In May 2005, branches originating from five separate whorls below the terminal on a single California red fir (Abies magnifica) in a mixed grand fir (Abies grandis) and Douglas-fir (Pseudotsuga menziesii) Christmas tree plantation near Los Gatos, CA displayed wilting and dieback of new shoot growth. Brown dieback, delineated by needle loss, extended 6 to 8 cm into 1-year-old and sometimes 2-year-old growth. The ~7-year-old, 1-m tall tree was located near the edge of the plantation, beneath an overstory of California bay laurel (Umbellularia californica) trees that were infected with Phytophthora ramorum. Isolations from dieback margins onto corn meal agar amended with ampicillin, rifamycin, and pimaricin (CARP) yielded hyphae and large, dark brown chlamydospores that were morphologically consistent with P. ramorum (1). Microsatellite analysis confirmed that isolates were of the NA1 lineage of P. ramorum. Isolates were deposited in the Washington State University Puyallup Phytophthora Master Collection. Dormant bareroot California red fir seedlings were obtained from the USDA Forest Service Placerville Nursery (Camino, CA) in February 2006 and planted in SC-10 super cell cones (Stuewe & Sons, Inc., Tangent, OR) in a standard greenhouse potting mix. Seedlings (average height 11 cm) were then forced to initiate bud break and new shoot elongation (0.5 to 1.5 cm) in a greenhouse at 21°C. Eight unwounded seedlings were inoculated with a zoospore suspension (4.185 × 105 zoospores/ml of sterile water) produced from 3- to 4-week-old V8 juice agar cultures of isolate WSU#106-0021 using an artist's airbrush powered by Badger Propel canned propellant. Eight control seedlings were sprayed with water alone. Seedlings were placed in plastic tubs with ~2.5 cm of warm water in the bottom to provide humidity. A plastic bag supported by a wire frame was used to cover each tub. Tubs were placed in a biocontainment unit at 15 to 16°C under 24 h of fluorescent light. The plastic was removed after 5 days and seedlings were left under the same conditions. Seven days after inoculation, 25 to 100% (average 68%) of the new shoots on each of the eight inoculated seedlings were wilted and 100% of these seedlings exhibited dark brown dieback into the 1-year-old stems (range of 1.0 to 2.3 cm, average 1.6 cm). Tissues from shoots and dieback edges were plated onto CARP media. All of these attempts resulted in successful isolation of P. ramorum, and cultures exhibited the same hyphal morphology and chlamydospore characteristics when compared with the isolate tested. Control plants did not develop symptoms. This trial completes Koch's postulates to establish California red fir as a host of Phytophthora ramorum. To our knowledge, this site contains the only reported infection of California red fir by P. ramorum. The potential for infection within its native range is unknown.
Reference: (1) S. Werres et al. Mycol. Res. 105:1155, 2001.
