title: Simple detection of Pythium irregulare using loop-mediated isothermal amplification assay.
abstract: Pythium irregulare is an important soil-borne pathogen that causes seed, stem and root rot, and seedling damping-off in various crops. Here, we have developed a rapid and reliable approach for detecting the pathogen using loop-mediated isothermal amplification (LAMP) in combination with primers designed from the sequences of the P. irregulare ribosomal DNA internal transcribed spacer region. The specificity of the primers for P. irregulare was tested using 50 isolates of 40 Pythium species, 11 Phytophthora isolates and 8 isolates of 7 other soil-borne pathogens. The assay showed that the limit of sensitivity of the LAMP method was 100 fg of pure DNA, a similar level to that of a polymerase chain reaction. LAMP detected P. irregulare from the supernatant after mixing culture medium (template DNA source) with distilled water. Similarly, positive results were obtained using a 'Plant-LAMP' method applied to a suspension rotted roots in water. A 'Bait-LAMP' method using the supernatant of autoclaved perilla seeds incubated in a soil/water mixture for 1 week at 25°C successfully detected P. irregulare from the soil. The LAMP assay described in this study is therefore a simple and effective way for practical detection of P. irregulare.
authors.affiliation: Graduate School of Applied Biological Sciences, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan feng@green.gifu-u.ac.jp wenzhuofeng@126.com.
authors.forename: Wenzhuo
authors.initials: W
authors.lastname: Feng
authors.affiliation: River Basin Research Center, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan.
authors.forename: Yasushi
authors.initials: Y
authors.lastname: Ishiguro
authors.affiliation: River Basin Research Center, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan.
authors.forename: Keisuke
authors.initials: K
authors.lastname: Hotta
authors.affiliation: Gifu Prefectural Agricultural Technology Center, Matamaru, Gifu 501-1152, Japan.
authors.forename: Hideki
authors.initials: H
authors.lastname: Watanabe
authors.affiliation: Life Science Research Center, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan.
authors.forename: Haruhisa
authors.initials: H
authors.lastname: Suga
authors.affiliation: River Basin Research Center, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan.
authors.forename: Koji
authors.initials: K
authors.lastname: Kageyama
mesh_terms: D004247:DNA; D017931:DNA Primers; D021903:DNA, Ribosomal Spacer; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D010838:Phytophthora / Q000235:genetics; D010935:Plant Diseases / Q000469:parasitology; D018517:Plant Roots / Q000382:microbiology / Q000469:parasitology; D011775:Pythium / Q000302:isolation & purification*; D012680:Sensitivity and Specificity; D012987:Soil / Q000469:parasitology*
chemical_list: D017931:DNA Primers; D021903:DNA, Ribosomal Spacer; D012987:Soil; D004247:DNA
keywords: Pythium irregulare; detection; diagnosis; loop-mediated isothermal amplification