Phytophthora ramorum and Sudden Oak Death in California: I. Host Relationships

A new canker disease of Lithocarpus densiflorus, Quercus agrifolia, Q. kellogii, and Q. parvula var. shrevei in California is shown to be caused by Phytophthora ramorum. The pathogen is a recently described species that was previously known only from Germany and The Netherlands on Rhododendron and Viburnum. This disease has reached epidemic proportions in mixed evergreen and redwood forests over an area approximately 300 km long along the central coast of California. The most consistent and diagnostic symptoms on larger trees are the cankers that develop before foliage symptoms become evident. Cankers have brown or black discolored bark, seep dark red sap and occur on the trunk at the root crown up to 20 m above the ground. Cankers do not enlarge below the soil line into the roots. Cankers can be over 2 m in length and are delimited by thin black zone lines in the inner bark. Foliage on affected trees often turns from a healthy green color to brown over a period of several weeks. In L. densiflorus saplings, P. ramorum was isolated from branches as small as 5 mm diameter. Lithocarpus densiflorus and Q. agrifolia inoculated with P. ramorum in the field and greenhouse developed symptoms similar to those of natural infections. The pathogen was re-isolated from inoculated plants, thereby confirming pathogenicity. Based on field observations and greenhouse inoculations, the host range of P. ramorum in California has been expanded and now includes Rhododendron spp., madrone (Arbutus menziesii), huckleberry (Vaccinium ovatum), manzanita (Arctostaphylos spp.), California bay laurel (Umbellularia californica), buckeye (Aesculus californica), bigleaf maple (Acer macrophyllum), toyon (Heteromeles arbutifolia), California coffeeberry (Rhamnus californica), and California honeysuckle (Lonicera hispidula). On these hosts, P. ramorum causes a variety of foliar and branch symptoms. Introduction Over the past 7 years, a disease of tanoak (Lithocarpus densiflorus), coast live oak (Quercus agrifolia) and California black oak (Q. kelloggii) has caused considerable mortality in central California (Garbelotto and others 2001, McPherson and others 2000). Named “Sudden Oak Death” in the popular press, the whole crown 1 An abbreviated version of this paper was presented at the Fifth Symposium on Oak Woodlands: Oaks in California’s Changing Landscapes, October 22-25, 2001, San Diego, California. 2 Associate Professor, Post-doctoral Associate, and Staff Research Associate, respectively, Department of Plant Pathology, One Shields Ave., University of California, Davis, CA 95616 (email: [email protected]) 3 Cooperative Extension Specialist, Department of Environmental Science, Policy and Management, Ecosystem Science Division, 151 Hilgard Hall, University of California, Berkeley, CA 94720. 4 Farm Advisor, University of California Cooperative Extension, 1432 Abbott Street, Salinas, CA 93901. USDA Forest Service Gen. Tech. Rep. PSW-GTR-184. 2002. 733 Phytophthora ramorum Host Relationships—Rizzo, Garbelotto, Davidson, Slaughter, and Koike of affected trees often appears to die rapidly, and the foliage may turn from an apparently healthy green to brown within a few weeks. The most consistent and diagnostic symptom of the disease on larger trees is the development of cankers that have brown or black discolored bark on the lower trunk and seep dark red sap. The cankers do not extend into the roots. These cankers develop before foliage symptoms become evident. Such discoloration and bleeding are common symptoms associated with infection by Phytophthora species on oaks (Brasier and others 1993, Mircetich and others 1977, Tainter and others 2000). In agreement with this observation, we have consistently isolated the recently described Phytophthora ramorum from diseased L. densiflorus and Quercus spp. (Rizzo and others [In press]). The current range of the pathogen is considered to be from Big Sur in Monterey County to southern Oregon; the furthest inland sites are in western Solano County (Kelly 2002). Previously, P. ramorum had only been reported on ornamental rhododendron and viburnum in Germany and the Netherlands (Werres and others 2001). Field and greenhouse inoculations of tanoak and coast live oak seedlings, saplings, and mature trees produced symptoms similar to those observed with natural infections and confirmed the pathogenicity of P. ramorum (Rizzo and others [In press]). To date, most work on this disease in California has concentrated on oaks. However, the finding of P. ramorum on rhododendron in Europe suggested that the host range of the pathogen in California needed further investigation. To understand the potential impact on oak trees we must have a thorough understanding of the biology of this pathogen including the complete host range, transmission dynamics and population biology of the pathogen. In this paper, we describe our efforts to determine the host range of P. ramorum in California including field isolations and inoculations of non-oak hosts. We also report on inoculations of two California white oak species from which the pathogen has not been found in the field, valley oak (Q. lobata) and blue oak (Q. douglasii). Two red oak species from eastern North American, northern red oak (Quercus rubra) and pin oak (Quercus palustris), were also included in these inoculations. In parts II (Davidson and others 2002) and III (Garbelotto and others 2002), we describe preliminary results on the transmission mechanisms and genetics of the pathogen, respectively. Finally, in part IV (Garbelotto and others 2002), we discuss initial results from chemical control studies of P. ramorum. Methods Field Survey Symptomatic and dead trees and shrubs were examined throughout the known range of P. ramorum. At each site, samples were taken from trees that matched symptoms of Phytophthora infection and returned to the laboratory for isolation. Specific locations of sampled hosts were marked using global positioning equipment (GPS, Trimble Corp., Sunnyvale, California) and entered into a monitoring database maintained by Dr. Maggi Kelly, University of California, Berkeley (http://camfer.cnr.berkeley.edu/oaks/). Additional data collected with each sample included host species, host symptoms, tree diameter, forest type, overall stand health, and relationship to other symptomatic trees. Where possible, samples were collected from multiple hosts at each location. Isolation methods for P. ramorum on oaks are described elsewhere (Rizzo and others [In press]). For hosts with foliar lesions, discolored and necrotic areas were USDA Forest Service Gen. Tech. Rep. PSW-GTR-184. 2002. 734 Phytophthora ramorum Host Relationships—Rizzo, Garbelotto, Davidson, Slaughter, and Koike excised from leaves and placed in petri dishes (either in the laboratory or in the field) containing pimaricin-ampicillin-rifampicin-PCNB agar (PARP), a selective medium for Phytophthora species (Erwin and Ribeiro 1996). Plates were incubated in the dark at 20 to 22°C and examined within 2 to 5 days. We have also developed PCR (polymerase chain reaction) primers based on the nucleotide sequences of the internal transcribed spacer (ITS) region of the nuclear ribosomal DNA that are specific for P. ramorum (Garbelotto and others 2002). We have used these primers to successfully amplify DNA from infected leaves of multiple hosts and have used this methodology to pre-screen most foliar hosts (Garbelotto and others 2002). Inoculation Trials Greenhouse trials were conducted on seedlings of potential host species to evaluate the pathogenicity of P. ramorum. Inoculum was prepared by growing isolates on V-8 juice agar for 7 days, then cutting out 5 mm diameter plugs using a sterilized cork borer. Three isolates of P. ramorum were used for all tests: Pr-5 from tanoak, Pr-6 from coast live oak, and Pr-52 from rhododendron. Methods for stem inoculations of oak seedlings are described elsewhere (Rizzo and others [In press]). Foliar hosts were selected based on results of positive field isolations; a complete range of all woody plants found in oak forests have not been tested. Plants were purchased from native plant nurseries and were typically 2-3 years old. Foliage inoculations were conducted by misting leaves with sterile distilled water (SDW) and then pinning inoculum plugs to the upper surface of leaves. Sterile agar plugs were used as controls. A zip-loc plastic bag was then placed over the individual leaves; prior to sealing the bag the leaves were misted again with SDW. Each trial consisted of 10 leaves per host species per isolate plus controls. For most trials, the three isolates of P. ramorum listed above were used. On each of five seedlings, two leaves per isolate plus two control leaves were inoculated. Seedlings were incubated for 2 weeks in a greenhouse that was maintained at 22 to 24°C. For each trial, symptoms were recorded, lesion length and width was measured and pieces of stems or leaves were plated on PARP to verify presence or absence of P. ramorum. All leaf inoculations were conducted twice. Results Field Isolations We have recovered P. ramorum via isolation and direct PCR from symptomatic plant tissue of the following tree species: madrone (Arbutus menziesii), California bay laurel (Umbellularia californica), buckeye (Aesculus californica), huckleberry (Vaccinium ovatum) and Rhododendron spp. (cultivars “Gomer Waterer” and “Colonel Coen,” R. macrophyllum) (table 1). Using the direct PCR method on symptomatic leaf tissue, we have also detected P. ramorum DNA from field samples of manzanita (Arctostaphylos manzanita), bigleaf maple (Acer macrophyllum), toyon (Heteromeles arbutifolia), California coffeeberry (Rhamnus californica), and California honeysuckle (Lonicera hispidula). However, at the present time, we have been unable to isolate P. ramorum from these latter hosts. In all cases, P. ramorum was recovered from aboveground plant parts such as leaves or branches (table 1). Of the non-oak hosts, madrone, manzanita, rhododendron, and huckleberry appear to show the most dieback associated with the disease in the field. USDA Forest Service Gen. Tech. Rep. PSW-GTR-184. 2002. 735 Phytophthora ramorum Host Relationships—Rizzo, Garbelotto, Davidson, Slaughter, and Koike Table 1—Known hosts of Phytophthora ramorum in California and their method of detection in the field.