2012 Annual Report
1a.Objectives (from AD-416):
Determine the potential for seed transmission of the sudden oak death pathogen, Phytophthora ramorum.
1b.Approach (from AD-416):
A variety of plant species will be examined for seed transmission of P. ramorum. We will target those species for which seeds are commonly shipped from the West Coast, where P. ramorum is established, to the Eastern US where it is not established. Seeds will be inoculated by immersion in a suspension of P. ramorum sporangia for 24 h at 20 degrees C in darkness. Seeds will then be sown in seed cavity trays in Fafard 52 soil mix and incubated on a bench in a biosafety level BSL-GH3 containment greenhouse with overhead irrigation applied three times a day. We will also inoculate seeds by incubating them with P. ramorum-infected leaves of Rhododendron ‘Cunningham’s White’ which will be produced following established protocols. Seeds will be placed in plastic containers containing a layer of infected leaves, inside a mist tent programmed for 12 seconds of mist every 4 minutes. Seeds will be allowed to incubate with the infected leaves for seven days, after which time they will be planted and observed as above. Duplicate sets of untreated seeds will be planted alongside the treated ones as controls. At periodic intervals, subsamples of the inoculated, planted seeds will be surface disinfested in 1 percent sodium hypochlorite for 5 minutes, then cut up with a sterile scalpel and plated on selective medium to determine the presence of P. ramorum. We will take note of visible signs of seed infection observable with the naked eye and under a dissecting microscope. Symptoms will be recorded photographically. The percentage of seeds infected by P. ramorum will be recorded. Germination of the inoculated seeds will be observed over time and resulting plants observed for symptoms of P. ramorum infection. Areas of suspected infection will be plated on selective agar medium to confirm presence of P. ramorum in the tissue. We will thus determine the percentage of inoculated seeds that give rise to infected plants. Experiments will be repeated at least three times and data analyzed using the SAS statistical package.
This is a final report. Several known and potential species with both fleshy and hard seeds were selected for study. Species tested include dogwood (Cornus florida), Cornus kousa, Pyrocantha, Euonomous, crabapple, Camellia, white oak, northern red oak, chestnut oak, and barberry. Seeds were cleaned of debris and stored at 4 C in ziploc bags until ready to use. Sporangia suspension (1500 to 2000 sporangia/ml) was prepared from six P. ramorum isolates using V-8 juice agar plugs placed in 1 percent soil extract for 72 h. Seed were soaked in sporangia suspension for one hour, drained, and transferred to ziploc bags containing moistened vermiculite. Some inoculated seed was subject to an extended cold period to allow vernalization of the seed. Recovery of P. ramorum from inoculated seed was determined by plating on selective agar medium. In addition, the germination percentages of seeds inoculated with P. ramorum, incubated, and then sown in pots in the greenhouse was determined. For most hosts tested, a very low percentage of seed was determined to have become infected by P. ramorum and very few infected plants were identified as the result of seed inoculation.