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Development of conventional and quantitative real‐time PCR assays for the detection and identification of Rhizoctonia solani AG‐3 in potato and soil
Author(s) -
Lees A. K.,
Cullen D. W.,
Sullivan L.,
Nicolson M. J.
Publication year - 2002
Publication title -
plant pathology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.928
H-Index - 85
eISSN - 1365-3059
pISSN - 0032-0862
DOI - 10.1046/j.1365-3059.2002.00712.x
Subject(s) - rhizoctonia solani , biology , taqman , internal transcribed spacer , polymerase chain reaction , dna extraction , canker , ribosomal dna , primer (cosmetics) , rhizoctonia , real time polymerase chain reaction , microbiology and biotechnology , horticulture , ribosomal rna , genetics , gene , phylogenetics , chemistry , organic chemistry
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 × 10 −4 g sclerotia/g soil. In addition, specific primers RsTqF1 (based on the Rs1F2 sequence) and RsTqR1, and a TaqMan™ 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.