Molecular taxonomy of recently described
       Phytophthora and Pythium




                           Dr. Lassaad Belbah...
Declining Polish forests


Alnus glutinosa, Kolo, Poland.
Direct PCR, DNA barcoding
Collaboration with FASTERIS SA (Geneva)
Single tube nested PCR for
sensitive detection of oomycetes
ARISA Analysis of oomycete infected
      Rhododendron leaves
ITS rDNA PCR Amplification of oomycete
       community from soil DNA
ARISA analysis of oomycete community
           from soil DNA
Evolution rate of new species discovery during
                     time
Genomic assisted Phylogeny in the genus Phytophthora
Blair et al. (2007) Fungal Genetics and Biology 45 266-277
Morphological description : Ph. polonica
Morphological description: Py. recalcitrans




  Hypha and hyphal swellings   Appressoria




           Antheridia      ...
Phylogenetic analysis of Phytophthora spp.



Phylogenetic analysis based
on four concatenated genes
(ß-tubuline, EF1-α, N...
Phylogenetic analysis of Pythium spp.

Unrooted
phylogenetic analysis




                                                ...
Towards a redefinition of oomycete genera
Ph. hedraiandra




Ph. hedraiandra, a
species with high
hybridization
potential spreading
on multiple hosts.
Ph. ramorum detection by real time PCR
Ph. alni detection by real time PCR
                Ph. alni subsp. alni
                Ph. alni subsp. multiformis
     ...
Detection of Py. spp. and Ph. spp. by microarray
Ph. ramorum, tissue colonization


                                 Tissue of infected Rhododendron
                      ...
Ph. ramorum, Genetic transformation




                                          Transgenic Ph. ramorum strain BBA9/95_6G...
Genomic organisation of virulence factors
        in oomycete genomes
Evolutionary lines of oomycete virulence factors
Outlook



Diversity of oomycete species using non culture based techniques

Reinforcing environmental monitoring tools (M...
Special Thanks To:



Steve Woodward
Eduardo Moralejo
Enrique Descals
Jozef Bakonyi
Thanks for your attention
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O14 Belbahri

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O14 Belbahri

  1. 1. Molecular taxonomy of recently described Phytophthora and Pythium Dr. Lassaad Belbahri
  2. 2. Declining Polish forests Alnus glutinosa, Kolo, Poland.
  3. 3. Direct PCR, DNA barcoding Collaboration with FASTERIS SA (Geneva)
  4. 4. Single tube nested PCR for sensitive detection of oomycetes
  5. 5. ARISA Analysis of oomycete infected Rhododendron leaves
  6. 6. ITS rDNA PCR Amplification of oomycete community from soil DNA
  7. 7. ARISA analysis of oomycete community from soil DNA
  8. 8. Evolution rate of new species discovery during time
  9. 9. Genomic assisted Phylogeny in the genus Phytophthora Blair et al. (2007) Fungal Genetics and Biology 45 266-277
  10. 10. Morphological description : Ph. polonica
  11. 11. Morphological description: Py. recalcitrans Hypha and hyphal swellings Appressoria Antheridia Oospore
  12. 12. Phylogenetic analysis of Phytophthora spp. Phylogenetic analysis based on four concatenated genes (ß-tubuline, EF1-α, NADH1 and COX1), induced by Maximum Likelihood (PhyML) Ph. sylvatica sp. nov Ph. hungarica sp. nov
  13. 13. Phylogenetic analysis of Pythium spp. Unrooted phylogenetic analysis Py.attra Py.debaryanu Py.helic P Py.dimorphu Py.inte Py.sylvaticum Py.undulatum Py.p y.ma Py Py.a Py.pro ntheridiu Py. aro based on Internal cros Py .hy rm andrum olig ec na nd .am dno edium poru latum an d Py an d as spo m Py m m .pe m r um Py cu rum ru m m .ac .a rip Transcribed Spacers rum linu r an Py rh loc m en th .a ue ale Py um r icu s om rum ist um .v s re de os an an Py .bo rc Py co po te 1 and 2 induced by e m .p rp Pythium quercum Py tra ilu .q yr ilo ooli os ch Py bu i y. edo .in m P .o m on fla ilu ph Py .pe tum Py s t er hy ae Bayesian Inference Py riil y. .gr u P am es Py. pl u am risp inic m ola Py P .ch y.h elic oid bita cea rum Pythium sterilum Py.s oru m cu cur (MrBayes v.3.1.2) Py. ae ulca ofer tum ndig Py.d issim Py.i s Py.sc ile exan lerote Py.v ichum nn Py.zingib Py.nu eris onon Py.orthog Py.myriotylum n Py.acanthophoro Py.tracheiphilum Py.perplexum Py.salpingophorum Py.buismaniae horum Py.polym Py.conidiop astum losum Py.unc Py .follicu inulatu m um Py.m rulos astop Py.to m Py.c horu m u latu ylind at en P y. rosp Py.c atu m irre orum ust Py gula ang tum .sp re Py. olu Py i cu .v e lum Py ns .m v oe Py am .fle um . Py kunm atum ill Py clin .di m Py .spin Py oto iss arin cu um m Py .ac .h ro os um ing en se Pythium spiculum .d riu yp gyn Py y. m ta Py .erin sum m P og um .lu ch ratu .e Py yn .aq aule Py ch um Py tile o in ol yc Py .ra um ul .c ua Py ac at phy a Py .vio amai dio Py. rm l um rae eu a rico .iw Py.o .p s Py.d Py.o pe lae Py m Py.ap e r en Py.hete pad Py Py.insidios ag Py.glomer Py.ultimumvar.u Py.splendens Py.ultimumvar.sporangiiferum on d os kan elien dicu on kano por dha .ch hanide .m oga P y. Py m rothalli Py.a se Py gane nen atum um rmatu seC nseC cum ltimum BS3 m BS70 15.8 1.83 1
  14. 14. Towards a redefinition of oomycete genera
  15. 15. Ph. hedraiandra Ph. hedraiandra, a species with high hybridization potential spreading on multiple hosts.
  16. 16. Ph. ramorum detection by real time PCR
  17. 17. Ph. alni detection by real time PCR Ph. alni subsp. alni Ph. alni subsp. multiformis Ph. alni subsp. uniformis Ph. alni subsp. alni Ph. alni subsp. multiformis Ph. alni subsp. alni Ph. alni subsp. uniformis
  18. 18. Detection of Py. spp. and Ph. spp. by microarray
  19. 19. Ph. ramorum, tissue colonization Tissue of infected Rhododendron leaves with Ph. ramorum structures, hyphae with zoosporangia growing out of stomata on discolored leaf surface (vital staining with FUN® 1, Molecular Probes), zoospore production is induced, when infected P. ramorum in Rhododendron leaves were moistened and kept at low cortex tissue, chlamydospores temperatures (7°C) with white-yellow autofluorescence in cortex of necrotic Rhododendron stem
  20. 20. Ph. ramorum, Genetic transformation Transgenic Ph. ramorum strain BBA9/95_6G: Transgenic Ph. ramorum strain BBA26/02- CLSM micrographs showing GFP expression in 4: CLSM micrographs indicating GFP protoplasm of zoosporangia and hyphae, yellow expression in germinating cysts. signals: natural Ph. ramorum autofluorescence Left: transmission image in some hyphae, sporangium wall, base and pedicel, GFP signals were amplified with Alexa Right: GFP signals Fluor 488®labelled anti GFP antibodies (Molecular Probes)
  21. 21. Genomic organisation of virulence factors in oomycete genomes
  22. 22. Evolutionary lines of oomycete virulence factors
  23. 23. Outlook Diversity of oomycete species using non culture based techniques Reinforcing environmental monitoring tools (Molecular toolbox) Evolutionary history of main virulence factors Linking genetic phylogeny to morphology
  24. 24. Special Thanks To: Steve Woodward Eduardo Moralejo Enrique Descals Jozef Bakonyi
  25. 25. Thanks for your attention

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