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1.
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 14α‐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, ΔY459/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 © 2012 Society of Chemical Industry  相似文献   

2.
Although fungicide resistance in crop pathogens is a global threat to food production, surprisingly little is known about the evolutionary processes associated with the emergence and spread of fungicide resistance. Early stages in the evolution of fungicide resistance were evaluated using the wheat pathogen Zymoseptoria tritici, taking advantage of an isolate collection spanning 20 years in Oregon, USA, and including two sites with differing intensity of fungicide use. Sequences of the mitochondrial cytb protein conferring single‐mutation resistance to QoI fungicides and the nuclear CYP51 gene implicated in multiple‐mutation resistance to azole fungicides were analysed. Mutations associated with resistance to both fungicides were absent in the 1992 isolates, but frequent in the 2012 collection, with higher frequencies of resistance alleles found at the field site with more intensive fungicide use. Results suggest that the QoI resistance evolved independently in several lineages, and resulted in significant mitochondrial genome bottlenecks. In contrast, the CYP51 gene showed signatures of diversifying selection and intragenic recombination among three phylogenetic clades. The findings support a recent emergence of resistance to the two fungicide classes in Oregon, facilitated by selection for mutations in the associated resistance genes.  相似文献   

3.
Prior to the use of fungicides, the baseline sensitivity of individuals in a pathogen population may already differ by a factor of 10 to 100 between the least and the most sensitive isolates. In Mycosphaerella graminicola populations, this factor, measured in vitro, was 5 to 20 for both the strobilurin analogue azoxystrobin (baseline) and the triazole cyproconazole which has been in use for several years. In Phytophthora infestans populations, this factor, measured in a leaf disc assay, was about 100 for azoxystrobin (baseline), up to 1000 for the cyanoacetamide cymoxanil and >10000 for the phenylamide oxadixyl; both of the latter have been used for many years. In M. graminicola, cross-sensitivity was present between all azole fungicides for the majority of the isolates, whereas no correlation was found between triazoles and azoxystrobin. Despite the existence of cross-sensitivity between azoles, ‘box-and-whiskers’ plots revealed large variations in the sensitivity profiles of some triazoles; isolates resistant to triazoles have not been detected in M. graminicola populations. In P. infestans populations, the proportion of the phenylamide-resistant sub-population increased during the season more rapidly in treated than in untreated fields, but it was low at the beginning of the next season in all fields. During disease epidemics, the fitness of phenylamide-resistant P. infestans isolates, as characterised by lesion size, was higher than that of the sensitive isolates, but after the overwintering period, the recovery of resistant isolates was apparently lower. The presence of both A1 and A2 mating types of P. infestans in European populations, although at different frequencies, allows sexual recombination and increased genetic diversity, affecting sensitivity and fitness. Such mixed populations can still be adequately controlled by using sound anti-resistance strategies. ©1997 SCI  相似文献   

4.
Field experiments, involving various fungicide strategies with pyraclostrobin and/or epoxiconazole were carried out in 2004 and 2005, with the overall purpose of monitoring the evolution of fungicide sensitivity in Mycosphaerella graminicola on different isolates per leaf, leaf levels at different points of time, and points in the field. Sensitivity was assessed on single isolates by means of epoxiconazole EC50-values, and monitoring of the G143A-mutation, which confers strobilurin resistance. In both years, fungicide application strategies did not cause any significant shifts in epoxiconazole sensitivity of the population median or variance over time compared to the starting population. In 2004, the end-population median was the same for all sprayed strategies, although compared to untreated median sensitivities were higher. In 2005, epoxiconazole sensitivity levels were similar on individual flag leaves and different points in the field. Measured on all isolates the EC50-values ranged from 0.007–1.15 mg l−1. In 2004, due to the high initial level of pyraclostrobin resistance, stabilisation of pyraclostrobin resistance was observed following the various combination treatments. No correlation between epoxiconazole sensitivities and pyraclostrobin resistance were observed. High input strategies using a mixture of epoxiconazole and pyraclostrobin resulted in the best control and yield response. A subpopulation of the isolates from 2004 was also screened for sensitivity towards five different triazoles of which tebuconazole proved to be least sensitive, and this could further be split into two subpopulations.  相似文献   

5.
Wheat blast is one of the most important and devastating fungal diseases of wheat in South America, South-east Asia, and now in southern Africa. The disease can reduce grain yield by up to 70% and is best controlled using integrated disease management strategies. The difficulty in disease management is compounded by the lack of durable host resistance and the ineffectiveness of fungicide sprays. New succinate dehydrogenase inhibitor (SDHI) fungicides were recently introduced for the management of wheat diseases. Brazilian field populations of the wheat blast pathogen Pyricularia oryzae Triticum lineage (PoTl) sampled from different geographical regions in 2012 and 2018 were shown to be resistant to both QoI (strobilurin) and DMI (azole) fungicides. The main objective of the current study was to determine the SDHI baseline sensitivity in these populations. Moderate levels of SDHI resistance were detected in five out of the six field populations sampled in 2012 and in most of the strains isolated in 2018. No association was found between target site mutations in the sdhB, sdhC, and sdhD genes and the levels of SDHI resistance, indicating that a pre-existing resistance mechanism not associated with target site mutations is probably present in Brazilian wheat blast populations.  相似文献   

6.
This review summarises recent investigations into the molecular mechanisms responsible for the decline in sensitivity to azole (imidazole and triazole) fungicides in European populations of the Septoria leaf blotch pathogen, Mycosphaerella graminicola. The complex recent evolution of the azole target sterol 14α‐demethylase (MgCYP51) enzyme in response to selection by the sequential introduction of progressively more effective azoles is described, and the contribution of individual MgCYP51 amino acid alterations and their combinations to azole resistance phenotypes and intrinsic enzyme activity is discussed. In addition, the recent identification of mechanisms independent of changes in MgCYP51 structure correlated with novel azole cross‐resistant phenotypes suggests that the further evolution of M. graminicola under continued selection by azole fungicides could involve multiple mechanisms. The prospects for azole fungicides in controlling European M. graminicola populations in the future are discussed in the context of these new findings. Copyright © 2012 Society of Chemical Industry  相似文献   

7.
Kansas and California wheat-growing regions differ dramatically in soils, climate, wheat cultivars, crop rotation patterns, and cultural practices, which could select for different fungal populations of Mycosphaerella graminicola. Our objective in this study was to use amplified fragment length polymorphism (AFLP) loci to assess the genetic diversity of M. graminicola populations within single fields in two widely separated, and geographically isolated sites in Kansas and California. Three primer-pair combinations were used to resolve polymorphism at 177 loci in 67 and 63 isolates from Kansas and California, respectively. Genotypic variability was high, which is consistent with a genetically diverse initial inoculum. There was no evidence of genetic disequilibrium in either population, with only 4.6% of the locus pairs in Kansas, and 5.4% of the locus pairs in California in detectable disequilibrium. The migration rate calculated between the two sites was as low as 1.8 individuals per generation, and significant differences in allele frequencies were observed. Therefore, these two populations do not represent mere subsamples of a larger, randomly mating population. This is a rare report of isolation by distance occurring between two North American populations of M. graminicola, indicating that at least some of these populations may be differentiating. Although genetic isolation by distance may occur, we cannot exclude movement of new gene combinations such as fungicide resistance or virulence between these two locations.  相似文献   

8.
To provide insight into the genetic structure of Mycosphaerella graminicola populations in Iran, a total of 221 isolates were collected from naturally infected wheat fields of five major wheat‐growing provinces and analysed using AFLP markers and mating‐type loci. All populations showed intermediate to high genotypic diversity. In the Golestan and Ardabil populations two mating types were found at near‐equal frequencies, whilst all populations were in gametic disequilibrium. Moreover, clonal haplotypes were identified in different sampling sites within a field in both the Khuzestan and Fars provinces, demonstrating that pycnidia are probably the primary source of inoculum. All five populations had low levels of gene diversity and had private bands. Low levels of gene flow and high genetic differentiation were observed among populations and different clustering methods revealed five genetically distinct groups in accordance with the sampling areas. The Golestan and East Azarbaijan populations were more genetically differentiated than the others. Random genetic drift, selection and geographic barriers may account for the differentiation of the populations. The results of this study indicate a population structure of M. graminicola in Iran contrasting to that of most other countries studied.  相似文献   

9.
The population structure and genotypic diversity of Mycosphaerella graminicola from six natural field populations in Germany were studied with molecular markers. To reveal the potential effects of plant host resistance on the pathogen population, hierarchical samples were taken from susceptible and resistant cultivars. A total of 203 single spore isolates was subjected to molecular marker analysis using the amplified fragment length polymorphism technique (AFLP). Among the 203 isolates analyzed, 142 different multilocus haplotypes (MLH) were identified revealing a high degree of genotypic diversity of the M. graminicola population. On average, a F ST value of 0.04 was found, indicating a low genetic differentiation with only 4% of the genetic variation between the local populations but leaving 96% of the genetic variation within the populations. According to the low F ST value, a high migration rate of Nm 12 was found. The observed high within-population diversity, and the significant migration between populations, prevented genetic isolation and differentiation of putative geographically separated populations. Furthermore, plant host resistance had no obvious effect on the population structure and diversity of M. graminicola. Genotypic variability can be attributed to sexual recombination which appears to have a considerably larger influence on the population structure. Gene flow on this scale could have significant implications for plant breeding and fungicide spraying programmes.  相似文献   

10.
The effect of the quinone outside inhibitors (QoI) azoxystrobin and pyraclostrobin on yields of winter wheat where QoI resistant Mycosphaerella graminicola isolates were dominant was investigated in field trials in 2006 and 2007. Pyraclostrobin significantly increased yields by 1·57 t ha?1 in 2006 and 0·89 t ha?1 in 2007 when compared to the untreated controls, while azoxystrobin only provided a significant increase of 1·28 t ha?1 in 2006. These yield increases were associated with reduction in septoria tritici blotch (STB) development as determined by weekly disease assessments over a 7 week interval. The effect of pyraclostrobin on STB was studied in controlled environment experiments using wheat seedlings inoculated with individual M. graminicola isolates. Pyraclostrobin significantly reduced STB symptoms by up to 62%, whether applied 48 h pre‐ or post‐ inoculation with resistant M. graminicola isolates containing the cytochrome b mutation G143A. Extremely limited disease (<1%) was observed on similarly treated seedlings inoculated with an intermediately resistant isolate containing the cytochrome b mutation F129L, while no disease was observed on seedlings inoculated with a wild‐type isolate. Germination studies of pycnidiospores of M. graminicola on water agar amended with azoxystrobin or pyraclostrobin showed that neither fungicide inhibited germination of spores of resistant isolates containing the mutation G143A. However, pyraclostrobin significantly reduced germ tube length by up to 46% when compared with the untreated controls. Although the QoIs can no longer be relied upon to provide effective M. graminicola control, this study provides an insight into why QoIs still provide limited STB disease control and yield increases even in situations of high QoI resistance.  相似文献   

11.
Green fluorescent protein (GFP)‐expressing transformants were used to investigate the effects of strobilurin fungicide azoxystrobin on Mycosphaerella graminicola infection. Azoxystrobin treatments (125 or 250 g AI ha?1) were applied at various stages of the infection process under controlled conditions. GFP transformants showed conserved in vitro sensitivity to azoxystrobin and pathogenicity. Azoxystrobin controlled over 90% of M graminicola infections when applied before or during penetration of the pathogen (15% of the incubation phase). Azoxystrobin also impaired the growth of intercellular hyphae in M graminicola post‐penetration infection stages when applied at up to 50% of the incubation phase. Incubating infections observed in treated leaves were viable, but their growth was impaired and they did not induce necrosis under controlled conditions. Reduction by half of azoxystrobin dosage had little or no effect on azoxystrobin efficiency in controlling M graminicola. The contribution of post‐penetration fungistatic effect to azoxystrobin curative properties toward M graminicola in a field situation is discussed. © 2001 Society of Chemical Industry  相似文献   

12.
BACKGROUND: It is possible that a single nucleotide polymorphism (SNP) (G143A mutation) in the cytochrome b gene could confer resistance to quinone outside inhibiting (QoI) fungicides (strobilurins) in rice blast fungus because this mutation caused a high level of resistance to fungicides such as azoxystrobin in Pyricularia grisea Sacc. and other fungal plant pathogens. The aim of this study was to survey Magnaporthe oryzae B Couch sp. nov. isolates in Japan for resistance to QoIs, and to try to develop molecular detection methods for QoI resistance. RESULTS: A survey on the QoI resistance among M. oryzae isolates from rice was conducted in Japan. A total of 813 single‐spore isolates of M. oryzae were tested for their sensitivity to azoxystrobin using a mycelial growth test on PDA. QoI fungicide resistance was not found among these isolates. The introduction of G143A mutation into a plasmid containing the cytochrome b gene sequence of rice blast fungus was achieved by site‐directed mutagenesis. Molecular diagnostic methods were developed for identifying QoI resistance in rice blast fungus using the plasmid construct. CONCLUSION: As the management of rice blast disease is often dependent on chemicals, the rational design of control programmes requires a proper understanding of the fungicide resistance phenomenon in field populations of the pathogen. Mutation of the cytochrome b gene of rice blast fungus would be specifically detected from diseased leaves and seeds using the molecular methods developed in this study. Copyright © 2009 Society of Chemical Industry  相似文献   

13.
Damage caused by the wheat pathogen Mycosphaerella graminicola increased rapidly during the last two decades in the Czech Republic. We collected isolates from naturally infected fields in seven wheat-growing locations and analysed these using eight microsatellite markers. All markers were highly polymorphic. We found a high degree of genetic diversity and low clonality within all sampled Czech populations. We identified 158 unique multilocus haplotypes among 184 isolates. Field populations showed weak genetic structure but we detected more differentiation between climatic regions within the Czech Republic. We compared the Czech field populations to populations from the United Kingdom, Germany and Switzerland and found a marked differentiation between Czech populations and Western European populations. We hypothesize that decades of different agricultural practices, including the use of different wheat cultivars, may explain this genetic differentiation. We detected a rapid increase in QoI fungicide resistance during the sampling period from 2005 to 2011, coinciding with the widespread application of this class of fungicides in the Czech Republic. M. graminicola populations in the Czech Republic underwent a rapid adaptive evolution from sensitivity to resistance similar to what was described earlier in Western Europe.  相似文献   

14.
Fungicides have not been effective in controlling the wheat blast disease in Brazil. An earlier analysis of 179 isolates of Pyricularia oryzae Triticum lineage (PoTl) sampled from wheat fields across six populations in central-southern Brazil during 2012 discovered a high level of resistance to strobilurin fungicides. Here we analysed azole resistance in the same strains based on EC50 measurements for tebuconazole and epoxiconazole. All six Brazilian populations of PoTl exhibited high resistance to both azoles, with in vitro EC50 values that were at least 35 to 50 times higher than the recommended field doses. We sequenced the CYP51A and CYP51B genes to determine if they were likely to play a role in the observed azole resistance. Although we found five distinct haplotypes in PoTl carrying four nonsynonymous substitutions in CYP51A, none of these substitutions were correlated with elevated EC50. CYP51B was sequenced for nine PoTl isolates, three each representing low, medium, and high tebuconazole EC50. Both PoTl CYP51A and CYP51B could complement yeast CYP51 function. All PoTl CYP51A-expressing yeast transformants were less sensitive to triazoles than the PoTl CYP51B ones. Transformants expressing PoTl CYP51A haplotype H1 carrying the R158K substitution were not more resistant than those expressing PoTl CYP51A haplotype H5, which is synonymous to haplotype H6, found in triazole-sensitive P. oryzae Oryza isolates from rice blast. Therefore, the reduced triazole sensitivity of wheat blast isolates compared to rice blast isolates appears to be associated with a non-target-site related resistance mechanism acquired after higher exposure to triazoles.  相似文献   

15.
A novel, high‐resolution melting (HRM) analysis was developed to detect single nucleotide polymorphisms (SNPs) associated with resistance to fenhexamid (hydroxyanilides) and boscalid (succinate dehydrogenase inhibitors) in Botrytis cinerea isolates. Thirty‐six single‐spore isolates arising from 13 phenotypes were selected and tested for fungicide sensitivity. Germ tube elongation assays showed two distinct sensitivity levels for each fungicide. Sequencing revealed that resistance to fenhexamid was due to a nucleotide change in the erg27 gene, resulting in an amino acid replacement of phenylalanine (F) with serine (S) or valine (V) at position 412 of the protein, whereas in isolates resistant to boscalid, a nucleotide change in the sdhB gene resulted in the replacement of histidine (H) with arginine (R) or tyrosine (Y) at position 272 of the respective protein. In each case, melting curve analysis generated three distinct profiles corresponding to the presence of each nucleotide in the targeted areas. HRM analysis successfully detected and differentiated the substitutions associated with resistance to both fungicides. In vitro bioassays, direct sequencing and high‐resolution melting analysis showed a 100% correlation with detection of resistance. The results demonstrate the utility of HRM analysis as a potential molecular tool for routine detection of fungicide resistance using known polymorphic genes of B. cinerea populations.  相似文献   

16.

BACKGROUND

Fenpicoxamid is a new fungicide for control of Zymoseptoria tritici, and is a derivative of the natural product UK‐2A. Its mode of action and target site interactions have been investigated.

RESULTS

UK‐2A strongly inhibited cytochrome c reductase, whereas fenpicoxamid was much less active, consistent with UK‐2A being the fungicidally active species generated from fenpicoxamid by metabolism. Both compounds caused rapid loss of mitochondrial membrane potential in Z. tritici spores. In Saccharomyces cerevisiae, amino acid substitutions N31K, G37C and L198F at the Qi quinone binding site of cytochrome b reduced sensitivity to fenpicoxamid, UK‐2A and antimycin A. Activity of fenpicoxamid was not reduced by the G143A exchange responsible for strobilurin resistance. A docking pose for UK‐2A at the Qi site overlaid that of antimycin A. Activity towards Botrytis cinerea was potentiated by salicylhydroxamic acid, showing an ability of alternative respiration to mitigate activity. Fungitoxicity assays against Z. tritici field isolates showed no cross‐resistance to strobilurin, azole or benzimidazole fungicides.

CONCLUSION

Fenpicoxamid is a Qi inhibitor fungicide that provides a new mode of action for Z. tritici control. Mutational and modeling studies suggest that the active species UK‐2A binds at the Qi site in a similar, but not identical, fashion to antimycin A. © 2017 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.  相似文献   

17.
The economically most damaging disease to winter rye is brown rust (Puccinia recondita f.sp.secalis Rob.ex Desm.). There are fungicides of different drug groups in order to fight brown rust in agriculture. Usually mixtures of different active ingredients with different modes of action can be used for the treatment of fungal pathogens. In experiments single agents like pyrazole-carboxamides, azoles and strobilurines were compared to drug combination products like azole strobilurin, azole-carboxamide, or azole carboxamide-strobilurin mixture and their fungicide performance was determined. The active ingredients Epoxiconazole, Pyraclostrobin, and Fluxapyroxad were tested. In eight trials from 2012 to 2013 Fluxapyroxad reached the highest levels of efficiency for P. recondita. Pyraclostrobin or the combination of Pyraclostrobin and Epoxiconazole showed higher efficiencies than Epoxiconazole or the combination of Epoxiconazole and Metconazole in all trials. The thousand seed weight and income from the harvest reflect the efficiency against P. recondita. Both azole strobilurin mixtures as well as the azole carboxamide mixtures are suitable for the control of leaf rust in agriculture. In order to minimize the risk of resistance to strobilurin and azole carboxamide mixtures should be used alternately.  相似文献   

18.
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19.
Control of the potato late blight pathogen Phytophthora infestans relies heavily on chemicals. The fungicide metalaxyl‐M (Mefenoxam) has played an important role in controlling the disease, but insensitivity to the fungicide in certain isolates is now of major concern. A genetic basis for resistance to metalaxyl suggests the possibility for linking resistance phenotypes to specific population genetic markers, but in order to do this, the population genetic structure and mode of reproduction in a population must first be well described. The dynamics of metalaxyl‐M resistance in the Danish population of P. infestans was characterized over the course of the 2013 growing season, as was the population genetic structure, using simple sequence repeat (SSR) genotypes and single nucleotide polymorphism (SNP)‐based mitochondrial haplotyping of over 80 isolates. Both mating types A1 and A2 were present in most fields, but tests for recombination showed that clonal reproduction dominates in Danish populations. Genotype was not linked to haplotype and no differentiation was observed at the haplotype level, but rather between fields. Resistance phenotypes were linked to specific SSR alleles, demonstrating the potential for a more precise SNP‐based marker system for predicting resistance to metalaxyl‐M.  相似文献   

20.
Restriction fragment length polymorphism (RFLP) markers were used to assess the genetic structure of populations of Mycosphaerella graminicola collected from wheat fields. A total of 585 isolates representing 10 field populations were sampled from Iran, Argentina and Australia. The genetic structure of M. graminicola populations from Iran and Argentina is described for the first time. Results were compared to previously investigated populations from Israel, Uruguay and Australia. Populations from Iran exhibited high clonality and low gene diversity, suggesting an inoculation event. Populations from uninoculated fields in Argentina had gene and genotype diversities similar to previously described European and North American populations. Genotype diversity was high for populations from Australia and tests for multilocus associations were consistent with sexual recombination in these populations. Gene diversity was low and fixed alleles were found for several loci. These findings are consistent with a relatively small founding population for Australia. These 10 new populations were integrated into a genetic distance comparison with 13 global populations that were characterized earlier.  相似文献   

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