Survival of Phytophthora ramorum in Rhododendron root balls and in rootless substrates

This study assesses the survival of Phytophthora ramorum in the root ball of Rhododendron container plants as well as in different rootless forest substrates and a horticultural potting medium. Following inoculation of the root balls, the aboveground plant parts stayed symptomless, whilst the pathogen could be recovered with a novel non‐destructive baiting assay from the root balls until at least 8 months post‐inoculation. Plating of surface‐sterilized roots and direct microscopic analysis confirmed the presence of P. ramorum in the roots. Phytophthora ramorum could also be baited from the root balls of symptomless Rhododendron plants from commercial nurseries, even 2 years after acquisition. Survival of P. ramorum in rootless media was assessed after burying disks of infected leaf material below the soil surface in columns filled with four different undisturbed forest substrates or a potting medium, and incubated at an outdoor quarantine facility. Phytophthora ramorum could be recovered at least 33 months after burial from all substrates, with a significant increase in recovery after the winter period. These data suggest the possibility for long‐term symptomless presence of P. ramorum in root balls of commercial Rhododendron plants as well as survival in potting medium and different forest substrates under western European climate conditions. Symptomless presence in root balls can contribute to latent spread of this pathogen between nurseries. The novel baiting test, being non‐destructive, simple and applicable to a relatively large number of plants, can offer a valuable tool to test plants for the presence of Phytophthora species in root balls.     

Managing Phytophthora ramorum in the Netherlands1

Phytophthora ramorum came to the Netherlands in 1993. Despite initially not seeming to pose a high risk, findings in California showed its potential destructive impact on ecosystems. A programme began in the Netherlands to eliminate P. ramorum from nurseries and surveys in the natural environment were held to obtain information to determine a strategy for dealing with the disease. About 1100 nurseries are inspected annually by NAKtuinbouw under the auspices of the Plant Protection Service and measures are taken according to EC directives. The percentage of infected nurseries decreased steadily during recent years, from 4% in 2002/2003 to 0.5% in 2004/2005. Surveys in the natural environment show that P. ramorum occurs on 2% of the sites with Rhododendron and therefore it was concluded that an elimination scenario is not realistic. A programme based on containment measures supported by an extension programme was put into place with its effects being monitored by the Plant Protection Service. 12 years of observing P. ramorum show that the risk for indigenous trees and shrubs in the Netherlands is very limited. Spread from infected rhododendrons to other potential hosts, even at heavily infected Rhododendron sites, hardly takes place although some infected Quercus rubra trees have been found. Recently several new Phytophthora species were found in natural environments in Europe and California, mainly as a result of intensive P. ramorum surveys. As well as P. ramorum, the Phytophthora spp. P. kernoviae, P. numerosa and P. pseudosyringae pose risks, indicating the need for a more general approach against Phytophthora diseases. As a result, a new protocol for detection and identification of Phytophthora spp. both as a group and individually is being developed and workers are asking whether these Phytophthora species could be managed together.     

Effect of host factors on the susceptibility of Rhododendron to Phytophthora ramorum

Phytophthora ramorum causes sudden oak death (SOD) in western coastal forests of the USA. In Europe, the pathogen is mainly present in the nursery industry, particularly on Rhododendron. Because of the primary role of Rhododendron as a host and potentially as a vector, the effect of Rhododendron host factors on P. ramorum susceptibility and sporulation was investigated. Inoculation methods using either wounded or non‐wounded detached leaves were applied to 59 Rhododendron cultivars and 22 botanical species, replicated in three separate years. All Rhododendron species and cultivars were susceptible when using wounded leaves, but not when using non‐wounded leaves, suggesting a resistance mechanism operating at the level of leaf penetration. Using a regression tree analysis, the cultivars and species were split into four susceptibility classes. Young leaves were more susceptible than mature leaves when wounded, but less susceptible when non‐wounded. This effect was not correlated with leaf hydrophobicity or the number of leaf hairs. The presence or the type of rootstock did not affect the cultivar susceptibility level. Sporangia and chlamydospore production in the leaf lesions varied widely among Rhododendron cultivars and was not correlated with the susceptibility level. The susceptibility to P. ramorum correlated well with the susceptibility to P. citricola and P. hedraiandra × cactorum, suggesting that the resistance mechanisms against these species are non‐specific. Susceptibility to P. kernoviae was low for most cultivars. These findings have implications for detection, spread and disease control, and are therefore important in pest risk assessment.     

Diagnosis of Phytophthora ramorum– evaluation of testing methods1

Phytophthora ramorum is a recently described pathogen that has resulted in the introduction of emergency European Community phytosanitary legislation in November 2002. The Department of Environment, Food and Rural Affairs Plant Health and Seeds Inspectorate (PHSI) initiated a survey in England and Wales in autumn 2001 to ascertain whether the organism was present. In April 2002, P. ramorum was found for the first time in the UK and for the first time in Europe since the initial reports in the Netherlands and Germany. Following this finding, P. ramorum has been reported mainly on rhododendrons and viburnums but also on a wide range of other ornamental plants in both the UK and other European countries. Effective disease management and implementation of plant health legislation is reliant upon rapid and accurate disease diagnosis based upon recognition of symptoms in the field and identification of the cause by testing. Other diseases and disorders prevent reliable identification of the problem at the time of inspection with typically only 20–30% of samples submitted testing positive for Phytophthora species. This paper describes the evaluation of a direct real‐time PCR and a rapid serological assay (lateral flow device) developed by CSL for testing for P. ramorum and Phytophthora spp., respectively.     

A Phytophthora conserved transposon‐like DNA element as a potential target for soyabean root rot disease diagnosis

A transposon‐like element, A3aPro, with multiple copies in the Phytophthora sojae genome, was identified as a suitable detection target for this devastating soyabean root rot pathogen. The PCR primers TrapF1/TrapR1 were designed based on unique sequences derived from the transposon‐like sequence. A 267‐bp DNA fragment was amplified using this primer pair, the specificity of which was evaluated against 118 isolates of P. sojae, 72 isolates of 25 other Phytophthora spp., isolates of Pythium spp. and isolates of true fungi. In tests with P. sojae genomic DNA, detection sensitivities of 10 pg and 10 fg DNA were achieved in standard PCR (TrapF1/TrapR1) and nested PCR (TrapF1/TrapR1 and TrapF2/TrapR2), respectively. Meanwhile, PCR with TrapF1/TrapR1 primers detected the pathogen at the level of a single oospore, and even one zoospore. These primers also proved to be efficient in detecting pathogens from diseased soyabean tissues, residues and soils. In addition, real‐time quantitative PCR (qPCR) assays coupled with the TrapF1/TrapR1 primers were developed to detect and quantify the pathogen. The results demonstrated that the TrapF1/TrapR1 and TrapF2/TrapR2 primer‐based PCR assay provides a rapid and sensitive tool for the detection of P. sojae in plants and in production fields.     

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.     

Improved detection and identification of the sudden oak death pathogen Phytophthora ramorum and the Port Orford cedar root pathogen Phytophthora lateralis

Early detection provides the best way to prevent introduction and establishment of alien plant pathogens. Amplification of DNA by PCR has revolutionized the detection and monitoring of plant pathogens. Most of those assays rely on the amplification of a fraction of the genome of the targeted species. With the availability of whole genomes for a growing number of fungi and oomycetes it is becoming possible to compare genomes and discover regions that are unique to a target organism. This study has applied this pipeline to develop a set of hierarchical TaqMan real-time PCR detection assays targeting DNA of all four Phytophthora ramorum lineages, and a closely related species, P. lateralis. Nine assays were generated: three targeting DNA of all P. ramorum lineages, one for each lineage of P. ramorum, one for P. lateralis and one targeting DNA of P. ramorum and P. lateralis. These assays were very accurate and sensitive, ranging from 98.7% to 100% detection accuracy of 2–10 gene copies of the targeted taxa from pure cultures or inoculated tissues. This level of sensitivity is within the lowest theoretical limit of detection of DNA. It is expected that these assays will be useful because of their high level of specificity and the ease with which they can be multiplexed because of the inherent flexibility in primer and probe design afforded by their lack of conservation in non-target species.     

Development of PCR-based Detection Methods for the Quarantine Phytopathogen <Emphasis Type="Italic">Synchytrium endobioticum</Emphasis>, Causal Agent of Potato Wart Disease

PCR-based methods were developed for the detection and quantification of the potato pathogen Synchytrium endobioticum in soil extracts and in planta. PCR primers, based on the internal transcribed spacer region of the multi-copy gene rDNA were tested for specificity, sensitivity and reproducibility in conventional and real-time PCR assays. Soil extraction procedures compared included the Hendrickx centrifugation (HC) procedure, nested wet sieving (NWS) and a method used by the Plant Protection Service (PPS). The primers amplified a 472 bp product from S. endobioticum DNA, but did not amplify DNA from other potato pathogens, other plant pathogens, and related species. Standard cell disruption and DNA extraction and purification methods were optimized for amplification of S. endobioticum DNA from resting sporangia. DNA was successfully amplified from a single sporangium and equivalent DNA preparations from soil extracts. Low levels of target DNA in water did not amplify, possibly due to DNA loss during final purification steps. A real-time PCR assay, developed for soil-based extracts using primers and probe based on the rDNA gene sequences, involved co-amplification of target DNA along with an internal DNA fragment. Both conventional and real-time PCR methods performed well with HC- and NWS-extracts having a threshold sensitivity of 10 sporangia per PCR assay. Of the three soil extraction methods, only with the HC method could 100 g soil samples be efficiently processed in one single PCR assay. Such a high capacity assay could be useful for routine soil analysis in respect to disease risk assessments and to secure de-scheduling according to EPPO guidelines.     

Development of a real‐time PCR method for the detection of the dagger nematodes Xiphinema index,X. diversicaudatum,X. vuittenezi and X. italiae,and for the quantification of X. index numbers

The ectoparasitic dagger nematodes Xiphinema index and Xiphinema diversicaudatum, often at low numbers in the soil, are vectors of grapevine nepoviruses, which cause huge agronomical problems for the vineyard industry. This study reports a method, based on real‐time PCR, for the specific detection of these species and of the closely related non‐vector species Xiphinema vuittenezi and Xiphinema italiae. Specific primers and TaqMan probes were designed from the ribosomal DNA internal transcribed spacer 1 (ITS1), enabling the specific detection of single individuals of each of the X. index, X. diversicaudatum, X. italiae and X. vuittenezi species whatever the nematode population. The specificity of detection and absence of false positive reaction were confirmed in samples of each species mixed with the three other Xiphinema species or mixed with nematodes representative from other genera (non‐plant‐parasitic Dorylaimida, Longidorus sp., Meloidogyne spp., Globodera spp. and Pratylenchus sp.). The method was shown to be valid for the relative quantification of X. index numbers through its use, from crude nematode extracts of soil samples, in a greenhouse assay of grapevine accessions ranging from highly susceptible to resistant. As an alternative to time‐consuming microscopic identification and counting, this real‐time PCR method will provide a fast, sensitive and reliable diagnostic and relative quantification technique for X. index nematodes extracted from fields or controlled conditions.     

In‐field distribution of Plasmodiophora brassicae measured using quantitative real‐time PCR

A protocol using real‐time polymerase chain reaction (PCR) for the direct detection and quantification of Plasmodiophora brassicae in soil samples was developed and used on naturally and artificially infested soil samples containing different concentrations of P. brassicae. Species‐specific primers and a TaqMan fluorogenic probe were designed to amplify a small region of P. brassicae ribosomal DNA. Total genomic DNA was extracted and purified from soil samples using commercial kits. The amount of pathogen DNA was quantified using a standard curve generated by including reactions containing different amounts of a plasmid carrying the P. brassicae target sequence. The PCR assay was optimized to give high amplification efficiency and three to four copies of the target DNA sequence were detected. Regression analysis showed that the standard curve was linear over at least six orders of magnitude (R² > 0·99) and that the amplification efficiency was >92%. The detection limit in soil samples corresponded to 500 resting spores g^?1 soil. The intersample reproducibility was similar to, or higher than, that of assays for other pathogens quantified in soil samples. Bait plants were used to validate the real‐time PCR assay. The protocol developed was used to investigate the spatial distribution of P. brassicae DNA in different fields and a significant difference was found between in‐field sampling points. The reproducibility of soil sampling was evaluated and showed no significant differences for samples with low levels of inoculum, whereas at higher levels differences occurred. Indicator kriging was used for mapping the probability of detecting P. brassicae within a 2‐ha area of a field. A threshold level of 5 fg plasmid DNA g^?1 soil, corresponding to approximately 3 × 10³P. brassicae resting spores g^?1 soil, is suggested for growing resistant cultivars. The results provide a robust and reliable technique for predicting the risk of disease development and for assessing the distribution of disease within fields.     

Rapid detection and quantification of viable potato cyst nematodes using qPCR in combination with propidium monoazide

Potato cyst nematodes (PCN), Globodera pallida and Globodera rostochiensis, are obligate parasites of solanaceous plants, causing severe losses in several potato growing areas throughout the world. To date, management of PCN is related to nematode population densities estimated as eggs per gram of soil, without considering the actual number of viable juveniles within the cysts. In classical nematology, the standard method to determine PCN viability is based on a staining assay, using Meldola's blue dye (MB) followed by microscopic visualization of MB‐treated nematodes. Although MB is considered to be reliable in staining embryonated juveniles within eggs and cysts, it is a time‐ and labour‐consuming assay. In the present work, a real‐time PCR (qPCR)‐based method combined with propidium monoazide (PMA), a photoreactive DNA‐intercalating dye, was developed for the quantification of viable PCN. This dye renders exposed DNA of dead cells unable to be amplified by PCR, and thus only DNA from viable/intact PCN juveniles is amplified and detected. The novelty of the present method lies in the simultaneous quantitative and qualitative estimation of viable PCN inocula using species‐specific primers and TaqMan probes. The PMA–qPCR viability method (v‐PCR) developed for the two Globodera species successfully discriminated dead from living specimens in heat‐treated samples and eggs in old and newly formed cysts. Interestingly, the detection of DNA from 34‐year‐old nematode cysts stored at room temperature was observed. In conclusion, the proposed v‐PCR method should prove to be very useful for the routine determination of PCN viability from field samples.     

A real‐time (TaqMan) PCR assay to differentiate Monilinia fructicola from other brown rot fungi of fruit crops

To prevent the entry and spread of the brown rot fungus Monilinia fructicola in Europe, a fast and reliable method for detection of this organism is essential. In this study, an automated DNA extraction method combined with a multiplex real‐time PCR based on TaqMan chemistry was developed for fast, convenient and reliable detection of both the EU quarantine organism Monilinia fructicola and the three other brown rot fungi M. fructigena, M. laxa and Monilia polystroma. Using the internal transcribed spacer (ITS) region of the nuclear ribosomal RNA gene repeat, a Monilinia genus‐specific primer pair and two differently labelled fluorogenic probes specific for M. fructicola and the group M. fructigena/M. laxa/Monilia polystroma were developed. The analytical specificity of the assay was assessed by testing 33 isolates of the four brown rot fungi and 13 isolates of related fungal species or other fungal species that can be present on stone and pome fruit. No cross‐reactions were observed. The assay was found to have a detection limit of 0·6 pg of DNA, corresponding to 27 haploid genomes or four conidia. Comparison of a manual DNA isolation followed by a conventional PCR with an automated DNA isolation combined with the presently developed real‐time PCR showed that the latter method gave improved results when tested with 72 naturally infected stone fruit samples. The detection rate increased from 65 to 97%.     

Tissue‐print and squash real‐time PCR for direct detection of ‘Candidatus Liberibacter’ species in citrus plants and psyllid vectors

Huanglongbing (HLB) disease is seriously threatening and/or damaging the citrus industry worldwide. Accurate detection of the three species associated with HLB disease, ‘Candidatus Liberibacter asiaticus’, ‘Candidatus Liberibacter africanus’ and ‘Candidatus Liberibacter americanus’, is essential for the preventive control of the disease. Real‐time PCR is a useful tool for bacterial detection. However, nucleic acid purification steps limit the number of samples that can be processed by PCR. Universal detection of ‘Ca. Liberibacter’ species was achieved by direct tissue‐printing and spotting of plant leaf petiole extracts or squashing of individual psyllids onto paper or nylon membranes. Primers were designed and used with TaqMan chemistry for accurate detection of the bacterium in immobilized targets (prints of 10 overlapping leaf pedicels per tree, or squashed single vectors), by extraction with water and direct use for real‐time PCR. This simplified method was validated and could detect HLB‐liberibacters in 100% of leaves with symptoms and 59% of symptomless leaves collected from HLB‐infected trees. The use of direct assays as template showed good agreement with use of purified DNA (κ = 0·76 ± 0·052). The squash assay allowed detection of the bacterium in 40% of mature Diaphorina citri that fed on leaves of HLB‐infected trees with or without symptoms. A commercial ready‐made kit based on this technology showed 96% accuracy in intra‐laboratory performance studies. The simplified direct methods of sample preparation presented herein can be effectively adopted for use in rapid screening of HLB agents in extensive surveys, certification schemes or for epidemiological and research studies.     

Diversity and detections of Phytophthora species from trade and non‐trade environments in Ireland

The genus Phytophthora is one of the genera of organisms that poses the most threat to plant health worldwide. Statutory monitoring for Phytophthora species focuses on the species regulated in the European Union and recommended for regulation by EPPO (Plant Health Directive 2000/29 EC and the EPPO A2 List). This research provides details of the Phytophthora species detected from trade and non‐trade environments in Ireland between 2013 and 2015. The results of statutory surveys for the regulated species Phytophthora ramorum, Phytophthora kernoviae and Phytophthora lateralis from 2003 to 2015 are also presented. Testing of more than 11 000 samples was carried out using morphological and/or DNA identification with specifically designed Phytophthora conserved primers. This led to the detection of 19 species and 3 informally designated taxa of Phytophthora, including 8 new records for Ireland. Eight species were found in both trade and non‐trade locations, and three informally designated taxa were also detected. Phytophthora ramorum was found on the most hosts (30 hosts), followed by Phytophthora syringae (6 hosts) and Phytophthora kernoviae (3 hosts). Rhododendron was the host on which Phytophthora species were most frequently detected (12 Phytophthora species). The role of the plant trade in spreading invasive Phytophthora species is discussed.     

Quantification of potato common scab pathogens in soil by quantitative competitive PCR with fluorescent quenching‐based probes

Common scab of potato tubers caused by pathogenic Streptomyces spp. is a cause of serious economic loss worldwide. For the rapid and accurate quantification of pathogenic Streptomyces spp. residing in soil, a new competitive real‐time PCR method using fluorescent quenching‐based probes (quantitative competitive quenching probe PCR: QCQP‐PCR) was developed. The virulence gene of pathogenic Streptomyces spp., nec1, was selected as the target for QCQP‐PCR. A specific primer set to amplify the nec1 gene, and a fluorescently labelled probe that specifically hybridizes with the nec1 amplicon were designed. For QCQP‐PCR, an internal standard DNA (IS DNA) that is identical to the nec1 amplicon but has a 4‐base mismatch in the probe‐hybridizing region, and a fluorescently labelled probe IS, which specifically hybridizes with IS DNA at the mutagenized region, were PCR‐synthesized. The target nec1 gene was co‐amplified with the known copy number of IS DNA by PCR using the same primer set in the presence of the specific probes. The PCR products were monitored in real‐time by measuring the fluorescence intensity (quenching) of each probe. The initial amount of the nec1 gene was quantified based on the ratio of the PCR products of the same PCR cycle. The results revealed that QCQP‐PCR could be used to precisely quantify the nec1 gene, even in the presence of PCR inhibitors in the soil samples examined. The lower limit of quantification was 20 copies per tube, which corresponded to 1500 copies per g dry soil. The quantification achieved by this method was completed within 5 h, i.e. the duration of the entire analysis. These results demonstrate the usefulness of the present method for monitoring pathogenic Streptomyces species in soil.     

Improved real-time PCR assay for detection of the quarantine potato pathogen, <Emphasis Type="Italic">Synchytrium endobioticum</Emphasis>, in zonal centrifuge extracts from soil and in plants

Real-time PCR was used for quantitative detection of the potato pathogen, Synchytrium endobioticum, in different substrates: zonal centrifuge extracts, warts and different plant parts of potato. Specific primers and a TaqMan probe, designed from the internal transcribed spacer region of the multi-copy rDNA gene were tested in extracts from artificially and naturally infested soil. Co-amplification of target DNA along with an internal competitor DNA fragment made the diagnostic assay more reliable by guarding against false negative results. A calibrations curve was created by spiking zonal centrifuge fractions of clean soil samples with a dilution series of winter spores. The Taqman assay was also performed on infected potato plant material (stolons) along with the detection of the cytochrome oxidase gene as a potato endogenous control. Sensitivity of the TaqMan assay was improved at least 100-fold and proved to be reliable for accurate diagnosis of the disease.     

Development of a real‐time PCR assay for Pseudomonas cichorii,the causal agent of midrib rot in greenhouse‐grown lettuce,and its detection in irrigating water

Midrib rot is an emerging disease in greenhouse production of lettuce caused by Pseudomonas cichorii, and probably introduced through contaminated irrigation water. Concentrations of 100 CFU mL^?1 are enough to induce the typical midrib rot symptoms. A sensitive real‐time PCR assay was developed, based on a 90‐bp amplicon from the pathogenicity gene cluster hrcRST and a Taqman Minor Groove Binding probe. Specificity of the assay was tested with 39 P. cichorii strains, including the type strain, and 89 strains from 83 other Pseudomonas species. The relationship between detection signals and P. cichorii DNA concentrations was linear over 6‐logs. Detection threshold with excellent reproducibility was 500 fg of DNA or about 70 genome copies. Sample preparation and DNA isolation were optimized to allow detection in 1 L water samples. The assay was first evaluated with greenhouse irrigation water spiked with serial dilutions of P. cichorii. The calculated cell numbers obtained with real‐time PCR were 10‐fold lower than plate counts of actual spiked cells. However, the assay consistently detected 100 CFU per reaction, corresponding to the detection of 1 CFU mL^?1 of irrigation water, which is well below the concentration needed for midrib rot infection. Finally, the assay proved to be valuable for detecting infective P. cichorii concentrations in the irrigation water of a commercial lettuce production greenhouse.     

Potential for eradication of the exotic plant pathogens Phytophthora kernoviae and Phytophthora ramorum during composting

Temperature and exposure time effects on Phytophthora kernoviae and Phytophthora ramorum viability were examined in flasks of compost and in a large‐scale composting system containing plant waste. Cellophane, rhododendron leaf and peat‐based inoculum of P. kernoviae and P. ramorum isolates were used in flasks; naturally infected leaves were inserted into a large‐scale system. Exposures of 5 and 10 days respectively at a mean temperature of 35°C in flask and large‐scale composts reduced P. kernoviae and P. ramorum inocula to below detection limits using semi‐selective culturing. Although P. ramorum was undetectable after a 1‐day exposure of inoculum to compost at 40°C in flasks, it survived on leaves exposed to a mean temperature of 40·9°C for 5 days in a large‐scale composting system. No survival of P. ramorum was detected after exposure of infected leaves for 5 days to a mean temperature of ≥41·9°C (32·8°C for P. kernoviae) or for 10 days at ≥31·8°C (25·9°C for Phytophthora pseudosyringae on infected bilberry stems) in large‐scale systems. Fitted survival probabilities of P. ramorum on infected leaves exposed in a large‐scale system for 5 days at 45°C or for 10 days at 35°C were <3%, for an average initial infection level of leaves of 59·2%. RNA quantification to measure viability was shown to be unreliable in environments that favour RNA preservation: high levels of ITS1 RNA were recovered from P. kernoviae‐ and P. ramorum‐infected leaves exposed to composting plant wastes at >53°C, when all culture results were negative.     

A duplex PCR method for the simultaneous identification of Phytophthora ramorum and Phytophthora kernoviae

Phytophthora ramorum and Phytophthora kernoviae are two fungus‐like organisms affecting a wide range of hardy ornamental plants and trees. Emergency measures are implemented in the European Union for P. ramorum and aim to eradicate, or at least prevent the further spread of this harmful pathogen. Phytophthora kernoviae has so far been found only in New Zealand, the UK and Ireland, and is regulated on a UK level using the same measures as for P. ramorum. Both Phytophthora species have a similar host range and can be diagnosed using similar methods. Therefore a duplex PCR detection, based on the internal transcribed spacer (ITS) regions of the ribosomal DNA, was developed to enable simultaneous testing to reduce diagnostic times. The method was tested for its specificity and sensitivity, and on plant samples, and was shown to be reliable for identification of the two organisms.     

Detection of Colletotrichum coccodes from soil and potato tubers by conventional and quantitative real-time PCR

Colletotrichum coccodes is the causal agent of the potato blemish disease black dot. Two PCR primer sets were designed to sequences of the ribosomal internal transcribed spacer (ITS1 and ITS2) regions for use in a nested PCR. The genus-specific outer primers (Cc1F1/Cc2R1) were designed to regions common to Colletotrichum spp., and the species-specific nested primers (Cc1NF1/Cc2NR1) were designed to sequences unique to C . coccodes . The primer sets amplified single products of 447 bp (Cc1F1/Cc2R1) and 349 bp (Cc1NF1/Cc2NR1) with DNA extracted from 33 European and North American isolates of C. coccodes. The specificity of primers Cc1NF1/Cc2NR1 was confirmed by the absence of amplified product with DNA of other species representing the six phylogenetic groups of the genus Colletotrichum and 46 other eukaryotic and prokaryotic plant pathogenic species. A rapid procedure for the direct extraction of DNA from soil and potato tubers was used to verify the PCR assay for detecting C. coccodes in environmental samples. The limit of sensitivity of PCR for the specific detection of C. coccodes when inoculum was added to soils was 3·0 spores per g, or the equivalent of 0·06 microsclerotia per g soil, the lowest level of inoculum tested. Colletotrichum coccodes was also detected by PCR in naturally infested soil and from both potato peel and peel extract from infected and apparently healthy tubers. Specific primers and a TaqMan fluorogenic probe were designed to perform quantitative real-time (TaqMan) PCR to obtain the same levels of sensitivity for detection of C. coccodes in soil and tubers during a first-round PCR as with conventional nested PCR and gel electrophoresis. This rapid and quantitative PCR diagnostic assay allows an accurate estimation of tuber and soil contamination by C. coccodes .