title: Multilaboratory Comparison of Quantitative PCR Assays for Detection and Quantification of Fusarium virguliforme from Soybean Roots and Soil.
abstract: The ability to accurately detect and quantify Fusarium virguliforme, the cause of sudden death syndrome (SDS) in soybean, in samples such as plant root tissue and soil is extremely valuable for accurate disease diagnoses and to address research questions. Numerous quantitative real-time polymerase chain reaction (qPCR) assays have been developed for this pathogen but their sensitivity and specificity for F. virguliforme have not been compared. In this study, six qPCR assays were compared in five independent laboratories using the same set of DNA samples from fungi, plants, and soil. Multicopy gene-based assays targeting the ribosomal DNA intergenic spacer (IGS) or the mitochondrial small subunit (mtSSU) showed relatively high sensitivity (limit of detection [LOD] = 0.05 to 5 pg) compared with a single-copy gene (FvTox1)-based assay (LOD = 5 to 50 pg). Specificity varied greatly among assays, with the FvTox1 assay ranking the highest (100%) and two IGS assays being slightly less specific (95 to 96%). Another IGS assay targeting four SDS-causing fusaria showed lower specificity (70%), while the two mtSSU assays were lowest (41 and 47%). An IGS-based assay showed consistently highest sensitivity (LOD = 0.05 pg) and specificity and inclusivity above 94% and, thus, is suggested as the most useful qPCR assay for F. virguliforme diagnosis and quantification. However, specificity was also above 94% in two other assays and their selection for diagnostics and research will depend on objectives, samples, and materials used. These results will facilitate both fundamental and disease management research pertinent to SDS.
authors.affiliation: First, twelfth, and thirteenth authors: Department of Plant Pathology and Microbiology, Iowa State University, Ames 50011; second and ninth authors: United States Department of Agriculture-Agricultural Research Service, Urbana, IL 61801; third, fourth, and fifth authors: Department of Plant, Soil and Ag. Systems, Southern Illinois University, Carbondale 62901; sixth author: Department of Biochemistry and Molecular Biology, UNR 1664, N. Virginia St. MS 330, Reno, NV; seventh, and eighth authors: Department of Plant, Soil and Microbial Sciences, Michigan State University, East Lansing 48824; and tenth and eleventh authors: Department of Plant Pathology, University of Minnesota, St. Paul 55108.
authors.forename: Yuba R
authors.initials: YR
authors.lastname: Kandel
authors.affiliation: First, twelfth, and thirteenth authors: Department of Plant Pathology and Microbiology, Iowa State University, Ames 50011; second and ninth authors: United States Department of Agriculture-Agricultural Research Service, Urbana, IL 61801; third, fourth, and fifth authors: Department of Plant, Soil and Ag. Systems, Southern Illinois University, Carbondale 62901; sixth author: Department of Biochemistry and Molecular Biology, UNR 1664, N. Virginia St. MS 330, Reno, NV; seventh, and eighth authors: Department of Plant, Soil and Microbial Sciences, Michigan State University, East Lansing 48824; and tenth and eleventh authors: Department of Plant Pathology, University of Minnesota, St. Paul 55108.
authors.forename: James S
authors.initials: JS
authors.lastname: Haudenshield
authors.affiliation: First, twelfth, and thirteenth authors: Department of Plant Pathology and Microbiology, Iowa State University, Ames 50011; second and ninth authors: United States Department of Agriculture-Agricultural Research Service, Urbana, IL 61801; third, fourth, and fifth authors: Department of Plant, Soil and Ag. Systems, Southern Illinois University, Carbondale 62901; sixth author: Department of Biochemistry and Molecular Biology, UNR 1664, N. Virginia St. MS 330, Reno, NV; seventh, and eighth authors: Department of Plant, Soil and Microbial Sciences, Michigan State University, East Lansing 48824; and tenth and eleventh authors: Department of Plant Pathology, University of Minnesota, St. Paul 55108.
authors.forename: Ali Y
authors.initials: AY
authors.lastname: Srour
authors.affiliation: First, twelfth, and thirteenth authors: Department of Plant Pathology and Microbiology, Iowa State University, Ames 50011; second and ninth authors: United States Department of Agriculture-Agricultural Research Service, Urbana, IL 61801; third, fourth, and fifth authors: Department of Plant, Soil and Ag. Systems, Southern Illinois University, Carbondale 62901; sixth author: Department of Biochemistry and Molecular Biology, UNR 1664, N. Virginia St. MS 330, Reno, NV; seventh, and eighth authors: Department of Plant, Soil and Microbial Sciences, Michigan State University, East Lansing 48824; and tenth and eleventh authors: Department of Plant Pathology, University of Minnesota, St. Paul 55108.
authors.forename: Kazi Tariqul
authors.initials: KT
authors.lastname: Islam
authors.affiliation: First, twelfth, and thirteenth authors: Department of Plant Pathology and Microbiology, Iowa State University, Ames 50011; second and ninth authors: United States Department of Agriculture-Agricultural Research Service, Urbana, IL 61801; third, fourth, and fifth authors: Department of Plant, Soil and Ag. Systems, Southern Illinois University, Carbondale 62901; sixth author: Department of Biochemistry and Molecular Biology, UNR 1664, N. Virginia St. MS 330, Reno, NV; seventh, and eighth authors: Department of Plant, Soil and Microbial Sciences, Michigan State University, East Lansing 48824; and tenth and eleventh authors: Department of Plant Pathology, University of Minnesota, St. Paul 55108.
authors.forename: Ahmad M
authors.initials: AM
authors.lastname: Fakhoury
authors.affiliation: First, twelfth, and thirteenth authors: Department of Plant Pathology and Microbiology, Iowa State University, Ames 50011; second and ninth authors: United States Department of Agriculture-Agricultural Research Service, Urbana, IL 61801; third, fourth, and fifth authors: Department of Plant, Soil and Ag. Systems, Southern Illinois University, Carbondale 62901; sixth author: Department of Biochemistry and Molecular Biology, UNR 1664, N. Virginia St. MS 330, Reno, NV; seventh, and eighth authors: Department of Plant, Soil and Microbial Sciences, Michigan State University, East Lansing 48824; and tenth and eleventh authors: Department of Plant Pathology, University of Minnesota, St. Paul 55108.
authors.forename: Patricia
authors.initials: P
authors.lastname: Santos
authors.affiliation: First, twelfth, and thirteenth authors: Department of Plant Pathology and Microbiology, Iowa State University, Ames 50011; second and ninth authors: United States Department of Agriculture-Agricultural Research Service, Urbana, IL 61801; third, fourth, and fifth authors: Department of Plant, Soil and Ag. Systems, Southern Illinois University, Carbondale 62901; sixth author: Department of Biochemistry and Molecular Biology, UNR 1664, N. Virginia St. MS 330, Reno, NV; seventh, and eighth authors: Department of Plant, Soil and Microbial Sciences, Michigan State University, East Lansing 48824; and tenth and eleventh authors: Department of Plant Pathology, University of Minnesota, St. Paul 55108.
authors.forename: Jie
authors.initials: J
authors.lastname: Wang
authors.affiliation: First, twelfth, and thirteenth authors: Department of Plant Pathology and Microbiology, Iowa State University, Ames 50011; second and ninth authors: United States Department of Agriculture-Agricultural Research Service, Urbana, IL 61801; third, fourth, and fifth authors: Department of Plant, Soil and Ag. Systems, Southern Illinois University, Carbondale 62901; sixth author: Department of Biochemistry and Molecular Biology, UNR 1664, N. Virginia St. MS 330, Reno, NV; seventh, and eighth authors: Department of Plant, Soil and Microbial Sciences, Michigan State University, East Lansing 48824; and tenth and eleventh authors: Department of Plant Pathology, University of Minnesota, St. Paul 55108.
authors.forename: Martin I
authors.initials: MI
authors.lastname: Chilvers
authors.affiliation: First, twelfth, and thirteenth authors: Department of Plant Pathology and Microbiology, Iowa State University, Ames 50011; second and ninth authors: United States Department of Agriculture-Agricultural Research Service, Urbana, IL 61801; third, fourth, and fifth authors: Department of Plant, Soil and Ag. Systems, Southern Illinois University, Carbondale 62901; sixth author: Department of Biochemistry and Molecular Biology, UNR 1664, N. Virginia St. MS 330, Reno, NV; seventh, and eighth authors: Department of Plant, Soil and Microbial Sciences, Michigan State University, East Lansing 48824; and tenth and eleventh authors: Department of Plant Pathology, University of Minnesota, St. Paul 55108.
authors.forename: Glen L
authors.initials: GL
authors.lastname: Hartman
authors.affiliation: First, twelfth, and thirteenth authors: Department of Plant Pathology and Microbiology, Iowa State University, Ames 50011; second and ninth authors: United States Department of Agriculture-Agricultural Research Service, Urbana, IL 61801; third, fourth, and fifth authors: Department of Plant, Soil and Ag. Systems, Southern Illinois University, Carbondale 62901; sixth author: Department of Biochemistry and Molecular Biology, UNR 1664, N. Virginia St. MS 330, Reno, NV; seventh, and eighth authors: Department of Plant, Soil and Microbial Sciences, Michigan State University, East Lansing 48824; and tenth and eleventh authors: Department of Plant Pathology, University of Minnesota, St. Paul 55108.
authors.forename: Dean K
authors.initials: DK
authors.lastname: Malvick
authors.affiliation: First, twelfth, and thirteenth authors: Department of Plant Pathology and Microbiology, Iowa State University, Ames 50011; second and ninth authors: United States Department of Agriculture-Agricultural Research Service, Urbana, IL 61801; third, fourth, and fifth authors: Department of Plant, Soil and Ag. Systems, Southern Illinois University, Carbondale 62901; sixth author: Department of Biochemistry and Molecular Biology, UNR 1664, N. Virginia St. MS 330, Reno, NV; seventh, and eighth authors: Department of Plant, Soil and Microbial Sciences, Michigan State University, East Lansing 48824; and tenth and eleventh authors: Department of Plant Pathology, University of Minnesota, St. Paul 55108.
authors.forename: Crystal M
authors.initials: CM
authors.lastname: Floyd
authors.affiliation: First, twelfth, and thirteenth authors: Department of Plant Pathology and Microbiology, Iowa State University, Ames 50011; second and ninth authors: United States Department of Agriculture-Agricultural Research Service, Urbana, IL 61801; third, fourth, and fifth authors: Department of Plant, Soil and Ag. Systems, Southern Illinois University, Carbondale 62901; sixth author: Department of Biochemistry and Molecular Biology, UNR 1664, N. Virginia St. MS 330, Reno, NV; seventh, and eighth authors: Department of Plant, Soil and Microbial Sciences, Michigan State University, East Lansing 48824; and tenth and eleventh authors: Department of Plant Pathology, University of Minnesota, St. Paul 55108.
authors.forename: Daren S
authors.initials: DS
authors.lastname: Mueller
authors.affiliation: First, twelfth, and thirteenth authors: Department of Plant Pathology and Microbiology, Iowa State University, Ames 50011; second and ninth authors: United States Department of Agriculture-Agricultural Research Service, Urbana, IL 61801; third, fourth, and fifth authors: Department of Plant, Soil and Ag. Systems, Southern Illinois University, Carbondale 62901; sixth author: Department of Biochemistry and Molecular Biology, UNR 1664, N. Virginia St. MS 330, Reno, NV; seventh, and eighth authors: Department of Plant, Soil and Microbial Sciences, Michigan State University, East Lansing 48824; and tenth and eleventh authors: Department of Plant Pathology, University of Minnesota, St. Paul 55108.
authors.forename: Leonor F S
authors.initials: LF
authors.lastname: Leandro
mesh_terms: D005670:Fusarium / Q000235:genetics / Q000302:isolation & purification*; D018517:Plant Roots / Q000382:microbiology; D016133:Polymerase Chain Reaction / Q000379:methods / Q000706:statistics & numerical data*; D012988:Soil Microbiology; D013025:Glycine max / Q000382:microbiology*