Evaluating the risk of fungicide resistance evolution to succinate dehydrogenase inhibitors in Ramularia collo-cygni

Abstract

Ramularia collo-cygni (Rcc) is a damaging fungal pathogen of barley (Hordeum vulgare). It is a causal agent of Ramularia Leaf Spot (RLS), which contributes to significant economic yield losses worldwide. Protection against the disease has been, and is currently, based on foliar fungicide applications as available seed treatments are not effective and there are no fully resistant barley varieties. Two groups of systemic fungicides, Succinate Dehydrogenase Inhibitors (SDHIs) and DeMethylation Inhibitors in sterol biosynthesis (DMIs), are used and in addition a protectant multisite inhibitor chlorothalonil has been used to minimise the damaging effects of RLS in barley. SDHI fungicides have been extensively used in cereals since 2005 in the UK. Resistance outbreaks to SDHIs have been reported in several plant pathogenic fungi. The risk of resistance in Rcc was unstudied and so this study aimed to develop the methodologies needed to screen populations, establish baseline sensitivity data, evaluate the risk of evolution of fungicide resistance to SDHIs in the population of Rcc and make recommendations on appropriate anti-resistance strategies to minimise the risk. A combined approach of SDHIs’ field performance under different application regimes, sensitivity testing in vitro, molecular analysis of resistance mutations and studies of the genetic structure of populations was used. The results in this study demonstrated that currently SDHI foliar applications remain effective in controlling RLS in barley. The most consistent control was observed when applying them in mixtures with DMIs. All of the tested field isolates were in the range of baseline sensitivity to SDHIs and no shifts between years and different applications regimes were observed. The molecular characterisation of Rcc resistant mutants developed in UV mutagenesis studies revealed that in four out of five mutants a single amino acid change in a target succinate dehydrogenase (Sdh) protein was associated with decrease in sensitivity to SDHIs. All of these mutations, with the exception of one mutant, were stable in the absence of SDHI fungicide. The analysis of fitness components indicated that mutated strains did not confer a fitness penalty associated with the mutation when measuring the growth in vitro. Additionally a detached leaf assay performed for two selected mutants with high or moderate resistant factors showed that the resistant phenotypes were able to colonise the leaf surface and reproduce successfully. Thus if the resistance was to develop due to these point mutations in the target Sdh gene, mutated phenotypes are likely to be fit enough to outcompete the sensitive population. Analysis of the genetic structure of the Rcc populations demonstrated that the pathogen is highly diverse, is likely to undergo sexual reproduction over the growing season (the sexual stage remains undiscovered) and has a potential for extensive spore dispersal across the field. Thus Rcc has a high evolutionary potential and could adapt to different control measures relatively quickly. In the 2012 growing season, two field phenotypes with reduced sensitivity to SDHIs were identified as a consequence of sensitivity monitoring in vitro. Neither of the isolates had any nucleotide or amino acid changes in the target Sdh gene and the mechanism responsible for the resistance in these two strains remains unknown. One of the field isolates was tested in planta and exhibited abnormal growth on the leaf surface. This could result either from fitness costs incurred due to resistance or the fact that this isolate does not belong to the Rcc species, a possibility which must be further investigated. In conclusion, SDHI fungicides remain effective in controlling RLS in barley, however this study demonstrated that there is a risk of measurable loss of efficacy in field conditions. Thus monitoring studies should be intensified and integrated crop management practices applied to prolong the life span of SDHI fungicide treatments.