Detection of Clavibacter michiganensis subsp. sepedonicus in Potato Tubers by BIO-PCR and an Automated Real-Time Fluorescence Detection System
Open Access
- 1 December 1999
- journal article
- research article
- Published by Scientific Societies in Plant Disease
- Vol. 83 (12), 1095-1100
- https://doi.org/10.1094/pdis.1999.83.12.1095
Abstract
Ring rot of potato, caused by Clavibacter michiganensis subsp. sepedonicus, is one of the most regulated diseases of potatoes world wide. The organism is often difficult to detect in symptomless tubers because of low populations and slow competitive growth on available media. Polymerase chain reaction (PCR) primers and a fluorescent probe for use in the Perkin Elmer 7700 automated real time PCR detection system (TaqMan) were designed from a C. michiganensis subsp. sepedonicus-specific genomic DNA fragment for development of a BIO-PCR assay for C. michiganensis subsp. sepedonicus in potato tubers. Results of screening the primers with strains of C. michiganensis subsp. sepedonicus and other bacteria showed the primers to be specific. A total of 30 naturally infected ring rot suspect tubers were sampled by the core extract, shaker incubation procedure and assayed by (i) plating aliquots onto agar media, (ii) classical PCR, and (iii) BIO-PCR. In all, 4 tubers were positive by agar plating and pathogenicity tests, 8 by classical TaqMan PCR, and 26 by TaqMan BIO-PCR. We conclude that BIO-PCR combined with the TaqMan automated closed detection system is a rapid, reliable method of assaying large numbers of potato tuber extracts for C. michiganensis subsp. sepedonicus. Furthermore, for a large central laboratory running large numbers of PCR assays, the high-throughput TaqMan system can reduce costs per sample over the more labor-intensive classical PCR.Keywords
This publication has 17 references indexed in Scilit:
- Specific Detection of Clavibacter michiganensis subsp. sepedonicus by Amplification of Three Unique DNA Sequences Isolated by Subtraction HybridizationPhytopathology®, 1997
- Comparison of PCR, ELISA, and DNA Hybridization for the Detection ofClavibacter michiganensissubsp.sepedonicusin Field-Grown PotatoesPlant Disease, 1996
- Oligonucleotides with fluorescent dyes at opposite ends provide a quenched probe system useful for detecting PCR product and nucleic acid hybridization.Genome Research, 1995
- A Combined Biological and Enzymatic Amplification (BIO-PCR) Technique to DetectPseudomonas syringaepv.phaseolicolain Bean Seed ExtractsPhytopathology®, 1995
- Identification of Clavibacter michiganensis subsp. sepedonicus using the polymerase chain reactionCanadian Journal of Microbiology, 1994
- Specific Detection ofPseudomonas syringaepv.phaseolicolaDNA in Bean Seed by Polymerase Chain Reaction-Based Amplification of a Phaseolotoxin Gene RegionPhytopathology®, 1993
- Serological Detection of Nonmucoid Strains ofClavibacter michiganensissubsp.sepedonicusin PotatoPhytopathology®, 1993
- A Semiselective Agar Medium for Isolation ofClavibacter michiganensissubsp.sepedonicusfrom Potato TissuesPlant Disease, 1992
- An ELISA Test for Bacterial Ring Rot of Potato with a New Monoclonal AntibodyPlant Disease, 1988
- Current Status and Prospects for Detecting and Controlling Bacterial Ring Rot of Potatoes in North AmericaPlant Disease, 1984