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 lin gam) 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 abilit y to form sirodesmins, (iii) for cellulolytic enzymes, and (iv) for pathoty pe-differentiating molecular markers generated by RAPD-PCR, PCR analysis wi th pathotype-specific primer pairs and PFGE. With two exceptions all Polish isolates do not form sirodesmins, grow rapid ly without penetrating into the substrate and form in most cases yellow or brown pigments in Czapek-Dox liquid cultures. With respect to cellulase sec retion and molecular fingerprinting Polish A strains (aggressive) fit into the general picture of the aggressive pathotype group, whereas the NA isola tes (non-aggressive) display a higher degree of heterogeneity. This matches with inoculation tests on rape seedlings, which revealed a considerable nu mber of isolates ranging in aggressivity between the conventional A and NA pathotype group. Molecular fingerprinting techniques unequivocally sorted i ntermediately aggressive isolates into the NA pathotype group. Isolate Ph B ial, which produces sirodesmin but groups within NA isolates according to m olecular and physiological markers, may represent a novel third group besid es A and NA strains with intermediate aggressivity (IA). We hybridized Southern blots of electrophoretically separated chromosomes w ith 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 e xclusively in A strains. The NA probe reveals several chromosomes and is sp ecific for the NA pathotype group. Chromosomes from intermediately aggressi ve strains are equally well recognized by the NA probe as are Polish isolat es with low aggressivity and give no signal with the A probe. Both diagnost ic DNA sequences are highly specific for the pathotype group they were deri ved 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 homogeneou s 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.