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O6 De Dobbelaere
O6 De Dobbelaere
O6 De Dobbelaere
O6 De Dobbelaere
O6 De Dobbelaere
O6 De Dobbelaere
O6 De Dobbelaere
O6 De Dobbelaere
O6 De Dobbelaere
O6 De Dobbelaere
O6 De Dobbelaere
O6 De Dobbelaere
O6 De Dobbelaere
O6 De Dobbelaere
O6 De Dobbelaere
O6 De Dobbelaere
O6 De Dobbelaere
O6 De Dobbelaere
O6 De Dobbelaere
O6 De Dobbelaere
O6 De Dobbelaere
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O6 De Dobbelaere

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  • 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 ILVO - Plant (Crop Protection) http://www.ilvo.vlaanderen.be/
  • 2. Northern part of Belgium (Flanders) is one of the largest producers of Rhododendron in Europe ILVO - Plant (Crop Protection) http://www.ilvo.vlaanderen.be/
  • 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 ILVO - Plant (Crop Protection) 3 http://www.ilvo.vlaanderen.be/
  • 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 ILVO - Plant (Crop Protection) 4 http://www.ilvo.vlaanderen.be/
  • 5. Objective Determine the genetic diversity within the population of Belgian P. ramorum isolates ILVO - Plant (Crop Protection) 5 http://www.ilvo.vlaanderen.be/
  • 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) ILVO - Plant (Crop Protection) 6 http://www.ilvo.vlaanderen.be/
  • 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 ILVO - Plant (Crop Protection) 7 http://www.ilvo.vlaanderen.be/
  • 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 ILVO - Plant (Crop Protection) 8 http://www.ilvo.vlaanderen.be/
  • 9. I (71) II (1) III (1) VI (2) VII (1) V (1) IV (2) 0.1 Neighbour-joining cluster analysis ILVO - Plant (Crop Protection) http://www.ilvo.vlaanderen.be/
  • 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 ILVO - Plant (Crop Protection) 10 http://www.ilvo.vlaanderen.be/
  • 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 http://www.ilvo.vlaanderen.be/ 137 246 186 202 217 265 156
  • 12. 1 Neighbour-joining cluster analysis ILVO - Plant (Crop Protection) http://www.ilvo.vlaanderen.be/
  • 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) ILVO - Plant (Crop Protection) http://www.ilvo.vlaanderen.be/ 2007 (33)
  • 14. Resistance to fungicide metalaxyl-M Year Percentage resistant isolates 2002 29 2003 17 2004 56 2005 95 2006 84 2007 59 ILVO - Plant (Crop Protection) http://www.ilvo.vlaanderen.be/
  • 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) ILVO - Plant (Crop Protection) http://www.ilvo.vlaanderen.be/ 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 ILVO - Plant (Crop Protection) http://www.ilvo.vlaanderen.be/
  • 17. 2004-2005: 1399 samples were collected → 331 P. ramorum isolates found ILVO - Plant (Crop Protection) 17 http://www.ilvo.vlaanderen.be/
  • 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 http://www.ilvo.vlaanderen.be/
  • 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 ILVO - Plant (Crop Protection) 20 http://www.ilvo.vlaanderen.be/
  • 21. Thanks to: • The Belgian Plant Inspection Service (FAVV/AFSCA) • FOD (grant RT-05/04-PHYRAM-1 ) • IWT (grant 040692) • Tineke Van Vooren ILVO - Plant (Crop Protection) 21 http://www.ilvo.vlaanderen.be/

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