title: Genome Analysis and Development of a Multiplex TaqMan Real-Time PCR for Specific Identification and Detection of Clavibacter michiganensis subsp. nebraskensis.
abstract: The reemergence of the Goss's bacterial wilt and blight disease in corn in the United States and Canada has prompted investigative research to better understand the genome organization. In this study, we generated a draft genome sequence of Clavibacter michiganensis subsp. nebraskensis strain DOAB 395 and performed genome and proteome analysis of C. michiganensis subsp. nebraskensis strains isolated in 2014 (DOAB 397 and DOAB 395) compared with the type strain, NCPPB 2581 (isolated over 40 years ago). The proteomes of strains DOAB 395 and DOAB 397 exhibited a 99.2% homology but had 92.1 and 91.8% homology, respectively, with strain NCPPB 2581. The majority (99.9%) of the protein sequences had a 99.6 to 100% homology between C. michiganensis subsp. nebraskensis strains DOAB 395 and DOAB 397, with only four protein sequences (0.1%) exhibiting a similarity <70%. In contrast, 3.0% of the protein sequences of strain DOAB 395 or DOAB 397 showed low homologies (<70%) with the type strain NCPPB 2581. The genome data were exploited for the development of a multiplex TaqMan real-time polymerase chain reaction (PCR) tool for rapid detection of C. michiganensis subsp. nebraskensis. The specificity of the assay was validated using 122 strains of Clavibacter and non-Clavibacter spp. A blind test and naturally infected leaf samples were used to confirm specificity. The sensitivity (0.1 to 1.0 pg) compared favorably with previously reported real-time PCR assays. This tool should fill the current gap for a reliable diagnostic technique.
authors.affiliation: First, second, and eighth authors: Ottawa Research and Development Centre, Agriculture and Agri-Food Canada, Ottawa, Ontario K1A 0C6, Canada; third author: Department of Plant Science, University of Manitoba, Winnipeg, Manitoba, Canada; fourth, fifth, sixth, and seventh authors: Canadian Food Inspection Agency, Ottawa, Ontario, Canada; ninth and tenth authors: Manitoba Corn Growers Association, Carman, Manitoba, Canada; and eleventh author: Department of Plant Pathology, University of Nebraska, Lincoln.
authors.forename: James T
authors.initials: JT
authors.lastname: Tambong
authors.affiliation: First, second, and eighth authors: Ottawa Research and Development Centre, Agriculture and Agri-Food Canada, Ottawa, Ontario K1A 0C6, Canada; third author: Department of Plant Science, University of Manitoba, Winnipeg, Manitoba, Canada; fourth, fifth, sixth, and seventh authors: Canadian Food Inspection Agency, Ottawa, Ontario, Canada; ninth and tenth authors: Manitoba Corn Growers Association, Carman, Manitoba, Canada; and eleventh author: Department of Plant Pathology, University of Nebraska, Lincoln.
authors.forename: Renlin
authors.initials: R
authors.lastname: Xu
authors.affiliation: First, second, and eighth authors: Ottawa Research and Development Centre, Agriculture and Agri-Food Canada, Ottawa, Ontario K1A 0C6, Canada; third author: Department of Plant Science, University of Manitoba, Winnipeg, Manitoba, Canada; fourth, fifth, sixth, and seventh authors: Canadian Food Inspection Agency, Ottawa, Ontario, Canada; ninth and tenth authors: Manitoba Corn Growers Association, Carman, Manitoba, Canada; and eleventh author: Department of Plant Pathology, University of Nebraska, Lincoln.
authors.forename: Fouad
authors.initials: F
authors.lastname: Daayf
authors.affiliation: First, second, and eighth authors: Ottawa Research and Development Centre, Agriculture and Agri-Food Canada, Ottawa, Ontario K1A 0C6, Canada; third author: Department of Plant Science, University of Manitoba, Winnipeg, Manitoba, Canada; fourth, fifth, sixth, and seventh authors: Canadian Food Inspection Agency, Ottawa, Ontario, Canada; ninth and tenth authors: Manitoba Corn Growers Association, Carman, Manitoba, Canada; and eleventh author: Department of Plant Pathology, University of Nebraska, Lincoln.
authors.forename: Stephan
authors.initials: S
authors.lastname: Brière
authors.affiliation: First, second, and eighth authors: Ottawa Research and Development Centre, Agriculture and Agri-Food Canada, Ottawa, Ontario K1A 0C6, Canada; third author: Department of Plant Science, University of Manitoba, Winnipeg, Manitoba, Canada; fourth, fifth, sixth, and seventh authors: Canadian Food Inspection Agency, Ottawa, Ontario, Canada; ninth and tenth authors: Manitoba Corn Growers Association, Carman, Manitoba, Canada; and eleventh author: Department of Plant Pathology, University of Nebraska, Lincoln.
authors.forename: Guillaume J
authors.initials: GJ
authors.lastname: Bilodeau
authors.affiliation: First, second, and eighth authors: Ottawa Research and Development Centre, Agriculture and Agri-Food Canada, Ottawa, Ontario K1A 0C6, Canada; third author: Department of Plant Science, University of Manitoba, Winnipeg, Manitoba, Canada; fourth, fifth, sixth, and seventh authors: Canadian Food Inspection Agency, Ottawa, Ontario, Canada; ninth and tenth authors: Manitoba Corn Growers Association, Carman, Manitoba, Canada; and eleventh author: Department of Plant Pathology, University of Nebraska, Lincoln.
authors.forename: Raymond
authors.initials: R
authors.lastname: Tropiano
authors.affiliation: First, second, and eighth authors: Ottawa Research and Development Centre, Agriculture and Agri-Food Canada, Ottawa, Ontario K1A 0C6, Canada; third author: Department of Plant Science, University of Manitoba, Winnipeg, Manitoba, Canada; fourth, fifth, sixth, and seventh authors: Canadian Food Inspection Agency, Ottawa, Ontario, Canada; ninth and tenth authors: Manitoba Corn Growers Association, Carman, Manitoba, Canada; and eleventh author: Department of Plant Pathology, University of Nebraska, Lincoln.
authors.forename: Allison
authors.initials: A
authors.lastname: Hartke
authors.affiliation: First, second, and eighth authors: Ottawa Research and Development Centre, Agriculture and Agri-Food Canada, Ottawa, Ontario K1A 0C6, Canada; third author: Department of Plant Science, University of Manitoba, Winnipeg, Manitoba, Canada; fourth, fifth, sixth, and seventh authors: Canadian Food Inspection Agency, Ottawa, Ontario, Canada; ninth and tenth authors: Manitoba Corn Growers Association, Carman, Manitoba, Canada; and eleventh author: Department of Plant Pathology, University of Nebraska, Lincoln.
authors.forename: Lana M
authors.initials: LM
authors.lastname: Reid
authors.affiliation: First, second, and eighth authors: Ottawa Research and Development Centre, Agriculture and Agri-Food Canada, Ottawa, Ontario K1A 0C6, Canada; third author: Department of Plant Science, University of Manitoba, Winnipeg, Manitoba, Canada; fourth, fifth, sixth, and seventh authors: Canadian Food Inspection Agency, Ottawa, Ontario, Canada; ninth and tenth authors: Manitoba Corn Growers Association, Carman, Manitoba, Canada; and eleventh author: Department of Plant Pathology, University of Nebraska, Lincoln.
authors.forename: Morgan
authors.initials: M
authors.lastname: Cott
authors.affiliation: First, second, and eighth authors: Ottawa Research and Development Centre, Agriculture and Agri-Food Canada, Ottawa, Ontario K1A 0C6, Canada; third author: Department of Plant Science, University of Manitoba, Winnipeg, Manitoba, Canada; fourth, fifth, sixth, and seventh authors: Canadian Food Inspection Agency, Ottawa, Ontario, Canada; ninth and tenth authors: Manitoba Corn Growers Association, Carman, Manitoba, Canada; and eleventh author: Department of Plant Pathology, University of Nebraska, Lincoln.
authors.forename: Tammy
authors.initials: T
authors.lastname: Cote
authors.affiliation: First, second, and eighth authors: Ottawa Research and Development Centre, Agriculture and Agri-Food Canada, Ottawa, Ontario K1A 0C6, Canada; third author: Department of Plant Science, University of Manitoba, Winnipeg, Manitoba, Canada; fourth, fifth, sixth, and seventh authors: Canadian Food Inspection Agency, Ottawa, Ontario, Canada; ninth and tenth authors: Manitoba Corn Growers Association, Carman, Manitoba, Canada; and eleventh author: Department of Plant Pathology, University of Nebraska, Lincoln.
authors.forename: Irina
authors.initials: I
authors.lastname: Agarkova
mesh_terms: D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004275:DNA, Ribosomal / Q000737:chemistry / Q000235:genetics; D016680:Genome, Bacterial / Q000235:genetics*; D008835:Micrococcaceae / Q000235:genetics* / Q000302:isolation & purification; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods; D010802:Phylogeny; D010935:Plant Diseases / Q000382:microbiology*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D003313:Zea mays / Q000382:microbiology*
chemical_list: D004269:DNA, Bacterial; D004275:DNA, Ribosomal
keywords: pseudogenes; stacking energy