title: Detection and Quantification of Bremia lactucae by Spore Trapping and Quantitative PCR.
abstract: Bremia lactucae is an obligate, oomycete pathogen of lettuce that causes leaf chlorosis and necrosis and adversely affects marketability. The disease has been managed with a combination of host resistance and fungicide applications with success over the years. Fungicide applications are routinely made under the assumption that inoculum is always present during favorable environmental conditions. This approach often leads to fungicide resistance in B. lactucae populations. Detection and quantification of airborne B. lactucae near lettuce crops provides an estimation of the inoculum load, enabling more judicious timing of fungicide applications. We developed a quantitative polymerase chain reaction (qPCR)-based assay using a target sequence in mitochondrial DNA for specific detection of B. lactucae. Validation using amplicon sequencing of DNA from 83 geographically diverse isolates, representing 14 Bremia spp., confirmed that the primers developed for the TaqMan assays are species specific and only amplify templates from B. lactucae. DNA from a single sporangium could be detected at a quantification cycle (Cq) value of 32, and Cqvalues >35 were considered to be nonspecific. The coefficient of determination (R2) for regression between sporangial density derived from flow cytometry and Cqvalues derived from the qPCR was 0.86. The assay was deployed using spore traps in the Salinas Valley, where nearly half of U.S. lettuce is produced. The deployment of this sensitive B. lactucae-specific assay resulted in the detection of the pathogen during the 2-week lettuce-free period as well as during the cropping season. These results demonstrate that this assay will be useful for quantifying inoculum load in and around the lettuce fields for the purpose of timing fungicide applications based on inoculum load.
authors.affiliation: First and tenth authors: Department of Plant Pathology, University of California-Davis, 1636 E. Alisal St. Salinas 93901; second, third, and eleventh authors: United States Department of Agriculture-Agricultural Research Service (USDA-ARS), 1636 E. Alisal St., Salinas, CA 93905; fourth author: Kunsan National University, Department of Biology, Gunsan 54150, Republic of Korea; fourth and fifth authors: Biodiversity and Climate Research Center (BiK-F) Senckenberg Gesellscharft für Naturforschung, D-60325 Frankfurt am Main, and Goethe University, Department of Biological Sciences, Institute of Ecology, Evolution and Diversity, D-60325 Frankfurt am Main, Germany; sixth author: The Genome Center and Department of Plant Sciences, University of California, Davis 95616; seventh author: University of California Cooperative Extension, Monterey County, Salinas; eighth author: The Genome Center and Department of Plant Pathology, University of California, Davis; and ninth author: USDA-ARS, Corvallis, OR.
authors.forename: Sridhara G
authors.initials: SG
authors.lastname: Kunjeti
authors.affiliation: First and tenth authors: Department of Plant Pathology, University of California-Davis, 1636 E. Alisal St. Salinas 93901; second, third, and eleventh authors: United States Department of Agriculture-Agricultural Research Service (USDA-ARS), 1636 E. Alisal St., Salinas, CA 93905; fourth author: Kunsan National University, Department of Biology, Gunsan 54150, Republic of Korea; fourth and fifth authors: Biodiversity and Climate Research Center (BiK-F) Senckenberg Gesellscharft für Naturforschung, D-60325 Frankfurt am Main, and Goethe University, Department of Biological Sciences, Institute of Ecology, Evolution and Diversity, D-60325 Frankfurt am Main, Germany; sixth author: The Genome Center and Department of Plant Sciences, University of California, Davis 95616; seventh author: University of California Cooperative Extension, Monterey County, Salinas; eighth author: The Genome Center and Department of Plant Pathology, University of California, Davis; and ninth author: USDA-ARS, Corvallis, OR.
authors.forename: Amy
authors.initials: A
authors.lastname: Anchieta
authors.affiliation: First and tenth authors: Department of Plant Pathology, University of California-Davis, 1636 E. Alisal St. Salinas 93901; second, third, and eleventh authors: United States Department of Agriculture-Agricultural Research Service (USDA-ARS), 1636 E. Alisal St., Salinas, CA 93905; fourth author: Kunsan National University, Department of Biology, Gunsan 54150, Republic of Korea; fourth and fifth authors: Biodiversity and Climate Research Center (BiK-F) Senckenberg Gesellscharft für Naturforschung, D-60325 Frankfurt am Main, and Goethe University, Department of Biological Sciences, Institute of Ecology, Evolution and Diversity, D-60325 Frankfurt am Main, Germany; sixth author: The Genome Center and Department of Plant Sciences, University of California, Davis 95616; seventh author: University of California Cooperative Extension, Monterey County, Salinas; eighth author: The Genome Center and Department of Plant Pathology, University of California, Davis; and ninth author: USDA-ARS, Corvallis, OR.
authors.forename: Frank N
authors.initials: FN
authors.lastname: Martin
authors.affiliation: First and tenth authors: Department of Plant Pathology, University of California-Davis, 1636 E. Alisal St. Salinas 93901; second, third, and eleventh authors: United States Department of Agriculture-Agricultural Research Service (USDA-ARS), 1636 E. Alisal St., Salinas, CA 93905; fourth author: Kunsan National University, Department of Biology, Gunsan 54150, Republic of Korea; fourth and fifth authors: Biodiversity and Climate Research Center (BiK-F) Senckenberg Gesellscharft für Naturforschung, D-60325 Frankfurt am Main, and Goethe University, Department of Biological Sciences, Institute of Ecology, Evolution and Diversity, D-60325 Frankfurt am Main, Germany; sixth author: The Genome Center and Department of Plant Sciences, University of California, Davis 95616; seventh author: University of California Cooperative Extension, Monterey County, Salinas; eighth author: The Genome Center and Department of Plant Pathology, University of California, Davis; and ninth author: USDA-ARS, Corvallis, OR.
authors.forename: Young-Joon
authors.initials: YJ
authors.lastname: Choi
authors.affiliation: First and tenth authors: Department of Plant Pathology, University of California-Davis, 1636 E. Alisal St. Salinas 93901; second, third, and eleventh authors: United States Department of Agriculture-Agricultural Research Service (USDA-ARS), 1636 E. Alisal St., Salinas, CA 93905; fourth author: Kunsan National University, Department of Biology, Gunsan 54150, Republic of Korea; fourth and fifth authors: Biodiversity and Climate Research Center (BiK-F) Senckenberg Gesellscharft für Naturforschung, D-60325 Frankfurt am Main, and Goethe University, Department of Biological Sciences, Institute of Ecology, Evolution and Diversity, D-60325 Frankfurt am Main, Germany; sixth author: The Genome Center and Department of Plant Sciences, University of California, Davis 95616; seventh author: University of California Cooperative Extension, Monterey County, Salinas; eighth author: The Genome Center and Department of Plant Pathology, University of California, Davis; and ninth author: USDA-ARS, Corvallis, OR.
authors.forename: Marco
authors.initials: M
authors.lastname: Thines
authors.affiliation: First and tenth authors: Department of Plant Pathology, University of California-Davis, 1636 E. Alisal St. Salinas 93901; second, third, and eleventh authors: United States Department of Agriculture-Agricultural Research Service (USDA-ARS), 1636 E. Alisal St., Salinas, CA 93905; fourth author: Kunsan National University, Department of Biology, Gunsan 54150, Republic of Korea; fourth and fifth authors: Biodiversity and Climate Research Center (BiK-F) Senckenberg Gesellscharft für Naturforschung, D-60325 Frankfurt am Main, and Goethe University, Department of Biological Sciences, Institute of Ecology, Evolution and Diversity, D-60325 Frankfurt am Main, Germany; sixth author: The Genome Center and Department of Plant Sciences, University of California, Davis 95616; seventh author: University of California Cooperative Extension, Monterey County, Salinas; eighth author: The Genome Center and Department of Plant Pathology, University of California, Davis; and ninth author: USDA-ARS, Corvallis, OR.
authors.forename: Richard W
authors.initials: RW
authors.lastname: Michelmore
authors.affiliation: First and tenth authors: Department of Plant Pathology, University of California-Davis, 1636 E. Alisal St. Salinas 93901; second, third, and eleventh authors: United States Department of Agriculture-Agricultural Research Service (USDA-ARS), 1636 E. Alisal St., Salinas, CA 93905; fourth author: Kunsan National University, Department of Biology, Gunsan 54150, Republic of Korea; fourth and fifth authors: Biodiversity and Climate Research Center (BiK-F) Senckenberg Gesellscharft für Naturforschung, D-60325 Frankfurt am Main, and Goethe University, Department of Biological Sciences, Institute of Ecology, Evolution and Diversity, D-60325 Frankfurt am Main, Germany; sixth author: The Genome Center and Department of Plant Sciences, University of California, Davis 95616; seventh author: University of California Cooperative Extension, Monterey County, Salinas; eighth author: The Genome Center and Department of Plant Pathology, University of California, Davis; and ninth author: USDA-ARS, Corvallis, OR.
authors.forename: Steven T
authors.initials: ST
authors.lastname: Koike
authors.affiliation: First and tenth authors: Department of Plant Pathology, University of California-Davis, 1636 E. Alisal St. Salinas 93901; second, third, and eleventh authors: United States Department of Agriculture-Agricultural Research Service (USDA-ARS), 1636 E. Alisal St., Salinas, CA 93905; fourth author: Kunsan National University, Department of Biology, Gunsan 54150, Republic of Korea; fourth and fifth authors: Biodiversity and Climate Research Center (BiK-F) Senckenberg Gesellscharft für Naturforschung, D-60325 Frankfurt am Main, and Goethe University, Department of Biological Sciences, Institute of Ecology, Evolution and Diversity, D-60325 Frankfurt am Main, Germany; sixth author: The Genome Center and Department of Plant Sciences, University of California, Davis 95616; seventh author: University of California Cooperative Extension, Monterey County, Salinas; eighth author: The Genome Center and Department of Plant Pathology, University of California, Davis; and ninth author: USDA-ARS, Corvallis, OR.
authors.forename: Cayla
authors.initials: C
authors.lastname: Tsuchida
authors.affiliation: First and tenth authors: Department of Plant Pathology, University of California-Davis, 1636 E. Alisal St. Salinas 93901; second, third, and eleventh authors: United States Department of Agriculture-Agricultural Research Service (USDA-ARS), 1636 E. Alisal St., Salinas, CA 93905; fourth author: Kunsan National University, Department of Biology, Gunsan 54150, Republic of Korea; fourth and fifth authors: Biodiversity and Climate Research Center (BiK-F) Senckenberg Gesellscharft für Naturforschung, D-60325 Frankfurt am Main, and Goethe University, Department of Biological Sciences, Institute of Ecology, Evolution and Diversity, D-60325 Frankfurt am Main, Germany; sixth author: The Genome Center and Department of Plant Sciences, University of California, Davis 95616; seventh author: University of California Cooperative Extension, Monterey County, Salinas; eighth author: The Genome Center and Department of Plant Pathology, University of California, Davis; and ninth author: USDA-ARS, Corvallis, OR.
authors.forename: Walt
authors.initials: W
authors.lastname: Mahaffee
authors.affiliation: First and tenth authors: Department of Plant Pathology, University of California-Davis, 1636 E. Alisal St. Salinas 93901; second, third, and eleventh authors: United States Department of Agriculture-Agricultural Research Service (USDA-ARS), 1636 E. Alisal St., Salinas, CA 93905; fourth author: Kunsan National University, Department of Biology, Gunsan 54150, Republic of Korea; fourth and fifth authors: Biodiversity and Climate Research Center (BiK-F) Senckenberg Gesellscharft für Naturforschung, D-60325 Frankfurt am Main, and Goethe University, Department of Biological Sciences, Institute of Ecology, Evolution and Diversity, D-60325 Frankfurt am Main, Germany; sixth author: The Genome Center and Department of Plant Sciences, University of California, Davis 95616; seventh author: University of California Cooperative Extension, Monterey County, Salinas; eighth author: The Genome Center and Department of Plant Pathology, University of California, Davis; and ninth author: USDA-ARS, Corvallis, OR.
authors.forename: Krishna V
authors.identifier: 0000-0002-2075-1835
authors.initials: KV
authors.lastname: Subbarao
authors.affiliation: First and tenth authors: Department of Plant Pathology, University of California-Davis, 1636 E. Alisal St. Salinas 93901; second, third, and eleventh authors: United States Department of Agriculture-Agricultural Research Service (USDA-ARS), 1636 E. Alisal St., Salinas, CA 93905; fourth author: Kunsan National University, Department of Biology, Gunsan 54150, Republic of Korea; fourth and fifth authors: Biodiversity and Climate Research Center (BiK-F) Senckenberg Gesellscharft für Naturforschung, D-60325 Frankfurt am Main, and Goethe University, Department of Biological Sciences, Institute of Ecology, Evolution and Diversity, D-60325 Frankfurt am Main, Germany; sixth author: The Genome Center and Department of Plant Sciences, University of California, Davis 95616; seventh author: University of California Cooperative Extension, Monterey County, Salinas; eighth author: The Genome Center and Department of Plant Pathology, University of California, Davis; and ninth author: USDA-ARS, Corvallis, OR.
authors.forename: Steven J
authors.initials: SJ
authors.lastname: Klosterman
mesh_terms: D000391:Air Microbiology; D017931:DNA Primers / Q000235:genetics; D005659:Fungicides, Industrial; D005843:Geography; D018545:Lactuca / Q000469:parasitology*; D009868:Oomycetes / Q000235:genetics / Q000302:isolation & purification*; D010935:Plant Diseases / Q000469:parasitology*; D018515:Plant Leaves / Q000469:parasitology; D060888:Real-Time Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D013170:Spores
chemical_list: D017931:DNA Primers; D005659:Fungicides, Industrial