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ARS Home » Southeast Area » Fort Pierce, Florida » U.S. Horticultural Research Laboratory » Subtropical Plant Pathology Research » Research » Publications at this Location » Publication #366171
Research Project: Mitigating High Consequence Domestic, Exotic, and Emerging Diseases of Fruits, Vegetables, and Ornamentals

Location: Subtropical Plant Pathology Research

Title: A Viability-qPCR Test for Quantifying Living Pathogens of Bacterial Spot in Tomato Seed

Author
item WANG, HEHE - Clemson University
item VALLAD, GARY - University Of Florida
item JONES, JEFF - University Of Florida
item Turechek, William

Submitted to: American Phytopathological Society Abstracts
Publication Type: Abstract Only
Publication Acceptance Date: 7/15/2019
Publication Date: 8/2/2019
Citation: Wang, H., Vallad, G., Jones, J., Turechek, W. 2019. A Viability-qPCR Test for Quantifying Living Pathogens of Bacterial Spot in Tomato Seed. American Phytopathological Society Abstracts. 2019.

Interpretive Summary: Bacterial spot is one of the most serious diseases of tomato. 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. 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. The assay was shown to accurately quantify viable cells within cell mixtures. This assay provides a sensitive, specific, and cost-effective way to certify tomato seed free of the pathogens causing bacteria spot.

Technical Abstract: 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 as 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 pathogens causing bacteria spot.