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.
Clavibacter michiganensis ssp. sepedonicus (Cms), causal agent of bacterial ring rot (BRR) of potato, is a globally important quarantine pathogen that is managed in North America using zero tolerance regulations in the certified seed industry. Cms is well documented to cause symptomless infections in potato, contributing to its persistence in certified seed stocks. Reliable laboratory methods to detect symptomless infections of Cms 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) pathogenicity gene as the basis for primer development. CelA primers were specific to Cms grown in vitro and did not detect any other coryneform bacteria or potato pathogenic bacteria but did detect 69 isolates of Cms. The CelA PCR primers were determined to be more sensitive than Cms50/72a primers in detecting in vitro grown Cms cells in both classical and real-time PCR but no differences in detection sensitivity were found between these two primer sets when purified DNA was used. The CelA PCR assay was more sensitive than other methods in detecting Cms in infected potato tuber cores blended with healthy tuber cores in simulated seed lot contamination experiments. In preliminary experiments using real-time PCR, CelA PCR assays were capable of detecting two asymptomatic Cms-infected tuber cores blended with 198 healthy cores while the limit of detection in these experiments for immunofluorescence (IFA) and Cms50/72a PCR primers were 10 infected cores in 190 healthy cores. In additional experiments, CelA primers detected one asymptomatic Cms-infected tuber in 400 as efficiently as Cms50/72a primers detected one infected tuber in 100 tubers. CelA primers detected nonmucoid and mucoid strains of Cms with equivalent sensitivity. In naturally infected seed lots, CelA PCR assay was also more sensitive in detecting symptomless infections of Cms 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 Cms and has an added advantage of detecting only virulent isolates of the ring rot bacterium. Additional studies will concentrate on developing a multiplex PCR reaction using the CelA primers with Cms50/72a primers as well as the potential use of an internal reaction control that can be integrated into the TaqMan PCR assay.
Progress on this project was monitored by the ADODR via phone and email correspondence with North Dakota State University's lead researcher.