2007 Annual Report 1a.Objectives (from AD-416) 1. To monitor the migration, population structure and evolution of Phytophthora ramorum in North America using microsatellites and multiple sequence loci. 2. To maintain an open-access, web-based database and culture collection with genotypic information. 3. To develop more powerful molecular markers for genotyping useful in monitoring migration and contemporary evolution of P. ramorum. 1b.Approach (from AD-416) Isolate collection, mating type determination and DNA extraction: P. ramorum is a pathogen currently regulated by USDA APHIS. Thus findings of P. ramorum in nurseries and certain urban or natural forest ecosystems are usually eradicated under a federal quarantine. Isolates will be obtained from federal and/or state agencies from Canada and the US as SOD is detected. Select isolates from Europe will be included as references isolates. Microsatellite analysis: Microsatellite genotypes will be determined using four rapidly evolving tetranucleotide loci. Marker development: Additional markers will be developed that include both SSR loci and single nucleotide polymorphism (SNP) markers. SSR loci that we have characterized in the genome of P. ramorum will be screened for with select isolates to find more polymorphic loci. Data analysis: Standard population genetic analysis based on genotype and allele frequencies. Web-based database: A web-based, relational and searchable database will be developed and maintained at the Horticultural Crops Research Laboratory using BioNumerics. Documents Trust with USDA/Forest Service. Log 31120. 3.Progress Report This report serves to document research conducted under an interagency reimbursable agreement between ARS and the Forest Service. Additional details of research can be found in the report for the parent project 5358-12220-003-00D, Biology and Management of Soilborne Diseases of Horticultural Crops. Dr. Niklaus Grunwald and collaborators conducted the following research towards the agreement’s objectives: Phytophthora ramorum is considered to be an emerging pathogen that has received worldwide attention as the causal agent of Sudden Oak Death (SOD). P. ramorum has received wide attention because (i) it has a large host range, (ii) it causes severe mortality of tanoak and coast live oak in natural ecosystems, and (iii) it affects many nursery hosts thus providing this pathogen a means of dispersal throughout the United States. The host range currently includes over 41 plant species and continues to increase. Most importantly, nursery crops offer a very effective means of dispersing the pathogen across the country. Once a nursery location is found to be infected with P. ramorum, state and federal government agencies generally try to trace infection back to an infected nursery of origin, or, if shipments have been made, attempt to trace the infection forward to nursery or retail operations that received shipments from an infected site. This provides a unique opportunity to further monitor the evolution, migration and population structure of P. ramorum using a range of highly polymorphic to conserved molecular markers that can detect genotypes and strain evolution. This research proposal attempts to develop further markers and implement techniques to monitor migration, evolution and population structure of P. ramorum in North America. We continue to follow up on every new outbreak in U.S. nurseries and have obtained isolates from 18 different states. All isolates have been genotyped using microsatellites. We have been able to modify existing protocols for genotyping P. ramorum based on SSR. Currently, we use a modified multiplexed protocol to identify genotypes and place them into one of three known clonal lineages. Applying our protocols, all new finds for which we were able to obtain cultures were determined to belong to lineage 1, 2, or 3. The most important finding was the discovery of coexistence of A1 and A2 mating type isolates in a Humboldt County, CA nursery. This finding has implications for disease management as it highlights the potential for sexual reproduction. We have also developed a searchable, relational database that maintains comprehensive, web-based information on distribution of multilocus microsatellite genotypes. This website is regularly updated. Information on distribution of multilocus microsatellite genotypes in the United States has been transferred to state departments of agriculture (CDFA, ODA, WSDA), USDA APHIS and other interested parties via a web-based database updated regularly. ADODR Statement: The ADODR met with the cooperating PI and project personnel at meetings during the year and discussed results through in person, via phone calls and e-mail.