Preventing multi-resistance: New insights for managing fungal adaptation.

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.

Antifungal alternation can be beneficial for durability but at the cost of generalist resistance.

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 overall resistance evolution rate, relative to continuous fungicide use, but results in higher levels of generalism in evolved lines. We demonstrate that the nature of the fungicides, and therefore their relative intrinsic risk of resistance may underly this trade-off, more so than the number of fungicides and the rhythm 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.

Directed evolution predicts cytochrome b G37V target site modification as probable adaptive mechanism towards the QiI fungicide fenpicoxamid in Zymoseptoria tritici.

Acquired resistance is a threat to antifungal efficacy in medicine and agriculture. The diversity of possible resistance mechanisms and highly adaptive traits of pathogens make it difficult to predict evolutionary outcomes of treatments. We used directed evolution as an approach to assess the resistance risk to the new fungicide fenpicoxamid in the wheat pathogenic fungus Zymoseptoria tritici. Fenpicoxamid inhibits complex III of the respiratory chain at the ubiquinone reduction site (Qi site) of the mitochondrially encoded cytochrome b, a different site than the widely used strobilurins which inhibit the same complex at the ubiquinol oxidation site (Qo site). We identified the G37V change within the cytochrome b Qi site as the most likely resistance mechanism to be selected in Z. tritici. This change triggered high fenpicoxamid resistance and halved the enzymatic activity of cytochrome b, despite no significant penalty for in vitro growth. We identified negative cross-resistance between isolates harbouring G37V or G143A, a Qo site change previously selected by strobilurins. Double mutants were less resistant to both QiIs and quinone outside inhibitors compared to single mutants. This work is a proof of concept that experimental evolution can be used to predict adaptation to fungicides and provides new perspectives for the management of QiIs.

Are Efficient-Dose Mixtures a Solution to Reduce Fungicide Load and Delay Evolution of Resistance? An Experimental Evolutionary Approach.

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.

Population Genomics Reveals Molecular Determinants of Specialization to Tomato in the Polyphagous Fungal Pathogen Botrytis cinerea in France.

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.

Large-scale study validates that regional fungicide applications are major determinants of resistance evolution in the wheat pathogen Zymoseptoria tritici in France.

In modern cropping systems, the near-universal use of plant protection products selects for resistance in pest populations. The emergence and evolution of this adaptive trait threaten treatment efficacy. We identified determinants of fungicide resistance evolution and quantified their effects at a large spatiotemporal scale. We focused on Zymoseptoria tritici, which causes leaf blotch in wheat. Phenotypes of qualitative or quantitative resistance to various fungicides were monitored annually, from 2004 to 2017, at about 70 sites throughout 22 regions of France (territorial units of 25 000 km2 on average). We modelled changes in resistance frequency with regional anti-Septoria fungicide use, yield losses due to the disease and the regional area under organic wheat. The major driver of resistance dynamics was fungicide use at the regional scale. We estimated its effect on the increase in resistance and relative apparent fitness of each resistance phenotype. The predictions of the model replicated the spatiotemporal patterns of resistance observed in field populations (R2 from 0.56 to 0.82). The evolution of fungicide resistance is mainly determined at the regional scale. This study therefore showed that collective management at the regional scale could effectively complete local actions.

The polyphagous plant pathogenic fungus Botrytis cinerea encompasses host-specialized and generalist populations.

The host plant is often the main variable explaining population structure in fungal plant pathogens, because specialization contributes to reduce gene flow between populations associated with different hosts. Previous population genetic analysis revealed that French populations of the grey mould pathogen Botrytis cinerea were structured by hosts tomato and grapevine, suggesting host specialization in this highly polyphagous pathogen. However, these findings raised questions about the magnitude of this specialization and the possibility of specialization to other hosts. Here we report specialization of B. cinerea populations to tomato and grapevine hosts but not to other tested plants. Population genetic analysis revealed two pathogen clusters associated with tomato and grapevine, while the other clusters co-occurred on hydrangea, strawberry and bramble. Measurements of quantitative pathogenicity were consistent with host specialization of populations found on tomato, and to a lesser extent, populations found on grapevine. Pathogen populations from hydrangea and strawberry appeared to be generalist, while populations from bramble may be weakly specialized. Our results suggest that the polyphagous B. cinerea is more accurately described as a collection of generalist and specialist individuals in populations. This work opens new perspectives for grey mould management, while suggesting spatial optimization of crop organization within agricultural landscapes.

Spatiotemporal dynamics of fungicide resistance in the wheat pathogen Zymoseptoria tritici in France.

BACKGROUND: Management of pesticide resistance is a major issue in modern agricultural systems, particularly in the context of the broader challenge of reducing pesticide use. However, such management must be adapted to resistance dynamics, which remains challenging to predict due to its dependence on many biological traits of pests, interactions with the environment and pesticide use. We retrospectively studied the evolution of reported resistances to four modes of action (benzimidazoles, quinone outside inhibitors, sterol demethylation inhibitors and succinate dehydrogenase inhibitors), in French populations of the wheat pathogen Zymoseptoria tritici. RESULTS: We used statistical models to analyse the Performance trial network data set (2004-2017; ∼ 70 locations in France yearly). They highlighted contrasting behaviours between phenotypes, for example: (i) stable spatial distributions and colonization front structures over time, and (ii) different frequency growth rates at the national scale and between regions. CONCLUSION: We provide a quantitative description of the spatiotemporal patterns of resistance evolution for fungicides with several modes of action. Moreover, we highlight some unexpected resistance dynamics in France, with major differences between the north and south. This complex pattern of resistance evolution in French populations is consistent with previous descriptions of dynamics at the European scale. These results should make it easier to anticipate evolution locally and to improve the management of resistance. © 2019 Society of Chemical Industry.

Biosynthesis of abscisic acid in fungi: identification of a sesquiterpene cyclase as the key enzyme in Botrytis cinerea.

While abscisic acid (ABA) is known as a hormone produced by plants through the carotenoid pathway, a small number of phytopathogenic fungi are also able to produce this sesquiterpene but they use a distinct pathway that starts with the cyclization of farnesyl diphosphate (FPP) into 2Z,4E-α-ionylideneethane which is then subjected to several oxidation steps. To identify the sesquiterpene cyclase (STC) responsible for the biosynthesis of ABA in fungi, we conducted a genomic approach in Botrytis cinerea. The genome of the ABA-overproducing strain ATCC58025 was fully sequenced and five STC-coding genes were identified. Among them, Bcstc5 exhibits an expression profile concomitant with ABA production. Gene inactivation, complementation and chemical analysis demonstrated that BcStc5/BcAba5 is the key enzyme responsible for the key step of ABA biosynthesis in fungi. Unlike what is observed for most of the fungal secondary metabolism genes, the key enzyme-coding gene Bcstc5/Bcaba5 is not clustered with the other biosynthetic genes, i.e., Bcaba1 to Bcaba4 that are responsible for the oxidative transformation of 2Z,4E-α-ionylideneethane. Finally, our study revealed that the presence of the Bcaba genes among Botrytis species is rare and that the majority of them do not possess the ability to produce ABA.

Plasticity of the MFS1 Promoter Leads to Multidrug Resistance in the Wheat Pathogen Zymoseptoria tritici.

The ascomycete Zymoseptoria tritici is the causal agent of Septoria leaf blotch on wheat. Disease control relies mainly on resistant wheat cultivars and on fungicide applications. The fungus displays a high potential to circumvent both methods. Resistance against all unisite fungicides has been observed over decades. A different type of resistance has emerged among wild populations with multidrug-resistant (MDR) strains. Active fungicide efflux through overexpression of the major facilitator gene MFS1 explains this emerging resistance mechanism. Applying a bulk-progeny sequencing approach, we identified in this study a 519-bp long terminal repeat (LTR) insert in the MFS1 promoter, a relic of a retrotransposon cosegregating with the MDR phenotype. Through gene replacement, we show the insert as a mutation responsible for MFS1 overexpression and the MDR phenotype. Besides this type I insert, we found two different types of promoter inserts in more recent MDR strains. Type I and type II inserts harbor potential transcription factor binding sites, but not the type III insert. Interestingly, all three inserts correspond to repeated elements present at different genomic locations in either IPO323 or other Z. tritici strains. These results underline the plasticity of repeated elements leading to fungicide resistance in Z. tritici and which contribute to its adaptive potential. IMPORTANCE Disease control through fungicides remains an important means to protect crops from fungal diseases and to secure the harvest. Plant-pathogenic fungi, especially Zymoseptoria tritici, have developed resistance against most currently used active ingredients, reducing or abolishing their efficacy. While target site modification is the most common resistance mechanism against single modes of action, active efflux of multiple drugs is an emerging phenomenon in fungal populations reducing additionally fungicides' efficacy in multidrug-resistant strains. We have investigated the mutations responsible for increased drug efflux in Z. tritici field strains. Our study reveals that three different insertions of repeated elements in the same promoter lead to multidrug resistance in Z. tritici. The target gene encodes the membrane transporter MFS1 responsible for drug efflux, with the promoter inserts inducing its overexpression. These results underline the plasticity of repeated elements leading to fungicide resistance in Z. tritici.

Fashionably late partners have more fruitful encounters: Impact of the timing of co-infection and pathogenicity on sexual reproduction in Zymoseptoria tritici.

The wheat pathogen Zymoseptoria tritici is a relevant fungal model organism for investigations of the epidemiological determinants of sexual reproduction. The objective of this experimental study was to determine which intrinsic factors, including parental fitness and timing conditions of infection, affect the numbers of ascospores produced. We first performed 28 crosses on adult wheat plants in semi-controlled conditions, with 10 isolates characterized for their fitness traits. We validated the efficiency of the crossing method, opening up new perspectives for epidemiological studies. We found that the ability to reproduce sexually was determined, at least partly, by the parental genotypes. We also found that the number of ascospores released was correlated with the mean size of the sporulating lesions of the parental isolates on the one hand, and the absolute difference in the latent periods of these isolates on the other. No functional trade-off between the two modes of reproduction in Z. tritici was revealed: there was no adaptive compromise between pathogenicity (asexual multiplication on leaves) and transmission (intensity of sexual reproduction on wheat debris). Moreover, a few days' difference in the latent periods of the two parental isolates, such that one progressed more rapidly in the host tissue than the other, seemed to be slightly beneficial to ascosporogenesis. This may be because the first parental isolate breaks down host defenses, thereby facilitating infection for the other parental isolate. However, a larger difference (a few weeks), generated by leaving two to three weeks between the inoculations of the plant with the parental isolates, was clearly detrimental to ascosporogenesis. In this case, the host tissues were likely colonized by the first isolate, leaving less host resources available for the second, consistent with a competition effect during the asexual stage.

Proposal for a unified nomenclature for target-site mutations associated with resistance to fungicides.

Evolved resistance to fungicides is a major problem limiting our ability to control agricultural, medical and veterinary pathogens and is frequently associated with substitutions in the amino acid sequence of the target protein. The convention for describing amino acid substitutions is to cite the wild-type amino acid, the codon number and the new amino acid, using the one-letter amino acid code. It has frequently been observed that orthologous amino acid mutations have been selected in different species by fungicides from the same mode of action class, but the amino acids have different numbers. These differences in numbering arise from the different lengths of the proteins in each species. The purpose of the present paper is to propose a system for unifying the labelling of amino acids in fungicide target proteins. To do this we have produced alignments between fungicide target proteins of relevant species fitted to a well-studied 'archetype' species. Orthologous amino acids in all species are then assigned numerical 'labels' based on the position of the amino acid in the archetype protein. © 2016 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Fungicide efflux and the MgMFS1 transporter contribute to the multidrug resistance phenotype in Zymoseptoria tritici field isolates.

Septoria leaf blotch is mainly controlled by fungicides. Zymoseptoria tritici, which is responsible for this disease, displays strong adaptive capacity to fungicide challenge. It developed resistance to most fungicides due to target site modifications. Recently, isolated strains showed cross-resistance to fungicides with unrelated modes of action, suggesting a resistance mechanism known as multidrug resistance (MDR). We show enhanced prochloraz efflux, sensitive to the modulators amitryptiline and chlorpromazine, for two Z. tritici strains, displaying an MDR phenotype in addition to the genotypes CYP51(I381V Y461H) or CYP51(I381V ΔY459/) (G460) , respectively, hereafter named MDR6 and MDR7. Efflux was also inhibited by verapamil in the MDR7 strain. RNA sequencing lead to the identification of several transporter genes overexpressed in both MDR strains. The expression of the MgMFS1 gene was the strongest and constitutively high in MDR field strains. Its inactivation in the MDR6 strain abolished resistance to fungicides with different modes of action supporting its involvement in MDR in Z. tritici. A 519 bp insert in the MgMFS1 promoter was detected in half of the tested MDR field strains, but absent from sensitive field strains, suggesting that the insert is correlated with the observed MDR phenotype. Besides MgMfs1, other transporters and mutations may be involved in MDR in Z. tritici.

Inflorescences vs leaves: a distinct modulation of carbon metabolism process during Botrytis infection.

Plant growth and survival depends critically on photo assimilates. Pathogen infection leads to changes in carbohydrate metabolism of plants. In this study, we monitored changes in the carbohydrate metabolism in the grapevine inflorescence and leaves using Botrytis cinerea and Botrytis pseudo cinerea. Fluctuations in gas exchange were correlated with variations in chlorophyll a fluorescence. During infection, the inflorescences showed an increase in net photosynthesis (Pn) with a stomatal limitation. In leaves, photosynthesis decreased, with a non-stomatal limitation. A decrease in the effective photosystem II (PSII) quantum yield (ΦPSII) was accompanied by an increase in photochemical quenching (qP) and non-photochemical quenching (qN). The enhancement of qP and ΦPSII could explain the observed increase in Pn. In leaves, the significant decline in ΦPSII and qP, and increase in qN suggest that energy was mostly oriented toward heat dissipation instead of CO2 fixation. The accumulation of glucose and sucrose in inflorescences and glucose and fructose in the leaves during infection indicate that the plant's carbon metabolism is differently regulated in these two organs. While a strong accumulation of starch was observed at 24 and 48 hours post-inoculation (hpi) with both species of Botrytis in the inflorescences, a significant decrease with B. cinerea at 24 hpi and a significant increase with B. pseudo cinerea at 48 hpi were observed in the leaves. On the basis of these results, it can be said that during pathogen attack, the metabolism of grapevine inflorescence and leaf is modified suggesting distinct mechanisms modifying gas exchange, PSII activity and sugar contents in these two organs.

Population structure and temporal maintenance of the multihost fungal pathogen Botrytis cinerea: causes and implications for disease management.

Understanding the causes of population subdivision is of fundamental importance, as studying barriers to gene flow between populations may reveal key aspects of the process of adaptive divergence and, for pathogens, may help forecasting disease emergence and implementing sound management strategies. Here, we investigated population subdivision in the multihost fungus Botrytis cinerea based on comprehensive multiyear sampling on different hosts in three French regions. Analyses revealed a weak association between population structure and geography, but a clear differentiation according to the host plant of origin. This was consistent with adaptation to hosts, but the distribution of inferred genetic clusters and the frequency of admixed individuals indicated a lack of strict host specificity. Differentiation between individuals collected in the greenhouse (on Solanum) and outdoor (on Vitis and Rubus) was stronger than that observed between individuals from the two outdoor hosts, probably reflecting an additional isolating effect associated with the cropping system. Three genetic clusters coexisted on Vitis but did not persist over time. Linkage disequilibrium analysis indicated that outdoor populations were regularly recombining, whereas clonality was predominant in the greenhouse. Our findings open up new perspectives for disease control by managing plant debris in outdoor conditions and reinforcing prophylactic measures indoor.

Development of a rapid multiplex SSR genotyping method to study populations of the fungal plant pathogen Zymoseptoria tritici.

BACKGROUND: Zymoseptoria tritici is a hemibiotrophic ascomycete fungus causing leaf blotch of wheat that often decreases yield severely. Populations of the fungus are known to be highly diverse and poorly differentiated from each other. However, a genotyping tool is needed to address further questions in large collections of isolates, regarding regional population structure, adaptation to anthropogenic selective pressures, and dynamics of the recently discovered accessory chromosomes. This procedure is limited by costly and time-consuming simplex PCR genotyping. Recent development of genomic approaches and of larger sets of SSRs enabled the optimization of microsatellite multiplexing. FINDINGS: We report here a reliable protocol to amplify 24 SSRs organized in three multiplex panels, and covering all Z. tritici chromosomes. We also propose an automatic allele assignment procedure, which allows scoring alleles in a repeatable manner across studies and laboratories. All together, these tools enabled us to characterize local and worldwide populations and to calculate diversity indexes consistent with results reported in the literature. CONCLUSION: This easy-to-use, accurate, repeatable, economical, and faster technical strategy can provide useful genetic information for evolutionary inferences concerning Z. tritici populations. Moreover, it will facilitate the comparison of studies from different scientific groups.

Site-directed mutagenesis of the P225, N230 and H272 residues of succinate dehydrogenase subunit B from Botrytis cinerea highlights different roles in enzyme activity and inhibitor binding.

Carboxamide fungicides target succinate dehydrogenase (SDH). Recent field monitoring studies have identified Botrytis cinerea isolates resistant to one or several SDH inhibitors (SDHIs) with amino acid substitutions in the SDH B subunit. We confirmed, by site-directed mutagenesis of the sdhB gene, that each of the mutations identified in field strains conferred resistance to boscalid in B.cinerea, and in some cases cross-resistance to other SDHIs (fluopyram, carboxin). Enzyme inhibition studies showed that the studied modifications (SdhB_P225T/L/F, N230I, H272Y/R/L) affected the inhibition of SDH activity by SDHIs, directly contributing to resistance. Our results confirm the importance of H272, P225 and N230 for carboxamide binding. Modifications of P225 and N230 conferred resistance to the four carboxamides tested (boscalid, fluopyram, carboxin, bixafen). Modifications of H272 had differential effects on the susceptibility of SDH to SDHIs. SdhB(H272L) , affected susceptibility to all SDHIs, SdhB(H272R) conferred resistance to all SDHIs tested except fluopyram, and SdhB(H272Y) conferred fluopyram hypersensitivity. Affinity-binding studies with radiolabelled fluopyram revealed strong correlations among the affinity of SDHIs for SDH, SDH inhibition and in vivo growth inhibition in the wild type. The sdhB(H272Y) mutation did not affect SDH and respiration activities, whereas all the other mutations affected respiration by decreasing SDH activity.

French vineyards provide information that opens ways for effective resistance management of Botrytis cinerea (grey mould).

Resistance to fungicides is an evolutionary process resulting from the selection of advantageous genotypes in naturally diverse populations. Seven fungicide modes of action are authorised to control grey mould caused by Botrytis cinerea on grapevine in France, and five of them have encountered specific resistance, with variable frequencies in populations and possible consequences for field fungicide efficacy. Moreover, multidrug resistance is caused by fungicide efflux and allows a weak resistance towards six unrelated modes of action. Here, a review is given of the fungicide resistance status of B. cinerea in France, particularly in the vineyards of Champagne, which are the most affected. Recently developed resistance and recent findings concerning the associated resistance mechanisms are focused upon in particular. Finally, antiresistance strategies are presented, and examples of managed resistance are discussed in a more general manner with the aim of extending this knowledge to other crops and countries undergoing similar resistance problems.

Fungicide resistance status in French populations of the wheat eyespot fungi Oculimacula acuformis and Oculimacula yallundae.

Eyespot, caused by Oculimacula acuformis and Oculimacula yallundae, is the major foot disease of winter wheat in several European countries, including France. It can be controlled by chemical treatment between tillering and the second node stage. The fungicides used include antimicrotubule toxicants (benzimidazoles), inhibitors of sterol 14α-demethylation (DMIs) or of succinate dehydrogenase (SDHIs), the anilinopyrimidines cyprodinil and the benzophenone metrafenone. Since the early 1980s, a long-term survey has been set up in France to monitor changes in the sensitivity of eyespot populations to fungicides. Resistance to benzimidazoles has become generalised since the early 1990s, in spite of the withdrawal of this class of fungicides. In the DMI group, resistance to triazoles is generalised, whereas no resistance to the triazolinethione prothioconazole has yet developed. Resistance to the imidazole prochloraz evolved successively in O. acuformis and O. yallundae and is now well established. Specific resistance to cyprodinil has also been detected, but its frequency has generally remained low. Finally, since the early 2000s, a few strains of O. yallundae displaying multidrug resistance (MDR) have been detected. These strains display low levels of resistance to prothioconazole and SDHIs, such as boscalid. Knowledge of the spatiotemporal distribution in France of O. acuformis and O. yallundae field strains resistant to fungicides allows resistance management strategies for eyespot fungi in winter wheat to be proposed.