Investigation of the sensitivity of Plasmopara viticola to amisulbrom and ametoctradin in French vineyards using bioassays and molecular tools

Abstract

BACKGROUND: Complex III inhibitors are key compounds in the control of Plasmopara viticola. They are prone to the development of resistance, as demonstrated by the emergence of resistance to quinone-outside inhibitors. By using a combination of bioassays and molecular methods, we monitored sensitivity to amisulbrom and ametoctradin in P. viticola populations in French vineyards from 2012 to 2017. RESULTS:We found that the alternative oxidase (AOX)-related resistance mechanism was common in French P. viticola populations. Target-site resistance to ametoctradin was first detected in 2015 and is likely caused by a single point mutation in the cytochrome b gene, leading to the S34L substitution. The role of this substitution in resistance to ametoctradin was corroborated by another study using an experimental model. A molecular biology method has been developed to detect the mutant allele. To date, the frequency of this mutation is low in French P. viticola populations and it is often co-detected with the wild-type allele. CONCLUSION: Populations of P. viticola displaying evidence of AOX-related resistance were detected for every surveyed year, and their occurrence in French vineyards seems to be increasing over time. This resistance mechanism is currently threatening the efficacy of complex III inhibitors in the field. The low frequency of the S34L allele conferring resistance to ametoctradin, and the instability of resistant phenotypes in some populations, suggest that a fitness cost may be associated with the mutation.

The cytochrome bc1 complex as an antipathogenic target

“…Further spectroscopic studies indicated that the compound could target both Q o and Q i sites [31] and the fungicide was re‐classified as QioI (https://osf.io/qwg42/). However, the appearance of the ametoctradin resistance mutation S34L in the Q i site of Plasmopara viticola ‐resistant isolates showed that the fungicide preferentially targets the Q i site, which was confirmed by the study of the mutation in the yeast model [51,65]. Analysis of in silico docking of ametoctradin into a homology model of the Q i site of Plasmopara viticola suggests a binding mode similar to that of ubiquinol in yeast cyt bc 1 .…”

“…This represents a significant decrease from the 10H + /2e − yield expected for cyt bc 1 /cytochrome c oxidase‐linked respiration. Putatively AOX‐linked fungicide resistance was first noted in laboratory‐generated strains of Z. tritici , that were resistant to the (then) newly developed QoI azoxystrobin [77], and has subsequently has been observed in QoI/QiI‐treated field isolates of the phytopathogens Plasmopara viticola , Z. tritici , Magnaporthe grisea and Mycosphaerella fijensis [65,78–80]. In vitro studies suggest that, in the absence of selective pressure, AOX overexpression in Plasmopara viticola sporangia comes with an associated fitness penalty [6], presumably due to the decreased energetic efficiency of mitochondrial respiration as outlined above.…”

“…SHAM is a relatively poor inhibitor of the AOX, with an inhibition constant ( K i ) of 21 μ m against the Trypanosoma brucei enzyme (as measured by glycerophosphate‐linked oxygen uptake assay) [81]. As an antifungal agent, SHAM has also been observed to act synergistically with QoIs in vitro , but this response is species (and QoI)‐dependent [65,70,82]. Unfortunately, SHAM is a relatively nonspecific inhibitor and poorly taken up by plants, and so is unsuitable for use in the field [70].…”

“…The significance of phytopathogen AOX activity with regard to QoI/QiI fungicide resistance in the field has been debated [6,70]. Its increasing prevalence, however, warrants close attention, particularly as this respiratory shunt (and MDR activity, as described above) may facilitate the development of QoI/QiI‐resistant cyt b mutants [65].…”

Plasmopara viticola infection affects mineral elements allocation and distribution in Vitis vinifera leaves

“…Downey mildew symptoms are characterised by initial yellow and oily spots on leaves and other green parts of the plant that evolve in necrosis and death of infected tissues, including bunches, possibly resulting in a total crop loss 4 . In order to control the disease, several fungicide treatments, including the copper (Cu)-based ones, are commonly applied in vineyards 3 , 5 , 6 . However, there are growing concerns about the possible negative impact of synthetic chemical fungicides and Cu on human health and the environment 7 – 9 .…”