitivity  of  different  methods  for  the
detection  of  Pseudomonas  solanacearum  (Smith)  Smith  in  potato  tuber  extracts.  EPPO  Bulletin  26.

Engelbrecht,  M.C.  1994.  Modification  of  a  semi-selective  medium  for  the  isolation  and  quantification  of
Pseudomonas  solanacearum.  ACIAR  Bacterial  Wilt  Newsletter  10,  3—5.

Hayward,  A.C.  1964.  Characteristics  of  Pseudomonas  solanacearum.  Journal  of  Applied  Bacteriology  27,
265—277.

Hayward,  A.C.  1994.  Systematic  and  phylogeny  of  Pseudomonas  solanacearum  and  related  bacteria.  In:
Bacterial  Wilt:  the  disease  and  its  causative  agent,  Pseudomonas  solanacearum  (eds.  A.C.  Hayward  and
G.L.  Hartman)  CAB  International  Oxford,  127—135.

Janse,  J.D.  1988.  A  detection  method  for  Pseudomonas  solanacearum  in  symptomless  potato  tubers  and
some  data  on  its  sensitivity  and  specificity.  EPPO  Bulletin  18,  343—351.

Janse,  J.D.  1991.  Infra-  and  intraspecific  classification  of  Pseudomonas  solanacearum  strains  using  whole
cell  fatty  acid  analysis.  Systematic  and  Applied  Microbiology  14,  335—345.

Kelman, A. 1954. The relationship of pathogenicity in Pseudomonas solanacearum to colony appearance on
a  tetrazolium  medium.  Phytopathology  64,  293—695.

Lelliot,  R.A.  and  Stead,  D.E.,  1987.  Methods  for  the  diagnosis  of  bacterial  diseases  of  plants.  (T.F.  Preece
ed.)  Blackwell  Scientific  Publications,  Oxford.  216  pp.

Louws, F.J.; Fulbright, D.W.; Stephens, C.T. and De Bruijn, F.J., 1995. Differentiation of genomic structure
by  rep-PCR  fingerprinting  to  rapidly  classify  Xanthomonas  campestris  pv.  vesicatoria.  Phytopathology  85,
528—536.

Lozano,  J.C.  and  Sequeira,  L.,  1970.  Differentiation  of  races  of  Pseudomonas  solanacearum  by  a  leaf
infiltration  technique.  Phytopathology  60,  838.

Mirza,  M.S.;  Rademaker,  J.W.L.;  Janse,  J.D.  and  Akkermans,  A.D.L.  1993,  Specific  16S  ribosomal  RNA
targeted  oligonucleotide  probe  against  Clavibacter  michiganensis  subsp.  sepedonicus.  Canadian  Journal  of
Microbiology 39,  1029—1034.

Robinson-Smith,  A.;  Jones,  P.;  Elphinstone,  J.G.  and  Forde,  S.M.D.,  1995.  Production  of  antibodies  to
Pseudomonas  solanacearum,  the  causative  agent  of  bacterial  wilt.  Food  and  Agricultural  Immunology  7,
67—79.

Seal,  S.E.;  Jackson,  L.A.;  Young,  J.P.W.  and  Daniels,  M.J.,  1993.  Differentiation  of  Pseudomonas
solanacearum,  P.  syzygii,  P.  picketti  and  the  blood  disease  bacterium  by  partial  16S  rRNA  sequencing:
construction  of  oligonucleotide  primers  for  sensitive  detection  by  polymerase  chain  reaction.  Journal  of
General  Microbiology  139,  1587—1594.

Smith,  J.J.;  Offord,  L.C.;  Holderness,  M.  and  Saddler,  G.S.,  1995.  Genetic  diversity  of  Burkholderia
solanacearum  (synonym  Pseudomonas  solanacearum)  race  3  in  Kenya.  Applied  and  Environmental
Microbiology 61,  4262—4268.

Stead,  D.E.,  1992a.  Techniques  for  detecting  and  identifying  plant  pathogenic  bacteria.  In:  Techniques  for
rapid  detection  of  plant  pathogens  (eds.  J.M.  Duncan  and  L.  Torrance).  Blackwell  Scientific  Publications,
Oxford,  76—111.

Stead,  D.E.,  1992b.  Grouping  of  plant  pathogenic  and  some  other  Pseudomonas  spp.  using  cellular  fatty
acid  profiles.  International  Journal  of  Systematic  Bacteriology  42,  281—295.

Van  Beuningen,  A.;  Derks,  H.  and  Janse  J.D.,  1995.  Detection  and  identification  of  Clavibacter
michiganensis  subsp.  sepedonicus  with  special  attention  to  fluorescent  in-situ  hybridisation  (FISH)  using  a
16S  rRNA  targeted  oligonucleotide  probe.  Züchtungsforschung  2,  266—269.

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ANNEX  III

1. For  each  suspected  occurrence  for  which  a  positive  result  in  the  screening  test(s)  has  been  identified
according to, for the listed plant material, the relevant method set out in Annex II or, in all other cases,
any other officially approved method, and confirmation or refutation by completion of the said method
is  awaited,  there  should  be  retention  and  appropriate  conservation  of:

— wherever  possible,  the  lot,  or  part  thereof  (from  which  the  sample  has  been  taken)  in  its  original

packaging with label,

— wherever  possible,  the  remaining  part  of  the  samples,

— any  remaining  extract  and  additional  prepared  material 

for 

the  screening 

test(s)  e.g.

immunofluorescence  slides,  and,

— all  relevant  documentation,

until  the  completion  of  the  said  method.

2.

In  the  case  of  confirmation  of  the  organism,  there  should  be  retention  and  appropriate  conservation
of:

— the  material  specified  in  paragraph  1,  and

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

where  appropriate,  and

— the  isolated  culture  of  the  organism,

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

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ANNEX  IV

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

(i) places  of  production,

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

the  organism,

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

infected  with  the  organism,

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

because  of  the  suspected  occurrence  of  the  organism,

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

places  of  production  found  to  be  infested  with  the  organism,

— growing  potatoes  or  tomatoes  and  located  in  the  neighbourhood  of  infested  places  of  production,
including such places of production sharing production equipment and facilities directly or through
a  common  contractor,

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

with  the  organism,

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

production  confirmed  or  suspected  to  be  infested  with  the  organism,

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

organism; and,

(ii)

surface water used for irrigation or spraying of, or which has flooded field(s) or place(s) of production
confirmed  to  be  infested  with  the  organism.

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ANNEX  V

1. The elements in the determination of the extent of probable contamination under Article 5(1)(a)(iii) and

5(1)(c)(iii),  shall  include  where  relevant:

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

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

— place(s) of production with a production link to the listed plant material designated as contaminated
under  Article  5(1)(a)(ii),  including  those  sharing  production  equipment  and  facilities  directly  or
through  a  common  contractor,

— the listed plant material produced in the place(s) of production referred to in the previous indent, or
present in such place(s) of production during the period when the listed plant material designated as
contaminated under Article 5(1)(a)(ii), was present on the places of production referred to in the first
indent,

— stores  handling  the  listed  plant  material  from  the  above  places  of  production,

— any  machinery,  vehicle,  vessel,  store,  or  units  thereof,  and  any  other  objects  including  packaging
material, that may have come into contact with the listed plant material designated as contaminated
under  Article  5(1)(a)(ii),

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

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

— as a result of the investigation and testing under Article 5(1)(a)(i), in the case of potato, those tubers
or plants with a sister or parental clonal relationship to, and in the case of tomato, those plants with
the  same  source  as,  the  listed  plant  material  designated  to  be  contaminated  under  Article  5(1)(a)(ii)
and  for  which,  although  they  may  have  tested  negative  for  the  organism,  it  appears  that
contamination  is  probable  through  a  clonal  link,

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

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

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

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

organism.

2. The  determination  of  the  possible  spread  under  Article  5(1)(a)(iv)  and  5(1)(c)(iii)  shall  include:

(i)

in  cases  under  Article  5(1)(a)(iv),  a  consideration  of  the  following  elements,

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

— the  common  production  and  use  of  seed  potato  stocks,

— places  of  production  using  surface  water  for  irrigation  or  spraying  of  listed  plant  material  in
cases  where  there  is  or  has  been  risk  of  surface  water  run-off  from,  or  flooding  of,  place(s)  of
production  designated  to  be  contaminated  under  Article  5(1)(a)(ii);

(ii)

in  cases  where  surface  water  has  been  designated  as  contaminated  under  Article  5(1)(c)(ii):

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

the  surface  water  designated  as  contaminated,

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

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3. The  details  of  the  notification  referred  to  in  the  first  subparagraph  of  Article  5(2)  shall  include:

— date of reporting of suspected occurrence under Article 4 and dates of sampling and confirmation of

the  presence  of  the  organism  under  Article  5,  as  appropriate,

— a  description  of  the  elements  of  the  designated  contamination  and  zone  demarcation.

4. The  details  of  the  additional  notification  referred  to  in  the  second  subparagraph  of  Article  5(2)  shall

include:

— for  any  potato  consignment  or  lot  designated  as  contaminated,  the  certificates  prescribed  in
Articles  7  and  8  of  Directive  77/93/EEC,  the  passport  number  or  registration  number  of  potato
producers,  collective  warehouses  and  dispatching  centres,  as  appropriate,

— for  any  tomato  plant  consignment  or  lot  designated  as  contaminated  the  crtificates  prescribed  in
Articles  7  or  8  of  Directive  77/93/EEC  and  passport  number,  in  accordance  with  the  listing  in
Annex  V,  Part  A,  Section  I.2.2  to  Directive  77/93/EEC,

— the  variety  name  and  category  for  seed  potato  stocks,  and  where  possible  in  all  other  cases,

— such  other  information  relating  to  the  confirmed  outbreak  as  the  Commission  may  require.

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ANNEX  VI

1. With  refrence  to  Article  6(1)  the  provisions  shall  be:

— incineration,  or

— use  as  animal  feed  after  heat  treatment  such  that  there  is  no  risk  of  the  organism  surviving,  or

— deep  burial,  at  a  disposal  site  at  which  there  is  no  risk  of  seepage  to  agricultural  land  or  contact

with  water  sources  which  could  be  used  for  irrigation  of  agricultral  land,  or

— industrial  processing  through  direct  and  immediate  delivery  to  a  processing  plant  with  officially
approved waste disposal facilities which conform to the provisions laid down in Annex VII to this
Directive, or

— other  measures,  provided  that  it  has  been  established  that  there  is  no  identifiable  risk  of  the
organism spreading; such measures to be immediately notified to the Commission and to the other
Member  States.

2.

The  appropriate  use  or  disposal  of  the  listed  plant  material  referred  to  in  Article  6(2),  under  the
control  of  the  responsible  official  bodies  of  the  Member  State(s)  concerned,  with  appropriate
communication between responsible official bodies to ensure such control at all times and approval by
the responsible official body of the Member State where the potatoes are to be packed or processed in
respect  of  the  waste  disposal  facilities  referred  to  in  the  first  and  second  indents,  shall  be:

(i)

for  potato  tubers,

— use  as  ware  potatoes  intended  for  consumption  and  packed  at  sites  with  appropriate  waste
disposal  facilities,  ready  for  direct  delivery  and  use  without  repacking,  and  intended  for  such
direct  delivery  and  use,  or

— use as ware potatoes intended for industrial processing, and intended for direct and immediate

delivery  to  a  processing  plant  with  appropriate  waste  disposal  facilities,  or

— some  other  use  or  disposal,  provided  that  it  is  established  that  there  is  no  identifiable  risk  of
the  organism  spreading,  and  subject  to  approval  by  the  said  responsible  official  bodies.  Such
measures  to  be  immediately  notified  to  the  Commission  and  to  the  other  Member  States.

(ii)

for  other  plant  parts  including  stem  and  foliage  debris,

— destruction,  or

— some  other  use  or  disposal,  provided  that  it  is  established  that  there  is  no  identifiable  risk  of
the  organism  spreading;  such  measures  shall  be  notified  to  the  Commission  and  to  other
Member  States.

3.

The  appropriate  methods  for  decontamination  of  the  objects  referred  to  in  Article  6(3)  shall  be
cleansing  and,  where  appropriate,  disinfection,  such  that  there  is  no  identifiable  risk  of  the  organism
spreading  and  shall  be  employed  under  the  supervision  of  the  responsible  official  bodies  of  the
Member  States.

4.

The series of measures to be implemented by Member States within the demarcated zone(s) established
under  Article  5(1)(a)(iv)  and  (c)(iii)  and  referred  to  in  Article  6(4)  shall  include:

4.1.

In  cases  where  places  of  production  have  been  designated  as  contaminated  under  Article  5(1)(a)(ii):

(a)

in  a  field  or  unit  of  protected  crop  production  designated  to  be  contaminated  under
Article  5(1)(a)(ii),  either

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

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

host  plants  of  the  organism  including  solanaceous  weeds,  and

— the  following  shall  not  be  planted:

— potato  tubers  or  plants,

— tomato  plants  and  seeds,

— taking  into  account  the  biology  of  the  organism,

— other  host  plants,

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

surviving,

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

— in  the  first  potato  or  tomato  cropping  season  following  the  period  specified  in  the
preceding  indent,  and  on  the  condition  that  the  field  has  been  found  free  from  volunteer
potato  and  tomato  plants  and  other  host  plants  including  solanaceous  weeds  for  at  least
the  two  consecutive  growing  years  prior  to  planting,

— in  the  case  of  potatoes,  officially  certified  seed  potatoes  shall  be  planted  for  ware

production  only  and,

— an  official  survey  including  testing,  as  detailed  in  Article  2(1),  shall  be  conducted,

— in the potato or tomato cropping season succeeding that referred to in the previous indent
and following an appropriate rotation cycle, in the case of potatoes officially certified seed
potatoes  shall  be  planted  for  either  seed  or  ware  production  and  in  the  case  of  potatoes
and  tomatoes  an  official  survey  as  detailed  in  Article  2(1),  shall  be  conducted;  or 

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

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

host  plants  of  the  organism  including  solanaceous  weeds,  and

— the  field  shall  be  established  and  maintained  during  the  first  three  years  either,  in  bare
fallow  or,  in  cereals  according  to  the  risk  identified,  or,  in  permanent  pasture  with
frequent  close  cutting  or  intensive  grazing  or,  as  grass  for  seed  production,  followed  by
planting in the succeeding two years with non-host plants of the organism for which there
is  no  identified  risk  of  the  organism  surviving  or  spreading,

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

preceding  indent,

— in the case of potatoes, officially certified seed potatoes shall be planted for either seed

or  ware  production,

and  an  official  survey  including  testing,  as  detailed  in  Article  2(1),  shall  be  conducted;

(b)

in  other  fields:

— in  the  growing  year  following  the  designated  contamination:

— either  no  potato  tubers  or  plants,  or  other  host  plants  of  the  organism  shall  be  planted,
and  measures  shall  be  taken  to  eliminate  volunteer  potato  and  tomato  plants  and  other
host  plants  including  solanaceous  weeds  as  appropriate,  or

— in  the  case  of  potato  tubers,  officially  certified  seed  potatoes  may  be  planted  for  ware
production only, on the condition that the responsible official bodies are satisfied that the
risks  of  volunteer  potato  and  tomato  plants  and  other  host  plants  of  the  organism

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including solanaceous weeds, have been eliminated. The growing crop shall be inspected at
appropriate times and volunteer potato plants shall be tested for the organism; in addition,
for  potatoes,  the  harvested  tubers  shall  be  inspected;

— for  the  first  growing  year  following  that  specified  in  the  first  indent,

— in  the  case  of  potatoes,  only  officially  certified  seed  potatoes  shall  be  planted  for  either

seed  or  ware  production,

— for  at  least  the  second  growing  year  following  that  in  the  first  indent,

— in the case of potatoes, only officially certified seed potatoes or seed potatoes grown under
official  control  from  officially  certified  seed  potatoes  shall  be  planted  for  either  seed  or
ware  production,

— in  each  of  the  growing  years  referred  to  in  the  previous  indents  measures  shall  be  taken  to
eliminate volunteer potato and tomato plants and other host plants of the organism including
solanaceous weeds, and an official survey as detailed in Article 2(1), shall be conducted, and in
the cases where seed potatoes are planted for seed production testing of tubers shall be carried
out;

(c)

immediately  following  the  designation  of  contamination  under  Article  5(1)(a)(ii)  and  in  each  of
the subsequent growing years up to and including the first permissible potato or tomato cropping
season  on  the  field(s)  designated  as  contaminated,  as  detailed  in  paragraph  (a):

— all  machinery  and  storage  facilities  on  the  place  of  production  and  involved  in  potato  or
tomato  production  shall  be  cleansed  and,  where  appropriate,  disinfected  using  appropriate
methods,  as  specified  in  point  3,

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

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

(d)

in a unit of protected crop production designated as contaminated under Article 5(1)(a)(ii) where
complete  replacement  of  the  growing  medium  is  possible,

— no  potato  tubers  or  plants,  or  other  host  plants  of  the  organism  including  tomato  plants  and
seeds shall be planted unless the said unit has been subjected to officially supervised measures
to eliminate the organism and to remove all host plant material, including, at least, a complete
change in growing medium and cleansing and, where appropriate, disinfection of the said unit
and  all  equipment,  and  subsequently  has  been  granted  approval  for  potato  or  tomato
production  by  the  responsible  official  bodies,  and

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

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

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

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

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

shall:

(a)

immediately,  and  for  at  least  three  growing  years,  after  the  designated  contamination:

(aa)

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

— ensure  supervision  by  their  responsible  official  bodies  of  premises  growing,  storing  or
handling potato tubers or tomatoes, together with premises which operate machinery for
potato  or  tomato  production  under  contract,

— require  cleansing  and,  where  appropriate,  disinfection  of  machinery  and  stores  on  such

premises,  using  appropriate  methods,  as  specified  under  point  3,

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21.8.98

— require  the  planting  of  only  certified  seed  or  seed  grown  under  official  control  for  all
potato  crops  within  that  zone,  and  testing  after  harvest  of  seed  potato  crops  grown  in
places  of  production  determined  as  probably  contaminated  under  Article  5(1)(a)(iii),

— require  the  separate  handling  of  harvested  potato  seed  stocks  to  those  of  ware  on  all

premises  within  the  zone,

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

(ab)

in cases where surface water has been designated as contaminated under Article 5(1)(c)(ii) or
included  in  the  elements  for  the  possible  spread  of  the  organism  in  accordance  with
Annex  V  point  2,

— conduct  an  annual  survey  at  appropriate  times  including  sampling  of  surface  water  and
appropriate  solanaceous  host  plants  in  the  relevant  water  sources  and  testing  in
accordance  with

— for  listed  plant  material,  the  relevant  method  set  out  in  Annex  II,  or

— in  other  cases,  any  other  officially  approved  method,

— introduce  official  controls  on  irrigation  and  spraying  programmes,  including  a  ban  on
the use of the water designated as contaminated for the irrigation and spraying of listed
plant  material,  and,  where  appropriate,  other  host  plants  in  order  to  prevent  the  spread
of  the  organism.  This  ban  may  be  reviewed  on  the  basis  of  the  results  obtained  in  the
said  annual  survey,

— in  cases  where  liquid  waste  discharges  are  contaminated,  introduce  official  controls  on
the  disposal  of  waste  from  industrial  processing  or  packaging  premises  handling  listed
plant material;

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

appropriate  period  of  time.

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ANNEX  VII

The  officially  approved  waste  disposal  facilities  referred  to  in  Annex  VI  paragraph  1,  fourth  indent  shall
conform  to  the  following  provisions  such  that  the  risk  of  spreading  the  organism  is  obviated:

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

other  solid  waste  associated  with  the  potatoes  and  tomatoes  shall  be  disposed  by  either,

— deep burial at disposal site at which there is no risk of seepage to agricultural land or contact with
water  sources  which  could  be  used  for  irrigation  of  agricultural  land.  The  waste  shall  be  conveyed
directly  to  the  site  under  containment  conditions  such  that  there  is  no  risk  of  loss  of  the  waste,
or

— incineration;

(ii)

liquid processing waste: prior to disposal, liquid waste containing suspended solids shall be subjected to
filtration  or  settlement  processes  to  remove  such  solids.  These  solids  shall  be  disposed  of  as  set  out  in
subparagraph  (i).

The  liquid  waste  shall  then  be  either:

— heated  to  a  minimum  of  70 °C  during  at  least  30  minutes,  prior  to  disposal,  or

— otherwise  disposed  of  subject  to  official  approval  and  under  official  control  such  that  there  is  no
risk that the waste could come into contact with agricultural land or water sources which could be
used  for  irrigation  of  agricultural  land.  The  details  thereof  shall  be  notified  to  the  other  Member
States  and  to  the  Commission.