2010 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.
Bare-root seedlings (2-3 years old) were received in April of 2009. Soon after the trees leafed out and hardened off, experiments were performed in dew chambers inside a bio-safety level 3 greenhouse with Acer rubrum, Quercus rubra, and Q. prinus. Plants were dip inoculated with a spore suspension (0, 100, 500, 1000, 3000 sporangia ml-1), and arranged in a large dew chamber programmed for 20 degrees C (without lights) in a randomized block design. Two trees/concentration/species were included in each run. Trees were removed following five days incubation, and symptomatic leaves were collected and scanned. A representative sampling of diseased leaves were plated on selective agar medium to confirm infection by P. ramorum. Trees were also inoculated with 50 and 2000 sporangia/ml and incubated in smaller dew chambers. Kalmia latifolia ‘Hoffman’s K’ plants were screened in 3 runs utilizing sporangia concentrations of 0, 50, 100, 250, 500, 1000, and 2000 ml-1, and 4 runs with 0, 10, 25, and 50 sporangia/ml. Detached leaves of all 5 species were also dip inoculated with sporangia and incubated in humid chambers for 5 days prior to evaluation. Nine leaves/concentration/species were inoculated in each of 5 runs. Determinations of the lowest concentration of sporangia to produce symptoms were recorded. Sporangia deposition on leaf surfaces was also determined in 8 runs with 3 leaves/concentration/species. Leaves were dip inoculated in sporangia suspensions of varying concentration. Leaf surfaces were rinsed with sterile distilled water, and the number of sporangia present in the rinsate determined by direct counting. We used e-mail for monitoring.