On‐site real‐time PCR detection of Phytophthora ramorum causing dieback of Parrotia persica in the UK

Abstract

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.

Evaluation of a rapid diagnostic field test kit for identification of Phytophthora species, including P. ramorum and P. kernoviae at the point of inspection

“…They have been of considerable value in instructing new plant health inspectors in disease recognition, helping to convince growers and land‐owners of the need to sample and hold plants and have led to the discovery of several new host species (e.g. P. ramorum on Parrotia ) (Hughes et al ., 2006b). The simplicity and robustness of these kits makes them ideally suited for all skill levels and their size and weight ideal for varying sites and conditions to obtain a rapid assessment of whether Phytophthora may be present.…”

“…For these reasons many negative samples are submitted which must undergo labour and time‐intensive laboratory testing including isolation onto semi‐selective agar medium and morphological characterization, screening by enzyme‐linked immunosorbent assay (ELISA) and molecular methods based on conventional (Lane et al ., 2003a; Martin et al ., 2004; Anonymous, 2006; Schena et al ., 2006;Tooley et al ., 2006) and real‐time polymerase chain reaction (PCR) (Hayden et al ., 2004; Hughes et al ., 2006a). At the Central Science Laboratory (CSL), direct testing of the same plant material by real‐time PCR when compared with the combination of isolation and real‐time PCR of suspect positive cultures has resulted in a very accurate and reliable diagnostic test for P. ramorum with a diagnostic specificity of 99·3% and diagnostic sensitivity of 92·3% (Hughes et al ., 2006a,b). However, these techniques are not suitable for identification at the time of inspection.…”

Multispecies Phytophthora disease patterns in declining beech stands

“…The present survey also emphasized monitoring for P. ramorum, a potential cause of bleeding cankers on beech (Brasier, Denman, Brown, & Webber, 2004). In order to check for the presence of P. ramorum, all 131 bark samples and all 212 rhododendron leaf baits from soil baiting were tested using P. ramorum-specific qPCR (Hughes et al, 2006). An ABI 7500 Fast Real-Time System (Applied Biosystems) cycler was used and data analyzed with the 7500 Software 2.3 (Applied Biosystems).…”

Plant and veterinary disease diagnosis: a generic approach to the development of field tools for rapid decision making?

“…For FMDV, portable real‐time PCR equipment could be rapidly deployed close to infectious foci, abattoirs, veterinary clinics or milk processing plants. For P. ramorum, the assays have already been successfully deployed in the field (Hughes et al. , 2006b).…”

“…For FMDV, portable real-time PCR equipment could be rapidly deployed close to infectious foci, abattoirs, veterinary clinics or milk processing plants. For P. ramorum, the assays have already been successfully deployed in the field (Hughes et al, 2006b). However, unlike Phytophthora LFDs, which are widely deployed across the inspection service, the use of Cepheid SmartCyclers (Cephied, Sunnyvale, CA, US) for onsite real-time PCR has been confined to selected trained inspectors at specific sites.…”