Zymoseptoria Tritici Populations Research Articles

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.

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

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

Screening the Zymoseptoria tritici population in Turkey for resistance to azole and strobilurin fungicides

Screening the Zymoseptoria tritici population in Turkey for resistance to azole and strobilurin fungicides

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

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

Changes in DMI, SDHI, and QoI Fungicide Sensitivity in the Estonian Zymoseptoria tritici Population between 2019 and 2020.

Zymoseptoria tritici (Zt) populations adapt under the selection pressure of fungicides applied for disease control. The primary objective of this study was to assess fungicide sensitivity in the Estonian Zt population. A total of 282 Zt isolates from 2019 and 2020 were tested for sensitivity to azoles (DMIs; prothioconazole-desthio, epoxiconazole, mefentrifluconazole) and succinate dehydrogenase inhibitors (SDHIs; boscalid, fluxapyroxad). The efficacy of the tested fungicides varied considerably between the Estonian counties, but the Zt population is mainly sensitive to DMIs. Additionally, the frequencies of CYP51 gene alterations varied; D134G, V136C, A379G, and S524T had increased, but V136A and I381V showed a moderate decrease in 2020 in comparison to 2019. Sensitivity to SDHIs was stable, but boscalid was less effective than fluxapyroxad. SdhC gene mutations C-T33N, C-T34N, and C-N86S were common, but not linked with SDHI fungicide sensitivity assay results. Otherwise, mutation B-N225I in the SdhB subunit occurred in isolates with reduced sensitivity to SDHIs. Sensitivity to strobilurins was evaluated by the mutation G143A in the CytB gene, which was present in nearly half of the population. The data presented confirm the ongoing evolution of fungicide sensitivity in the Zt population in Estonia and highlight the importance of knowledge-based decisions for optimizing anti-resistance strategies in the field.

Reduced field efficacy and sensitivity of demethylation inhibitors in the Danish and Swedish Zymoseptoria tritici populations

Zymoseptoria tritici causes septoria tritici blotch (STB), the predominant fungal disease in wheat in Denmark and Sweden. Disease control is highly reliant on fungicides in the group of demethylation inhibitors (DMI). The use of DMIs has increased steadily since their introduction in the 1970s. Epoxiconazole and prothioconazole were the most widely used active ingredients in the last ten years. The goal of this investigation was to survey the resistance development of Z. tritici towards these two compounds. In total, EC50 values were determined for 3472 Z. tritici isolates from 2012 to 2019. Also, the field performance of the most used DMI compounds was tested in field trials. EC50 values of epoxiconazole and prothioconazole increased in the testing period. A significant shift was observed for epoxiconazole in 2016 and again 2018 with average EC50 values >1 ppm in Denmark. In Sweden, average EC50 values for epoxiconazole reached 1 ppm in 2017. The sensitivity towards prothioconazole remained stable at a high level. Following the decline in sensitivity in vitro, field efficacies of epoxiconazole and prothioconazole decreased from 80 - 90% to 30 - 40% in Denmark and Sweden. Currently, the Danish and Swedish Z. tritici populations are highly adapted to epoxiconazole and prothioconazole. At the same time, a recovery in the sensitivity of tebuconazole and metconazole is observed. Our studies reinforce the view that the principles of anti-resistance management should be applied upon the launch of a new active ingredient to prolong the effective life of both the old and new products.

A dispensable paralog of succinate dehydrogenase subunit C mediates standing resistance towards a subclass of SDHI fungicides in Zymoseptoria tritici.

Succinate dehydrogenase inhibitor (SDHI) fungicides are widely used for the control of a broad range of fungal diseases. This has been the most rapidly expanding fungicide group in terms of new molecules discovered and introduced for agricultural use over the past fifteen years. A particular pattern of differential sensitivity (resistance) to the stretched heterocycle amide SDHIs (SHA-SDHIs), a subclass of chemically-related SDHIs, was observed in naïve Zymoseptoria tritici populations not previously exposed to these chemicals. Subclass-specific resistance was confirmed at the enzyme level but did not correlate with the genotypes of the succinate dehydrogenase (SDH) encoding genes. Mapping and characterization of the molecular mechanisms responsible for standing SHA-SDHI resistance in natural field isolates identified a gene paralog of SDHC, termed ZtSDHC3, which encodes for an alternative C subunit of succinate dehydrogenase, named alt-SDHC. Using reverse genetics, we showed that alt-SDHC associates with the three other SDH subunits, leading to a fully functional enzyme and that a unique Qp-site residue within the alt-SDHC protein confers SHA-SDHI resistance. Enzymatic assays, computational modelling and docking simulations for the two SQR enzymes (altC-SQR, WT_SQR) enabled us to describe enzyme-inhibitor interactions at an atomistic level and to propose rational explanations for differential potency and resistance across SHA-SDHIs. European Z. tritici populations displayed a presence (20–30%) / absence polymorphism of ZtSDHC3, as well as differences in ZtSDHC3 expression levels and splicing efficiency. These polymorphisms have a strong impact on SHA-SDHI resistance phenotypes. Characterization of the ZtSDHC3 promoter in European Z. tritici populations suggests that transposon insertions are associated with the strongest resistance phenotypes. These results establish that a dispensable paralogous gene determines SHA-SDHIs fungicide resistance in natural populations of Z. tritici. This study paves the way to an increased awareness of the role of fungicidal target paralogs in resistance to fungicides and demonstrates the paramount importance of population genomics in fungicide discovery.

Prevalence of QoI resistance and mtDNA diversity in the Irish Zymoseptoria tritici population

Abstract The emergence and spread of Quinone outside Inhibitor (QoI) fungicide resistance in the Irish Zymoseptoria tritici population in the early 2000s had immediate impacts on the efficacy of the entire group of fungicides for the control of septoria tritici blotch. As a result, a dramatic reduction in the quantities applied to winter wheat occurred in the following seasons. Even in the absence of these fungicides, the frequency of the resistance allele, G143A in the pathogens mtDNA has remained exceptionally high (>97%), and as such, it can be anticipated that continued poor efficacy of current QoI fungicides will be observed. Amongst the isolates with G143A, differences in sensitivity to the QoI pyraclostrobin were observed in vitro. The addition of the alternative oxidase (AOX) inhibitor salicylhydroxamic acid increased sensitivity in these isolates, suggesting some continued impairment of respiration by the QoI fungicides, albeit weak. Interestingly, amongst those tested, the strains from a site with a high frequency of inserts in the MFS1 transporter gene known to enhance QoI efflux did not exhibit this increase in sensitivity. A total of 19 mtDNA haplotypes were detected amongst the 2017 strain collection. Phylogenetic analysis confirmed the suggestion of a common ancestry of all the haplotypes, even though three of the haplotypes contained at least one sensitive strain.

Overall stability in the genetic structure of a Zymoseptoria tritici population from epidemic to interepidemic stages at a small spatial scale

Subpopulations of the wheat pathogen Zymoseptoria tritici (26 sample groups composed of 794 isolates) were collected in two nearby wheat fields in the Paris basin, during both epidemic and inter-epidemic periods of three successive years (2009–2013). In addition to the type of inoculum (ascospores vs. pycnidiospores), the alternative presence of wheat debris allowed taking into account its putative origin (local vs. distant). We used a molecular epidemiology approach, based on population genetic indices derived from SSR marker analysis, to describe putative changes in the structure and genotypic diversity of these subpopulations over 3 years, at a spatiotemporal scale consistent with epidemiological observations. Genetic structure was broadly stable over time (within and between years) and between fields, however with weak population differentiation over time. All subpopulations displayed very high diversity and the occurrence of regular sexual reproduction was confirmed in the two fields. A significant increase of the MAT1–1/MAT1–2 ratio was observed over the course of the epidemics. This original finding suggests a competitive advantage of MAT1–1 strains consistently with their greater pathogenicity reported in the literature and may reveal undescribed adaptation. Finally, we found that the period, the type of inoculum and its putative origin had little effect on the short term evolution of the local population of Z. tritici. Fungal population size and diversity are apparently large enough to prevent genetic drift at this fine spatiotemporal scale, and more likely short distance migration contributes strongly to the stabilization of genetic diversity among and within plots.

A combination of target-site alterations, overexpression and enhanced efflux activity contribute to reduced azole sensitivity present in the Irish Zymoseptoria tritici population

Control of septoria tritici blotch in Irish winter wheat crops has been reliant on the application of azoles fungicides for over a decade. The resulting intensive applications of azole fungicides have placed the Irish Zymoseptoria tritici populations under immense pressures to adapt. Evidence of this adaptation is observed in the decreasing sensitivity of the Irish population to the azole fungicide epoxiconazole, where the mean sensitivity has decreased almost 16x over the period 2006–2015. This decrease in sensitivity has occurred in a stepwise manner, with the sensitive proportion of the population being gradually replaced by those more resistant. The decrease in sensitivity was also accompanied by an increase in frequency of Z. tritici strains with an insert (120, 862 or 866 bp) in their CYP51 promoter region. Neither the 862 bp nor 866 bp inserts impacted the expression of CYP51 in either absence or presence of epoxiconcazole. Sequencing of the CYP51 gene in a sub-sample of the 2015 collection identified 25 different CYP51 haplotypes, the majority of which combined previously identified mutations, including V136A, I381V and S524 T. The presence of the 862 or 866 bp insert in the different strains, whilst not exclusive was strongly associated with specific CYP51 haplotypes. This was also reflected in their sensitivity to a range of azoles, most notably metconazole and tebuconazole. Strains with inserts in their MFS1 gene promoter region and identical in size to those known confer multiple drug resistance (MDR) phenotypes were also identified in the 2015 sub-collection. Although the frequencies of strains with those inserts known to confer moderate-high levels of MDR were extremely low, their presence further highlights the adaptability and continued erosion of azole sensitivity in the Irish Z. tritici population.

Population structure and frequency differences of CYP51 mutations in Zymoseptoria tritici populations in the Nordic and Baltic regions

Septoria tritici blotch caused by the fungus Zymoseptoria tritici (formerly Mycosphaerella graminicola) is one of the most yield-reducing diseases worldwide. Effective disease management involves the use of resistant cultivars and application of fungicides. In this study, the population structure and genetic diversity of 183 Z. tritici isolates from Denmark, Sweden, Finland and the Baltic countries were analysed by molecular markers. In population structure analysis, isolates from Denmark and Sweden were grouped together, whereas isolates from the Baltics and Finland were grouped together. Analysis of genetic diversity and ϕ-values confirmed the division of Nordic and Baltic regions. Danish isolates sampled from different regions and different varieties were not genetically different. However, significant genetic differences were detected between isolates sampled from different years in Denmark and for isolates sampled from specific cultivars in different years. Additionally, the frequency of several known point mutations in the gene cyp51, conferring decreased sensitivity to DMI fungicides, was investigated. Several of the examined mutations were detected at a lower frequency in Baltic isolates compared to Danish and Swedish isolates. Analysis of the Danish population revealed a significant increase in specific mutations over the years. Lastly, some mutations were significantly more frequent in isolates derived from certain varieties. By using different resistance sources in breeding programmes and application of a wide range of fungicides, a sustainable and efficient disease management can be obtained.

Epidemiological trade-off between intra- and interannual scales in the evolution of aggressiveness in a local plant pathogen population.

The efficiency of plant resistance to fungal pathogen populations is expected to decrease over time, due to their evolution with an increase in the frequency of virulent or highly aggressive strains. This dynamics may differ depending on the scale investigated (annual or pluriannual), particularly for annual crop pathogens with both sexual and asexual reproduction cycles. We assessed this time‐scale effect, by comparing aggressiveness changes in a local Zymoseptoria tritici population over an 8‐month cropping season and a 6‐year period of wheat monoculture. We collected two pairs of subpopulations to represent the annual and pluriannual scales: from leaf lesions at the beginning and end of a single annual epidemic and from crop debris at the beginning and end of a 6‐year period. We assessed two aggressiveness traits—latent period and lesion size—on sympatric and allopatric host varieties. A trend toward decreased latent period concomitant with a significant loss of variability was established during the course of the annual epidemic, but not over the 6‐year period. Furthermore, a significant cultivar effect (sympatric vs. allopatric) on the average aggressiveness of the isolates revealed host adaptation, arguing that the observed patterns could result from selection. We thus provide an experimental body of evidence of an epidemiological trade‐off between the intra‐ and interannual scales in the evolution of aggressiveness in a local plant pathogen population. More aggressive isolates were collected from upper leaves, on which disease severity is usually lower than on the lower part of the plants left in the field as crop debris after harvest. We suggest that these isolates play little role in sexual reproduction, due to an Allee effect (difficulty finding mates at low pathogen densities), particularly as the upper parts of the plant are removed from the field, explaining the lack of transmission of increases in aggressiveness between epidemics.

Effect of host resistance on genetic structure of core and accessory chromosomes in Irish Zymoseptoria tritici populations

In agricultural pathosystems resistant cultivars are typically only temporarily effective, as widespread growth of said cultivars drives selection for pathogen genotypes capable of infecting them. A gene-for-gene interaction between Z. tritici and wheat has been demonstrated for one cultivar; however results of studies into the relevance of these interactions in the field remain inconsistent. Because genetic drift does not appear to occur between Z. tritici populations that are not widely geographically separated, according to neutral genetic theory if adaptation to different host cultivars is occurring, reduced genetic variation, and some differentiation between populations sourced from different cultivars should be observed. Selectively neutral microsatellite markers were used to genotype 260 isolates of Z. tritici taken from two naturally infected randomized block trials of four different cultivars, representing a spectrum of resistance to Z. tritici from susceptible to resistant. By calculating genetic parameters such as overall heterozygosity and F ST from this genotypic data, the presented study aimed to determine if genetic drift or host selection is impacting on the genetic structure of the Irish Z. tritici population. Results indicated that diversity was distributed almost entirely within, rather than among populations, with little or no differentiation, and almost no clone isolates were present in the dataset. However this result was not reflected in the accessory chromosomes, where evidence of minor but significant genetic structure was found. This lack of structure in the core chromosomes and weak structure in the accessory chromosomes confirms that forces of genetic drift and selection are minor compared to sexual reproduction, in concurrence with multiple previous studies on other populations worldwide.

Reduced Virulence of Azoxystrobin-Resistant Zymoseptoria tritici Populations in Greenhouse Assays.

The development of resistance to multiple fungicide classes is currently limiting disease management options for many pathogens, while the discovery of new fungicide classes may become less frequent. In light of this, more research is needed to quantify virulence trade-offs of fungicide resistance in order to more fully understand the implications of fungicide resistance on pathogen fitness. The purpose of this study was to measure the virulence of azoxystrobin-resistant and -sensitive Zymoseptoria tritici populations collected from North and South Willamette Valley, Oregon, in 2012 and 2015. Inoculum mixtures of known fungicide-resistant phenotypes were used to simulate natural field conditions, where multiple genotypes exist and interact in close proximity. Six greenhouse inoculations were conducted over 2 years, and virulence of the isolate mixtures was evaluated in planta. We considered virulence to be "the degree of pathology caused by the organism" and visually estimated the percent area of leaf necrosis as a measure of virulence. In greenhouse conditions, a consistent association of reduced virulence with azoxystrobin-resistant Z. tritici isolate mixtures was observed. North Willamette Valley and South Willamette Valley populations did not differ in virulence.

First Report of Resistance to QoI Fungicides in North American Populations of Zymoseptoria tritici, Causal Agent of Septoria Tritici Blotch of Wheat.

The G143A mutation in cytb (cytochrome b gene) is associated with high levels of resistance to quinone outside inhibitor (QoI or strobilurin) fungicides that disrupt electron transport during cellular respiration (1). The G143A mutation in Zymoseptoria tritici (synonyms: Mycosphaerella graminicola and Septoria tritici), the causal agent of septoria tritici blotch of wheat (Triticum aestivum), was first reported in Europe in 2001 (1). Although Z. tritici has a global distribution (3), G143A mutants of Z. tritici have not been reported outside of Europe. We used PCR-RFLP (4) to estimate the frequencies of G143A mutants in Z. tritici populations at two locations in the Willamette Valley of western Oregon: the Hyslop Crop Science Field Research Laboratory (Hyslop Farm, HF), Benton County (44°37'52.85″ N, 123°11'55.19″ W) and research plots planted in a commercial wheat field in Washington County (45°33'58.53″ N, 123°00'11.78″ W) (North Valley Farm, NVF). Isolates originated from flag leaf collections from two cultivars ('Bobtail' and 'Tubbs 06') made in April and June of 2012 from plants in a replicated fungicide-treatment experiment, with isolates collected from both sprayed and unsprayed plots. Sixteen of the 169 isolates (9.5%) from HF possessed the G143A mutation (7 of 132 isolates from plots not receiving a QoI fungicide and 9 of 37 isolates collected from plots receiving two applications of the QoI azoxystrobin). One hundred forty six of the 175 isolates (83.4%) from NVF were G143A mutants (101 of 129 isolates from plots receiving no QoI fungicide and 45 of 46 isolates from plots receiving two applications of azoxystrobin). Results of phenotypic assays of a subset of 10 isolates from each location (5 mutants, 5 wild types from each location; 20 isolates altogether) supported a high level of resistance to azoxystrobin only in the G143A mutants. All 10 G143A mutants developed colonies after 8 days of growth on YMA plates amended with SHAM (2) and 1 ppm or 10 ppm azoxystrobin, with nine and eight G143A mutant isolates developing colonies on plates amended with 1 ppm and 10 ppm azoxystrobin, respectively. None of the wild-type isolates developed colonies on plates amended with SHAM and 1 ppm azoxystrobin, nor on plates amended with SHAM and 10 ppm azoxystrobin. All 20 isolates developed colonies on YMA plates lacking azoxystrobin, and treatments produced identical results across three replicates. These results are consistent with findings of higher levels of azoxystrobin resistance in G143A mutants compared to wild types in European populations (1). Isolates from HF and NVF differ in their previous exposure to QoI fungicides. The majority of the wheat area at HF is planted to breeding plots that are not sprayed with fungicide. Plots at NVF were planted in a commercial wheat field in a county where most wheat fields were treated with two to three applications of strobilurins each year over the past 4 years. Future monitoring for G143A mutants of Z. tritici throughout its range in North America will be necessary to assess whether strobilurin resistance will spread via wind-dispersal of ascospores or emerge de novo in treated fields. In Europe, stobilurins were first applied to wheat in 1996. G143A mutants of Z. tritici emerged de novo several times (4) and were widespread by 2007.

Invasion thresholds for fungicide resistance: deterministic and stochastic analyses

Fungicide resistance is an important practical problem, but one that is poorly understood at the population level. Here we introduce a simple nonlinear model for fungicide resistance in botanical epidemics which includes the dynamics of the chemical control agent and the host population, while also allowing for demographic stochasticity in the host-parasite dynamics. This provides a mathematical framework for analysing the risk of fungicide resistance developing by including the parameters for the amount applied, longevity and application frequency of the fungicide. The model demonstrates the existence of thresholds for the invasion of the resistant strain in the parasite population which depend on two quantities: the relative fitness of the resistant strain and the effectiveness of control. This threshold marks a change from definite elimination of the resistant strain below the threshold to a finite probability of invasion which increases above the threshold. The fungicide decay rate, the amount of fungicide applied and the period between applications affect the effectiveness of control and, consequently, they influence whether or not resistance develops and the time taken to achieve a critical frequency of resistance. All three parameters are amenable to control by the grower or by coordinating the activity of a population of growers. Providing crude estimates of the effectiveness of control and relative fitness are available, the results can be used to predict the consequences of changing these parameters for the risk of invasion and the proportion of sites at which this might be expected to occur. Although motivated for fungicide resistance, the model has broader application to herbicide, antibiotic and antiviral resistance. The modelling approach and results are discussed in the context of resistance to chemical control in general.