Development of PCR based diagnostic techniques for the two mating types of Pyrenopeziza brassicae (light leaf spot) on winter oilseed rape (Brassica napus ssp oleifera)

Two sets of PCR primers were designed from DNA sequence which flanked the 3' end of the Pyrenopeziza brassicae mating type idiomorphs (MAT-1 and MAT-2). The first set of primers facilitated the amplification of a 750 bp product from genomic DNA of 50 isolates of P. brassicae, which included both mating types. The 750 bp PCR product was also amplified from DNA extracted from Brassica napus tissue infected with P. brassicae. The 750 bp PCR product was not produced when these diagnostic primers were used in PCR reactions containing DNA from a number of other fungal species, including fungal pathogens of oilseed rape and Tapesia yallundae, which is closely related to P. brassicae. The second set of primers were designed to amplify different sized products from the two mating types of P. brassicae. These mating type specific primers facilitated the amplification of a 440 bp product from all MAT-1 isolates tested and a 535 bp product from 30% of the MAT-2 isolates tested. The value of molecular diagnostic techniques for the identification of P. brassicae in oilseed rape crops, for distinguishing between mating types of the fungus and in disease forecasting is discussed. (C) 1999 Academic Press.