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

Abstract

Downy mildew is caused by Plasmopara viticola, an obligate oomycete plant pathogen, a devasting disease of grapevine. To protect plants from the disease, complex III inhibitors are among the fungicides widely used. They specifically target the mitochondrial cytochrome b (cytb) of the pathogen to block cellular respiration mechanisms. In the French vineyard, P. viticola has developed resistance against a first group of these fungicides, the Quinone outside Inhibitors (QoI), with a single amino acid substitution G143A in its cytb mitochondrial sequence. The use of QoI was limited and another type of fungicide, the Quinone inside Inhibitors, targeting the same gene and highly effective against oomycetes, was used instead. Recently however, less sensitive P. viticola populations were detected after treatments with some inhibitors, in particular ametoctradin and cyazofamid. By isolating single-sporangia P. viticola strains resistant to these fungicides, we characterized new variants in the cytb sequences associated with cyazofamid resistance: a point mutation (L201S) and more strikingly, two insertions (E203-DE-V204, E203-VE-V204). In parallel with the classical tools, pyrosequencing and qPCR, we then benchmarked short and long-reads NGS technologies (Ion Torrent, Illumina, Oxford Nanopore Technologies) to sequence the complete cytb with a view to detecting and assessing the proportion of resistant variants of P. viticola at the scale of a field population. Eighteen populations collected from French vineyard fields in 2020 were analysed: 12 showed a variable proportion of G143A, 11 of E203-DE-V204 and 7 populations of the S34L variant that confers resistance to ametoctradin. Interestingly, the long reads were able to identify variants, including SNPs, with confidence and to detect a small proportion of P. viticola with multiple variants along the same cytb sequence. Overall, NGS appears to be a promising method for assessing fungicide resistance of pathogens linked to cytb modifications at the field population level. This approach could rapidly become a robust decision support tool for resistance management in the future.

The widely used cymoxanil fungicide impairs respiration inSaccharomyces cerevisiaevia cytochromecoxidase inhibition

“…In both cases, the electron transfer process is blocked, leading to the inhibition of mitochondrial respiration [44–46]. Indeed, the quinone inside inhibitors used in agriculture are highly specific against oomycete pathogens [47,48]. Furthermore, it has previously been demonstrated that P. infestans mitochondria exhibit sensitivity to cyanide, a known inhibitor of C c O activity in yeast cells [49].…”

Exploiting long read sequencing to detect azole fungicide resistance mutations in Pyrenophora teres using unique molecular identifiers

“…Recently, ONT sequencing was used alongside pyrosequencing, qPCR, Ion Torrent, and Illumina sequencing to profile cytochrome b in single spored isolates and field populations of Plasmopara viticola, a pathogen of grapes 33 . The use of multiple technologies alongside biological assays allowed the authors to produce a comparative analysis highlighting the advantages and limitations of each.…”

“…The use of multiple technologies alongside biological assays allowed the authors to produce a comparative analysis highlighting the advantages and limitations of each. Unlike other sequencing technologies, ONT sequencing detected multiple SNPs in one gene from mixed field samples; however, the main drawback was sequencing errors 33 . The ONT assay had a sensitivity limit of 5% based on detection of unexpected variants 33 .…”

“…Unlike other sequencing technologies, ONT sequencing detected multiple SNPs in one gene from mixed field samples; however, the main drawback was sequencing errors 33 . The ONT assay had a sensitivity limit of 5% based on detection of unexpected variants 33 . Only reads greater than Q7 were used in the analysis and 5000 reads from each sample were aligned to the reference sequence and variable regions searched using either BLAST or Nanopolish analysis.…”

“…This study also used a quality score cutoff of 7. Frequency of each variant was computed as percentage of total reads 33 .…”