Genomic surveillance and molecular evolution of fungicide resistance in European populations of wheat powderymildew

Key results

1. ShownG143A in cytb, the quinone outside inhibitor (QoI) resistance substitution, is common but not fixed. It is near-universal in Northwestern Europe, intermediate in Central and Eastern Europe, rare in the south and absent in the Middle East, and it has increased over time in Western Europe.

   G143A (nucleotide G428C) was found in 146 of 368 (39.7%) Europe+_recent isolates, and every sample from Ireland, England, Northern France and the Netherlands carried it. In the temporal dataset (UK, France, Switzerland), Fisher's exact test gave p < 0.001 comparing 1990-2001 with 2022-2023. · cytb – QoI resistance; Discussion

2. ShownMultiple cyp51 copies and Y136F, a mutation linked to demethylation inhibitor (DMI) resistance, are widespread in Northern Europe, Spain and Turkey. Y136F occurs only in multi-copy isolates and rose in frequency, but mean copy number showed no significant increase between pre-2001 and 2022-2023 samples.

   Isolates carried 1 to 7 cyp51 copies; 88% (324/368) had more than one, and single-copy isolates came mostly from Southern Europe and the Middle East. Y136F was in 298/368 (81%), Fisher p < 0.001 (1980-2001 vs 2022-2023). For copy number, Welch t-test p = 0.351. Estimates agreed with a PacBio assembly (CHVD042201: four identical tandem copies) and with ddPCR for two isolates (two copies each). · Copy number variation; Amino acid mutations in cyp51; cyp51 - DMI resistance

3. SuggestedIdentity-by-descent (IBD) clustering at cyp51 points to at least two independent origins of DMI resistance that then spread. Cluster i has more copies, a short repeat unit and homoallelic Y136F with S79T and K175N; cluster ii has fewer copies, a longer unit and heteroallelic Y136F and S509T.

   isoRelate on a 3 Mb window around cyp51 in 368 isolates gave two large clusters of 129 and 75 isolates (Europe and Turkey). Cluster i had more copies (Welch p < 0.001), and all its members shared a repeat unit of about 2.5 Kb. In cluster ii, 72/75 isolates were heteroallelic at position 136 and 65 at position 509. S509T (62 isolates, 16%) was found, among the two largest clusters, only in cluster ii. · Amino acid mutations in cyp51; Recent evolution of cyp51 in Europe; cyp51 - DMI resistance; IsoRelate and haplotype networks

4. SuggestedIn erg24 (a morpholine target), V295L is common but almost entirely limited to Northern Europe. Two nearby mutations, D291N (first report in Bgt) and the new F289H, were seen only in recent northern samples, and the authors propose both as likely morpholine-resistance mutations.

   Eight amino acid mutations were found. V295L was in 141/368 (38%), with a temporal trend of Fisher p = 0.075. D291N was in 8 isolates from 2022-2023; F289H was in 36 N_EUR isolates and absent before 2007 (p = 0.195). Y165F did not change over time (p = 0.732). In the haplotype network, the wildtype was the most common haplotype overall, especially abundant in Southern Europe, Turkey and the Middle East. · erg24 – morpholine resistance; erg24 - Morpholine resistance

5. ShownThe targets of succinate dehydrogenase inhibitors (sdhB, sdhC, sdhD), of morpholines (erg2) and of methyl benzimidazole carbamates (beta tubulin) show almost no variation, so no candidate resistance signal was found in these genes.

   There were only a few amino acid mutations, at frequencies between 0.3% and 2.5%, and virtually no copy number variation across these five genes. · Results; Other target genes