Validation of a PCR method for detection and identification of Phytophthora ramorum
Linda Kox1, Hans de Gruyter1, Matteo Garbelotto2, Ilse van Brouwershaven1, Joke Admiraal1, and Robert Baayen1
Identification of Phytophthora ramorum Werres, De Cock & Man in 't Veld, the causal agent of Sudden Oak Death in California and associated in Europe with disease symptoms on Rhododendron spp. and Viburnum spp., is commonly based on microscopical examination of the morphological structures. Routinely this takes 5-10 days. Recently Matteo Garbelotto, University of Berkeley, California, developed a PCR method which is less time-consuming. We implemented this PCR method in our laboratory. The PCR method is based on sequences in the internal transcribed spacer (ITS) region of the nuclear ribosomal DNA gene repeat. This region is broadly used in diagnostic and evolutionary studies because it is conserved within species and generally variable between species, and is present in multiple copies in each cell.
For the validation of the PCR method, we investigated 129 samples possibly infected with P. ramorum. These samples were received as part of a survey held to obtain information concerning the distribution of P. ramorum in The Netherlands. The samples consisted of mainly Rhododendron spp. (93) as well as Viburnum spp. (23), Quercus spp. (6), Aesculus sp. (2), Buxus sp. (1), Castanea sativa (1), Larix sp. (1), Sambucus sp. (1), and Vaccinium sp. (1). The samples from Aesculus, Buxus, Castanea, Quercus, Larix, and Sambucus were taken from shrubs or trees in the vicinity of diseased Rhododendron plants. Plant parts were cut in 32 small pieces and divided randomly for culturing and PCR. DNA for PCR was isolated using the DNAeasy Plant Kit (Qiagen) and further purified using polyvinylpolypyrrolidone (PVPP) columns. Fifty-three samples were positive both after culturing and with PCR, and 51 samples (including those from Aesculus, Buxus, Castanea, Larix, Sambucus, Quercus, and Vaccinium) were negative with both methods. This results in a concordance of 80% between both methods (104/129). Five samples were positive by culturing only, and 20 samples were positive by PCR only. When the total of positives is used as the golden standard, the sensitivity of PCR and culturing is 94 % and 72 %, respectively. Samples of healthy control plants of the aforementioned trees and shrubs were all negative. In several cases where PCR testing was positive but culturing was not successful, sporangia of P. ramorum were observed with the microscope but the plant material was apparently too dry. Further investigations should be directed at explaining the remaining differences between culturing and PCR.
1Plant Protection Service, P.O. Box 9102, 6700 HC Wageningen, The Netherlands
2Department of Environmental Science, Policy, and Management, Ecosystem Sciences Division, 151 Hilgard Hall, University of California, Berkeley, CA 94720, USA
|
