RAPD-based Molecular Probes for the Blackleg Fungus Leptosphaeria maculans (Phoma lingam): Evidence for Pathogenicity Group-specific Sequences in tbe Fungal Genomes

Polymerase chain reaction (PCR) primers, specific for the aggressive pathogenicity group of the rape seed pathogen Leptosphaeria maculans, were obtained according to the following experimental outline: DNA bands that differentiate between aggressive (A) and nonaggressive (NA) isolates were generated by the random amplified polymorphic DNA approach (RAPD). Differential RAPD bands were checked for pathogenicity group-specificity and sequenced by a combination of chemical and dideoxynucleotide chain termination methods. Based on the DNA sequence longer and highly specific primer pairs for conventional PCR under stringent conditions were designed, which were tested on 97 isolates of L, maculans belonging to different pathogenicity groups. The primer pair specific for the aggressive pathogenicity group unequivocally identifies A-isolates. The primer pair designed for the NA-isolates. cross-reacts with A-isolates showing positive signals in 28 out of 63 cases. The combined application of A- and NA-specific primers identifies all isolates unambiguously. The method does not require pure fungal cultures. L. macuians can be detected directly in infected plant material within a single working day. The experimental outhne presented in this communication is proposed as a general method for obtaining taxon-specific PCR primer pairs.