Status from monitoring for Fungicide Resistance in cereals 2017

Transcription

1 1 Status from monitoring for Fungicide Resistance in cereals 2017 Including data from NORBARAG trials Created by: Thies Marten Heick and Lise Nistrup Jørgensen Aarhus University Science and Technology Department of Agroecology Research Centre Flakkebjerg DK-4200 Slagelse Denmark Date: 25 May 2018 Flakkebjerg AU Flakkebjerg Forsøgsvej 1 DK-4200 Slagelse

2 1 Foreword This report summarises data from resistance testing in cereal crops carried out in 2017 in the NORBARAG region. The sampling plan for the activity was agreed upon at the NORBARAG meeting in Vilnius in 2016 and supported by activities carried out by BASF, Bayer, DowDupont, Syngenta and Adama. Sampling was coordinated by Aarhus University Flakkebjerg, and samples have locally been organised by the following institutions: Sweden, Jordbrugsverket, Alnarp; Gunilla Berg Finland, Luke (MTT) Joikioinen; Marja Jalli Lithuania, Institute of Agriculture, Akademija; Roma Semaskiene Latvia, Latvian Plant Protection Research Centre Ltd; Olga Treikale Estonia, Estonian Crop Research Institute; Veiko Kastanje Norway, NIBIO; Andrea Ficke Thanks to everybody who contributed constructively to the generation of these data and were willing to share data with partners being involved in the North zone.

3 2 Contents SUMMARY... 3 METHOD FOR AZOLE MONITORING... 5 MONITORING FOR AZOLE SENSITIVITY TO ZYMOSEPTORIA TRITICI IN DENMARK... 6 CYP51 MUTATIONS... 9 FIELD PERFORMANCES MONITORING FOR AZOLE SENSITIVITY TO ZYMOSEPTORIA TRITICI IN SWEDEN FIELD CONTROL OF SEPTORIA TRITICI BLOTCH IN SWEDEN SENSITIVITY OF TEBUCONAZOLE AND DIFENOCONAZOLE ZYMOSEPTORIA TRITICI DATA FROM OTHER NORBARAG COUNTRIES Z. TRITICI SENSITIVITY TO FOLPET AND BOSCALID EURO-RES TRIALS IN NORBARAG REGION STROBILURIN RESISTANCE TO TAN SPOT (PYRENOPHORA TRITICI-REPENTIS) IN VITRO FUNGICIDE RESISTANCE TO NET BLOTCH... 22

4 3 Summary Sensitivity in the Septoria population to azoles in the NORBARAG region 2017 Monitoring for azole resistance in the Nordic-Baltic Zymoseptoria tritici population was conducted by Aarhus University, Department of Agroecology, Flakkebjerg. The project was carried out in collaboration with SEGES in Denmark, the Swedish Board of Agriculture in Sweden, LUKE in Finland and partners in Norway (NIBIO), Lithuania (LIZ), Latvia (LAAPC) and Estonia (EKTI). Leaf samples with septoria tritici blotch (Zymoseptoria tritici) were collected from field trials or farmers' fields. Sensitivity testing was carried out as a bioassay analysing single spore isolates on different concentrations of fungicides. This report also provides data from field performances using azoles for control of septoria leaf blotch in Denmark and Sweden as well as data on mutation frequencies (cyp51 and Sdh) and data from common field trials carried out in In Denmark, epoxiconazole and prothioconazole are the most commonly used for control of septoria tritici blotch, mostly in combinations with other azoles or SDHI fungicides (boscalid and fluopyram). In 2017, a total number of 272 isolates from 40 sites were tested for sensitivity to epoxiconazole and 270 isolates from 40 sites for sensitivity to prothioconazole-desthio, the active compound in prothioconazole responsible for inhibiting fungal growth. In recent years, sensitivity of Z. tritici towards azoles has declined significantly. This trend has continued in 2017 and is seen for epoxiconazole and prothioconazole-desthio. The average EC50 values in 2017 in Denmark were 1.81 ppm for epoxiconazole (2016: 1.31 ppm). Prothioconazole has been discontinued in The average EC50 value for prothioconazole-desthio was 0.32 ppm and higher than preliminary results from This confirms the ongoing shift in azole sensitivity in Denmark. The field performances of epoxiconazole and prothioconazole accordingly continued to decline in In Sweden, the main azole used for the control of septoria tritici blotch is prothioconazole as epoxiconazole is not authorised. In 2017, a total of 170 isolates from 18 sites were screened for sensitivity shift. The average EC50 values in 2016 in Sweden were 3.17 ppm (2016: 0.52 ppm; 2015: 0.33 ppm) and 0.58 ppm (first year tested) for epoxiconazole and prothioconazole-desthio, respectively. EC50 values were higher than in previous years and in Denmark indicate that a major shift in sensitivity towards azole fungicides has taken place. The field performances in trials carried out in Sweden in 2017 showed lower control level from prothioconazole than in previous years. A limited number of STB samples from Finland (27) and Norway (2) was tested for epoxiconazole and prothio-desthio. The average EC50 values for epoxiconazole were 0.48 and 2.60 ppm, respectively, and for prothio-desthio 0.05 and 0.27 ppm, respectively. Compared to 2016, DMI sensitivity values from 2017 remain in the same level, after a general shift during the previous years.

5 4 Sensitivity to boscalid and folpet Sixty-three Z. tritici isolates from Denmark (n = 29) and Sweden (n = 34) were tested for sensitivity to folpet and boscalid. All isolates showed high levels of sensitivities with an average resistance factor (RF) of 1 for folpet and 2 for boscalid. For the first time some outliers were found for Boscalid. Monitoring for resistance in the population of Pyrenophora teres For ten years now, the level of strobilurin resistance (F129L) in the population of P. teres was tested by BASF. As in previous years, the data showed that the level of F129L in the population of Pyrenophora teres remains stable with no dramatic changes. No cases of G137R were found. In Sweden, 6 out of 14 samples were tested positively, whereas none of the Norwegian, Finnish or Lithuanian samples had the F129L mutation. A total of 61 Danish P. teres isolates were tested for resistance to prothioconazole-desthio, fluxapyraxad and azoxystrobin, showing generally high sensitivity and low resistance factors to all three actives. Monitoring for resistance in Ramularia collo-cygni In most recent years, mutations associated with decrease in sensitivity to azoles and SDHI have been described. In 2017, a total of 61 leaf samples from Northern Europe and the United Kingdom were analysed for CYP51 mutations and c-sdh mutations. The frequencies of CYP51 mutations I325T and I328L were moderate to high, mostly in Denmark and Sweden. However, now sdh mutations were also present in the North European samples, but at a low level.

6 5 Method for azole monitoring Zymoseptoria tritici Twenty to thirty leaves with symptoms of septoria tritici blotch (STB) were sampled around GS at each site, dried at room temperature and sent to AU Flakkebjerg. To promote sporulation the leaf samples were placed in petri dishes with moist filter paper. After 18 hours. the leaves were checked for cirri. If no sporulation had occurred, the leaves were incubated for a further hours. The individual pycnidium spores were transferred unto PDA plates amended with 0.01% streptomycin and 0.01% rifampicin for propagation. The aim was to isolate ten isolates per site. In vitro test Sensitivity towards epoxiconazole and prothioconazole-desthio was estimated on the basis of pure cultured isolates aiming at ten isolates per locality. A spore suspension containing 2.5 x 10 4 spores ml -1 of each fungal isolate was produced from 5-day-old potato dextrose agar-grown vegetative Zymoseptoria tritici spores and transferred to a microtitre plate with seven different concentrations of epoxiconazole a.i. (10, 3.333, 1.111, 0.37, 0.123, 0.041, and 0 ppm) or prothioconazole-desthio a.i. (3, 1, 0.33, 0.11, 0.037, 0.012, and 0 ppm) mixed with 2x potato dextrose broth. Two technical replicates of each isolate were tested. The reference isolates IPO 323, S27 (both highly sensitive) and OP15.1 (moderately resistant) were included on one plate per run in order to ensure correct and similar conditions. The microtitre plates were incubated at 22 C in the dark for six days, after which per cent growth was assessed relative to control by first measuring absorbance on a Labsystems IEMS ELISA reader and then converting into per cent inhibition. EC50 values were estimated by means of Graphpad prism software. Microtitre plate with fungicide gradient and spore growth.

7 6 Monitoring for azole sensitivity to Zymoseptoria tritici in Denmark Sensitivity to epoxiconazole In 2017, the disease level of septoria tritici blotch was moderate to severe. STB attacks were generally more pronounced in Jutland than they were on the island of Sjælland. Isolation of single isolates was a challenge as pycnidia already had oozed in the field due to frequent rainy events. In 2017, samples were collected from 40 different localities and in total 272 isolates were tested for epoxiconazole and 270 isolates for prothioconazole-desthio (Table 1). The average EC50 value for epoxiconazole was 1.81 ppm compared to 1.39 ppm (2015: 0.45 ppm; 2014: 0.50 ppm), respectively. The average sensitivity per locality varied from 0.29 to 7.67 ppm. The reference isolate IPO 323 had an EC50 value of 0.02 ppm in this year s testing, given a variation in resistance factors between 14 and 396 for the tested localities. The average resistance factor for 2017 isolates was similar to previous years. In general, sensitivity for Danish Z. tritici isolates in 2017 was the same as in 2016; however, there were many more outliers with EC50 > 10 ppm (Figure 1). Those outliers were found in all the growing area samples. These results indicate that the general shift in sensitivity towards azoles is continuing. The variation across most sites was higher compared to previous years (Table 2). Sensitivity to prothioconazole-desthio After initial tests in 2016, prothioconazole-desthio was included in the annual sensitivity testing. In recent years, several laboratories have changed their protocol to prothioconazoledesthio. A baseline sensitivity was elaborated in 2016, analysing 26 isolates from that year. In 2017, 270 Danish Z. tritici isolates were analysed for sensitivity towards prothioconazoledesthio. The average EC50 for this a.i. was 0.32 ppm in 2017, with resistance factors ranging from 5 to 226 (Figure 3). In comparison to 2016, EC50 were twice as high, indicating a slight shift in sensitivity. Prothioconazole was discontinued. In their survey Bayer similarly found an increase in EC50 values for prothioconazole in 2017.

8 7 Table 1. Results from Danish samples screened for sensitivity of Zymoseptoria tritici to epoxiconazole and prothioconazole-desthio in Location Number Epoxiconazole Prothio-Desthio Average RF Average RF 17-ZT-DK-01 Flakkebjerg ZT-DK-02 Flakkebjerg ZT-DK-03 Flakkebjerg ZT-DK-04 Horsens,LMO ZT-DK-05 Horsens ZT-DK-06 Kolding ZT-DK-07 Rønde ZT-DK-08 Thisted ZT-DK-09 Åbenrå ZT-DK-10 Syd ZT-DK-11 Åbenrå ZT-DK-12 Odense ZT-DK-14 LMO ZT-DK-15 Ringsted ZT-DK-16 Ringsted ZT-DK-18 Lolland ZT-DK-19 Brønderslev ZT-DK-22 Nr.Kongerslev ZT-DK-23 Hjerm ZT-DK-24 Hjerm ZT-DK-25 Rønnede ZT-DK-26 Ørbæk ZT-DK-27 Odder ZT-DK-28 Hinnerup ZT-DK-29 Skive ZT-DK-30 Jyndevad ZT-DK-31 Eltang ZT-DK-32 Flakkebjerg ZT-DK-33 Flakkebjerg ZT-DK-34 Flakkebjerg ZT-DK-35 Flakkebjerg ZT-DK-37 Djursland ZT-DK-38 Djursland ZT-DK-39 Djursland ZT-DK-40 Abildgård ZT-DK-41 Holstebro ZT-DK-42 Holstebro ZT-DK-43 Bramstrup ZT-DK-44 Brønderslev ZT-DK-45 Brønderslev Reference IPO Total Average

9 Frequency EC50 epoxiconazole Denmark Figure 1. EC50 values with epoxiconazole measured in microtitre plates. Cumulative frequencies (%) of isolate sensitivity to epoxiconazole from 2013 to Table 2. Summary of measured EC50 (ppm) values for epoxiconazole and prothioconazoledesthio assessed for Z. tritici in Denmark. Total numbers of tested isolates are given in brackets. Year Epoxiconazole RF Prothioconazole RF Prothiodesthio RF (47) (180) (140) (88) (96) (96) (54) (54) (85) (85) (40) (40) (133) (98) (290) 25* 9.90 (290) 66* (262) (192) (220) (124) (26) (272) 94 Discontinued (270) 32 Ref. IPO

10 9 CYP51 mutations Since 2009, the presence of the most important CYP51 mutations (D134G, V136A/C, A379G, I381V and S524T) is investigated in the Danish Z. tritici population using qpcr or pyrosequencing, carried out in collaboration with BASF. In the past eight years, a continuing increase in frequencies of those mutations has been witnessed. In this period the frequencies of D134G and V136A have increased from 0 to 61% and 8% to 72%. Mutation S524T, regarded as a relatively new mutation in our region, was present in approximately 15% of the population. I381V continues to be wide established in Denmark; the decline of mutation A379G, known to be linked to I381V, continues (Table 3). Table 3. Frequency of CYP51 mutations in the Danish Z. tritici population. Assessed using qpcr (KASP) or pyrosequencing. Year D134G V136A V136C A379G I381V S524T % 8% 6% 42% 83% 0% % 8% 3% 46% 88% 2% % 22% 3% 35% 87% 3% % 33% 4% 22% 93% 5% % 57% 6% 25% 98% 9% % 72% 3% 15% 98% 14% In 2017, samples from several Danish localities were analysed for CYP51 mutations D134G, V136A/C, A379G, I381V and S524T. Table 4 shows the variation in mutation frequencies across the country. Compared to the years before, the Danish Z. tritici population seems to be more similar than in previous years. Especially mutations D134G and V136A have increased in the population, whereas A379G continues to decline. Table 4. CYP51 mutation in Z. tritici samples from Danish localities collected in Denmark 2017 CYP51 mutations D134G V136A V136C A379G I381V S524T Thisted 51% 65% 0% 23% 99% 3% Åbenrå 91% 96% 0% 0% 99% 0% Bornholm 54% 66% 0% 14% 99% 2% Brønderslev 51% 59% 0% 0% 99% 8% Hjerm 51% 59% 0% 0% 100% 18% Rønnede 78% 89% 0% 0% 99% 3% Lolland 65% 69% 0% 0% 99% 2% Flakkebjerg 50% 62% 12% 15% 99% 9% Horsens 51% 66% 0% 23% 96% 5% Trend

11 % control 10 Field performances Per cent control of septoria tritici blotch under field conditions was tested in several trials in Figure 2 shows specific control from applications of 2 x ½ rates from the two main azoles used for STB control applied at GS & Over the years, there has been a clear drop in performance. The efficacy of azoles has been declining since 2011, and used as a solo treatment they do not provide sufficient control against STB. However, the efficacy of Folicur (tebuconazole) and Armure (difenoconazole + propiconazole) increased in 2017, a trend that has been seen in several parts of Europe. One explanation for this development is the change within the European Z. tritici population: in recent years a selection to more insensitive isolates towards epoxiconazole and prothioconazole is observed, whereas those isolates are more sensitive towards tebuconazole and difenoconazole (D134G; V136A) Control of Septoria on flag leaf epoxi. prothio met tebu propi difeno + propi Figure 2. Control (%) of septoria tritici blotch from treatments with different azoles - 2 treatments with ½ rates. Average of two trials from different years. Assessed on flag leaf at GS Applied at GS 33 and

12 11 Monitoring for azole sensitivity to Zymoseptoria tritici in Sweden Sensitivity to epoxiconazole The disease level of STB in 2017 was moderate to high, but varied across the country with generally more severe attacks in the southern part of the country than in the centre of the country. In 2017, samples were collected from 18 different localities, and in total 170 isolates were tested for sensitivity to epoxiconazole and prothioconazole-desthio (Table 5). The average EC50 value to epoxiconazole was 3.17 ppm, compared to 0.52 ppm in 2016 and 0.33 ppm in The average EC50 values varied for the localities between 0.66 and 5.92 ppm. The reference isolates IPO 323 had a sensitivity of approximately 0.02 ppm and accordingly resistance factors varied between 44 and 305 for the different localities. In contrast to the Danish Z. tritici population, the shift in the Swedish population had been slower. Results from 2017 show that a major sensitivity shift has taken placed that year as all localities but one had average EC50 values over 1.00 ppm (Table 6). Figure 5 illustrates the frequency of EC50 values in Sweden from 2014 to Sensitivity to prothioconazole-desthio The average EC50 value to prothioconazole-desthio in 2017 was 0.58 ppm. This was the first year of testing and therefore there is no reference to previous years. EC50 values for specific localities varied from 0.03 to 1.61 ppm. The reference isolate (IPO323) had a sensitivity of 0.01 ppm, giving resistance factors per locality between 4 and 197, indicating that a large variation in the populations sensitivity exist. Isolates with high levels of EC50 values could be found in all regions of Sweden (Table 4). Sensitivity data from 2017 indicate that a major shift for azole fungicides has taken place in Sweden

13 Cumulative frequency EC50 prothioconazole-desthio Denmark Sweden Figure 3. Cumulative frequencies (%) of EC50 values of prothioconazole-desthio (ppm) for Z. tritici populations in Denmark and Sweden Table 5. Summarising results from Sweden from EC50 for epoxiconazole and prothioconazole for Swedish localities in Prothio- Epoxiconazole Location Number Desthio Average RF Average RF 17-ZT-SW-01 Marstad, Skänninge ZT-SW-02 Sjökumla, Motala ZT-SW-03 Sjökumla, Motala ZT-SW-04 Uppåkra, Lund ZT-SW-05 Gislöv, Klagstorp ZT-SW-06 Vessigebro, Halmstad ZT-SW-07 Hviderup, Eslöv ZT-SW-08 Wirblad, Rinkaby, Kalmar ZT-SW-09 Winström Hagby, Borgholm, Öland ZT-SW-10 Åstorp, Ängelholm ZT-SW-11 Tegelvreten, Falun ZT-SW-12 Tun, Grästorp ZT-SW-13 Skofteby, Norra Härene ZT-SW-14 Glättestorp, Kvänum ZT-SW-15 Kampetorp, Grästorp ZT-SW-16 Rutekvarn, Vara ZT-SW-17 Gunnarstorp, Grästorp ZT-SW-18 Nolebo Reference IPO Total Average

14 Per cent control 13 Table 6. Swedish EC50 values for epoxiconazole and prothioconazole assessed on Zymoseptoria tritici in Sweden. Number of isolates are given in brackets. Year Epoxiconazole RF Prothioconazole RF Prothiodesthio RF (55) (101) (131) (131) (166) (166) (211) (211) (170) (170) 63* (337) 35* 7.76 (337) 89* (227) (225) (212) (173) (170) 159 Discontinued (150) 71 Ref. IPO Field control of septoria tritici blotch in Sweden Per cent control of STB under field conditions was tested in several field trials in Figure 4 shows the average control of STB at GS in trials carried out with fungicides applied at GS & The field performance of prothioconazole remained stable until 2013; however, the field performance has been declining since Figure 4. Per cent control of Z. tritici under field conditions from Southern Sweden. Average of trials with two fungicides applied at GS & Source: Gunilla Berg, Jordbruksverket.

15 Frequency Log 10 EC50 epoxiconazole Sweden Figure 5. Cumulative frequencies (%) of EC50 values of Swedish Z. tritici isolates to epoxiconazole from 2014 to CYP51 mutation Ongoing studies are investigating for CYP51 mutations in selected numbers of isolates. There is a clear difference between the development in Middle Sweden compared with southern Sweden (Table 7). An increasing number of isolates with more CYP51 mutations has been found since Especially S524T is only seen in the south and also D134G is not very common in Middle Sweden. Table 7. Frequency of CYP51 mutations in Swedish populations. Assessed using either qpcr (KASP) genotyping or pyrosequencing (by BASF). Year Region D134G V136A V136C A379G I381V S524T 2009 All Sweden 0% 19% 0% 17% 77% 0% 2013 Middle 2% 8% 0% 9% 90% 0% 2015 Middle 0% 15% 0% 15% 90% 0% 2016 Middle 3% 11% 6% 77% 96% 3% 2017 Middle 6% 8% 0& 28% 98% 0% 2013 South 19% 24% 0% 6% 85% 15% 2015 South 49% 58% 0% 19% 84% 11% 2016 South 49% 64% 5% 20% 91% 12% 2017 South 54% 67% 0% 20% 96% 9%

16 15 Sensitivity of tebuconazole and difenoconazole A total of 63 Z. tritici isolates from Denmark and Sweden were tested for their sensitivity to tebuconazole and difenoconazole. Resistance level for tebuconazole has been at a high level for many years now. In 2017, the average EC50 value was 3.09 ppm with single isolates ranging from 0.10 to 30.oo ppm. One third of the isolates tested had EC50 values higher than 1 ppm. The average EC50 was higher in Denmark (4.46 ppm) than in Sweden (1.91 ppm). The average RF for tebuconazole was 772 (reference isolate IPO323: ppm). Those values are in line with results from 2014, where the average EC50 for Z. tritici from Denmark and Sweden was 2.95 ppm ( ppm) and an average RF of 737. The in vitro sensitivity of tebuconazole has since remained stable. From other European countries, a slow recovery of tebuconazole has been reported (e.g. in Ireland and Belgium). This is partly explained by a selection of isolates which are less sensitive towards epoxiconazole and prothioconazole, but again more sensitive towards tebuconazole. In Denmark, the field performance of tebuconazole was for the first time better than this of epoxiconazole and prothioconazole, indicating that a similar development is ongoing in Denmark (Figure 2). EC50 values for difenoconazole ranged from 0.01 to 3.00 ppm, with an average EC50 value of 0.33 ppm and a resistance factor of 26, indicating the presence of slightly adapted isolates in the Scandinavian Z. tritici population. No difference was found between Danish and Swedish isolates. Zymoseptoria tritici data from other NORBARAG countries Each year, it is the goal to test for fungicide sensibility in Z. tritici isolates from the Baltic countries, Finland and Norway, in addition to Danish and Swedish Z. tritici samples. As disease pressure is normally lower than in Denmark and Sweden, only a limited number of Z. tritici isolates could be recovered (Table 7). In 2017, 27 Z. tritici from Finland were tested. The average EC50 for epoxiconazole was 0.48 ppm and at the same level as in previous years (0.74 ppm in 2016; 0.52 ppm in 2015), with five outliers with EC50 values over 1 ppm. Only two isolates from Norway were isolated in 2017, which had an average EC50 value of 2.60 ppm, also at the same (high) level as the year before (1.67 ppm in 2016). EC50 values for prothio-desthio were at the same level as the year before for both countries (Table 8). Disease pressure for STB in Lithuania and Latvia was relatively low in 2017 (2-6%), and no specific isolates were recovered and tested. As seen in previous years, a gradient of sensitivity for azoles in North European countries still exists, but a shift is ongoing. The CYP51 mutation pattern in the Baltic countries was quite different from other NORBARAG countries. This is also seen in the variation of CYP51 frequencies within the region as shown in Table 9.

17 16 Table 8. EC50 values for epoxiconazole and prothio-desthio 2017 for isolates from Finland and Norway. Country Number Epoxiconazole Prothio-Desthio Average RF Average RF Finland Norway Table 9. CYP51 mutation frequencies (%) in bulked Z. tritici samples from Norway, Latvia and Lithuania in Baltic Sea Area 2017 CYP51 D134G V136A V136C A379G I381V S524T Norway 73% 91% 0% 9% 100% 14% Finland 0% 9% 0% 14% 97% 0% Lithuania 15% 20% 0% 22% 100% 5% Latvia 67% 82% 0% 11% 100% <1% Z. tritici sensitivity to folpet and boscalid A total of 63 Z. tritici isolates from Denmark (n=29) and Sweden (n=34) were tested for sensitivity towards the multisite inhibitor folpet and SDHI boscalid. Folpet had EC50 values between 0.07 and 0.81 ppm (average 0.29 ppm; RF = 2), which was at the same level as in the period from 2014 to For boscalid the EC50 varied from 0.13 to 10 ppm; on average the EC50 was 1.98 giving a RF of 6, which was slightly higher than in previous years (Table 10). Twelve out of 63 isolates with EC50 > 1 ppm were found in this test. In some European countries (the United Kingdom, Ireland, the Netherlands) isolates with mutations associated with a decrease sensitivity to SDHIs have been observed since Until 2016, SDHIs were not largely used in Scandinavia; however, with the registration of several new compounds of this fungicide class in 2017, the use pattern is currently changing. The new SDHIs are authorised in all countries in the North Zone, with an exception of Denmark where boscalid and fluopyram are the only SDHIs. Table 10. EC50 values of Z. tritici for boscalid and folpet in 2017, 2016, 2015 and Year Boscalid Boscalid Folpet Folpet RF EC50 range EC50 range RF

18 17 SDHI mutations in Zymoseptoria tritici in 2017 In 2017, bulked Zymoseptoria tritici samples from Denmark, Sweden, Finland, Norway and Latvia were tested for the following SDHI mutations by BASF using qpcr or pyrosequencing: B-N225I/T, B-H267X, B-T268I, B-I269V,C-N86S, C-N86K, C-T79N, C- T79I, C-W80S, C-G90R, C-H152R. None of these mutations was found in the NORBARAG samples Results from AU Flakkebjerg and Syngenta, however, indicated the presence of C-T79N in few isolates of the Danish Z. tritici population in Strobilurin resistance (G143A) in Zymoseptoria tritici The frequency of mutation G143A conferring strobilurin resistance of bulked samples from Denmark, Sweden, Finland, Norway, Latvia and Lithuania was determined by BASF using qpcr. The frequencies were at a high level throughout the NORBARAG region: Denmark 99%, Sweden 96%, Norway 100%, Finland 98% and a little low in Latvia (71%) and Lithuania (62%). Whereas the level of G143A was already high in the previous years in Denmark, Sweden and Norway, the frequencies have increased compared to 2014 in Finland (63%), Latvia (26%) and Lithuania (19%). Euro-Res trials in NORBARAG region 2017 In 2017, a new European C-IPM project was launched under the name Euro-Res. The aims of this project are (1) to determine the level of resistance in Z. tritici populations in Europe and analyse the dynamics of fungicide resistance based on molecular methods and (2) to develop and test robust and sustainable IPM control strategies for the control of Z. tritici that minimise the risk of fungicide resistance development. The Swedish Agricultural University SLU and Jordbruksverket are active partners in the Euro-Res consortium; Aarhus Universitet contributes own field trials on behalf of Denmark. Inspired by this project, additional field trials with a modified trial plan were carried out in Finland (LUKE), Norway (NIBIO), Latvia (LAPPC) and Lithuania, with the goal to visualise the effect of different treatments on resistance in a diverse resistance environment. More information on the project can be found under the following link:

19 18 Table 11. Attack STB (%) and yield (tonnes ha -1 ) in field trials in Denmark, Sweden, Finland, Latvia and Norway. Treatment DK SWE FIN LA N SEPTTR Yield SEPTTR Yield SEPTTR Yield SEPTTR Yield SEPTTR Yield % GS 75 tonnes ha -1 % GS 79 tonnes ha -1 % GS 78 tonnes ha -1 % GS 77 tonnes ha -1 % GS 75 tonnes Untreated l Proline EC250 GS l Elatus Era GS ha -1 1 l Elatus Era + 1 l Bravo GS l Proline EC250 GS 55 1 l Elatus Era GS 55 1 l Elatus Era + 1 l Bravo GS l Elatus Era GS37/ 0.4 Armure 300 EC GS 55 1 l Inatreq GS 37/ 0.5 Elatus Era GS l Proline EC250 GS l Elatus Era GS l Armure GS l Elatus Era GS37/ 0.5 Ascra Xpro GS In the NORBARAG Euro-Res trials, disease pressure was highest in Denmark and Sweden with 92.2% and 52.5% (Table 11) STB in the untreated, respectively. In Finland, Latvia and

20 CYP51 mutations 19 Estonia only 5.5%, 3.0% and 2.5% was registered in the untreated. In Denmark and Sweden, all treatments reduced STB. Two treatments with fungicide applications at GS 37 and GS 55 controlled STB better than a solo treatment at either GS 37 or GS 55. Leaf samples were collected at GS 75. DNA was extraced from bulked samples comprised of 20 leaves and subsequently analysed by BASF using pyrosequencing or qpcr (Figures 6 10). Frequencies of mutations D134G and V136A were high (40-50%) in all samples in Denmark and Sweden. V136C and S524T were only detected at a low frequency in both countries. These data confirmed the level seen at national level (Tables 3, 4 and 7). No trend was seen following different treatments. Levels of the mutations were much lower in Finnish samples, followed by Lithuanian and Latvian samples, again confirming the diversity in the NORBARAG Z. tritici population (Table 9). 180% 160% 140% 120% 100% 80% 60% 40% 20% 0% DK-1 DK-2 DK-3 DK-4 DK-5 DK-6 DK-7 DK-8 DK-9 DK-10 DK-11 D134G V136A V136C S524T Figure 6. Frequencies of CYP51 mutations (D134G, V136A/C and S524T) post-treatment measured using pyrosequencing in the Danish field trial (all entries).

21 CYP51 mutations CYP51 mutations % 160% 140% 120% 100% 80% 60% 40% 20% 0% D134G V136A V136C S524T Figure 7. Frequencies of CYP51 mutations (D134G, V136A/C and S524T) post-treatment measured using pyrosequencing in the Swedish field trial (all entries). 180% 160% 140% 120% 100% 80% 60% 40% 20% 0% FIN-1 FIN-2 FIN-3 FIN-4 FIN-5 FIN-6 FIN-7 FIN-11 D134G V136A V136C S524T Figure 8. Frequencies of CYP51 mutations (D134G, V136A/C and S524T) post-treatment measured using pyrosequencing in the Finnish field trial (entries 1 7 and 11).

22 CYP51 mutations CYP51 mutations % 160% 140% 120% 100% 80% 60% 40% 20% 0% LA-1 LA-2 LA-3 LA-4 LA-5 LA-6 LA-7 LA-11 D134G V136A V136C S524T Figure 9. Frequencies of CYP51 mutations (D134G, V136A/C and S524T) post-treatment measured using pyrosequencing in the Latvian field trial (entries 1 7 and 11; no data for entry 6). 180% 160% 140% 120% 100% 80% 60% 40% 20% 0% LT-1 LT-2 LT-3 LT-4 LT-5 LT-6 LT-7 LT-11 D134G V136A V136C S524T Figure 10. Frequencies of CYP51 mutations (D134G, V136A/C and S524T) post-treatment measured using pyrosequencing in the Lithuanian field trial (entries 1 7 and 11).

23 22 Strobilurin resistance to tan spot (Pyrenophora tritici-repentis) Nine samples from the NORBARAG region were tested for resistance to strobilurin: one Danish, four Swedish, one Latvian and three Finnish samples. None of the samples had mutations F129L and G137R. All but one Finnish sample had G143A at frequencies ranging from 84 to 100%. In vitro fungicide resistance to net blotch Sixty-one Danish Pyrenosphora teres isolates were screened at Flakkebjerg and tested for sensitivity to azoxystrobin, fluxapyraxad and prothioconazole-desthio, using a range of concentrations. As in previous years, low EC50 values for all three actives were detected in 2017 (Table 12). Seven isolates were found with higher EC50 for fluxapyroxad. Table 12. Screening of Danish P. teres isolates to azoxystrobin, fluxapyraxad and prothio-desthio in Location No. Azoxystrobi n Fluxapyroxa d Prothio- Desthio Average RF Average RF Average RF 17-PT-DK-01 Møn PT-DK-02 Flakkebjerg PT-DK-03 Randers, Stampen PT-DK-06 Grenå, Homå PT-DK-08 Lemvig PT-DK-10 Hinnerup PT-DK-12 Horsens PT-DK-13 Everdrup, Tappernøje PT-DK-14 Askov PT-DK-15 Gørløse PT-DK-16 Gørløse PT-DK-18 Abildgård Reference Total 61 Average

24 23 Strobilurin resistance to net blotch In 2017, ten samples with net blotch were investigated for their frequency of mutation F129L and G137R, conferring resistance to QoI fungicides. The mutation F129L is known to be a mutation which only partly influences the field performances of strobilurins. The samples were collected from field trials by AU Flakkebjerg, SEGES and the Tystofte Foundation and originate mainly from untreated plots in field trials. As in previous years, the investigation for mutations was carried out by BASF. The data from 2017 showed that the level of F129L in the population of Pyrenophora teres remains stable with no dramatic changes. No cases of G137R were found. Data showed that F129L was found in 80% of the tested Danish samples. Data from the last 10 years monitoring are illustrated in Table 13. The localities where no resistance was found were in the island of Funen and Southern Jutland. Field data from Flakkebjerg where the level of F129L is quite high have shown that the different strobilurins perform differently. Amistar has been seen to be more influenced by F129L than Comet and Aproach/Acanto. Although the number of positive samples is moderate, it can unfortunately not be verified which fields are affected with F129L mutations before treatments, so farmers generally have to go for the most effective products. Table 13. Summing up of results from the strobilurin resistance investigation, F129L incidence in the net blotch fungus (Pyrenophora teres) in Denmark. Year No. of samples No. without F129L No. with 1-20% No. with >20-61% No. with >60% % samples with F129L

25 24 Samples from other Scandinavian countries were also investigated for F129L. A total of 8 out of 14 Swedish samples were tested negative; the amount in six positive samples ranged from 11% to 78%. No F129L was found in Norway (two samples), Finland (ten samples) and Lithuania (one sample). Mutations conferring resistance to azoles and SDHI fungicides in the Ramularia collo-cygni populations in Northern Europe 2017 In recent years, ramularia leaf spot (Ramularia collo-cygni; Rcc) has gained more interest as cases of multiple resistance to QoI, azole and SDHI fungicides have been reported from Germany and other countries. In the NORBARAG region, medium to severe attacks of the disease were observed in Several target site mutations have been identified conferring resistance to azoles and SDHIs. In order to get an idea of the resistance status in the NORBARAG region leaf samples with symptoms of Rcc were analysed for frequencies of CYP51 mutations I325T and I328L and sdhc mutations H146R and H153R. DNA of leaf samples provided by several research institutions throughout Northern Europe and the United Kingdom was extracted at AU Flakkebjerg. Analyses of mutations were carried out by qpcr by BayerCropScience and Syngenta. In total, leaf samples of 61 sites were tested (Table 14). Table 14. Average frequencies (%) of mutations conferring resistance to SDHI fungicides (sdhc) or triazoles (CYP51) in Scandinavia, the Baltic countries, Ireland, Scotland, England and Northern Ireland. Country sdhc CYP51 H146R H153R I325T I328L Sites Denmark Sweden Norway Finland Latvia Estonia England Scotland Northern Ireland Ireland In Denmark high frequencies of CYP51 mutations were seen in 26 of 27 samples; in Sweden, only two sites showed high frequencies. SDHI mutations were found at a low level in both

26 25 countries. Those findings are in line with samples from the other Northern European samples: whereas CYP51 mutations are present in the NORBARAG region, SDHI mutations do not occur as frequent. This is in contrast to the situation in the UK and Ireland, where high levels of both CYP51 and SDHI mutations are found (Table 14?). So far, no field failure has been seen in the NORBARAG region. However, a decreased dose response of both azoles and SDHIs has been reported in Germany and the UK (see FRAC).

27 0 DK SWE FIN N LA LT EE Wheat Septoria azoles SDHI/ Flakkebjerg/BASF/Bayer Stagonospora Bioforsk - N Mildew metrafenon BASF BASF Leaf samples to Epilogic Tan spot BASF Yellow rust Fusarium Bioforsk - N QoI resistance few samples from field trials To be decided Prepared to analysed some samples Barley Net blotch BASF/Bayer Ramularia Mildew QoI EPilogic Rhyncho, Dupont Potato Alternaria solani 30 isolates in total contact Bent Nielsen AU