Strain typing of Polish Leptosphaeria maculans isolates supports at the genomic level the multi-species concept of aggressive and non-aggressive strains

47 Polish isolates of the blackleg fungus Leptosphaeria maculans (Phoma lingam) were compared with eight well-defined reference strains from Germany, France, Denmark, Australia and one Polish isolate of Phoma nigrificans. The isolates were tested (i) for growth characteristics, (ii) for their ability to form sirodesmins, (iii) for cellulolytic enzymes, and (iv) for pathotype-differentiating molecular markers generated by RAPD-PCR, PCR analysis with pathotype-specific primer pairs and PFGE. With two exceptions all Polish isolates do not form sirodesmins, grow rapidly without penetrating into the substrate and form in most cases yellow or brown pigments in Czapek-Dox liquid cultures. With respect to cellulase secretion and molecular fingerprinting Polish A strains (aggressive) fit into the general picture of the aggressive pathotype group, whereas the NA isolates (non-aggressive) display a higher degree of heterogeneity. This matches with inoculation tests on rape seedlings, which revealed a considerable number of isolates ranging in aggressivity between the conventional A and NA pathotype group. Molecular fingerprinting techniques unequivocally sorted intermediately aggressive isolates into the NA pathotype group. Isolate Ph Bial, which produces sirodesmin but groups within NA isolates according to molecular and physiological markers, may represent a novel third group besides A and NA strains with intermediate aggressivity (IA). We hybridized Southern blots of electrophoretically separated chromosomes with radioactively labelled PCR fragments used for differentiation between A and NA isolates. The specificity of diagnostic PCR amplicons is reflected at the genomic level, The A probe reveals a single hybridizing chromosome exclusively in A strains. The NA probe reveals several chromosomes and is specific for the NA pathotype group. Chromosomes from intermediately aggressive strains are equally well recognized by the NA probe as are Polish isolates with low aggressivity and give no signal with the A probe. Both diagnostic DNA sequences are highly specific for the pathotype group they were derived from. The lack of correspondence of both genetic elements between A and NA strains strongly supports the idea of ascribing the pathotype groups to different species. Whereas the A pathotype group is genetically homogeneous and congruent with the species Leptosphaeria maculans, the NA group needs to be revised taxonomically. NA isolates will presumably have to be split into several independent species.