GENERAL TECHNICAL REPORT PSW-GTR-196
Low Genotypic Diversity of Phytophthora
ramorum in North America Revealed by
Microsatellite Markers1
S. Prospero2, E.M. Hansen2, and L.M. Winton3
Key words: Phytophthora ramorum, microsatellites
Abstract
The newly discovered pathogen Phytophthora ramorum causes an emerging forest disease in California
and Oregon. P. ramorum affects a broad range of hosts, including tanoak (Lithocarpus densiflorus) and
several species of oak (Quercus spp.), on which it causes stem cankers, leaf blight, and/or twig dieback.
Knowledge of the population diversity of P. ramorum may provide important information on the origin
of the pathogen (native vs. introduced), its reproductive mode (recombinant vs. clonal), and its
evolutionary history. In this study we investigated the population structure of P. ramorum in North
America by using microsatellite markers.
A total of 350 isolates of P. ramorum were used for our study. Most of these isolates (189) were
recovered from 2001 to 2004 from different hosts in forests of Oregon and California (10 isolates).
Seven isolates were obtained from soil samples and 22 were obtained from stream baits. The remaining
132 isolates were recovered from infested nurseries in Oregon and Washington. DNA was extracted
from pure cultures using standard protocols.
All isolates of P. ramorum were screened at 10 microsatellite loci. The di-nucleotide and tri-nucleotide
loci (CA15, CGA8, TCG9, TTG6, TCG7, and CAC8) were isolated from an enriched genomic library as
described by Prospero and others (2004). The four tetra-nucleotide loci (AAAC6, TATC32, GTGA8, and
TCCG11) were identified by screening the genome of P. ramorum (available online at:
http://genome.jgi-psf.org) for the most common tetra-nucleotide repeats according to literature. For
each locus, specific primers were designed using the program Primer3. In each primer pair, the forward
primer was labeled with a fluorescent dye (either 6-FAM or HEX) for detection on a capillary
sequencer. The products obtained by PCR amplification of the loci were sized on an ABI 3100
sequencer. Results were analyzed using GeneScan and Genotyper software.
Our results show that the field isolates (in other words, from forests, soil and streams) of P. ramorum
belong to seven different North American genotypes, whereas eight nursery isolates belong to a
European genotype. All ten microsatellite loci differentiate North American and European genotypes
but only two tetra-nucleotide loci (TATC32 and TCCG11) show variation within North American
isolates. About 95% of the field isolates from Oregon belong to the same genotype. Rare genotypes
1
An abstract of a poster presented at the Sudden Oak Death Second Science Symposium: The State of Our
Knowledge, January 18 to 21, 2005, Monterey, California.
2
3
Department of Botany and Plant Pathology, Oregon State University, Corvallis, Oregon 97331
USDA, ARS Subartic Research Unit, University of Alaska, Fairbanks, AK, USA 99775
536
The proceedings of the sudden oak death second science symposium: the state of our knowledge
were found both in the field and in nurseries. The ten isolates from California belong to a genotype that
is very rare in Oregon. The index of association value for all P. ramorum isolates is significantly higher
(P < 0.001) than the value calculated from 1000 artificially recombined datasets.
Our study shows a low genotypic diversity of P. ramorum in North America, confirming the results of
AFLP analysis (Ivors and others 2004). This strongly clonal population structure may suggest a recent
introduction of the pathogen. The observed index of association value indicates that, despite the
presence of both mating types, the North American population of P. ramorum is not recombining. We
are now screening more isolates to determine: (1) if the North American population of P. ramorum is
geographically subdivided (for example, Oregon vs. California), and (2) the origin of the rare nursery
genotypes.
References
Ivors, K.L.; Hayden, K.J.; Bonants, P.J.M.; Rizzo, D.M.; and Garbelotto, M. 2004. AFLP and
phylogenetic analyses of North American and European populations of Phytophthora
ramorum. Mycological Research 108: 378-392.
Prospero, S.; Black, J.A.; and Winton, L.M. 2004. Isolation and characterization of microsatellite
markers in Phytophthora ramorum, the causal agent of sudden oak death. Molecular Ecology
Notes 4: 672-674.
537