Several studies reported the rapid evolution of avirulence (Avr) genes to escape R-mediated plant immunity and identified a variety of mechanisms leading to virulence. The poplar rust fungus Melampsora larici-populina is the most damaging pathogen of poplars. A major adaptive event occurred in 1994 with the breakdown of RMlp7 resistance gene in poplar. Population genomics studies identified a locus in the genome of M. larici-populina, which likely corresponds to the AvrMlp7 candidate avirulence gene. We used a population genetics approach combined with dedicated qPCR assays on a comprehensive set of 281 isolates, covering 27 years (encompassing the resistance breakdown event), to validate the candidate locus and to assess its polymorphism. We found two mechanisms, a point mutation and a deletion, that allowed the pathogen to escape RMlp7-mediated resistance. Six diploid genotypes were thus characterized at the candidate locus (three avirulent and three virulent). In addition, a temporal analysis revealed that the two virulence alleles pre-existed (harboured as avirulent heterozygous genotypes) since the early samplings and were found in association (as virulent genotypes) at the time of the resistance breakdown.These molecular analyses were complemented by a population genetic analysis of those temporal samples, using 22 microsatellite markers.

281 isolates were chosen from a historical collection maintained at INRAE Grand-Est – Nancy, France, which ranges from 1989 to 2016. This collection included most samples from a previous study of Persoons et al. (2017) but extended the time-period covered by the analysis (see Table S1, Supporting information).

Virulence factor of all isolates were confirmed upon inoculation of a poplar cultivar carrying RMlp7 resistance gene (P. × interamericana ‘Beaupré’) and a universal cultivar susceptible to all poplar rust isolates (P. × euramericana ‘Robusta’), as a positive control.

Genotyping was performed using 22 microsatellite markers (Xhaard et al., 2011).

Two assays based on TaqMan® real-time PCR were used to detect two types of mutations at the AvrMlp7 locus: a deletion of the locus containing the gene and a non-synonymous SNP in the coding sequence.