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Map-based cloning of the avirulence gene AvrLm1 of Leptosphaeria maculans was initiated utilizing a genetic map of the fungus and a BAC library constructed from an AvrLm1 isolate. Seven polymorphic DNA markers closely linked to AvrLm1 were identified. Of these, two were shown to border the locus on its 5′ end and were present, with size polymorphism, in both the virulent and the avirulent isolates. In contrast, three markers, J19-1.1, J53-1.3 (in coupling phase with avirulence), and Vir1 (in repulsion phase with avirulence), cosegregated with AvrLm1 in 312 progeny from five in vitro crosses. J19-1.1 and J53-1.3 were never amplified in the virulent parents or progeny, whereas Vir1 was never amplified in the avirulent parents or progeny. J19-1.1 and J53-1.3 were shown to be separated by 40 kb within a 184-kb BAC contig. In addition, the 1.6-cM genetic distance between J53-1.3 and the nearest recombinant marker corresponded to a 121-kb physical distance. When analyzing a European Union-wide collection of 192 isolates, J53-1.3, J19-1.1, and Vir1 were found to be closely associated with the AvrLm1 locus. The results of polymerase chain reaction amplification with primers for the three markers were in accordance with the interaction phenotype for 92.2% (J53-1.3), 90.6% (J19-1.1), and 88.0% (Vir1) of the isolates. In addition, genome organization of the AvrLm1 region was highly conserved in field isolates, because 89.1% of the avirulent isolates and 79.0% of the virulent isolates showed the same association of markers as that of the parents of in vitro crosses. The large-scale analysis of field isolates with markers originating from the genetic map therefore confirms (i) the physical proximity between the markers and the target locus and (ii) that AvrLm1 is located in (or close to) a recombination-deficient genome region. As a consequence, map-based markers provided us with high-quality markers for an overview of the occurrence of race “AvrLm1” at the field scale. These data were used to propose hypotheses on evolution towards virulence in field isolates.

Specificity of interaction at the cotyledon stage was recently demonstrated between the blackleg pathogen, Leptosphaeria maculans, and Brassica napus. Three pathogenicity groups were distinguished, PG2 avirulent towards 'Quinta' and 'Glacier', PG3 avirulent towards 'Quinta', and PG4 virulent on the two cultivars. The genetic control of the interactions was investigated on both the pathogen and the plant. Tetrad analysis was performed following PG3 × PG4 and PG2 × PG4 crosses. 'Quinta' and 'Glacier' were crossed with the susceptible winter oilseed rape cultivar 'Score'. The analysis of F 1 , F 2 and testcross populations suggested that the incompatible interaction between 'Quinta' and PG3 isolates is conditioned by the presence of the dominant single resistance allele Rlm1 in 'Quinta' and the matching avirulence gene Avr Lm1 in L. maculans. Race-specific resistance of 'Glacier' to PG2 isolates was conditioned by the matching gene pair Rlm2/Avr Lm2. Finally, the data suggest that two avirulence genes matching two dominant loci control the 'Quinta'-PG2 interaction. The consequences of the occurrence of race-specific resistance in B. napus are discussed with respect to future breeding for blackleg resistance.

All Leptosphaeria maculans field isolates displayed a minichromosome (MC) clearly separated from the overall electrokaryotype following pulsed-field gel electrophoresis. MCs exhibited a length polymorphism ranging from 650 to 950 kb. Tetrad analyses revealed the parental inheritance of MC length polymorphism (50% of the tetrads) or else the generation of novel-sized MCs (27%), which suggested that recombination occurred between MCs. Nineteen percent of the tetrads displayed a lack of the MC band in the electrokaryotype for one or two of the four resulting genotypes. Crosses between isolates carrying or lacking MCs revealed non-Mendelian segregation and suggested that some isolates could display at least two copies of the MC. Only repeated sequences hybridising to all chromosomes were isolated from the MC. Finally, saprophytic or parasitic fitness was not modified when isolates apparently lacked the MC. All these data suggested that the L. maculans MC behaves like a 'B' chromosome.

Specific interactions of the fungal pathogen Leptosphaeria maculans with Brassica napus cultivars were observed when the cultivars were inoculated with isolates belonging to pathogenicity groups (PG) PG3 and PG4. PG3 isolates induced resistance responses on cotyledons or leaves of cv. Quinta, whereas PG4 isolates caused sporulating lesions on this cultivar. In contrast, both pathotypes caused disease symptoms on cvs. Westar and Glacier. The genetic basis of cultivar specificity was studied using tetrad analysis after in vitro crosses between one PG3 and one PG4 isolate. For the genetic study, the use of random amplified polymorphic DNA (RAPD) as genetic markers was assessed. Of 61 primers, 10 generated reproducible polymorphisms. Of these, 9 generated 18 RAPD markers displaying a 2 :2 segregation ratio within the 10 analyzed tetrads. A 2 :2 segregation ratio for avirulence/virulence to cv. Quinta also was observed in the progeny. Consequently, the single genetic locus controlling cultivar specificity on Quinta was considered the first avirulence gene described in L. maculans and was designated AvrLM1.