Monitoring of plant and airborne inoculum of Sclerotinia sclerotiorum in spring oilseed rape using real‐time PCR

Abstract

Sclerotinia stem rot of spring oilseed rape (Brassica napus) is caused by Sclerotinia sclerotiorum. In Sweden, the disease leads to severe crop damage that varies from year to year. A real‐time PCR assay was developed and used to determine the incidence of S. sclerotiorum DNA on petals and leaves of spring oilseed rape as well as in air samples, with the aim of finding tools to improve precision in disease risk assessment. Five field experiments were conducted from 2008 to 2010 to detect and study pathogen development. Assessments of stem rot showed significant differences between experimental sites. The real‐time PCR assay proved fast and sensitive and the relationship between percentage of infected petals determined using a conventional agar test and the PCR assay was linear (R2 > 0·76). There were significant differences in S. sclerotiorum incidence at different stages of flowering. The incidence of S. sclerotiorum DNA on the leaves varied (0–100%), with significantly higher incidence on leaves at lower levels. In one field experiment, S. sclerotiorum DNA was not detected on petals during flowering, whereas the pathogen was detected on leaves, with a corresponding stem rot incidence of 7%. The amount of S. sclerotiorum DNA in sampled air revealed that spore release did not coincide with flowering on that experimental site. Thus, using a real‐time PCR assay to determine the incidence of S. sclerotiorum on oilseed rape leaves, rather than on petals, could potentially improve disease risk assessment.

The role of precipitation, and petal and leaf infections in Sclerotinia stem rot of spring oilseed Brassica crops in Norway

“…Infection of leaf three sampled at full flowering showed the highest explanation of variation in later SSR incidence. Almquist and Wallenhammar (2015) reported significantly higher incidences of SSR on leaves at lower canopy levels, than on higher levels. Moreover, they detected the pathogen on leaves and not on petals in one field experiment, with a corresponding stem rot incidence of 7%, which suggested that leaves could be an important entry port for Sclerotinia spp.…”

“…The use of rapid petal and leaf testing kits to determine petal infections, such as commercialized in Australia (Derbyshire and DentonGiles 2016) are based on culture plates and might need to be substituted by a quantitative or real-time PCR based systems, such as those that detect aerial inoculum levels caught by spore traps in the SYield sensor system from Syngenta (Derbyshire and Denton-Giles 2016). PCR methods for petal and leaf infections, such as published by Ziesman et al (2016), Reich et al (2016) and Almquist and Wallenhammar (2015), should be adapted for large scale use to improve future SSR prediction in the field. Pre-infection treatment before full flowering with the commonly recommended protectant fungicides might be more effective against SSR and more feasible than applying the fungicides during full or late flowering, after determining the risk of infection based on leaf and/ or petal testing.…”

“…in the samples was tested by a Taqman-real-time assay, essentially as described by Almquist and Wallenhammar (2015). The reaction mixture consisted of SsoAdvanced Universal Probes Supermix (Biorad), 0.2 μM of each primer, 0.2 μM of TaqMan probe and 2 μL of DNA in a 25 μl reaction volume.…”

“…The reaction mixture consisted of SsoAdvanced Universal Probes Supermix (Biorad), 0.2 μM of each primer, 0.2 μM of TaqMan probe and 2 μL of DNA in a 25 μl reaction volume. The real-time PCR was run on a C1000 Touch™ CFX96™ Real-Time System (Biorad) with cycling conditions as described by Almquist and Wallenhammar (2015). Three replicates of each DNA (2) 100 (4) 50 (4) 100 (4) 28 24.06.14 65 100 (3) 100 (4) 50 (4) 100 (4) 30.06.14 65 67 (3) 100 (3) 75 (4) 50 (4) 07.07.14 69 100 (3) 100 (4) 100 (3) 25 ( sample were tested.…”

Biological control of the soil-borne fungal pathogen Sclerotinia sclerotiorum –– a review

“…Ascospores can germinate on the surface of healthy tissue but cannot infect plants without an exogenous nutrient source (Bolton et al 2006),which often is provided by senescing leaves and petioles or juices flowing from the damaged plants (Kora et al 2003). Thus, flowering is a particularly dangerous moment because senescing flowers serve as nutrient source for the pathogen (Turkington and Morrall 1993;Almquist and Wallenhammar 2015). Direct penetration of fungal hyphae was observed through the cuticle within 12 h from inoculation.…”