ABSTRACT While transposable elements (TEs) are recognized as major drivers of fungal genome structure, evidence of their direct involvement in the interaction with host plants and the environment is only beginning to emerge. Retrotransposons can generate small RNA (sRNA) that act through cross-kingdom RNA interference, while giant DNA TEs called Starships carry dozens of cargo genes that enrich the accessory gene compartment of fungal genomes. In the polyphagous pathogen Botrytis cinerea, the Vv3 strain and other strains specialized on grapevine display a specific repertoire of TEs, including the retrotransposons BcCopia4, BcGypsy6 , and BcGypsy7 . This study first explored the putative role of sRNA generated from these retrotransposons in the interaction between the Vv3 strain and its host of origin, grapevine. Putative targets were identified among the host mRNAs, but predicted cleavage sites could not be experimentally validated. Moreover, Dicer mutants unable to produce retrotransposons-derived sRNA remained fully pathogenic on grapevine, indicating that these sRNAs do not act as virulence factors on this host. In parallel, this study provides an updated RNA-seq-based annotation of the accessory genes of the Vv3 strain, which revealed a new 93 kb- Starship harboring 43 cargo genes, some of which are related to arsenic resistance. A formal genetic approach confirmed that this locus confers resistance to this metalloid. This giant TE, named Ariane, was also detected in additional grapevine-specialized strains resistant to arsenic but not in strains isolated from other hosts such as tomato. In conclusion, this study highlights how a Starship giant transposon shaped the accessory genes compartment of the polyphagous fungus B. cinerea and may have contributed to its adaptation to vine cultivation by conferring resistance to arsenic, a compound widely used in vineyards during the last century. IMPACT STATEMENT Fungal genomes contain many families of transposons whose functional role in adaptation to the environment and in biotic interactions remained hidden for a long time. In the grey mold fungus Botrytis cinerea , strains specialized on grapevine, such as Vv3, carry a specific repertoire of transposons which provides a valuable opportunity to investigate their role in niche adaptation. In this study, we first investigated retrotransposon-derived small RNA, previously described as effectors capable of manipulating the immunity of the model plant Arabidopsis thaliana . Although in silico analysis of the specific repertoire of small RNAs of the Vv3 strain suggested that some could target the expression of grapevine genes, a genetic approach demonstrated that they do not play a significant role in virulence on this host. In contrast, this study identified a new transposon, named Ariane, that carries 43 cargo genes and confers a selective advantage to the Vv3 strain. Ariane belongs to a family of giant transposons called Starships , recently discovered in fungi and considered to be responsible for horizontal genes transfers between unrelated species. Ariane was detected only in some B. cinerea strains isolated from grapevine, and a genetic cross showed that it provides these strains with the ability to grow in presence of arsenic. Arsenic was used in vineyards until the beginning of the 21 st century to control fungal trunk diseases and insect pests. Therefore, Ariane appears to have played an important role in the adaptation of B. cinerea strains to cultivated grapevine. Overall, these results underline the importance of considering Starships when predicting emergence of resistance to antifungal compounds. DATA SUMMARY The novel data described in this study, i.e., RNA-Seq data and the Starship element are accessible under NCBI GEO accession GSE327899 and at https://doi.org/10.57745/HYWRNM , respectively. All information related to Botrytis cinerea genomes used in this study are centralized and kept up to date at the Bioinfo Bioger genomic web portal: https://bioinfo.bioger.inrae.fr/portal/genome-portal/ . Direct links to individual portals are respectively https://bioinfo.bioger.inrae.fr/portal/genome-portal/3/ for B. cinerea Vv3 genome, https://bioinfo.bioger.inrae.fr/portal/genome-portal/2/ for B. cinerea Sl3 genome, and https://bioinfo.bioger.inrae.fr/portal/genome-portal/4/ for B. cinerea populations isolated on tomato or grapevine. Each portal provides: (i) a centralized access to public genomic resources, including the genome, transposon, and RNA repositories; (ii) a data browser to download the genomic files; (iii) a genome browser that enables visualization of features within their genomic context, along with associated expression data. As a summary, the prior main public B. cinerea genomic accessions and resources used in this study are: GCA_039644125 for VV3 genome, GCA_022560135 for Sl3 genome, GCA_000143535 for B05.10 genome, PRJNA624742 for populations, https://doi.org/10.57745/HYWRNM for transposons, and GSE181592 for small RNAs. Furthermore, table S1 summarizes the list and characteristics of the 64 B. cinerea genomes publicly available to date. The genomic data for Vitis vinifera genome PN40024.v4 used in this study are available at: https://integrape.eu/resources/genes-genomes/genome-accessions/ .

Sustainable crop protection is vital for food security, yet it is under threat due to the adaptation of a diverse and evolving pathogen population. Resistance can be managed by maximising the diversity of selection pressure through dose variation and the spatial and temporal combination of active ingredients. This study explores the interplay between operational drivers for maximising the sustainability of management strategies in relation to the resistance status of fungal populations. We applied an experimental evolution approach to three artificial populations of Zymoseptoria tritici, an economically significant wheat pathogen, each differing in initial resistance status. Our findings reveal that diversified selection pressure curtails the selection of resistance in naïve populations and those with low frequencies of single resistance. Increasing the number of modes of action most effectively delays resistance development, surpassing the increase in the number of fungicides, fungicide choice based on resistance risk, and temporal variation in fungicide exposure. However, this approach favours generalism in the evolved populations. The prior presence of multiple resistant isolates and their subsequent selection in populations override the effects of diversity in management strategies, thereby invalidating any universal ranking. Therefore, the initial resistance composition must be specifically considered in sustainable resistance management to address real-world field situations.

The European agricultural protests of 2023/2024 have prompted a reassessment of public policies at the EU level with the SUR put on a stand still. In France, in early 2024, the government replaced the historical monitoring indicator "NoDU'' with the European Harmonized Risk Indicator HRI1 to monitor the progress in the use of Plant Protection Products (PPP) in its National Action Plan (NAP). This study aimed to assess the relevance of these two indicators regarding the objectives outlined in the Sustainable Use of pesticides Directive ("SUD") that defines the framework within which they operate. To this effect, we analyzed the PPPs they consider, the official calculation formulas, but also their past evolution and possible future evolutions through putative scenarios of changes in PPP use and regulation: ban or not; substitution or not; for the top 5 synthetic insecticides; all synthetic insecticides; glyphosate; mancozeb; and the top five active ingredients from HRI1 risk group 3 (candidates to substitution). French PPP sales data from 2011 to 2021 were used for past evolutions and we used the last available figures in France, 2021, for simulations. Designed to monitor the use of PPPs by farmers, the NoDU mostly monitors achievement with regards to one aim of the SUD: to "reduce dependency on the use of pesticides" and promote "the use of Integrated Pest Management". Its value does not change with the mere substitution of a product by another, even if the second is deemed less toxic. This limits its ability to assess the "risks and impacts of pesticide use on human health and the environment", the first aim of the directive. The HRI1 indicator is not supposed to strictly quantify the use of PPP and was found to inadequately reflect significant changes in PPP use, but also in the induced risk: 1) changes of use without a ban on a substance are limited and as such, past correlation of HRI1 with the QAI of the fourth group (banned PPPs) is very high (0.90); 2) the impact of variations of low-dose active ingredients is minimal, independently of their toxicity/ecotoxicity, even in the case of a ban. Thus, the putative withdrawal of all insecticides sold, even without substitution by other PPPs, would reduce HRI1 by only 4 in percentage points (from 67.1% to 63.1%), while NoDU would drop by 16 percentage points (from 94.5% to 78.5%), thus better reflecting both the paradigm shift in PPP use for farmers and the large diminution of the risk induced for the environment. On the opposite end, banning only glyphosate, a high dose active ingredient, even with full substitution with another herbicide, would bring the HRI1 down to 43.8% of its value in 2011-2013 and sells of glyphosate in 2021 would represent 47.8% of the HRI1 before the ban, when it is only 4.3% of the use of PPPs as measured by the NoDU. Finally, 2022 ban of mancozeb, even if fully substituted by other PPPs, might be enough to bring the HRI1 very near to the 50% target of the Ecophyto plan (56.9%), while the NoDU would remain at 94.5% of its average value in 2011-2013. Our results strongly suggest that HRI1 fails to adequately monitor the objectives of the SUD. We accordingly recommend that the European Commission reconsider the design of Harmonized Risk Indicators. NAP monitoring indicators at national and European level should encompass three primary features. First, the indicators should be founded on robust scientific and technical evidence. Second, they should consider toxicity and ecotoxicity profiles of PPPs. Last, they should be computable for each member state and allow comparisons in absolute values between member states to account for "the risk or use reduction" targets already achieved prior to the application of this Directive.

Background Multidrug resistance has been identified in the fungal pathogen responsible for Septoria leaf blotch, Zymoseptoria tritici , since 2011. It has been linked to the overexpression of the gene encoding the MFS1 transporter due to inserts in the promoter region of MFS1 (P MFS1 ), namely types I-III. Recently, two new inserts were discovered in P MFS1 that were not linked to MDR, interrogating about whether P MFS1 inserts are the only drivers of MDR in Z. tritici . The goal of our study was to gain a more complete view of MDR in Z. tritici by examining the genotypic diversity associated with the MDR phenotype in a large sample of the modern population. Results We isolated 384 potential MDR strains between 2020 and 2021 in northern Europe for P MFS1 genotype and MDR assessment. We discovered six new inserts in P MFS1 , bringing the total count to 13 including one insertion-deletion in the 5’ UTR region. Of these, 11 display similarities with transposable elements, and 3 are not linked to MDR. Some field strains were significantly more resistant than their respective reference of the same P MFS1 genotype and some strains without insert displayed MDR phenotype. Conclusion We described the landscape of the MDR in modern Z. tritici population and postulate that P MFS1 is a hot-spot for insertions involving transposition events. Our study shows that MDR cannot be solely explained by inserts found in P MFS1 , and that additional mechanisms might be at work.

The fungal pathogen Zymoseptoria tritici is the causal agent of Septoria tritici blotch (STB), a major wheat disease in Western Europe. Microorganisms inhabiting wheat leaves might act as beneficial, biocontrol, or facilitating agents that could limit or stimulate the development of Z. tritici. Improving our understanding of microbial communities in the wheat phyllosphere would lead to new insights into STB management. This article provides fungal and bacterial metabarcoding datasets obtained by sampling wheat leaves with and without symptoms caused by Z. tritici. Tissues were sampled from three commercial wheat varieties on three sampling dates during a cropping season. Weeds around wheat fields were sampled as well. In total, more than 450 leaf samples were collected. The pathogen Z. tritici was quantified using quantitative PCR. We provide the raw metabarcoding datasets, the amplicon sequence variant tables obtained after bioinformatic processing, the metadata associated with each sample (sampling date, wheat variety and tissue health condition), a preliminary descriptive analysis of the data, and the code used for bioinformatic and descriptive statistical analysis. [Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Pesticide resistance poses a critical threat to agriculture, human health and biodiversity. Mixtures of fungicides are recommended and widely used in resistance management strategies. However, the components of the efficiency of such mixtures remain unclear. We performed an experimental evolutionary study on the fungal pathogen Z. tritici to determine how mixtures managed resistance. We compared the effect of the continuous use of single active ingredients to that of mixtures, at the minimal dose providing full control of the disease, which we refer to as the "efficient " dose. We found that the performance of efficient-dose mixtures against an initially susceptible population depended strongly on the components of the mixture. Such mixtures were either as durable as the best mixture component used alone, or worse than all components used alone. Moreover, efficient dose mixture regimes probably select for generalist resistance profiles as a result of the combination of selection pressures exerted by the various components and their lower doses. Our results indicate that mixtures should not be considered a universal strategy. Experimental evaluations of specificities for the pathogens targeted, their interactions with fungicides and the interactions between fungicides are crucial for the design of sustainable resistance management strategies.

Many fungal plant pathogens encompass multiple populations specialized on different plant species. Understanding the factors underlying pathogen adaptation to their hosts is a major challenge of evolutionary microbiology, and it should help to prevent the emergence of new specialized pathogens on novel hosts. Previous studies have shown that French populations of the gray mold pathogen Botrytis cinerea parasitizing tomato and grapevine are differentiated from each other, and have higher aggressiveness on their host of origin than on other hosts, indicating some degree of host specialization in this polyphagous pathogen. Here, we aimed at identifying the genomic features underlying the specialization of B. cinerea populations to tomato and grapevine. Based on whole genome sequences of 32 isolates, we confirmed the subdivision of B. cinerea pathogens into two genetic clusters on grapevine and another, single cluster on tomato. Levels of genetic variation in the different clusters were similar, suggesting that the tomato-specific cluster has not recently emerged following a bottleneck. Using genome scans for selective sweeps and divergent selection, tests of positive selection based on polymorphism and divergence at synonymous and nonsynonymous sites, and analyses of presence and absence variation, we identified several candidate genes that represent possible determinants of host specialization in the tomato-associated population. This work deepens our understanding of the genomic changes underlying the specialization of fungal pathogen populations.

The evolution of resistance to pesticides is a major burden in agriculture. Resistance management involves maximizing selection pressure heterogeneity, particularly by combining active ingredients with different modes of action. We tested the hypothesis that the temporal alternation of active ingredients may delay the build-up of resistance not only by spreading selection pressure over longer periods, but also by decreasing the rate of evolution of resistance to alternated fungicides. Here, we applied an original experimental evolution approach to the economically important crop pathogen Zymoseptoria tritici. We observed the dynamics of Z. tritici resistance in 56 independent lines subjected to 14 continuous or alternation regimes of 3 fungicides contrasting for their mode of action, at their EC95 selection doses. For the first time in a plant pathogen, our results show that alternation is either neutral or slows the evolution of resistance, relative to continuous fungicide use, but results in higher levels of generalism in evolved lines. We demonstrate that the mode of action of resistance of fungicides drivingly underlies this trade-off, more so than the number of fungicides and the frequency of alternation. This trade-off is also dynamic over the course of resistance evolution, as shown by the change in population phenotype structure and the relative impact of selection drivers. These findings open up new possibilities for tailoring resistance management effectively while optimizing smart interplay between alternation components. They also confirm experimental evolution as an untapped but promising approach to dissect adaptation in phytopathogenic fungi. Author summary The efficacy of pesticides has been compromised by the generalization of their use, leading to the rapid and widespread evolution of resistance. This constitutes a major economic and environmental burden in agriculture. The temporal alternation (or cycling) of active ingredients is a management strategy that induces temporal variation of selection of pathogens. Here, we dissected how it can be optimized according to the number and nature of alternated modes of action and to the rhythm of their application and sought to understand how these drivers determine the performance of alternation strategies. We used an approach original in plant pathology, experimental evolution, applied on an economically important fungus, Zymoseptoria tritici. We concluded that alternation can delay the rate of resistance selection with performance depending most likely on the mode of action of alternated antifungals, more so than on other drivers. But we also highlighted that alternation regimes select generalist resistance, that is the ability to resist a large diversity of antifungals. This trade-off was dynamic over time. Our study provides new insights for the informed management of pesticides and the reduction of their side effects.

The evolution of resistance to pesticides is a major burden in agriculture. Resistance management involves maximizing selection pressure heterogeneity, particularly by combining active ingredients with different modes of action. We tested the hypothesis that alternation may delay the build-up of resistance not only by spreading selection pressure over longer periods, but also by decreasing the rate of evolution of resistance to alternated fungicides, by applying an experimental evolution approach to the economically important crop pathogen Zymoseptoria tritici. Our results show that alternation is either neutral or slows the evolution of resistance, relative to continuous fungicide use, but results in higher levels of generalism in evolved lines. We demonstrate that the relative risk of resistance intrinsic to fungicide alternation probably underlies a trade-off between the number of fungicides and the frequency of alternation. This trade-off is also dynamic over the course of resistance evolution. These findings open up new possibilities for tailoring resistance management effectively while optimizing interplay between alternation components.

BACKGROUND Monitoring resistance to plant protection products (PPPs) is crucial for understanding the evolution of resistances in bioagressors, thereby allowing scientists to design sound bioagressor management strategies. Globally, resistance monitoring is implemented by a wide range of actors that fall into three distinct categories: academic, governmental, and private. The purpose of this study was to investigate worldwide diversity in PPP resistance monitoring systems and to shed light on their different facets. RESULTS A large survey involving 162 experts from 48 countries made it possible to identify and analyze 250 resistance monitoring systems. Through an in-depth analysis, the features of the different monitoring systems were identified. The main factor differentiating monitoring systems was essentially the capabilities (funding, manpower, technology, etc.) of the actors involved in each system. In most countries, and especially in those with a high Human Development Index, academic, governmental, and private monitoring systems coexist. Overall, systems focus far more on monitoring established resistances than on the detection of emerging resistances. Governmental and private resistance monitoring systems generally have considerable capacities to generate data, whereas academic resistance monitoring systems are more specialized. Governmental actors federate and enroll a wider variety of stakeholders. CONCLUSION The results show functional complementarities between the coexisting actors in countries where they coexist. We suggest PPP resistance monitoring might be enhanced if the different actors focus more on detecting emerging resistances (and associated benefits) and increase collaborative and collective efforts and transparency.