GWAS reveals a rapidly evolving candidate avirulence effector in the Cercospora leaf spot pathogen

The major resistance gene BvCR4 recently bred into sugar beet hybrids provides a high level of resistance to Cercospora leaf spot caused by the fungal pathogen Cercospora beticola. The occurrence of pathogen strains that overcome BvCR4 was studied using field trials in Switzerland conducted under natural disease pressure. Virulence of a subset of these strains was evaluated in a field trial conducted under elevated artificial disease pressure. We created a new C. beticola reference genome and mapped whole genome sequences of 256 isolates collected in Switzerland and Germany. These were combined with virulence phenotypes to conduct three separate genome-wide association studies (GWAS) to identify candidate avirulence genes. We identified a locus associated with avirulence containing a putative avirulence effector gene named AvrCR4. All virulent isolates either lacked AvrCR4 or had nonsynonymous mutations within the gene. AvrCR4 was present in all 74 isolates from non-BvCR4 hybrids, whereas 33 of 89 isolates from BvCR4 hybrids carried a deletion. We also mapped genomic data from 190 publicly available US isolates to our new reference genome. The AvrCR4 deletion was found in only one of 95 unique isolates from non-BvCR4 hybrids in the United States. AvrCR4 presents a unique example of an avirulence effector in which virulent alleles have only recently emerged. Most likely these were selected out of standing genetic variation after deployment of BvCR4. Identification of AvrCR4 will enable real-time screening of C. beticola populations for the emergence and spread of virulent isolates.