Development of PCR-based detection methods for the quarantine phytopathogen Synchytrium endobioticum, causal agent of wart disease

P.H.J.F. van den Boogert, M.P.E. van Gent-Pelzer, P.J.M. Bonants, S.H. de Boer, J.G.N. Wander, C.A. Lévesque, G.C.M. van Leeuwen, R.P. Baayen

    Research output: Contribution to journalArticleAcademicpeer-review

    22 Citations (Scopus)

    Abstract

    Abstract 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
    Original languageEnglish
    Pages (from-to)47-57
    JournalEuropean Journal of Plant Pathology
    Volume113
    Issue number1
    DOIs
    Publication statusPublished - 2005

    Fingerprint

    Synchytrium endobioticum
    warts
    quarantine
    plant pathogens
    DNA
    sporangia
    centrifugation
    assays
    methodology
    quantitative polymerase chain reaction
    sieving
    extracts
    soil
    potatoes
    purification methods
    soil analysis
    Plantae
    plant protection
    reproducibility
    probes (equipment)

    Keywords

    • real-time pcr
    • spongospora-subterranea
    • resting sporangia
    • soil
    • tubers
    • quantification
    • primers
    • solani
    • dna

    Cite this

    van den Boogert, P.H.J.F. ; van Gent-Pelzer, M.P.E. ; Bonants, P.J.M. ; de Boer, S.H. ; Wander, J.G.N. ; Lévesque, C.A. ; van Leeuwen, G.C.M. ; Baayen, R.P. / Development of PCR-based detection methods for the quarantine phytopathogen Synchytrium endobioticum, causal agent of wart disease. In: European Journal of Plant Pathology. 2005 ; Vol. 113, No. 1. pp. 47-57.
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    abstract = "Abstract 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",
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    Development of PCR-based detection methods for the quarantine phytopathogen Synchytrium endobioticum, causal agent of wart disease. / van den Boogert, P.H.J.F.; van Gent-Pelzer, M.P.E.; Bonants, P.J.M.; de Boer, S.H.; Wander, J.G.N.; Lévesque, C.A.; van Leeuwen, G.C.M.; Baayen, R.P.

    In: European Journal of Plant Pathology, Vol. 113, No. 1, 2005, p. 47-57.

    Research output: Contribution to journalArticleAcademicpeer-review

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    AU - van Gent-Pelzer, M.P.E.

    AU - Bonants, P.J.M.

    AU - de Boer, S.H.

    AU - Wander, J.G.N.

    AU - Lévesque, C.A.

    AU - van Leeuwen, G.C.M.

    AU - Baayen, R.P.

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    AB - Abstract 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

    KW - real-time pcr

    KW - spongospora-subterranea

    KW - resting sporangia

    KW - soil

    KW - tubers

    KW - quantification

    KW - primers

    KW - solani

    KW - dna

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    DO - 10.1007/s10658-005-0297-x

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