Rapid PCR-based assay for Sclerotinia sclerotiorum detection on soybean seeds

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.

Quick and accurate detection of Sclerotiniasclerotiorum and Phomopsis spp. in soybean seeds using qPCR and seed‐soaking method

“…Using primers developed by Freeman, Ward, Calderon, and McCartney (), S. sclerotiorum was detected in artificially inoculated soybean seeds, up to an incidence of 10% by conventional PCR and up to 1% by qPCR (Botelho et al, ). Until now, the minimum limit detected for this pathogen in soybean seeds was an incidence of 0.25% for conventional PCR, using the same primers (Grabicoski et al, ). In this study, using the TaqMan system proposed by Chen et al (), we obtained a higher sensitivity for the detection of the pathogen, up to an incidence level of 0.0625%, with good repeatability.…”

“…The molecular detection of Phomopsis spp. and S. sclerotiorum was done by the seed‐soaking method, in which 400 inoculated seeds, at the above‐mentioned ratios, were incubated at 20°C in closed bottles with 400 ml sterile water for 4 hr (Grabicoski et al, ). From the soaking solution, 1 ml aliquots was stored at −20°C and subjected to qPCR, under the same conditions as described above, separately for each pathogen.…”

Diagnosis of Soybean Diseases Caused by Fungal and Oomycete Pathogens: Existing Methods and New Developments

“…This fungus can infect over 400 species of plants, including sunflower, soybean, and oilseed rape [33]. On soybean S. sclerotiorum causes white mold, which reduces yield by more than 40% in wet and mild weather [34]. Since the pathogen disseminates via seeds, sowing seeds with certified health quality is the first step to avoid this disease.…”

“…Variability among isolates from this species from different regions or hosts can lead to these differences. In a separate study, the primer pair SSFWD/SSREV [35] was tested to detect S. sclerotiorum in inoculated and in naturally infected soybean seeds using the seed soaking procedure [34]. Since the ITS region did not allow for fully reliable diagnosis the mitochondrial small rRNA and SS1G_00263, coding for a hypothetical secreted protein was used [39,40].…”