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A robust and specific real-time PCR tool for the detection of Phytophthora lateralis in plant tissues

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Abstract

Phytophthora lateralis is an oomycete responsible for Port-Orford Cedar (Chamaecyparis lawsoniana) dieback and mortality since the 1920s in western North America. It is recommended for quarantine regulation by EPPO (A2 list) and has been recently detected in Europe. In order to implement efficient control of the disease, a sensitive real-time PCR assay was developed to detect the presence of P. lateralis in plant tissues. In this study, we used the species-specific polymorphisms within the RAS-related protein gene Ypt1 to design a primer pair and a hydrolysis probe targeting the pathogen. The tool proved to be specific and inclusive, based on in silico and in vitro assessments, and could detect as few as 47 copies of target DNA. In addition, the test demonstrated its robustness and remained highly sensitive and specific under deliberately modified experimental conditions such as the hybridization temperature, or the reaction and DNA template volumes. The DNA extraction step from diseased tissues was also optimized, and the reliability of the results was ensured by a set of controls and a test targeting plant DNA that enabled the assessment of the quality of the extracts.

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Acknowledgments

This research was supported financially by the French Agency for Food, Environmental and Occupational Health & Safety (ANSES).

We thank Gilbert Douzon, Dominique Sage (Pôle Santé des Forêts, Orléans, France), Maurice Nicolas, Xavier Grenie and Laurence Roche (Office National des Forêts, France) for their collaboration in the collection of the Port-Orford Cedar samples in Bretagne ; Cécile Robin (Institut National de la Recherche Agronomique, Bordeaux, France), Everett E. Hansen (Oregon University, USA), Karin Rosendahl (Nieuwe Voedsel en Waren Autoriteit, Wageningen, the Netherlands) and Alexandra Schlenzig (Science and Advice for Scottish Agriculture, Edinburgh, Scotland) for supplying numerous P. lateralis isolates used in this study; Claude Husson (INRA Champenoux, France) for providing healthy C. lawsoniana material.

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Authors and Affiliations

  1. ANSES Laboratoire de la Santé des Végétaux, Unité de mycologie, Domaine de Pixérécourt, 54220, Malzéville, France

    Nathalie Schenck, Celine Fourrier-Jeandel & Renaud Ioos

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  1. Nathalie Schenck
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  2. Celine Fourrier-Jeandel
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  3. Renaud Ioos
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Correspondence to Nathalie Schenck.

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Schenck, N., Fourrier-Jeandel, C. & Ioos, R. A robust and specific real-time PCR tool for the detection of Phytophthora lateralis in plant tissues. Eur J Plant Pathol 146, 231–244 (2016). https://doi.org/10.1007/s10658-016-0909-7

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  • DOI: https://doi.org/10.1007/s10658-016-0909-7

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