title: Detection and differentiation of Fusarium oxysporum f. sp. lycopersici race 1 using loop-mediated isothermal amplification with three primer sets.
abstract: Fusarium oxysporum f. sp. lycopersici (Fol) causes tomato wilt. Based on the difference in pathogenicity towards tomato cultivars, Fol is classified into three races. In this study, a rapid method is developed for the detection and discrimination of Fol race 1 using a loop-mediated isothermal amplification (LAMP) assay with two primer sets targeting a region of the nucleotide sequence of the SIX4 gene specific for race 1 and a primer set targeting the SIX5 gene, conserved in all known Fol isolates. Upon LAMP reaction, amplification using all three primer sets was observed only when DNA of Fol race 1 was used as a template, and not when DNA of other Fol races or other fungal species was used. This method could detect 300 fg of Fol race 1 DNA, a 100-fold higher sensitivity than that obtained by conventional PCR. The method can also detect DNA extracted from soil artificially infested with Fol race 1. It is now possible to detect Fol race 1 in colonies and infected tomato stems without DNA isolation. This method is a rapid and simple tool for discrimination of Fol race 1.
  
  
  CONCLUSIONS
  This study developed a loop-mediated isothermal amplification (LAMP) assay for detection and differentiation of Fusarium oxysporum f. sp. lycopersici (Fol) race 1 by using three primer sets targeting for the SIX4 and SIX5 genes. These genes are present together only in Fol race 1. This method can detect Fol race 1 in infected tomato stems without DNA extraction, affording an efficient diagnosis of Fusarium wilt on tomatoes in the field.
authors.affiliation: United Graduate School of Agricultural Science, Tokyo University of Agriculture and Technology (TUAT), Fuchu, Tokyo, Japan.|Laboratory of Plant Pathology, Graduate School of Agriculture, Tokyo University of Agriculture and Technology (TUAT), Fuchu, Tokyo, Japan.
authors.forename: Y
authors.initials: Y
authors.lastname: Ayukawa
authors.affiliation: Laboratory of Plant Pathology, Graduate School of Agriculture, Tokyo University of Agriculture and Technology (TUAT), Fuchu, Tokyo, Japan.
authors.forename: K
authors.initials: K
authors.lastname: Komatsu
authors.affiliation: Chemical Biology Research Group, RIKEN Center for Sustainable Resource Science (CSRS), Wako, Saitama, Japan.
authors.forename: T
authors.initials: T
authors.lastname: Kashiwa
authors.affiliation: Laboratory of Plant Pathology, Graduate School of Agriculture, Tokyo University of Agriculture and Technology (TUAT), Fuchu, Tokyo, Japan.
authors.forename: K
authors.initials: K
authors.lastname: Akai
authors.affiliation: Laboratory of Plant Pathology, Graduate School of Agriculture, Tokyo University of Agriculture and Technology (TUAT), Fuchu, Tokyo, Japan.
authors.forename: M
authors.initials: M
authors.lastname: Yamada
authors.affiliation: Laboratory of Plant Pathology, Graduate School of Agriculture, Tokyo University of Agriculture and Technology (TUAT), Fuchu, Tokyo, Japan.
authors.forename: T
authors.initials: T
authors.lastname: Teraoka
authors.affiliation: Laboratory of Plant Pathology, Graduate School of Agriculture, Tokyo University of Agriculture and Technology (TUAT), Fuchu, Tokyo, Japan.
authors.forename: T
authors.initials: T
authors.lastname: Arie
mesh_terms: D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D005670:Fusarium / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification*; D018551:Solanum lycopersicum / Q000382:microbiology*; D021141:Nucleic Acid Amplification Techniques; D010935:Plant Diseases / Q000382:microbiology*; D012988:Soil Microbiology
chemical_list: D017931:DNA Primers
keywords: Fusarium oxysporum f. sp. lycopersici race 1; SIX4; SIX5; annealing curve analysis; loop-mediated isothermal amplification; tomato wilt