Recently introduced, exotic plant pathogens may exhibit low genetic diversity and be limited to clonal reproduction. However, rapidly mutating molecular markers such as microsatellites can reveal genetic variation within these populations and be used to model putative migration patterns. Phytophthora ramorum is the exotic pathogen, discovered in the late 1990s, that is responsible for sudden oak death in California forests and ramorum blight of common ornamentals. The nursery trade has moved this pathogen from source populations on the West Coast to locations across the United States, thus risking introduction to other native forests. We examined the genetic diversity of P. ramorum in United States nurseries by microsatellite genotyping 279 isolates collected from 19 states between 2004 and 2007. Of the three known P. ramorum clonal lineages, the most common and genetically diverse lineage in the sample was NA1. Two eastward migration pathways were revealed in the clustering of NA1 isolates into two groups, one containing isolates from Connecticut, Oregon, and Washington and the other isolates from California and the remaining states. This finding is consistent with trace forward analyses conducted by the US Department of Agriculture's Animal and Plant Health Inspection Service. At the same time, genetic diversities in several states equaled those observed in California, Oregon, and Washington and two-thirds of multilocus genotypes exhibited limited geographic distributions, indicating that mutation was common during or subsequent to migration. Together, these data suggest that migration, rapid mutation, and genetic drift all play a role in structuring the genetic diversity of P. ramorum in US nurseries. This work demonstrates that fast-evolving genetic markers can be used to examine the evolutionary processes acting on recently introduced pathogens and to infer their putative migration patterns, thus showing promise for the application of forensics to plant pathogens.

Contemporary species identification relies strongly on sequence-based identification, yet resources for identification of many fungal and oomycete pathogens are rare. We developed two web-based, searchable databases for rapid identification of Phytophthora spp. based on sequencing of the internal transcribed spacer (ITS) or the cytochrome oxidase (cox) 1 and 2 spacer region, followed by BLAST searching the databases. Both databases are highly selective. For ITS, only sequences associated with published Phytophthora spp. descriptions or classic Phytophthora phylogenetics references are included. For the cox spacer region, only data obtained by resequencing select isolates reported in published work were included. Novel taxa tentatively named are selectively included in the database and labeled as Phytophthora taxon “X”; as in, for example, P. taxon “asparagi”. The database was validated with 700 Phytophthora isolates collected from nursery environments during 2006 to 2009. This resource, found at www.Phytophthora-ID.org , is a robust and validated tool for molecular identification of Phytophthora spp. and is regularly being updated.

Phytophthora ramorum, the cause of sudden oak death on oak and ramorum blight on woody ornamentals, has been reported in ornamental nurseries on the West Coast of North America from British Columbia to California. Long-distance migration of P. ramorum has occurred via the nursery trade, and shipments of host plants are known to have crossed the U.S.-Canadian border. We investigated the genotypic diversity of P. ramorum in Canadian nurseries and compared the Canadian population with U.S. and European nursery isolates for evidence of migration among populations. All three of the P. ramorum clonal lineages were found in Canada but, unexpectedly, the most common was the NA2 lineage. The NA1 clonal lineage, which has been the most common lineage in U.S. nurseries, was found relatively infrequently in Canada, and these isolates may have been the result of migration from the United States to Canada. The EU1 lineage was observed almost every year and shared multilocus genotypes with isolates from Europe and the United States. Estimation of migration rates between Europe and North America indicated that migration was higher from Europe to North America than vice versa, and that unidirectional migration from Europe to North America was more likely than bidirectional migration.

Sudden oak death caused by the oomycete Phytophthora ramorum was first discovered in California toward the end of the 20th century and subsequently emerged on tanoak forests in Oregon before its first detection in 2001 by aerial surveys. The Oregon Department of Forestry has since monitored the epidemic and sampled symptomatic tanoak trees from 2001 to the present. Populations sampled over this period were genotyped using microsatellites and studied to infer the population genetic history. To date, only the NA1 clonal lineage is established in this region, although three lineages exist on the North American west coast. The original introduction into the Joe Hall area eventually spread to several regions: mostly north but also east and southwest. A new introduction into Hunter Creek appears to correspond to a second introduction not clustering with the early introduction. Our data are best explained by both introductions originating from nursery populations in California or Oregon and resulting from two distinct introduction events. Continued vigilance and eradication of nursery populations of P. ramorum are important to avoid further emergence and potential introduction of other clonal lineages.

Phytophthora ramorum S. Werres & A.W.A.M. de Cock is the causal agent of sudden oak death in California and Oregon forests and ramorum blight on a broad range of host species in wildlands and nurseries. It is thought to be an introduced pathogen and only three clonal lineages are known (3). The North American lineage (lineage NA1, mating type A2) is responsible for infections in California and Oregon forests. The European lineage (lineage EU1, predominantly A1) is responsible for infections in Europe, but has also been found in nurseries in Oregon and Washington. A third lineage (NA2) has only been isolated in a few instances from nurseries in Washington and California. In June 2006, P. ramorum was isolated from diseased Viburnum tinus, Osmanthus heterophyllus, and O. fragrans cultivars from a Humboldt County retail nursery in northern California. We genotyped isolates and placed them into clonal lineages using microsatellite markers developed for P. ramorum (3,4). Genomic DNA was extracted from mycelia with the FastDNA SPIN kit (Q-Biogene, Morgan, Irvine, CA). Primers used were PrMS6, Pr9C3, PrMS39, PrMS43a, PrMS43b, and PrMS45 (3) and 18, 64, and 82 (4). We sized fluorescently labeled amplicons using capillary electrophoresis (3100 Avant Genetic Analyzer, Applied Biosystems, Foster City, CA). Isolate genotypes were compared with control isolates of known clonal lineage, including BBA9/95 (EU1), Pr102 (NA1), and WSDA3765 (NA2). Three of four isolates belonged to genotype EU1. The fourth isolate, obtained from O. fragrans, belonged to genotype NA1. We repeated genotyping on independent genomic DNA extractions and obtained identical results. Two EU1 isolates and the single NA1 isolate were tested for mating type (1) and found to be of A1, A1, and A2 mating type, respectively. The coexistence of A1 and A2 mating types in the same retail nursery suggests the potential for sexual reproduction, as is the case in P. infestans where clonal and sexual populations exist (2), although to date, sexual reproduction in nature has not been documented in P. ramorum. The California retail nursery infestation highlights the risks associated with the unintentional transport of host nursery stock infested with P. ramorum. References: (1) C. M. Brasier and S. Kirk. Mycol. Res. 108:823, 2004. (2) N. J. Grünwald and W. G. Flier. Ann. Rev. Phytopathol. 43:171, 2005. (3) K. Ivors et al. Mol. Ecol. 15:1493, 2006. (4) S. Prospero et al. Mol. Ecol. 16:2958, 2007.