The mycovirus Fusarium culmorum phenuivirus 1 (FcPV1) infects the wheat pathogen Fusarium culmorum, a causal agent of Fusarium head blight (FHB). FHB remains a major threat to cereal cultivation, and the potential of biological control approaches, including the use of mycoviruses, has been suggested for its mitigation. In this study, we describe the biological characterisation of FcPV1, which is a single-stranded, negative-sense RNA mycovirus from the Phenuiviridae family. FcPV1 was detected in our previous study, and here we examine its impact on fungal growth and virulence by assessing the pathogenicity of infected F. culmorum on wheat under laboratory to semifield conditions. In laboratory experiments, the presence of FcPV1 resulted in no discernible phenotypic changes, but it was readily transmitted vertically to conidia and horizontally to Fusarium graminearum and other F. culmorum strains. In a greenhouse pot experiment with FHB-susceptible and -resistant wheat, FcPV1 infection appeared to induce hypervirulence, while under semifield conditions, it seemed to result in hypovirulence. In the susceptible cultivar, FcPV1 infection decreased biomass-adjusted nivalenol concentrations by approximately 40%, whereas deoxynivalenol increased by 28%. To our knowledge, this is the first study comparing mycovirus effects in laboratory, greenhouse and semifield environments. Our findings highlight the complexity of mycovirus-host interactions and the importance of environmental context, emphasising that potential biological control using mycoviruses should be assessed across varying complexities, including semifield and field conditions. This study challenges the notion that mycoviruses are typically cryptic, emphasising that their effects may only manifest under specific environmental conditions and measured parameters.

Sugar beet (Beta vulgaris) is an important cash crop grown in temperate regions to produce sugar. Fungal leaf diseases are known to infect the sugar beet crop during the season, causing significant yield reductions. This study aimed to investigate the effect of biological control agents and sulphur on disease control and yield, regarded as alternatives to conventional synthetic fungicides. Ten field trials were carried out between 2016 and 2020. The study focused on control of powdery mildew (Erysiphe betae) and beet rust (Uromyces betae). In six field trials, the impact of the BCA product (Serenade (R) ASO) was compared using two to four applications. The BCA was applied either repeatedly as a solo product or within an IPM strategy combined with conventional fungicides. A second series of four field trials tested sulphur and Serenade (R) ASO as solo solutions compared with chemical solutions and in combination with the chemical solutions. Traditional fungicides using two applications gave 76-99 % (avg. 88 %) control of powdery mildew and 59-100 % (avg. 78 %) control of leaf rust (based on AUDPC) and 3.1-9.8 t/ha root yield increases. Serenade (R) ASO applied as a solo product at 1-4 timings gave a lower and more variable effect of 0-66 % (avg. 20 %) against powdery mildew and 0-30 % (avg. 14 %) against rust, but these were not statistically significant. Root yield increases ranging from 0-6.6 t/ha were observed with Serenade (R) ASO, though these were not statistically significant. No substantial effect was seen from changing the dose or number of treatments with Serenade (R) ASO. When applied as part of a spraying programme in conjunction with traditional chemistry, no clear benefit could be measured in terms of either disease control or yield responses compared to using traditional fungicides alone. A greenhouse trial indicated verification that the earlier timing using Serenade (R) ASO was more effective and gave indications of a dose effect. Sulphur used as a solo solution gave significant control of powdery mildew in line with chemical substances (avg. 78 %), but statistically non-significant root yield increases of 2.4-11.5 t/ha, and inferior control of rust compared to chemical solutions. In an IPM context, sulphur could replace chemical treatments for control of powdery mildew while this was not the case for Serenade (R) ASO. Neither could replace rust control with traditional chemistries.

BACKGROUNDThe hemibiotrophic fungus Zymoseptoria tritici causing Septoria tritici blotch (STB), is a devastating foliar pathogen of wheat worldwide. A common group of fungicides used to control STB are the demethylation inhibitors (DMIs). DMI fungicides restrict fungal growth by inhibiting the sterol 14-alpha-demethylase, a protein encoded by CYP51 gene and essential for maintaining fungal cell permeability. However, the adaptation of Z. tritici populations in response to intensive and prolonged DMI usage has resulted in a gradual shift towards reduced sensitivity to this group of fungicides. In this study, 311 isolates were collected pre-treatment from nine wheat-growing regions in Europe in 2019. These isolates were analysed by high-throughput amplicon-based sequencing of nine housekeeping genes and the CYP51 gene.RESULTSAnalyses based on housekeeping genes and the CYP51 gene revealed a lack of population structure in Z. tritici samples irrespective of geographical origin. Minimum spanning network (MSN) analysis showed clustering of multilocus genotypes (MLGs) based on CYP51 haplotypes, indicating an effect of selection due to DMI fungicide use. The majority of the haplotypes identified in this study have been reported previously. The diversity and frequencies of mutations varied across regions.CONCLUSIONUsing a high-throughput amplicon-sequencing approach, we found several mutations in the CYP51 gene combined in different haplotypes that are likely to cause fungicide resistance. These mutations occurred irrespective of genetic background or geographical origin. Overall, these results contribute to the development of effective and sustainable risk monitoring for DMI fungicide resistance. (c) 2024 The Author(s). Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Food and feed production worldwide heavily relies on wheat ( Triticum aestivum ). However, current agricultural practices face numerous challenges including a shortage of land for cultivation, a desire to reduce the use of chemical pesticides and fertilizers, and the development of resistance towards employed pesticides and virulence towards host resistance in the most widely grown varieties. In this paper, we demonstrate based on the literature that cultivating wheat variety mixtures generally leads to increased yield and yield stability across years and environments, reduced severity of multiple diseases, and a decreased risk of lodging before harvest compared to the cultivation of pure stands. Moreover, mixtures may delay fungicide resistance development and increase genetic diversity, ultimately prolonging the durability of resistance genes. Furthermore, growing mixtures may lower the risk of crop failure due to more extreme weather events and lead to better utilization of water and nutrients. We discuss a Danish case study advocating the cropping of wheat varieties in mixtures as an example of how variety mixtures can be utilized in integrated pest management strategies. The study shows that if all involved stakeholder groups promote the adoption of variety mixtures, a major uptake by farmers can be reached, potentially reducing the dependency on pesticides in current cropping schemes.

Fusarium head blight (FHB) continues to be a major problem in wheat production and is considered a disease complex caused by several fungal pathogens including Fusarium culmorum, F. graminearum and F. equiseti. With the objective of investigating diversity of mycoviruses in FHB-associated pathogens, we isolated Fusarium spp. from six wheat (Triticum aestivum) cultivars. In total, 56 Fusarium isolates (29 F. culmorum, 24 F. graminearum, one F. equiseti) were screened for mycoviruses by extracting and sequencing double-stranded RNA. We found that a large proportion of Fusarium isolates (46 %) were infected with mycoviruses. F. culmorum, previously described to harbor only one mycovirus, tended to host more viruses than F. graminearum, with a few isolates harboring seven mycoviruses simultaneously. Based on the RNA-dependent RNA polymerase domain analysis, ten were positive-sense single-stranded RNA viruses (related to viruses from families Mitoviridae, Botourmiaviridae, Narnaviridae, Tymoviridae, Gammaflexiviridae, as well as proposed Ambiguiviridae and ormycovirus viral group), one was double-stranded RNA virus (Partitiviridae), and five were negative-sense single-stranded RNA viruses (related to members in the families of Yueviridae, Phenuiviridae, Mymonaviridae, as well as proposed Mycoaspiviridae). Five mycoviruses were shared between F. graminearum and F. culmorum. These results increase our general understanding of mycovirology. To our knowledge, this is the first in-depth report of the mycovirome in F. culmorum and the first report on the diversity of mycoviruses from Danish isolates of FHB-causing fungi in general.

Overall, there is a major wish that European farmers implement integrated pest management (IPM), particularly to reduce dependence on pesticides. In the European Rustwatch project, partners conducted nineteen trials across nine different countries during 2020 and 2021 to investigate different IPM strategies, focusing on controlling rust diseases in winter wheat. The trials included the use of varieties with contrasting levels of resistance, variety mixtures, reduced fungicide rates, thresholds, and Decision Support Systems (DSSs), and testing alternative products to fungicides. Sixteen trials developed yellow rust (Puccinia striiformis f. sp. tritici) infections, and six trials developed brown rust (Puccinia triticina) infections. Resistant varieties proved highly effective in keeping down yellow rust infection, and variety mixtures also effectively reduced infection levels and stabilized yields. Rust was fully controlled using 25% of standard fungicide rates, even under high disease pressure. Using DSSs provided sufficient control of rust diseases and resulted in competitive net economic returns due to fewer fungicide applications. The alternative products tested included two biological control agents and four alternative chemistries, which all gave inferior and insufficient control against rust compared with chemical fungicides. The trial work demonstrated that there are good and reliable options for including IPM into disease control in wheat.

Barley crops are at risk of being attacked by several leaf diseases. Net blotch, brown rust, Rhynchosporium and Ramularia leaf spot are among the most widespread and can cause severe attack and yield losses. Two trial protocols targeting Ramularia and net blotch, respectively, have been tested in several countries in 2021 and 2022. Ramularia trials were situated in Germany, Ireland, Scotland, and Denmark. The net blotch trials were placed in Denmark, Belgium, the UK, Germany, Finland, and France. In the two protocols, 12–13 different fungicide solutions including co-formulations of DMIs, SDHIs, QoIs, and multi-site inhibitors have been tested to compare efficacy and yield responses. Against Ramularia leaf spot, the fungicides were applied at GS 47–51 and against net blotch at GS 37–45. In six trials, the efficacy against Ramularia leaf spot was scored. The results showed a superior control from the co-formulation fluxapyroxad + metyltetraprole (78–100% control), but also solo mefentrifluconazole and the mixtures fluxapyroxad + mefentrifluconazole performed well (average 74–76% control). The mixture fluxapyroxad + metyltetraprole provided the best yield increase followed by Ascra Xpro. Folpet as a solo solution was inferior. Following the net blotch protocol, only three trials developed enough disease to rank the different fungicides; however, in five trials ranking against brown rust was also possible. Most treatments gave very good control of net blotch, and brown rust (> 80% control). The mixture fluxapyroxad + metyltetraprole delivered the best control against all diseases overall. Average yield responses from eight trials showed very similar increases from the tested fungicides.

Decision support systems (DSSs) enable the optimisation of pesticide application timing to increase pesticide efficacy and thus reduce pesticide use without compromising yield quality and quantity. Limited access to information about available DSSs for use in integrated pest management (IPM) is a major barrier to the uptake of DSSs for IPM across Europe. To overcome this barrier, a typology for DSSs for IPM in Europe was developed, introducing a systematic approach to describe the ever-growing number of DSSs for IPM. The developed IPM-DSS typology was implemented in the free web tool "IPM Adviser", where currently 79 IPM DSSs are described with over 50 attributes describing their structural and performance characteristics. The information about IPM DSSs, which was previously scattered on different websites and difficult to compare, is now standardised and presented in a uniform way, so that it is possible to compare different IPM DSSs on the basis of all the attributes described. The presented IPM-DSS typology implemented in the web tool IPM Adviser facilitates the dissemination and uptake of DSSs for IPM and thus contributes to the achievement of the EU targets for the sustainable use of pesticides.

Azole-resistant Aspergillus fumigatus (ARAf) Af ) 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 34 /L98H or TR46/Y121F/T289A 46 /Y121F/T289A resistance mutations in 3.6% of isolates, prompting environmental sampling for ARAf Af and azole fungicides and investigation for selection of ARAf Af in field and microcosmos experiments. ARAf Af was ubiquitous (20% of 366 samples; 16% TR34/ 34 / L98H- and 4% TR46/Y121F/T289A-related 46 /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 Af and overlaps with clinical isolates in Denmark. A. fumigatus fungi grew poorly in the field experiment with no postapplication change in ARAf Af proportions. However, in microcosmos experiments, a sustained complete (tebuconazole) or partial (prothioconazole) inhibition against wild-type A. fumigatus but not ARAf Af indicated that, under some conditions, azole fungicides may favor growth of ARAf Af in soil.

Disease management decision support systems (DSS) are typically prediction algorithms that help farmers assess the risk of an epidemic, to guide whether, and to what extent, fungicide treatment is needed. However, there is frequently little information presented to quantify the value of using the DSS, i.e. the likely increased profit or reduced impact to the environment, and the risks of failing to control the pest. Validation of DSS is often limited to a small number of sites and seasons, as extensive field testing is prohibitively expensive. It would therefore be beneficial to have a method to estimate the value of a DSS using existing data sets gathered for other purposes. We present a theoretical framework for evaluating the value of DSS, and then describe how this can be applied in practice using four case studies of contrasting DSS under different data constraints. The four case studies include DSS that guide (i) the total dose of pesticide applied; (ii) the number of sprays required; (iii) the timing of the first fungicide application in a spray programme; and (iv) infection risk alerts. We demonstrate how our theoretical framework can be used to evaluate DSS, using existing field and literature data to infer the benefits and risks associated with their use. The limitations of using existing data are explored.

Zymoseptoria tritici, the causal agent of septoria tritici blotch (STB), is one of the most destructive fungal pathogens on wheat. To control the disease and limit yield losses, farmers rely mainly on uni-site fungicides belonging to two modes of action: demethylase inhibitors (DMIs) and succinate dehydrogenase inhibitors (SDHIs). DMIs have been used extensively for STB control for the last 40 years, resulting in a gradual sensitivity shift, which has been observed worldwide, due to the stepwise accumulation of resistance mutations in the CYP51 gene. SDHI fungicides have been used for just over ten years to control STB. Several mutations conferring resistance to this group have been detected in several countries to varying degrees. Fungicide resistance in Sweden and Denmark has generally been less prominent because of the lower fungicide input and restricted fungicide availability. Microtiter plate assays and allele-specific qPCRs (targeting C-T79N and C-N86S in SdhC and S524T in CYP51) confirmed that the Danish and Swedish Z. tritici populations still remain sensitive to SDHI and DMI fungicides, but that the frequency of resistant isolates is increasing. The target site alterations were further used as markers to evaluate the selection potential imposed by different control strategies in the field. Results indicated that efficacious SDHIs, including fluxapyroxad, imposed a higher selection of resistance mutations compared to less effective SDHIs. Treatments including prothioconazole increased the frequency of S524T more than the ones containing mefentrifluconazole. Splitting the dose into two treatments also selected for more resistant isolates compared to single-treatment strategies. This investigation also tackles the impacts of current anti-resistance strategies, including the use of lower doses, limiting the number of treatments, and mixing and alternating fungicides of a different mode of action under field conditions. These elements are essential to prolong the efficacy of current and future fungicides and delay resistance development in the Z. tritici population.

All plant tissues from leaves, stems, and roots are hosting a wide diversity of fungal species. Our understanding of the assembly of this diversity of fungi during the plant growth cycle is limited. Here, we characterized the mycobiome of three spring barley cultivars grown in Zealand, Denmark, at weekly intervals during a growth season from seedling emergence to senescence and seed maturity. A notable proportion of members of the fungal communities were shared among different plant organs, but community dynamics were tissue-specific. A severe attack of Puccinia hordei occurring during the vegetative stage had profound effects on the mycobiome, and P. hordei biomass displaced that of other taxa. Plant tissue type was the most important factor determining the mycobiome, but also plant age was contributing significantly. Using a random forest model, we found that specific members of the mycobiome were responding differently to plant age, for instance, Olpidium and Articulospora in roots, Dioszegia and Sporobolomyces in leaves, Pyrenophora in stems, and Epicoccum in heads. A co-occurrence network analysis revealed complex interactions among fungal OTUs, and network connectivity was changing as per plant growth stage and plant tissue type. This study contributes to the understanding of assembly of fungal communities in cereals by providing a detailed description of fungal communities associated with barley. This knowledge will be vital for microbiome assisted plant health management and our study will serve as an important baseline for future efforts to harness microbiota in cereal health.

During the project Rustwatch 19 field trials in winter wheat was carried out in 9 European countries during 2 seasons collecting disease data and yield data from the use of different cultivars and fungicide treatments.

Septoria tritici blotch (STB) is among the most devastating diseases in European wheat production. In recent years, there has been increased interest in using cultivar mixtures as part of an integrated control strategy against diseases. This study investigated different cultivar mixtures for their ability to control STB across three years and at seven trial sites in Denmark with a range of fungicide strategies, yielding a total of 194 individual cultivar mixture combinations. The mixtures were composed of two, three, or four cultivars that were either similar or contrasting in their susceptibility to STB. Across all trials, the cultivar mixtures reduced disease severity significantly, by 14% compared with the component cultivars grown in monoculture. The reductions were larger when the disease pressure was high and when the mixtures included more cultivars. Mixtures composed of four cultivars reduced disease severity significantly, by 24%. Across all trials, cultivar mixtures significantly increased yield by 2% compared with the component cultivars grown in monoculture. The yield increase was significant for plots treated with one or two fungicide applications, and cultivar mixtures increased yield significantly, by 4.4% in untreated plots. The yield increase was smaller for mixtures with a high proportion of resistant cultivars. Based on the results from this study, cultivar mixtures can contribute positively to an integrated pest management (IPM) strategy, by reducing disease severity for STB and increasing yield. The most pronounced benefits from cultivar mixtures were found in fields with moderate to low fungicide input, under conditions with high disease pressure, when combining four cultivars with varying susceptibilities.

The adaptive potential of plant fungal pathogens is largely governed by the gene content of a species, consisting of core and accessory genes across the pathogen isolate repertoire. To approximate the complete gene repertoire of a globally significant crop fungal pathogen, a pan genomic analysis was undertaken for Pyrenophora tritici-repentis (Ptr), the causal agent of tan (or yellow) spot disease in wheat. In this study, 15 new Ptr genomes were sequenced, assembled and annotated, including isolates from three races not previously sequenced. Together with 11 previously published Ptr genomes, a pangenome for 26 Ptr isolates from Australia, Europe, North Africa and America, representing nearly all known races, revealed a conserved core -gene content of 57 % and presents a new Ptr resource for searching natural homologues (orthologues not acquired by horizontal transfer from another species) using remote protein structural homology. Here, we identify for the first time a non -synonymous mutation in the Ptr necrotrophic effector gene ToxB, multiple copies of the inactive toxb within an isolate, a distant natural Pyrenophora homologue of a known Parastagonopora nodorum necrotrophic effector (SnTox3), and clear genomic break points for the ToxA effector horizontal transfer region. This comprehensive genomic analysis of Ptr races includes nine isolates sequenced via long read technologies. Accordingly, these resources provide a more complete representation of the species, and serve as a resource to monitor variations potentially involved in pathogenicity.

Validation of models for plant disease management is a crucial part in the development of decision support systems in plant protection. Bespoke field trials are usually conducted to determine the performance of a model under practical conditions. However, field trials are very resource-demanding, and the use of already existing field trial data could significantly reduce costs for model validation. In this study, we took this novel approach to verify the performance of models for determining the need of fungicide applications against leaf blotch diseases in wheat by utilising historical weather data and yield data available from fungicide efficacy field trials. Two models based on humidity factors were used in the study. To estimate how specific humidity settings in the two models affect the number of recommended fungicide treatments per season, historical weather data from a 5-year period from weather stations in Denmark, Sweden, Norway, Finland, and Lithuania was used. The model output shows major differences between seasons and regions, typically recommending between one and three treatments per season. To determine the prediction potential of the models, data on yield gains from either one or two fungicide applications in fungicide efficacy trials conducted in wheat over a 5-year period in the five countries was utilised. The yield responses from fungicide treatments in the efficacy trials varied considerably between years and countries, as did the proportion of predictions of profitable treatments. In general, there was a tendency for the models to overestimate the need to apply fungicides (low specificity), but they rarely failed to recommend an application that was needed (high sensitivity). Despite the importance of having specific trials across regions in order to adjust models to local cropping and weather conditions, our study shows that historical weather data and existing field trial data have the potential to be used in model validation.

Septoria tritici blotch (STB; Zymoseptoria tritici) is a severe leaf disease on wheat in Northern Europe. Fungicide resistance in the populations of Z. tritici is increasingly challenging future control options. Twenty-five field trials were carried out in nine countries across Europe from 2019 to 2021 to investigate the efficacy of specific DMI and SDHI fungicides against STB. During the test period, two single DMIs (prothioconazole and mefentrifluconazole) and four different SDHIs (fluxapyroxad, bixafen, benzovindiflupyr and fluopyram) along with different co-formulations of DMIs and SDHIs applied at flag leaf emergence were tested. Across all countries, significant differences in azole performances against STB were seen; prothioconazole was outperformed in all countries by mefentrifluconazole. The effects also varied substantially between the SDHIs, with fluxapyroxad providing the best efficacy overall, while the performance of fluopyram was inferior to other SDHIs. In Ireland and the UK, the efficacy of SDHIs was significantly lower compared with results from continental Europe. This reduction in performances from both DMIs and SDHIs was reflected in yield responses and also linked to decreased sensitivity of Z. tritici isolates measured as EC50 values. A clear and significant gradient in EC50 values was seen across Europe. The lower sensitivity to SDHIs in Ireland and the UK was coincident with the prevalence of SDH-C-alterations T79N, N86S, and sporadically of H152R. The isolates' sensitivity to SDHIs showed a clear cross-resistance between fluxapyroxad, bixafen, benzovindiflupyr and fluopyram, although the links with the latter were less apparent. Co-formulations of DMIs + SDHIs performed well in all trials conducted in 2021. Only minor differences were seen between fluxapyroxad + mefentrifluconazole and bixafen + fluopyram + prothioconazole; the combination of benzovindiflupyr + prothioconazole gave an inferior performance at some sites. Fenpicoxamid performed in line with the most effective co-formulations. This investigation shows a clear link between reduced field efficacy by solo SDHIs as a result of increasing problems with sensitivity shifting and the selection of several SDH-C mutations. The presented data stress the need to practice anti-resistance strategies to delay further erosion of fungicide efficacy.

This paper presents and discusses the “PesticideLoadIndicator” package, a new R-package to compute potential environmental and health effects of pesticide applications using the Danish ‘Pesticide Load’ indicator. The implementation in the R Statistical Language makes it easy for researchers, practitioners and institutions to compare potential pesticide risks for a wide range of applications and compute risk indicators at field-, crop-, farm-, regional- or national level. The tool is publicly available. It provides a possibility for the direct integration of pesticide properties needed for indicator computation (ecotoxicity, environmental fate and human health), from the Pesticide Properties Database or other sources and allows users to change default reference values and weights.

Wheat ( Triticum aestivum L.) is one of the world’s staple food crops and one of the most devastating foliar diseases attacking wheat is powdery mildew (PM). In Denmark only a few specific fungicides are available for controlling PM and the use of resistant cultivars is often recommended. In this study, two Chinese wheat landraces and two synthetic hexaploid wheat lines were used as donors for creating four multi-parental populations with a total of 717 individual lines to identify new PM resistance genetic variants. These lines and the nine parental lines (including the elite cultivars used to create the populations) were genotyped using a 20 K Illumina SNP chip, which resulted in 8,902 segregating single nucleotide polymorphisms for assessment of the population structure and whole genome association study. The largest genetic difference among the lines was between the donors and the elite cultivars, the second largest genetic difference was between the different donors; a difference that was also reflected in differences between the four multi-parental populations. The 726 lines were phenotyped for PM resistance in 2017 and 2018. A high PM disease pressure was observed in both seasons, with severities ranging from 0 to >50%. Whole genome association studies for genetic variation in PM resistance in the populations revealed significant markers mapped to either chromosome 2A, B, or D in each of the four populations. However, linkage disequilibrium between these putative quantitative trait loci (QTL) were all above 0.80, probably representing a single QTL. A combined analysis of all the populations confirmed this result and the most associated marker explained 42% of the variation in PM resistance. This study gives both knowledge about the resistance as well as molecular tools and plant material that can be utilised in marker-assisted selection. Additionally, the four populations produced in this study are highly suitable for association studies of other traits than PM resistance.