© 2006 Plant Management Network.
The Occurrence of Phytophthora ramorum in Nursery Stock in California, Oregon, and Washington States
K. M. Tubajika, USDA APHIS, Center for Plant Health Science and Technology, Otis Pest Survey, Detection and Exclusion Laboratory, Cape Cod, MA 02542; R. Bulluck and P. J. Shiel, USDA APHIS, Center for Plant Health Science and Technology, Raleigh, NC 27606; S. E. Scott, and USDA APHIS, Center for Plant Health Science and Technology, Riverdale, MD 27606; and A. J. Sawyer, USDA APHIS, Center for Plant Health Science and Technology, Otis Pest Survey, Detection and Exclusion Laboratory, Cape Cod, MA 02542
Tubajika, K. M., Bulluck, R., Shiel, P. J., Scott, S. E., and Sawyer, A. J. 2006. The occurrence of Phytophthora ramorum in nursery stock in California, Oregon, and Washington states. Online. Plant Health Progress doi:10.1094/PHP-2006-0315-02-RS.
Data from nursery surveys conducted in 2003, 2004, and 2005 for ramorum blight and dieback caused by Phytophthora ramorum were analyzed to determine which plant cultivars were likely to be infected with P. ramorum. Susceptible plant host genera, species, and cultivars were identified using culture and nested-polymerase chain reaction analyses, and disease incidence was calculated. For the genus, species, or cultivar analyses at least 20 samples were utilized in this study. The results indicated that the genus Camellia had the highest disease incidence, followed by Rhododendron and Viburnum. Camellia cvs. Setsugekka, Jean May, April Remembered, Winter’s Fire, Kramer’s Supreme, Bonanza, Magnoliaeflora, Apple Blossom, Silver Waves, Yuletide, and Nuccio’s Gem showed the highest risk of P. ramorum infection. Four Rhododendron cvs. Purple Splender, Cunningham’s White, Unique, and Nova Zembla were also grouped into the high risk category. All other Rhododendron and Viburnum plant species were at medium or low risk of infection by the pathogen. This study indicated that there is a large variation in disease incidence among genera and specific cultivars within a genus. A series of controlled, replicated, and well designed experiments involving a large sample size of plant cultivars is essential in order to identify the underlying reasons for the observed variation in infection risks of the surveyed data.
The oomycete Phytophthora ramorum (S. Werres, A.W.A.M. de Cock & W.A. Man in’t Veld) is the causal agent of ramorum blight and dieback disease, which is one of the most devastating diseases of forest plants, horticultural crops, ornamental trees, and shrubs (6,12,20). The infection of several plant species by P. ramorum has been described (6,12,20). A large number of these plants are associated with the nursery trade. From the recent outbreak of ramorum blight and dieback in different nurseries in California, Oregon, Washington, and British Columbia, along with recent research reports of additional host range of P. ramorum (2,3,6,8,11,12,13,16,17,18,19), it has become apparent that the intensity of the disease varies greatly in nursery settings. This outbreak has been a national concern since it was reported that a large nursery in California shipped nursery stock infested or infected with P. ramorum to nurseries in 40 states (7,12,16,17). Therefore, the monitoring of plant health in nurseries is important in order to prevent the spread and distribution of the pathogen.
Plant Protection and Quarantine (PPQ) records currently have at least 83 plant taxa listed as proven hosts of P. ramorum, or associated hosts (14). More taxa have been recently added to this list. Some of these taxa are only reported infected in US wildlands, some exclusively in European nurseries, public gardens, or wooded areas, whereas other taxa were found only in US nurseries (3,4,18). Furthermore, some Camellia and Rhododendron host species are known to have hundreds of cultivars that are commercially produced. The identification of plant cultivars in US nurseries that have demonstrated risk of infection can be useful in developing science-based disease mitigation and management strategies.
Research on the infection potential of P. ramorum have been conducted (11,14,15,20) in which some Camellia, Rhododendron, and Viburnum species were tested alone or in combination, however, the frequency of these species infected or infested with P. ramorum in nursery settings remains unknown. This information would be beneficial to the nursery industry, policy makers, and regulators in order to manage limited resources. The information generated from this study will also provide evidence on potential risks of spread and establishment of P. ramorum posed by various cultivars of Rhododendron and Camellia species in nursery settings. In addition, the results of this study will help policy makers and regulators target resources to risk mitigations of cultivars demonstrating the greatest risk, thereby providing effective disease management. Therefore, the objective of this study is to determine from disease survey data which plant cultivars are likely to be carriers of P. ramorum in common nursery stock.
Data Collection and Intergration
The data in this study were compiled from several types of nursery surveys conducted in California, Oregon, and Washington during 2003, 2004, and 2005. The Departments of Agriculture of each state, in collaboration with federal inspectors, conducted these surveys because of the requirements of a national nursery survey for P. ramorum. Following the widespread distribution of P. ramorum infected plants by a large southern California production nursery in March 2004, the survey was expanded to all West Coast states and California, Washington State, and Oregon were placed under a federal order requiring inspection, and certification of every nursery being shipped interstate. The survey data collected in 2003, 2004, and 2005 production years are summarized in Table 1. APHIS developed inspection and sampling protocols for each survey type (19). In general, these protocols required visual inspection of plants (dark brown lesions, necrosis, leaf wilting, branch dieback, stem and twig blight) and sampling of symptomatic plant tissue, followed by subsequent pathogen detection using laboratory assays (5,6,9). To the best of our knowledge, these protocols were always utilized by participating states during their surveys.
Table 1. Occurrence of P. ramorum in nurseries which tested positive for infection in the West Coast states during 2003, 2004, and 2005 production years.
v Total number of nurseries with positive test results for infection by P. ramorum.
w Positive samples were confirmed by nested PCR by the National Plant Germplasm and Biotechnology Laboratory, Riverdale, MD; the California Department of Food and Agriculture Plant Pest Diagnostics Center, Sacramento, CA; the Oregon Department of Agriculture, Salem, OR; and the Washington State Department of Agriculture, Olympia, WA.
x Percent incidence of P. ramorum sample was calculated by dividing the number of positive samples by the total number of samples and multiplying by 100.
y Number in parenthesis refers to the proportion of positives samples in each state.
z Data not available.
Based on visual assessment, a minimum of 40 plants per nursery was sampled and tested for P. ramorum. In the absence of adequate plants with symptoms, 40 plants were sampled, including asymptomatic plants. A sample consisted of between 7 to 15 leaves or leaves with stems, but a single sample was obtained from at least one plant. Samples were taken from known hosts of P. ramorum, associated plants as described by the federal order, and occasionally nearby plants. Plant samples were bagged, kept on ice, and sent to State Department of Agriculture laboratories for analysis. Plant samples were placed in the temperature controlled chambers until pathogen detection. The samples were then screened for the presence of Phytophthora species by ELISA (Agdia Inc., Elkhart, IN). Samples positive by ELISA were either plated on PARP or DNA was extracted and subjected to the validated nested PCR protocol. Confirmation of P. ramorum in a sample was accomplished at the National Plant Germplasm and Biotechnology Laboratory (NPGBL) or the National Identification Service in Beltsville, MD (5,6,9). A negative culture would require DNA extraction and submission to the NPGBL. The California Department of Food and Agriculture, Plant Pest and Diagnostics Center conducted screening and confirmatory testing for all samples collected in California, with submission of numerous samples to NPGBL for confirmation. Samples from Oregon and Washington States were confirmed as positive by the USDA APHIS PPQ National Germplasm and Biotechnology Laboratory (9).
Each state recorded survey data, plant identity, and lab results in a database. Identification of plant genus, species, and cultivar was made by field inspectors or laboratory personnel. Data collected in California was obtained from a state maintained database. Data from Oregon and Washington States were obtained from the National Agriculture Pest Information System (NAPIS) maintained by APHIS, PPQ, and Purdue University. All data from Camellia, Rhododendron, and Viburnum plants were analyzed by state and then combined across states for analysis.
Data were retrieved from the CDFA and NAPIS databases using query SORT of Microsoft Excel. New datasets were generated and used in subsequent analyses using the GENOMOD procedure and PROC RANK of SAS (version 2003, SAS Institute Inc., Cary, NC). The mean infection was calculated using PROC LSMEANS of SAS and the probability of infection determined using the binomial distribution. Analysis of mean infection rate was done by state, genus, species, and cultivar when the sample size exceeded 20. Categories of high, medium, and low risk of infections were constructed based on the 75th, 50th, and 25th percentile, respectively.
During 2003, 2004, and 2005 California, Oregon, and Washington collected 31,473 samples in which 1,243 were confirmed to be infected with P. ramorum for an overall infection rate of 3.9% (Table 1). In 2003, there were 685 samples collected in California and Oregon in which 27 samples were confirmed to be positive with an infection rate of 3.9%. California accounted for 75% of samples and 67% of the confirmed positives. Oregon accounted for 37% of samples and 33% of confirmed positives (Table 1). In 2004, there were 16,241 samples collected in the three states in which 808 samples tested positive for P. ramorum with an infection rate of 5.0%. California accounted for 65% of the samples and 76% of the confirmed positives. Oregon reported 7% of samples and 3% confirmed positives. Washington accounted for 28% of the samples and 21% of the confirmed positives (Table 1). In 2005, there were 14,547 samples collected in the three states in which 408 samples tested positive for P. ramorum with a prevalence rate of 3%. California accounted for 63% of the samples and 62% of the confirmed positives. Oregon reported 21% of the samples and 17% of the confirmed positives. Washington reported 20% of the samples and 21% of the confirmed positives (Table 1). P. ramorum incidence among states and years ranged from 2.5% in Oregon samples in 2004 to 5.8% in California samples in 2004. P. ramorum incidence among states was lower in 2005 than that in 2003 and 2004. California had the most number of infested plant samples (808 positive samples) followed by Washington (253 samples), and Oregon (96 samples). An analysis of P. ramorum incidence is presented in Table 1.
P. ramorum incidence varied among states, nurseries, and years (Table 2). In California, disease incidence ranged from 2.1 to 85.0% in 2004 and 2.3% to 22.5% in 2005. In Washington State, the incidence ranged from 1 to 5% and 1.5% to 5.5% in 2004 and 2005, respectively. In Oregon however, disease incidence was < 3% in both years (data not shown).
Table 2. Phytophthora ramorum incidence in nursery stock in California
x Data from Oregon State was dropped because of low disease
y Total number of positive nurseries in California was 53 in 2004, and
z Total number of positive nurseries in Washington State was 53 in
In 2004, the number of positive nurseries was 53, 24, and 25 in California, Oregon, and Washington State, respectively (Table 3). These positive nurseries were found through National Survey, Compliance Agreement Inspection [7CFR301.92], trace-forward and trace-back investigations, and delimiting surveys of confirmed infested nurseries. In 2005, however, 55 (3% of all nurseries), 20 (6% of all nurseries), and 16 positive nurseries (6% of all nurseries) were found in California, Oregon, and Washington State, respectively. Of 55 nurseries in California, 27 nurseries were found during trace-forward survey, 4 by Nursery Stock Cleanliness Inspection, 22 nurseries during Compliance Agreement Inspection, and 2 during trace-back survey. In Oregon, of 20 positive nurseries, 6 were found positives during trace-forward survey, 3 during trace-back survey, and 11 during the National Survey. In Washington State, 16 positive nurseries were found positives during the National Nursery Survey (Table 3). The genus Camellia was the most frequently infected with P. ramorum during the trace-forward and trace-back surveys, annual compliance inspection, and national survey (Table 3). In 2004, Camellia was observed in 98 and 61% of all positive samples in California and Oregon, respectively, followed by Rhododendron and Viburnum (Table 3). In 2005 however, a high number of Camellia was observed in California (81%), but not in Washington State. Rhododendron positive samples were in high number in Oregon (75%) and Washington State (98%) (Table 3).
Table 3. Camellia, Rhododendron, and Viburnum samples infected by Phytophthora ramorum in nurseries with positive test results in California, Oregon, and Washington State during 2004 and 2005.
x Positive samples were those where the presence of the pathogen was detected based on ELISA and PCR analysis.
y Percentage of Camellia, Rhododendron, and Viburnum infected with P. ramorum (expressed as percent of the total number of positive samples).
z Data not available.
Overall, across the West Coast states, 6,471 Camellia samples were collected and of those 1,098 (17.0%) were positive for P. ramorum. Rhododendron and Viburnum were less likely to test positive, 3.9% and 0.4% respectively (Table 4). Camellia samples were found to be positive in 85% of all positive nurseries (105). Rhododendron and Viburnum samples accounted for 38 (40 nurseries) and 7% (7 nurseries) of all positive nurseries, respectively (Table 4).
When analyzed by plant cultivars, eleven plant cultivars of Camellia and four Rhododendron most frequently observed infected by P. ramorum were: ‘Setsugekka’ (10 locations, 56%), ‘Jean May’ (10 locations, 56%), ‘April Remembered’ (6 locations, 43%), ‘Winter’s Fire’ (4 locations,42%), ‘Kramer’s Supreme’ (12 locations, 41%), ‘Bonanza’ (16 locations, 40%), ‘Magnoliaeflora’ (4 locations, 38%), ‘Apple Blossom’ (12 locations, 37%), ‘Silver Wave’ (8 locations, 35%), ‘Yuletide’ (6 locations, 35%), and ‘Nuccio’s Gem’ (15 locations, 33%) for Camellia; and ‘Purple Splendor’(4 locations, 24%), ‘Cunningham’s White’ (8 locations, 21%), ‘Baden Baden’(3 locations, 22%), ‘Unique’ (8 locations, 21%), and ‘Nova Zembla’ (3 locations, 20%) for Rhododendron (Table 5). Other Camellia and Rhododendron plant cultivars presenting relatively medium to lower risk for P. ramorum are shown in Table 5.
Table 4. Phytophthora ramorum incidence on Camellia, Rhododendron, and Viburnum in the positive nurseries in the three West Coast states during 2003, 2004, and 2005.
x Total number of number of nurseries in which P. ramorum was found (total of 105; CA = 55, OR = 24, and WA = 26).
y Positive samples were confirmed by nested-PCR by the National Plant Germplasm and Biotechnology Laboratory, Riverdale, MD; the California Department of Food and Agriculture Plant Pest Diagnostics Center, Sacramento, CA; the Oregon Department of Agriculture, Salem, OR; and the Washington State Department of Agriculture, Olympia, WA.
z Incidence of P. ramorum was calculated by dividing the number of positive samples by the total number of samples and multiplying by 100.
Table 5. Camellia, Rhododendron, and Viburnum cultivars affected by Phytophthora ramorum in the positive nurseries and associated risk level in the three West Coast states during 2003, 2004, and 2005 production years.
w Total number of number of nurseries in which P. ramorum was found (total of 105; CA=55, OR=24, and WA=26).
x Positive samples were confirmed by nested-PCR by the National Plant Germplasm and Biotechnology Laboratory, Riverdale, MD.
y Incidence of P. ramorum in sample was calculated by dividing the number of positive samples by the total number of samples and multiplying by 100 (refer to number of collected samples for genus).
z Categorized based on ranked disease incidence of cultivars within genera, with the top 75% of cultivars considered high risk [H], 50% of cultivars in the middle considered medium risk [M] and the lowest 25% of cultivars considered low risk [L].
The results reported here indicate that P. ramorum varied among states, nurseries, and years. This was expected because the origin of the infected plant materials was known. Through trace-forward and trace-back surveys, the number of shipments of nursery stock, as well as number of locations, were identified and inspected. The difference in disease incidence observed among Camellia and Rhododendron cultivars may probably be due in part to genetic and phenotypic variation and other unknown factors than to the number of the infected plants observed at few production nurseries. For each genus used in this study, a sample size ≥ 20 was used to calculate the infection frequency. There is limited information on the risk of Camellia, Rhododendron, and Viburnum plant cultivars presented in this study. Results also show that the genus Camellia and specific species and cultivars within the genus had a higher risk of infection by P. ramorum when compared to Rhododendron and Viburnum samples under natural inoculation conditions. This suggests that certain cultivars of Camellia may contribute to pathogen spread when the plant species is transported in trade. Therefore, these cultivars of Camellia may pose a risk to nursery infestation and may also serve as a source of inoculum for disease spread within a nursery. Tooley et al. (14) conducted a study on susceptibility of selected Ericaceous ornamental host species to P. ramorum, and the results from their study showed that Rhododendron cv. Nova Zembia and Rhododendron cv. Cunningham’s White were highly susceptible to BBA 995, and Pr-42 isolates of P. ramorum under artificial inoculation conditions. Results from our study show that two of their Rhododendron plant cultivars Nova Zembla and Cunningham’s White were susceptible to P. ramorum under natural inoculation conditions. Plant culivars used in Tooley et al’s study (14) were chosen to represent the diversity within the family of Ericaceae while focusing on hosts of commercial importance (14). Our study provides evidence on potential risks of spread and establishment of P. ramorum posed by these cultivars of Rhododendron and Camellia species in nursery settings. In addition, the results of this study will help policy makers and regulators target resources to risk mitigations of cultivars demonstrating the greatest risk, thereby providing effective disease management.
The information on P. ramorum infection obtained from this study provides a preliminary estimate of P. ramorum incidence and occurrence in several plant taxa and supports the need of a series of controlled, replicated, and repeated experiments involving a large number of plant cultivars. In order to fully define the risks associated with the taxa analyzed in this study, they need to be screened for resistance and susceptibility to P. ramorum in growth chambers and greenhouses. Additional research on the disease epidemiology and nursery transmission is also required.
We would like to thank Nancy K. Osterbauer of Oregon Department of Agriculture (OSDA), Casey Estep of California Department of Food and Agriculture (CDFA), Jennifer Falacy of Washington State Department of Agriculture (WSDA), and Julie Golod of National Pest Information System (NAPIS) in West Lafayette, IN, for their assistance with Phytophthora ramorum survey databases and two anonymous reviewers for helpful comments. We are grateful to Jami L. Nydam (USDA CPHST Otis Lab, MA) for her technical assistance with this project and to P. ramorum National Program staff for critical review of this manuscript.
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