title: Development of a TaqMan real-time PCR assay for quantification of airborne conidia of Botrytis squamosa and management of botrytis leaf blight of onion.
abstract: The use of a DNA-based method for quantifying airborne inoculum of Botrytis squamosa, a damaging pathogen of onion, was investigated. A method for purifying DNA from conidia collected using rotating-arm samplers and quantifying it using a TaqMan real-time quantitative polymerase chain reaction (qPCR) assay is described. The sensitivity of the qPCR assay was high, with a detection limit of 2 conidia/rod. A linear relationship between numbers of conidia counted with a compound microscope and those determined with the qPCR assay was obtained. Receiver operating characteristic curve analysis was used to evaluate the reliability of the two methods of conidia quantification (microscope examination and qPCR assay) to predict the risk of disease being below or above a damage threshold (D(th)). In total, 142 field samples from commercial onion fields were analyzed. At damage thresholds of 5 or 10 lesions/leaf, conidia quantification with the qPCR assay was more reliable at predicting disease risk than conidia quantification based on microscope counts. The proportion of decisions where the disease was present and predicted was higher for the qPCR assay than for the microscope counts, with values of 0.95 and 0.89 compared with 0.79 and 0.81 for D(th) of 5 and 10 lesions/leaf, respectively. The proportion of decisions where the disease was present but not predicted was lower for the qPCR assay than for microscope counts, with values of 0.05 and 0.11 compared with 0.20 and 0.19 for D(th) of 5 and 10 lesions/leaf, respectively. The results demonstrated that this new qPCR assay was reliable for quantifying B. squamosa airborne inoculum in commercial onion fields and that molecular conidia quantification could be used as a component of a risk management system for Botrytis leaf blight.
authors.affiliation: Agriculture and Agri-Food Canada, Saint-Jean-sur-Richelieu, Québec, Canada.
authors.forename: O
authors.initials: O
authors.lastname: Carisse
authors.forename: D M
authors.initials: DM
authors.lastname: Tremblay
authors.forename: C A
authors.initials: CA
authors.lastname: Lévesque
authors.forename: K
authors.initials: K
authors.lastname: Gindro
authors.forename: P
authors.initials: P
authors.lastname: Ward
authors.forename: A
authors.initials: A
authors.lastname: Houde
mesh_terms: D000391:Air Microbiology; D020171:Botrytis / Q000502:physiology*; D004271:DNA, Fungal / Q000302:isolation & purification*; D019697:Onions / Q000382:microbiology*; D010935:Plant Diseases / Q000382:microbiology; D016133:Polymerase Chain Reaction; D013172:Spores, Fungal / Q000235:genetics / Q000302:isolation & purification*
chemical_list: D004271:DNA, Fungal