Fiona Burnett

Proposal for a unified nomenclature for target-site mutations associated with resistance to fungicides

Abstract Evolved resistance to fungicides is a major problem limiting our ability to control agricultural, medical and veterinary pathogens and is frequently associated with substitutions in the amino acid sequence of the target protein. The convention for describing amino acid substitutions is to cite the wild‐type amino acid, the codon number and the new amino acid, using the one‐letter amino acid code. It has frequently been observed that orthologous amino acid mutations have been selected in different species by fungicides from the same mode of action class, but the amino acids have different numbers. These differences in numbering arise from the different lengths of the proteins in each species. The purpose of the present paper is to propose a system for unifying the labelling of amino acids in fungicide target proteins. To do this we have produced alignments between fungicide target proteins of relevant species fitted to a well‐studied ‘archetype’ species. Orthologous amino acids in all species are then assigned numerical ‘labels’ based on the position of the amino acid in the archetype protein.

Development and use of microsatellite markers to study diversity, reproduction and population genetic structure of the cereal pathogen Ramularia collo-cygni.

Ramularia collo-cygni (Rcc) is a major pathogen of barley that causes economically serious yield losses. Disease epidemics during the growing season are mainly propagated by asexual air-borne spores of Rcc, but it is thought that Rcc undergoes sexual reproduction during its life cycle and may also disperse by means of sexual ascospores. To obtain population genetic information from which to infer the extent of sexual reproduction and local genotype dispersal in Rcc, and by implication the pathogens ability to adapt to fungicides and resistant cultivars, we developed ten polymorphic microsatellite markers, for which primers are presented. We used these markers to analyse the population genetic structure of this cereal pathogen in two geographically distant populations from the Czech Republic (n=30) and the United Kingdom (n=60) that had been sampled in a spatially explicit manner. Genetic diversity at the microsatellite loci was substantial, Ht=0.392 and Ht=0.411 in the Czech and UK populations respectively, and the populations were moderately differentiated at these loci (Θ=0.111, P<0.01). In both populations the multilocus genotypic diversity was very high (one clonal pair per population, resulting in >96% unique genotypes in each of the populations) and there was a lack of linkage disequilibrium among loci, strongly suggesting that sexual reproduction is an important component of the life cycle of Rcc. In an analysis of spatial genetic structure, kinship coefficients in all distance classes were very low (-0.0533 to 0.0142 in the Czech and -0.0268 to 0.0042 in the Scottish population) and non-significant (P>0.05) indicating lack of subpopulation structuring at the field scale and implying extensive dissemination of spores. These results suggest that Rcc possesses a high evolutionary potential for developing resistance to fungicides and overcoming host resistance genes, and argue for the development of an integrated disease management system that does not rely solely on fungicide applications.

Changes in field dose–response curves for demethylation inhibitor (DMI) and quinone outside inhibitor (QoI) fungicides against Zymoseptoria tritici, related to laboratory sensitivity phenotyping and genotyping assays

BACKGROUND Insensitivity of Zymoseptoria tritici to demethylation inhibitor (DMI) and quinone outside inhibitor (QoI) fungicides has been widely reported from laboratory studies, but the relationships between laboratory sensitivity phenotype or target site genotype and field efficacy remain uncertain. This article reports field experiments quantifying dose-response curves, and investigates the relationships between field performance and in vitro half maximal effective concentration (EC50 ) values for DMIs, and the frequency of the G143A substitution conferring QoI resistance. RESULTS Data were analysed from 83 field experiments over 21 years. Response curves were fitted, expressed as percentage control, rising towards an asymptote with increasing dose. Decline in DMI efficacy over years was associated with a decrease in the asymptote, and reduced curvature. Field ED50 values were positively related to in vitro EC50 values for isolates of Z. tritici collected over a 14-year period. Loss of QoI efficacy was expressed through a change in asymptote. Increasing frequency of G143A was associated with changes in field dose-response asymptotes. CONCLUSION New resistant strains are often detected by resistance monitoring and laboratory phenotyped/genotyped before changes in field performance are detected. The relationships demonstrated here between laboratory tests and field performance could aid translation between laboratory and field for other fungicide groups.

Characterisation of Ramularia collo-cygni laboratory mutants resistant to succinate dehydrogenase inhibitors.

BACKGROUND Ramularia collo-cygni (Rcc) is responsible for Ramularia leaf spot (RLS), a foliar disease of barley contributing to serious economic losses. Protection against the disease has been almost exclusively based on fungicide applications, including succinate dehydrogenase inhibitors (SDHIs). In 2015, the first field isolates of Rcc with reduced sensitivity to SDHIs were recorded in some European countries. In this study we established baseline sensitivity of Rcc to SDHIs in the United Kingdom and characterised mutations correlating with resistance to SDHIs in UV-generated mutants. RESULTS Five SDHI-resistant isolates were generated by UV mutagenesis. In four of these mutants a single amino acid change in a target succinate dehydrogenase (Sdh) protein was associated with decrease in sensitivity to SDHIs. Three of these mutations were stably inherited in the absence of SDHI fungicide, and resistant isolates did not demonstrate a fitness penalty. There were no detectable declines in sensitivity in field populations in the years 2010-2012 in the United Kingdom. CONCLUSIONS SDHIs remained effective in controlling Rcc in the United Kingdom in the years 2010-2012. However, given that the first isolates of Rcc with reduced sensitivity appeared in other European countries in 2015, robust antiresistance strategies need to be continuously implemented to maintain effective disease control.