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

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.     

Development of a One-Step Real-Time Polymerase Chain Reaction Assay for Diagnosis of Phytophthora ramorum

ABSTRACT 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.     

Molecular Detection of Phytophthora ramorum by Real-Time Polymerase Chain Reaction Using TaqMan, SYBR Green, and Molecular Beacons

ABSTRACT Sudden oak death, caused by Phytophthora ramorum, is a severe disease that affects many species of trees and shrubs. This pathogen is spreading rapidly and quarantine measures are currently in place to prevent dissemination to areas that were previously free of the pathogen. Molecular assays that rapidly detect and identify P. ramorum frequently fail to reliably distinguish between P. ramorum and closely related species. To overcome this problem and to provide additional assays to increase confidence, internal transcribed spacer (ITS), beta-tubulin, and elicitin gene regions were sequenced and searched for polymorphisms in a collection of Phytophthora spp. Three different reporter technologies were compared: molecular beacons, TaqMan, and SYBR Green. The assays differentiated P. ramorum from the 65 species of Phytophthora tested. The assays developed were also used with DNA extracts from 48 infected and uninfected plant samples. All environmental samples from which P. ramorum was isolated by PARP-V8 were detected using all three real-time PCR assays. However, 24% of the samples yielded positive real-time PCR assays but no P. ramorum cultures, but sequence analysis of the coxI and II spacer region confirmed the presence of the pathogen in most samples. The assays based on detection of the ITS and elicitin regions using TaqMan tended to have lower cycle threshold values than those using beta-tubulin and seemed to be more sensitive.     

Real-Time Fluorescent Polymerase Chain Reaction Detection of Phytophthora ramorum and Phytophthora pseudosyringae Using Mitochondrial Gene Regions

ABSTRACT A real-time fluorescent polymerase chain reaction (PCR) detection method for the sudden oak death pathogen Phytophthora ramorum was developed based on mitochondrial DNA sequence with an ABI Prism 7700 (TaqMan) Sequence Detection System. Primers and probes were also developed for detecting P. pseudosyringae, a newly described species that causes symptoms similar to P. ramorum on certain hosts. The species-specific primer-probe systems were combined in a multiplex assay with a plant primer-probe system to allow plant DNA present in extracted samples to serve as a positive control in each reaction. The lower limit of detection of P. ramorum DNA was 1 fg of genomic DNA, lower than for many other described PCR procedures for detecting Phytophthora species. The assay was also used in a three-way multiplex format to simultaneously detect P. ramorum, P. pseudosyringae, and plant DNA in a single tube. P. ramorum was detected down to a 10(-5) dilution of extracted tissue of artificially infected rhododendron 'Cunningham's White', and the amount of pathogen DNA present in the infected tissue was estimated using a standard curve. The multiplex assay was also used to detect P. ramorum in infected California field samples from several hosts determined to contain the pathogen by other methods. The real-time PCR assay we describe is highly sensitive and specific, and has several advantages over conventional PCR assays used for P. ramorum detection to confirm positive P. ramorum finds in nurseries and elsewhere.     

TaqMan Chemistry for Phytophthora ramorum Detection and Quantification, with a Comparison of Diagnostic Methods

ABSTRACT The choice of detection method for phytopathogens can be critically important in determining the success or failure of pest regulation systems. We present an assay for Phytophthora ramorum that uses 5' fluorogenic exonuclease (TaqMan) chemistry to detect and quantify the pathogen from diseased tissue, and include a universal primer and probe set for an internal positive control. This method is sensitive, detecting as little as 15 fg of target DNA when used in a nested design or 50 fg when used in a single round of polymerase chain reaction. None of the 17 other Phytophthora spp. tested was amplified by this assay. A comparison of the nested and non-nested TaqMan assays, and of one other nested assay, showed nested methods to be significantly more sensitive than nonnested and showed that host substrate significantly affected sensitivity of all assays. The nested TaqMan protocol was successfully field-tested; P. ramorum was detected in 255 of 874 plants in California woodlands, whereas the single-round TaqMan protocol detected significantly fewer positive samples. Finally, we documented increases in the quantity of pathogen DNA in Umbellularia californica leaves in initial stages of infection.     

Diagnostic sensitivity and specificity of different methods used by two laboratories for the detection of Phytophthora ramorum on multiple natural hosts

Five detection methods were comparatively tested on putative Phytophthora ramorum field samples from 41 wild plant species. The tested methods included two culture‐based assays, a DAS‐ELISA‐based polyclonal assay, a nested PCR‐based assay, and a TaqMan real‐time PCR assay. Diagnostic values including sensitivity, specificity, positive predictive value and negative predictive value were calculated for each method. The effects of host species, seasonality and host location were analysed and compared between two laboratories. Significant effects of season, host species and laboratory were detected. It is concluded that a combination of either culturing and molecular diagnosis or of two molecular assays is the most promising approach to diagnose this pathogen. Based on the results of this and other studies, diagnosis should occur as much as possible during wet and warm periods favourable to the pathogen, and proficiency tests should be performed to compare results obtained with molecular approaches in different laboratories. Furthermore, length of time lapsed between sample collection and processing strongly affected the diagnostic sensitivity of culture‐based methods, and therefore needs to be taken into account when comparing results from different laboratories.     

Molecular Detection of Phytophthora ramorum, the Causal Agent of Sudden Oak Death in California, and Two Additional Species Commonly Recovered from Diseased Plant Material

ABSTRACT Sudden oak death is a disease currently devastating forest ecosystems in several coastal areas of California. The pathogen causing this is Phy-tophthora ramorum, although species such as P. nemorosa and P. pseudo-syringae often are recovered from symptomatic plants as well. A molecular marker system was developed based on mitochondrial sequences of the cox I and II genes for detection of Phytophthora spp. in general, and P. ramorum, P. nemorosa, and P. pseudosyringae in particular. The first-round multiplex amplification contained two primer pairs, one for amplification of plant sequences to serve as an internal control to ensure that extracted DNA was of sufficient quality to allow for polymerase chain reaction (PCR) amplification and the other specific for amplification of sequences from Phytophthora spp. The plant primers amplified the desired amplicon size in the 29 plant species tested and did not interfere with amplification by the Phytophthora genus-specific primer pair. Using DNA from purified cultures, the Phytophthora genus-specific primer pair amplified a fragment diagnostic for the genus from all 45 Phytophthora spp. evaluated, although the efficiency of amplification was lower for P. lateralis and P. sojae than for the other species. The genus-specific primer pair did not amplify sequences from the 30 Pythium spp. tested or from 29 plant species, although occasional faint bands were observed for several additional plant species. With the exception of one plant species, the resulting amplicons were smaller than the Phytophthora genus-specific amplicon. The products of the first-round amplification were diluted and amplified with primer pairs nested within the genus-specific amplicon that were specific for either P. ramorum, P. nemorosa, or P. pseudo-syringae. These species-specific primers amplified the target sequence from all isolates of the pathogens under evaluation; for P. ramorum, this included 24 isolates from California, Germany, and the Netherlands. Using purified pathogen DNA, the limit of detection for P. ramorum using this marker system was approximately 2.0 fg of total DNA. However, when this DNA was spiked with DNA from healthy plant tissue extracted with a commercial miniprep procedure, the sensitivity of detection was reduced by 100- to 1,000-fold, depending on the plant species. This marker system was validated with DNA extracted from naturally infected plant samples collected from the field by comparing the sequence of the Phytophthora genus-specific amplicon, morphological identification of cultures recovered from the same lesions and, for P. ramorum, amplification with a previously published rDNA internal transcribed spacer species-specific primer pair. Results were compared and validated with three different brands of thermal cyclers in two different laboratories to provide information about how the described PCR assay performs under different laboratory conditions. The specificity of the Phytophthora genus-specific primers suggests that they will have utility for pathogen detection in other Phytophthora pathosystems.     

Development and application of a PCR-based 'molecular tool box' for the identification of Phytophthora species damaging forests and natural ecosystems

A PCR-based 'molecular tool box', based on a region of the ras-related protein gene Ypt 1, was developed for the identification of 15 Phytophthora species that damage forests and trees: P. cactorum , P. cambivora , P. cinnamomi , P. citricola , P. europaea , P. inundata , P. lateralis , P. megasperma , P. nemorosa , P. kernoviae , P. pseudosyringae , P. psychrophila , P. quercina , P. ramorum and P. ilicis . Most primers proved highly specific in blast analyses and in tests with DNA from 72 isolates of 35 species of Phytophthora and nine species representative of Pythium . Exceptions were primers designed for P. cactorum and P. ilicis , which cross-reacted with P. idaei and P. nemorosa , respectively. Amplification with Phytophthora -genus-specific primers before amplification with the various species-specific primers (nested PCR) increased the sensitivity of detection over amplification with species-specific primers only: detection limits ranged between 100 and 10 pg target DNA µ L⁻¹ in the latter, compared with 100 fg µ L⁻¹ in nested PCR. Using existing methods for rapid extraction and purification of DNA, single-round amplification was appropriate for detection of target Phytophthora species in leaves, but nested PCR was required for soil and water samples. The quarantine pathogens P. ramorum and P. kernoviae were detected in a number of naturally infected leaves collected in England and Wales, whereas P. citricola was commonest in water and soil samples from natural Scottish ecosystems.     

非洲菊疫霉根腐病的快速分子诊断

 隐地疫霉引起的根腐病是非洲菊生产上的主要病害,为发展该病的快速诊断技术,本文比较了卵菌核糖体基因ITS的序列,在此基础上设计了2条针对隐地疫霉的特异性PCR引物PC1和PC2。供试的23种不同真菌和疫霉菌的46个菌株中,利用这对引物能从隐地疫霉基因组DNA中扩增出一条分子量为620bp的特异性条带,该引物的检测灵敏度可达10pg。采用快速组织碱裂解法提取发病植物组织的DNA,结合PCR检测技术,4h内可从发病的非洲菊根部组织中特异性地检测到隐地疫霉菌。结果表明,建立的非洲菊疫霉根腐病菌分子检测方法可用于该病害的快速分子诊断。     

Phytophthora ramorum Colonizes Tanoak Xylem and Is Associated with Reduced Stem Water Transport

ABSTRACT Isolation, detection with diagnostic polymerase chain reaction (PCR), and microscopy demonstrated the presence of Phytophthora ramorum in the sapwood of mature, naturally infected tanoak (Lithocarpus densiflorus) trees. The pathogen was strongly associated with discolored sapwood (P < 0.001), and was recovered or detected from 83% of discolored sapwood tissue samples. Hyphae were abundant in the xylem vessels, ray parenchyma, and fiber tracheids. Chlamydospores were observed in the vessels. Studies of log inoculation indicated that P. ramorum readily colonized sapwood from inoculum placed in the bark, cambium, or sapwood. After 8 weeks, radial spread of P. ramorum in sapwood averaged 3.0 to 3.3 cm and axial spread averaged 12.4 to 18.8 cm. A field study was conducted to determine if trees with infected xylem had reduced sap flux and reduced specific conductivity relative to noninfected control trees. Sap flux was monitored with heat-diffusion sensors and tissue samples near the sensors were subsequently tested for P. ramorum. Adjacent wood sections were excised and specific conductivity measured. Both midday sap flux and specific conductivity were significantly reduced in infected trees versus noninfected control trees. Vessel diameter distributions did not differ significantly among the two treatments, but tyloses were more abundant in infected than in noninfected trees. Implications for pathogenesis, symptomology, and epidemiology are discussed.     

Detection and Quantification of Phytophthora ramorum from California Forests Using a Real-Time Polymerase Chain Reaction Assay

ABSTRACT The timely and accurate detection of pathogens is a critical aid in the study of the epidemiology and biology of plant diseases. In the case of regulated organisms, the availability of a sensitive and reliable assay is essential when trying to achieve early detection of the pathogen. We developed and tested a real-time, nested polymerase chain reaction (PCR) assay for the detection of Phytophthora ramorum, causal agent of sudden oak death. This technique then was implemented as part of a widespread environmental screen throughout California. The method here described is sensitive, detecting less than 12 fg of pathogen DNA, and is specific for P. ramorum when tested across 21 Phytophthora spp. Hundreds of symptomatic samples from 33 sites in 14 California counties were assayed, resulting in the discovery of 10 new host species and 23 infested areas, including 4 new counties. With the exception of a single host, PCR-based discovery of new hosts and infested areas always was confirmed by traditional pathogen isolations and inoculation studies. Nonetheless, molecular diagnostics were key in early pathogen detection, and steered the direction of further research on this newly discovered and generalist Phytophthora species.     

Development and evaluation of specific PCR and LAMP assays for the rapid detection of Phytophthora melonis

Phytophthora melonis is a widespread and devastating pathogen for the Cucurbitaceae family. Early and accurate detection of P. melonis is essential to control the disease in the field. To establish a simple, visual, and rapid detection system for P. melonis, we developed nested polymerase chain reaction (PCR) and loop-mediated isothermal amplification (LAMP) systems based on the Ras-related protein (Ypt1) gene. All 36 isolates of P. melonis, from geographically distinct counties in China, yielded positive detection results on LAMP or nested PCR assays. No cross reaction was observed with other oomycetes or fungal pathogens. A sensitivity assay showed that both methods had a detection limit of 10 fg genomic DNA. We also detected P. melonis in diseased cucumber tissues and soils, and evaluated positive detection rates using LAMP, nested PCR, and conventional isolation methods. The results suggest that the LAMP assay has the greatest potential for active detection of P. melonis in regions that are at risk of contracting the disease, and for use in resource-poor settings.     

Molecular detection of Phytophthora capsici in infected plant tissues, soil and water

A species-specific PCR assay was developed for rapid and accurate detection of the pathogenic oomycete Phytophthora capsici in diseased plant tissues, soil and artificially infested irrigation water. Based on differences in internal transcribed spacer (ITS) sequences of Phytophthora spp. and other oomycetes, one pair of species-specific primers, PC-1/PC-2, was synthesized. After screening 15 isolates of P. capsici and 77 isolates from the Ascomycota, Basidiomycota, Deuteromycota and Oomycota, the PC-1/PC-2 primers amplified only a single PCR band of c . 560 bp from P. capsici . The detection sensitivity with primers PC-1/PC-2 was 1 pg genomic DNA (equivalent to half the genomic DNA of a single zoospore) per 25- µ L PCR reaction volume; traditional PCR could detect P. capsici in naturally infected plant tissues, diseased field soil and artificially inoculated irrigation water. Using ITS1/ITS4 as the first-round primers and PC-1/PC-2 in the second round, nested PCR procedures were developed, increasing detection sensitivity to 1 fg per 25- µ L reaction volume. The results suggested that the assay detected the pathogen more rapidly and accurately than standard isolation methods. The PCR-based methods developed here could simplify both plant disease diagnosis and pathogen monitoring, as well as guiding plant disease management.     

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

Rapid and accurate diagnostic tests make an important contribution to programmes to monitor and eradicate infectious diseases that impact animal and plant health. Using foot-and-mouth disease (FMD) and sudden oak death as examples, this review outlines recent progress to develop new field tools for detection of the infectious agents that cause high-impact livestock and plant diseases. The principal driver for this work is to develop tools that can be used locally to assist in decision making. Advances in this area have developed simple-to-use lateral-flow devices for the detection of FMD virus and the genus Phytophthora (including Phytophthora ramorum , the causal agent of sudden oak death and the related pathogen P. kernoviae ), as well as new hardware platforms to allow PCR testing for these agents by non-specialists in the field. Although developed for different diseases, the user requirements for rapid diagnostic tools for FMD and sudden oak death share many similarities. Using generic solutions to these challenging problems, it is now possible to imagine a new paradigm for how the collection and testing of samples to monitor the spread of important livestock and plant diseases might be achieved.     

European collaborative studies for the validation of PCR-based detection tests targeting regulated fungi and oomycetes

In 2007, the mycology unit of the French plant protection laboratory (LNPV-UMAF) organized and launched four collaborative studies for the validation of detection protocols targeting the regulated oomycetes Phytophthora ramorum , P. fragariae / P . rubi , Plasmopara halstedii and the fungus Monilia fructicola . The participants were recruited through the European Mycological Network (EMN). All four protocols were based on species-specific PCR tests already published in the scientific literature and, except for Pl. halstedii , combined a detection test and a confirmation of detection test. For each target organism, we evaluated the performance of protocols, i.e. accuracy, qualitative repeatability and qualitative reproducibility, by a statistical analysis of the results obtained by the 16 participant laboratories with a series of 10 blinded samples. As demonstrated by the collaborative trials results, all four detection protocols were shown to be fit for the purpose of regulatory compliance. The collaborative trial appears a powerful tool to evaluate the performance of a detection method, and is of special interest to laboratories employing a quality assurance system.     

Detection, Distribution, Sporulation, and Survival of Phytophthora ramorum in a California Redwood-Tanoak Forest Soil

ABSTRACT Recovery of Phytophthora ramorum from soils throughout sudden oak death-affected regions of California illustrates that soil may serve as an inoculum reservoir, but the role of soil inoculum in the disease cycle is unknown. This study addresses the efficacy of soil baiting, seasonal pathogen distribution under several epidemiologically important host species, summer survival and chlamydospore production in soil, and the impact of soil drying on pathogen survival. The efficacy of rhododendron leaves and pears as baits for detection of soilborne propagules were compared. Natural inoculum associated with bay laurel (Umbellularia californica), tanoak (Lithocarpus densiflorus), and redwood (Sequoia sempervirens) were determined by monthly baiting. Summer survival and chlamydospore production were assessed in infected rhododendron leaf disks incubated under bay laurel, tanoak, and redwood at either the surface, the litter/soil interface, or in soil. Rhododendron leaf baits were superior to pear baits for sporangia detection, but neither bait detected chlamydospores. Most inoculum was associated with bay laurel and recovery was higher in soil than litter. Soil-incubated inoculum exhibited over 60% survival at the end of summer and also supported elevated chlamydospore production. P. ramorum survives and produces chlamydospores in forest soils over summer, providing a possible inoculum reservoir at the onset of the fall disease cycle.     

A One-Step, Immunochromatographic Lateral Flow Device Specific to Rhizoctonia solani and Certain Related Species, and Its Use to Detect and Quantify R. solani in Soil

ABSTRACT A murine hybridoma cell line GD2 secreting an immunoglobulin (Ig)M monoclonal antibody (MAb) was produced against surface antigens from an anastomosis group (AG) 4 isolate of Rhizoctonia solani (teleomorph: Thanatephorus cucumeris). Ascites were produced in mice using GD2 hybridoma cells and used to develop a rapid immunochromatographic lateral flow device (LFD) for the detection of antigens from R. solani and certain related Rhizoctonia spp. The LFD was tested for specificity against surface antigens from related and unrelated soil fungi. Antigens from representative isolates of R. solani AGs 1, 2-1, 2-3, 2-t, 3, 4, 5, 6, 7, 8, 9, 10, 11, and BI gave a positive response in LFD tests, as did antigens from Thanatephorus orchidicola, T. praticola, R. fragariae (teleomorph: Ceratorhiza fragariae), Ceratorhiza goodyerae-repentis, Ceratobasidium cornigerum, and binucleate AGE. Antigens from R. solani AGs 2-2, 2-2IIIB, and 2-2IV and from the related fungi R. carotae, R. cerealis (teleomorph: Ceratobasium cereale), R. crocorum (teleomorph: Helicobasidium brebissonii), R. oryzae (teleomorph Waitea circinata), and R. zeae gave negative responses, as did antigens from a range of unrelated fungi and oomycetes including Fusarium, Gliocladium, Trichoderma, Pythium, and Phytophthora spp. The usefulness of the LFD to detect R. solani was demonstrated in soils naturally infested with R. solani AG3. There was close agreement between results of LFD tests and conventional plate enrichment tests employing selective medium. The specificity of the technique was confirmed by polymerase chain reaction PCR using R. solani AG3-specific primers and by analyses based on sequences of the internal transcribed spacer (ITS)1-5.8S-ITS2 rRNA-encoding regions of unrelated fungi recovered from soil samples. The LFD was used to quantify R. solani AG4 in artificially infested soil samples (chopped potato soil inoculum). Estimates of CFU per gram of soil were derived using a most-probable number technique, which was based on the presence or absence of a detectable signal in the LFD. Estimates of CFU obtained in LFD tests and those obtained in a plate-trapped antigen enzyme-linked immunosorbent assay incorporating MAb GD2 were identical (449 CFU g(-1) of soil).     

Efficacy of phosphonic acid, metalaxyl-M and copper hydroxide against Phytophthora ramorum in vitro and in planta

The ability of metalaxyl-M, phosphonic acid in the form of phosphonate, and copper hydroxide to inhibit different stages in the life cycle of Phytophthora ramorum , the causal agent of sudden oak death (SOD), was tested in vitro using 12 isolates from the North American forest lineage. In addition, experiments were conducted in planta to study the ability of phosphonic acid injections and metalaxyl-M drenches to control pathogen growth on saplings of California coast live oak ( Quercus agrifolia ), and of copper hydroxide foliar sprays to control infection of California bay laurel ( Umbellularia californica ) leaves. Phytophthora ramorum was only moderately sensitive to phosphonic acid in vitro , but was highly sensitive to copper hydroxide. In planta experiments indicated the broad efficacy of phosphonic acid injections and of copper hydroxide sprays in preventing growth of P. ramorum in oaks and bay laurels, respectively. Finally, although metalaxyl-M was effective in vitro , drenches of potted oak trees using this active ingredient were largely ineffective in reducing the growth rate of the pathogen in planta .     

Enhanced Polymerase Chain Reaction Methods for Detecting and Quantifying Phytophthora infestans in Plants

ABSTRACT Sensitive and specific primer sets for polymerase chain reaction (PCR) for Phytophthora infestans, the oomycete that causes late blight of potato and tomato, were developed based on families of highly repeated DNA. The performance of these primers was compared to those developed previously for the internal transcribed spacer (ITS) of ribosomal DNA. The detection limit using the new primers is 10 fg of P. infestans DNA, or 0.02 nuclei. This is about 100 times more sensitive than ITS-directed primers. Nested polymerase chain reaction (PCR) allows the measurement of down to 0.1 fg of DNA using the new primers. To enhance the reliability of diagnostic assays, an internal positive control was developed using an amplification mimic. The mimic also served as a competitor for quantitative PCR, which was used to assess the growth of P. infestans in resistant and susceptible tomato. A key dimension of this study was that two laboratories independently checked the specificity and sensitivity of each primer set; differences were noted that should be considered when PCR is adopted for diagnostic applications in any system.     

Phytophthora ramorum and Phytophthora kernoviae on naturally infected asymptomatic foliage

Phytophthora ramorum and Phytophthora kernoviae are recently discovered invasive Phytophthoras causing leaf necrosis and shoot tip dieback mostly on ornamental and forest understorey species, but also cause bleeding cankers on stems of a wide range of tree species. Sporulation occurs only on infected shoots or fruits and foliage so foliar hosts are central to the disease epidemiology. In field trials to assess infection in trap plants exposed to natural inoculum of P. ramorum and P. kernoviae on rhododendron in south west England, it was discovered that leaves of the trap plants ( Rhododendron 'Cunninghams White') and holm oak ( Quercus ilex ) were asymptomatically infected and supported sporulation of both pathogens. More than half the rhododendron trap plants exposed to inoculum of P. kernoviae became infected compared with approximately a third of those exposed to P. ramorum in a natural situation. Approximately one third of the infections were detected from asymptomatic foliage for both pathogens. The significance of these findings for plant health regulation based on visual inspection as a measure to prevent introduction and dissemination of both these pathogens is explored and research gaps identified.