2009 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 Funds were awarded after the possibility of obtaining young trees from the Maryland State Nursery in 2008, so trees were ordered in Fall 2008 for a Spring 2009 delivery. One hundred each of Quercus rubra, Quercus prinus, and Acer rubrum, were received in March 2009 as 1 yr-old, bare-root seedlings. We also obtained 125 Kalmia latifolia ‘Hoffman’s K’ plants in one- gallon containers from a commercial nursery in Connecticut. This is the commercially available K. latifolia genotype which most closely resembles the native species. Inoculations are proceeding using seedlings of Quercus rubra, Quercus prinus, and Acer rubrum, and inoculating them with combined inoculum of six P. ramorum isolates, four of the NA1 clonal lineage and two of the EU1 clonal lineage. Thus, our results will represent disease reactions against a mix of P. ramorum genotypes rather than a single genotype. Both the NA1 as well as the EU1 clonal lineage are represented in the inoculum mix. Sporangia of all isolates were produced weekly by incubating agar plugs in 1% soil extract, concentrations are adjusted, and sporangial suspensions combined for inoculation. All work with whole plants has been performed inside BSL-3 level containment facilities at Ft. Detrick. Inoculated trees were placed in dew chambers at 20 degrees C for 5 days, removed and infected leaves scanned on a flatbed scanner for analysis of leaf and lesion areas using the ASSESS software program. 'Cunningham's White' rhododendron is used in all experiments as a positive control. We performed experiments to determine the range of inoculum densities over which infection may be obtained with young seedlings. We are now using concentrations ranging from 3000 sporangia/ml to 100 sporangia/ml, plus a control of soil extract alone. In our first few replicated inoculum density experiments with northern red oak, chestnut oak, and red maple seedlings we have observed some disease at all concentrations tested including the lowest concentration of 100 sporangia/ml. If disease is consistently observed at the lowest concentration tested, we will perform additional studies to go below this concentration to determine the lowest number of P. ramorum spores able to cause disease on these host species. Differences will be noted in plant age as the studies progress through the summer and into the fall. We are also determining the actual numbers of spores applied to leaves during the inoculation process. Spore concentrations are determined as sporangia/ml and plants are then dipped in selected concentrations. We are also performing experiments to determine how many spores are actually applied to leaves at each concentration. We have performed dip inoculation on a known size area of host tissue, then washed the applied spores onto a 15 um-mesh nylon screen and counted them under a dissecting microscope to determine the actual number of spores applied. We used e-mail for monitoring.