Development of conventional and quantitative real-time PCR assays for the detection and identification of Rhizoctonia solani AG-3 in potato and soil

A specific and sensitive PCR assay was developed for the detection and identification of Rhizoctonia solani AG-3, the main causal pathogen of stem canker and black scurf of potato. A conventional primer set (Rs1F2 and Rs2R1) was designed from the nuclear ribosomal internal transcribed spacer (ITS1 and ITS2) regions of R. solani. Following PCR amplification, a 0.5-kb product was amplified from DNA of all isolates of AG-3 using primers Rs1F2 and Rs2R1. No product was amplified when DNA from isolates belonging to a range of other R. Solani anastomosis groups or from a selection of other potato pathogens was tested, confirming the specificity of the primers for AG-3 only. Rhizoctonia solani AG-3 was also detected in potato tissue with varying black scurf severity, and in soil inoculated with sclerotia of R. solani to a minimum detection level of 5 X 10(-4) g sclerotia/g soil. In addition, specific primers RsTqF1 (based on the Rs1F2 sequence) and RsTqR1, and a TaqMan(TM) fluorogenic probe RQP1, were designed to perform real-time quantitative (TaqMan) PCR. The conventional PCR and real-time PCR assays were compared and combined with direct DNA extraction from soil and a seed-baiting method to determine the most reliable method for the detection and quantification of AG-3 in both artificially inoculated field soil and naturally infested soils. It was shown that direct DNA extractions from soil could be problematic, although AG-3 was detectable using this method combined with the real-time PCR assay. The amplification of Rhizoctonia solani by seed baiting increased the sensitivity of the assay compared with direct extraction of DNA from the soil, and AG-3 was detectable in artificially inoculated and naturally infested soils when seed baiting was combined with either the conventional PCR or the real-time PCR assay. The potential for using these rapid and quantitative AG-3-specific assays to address epidemiological questions and as tools for decision-making in disease management is discussed.