[show abstract][hide abstract] ABSTRACT: 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 liter-ature. It was not possible to detect oospores in soil samples due to problems with DNA extraction from the oospores them-selves. 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 dis-parate: 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.
[show abstract][hide abstract] ABSTRACT: Skin spot disease of potato caused by the pathogen Polyscytalum pustulans is likely to become more important with the withdrawal of 2-aminobutane as a fungicide, and new methods of control will need to be found. As part of a disease control strategy, it will be necessary to study the disease in more detail, to utilize host resistance and to identify stocks where problems are likely to arise. Existing methods for the detection and quantification of P. pustulans are time-consuming and require specific expertise. Real-time PCR assays have been developed for many pathogens of potato and have subsequently been used as tools for the study of the epidemiology and control of disease. The development of a real-time PCR assay for the detection and quantification of P. pustulans is described. The specificity of the assay was demonstrated and detection was shown to be reliable at levels as low as 20–250 fg/μl DNA, (equivalent to 60–680 pg DNA/g) in soil and on symptomless tubers at attogram (ag) levels. These values are in line with previously developed tests.
[show abstract][hide abstract] ABSTRACT: A 3-year survey was undertaken to establish the relative frequency of different Fusarium spp. present as inoculum on potato tubers collected from four regions of Great Britain. A total of 219 samples (comprising 10 950 tubers) were collected from the 2000, 2001 and 2002 crops and processed to recover dry rot-producing isolates. In total, 228 isolates of Fusarium spp. were recovered. Most (94·7%) of these isolates were attributed to one of four Fusarium species: F. coeruleum, F. avenaceum, F. culmorum and F. sambucinum (formerly F. sulphureum). The incidence of the combined Fusarium spp. increased the further south the crops had been grown. Fusarium coeruleum was the most commonly isolated species in each survey year, comprising 37 to 52% of the total Fusarium species. Selected isolates of each species were evaluated for their ability to produce rots in potato tubers. Fusarium sambucinum was a more aggressive pathogen than the other Fusarium species in eight out of 10 cultivars. Fusarium avenaceum and F. culmorum were relatively weaker pathogens. However, these species were aggressive on some cultivars, notably Hermes. The selected isolates were also assessed for their sensitivity to the fungicides thiabendazole and imazalil. Using in vitro tests, 65% of F. sambucinum isolates were resistant to thiabendazole and 7% of F. avenaceum isolates were resistant to imazalil. Tubers treated with imazalil yielded a higher proportion of isolates of F. avenaceum than those that were untreated. Similarly, a higher proportion of F. sambucinum isolates were recovered from tubers treated with thiabendazole than from those that were not treated.
[show abstract][hide abstract] ABSTRACT: The diseases pink rot, watery wound rot and gangrene are important storage rot diseases of potato associated predominantly with Phytophthora erythroseptica (P.), Pythium ultimum (Py.) and Phoma exigua (Phoma) var. foveata respectively. Reliable molecular-based diagnostic tests are required that will not only allow unequivocal identification of symptoms but will further advance epidemiological studies of these potato diseases to increase our understanding and contribute to more effective management and control strategies to the potato industry. Primers and probes were designed in specific regions of the internal transcribed spacer (ITS) regions to develop conventional and real-time quantitative polymerase chain reaction (PCR) assays able to detect all possible fungal and oomycete pathogens causing pink rot, watery wound rot and gangrene. The specificity of each diagnostic assay was rigorously tested with over 500 fungal/oomycete plant pathogen isolates from potato and reference culture collections, and both conventional and real-time PCR methods produced similar results. In terms of sensitivity, the detection limits for real-time PCR went below ag DNA levels compared with pg DNA levels with conventional PCR. The real-time PCR assays developed to detect Phoma foveata and Py. ultimum on tubers were suitable for the comparative Ct method (ΔΔCt) of quantification using the cytochrome oxidase gene of potato as a normalizer assay; an advantage as the need for a standard curve is eliminated. Each assay detected Phoma species (var. foveata or exigua) from naturally infected tubers showing symptoms of gangrene, and P. erythroseptica or Py. ultimum were also detected following inoculation of Russet Burbank tubers. Each diagnostic assay developed could reliably detect and distinguish between the pink rot, watery wound rot and gangrene-causing potato pathogens.
[show abstract][hide abstract] ABSTRACT: Aims: To evaluate the virulence gene nec1 as a reliable marker for the detection of pathogenic Streptomyces species on potato tubers and in soil samples using conventional and real-time quantitative PCR assays.Methods and Results: Two pairs of conventional primers (outer and nested) and one set of primers/probe for use in real-time PCR were designed to detect the necrogenic protein encoding nec1 gene of Streptomyces scabiei strain ATCC 49173T. The conventional PCR primers were also incorporated into a multiplex PCR assay to simultaneously detect the nec1 gene in conjunction with the potato pathogens Helminthosporium solani and Colletotrichum coccodes. The specificity of each PCR assay was confirmed by testing 32 pathogenic and nonpathogenic reference strains of Streptomyces representing 12 different species and 74 uncharacterized streptomycete strains isolated from diseased tubers. A clear correlation between pathogenicity and the detection of nec1 by PCR was demonstrated. The sensitivity and specificity of both the conventional and real-time PCR assays allowed the detection of nec1 on potato tubers in the absence of visible symptoms of common scab, and in seeded soil down to a level equivalent to three S. scabiei spores per gram soil.Conclusions: Reliable and quantitative PCR techniques were developed in this study for the specific detection of the virulence gene nec1 of pathogenic Streptomyces species on potato tubers and in soil samples, and the data demonstrated a clear correlation between pathogenicity in Streptomyces species and the presence of the nec1 gene.Significance and Impact of the Study: Together with the DNA extraction protocols, these diagnostic methods will allow a rapid and accurate assessment of tuber and soil contamination by pathogenic Streptomyces species.
[show abstract][hide abstract] ABSTRACT: ABSTRACT Specific and sensitive quantitative diagnostics, based on real-time (TaqMan) polymerase chain reaction (PCR) and PCR enzyme-linked immunosorbent assay, were developed to detect dry-rot-causing Fusarium spp. (F. avenaceum, F. coeruleum, F. culmorum, and F. sulphureum). Each assay detected Fusarium spp. on potato seed stocks with equal efficiency. Four potato stocks, sampled over two seed generations from Scottish stores, were contaminated with F. avenaceum, F. sulphureum, F. culmorum, F. coeruleum or a combination of species, and there was a general trend towards increased Fusarium spp. contamination in the second generation of seed sampled. F. sulphureum and F. coeruleum caused significantly (P < 0.05) more disease in storage than the other species when disease-free tubers of potato cvs. Spunta and Morene were inoculated at a range of inoculum concentrations (0, 10(4), 10(5), and 10(6) conidia/ml). Increased DNA levels were correlated with increased disease severity between 8 and 12 weeks of storage. The threshold inoculum levels resulting in significant disease development on both cultivars were estimated to be 10(4) conidia/ml for F. sulphureum and 10(5) conidia/ml for F. coeruleum. To study the effect of soil infestation and harvest date on disease incidence, seed tubers of cvs. Morene and Spunta were planted in a field plot artificially infested with the four Fusarium spp. F. culmorum and F. sulphureum were detected in soil taken from these plots at harvest, and F. sulphureum DNA levels increased significantly (P < 0.05) at the final harvest. All four Fusarium spp. were detected in progeny tubers. There was a trend toward higher levels of F. culmorum detected in progeny tubers at the earliest harvest date, and higher levels of F. sulphureum at the final harvest. The use of diagnostic assays to detect fungal storage rot pathogens and implications for disease control strategies are discussed.
[show abstract][hide abstract] ABSTRACT: A specific and sensitive PCR assay was developed for the detection and identification of Rhizoctonia solani AG-3, the main causal pathogen of stem canker and black scurf of potato. A conventional primer set (Rs1F2 and Rs2R1) was designed from the nuclear ribosomal internal transcribed spacer (ITS1 and ITS2) regions of R. solani. Following PCR amplification, a 0·5-kb product was amplified from DNA of all isolates of AG-3 using primers Rs1F2 and Rs2R1. No product was amplified when DNA from isolates belonging to a range of other R. solani anastomosis groups or from a selection of other potato pathogens was tested, confirming the specificity of the primers for AG-3 only. Rhizoctonia solani AG-3 was also detected in potato tissue with varying black scurf severity, and in soil inoculated with sclerotia of R. solani to a minimum detection level of 5 × 10−4 g sclerotia/g soil. In addition, specific primers RsTqF1 (based on the Rs1F2 sequence) and RsTqR1, and a TaqMan™ fluorogenic probe RQP1, were designed to perform real-time quantitative (TaqMan) PCR. The conventional PCR and real-time PCR assays were compared and combined with direct DNA extraction from soil and a seed-baiting method to determine the most reliable method for the detection and quantification of AG-3 in both artificially inoculated field soil and naturally infested soils. It was shown that direct DNA extractions from soil could be problematic, although AG-3 was detectable using this method combined with the real-time PCR assay. The amplification of Rhizoctonia solani by seed baiting increased the sensitivity of the assay compared with direct extraction of DNA from the soil, and AG-3 was detectable in artificially inoculated and naturally infested soils when seed baiting was combined with either the conventional PCR or the real-time PCR assay. The potential for using these rapid and quantitative AG-3-specific assays to address epidemiological questions and as tools for decision-making in disease management is discussed.
[show abstract][hide abstract] ABSTRACT: 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.
[show abstract][hide abstract] ABSTRACT: We have developed polymerase chain reaction (PCR)-based diagnostic assays for fungal and bacterial diseases of potato together with rapid methods of DNA extraction and purification from soil and plant samples. Pathogen detection and quantification is a crucial step in the control of crop diseases. PCR primers were designed to unique sequences for specific detection of the potato blemish complex of pathogens: Spongospora subterranea (powdery scab), Streptomyces scabies (common scab), Helminthosporium solani (silver scurf), Rhizoctonia solani AG-3 (black scurf) and Colletotrichum coccodes (black dot). A multiplex PCR test was designed with a high level of sensitivity to detect H. solani, C. coccodes, and S. scabies simultaneously in one reaction, and individual quantitative PCR assays were also developed to detect these pathogens and S. subterranea in soil and tubers. PCR-based detection systems for the major storage-rot pathogens of potato (Fusarium spp., Phoma foveata, Pythium ultimum and Phytophthora erythroseptica) are also under development. The potential of these techniques to detect and monitor these pathogen populations in soil and potato tubers for epidemiological studies is discussed.
[show abstract][hide abstract] ABSTRACT: A simple and rapid procedure for direct extraction of DNA from soils was developed to yield DNA of a high purity and quality suitable for amplification using the polymerase chain reaction (PCR). Co-extracted humic material from soil was a major contaminant of DNA and methods were devised to overcome this problem. Oligonucleotide PCR primers were designed to detect and monitor a genetically-modified (GM) Rhizobium leguminosarum bv. viciae strain RSM2004 (marked with Tn5) which had become established in Rothamsted field soils. The key steps of the procedure were alkaline-SDS buffer assisted lysis of indigenous soil bacteria in a bead-beater and the purification of extracted DNA by separate PVPP and Sephadex G-75 spin-column chromatography. The mean yield from Rothamsted soil was 25±1.7 μg crude DNA g−1 wet soil (i.e. 20 μg g−1 dry soil), sheared to fragment sizes of about 22–25 kb. The recovered DNA was easier to purify and of a higher quality, as verified by PCR amplification of a 442 bp target sequence of Tn5, than DNA extracted by a hot-SDS lysis method. The detection limit was demonstrated to be one culturable cell of RSM2004 (i.e. a single copy of Tn5) 10 mg−1 soil against a background of 107 diverse non-target bacteria.
[show abstract][hide abstract] ABSTRACT: Monitoring genetically modified (GM) bacterial inoculants after field release using conventional culture methods can be difficult. An alternative is the detection of marker genes in DNA extracted directly from soil, using specific oligonucleotide primers with the polymerase chain reaction (PCR). The PCR was used to monitor survival of two GM Rhizobium leguminosarum bv. viciae inoculants after release in the field at Rothamsted. One strain, RSM2004, is marked by insertion of transposon Tn5; the second strain, CT0370, released at the same site, is modified by chromosomal integration of a single copy of the gene from E. coli conferring GUS activity. Both GM strain provide a realistic case study for the development of PCR-based detection techniques. Specific primers were developed to amplify regions of the Tn5 and GUS genetic markers using PCR and conditions optimized for each primer set to routinely detect a signal from 10 fg of purified template DNA, the equivalent of one cell per reaction. Procedures to improve the sensitivity of detection are described, to detect fewer than 50 cells g-1 soil in soil-extracted DNA.
Journal of Applied Microbiology 07/1998; 84(6):1025-34. · 2.20 Impact Factor
[show abstract][hide abstract] ABSTRACT: A simple and rapid procedure for direct extraction of DNA from soils was developed to yield DNA of a high purity and quality suitable for amplification using the polymerase chain reaction (PCR). Go-extracted humic material from soil was a major contaminant of DNA and methods were devised to overcome this problem. Oligonucleotide PCR primers were designed to detect and monitor a genetically-modified (GM) Rhizobium leguminosarum by, vicine strain RSM2004 (marked with Tn5) which had become established in Rothamsted field soils. The key steps of the procedure were alkaline-SDS buffer assisted lysis of indigenous soil bacteria in a bead-beater and the purification of extracted DNA by separate PVPP and Sephadex G-75 spin-column chromatography. The mean yield from Rothamsted soil was 25 +/- 1.7 mu g crude DNA g(-1) wet soil (i.e. 20 mu g g(-1) dry soil), sheared to fragment sizes of about 22-25 kb. The recovered DNA was easier to purify and of a higher quality, as verified by PCR amplification of a 442 bp target sequence of Tn5, than DNA extracted by a hot-SDS lysis method. The detection limit was demonstrated to be one culturable cell of RSM2004 (i.e. a single copy of Tn5) 10 mg(-1) soil against a background of 10(7) diverse non-target bacteria. (C) 1998 Elsevier Science Ltd. All rights reserved.
Soil Biology and Biochemistry 01/1998; 30(8-9):983-993. · 3.65 Impact Factor