Data and methods applied

How are those data generated?

Each year, we collected diseased leaf samples from Scandinavia, the Baltic area and other countries. Single pycnidium isolates are produced and analysed by sequencing or qPCR. In some cases, leaf samples are bulked and analyzed using a pyrosequencing approach (Sierotzki et al. 2019). This service is either provided by the industry (BASF) or by collaborating institutes. The outcome of all of the methods mentioned above is the information to what extent (in per cent) the target site mutations associated with fungicide resistance are present in different European areas. This investigation aims to follow the evolution of CYP51 (azole) and SDH mutations over time and space.

Azole resistance to Zymoseptoria tritici

After a drop in-field performance of the currently widely used azole fungicides in many European countries, investigations for specific target site mutations have been carried out to see how variable the situation of CYP51 mutations is across a range of countries. Leaf samples or single isolates are analysed for CYP51 mutations D134G, V136A/C, A379G, I381V by pyrosequencing and S524T by qPCR or by sequencing. In the natural Z. tritici population, more mutations occur, however, the mutations included in this investigation are considered to give a good indication of the resistance situation for the currently most widely used azoles epoxiconazole, prothioconazole, tebuconazole and difenoconazole (Cools and Fraaije 2013). Furthermore, due to the complexity of the CYP51 gene, several mutations may occur in one single Z. tritici strain; consequently, numerous haplotypes are found at different localities (Huf et al. 2018). The results from this network have shown that some general trends in evolution in the current European Z. tritici population are seen, e.g., an overall increase of D134G and V136A, whereas S524T remains more widespread in Ireland and the U.K., reflecting a more intensive use of azoles during many seasons in these parts of Europe. Field trials have shown that strategies, where the same azole is used repeatedly, lead to an increase in point mutations like D134G, V136A and S524T (Heick et al. 2017).

SDHI resistance to Zymoseptoria tritici

SDHI (succinate dehydrogenase inhibitor) are widely used for control of septoria tritici blotch in most regions of Europe. Currently, fungicide resistance development is also observed for this key fungicide group. Several point mutations in the subunits of the target gene Sdh have been associated with high EC₅₀ values (Rehfus et al. 2018).  The most critical mutations are now included on this platform (C-T79N, C-T79I, C-W80S, C-N86S, C-G90R, C-H152R). Among those a rapid increase in frequencies is seen for C-T79N, which is considered a ‘moderate’ mutation, giving a ‘moderate’ resistance level. However, when present in the field in high frequencies, it has been seen that this mutation can lead to field failure. Mutation C-H152R, which through in-vitro evolutionary studies, was identified as causing resistance to all the major SDHI, has been detected in European field Z. tritici populations, first in Ireland in 2015 (Dooley et al. 2016; Gutiérrez-Alonso et al. 2017; Rehfus et al. 2018). The occurrence of this allele, which appeared to be relatively low and restricted to Ireland and the U.K., has recently appeared sporadically in continental Europe, including Northern France, Germany and the Netherlands (FRAC, 2018). In 2017 and 2018, the first isolates harboring the C-T79N mutation were found in Denmark and Estonia, and a few cases of the presence of the C-N86S mutation was found in Sweden. As seen for the evolution of azole resistance of Z. tritici, a gradient for development and spread of SDHI resistance throughout Europe seem to exist.

The formulated guidelines based on field studies and current literature to reduce fungicide resistance development is to keep the total number of spraying with azoles down and not to exceed three in one season, not to use the same azole more than twice, and to include products containing other modes of action, like SDHIs and multisite inhibitors. Field trials have also shown that by diversifying the spray programme, including different azoles, SDHI and the multisite inhibitor (e.g., chlorothalonil and Folpet), the selections for specific mutations can be lowered. Similar guidelines also applied for SDHI. However, currently, the restricted availability of fungicides may constrain exploiting this approach. As an example, chlorothalonil will not be available anymore from 2021. Furthermore, including I.P.M. elements like resistant cultivars are also seen as essential steps to minimise the need for treatments, which again can help to hold down the resistance levels.

Scientists, advisory services and chemical companies should help to raise the awareness of the current situation and in communicating the recommendations to the farmers.

Data included on this platform originate from the annual sensitivity testing carried out by A.U. and collaborating institutes in the Nordic-Baltic region, the C-IPM project Eurores and Eurowheat trials carried out in recent years.

Literature

Cools, H.J., Fraaije, B.A. (2013). Update on mechanisms of azole resistance in Mycosphaerella graminicola and implications for future control. Pest Management Science 69 (2):150-5.

Dooley, H., Shaw, M.W., Mehenni-Ciz, J., Spink, J., Kildea, S. (2016). Detection of Zymoseptoria tritici SDHI insensitive field isolates carrying the SdhC-H152R and SdhD-R47W substitutions. Pest Management Science 72 (12):2203-2207.

FRAC (2018). www.frac.info/home/news/2018/04/13/minutes-and-recommendations-of-the-sdhi-working-group-are-now-available. Accessed 27.03.2020.

Gutiérrez‐Alonso, O., Hawkins, N.J., Cools, H.J., Shaw, M.W., Fraaije, B.A. (2017). Dose‐dependent selection drives lineage replacement during the experimental evolution of SDHI fungicide resistance in Zymoseptoria tritici. Evolutionary Applications 10 (10):1055–1066.

Heick, T.M., Justesen, A.F., Jørgensen, L.N. (2017). Anti-resistance strategies for fungicides against wheat pathogen Zymoseptoria tritici with focus on D.M.I. fungicides. Crop Protection 99:108-117

Huf, A., Rehfus, A., Lorenz, K.H., Bryson, R., Voegele, T.R., Stammler, G. (2018) Proposal for a new nomenclature for CYP51 haplotypes in Zymoseptoria tritici and analysis of their distribution in Europe. Plant Pathology 67 (8):1706-1712.

Rehfus, A., Strobel, D., Bryson, R., Stammler G. (2018). Mutations in sdh genes in field isolates of Zymoseptoria tritici and impact on the sensitivity to various succinate dehydrogenase inhibitors. Plant Pathology 67 (1):175-180.

Sierotzki, H., Mehl, A., and Stammler, G. (2019). Molecular Detection Methods for Fungicide Resistance. In Stevenson, K. L., McGrath, M. T., and Wyenandt, C. A., editors, Fungic. Resist. North. Am., pages 175–193. American Phytopathological Society, St. Paul, Minnesota 55121, U.S.A., second edition.