© 2005 Plant Management Network.
Comparing Diagnostic Protocols for Phytophthora ramorum in Rhododendron Leaves
Nancy Osterbauer, Senior Plant Pathologist, and Aaron Trippe, Plant Health Program Specialist, Oregon Department of Agriculture, Salem 97301-2532
Corresponding author: Nancy Osterbauer. firstname.lastname@example.org
Osterbauer, N., and Trippe, A. 2005. Comparing diagnostic protocols for Phytophthora ramorum in rhododendron leaves. Online. Plant Health Progress doi:10.1094/PHP-2005-0314-01-HN.
The federally regulated pathogen Phytophthora ramorum (Werres, de Cock & Man in’t Veld) attacks many plant species important to the nursery industry and to the natural environment (7). In 2003, this pathogen was reported for the first time infecting plants in a nursery in the United States (3) (Fig. 1). Despite a strong regulatory response, P. ramorum was reported infecting plants in a large, wholesale nursery in 2004 (1). Because of this and similar nursery detections on the West Coast, the USDA Animal and Plant Health Inspection Service (APHIS) adopted a federal order requiring all West Coast (California, Oregon, and Washington) nursery stock to be inspected and, if necessary, tested for P. ramorum prior to export (6). The plants must be tested using a diagnostic protocol validated by USDA-APHIS. First, the plants are screened for the presence of a Phytophthora species by a commercial DAS-ELISA kit. The samples confirmed as positive by DAS-ELISA are then tested for P. ramorum using the species-specific methods of pathogen isolation onto the selective medium PARP (culture plating) (5) and nested polymerase chain reaction (PCR) (2,4). This paper presents a comparison of these diagnostic methods for the detection of P. ramorum in Rhododendron (L.) nursery stock.
P. ramorum was recovered from infected Rhododendron ‘Anah Krushke’ plants at a Columbia County, Oregon nursery in May 2004 by culture plating. As the block of infected plants were being loaded into a truck for destruction by incineration, 24 leaves that had fallen off the plants while being moved were collected, placed in a sealed plastic bag and shipped in a cooler to the Oregon Department of Agriculture (ODA) Plant Health Laboratory. All of the leaves were exhibiting symptoms typical of a foliar Phytophthora infection. In the laboratory, each leaf was washed in tap water and then numbered using a permanent marker. Using a flame-sterilized paper punch, two 6-mm discs were taken from the disease margin of the suspicious lesion on each leaf. One leaf disc was plated onto PARP. The other leaf disc was macerated in a sodium azide-free DAS-ELISA extraction buffer for 3-min at 30 MHz using a Reisch Mixer Mill 300 (Retsch, Inc., Newtown, PA). DAS-ELISA was performed on 100 ml of the plant-buffer mixture according to the manufacturer’s directions (PathoScreen kit for Phytophthora, Agdia Inc., Elkhart, IN). DNA was extracted from the remaining plant-buffer mixture using spin column chromatography (DNeasy Plant Mini Kit, Qiagen Inc., Valencia, CA). Extracted DNA was then diluted 1:10 and amplified by nested PCR as directed in the USDA-validated protocol (4).
The DAS-ELISA detected a Phytophthora on 21 of the 24 leaves (Table 1). Results from both species-specific tests indicated that P. ramorum was detected on 22 of the 24 leaves. Of those 22 positive detections, 13 were detected by both culture plating and nested PCR, five by nested PCR alone, and four by culture plating alone. Of the five leaves tested positive by nested PCR alone, two were negative by both DAS-ELISA and culture plating. One sample was Phytophthora-free by all three methods.
Table 1. A comparison of USDA-validated Phytophthora ramorum
* Detects all Phytophthora species present.
** Species-specific diagnostic protocol for P. ramorum.
In November 2004, leaves exhibiting symptoms typical of a foliar Phytophthora infection were collected from eight Rhododendron plants (cultivars Kluis Sensation, Roseum Elegans, Roseum Pink, and Catawbiense Boursault) located within an infected block in a Washington Co., Oregon nursery. Five leaves were collected per plant. The leaves were placed in sealed plastic bags and shipped in a cooler to the ODA Plant Health Laboratory for testing as described above. The DAS-ELISA detected Phytophthora from all eight of the plants; P. ramorum was detected on four of the plants. Of the four P. ramorum-positive plants, two were positive by both nested PCR and culture plating, one by nested PCR alone, and one by culture plating alone.
All together, 81.2% of the samples tested were positive for P. ramorum while 18.8% were negative using the validated testing protocols (Fig. 2). Of the positive samples, 57.7% were positive by both species-specific methods (culture plating and nested PCR) while 42.3% were confirmed by only one of the two methods.
Although a limited study, our results suggest that further research is needed to improve the level of detection provided particularly by the species-specific diagnostic protocols. At present, the federal order requires all DAS-ELISA-positive samples be tested by nested PCR as the final confirmatory test. Culture plating is suggested for those samples, but is not required. In our study, several samples that were P. ramorum-positive by culture plating alone were negative by nested PCR and several samples that were P. ramorum-positive by nested PCR were negative by culture (Table 1, Fig. 2). Further investigations showed the DNA was amplifiable from the culture-positive samples (data not shown), but the P. ramorum was not detected by nested PCR. Although the 6-mm disks of infected plant tissue were collected side by side from the same lesion on each leaf tested, this may have accounted for the differing results using the two species-specific diagnostic methods. Thus, neither species-specific diagnostic protocol provided perfect accuracy in detecting P. ramorum. Because the DAS-ELISA will detect any Phytophthora in the plant tissue, it is critically important that the species-specific diagnostic protocol reliably detect P. ramorum amongst other Phytophthora species that may be present in the infected plants. Until official validation of an improved species-specific diagnostic protocol, our results suggest that all DAS-ELISA-positive samples should be further tested by both culture plating and nested PCR.
2. Davidson, J. M., Werres, S., Garbelotto, M., Hansen, E. M., and Rizzo, D. M. 2003. Sudden oak death and associated diseases caused by Phytophthora ramorum. Online. Plant Health Progress doi:10.1094/PHP-2003-0707-01-DG.
3. Hansen, E. M., Reeser, P. W., Sutton, W., Winton, L. M., and Osterbauer, N. 2003. First report of A1 mating type of Phytophthora ramorum in North America. Plant Dis. 87:1267.
4. USDA-APHIS. 2004. PCR detection and DNA isolation methods for use in the Phytophthora ramorum national program, August 2004. Online. L. Levy and V. Mabrodieva, eds. U.S. Dept Agric., Animal Plant Health Insp. Serv., Plant Prot. Quarant.
5. USDA-APHIS. 2004. Guidelines for isolation by culture and morphological identification of Phytophthora ramorum, 26 October 2004. Online. U.S. Dept Agric., Animal Plant Health Insp. Serv., Plant Prot. Quarant.
6. USDA-APHIS. 2004. Emergency Federal Order restricting movement of nursery stock from California, Oregon and Washington nurseries, 21 December 2004. Online. R. Dunkle, ed. U.S. Dept Agric., Animal Plant Health Insp. Serv., Plant Prot. Quarant.