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

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 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.

Detection of Sclerotinia sclerotiorum in soybean seeds by conventional and quantitative PCR techniques

“…It is important to consider that the cPCR technique only indicates the presence or absence of the pathogen in a sample, which may be a limiting factor for the healthy tests in seed pathology. The detection and quantification of the pathogen in a sample of seeds have been important for some epidemiological studies of view have been used to examine diverse seedborne pathogens such as bacteria (Becker et al, 2011;Cottyn et al, 2011), fungi (Chen et al,2013;Duressa et al, 2012;Montes-Borrego et al, 2012;Ioos et al, 2012;Kuan et al, 2011) andviruses (Ling et al, 2011), including in detection for many seed borne pathogens in the same time (Feng et al, 2014). For molecular detection of S. sclerotiorum in soybean seeds in Brazil, where the presence of this fungus in seeds lots isn´t accepted, the test may be recommended as a screening procedure, which aims to inform if the lot is infected or not.…”

Scientific opinion on the pest categorisation of Verticillium dahliae Kleb

“…dahliae can spread over short or long distances by human-assisted means and particularly through the movement of infected host plants for planting (grafts, buds, shoots, cuttings, bulbs, rhizomes, potato seed tubers, etc. ), especially asymptomatic ones (Woolliams, 1967;Sinclair et al, 1987;Thanassoulopoulos, 1993;Karajeh and Masoud, 2006;Sanei et al, 2008), and seeds of host plants (Hawksworth and Talboys, 1970;du Toit et al, 2005;Vallad et al, 2005;Karajeh, 2006;EPPO, 2007;Athallah, et al, 2011Athallah, et al, , 2012Göre et al, 2011;Duressa et al, 2012;Maruthachalam et al, 2013).…”

“…Transmission of the pathogen by infected or contaminated seed of host plants has been, so far, demonstrated for cotton, aubergine, lettuce, linseed, olive, safflower, soybean, spinach, sunflower and tomato (Sackston and Martens, 1959;Hawksworth and Talboys, 1970;Van der Spek, 1972;Klisiewicz, 1975;Richardson, 1990;Karajeh, 2006;Göre et al, 2011;Athallah, et al, 2011Athallah, et al, , 2012Duressa et al, 2012;Maruthachalam et al, 2013). Seed transmission may play an important role in the epidemiology of Verticillium wilt by facilitating the immigration of exotic strains of the pathogen to new production areas.…”

“…Hot water treatment of tubers may be used to eliminate the pathogen in stocks of some high-value ornamental crops (Gilad and Borochov, 1993). Seed transmission of V. dahliae is very important in several crops, in terms of increasing the size of inoculum in soil in infested areas, as well as spreading exotic strains of the pathogen into new areas (Goethal, 1971;Bejarano-Alcazar et al, 1996;Athallah et al, 2011Athallah et al, , 2012Gore et al, 2011;Duressa et al, 2012;Maruthachalam et al, 2013). Fungicidal seed treatments may have some value, but only in eliminating the pathogen on seed surfaces.…”

Evaluation of inoculum potential of pathogens in seeds: relation to physiological quality and DNA quantification by qPCR

“…The most common method used for detection of necrotrophic fungi in general has been the blotter test. However, currently, the real-time polymerase chain reaction (qPCR) technique has been an important tool to assist in these methods because of its precision and specificity (Konstantinova et al, 2002;Gao et al, 2004;Barros et al, 2008;Duressa et al, 2012;Botelho et al, 2015;Sousa et al, 2015).…”