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POSTERS: Pathogen detection, quantification and diagnosis

A viability-qPCR test for quantifying living pathogens of bacterial spot in tomato seed
Hehe Wang - Clemson University. William Turechek- USDA, ARS, U.S. Horticultural Research Laboratory, Jeffrey Jones- University of Florida, Gary Vallad- Gulf Coast Research and Education Center, University of Florida

Bacterial spot is one of the most serious diseases of tomato. It is caused by four different species of Xanthomonas: X. euvesicatoria, X. gardneri, X. perforans, and X. vesicatoria. Contaminated and/or infected seed serve as a major inoculum source for this disease. The use of certified pathogen-free seed is one of the primary management practices to reduce the inoculum load in commercial production. However, current seed certification protocols rely mainly on culturing and PCR, and both methods have limited capability to accurately quantify viable pathogen cells. To improve the seed certification process, a viability-qPCR assay in conjunction with a crude DNA extraction procedure was developed to selectively quantify viable xanthomonads in tomato seed. A DNA-binding dye, propidium monoazide (PMA), was used to treat seed extract where it selectively binds to the DNA of dead cells, thus preventing the DNA from dead cells from being amplified in downstream qPCR. The procedure was tested with tomato seed samples spiked with mixtures of viable and dead cells in different concentrations and proportions. The assay accurately quantified viable cells within mixtures of 1:10,000 (live:dead), and use of the crude DNA extract provided results similar to those obtained using purified DNA prepared from commercial kits. This assay will provide a sensitive, specific, and cost-effective way to certify tomato seed free of the bacteria spot pathogens.