Quantification of seedborne infection by Rhynchosporium secalis in barley using competitive PCR

• Published in: Plant pathology Vol. 51; no. 2; pp. 217 - 224
• Main Authors: Lee, H. K., Tewari, J. P., Turkington, T. K.
• Format: Journal Article
• Language: English
• Published: Oxford, UK Blackwell Science Ltd 01-04-2002; Blackwell; Wiley Subscription Services, Inc
• Subjects: Biological and medical sciences; Biotechnology; competitive PCR; Fundamental and applied biological sciences. Psychology; Fungal plant pathogens; Generalities. Techniques; Hordeum vulgare; internal transcribed spacer; Phytopathology. Animal pests. Plant and forest protection; Rhynchosporium secalis; seed health testing; visual disease assessment; Biotechnology; Monocotyledones; Plant pathology; Plant pathogen; Mycosis; Ribosomal DNA; Transmission; Cereal crop; Fungi; Assay; Gramineae; Ribosomal RNA; Angiospermae; Fungi Imperfecti; Thallophyta; Quantitative analysis; Method study; Rhynchosporium secalis; Hordeum vulgare; Mycology; Competitive reaction; Seedborne transmission; Spacer DNA; Experimental study; Infection; Polymerase chain reaction; Analysis method; Seed; Spermatophyta; Molecular biology
• ISSN: 0032-0862; 1365-3059
• DOI: 10.1046/j.1365-3059.2002.00685.x

The potential of the competitive polymerase chain reaction (PCR) assay for quantification of seedborne infection by Rhynchosporium secalis in barley was examined using a primer set (RS1 and RS3) derived from the internal transcribed spacer (ITS) regions of ribosomal RNA genes of this pathogen. Introduction of a heterologous internal control, which competes for the same primer set in the conventional PCR assay, allowed for detection and quantification of R. secalis fungal biomass. In order to generate a standard calibration curve, DNA prepared from infected seeds with different levels of R. secalis infection was subjected to competitive PCR assay. The resulting PCR product ratio for each PCR reaction (R. secalis‐amplified DNA/internal control template‐amplified DNA) increased proportionally with increasing levels of infected seed DNA in the reaction mixture. Naturally infected seed lots collected from 1995 to 1999 were used to demonstrate the potential of the competitive PCR assay as an alternative seed health testing method. The results from this competitive PCR assay were compared with those from conventional visual disease assessment and an agar plate assay. Although relatively good correlation between visual disease assessment and the competitive PCR was found in the case of artificially mixed seed samples, there was poor correlation in the experiments using naturally infected seed samples.