3.3.42
MULTIPLEX PCR-BASED IMMUNODETECTION OF THE BACTERIAL RING ROT PATHOGEN CLAVIBACTER MICHIGANENSIS SUBSP. SEPIDONICUS

D MILLS and BW RUSSELL

Department of Botany and Plant Pathology, Oregon State University, Corvailis, OR 97330-2902 USA

Background and objectives
Bacterial ring rot disease of potato is caused by Clavibacter michiganensis subsp. sepedonicus (Cms), and is a major concern of producers of seed potatoes worldwide because of a zero tolerance policy. Current methods for its detection rely primarily upon the occurrence of disease symptoms in the field and the use of serological tests. Accurate identification of Cms is hindered by asymptomatic infection or atypical symptomatology, and serological procedures are compromised when population levels are below 10,000 cfu/gm of tissue. Moreover, certain nonmucoid variant cells of Cms may exhibit antigenic variation and escape serological detection. To develop a verifiable DNA-based detection system, three single copy, unique DNA fragments, Cms50, Cms72 and Cms85, were isolated from Cms strain Cs3 and shown by Southern analysis to be specific to 30 North American Cms strains tested, including plasmidless and nonmucoid strains [1]. Internal primer sets permitted PCR amplification of each fragment only from Cms DNA, regardless of the source. The objectives of this research were to: 1. Develop a multiplex PCR (MPCR) system of detection based on Cms50, Cms72 and Cms85; 2. Analyze abberrant strains whose classification as Cms was ambiguous by other methods; 3. Determine the efficacy of the MPCR system with infected field-grown potatoes and; 4. Develop a verifiable MPCRimmunodetection system for Cms-infected plants.

Results and conclusions
Optimal MPCR conditions were established for amplification of Cms-specific fragments, Cms50, Cms72, and Cms85. The conditions were as published for the individual primer sets [1] except that the reaction requires 2.5 mM magnesium chloride and an annealing temperature of 63 C. The three fragments were amplified by MPCR when frozen cells from each of 41 diverse Cms strains representing both mucoid and nomucoid cell types were added to the reaction mixture. To examine the efficacy of detecting Cms in field samples by MPCR, 30 stems were removed from mature plants derived from seed pieces inoculated with 10,000 Cms cells. In a reaction containing lnstagene TM matrix (Bio-Rad) prepared template to relieve PCR inhibition from plant components, all three fragments were amplified in 11/30 stem samples, one or two fragments were amplified in 6/30, and no fragments were amplified in 13/30 samples. However, the addition of acid hydrolysed casein protein at 0.05% (w/v) to the reaction mix completely relieved MPCR inhibition, and all three fragments were amplified in the 30 stem samples. The efficacy of this MPCR detection system was then determined for 10 strains whose identity as Cms was questionable based on serological tests and/or other analyses. Cms50, Cms72, and Cms85 fragments were amplified by MPCR in 8 of 10 strains that scored negative by one of the tests. Two strains were determined not to be Cms with MPCR, and these also were determined not to be Cms by more than one currently used Cms tests.

A multiplex MPCR-ELISA detection system was developed that uses Digoxigenin-li-dUTP to label Cms50, Cms72, and Cms85 fragments. These fragments were subsequently detected in a solution containing peroxidase-conjugated anti-digoxigenin and ABTS calorimetric substrate in separate tubes by the three biotinylated capture probes (Cp50, Cp72 and Cp85) which were anchored to a streptavidin-coated microtiter well. As little as 330 picograms of Dig-labeled DNA has been detected by Cp85, and all three PCR products have been detected in Cms-infected stems and tubers. This MPCR-based immunodetection detection system is verifiable at three loci in the Cms genome, it has uncompromised specificity and superior sensitivity.

References
1. Mills D, Russell BW, Hanus JW, 1997. Phytopathology 87,853-61.