title: Simultaneous detection and quantification of Phytophthora nicotianae and P. cactorum, and distribution analyses in strawberry greenhouses by duplex real-time PCR.
abstract: Phytophthora nicotianae and P. cactorum cause Phytophthora rot of strawberry. A duplex real-time PCR technique for simultaneous detection and quantification of the two pathogens was developed. Species-specific primers for P. nicotianae and P. cactorum were designed based on the internal transcribed spacer regions (ITS) of rDNA and the ras-related protein gene Ypt1, respectively. TaqMan probes were labeled with FAM for P. nicotianae and HEX for P. cactorum. Specificities were demonstrated using 52 isolates, including various soil-borne pathogens. Sensitivities for P. nicotianae and P. cactorum DNAs were 10 fg and 1 pg, respectively. The technique was applied to naturally infested soil and root samples; the two pathogens were detected and the target DNA concentrations were quantified. Significant correlations of DNA quantities in roots and the surrounding soils were found. The minimum soil DNA concentration predicting the development of disease symptoms was estimated as 20 pg (g soil)(-1). In three strawberry greenhouses examined, the target DNA concentrations ranged from 1 to 1,655 pg (g soil)(-1) for P. nicotianae and from 13 to 233 pg (g soil)(-1) for P. cactorum. The method proved fast and reliable, and provides a useful tool to monitor P. nicotianae and P. cactorum in plants or soils.
authors.affiliation: River Basin Research Center, Gifu University, Gifu 501–1193, Japan.
authors.forename: Mingzhu
authors.initials: M
authors.lastname: Li
authors.forename: Minoru
authors.initials: M
authors.lastname: Inada
authors.forename: Hideki
authors.initials: H
authors.lastname: Watanabe
authors.forename: Haruhisa
authors.initials: H
authors.lastname: Suga
authors.forename: Koji
authors.initials: K
authors.lastname: Kageyama
mesh_terms: D015169:Colony Count, Microbial / Q000379:methods*; D017931:DNA Primers / Q000235:genetics; D004271:DNA, Fungal / Q000235:genetics; D021903:DNA, Ribosomal Spacer / Q000235:genetics; D031985:Fragaria / Q000254:growth & development / Q000382:microbiology*; D005656:Fungal Proteins / Q000235:genetics; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D010838:Phytophthora / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D010935:Plant Diseases / Q000382:microbiology; D018517:Plant Roots / Q000382:microbiology; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D012988:Soil Microbiology; D013997:Time Factors
chemical_list: D017931:DNA Primers; D004271:DNA, Fungal; D021903:DNA, Ribosomal Spacer; D005656:Fungal Proteins