Advertisement

European Journal of Plant Pathology

, Volume 153, Issue 1, pp 135–152 | Cite as

Early detection of Cryphonectria parasitica by real-time PCR

  • Anne ChandelierEmail author
  • Marie Massot
  • Olivier Fabreguettes
  • Fabian Gischer
  • Felix Teng
  • Cécile Robin
Article

Abstract

The development and validation of a real-time PCR method for the detection of Cryphonectria parasitica in bark tissues is described. The selected region in the genome was a fragment of the internal transcribed spacer region. The DNA extraction and PCR conditions were optimized to be routinely applicable to fresh and dried bark. The sensitivity of the assay allowed the detection of 2 fg of genomic DNA, equivalent to one spore of the pathogen. There was no cross-reaction with closely related Cryphonectria species. The use of droplet digital PCR (ddPCR) confirmed the high sensitivity of the real-time PCR method, and its capacity to be used as an early detection method. A survey was conducted in Belgium on chestnut trees displaying cankers using isolation and the real-time PCR method. Both methods provided the same results for 83% of the samples (either negative or positive results). For the remaining samples, C. parasitica was not isolated, while amplifications close to the end of the PCR run (resulting in late Cycle threshold (Ct) values) were observed with the real-time PCR. Some of these samples were collected in stands where C. parasitica had already been detected on other chestnut trees, or in sites close to the infected stands. The application of this method may help plant protection services to detect new introductions of the pathogen within areas still free of chestnut blight and to prevent its establishment. It may also be useful for scientists involved in the epidemiology of the disease.

Keywords

Castanea Chestnut blight Droplet digital PCR Latent pathogens 

Notes

Acknowledgements

The authors wish to thank Dr. Rigling (WSL, Switzerland), Dr. Bragança (INIAV, Portugal), Dr. Edwards (AgriBio, Australia), Dr. Halász (Nébih, Hungary), Dr. Vettraino (University of Tuscia, Viterbo, Italy), Dr. Palovčíková (MZLU Brno, Czech Republic) and Dr. Marçais (INRA, Nancy, France) for providing fungal isolates used in this study. They are also grateful to Dr. Heungens (ILVO, Belgium) for fruitful discussion about the comparison of PCR kits for the detection of C. parasitica in chestnut bark, Dr. Blancquaert (PCS, Belgium) for his participation to the survey, and Amélien Jeanjot (CRAW, Belgium) for his technical assistance. This work was supported by Euphresco (project CERACRY) and by the ANR (Project ANR-13-BSV7-0011 FunFit).

Funding

The research was carried out in the framework of a project (contract RT 15/6 FUNGIFOR 1) funded by a grant from the Belgian Federal Public Service Health, Food Chain Safety and Environment and a project funded by a grant from the French National Research agency (Projet ANR-13-BSV7–0011 FunFit). Some of the experiments (PCR quantitative and Droplet Digital PCR) were performed at the Genome Transcriptome Facility of Bordeaux (with grants from the Conseil Régional d’Aquitaine n°20030304002FA and 20040305003FA, from the European Union FEDER n°2003227 and from Investissements d’Avenir ANR-10-EQPX-16-01).

Compliance with ethical standards

Conflict of interest

A. Chandelier declares that she has no conflict of interest. Marie Massot declares that she has no conflict of interest. Olivier Fabreguettes declares that he has no conflict of interest. Fabian Gischer declares that he has no conflict of interest. Felix Teng declares that he has no conflict of interest. Cécile Robin declares that she has no conflict of interest.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

References

  1. Abreu, C. G. (1996). Recent outbreak of bark canker caused by Melanconis modonia on European chestnut in northern Portugal. Plant Disease, 80, 1301.CrossRefGoogle Scholar
  2. Adamčíková, K., Kobza, M., & Juhásová, G. (2010). Characteristics of the Cryphonectria parasitica isolated from Quercus in Slovakia. Forest Pathology, 40, 443–449.CrossRefGoogle Scholar
  3. Adamčíková, K., Juhásová, G., Kobza, M., & Ondruškova, E. (2013). Diversity of microfungi on branches of Castanea sativa in Slovakia. Polish Botanical Journal, 58, 741–746.CrossRefGoogle Scholar
  4. Anagnostakis, S. L. (1987). Chestnut blight: The classical problem of an introduced pathogen. Mycologia, 79, 23–37.CrossRefGoogle Scholar
  5. Anagnostakis, S. L., Hau, B., & Kranz, J. (1986). Diversity of vegetative compatibility groups of Cryphonectria parasitica in Connecticut and Europe. Plant Disease, 70, 536–538.CrossRefGoogle Scholar
  6. Atkins, S. D., & Clark, I. M. (2004). Fungal molecular diagnostics: a mini review. Journal of Applied Genetics, 45, 3–15.Google Scholar
  7. Biraghi, A. (1946). Il cancro del Castagno causato da Endothia parasitica. Italian Agriculture, 7, 1–9.Google Scholar
  8. Bissegger, M., & Sieger, T. N. (1994). Assemblages of endophytic fungi in coppice shoots of Castanea sativa. Mycologia, 86, 648–655.CrossRefGoogle Scholar
  9. Bodles, W. J. A., Fossdal, C. G., & Woodward, S. (2006). Multiplex real-time PCR detection of pathogen colonization in the bark and wood of Picea sitchensis clones differing in resistance to Heterobasidion annosum. Tree Physiology, 26, 775–782.CrossRefGoogle Scholar
  10. Bragança, H., Rigling, D., Diogo, E., Capelo, J., Phillips, A., & Tenreiro, R. (2011). Cryphonectria naterciae: A new species in the Cryphonectria-Endothia complex and diagnostic molecular markers based on microsatellite-primed PCR. Fungal Biology, 115, 852–861.CrossRefGoogle Scholar
  11. Broeders, S., Huber, I., Grohmann, L., Berben, G., Taverniers, I., Mazzara, M., Roosens, N., & Morisset, D. (2014). Guidelines for validation of qualitative real-time PCR methods. Trends in Food Science & Technology, 37, 115–126.CrossRefGoogle Scholar
  12. Chandelier, A., André, F., & Laurent, F. (2010). Detection of Chalara fraxinea by real time PCR. Forest Pathology, 40, 87–95.CrossRefGoogle Scholar
  13. Chen, C., Verkley, G. J. M., Sun, G., Groenewald, J. Z., & Crous, P. W. (2016). Redefining common endophytes and plant pathogens in Neofabraea, Pezicula, and related genera. Fungal Biology, 120, 1291–1322.CrossRefGoogle Scholar
  14. Dolezel, J., Bartos, J., Vogimar, H., & Guilhuber, J. (2003). Nuclear DNA content and genome size of trout and human. Cytometry, 51, 127–128.CrossRefGoogle Scholar
  15. Dutech, C., Fabreguettes, O., Capdevielle, X., & Robin, C. (2010). Multiple introductions of divergent genetic lineages in an invasive fungal pathogen, Cryphonectria parasitica, in France. Heredity, 105, 220–228.CrossRefGoogle Scholar
  16. Dutech, C., Barrès, B., Bridier, J., Robin, C., Milgroom, M. G., & Ravigné, V. (2012). The chestnut blight fungus world tour: Successive introduction events from diverse origins in an invasive plant fungal pathogen. Molecular Ecology, 21, 3931–3946.CrossRefGoogle Scholar
  17. EFSA PLH Panel (EFSA Panel on Plant Health). (2014). Scientific opinion on the pest categorization of Cryphonectria parasitica (Murrill) Barr. EFSA Journal, 12(10), 3859, 42.  https://doi.org/10.2903/j.efsa.2014.3859.CrossRefGoogle Scholar
  18. EFSA PLH Panel (EFSA Panel on Plant Health). (2016). Scientific opinion on the Risk assessment and reduction options for Cryphonectria parasitica in the EU. EFSA Journal, 14(12), 4641, 54.  https://doi.org/10.2903/j.efsa.2016.4641.Google Scholar
  19. Gambino, G., Perrone, I., & Gribaudo, I. (2008). A rapid and effective method for RNA extraction from different tissues of grapevine and other woody plants. Phytochemical Analysis, 19, 520–525.CrossRefGoogle Scholar
  20. Ghasemkhani, M., Holefors, A., Marttila, S., Dalman, K., Zborowska, A., Rur, M., Rees-George, J., Nybom, H. et al. (2016). Real-time PCR for detection and quantification, and histological characterization of Neonectria ditissima in appel trees. Trees.  https://doi.org/10.1007/s00468-015-1350-9.
  21. Gonthier, P., Guglielmo, F., Sillo, F., Giordano, L., & Garbelotto, M. (2015). A molecular diagnostic assay for the detection and identification of wood decay fungi of conifers. Forest Pathology, 45, 89–101.CrossRefGoogle Scholar
  22. Gryzenhout, M., Myburg, H., Hodges, C. S., Wingfield, B. D., & Wingfield, M. J. (2006a). Microthia, Holocryphia and Ursicollum, three new genera on Eucalyptus and Coccoloba for fungi previously known as Cryphonectria. Studies in Mycology, 55, 35–52.CrossRefGoogle Scholar
  23. Gryzenhout, M., Wingfield, B. D., & Wingfield, M. J. (2006b). New taxonomic concepts for the important forest pathogen Cryphonectria parasitica and related fungi. FEMS Microbiology Letters, 258, 161–172.CrossRefGoogle Scholar
  24. Gryzenhout, M., Vermeulen, M., Dick, M., & Wingfield, M. J. (2010). The eucalyptus canker pathogen Holocryphia eucalypti on Eucalyptus in New Zealand. Australasian Plant Disease Notes, 5, 5–8.CrossRefGoogle Scholar
  25. Guerin, L., & Robin, C. (2003). Seasonal effect on infection and development of lesions caused by Cryphonectria parasitica in Castanea sativa. Forest Pathology, 33, 223–235.CrossRefGoogle Scholar
  26. Haltofová, P., Jankovsky, L., & Palovčiková, D. (2005). New findings of Cryphonectria parasitica and the first record of chestnut blight on read oak Quercus rubra L. in the Czech Republic. Journal of Forest. Sciences, 51, 256–258.Google Scholar
  27. Heiniger, U., & Rigling, D. (1994). Biological control of chestnut blight in Europe. Annual Review of Phytopathology, 32, 581–599.CrossRefGoogle Scholar
  28. Hoegger, P. J., Rigling, D., Holdenrieder, O., & Heiniger, U. (2002). Cryphonectria radicalis: Rediscovery of a lost fungus. Mycologia, 94, 105–115.CrossRefGoogle Scholar
  29. Johnson, S. M., Carlson, E. L., & Pappagianis, D. (2015). Determination of ribosomal DNA copy number and comparison among strains of Coccidioides. Mycopathologia, 179, 45–51.CrossRefGoogle Scholar
  30. Kehr, R. D. (1991). Pezicula canker of Quercus rubra L., caused by Pezicula cinnamomea (DC.) Sacc. I. Symptoms and pathogenesis. European Journal of Forest Pathology, 21, 218–233.CrossRefGoogle Scholar
  31. Koonjul, P. K., Brandt, W. F., Farrant, J. M., & Lindsey, G. (1999). Inclusion of polyvinylpyrrolidone in the polymerase chain reaction reverses the inhibitory effects of polyphenolic contamination of RNA. Nucleic Acids Research, 27, 915–916.CrossRefGoogle Scholar
  32. Langrell, S. R. H. (2005). Development of a nested PCR detection procedure for Nectria fuckeliana direct from Norway spruce bark extracts. FEMS Microbiology Letters, 242, 185–193.CrossRefGoogle Scholar
  33. Longo, A. V., Rodriguez, D., da Silva Leite, D., Toledo, L. F., Mendoza Almeralla, C., Burrowes, P. A., & Zamudio, K. R. (2013). ITS1 copy number varies among Batrachochytrium dendrobatidis strains: Implications for qPCR estimates of infection intensity from field-collected amphibian skin swabs. PLoS One, 8(3), e59499.CrossRefGoogle Scholar
  34. Luchi, N., Ghelardini, L., Belbahri, L., Quartier, M., & Santini, A. (2013). Rapid detection of Ceratocystis platani inoculum by quantitative real-time PCR assay. Applied and Environmental Microbiology, 79, 5394–5404.CrossRefGoogle Scholar
  35. Meyer, J. B., Trapiello, E., Senn-irlet, B., Sieber, T. N., Cornejo, C., Aghayeva, D., Gonzalez, A. J., & Prospero, S. (2017). Phylogenetic and phenotypic characterization of Sirococcus castanea comb. Nov. (synonym Diplodina castanea), a fungal endophyte of European chestnut. Fungal Biology, 121, 625–637.CrossRefGoogle Scholar
  36. Milgroom, M. G., & Cortesi, P. (2004). Biological control of chestnut blight with hypovirulence: A critical analysis. Annual Review of Phytopathology, 42, 311–338.CrossRefGoogle Scholar
  37. Myburg, H., Gryzenhout, M., Wingfield, B. D., Milgroom, M. G., Kaneko, S., & Wingfield, M. J. (2004). DNA sequence data and morphology define Cryphonectria species in Europe, China, and Japan. Canadian Journal of Botany, 82, 1730–1743.CrossRefGoogle Scholar
  38. Neuhauser, S., Huber, L., & Kirchmair, M. (2009). A DNA based method to detect the grapevine root-rotting fungus Roesleria subtterranea in soil and root samples. Phytopathologia Mediterranea, 48, 59–72.Google Scholar
  39. Pasche, S., Calmin, G., Auderset, G., Crovadore, J., Pelleteret, P., Mauch-Mani, B., Barja, F., Paul, B., Jermini, M., & Lefort, F. (2016). Gnomoniopsis smithogilvyi causes chestnut canker symptoms in Castanea sativa shoots in Switzerland. Fungal Genetics and Biology, 87, 9–21.CrossRefGoogle Scholar
  40. Peters, F. S., Bußkamp, J., Prospero, S., Rigling, D., & Metzler, B. (2014). Genetic diversification of the chestnut blight fungus Cryphonectria parasitica and its associated hypovirus in Germany. Fungal Biology, 118, 193–210.CrossRefGoogle Scholar
  41. Prospero, S., & Rigling, D. (2012). Invasion genetics of the chestnut blight fungus Cryphonectria parasitica in Switzerland. Phytopathology, 102, 73–82.CrossRefGoogle Scholar
  42. Prospero, S. & Rigling, D. (2013). Chestnut blight. (In: P. Gonthier & G. Nicolotti (Eds), Infectious Forest Diseases (pp. 318–339). CABI International).Google Scholar
  43. Prospero, S., Conedera, M., Heiniger, U., & Rigling, D. (2006). Saprophytic activity and sporulation of Cryphonectria parasitica on dead chestnut wood in forests with naturally established hypovirulence. Phytopathology, 96, 1337–1344.CrossRefGoogle Scholar
  44. Robin, C., Andanson, A., Saint-Jean, G., Fabreguettes, O., & Dutech, C. (2017). What was old is new again: Thermal adaptation within clonal lineages during range expansion in a fungal pathogen. Molecular Ecology, 26, 1952–1963.CrossRefGoogle Scholar
  45. Rubio, S., Barnes, A., Webb, K., & Hodgetts, J. (2017). A real-time PCR assay for improved rapid, specific detection of Cryphonectria parasitica. Annals of Applied Biology, 171, 52–61.  https://doi.org/10.1111/aab.12354.CrossRefGoogle Scholar
  46. Schaad, N. W., & Frederick, R. D. (2002). Real-time PCR and its application for rapid plant disease diagnostics. Canadian Journal of Plant Pathology, 24, 250–258.CrossRefGoogle Scholar
  47. Shuttleworth, L. A., Walker, D. M., & Guest, D. I. (2015). The chestnut pathogen Gnomoniopsis smithogilvyi (Gnomoniaceae, Diaporthales) and its synonyms. Mycotaxon, 130, 929–940.CrossRefGoogle Scholar
  48. Svec, D., Tichopad, A., Novosadova, V., Pfaffl, M. W., & Kubista, M. (2015). How good is a PCR efficiency estimate: Recommendations for precise and robust qPCR efficiency assessments. Biomolecular Detection and Quantification, 3, 9–16.CrossRefGoogle Scholar
  49. Tziros, G. T., Nakopoulou, Z. G., & Diamandis, S. (2015). Cryphonectria parasitica, the chestnut blight fungus, causes cankers on Quercus frainetto in Greece. Australasian Plant Disease Notes, 10, 19–22.CrossRefGoogle Scholar
  50. Vettraino, A. M., Morel, O., Perlerou, C., Robin, C., Diamandis, S., & Vannini, A. (2005). Occurrence and distribution of Phytophthora species in European chestnut stands, and their association with ink disease and crown decline. European Journal of Plant Pathology, 111, 169–180.CrossRefGoogle Scholar
  51. 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). New York: Academic Press, Inc..Google Scholar
  52. Zhao, Y., Xia, Q., Yin, Y. & Wang, Z. (2016). Comparison of droplet digital PCR and quantitative PCR assays for quantitative detection of Xanthomonas citri subsp. citri. Plos One.  https://doi.org/10.1371/journal.pone.0159004.

Copyright information

© Koninklijke Nederlandse Planteziektenkundige Vereniging 2018

Authors and Affiliations

  • Anne Chandelier
    • 1
    Email author
  • Marie Massot
    • 2
  • Olivier Fabreguettes
    • 2
  • Fabian Gischer
    • 1
  • Felix Teng
    • 1
  • Cécile Robin
    • 2
  1. 1.Department Life Sciences, Walloon Agricultural Research CentreGemblouxBelgium
  2. 2.BIOGECO, INRAUniversity of BordeauxCestasFrance

Personalised recommendations