Molecular diagnosis of rape seed pathogens

Abstract

Conventional methods for the precise determination of pathogenic fungi depend largely on morphological criteria and thus require axenic cultures. This process is time-consuming and needs appreciable mycological expertise. The analysis of mixed infections is especially complicated. The identification of fungi is highly facilitated by exploiting molecular markers directly at the DNA level. This approach does not depend on the development of micro-morphological characteristics of the fungi. For this purpose Restriction Fragment Length Polymorphism (RFLP)-analysis, various conventional Polymerase Chain Reaction (PCR)-approaches including Random Amplification of Polymorphic DNA (RAPD)-PCR has been proposed (Hassan et al., 1991; Voigt et al., 1995). All of these methods have their specific drawbacks. RFLP analysis depends on considerable amounts of purified, restriction grade DNA. Conventional PCR works only within highly conserved genomic regions, which may not offer enough variability for all differential purposes. RAPD-PCR has certainly enough resolution but needs an exact control of the reaction conditions for reproducible results. Here we propose a generally applicable experimental approach that makes use of RAPD-PCR for generating taxon-specific probes. In a second step, these probes are used as the basis for synthesis of highly specific PCR primer pairs that can be employed under conventional reaction conditions. This strategy combines the high resolution of RAPD with the ease of applicability and reproducibility of PCR diagnosis. Even mixed infections can be identified within a single working day with simple plant extracts as starting material. Plants can be analysed at early stages of infections, before manifestation of visible symptoms.