title: Development and application of a loop-mediated isothermal amplification assay for rapid detection of Pythium helicoides.
abstract: Root rot of poinsettia, caused by Pythium helicoides at high temperatures in hydroponic cultures, has become a serious problem in many parts of the world. We have developed a species-specific, loop-mediated isothermal amplification (LAMP) assay for the rapid diagnosis of this pathogen. The primers were designed using the ribosomal DNA internal transcribed spacer sequence. Primer specificity was established using 40 Pythium species including P. helicoides, 11 Phytophthora species, and eight other soil-borne pathogens. A sensitivity test was carried out using genomic DNA extracted from P. helicoides, and the detection limit was c. 100 fg which is comparable to that of the polymerase chain reaction (PCR). In addition, we tested the ease of pathogen detection in poinsettia roots. The LAMP results were consistent with those from the conventional plating method and showed more sensitivity than the PCR results. Consequently, the LAMP method developed in this study is effective for the rapid and easy detection of P. helicoides.
authors.affiliation: Agri-environmental Division, Aichi Agricultural Research Center, Nagakute, Aichi, Japan.
authors.forename: Reiko
authors.initials: R
authors.lastname: Takahashi
authors.forename: Shiro
authors.initials: S
authors.lastname: Fukuta
authors.forename: Satoru
authors.initials: S
authors.lastname: Kuroyanagi
authors.forename: Noriyuki
authors.initials: N
authors.lastname: Miyake
authors.forename: Hirofumi
authors.initials: H
authors.lastname: Nagai
authors.forename: Koji
authors.initials: K
authors.lastname: Kageyama
authors.forename: Yasushi
authors.initials: Y
authors.lastname: Ishiguro
mesh_terms: D017931:DNA Primers / Q000235:genetics; D021903:DNA, Ribosomal Spacer / Q000235:genetics; D028481:Euphorbia / Q000382:microbiology; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D009868:Oomycetes / Q000235:genetics / Q000302:isolation & purification*; D010935:Plant Diseases / Q000382:microbiology; D018517:Plant Roots / Q000382:microbiology; D012680:Sensitivity and Specificity
chemical_list: D017931:DNA Primers; D021903:DNA, Ribosomal Spacer
keywords: Pythium helicoides; hydroponic culture; loop-mediated isothermal amplification