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.
Combined sporangia harvested from six different P. ramorum isolates representing the NA-1 and EU-1 clonal lineages were used to inoculate stems of 2-3 year old seedlings; four concentrations of spores were painted on each stem comparing abrasion with sandpaper versus no abrasion. Treated areas were covered with moistened cheesecloth and wrapped with parafilm. Trees were maintained in a climate-controlled greenhouse for 2 months after which stem sections were excised and examined for symptoms of infection. Lesion areas were determined, and stem segments were plated on PARP-V8 selective medium to confirm infection by P. ramorum. Infection was only obtained in treatments where abrasion of stems with sandpaper was performed prior to inoculation. For abrasion treatments, disease was obtained over the whole range of inoculum densities employed (100-3000 sporangia/ml) with lower percentages occurring at 100 and 250 sporangia/ml. The results indicate that wounds may be necessary for infection of young trees by P. ramorum to occur in nature.