Detection and quantification of airborne ascospores of Sclerotinia sclerotiorum by quantitative-PCR

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Detection and quantification of airborne ascospores of Sclerotinia sclerotiorum by quantitative-PCR

Description

Abstract: A new SYBR-green quantitative PCR (qPCR) method was developed to quantify airborne inoculum of Sclerotinia sclerotiorum. This was tested on DNA extracted from spores deposited onto wax-coated plastic tapes, such as those used in Burkard (Hirst-type) spore traps and rotating-arm traps. A linear relationship between ascospore numbers and S. sclerotiorum DNA indicated a mean of 0.35 pg DNA per spore. The method could detect DNA representing as few as 2 ascospores. The technique was insensitive to DNA of the host plant, Brassica napus, and other plant pathogens, including S. minor, S. trifoliorum, Botrytis cinerea, Leptosphaeria maculans and Pyrenopeziza brassicae, and common airborne fungal genera such as Cladosporium and Penicillium. Specific quantification of S. sclerotiorum was achieved in the qPCR method by including a heating step to 79 °C to melt off any exogenous products such as primer dimers that would otherwise falsely contribute to the calculation of target DNA present. This step also eliminated products amplified from any DNA of B. cinerea, if present in the sample. The feasibility of using the method in disease forecasting schemes was tested using archived DNA from air samplers that operated at Rothamsted in April-May in three different seasons, which had contrasting Sclerotinia stem rot (SSR) epidemics; 2007 had a severe SSR epidemic in England and high numbers of airborne ascospores were trapped at Rothamsted; while both 2003 and 2004 had a very low incidence of SSR in England and low numbers of airborne ascospores trapped at Rothamsted. The severe SSR season of 2007 occurred throughout a large part of northern Europe and was not predicted in the UK by climate-based disease-forecasts. This study showed that there was no relationship between rainfall and numbers of airborne ascospores of S. sclerotiorum present at Rothamsted during the period of infection in the severe SSR season (2007). In addition to the example application tested in this study, the qPCR method reported here has potential to evaluate the presence and quantity of S. sclerotiorum in a wider range of environmental samples such as soil, seed or plant tissues such as petals or stems. In the case of airborne inoculum, further research is required to develop methods to rapidly apply the S. sclerotiorum-specific qPCR to air samples and to confirm that airborne inoculum is a reliable indicator of SSR risk by testing over a wider geographical range and number of growing seasons.

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