Comparison of Dig-Labeled PCR, Nested PCR, and ELISA for the Detection of Clavibacter michiganensis subsp. sepedonicus in Field-Grown Potatoes

Bacterial ring rot disease, caused by the gram-positive bacterium C. michiganensis subsp. sepedonicus, is readily spread by planting infected seed potatoes. Zero tolerance levels have been set in the United States, Canada, and the European Union in an effort to eradicate the disease (26). Despite zero tolerance levels that have existed for 50 years in the United States, potato ring rot remains one of the most important pathogens in U.S. seed potato lots. The difficulty in controlling the spread of C. michiganensis subsp. sepedonicus may be due to latent infection that escapes detection (8,9,16,27,32). Strong efforts have been made to develop efficient and sensitive assays for commercial detection of C. michiganensis subsp. sepedonicus in seed potato. Serological assays, particularly enzyme-linked immunosorbent assay (ELISA), are the most commonly used methods for indexing of potato seed lots. The popularity of serological assays for the indexing of potatoes is largely due to their simplicity of operation and their applicability in largescale indexing (10,12,16,33). One major drawback of serological assays, however, is false positives caused by cross reaction of antibodies with plant debris or nonpathogenic bacteria (6,7,10,21). Although cross reactions can be greatly reduced by use of specific monoclonal antibodies, they remain a problem (11,16,20,22). Furthermore, antigenic variation among the strains of C. michiganensis subsp. sepedonicus has increased the difficulty in developing an antibody with the same detection efficiency for all strains (2,10,11). An additional problem inherent with ELISA is the need to set an appropriate threshold level to compensate for the nonspecific reactions, which can lessen the sensitivity of the test (16,33). Confirmation of C. michiganensis subsp. sepedonicus in positive samples from serological assays with an eggplant bioassay is often necessary (3). Because eggplant bioassay is laborious and time-consuming, it is unsuitable for largescale use. In addition, some eggplants may be subject to the same latent infection with C. michiganensis subsp. sepedonicus as potato (10). Currently, confirmation is often carried out by the more specific immunofluorescence assay (IFA) using monoclonal antibodies to a somatic antigen of the bacteria (10,11). Although significantly improved sensitivity and specificity have been reported, IFA is not completely free of the problems of cross-reactivity with saprophytic bacteria (16,20). DNA hybridization has been suggested as an alternative method to index seed potato lots (15,17,22,23,28,33). Although highly specific probes have been developed, the sensitivity of the assay remains a problem. DNA-based polymerase chain reaction (PCR) assays developed recently are a promising alternative to current assays (14,28–31,33,34). C. michiganensis subps. sepedonicus-specific oligonucleotide primers have been designed from 16S/23S rRNA intergenic spacer region (20), selected chromosomal regions and the 1.3-kb insertion element IS1121, a highly repetitive segment of DNA that is present in plasmid pCS1 and integrated into the chromosome of C. michiganensis subsp. sepedonicus (4,5,24,25). In a previous study, nested PCR with two sets of primer pairs designed from the IS1121 insertion element was used to detect C. michiganensis subsp. sepedonicus in infected field potato tubers and symptomless tubers from supermarkets (18). The nested PCR assay proved to be a highly sensitive method for detecting very low titers of C. michiganensis subsp. sepedonicus (18). The tediousness of nested PCR, however, makes largescale use difficult. A practical, ultrasensitive method for the detection of C. michiganensis subsp. sepedonicus is needed. In this study, a simple detection method was developed involving an alkaline extraction of C. michiganensis subsp. sepedonicus DNA (36), digoxigenin(dig-) labeled PCR, and colorimetric detection. The method is simple, specific, and highly sensitive. As such, dig-labeled PCR is highly suitable to confirm serological tests for detecting ring rot bacteria and has the potential to be adapted for large-scale indexing of potato seed lots. This method was compared with ELISA and nested PCR using identical tuber and stem samples. A preliminary report on this study was published previously (19).