title: PCR-Mediated Detection and Quantification of the Goss's Wilt Pathogen Clavibacter michiganensis subsp. nebraskensis Via a Novel Gene Target.
abstract: Goss's leaf blight and wilt of maize (corn) is a significant and reemerging disease caused by the bacterium Clavibacter michiganensis subsp. nebraskensis. Despite its importance, molecular tools for diagnosing and studying this disease remain limited. We report the identification of CMN_01184 as a novel gene target and its use in conventional PCR (cPCR) and SYBR green-based quantitative PCR (qPCR) assays for specific detection and quantification of C. michiganensis subsp. nebraskensis. The cPCR and qPCR assays based on primers targeting CMN_01184 specifically amplified only C. michiganensis subsp. nebraskensis among a diverse collection of 129 bacterial and fungal isolates, including multiple maize bacterial and fungal pathogens, environmental organisms from agricultural fields, and all known subspecies of C. michiganensis. Specificity of the assays for detection of only C. michiganensis subsp. nebraskensis was also validated with field samples of C. michiganensis subsp. nebraskensis-infected and uninfected maize leaves and C. michiganensis subsp. nebraskensis-infested and uninfested soil. Detection limits were determined at 30 and 3 ng of pure C. michiganensis subsp. nebraskensis DNA, and 100 and 10 CFU of C. michiganensis subsp. nebraskensis for the cPCR and qPCR assays, respectively. Infection of maize leaves by C. michiganensis subsp. nebraskensis was quantified from infected field samples and was standardized using an internal maize DNA control. These novel, specific, and sensitive PCR assays based on CMN_01184 are effective for diagnosis of Goss's wilt and for studies of the epidemiology and host-pathogen interactions of C. michiganensis subsp. nebraskensis.
authors.affiliation: First, second, and third authors: Department of Plant Pathology, University of Minnesota, St. Paul 55108; and second and third authors: Stakman-Borlaug Center for Sustainable Plant Health, University of Minnesota.
authors.forename: R Ryan
authors.initials: RR
authors.lastname: McNally
authors.affiliation: First, second, and third authors: Department of Plant Pathology, University of Minnesota, St. Paul 55108; and second and third authors: Stakman-Borlaug Center for Sustainable Plant Health, University of Minnesota.
authors.forename: Carol A
authors.initials: CA
authors.lastname: Ishimaru
authors.affiliation: First, second, and third authors: Department of Plant Pathology, University of Minnesota, St. Paul 55108; and second and third authors: Stakman-Borlaug Center for Sustainable Plant Health, University of Minnesota.
authors.forename: Dean K
authors.initials: DK
authors.lastname: Malvick
mesh_terms: D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000032:analysis / Q000235:genetics; D008835:Micrococcaceae / Q000254:growth & development / Q000302:isolation & purification*; D010935:Plant Diseases / Q000382:microbiology*; D018515:Plant Leaves / Q000382:microbiology; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D003313:Zea mays / Q000382:microbiology*
chemical_list: D017931:DNA Primers; D004269:DNA, Bacterial