Comparison of PCR, ELISA, and DNA Hybridization for the Detection ofClavibacter michiganensissubsp.sepedonicusin Field-Grown Potatoes

Abstract

Clavibacter michiganensis subsp. sepedonicus, the causal agent of bacterial ring rot, was specifically detected in field-grown potatoes by means of the polymerase chain reaction (PCR). A 20-bp synthetic oligomer, derived from an inverted repeat region of the repeated sequence of the bacterial plasmid pCS1, was used as the PCR primer. In assays of pure bacterial cultures, this system gave positive reactions with all strains of C. michiganensis subsp. sepedonicus tested and had a sensitivity of <10 CFU per PCR reaction. With the exception of 4 out of 12 strains of C. michiganensis subsp. insidiosus, no other species of bacteria tested produced a specific PCR product. The ability of PCR to detect C. michiganensis subsp. sepedonicus in field material was evaluated by testing sections of potato stems that were grown in New York and North Dakota from seed pieces of potato cultivars BelRus and Russet Burbank inoculated with 0, 10 2 , or 10 9 CFU C. michiganensis subsp. sepedonicus and destructively sampled at 35 and 90 days after planting (DAP). Parallel tests of these samples by enzyme-linked immunosorbent assay (ELISA) and DNA hybridization assay (DHA) were conducted for comparative purposes. Overall, 36.2, 35.8, and 29.1% of inoculated samples tested positive by PCR, ELISA, and DHA, respectively. Each assay was affected significantly by inoculum dose, cultivar, and sampling date (P = 0.0001), with detection approaching 100% for the combination of the following parameters: 90 DAP, susceptible cv. Russet Burbank, and 10 9 CFU inoculum level. None of the buffer-inoculated plants tested positive with either PCR or DHA, whereas ELISA results were highly dependent upon the positive-negative threshold used.