2012 Annual Report 1a.Objectives (from AD-416): The objectives of this proposal are to characterize inoculum density relationships between P. ramorum and selected Eastern US forest and nursery hosts. We will determine the number of sporangia required to cause infection on several major Eastern forest species. Species to be evaluated include chestnut oak (Quercus prinus), northern red oak (Quercus rubra), red maple (Acer rubrum), native mountain laurel (Kalmia latifolia), and Rhododendron 'Cunningham's White'. For each host, we plan to determine the minimum amount of inoculum necessary for infection and characterize the relationship between inoculum density and symptom development in forest hosts. Results will help workers predict the likelihood of P. ramorum spreading from potentially infected ornamental nursery hosts to Eastern forests and be useful in developing pest risk assessments. 1b.Approach (from AD-416): We will use our specialized containment facilities to investigate the relationship between inoculum density and disease for P. ramorum on five selected host species. All plant inoculations with P. ramorum will be conducted inside level BSL-3P plant disease quarantine containment greenhouses and laboratories. We will perform whole-plant and detached leaf experiments to determine the minimum number of sporangia required to cause disease on stems and foliage of three important Eastern forest species as well as the important understory species Kalmia latifolia (mountain laurel) and rhododendron 'Cunningham's White'. Stems and foliage will be inoculated using a range of inoculum concentrations from 0 to 5000 sporangia per ml, and plants placed in dew chambers for 7 days at 20ºC. Following incubation in dew chambers, lesion areas will be assessed by scanning leaves and using image analysis software. Detached leaf experiments will also be performed to allow use of a wider range of inoculum densities than would be feasible using whole plants. 3.Progress Report: Detached leaves of selected Eastern forest species, not included in earlier studies, were submerged in P. ramorum sporangia suspensions until fully wetted, then transferred to a 9 x 13-inch Rubbermaid container with a piece of nylon screen material on the bottom. Leaves were placed in the Rubbermaid container between layers of wet paper towels, the lid secured, and the containers placed at 20 C in darkness for 7 days. Following incubation, symptoms were recorded by scanning the leaves, and leaf and lesion areas determined using the ASSESS 2.0 software package. Inoculum density relationships will be analyzed and calibration threshold estimates obtained to provide estimates of the number of sporangia required to produce given levels of disease on the host species. We will also analyze whether the response to increasing inoculum levels varies among species, and note any variation in susceptibility observed among the species.