2011 Annual Report 1a.Objectives (from AD-416) The goal of this project is to isolate and characterize strains of fungi (Ambrosiella spp.) associated with ambrosia beetles. Objectives include. 1)Isolate strains of Ambrosiella from ambrosia beetles (Xylosandrus spp.),. 2)Develop molecular characterization of these strains, and. 3)Determine their susceptibility to commercially available fungal antagonists. 1b.Approach (from AD-416) Ambrosia beetles (Xylosandrus spp. and others) are pests of a wide array of woody plants used in the nursery industry. They depend for their survival on their own cultivation of a fungus (Ambrosiella spp. and others). Fungi will be isolated from available Xylosandrus spp., including X. crassiusculus and X. germanus. RAPD-based techniques and others will be used to characterize strains genetically. Each strain will be tested for susceptibility to commercially available fungal antagonists, including Trichoderma spp. and others. Survival of Ambrosiella spp. and beetles within galleries will be evaluated. 3.Progress Report Additional ambrosia fungi were isolated from field-collected X. crassiusculus and X. germanus female adults from OH, NY, TN and VA in late spring to early summer. A total of 60 and 124 isolates have been established from X. crassiusculus and X. germanus, respectively. All isolates are maintained at USDA-ARS in Ithaca, NY. Initial characterization assays using arbitrary primers against 88 isolates from X. germanus revealed the presence of two or more genotypes from each sampling site, with one common genotype present in all four states. Sequencing of the 18S, 28S and ITS of the ribosomal DNA of representatives of the more common genotypes identified these fungi in the genus Ambrosiella and closely related to A. xylobori. Further testing will be conducted by comparing obtained sequences with those of other Ambrosiella spp. Monitoring done through weekly meetings and periodic written summaries.