Thies Marten Heick

BACKGROUND: Fenpicoxamid is a recently developed fungicide belonging to the quinone inside inhibitor (QiI) group. This is the first fungicide within this group to be active against the Zymoseptoria tritici, which causes Septoria tritici blotch on wheat. The occurrence of pre‐existing resistance mechanisms was monitored, using sensitivity assays and Illumina sequencing, in Z. tritici populations sampled in multiple European countries before the introduction of fenpicoxamid.RESULTS: Although differences in sensitivity to all three fungicides tested (fenpicoxamid, fentin chloride and pyraclostrobin) existed between the isolate collections, no alterations associated with QiI resistance were detected. Among the isolates, a range in sensitivity to fenpicoxamid was observed (ratio between most sensitive/least sensitive = 53.1), with differences between the most extreme isolates when tested in planta following limited fenpicoxamid treatment. Sensitivity assays using fentin chloride suggest some of the observed differences in fenpicoxamid sensitivity are associated with multi‐drug resistance. Detailed monitoring of the wider European population using Illumina‐based partial sequencing of the Z. tritici also only detected the presence of G143A, with differences in frequencies of this alteration observed across the region.CONCLUSIONS: This study provides a baseline sensitivity for European Z. tritici populations to fenpicoxamid. Target‐site resistance appears to be limited or non‐existing in European Z. tritici populations prior to the introduction of fenpicoxamid. Non‐target site resistance mechanisms exist, but their impact in the field is predicted to be limited. © 2022 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Cercospora leaf spot (CLS) is a globally important disease of sugar beet (Beta vulgaris) caused by the fungus Cercospora beticola. Long-distance movement of C. beticola has been indirectly evidenced in recent population genetic studies, suggesting potential dispersal via seed. Commercial sugar beet “seed” consists of the reproductive fruit (true seed surrounded by maternal pericarp tissue) coated in artificial pellet material. In this study, we confirmed the presence of viable C. beticola in sugar beet fruit for 10 of 37 tested seed lots. All isolates harbored the G143A mutation associated with quinone outside inhibitor resistance, and 32 of 38 isolates had reduced demethylation inhibitor sensitivity (EC₅₀> 1 µg/ml). Planting of commercial sugar beet seed demonstrated the ability of seedborne inoculum to initiate CLS in sugar beet. C. beticola DNA was detected in DNA isolated from xylem sap, suggesting the vascular system is used to systemically colonize the host. We established nuclear ribosomal internal transcribed spacer region amplicon sequencing using the MinION platform to detect fungi in sugar beet fruit. Fungal sequences from 19 different genera were identified from 11 different sugar beet seed lots, but Fusarium, Alternaria, and Cercospora were consistently the three most dominant taxa, comprising an average of 93% relative read abundance over 11 seed lots. We also present evidence that C. beticola resides in the pericarp of sugar beet fruit rather than the true seed. The presence of seedborne inoculum should be considered when implementing integrated disease management strategies for CLS of sugar beet in the future.

BACKGROUND: Over the past decade, demethylation inhibitor (DMI) and succinate dehydrogenase inhibitor (SDHI) fungicides have been extensively used to control to septoria tritici blotch, caused by Zymoseptoria tritici on wheat. This has led to the development and selection of alterations in the target‐site enzymes (CYP51 and SDH, respectively).RESULTS: Taking advantage of newly and previously developed qPCR assays, the frequency of key alterations associated with DMI (CYP51‐S524T) and SDHI (SDHC‐T79N/I, C‐N86S and C‐H152R) resistance was assessed in Z. tritici‐infected wheat leaf samples collected from commercial crops (n = 140) across 14 European countries prior to fungicide application in the spring of 2019. This revealed the presence of a West to East gradient in the frequencies of the most common key alterations conferring azole (S524T) and SDHI resistance (T79N and N86S), with the highest frequencies measured in Ireland and Great Britain. These observations were corroborated by sequencing (CYP51 and SDH subunits) and sensitivity phenotyping (prothioconazole‐desthio and fluxapyroxad) of Z. tritici isolates collected from a selection of field samples. Additional sampling made at the end of the 2019 season confirmed the continued increase in frequency of the targeted alterations. Investigations on historical leaf DNA samples originating from different European countries revealed that the frequency of all key alterations (except C‐T79I) has been gradually increasing over the past decade.CONCLUSION: Whilst these alterations are quickly becoming dominant in Ireland and Great Britain, scope still exists to delay their selection throughout the wider European population, emphasizing the need for the implementation of fungicide antiresistance measures. © 2021 Society of Chemical Industry.

Zymoseptoria tritici (Zt) populations adapt under the selection pressure of fungicides applied for disease control. The primary objective of this study was to assess fungicide sensitivity in the Estonian Zt population. A total of 282 Zt isolates from 2019 and 2020 were tested for sensitivity to azoles (DMIs; prothioconazole-desthio, epoxiconazole, mefentrifluconazole) and succinate dehydrogenase inhibitors (SDHIs; boscalid, fluxapyroxad). The efficacy of the tested fungicides varied considerably between the Estonian counties, but the Zt population is mainly sensitive to DMIs. Additionally, the frequencies of CYP51 gene alterations varied; D134G, V136C, A379G, and S524T had increased, but V136A and I381V showed a moderate decrease in 2020 in comparison to 2019. Sensitivity to SDHIs was stable, but boscalid was less effective than fluxapyroxad. SdhC gene mutations C-T33N, C-T34N, and C-N86S were common, but not linked with SDHI fungicide sensitivity assay results. Otherwise, mutation B-N225I in the SdhB subunit occurred in isolates with reduced sensitivity to SDHIs. Sensitivity to strobilurins was evaluated by the mutation G143A in the CytB gene, which was present in nearly half of the population. The data presented confirm the ongoing evolution of fungicide sensitivity in the Zt population in Estonia and highlight the importance of knowledge-based decisions for optimizing anti-resistance strategies in the field.

Septoria tritici blotch (STB;Zymoseptoria tritici) is the most important leaf disease of wheat in Northern and Western Europe. The problem of fungicide resistance inZ. triticipopulations is challenging future control options. In order to investigate differences in azole performances against STB, 55 field trials were carried out during four seasons (2015–2018). These trials were undertaken in ten different countries across Europe covering a diversity of climatic zones and agricultural practices. During all four seasons, four single azoles (epoxiconazole, prothioconazole, tebuconazole and metconazole) were tested. Increasing variability in the performances of these azoles against STB was observed across Europe. The efficacy of the tested azoles varied considerably across the continent and between countries. The shifts in disease control from these commonly used azoles were confirmed by increasing EC₅₀values for epoxiconazole, prothioconazole-desthio and metconazole. The sensitivity towards tebuconazole remained relatively low across the four years. The frequencies ofCYP51mutations varied substantially across Europe, with a clear pattern of significantly decreasing frequencies of D134G, V136A and S524T in the localZ. triticipopulations from west to east. In contrast, no major differences were seen forCYP51mutations V136C, A379 and I381V. The four azoles showed different levels of cross-resistance, which again depended on specificCYP51mutations. Across the four seasons, the single azoles increased the yields between 9 and 11% on average.

Demethylation inhibitor (DMI) and succinate dehydrogenase inhibitor (SDHI) fungicides are currently relied upon for the control of septoria tritici blotch (STB) in European wheat fields. However, multiple mutations have occurred over time in the genes encoding the targeted proteins that have led to a practical loss of fungicide efficacies. Among the different amino acid substitutions in Zymoseptoria tritici associated with resistance to these fungicides, S524T in CYP51 (DMI target) and H152R in SdhC (SDHI target) are regarded as conferring the highest resistance factors to DMI and SDHI, respectively. To facilitate further studies on the monitoring and selection of these substitutions in Z . tritici populations, a multiplex allele‐specific quantitative PCR (qPCR) assay allowing for estimation of both allele frequencies in bulk DNA matrices was developed. The assay was then used on complex DNA samples originating from a spore trap network set up in Belgium, Denmark, Sweden, and Ireland in 2017 and 2018, as well as on leaf samples with symptoms. The S524T allele was present in all field samples and its proportion was significantly higher in Ireland than in Belgium, whereas the proportion of H152R was only sporadically present in both countries. The frequency of S524T varied greatly in the airborne inoculum of all four countries; however, the H152R allele was never detected in the airborne inoculum. The method developed in this study can be readily adopted by other laboratories and used for multiple applications including resistance monitoring in field populations of Z . tritici .