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An Optimized Duplex Real-Time PCR Tool for Sensitive Detection of the Quarantine Oomycete Plasmopara halstedii in Sunflower Seeds

First, second, and third authors: ANSES, Laboratoire de la Santé des Végétaux, Unité de mycologie, IFR 110, Domaine de Pixérécourt, F-54220 Malzéville, France; fourth author: FERA, 04GA08/09, Sand Hutton, York, YO41 1LZ, United Kingdom; fifth author: ANSES, Laboratoire de la Rage et de la Faune Sauvage de Nancy, Domaine de Pixérécourt; and sixth author: INRA, Plate-Forme IPM, UMR 1095, Domaine de la Crouelle, 234 avenue du Brézet, F-63100 Clermont-Ferrand, France.

Plasmopara halstedii, the causal agent of downy mildew of sunflower, is an oomycete listed as a quarantine pathogen. This obligate parasite resides in a quiescent state in seeds of sunflower and can be spread from seed production areas to areas of crop production by international seed trade. To prevent the spread or the introduction of potentially new genotypes or fungicide-tolerant strains, an efficient method to detect P. halstedii in sunflower seed is required. This work reports the optimization of a real-time detection tool that targets the pathogen within sunflower seeds, and provides statistically validated data for that tool. The tool proved to be specific and inclusive, based on computer simulation and in vitro assessments, and could detect as few as 45 copies of target DNA. A fully optimized DNA extraction protocol was also developed starting from a sample of 1,000 sunflower seeds, and enabled the detection of <1 infected seed/1,000 seeds. To ensure reliability of the results, a set of controls was used systematically during the assays, including a plant-specific probe used in a duplex quantitative polymerase chain reaction that enabled the assessment of the quality of each DNA extract.