Early detection of Cryphonectria parasitica by real-time PCR
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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.
KeywordsCastanea Chestnut blight Droplet digital PCR Latent pathogens
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).
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.
This article does not contain any studies with human participants or animals performed by any of the authors.
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