Gabriel Scalliet

Fungicide resistance in crop pathogens poses severe challenges to sustainable agriculture. Demethylation inhibitors (DMIs) are critical for controlling crop diseases but face rapid resistance gains. Even though the main molecular basis of resistance is well established, field surveys have repeatedly revealed alternative resistance mechanisms. The European continent has seen rapid and heterogeneous gains in azole resistance. Here, we establish a large genome panel to dissect the genetic architecture of emerging resistance in the major wheat pathogen Zymoseptoria tritici . The European diversity panel spans 15 sampling years and 27 countries for a total of 1,394 sequenced and phenotyped strains. Using two complementary assays to quantify resistance levels of each strain, we captured fine-grained shifts in DMI resistance over space and time. We conducted genome-wide association studies based on a comprehensive set of genotyping approaches for six DMIs. We mapped a vast array of loci encoding target functions, but also diverse channel, kinase, phosphotransferase, oxidoreductase, and monooxygenase functions. The substantial scope in genetic mechanisms underpinning DMI resistance significantly expands our mechanistic understanding of how continent-wide resistance arises in fungal pathogens over time. Diversification of the Cyp51 coding sequence was particularly striking with new resistant haplotypes emerging with complex configurations and geographic patterns. Finally, we functionally assessed the impact of resistance-associated synonymous mutations in Cyp51 in a sensitive and resistant background. However, no significant contribution was detectable, suggesting that associations were caused by linkage disequilibrium. This study provides expansive new insights into fungicide resistance gains of crop pathogens relevant for future resistance management strategies. IMPORTANCE: Sustainable food production requires the management of disease agents attacking crops. Application of antifungal compounds is among the key elements of pathogen containment; however, resistance can rise rapidly, leaving crop plants vulnerable to resurgent pathogen attacks. Most research has focused on establishing the molecular basis of fungicide resistance, studying mutant strains under laboratory conditions. How fungicide resistance is gained in agricultural fields over space and time remains poorly understood. Our work introduces the largest yet systematic collection of strains of a crop pathogen to assess how resistance mutations arose against a major class of fungicides. Tracking the fungal wheat pathogen, Zymoseptoria tritici , reveals heterogeneous gains in resistance over the past decades across the European continent. Using association mapping techniques and functional validation, we disentangled a complex set of mutations contributing to resistance. Unraveling the complexity of concurrent resistance mutation gains helps focus resistance management practices in agriculture.

The rise of antifungal resistance is a global challenge for both human health and food security, because resistance emergence easily outpaces the antifungal development pipeline. Furthermore, resistance arises often in parallel and through alternative mechanisms creating challenges to predict emergence. In agriculture, where vast areas are sprayed by diverse cocktails, antifungal resistance gains are particularly complex. Despite broad efforts, knowledge of resistance mechanisms is often limited to model genotypes and empirical evidence from the field is lacking. Here, we define and validate a high- throughput pipeline for antifungal resistance discovery informed by emerging resistance gains at continental scale. We analyzed a thousand-genome European diversity panel of the major wheat pathogen Zymoseptoria tritici and assessed resistance levels against over 29 fungicides covering all major classes. We optimized high-throughput phenotyping assays to comprehensively capture emerging resistance phenotypes. Pangenome-informed genotyping techniques revealed a total of 2192 genes associated with antifungal resistance. This expands by an order of magnitude the current knowledge and establishes a refined atlas of resistance mutations. We generated mutants to recapitulate several of the discovered resistance factors. Hence, our approach captures in-field resistance gains across Europe for all major fungicide classes and can define exact molecular targets. Broad knowledge of resistance gains will guide more sustainable fungicide development pipelines.

Directed evolution (DE) optimizes biomolecules through natural selection principles, revolutionizing the development of proteins, nucleic acids, and strains for various applications. However, conventional DE methods face limitations in screening throughput, which can prevent the identification of rare but optimal variants within a population. Droplet-based microfluidics enable the transfer of conventional screening methods into nanolitre- scale droplets, enabling high-throughput screening while preserving genotype-phenotype connections. This technology allows rapid screening of millions of variants, opening new possibilities for microbial strain engineering and metabolite production optimization. We discuss the integration of microfluidics into DE workflows and reflect on its potential applications in agrochemical research, including enzyme evolution, crop trait improvement, and natural product biosynthesis.

Fungicide resistance in crop pathogens poses severe challenges to sustainable agriculture. Demethylation inhibitors (DMIs) are critical for controlling crop diseases but face rapid resistance gains in the field. Even though the main molecular basis of resistance is well established, field surveys have repeatedly revealed alternative resistance mechanisms. The European continent in particular has seen rapid and heterogeneous gains in azole resistance in the past decades. Here, we establish a large genome panel to dissect the genetic architecture of emerging resistance in the major wheat pathogen Zymoseptoria tritici . The European diversity panel spans 15 sampling years and 27 countries for a total of 1394 sequenced and phenotyped strains. Using two complementary assays to quantify resistance levels of each strain, we captured fine-grained shifts in DMI resistance over space and time. We conducted genome-wide association studies based on a comprehensive set of genotyping approaches for six DMIs. We mapped a total of 21,220 genetic variants and 158 genes linked to resistance. The substantial scope in genetic mechanisms underpinning DMI resistance significantly expands our mechanistic understanding how continent-wide resistance arises in fungal pathogens over time. Diversification of the Cyp51 coding sequence was particularly striking with new resistant haplotypes emerging with complex configurations and geographic patterns. This study provides expansive new insights into fungicide resistance gains of crop pathogens relevant for future resistance management strategies.

Multidrug-resistant fungal pathogens are a serious threat to public health and global food security. Mitigation requires the discovery of antifungal compounds with novel modes of action, along with a comprehensive understanding of the molecular mechanisms governing antifungal resistance. Here, we apply SAturated Transposon Analysis in Yeast (SATAY), a powerful transposon sequencing method in Saccharomyces cerevisiae, to uncover loss- and gain-of-function mutations conferring resistance to 20 different antifungal compounds. These screens identify a diverse array of novel resistance mechanisms and multiple modes of action. SATAY is performed in drug-sensitive strains to generate chemogenomic profiles for compounds that lack activity against conventional laboratory strains. This study therefore provides a significant resource for exploring cellular responses to chemical stresses. We discover that the natural antifungal Chitosan electrostatically interacts with cell wall mannosylphosphate, and that the transporter Hol1 concentrates the novel antifungal ATI-2307 within yeast. This latter finding presents an avenue for drug design initiatives, but also unveils a straightforward evolutionary path to ATI-2307 resistance with minimal fitness cost.

The gray mold (Botrytis cinerea; Botrytis) is the main disease affecting grapevine production in Chile. Succinate dehydrogenase inhibitors belonging to the carboxamide fungicide family are a key tool for the control of Botrytis in grapevines from the Chilean Central Valley. This study aimed to determine the sensitivity of the Chilean Botrytis population to the new generation carboxamide pydiflumetofen. Conidial germination (CG) and germ-tube elongation (GTE) sensitivity assays were conducted on 200 single-spore isolates collected during the 2016 to 2017 season. The mean effective concentration that inhibited 50% (EC₅₀) of CG in the Botrytis population was 0.0545 μg/ml, with mean values of 0.066 and 0.042 μg/ml for table and wine grapes, respectively. The mean EC₅₀ value of GTE was 0.000245, 0.0003, and 0.0019 μg/ml for the total, table grape, and wine grape populations, respectively. The comparison between pydiflumetofen and fludioxonil, a highly efficient fungicide carrying a different mode of action, showed 87.5 and 97.5% of Botrytis control with an EC₅₀ threshold of 0.1 μg/ml in table grape and wine grape populations, respectively. No cross-resistance between pydiflumetofen and fludioxonil was detected. For nine isolates with reduced pydiflumetofen sensitivity, we evaluated SdhB mutations using a qPCR-HRM diagnostic system. Two isolates carried the sdhB^P225/H272R genotype, and two isolates carried the sdhB^P225/H272Y genotype. Additional analysis of SdhB mutant isolates determined that pydiflumetofen controls wild-type as well as sdhB^P225/H272R and sdhB^P225H/H272 mutants. Pydiflumetofen does not control CG in the sdhB^P225/H272Y mutant but is effective in the GTE control. Pydiflumetofen significantly controls Botrytis independently of the SdhB genotype in wounded berry assays. This condition resembles the berry cracking due to heavy rainfall right before harvest, as seen in recent years in the Chilean Central Valley. The findings demonstrate that pydiflumetofen effectively controls the grapevine Botrytis population, suggest a moderate risk of pydiflumetofen resistance, and highlight the significance of incorporating genetic data into the design of control programs.

Despite large omics datasets, the prediction of eukaryotic genes is still challenging. We have developed a new method to improve the prediction of eukaryotic genes and demonstrate its utility using the genome of the fungal wheat pathogen Zymoseptoria tritici. From 10,933 to 13,260 genes were predicted by four previous annotations, but only one third were identical. A novel bioinformatics suite, InGenAnnot, was developed to improve Z. tritici gene annotation using Iso-Seq full-length transcript sequences. The best gene models were selected among different ab initio gene predictions, according to transcript and protein evidence. Overall, 13,414 reannotated gene models (RGMs) were predicted, improving previous annotations. Iso-Seq transcripts outlined 5′ and 3′ untranslated regions for 73% of the RGMs and alternative transcripts mainly due to intron retention. Our results showed that the combination of different ab initio gene predictions and evidence-driven curation improved gene annotation of a eukaryotic genome. It also provided new insights into the transcriptional landscape of this fungus. [ ] Copyright © 2025 The Author(s). This is an open access article distributed under the CC BY 4.0 International license .

Multidrug-resistant fungal pathogens are a serious threat to public health and global food security. Mitigation requires the discovery of antifungal compounds with novel modes of action, along with a comprehensive understanding of the molecular mechanisms governing antifungal resistance. Here, we apply SAturated Transposon Analysis in Yeast (SATAY), a powerful transposon sequencing method in Saccharomyces cerevisiae , to uncover loss- and gain-of-function mutations conferring resistance to 20 different antifungal compounds. These screens identify a diverse array of novel resistance mechanisms and multiple modes of action. SATAY is performed in drug-sensitive strains to generate chemogenomic profiles for compounds that lack activity against conventional laboratory strains. This study therefore provides a significant resource for exploring cellular responses to chemical stresses. We discover that the natural antifungal Chitosan electrostatically interacts with cell wall mannosylphosphate, and that the transporter Hol1 concentrates the novel antifungal ATI-2307 within yeast. This latter finding presents an avenue for drug design initiatives, but also unveils a straightforward evolutionary path to ATI-2307 resistance with minimal fitness cost.

With >7000 species the order of rust fungi has a disproportionately large impact on agriculture, horticulture, forestry and foreign ecosystems. The infectious spores are typically dikaryotic, a feature unique to fungi in which two haploid nuclei reside in the same cell. A key example is Phakopsora pachyrhizi, the causal agent of Asian soybean rust disease, one of the world’s most economically damaging agricultural diseases. Despite P. pachyrhizi’s impact, the exceptional size and complexity of its genome prevented generation of an accurate genome assembly. Here, we sequence three independent P. pachyrhizi genomes and uncover a genome up to 1.25 Gb comprising two haplotypes with a transposable element (TE) content of ~93%. We study the incursion and dominant impact of these TEs on the genome and show how they have a key impact on various processes such as host range adaptation, stress responses and genetic plasticity.

Human activity impacts the evolutionary trajectories of many species worldwide. Global trade of agricultural goods contributes to the dispersal of pathogens reshaping their genetic makeup and providing opportunities for virulence gains. Understanding how pathogens surmount control strategies and cope with new climates is crucial to predicting the future impact of crop pathogens. Here, we address this by assembling a global thousand-genome panel of Zymoseptoria tritici, a major fungal pathogen of wheat reported in all production areas worldwide. We identify the global invasion routes and ongoing genetic exchange of the pathogen among wheat-growing regions. We find that the global expansion was accompanied by increased activity of transposable elements and weakened genomic defenses. Finally, we find significant standing variation for adaptation to new climates encountered during the global spread. Our work shows how large population genomic panels enable deep insights into the evolutionary trajectory of a major crop pathogen.