Aphanomyces astaci is an invasive pathogenic oomycete responsible for the crayfish plague, a disease that has devastated European freshwater crayfish. So far, five genotype groups of this pathogen have been identified by applying random amplified polymorphic DNA analysis on axenic cultures. To allow genotyping of A. astaci in host tissue samples, we have developed co-dominant microsatellite markers for this pathogen, tested them on pure cultures of all genotype groups, and subsequently evaluated their use on tissues of (1) natural A. astaci carriers, i.e., North American crayfish species, and (2) A. astaci-infected indigenous European species from crayfish plague outbreaks. Out of over 200 potential loci containing simple sequence repeat (SSR) motifs identified by 454 pyrosequencing of SSR-enriched library, we tested 25 loci with highest number of repeats, and finally selected nine that allow unambiguous separation of all known RAPD-defined genotype groups of A. astaci from axenic cultures. Using these markers, we were able to characterize A. astaci strains from DNA isolates from infected crayfish tissues when crayfish had a moderate to high agent level according to quantitative PCR analyses. The results support the hypothesis that different North American crayfish hosts carry different genotype groups of the pathogen, and confirm that multiple genotype groups, including the one originally introduced to Europe in the 19th century, cause crayfish plague outbreaks in Central Europe. So far undocumented A. astaci genotype seems to have caused one of the analysed outbreaks from the Czech Republic. The newly developed culture-independent approach allowing direct genotyping of this pathogen in both axenic cultures and mixed genome samples opens new possibilities in studies of crayfish plague pathogen distribution, diversity and epidemiology.

Charles University Prague Faculty of Science Department of Ecology Viničná 7 CZ 12844 Prague Czech Republic

Departamenta de Micologia Real Jardín Botánico CSIC Plaza Murillo 2 28014 Madrid Spain

Laboratoire Ecologie et Biologie des Interactions Equipe Ecologie Evolution Symbiose Université de Poitiers UMR CNRS 7267 40 av du Recteur Pineau F 86022 Poitiers Cedex France

Laboratoire Signalisation et Transports Ioniques Membranaires STIM ERL 7368 1 rue George Bonnet F 86022 Poitiers France

Microbial Evolution Research Group Department of Biosciences University of Oslo N 0316 Oslo Norway

Norwegian Veterinary Institute N 0106 Oslo Norway

OIE Reference Laboratory for Crayfish Plague Finnish Food Safety Authority Evira Neulaniementie 4 FI 70210 Kuopio Finland

University of Zagreb Faculty of Science Division of Biology Department of Zoology Rooseveltov trg 6 10000 Zagreb Croatia

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