Location: Crop Improvement and Protection ResearchTitle: A real-time PCR assay for detection and quantification of verticillium dahliae in spinach seed Author
Submitted to: Phytopathology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/26/2011
Publication Date: 3/1/2012
Citation: Duressa, D.O., Rauscher, G.M., Koike, S.T., Mou, B., Hayes, R.J., Maruthachalam, K., Subbarao, K.V., Klosterman, S.J. 2012. A real-time PCR assay for detection and quantification of verticillium dahliae in spinach seed. Phytopathology. 102:443-451. Interpretive Summary: Verticillium dahliae is a soilborne fungus that causes the disease known as Verticillium wilt on many types of plants. Spinach seeds are known to harbor this fungal pathogen, and therefore may transmit the pathogen to other crops that are grown in rotation with spinach, such as lettuce in central coastal California. Establishment of allowable limits of seed infection may help to slow the spread of V. dahliae on spinach seeds. Currently, Mexico requires that imported seed have less than 10 percent infestation with V. dahliae. In this study, a DNA-based assay was developed and employed to specifically detect and quantify the fungus V. dahliae in commercial spinach seed lots. The sensitivity limit of this assay was determined to correlate with approximately 1 infected seed per 100 (1%). This method to quickly assess the level of infection of spinach seed lots can be used to determine which seed lots may require treatment, if threshold levels of seed infection are established. Therefore, the application of the DNA-based assay described herein may help to slow the spread of the pathogen in central coastal California and elsewhere in the U.S. The assay may also be useful to assist in breeding spinach with resistance to Verticillium wilt.
Technical Abstract: 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 seed produced in the U.S. 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 infested lots, curtail their planting and minimize the spread of exotic strains via spinach seed in a given area. In this study, a quantitative real-time PCR (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 seeds 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% 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 percentage seed infection of =1%. The assay is useful in typing the seed lots for V. dahliae infection levels, and potentially in decisions on the application of seed treatments.