The use of long-read PCR amplicon sequencing to study the evolution of resistance to zoxamide, oxathiapiprolin and complex III inhibitors in French Plasmopara viticola field populations

Molecular methods are one of the most effective tools to monitor fungicide resistance. Long-read sequencing is an emerging technology in the field of plant pathology. We developed a PCR-based Oxford Nanopore Technologies amplicon sequencing method allowing the simultaneous detection and quantification of Plasmopara viticola variants conferring fungicide resistance to complex III inhibitors, zoxamide and oxathiapiprolin in the same vineyard population. Analysis of cyt b gene variants in natural P. viticola populations showed that almost all samples (23 out of 24 populations) collected in France contain variants G143A, S34L and/or E203-DE-V204 insertion. In the analysed populations, only cyt b reads with both substitutions S34L and G134A were detected at significant levels, suggesting the selection of resistant strains to both QoI fungicides and ametoctradin. French P. viticola population P36 with low sensitivity to oxathiapiprolin did not contain oxysterol binding protein sequences with both variants G770V and N837I, suggesting the presence of two different genotypes of P. viticola strain in this population. Zoxamide insensitivity associated with β-tubulin variants carrying the C239S substitution was detected in Italian vineyard populations but not in France.