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European Journal of Plant Pathology

, Volume 125, Issue 2, pp 329–335 | Cite as

Rapid in planta detection of Chalara fraxinea by a real-time PCR assay using a dual-labelled probe

  • Renaud Ioos
  • Tadeusz Kowalski
  • Claude Husson
  • Ottmar Holdenrieder
Article

Abstract

Chalara fraxinea is a fungus currently threatening ash trees (Fraxinus excelsior) in several European countries. This emerging pathogen was assigned to the EPPO’s alert list and therefore accurate detection and identification tools are needed. Because of its slow growth rate on agar media and the frequent presence of fast-growing saprotrophic fungi within the host tissue, classical isolation techniques are time-consuming and sometimes inefficient. In this study, we used species-specific polymorphisms observed within the internal transcribed spacer region to design a primer pair and a dual-labelled probe to be used in a real-time PCR assay for the detection of C. fraxinea. The test proved to be specific, based on in silico and in vitro assessments, and could detect as little as 20 fg of C. fraxinea DNA. A protocol was developed in order to detect the pathogen directly in plant tissue and proved to be more efficient and rapid than isolation on agar plates. This new tool should be useful both for monitoring and to conduct epidemiology research on this emerging pathogen.

Keywords

Diagnosis Fraxinus excelsior Disease 

Notes

Acknowledgements

The authors are very grateful to P. Loevenbruck, and C. Saurat for providing C. fraxinea isolates and the French forest health service (DSF) for collecting naturally infected ash samples.

References

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Copyright information

© KNPV 2009

Authors and Affiliations

  • Renaud Ioos
    • 1
  • Tadeusz Kowalski
    • 2
  • Claude Husson
    • 3
  • Ottmar Holdenrieder
    • 4
  1. 1.Laboratoire National de la Protection des Végétaux, Station de Mycologie, IFR 110MalzévilleFrance
  2. 2.Department of Forest Pathology, Faculty of ForestryAgricultural UniversityKrakówPoland
  3. 3.INRA, Nancy Université, UMR 1136 Interaction Arbres/Micro-organismes, IFR 110ChampenouxFrance
  4. 4.Forest Pathology and DendrologyInstitute of Integrative Biology (IBZ), ETH ZurichZurichSwitzerland

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