A multiplex PCR assay for the detection and quantification of Sclerotinia sclerotiorum and Botrytis cinerea

Traditional culture methods for identifying the plant fungal pathogens Sclerotinia sclerotiorum (Lib.) de Bary and Botrytis cinerea Pers.:Fr. are slow and laborious. The goal of this study was to develop a multiplex real-time PCR (qPCR) assay to detect and quantify DNA from S.sclerotiorum and B.cinerea. A primer set (SsIGS_5) for S.sclerotiorum was designed that targeted the intergenic spacer (IGS) regions of the ribosomal DNA. Addition of a probe to the assay increased its specificity: when the primer/probe set was tested against 21 fungal species (35 strains), amplification was detected from all S.sclerotiorum strains and no other species. For qPCR, the SsIGS_5 primer and probe set exhibited a linear range from 7.0ng to 0.07pg target DNA (R-2=0.99). SsIGS_5 was then multiplexed with a previously published primer/probe set for B.cinerea to develop a high-throughput method for the detection and quantification of DNA from both pathogens. When multiplexed, the sensitivity and specificity of both assays were not different from individual qPCR reactions. The multiplex assay is currently being used to detect and quantify S.sclerotiorum and B.cinerea DNA from aerosol samples collected in commercial seed alfalfa fields.