Environmental Hot Spots and Resistance-Associated Application Practices for Azole-Resistant Aspergillus fumigatus, Denmark, 2020-2023.

Azole-resistant Aspergillus fumigatus (ARAf) fungi have been found inconsistently in the environment in Denmark since 2010. During 2018-2020, nationwide surveillance of clinical A. fumigatus fungi reported environmental TR34/L98H or TR46/Y121F/T289A resistance mutations in 3.6% of isolates, prompting environmental sampling for ARAf and azole fungicides and investigation for selection of ARAf in field and microcosmos experiments. ARAf was ubiquitous (20% of 366 samples; 16% TR34/L98H- and 4% TR46/Y121F/T289A-related mechanisms), constituting 4.2% of 4,538 A. fumigatus isolates. The highest proportions were in flower- and compost-related samples but were not correlated with azole-fungicide application concentrations. Genotyping showed clustering of tandem repeat-related ARAf and overlaps with clinical isolates in Denmark. A. fumigatus fungi grew poorly in the field experiment with no postapplication change in ARAf proportions. However, in microcosmos experiments, a sustained complete (tebuconazole) or partial (prothioconazole) inhibition against wild-type A. fumigatus but not ARAf indicated that, under some conditions, azole fungicides may favor growth of ARAf in soil.

Mefentrifluconazole sensitivity amongst European Zymoseptoria tritici populations and potential implications for its field efficacy.

BACKGROUND: Septoria tritici blotch caused by Zymoseptoria tritici continues to be one of the most economically destructive diseases of winter wheat in north-western Europe. Control is heavily reliant on the application of fungicides, in particular those belonging to the azole group. Here we describe the sensitivity of European Z. tritici populations to the novel azole mefentrifluconazole and the analysis of associated mechanisms of resistance. RESULTS: A wide range of sensitivity to mefentrifluconazole was observed amongst the Z. tritici collections examined, with strong cross-resistances also observed between mefentrifluconazole, difenoconazole and tebuconazole. Overall, the Irish population displayed the lowest sensitivity to all azoles tested. Further detailed analysis of the Irish population in 2021 demonstrated differences in sensitivity occurred between sampling sites, with these differences associated with the frequencies of key resistance mechanisms (CYP51 alterations and MFS1 promoter inserts linked to overexpression). Under glasshouse conditions reductions in the efficacy of mefentrifluconazole were observed towards those strains exhibiting the lowest in vitro sensitivities. CONCLUSIONS: This study demonstrates that a large range of sensitivity to mefentrifluconazole exists in European Z. tritici populations. Those strains exhibiting the lowest sensitivity to the azoles tested had the most complex CYP51 haplotypes in combination with the 519 bp insert, associated with enhanced activity of MFS1. The future use of mefentrifluconazole should take these findings into consideration to minimise the selection of these strains. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Baseline sensitivity of European Zymoseptoria tritici populations to the complex III respiration inhibitor fenpicoxamid.

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.

Seedborne Cercospora beticola Can Initiate Cercospora Leaf Spot from Sugar Beet (Beta vulgaris) Fruit Tissue.

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.

Fungicide sensitivity levels in the Lithuanian Zymoseptoria tritici population in 2021.

Zymoseptoria tritici causes the disease known as septoria leaf blotch in winter wheat and is a major factor in yield loss worldwide. Farmers are inclined to use fungicides to protect their crops; however, the efficacy of these measures is rapidly decreasing due to the natural mechanisms of mutation emergence in pathogen populations. Increasing fungicide resistance is being recorded worldwide, therefore, screening of the current situation in Lithuania is essential to determine the subsequent steps of crop protection strategies. In this study, in vitro fungicide sensitivity tests, mutation detection, and field experiments were carried out. The mean EC50 values for prothioconazole-desthio and mefentrifluconazole were 0.14 and 0.28 mg/l, respectively. Increased frequency of the mutation S524T, linked to DMIs resistance, was observed. Results revealed that the dominant point mutation in the gene CYP51 was I381V, and the most frequent CYP51 haplotype was D13 (V136C, I381V, Y461H, S524T). The mutation G143A, linked to QoI resistance, was detected in ¾ of the population. Mutations conferring resistance to SDHIs were not detected in single pycnidium isolates. Two-year field experiments likewise showed no decline in field efficacy of SDHI fungicide in Lithuania. Moreover, the baseline sensitivity of the Lithuanian Z. tritici population to QiI fungicide fenpicoxamid was established. The findings of this study provide an update on the current status of fungicide resistance in the Lithuanian Z. tritici population.

Spatio-temporal distribution of DMI and SDHI fungicide resistance of Zymoseptoria tritici throughout Europe based on frequencies of key target-site alterations.

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.

Development of a PacBio Long-Read Sequencing Assay for High Throughput Detection of Fungicide Resistance in Zymoseptoria tritici.

Fungicide resistance has become a challenging problem in management of Septoria tritici blotch (STB), caused by Zymoseptoria tritici, the most destructive disease of winter wheat throughout western and northern Europe. To ensure the continued effectiveness of those fungicides currently used, it is essential to monitor the development and spread of such resistance in field populations of the pathogen. Since resistance to the key families of fungicides used for STB control (demethyalation inhibitors or azoles, succinate dehydrogenase inhibitors or SDHIs and Quinone outside Inhibitors or QoIs) is conferred through target-site mutations, the potential exists to monitor resistance through the molecular detection of alterations in the target site genes. As more efficient fungicides were developed and applied, the pathogen has continuously adapted through accumulating multiple target-site alterations. In order to accurately monitor these changes in field populations, it is therefore becoming increasingly important to completely sequence the targeted genes. Here we report the development of a PacBio assay that facilitates the multiplex amplification and long-read sequencing of the target gene(s) for the azole (CYP51), SDHI (Sdh B, C, and D), and QoI (cytochrome b) fungicides. The assay was developed and optimised using three Irish Z. tritici collections established in spring 2017, which capture the range of fungicide resistance present in modern European populations of Z. tritici. The sequences obtained through the PacBio assay were validated using traditional Sanger sequencing and in vitro sensitivity screenings. To further exploit the long-read and high throughput potential of PacBio sequencing, an additional nine housekeeping genes (act, BTUB, cal, cyp, EF1, GAPDH, hsp80-1, PKC, TFC1) were sequenced and used to provide comprehensive Z. tritici strain genotyping.