Large‐scale population survey of Leptosphaeria maculans in France highlights both on‐going breakdowns and potentially effective resistance genes in oilseed rape

Abstract

BACKGROUNDLeptosphaeria maculans, the cause of stem canker of oilseed rape, develops gene‐for‐gene interactions with its host and shows a high evolutionary potential to ‘break down’ novel resistance genes (R, Rlm) deployed in cultivars over large areas. For optimal management of R genes, updated knowledge of the population structure of the pathogen is needed. In France, large‐scale surveys have been done at 10‐year intervals since 2000. Here we report the characterization of a large L. maculans population collected in France in 2019–2020.RESULTSA total of 844 isolates were collected from 11 sites in ten French departments and were phenotyped for their virulence against nine Brassica napus R genes. All isolates were virulent toward Rlm2 and Rlm9. Very few isolates were avirulent on Rlm1 (1.8%) and Rlm4 (0.6%). Avirulent isolates toward Rlm7 (‘AvrLm7’) varied from 67% to 11.3%, depending on the site sampled, illustrating the ongoing breakdown of Rlm7. The decrease of AvrLm7 isolates (29.2% at the national level) compared to the 2010 survey (96.5%) was accompanied by an increase of avirulent isolates on Rlm3 (0% in 2010; 54% in 2019–2020). However, virulent isolates on both Rlm3 and Rlm7, previously rarely detected, were found in all sites with a frequency of 17.3%. Finally, most or all isolates were avirulent on Rlm11 (96.1%), LepR2 (RlmS, 99.8%), and Rlm6 (100%), suggesting these three genes still effectively control the disease.CONCLUSIONThese data will help guide strategies for breeding and deploying resistant oilseed rape varieties against L. maculans in France. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

MPSeqM, a tool combining multiplex PCR and high-throughput sequencing to study the polymorphism of eightLeptosphaeria maculansavirulence genes and its application to field surveys in France

“…The two others displayed only one AA change each (R63L and K101Q, respectively), whose impact on the phenotype cannot be determined. Among the two alleles encoding avirulent isoforms on Rlm9 , isoform AvrLm5-9_4 was never detected in any of the 42 pools, and AvrLm5-9_1 was found in only nine pools with very low proportions (<0.27%, close to the 0.2% detection limit, 0.06% on average over all pools), confirming the near-fixation of the virulence phenotype toward Rlm9 in French L. maculans populations (Balesdent et al, 2023 Gautier et al, 2023). Similarly, alleles with stop codons in the coding sequence were never detected.…”

“…These two new alleles were considered as virulent, as were deleted isolates whose proportions were estimated using the deletion curve. Altogether, the MPSeqM analysis of environmental samples correlated well with pathotyping; 1.8% of the population was found avirulent on Rlm1 by pathotyping (Balesdent et al, 2023), while the MPSeqM analysis of populations from the same sites detected 2.14% of such isolates. With both approaches, the site with the highest proportion of avirulent isolates was that of Le Rheu (Supplementary Figure S3).…”

“…Using leaf samples from the same site and the same sampling date, we compared the percentages of avirulent isolates in L. maculans populations as estimated following inoculation tests on a plant differential set (Balesdent et al, 2023) or using the MPSeqM tool (this study). For most avirulence loci, the inference of the phenotype (virulence or avirulence) from the allele sequences or deletion proportions was possible (see above).…”

“…For most avirulence loci, the inference of the phenotype (virulence or avirulence) from the allele sequences or deletion proportions was possible (see above). As regards AvrLm3 , the situation is more complex since the avirulence phenotype is masked whenever AvrLm4-7 is present, at least for some AvrLm4-7 alleles (Balesdent et al, 2023). To determine the proportion of avirulent isolates on Rlm3 , we multiplied, for each pool, the percentage of AvrLm3 avirulent alleles by the percentage of AvrLm4-7 alleles unable to mask the AvrLm3-Rlm3 interaction.…”

“…Monitoring virulent isolates is particularly important for the fungal ascomycete Leptosphaeria maculans , responsible for the stem canker disease of oilseed rape ( Brassica napus ), for the following reasons; (i) the main control measure deployed to limit the impact of the disease is genetic control, including the use of specific resistance ( Rlm ) genes; (ii) L. maculans populations have the potential to rapidly evolve toward virulence, and this has been reported in many instances (Rouxel et al, 2003; Sprague et al, 2006, Zhang et al, 2016); (iii) potentially effective resistance sources are scarce in oilseed rape and the few currently available Rlm genes must be carefully managed to avoid new breakdowns (Borhan et al, 2022; Rouxel and Balesdent, 2017). Until recently, the monitoring of L. maculans populations consisted of large-scale isolate samplings from diseased B. napus plants, followed by inoculation of each isolate on a plant differential set (for instance, Alnajar et al, 2022; Balesdent et al, 2006, 2023; Stachowiak et al, 2006; Zhang et al, 2016). These biological tests are however inadequate for high-throughput analyses and for the detection of rare mutation events in populations.…”