Skip to main content
SpringerLink
Log in

The development of a validated real-time (TaqMan) PCR for detection of Stagonosporopsis andigena and S. crystalliniformis in infected leaves of potato and tomato

  • Original Research
  • Published:
European Journal of Plant Pathology Aims and scope Submit manuscript

Abstract

Stagonosporopsis andigena and S. crystalliniformis are serious foliage pathogens on potato (Solanum tuberosum) and tomato (Solanum lycopersicum). As both species have been recorded only in the Andes area, S. andigena is listed as an A1 quarantine organism in Europe. The actin region of isolates of Stagonosporopsis and allied species of Boeremia, Didymella, Peyronellaea and Phoma was amplified using generic primers. DNA sequence differences of the actin gene were utilised to develop species-specific real-time (TaqMan) PCR assays for the detection of S. andigena and S. crystalliniformis in leaves of potato or tomato. The specificity of the TaqMan PCR assays was determined on genomic DNA extracted from two S. andigena and two S. crystalliniformis isolates and 16 selected isolates of Stagonosporopsis, Phoma and Boeremia, which are the closest relatives. The validation of the methods developed included the DNA extraction and the TaqMan PCR assays. The performance criteria specificity, analytical sensitivity, reproducibility, repeatability and robustness of the TaqMan PCR assays demonstrated the reliability of both methods for the detection of S. andigena and S. crystalliniformis in leaf material. The TaqMan PCR assays were tested on symptomatic leaves of potato and tomato that were obtained after artificial inoculation of detached leaves with both pathogens under quarantine conditions. In the artificial inoculation experiments both S. andigena and S. crystalliniformis caused leaf infections on potato and tomato.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1

Similar content being viewed by others

References

  • Arzanlou, M., Kema, G. H. J., Waalwijk, C., Carlier, J., De Vries, I., Guzman, M., Vargas, M. A., Helder, J., & Crous, P. W. (2009). Molecular diagnostics in the Mycosphaerella leaf spot disease complex of banana and for Radopholus similis. Acta Horticulturae, 828, 237–244.

    CAS  Google Scholar 

  • Aveskamp, M. M., De Gruyter, J., & Crous, P. W. (2008). Biology and systematics of the genus Phoma: a complex genus of major quarantine significance. Fungal Diversity, 31, 1–18.

    Google Scholar 

  • Aveskamp, M. M., Verkley, G. J. M., De Gruyter, J., Murace, M. A., Perelló, A., Woudenberg, J. H. C., Groenewald, J. Z., & Crous, P. W. (2009a). DNA phylogeny reveals polyphyly of Phoma section Peyronellaea and multiple taxonomic novelties. Mycologia, 101, 363–382.

    Article  CAS  Google Scholar 

  • Aveskamp, M. M., Woudenberg, J. H. C., De Gruyter, J., Turco, E., Groenewald, J. Z., & Crous, P. W. (2009b). Development of taxon-specific SCAR markers based on actin sequences and DAF: a case study in the Phoma exigua species complex. Molecular Plant Pathology, 10, 403–414.

    Article  CAS  Google Scholar 

  • Aveskamp, M. M., De Gruyter, J., Woudenberg, J. H. C., Verkley, G. J. M., & Crous, P. W. (2010). Highlights of the Didymellaceae: a polyphasic approach to characterise Phoma and related pleosporalean genera. Studies in Mycology, 65, 1–60.

    Article  PubMed  CAS  Google Scholar 

  • Balmas, V., Scherm, B., Ghignone, S., Salem, A. O. M., Cacciola, S. O., & Migheli, Q. (2005). Characterisation of Phoma tracheiphila by RAPD-PCR, microsatellite-primed PCR and ITS rDNA sequencing and development of specific primers for in planta PCR detection. European Journal of Plant Pathology, 111, 235–247.

    Article  CAS  Google Scholar 

  • Boerema, G. H., De Gruyter, J., & Noordeloos, M. E. (1995). New names in Phoma. Persoonia, 16, 131.

    Google Scholar 

  • Boerema, G. H., De Gruyter, J., & Noordeloos, M. E. (1997). Contributions towards a monograph of Phoma (Coelomycetes)—IV. Section Heterospora: Taxa with large sized conidial dimorphs, in vivo sometimes as Stagonosporopsis synanamorphs. Persoonia, 16, 335–371.

    Google Scholar 

  • Boerema, G. H., De Gruyter, J., Noordeloos, M. E., & Hamers, M. E. C. (2004). Phoma identification manual. Differentiation of specific and infra-specific taxa in culture. Wallingford: CABI Publishing.

    Book  Google Scholar 

  • Carbone, I., & Kohn, L. M. (1999). A method for designing primer sets for speciation studies in filamentous ascomycetes. Mycologia, 91, 553–556.

    Article  CAS  Google Scholar 

  • Cline, E. (2011). Phoma andigena. Systematic Mycology and Microbiology Laboratory, ARS, USDA. Retrieved July 1, 2011, from http://nt.ars-grin.gov/sbmlweb/fungi/index.cfm.

  • Crous, P. W., Verkley, G. J. M., Groenewald, J. Z., & Samson, R. A. (2009). Fungal biodiversity. CBS Laboratory Manual Series. Utrecht: Centraalbureau voor Schimmelcultures.

    Google Scholar 

  • Cullen, D. W., Toth, I. K., Boonham, N., Walsh, K., Barker, I., & Lees, A. K. (2007). Development and validation of conventional and quantitative polymerase chain reaction assays for the detection of storage rot potato pathogens, Phytophthora erythroseptica, Pythium ultimum, Phoma foveata. Journal of Phytopathology, 155, 309–315.

    Article  CAS  Google Scholar 

  • De Gruyter, J., Aveskamp, M. M., Woudenberg, J. H. C., Verkley, G. J. M., Groenewald, J. Z., & Crous, P. W. (2009). Molecular phylogeny of Phoma and allied anamorph genera: towards a reclassification of the Phoma complex. Mycological Research, 113, 508–519.

    Article  PubMed  Google Scholar 

  • De Gruyter, J., Woudenberg, J. H. C., Aveskamp, M. M., Verkley, G. J. M., Groenewald, J. Z., & Crous, P. W. (2010). Systematic reappraisal of species in Phoma section Paraphoma, Pyrenochaeta and Pleurophoma. Mycologia, 102, 1066–1081.

    Article  PubMed  Google Scholar 

  • Demontis, M. A., Cacciola, S. O., Orrù, M., Balmas, V., Chessa, V., Maserti, B. E., Mascia, L., Raudino, F., di San, M., Lio, G., & Migheli, Q. (2008). Development of real-time PCR systems based on SYBR® Green I and TaqMan® technologies for specific quantitative detection of Phoma tracheiphila in infected Citrus. European Journal of Plant Pathology, 120, 339–351.

    Article  CAS  Google Scholar 

  • Dutch guideline for the validation of detection and identification methods for plant pathogens and pests. (2010) Retrieved August 31, 2010, from http://www.naktuinbouw.nl/sites/naktuinbouw.eu/files/Dutch%20Validation%20Guideline%20v1.0.pdf.

  • Huelsenbeck, J. P., & Ronqvist, F. (2001). MRBAYES: Bayesian inference of Phylogenic trees. Bioinformatics, 17, 754–755.

    Article  PubMed  CAS  Google Scholar 

  • Katoh, K., Asimenos, G., & Toh, H. (2009). Multiple alignment of DNA sequences with MAFFT. In D. Posada (Ed.), Bioinformatics for DNA sequence analysis. Methods in Molecular Biology, 537, 39–64.

  • Koch, C. A., & Utkhede, R. S. (2004). Development of a multiplex classical polymerase chain reaction technique for detection of Didymella bryoniae in infected cucumber tissues and greenhouse air samples. Canadian Journal of Plant Pathology, 26, 291–298.

    Article  CAS  Google Scholar 

  • Lartey, R. T., Weiland, J. J., Caesar-TonThat, T., & Bucklin-Comiskey, S. (2003). A PCR protocol for rapid detection of Cercospora beticola in sugarbeet tissues. Journal of Sugar Beet Research, 40, 1–10.

    Article  Google Scholar 

  • Licciardella, G., Grasso, F. M., Bella, P., Cirvilleri, G., Grimaldi, V., & Catara, V. (2006). Identification and detection of Phoma tracheiphila, causal agent of Citrus Mal Secco disease, by real-time polymerase chain reaction. Plant Disease, 90, 1523–1530.

    Article  Google Scholar 

  • Loerakker, W. M., Navarro, R., Lobo, M., & Turkensteen, L. J. (1986). Phoma andina var. crystalliniformis var. nov., un patógeno nuevo del tomate y de la papa en los Andes. Fitopatología, 21, 99–102.

    Google Scholar 

  • MacDonald, J. E., White, G. P., & Coté, M. J. (2000). Differentiation of Phoma foveata from P. exigua using a RAPD generated PCR-RFLP marker. European Journal of Plant Pathology, 106, 67–75.

    Article  CAS  Google Scholar 

  • Mumford, R. A., Skelton, A. L., Boonham, N., Postuma, K. I., Kirby, M. J., & Adams, A. N. (2004). The improved detection of Strawberry Crinkle Virus using Real-Time RT-PCR (Taqman®). In R. R. Martin (Ed.), ISHS Xth international symposium on small fruit virus diseases. Valencia, Spain, Acta Horticulturae, 656, 81–86.

  • Navarro, R. A., & Puerta, O. D. (1981). Presencia de Phoma sp. en tomate (Lycopersicon esculentum). Ascolfi Informa, 7, 32.

    Google Scholar 

  • Noordeloos, M. E., De Gruyter, J., Van Eijk, G. W., & Roeijmans, H. J. (1993). Production of dendritic crystals in pure cultures of Phoma and Ascochyta and its value as a taxonomic character relative to morphology, pathology and cultural characteristics. Mycological Research, 97, 1343–1350.

    Article  Google Scholar 

  • OEPP/EPPO. (1984). Data sheets on quarantine organisms No. 141, Phoma andina. Bulletin OEPP/EPPO Bulletin, 14, 45–48.

    Google Scholar 

  • OEPP/EPPO. (2010). PM 7/98 (1): specific requirements for laboratories preparing accreditation for a plant pest diagnostic activity. Bulletin OEPP/EPPO Bulletin, 40, 5–22.

    Google Scholar 

  • Otazú, V., Boerema, G. H., Mooi, J. C., & Salas, B. (1979). Possible geographical origin of Phoma exigua var. foveata, the principal causal organism of potato gangrene. Potato Research, 22, 333–338.

    Article  Google Scholar 

  • Page, R. D. M. (1996). Treeview: an application to display phylogenetic trees on personal computers. Computer Application in the Biosciences, 12, 357–358.

    CAS  Google Scholar 

  • Somai, B. M., Keinath, A. P., & Dean, R. A. (2002). Development of PCR-ELISA for detection and differentiation of Didymella bryoniae from related Phoma species. Plant Disease, 86, 710–716.

    Article  CAS  Google Scholar 

  • Turkensteen, L. J. (1978). Tizón foliar de la papa en el Perú. I, Especies de Phoma asociadas. Fitopatología, 13, 67–69.

    Google Scholar 

  • Turkensteen, L. J., & Pinedo, H. M. (1982). Didymella bryoniae como patógeno de la papa en las regiones Cálidas del Perú. Fitopatología, 17, 54–55.

    Google Scholar 

  • Weller, S. A., Elphinstone, J. G., Smith, N. C., Boonham, N., & Stead, D. E. (2000). Detection of Ralstonia solanacearum strains with a quantitative, multiplex, real-time, fluorogenic PCR (TaqMan) assay. Applied Environmental Microbiology, 66, 2853–2558.

    Article  CAS  Google Scholar 

  • White, T. J., Bruns, T., Lee, S., & Taylor, J. (1990). Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In M. A. Innis, D. H. Gelfand, J. J. Sninsky, & T. J. White (Eds.), PCR protocols: a guide to methods and applications (pp. 315–322). San Diego: Academic.

    Google Scholar 

Download references

Acknowledgments

The Dutch Ministry of Economics, Innovation and Agriculture has supported this research through an endowment of the FES program ‘Reinforcement Infrastructure Plant Health’.

Author information

Authors and Affiliations

  1. CBS-KNAW Fungal Biodiversity Centre, P.O. Box 85167, 3508 AD, Utrecht, The Netherlands

    Johannes de Gruyter, Joyce H. C. Woudenberg & Pedro W. Crous

  2. National Reference Centre, National Plant Protection Organization, P.O. Box 9102, 6700 HC, Wageningen, The Netherlands

    Johannes de Gruyter & Patricia C. J. van Rijswick

  3. Plant Research International BV, P.O. Box 16, 6700 AA, Wageningen, The Netherlands

    Marga P. E. van Gent-Pelzer & Peter J. M. Bonants

  4. Naktuinbouw, P.O. Box 40, 2370 AA, Roelofarendsveen, The Netherlands

    Ellis T. M. Meekes

  5. Wageningen University and Research Centre (WUR), Laboratory of Phytopathology, Droevendaalsesteeg 1, 6708 PB, Wageningen, The Netherlands

    Pedro W. Crous

Authors
  1. Johannes de Gruyter
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Marga P. E. van Gent-Pelzer
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Joyce H. C. Woudenberg
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Patricia C. J. van Rijswick
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Ellis T. M. Meekes
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Pedro W. Crous
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Peter J. M. Bonants
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Johannes de Gruyter.

Rights and permissions

Reprints and permissions

About this article

Cite this article

de Gruyter, J., van Gent-Pelzer, M.P.E., Woudenberg, J.H.C. et al. The development of a validated real-time (TaqMan) PCR for detection of Stagonosporopsis andigena and S. crystalliniformis in infected leaves of potato and tomato. Eur J Plant Pathol 134, 301–313 (2012). https://doi.org/10.1007/s10658-012-9990-8

Download citation

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10658-012-9990-8

Keywords

Search

Navigation