Development of diagnostic techniques for Pyrenopeziza brassicae on winter oilseed rape

• Main Author: Foster, S. J.
• Published: University of Cambridge 2000
• Subjects: 579.5
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.599134

Using sequence information from the mating type idiomorphs (<I>MAT-1</I> and <I>MAT-2</I>) of <I>P. brassicae</I>, the causal agent of light leaf spot of winter oilseed rape (<I>Brassica napus </I>ssp. <I>oleifera</I>), two PCR-based diagnostic assays were developed. The first assay was designed as a general diagnostic tool for <I>P. brassicae</I> and was shown to be specific, amplifying a 750 base pair (bp) product from genomic DNA from over 50 isolates of <I>P. brassicae</I> and not from DNA from a number of other fungal species pathogenic on oilseed rape or from the closely related discomycete <I>Tapesia yallundae</I>. The second assay was designed to differentiate between the two mating types (<I>MAT-1</I> and <I>MAT-2</I>) of <I>P. brassicae</I>, and was also specific to <I>P. brassicae</I>. Both assays were ale to detect the presence of <I>P. brassicae</I> from DNA extracted from oilseed rape leaves with light leaf spot symptoms. The sensitivities of both of the assays were increased by using templates consisting of mixtures of NDA from <I>P. brassicae</I> and <I>B. napus</I> compared with DNA from <I>P. brassicae</I> alone. Using a nested PCR approach, the sensitivity of the general diagnostic assay was increased by two orders of magnitude and it was able to detect the presence of <I>P. brassicae</I> prior to the development of visible symptoms on oilseed rape leaves. As a first step towards the development of an immuno-diagnostic tool for light leaf spot, the feasibility of adopting a targeted approach, combining molecular and immunological techniques, was investigated.