Parameters for specific detection of Clavibacter michiganensis subsp. sepedonicus in potato stems and tubers by multiplexed PCR-ELISA

American Journal of Potato Research, Jul/Aug 2003 by Mills, Dallice, Russell, Brian W

Accepted for publication 31 March 2003.

ABSTRACT

A multiplex PCR-ELISA protocol for detection of Clavibacter michiganensis subsp. sepedonicus (Cms) was developed that is based on primers for amplification of three single-copy, unique DNA sequences, Cms50, Cms72, and Cms85. The three sequences were simultaneously amplified from the genomes of all 42 strains of Cms that were tested including variant mucoid forms, but not from strains representing five related subspecies, and Rathayibacter rathayi and Rhodococcus faciens. The lowest limit of detection by gel electrophoresis was estimated to be approximately 300 CFU per mL when cells were spiked into potato core fluid, but sensitivity increases approximately 10-fold using PCR-ELISA. Inclusion of a sea anemone DNA fragment engineered so it could be amplified from the Cms72 primer set provided the simultaneous signal that the system functioned properly when any sample was free of the pathogen. The addition of hydrolyzed casein to the reaction mix was demonstrated to markedly reduce or eliminate inhibition of PCR by plant cell components or contaminants. Multiplex PCR-ELISA detection of Cms was determined to be verifiable for analysis of both stems and tubers based on the amplification of multiple sites in its genome, it provides absolute specificity, and it was more sensitive than detection based on gel electrophoresis of PCR products and serological approaches.

ADDITIONAI, KEY WORDS: Bacterial ring rot, Cms-specific DNA, multiple-site PCR amplification.

INTRODUCTION

A worldwide policy of zero tolerance for the presence of Clavibacter michiganensis subsp. sepedonicus (Cms) in seed stock is presently in effect to reduce the spread of bacterial ring rot disease of potato. Proper implementation of this policy requires methods of detection that are both highly sensitive and absolutely specific for Cms. Detection of Cms has been difficult because it is frequently present in potato tubers and stems in low numbers and variant morphological forms can compromise certain serological methods of detection. The indirect fluorescent antibody staining assay (IFAS) (De Boer and Copeman 1980; De Boer and Hall 1988; Slack et al. 1979) and an enzyme-linked immunosorbent assay (ELISA) (De Boer et al. 1988) are typically used for detection. The MAb 9A1 antibody used in IFAS assays is highly specific (De Boer and Wieczorek 1984) and the detection sensitivity is estimated to no less than 10^sup 4^ colony-forming units/mL (cfu/mL) (Baer and Gudmestad 1993). However, detection by ELISA has shown cross-reactivity with other unidentified microbes (Crowley and De Boer 1982) and an inability to detect nonmucoid Cms strains (Baer and Gudmestad 1993).

DNA-based methods of Cms detection have recently been examined in an effort to increase sensitivity and retain high specificity. PCR primers have been developed to amplify sequences of an indigenous plasmid of Cms (Schneider et al., 1993; Firrao and Locci 1994) Rademaker and Janse 1994), as well as sequences within the RNA intergenic transcribed spacer region (Li and De Boer 1995) and single-copy DNA sequences (Mills et al. 1997). PCR primers for amplification of three single-copy Cms DNA sequences, designated Cms50, Cms72 and Cms85 (Mills et al. 1997) were previously shown by Southern hybridization to have no homology with DNA from other species of bacteria tested, including Clavibacter subsp. insidiosus, michiganensis and tessellarius, Pseudomonas syringae, pv. syringae, Erwinia carotovora subsp. carotovora, and Agrobacterium tumefaciens, nor with DNA from other microbes that have been shown to cross-react with antisera made to Cms. Moreover, PCR detection of Cms in potato core fluid was also highly sensitive and approximately 100 cfu of potato core fluid were detected with the Cms85 primers.

Amplification of single-copy, unique DNA sequences provides an opportunity to verify the presence or absence of the pathogen with molecular probes. DNA probes constructed to have homology with one of the DNA strands of the PCR product will not form hybrids with spuriously amplified DNA from other species. Using the primer sets for amplification of Cms50 and Cms72, Baer et al. (2001) applied previously reported protocols (Mills and Russell 1997) to compare the efficacy of PCR-ELISA (enzyme-linked oligonucleosorbent assay) with serological approaches. They showed that this system has greater sensitivity than serological methods of testing, and that its specificity was uncompromised when challenged with strains from other subspecies of Clavibacter and strains of other species that are problematic in serological tests. However, their results also revealed certain inconsistencies in the ability of the system to detect Cms that could be attributed to problems with PCR inhibition rather than the limits of detection by this system. In certain cases where a dilution series was examined, cells were detected at both high and low dilution, but not at intermediate levels. These results are characteristic of a system that is lacking a component for the identification of samples where the PCR has failed for a variety of reasons.