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  • Rapid nested PCR-based detection of Ramularia collo-cygni direct from barley.

Rapid nested PCR-based detection of Ramularia collo-cygni direct from barley.

FEMS microbiology letters (2006-02-28)
Neil D Havis, Simonj P Oxley, Stephen R Piper, Stephen R H Langrell
ABSTRACT

Ramularia collo-cygni is a barley pathogen of increasing importance in Northern and Central Europe, New Zealand and South America. Accurate visual and microscopic identification of the pathogen from diseased tissue is difficult. A nested PCR-based diagnostic test has been developed as part of an initiative to map the distribution of the pathogen in Scotland. The entire nuclear ribosomal internal transcribed spacer and 5.8S rRNA gene regions from 14 isolates of diverse global origin exhibited complete homology following sequence characterization. Two pairs of species-specific primers, based on inter-specific sequence divergence with closely related species, were designed and empirically evaluated for diagnostic nested PCR. Nested primers Rcc3 and Rcc4 consistently amplified a single product of 256 bp from DNA of 24 R. collo-cygni isolates of diverse global provenance, but not from other Ramularia species, or other fungi commonly encountered in cereal pathosystems, as well as Hordeum or Secale DNA preparations. Using this approach, R. collo-cygni was successfully identified from naturally infected barley leaf, awn and grain samples of diverse geographical provenance, in particular from symptoms that lacked the presence of characteristic conidiophores. It is envisaged that this assay will become established as an important tool in continuing studies into the ecology, aetiology and epidemiology of this poorly understood yet economically damaging plant pathogen.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
REDExtract-N-Amp Plant PCR Kit, sufficient for 10 extractions, sufficient for 10 amplifications
Sigma-Aldrich
REDExtract-N-Amp Plant PCR Kit, sufficient for 100 extractions, sufficient for 100 amplifications
Sigma-Aldrich
REDExtract-N-Amp Plant PCR Kit, sufficient for 1000 extractions, sufficient for 1000 amplifications