Search

Background Over the past decade, demethylation inhibitors (DMIs) and succinate dehydrogenase inhibitors (SDHIs) fungicides have been extensively used to control to septoria tritici blotch, caused by Zymoseptoria tritici on wheat. This has led to the development and selection of alterations in the target-site enzymes (CYP51 and SDH, respectively). Results Taking advantage of newly and previously developed qPCR assays, the frequency of key alterations associated with DMI (CYP51-S524T) and SDHI (SDHC-T79N/I, C-N86S and C-H152R) resistance was assessed in Z. tritici infected wheat leaf samples collected from commercial crops (n = 140) across 14 European countries prior to fungicide application in the spring of 2019. This revealed the presence of a West to East gradient in the frequencies of the most common key alterations conferring azole (S524T) and SDHI resistance (T79N and N86S), with the highest frequencies measured in Ireland and Great Britain. These observations were corroborated by sequencing (CYP51 and SDH subunits) and sensitivity phenotyping (prothioconazole-desthio and fluxapyroxad) of Z. tritici isolates collected from a selection of field samples. Additional sampling made at the end of the 2019 season confirmed the continued increase in frequency of the targeted alterations. Investigations on historical leaf DNA samples originating from different European countries revealed that the frequency of all key alterations (except C-T79I) has been gradually increasing over the past decade. Conclusion Whilst these alterations are quickly becoming dominant in Ireland and Great Britain, scope still exists to delay their selection throughout the wider European population, emphasizing the need for the implementation of fungicide anti-resistance measures. This article is protected by copyright. All rights reserved.

Azole resistance is a concern for the management of diseases caused by Aspergillus fumigatus in humans. Azole fungicide use in the environment has been identified as a possible cause for development of resistance, which increases the complexity and number of stakeholders involved in this emerging problem. A workshop was held in Amsterdam early 2019 in which stakeholders, including medical and agricultural researchers, representatives from the government, public health, fungicide producers and end-users, reviewed the current evidence supporting environmental selection for resistance and to discuss which research and measures are needed to retain the effectiveness of the azole class for environmental and medical applications. This paper provides an overview of the latest insights and understanding of azole resistance development in the clinical setting and the wider environment. A One Health problem approach was undertaken to list and prioritize which research will be needed to provide missing evidence and to enable preventive interventions.

Light leaf spot, caused by the ascomycete Pyrenopeziza brassicae, is an established disease of Brassicaceae in the United Kingdom (UK), continental Europe, and Oceania (OC, including New Zealand and Australia). The disease was reported in North America (NA) for the first time in 2014 on Brassica spp. in the Willamette Valley of western Oregon, followed by detection in Brassica juncea cover crops and on Brassica rapa weeds in northwestern Washington in 2016. Preliminary DNA sequence data and field observations suggest that isolates of the pathogen present in NA might be distinct from those in the UK, continental Europe, and OC. Comparisons of isolates from these regions using genetic (multilocus sequence analysis, MAT gene sequences, and rep-PCR DNA fingerprinting), pathogenic (B. rapa inoculation studies), biological (sexual compatibility), and morphological (colony and conidial morphology) analyses demonstrated two genetically distinct evolutionary lineages. Lineage 1 comprised isolates from the UK, continental Europe, and OC, and included the P. brassicae type specimen. Lineage 2 contained the NA isolates associated with recent disease outbreaks in the Pacific Northwest region of the USA. Symptoms caused by isolates of the two lineages on B. rapa and B. juncea differed, and therefore "chlorotic leaf spot" is proposed for the disease caused by Lineage 2 isolates of P. brassicae. Isolates of the two lineages differed in genetic diversity as well as sensitivity to the fungicides carbendazim and prothioconazole.

Summary Background Loop‐mediated isothermal amplification (LAMP) assays, which operate at a single temperature and require no postreaction processing, have been described for rapid species‐specific detection of numerous fungi. The technology has much less commonly been applied to identification of other key genetic traits such as fungicide resistance, and has not yet been applied to mating‐type determination in any fungus. Objectives To develop first LAMP assays for mating‐type identification in a fungus, in this instance with the saprophytic mould and human opportunistic pathogen Aspergillus fumigatus, a heterothallic ascomycete requiring isolates of opposite mating type (MAT1‐1, MAT1‐2) for sexual reproduction. Methods New LAMP primer sets, targeted to MAT gene sequences, were screened against 34 A fumigatus isolates (of known mating type) from diverse clinical, environmental and geographic sources to establish whether they could distinguish MAT1‐1 or MAT1‐2 genotypes. Results and conclusions The new assays, operating at a single temperature of 65°C, correctly identified the mating type of A fumigatus isolates in

Sustainable disease management depends on the ability to monitor the development of fungicide resistance in pathogen populations. A point mutation resulting in an alteration (F200Y) at codon 200 of the target protein β‐tubulin leads to a moderate level of resistance to carbendazim in Botrytis cinerea. Although traditional methods remain a cornerstone in detection of fungicide resistance, molecular methods that do not require the isolation of pathogens, can detect the presence of resistance alleles at low frequencies, and require less time and labour than traditional methods. In this study, we present an efficient, rapid, and highly specific method for detecting the moderately carbendazim‐resistant isolates in B. cinerea based on loop‐mediated isothermal amplification (LAMP). By using specific LAMP primers, we detected the resistance‐conferring mutation underlying β‐tubulin F200Y. The concentrations of LAMP components and LAMP parameters were optimised, resulting in reaction temperatures and times of 61–65°C and 45 min, respectively. The feasibility of the LAMP assay was verified by assaying the diseased samples with artificial inoculation in the different hosts. The LAMP assay developed in the current study was specific, stable, repeatable and sensitive, and was successfully applied for detection of moderately carbendazim‐resistant isolates of B. cinerea in plant samples.

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

Optimal disease management depends on the ability to monitor the development of fungicide resistance in plant pathogen populations. Benzimidazole resistance is caused by the point mutations of the β-tubulin gene in Botrytis cinerea, and three mutations (E198A, E198K, and E198V) at codon 198 account for more than 98% of all resistant strains. Although traditional methods remain a cornerstone in monitoring fungicide resistance, molecular methods that do not require the isolation of pathogens can detect resistance alleles present at low frequencies, and require less time and labor than traditional methods. In this study, we present an efficient, rapid, and highly specific method for detecting highly benzimidazole-resistant B. cinerea isolates based on loop-mediated isothermal amplification (LAMP). By using specific primers, we could simultaneously detect all three resistance-conferring mutations at codon 198. The LAMP reaction components and conditions were optimized, and the best reaction temperatures and times were 60 to 62°C and 45 min, respectively. When B. cinerea field isolates were assessed for benzimidazole resistance, similar results were obtained with LAMP, minimal inhibition concentration, and sequencing. The LAMP assay developed in the current study was highly suitable for detection of highly benzimidazole-resistant field isolates of B. cinerea.

BACKGROUND Methyl benzimidazole carbamate (MBC) fungicides are used to control the oilseed rape pathogen Pyrenopeziza brassicae. Resistance to MBCs has been reported in P. brassicae, but the molecular mechanism(s) associated with reductions in sensitivity have not been verified in this species. Elucidation of the genetic changes responsible for resistance, hypothesised to be target-site mutations in β-tubulin, will enable resistance diagnostics and thereby inform resistance management strategies. RESULTS P. brassicae isolates were classified as sensitive, moderately resistant or resistant to MBCs. Crossing P. brassicae isolates of different MBC sensitivities indicated that resistance was conferred by a single gene. The MBC-target encoding gene β-tubulin was cloned and sequenced. Reduced MBC sensitivity of field isolates correlated with β-tubulin amino acid substitutions L240F and E198A. The highest level of MBC resistance was measured for isolates carrying E198A. Negative cross-resistance between MBCs and the fungicides diethofencarb and zoxamide was only measured in E198A isolates. PCR-RFLP was used to screen isolates for the presence of L240F and E198A. The substitutions E198G and F200Y were also detected in DNA samples from P. brassicae populations after cloning and sequencing of PCR products. The frequencies of L240F and E198A in different P. brassicae populations were quantified by pyrosequencing. There were no differences in the frequencies of these alleles between P. brassicae populations sampled from different locations or after fungicide treatment regimes. CONCLUSIONS The molecular mechanisms affecting sensitivity to MBCs in P. brassicae have been identified. Pyrosequencing assays are a powerful tool for quantifying fungicide-resistant alleles in pathogen populations.

Due to the repeated discovery of new members of the Fusarium graminearum species complex (FGSC), some of the F. graminearum sensu stricto (s.s.)-specific qPCR assays developed to date have since been shown to be non-specific. In this study, a probe-based qPCR method was developed, targeting a sterol 14-alpha demethylase (CYP51) paralogue, CYP51C unique to the genus Fusarium, for the simultaneous detection of F. asiaticum, F. ussurianum and F. vorosii. Specificity of the assay was demonstrated for a wide range of Fusarium species, including all tested FGSC members (n = 6), originating from different hosts and geographic regions. Alongside a previously published assay for detection of F. graminearum, we were able to show that members of the Asian clade of the FGSC (i.e. F. asiaticum, F. ussurianum and F. vorosii) were the primary etiological agent in wheat seeds samples originating from Central-East China. The grain samples from the UK tested negative for the presence of the FGSC's Asian clade and positive for presence of F. graminearum. It is likely that only F. graminearum s.s. is present in the UK, but the presence of other FGSC members cannot be ruled out and need further investigation.

Early establishment of endophytes can play a role in pathogen suppression and improve seedling development. One route for establishment of endophytes in seedlings is transmission of bacteria from the parent plant to the seedling via the seed. In wheat seeds, it is not clear whether this transmission route exists, and the identities and location of bacteria within wheat seeds are unknown. We identified bacteria in the wheat (Triticum aestivum) cv. Hereward seed environment using embryo excision to determine the location of the bacterial load. Axenic wheat seedlings obtained with this method were subsequently used to screen a putative endophyte bacterial isolate library for endophytic competency. This absence of bacteria recovered from seeds indicated low bacterial abundance and/or the presence of inhibitors. Diversity of readily culturable bacteria in seeds was low with 8 genera identified, dominated by Erwinia and Paenibacillus. We propose that anatomical restrictions in wheat limit embryo associated vertical transmission, and that bacterial load is carried in the seed coat, crease tissue and endosperm. This finding facilitates the creation of axenic wheat plants to test competency of putative endophytes and also provides a platform for endophyte competition, plant growth, and gene expression studies without an indigenous bacterial background.

Following a sudden outbreak of light leaf spot disease in Polish winter oilseed rape crops at the beginning of the last decade, volumetric spore traps installed around the country were used for the detection of ascospores, which constitute the primary inoculum of Pyrenopeziza brassicae, the causal organism. In 2004–2008, weather conditions that were adverse to the development of the pathogen appeared to have hindered the macroscopic signs of light leaf spot disease symptoms on oilseed rape, even in Pomerania, a region in north Poland, which cultivates the crop intensively and is favourable for the disease. Recently, however, the development of β-tubulin-based primers and dual labelled fluorescent probes has enabled the detection of minute quantities of P. brassicae DNA that correlated highly and positively with low abundance of inoculum of the pathogen in air samples. Depending on the season, the Spearman's correlation rank between spore abundance obtained by light microscopy and P. brassicae DNA...

BACKGROUND: The recent evolution towards resistance to azole fungicides in European populations of the wheat pathogen Mycosphaerella graminicola has been caused by the progressive accumulation of mutations in MgCYP51 gene, encoding the azole target sterol 14a-demethylase. Particular combinations of mutations have been shown specifically to affect the interaction of the MgCYP51 protein with different members of the azole class. Although additional mechanisms, including increased MgCYP51 expression and enhanced active efflux, have been proposed, the genetic changes underlying these mechanisms are unknown. RESULTS: Analysis of the azole sensitivities of recent M. graminicola isolates identified a novel phenotype, seemingly independent of changes in MgCYP51 coding sequence. Characterised by a 7-16-fold reduction in in vitro sensitivity to all azoles tested and by growth on seedlings at higher doses of azoles in glasshouse tests compared with isolates carrying the same MgCYP51 variant (L50S, S188N, I381V, ?Y459/G460, N513K), isolates with this phenotype constitutively overexpress MgCYP51 by between 10- and 40-fold compared with the wild type. Analysis of sequences upstream of the predicted MgCYP51 translation start codon identified a novel 120 bp indel, considered to be an insertion, in isolates overexpressing MgCYP51. CONCLUSIONS: The identification of an insertion in the predicted MgCYP51 promoter in azole-resistant isolates overexpressing MgCYP51 is the first report of a genetic mechanism, other than changes in target-site coding sequence, affecting sensitivity to multiple azoles in field isolates of M. graminicola. The identification of recent isolates overexpressing MgCYP51 confirms the ongoing evolution and diversification of resistance mechanisms in European populations of M. graminicola. Copyright (c) 2012 Society of Chemical Industry

This review assesses the impacts, both direct and indirect, of man-made changes to the composition of the air over a 200 year period on the severity of arable crop disease epidemics. The review focuses on two well-studied UK arable crops, wheat and oilseed rape, relating these examples to worldwide food security. In wheat, impacts of changes in concentrations of SO2 in air on two septoria diseases are discussed using data obtained from historical crop samples and unpublished experimental work. Changes in SO2 seem to alter septoria disease spectra both through direct effects on infection processes and through indirect effects on soil S status. Work on the oilseed rape diseases phoma stem canker and light leaf spot illustrates indirect impacts of increasing concentrations of greenhouse gases, mediated through climate change. It is projected that, by the 2050s, if diseases are not controlled, climate change will increase yields in Scotland but halve yields in southern England. These projections are discussed in relation to strategies for adaptation to environmental change. Since many strategies take 10–15 years to implement, it is important to take appropriate decisions soon. Furthermore, it is essential to make appropriate investment in collation of long-term data, modelling and experimental work to guide such decision-making by industry and government, as a contribution to worldwide food security.

The effect of a novel mildew-specific quinazolinone fungicide, proquinazid, on the barley powdery mildew fungus, Blumeria graminis f.sp. hordei, has been studied using scanning electron microscopy and quantitative RT-PCR. Proquinazid has previously been shown to perturb conidial morphogenesis, similar to quinoxyfen, a currently widely used mildewicide. In this study, we confirm an effect of proquinazid on appressorial differentiation. By comparison to quinoxyfen, however, proquinazid affects this highly coordinated process differently, with more deformed appressorial germ tubes observed, often growing away from the leaf surface. Comparison of the expression of genes involved in the transduction of signals directing conidial development has also suggested differences in the affects of proquinazid and quinoxyfen. In particular, the expression of the Ras-type GTPase activating gene, previously implicated in quinoxyfen resistance, is distinctly affected by proquinazid treatment at time points critical to normal conidial morphogenesis. Together, these data indicate differences in the mechanisms by which proquinazid perturbs appressorial differentiation in comparison with quinoxyfen.

SUMMARY The recent reduction in the efficacy of azole fungicides in controlling Septoria leaf blotch of wheat, caused by Mycosphaerella graminicola, has prompted concerns over possible development of resistance, particularly in light of the recent emergence of widespread resistance to quinone outside inhibitors (QoIs). We have recently implicated alterations in the target-encoding sterol 14alpha-demethylase protein (CYP51), and over-expression of genes encoding efflux pumps, in reducing sensitivity to the azole class of sterol demethylation inhibitors (DMIs) in M. graminicola. Here we report on the prevalence and selection of two CYP51 alterations, substitution I381V and deletion of codons 459 and 460 (DeltaY459/G460), in populations of M. graminicola. Neither alteration has previously been identified in human or plant pathogenic fungi resistant to azoles. The presence of DeltaY459/G460 showed a continuous distribution of EC(50) values across isolates with either I381 or V381, and had no measurable effect on azole sensitivity. Data linking fungicide sensitivity with the presence of I381V in M. graminicola show for the first time that a particular CYP51 alteration is differentially selected by different azoles in field populations of a plant pathogen. Substitution I381V although not an absolute requirement for reduced azole sensitivity, is selected by tebuconazole and difenoconazole treatment, suggesting an adaptive advantage in the presence of these two compounds. Prochloraz treatments appeared to select negatively for I381V, whereas other azole treatments did not or only weakly impacted on the prevalence of this substitution. These findings suggest treatments with different members of the azole class of fungicides could offer a resistance management strategy.

Strobilurin-resistant isolates of Blumeria (Erysiphe) graminis f.sp. tritici, the cause of wheat powdery mildew, were more than 10-fold less sensitive to azoxystrobin than sensitive isolates. In all resistant isolates, a mutation resulting in the replacement of a glycine by an alanine residue at codon 143 (G143A) in the mitochondrial cytochrome b gene was found. Allele-specific primers were designed to detect this point mutation in infected wheat leaves. Using quantitative fluorescent allele-specific real-time polymerase chain reaction (PCR) measurements, strobilurin-resistant A143 alleles could be detected amongst strobilurin-sensitive G143 alleles at a frequency of at least 1 in 10 000, depending on the amount of target and nontarget DNA. Most isolates tested were dominant homoplasmic for either the A143 or G143 allele, although mixed populations of alleles could be detected in some isolates. In some of these isolates, strobilurin resistance was not always stable when they were maintained for many generations in the absence of selection. The allele-specific real-time PCR assay was also used to follow the dynamics of A143 alleles in field populations of B. graminis f.sp. tritici before and after application of fungicides. As expected, the A143 allele frequency only increased under selection pressure from a strobilurin fungicide. After three sprays of azoxystrobin, a pronounced selection for the strobilurin-resistant allele, with an increase in average frequency from 2·2 to 58%, was measured. The use of quantitative real-time PCR diagnostics for early detection of fungicide resistance genes at low frequency, coupled with risk evaluation, will be invaluable for further resistance risk assessment and validation of antiresistance strategies.

Several techniques for detection of Erwinia carotovora ssp. atroseptica (Eca) on potato tubers were used to study the transmission of Eca from inoculated mother tubers into progeny tubers for 12 different potato cultivars. The sensitivity of the polymerase chain reaction (PCR) for a direct detection of Eca in crude peel extracts was only 10 7 -10 8 cells/ml, due to inhibition of PCR amplification by potato tuber-derived compounds. With dilution plating and ELISA, 10 5 and 10 7 cells/ml were detected, respectively. Immunofluorescence cell staining (IF), with a detection threshold of 10 4 -10 5 cells/ml, was shown to be most sensitive for direct detection, and transmission of Eca was found for six potato cultivars, whereas with the other techniques tested no transmission was found. After 48 h enrichment of peel extract in polypectate medium at 20°C, IF and PCR detected an initial inoculum concentration of 10 Eca cells/ml, whereas 10 2 , 10 4 and 10 4 cells/ml were detected in dilution plating, ELISA and conductimetry, respectively. With dilution plating, PCR and IF, performed after enrichment, transmission of Eca was found for, respectively, four, six and seven out of 12 potato cultivars tested. No transmission was found using ELISA and conductimetry, due to their lower sensitivity. Erwinia carotovora ssp. carotovora (Ecc) was only detected in the progeny tubers of control plants. No relation was found between the transmission of Eca to progeny tubers and blackleg incidence in the field.

Four media were tested for their ability to detect the soft rot potato pathogens Erwinia chrysanthemi (Ech) and Erwinia carotovora ssp. atroseptica (Eca) in potato tubers by means of automated conductance measurements. The specificity of the conductimetric assays was determined by testing a set of different Erwinia spp. and potato-associated saprophytes, including the genera Pseudomonas, Bacillus, Enterobacter and Flavobacterium. All bacteria tested produced conductance responses in Special Peptone Yeast Extract, whereas in minimal medium with L-asparagine only Erwinia spp. and Pseudomonas spp. were able to generate large conductance responses. In minimal medium supplemented with glucose and trimethylamine-N-oxide only Enterobacteriaceae, Erwinia spp. included, generated conductance responses, while with pectate as sole carbon source only Erwinia spp. produced distinct conductance responses. The pectate medium proved to be particularly useful for specific automated conductimetric detection of Erwinia spp. in potato peel extracts. Within 48 h, the detection threshold of the conductimetric assay for Eca varied between 10 2 and 10 3 cfu per ml peel extract at both incubation temperatures of 20° and 26°C. Ech was detected at concentrations of 10 4 -10 5 or 10 3 -10 4 cfu mL -1 at 20° and 26°C, respectively. To eliminate 'false'-positive reactions in conductimetric caused by Erwinia carotovora ssp. carotovora, results of the conductance measurements have to be confirmed by other techniques, like serology or DNA assays.

Methyl benzimidazole carbamate (MBC) fungicides are used to control the oilseed rape pathogen Pyrenopeziza brassicae. Resistance to MBCs has been reported in P. brassicae, but the molecular mechanism(s) associated with reductions in sensitivity have not been verified in this species. Elucidation of the genetic changes responsible for resistance, hypothesised to be target-site mutations in β-tubulin, will enable resistance diagnostics and thereby inform resistance management strategies.P. brassicae isolates were classified as sensitive, moderately resistant or resistant to MBCs. Crossing P. brassicae isolates of different MBC sensitivities indicated that resistance was conferred by a single gene. The MBC-target encoding gene β-tubulin was cloned and sequenced. Reduced MBC sensitivity of field isolates correlated with β-tubulin amino acid substitutions L240F and E198A. The highest level of MBC resistance was measured for isolates carrying E198A. Negative cross-resistance between MBCs and the fungicides diethofencarb and zoxamide was only measured in E198A isolates. PCR-RFLP was used to screen isolates for the presence of L240F and E198A. The substitutions E198G and F200Y were also detected in DNA samples from P. brassicae populations after cloning and sequencing of PCR products. The frequencies of L240F and E198A in different P. brassicae populations were quantified by pyrosequencing. There were no differences in the frequencies of these alleles between P. brassicae populations sampled from different locations or after fungicide treatment regimes.The molecular mechanisms affecting sensitivity to MBCs in P. brassicae have been identified. Pyrosequencing assays are a powerful tool for quantifying fungicide-resistant alleles in pathogen populations.

Accurate identification of fungal phytopathogens is essential for virtually all aspects of plant pathology, from fundamental research on the biology of pathogens to the control of the diseases they cause. Although molecular methods, such as polymerase chain reaction (PCR), are routinely used in the diagnosis of human diseases, they are not yet widely used to detect and identify plant pathogens. Here we review some of the diagnostic tools currently used for fungal plant pathogens. and describe some novel applications. Technological advances in PCR-based methods, such as real-time PCR, allow fast, accurate detection and quantification of plant pathogens and are now being applied to practical problems. Molecular methods have been used to detect several pathogens simultaneously in wheat, and to study the development of fungicide resistance in wheat pathogens. Information resulting from such work could be used to improve disease control by allowing more rational decisions to be made about the choice and use of fungicides and resistant cultivars. Molecular methods have also been applied to the study of variation in plant pathogen populations, for example detection of different mating types or virulence types. PCR-based methods can provide new tools to monitor the exposure of a crop to pathogen inoculum that are more reliable and faster than conventional methods. This information can be used to improve disease control decision making. The development and application of molecular diagnostic methods in the future is discussed and we expect that new developments will increase the adoption of these new technologies for the diagnosis and study of plant disease. (C) 2003 Society of Chemical Industry.

Fungicide resistance is a constant threat to agricultural production worldwide. Molecular\ud mechanisms of fungicide resistance have been studied extensively in the wheat pathogen\ud Zymoseptoria tritici. However, less is known about the evolutionary processes driving resistance\ud development. In vitro evolutionary studies give the opportunity to investigate this. Here, we\ud examine the adaptation of Z. tritici to fluxapyroxad, a succinate dehydrogenase (Sdh) inhibitor.\ud Replicate populations of Z. tritici derived from the sensitive isolate IPO323 were exposed to\ud increasing concentrations of fluxapyroxad with or without UV mutagenesis. After ten increases in\ud fungicide concentration, sensitivity had decreased dramatically, with replicate populations showing similar phenotypic trajectories. Sequencing the Sdh subunit B, C and D encoding genes identified seven mutations associated with resistance to fluxapyroxad. Mutation frequency over time was measured with a pyrosequencing assay, revealing sequential lineage replacement in the UV mutagenized populations but not in the untreated populations. Repeating selection from set timepoints with different fungicide concentrations revealed that haplotype replacement of Sdh variants was driven by dose-dependent selection as fungicide concentration changed, and was not mutation limited. These findings suggest that fungicide field applications may select for highly insensitive Sdh variants with higher resistance factors if the fungicide concentration is increased to achieve a better disease control. However, in the absence or presence of lower fungicide concentrations, the spread of these strains might be restricted if the underlying Sdh mutations carry fitness penalties.