Detection and quantification of airborne inoculum of Sclerotinia sclerotiorum using quantitative PCR

Abstract

This paper reports the development of a new specific diagnostic technique to accurately quantify airborne inoculum of Sclerotinia sclerotiorum and discusses its potential use in disease-forecasting schemes, using examples of three contrasting epidemic seasons: 2007, when there was a severe epidemic of sclerotinia stem rot (SSR) in England and high numbers of airborne ascospores were trapped at Rothamsted, and, in contrast, 2003 and 2004, when the incidence of SSR in England was low and low numbers of airborne ascospores were trapped at Rothamsted. DNA was extracted from wax-coated plastic tapes, such as those used in Burkard (Hirst-type) spore traps and rotating-arm traps. A SYBR-green quantitative PCR (qPCR) method produced a linear relationship between ascospore numbers and S. sclerotiorum DNA (mean 0·35 pg DNA per spore) and was able to detect DNA representing as few as two ascospores. The technique was insensitive to DNA of the host plant, Brassica napus, and other plant pathogens, including Sclerotinia minor, S. trifoliorum and Botrytis cinerea, and common airborne fungal genera such as Cladosporium and Penicillium. 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).