Abstract
Caused by Sclerotinia sclerotiorum, white mold is an important seed-transmitted disease of soybean (Glycine max). Incubation-based methods available for the detection and quantification of seed-borne inoculum such as the blotter test, paper roll and Neon-S assay are time-consuming, laborious, and not always sensitive. In this study, we developed and evaluated a molecular assay for the detection of S. sclerotiorum in soybean seeds using a species-specific PCR (polymerase chain reaction) primer set and seed soaking (without DNA extraction) for up to 72 h. The PCR products were amplified in all the samples infected with the pathogen, but not in the other samples of plant material or the other seed-borne fungi DNA. The minimum amount of DNA detected was 10 pg, or one artificially infested seed in a 400-seed sample (0.25 % fungal incidence) and one naturally infected seed in a 300-seed sample (0.33 % incidence). The PCR-based assay was rapid (< 9 h), did not require DNA extraction and was very sensitive.
Highlights
Sclerotinia sclerotiorum (Lib.) de Bary is one of the most devastating and widespread fungal pathogens, affecting over 400 plant species worldwide
We developed and evaluated a molecular assay for the detection of S. sclerotiorum in soybean seeds using a species-specific PCR primer set and seed soaking for up to 72 h
The pathogen is transmitted by seeds, which are considered to be the main source of inoculum and an important dissemination agent (Adams and Ayers, 1979; Steadman, 1983; Yang et al, 1998)
Summary
Sclerotinia sclerotiorum (Lib.) de Bary is one of the most devastating and widespread fungal pathogens, affecting over 400 plant species worldwide. Seed health tests available for the detection of S. sclerotiorum in soybean seeds are based on seed incubation (Carvalho-Vieira and Machado, 2002; Napoleão et al, 2006; Brasil, 2009), which requires a large space, is not time-effective, and may lack sensitivity (Blakemore and Reeves, 2002; Carvalho-Vieira and Machado, 2002; Jaccoud-Filho et al, 2002; ISTA, 2008; Henneberg et al, 2011) Alternatives, such as PCR-based assays, are time-effective, versatile, and sensitive in identifying low quantities of DNA and amplifying specific products (Mullis and Faloona, 1987); they have been used to detect several seed-borne pathogens (Audy et al, 1996; Blakemore and Reeves, 2002; Jaccoud-Filho et al, 2002; Taylor et al, 2006; Landa et al, 2007; Kulik, 2008; Jaccoud-Filho and Dabul, 2011). Partial detection of the pathogen population is undesirable for a species-specific detection test, i.e. it detects only some races, isolates or strains (Carvalho-Vieira and Machado, 2002)
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