1. Genetic diversity of Belgian
Phytophthora ramorum isolates
I. De Dobbelaere, A. Vercauteren, K. Heungens, M. Maes
Phytophthora & Pythium Workshop
ICPP – Torino – August 23-24, 2008
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2. Northern part of Belgium
(Flanders) is one of the
largest producers of
Rhododendron in Europe
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3. Results of the annual federal survey (FAVV)
• ± 50 major nurseries sites inspected
• 100-500 samples annually
• 20-25% of Belgian samples positive
• Main host plants:
Rhododendron (>80%) and Viburnum
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4. Global research genotyping P. ramorum
Number Primer Different Dominant
of isolates pairs genotypes genotype
Ivors et al. 2004 AFLP 67 USA 4 16 USA USA: 1
18 EU 15 EU
Ivors et al. 2006 SSLP 71 USA 12 4 USA USA: 1
80 EU 7 EU EU: 1
Prospero et al. SSLP 323 USA 10 33 USA USA: 1 (OR)
2007
Mascheretti et al. SSLP 292 USA 9 35 USA USA: 3 (CA)
2008
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5. Objective
Determine the genetic diversity within the population
of Belgian P. ramorum isolates
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6. Materials and methods
1. Amplified Fragment Length Polymorphism (AFLP)
• 79 isolates (2002-2006)
• Protocol:
Adapted from Ivors et al. (2004)
Five pairs of selective primers
2-3 replicates per P. ramorum isolate (reproducibility)
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7. Materials and methods
2. SSLP (microsatellite polymorphisms)
• 314 isolates (2002-2007)
• Protocol:
Screening of isolates with 6 selected SSR markers:
3 from Ivors et al. (2006)
3 from A. Vercauteren: screening of 132 candidate polymorphic SSR
(screening based on polymorphisms within selected EU isolates)
Confirmation of polymorphic status of isolates
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8. Results AFLP
• 5 primer pairs → 222 fragments →13 polymorphic
• 71 isolates (90%) belong to a single genotype
• 7 genotypes identified
• no correlation between genotype and place, year
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9. I (71)
II (1)
III (1)
VI (2)
VII (1)
V (1)
IV (2)
0.1
Neighbour-joining cluster analysis
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10. Results microsatellites
• 5 out of 6 EU-polymorphic primer pairs show polymorphisms
within BE isolates
• 233 out of 314 isolates (74%) belong to one genotype
• 26 genotypes identified
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11. Microsatellite primer pair code
Ivors et al. Vercauteren
18 64 82 2199 512
A 262 398 137 242 186 202 217 265 156
D 262 400 137 242 186 202 217 265 156
H 262 398 137 248 186 202 217 265 156
I 262 398 137 242 186 202 217 249 156
J 262 398 137 242 186 202 217 260 156
G K 262 398 137 238 186 202 217 265 156
e L
M
262
262
398
398
137
137
244
242
186
-
202
-
217
217
260
265
156
156
n N 262 398 137 242 186 202 212 249 156
O 262 400 137 242 186 202 217 260 156
o P 262 400 137 244 186 202 217 260 156
t Q
R
262
262
398
398
137
137
244
244
186
186
202
202
217
217
270
265
156
156
y S 262 398 137 242 186 202 217 265 167
p T
U
262
262
398
398
133
137
242
242
186
186
202
202
217
217
270
254
156
156
e V 260 398 137 242 186 202 222 265 156
s
W 262 398 137 242 186 202 217 270 156
X 262 398 137 242 186 202 212 265 156
Y 262 398 137 242 186 202 217 265 171
Z 262 398 137 228 186 202 217 260 156
AA 262 398 137 242 186 202 222 265 156
AB 262 398 137 246 186 202 212 249 156
AC 262 402 137 242 186 202 212 249 156
AD 262 398 ILVO137- Plant (Crop Protection)
242 186 202 217 243 156
AE 262 398
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137 246 186 202 217 265 156
13. Genotype composition of Belgian P. ramorum population
L M RQS VWXY
K O
P
LQU
T
J N J
L
I
J D
I I
D
H
D
A
A
A
2002 (29)
jaar: 2002
2003 (49)
jaar: 2003 jaar:
(111)
2004 2004
O R R AA AA
N
J R
J
I
I D N
Z
J
AC
A
I
AB A
AE
A AD
2005 (55) 2006 (37)
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2007 (33)
15. Genotype composition of Belgian P. ramorum population
L M RQS VWXY
K O
P
LQU
T
J N J
L
I
J D
I I
D
H
D
A
A
A
2002 (29)
jaar: 2002
2003 (49)
jaar: 2003 jaar:
(111)
2004 2004
O R R AA AA
N
J R
J
I
I D N
Z
J
AC
A
I
AB A
AE
A AD
2005 (55) 2006 (37)
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2007 (33)
16. Local occurence of genotypes
Q R
S
AD
Q
site 1 P A
A
A A A A
(1) (8) (21) (14) (2) (13)
J J A
AB
site 2 O
L J N A
I N
L
I
(5) N (2) (2) (8) AC (5)
WX R AA
J R
site 3
AA A
A A A
(46) (14) (17) (2)
V
site 4
A
(7) (2) A (2) A
2002 2003 2004 2005 2006 2007
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17. 2004-2005: 1399 samples were collected → 331 P. ramorum isolates found
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18. 2004 2005
WX R
site 1 J
Federal survey
site 2
A A
(46) (14)
K R AFAE
W KRAE
JWK R AF
R AF J JW AF
JW K
Project
A
ILVO - Plant (Crop Protection)
A A
A
(157) http://www.ilvo.vlaanderen.be/ (174)
19. Field surveyed in 2004
Genotypes
A
J
W
K
R
AF
X
10m ILVO - Plant (Crop Protection) 19
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20. Conclusions
• Multiple P. ramorum genotypes in Belgium (based on AFLP and SSLP)
• One genotype is dominant in Belgium (AFLP: 90% – SSLP: 74%)
→ founder genotype
• Other genotypes (6/79 isolates with AFLP – 25/314 isolates with SSLP)
→ mostly single isolates
• Evolution: some genotypes disappear, new genotypes arise
• Bottleneck in 2004-2005 : metalaxyl-M resistance
• Local specialization: 15/26 genotypes found at single nurseries
• Application to study of epidemiology → dispersal of genotypes in a field
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21. Thanks to:
• The Belgian Plant Inspection Service (FAVV/AFSCA)
• FOD (grant RT-05/04-PHYRAM-1 )
• IWT (grant 040692)
• Tineke Van Vooren
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