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Multiple mechanisms account for resistance to sterol 14α-demethylation inhibitors in field isolates of Mycosphaerella graminicola
Pest Management Science
Abstract
Sterol 14α-demethylation inhibitors (DMIs) have been widely used in many European countries to control septoria leaf blotch, which is caused by Mycosphaerella graminicola (Fückel) J Schrot (anamorph Septoria tritici Berk & MA Curtis). However, treatment efficacy has declined, and significant shifts in population susceptibility have occurred in recent years, with the isolation of particularly highly resistant strains from French, English and Irish populations. The present aim was to determine the phenotypic characteristics of these field isolates and to identify the possible resistance mechanisms.
Target alteration, linked to 11 possible changes in the gene encoding 14α-demethylase (Cyp51), was the basic resistance mechanism in weakly, moderately and highly resistant strains. Changes in Cyp51 combined with the overexpression of drug efflux transporters probably result in multidrug resistance in some of the most resistant phenotypes. Finally, some moderately or highly resistant isolates were found to harbour an insertion in the Cyp51 promoter and/or new combinations of known mutations in the target gene.
An updated overview of M. graminicola field strains displaying low to high resistance to DMIs is provided here. The management of field resistance and efficacy should be adapted to take these findings into account.
... The combination of multiple mutations in the same isolate, resulting in multiple resistance, is favoured by the high rates of sexual reproduction in this pathogen (Singh et al., 2021). Generalist resistance may result from non-target-site resistance (NTSR), as in the multidrug resistance (MDR) caused by the enhanced efflux of unrelated fungicides (Leroux & Walker, 2011;Omrane et al., 2015). The diversity of resistant phenotypes observed in Z. tritici and their contrasting local frequencies in the field renders this pathogen a relevant biological model for investigations of the interplay between the drivers of the performance of anti-resistance strategies. ...
... Finally, for the detection of generalist resistance, we also included fungicides not used for experimental evolution (tolnaftate and azoxystrobin although this fungicide is of limited interest for PopRR lines where the cytb G143A was introduced with the R SDHI/DMI isolate). Tolnaftate, in particular, identifies individuals displaying enhanced fungicide efflux leading to non-specific multidrug resistance (MDR), as described in field isolates of Z. tritici (Leroux & Walker, 2011). Ancestral isolates and an MDR isolate collected in the field were included as control strains. ...
... Complementary genotyping revealed that the R SDHI , R DMI and R SDHI/DMI isolates had inherited resistance to benzimidazoles from their parental field isolate, precluding any further use of these fungicides in selection regimes (Hawkins & Fraaije, 2016), and that only the multipleresistant R SDHI/DMI isolate had inherited resistance to QoIs due to the cytb G143A change (Torriani et al., 2015). All isolates were equally susceptible to the multisite agent chlorothalonil and tolnaftate, an inhibitor of squalene epoxidase used to reveal multidrug resistance in Z. tritici (Leroux & Walker, 2011). ...
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.
... In Z. tritici MDR confers moderate resistance to SDHIs, QoIs, and DMIs (Leroux & Walker, 2011;Omrane et al., 2015), but combined to target-site resistance MDR leads to particularly high resistance factors as in the case of DMI resistance. In vitro, the selection of multidrug resistant strains of Z. tritici was favored by alternation or mixture of fungicides (Ballu et al., 2021). ...
... Most of the populations used for this study were collected from fields in France and provided by the "Performance network" (Garnault et al. 2019). To isolate potential MDR strains we inoculated spore-solutions from wheat leaves on Petri dishes containing the medical fungicides tolnaftate or terbinafine at their respective discriminating concentrations (adapted after Leroux & Walker, 2011;Experimental procedures). Both molecules are inhibitors of squalene epoxidase (Ryder et al. 1992). ...
... CC-BY-NC-ND 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in Among the 23 field strains with PMFS1-type I genotype, all isolates showed significant differences in EC50 values compared to the sensitive reference (Fig. 5) including the PMFS1type I reference and the previously published strain 09-ASA-3apz (Omrane et al. 2015;Leroux et Walker, 2011). Resistance factors were heterogenic among strains. ...
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 (PMFS1), namely types I-III. Recently, two new inserts were discovered in PMFS1 that were not linked to MDR, interrogating about whether PMFS1 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 PMFS1 genotype and MDR assessment. We discovered six new inserts in PMFS1, 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 PMFS1 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 PMFS1 is a hotspot for insertions involving transposition events. Our study shows that MDR cannot be solely explained by inserts found in PMFS1, and that additional mechanisms might be at work.
... Z. tritici exhibits TSR, determined by various specific target-site mutations, that can be associated with target overexpression 40,43-45 but also with NTSR via enhanced efflux. This latter generalist mechanism causes weak multidrug resistance (MDR) when not associated with TSR and affects at least three modes of action 43,46 . These biological features and field background make Z. tritici a textbook model for dissecting the adaptation of populations under contrasting selection pressures and reasoning about general principles. ...
... The medium-risk fungicide selected patterns of resistance to a mean of 2 AIs. The patterns selected by the lowrisk fungicide were the most generalist (resistance to a mean of 4 AIs) and often included resistance to tolnaftate, an indicator of multidrug resistance in several pathogens 43 . In alternating lines, such contrasting patterns of adaptation to a single AI should recur regularly as AIs succeed each other over the generations, thereby resulting in even more complex trajectories. ...
... As these weak variations included a susceptible isolate collected from the control line, we assumed that overexpression of these three target genes did not operate in the dataset or was rather weak. Enhanced efflux is a non-target site resistance mechanism associated with low RFs per se but causing cross-resistance to unrelated compounds in Z. tritici 43 . In field strains, it is determined by insertions in the promoter region of the MFS1 transporter gene 52 . ...
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.
... 8 Differences between QoI and QiI fungicides in their sites of action in the bc complex are significant, and fenpicoxamid is not impacted by the cytochrome b alteration G143A. 9,10 Other non-target site resistance mechanisms can influence the sensitivity to QoI in Z. tritici, such as multi-drug resistance (MDR), mediated by the overexpression of the MFS1 transporter, 11,12 or the existence of an alternative oxidase (AOX) that by-passes the function of cytochrome b. 13,14 Although these mechanisms are present in Z. tritici populations, 15,16 their effect on the field performance of QoI and QiI are believed to be low. 9,10,12 Given the current importance of fungicides in the control of STB, ensuring their continued efficacy is essential. ...
... 9,10 Other non-target site resistance mechanisms can influence the sensitivity to QoI in Z. tritici, such as multi-drug resistance (MDR), mediated by the overexpression of the MFS1 transporter, 11,12 or the existence of an alternative oxidase (AOX) that by-passes the function of cytochrome b. 13,14 Although these mechanisms are present in Z. tritici populations, 15,16 their effect on the field performance of QoI and QiI are believed to be low. 9,10,12 Given the current importance of fungicides in the control of STB, ensuring their continued efficacy is essential. Therefore, the commercialization of fenpicoxamid and incorporation into STB control programmes is a welcome development. ...
... 23,24 Overexpression of the MFS1 transporter in Z. tritici has been known to provide low/moderate to high RF values depending on the fungicide. 11,12 Such increases in efflux activity have been shown to confer a moderate reduction in sensitivity to fenpicoxamid, with an RF of about 10 detected previously. 10 To test whether the differences in sensitivity observed in the current isolate collections were related to increased efflux activity, the isolates were tested for sensitivity to fentin chloride. Fentin chloride, although also inhibiting respiration, is unrelated to either the QoIs or QiIs and has previously been used to identify efflux activity in Z. tritici. ...
BACKGROUND
Fenpicoxamid is a recently developed fungicide belonging to the quinone inside inhibitor (QiI) group. This is the first fungicide within this group to be active against the Zymoseptoria tritici, which causes Septoria tritici blotch on wheat. The occurrence of pre‐existing resistance mechanisms was monitored, using sensitivity assays and Illumina sequencing, in Z. tritici populations sampled in multiple European countries before the introduction of fenpicoxamid.
RESULTS
Although differences in sensitivity to all three fungicides tested (fenpicoxamid, fentin chloride and pyraclostrobin) existed between the isolate collections, no alterations associated with QiI resistance were detected. Among the isolates, a range in sensitivity to fenpicoxamid was observed (ratio between most sensitive/least sensitive = 53.1), with differences between the most extreme isolates when tested in planta following limited fenpicoxamid treatment. Sensitivity assays using fentin chloride suggest some of the observed differences in fenpicoxamid sensitivity are associated with multi‐drug resistance. Detailed monitoring of the wider European population using Illumina‐based partial sequencing of the Z. tritici also only detected the presence of G143A, with differences in frequencies of this alteration observed across the region.
CONCLUSIONS
This study provides a baseline sensitivity for European Z. tritici populations to fenpicoxamid. Target‐site resistance appears to be limited or non‐existing in European Z. tritici populations prior to the introduction of fenpicoxamid. Non‐target site resistance mechanisms exist, but their impact in the field is predicted to be limited. © 2022 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.
... DeMethylation Inhibitors (DMIs) inhibits the sterol 14α-demethylatase (a cytochrome P450 encoded by cyp51) within the sterol biosynthesis pathway and therefore affect the fungal cell wall (Leadbeater, 2011;Leroux and Walker, 2011;Leroux et al., 2007;Torriani et al., 2015). ...
... But globally more than 30 different mutations and 100 haplotypes have been identified (Cools and Fraaije, 2013;Huf et al., 2018;Lucas et al., 2015). These genotypes are associated to varying RFs and partial crossresistance, depending on the DMI considered (Cools et al., 2011;Heick et al., 2020;Leroux and Walker, 2011;Lucas et al., 2015). For example, the changes V136A and I381V are respectively providing low and high RFs to tebuconazole and conversely for prochloraz (Huf et al., 2018;Leroux and Walker, 2011). ...
... These genotypes are associated to varying RFs and partial crossresistance, depending on the DMI considered (Cools et al., 2011;Heick et al., 2020;Leroux and Walker, 2011;Lucas et al., 2015). For example, the changes V136A and I381V are respectively providing low and high RFs to tebuconazole and conversely for prochloraz (Huf et al., 2018;Leroux and Walker, 2011). Patterns of incomplete cross-resistance are also quite apparent. ...
The evolution of resistance to pesticides is a major economic and environmental burden in agriculture. Resistance management involves maximizing selection pressure heterogeneity, particularly by combining active ingredients (AIs) with different modes of action. Yet, there is still no consensus on the best strategy to delay the emergence and selection of resistance in pathogen populations. Here, we aimed at contributing to this debate while addressing two questions: (i) How can we enhance the performance of anti-resistance strategies and what are the drivers of their optimization? (ii) How the initial status of resistance in populations modulates the performance of strategies? We used Zymoseptoria tritici, the causal agent of the major wheat disease, which is resistant in Europe to most fungicides to varying degrees, to exemplify this debate. We applied an innovative approach for fungi, experimental evolution, to mimic and accelerate, in laboratory-controlled conditions, fungicide resistance usually occurring in the field. In particular, we disentangled the relative impact in reducing resistance selection of multiple drivers of alternation, mixture and dose modulation strategies by submitting a susceptible strain to heterogeneous selection regimes as compared to the single use of the same AIs. In a first experiment, alternation was found a beneficial or neutral strategy to decrease the rate of evolution of resistance, relative to continuous fungicide use, but resulted in higher levels of generalism. We demonstrated that the relative intrinsic resistance risk of AI probably underpinned such trade-off, more than the number of fungicides used or alternation frequency. In a second experiment, we dissected how an efficient-dose mixture, i.e. a mixture with reduced doses of its components but still allowing a disease control similar to that provided by these components used alone, may delay resistance evolution of a naïve population. We concluded that the durability of such mixtures was detrimental to beneficial, strongly depending on its components. Such mixtures possibly favoured generalist or multiple resistance patterns, depending on the combined AIs. Dose reduction in sequence strategies didn’t prevent the selection of highly resistant specialist strains, nor of generalist resistance in some lines. A final experiment was designed to validate the previous findings, while submitting a susceptible isolate and two artificial populations containing low frequencies of single vs. multiple field resistances to selection regimes displaying patterns differing for four possible sources of heterogeneity. Preliminary results concluded that increasing the number of AIs and varying their modes of actions were efficient to delay resistance build-up, either in mixture or alternation regimes, although it facilitated the selection of some generalism in addition to the initially introduced specialist resistances. However, none of these sources of heterogeneity were found efficient at mitigating resistance evolution when multiple resistance was introduced in populations, suggesting that recombination, occurring during sexual reproduction in field populations, should be considered for fungicide sustainability management. At last, fitness cost associated to some resistance alleles shortly delayed resistance evolution and may reshaped population structure in some situations. Experimental evolution has proven to be a powerful tool to enrich the debate on anti-resistance strategies whilst dissecting the interplay between their drivers in realistic biological conditions. These findings open up new possibilities for tailoring resistance management and suggest that the prevention of resistance emergence would be more sustainable than the mitigation of selection.
... By contrast, lines exposed to P had specific profiles in each of the four repeats, suggesting distinct genotypes, all broader than that for lines exposed to C (on average, P lines were resistant to 3.25 of eight discriminatory doses, whereas C lines were resistant to two) and including various degrees of resistance to P and to CP, but also to tolnaftate (for 3 of 4 lines). Tolnaftate resistance is considered an indicator of multidrug resistance due to enhanced efflux in Z. tritici [21,25]. Such patterns are consistent with the evolution of multiple and/or generalist resistance mechanisms. ...
... Indeed, lines evolved under mixture regimes often displayed broader resistance spectra than those exposed to a single AI, with lower resistance intensity, and growth on tolnaftate. As tolnaftate resistance is considered to be an indicator of MDR [25], we assume that generalist resistance was more likely to occur than multiple specific resistances, although we cannot rule out the possibility of such specialist resistance. Indeed, both types of resistance may coexist within an individual or within a population, as previously described [33] in the "bet-hedging" hypothesis, according to which, in an isogenic population, differently specialized phenotypes with fitnesses varying between conditions, may co-exist in a dynamic equilibrium in a heterogeneous environment. ...
... Genetic analysis (e.g., of the promoter of the mfs1 gene, variants of which are associated with MDR in field isolates of Z. tritici; [34]) could be performed to determine the resistance structure of evolved populations, although nontarget-site resistance could also be acquired by epigenetic mechanisms [35]. MDR might also be determined in our evolved strains by multiple mechanisms different from those already described for Z. tritici, as resistance to B was not observed in all isolates resistant to tolnaftate, as observed in field strains [25]. ...
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.
... The potential involvement of efflux pumps as a mechanism of reduced sensitivity to azoles and fengycin was examined phenotypically and genetically in both strains of V. inaequalis. Tolnaftate is an antifungal agent used to treat dermatophytic fungi and is also an indicator of multidrug resistance (MDR) in plant pathogenic fungi overexpressing efflux pumps (Leroux and Walker, 2010;Omrane et al., 2015). Interestingly, tolnaftate significantly inhibited the growth of the sensitive strain S755 at different concentrations (IC 50 value of 0.66 mg/L), whereas no inhibitory activity was observed on the strain Rs552 (data not shown). ...
... Chlorpromazine has previously shown synergistic activity with tebuconazole on sensitive and resistant strains of B. cinerea, and IR values appear to be linked to the level of expression of BcatrD (gene encoding an ABC transporter) (Leroux and Walker, 2013). Furthermore, synergy with tolnaftate was also observed with chlorpromazine, with a high IR on Rs552, which was only noticed on MDR strains of Z. tritici strains (Leroux and Walker, 2010). In contrast to chlorpromazine, the combination of verapamil with tebuconazole and tolnaftate resulted in additive interactions. ...
... As mentioned in the Introduction, fungicide resistance is mainly driven by specific target site mutations, but there is also a non-specific mechanism associated with the loss of fungicide sensitivity, namely increased efflux, referred to as multidrug resistance (MDR) [39]. It is known that MDR leads to moderate resistance to all three of the main fungicide groups mentioned previously (DMI, SDHI, and QoI) [39,40]. ...
... As mentioned in the Introduction, fungicide resistance is mainly driven by specific target site mutations, but there is also a non-specific mechanism associated with the loss of fungicide sensitivity, namely increased efflux, referred to as multidrug resistance (MDR) [39]. It is known that MDR leads to moderate resistance to all three of the main fungicide groups mentioned previously (DMI, SDHI, and QoI) [39,40]. MDR is caused by insertions in the promoter region of the major facilitator gene (MFS1). ...
Septoria tritici blotch (STB) ais one of the most damaging winter wheat diseases worldwide, presenting a significant threat to its yields. The causal STB agent, Zymoseptoria tritici, also presents a challenge to control due to its rapid adaptation to fungicides. This requires researchers to continuously monitor the pathogen and investigate and explore strategies to manage the spread of the disease and the development of resistance in the pathogen. Therefore, this study presents the current situation and describes changes in the sensitivity of Z. tritici isolates from Lithuania to quinone outside inhibitors (QoIs) and succinate dehydrogenase inhibitors (SDHIs) for the years 2019–2022. The isolates were tested at five different concentrations of two QoI fungicides (azoxystrobin and pyraclostrobin) and three SDHI fungicides (fluxapyroxad, benzovindiflupyr, and bixafen). During the test period, the EC50 values of the tested QoIs increased, while no clear changes were observed in the SDHIs. The most pronounced shift was observed for the active QoI substance pyraclostrobin. The distribution of the EC50 values of the SDHI fungicides showcased one isolate with an outstandingly high EC50 value of 2.6 mg L⁻¹. The results of this study did not reveal any strong patterns of cross-resistance between the fungicides tested. However, a significant positive, moderate correlation (r = 0.55) was found between fluxapyroxad and benzovindiflupyr. Overall, the results of this study contribute to the understanding of the fungicide-resistance situation of Z. tritici in Lithuania and may complement management strategies for the pathogen and its fungicide resistance.
... The phenotypes of the plants were recorded when the control plants had full Pst sporulating, about 14 days post-Pst inoculation (dpi). DNA was extracted from 4 cm segments of MX169 leaves collected at 6,12,18,24,36,48,72,96, and 120 h post-treatment (hpt) with flubeneteram. The total DNA obtained at the different time points were quantified through quantitative real-time PCR (qRT-PCR). ...
... Triadimefon is an inhibitor of ergosterol synthesis, and ergosterol is a main component of cell membranes. 36 Inhibition of ergosterol synthesis will lead to changes in the structures and biological functions of membranes, which are specifically observed in the present study. Flubeneteram, as an SDH fungicide, can also affect the synthesis of membranes and the structures and biological functions of Pst due to the blockage of energy supply. ...
Stripe rust caused by Puccinia striiformis f. sp. tritici (Pst) is a serious threat to wheat production, and the application of fungicides is one of the most important means for controlling the disease. The purpose of this study is to determine the effects of a new succinate dehydrogenase inhibitor (SDHI) fungicide, flubeneteram, on reducing stripe rust. The baseline sensitivity of 173 Pst isolates from 13 provinces of China to flubeneteram was determined. Flubeneteram displayed significant effects on inhibiting SDH enzymes of Pst. Histological observations showed that after flubeneteram application, the formation and development of Pst hyphae and haustoria were significantly inhibited, and the structures were destroyed. Flubeneteram primed wheat for salicylic acid-induced defenses via upregulating pathogenesis-related genes (PR1 and PR2). Altogether, our study is the first to provide evidence that flubeneteram induces wheat defense against Pst infection. The findings indicate that flubeneteram could be an effective fungicide for managing stripe rust.
... Compared to earlier periods of this long-term study, no significant control of this pathogen was achieved in the last years. This development must concern the agricultural practice, because an obvious change in the pathogen has taken place as a consequence of the fungicidal disease control [70][71][72][73][74]. The decreased fungicide efficiency could be caused by the sensitivity of the pathogen towards the different fungicide classes. ...
... In contrast, Klink et al. [46] were able to demonstrate a stable sensitivity over the last decades of Z. tritici towards mefentrifluconazole with its flexible isopropanol group. It seems to be less affected by the mutation in the CYP51 gene of the pathogen [73,74], which is of major importance in for resistance management in current farm practices. Z. tritici achieved a complete resistance for the fungicide class of strobilurins [80,81]. ...
Foliar diseases are a major threat to worldwide wheat production, especially during the vegetative period in maritime climates. Despite advancements in agronomic practices, infestations by foliar diseases are possible under favourable weather conditions, thus, fungicides are essential for maintaining control. Stage-oriented applications are therefore common in farm practices. The optimization of fungicide use according to biological–epidemiological thresholds reduces the total amount of fungicides used, which is of political interest, especially in the European Union. Therefore, the efficiency and effectivity of the fungicides used to control the six major foliar diseases (Septoria tritici blotch, glume blotch, tans spot, powdery mildew, stripe rust, and leaf rust) were analysed in a long-term study of 26 years in northern Germany under favourable maritime conditions. Of those diseases, Septoria tritici blotch was the most dominant recurring disease, with high severity noted in every year of the study. The threshold-based disease management system was compared to a fungicide untreated control and a healthy-standard fungicide treatment (according to growth stages). The usage of the threshold-based system reduced the disease severities significantly compared to the fungicide untreated control, without any loss of yield compared to the healthy-standard fungicide treatment. Thereby, the use of fungicides was reduced by two thirds compared to the stage-oriented healthy-standard treatment. Thus, the advantages of the threshold-based system were obvious, and this approach will be an important tool for future evaluations of current farm practices.
... However, DMI resistance has been found in a variety of phytopathogenic fungi 16,17,20 . The resistance mechanisms of DMIs have been reported to be diverse: (I) point mutations in the target gene 14α-demethylase (CYP51) [22][23][24] ; (II) CYP51 gene overexpression 16,[25][26][27][28][29] ; and (III) overexpression of efflux proteins 30,31 . In this study, we established the baseline sensitivity of L. theobromae to difenoconazole using 121 isolates from five major mango-producing regions in Hainan, China. ...
... Some studies reported mutations and overexpression of the CYP51 gene simultaneously in some DMI-resistant isolates of plant pathogens 17,20,45 . Furthermore, in L. theobromae of papaya, Mycosphaerella graminicola of wheat, Blumeriella jaapii of cherry and Neophysopella meliosmae-myrianthae of grapevine no CYP51 gene point mutation linked to DMI resistance has been identified, but CYP51 overexpression has been observed 18,30,46,47 . A similar pattern of results was obtained in this study. ...
Stem-end rot (SER) caused by Lasiodiplodia theobromae is an important disease of mango in China. Demethylation inhibitor (DMI) fungicides are widely used for disease control in mango orchards. The baseline sensitivity to difenoconazole of 138 L. theobromae isolates collected from mango in the field in 2019 was established by the mycelial growth rate method. The cross-resistance to six site-specific fungicides with different modes of action were investigated using 20 isolates randomly selected. The possible mechanism for L. theobromae resistance to difenoconazole was preliminarily determined through gene sequence alignment and quantitative real-time PCR analysis. The results showed that the EC 50 values of 138 L. theobromae isolates to difenoconazole ranged from 0.01 to 13.72 µg/mL. The frequency of difenoconazole sensitivity formed a normal distribution curve when the outliers were excluded. Difenoconazole showed positive cross-resistance only with the DMI tebuconazole but not with non-DMI fungicides carbendazim, pyraclostrobin, fludioxonil, bromothalonil, or iprodione. Some multifungicide-resistant isolates of L. theobromae were found. Two amino acid substitutions (E209k and G207A) were found in the CYP51 protein, but they were unlikely to be related to the resistance phenotype. There was no alteration in the promoter region of the CYP51 gene. However, difenoconazole significantly increased the expression of the CYP51 gene in the resistant isolates compared to the susceptible isolates. These results are vital to develop effective mango disease management strategies to avoid the development of further resistance.
... Yet, after decades of such input-intensive farming, many negative effects of pesticides are evident: contamination of groundwater and rivers (Patinha et al. 2018), air pollution (ANSES 2020), human health problems (Weber et al. 2019), and loss of biodiversity (Cadillo-Benalcazar et al. 2020). Moreover, the efficiency of these intensive agricultural practices has decreased due to the increase of pest resistances (Aves et al. 2020), fast pathogen adaptations (Leroux and Walker 2011), and resistance breakdown of highly resistant cultivars sown on large areas (de Vallavieille-Pope et al. 2012). Currently farmers have to face the removal of numerous pesticides from the market, due to environmental and health risks recently reconsidered. ...
To ensure regular and high yields, current agriculture is based on intensive use of pesticides and fertilizers, which are detrimental to the environment and human health. Moreover, as pest resistance to pesticides increases, and more and more pesticides are taken off the market, national and European policies are becoming powerful drivers to deliver pesticide-free farming systems. Whereas numerous studies have compared organic versus conventional systems, our study assessed, for the first time, the performances of a pesticide-free arable cropping system (No-Pesticide), using synthetic fertilizers, specifically designed to produce high yields and meet environmental goals. This system was compared with an input-based cropping system designed with the same environmental targets (PHEP: productive with high environmental performances) in an 11-year field trial in France (Paris Basin). Banning pesticides did not result in a significant average yield gap (in GJ.ha ⁻¹ .year ⁻¹ or in kg N.ha ⁻¹ .year ⁻¹ ) calculated over the crop sequence. Yet, some crops’ yields significantly decreased, due either to pest damages, or to limited nitrogen nutrition. In the No-Pesticide system, the mycotoxin content of cereal grains was lower than the regulatory threshold, and the average wheat protein content was higher than the required standard for baking. Indirect energy consumption, total greenhouse gas emissions, number of technical operations, nitrogen fertilizer amounts, and treatment frequency indexes were significantly lower compared to the PHEP system. Conversely, results showed significantly higher direct energy consumption, direct greenhouse gas emissions, and number of work hours for weed control. We identify highly effective agricultural strategies to avoid pesticide use (e.g., widely diverse and long crop sequence; introduction of hemp) and pinpoint several technical lock-ins hampering steady production in pesticide-free systems. We argue that more experiments should be undertaken to deliver technical knowledge for managing major or orphan species within pesticide-free systems, and to provide supplementary results, including economic and social performances.
... Again, azole fungicide resistance developed in Z. tritici populations, field performance of several products has been negatively impacted (Clark 2006). Therefore, further changes in susceptibility to various azoles due to the ongoing evolution of novel CYP51 (sterol 14ademethylase) variations (Brunner et al. 2008;Cools and Fraaije 2008;Cools et al. 2011;Leroux and Walker 2011), and guarantee long-term sustainable disease management, new modalities of intervention are urgently required. Moreover, sensitivity in Z. tritici populations has been declining over time (Clark 2006). ...
Septoria tritici blotch or Septoria leaf blotch has been used for long time, but leaf blotch is a correct disease name. Moreover, Lb resistant gene is the correct name, but, not Stb gene. It has sexual and asexual parts on the mycelia, known as heterothallic fungi. Its pathogenic diversity ranged from 40% to 93% and has produced a wide variety of AvrLb6 haplotypes. M. graminicola has a plasmogamy and karyogamy sexual process. The pathogen can use macropycnidiospores, micropycnidiospores, and pycnidia vegetative growths for infection and overwintering. Synthetic M3, Kavkaz-K4500, Synthetic 6×, and TE9111 wheat genotypes have horizontal resistance. Avirulence (Avr) genes in Z. tritici and their matching wheat (R) genes indicate gene for gene mechanisms of resistance. Twenty-two R genes (vertical resistance) have been identified. In both horizontal and vertical resistance, different Lb genes have been broken down due to new Z.tritici virulent gene and currently Lb19 resistant gene is being recommended. Mixing of resistant and susceptible cultivars is also the most effective management strategy. Moreover, different cultural practices and biological control have been proposed. Lastly, different fungicides are also available. However, in developing countries cultivar mixture, isolates diversity, biological control, and epidemic studies have been greatly missed.
... Overexpression of ABC transporterencoding gene atrB is responsible for MDR1 phenotype in B. cinerea and shows considerable resistance towards fludioxonil (osmotic signal transduction inhibitor), cyprodinil (methionine biosynthesis inhibitor) and tolnaftate (squalene epoxidase inhibitor, belonging to sterol biosynthesis inhibitor), while MDR2 phenotype caused by overexpression of MFS transporter-encoding gene mfsM2 is resistant to fenhexamid (ketoreductase inhibitor, belonging to sterol biosynthesis inhibitor), tolnaftate, cycloheximide (protein synthesis inhibitor) and cyprodinil (Kretschmer et al., 2009). MDR induced by increased expression of MFS transporter-encoding gene MgMFS1 has developed resistance to most categories of fungicides including demethylation inhibitors (DMIs, belonging to sterol biosynthesis inhibitor), succinatedehydrogenase inhibitors (SDHIs), the multi-site fungicides, and other unrelated compounds, such as tolnaftate and terbinafine (Leroux and Walker, 2011). An MDR phenotype in S. homoeocarpa associated with overexpression of an efflux transporter ShPDR1 displays resistance to propiconazole (DMI), iprodione (osmotic signal transduction inhibitor) and boscalid (SDHI) (Sang et al., 2015). ...
Plant pathogens can develop multidrug resistance (MDR) through metabolomic and efflux activities. Although MDR has been observed in the field, its mechanisms are yet to be further studied. MDR in Rhizoctonia solani induced by the uncoupler SYP-14288, which involved efflux transporters including ATP binding cassette (ABC) and major facilitator superfamily (MFS) have been reported in our previous study. To confirm this, corresponding genes of the wild-type R. solani X19 and its derived MDR mutant X19-7 were compared through transcriptomics, RNA-Seq data validation, and heterologous expression. Genes encoding six ABC transporters and seven MFS transporters were identified to be associated with MDR and mostly showed a constitutively higher expression in X19-7 than in X19 regardless of SYP-14288 treatment. Eight ABC transporter-encoding genes and eight MFS transporter-encoding genes were further characterized by transferring into Saccharomyces cerevisiae. The sensitivity of transformants containing either ABC transporter-encoding gene AG1IA_06082 and MFS transporter-encoding gene AG1IA_08645 was significantly decreased in responses to fungicides having various modes of action including SYP-14288, fluazinam, chlorothalonil, and difenoconazole, indicating that these two genes were related to MDR. The roles of two genes were further confirmed by successfully detecting their protein products and high accumulation of SYP-14288 in yeast transformants. Thus, ABC and MFS transporters contributed to the development of MDR in R. solani. The result helps to understand the cause and mechanisms that influence the efficient use of fungicide.
... Several amino acid alterations have been correlated with fungicide resistance in Z. tritici. The gradual decrease in sensitivity observed for the DMIs is explained by multiple haplotypes of Z. tritici harboring several target site mutations (Huf et al., 2018;Kildea et al., 2019;Leroux & Walker, 2011). In this study, we investigated the presence of amino acid substitutions using S524T, C-T79N, and C-N86S as markers to evaluate the status of DMI and SDHI resistance, respectively. ...
Zymoseptoria tritici, the causal agent of septoria tritici blotch (STB), is one of the most destructive fungal pathogens on wheat. To control the disease and limit yield losses, farmers rely mainly on uni-site fungicides belonging to two modes of action: demethylase inhibitors (DMIs) and succinate dehydrogenase inhibitors (SDHIs). DMIs have been used extensively for STB control for the last 40 years, resulting in a gradual sensitivity shift, which has been observed worldwide, due to the stepwise accumulation of resistance mutations in the CYP51 gene. SDHI fungicides have been used for just over ten years to control STB. Several mutations conferring resistance to this group have been detected in several countries to varying degrees. Fungicide resistance in Sweden and Denmark has generally been less prominent because of the lower fungicide input and restricted fungicide availability. Microtiter plate assays and allele-specific qPCRs (targeting C-T79N and C-N86S in SdhC and S524T in CYP51) confirmed that the Danish and Swedish Z. tritici populations still remain sensitive to SDHI and DMI fungicides, but that the frequency of resistant isolates is increasing. The target site alterations were further used as markers to evaluate the selection potential imposed by different control strategies in the field. Results indicated that efficacious SDHIs, including fluxapyroxad, imposed a higher selection of resistance mutations compared to less effective SDHIs. Treatments including prothioconazole increased the frequency of S524T more than the ones containing mefentrifluconazole. Splitting the dose into two treatments also selected for more resistant isolates compared to single-treatment strategies. This investigation also tackles the impacts of current anti-resistance strategies, including the use of lower doses, limiting the number of treatments, and mixing and alternating fungicides of a different mode of action under field conditions. These elements are essential to prolong the efficacy of current and future fungicides and delay resistance development in the Z. tritici population.
... MDR is explained as the overexpression of the "major facilitator gene" (MFS1). So far, 3 types of inserts (type I, II, and III) in the promoter region were identified concerning MFS1 overexpression (Leroux and Walker, 2011;Omrane et al., 2017). In the present study, none of these were detected; however, we found one insertion that had not been reported previously. ...
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.
... The in vitro sensitivity assays such as measurement of EC50 is a traditional method to determine fungicide tolerance and its association with CYP51 mutations in Z. tritici known to lead to fungicide resistance (Siah et al., 2010). In previous years, PCR assays combined with RFLP (restriction fragment length polymorphisms) and Sanger sequencing have been used to identify and detect mutations in fungicide gene targets, including the CYP51 and cytochrome b genes in Z. tritici isolates (Zhan et al., 2006, Leroux & Walker, 2011, Estep et al., 2015, Huf et al., 2018, Kildea et al., 2019, Stammler et al., 2008. The performance of these methods is limited due to lack of accuracy, required time scale and costs. ...
The main questions of the project were (i) Can Septoria tritici blotch and Fusarium head blight i wheat be efficiently controlled by microbial biological control agents (BCAs) alone or combined with traditional chemical fungicides (ii) Can the development of fungicide resistance in the Zymoseptoria tritici pathogen population be reduced by the combined use of BCAs and fungicides as compared to repeated chemical fungicide applications? And (iii) can accumulation of mycotoxins in harvested grain be reduced by the use of (BCAs)?
... The strain with S312T/F511S had a higher EC 50 value for prochloraz than the strain with F511S (Fig. 2, Table S3). This is consistent with the suggestion that the population shifts to a higher resistance to specific DMI under the frequent use of the DMI fungicide (Becher and Wirsel, 2012;Brunner et al., 2008;Leroux and Walker, 2011). Although frequencies of F511S and S312T/F511S in Japanese prochloraz-resistant strains have not been clarified yet, monitoring of these mutations contributes to basic understanding behind the conceivable population shifts to a higher resistance to prochloraz. ...
Prochloraz has been used to control Fusarium fujikuroi, the causative pathogen of rice bakanae disease. Linkage analysis of FfCYP51 genes in the progenies obtained from crossing prochloraz moderately resistant and sensitive strains suggested that the FfCYP51B gene is involved in prochloraz resistance. Sequence comparison revealed that the prochloraz-resistant strain had an F511S or S312T/F511S substitution in FfCYP51B compared with the sensitive strains. The contribution of the S312T and F511S substitutions in FfCYP51B to prochloraz resistance was investigated by creating S/F-, T/F-, or T/S- types at 312/511 codons from the S/S-type, which is a natural moderately resistant strain, using a gene-editing technique. T/S exhibited the highest prochloraz resistance, followed by S/S-, T/F-, and S/F-types. These results indicated that the S312T and F511S substitutions in FfCYP51B had a synergistic effect on prochloraz resistance in F. fujikuroi.
... Mutations within the CYP51 coding region, lead to conformational changes referred to as target site resistance (TSR) (Leroux et al., 2007;Stammler et al., 2008;Cools and Fraaije, 2013). Upregulation of CYP51 due to inserts in the promoter or upregulation of membrane-bound transporters and intragenic recombination of CYP51 lead to non-target site resistance (NTSR) (Leroux and Walker, 2011;Cools and Fraaije, 2013;Kildea et al., 2019). ...
Zymoseptoria tritici is the causal agent of Septoria tritici blotch, the most important disease affecting wheat crops and responsible for up to 50% loss in yield. Azoles and SDHIs are the main groups of fungicides used to control the disease. The efficacy of these fungicides has decreased in recent years due to resistance development. Fungicide resistance can be a result of mutations in the target genes, mutations in upstream regulatory elements that result in over-expression of target proteins and the overexpression of transporters that remove toxic compounds from cells. In the current study nanopore sequencing was used to analyse isolates, mock communities and field samples to determine the pathogen population composition related to azole resistance. Despite the presence of sequencing errors, the method was able to effectively differentiate the sequences of different haplotypes present in both mock and field populations for haplotypes that were relatively common in the overall population. However, haplotypes present at a frequency lower than 5% of the total population, could not be accurately distinguished from haplotypes generated through background sequencing errors. The nanopore protocols are rapid and inexpensive, enabling the routine profiling of haplotypes within pathogen populations at the start of the season or between sprays, facilitating the selection of the most appropriate fungicide to control them, yet limit further selection for azole resistance.
... Previous studies have indicated that overexpression of CYP51, ABC transporter, and/or MFS in fungi enhances drug resistance, including DMI-resistance, and thus reduces fungal sensitivity to fungicides, including DMIs [26][27][28]. In our study, one DEG-encoding CYP51 was significantly up-regulated in TJ-NH-51S-4, while one DEG related to ABC transporter and six DEGs related to MFS transporters were all down-regulated. ...
Alternaria alternata botybirnavirus 1 (AaBRV1) was isolated from a strain of Alternaria alternata, causing watermelon leaf blight in our previous research. The effect of AaBRV1 on the phenotype of its host fungus, however, was not determined. In the present study, a novel strain of AaBRV1 was identified in A. tenuissima strain TJ-NH-51S-4, the causal agent of cotton Alternaria leaf spot, and designated as AaBRV1-AT1. A mycovirus AaBRV1-AT1-free strain TJ-NH-51S-4-VF was obtained by protoplast regeneration, which eliminated AaBRV1-AT1 from the mycovirus AaBRV1-AT1-infected strain TJ-NH-51S-4. Colony growth rate, spore production, and virulence of strain TJ-NH-51S-4 were greater than they were in TJ-NH-51S-4-VF, while the sensitivity of strain TJ-NH-51S-4 to difenoconazole, as measured by the EC50, was lower. AaBRV1-AT1 was capable of vertical transmission via asexual spores and horizontal transmission from strain TJ-NH-51S-4 to strain XJ-BZ-5-1hyg (another strain of A. tenuissima) through hyphal contact in pairing cultures. A total of 613 differentially expressed genes (DEGs) were identified in a comparative transcriptome analysis between TJ-NH-51S-4 and TJ-NH-51S-4-VF. Relative to strain TJ-NH-51S-4-VF, the number of up-regulated and down-regulated DEGs in strain TJ-NH-51S-4 was 286 and 327, respectively. Notably, the expression level of one DEG-encoding cytochrome P450 sterol 14α-demethylase and four DEGs encoding siderophore iron transporters were significantly up-regulated. To our knowledge, this is the first documentation of hypervirulence and reduced sensitivity to difenoconazole induced by AaBRV1-AT1 infection in A. tenuissima.
... DMI resistance has been reported in many phytopathogenic fungi, including C. gloeosporioides, 3 C. truncatum, 35 Fusarium fujikuroi, 36 Fusarium asiaticum, 37 Fusarium graminearum, 37,38 Aspergillus fumigatus, 39 Mycosphaerella graminicola, 40,41 Mycosphaerella fijiensis, 42 Venturia inaequalis, 43 Botrytis cinerea, 44 and Cercospora beticola. 45 To date, however, the DMI resistance is elusive in C. acutatum. ...
Colletotrichum acutatum, the main pathogen causing anthracnose on chili worldwide, is controlled by tebuconazole [a sterol C14-demethylation inhibitor (DMI) fungicide, abbreviated as Teb] with excellent efficacy. Our previous study exhibited that all C. acutatum isolates were sensitive to Teb while the Colletotrichum gloeosporioides population had developed resistance to Teb on the same fungicide-pressure selection. Therefore, the assessment of Teb-resistance in C. acutatum is impending. Twenty Teb-resistant (TebR) mutants obtained by fungicide domestication and ultraviolet (UV)-mutagenesis displayed similar fitness compared to parental isolates. Data in the current study exhibited that mutations at CaCYP51A and/or overexpression of CaCYP51s were responsible for Teb-resistance. Furthermore, the deletion mutants ΔCaCYP51A and ΔCaCYP51B played different roles in sensitivities to DMIs. Taken together, this study first reported that mutations at CaCYP51A and/or overexpression of CaCYP51s conferred resistance to Teb in C. acutatum, CaCYP51A and CaCYP51B are functionally redundant, but differentially regulated in DMI resistance.
... Chemically unrelated antifungal compounds have been used as efflux pump substrates in order to test hypotheses about the role of MDR mechanisms and to distinguish different MDR phenotypes caused by enhanced activity of MFS and/or ABC transporters. Triphenyltin chloride, tolnaftate, rhodamine 6G and cycloheximide are amongst the most common efflux pump substrates used as indicators of MDR phenotypes [14,[30][31][32]. In our study, we tested the hypothesis that resistance to DMI and SDHI fungicides detected in PoTl was due to efflux pump mediated MDR mechanism(s) by characterizing the sensitivity to these four antifungal efflux pump substrates. ...
Widespread resistance to QoIs, DMI and SDHIs fungicides has been reported for Brazilian populations of the wheat blast pathogen Pyricularia oryzae Triticum lineage (PoTl). A pre-existing resistance mechanism not associated with target site mutations has been indicated for resistance to DMIs and SDHIs, with strong indication that PoTl has multidrugresistance (MDR). Therefore, the main objective of this study was to test the hypothesis that resistance to DMI and SDHI fungicides detected in PoTl was due to efflux pump mediated MDR mechanism(s) by characterizing the sensitivity to antifungal efflux pump substrates. Four antifungal substrates were tested: tolnaftate (TOL), cycloheximide (CHX), rhodamine 6G (RH6G) and triphenyltin chloride (TPCL). TPCL and RH6G were considered the most relevant indicators for enhanced MDR activity. Among the 16 PoTl isolates tested, 9 were insensitive to TPCL, 1 to TOL, 16 to RH6G and 1 to CHX. The PoTl isolates were grouped into four distinct multidrug resistance phenotypes (MDRPs) based on resistance to combinations of fungicides and antifungal efflux pump substrates. Insensitivity to TPCL, RH6G and or TOL correlated well with DMI insensitivity, but MDR was not associated with SDHI resistance. The identification of multiple MDRP phenotypes associated with DMI resistance in our study warrants further research aimed at revealing the exact mechanisms of multidrug resistance in the wheat blast pathogen, including efflux pumps overexpression via transcriptomic analyses of differentially expressed genes; identification and discovery of mutations associated with changes in promoter regions or transcription factors of efflux transporters associated with multidrug resistance.
... Similarly, MDR strains of Mycosphaerella graminicola from wheat grown in France and UK were identified with increased resistance against DMI fungicides as also with reduced sensitivity to boscalid, tolnaflate and terbinafine, having a frequency of 13% of the collected strains in France (Leroux and Walker 2011). The efflux transporter involved in this case has not been identified, though it is assumed that PDR5 may be associated. ...
Development of fungicide resistance in plant pathogens is a challenging problem that has affected the performance of several site-specific fungicide groups including benzimidazoles, phenylamides, demethylation inhibitors, quinone outside inhibitors and succinate dehydrogenase inhibitors. Over the past years, research efforts have led to a better understanding of the emergence, spread, behaviour, diagnostics and mechanisms of resistance to different groups of fungicides in diverse types of pathogens. Molecular tools have proved handy in rapid detection of resistance that has greatly helped in monitoring the evolution of resistance in pathogen populations. The recently introduced unified system of labeling resistance-associated mutations has made it easier to determine novel changes in amino acids of the target protein. Knowledge of fitness cost of resistance and risk assessment is crucial in developing resistance management guidelines. Discovery of novel modes of action is an important aspect of resistance management. Use of different modes of action including conventional multi-site inhibitors in mixtures with at-risk fungicides still appears relevant to avoid resistance build up. There is a growing focus on the use of biologicals including biocontrol agents as a part of resistance management programmes.
... For example, the interaction of Prothioconazole, with the haem component of the target-site sterol 14α-demethylase (CYP51), was differently from other azoles [4], showing lower resistance but still effective control of some cereal diseases. In Mycosphaerella graminicola, the cause of wheat leaf blotch, azole resistance was shown to different target-site mutations alone, or in combination, generate different cross-resistance patterns [5] and indeed improved the performance of prochloraz [6]. ...
Fungicides have been used for over 200 years to protect plants from damage by fungi, but today fungicidal resistance is very common among potato pathogens and it is difficult to control. The best and intensively studied example is metalaxyl resistance in Pytophthora infestans. Causes are many to get pathogen resistance against the fungicides like intensive use or misuse of it, repeated application of same fungicides, etc. Hence, it is today’s need to find out the different strategies like different cultural practices, use of bio-agents, use of green chemicals, elimination of disease source, etc. to manage this fungicidal resistance. There are also alternative ways like increasing host resistance, use of new molecules, etc. that can be adopted to reduce the risk of fungicidal resistance.
... The DMI or azole fungicides, inhibitors of lanosterol 14α-demethylase in the fungal ergosterol biosynthesis pathway, were introduced in the 1970s and are still widely used for STB control (Clarke, 2006). Resistance toward DMIs occurs either through mutations of the CYP51 gene encoding the target and/or by mutations of its promoter leading to its overexpression (Leroux and Walker, 2011;Cools et al., 2012;Cools and Fraaije, 2013). A third resistance mechanism is based on an increased efflux through the overexpression of the MgMfs1 transporter (Omrane et al., 2015;Omrane et al., 2017;Maë et al. 2020). ...
... For metconazole, the opposite is the case, i.e., Y131H haplotype increased after treatment. Such a lack of cross-resistance for DMIs, even if partial, has already been described for several pathogens, especially Zymoseptoria tritici (Jørgensen et al. 2021;Leroux and Walker 2011). Differential selection of CYP51 haplotypes by different DMIs has also already been demonstrated for other pathogens (Fraaije et al. 2007;Stammler et al. 2008). ...
The generation of single spore isolates is usually the first approach to characterize plant fungal pathogens with respect to their specific resistance mechanisms to fungicides. The isolation procedure can be time-consuming and can carry the risk of contamination, but for fungal species with saprophytic growth (e.g., Zymoseptoria tritici) it is simple to perform in most cases. However, for biotrophic fungi such as Phakopsora pachyrhizi the situation is more difficult. This may be the reason why all published studies on fungicide resistance mechanisms of this fungus have been conducted with populations or monouredinial isolates. Monouredinial isolates, in contrast to single spore isolates, are generated from several spores of one uredinium, and thus, it is not ensured that these isolates are clones and therefore genetically identical. Since only dikaryotic uredospores of P. pachyrhizi have been found in the field so far, the mechanisms of fungicide resistance are more complex than for other pathogens. Therefore, the use of clones as a tool for further characterization is mandatory. We established a reliable method to generate single spore isolates of P. pachyrhizi and demonstrated that the isolates are clonal. For this verification, single spore isolates were used in detached leaf tests (DLTs) and treated with two demethylation inhibitors (DMIs) with different selection pattern. These isolates had different frequency values of three relevant mutations in CYP51. Additionally, a population was included in the DLT that represented a mixture of isolates with the respective CYP51 mutations under the detection limit (< 5%). In contrast to the population in which target site mutations were selected after DMI treatments in an expected manner, the single spore isolates showed no change in their mutation frequencies on untreated or treated leaves. This indicates that these isolates are genetically identical and thus valuable for further elucidation of the complex resistance mechanisms, especially against DMIs, in this fungal species.
... Les moyennes de chaque variété ont été ajustées de l'effet souche. Adaptation aux fongicides des populations de Z. tritici La diversité phénotypique (résistance aux fongicides) a été mesurée pour 4 modes d'action (benzimidazoles, strobilurines, triazoles et SDHI), en utilisant la méthode sur milieu solide (distinction des phénotypes résistants par doses discriminantes) et la nomenclature décrites dansLeroux et Walker (2011). Deux types de tests ont été menés : De tests en « bulk » réalisés sur 60 populations, Des tests sur 1493 souches isolées et monosporées.Les analyses microbiologiques effectuées pour caractériser les souches isolées de symptômes de type septoriose sur BT et BD et présentes dans les bassins de culture Nord, Sud-Ouest et Sud-Est ont permis d'isoler seulement l'espèce Zymoseptoria tritici. ...
La septoriose, causée par Zymoseptoria tritici et Parastagonospora nodorum est l’une des principalesmaladies des céréales à paille dans le monde. En France, cette maladie peut entrainer des pertes derendements jusqu’à 50% pour le blé tendre. Ces pertes sont principalement causées par Z. tritici.Cependant, très peu d’informations sont disponibles pour les épidémies de septoriose sur blé dur qui ontaugmenté en fréquence ces dernières années. Dans ce projet, une étude pluriannuelle a été menée pourcaractériser le complexe d’espèces de la septoriose du blé dur, étudier la structuration génétique despopulations selon l’hôte, évaluer la spécificité d’hôte, caractériser la sensibilité variétale et évaluer larésistance des populations aux fongicides, dans l’objectif d’apporter des solutions efficaces pour luttercontre cette maladie. Grâce à un large échantillonnage et différentes approches méthodologiques, laprésence significative de Z. tritici et P. nodorum dans les feuilles symptomatiques de blé dur et doncl’importance de prendre en compte ces deux espèces dans la lutte contre la septoriose du blé dur ont étémises en évidence. Les variétés de blé dur sont plus sensibles à P. nodorum que les variétés de blétendre. De même, ces variétés ont également montré, en conditions contrôlées, des niveaux derésistance assez faibles à Z. tritici. Les analyses de génomique et génétique des populations de Z. triticiont montré une structuration génétique des populations en fonction de l’hôte mais également en fonctionde la géographie pour les populations de blé dur. De plus, cette différenciation génétique est égalementassociée à la pathogénicité des isolats. Enfin, nous avons confirmé la généralisation de la résistance auxQoIs (Quinone outside Inhibitor) et benzimidazoles, et l’absence de résistance aux SDHI (SuccinateDeHydrogenase Inhibitor) en 2013. La résistance aux IDM (Inhibiteurs de la DéMéthylation) estgénéralisée dans toutes les populations mais les phénotypes majoritaires diffèrent en fréquence dans leNord et le Sud, ce qui confirme les efficacités contrastées de certains triazoles observées par ailleurs.
... The use of azoles in agriculture leads to resistance selection, in crop pathogens such as Penicillium digitatum destroying citrus or Mycosphaerella graminicola prevalent on wheat crops (Leroux and Walker 2011;de Ramón-Carbonell and Sánchez-Torres 2020), but also in non-target organisms such as A. fumigatus that co-exist in the same environments. This is probably even more favoured for azoles that are single site inhibitors, and above targeting pathogens that have very important spores production and short generation times. ...
... Compared to other single-site inhibitors such as QoIs or SDHIs, resistance development to DMIs is not so fast and substances within the group are slightly different in the development speed and selectivity of resistance (Fraaije et al., 2007). In recent years, reduced sensitivity to the DMIs have already been reported for the wheat pathogen Zymoseptoria tritici (Heick et al., 2017a(Heick et al., , 2017bLeroux & Walker, 2011). In the study by Rehfus et al. (2019), 12 different amino acid alterations in the coding region of the CYP51 enzyme of R. collo-cygni were found. ...
Winter barley (Hordeum vulgare L.) is the third most cultivated crop after corn and wheat in Austria but one of the most challenging for disease control. The foliar pathogen Ramularia collo-cygni B. Sutton and J.M. Waller, causing Ramularia leaf spots (RLS), is one of the most important diseases in barley. In the recent years, control has only been achieved using fungicide mixtures including the multi-site inhibitor chlorothalonil, however this compound is totally banned in the EU. The objective of this study was to assess fungicide dose-rates and spray mixtures for RLS control. Furthermore, a field monitoring within the main barley growing areas of Austria was carried out, to analyse the current resistance situation to DMI and SDHI fungicides, which are still the backbone in RLS control. The results indicate that only the mixture with chlorothalonil achieved a good RLS control. Prothioconazole or benzovindiflupyr (alone or additively) decrease the severity of RLS but increase the local frequency of Cyp51 and sdhC mutations, especially the high dose rates. Based on a low Cyp51 mutation frequency of 16% in untreated control this frequency increased over 3.8 times following an application with 300 g ha−1 prothioconazole. The cumulative-sdhC mutations were even more increased after an application with benzovindiflupyr. This study showed that Ramularia collo-cygni is present in 91% of barley fields presented in this field survey. Widespread use of chlorothalonil fungicide maintained a low to moderate mutation frequency (Cyp51-I325T, Cyp51-I328L, sdhC-H146R and sdhC-H153R) in Austrian barley regions with no increase between 2017 and 2019.
... The DMI or azole fungicides, inhibitors of lanosterol 14α-demethylase in the fungal ergosterol biosynthesis pathway, were introduced in the 1970s and are still widely used for STB control (Clarke, 2006). Resistance toward DMIs occurs either through mutations of the CYP51 gene encoding the target and/or by mutations of its promoter leading to its overexpression (Leroux and Walker, 2011;Cools et al., 2012;Cools and Fraaije, 2013). A third resistance mechanism is based on an increased efflux through the overexpression of the MgMfs1 transporter (Omrane et al., 2015;Omrane et al., 2017;Maë et al. 2020). ...
Zymoseptoria is a major fungal pathogen of wheat, responsible for the Septoria Tritici Blotch (STB) disease. Recently, STB has been the subject of intensive molecular studies. Notably, massive transcriptomic analyses have helped to explore this particular bi-phasic (asymptomatic/necrotrophic) infection process. Cytological analyses have also improved our understanding of the asymptomatic phase. These advances suggest that Zymoseptoria behaves as a hemi-biotrophic fungus, acting like an endophyte during its asymptomatic phase. STB is still difficult to control. The emergence of fungicide-resistant isolates has reduced the efficacy of many fungicides requiring the development of novel fungicides and methods to counteract/reduce fungicide resistance. Likewise, because Stb-resistant wheat cultivars have all been successively defeated by virulent isolates, there is a need to identify new resistance genes in wheat, and to develop better disease resistance management methods (pyramiding, mixture/alternation) to sustainably control this pathogen.
Fungicide resistance management principles recommend that farmers avoid splitting the total dose applied of a fungicidal mode of action (MoA) across multiple applications per season (‘dose splitting’). However, dose splitting may sometimes be needed to make another proven resistance management tactic—application in mixture with a different MoA—practically achievable, especially in cases where there are limited MoAs available for disease control. Variable effects of dose splitting on selection for resistance have been observed in field experiments, and its effect on selection for partial resistance in fungal pathogens is not well studied. An improved understanding of whether the effect of dose splitting depends on fungicide properties and the type of fungicide resistance is required. We developed a compartmental epidemiological model of Septoria leaf blotch (STB) ( Zymoseptoria tritici ) to investigate the effect of dose splitting on selection for both complete and partial target‐site and non‐target‐site resistance. To solely measure the effects of dose splitting, we restricted the analysis to solo fungicide application (solo use is not recommended in practice). Our results show variable effects of dose splitting: in general, it increased the selection for both target‐site and non‐target‐site resistance. Within the range of dose–response parameters expected for commercial fungicides, dose splitting increased the selection most for partial resistance mechanisms that result in a reduction in fungicide efficacy at low fungicide concentrations but not at high concentrations. We predict that dose splitting of a succinate dehydrogenase inhibitor (SDHI) fungicide (solo) will increase selection for target‐site and non‐target‐site resistance by between 20% and 35%, respectively.
Grain production serves as an important strategic resource of the Russian Federation, it is a fundamental branch of agricultural production. In order to get a high and stable yield, it is necessary to carry out protective measures for crops against various diseases. In recent years, leaf-stem diseases of grain crops stand out as the most harmful ones in agrocoenoses. They significantly decrease crop yield. Not only do they rapidly spread around multiple regions of the Russian Federation, but they encompass other grain-producing countries as well. Zymoseptoria tritici is a dangerous fungal phytopathogen that causes Septoria blotches among wheat, triticale, barley and rye. Within several decades, some significant progress has been made in the process of genetic control of wheat resistance to Z. tritici. However, due to the presence of favorable weather conditions contributing to the development of fungal infections, in order to prevent crop loss together with decrease in the quality of agricultural produce, from one to several fungicide treatments have to be implemented. Russian and foreign scientists have noted a tendency of Z. tritici to increase rersistance to some fungicides, which poses a problem with the successful implementation of efficient plant protection measures. Such classes as triazoles and strobilurins are no exception, and according to the FRAC rating, the risk of developing resistance to them is assessed as medium in the former and high in the latter, accordingly. Increasing problems caused by fungicide resistance in Z. tritici populations pose a threat to further wheat production. The purpose of the present research is to analyze modern literature data on the emergence of resistance to fungicides from the chemical classes of triazoles and strobilurins in Z. tritici. The given review examines the genetic mechanisms of resistance that appear in the phytopathogen; examples of monitoring studies of fungal resistance in various countries are provided alongside with practical recommendations on the implementation of anti-resistance strategies. The success of creating such strategies is impossible without knowledge of the pathogen population structure, cultivar resistance, regional agro-ecological peculiarities of the pathogen development and crop cultivation or the biological commercial and economic efficiency of protection means and methods.
Alternaria species are fungal pathogens that can infect maize, causing leaf blight disease and significant economic losses. This study aimed to determine the baseline sensitivity to prochloraz of A. alternata isolates obtained from diseased maize leaves collected from Heilongjiang province by assessing the half-maximal effective concentration (EC50) values. The EC50 values of prochloraz ranged from 0.0550 µg/mL to 2.3258 µg/mL, with an average of 0.9995 ± 0.5192 µg/mL. At EC50 (1.2495 µg/mL) and 2EC50 (2.4990 µg/mL), prochloraz increased the number of mycelial offshoots, disrupted the cell membrane integrity of conidia and mycelia, and resulted in a reduced ergosterol content in the mycelia. Prochloraz significantly affected the mycelial cell membrane permeability and increased the malondialdehyde (MDA) content and superoxide dismutase (SOD) activity. No cross-resistance was detected between prochloraz and other fungicides. These data demonstrate that prochloraz is a promising fungicide for managing maize leaf blight caused by A. alternata and provide novel insights into understanding the mechanism of prochloraz toxicity against A. alternata isolates.
Septoria tritici blotch (STB), caused by the pathogen Zymoseptoria tritici, is one of the most devastating diseases of wheat worldwide. Foliar disease management relies mainly on the application of fungicides. The demethylation inhibitors (DMIs) have been and continue to be one of the core components in STB control. A continuous shift of the Z. tritici population toward reduced sensitivity to DMIs is known for several years. This shift is mainly associated with the accumulation of target site mutations in the CYP51 gene, resulting in distinct CYP51 haplotypes, but also with CYP51 overexpression and increased efflux activity. The occurrence and distribution of the different DMI adaptation mechanisms were monitored in 2021 and compared in relation to previously published results from a survey in 2016 and 2017. In 2021, 54 different CYP51 haplotypes were identified across Europe; however, 15 CYP51 haplotypes represented 90% of the population. The frequency of different CYP51 haplotypes is dependent and variable between countries, with more complex CYP51 haplotypes found in Ireland and the United Kingdom carrying mostly the alteration S524T. In Germany, France, and Poland, a diverse pattern of moderately adapted CYP51 haplotypes together with complex haplotypes carrying S524T was identified, whereas the least complex haplotypes were found in the Eastern- and Southern-European countries. A comparison in the frequency of the different haplotypes for Germany, Poland, France, Ireland and the United Kingdom from 2016 to 2021 revealed a decrease in the frequency of less and moderately adapted haplotypes and an increase in the frequency of complex CYP51 haplotypes carrying the S524T alteration indicating an ongoing shift in DMI sensitivity. An increase in the occurrence and further distribution of CYP51 overexpression based on the ~ 120 bp promotor insert was found from 2017 to 2021. The frequency was shown to vary between countries, with higher frequencies found for example in Poland, Germany and France compared to the United Kingdom and Ireland. An increase in the frequency of increased efflux activity based on the 519 bp insert in the MFS1 promotor was mainly found in Ireland in 2021. In other countries, a stable situation or slight increase in the frequency of this insert was detected. In those countries, the frequency of increased efflux was found at lower levels comparable to findings from 2017. Altogether, the results imply that underlying mechanisms for DMI adaptation are getting more complex and diverse. Regular monitoring to understand the DMI adaptation mechanisms is crucial to implement appropriate anti-resistance strategies in order to slow down further shifts in sensitivity.
The use of pesticides in agriculture inevitably causes their amassment in food and different environmental compartments, mostly agroecosystems. Microbial enzymes could be a practical and eco-friendly option for efficient soil decontamination thanks to their diversity, high reaction and substrate specificities, and high catalytic activity. The major enzymatic activities involved in pesticide biodegradation include hydrolases (esterases, phosphotriesterases, OpdA, OpdB), oxidoreductases (oxygenases, laccases, peroxidases), transferase (glutathione S-transferase), and lyase (enolases). However, these enzymes can be subjected to inhibition by different compounds present in the environmental media. This chapter provides insight on the research works using microbial catabolic enzymes to partially (biotransformation) or totally (mineralization) degrade and counter the toxicity of some of the most currently applied pesticides in soil.
Background
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.
Results
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.
Conclusions
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.
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Botrytis cinerea is a broad-host-range necrotrophic phytopathogen responsible for serious diseases in leading crops worldwide. The novel sterol 14α-demethylase inhibitor (DMI) pyrisoxazole was recently registered for the control of tomato gray mold caused by B. cinerea in China. One hundred fifty-seven isolates of B. cinerea were collected from tomato greenhouses in fourteen cities of Liaoning Province from 2016 to 2021 and examined for sensitivity to pyrisoxazole, with a mean EC50 of 0.151 µg/mL. Three highly resistant isolates, XD-5, DG-4 and GQ-3, were screened, and the EC50 values were 0.734, 0.606 and 0.639 µg/mL with corresponding resistance factors of 12.88, 10.63 and 11.21, respectively. Compared to field-sensitive strains, the highly resistant isolate XD-5 exhibited fitness defects in traits including mycelial growth, conidial production, and pathogenicity, but the DG-4 and GQ-3 did not experience fitness costs. Positive cross-resistance was observed only between pyrisoxazole and the DMIs tebuconazole and prochloraz but not between pyrisoxazole and the none-DMIs iprodione, procymidone, pyrimethanil, fludioxonil, fluazinam and fluopyram. Sequence alignment of the CYP51 gene indicated that three point mutations were observed in the highly resistant mutant, namely, V24I in XD-5, G461S in GQ-3 and R464K in DG-4. When exposed to pyrisoxazole, the induced expression levels of the ABC transporter AtrD and MFS transporter Mfs1 increased in the resistant isolates compared to the sensitive isolates, while the expression level of the CYP51 gene did not change significantly. Molecular docking suggested that the G461S and R464K mutations both led to a decrease in the binding energy between CYP51 and pyrisoxazole, while no change was found with the V24I mutation. Thus, two point mutations in the CYP51 protein combined with induced expression of the Mfs1 and AtrD genes appeared to mediate the pyrisoxazole resistance of the highly resistant mutants DG-4 and GQ-3, while the overexpression of the Mfs1 and AtrD genes was responsible for the highly resistant mutant XD-5.
Tebuconazole, a demethylation-inhibitor (DMI) fungicide, is widely used on watermelon and muskmelon because it is inexpensive and has been effective against Stagonosporopsis citrulli, the primary causal agent of gummy stem blight in the southeastern United States. Most isolates (94% of 251) collected from watermelon in South Carolina in 2019 and 2021 were moderately resistant to tebuconazole at 3.0 mg/liter in vitro. Ninety isolates were identified as S. citrulli; no isolates of S. caricae were found in this study. On watermelon and muskmelon seedlings treated with the field rate of tebuconazole, sensitive, moderately resistant, and highly resistant isolates were controlled 99%, 74%, and 45%, respectively. In vitro, tebuconazole-sensitive isolates were moderately resistant to tetraconazole and flutriafol but sensitive to difenoconazole and prothioconazole, while highly resistant isolates were highly resistant to tetraconazole and flutriafol and moderately resistant to difenoconazole and prothioconazole. On watermelon seedlings treated with field rates of five DMI fungicides in the greenhouse, severity of gummy stem blight did not differ significantly from the nontreated control when seedlings were inoculated with a highly resistant isolate, while severity was lower with all DMIs on seedlings inoculated with a sensitive isolate, although severity was greater with tetraconazole than with the other four DMIs. In the field, tetraconazole rotated with mancozeb did not reduce severity of gummy stem blight caused by a tebuconazole-sensitive isolate when compared to the nontreated control, while the other four DMIs did. With a highly resistant isolate, all DMIs rotated with mancozeb reduced severity of gummy stem blight compared to the nontreated control, but severity with tetraconazole and tebuconazole was greater than with mancozeb alone, and severity with flutriafol, difenoconazole, prothioconazole, and difenoconazole plus cyprodinil did not differ from mancozeb applied alone. Results from in vitro, greenhouse, and field experiments with the five DMI fungicides were highly correlated with each other. Thus, determining relative colony diameters with a discriminatory dose of 3 mg/liter tebuconazole is an effective way to identify DMI-resistant isolates of S. citrulli highly resistant to tebuconazole.
Acclimatized fungal diseases in wheat crops pose a severe threat to world wheat production. Zymoseptoria tritici, which causes Septoria tritici blotch (STB), is known as a devastating latent necrotrophic fungal pathogen that causes a significant yield loss, especially in Europe and in other wheat-growing countries. This fungal pathogen developed rapid resistance against new and old fungicides and has become a major threat to future wheat crops. Strobilurines and Azoles are the main group of contact and systemic fungicides along with succinate dehydrogenase inhibitors with reliable results to control the Z. tritici all over the world. However, many reports from Europe described the presence of resistance in Z. tritici against these fungicides. In the present study, a total of 102 isolates of Z. tritici were collected from five regions (Central Anatolia, Southeastern Anatolia, Marmara, Aegean, and the Mediterranean) of 19 provinces of Turkey to check the presence of target-site mutation at a molecular level to these two main fungicides. Azole target-site mutation in the CYP51 gene was checked by sanger sequencing while the strobilurin resistance mutation in the Cytb gene was confirmed by using PCR–RFLP using the FnuHI restriction enzyme. The obtained results from the two genes revealed some target-site mutations, however no known amino acid changes that cause resistance against these two main group fungicides in Turkish isolates have been detected. We concluded that the severe infection of STB pathogen in Turkish wheat growing may be still manageable with these fungicides although the resistance tests must be conducted for certainity.
Fungicides play an important role in wheat blast management in Brazil. To determine whether the reported patterns of fungicide resistance are present in Pyricularia oryzae populations from wheat and signal grass grown in Minas Gerais state, Brazil, we characterized the sensitivity of a contemporary collection (2018 and 2019) of 64 fungal isolates to seven fungicides belonging to DMI (tebuconazole and epoxiconazole), QoI (azoxystrobin and pyraclostrobin and SDHI (bixafen, fluxapyroxad and benzovindiflupyr) groups. We further assessed if the differential sensitivity of selected isolates affected disease control using commercial doses of the fungicides applied protectively (prior to inoculation) on the heads of potted plants. Despite founding relatively low levels of in vitro sensitivity to some of the fungicides (e.g. azoxystrobin, fluxapyroxad and bixafen), control efficacies (percent reductions in head blast severity) were significantly lower than the untreated plant and not affected by the sensitivity of the strain to all fungicides but azoxystrobin. The levels of head blast control for some of the fungicides (>70%) in the greenhouse were similar to or higher than previously reported in the field. The molecular data for a set of isolates representing different fungicide sensitivity levels were generally not predictive of the phenotype. The wheat‐infecting isolates were less sensitive than signal grass‐infecting isolates to all fungicides including azoxystrobin. Our study confirms previous reports of low sensitivity to some fungicides, but it also suggests options among site‐specific fungicides, most notably tebuconazole and benzovindiflupyr, that can still be used under an anti‐resistance strategy such as co‐formulation with multi‐site fungicides.
Azole drugs target fungal sterol biosynthesis and are used to treat millions of human fungal infections each year. Resistance to azole drugs has emerged in multiple fungal pathogens including Candida albicans, Cryptococcus neoformans, Histoplasma capsulatum, and Aspergillus fumigatus. The most well-studied resistance mechanism in A. fumigatus arises from missense mutations in the coding sequence combined with a tandem repeat in the promoter of cyp51A, which encodes a cytochrome P450 enzyme in the fungal sterol biosynthesis pathway. Filamentous members of Ascomycota such as A. fumigatus have either one or two of three Cyp51 paralogs (Cyp51A, Cyp51B, and Cyp51C). Most previous research in A. fumigatus has focused on Cyp51A due to its role in azole resistance. We used the A. fumigatus Cyp51A protein sequence as the query in database searches to identify Cyp51 proteins across Fungi. We found 435 Cyp51 proteins in 295 species spanning from early-diverging fungi (Blastocladiomycota, Chytridiomycota, Zoopagomycota and Mucormycota) to late-diverging fungi (Ascomycota and Basidiomycota). We found these sequences formed 4 major Cyp51 groups: Cyp51, Cyp51A, Cyp51B, and Cyp51C. Surprisingly, we found all filamentous Ascomycota had a Cyp51B paralog, while only 50% had a Cyp51A paralog. We created maximum likelihood trees to investigate the evolution of Cyp51 in Fungi. Our results suggest Cyp51 is present in all fungi with three paralogs emerging in Pezizomycotina, including Cyp51C which appears to have diverged from the progenitor of the Cyp51A and Cyp51B groups.
Stem-end rot (SER) caused by Lasiodiplodia theobromae is one of the most critical diseases of mango in China. The demethylation inhibitor fungicide prochloraz has been widely used in China to control mango diseases. Isolates (n = 139) of L. theobromae were collected in 2019 from six mango-producing regions in Hainan Province, China. The fungicide sensitivity of L. theobromae isolates to prochloraz revealed that the EC 50 values ranged from 0.0006 to 16.4131 µg/ml. A total of 21 of the 139 isolates were categorized as resistant to prochloraz. The resistant isolates sprayed with prochloraz could not be effectively controlled in detached fruits. The mycelial growth, conidia germination and ability to grow at temperatures ranging from 12–35℃ of resistant isolates decreased, suggesting fitness penalties . The experiment showed that after treatment with 10 µg/ml prochloraz, the content of ergosterol in the mycelia of the sensitive isolate decreased by 80.23%, whereas the resistant strain decreased by only 57.52%. The damages of membranes in the sensitive isolates were more serious than for resistant isolates. The target gene CYP51 and the ATP-binding cassette subfamily ABCG gene were cloned, but no mutation was found. When treated with prochloraz, the expression of CYP51 and ABCG in resistant isolates was significantly higher than those in the sensitive isolates. Thus, induced expression of its target gene combined with the induction of expression drug efflux transporters appeared to mediate the prochloraz resistance of L. theobromae.
Alternaria alternata is among the most common fungi in our environments, such as indoor facilities, the soil, and outdoor air. It can cause diseases in >100 crop and ornamental plants.
Wheat stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), is a destructive disease of wheat that seriously threatens production safety in wheat-producing areas worldwide. In China, the disease has been largely controlled with fungicide triadimefon. Although high levels of fungicide resistance in other fungal pathogens have been reported, failure to control Pst with any fungicides has seldomly been reported and fungicide sensitivity of Pst has not been evaluated in China. The distribution of triadimefon-resistant Pst isolates was investigated in the present study. The baseline sensitivity of 446 Pst isolates across the country to triadimefon was determined, and the concentration for 50% of maximal effect (EC 50 ) showed a unimodal distribution curve, with a mean value of 0.19 μg mL ⁻¹ . The results indicated a wide range of sensitivity to triadimefon, with more insensitive isolates collected from Pst winter-increasing areas and northwest over-summering areas, whereas more sensitive isolates were collected from southwest over-summering areas and epidemic areas of Xinjiang and Tibet. The majority of the tested Pst isolates were sensitive to triadimefon; only 6.79% had developed varying degrees of resistance. Characterization of parasitic fitness revealed that the triadimefon-resistant isolates exhibited strong adaptive traits in urediniospore germination rate, latent period, sporulation intensity, and lesion expansion rate. Positive cross-resistance was observed between triadimefon and tebuconazole or hexaconazole, but not between pyraclostrobin or flubeneteram. The point mutation Y134F in the 14α-demethylase enzyme (CYP51) was detected in triadimefon-resistant isolates. A molecular method (Kompetitive Allele Specific PCR) was established for the rapid detection of Y134F mutants in the Pst population. Two genotypes with one point mutation Y134F conferred resistance to triadimefon in Pst. The risk of resistance to triadimefon in Pst may be low to moderate. This study provided important data for establishment of high throughput molecular detection methods, fungicide resistance risk management, and the development of new target fungicides.
Powdery mildew, caused by the ascomycete Podosphaera leucotricha, is an endemic disease found wherever apples are grown that negatively impacts both tree vigor and fresh market yield. In the absence of durable host resistance, chemical management is the primary means of disease control. Demethylation inhibitor (DMI) fungicides are widely used to manage apple powdery mildew, but members within this fungicide class have been observed to differ in efficacy with respect to disease control. Moreover, debate exists as to the optimal timing of DMI fungicide applications for management of apple powdery mildew. In this regard, the goal of this study was to determine the best-use practices for DMI fungicides to manage apple powdery mildew in New York State. Multi-year trials were conducted to evaluate the potential differential efficacy performance of four common DMI fungicides, as well as additional trials to assess optimal application timing. In all years, we observed that treatments of flutriafol and myclobutanil consistently had the lowest incidences of powdery mildew compared to difenoconazole and fenbuconazole. In the 2018 and 2021 trials, the newly registered mefentrifluconazole was more comparable to the difenoconazole program with respect to powdery mildew disease incidence. We hypothesize that differences in DMI efficacy may be due to each fungicides’ water solubility and lipophilicity characteristics, and thus their ability to move systemically in the host or more easily penetrate the surface of germinating conidia. Applications timed between petal fall and first cover resulted in the lowest incidence of powdery mildew on terminal leaves of apple shoots compared to applications timed prior to petal fall. These observations are contrary to previous studies conducted in regions with differing climates. We also found that the incidence of secondary powdery mildew observed two weeks after petal fall was influenced by applications of DMI fungicides during the previous season. For example, management programs consisting of applications of flutriafol or myclobutanil in the prior season tended to have lower incidence of apple powdery in the following spring, presumably owing to reductions in overwintering inoculum. Despite reports of DMI resistance in other apple pathosystems, the DMI fungicide class is still relevant for the successful management of apple powdery mildew in New York State.
Azole-resistant mutants of Botrytis cinerea have a multidrug resistance phenotype since they exhibit cross-resistance to unrelated chemicals. These mutants also display resistance to the new azole fungicide oxpoconazole. Resistance to oxpoconazole is associated with decreased accumulation of the fungicide, which is the result of energy-dependent efflux mediated by fungicide transporters. The ATP-binding cassette (ABC) transporter BcatrB (B. cinerea ABC transporter B), involved in efflux of phenylpyrrole fungicides, has no major role in efflux of oxpoconazole since accumulation of the fungicide by a replacement mutant of BcatrB showed a transient accumulation pattern similar to that of the wild-type isolate. The putative role of 10 additional ABC and 3 Major facilitator superfamily transporters in efflux of oxpoconazole was investigated by expression analysis of the corresponding genes. The basal transcription level o
The recent decline in the effectiveness of some azole fungicides in controlling the wheat pathogen Mycosphaerella graminicola has been associated with mutations in the CYP51 gene encoding the azole target, the eburicol 14α-demethylase (CYP51), an essential enzyme of the ergosterol biosynthesis pathway. In this study, analysis of the sterol content of M. graminicola isolates carrying different variants of the CYP51 gene has revealed quantitative differences in sterol intermediates, particularly the CYP51 substrate eburicol. Together with CYP51 gene expression studies, these data suggest that mutations in the CYP51 gene impact on the activity of the CYP51 protein.
This review provides an overview of members of the ATP-binding cassette (ABC) and major facilitator superfamily (MFS) of transporters identified in filamentous fungi. The most common function of these membrane proteins is to provide protection against natural toxic compounds present in the environment of fungi, such as antibiotics produced by other microorganisms. In plant pathogenic fungi, these transporters can also be an important determinant of virulence on host plants by providing protection against plant defence compounds or mediating the secretion of host-specific toxins. Furthermore, they play a critical role in determining base-line sensitivity to fungicides and other antimycotic agents. Overexpression of some of these transporters can lead to the development of resistance to chemically-unrelated compounds, a phenomenon described as multidrug resistance (MDR). This has been observed in a variety of organisms and can impose a serious threat to the effective control of pathogenic fungi.
A survey of fungicide resistance in Mycosphaerella graminicola and Tapesia acuformis, two major pathogens of winter wheat in France, respectively responsible for speckled leaf blotch and eyespot, led to the
characterization of two types of resistant strains to sterol 14α-demethylation inhibitors (DMIs). Most of the strains of M. graminicola collected in France in 1997–1998 were resistant to all DMIs, and only in a few strains was the resistance to several triazoles
associated with increased susceptibility to pyrimidine derivatives (i.e., fenarimol, nuarimol) and triflumizole. On the other
hand, in T. acuformis the most prevalent strains were those which exhibited negative-cross resistance between DMIs. In both fungi such a phenomenon
could be related to changes in cytochrome P450 sterol 14α-demethylase, the target site of these fungicides. For Botryotinia fuckeliana, the causal agent of grey mould, the extensive monitoring conducted in French vineyards before the marketing of fenhexamid
revealed the presence of highly resistant strains to this promising botryticide (only in tests involving mycelial growth measurements).
Negative cross-resistance to edifenphos and several sterol biosynthesis inhibitors, such as prochloraz and fenpropimorph,
was observed in fenhexamid resistant strains. Synergism of the antifungal action of fenhexamid by cytochrome P450 inhibitors,
such as the DMI fungicides, was only recorded in fenhexamid resistant strains. These data and those previously obtained with
edifenphos resistant strains of Magnaporthe grisea (rice blast pathogen) suggest that in fenhexamid resistant strains of B. fuckeliana the same cytochrome P450 monooxygenase could be involved in detoxification of fenhexamid and activation of edifenphos.
Seven phenotypes were identified among field isolates of Tapesia yallundae and Tapesia acuformis when tested for susceptibility to the benzimidazole fungicides carbendazim and thiabendazole and the N-phenylcarbamates diethofencarb, MDPC, and swep. PCR was used to amplify and sequence 627-bp fragments of the β-tubulin gene from 32 Tapesia spp. strains representing the seven field phenotypes and from six T. yallundae laboratory mutants. All benzimidazole-resistant field isolates analyzed had a punctual allelic mutation at codon 198, 200, or 240 of the β-tubulin gene fragment. Codon 198, which encodes glutamic acid in benzimidazole-sensitive strains (resistant to N-phenylcarbamates), was converted to a codon for alanine, glycine, lysine, or glutamine in benzimidazole-resistant strains exhibiting increased sensitivity toward the N-chlorophenylcarbamates MDPC and swep; the first two allelic mutations (alanine and glycine) also conferred susceptibility to diethofencarb. In T. yallundae, benzimidazole-resistant phenotypes, which remained resistant to all the tested N-phenylcarbamates, had a tyrosine instead of a phenylalanine at codon 200 or a phenylalanine instead of a leucine at codon 240. In T. acuformis, however, the change of a phenylalanine at codon 200 for a tyrosine conferred a weaker susceptibility to MDPC and swep as well as a reduced resistance to benzimidazoles compared to their T. yallundae counterparts. The same molecular analysis was performed with T. yallundae laboratory mutants obtained after UV mutagenesis and selection on carbendazim or diethofencarb of a former benzimidazole-sensitive or benzimidazole-resistant field strain. We found in two mutants a punctual change at codon 198, replacing the glutamic acid by a glycine or an aspartic acid, but multiple mutations were observed in the four other mutant strains: a double mutation in codon 198 resulting in the substitution of the glutamic acid by a threonine; a mutation at codon 198 (an alanine instead of a glutamic acid) and a mutation at codon 200 (a serine instead of a phenylalanine); a mutation at codon 198 (an alanine instead of a glutamic acid) and a mutation at codon 250 (a phenylalanine instead of a leucine); and one mutant had four codon changes: at codon 179 (a glycine substituting a valine), at codon 185 (a serine replacing an alanine), at codon 190 (an asparagine replacing a histidine), and at codon 198 (an alanine instead of a glutamic acid). We show here that each different phenotype could be correlated with particular mutations at the β-tubulin gene level.
Field strains of Botryotinia fuckeliana were isolated from diseased grape berries which were collected at the harvest, between 1993 and 1997, from French vineyards located in Alsace, Armagnac, Bordeaux, Champagne and Loire Valley. According to the inhibitory effects of fungicides towards spore germination, germ-tube elongation and mycelial growth, several phenotypes could be characterized. Thus, two types of benzimidazole (e.g. carbendazim, thiabendazole)-resistant strains were detected; negative cross-resistance towards diethofencarb, diphenylamine and dicloran was found only in one type. Most dicarboximide (e.g. iprodione, procymidone, vinclozolin)-resistant strains were also weakly resistant to aromatic hydrocarbon fungicides (e.g. chloroneb, dicloran, tolclofos-methyl) but remained sensitive to phenylpyrroles (e.g. fenpiclonil, fludioxonil). However, in some other dicarboximide-resistant strains, resistance was observed either as being restricted to dicarboximides or as extending weakly to phenylpyrroles. Towards anilinopyrimidines (e.g. cyprodinil, mepanipyrim, pyrimethanil), three resistant phenotypes have been identified. In the most resistant one, resistance was restricted to anilinopyrimidines, whereas in the others, resistance extended to various other groups of fungicides including dicarboximides, phenylpyrroles, sterol biosynthesis inhibitors (e.g. tolfanate, prochloraz, tebuconazole) and the hydroxyanilide derivative, fenhexamid. According to the pattern of cross-resistance, two phenotypes were characterized within these multifungicide-resistant strains. In vitro tests conducted with fenpropimorph, fenpropidin and spiroxamine, revealed the existence of strains highly susceptible to these sterol Δ14- reduction inhibitors. This property was correlated with a decreased sensitivity towards fenhexamid.
The recent decrease in the sensitivity of the Western European population of the wheat pathogen Mycosphaerella graminicola to azole fungicides has been associated with the emergence and subsequent spread of mutations in the CYP51 gene, encoding the azole target sterol 14α-demethylase. In this study, we have expressed wild-type and mutated M. graminicola CYP51 (MgCYP51) variants in a Saccharomyces cerevisiae mutant carrying a doxycycline-regulatable tetO7-CYC promoter controlling native CYP51 expression. We have shown that the wild-type MgCYP51 protein complements the function of the orthologous protein in S. cerevisiae. Mutant MgCYP51 proteins containing amino acid alterations L50S, Y459D, and Y461H and the two-amino-acid deletion ΔY459/G460,
commonly identified in modern M. graminicola populations, have no effect on the capacity of the M. graminicola protein to function in S. cerevisiae. We have also shown that the azole fungicide sensitivities of transformants expressing MgCYP51 variants with these alterations
are substantially reduced. Furthermore, we have demonstrated that the I381V substitution, correlated with the recent decline
in the effectiveness of azoles, destroys the capacity of MgCYP51 to complement the S. cerevisiae mutant when introduced alone. However, when I381V is combined with changes between residues Y459 and Y461, the function of
the M. graminicola protein is partially restored. These findings demonstrate, for the first time for a plant pathogenic fungus, the impacts
that naturally occurring CYP51 alterations have on both azole sensitivity and intrinsic protein function. In addition, we
also provide functional evidence underlying the order in which CYP51 alterations in the Western European M. graminicola population emerged.
The grey mould fungus Botrytis cinerea causes losses of commercially important fruits, vegetables and ornamentals worldwide. Fungicide treatments are effective for disease control, but bear the risk of resistance development. The major resistance mechanism in fungi is target protein modification resulting in reduced drug binding. Multiple drug resistance (MDR) caused by increased efflux activity is common in human pathogenic microbes, but rarely described for plant pathogens. Annual monitoring for fungicide resistance in field isolates from fungicide-treated vineyards in France and Germany revealed a rapidly increasing appearance of B. cinerea field populations with three distinct MDR phenotypes. All MDR strains showed increased fungicide efflux activity and overexpression of efflux transporter genes. Similar to clinical MDR isolates of Candida yeasts that are due to transcription factor mutations, all MDR1 strains were shown to harbor activating mutations in a transcription factor (Mrr1) that controls the gene encoding ABC transporter AtrB. MDR2 strains had undergone a unique rearrangement in the promoter region of the major facilitator superfamily transporter gene mfsM2, induced by insertion of a retrotransposon-derived sequence. MDR2 strains carrying the same rearranged mfsM2 allele have probably migrated from French to German wine-growing regions. The roles of atrB, mrr1 and mfsM2 were proven by the phenotypes of knock-out and overexpression mutants. As confirmed by sexual crosses, combinations of mrr1 and mfsM2 mutations lead to MDR3 strains with higher broad-spectrum resistance. An MDR3 strain was shown in field experiments to be selected against sensitive strains by fungicide treatments. Our data document for the first time the rising prevalence, spread and molecular basis of MDR populations in a major plant pathogen in agricultural environments. These populations will increase the risk of grey mould rot and hamper the effectiveness of current strategies for fungicide resistance management.
Molecular mechanisms that account for variation in base-line sensitivity to azole fungicides were examined in a collection of twenty field isolates, collected in France and Germany, of the wheat pathogen Mycosphaerella graminicola (Fuckel) Schroeter. The isolates tested represent the wide base-line sensitivity to the azole fungicide tebuconazole described previously. The isolates were cross-sensitive to other azoles tested, such as cyproconazole and ketoconazole, but not to unrelated chemicals like cycloheximide, kresoxim-methyl or rhodamine 6G. Progenies from a genetic cross between an isolate with an intermediate and a high sensitivity to azoles displayed a continuous range of phenotypes with respect to cyproconazole sensitivity, indicating that variation in azole sensitivity in this haploid organism is polygenic. The basal level of expression of the ATP-binding cassette transporter genes MgAtr1-MgAtr5 from M graminicola significantly varied amongst the isolates tested, but no clear increase in the transcript level of a particular MgAtr gene was found in the less sensitive isolates. Cyproconazole strongly induced expression of MgAtr4, but no correlation between expression levels of this gene and azole sensitivity was observed. One isolate with intermediate sensitivity to azoles over-expressed CYP51, encoding cytochrome P450 sterol 14-demethylase from M graminicola. Isolates with a low or high sensitivity to azoles were tested for accumulation of cyproconazole, but no clear correlation between reduced accumulation of the fungicide in mycelium and sensitivity to azoles was observed. Therefore, differences in accumulation cannot account exclusively for the variation in base-line sensitivity of the isolates to azoles. The results indicate that multiple mechanisms account for differences in base-line sensitivity to azoles in field isolates of M graminicola. Copyright © 2003 Society of Chemical Industry.
Sterol 14α-demethylase (14DM, the CYP51 family of cytochrome P450) is an essential enzyme in sterol biosynthesis in eukaryotes.
It serves as a major drug target for fungal diseases and can potentially become a target for treatment of human infections
with protozoa. Here we present 1.9 Å resolution crystal structures of 14DM from the protozoan pathogen Trypanosoma brucei, ligand-free and complexed with a strong chemically selected inhibitor N-1-(2,4-dichlorophenyl)-2-(1H-imidazol-1-yl)ethyl)-4-(5-phenyl-1,3,4-oxadi-azol-2-yl)benzamide that we previously found to produce potent antiparasitic
effects in Trypanosomatidae. This is the first structure of a eukaryotic microsomal 14DM that acts on sterol biosynthesis,
and it differs profoundly from that of the water-soluble CYP51 family member from Mycobacterium tuberculosis, both in organization of the active site cavity and in the substrate access channel location. Inhibitor binding does not
cause large scale conformational rearrangements, yet induces unanticipated local alterations in the active site, including
formation of a hydrogen bond network that connects, via the inhibitor amide group fragment, two remote functionally essential
protein segments and alters the heme environment. The inhibitor binding mode provides a possible explanation for both its
functionally irreversible effect on the enzyme activity and its selectivity toward the 14DM from human pathogens versus the human 14DM ortholog. The structures shed new light on 14DM functional conservation and open an excellent opportunity
for directed design of novel antiparasitic drugs.
Pathogens must be able to overcome both host defenses and antimicrobial treatment in order to successfully infect and maintain colonization of the host. One way fungi accomplish this feat and overcome intercellular toxin accumulation is efflux pumps, in particular ATP-binding cassette transporters and transporters of the major facilitator superfamily. Members of these two superfamilies remove many toxic compounds by coupling transport with ATP hydrolysis or a proton gradient, respectively. Fungal genomes encode a plethora of members of these families of transporters compared to other organisms. In this review we discuss the role these two fungal superfamilies of transporters play in virulence and resistance to antifungal agents. These efflux transporters are responsible not only for export of compounds involved in pathogenesis such as secondary metabolites, but also export of host-derived antimicrobial compounds. In addition, we examine the current knowledge of these transporters in resistance of pathogens to clinically relevant antifungal agents.
Molecular mechanisms of azole resistance in Candida albicans, including alterations in the target enzyme and increased efflux of drug, have been described, but the epidemiology of the
resistance mechanisms has not been established. We have investigated the molecular mechanisms of resistance to azoles inC. albicans strains displaying high-level fluconazole resistance (MICs, ≥64 μg/ml) isolated from human immunodeficiency virus (HIV)-infected
patients with oropharyngeal candidiasis. The levels of expression of genes encoding lanosterol 14α-demethylase (ERG11) and efflux transporters (MDR1 and CDR) implicated in azole resistance were monitored in matched sets of susceptible and resistant isolates. In addition,ERG11 genes were amplified by PCR, and their nucleotide sequences were determined in order to detect point mutations with a possible
effect in the affinity for azoles. The analysis confirmed the multifactorial nature of azole resistance and the prevalence
of these mechanisms of resistance in C. albicans clinical isolates exhibiting frank fluconazole resistance, with a predominance of overexpression of genes encoding efflux
pumps, detected in 85% of all resistant isolates, being found. Alterations in the target enzyme, including functional amino
acid substitutions and overexpression of the gene that encodes the enzyme, were detected in 65 and 35% of the isolates, respectively.
Overall, multiple mechanisms of resistance were combined in 75% of the isolates displaying high-level fluconazole resistance.
These results may help in the development of new strategies to overcome the problem of resistance as well as new treatments
for this condition.
The ability to develop fungicide resistance was assessed in Monilinia fructicola isolates with different fungicide sensitivity phenotypes by adapting mycelium and conidia to increasing concentrations of selective fungicides and UV mutagenesis. Results showed that adaptation to Quinone outside inhibitor (QoI) fungicide azoxystrobin and sterol demethylation inhibitor (DMI) fungicide propiconazole was more effective in conidial-transfer experiments compared to mycelial-transfer experiments. DMI-resistant (DMI-R) isolates adapted to significantly higher doses of azoxystrobin in both, mycelial- and conidial-transfer experiments compared to benzimidazole-resistant (BZI-R) and sensitive (S) isolates. Adaptation to propiconazole in conidial-transfer experiments was accelerated in BZI-R isolates when a stable, nonlethal dose of 50 microg/ml thiophanate-methyl was added to the selection medium. One of two azoxystrobin-resistant mutants from DMI-R isolates did not show any fitness penalties; the other isolate expired before further tests could be carried out. The viable mutant caused larger lesions on detached peach fruit sprayed with azoxystrobin compared to the parental isolate. The azoxystrobin sensitivity of the viable mutant returned to baseline levels after the mutant was transferred to unamended medium. However, azoxystrobin resistance recovered quicker in the mutant compared to the corresponding parental isolate after renewed subculturing on medium amended with 0.2 and 1 microg/ml azoxystrobin; only the mutant but not the parental isolate was able to adapt to 5 microg/ml azoxystrobin. In UV mutagenesis experiments, the DMI-R isolates produced significantly more mutants compared to S isolates. All of the UV-induced mutants showed stable fungicide resistance with little fitness penalty. This study indicates the potential for QoI fungicide resistance development in M. fructicola in the absence of a mutagen and provides evidence for increased mutability and predisposition to accelerated adaptation to azoxystrobin in M. fructicola isolates resistant to DMI fungicides.
The substrate recognition regions in cytochrome P450 family 2 (CYP2) proteins were inferred by group-to-group alignment of CYP2 sequences and those of bacterial P450s, including Pseudomonas putida P450 101A (P450cam), whose substrate-binding residues have been definitely identified by x-ray crystallography of a substrate-bound form (Poulos T. L., Finzel, B. C., and Howard, A. J. (1987) J. Mol. Biol. 195, 687-700). The six putative substrate recognition sites, SRSs, thus identified are dispersively located along the primary structure and constitute about 16% of the total residues. All the reported point mutations and chimeric fragments that significantly affect the substrate specificities of the parental CYP2 enzymes fell within or overlapped some of the six SRSs. Analysis of nucleotide substitution patterns in closely related members in four subfamilies, CYP2A, 2B, 2C, and 2D, consistently indicated that the SRSs have accumulated more nonsynonymous (amino acid-changing) substitutions than the rest of the sequence. This observation supports the idea that diversification of duplicate genes of drug-metabolizing P450s occurs primarily in substrate recognition regions to cope with an increasing number of foreign compounds.
We investigated the molecular basis of resistance of the obligate biotrophic grape powdery mildew fungus Uncinula necator to sterol demethylation-inhibiting fungicides (DMIs). The sensitivity of 91 single-spore field isolates of U. necator to triadimenol was assessed by using a leaf disc assay. Resistance factors (RF) ranged from 1.8 to 26.0. The gene encoding the target of DMIs (eburicol 14 alpha-demethylase) from five sensitive and seven resistant isolates was cloned and sequenced. A single mutation, leading to the substitution of a phenylalanine residue for a tyrosine residue at position 136, was found in all isolates exhibiting an RF higher than 5. No mutation was found in sensitive or weakly resistant (RF, < 5) isolates. An allele-specific PCR assay was developed to detect the mutation. Among the 91 isolates tested, only isolates with RF higher than 5 carried the mutation. Three of the 19 resistant isolates and all sensitive and weakly resistant isolates did not possess the mutation. The mutation at codon 136 is thus clearly associated with high levels of resistance to triadimenol.
We investigated the effects of combining tacrolimus and azole antifungal agents in azole-resistant strains of Candida albicans by comparing the accumulation of [3H]itraconazole. The CDR1-expressing resistant strain C26 accumulated less itraconazole than the CaMDR-expressing resistant strain C40 or the azole-sensitive strain B2630. A CDR1-expressing Saccharomyces cerevisiae mutant, DSY415, showed a marked reduction in the accumulation of both fluconazole and itraconazole. A CaMDR-expressing S. cerevisiae mutant, DSY416, also showed lower accumulation of fluconazole, but not of itraconazole. The addition of sodium azide, an electron-transport chain inhibitor, increased the intracellular accumulation of itraconazole only in the C26 strain, and not in the C40 or B2630 strains. Addition of tacrolimus, an inhibitor of multidrug resistance proteins, resulted in the highest increase in itraconazole accumulation in the C26 strain. The combination of itraconazole and tacrolimus was synergic in azole-resistant C. albicans strains. In the C26 strain, the MIC of itraconazole decreased from >8 to 0.5 mg/L when combined with tacrolimus. Our results showed that two multidrug resistance phenotypes (encoded by the CDR1 and CaMDR genes) in C. albicans have different substrate specificity for azole antifungal agents and that a combination of tacrolimus and azole antifungal agents is effective against azole-resistant strains of C. albicans.
Isolates of the obligately biotrophic fungus Uncinula necator cluster in three distinct genetic groups (groups I, II, and III). We designed PCR primers specific for these groups in order to monitor field populations of U. necator. We used the nucleotide sequences of the gene that encodes eburicol 14alpha-demethylase (CYP51) and of the ribosomal DNA internal transcribed spacer 1 (ITS1), ITS2, and 5. 8S regions. We identified four point mutations (three in CYP51 and one in ITS1) that distinguished groups I and II from group III based on a sample of 132 single-spore isolates originating from Europe, Tunisia, Israel, India, and Australia. We developed a nested allele-specific PCR assay in which the CYP51 point mutations were used to detect and distinguish groups I and II from group III in crude mildewed samples from vineyards. In a preliminary study performed with samples from French vineyards in which isolates belonging to genetic groups I and III were present, we found that a shift from a population composed primarily of group I isolates to a population composed primarily of group III isolates occurred during the grapevine growing season.
The cytochrome P450 14alpha-demethylase, encoded by the ERG11 (CYP51) gene, is the primary target for the azole class of antifungals. Changes in the azole affinity of this enzyme caused by amino acid substitutions have been reported as a resistance mechanism. Nine Candida albicans strains were used in this study. The ERG11 base sequence of seven isolates, of which only two were azole-sensitive, were determined. The ERG11 base sequences of the other two strains have been published previously. In these seven isolates, 12 different amino acid substitutions were identified, of which six have not been described previously (A149V, D153E, E165Y, S279F, V452A and G4655). In addition, 16 silent mutations were found. Two different biochemical assays, subcellular sterol biosynthesis and CO binding to reduced microsomal fractions, were used to evaluate the sensitivity of the cytochromes for fluconazole and itraconazole. Enzyme preparations from four isolates showed reduced itraconazole susceptibility, whereas more pronounced resistance to fluconazole was observed in five isolates. A three-dimensional model of C. albicans Cyp51p was used to position all 29 reported substitutions, 98 in total identified in 53 sequences. These 29 substitutions were not randomly distributed over the sequence but clustered in three regions from amino acids 105 to 165, from 266 to 287 and from 405 to 488, suggesting the existence of hotspot regions. Of the mutations found in the two N-terminal regions only Y132H was demonstrated to be of importance for azole resistance. In the C-terminal region three mutations are associated with resistance, suggesting that the non-characterized substitutions found in this region should be prioritized for further analysis.
After development of QoI resistance in Mycosphaerella graminicola in intensive European wheat growing areas, demethylation inhibitors (DMIs) have become the most important fungicides for control of this disease. The sensitivity of M. graminicola towards DMIs has been monitored in recent years and a shift in the population to slightly reduced sensitivities in vitro was determined, which has now reached a plateau. As one reason for this shift, mutations in the target protein 14α-demethylase (CYP51) have been discussed, mainly the amino acid exchanges V136A, A379G, I381V, and mutations or deletions at the amino acid positions 459–462 of CYP51. Comparison of the CYP51-haplotypes of 615 isolates from different European regions for their in vitro sensitivity towards the triazole epoxiconazole nevertheless showed that the influence of the CYP51-haplotypes on sensitivity is limited. There was no correlation between in vitro sensitivity or CYP51-haplotype pattern and field performance of epoxiconazole detectable at different trials sites. Equally high levels of efficacy were achieved at sites where the mutations I381V or A379G, i.e. mutations conferring the highest reduction of efficacy in vitro, were dominant as well as where they were less frequent. Consequently, epoxiconazole treatments did not differentiate between isolates with different mutations. The European survey provides an overview of the distribution of the frequency of different CYP51-haplotypes in Europe indicating a heterogeneous population in Europe and the different regions, and even in a single field.
Samples from 300 – 400 randomly selected winter wheat crops were taken annually at growth stage 73 – 75 from 1976 to 1988 with the exception of 1983 and 1984. The number of samples from each region was proportional to the area of wheat grown in each region. The percentage of the area of the top two leaves affected by diseases, the severity of ear and stem base diseases and, in 6 years, the severity of take-all were recorded. Septoria tritici and Septoria nodorum were, on average, the most severe of the foliar diseases and eyespot (Pseudocercosporella herpotrichoides) was the most severe of the stem base diseases. Regional differences in levels of S. tritici, brown rust, sharp eyespot and nodal fusarium were significant. Cultivar resistance affected disease severity, and previous cropping patterns particularly affected take-all and eyespot. Eyespot and sharp eyespot were less severe in late- than in early-sown crops. The percentage of crops treated with a fungicidal spray increased from 14% in 1976 to over 90% between 1983 and 1985. Use of benzimidazole fungicides applied at growth stage 31 declined, while use of morpholines from flag leaf emergence onwards increased between 1985 and 1988.
The sensitivity of Septoria tritici to the sterol biosynthesis inhibiting fungicide flutriafol was assessed using a quick and objective technique based on light absorbance to measure fungal growth. Microtitre plates were inoculated with suspensions of pycnidiospores taken directly from single pycnidia on leaves, after which glucose peptone broth containing different fungicide concentrations was added. After 10 days’ incubation in the dark at 17°C, growth was measured using a spectrophotometer at 405 nm. A dose-response curve fitted to the absorbance data was used to estimate the fungicide concentration reducing absorbance by one half (EC50). The method was precise, quick, reproducible and objective, with substantial advantages over conventional techniques.
The antimicrobial activity of the azole fungicides cyproconazole and propiconazole as single active ingredients and in mixtures with the ATP-Binding Cassette (ABC) transporter modulators rhodamine 6G, quercetin, quinidine, and verapamil and the strobilurin kresoxim-methyl was assessed against the wheat pathogen Mycosphaerella graminicola. Interactions amongst these compounds were evaluated on germination and germ tube growth of pycnidiospores using the Colby and Wadley method. Water agar proved to be the best test medium since all pycnidiospores germinated within 24 h of incubation and apical germ tube growth dominated over bud formation by intermediate cells. Analysis with the Colby method revealed that interactions between the compounds in all mixtures tested on germination of pycnidiospores were additive. With regard to germ tube growth, mixtures of cyproconazole and verapamil or kresoxim-methyl displayed a synergistic interaction. Analysis of mixtures of cyproconazole and kresoxim-methyl with the Wadley method revealed that the interaction between the two compounds was purely additive. These results indicate that the Colby method overestimated the interaction between these two compounds in a mixture.
Botrytis cinerea (anamorph of Botryotinia fuckeliana) is a filamentous ascomycete that causes grey mould especially on grapevine. Based on the presence or the absence of two transposable elements (Boty and Flipper) two sibling sympatric populations named transposa and vacuma have been described. Among the vacuma population, some strains (designated HydR1) were found to be resistant to fenhexamid (a sterol C-4 demethylase inhibitor) and to show an increased sensitivity to 14α-demethylase inhibitors (DMIs). In order to assess whether or not mutations at the target gene level (CYP51) could underlie increased sensitivity to DMIs in HydRl strains, we cloned the CYP51 gene and determined its DNA sequence in various B. cinerea strains. The gene was highly polymorphic, with mutations detected at 58 positions in the 35 strains analysed. The polymorphisms did not discriminate between transposa and vacuma strains, but did distinguish between HydRl and non-HydR1 ones. Two expressed mutations were present in all HydRl strains, namely phenylalanine to leucine at position 15 of the inferred protein, and serine to asparagine at position 105. These data, combined with the existence of morphological differences and somatic incompatibility between HydR1 and non-HydR1 strains, suggest that these two groups comprise distinct genetic entities.
Field isolates of the cereal eyespot pathogen can be divided into two groups which are now considered as two species: Tapesia yallundae and Tapesia acuformis. In both species the first case of acquired resistance was observed with benzimidazole fungicides in the early 1980s. At the same time, a number of sterol C-14 demethylation inhibitors (DMIs), such as the imidazole prochloraz and several triazoles, including flusilazole, were introduced. Surprisingly T. acuformis appeared intrinsically resistant to the triazole derivatives in comparison to T. yallundae, but both species were sensitive to prochloraz. The intensive use of these DMIs led to the development of acquired resistance towards triazoles in T. yallundae and towards prochloraz in T. acuformis. Today all the strains in both species appear equally sensitive to the anilinopyrimidine cyprodinil. ©1997 SCI
Cereal eyespot fungi Tapesia acuformis and Tapesia yallundae are closely related species which show different behaviours upon treatment with sterol 14-demethylase inhibitors (DMIs). T. acuformis is naturally resistant to DMIs belonging to the triazole family and susceptible to the imidazole ones, whilst T. yallundae is sensitive to both inhibitors. Cloning of the target enzyme gene, CYP51, from the two species revealed an important polymorphism between them. Further sequencing of CYP51 from sixteen T. acuformis and eleven T. yallundae strains with different phenotypes with regards to resistance to DMIs confirmed that at least eleven variations are species related. Among them, a conserved phenylalanine residue at position 180, found both in T. yallundae and in all known CYP51 proteins from filamentous fungi and yeast, was replaced in T. acuformis by a leucine. Therefore, a leucine at 180 could be possibly involved in natural resistance of T. acuformis to triazoles. Other mutations were observed in some resistant strains, sometimes simultaneously, but in contrast to what was reported for other filamentous fungi, where a mutation at the 136 position of the CYP51 gene product seemed to correlate with resistance to DMIs, we did not find a clear relationship between a given mutation and a particular phenotype. This result suggests that resistance to DMIs could have a polygenic nature in Tapesia. We took advantage of species-related variations to develop a PCR-based assay allowing rapid and easy discrimination between field strains of the two species.
The chemical control of Botrytis spp., and especially B. cinerea the causal agent of grey mould on many crops, can be achieved by several families of fungicides. Among those affecting fungal
respiration, the oldest ones are multi-site toxicants (e.g. dichlofluanid, thiram); newer ones are uncouplers (e.g. fluazinam),
inhibitors of mitochondrial complex II (e.g. boscalid) or complex III (e.g. strobilurins). Within anti-microtubule botryticides,
negative-cross resistance can occur between benzimidazoles (e.g. carbendazim) and phenylcarbamates (e.g. diethofencarb), a
phenomenon determined by a mutation in the gene encoding ??-tubulin. Aromatic hydrocarbon fungicides (e.g. dicloran), dicarboximides
(e.g. iprodione, procymidone, vinclozolin) and phenylpyrroles (e.g. fludioxonil) affect the fungal content of polyols and
resistance to these various compounds can be associated with mutations in a protein histidine kinase, probably involved in
osmoregulation. However, dicarboximide-resistant field strains of B. cinerea are sensitive to phenylpyrroles. Anilinopyrimidines (e.g. cyprodinil, mepanipyrim, pyrimethanil) inhibit methionine biosynthesis
but their primary target site remains unknown. In few situations, resistance of commercial significance has been recorded.
Among sterol biosynthesis inhibitors those inhibiting 14??- demethylase (DMIs) which are widely used against many fungal diseases
are of limited interest against Botrytis spp., whereas the hydroxyanilide fenhexamid, which inhibits the 3-keto reductase involved in sterol C4-demethylations, is
a powerful botryticide. Monitoring conducted in French vineyards revealed the presence of multi-drug resistant (MDR) strains,
a phenomenon probably determined by overproduction of ATP-binding cassette transporters. Resistance towards fungicides of
the different groups is described throughout the chapter.
Septoria tritici [Mycosphaerella graminicola], the causal agent of wheat leaf blotch can be controlled by various fungicides including multi-site compounds (e.g. chlorothalonil), benzimidazoles (e.g. carbendazim), DMIs (e.g. epoxiconazole, fluquinconazole and tebuconazole) and QoIs (e.g. azoxystrobin and pyraclostrobin). Since 1997, French populations of S. tritici have been monitored for their sensitivity towards these various fungicides. Although benzimidazoles are not yet used on wheat, highly resistant strains towards these antimicrotubule fungicides are widely spread. In most locations, the majority of strains are weakly to moderately resistant to DMIs, but this phenomenon generally has limited effects on the practical efficacy of this class of sterol biosynthesis inhibitors. Since 2003, strains of S. tritici highly resistant to strobilurins, the main group of QoI fungicides, have been found in France. To date, this has not led to practical resistance because their frequencies are low. In the future, it will be necessary to always apply QoIs in mixtures with non-cross resistant fungicides to control S. tritici.
In field strains of Septoria tritici [Mycosphaerella graminicola], resistance to benzimidazoles, strobilurins and inhibitors of sterol 14alpha-demethylation (DMIs) is determined by mutations in genes encoding their respective target sites. For benzimidazoles and strobilurins, resistance levels are high and result in a reduced field performance of these fungicides. Regarding DMIs, several mutations have been identified and determine low to medium resistance levels; moreover, cross resistance between DMIs is not always observed. The practical efficacy of DMIs, especially triazoles, remains good. Under field conditions, the highest selection pressures towards strains highly resistant to QoIs and moderately resistant to DMIs are recorded when strobilurins and triazoles, respectively, are applied at high doses. The combinations of members of group with reduced doses have a generally a less pronounced effect. Prochloraz was the only DMI that did not select strains moderately resistant to this class of fungicides.
Single applications of triazole fungicides propiconazole (125 g ha−1), tebuconazole (125 g ha−1) and cyproconazole (120 g ha−1) were equally effective in reducing Septoria nodorum and increasing yield on the spring wheat cultivar Aroona, which is moderately susceptible to the pathogen. A single application of propiconazole (125 g ha−1) at head emergence (GS 55) was as effective or more effective at increasing yield than application at full flag-leaf emergence (GS 39) or three applications of propiconazole (125 g ha−1) or mancozeb (1.6 kg ha−1) starting at stem elongation (GS 31). Application of propiconazole in either 154–2001 water ha−1 or 301 water ha−1 with 1% spray oil had similar effects. The response of two cultivars differing in resistance to S. nodorum and S. tritici varied, at least partly owing to the differing occurrence of the diseases at two locations. Resistance to only one Septoria species reduced, but did not prevent, yield losses because of occurrence of the other species. In the absence of combined resistance, fungicide sprays may offer a cost-effective means of reducing losses to S. tritici and S. nodorum in southern Western Australia when yield potentials are high and disease levels are moderate.
Seventeen fungicides were tested, singly and in mixtures, on Phytophthora cactorum and P. cinnamomi in vitro, and on the disease development by Phytophthora infestons on tomato or potato leaves and Plasmopara viticola on grape vine leaves under greenhouse and field conditions. Results indicated synergistic interactions of different intensities up to a ratio of 7. Ratios were calculated by comparing theoretical with observed EC90 values of the mixture. Among mixtures containing phenylamide fungicides like oxadixyl, metalaxyl or cyprofuram, mixtures of oxadixyl with mancozeb and/or cymoxanil showed the highest synergism. When tested on Phytophthora or Plasmopara diseases under greenhouse conditions, the highest synergism was observed in combinations of oxadixyl with mancozeb, cymoxanil, or phosetyl-Al against both diseases; with fentin acetate, folpet or thiram against Phytophthora; and with captan or maneb against Plasmopara. None of the tested oxadixyl mixtures showed antagonistic interactions.
The crystal structure of 14α-sterol demethylase from Mycobacterium tuberculosis (MTCYP51) [Proc. Natl. Acad. Sci. USA 98 (2001) 3068–3073] provides a template for analysis of eukaryotic orthologs which constitute the CYP51 family of cytochrome P450 proteins. Putative substrate recognition sites (SRSs) were identified in MTCYP51 based on the X-ray structures and have been compared with SRSs predicted based on Gotoh’s analysis [J. Biol. Chem. 267 (1992) 83–90]. While Gotoh’s SRS-4, 5, and 6 contribute in formation of the putative MTCYP51 substrate binding site, SRS-2 and 3 likely do not exist in MTCYP51. SRS-1, as part of the open BC loop, in the conformation found in the crystal can provide only limited contacts with the sterol. However, its role in substrate binding might dramatically increase if the loop closes in response to substrate binding. Thus, while the notion of SRSs has been very useful in leading to our current understanding of P450 structure and function, their identification by sequence alignment between distant P450 families will not necessarily be a good predictor of residues associated with substrate binding. Localization of CYP51 mutation hotspots in Candida albicans azole resistant isolates was analyzed with respect to SRSs. These mutations are found to be outside of the putative substrate interacting sites indicating the preservation of the protein active site under the pressure of azole treatment. Since the mutations residing outside the putative CYP51 active side can profoundly influence ligand binding within the active site, perhaps they provide insight into the basis of evolutionary changes which have occurred leading to different P450s.
The sensitivity of 73 isolates of Mycosphaerella graminicola collected over the period 1993–2002 from wheat fields in South England was tested in vitro against the triazole fluquinconazole, the strobilurin azoxystrobin and to the imidazole prochloraz. Over the sampling period, sensitivity of the population to fluquinconazole and prochloraz decreased by factors of approximately 10 and 2, respectively, but there was no evidence of changes in sensitivity to azoxystrobin. There was no correlation between sensitivity to fluquinconazole and prochloraz, but there was a weak negative cross-resistance between fluquinconazole and azoxystrobin.
SUMMARY Sterol demethylation inhibitors (DMIs) represent one of the largest groups of systemic fungicides that have been used to control agriculturally important fungal pathogens. Knowledge regarding the evolution of fungicide resistance in agricultural ecosystems is fragmentary and a better understanding of the processes driving the development of DMI resistance in populations of fungal pathogens is needed by plant pathologists and the agrochemical industry. We considered some of these processes using approaches based on molecular population and quantitative genetics. Five Mycosphaerella graminicola populations sampled from unsprayed wheat fields on four continents were assayed for eight restriction fragment length polymorphism (RFLP) markers and their level of tolerance to cyproconazole. DMI fungicides such as cyproconazole inhibit the enzyme eburicol 14-alpha-demethylase. The gene encoding this target, CYP51, was sequenced for all isolates. We found unimodal, continuous variations in cyproconazole tolerance among the M. graminicola isolates sampled from individual fields, consistent with a polygenic mode of inheritance. We also found that population differentiation for cyproconazole tolerance (Q(ST)) among the five M. graminicola populations was significantly higher than the corresponding population differentiation for neutral RFLP markers (G(ST)), suggesting that selection for cyproconazole tolerance in the Swiss population has already led to local adaptation that can be seen even in an unsprayed population. The Swiss population displayed the highest level of tolerance to cyproconazole, in addition to a lower than expected quantitative variation in fungicide tolerance and a skewed distribution, indicating that selection had increased the overall tolerance of this population. Further analysis with DNA sequencing showed that the population from Switzerland was dominated by isolates with several point mutations and a 6-bp deletion in CYP51. This deletion and one of the point mutations were previously related to increased resistance in field isolates. The fungal population from Oregon sampled from an unsprayed resistant host cultivar displayed the same gene diversity in RFLP loci but higher cyproconazole tolerance and quantitative variation in tolerance than the fungal population from the same field sampled from an unsprayed susceptible host cultivar.
The obligatory step in sterol biosynthesis in eukaryotes is demethylation of sterol precursors at the C14-position, which is catalyzed by CYP51 (sterol 14-alpha demethylase) in three sequential reactions. In mammals, the final product of the pathway is cholesterol, while important intermediates, meiosis-activating sterols, are produced by CYP51. Three crystal structures of human CYP51, ligand-free and complexed with antifungal drugs ketoconazole and econazole, were determined, allowing analysis of the molecular basis for functional conservation within the CYP51 family. Azole binding occurs mostly through hydrophobic interactions with conservative residues of the active site. The substantial conformational changes in the B' helix and F-G loop regions are induced upon ligand binding, consistent with the membrane nature of the protein and its substrate. The access channel is typical for mammalian sterol-metabolizing P450 enzymes, but is different from that observed in Mycobacterium tuberculosis CYP51. Comparison of the azole-bound structures provides insight into the relative binding affinities of human and bacterial P450 enzymes to ketoconazole and fluconazole, which can be useful for the rational design of antifungal compounds and specific modulators of human CYP51.
Invasive aspergillosis due to multi-azole-resistant Aspergillus fumigatus has emerged in the Netherlands since 1999, with 6.0-12.8% of patients harbouring resistant isolates. The presence of a single resistance mechanism (denoted by TR/L98H), which consists of a substitution at codon 98 of cyp51A and a 34-bp tandem repeat in the gene-promoter region, was found in over 90% of clinical A fumigatus isolates. This is consistent with a route of resistance development through exposure to azole compounds in the environment. Indeed, TR/L98H A fumigatus isolates were cultured from soil and compost, were shown to be cross-resistant to azole fungicides, and genetically related to clinical resistant isolates. Azoles are abundantly used in the environment and the presence of A fumigatus resistant to medical triazoles is a major challenge because of the possibility of worldwide spread of resistant isolates. Reports of TR/L98H in other European countries indicate that resistance might already be spreading.
Infections by opportunistic pathogenic fungi, especially Candida species, Cryptococcus neoformans, and Aspergillus fumigatus, are a serious medical problem in immunocompromised patients. Different classes of antimycotic drugs are available to treat fungal infections, but the pathogens can develop resistance to all these agents. A major mechanism of antifungal drug resistance is the overexpression of efflux pumps of the ABC transporter and major facilitator superfamilies, which confer resistance to many structurally and functionally unrelated toxic compounds. For some pathogenic fungi, like Candida albicans and Candida glabrata, the most important drug transporters, transcription factors controlling their expression, and mutations that cause the constitutive upregulation of the efflux pumps in drug-resistant clinical isolates have been identified. For other important pathogens comparatively little is known about the role of transporters in antimycotic resistance. This review summarizes our current knowledge about efflux pump-mediated drug resistance and its regulation in human-pathogenic fungi.
Mycosphaerella fijiensis Morelet causes black sigatoka, the most important disease in bananas and plantains. Disease control is mainly through the application of systemic fungicides, including sterol demethylation inhibitors (DMIs). Their intensive use has favoured the appearance of resistant strains. However, no studies have been published on the possible resistance mechanisms.
In this work, the CYP51 gene was isolated and sequenced in 11 M. fijiensis strains that had shown different degrees of in vitro sensitivity to propiconazole, one of the most widely used DMI fungicides. Six mutations that could be related to the loss in sensitivity to this fungicide were found: Y136F, A313G, Y461D, Y463D, Y463H and Y463N. The mutations were analysed using a homology model of the protein that was constructed from the crystallographic structure of Mycobacterium tuberculosis (Zoff.) Lehmann & Neumann. Additionally, gene expression was determined in 13 M. fijiensis strains through quantitative analysis of products obtained by RT-PCR.
Several changes in the sequence of the gene encoding sterol 14alpha-demethylase were found that have been described in other fungi as being correlated with resistance to azole fungicides. No correlation was found between gene expression and propiconazole resistance.
ABSTRACT We identified the cytochrome P450 sterol 14alpha-demethylase (CYP51A1) gene from Venturia inaequalis and optional insertions located upstream from CYP51A1 and evaluated their potential role in conferring resistance to the sterol demethylation-inhibitor (DMI) fungicide my-clobutanil. The CYP51A1 gene was completely sequenced from one my-clobutanil sensitive (S) and two myclobutanil-resistant (R) strains. No nucleotide variation was found when the three sequences were aligned. Allele-specific polymerase chain reaction (PCR) analysis indicated that a previously described single base pair mutation that correlated with resistance to DMI fungicides in strains of other filamentous fungi was absent in 19 S and 32 R strains of V. inaequalis from Michigan and elsewhere. The sequencing results and PCR analyses suggest that resistance in these strains was not due to a mutation in the sterol demethylase target site for DMI fungicides. Expression of CYP51A1 was determined for strains from an orchard that had never been sprayed with DMI fungicides (baseline orchard), and the data provided a reference for evaluating the expression of strains collected from a research orchard and from three commercial Michigan apple orchards with a long history of DMI use and a high frequency of R strains. Overexpression of CYP51A1 was significantly higher in 9 of 11 R strains from the research orchard than in S strains from the baseline orchard. The high expression was correlated with the presence of a 553-bp insertion located upstream of CYP51A1. Overexpression of the CYP51A1 gene was also detected in eight of eight, five of nine, and nine of nine R strains from three commercial orchards, but the insertion was not detected in the majority of these strains. The results suggest that overexpression of the target-site CYP51A1 gene is an important mechanism of resistance in some field resistant strains of V. inaequalis, but other mechanisms of resistance also appear to exist.
Recent findings are consistent with a slow but constant shift towards reduced sensitivity of Mycosphaerella graminicola to azole fungicides, which target the CYP51 gene. The goal of this study was to elucidate the evolutionary mechanisms through which CYP51-based mutations associated with altered sensitivity have evolved in M. graminicola over space and time. To accomplish this, we sequenced and compared a portion of the CYP51 gene encompassing the main mutations associated with altered sensitivity towards demethylation inhibitor fungicides. The CYP51 gene showed an extraordinary dynamic shift consistent with a selective haplotype replacement both in space and in time. No mutations associated with increased resistance to azoles were found in non-European populations. These mutations were also absent in the oldest collections from Europe, whereas they dominated in the recent European populations. Intragenic recombination was identified as an important evolutionary process in populations affected by high fungicide selection, suggesting the creation of novel alleles among existing mutations as a potential source of novel resistance alleles. We propose that CYP51 mutations giving resistance in M. graminicola arose only locally (perhaps in Denmark or the UK) and were then spread eastward across Europe through wind-dispersed ascospores. We conclude that recurring cycles of recombination coupled with selection due to the widespread use of azole fungicides will increase the frequency of novel mutants or recombinants with higher resistance. Long-distance gene flow due to wind dispersal of ascospores will move the resulting new alleles to new areas following the prevailing wind directions. A selective replacement favouring haplotypes with various coding mutations at the target site for azole fungicides during the last 5-10 years is the most likely cause of the decrease in sensitivity reported for many azole fungicides in the same period.
Mechanism of action of azole antifungal agents was studied by analyzing interaction of ketoconazole, itraconazole, triadimefon and triadimenol with a purified yeast cytochrome P-450 which catalyzes lanosterol 14 alpha-demethylation (P-45014DM). These antifungal agents formed low-spin complexes with P-45014DM, indicating the interaction of their azole nitrogens with the heme iron. Affinity of these antifungal agents for the cytochrome was extremely high compared with usual nitrogenous ligands. Upon reduction with sodium dithionite, the azole complexes of ferric P-45014DM were converted to the corresponding ferrous derivatives. Spectral analysis of these complexes suggested that geometric orientation of the azole moiety of an antifungal agent to the ferrous heme iron was regulated by the interaction between the N-1 substituent and the heme environment. CO could not readily replace ketoconazole or itraconazole co-ordinating to the heme iron of ferrous P-45014DM while triadimefon and triadimenol complexes of the cytochrome were promptly converted to the CO complexes. The inhibitory effects of ketoconazole and itraconazole on the P-45014DM-dependent lanosterol 14 alpha-demethylation were higher than that of triadimenfon. The substituents at N-1 of the azole moieties of ketoconazole and itraconazole are extremely large while those of triadimefon and triadimenol are relatively small. Accordingly, observations described above suggest that the N-1 substituent of an azole antifungal agent regulates the mobility of the molecule in the heme crevice of ferrous P-45014DM and determines the inhibitory effect of the compound.
Demethylation inhibitor (DMI)-resistant strains of the plant pathogenic fungus Penicillium digitatum were shown to be simultaneously resistant to cycloheximide, 4-nitroquinoline-N-oxide (4NQO), and acriflavine. A PMR1 (Penicillium multidrug resistance) gene encoding an ATP-binding cassette (ABC) transporter (P-glycoprotein) was cloned from a genomic DNA library of a DMI-resistant strain (LC2) of Penicillium digitatum by heterologous hybridization with a DNA fragment containing an ABC-encoding region from Botrytis cinerea. Sequence analysis revealed significant amino acid homology to the primary structures of PMR1 (protein encoded by the PMR1 gene) and ABC transporters of Saccharomyces cerevisiae (PDR5 and SNQ2), Schizosaccharomyces pombe (HBA2), Candida albicans (CDR1), and Aspergillus nidulans (AtrA and AtrB). Disruption of the PMR1 gene of P. digitatum DMI-resistant strain LC2 demonstrated that PMR1 was an important determinant of resistance to DMIs. The effective concentrations inhibiting radial growth by 50% (EC50s) and the MICs of fenarimol and bitertanol for the PMR1 disruptants (Deltapmr1 mutants) were equivalent to those for DMI-sensitive strains. Northern blot analysis indicated that severalfold more PMR1 transcript accumulated in the DMI-resistant strains compared with those in DMI-sensitive strains in the absence of fungicide. In both DMI-resistant and -sensitive strains, transcription of PMR1 was strongly enhanced within 10 min after treatment with the DMI fungicide triflumizole. These results suggested that the toxicant efflux system comprised of PMR1 participates directly in the DMI resistance of the fungus.