A rapid method for direct detection of Polymyxa DNA in soil

Polymyxa spp. are vectors for a number of economically important soilborne plant viruses. The development of a technique to detect virus and vectors directly in soil would be useful for epidemiological studies and assessment of disease risk prior to planting. A rapid method was developed to extract and quantify Polymyxa spp. DNA from soils. DNA was extracted from three soils infested with Polymyxa betae and three infested with P. graminis using an EDTA lysis buffer in combination with a MagneSil™ DNA extraction kit and Kingfisher™ magnetic particle processor. Primers and probes designed to correspond to sequences within the internal transcribed spacer region 2 (ITS2) of ribosomal DNA enabled recovery and amplification of P. betae and P. graminis DNA using real-time PCR and TaqMan chemistry. For the P. graminis- infested soils, the purity of DNA obtained was sufficient to allow Polymyxa DNA to be amplified without dilution to remove inhibitors, but with P. betae- infested soils, amplification was only achieved if the DNA was diluted 1:10. Using TaqMan PCR, a standard curve was constructed from uninfested soil spiked with known numbers of P. betae cystosori; the quantity of P. betae inoculum from naturally infested soil was then extrapolated from the curve. This technique offers a sensitive method of extracting, detecting and quantifying Polymyxa spp. DNA in soil.     

Detection and quantification of Rhizoctonia cerealis in soil using real-time PCR

Rhizoctonia cerealis E.P. van der Hoeven (anamorph of Ceratobasidium cereale D.I. Murray and Burpee), which causes sharp eyespot in wheat, is a major soilborne fungal pathogen that severely impairs yield and quality of winter wheat in China. Because the pathogen is difficult to identify and quantify in soil using conventional methods, a rapid and reliable method is needed to detect and quantify the fungus. In this study, we developed an SYBR Green-based quantitative real-time polymerase chain reaction assay for specific, sensitive detection and quantification of R. cerealis in soil samples. Using a specific primer pair based on the β-tubulin gene of the fungal DNA sequence, we could specifically detect R. cerealis at quantities as low as 100?fg of purified pathogen DNA. Using the real-time PCR assays, we were able to quantify R. cerealis in artificially and naturally infested soil samples. This new technique to quantify R. cerealis is rapid and accurate and will be a useful tool for future studies of pathogenic R. cerealis.     

PCR-based assays to detect and quantify <Emphasis Type="Italic">Phomopsis sclerotioides</Emphasis> in plants and soil

We developed polymerase chain reaction (PCR) assays to detect and quantify Phomopsis sclerotioides, the causal agent of black root rot of cucurbits. We used internal transcribed spacers 1 and 2 of the ribosomal DNA (rDNA) from representative isolates to search for target sequences. Primer pairs were selected after testing against 40 fungal isolates including 13 Ph. sclerotioides isolates, 9 Phomopsis isolates other than Ph. sclerotioides, and 18 soilborne fungi that were either pathogenic or nonpathogenic to cucurbits. Conventional PCR assays with the primer pair of CPs-1 (forward) and CPs-2 (reverse) produced target DNA amplicons from all Ph. sclerotioides isolates but none of the other isolates tested. From soil and root samples collected from fields naturally infested with black root rot of cucumber and melon, the CPs-1/CPs-2 primer pair successfully amplified target DNA fragments in conventional PCR assays. Moreover, we applied the CPs-1/CPs-2 primer pair in a real-time PCR assay with SYBR Green I, and PCR-amplified products were successfully quantified by reference to a standard curve generated by adding known amounts of target DNA. Target Ph. sclerotioides DNA fragments were similarly detected in artificially inoculated roots of cucumber, melon, pumpkin, and watermelon, but quantities of Ph. sclerotioides DNA in their hypocotyls of the hosts varied as follows: melon ≥ cucumber ≥ watermelon > pumpkin. These results suggest that Ph. sclerotioides infection is not species-specific but the rate of infection may differ among host species.     

Real-time PCR for Quantification of Toxigenic Fusarium Species in Barley and Malt

A real-time PCR technique was applied for the quantification of trichothecene-producing Fusarium species (TMTRI assay) as well as the highly toxigenic Fusarium graminearum (TMFg12 assay) present in barley grain and malt. PCR results were compared to the amounts of trichothecenes detected in the samples to find out if the PCR assays can be used for trichothecene screening instead of expensive and laborious chemical analyses. DNA was extracted from ground kernels using a commercial DNA extraction kit and analysed in a LightCycler® system using specific primers and fluorogenic TaqMan probes. Both naturally and artificially contaminated grains were analysed. The TMTRI assay and the TMFg12 assay enabled the quantification of trichothecene-producing Fusarium DNA and F. graminearum DNA present in barley grain and malt samples, respectively. Both TaqMan assays were considered to be sensitive and reproducible. Linearity of the assays was 4–5 log units when pure Fusarium DNAs were tested. The amount of Fusarium DNA analysed with the TMTRI-trichothecene assay could be used for estimation of the deoxynivalenol (DON) content in barley grain. Furthermore, the TMFg12 assay for F. graminearum gave a good estimation of the DON content in north American barley and malt samples, whilst the correlation was poor among Finnish samples. DON content and the level of F. graminearum DNA were found to be naturally low in Finnish barleys.     

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.     

Bait twig method for soil detection of <Emphasis Type="Italic">Rosellinia necatrix</Emphasis>, causal agent of white root rot of Japanese pear and apple,at an early stage of tree infection

Mulberry twigs were inserted into the soil as bait to detect Rosellinia necatrix at an early stage of tree infection in the orchard. R. necatrix was frequently trapped on twigs near the trunk base at soil depths of 6–20 cm within 10–20 days in May–July, suggesting that the incubation period was dependent on soil temperature. Subsequently, we inserted twig in the soil around healthy-looking trees in naturally infested orchards. R. necatrix was trapped from 80.0% of Japanese pear and 75.0% of apple trees that later proved to be infected. This bait twig method facilitated quicker diagnosis of white root rot on Japanese pear and apple at early stages of infection and can be used to detect recurrence of the fungus after fungicide treatment.     

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.     

Comparison of PCR assays for detection and quantification of Phomopsis sclerotioides in plant and soil

We developed a real-time PCR assay using a TaqMan probe (TM-qPCR) for specific detection and quantification of Phomopsis sclerotioides, causal agent of black root rot of cucurbit crops. The design of the primer sets and hybridization probe was based on the internal transcribed spacer region of the ribosomal DNA. The TM-qPCR assay was compared with a conventional, standard PCR (sPCR) assay and on a quantitative real-time PCR (SG-qPCR) assay based on SYBR Green I. The TM-qPCR assay had a detection limit of ca. 0.4 fg of P. sclerotioides DNA, which was approximately 100 times more sensitive than the sPCR assay and almost equivalent to the SG-qPCR assay. The TM-qPCR and SG-qPCR assays both were able to detect various quantities of P. sclerotioides DNA from diseased plants and infested soils, including DNA levels that were not detectable by the sPCR assay. However, the TM-qPCR was advantageous for samples containing PCR-inhibiting substances because its multiplex real-time PCR function allows the adjustment of cycle threshold values with an internal control. Based on the high specificity and sensitivity required for analyzing DNA in natural samples, the newly developed TM-qPCR assay was the most reliable tool for rapidly detecting and quantifying P. sclerotioides in plant and soil samples.     

Real-time detection of <Emphasis Type="Italic">Phytophthora nicotianae</Emphasis> and <Emphasis Type="Italic">P. citrophthora</Emphasis>in citrus roots and soil

Two primers, specific for Phytophthora nicotianae (Pn6) and P. citrophthora (Pc2B), were modified to obtain Scorpion primers for real-time identification and detection of both pathogens in citrus nursery soils and roots. Multiplex PCR with dual-labelled fluorogenic probes allowed concurrent identification of both species ofPhytophthora among 150 fungal isolates, including 14 species of Phytophthora. Using P. nicotianaespecific primers a delayed and lower fluorescence increase was also obtained from P. cactorumDNA. However, in separate real-time amplifications, the aspecific increase of fluorescence from P. cactorum was avoided by increasing the annealing temperature. In multiplex PCR, with a series of 10-fold DNA dilutions, the detection limit was 10 pg l^-1 for P. nicotianaeand 100 pg l^–1 for P. citrophthora, whereas in separate reaction DNA up to 1 pg l^-1 was detected for both pathogens.Simple and rapid procedures for direct DNA extraction from soil and roots were utilised to yield DNA whose purity and quality was suitable for PCR assays. By combining these protocols with a double amplification (nested Scorpion-PCR) using primers Ph2-ITS4 amplifying DNA from the main Phytophthora species (first round) and PnB5-Pn6 Scorpion and Pc2B Scorpion-Pc7 (second round), it was possible to achieve real-time detection of P. nicotianaeand P. citrophthora from roots and soil. The degree of sensitivity was similar to that of traditional detection methods based on the use of selective media. The analyses of artificially and naturally infested soil showed a high and significant correlation between the concentration of pathogen propagules and the real-time PCR cycle threshold.     

Quantification of the rice false smut pathogen <Emphasis Type="Italic">Ustilaginoidea virens</Emphasis> from soil in Japan using real-time PCR

Ustilaginoidea virens is the causal agent of false smut disease of rice. In this study, we developed a real-time polymerase chain reaction (PCR) assay to clarify the relationship between false smut occurrence on rice and quantification of U. virens from soil in Japan. The method here described is sensitive, detecting less than 50 fg of pathogen DNA, and specific to the nuclear ribosomal DNA for U. virens when tested across 27 rice-pathogenic fungi and bacteria, 26 other fungi and bacteria and four plant species. As few as eight chlamydospores of U. virens per gram soil were detected when added to sterilized Gley and Ando soils. The real-time PCR assay for the soil samples was at least 100-fold more sensitive than the conventional and nested-PCR assays tested. By quantification of U. virens with real-time PCR using DNA extracted from naturally contaminated Gley soils and visual assessment of the disease in agricultural fields, a linear correlation between cycle threshold (C_T) values and the number of false smut balls was revealed. Therefore, this specific quantitative assay could be a useful tool for optimization of disease control strategies, and for studying the ecology of U. virens.     

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.     

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.     

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.     

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.     

Identification and Detection of Rosellinia Necatrix by Conventional and Real-time Scorpion-PCR

Several polymerase chain reaction (PCR) primers were designed from the internal transcribed spacer (ITS) regions of the rDNA genes of Rosellinia necatrix to develop a PCR-based identification method. Screening the primers against two isolates of R. necatrix and six other Rosellinia species resulted in the amplification of a single specific product from R. necatrix for most of the primer pairs. Two primer pairs (R2-R8 and R10-R7) confirmed their specificity when tested against 72 isolates of R. necatrix and 93 other fungi from different hosts and geographic areas. The R10 primer was modified to obtain a Scorpion primer for detecting a specific 112bp amplicon by fluorescence emitted from a fluorophore in a self-probing PCR assay. This assay specifically recognised the target sequence of R. necatrix over a large number of other fungal species. In conventional PCR, with primer pairs R2-R8 and R10-R7, 10-fold dilutions of R. necatrix DNA indicated a detection limit of 10pgul^-1 using a single set of primers and 10fgl^-1 in nested-PCR. For Scorpion-PCR, the detection limit was 1pgl^-1 and 1fgl^-1 in nested Scorpion-PCR, i.e. 10 times more sensitive than conventional PCR. A simple and rapid procedure for DNA extraction directly from soil was modified and developed to yield DNA of purity and quality suitable for PCR assays. Combining this protocol with the nested Scorpion-PCR procedure it has been possible to specifically detect R. necatrix from artificially inoculated soils in approximately 6h.     

Conventional PCR and real time quantitative PCR detection of <Emphasis Type="Italic">Phytophthora cryptogea</Emphasis> on <Emphasis Type="Italic">Gerbera jamesonii</Emphasis>

A conventional PCR and a SYBR Green real-time PCR assays for the detection and quantification of Phytophthora cryptogea, an economically important pathogen, have been developed and tested. A conventional primer set (Cryp1 and Cryp2) was designed from the Ypt1 gene of P. cryptogea. A 369 bp product was amplified on DNA from 17 isolates of P. cryptogea. No product was amplified on DNA from 34 other Phytophthora spp., water moulds, true fungi and bacteria. In addition, Cryp1/Cryp2 primers were successfully adapted to real-time PCR. The conventional PCR and real-time PCR assays were compared. The PCR was able to detect the pathogen on naturally infected gerbera plants and on symptomatic artificially infected plants collected 21 days after pathogen inoculation. The detection limit was 5 × 10³ P. cryptogea zoospores and 16 fg of DNA. Real-time PCR showed a detection limit 100 times lower (50 zoospores, 160 ag of DNA) and the possibility of detecting the pathogen in symptomless artificially infected plants and in the re-circulating nutrient solution of closed soilless cultivation systems.     

Simultaneous one-tube quantification of host and pathogen DNA with real-time polymerase chain reaction

ABSTRACT Phaeocryptopus gaeumannii is a widespread foliar parasite of Douglas-fir. Although normally innocuous, the fungus also causes the defoliating disease Swiss needle cast in heavily infected needles. The extent of P. gaeumannii colonization in Douglas-fir foliage was estimated with real-time quantitative polymerase chain reaction (PCR) using TaqMan chemistry. In order to derive a normalized expression of colonization, both pathogen and host DNA were simultaneously amplified but individually detected by species-specific primers and TaqMan probes labeled with different fluorescent dyes. Detection of host DNA additionally provided an endogenous reference that served as both an internal positive control and adjusted for variation introduced by sample-to-sample differences in DNA extraction and PCR efficiencies. The genes employed for designing the TaqMan probes and primers were beta-tubulin for the pathogen and a LEAFY/FLORICAULA-like gene involved in floral development for the tree host. Both probe/primer sets exhibited high precision and reproducibility over a linear range of 4 orders of magnitude. This eliminated the need to analyze samples in multiple dilutions when comparing lightly with heavily infected needles. Quantification of the fungus within needles was successful as early as 1 month after initial infection. Real-time PCR is the only method currently available to quantify P. gaeumannii colonization early in the first year of the colonization process.     

Disease risk assessment of sugar beet root rot using quantitative real-time PCR analysis of <Emphasis Type="Italic">Aphanomyces cochlioides</Emphasis> in naturally infested soil samples

Sugar beet root rot, caused by the oomycete Aphanomyces cochlioides, is a serious and economically important disease of sugar beets world-wide. Today, disease risk assessment consists of a time-consuming greenhouse bioassay using bait plants. In the present study, a real-time quantitative PCR (qPCR) assay for determination of A. cochlioides DNA in field-infested soil samples was developed and validated using the standard bioassay. The qPCR assay proved to be species-specific and was optimized to give high amplification efficiency suitable for target copy quantification. A high correlation (R² > 0.98, p < 0.001) with pathogen inoculum density was shown, demonstrating the suitability for monitoring soil samples. The limit of detection (LOD) was evaluated in several different soil types and varied between 1 and 50 oospores/g soil, depending on clay content. Soils with a high LOD were characterised as having a low clay content and high content of sand. Varying levels of the A. cochlioides target sequence were detected in 20 of the 61 naturally infested soil samples. Discrepancies between the bioassay and the qPCR assay were found in soils from low- and medium-risk fields. However, the qPCR diagnostic assay provides a potentially valuable new tool in disease risk assessment, enabling sugar beet growers to identify high-risk fields.     

Multiplex real-time PCR assays for the detection and identification of Heterobasidion species attacking conifers in Europe

Four species of the destructive forest pathogen Heterobasidion annosum sensu lato (s.l.) are present in Europe: H. annosum sensu stricto (s.s.), H. abietinum and H. parviporum are native species, while H. irregulare is a non-native invasive species currently reported only in Italy, yet recommended for regulation throughout Europe. In this study, real-time PCR detection tests were developed for each of the four species, which can be used simultaneously or individually thanks to probes labelled with species-specific fluorescent dyes. The different performance criteria of each assay were evaluated, and it was determined that they were theoretically capable of detecting amounts of DNA corresponding to 311, 29 and 29 cell nuclei in H. annosum s.s., H. irregulare and H. parviporum, respectively. The specificity of each assay was assessed with a wide set of strains. Real-time PCR tests successfully detected Heterobasidion species from 36 fruiting bodies taken from the forest, as well as from artificially inoculated or naturally infected wood samples. The multiplex real-time PCR assays developed in this study could have practical applications both in forest management and in phytosanitary monitoring.     

A new large scale soil DNA extraction procedure and real-time PCR assay for the detection of Sclerotium cepivorum in soil

A real-time PCR assay specific for Sclerotium cepivorum, the causal agent of white rot in onions, was developed for use with a new DNA extraction method capable of processing up to 1?kg of soil in weight. The assay was specific for S. cepivorum when tested against 24 isolates representative of 14 closely related species and other pathogens of onion. The assay was highly sensitive when used with soil DNA extracted using the new DNA extraction procedure. In three different field soils tested, a good relationship between cycle threshold (Ct) and number of sclerotia was observed (R2?=?0.89). Twenty-nine soil samples from onion and leek crops were obtained and the pathogen was detected in four samples. All four positive samples were associated with current or past outbreaks of white rot of onion. Additional assays were also used on the 29 field soil samples, Botrytis aclada and Rhizoctonia solani AG8 were also detected, in one soil sample each. Rhizoctonia solani AG2-1 was more widespread and was detected in eight different soil samples. The method is therefore suitable to quantify levels of S. cepivorum in soil samples, with the added advantage that the technique allows other soil pathogens of interest to be assayed from the same DNA sample. The bulk soil DNA extraction method described here has the potential to be used to detect soil-borne pests and pathogens for other crops in a wide range of soil types.