Characterisation of metrafenone and succinate dehydrogenase inhibitor resistant isolates of the grapevine powdery mildew Erysiphe necator

Grape powdery mildew, Erysiphe necator, is one of the most significant plant pathogens, which affects grape growing regions world-wide. Because of its short generation time and the production of large amounts of conidia throughout the season, E. necator is classified as a moderate to high risk pathogen with respect to the development of fungicide resistance. The number of fungicidal mode of actions available to control powdery mildew is limited and for some of them resistances are already known. Aryl-phenyl-ketones (APKs), represented by metrafenone and pyriofenone, and succinate-dehydrogenase inhibitors (SDHIs), composed of numerous active ingredients, are two important fungicide classes used for the control of E. necator. Over the period 2014 to 2016, the emergence and development of metrafenone and SDHI resistant E. necator isolates in Europe was followed and evaluated. The distribution of resistant isolates was thereby strongly dependent on the European region. Whereas the north-western part is still predominantly sensitive, samples from east European countries showed higher resistance frequencies. Classical sensitivity tests with obligate biotrophs can be challenging regarding sampling, transport and especially the maintenance of the living strains. Whenever possible, molecular genetic methods are preferred for a more efficient monitoring. Such methods require the knowledge of the resistance mechanisms. The exact molecular target and the resistance mechanism of metrafenone is still unknown. Whole genome sequencing of metrafenone sensitive and resistant wheat powdery mildew isolates, as well as adapted laboratory mutants of Aspergillus nidulans, where performed with the aim to identify proteins potentially linked to the mode of action or which contribute to metrafenone resistance. Based on comparative SNP analysis, four proteins potentially associated with metrafenone resistance were identified, but validation studies could not confirm their role in metrafenone resistance. In contrast to APKs, the mode of action of SDHIs is well understood. Sequencing of the sdh-genes of less sensitive E. necator isolates identified four different target-site mutations, the B-H242R, B-I244V, C-G169D and C-G169S, in sdhB and sdhC, respectively. Based on this information it was possible to develop molecular genetic monitoring methods for the mutations B-H242R and C-G169D. In 2016, the B-H242R was thereby identified as by far the most frequent mutation. Depending on the analysed SDH compound and the sdh-genotype, different sensitivities were observed and revealed a complex cross-resistance pattern. Growth competition assays without selection pressure, with mixtures of sensitive and resistant E. necator isolates, were performed to determine potential fitness costs associated with fungicide resistance. With the experimental setups used, a clear fitness disadvantage associated with metrafenone resistance was not identified, although a strong variability of fitness was observed among the tested resistant E. necator isolates. For isolates with a reduced sensitivity towards SDHIs, associated fitness costs were dependent on the sdh-genotype analysed. Competition tests with the B-H242R genotypes gave evidence that there are no fitness costs associated with this mutation. In contrast, the C-G169D genotypes were less competitive, indicating a restricted fitness compared to the tested sensitive partners. Competition assays of field isolates, which exhibited several resistances towards different fungicide classes, indicated that there are no fitness costs associated with a multiple resistant phenotype in E. necator. Overall, these results clearly indicate the importance to analyse a representative number of isolates with sensitive and resistant phenotypes