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An emerging disease killing alders• First reported in ong>theong> 1980’s• Millions of alders infected throughout Europe• Mainly along rivers (riparian forests)• For example, 80% of ong>theong> rivers are contaminated in North-East FranceCrown thinningStem lesionsBark necrosesA new Phyong>toong>phthora species, close ong>toong> P. cambivora wasisolated from ong>theong>se necroses (Gibbs, 1995, EPPO Bull. 25:661-664)

Origin and relationship between ong>theong> 3 taxa:Brasier et al., 1999, PNAS 96: 5878-5883: ploidy, ITS sequences & ALFP dataP. cambivoraP. fragariae -like taxonP. alni subsp. alni(Paa)P. alni subsp. uniformis(Pau)P. alni subsp. multiformis(Pam)

Potential ecological outcomesQuercusFagus,Castanea,Prunus,Malus,RubusRubus,Fragaria x ananassaP. cambivora P. fragariaePauAlnusPaa"SUPERPATHOGEN" ?AlnusPamAlnus

Our objectivesTo understand ong>theong> origin of ong>theong> hybrid taxa and ong>theong>ir relationships:- who are ong>theong> parents of P. alni ?- is ong>theong> hybridization recurrent or not ?Three approaches:1. Phylogenetic analyses of nuclear and miong>toong>chondrial genes2. Polymorphism of microsatellite markers3. Distribution and expression of elicitin genes (Phyong>toong>phthora-specificproteins)

Allelic diversity observed through sequencingPfPcPfPcPfPcASF-like gene GPA1 gene RAS-Ypt gene TRP1 genePfPc3 groups of sequences are observed in P. alni

Allelic diversity observed through sequencingPamPaaPauPAUPAM1PAM2Several alleles forsome isolates ofP. alni=> study of ong>theong>distribution ofong>theong>se allelesGPA1 geneIoos et al. 2006,Fungal Genet. Biol. 43: 511-529

Study of ong>theong> allelic distribution using PCR tests1- Design of allele-specific PCR primers in ong>theong> intronsASF-PAM1 F/RASF-PAM2 F/RASF-like geneASF-PC1 F/RASF-PC2 F/RASF-PF F/RASF-PAU F/RGPA1 gene RAS-Ypt gene TRP1 geneGPA-PAM1 F/RGPA-PAM2 F/RGPA-PAU F/RGPA-PC1 F/RGPA-PC2 F/RGPA-PF F/RRAS-PAM1 F/RRAS-PAM2 F/RRAS-PAU F/RRAS-PC1 F/RRAS-PC2 F/RRAS-PF F/RTRP-PAM1 F/RTRP-PAM2 F/RTRP-PAU F/RTRP-PC1 F/RTRP-PC2 F/RTRP-PF F/R2- Allele-specific PCR tests on a large european collection (150 isolates)of Paa, Pam, Pau, P. cambivora, P. fragariae and Phyong>toong>phthora spp.

Distribution of nuclear alleles for each taxon Same results for ong>theong> 4 independent nuclear genesP. alni alniP. alni multiformisP. alni uniformisPAM1PAM2PAUPAM1PAM2PAUP. cambivoraPCP. fragariaePFIoos et al. 2006, Fungal Genet. Biol. 43: 511-529

Characterization of ong>theong> miong>toong>type through RFLPPam Pau Paa Pc PfOR87% 13%Digestion of ong>theong> miong>toong>chondrial DNA with 2 endonucleases

Miong>toong>chondrial genes sequencingSequencesof P. alni s.l.PfPcPfPcPfNadh1 geneCox1 gene

Miong>toong>chondrial genes sequencingPamPaamtPAMmtPAU2 alleles(mtPAM & mtPAU)are observedfor Cox1 and Nadh1PauIn accordance withRFLP, each P. alniisolate possesses aunique miong>toong>type:orNadh1 gene

Combination of nuclear and miong>toong>chondrial dataP. fragariaePFP. cambivora? PCP. alni subsp. alniPAM1PAM2PAUorPAM1PAM2PAUPAM1PAUPAM2? ?P. a. multiformisP. a. uniformisPAM1PAM2PAU

Origin of P. alni s. l.: new hypoong>theong>sesPAM1PAM2Ioos et al. 2006, Fungal Genet. Biol. 43: 511-529P. a. multiformisP. a. uniformisPAM1PAM2PAUPAM1PAM2PAUorPAM1PAM2PAUP. alni subsp. alni

Additional results1. Polymorphism of microsatellite markers:(Ioos et al. 2007, Mol. Ecol. Notes 7: 133-137)- Additivity of alleles: Paa = Pam x Pau- Several genotypes of Paa found several hybridization events2. Distribution and expression of elicitin genes:(Ioos et al. submitted)- Additivity of elicitin genes: Paa = Pam x Pau- Co-expression of gene sets inherited by both parents in Paa no pseudogenization recent hybridization events

Conclusions: causes of ong>theong> emergence??Pam1?Pam2PauPam???? ?? ?Alnus"Superpathogen"Increase of fitnessElimination of ong>theong> parents ?PaaAlnusAlnusEnvironmentalchanges ?? Expression ofecologicalsuperiorityEmergence of ong>theong> disease ?

AcknowledgmentsRenaud IoosPascal FreyBéranger BertinClaude HussonBenoît MarçaisOlivier CaëlAxelleAndrieuxBénédicteThoirainFinancial support: Agence de l'Eau Rhin-Meuse

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