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.

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.

Emergence of boscalid-resistant strains of Erysiphe necator in French vineyards.

The grapevine powdery mildew Erysiphe necator (E. necator) is an obligate pathogen. Powdery mildew-diseased vines show an important reduction in plant size, winter hardiness and grape yield. Even a low-level infection with powdery mildew was shown to taint wine and ultimately reduce wine quality. For many years, succinate dehydrogenase inhibitor (SDHI) fungicides, mainly the new generation active ingredients (AIs) boscalid, penthiopyrad and fluopyram, have been widely used to control powdery mildew in grapevines. The repeated use of fungicides (mainly boscalid) has resulted in the emergence of resistant microorganisms such as Botrytis cinerea (B. cinerea). However, boscalid resistance was never observed in E. necator. In this study, a large-scale survey of French grapevine field populations of E. necator revealed many field populations with low sensitivity to boscalid. Single spore strains originating from collected resistant populations showed Half maximal effective concentration (EC50) values greater than 100 mg L-1, and strains originating from boscalid sensitive populations showed EC50 values lower than 1 mg L-1. The complete nucleotide sequences of the EnSdhB succinate dehydrogenase of sensitive and resistant single spore strains revealed that H242R and H242Y substitutions in the EnSdhB succinate dehydrogenase subunit conferred E. necator resistance to boscalid. No cross-resistance of E. necator strains bearing H242R and H242Y substitutions in EnSdhB succinate dehydrogenase to fluxapyroxad and fluopyram was noticed. Therefore, our results highlight the emergence of resistance to boscalid activity in French vineyards and warrant the need of the implementation of risk assessment strategies to maintain effective grapevine protection against powdery mildew.