Phytophthora ramorum is a recently described pathogen causing bleeding cankers, dieback, and leaf blight on trees and shrubs in parts of Europe and North America, where the disease is commonly known as sudden oak death. This article describes the development of a single-round real-time polymerase chain reaction (PCR) assay based on TaqMan chemistry, designed within the internal transcribed spacer 1 region of the nuclear ribosomal (nr)RNA gene for detection of P. ramorum in plant material. Unlike previously described methods for the molecular detection of P. ramorum, this assay involves no post amplification steps or multiple rounds of PCR. The assay was found to have a limit of detection of 10 pg of P. ramorum DNA, and could detect P. ramorum in plant material containing 1% infected material by weight within 36 cycles of PCR. The assay also was used to test DNA from 28 other Phytophthora spp. to establish its specificity for P. ramorum. A quick and simple method was used to extract DNA directly from host plant material, and detection of P. ramorum was carried out in multiplex with an assay for a gene from the host plant in order to demonstrate whether amplifiable DNA had been extracted. Amplifiable DNA was extracted from 84.4% of samples, as demonstrated by amplification of host plant DNA. The real-time protocol was used to test 320 plant samples (from 19 different plant species) from which DNA extraction had been successful, and was shown to give results comparable with a traditional isolation technique for diagnosis of P. ramorum in plant material from common U.K. hosts.
Phytophthora ramorum is a recently described pathogen causing oak mortality (sudden oak death) in forests in coastal areas of California and southern Oregon and dieback and leaf blight in a range of tree, shrub, and herbaceous species in the United States and Europe. Due to the threat posed by this organism, stringent quarantine regulations are in place, which restrict the movement of a number of hosts. Fast and accurate diagnostic tests are required in order to characterize the distribution of P. ramorum, prevent its introduction into pathogen-free areas, and minimize its spread within affected areas. However, sending samples to a laboratory for testing can cause a substantial delay between sampling and diagnosis. A rapid and simple DNA extraction method was developed for use at the point of sampling and used to extract DNAs from symptomatic foliage and stems in the field. A sensitive and specific single-round real-time PCR (TaqMan) assay for P. ramorum was performed using a portable real-time PCR platform (Cepheid SmartCycler II), and a costeffective method for stabilizing PCR reagents was developed to allow their storage and transportation at room temperature. To our knowledge, this is the first description of a method for DNA extraction and molecular testing for a plant pathogen carried out entirely in the field, independent of any laboratory facilities.Phytophthora ramorum is the causal agent of extensive oak mortality (commonly known as sudden oak death) in coastal forests in California (27) and southern Oregon (12,25). This pathogen also causes ramorum leaf blight and dieback on a range of other plant species (9) and can have a profound and devastating effect on forest ecosystems. A distinct population of the same pathogen (6, 35) is found in a number of European countries (10,20,23,37), mostly causing dieback and leaf blight on a range of ornamental plants in nurseries and landscaped areas (2). There have also recently been a number of incidences of lethal bark cankers caused by P. ramorum in native and nonnative trees in Europe (7). P. ramorum has a broad and expanding host range (3,4,10,11,19,20,28,31), and as a result of the threat posed to forest ecosystems, the movement of a variety of its host species is subject to restrictions in Europe and the United States. Emergency European Community phytosanitary measures for P. ramorum were introduced in 2002 (1), and in the United States quarantine restrictions at both the federal and state levels control the movement of a variety of plant species from infested areas in California and Oregon (31). The availability of rapid and accurate detection methods for P. ramorum is critical to allow its prevalence to be monitored and to expedite management or eradication steps to prevent its introduction and minimize its spread.The identification of P. ramorum is not possible based on host symptoms alone due to the considerable variation in their expression and because a range of other causes can produce similar symptoms. These include infections by several other Phyt...
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