Fungicide resistance-related investigations in cereals

Transcription

1 Applied Crop Protection 2017 V Fungicide resistance-related investigations in cereals Thies Marten Heick, Lise Nistrup Jørgensen, Hanne-Birgitte Christiansen & Birgitte Boyer Frederiksen Azole resistance of Zymoseptoria tritici in Denmark and Sweden Since 2005 Zymoseptoria tritici (Z. tritici) isolates from Denmark and Sweden have been tested in vitro for azole resistance (epoxiconazole, later prothioconazole and prothioconazole-desthio) to survey shifting of sensibility of the North European Z. tritici populations. Each year, diseased leaf samples at growth stage are collected in collaboration with SEGES, Jordbruksverket in Sweden and local advisors and sent to Flakkebjerg. In 2017, a total of 278 Danish isolates from 40 sites and 170 Swedish isolates from 18 sites were investigated for sensitivity to epoxiconazole and prothioconazole-desthio. The aim was 10 isolates per site. The 58 sites were chosen to give a representative picture of resistance in both countries. Prothioconazole has been discontinued in 2017 due to severe changes in sensitivity and difficulties determining the EC 50 values with this active at higher doses. Instead, isolates were investigated for prothioconazole-desthio, responsible for the antifungal action of prothioconazole. Data from previous years testing are given in Heick et al. (2017 a, b). Resistance tests were carried out according to the FRAC protocol for DMI sensitivity testing of Z. tritici ( The individual pycnidia isolates were used to produce spore suspensions by scarping off six-day-old Z. tritici spores and transferring them into demineralised water. Spore suspensions were homogenised and adjusted to a spore concentration of 2.4 x 10 4 spores ml -1. Technical duplicates of each isolate were included in the study. Epoxiconazole and prothioconazole-desthio were mixed separately with 2 x potato dextrose broth (PDB) to obtain the following final microtitre plate fungicide concentrations (ppm): 30, 10, 3.3, 1.0, 0.3, 0.1, 0.33, 0 and 3.0, 1.0, 0.3, 0.1, 0.03, 0.01, , 0, respectively. A total of 100 µl of spore suspension and 100 µl of fungicide solution were added to a 96-deep well microtitre plate. Microtitre plates were wrapped in tin foil and incubated at 20 C for six days in the dark. Plates were visually analysed in an Elisa reader at 620 nm. Fungicide sensitivities were calculated as the concentration of a fungicidal compound, at which fungal growth in vitro is inhibited by 50% (EC 50 ) by non-linear regression (curve fit) using GraphPad Prism (GraphPad software, La Jolla, CA, USA). Isolates IPO323 and OP15.1 were used as reference isolates. Results - Denmark After a significant shift from 2015 to 2016 (2; 3), the average EC 50 values for epoxiconazole were once more higher in 2017, indicating that sensitivity shifting is ongoing (Figure 1). The average EC 50 f0r epoxiconazole was 1.81 ppm (2016: 1.39 ppm) (Table 1). A total of 112 of the 278 tested isolates had a EC 50 of > 1 ppm, of which 26 reached 10 ppm, with a potential of even higher EC 50. The average resistance factor (RF) for epoxiconazole, as compared to the reference isolate IPO 323, was 94, compared to 66 in 2016 (Table 1). Isolates with high EC 50 values were found at all sites (Table 2). These results support the trend towards more outliers in recent years and coincide with changes in mutations and ongoing decline in field efficacy of azoles seen since 2008 (see chapter II Disease control in cereals). After a preliminary test with prothioconazole-desthio in 2016, this active substance was included in 2017 to replace prothioconazole. The average EC 50 for Danish isolates was 0.32 ppm (2016: 0.13 ppm). The RF for prothioconazole-desthio was 32 almost two times of the RF in 2016; in that year, however, only 26 isolates were tested compared to 262 in Results from previous years with prothioconazole ought not to be compared to prothioconazole-desthio as it is not possible to correlate the two chemical 64

2 compounds. Furthermore, there is no clear cross-resistance between epoxiconazole and prothioconazole-desthio. However, the higher number of isolates having high EC 50 for prothioconazole-desthio confirms the findings of EC 50 for epoxiconazole. Table 1. Summary of measured EC 50 (ppm) values and resistance factors (RF) for epoxiconazole and prothioconazole and prothioconazole-desthio assessed for Z. tritici in Denmark. Total numbers of tested isolates are shown in brackets. Prothioconazole was discontinued in Year EC50 epoxiconazole RF EC50 prothioconazole RF EC50 prothio-desthio (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) (278) 94 Discontinued (262) 32 Ref. IPO *adjusted RF due to dose and ref. isolate change in Numbers in brackets indicate the total number of isolates tested. RF Figure 1. Cumulative frequencies of EC 50 values of epoxiconazole (ppm) for Danish Z. tritici populations

3 Table 2. Results from single sites with data from sensitivity testing for Zymoseptoria tritici screened on epoxiconazole and prothio-desthio using approximately 10 isolates per site. 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 Landboforening ZT-DK-38 Djursland Landboforening ZT-DK-39 Djursland Landboforening ZT-DK-40 Abildgård ZT-DK-41? ZT-DK-42 Holstebro ZT-DK-43 Bramstrup ZT-DK-44 Brønderslev ZT-DK-45 Brønderslev Reference IPO Total Average

4 Results - Sweden In 2016, a significant, but gradual shift in sensitivity towards epoxiconazole and prothiconazole of Z. tritici from 2014 to 2016 was observed, though not as pronounced as in Denmark (Table 3). In 2017, this development continued at a rapid pace. Consequently, the Swedish Z. tritici population has overtaken the Danish population. The average EC 50 values for epoxiconazole and prothioconazole-desthio were 3.17 pp, (2016: 0.52 ppm) and 0.58 ppm (no results from 2016), respectively. Figure 2 illustrates the shifting of EC 50 values for epoxiconazole from 2014 to Whereas in previous years there was a clear difference for locations in Middle and Southern Sweden, the shift that occurred in 2017 appears to have taken place in the entire country (Table 4). Accordingly, EC 50 values for prothioconazole-desthio were on average higher in Sweden than in Denmark with an average of 0.58 ppm (Figure 3). Table 3. Summary of measured EC 50 (ppm) values and resistance factors (RF) for epoxiconazole and prothioconazole assessed for Z. tritici in Sweden. Total numbers of tested isolates are shown in brackets. Year Epoxiconazole RF Prothioconazole RF Prothio-desthio RF (55) (101) (131) (131) (166) (166) (211) (211) (170) (170) 63* (337) 35* 7.76 (337) 89* (227) (225) (212) (173) (163) 170 Discontinued (150) 71 Ref. IPO *adjusted RF due to dose and ref. isolate change in Number in brackets indicate the total number of isolates tested. Table 4. Results from single sites with data from sensitivity testing for Z. tritici screened on epoxiconazole and prothioconazole using approximately 10 isolates per site. Location Number Epoxiconazole Prothio-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, Falkenberg Lilla Böslid, 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

5 Figure 2. Cumulative frequencies of EC 50 values of epoxiconazole (ppm) for Swedish Z. tritici populations Figure 3. Cumulative frequencies of EC 50 values of prothioconazole-desthio (ppm) for Z. tritici populations in Denmark and Sweden CYP51 mutations in the Z. tritici populations in the Baltic region 2017 The decline of triazoles has been associated with molecular changes in the fungus (Cools & Fraaije, 2013). Among those changes, the main focus has been on mutations in the CYP51 target gene of the azoles. In 2017, bulked leaf samples from diseased leaf samples from Denmark, Sweden, Norway, Finland, Latvia and Lithuania were analysed by sequencing, pyrosequencing and qpcr (performed at BASF A/S) for the frequency of the most important CYP51 mutations in Z. tritici: D134G, V136A/C, I381V and S524T (Table 5). As seen in previous years, mutation I381V was present throughout the region at frequencies of %. The frequencies for mutations D134G, V136A/C and S524T, all of which have only recently emerged in the North European Z. tritici population, varied greatly (Heick et al., 2017b). 68

6 Those mutations are now found in the majority of isolates in Denmark, Southern Sweden and Norway. Compared to 2016, the frequencies remain at the same level. In Finland and Latvia those mutations do not predominate as it was the case in Results indicate that a development has taken place in Lithuania, where frequencies resemble those in Denmark and Southern Sweden. Compared to previous years, a general trend towards higher frequencies of CYP51 mutations is seen, indicating the rapid development of less azole-sensitive Z. tritici strains in Northern Europe. The samples were also analysed for SDHI mutations (B-N225I/T, B-H267X, B-T268I, B-I269V, C-N86S/K, C-G90K); until now, no SDHI mutation has been found in the Nordic-Baltic region. Table 5. CYP51 mutation frequencies (%) in bulked Z. tritici samples from Denmark, Sweden (Southern and Middle), Norway, Lithuania, Latvia and Finland. n.a. = not available. Baltic Sea Area 2017 CYP51 D134G V136A V136C A379G I381V S524T Denmark 61% 72% 3% 15% 98% 14% Sweden (Southern) 54% 67% 0% 20% 96% n.a. Sweden (Middle) 0% 8% 0% 28% 98% n.a. Norway 55% 49% 0% 9% 100% n.a. Lithuania 67% 82% 0% 11% 100% n.a. Latvia 15% 20% 0% 22% 100% n.a. Finland 0% 9% 0% 99% 100% n.a. As also seen in previous seasons different azoles have differences in selection power for specific CYP51 mutations (Heick et al., 2017a). This trend was confirmed in two 2017 trials, in which treatments with difenoconazole (= Armure) in line with previous years again showed a different pattern in selection for particularly V136A and D134G (Figure 4). Other data from the trial correspond to Table 13 in chapter II. Figure 4. Summarised frequencies of 4 CYP51 mutations following 2 or 3 treatments with different fungicide solutions. Average of 2 trials (17328). Leaves with Septoria were sampled in July and analysed using pyrosequencing and QPCR. 69

7 SDHI mutations in the Z. tritici populations in the Baltic region 2017 In 2017, all samples tested for CYP51 mutations were also tested for presence of SDHI mutations. None of the tested samples showed any signs of mutations yet. This is in contrast to major parts of Europe where several SDHI mutations are found. In 2017 Norbarag samples were tested for the following SDHI mutations: B-N225I; B-N225T, B-H267X, B-T268I, B-I269V, C-N86K, C.T79N, C-T79I, C.W80S, C- G90R and C-H152R. Strobilurin resistance to net blotch In 2017, a total of ten samples with net blotch were investigated for the distribution of the QoI resistance mutations F129L and G137R. 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. Similarly to 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 6. The sites where no resistance was found were in the island of Funen and in 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 200 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 6. 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 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). 70

8 Mutations conferring resistance to azoles and SDHI fungicides in the Ramularia collocygni populations in Northern Europe 2017 Fungicide resistance in the ramularia leaf spot populations has become widespread in recent years. Resistance to strobilurins has been known for many years, but since 2015 resistance problems to both SDHI and azoles have been recorded particularly in Germany, France and the UK. In order to get an idea of the level of resistance in the Northern zone, it was decided to analyse samples in Barley leaf samples with ramularia leaf spot (Ramularia collo-cygni) were provided to AU Flakkebjerg by several research institutions throughout Northern Europe and the UK. Leaves were tested and DNA was extracted for further analysis of mutations. The testing for specific mutations was provided by Bayer Crop Science. The findings showed widespread occurrence of azole mutations in most areas, with the exception of Finland. The level of resistance found can compromise field performances. The level of SDHI mutations was more variable and still relatively low in the Northern zone. High levels were found in the UK and Ireland, where reports on poor field performances from these products also have been reported. Values below 10% reflects uncertain levels of resistance. (Table 7). Table 7. Mutations conferring resistance to SDHI fungicides (SdhC) or triazoles (CYP51) in Scandinavia, the Baltic countries, Ireland, Scotland, England and Northern Ireland. Country SdhC CYP51 Sites H146R H153R I325T I328L Denmark Sweden Norway Finland Latvia Estonia England Scotland Northern Ireland Ireland References Cools, H. J. and B. A. Fraaije (2013). Update on mechanisms of azole resistance in Mycosphaerella graminicola and implications for future control. Pest Management Science 69: Heick, T. M., A. F. Justesen and L. N. Jorgensen (2017a). Anti -resistance strategies for fungicides against wheat pathogen Zymoseptoria tritici with focus on DMI fungicides. Crop Protection 99: Heick, T. M., A. F. Justesen and L. N. Jorgensen (2017b). Resistance of wheat pathogen Zymoseptoria tritici to DMI and QoI fungicides in the Nordic-Baltic region - a status. European Journal of Plant Pathology 149: