CONFIRMATION OF CLAVIBACTER MICHIGANENSIS SUBSP. SEPEDONICUS INFECTIONS USING A MULTIPLEX REAL-TIME PCR ASSAY FOR RING ROT DIAGNOSIS (NDSU)
2009 Annual Report
1a.Objectives (from AD-416)
Confirmation of Clavibacter michiganensis subsp. sepedonicus infections using a multiplex real-time PCR assay for ring rot diagnosis and potato seed lot screening.
1b.Approach (from AD-416)
Real-time PCR primers and fluorescently labeled Taqman probes were previously developed for detection of three unique areas of the bacterial genome of C. michiganensis subsp. sepedonicus (Cms), the causal agent of bacterial ring rot (BRR) of potato. These genomic regions code for cellulose genes (CelA and CelB) that are primary virulence determinants for Cms. These reagents will be used for additional screening of bacterial potato endophytes by a multiplex real-time PCR to verify specificity and reliability of the procedure for Cms detection and identification. Documets SCA with N. Dakota State University. Formerly 5354-21220-002-20S (5/08).
Clavibacter michiganensis subsp. sepedonicus, causal agent of bacterial ring rot (BRR) of potato (Solanum tuberosum), is a globally important quarantine pathogen that is managed in North America using zero tolerance regulations in the certified seed industry. C. michiganensis subsp. sepedonicus is well documented to cause symptomless infections in potato, contributing to its persistence in certified seed stocks. Reliable laboratory methods to detect symptomless infections with a high degree of sensitivity could assist in the reduction of inoculum in certified seed potato stocks. A real-time polymerase chain reaction (PCR) assay was developed using the cellulase A (CelA) gene sequence as the basis for primer design. CelA primers were specific to C.michiganensis subsp. sepedonicus grown in vitro and did not detect any other coryneform bacteria or potato pathogenic bacteria but did detect 69 strains of C. michiganensis subsp. sepedonicus. The CelA real-time PCR assay was more sensitive than immunofluorescence (IFA) and Cms50/72a PCR assays in detecting C. michiganensis subsp. sepedonicus in infected potato tuber cores blended with healthy tuber cores in simulated seed lot contamination experiments. CelA primers detected nonmucoid and mucoid strains with equivalent sensitivity. In naturally infected seed lots, CelA PCR primers also were more sensitive in detecting symptomless infections of C. michiganensis subsp. sepedonicus in seed tubers prior to planting compared to Cms50/72a PCR primers, IFA, and enzyme-linked immunosorbent assay. A real-time PCR format using the newly developed CelA primers proved to be a very robust detection tool for C. michiganensis subsp. sepedonicus with the added advantage of detecting only virulent strains of the ring rot bacterium. Progress on this project was monitored by the ADODR via phone and email correspondence with North Dakota State University's lead researcher.