A molecular marker for the specific detection of new pathotype 5‐like strains of Plasmodiophora brassicae in canola

Clubroot of crucifers, caused by Plasmodiophora brassicae, is managed in canola (Brassica napus) by the deployment of resistant cultivars. Recently, however, new strains of P. brassicae have been detected in Alberta, Canada, that can overcome this resistance. Some of these strains are classified as pathotype 5 on the differential system of Williams, but are distinguished by their ability to overcome host resistance. In order to expedite the identification of these new pathotype 5‐like strains, three primer sets were developed based on the 18S‐ITS region of the pathogen. With primers P5XF3 and P5XR3, a 127 bp product was amplified from all new pathotype 5‐like strains following optimized PCR analysis. A TaqMan probe‐based quantitative assay was also developed. These protocols could be used to detect as little as 0.5 pg P. brassicae DNA, and as few as 10⁴ mL^?1 pathogen resting spores; infection of host tissues could be detected as soon as 4 days after inoculation. The PCR and qPCR assays described in this study represent useful tools for the rapid and reliable diagnosis and quantification of new pathotype 5‐like strains of P. brassicae.     

Effect of susceptible and resistant canola plants on Plasmodiophora brassicae resting spore populations in the soil

Clubroot, caused by Plasmodiophora brassicae, has become a serious threat to canola (Brassica napus) production in western Canada. Experiments were conducted to assess the effect of growing resistant and susceptible canola genotypes on P. brassicae soil resting spore populations under greenhouse, mini‐plot and field conditions. One crop of susceptible canola contributed 1·4 × 10⁸ spores mL^?1 soil in mini‐plot experiments, and 1 × 10¹⁰ spores g^?1 gall under field conditions. Repeated cropping of susceptible canola resulted in greater gall mass compared to resistant canola lines. It also resulted in reduced plant height, increased clubroot severity in susceptible canola, and increased numbers of resting spores in the soil mix.     

Dissemination of Plasmodiophora brassicae in livestock manure detected by qPCR

The present study was performed to investigate whether Plasmodiophora brassicae can be disseminated by livestock manure. A quantitative PCR (qPCR) assay was developed and used to detect and quantify P. brassicae in manure samples from naturally and artificially infested chickens and pigs. In naturally infested manure, quantifiable levels of infestation were observed in 7 out of the 28 samples, ranging from 10³ to 10⁷ resting spores per g of manure. The vast majority of the resting spores (76–91%) were viable, as determined by a dual fluorescence viability assay. Clubroot symptoms developed on plants inoculated with P. brassicae resting spores isolated from all seven qPCR‐positive samples. Artificially infested manure samples were produced by feeding chickens and pigs on P. brassicae‐contaminated feed. The levels of infestation were 10³ resting spores per g of manure for both chicken and pig manure sampled 24 and 48 h after feeding, respectively. Spore viability was >80% for both samples, and the disease severity indices were both >40 as indicated by bioassay. This showed that resting spores can survive the digestive tracts of chickens and pigs, and retain strong pathogenicity. The findings indicate that manure dissemination is possible for P. brassicae. Farmers should avoid feeding livestock on P. brassicae‐contaminated feed or applying infested manure as fertilizer on land intended for crucifer crops.     

Virulence and inoculum density‐dependent interactions between clubroot resistant canola (Brassica napus) and Plasmodiophora brassicae

To mitigate the impact and dissemination of clubroot in western Canada, canola (Brassica napus) producers have relied on clubroot resistance traits. However, in 2013 and 2014, new strains of the clubroot pathogen, Plasmodiophora brassicae, emerged that are virulent on most clubroot‐resistant (CR) canola genotypes. Novel strains of the pathogen were inoculated onto two susceptible canola cultivars, one resistant line and six CR cultivars. Although all cultivars/lines showed a susceptible response to inoculation with the new strains of P. brassicae, the severity of disease reaction, root hair infection rates and the amount of P. brassicae DNA present in each canola genotype varied depending on the strain. In addition, the effect of inoculum density on disease severity and gall formation was recorded for one of these new strains on a universally susceptible Chinese cabbage cultivar and one susceptible and 10 resistant canola genotypes. Although root galls were observed at an inoculum density of 10³ spores per mL of soil, clear differentiation of susceptible and resistant reactions among canola cultivars/lines was not observed until the inoculum density reached 10⁵ spores mL^?1. At a spore density of 10⁶ spores mL^?1 and above, all cultivars/lines developed susceptible reactions, although there was some differentiation in the degree of reaction. This study shows the potential to develop a unique disease profile for emergent clubroot pathotypes and shows a useful range of spore densities at which to study new P. brassicae strains.     

Quantitative PCR shows propagation of <Emphasis Type="Italic">Plasmodiophora brassicae</Emphasis> in Swedish long term field trials

Clubroot (Plasmodiophora brassicae) is a serious soil-borne disease in brassica crops world-wide. We report on a time series of soil samples from Swedish long-term fertility trials started in 1957, 1963 and 1966, which were analyzed for the amount of P. brassicae DNA. The crop rotations included a brassica crop every 4 or 6 years. All experimental sites with a 4-year rotation of oilseed rape, except one with calcium carbonate in the soil profile, showed high (>1000 fg DNA g^?1 soil) levels of P. brassicae DNA after 9, 11 and 12 rotations. In contrast, detectable levels (>5 fg DNA g^?1 soil) of P. brassicae were found only at one of five sites with a 6-year rotation of spring oilseed rape. In years with high levels of P. brassicae DNA, low yield was reported and a subsequent decline in P. brassicae DNA in soil was observed. Different NPK (nitrogen/phosphorus/potassium) fertiliser regimes resulted in similar P. brassicae DNA levels. The robustness and reliability of the method applied was verified by analyses of soil from individual plots compared with a mixture of plots and by repeated analyses of selected samples, which showed that P. brassicae DNA remained stable during dry storage.     

Comparison of techniques for estimation of resting spores of Plasmodiophora brassicae in soil

Clubroot (Plasmodiophora brassicae) is an important disease of canola (Brassica napus) and other brassica crops. Accurate estimation of inoculum load in soil is important for evaluating producer risk in planting a susceptible crop, but also for evaluation of management practices such as crop rotation. This study compared five molecular techniques for estimating P. brassicae resting spores in soil: quantitative polymerase chain reaction (qPCR), competitive positive internal control PCR (CPIC-PCR), propidium monoazide PCR (PMA-PCR), droplet digital PCR (ddPCR) and loop-mediated isothermal DNA amplification (LAMP). For ddPCR and LAMP, calibrations were developed using spiked soil samples. The comparison was carried out using soil samples collected from a long-term rotation study at Normandin, Québec, with replicated plots representing 0-, 1-, 2-, 3-, 5- and 6-year breaks following susceptible canola infested with clubroot. CPIC-PCR and ddPCR provided repeatable estimates of resting spore numbers in soil compared with estimates from qPCR or LAMP alone. CPIC-PCR provided the most robust measurement of spore concentration, especially in the 2 years following a crop of susceptible canola, because it corrected for effects of PCR inhibitors. PMA-PCR demonstrated that a large proportion of the DNA of P. brassicae detected in soil after the susceptible canola crop was derived from spores that were immature or otherwise not viable. Each assay provided a similar pattern of spore concentration in soil, which supported the conclusion of a previous study at this site that resting spore numbers declined rapidly in the first 2 years after a susceptible crop, but much more slowly subsequently.     

Direct evidence of surface infestation of seeds and tubers by Plasmodiophora brassicae and quantification of spore loads

Using quantitative PCR, DNA of Plasmodiophora brassicae, the causal agent of clubroot, was detected and quantified on canola, pea and wheat seeds, as well as on potato tubers, all harvested from clubroot‐infested fields in Alberta, Canada. Quantifiable levels of infestation were found on seven of the 46 samples analysed, and ranged from <1·0 × 10³ to 3·4 × 10⁴ resting spores per 10 g seeds; the vast majority (80–100%) of resting spores on these samples were viable, as determined by Evan’s blue vital staining. However, the levels of infestation found were generally lower than that required to cause consistent clubroot symptoms in greenhouse plant bioassays. While the occurrence of P. brassicae resting spores on seeds and tubers harvested from clubroot‐infested fields suggests that seedborne dissemination of this pathogen is possible, practices such as commercial seed cleaning may be sufficient to effectively mitigate this risk.     

Plasmodiophora brassicae resting spore dynamics in clubroot resistant canola (Brassica napus) cropping systems

The soilborne pathogen Plasmodiophora brassicae, causal agent of clubroot of canola (Brassica napus), is difficult to manage due to the longevity of its resting spores, ability to produce large amounts of inoculum, and the lack of effective fungicides. The cropping of clubroot resistant (CR) canola cultivars is one of the few effective strategies for clubroot management. This study evaluated the impact of the cultivation of CR canola on P. brassicae resting spore concentrations in commercial cropping systems in Alberta, Canada. Soil was sampled pre-seeding and post-harvest at multiple georeferenced locations within 17 P. brassicae-infested fields over periods of up to 4 years in length. Resting spore concentrations were measured by quantitative PCR analysis, with a subset of samples also evaluated in greenhouse bioassays with a susceptible host. The cultivation of CR canola in soil with quantifiable levels of P. brassicae DNA resulted in increased inoculum loads. There was a notable lag in the release of inoculum after harvest, and quantifiable P. brassicae inoculum peaked in the year following cultivation of CR canola. Rotations that included a ≥2-year break from P. brassicae hosts resulted in significant declines in soil resting spore concentrations. A strong positive relationship was found between the bioassays and qPCR-based estimates of soil infestation. Results suggest that CR canola should not be used to reduce soil inoculum loads, and crop rotations in P. brassicae infested fields should include breaks of at least 2 years away from B. napus, otherwise the risk of selecting for virulent pathotypes may increase.     

Detection and quantification of Ilyonectria spp. associated with black‐foot disease of grapevine in nursery soils using multiplex nested PCR and quantitative PCR

Three nursery fields and three rootstock mother fields from commercial nurseries located in Comunidad Valenciana region (central‐eastern Spain) were surveyed in July 2011 to detect the presence and to quantify Ilyonectria spp. in the soil. In each field, ten soil samples were taken randomly with a soil probe at a depth of 10–30 cm, and 10–20 cm from the base of the plant. Three replicate subsamples (10 g each) were taken from each soil sample. DNA was extracted and a multiplex nested PCR with species‐specific primer pairs (Mac1/MaPa2, Lir1/Lir2 and Pau1/MaPa2) was used to identify the species present. Among the 180 soil DNA samples analysed, Ilyonectria spp. were detected in 172 of them. Ilyonectria macrodidyma complex was the most frequently detected, being identified in 141 samples from all the fields evaluated. However, I. liriodendri was detected in only 16 samples, but was present in all open‐root field nurseries and in two rootstock mother fields. In addition, quantitative PCR (qPCR) assays were done to assess the levels of I. liriodendri and I. macrodidyma‐complex DNA in the soil samples. Detection of Ilyonectria spp. DNA using qPCR correlated with the fields found positive with the nested multiplex PCR. DNA concentrations of Ilyonectria spp. ranged from 0·004 to 1904·8 pg μL^?1. In general, samples from rootstock mother fields showed the highest DNA concentrations. The ability to detect and quantify Ilyonectria spp. genomic DNA in soil samples from nursery fields and rootstock mother fields confirms soils from both field types as important inoculum sources for black‐foot pathogens.     

Effects of R gene‐mediated resistance in Brassica napus (oilseed rape) on asexual and sexual sporulation of Pyrenopeziza brassicae (light leaf spot)

The phenotype of the R gene‐mediated resistance derived from oilseed rape (Brassica napus) cv. Imola against the light leaf spot plant pathogen, Pyrenopeziza brassicae, was characterized. Using a doubled haploid B. napus mapping population that segregated for resistance against P. brassicae, development of visual symptoms was characterized and symptomless growth was followed using quantitative PCR and scanning electron microscopy on leaves of resistant/susceptible lines inoculated with suspensions of P. brassicae conidia. Initially, in controlled‐environment experiments, growth of P. brassicae was unaffected; then from 8 days post‐inoculation (dpi) some epidermal cells collapsed (‘black flecking’) in green living tissue of cv. Imola and from 13 to 36 dpi there was no increase in the amount of P. brassicae DNA and no asexual sporulation (acervuli/pustules). By contrast, during this period there was a 300‐fold increase in P. brassicae DNA and extensive asexual sporulation in leaves of the susceptible cv. Apex. However, when leaf tissue senesced, the amount of P. brassicae DNA increased rapidly in the resistant but not in the susceptible cultivar and sexual sporulation (apothecia) was abundant on senescent tissues of both. These results were consistent with observations from both controlled condition and field experiments with lines from the mapping population that segregated for this resistance. Analysis of results of both controlled‐environment and field experiments suggested that the resistance was mediated by a single R gene located on chromosome A1.     

Relative importance of seed‐tuber and soilborne inoculum in causing black dot disease of potato

Controlled‐environment and field experiments were done to quantify the individual contribution of seed‐tuber and soilborne inoculum of Colletotrichum coccodes in causing black dot disease of potato tubers. Seed‐tuber and soilborne inocula of C. coccodes were quantified using an existing real‐time PCR assay and related to subsequent incidence and severity of disease. In four field trials, a controlled‐environment experiment and through the monitoring of 122 commercial crops, seed‐tuber inoculum was found to be relatively less important than soilborne inoculum in causing black dot, and the level of seed‐tuber inoculum did not significantly affect either the incidence or severity of disease or the percentage of progeny tubers deemed unmarketable. By contrast, soilborne inoculum had the potential to result in high levels of disease and the level of C. coccodes soil infestation (pg DNA g^?1 soil) was found to have a significant effect. At soil infestation levels below 100 pg DNA C. coccodes g^?1 soil, 7% of commercial crops had an incidence of black dot greater than 20%, increasing to 40% and 57% of crops at levels of 100–1000 pg g^?1 and >1000 pg g^?1 soil, respectively. These arbitrary threshold levels for soilborne inoculum related to disease risk are discussed. Interpretation of disease risk based on inoculum levels must, in the future, be informed by agronomic variables and potential control strategies.     

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.     

Detection and quantification of Fusarium commune in host tissue and infested soil using real‐time PCR

Fusarium wilt caused by Fusarium commune is a major limiting factor for Chinese water chestnut (Eleocharis dulcis) production in China. A SYBR Green I real‐time quantitative polymerase chain reaction (qPCR) assay was developed based on the mitochondrial small subunit rDNA of F. commune. Assay specificity of the FO1/FO2 primer set was tested on 41 fungal isolates, and only a single PCR band of c. 178 bp from F. commune was amplified. The detection limits of the assay were 1 fg μL^?1 pure F. commune genomic DNA, 1 pg μL^?1 F. commune genomic DNA mixed with host plant genomic DNA (0·5 ng μL^?1), and 1000 conidia/g soil (artificially inoculated). The amount of F. commune DNA in stem tissues detected by qPCR was significantly correlated with the disease severity (DS) ratings; however, the qPCR assay showed no significant positive correlation between spore densities in soil of different fusarium wilt DS groupings and the DS ratings. The qPCR assay was further applied to 76 soil samples collected from commercial fields of E. dulcis during the 2011 and 2012 growing seasons. The spore density of F. commune detected was positively correlated with disease index in the 2012 growing season but not in 2011. The qPCR method can be used for rapid and specific detection of F. commune in plant and soil samples, which will facilitate monitoring of the pathogen and improvement of disease management.     

Loop‐mediated isothermal amplification (LAMP) assays for rapid detection of Pyrenopeziza brassicae (light leaf spot of brassicas)

Pyrenopeziza brassicae (anamorph Cylindrosporium concentricum) is an ascomycete fungus that causes light leaf spot (LLS) disease of brassicas. It has recently become the most important pathogen of winter oilseed rape (Brassica napus) crops in the UK. The pathogen is spread by both asexual splash‐dispersed conidia and sexual wind‐dispersed ascospores. Such inoculum can be detected with existing qualitative and quantitative PCR diagnostics, but these require time‐consuming laboratory‐based processing. This study describes two loop‐mediated isothermal amplification (LAMP) assays, targeting internal transcribed spacer (ITS) or β‐tubulin DNA sequences, for fast and specific detection of P. brassicae isolates from a broad geographical range (throughout Europe and Oceania) and multiple brassica host species (B. napus, B. oleracea and B. rapa). Neither assay detected closely related Oculimacula or Rhynchosporium isolates, or other commonly occurring oilseed rape fungal pathogens. Both LAMP assays could consistently detect DNA amounts equivalent to 100 P. brassicae conidia per sample within 30 minutes, although the β‐tubulin assay was more rapid. Reproducible standard curves were obtained using a P. brassicae DNA dilution series (100 ng–10 pg), enabling quantitative estimation of amounts of pathogen DNA in environmental samples. In planta application of the β‐tubulin sequence‐based LAMP assay to individual oilseed rape leaves collected from the field found no statistically significant difference in the amount of pathogen DNA present in parts of leaves either with or without visible LLS symptoms. The P. brassicae LAMP assays described here could have multiple applications, including detection of symptomless host infection and automated real‐time monitoring of pathogen inoculum.     

Quantifying resistance to Plasmodiophora brassicae in Brassica hosts

Plasmodiophora brassicae causes clubroot of crucifers. A quantitative PCR (qPCR)‐based protocol was developed to measure P. brassicae DNA in the roots of susceptible, intermediately susceptible, intermediately resistant and resistant Brassica hosts, and the non‐host wheat, at 5, 10, 15, 20 and 42 days post‐inoculation (dpi). The final reaction of each plant genotype was recorded as an index of disease at 42 dpi. Plasmodiophora brassicae DNA showed an increase in susceptible and moderately resistant hosts from 5 to 42 dpi, in contrast to a decrease in a highly resistant host and the non‐host wheat over the same period. Index of disease was significantly positively correlated with the amount of P. brassicae DNA in the roots at 5, 15, 20 and 42 dpi in one experiment, and at 10, 15, 20 and 42 dpi in a repeated experiment. Significant positive correlations also existed between the amounts of P. brassicae DNA in the roots at 42 dpi and those at 5, 10, 15 and 20 dpi in one experiment, and those at 10, 15 and 20 dpi in a repeated experiment. The results generated by the qPCR assay were validated by microscopic examination of roots inoculated with P. brassicae. The qPCR‐based protocol developed in this study allows for the accurate quantification of P. brassicae DNA in host root tissues as early as 5 dpi, and may serve as a useful tool to evaluate pathogen proliferation and development in the roots.     

Biocontrol traits of two Paenibacillus polymyxa strains SQR‐21 and WR‐2 in response to fusaric acid,a phytotoxin produced by Fusarium species

The aim of the present study was to investigate the effects of the phytotoxin fusaric acid (FA) on the biocontrol traits of two biocontrol strains Paenibacillus polymyxa WR‐2 and SQR‐21. The results showed that the growth of both WR‐2 and SQR‐21 decreased with increasing FA concentration, and at 70 and 80 μg mL^?1 FA, respectively, the strains were unable to grow. The biocontrol traits of both strains were negatively affected by FA concentration higher than 2·5 μg mL^?1. However, at 2·5 μg mL^?1 FA, biofilm formation and root colonization were not affected and there was even a positive effect on the production of spores and hydrolytic enzymes (protease and β‐l,3‐glucanase). The production of fusaricidin‐type antifungal compounds was increased with an increase in FA concentration up to 50 and 60 μg mL^?1 for WR‐2 and SQR‐21, respectively. The production of antifungal volatile organic compounds by WR‐2 and SQR‐21 was increased only at 2·5 μg mL^?1 FA. The effect of FA on the overall metabolic activity of WR‐2 and SQR‐21 was also determined. This study will help to understand the response of P. polymyxa strains to FA and will help to improve their biocontrol efficiency.     

Detection of Plasmodiophora Brassicae By PCR in Naturally Infested Soils

A nested polymerase chain reaction (PCR) method was developed for detection of DNA from Plasmodiophora brassicae in naturally infested field soil samples. The target sequences 389 bp and 507 bp were amplified from Swedish populations of P. brassicae. The protocols described enabled detection of DNA in various soil classes with an inoculum level of P. brassicae corresponding to a disease severity index (DSI) higher than 21 in a greenhouse bioassay. Three sequenced Swedish P. brassicae isolates had identical sequence in the 18S/ITS 1 region, but differed by a few nucleotides from an isolate sequenced in the UK. The results indicate that the primers used are general for P. brassicae, and consequently the nested PCR assay has a potential to be developed as a routine diagnostic test.     

Pathogenicity and host‐parasite relationships of the root‐knot nematode Meloidogyne incognita on celery

Pathogenicity and host‐parasite relationships in root‐knot disease of celery (Apium graveolens ) caused by Meloidogyne incognita race 1 were studied under glasshouse conditions. Naturally and artificially infected celery cv. D’elne plants showed severe yellowing and stunting, with heavily deformed and damaged root systems. Nematode‐induced mature galls were spherical and/or ellipsoidal and commonly contained more than one female, males and egg masses with eggs. Feeding sites were characterized by the development of giant cells that contained granular cytoplasm and many hypertrophied nuclei. The cytoplasm of giant cells was aggregated along their thickened cell walls and consequently the vascular tissues within galls appeared disrupted and disorganized. The relationship between initial nematode population density (Pi) and growth of celery plants was tested in glasshouse experiments with inoculum levels that varied from 0 to 512 eggs and second‐stage juveniles (J₂) mL^?1 soil. Seinhorst's model y = m + (1 – m)z^P–T was fitted to height and top fresh weight data of the inoculated and control plants. The tolerance limit with respect to plant height and fresh top weight of celery to M. incognita race 1 was estimated as 0·15 eggs and J₂ mL^?1 soil. The minimum relative values (m) for plant height and top fresh weight were 0·37 and 0·35, respectively, at Pi ≥ 16 eggs and J₂ mL^?1 soil. The maximum nematode reproduction rate (Pf/Pi) was 407·6 at an initial population density (Pi) of 4 eggs and J₂ mL^?1 soil.     

Studies into primary and secondary infection processes by Plasmodiophora brassicae on canola

The early stages of infection of canola roots by the clubroot pathogen Plasmodiophora brassicae were investigated. Inoculation with 1 × 10⁵ resting spores mL^?1 resulted in primary (root hair) infection at 12 h after inoculation (hai). Secondary (cortical) infection began to be observed at 72 hai. When inoculated onto plants at a concentration of 1 × 10⁴ mL^?1, secondary zoospores produced primary infections similar to those obtained with resting spores at a concentration of 1 × 10⁵ mL^?1. Secondary zoospores caused secondary infections earlier than resting spores. When the plants were inoculated with 1 × 10⁷ resting spores mL^?1, 2 days after being challenged with 1 × 10⁴ or 1 × 10⁵ resting spores mL^?1, secondary infections were observed on the very next day, which was earlier than the secondary infections resulting from inoculation with 1 × 10⁷ resting spores mL^?1 alone and more severe than those produced by inoculation with 1 × 10⁴ or 1 × 10⁵ resting spores mL^?1 alone. Compared with the single inoculations, secondary infections on plants that had received both inoculations remained at higher levels throughout a 7‐day time course. These data indicate that primary zoospores can directly cause secondary infection when the host is under primary infection, helping to understand the relationship and relative importance of the two infection stages of P. brassicae.     

Development of a quantitative real‐time PCR assay for Phytophthora infestans and its applicability to leaf,tuber and soil samples

A sensitive real‐time polymerase chain reaction (PCR) assay was developed for the quantification of Phytophthora infestans, the cause of foliar and tuber late blight in potato. A primer pair (PinfTQF/PinfTQR) and a fluorogenic probe (PinfTQPR) were designed to perform a quantitative assay for the detection of P. infestans in leaves, tubers and soils. The assay was shown to be specific to P. infestans and the very closely taxonomically related non‐potato pathogen species P. mirabilis, P. phaseoli and P. ipomoea, but did not detect the potato pathogens P. erythroseptica and P. nicotianae. The assay was able to reliably detect P. infestans DNA at 100 fg per reaction and was effective in quantifying P. infestans in infected leaf tissue from 24 h after inoculation and also in infected symptomless tubers and diseased tubers. Attempts to detect oospores of P. infestans in naturally and artificially infested soil samples are described and compared with baiting tests and previous literature. It was not possible to detect oospores in soil samples due to problems with DNA extraction from the oospores themselves. However, the assay was shown to detect even very low levels of asexual inoculum (sporangia and mycelium) in soil. This work assembles all the necessary features of a quantitative P. infestans assay, which have previously been somewhat disparate: the sensitivity, specificity and quantitation are fully validated, the assay is shown to work in common applications in leaf and tuber tissue and the problems with P. infestans oospore detection are explored and tested experimentally.