Plant health regulations to prevent the introduction and spread of Phytophthora ramorum and P. kernoviae require rapid, cost effective diagnostic methods for screening large numbers of plant samples at the time of inspection. Current on-site techniques require expensive equipment, considerable expertise and are not suited for plant health inspectors. Therefore, an extensive evaluation of a commercially available lateral flow device (LFD) for Phytophthora species was performed involving four separate trials and 634 samples. The assay proved simple to use, provided results in a few minutes and on every occasion a control line reacted positively confirming the validity of the test. LFD results were compared with those from testing a parallel sample, using laboratory methods (isolation and real-time PCR). The diagnostic sensitivity of the LFD (87·6%) compared favourably with the standard laboratory methods although the diagnostic specificity was not as stringent (82·9%). There were a small number ( n = 28) of false negatives, but for statutory purposes where all positive samples must be identified to species level by laboratory testing, overall efficiency was 95·6% as compared with visual assessment of symptoms of between 20-30% for P. ramorum and P. kernoviae . This work demonstrates the value of the LFD for diagnosing Phytophthora species at the time of inspection and as a useful primary screen for selecting samples for laboratory testing to determine the species identification.
In Europe Phytophthora ramorum mainly causes dieback of Rhododendron and Viburnum , but in the UK it has also been reported on other ornamentals including Hamamelis (Giltrap et al., 2004) as well as on a limited number of tree species (Brasier et al ., 2004).In November 2004, Defra's PHSI collected samples from a public garden in south Wales where P. ramorum was under eradication. Each sample was tested on-site by CSL using real-time (TaqMan®) PCR for P. ramorum on a Cepheid SmartCycler (Tomlinson et al. , 2005). This identified P. ramorum on Parrotia persica (Persian ironwood; Hamamelidaceae), which was causing necrotic leaf lesions and twig dieback. Duplicate material was also sent to CSL where P. ramorum was consistently isolated from both stem and leaf tissue following surface decontamination and isolation onto semi-selective medium (Lane et al . , 2002). An ITS sequence was obtained from a culture of P. ramorum isolated from P. persica (GenBank DQ066919) and this was identical to other P. ramorum isolates on GenBank. Pathogenicity of the isolate was confirmed by wound-inoculating healthy leaves of P. persica with mycelial plugs and incubating these in a damp chamber at room temperature ( c . 20 ° C) in the laboratory for six days. Extensive lesions developed on the leaves and the pathogen was re-isolated from the leading edge; thus completing Koch's postulates. Healthy wounded leaves, inoculated with agar alone, did not develop symptoms.This is the first report of P. ramorum affecting P. persica . The infected plant was destroyed and measures were taken to eradicate the pathogen according to European Union phytosanitary legislation and the EU was notified.
Phytophthora kernoviae is a recently described pathogen causing leaf blight, aerial dieback and bleeding cankers on trees and shrubs in parts of Great Britain and Ireland and recently reported in New Zealand. This paper describes the development of a TaqMan real-time PCR assay based on internal transcribed spacer (ITS) sequence to aid diagnosis of this pathogen in culture and in plant material. The assay showed no cross reaction with 29 other Phytophthora species, including the closely related species P. boehmeriae, and detected at least 1.2 pg of P. kernoviae DNA per reaction. A rapid and simple method can be used to extract DNA prior to testing by real-time PCR, and a plant internal control assay can be used to aid interpretation of negative results. A comparison of real-time PCR and plating for 526 plant samples collected in the UK indicated that this assay is suitable for use in routine screening for P. kernoviae.
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