2006 Annual Report 4d.Progress report. This report serves to document research conducted under a Specific Cooperative Agreement between ARS and the American Farm School (AFS) in Thessaloniki, Greece. Additional details of research can be found in the report for the parent project 1920-22000-031-00D "Evaluation and Characterization of Exotic Plant Pathogens for Biological Control of Introduced, Invasive Weeds". This agreement was established to place scientific expertise at this USDA-ARS European Biological Control Laboratory satellite location for collaborative research measuring the efficacy of promising fungal pathogen biocontrol candidates on target invasive weeds in the field. This collaboration will lead to streamlined processes of collection, field evaluation, and release of safe biological control agents and sustainable control of invasive weeds in the U.S. During the course of the year, we inoculated, in replicated field tests, 12 non-target plant species with the smut fungus Microbotryum silybum Vanky & Berner and another 3 target plant species with the smut fungus Microbotryum cardui that we discovered on Carduus pycnocephalus (Italian thistle) in May of 2005. The target species (Silybum marianum (milk thistle) and Carduus pycnocephalus were included in each test. Seedlings of each plant were each inoculated in our greenhouses with 6 mg (1 mg more than previous years) of teliospores of the respective fungi. All plants were inoculated in non-heated greenhouses in early December 2005. All plants were transplanted to the field in late March, 2006. In addition, we conducted a test on Silybum marianum with different amounts of teliospore inoculum (from 0.125 mg to 4 mg in doubled increments). By mid May, many of the non-target plants had bolted. None of the non-targets showed any disease symptoms or signs of the fungi. These results are promising since several of the non-target plants were in the genus Carduus or closely related species, and lack of disease development on these plants indicates a high degree of host specificity of the fungi. On C. pycnocephalus, 18% of the plants inoculated with M. cardui were diseased while 90% of the S. marianum plants inoculated with M. silybum were diseased. Silyum marianum plants inoculated with less than 4 mg of teliospores were not diseased. In addition to research on host-range of the two smut fungi, another epidemiology test was set up in fields within American Farm School in October, 2005. This test supplements tests that were set up in 2004 in two nearby fields. In all tests teliospores of M. silybum were inoculated onto naturally occurring rosettes of Silybum marianum. The objective of the tests is to monitor the spread of diseased S. marianum plants in these fields over time. Results of our initial inoculation (2004) of these fields were an overall 12% (of 854) of the inoculated plants with systemic disease development and 11% (of 3956) of the seed heads completely filled with fungus teliospores. Results from our 2005 inoculation showed 9% (of 438) of the inoculated plants with systemic disease development and 7% (of 4322) of the seed heads completely filled with fungus teliospores. Achieving successful infection of S. marianum and disease development in the field is a major accomplishment of the project. The procedures used to achieve successful disease development can now be used with other plant species to determine the host range of M. silybum, and now M. cardui. Our initial results, in combination with the fact that both fungi are rare in nature, indicate that these fungi are likely to be very host specific and useable in a classical biological control programs in the U.S.