DEVELOPMENT OF A PCR-BASED DIAGNOSTIC TECHNIQUE FOR LIGHT LEAF SPOT (PYRENOPEZIZA BRASSICAE) ON WINTER OILSEED RAPE (BRASSICA NAPUS SUBSP. OLEIFERA)
SJ FOSTER1,2, BDL FITT2 and AM ASHBY1
1 Dept. Plant Sciences, University of Cambridge, Downing St, Cambridge, CB2 3EA, UK; 2IACR-Rothamsted, Harpenden, Herts AL5 2JQ, UK
Background and objectives
Light leaf spot, caused by the fungal pathogen Pyrenopeziza brassicae Sutton and Rawlinson (anamorph Cylindrosporium concentricum), is a major disease of winter oilseed rape (Brassica napus subsp. oleifera) in the UK, causing losses estimated at >£30 M per annum. Effective disease control can be achieved by fungicide applications in November/December when initial infection occurs. However, disease symptoms are often not visible until February/March and can be difficult to identify. In addition, epidemics have been found to vary in severity between seasons and between regions in the UK. Consequently, spray decisions are often taken without knowing if crops are affected by the disease. One of the components of a light leaf spot forecasting scheme currently under development  is the early detection of light leaf spot infections as this will enable more timely decisions to be made regarding the application of appropriate control measures. To achieve this, we are developing a molecular diagnostic technique for light leaf spot, based upon the polymerase chain reaction (PCR).
Materials and methods
DNA extractions and PCR reactions were performed according to standard protocols. Sequences from the P. brassicae genomic clones used for primer design were obtained from G. Singh, University of Cambridge .
Results and conclusions
Degenerate PCR primers, designed to amplify a region of the DNA coding sequence of the Sex Factor Induced (SFI1) protein of P. brassicae, were shown to amplify a 700 base pair (bp) product from DNA extracted from a range of P. brassicae isolates of mating type MAT 1-2. This PCR product was not produced upon amplification of DNA from other fungal pathogens of oilseed rape and was shown, by Southern blotting, to hybridise to 5 kb and 3 kb Sal 1 fragments from P. brassicae isolates of mating type MAT 1-1 and MAT 1-2, respectively. Sequence analysis of genomic clones from both mating types of P. brassicae, which included the 700 bp SFIl sequence, enabled the design of fully homologous primers which facilitated the amplification of 750 bp PCR products from 50 isolates of P. brassicae. In addition, a single primer pair was designed which could be used to differentiate between the two mating types of P. brassicae, generating 535 bp and 440 bp amplification products from isolates of mating type MAT 1-1 and MAT 1-2, respectively. These PCR products were not generated by amplification of DNA from other fungi pathogenic on oilseed rape.
The work presented here represents the initial stages in the development of a PCR based diagnostic tool for light leaf spot. Work is continuing to determine whether the technique is sensitive enough to identify the fungus in infected plant material and to determine the earliest stage in the infection process at which the fungus can be detected. Ultimately it is hoped that the technique will be able to identify infected plants prior to the onset of disease symptoms, thus enabling fungicide spray decisions to be made at the time when they will be most effective.
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Applied Biology 48, 135-142.
2. Singh G, Ashby AM, 1998. Proceedings of ICPP98.