New insights from short and long reads sequencing to explore cytochrome b variants of Plasmopara viticola populations collected in vineyard and related to resistance to complex III inhibitors

Abstract

Downy mildew is caused by Plasmopara viticola, an obligate oomycete plant pathogen, a devasting disease for grapevine. To preserve plants from the disease, complex III inhibitors are among the widely used fungicides that specifically target the mitochondrial cytochrome b (cytb) of the pathogen to block cellular respiration mechanisms. In French vineyard, P. viticola developed resistance against a first category of these fungicides, the Quinone outside inhibitors, by exhibiting a single amino acid substitution G143A in its cytb mitochondrial sequence. Their usage was restricted and another kind of fungicides, Quinone inside inhibitors, targeting the same gene and highly effective against oomycetes, were used instead. Recently however, less sensitive P. viticola populations were detected after treatments with some inhibitors, in particular ametoctradin and cyazofamid. By isolating resistant single-sporangia strains of P. viticola to these fungicides, we characterized new variants in cytb sequences associated with cyazofamid resistance: a point mutation (L201S) and more strikingly, two insertions (E203-DE-V204, E203-VE-V204). In parallel with classical tools, pyrosequencing and RT-PCR, we then benchmarked both short and long-reads NGS technologies (Ion Torrent, Illumina, Oxford Nanopore Technologies) to sequence the complete cytb with the prospect to detect and assess the proportion of resistant variants of P. viticola at a natural population scale. Eighteen populations collected from French vineyard fields in 2020 were analysed: 12 show a variable proportion of G143A, 11 of E203-DE-V204 and 7 populations of the S34L variant that confers resistance to ametoctradin. Interestingly, long reads were able to identify variants, including SNPs, with confidence and detect a small proportion of P. viticola showing several variants along the same cytb sequence. Altogether, NGS appear promising methods to evaluate pathogen resistance towards fungicides related to cytb modifications at a population scale in the field. This approach could be rapidly a robust decision-support management tool for vineyard in future.