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A Real-Time PCR Assay for Detection and Quantification of Verticillium dahliae in Spinach Seed

April 2012 , Volume 102 , Number  4
Pages  443 - 451

Dechassa Duressa, Gilda Rauscher, Steven T. Koike, Beiquan Mou, Ryan J. Hayes, Karunakaran Maruthachalam, Krishna V. Subbarao, and Steven J. Klosterman

First, second, fourth, fifth, and eighth authors: United States Department of Agriculture–Agricultural Research Service, 1636 E. Alisal St., Salinas, CA; third author: University of California Cooperative Extension, Salinas; and sixth and seventh authors: University of California, Davis, 1636 E. Alisal St., Salinas.

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Accepted for publication 26 December 2011.

Verticillium dahliae is a soilborne fungus that causes Verticillium wilt on multiple crops in central coastal California. Although spinach crops grown in this region for fresh and processing commercial production do not display Verticillium wilt symptoms, spinach seeds produced in the United States or Europe are commonly infected with V. dahliae. Planting of the infected seed increases the soil inoculum density and may introduce exotic strains that contribute to Verticillium wilt epidemics on lettuce and other crops grown in rotation with spinach. A sensitive, rapid, and reliable method for quantification of V. dahliae in spinach seed may help identify highly infected lots, curtail their planting, and minimize the spread of exotic strains via spinach seed. In this study, a quantitative real-time polymerase chain reaction (qPCR) assay was optimized and employed for detection and quantification of V. dahliae in spinach germplasm and 15 commercial spinach seed lots. The assay used a previously reported V. dahliae-specific primer pair (VertBt-F and VertBt-R) and an analytical mill for grinding tough spinach seed for DNA extraction. The assay enabled reliable quantification of V. dahliae in spinach seed, with a sensitivity limit of ≈1 infected seed per 100 (1.3% infection in a seed lot). The quantification was highly reproducible between replicate samples of a seed lot and in different real-time PCR instruments. When tested on commercial seed lots, a pathogen DNA content corresponding to a quantification cycle value of ≥31 corresponded with a percent seed infection of ≤1.3%. The assay is useful in qualitatively assessing seed lots for V. dahliae infection levels, and the results of the assay can be helpful to guide decisions on whether to apply seed treatments.

This article is in the public domain and not copyrightable. It may be freely reprinted with customary crediting of the source. The American Phytopathological Society, 2012.