Molecular taxonomy of recently described Phytophthora and Pythium

Dr. Lassaad Belbahri

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 infectedRhododendron leaves

ITS rDNA PCR Amplification of oomycetecommunity from soil DNA

ARISA analysis of oomycete community

from soil DNA

Evolution rate of new species discovery duringtime

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 Oospore

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

Phylogenetic analysis of Pythium spp.

Unrootedphylogenetic analysis based on Internal Transcribed Spacers 1 and 2 induced by Bayesian Inference (MrBayes v.3.1.2)

Py.bore

ale

Py.que

rcum

Py.ostr

acodes

Py.oedochilum

Py.sterilum

Py.chamaehyphon

Py.helicoides

Py.cucurbitacearum

Py.indigoferae

Py.vexans

Py.nunn

Py.orthogonon

Py.acanthophoron

Py.perplexumPy.buismaniaePy.polymastumPy.uncinulatumPy.mastophorumPy.cylindrosporum

Py.irregularePy.spiculumPy.mamillatumPy.kunmingense

Py.spinosum

Py.acrogynum

Py.hypogynum

Py.echinulatum

Py.erinaceum

Py.radiosum

Py.violae

Py.iwagamai

Py.paddicum

Py.okanoganenseCBS315.81

Py.okanoganenseCBS701.83

Py.heterothallicum

Py.glomeratum

Py.splendens

Py.u lti m

umvar.sp orangii fe rum

Py.ultim

umvar.ultim

umP

y.insidiosumPy.aphaniderm

atumPy.deliense

Py.a

dhae

rens

Py.p

orph

yrae

Py.c

hond

ricol

a

Py.m

onos

perm

um

Py.a

quat

ile

Py.pa

chyc

aule

Py.colo

ratum

Py.luta

rium

Py.marin

um

Py.diss

otocumPy.diclinum

Py.flevoensePy.volutumPy.angustatumPy.catenulatumPy.torulosumPy.folliculosum

Py.conidiophorumPy.salpingophorum

Py.tracheiphilum

Py.myriotylum

Py.zingiberis

Py.scleroteichum

Py.dissimile

Py.sulcatum

Py.plurisporum

Py.graminicola

Py.periilum

Py.inflatum

Py.pyrilobum

Py.vanterpoolii

Py.aristosporum

Py.arrhenomanes

Py.acanthicumPy.periplocum

Py.amasculinum

Py.hydnosporumPy.oligandrumPy.anandrumPy.prolatum

Py.helicandrumP

y.dimorphum

Py.undulatum

Py.sylvaticum

Py.debaryanum

Py.attrantheridium

Py.intermedium

Py.macrosporum

Py.paroecandrum

Pythium quercum

Pythium sterilum

Pythium spiculum

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

Ph. alni subsp. uniformis

Ph. alni subsp. alni

Ph. alni subsp. multiformis

Ph. alni subsp. alni

Ph. alni subsp. uniformis

Detection of Py. spp. and Ph. spp. by microarray

Ph. ramorum, tissue colonization

P. ramorum in Rhododendron cortex tissue, chlamydospores

with white-yellowautofluorescence in cortex of necrotic Rhododendron stem

Tissue of infected Rhododendron leaves with Ph. ramorum structures, hyphae with zoosporangia growing out of stomata on discoloredleaf surface (vital staining with FUN® 1, Molecular Probes), zoospore production is induced, when infectedleaves were moistened and kept at lowtemperatures (7°C)

Ph. ramorum, Genetic transformation

Transgenic Ph. ramorum strain BBA26/02-4: CLSM micrographs indicating GFP expression in germinating cysts. Left: transmission imageRight: GFP signals

Transgenic Ph. ramorum strain BBA9/95_6G: CLSM micrographs showing GFP expression in protoplasm of zoosporangia and hyphae, yellow signals: natural Ph. ramorum autofluorescencein some hyphae, sporangium wall, base and pedicel, GFP signals were amplified with AlexaFluor 488®labelled anti GFP antibodies (Molecular Probes)

Genomic organisation of virulence factorsin oomycete genomes

Evolutionary lines of oomycete virulence factors

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

Special Thanks To:

Steve WoodwardEduardo MoralejoEnrique DescalsJozef Bakonyi

Thanks for your attention