Molecular basis of fungicide resistance
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Molecular basis of fungicide resistance
Up-to-date resource of publications focused on the molecular basis of fungicide resistance and fungicide resistance management in crop pathogens.
Curated by Melvin Bolton
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Inherent Resistance to 14α-demethylation Inhibitor Fungicides in Colletotrichum truncatum is likely linked to CYP51A and/or CYP51B Gene Variants | Phytopathology

Inherent Resistance to 14α-demethylation Inhibitor Fungicides in Colletotrichum truncatum is likely linked to CYP51A and/or CYP51B Gene Variants | Phytopathology | Molecular basis of fungicide resistance | Scoop.it
Anthracnose disease, caused by Colletotrichum truncatum, affects marketable yield during preharvest production and postharvest storage of fruits and vegetables worldwide. Demethylation inhibitor fungicides (DMIs) are among the very few chemical classes of single-site mode of action fungicides that are effective in controlling anthracnose disease. However, some species are inherently resistant to DMIs and more information is needed to understand this phenomenon. Isolates of C. truncatum were collected from the USA and China from peach, soybean, citrus, and begonia and sensitivity to six DMIs (difenoconazole, propiconazole, metconazole, tebuconazole, flutriafol and fenbuconazole) was determined. Compared with DMI sensitive isolates of C. fructicola, C. siamense, and C. fioriniae (EC50 value ranging from 0.03 µg/ml to 16.2 µg/ml to six DMIs), C. truncatum and C. nymphaeae were resistant to flutriafol and fenbuconazole (with EC50 value of more 50 µg/ml). Moreover, C. truncatum was resistant to tebuconazole and metconazole (with resistance factor of 27.4 and 96.0), and displayed reduced sensitivity to difenoconazole and propiconazole (with resistance factor of 5.1 and 5.2). Analysis of the Colletotrichum spp. genome revealed two potential DMI targets, CYP51A and CYP51B, that putatively encode P450 sterol 14α-demethylases. Both genes were identified and sequenced from C. truncatum and other species and no correlation between CYP51 gene expression levels and fungicide sensitivity was found. Four amino acid variations L208Y, H238R, S302A, and I366L in CYP51A, and three variations H373N, M376L, and S511T in CYP51B correlated with the DMI resistance phenotype. CYP51A structure model analysis suggested the four alteration may reduce azole affinity. Likewise, CYP51B structure analysis suggested the H373N and M376L variants may change the conformation of the DMI binding pocket, thereby causing differential sensitivity to DMI fungicides in C. truncatum.
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Using epidemiological principles to explain fungicide resistance management tactics: why do mixtures outperform alternations? | Phytopathology

Using epidemiological principles to explain fungicide resistance management tactics: why do mixtures outperform alternations? | Phytopathology | Molecular basis of fungicide resistance | Scoop.it
Whether fungicide resistance management is optimised by spraying chemicals with different modes of action as a mixture (i.e. simultaneously) or in alternation (i.e. sequentially) has been studied by experimenters and modellers for decades. However results have been inconclusive. We use previously-parameterised and validated mathematical models of wheat septoria leaf blotch and grapevine powdery mildew to test which tactic provides better resistance management, using the total yield before resistance causes disease control to become economically-ineffective (“lifetime yield”) to measure effectiveness. We focus on tactics involving the combination of a low-risk and a high-risk fungicide, and the case in which resistance to the high-risk chemical is complete (i.e. in which there is no partial resistance). Lifetime yield is then optimised by spraying as much low-risk fungicide as is permitted, combined with slightly more high-risk fungicide than needed for acceptable initial disease control, applying these fungicides as a mixture. That mixture rather than alternation gives better performance is invariant to model parameterisation and structure, as well as the pathosystem in question. However if comparison focuses on other metrics, e.g. lifetime yield at full label dose, either mixture or alternation can be optimal. Our work shows how epidemiological principles can explain the evolution of fungicide resistance, and also highlights a theoretical framework to address the question of whether mixture or alternation provides better resistance management. It also demonstrates that precisely how spray tactics are compared must be given careful consideration.
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Slow and temperature‐mediated pathogen adaptation to a nonspecific fungicide in agricultural ecosystem

Slow and temperature‐mediated pathogen adaptation to a nonspecific fungicide in agricultural ecosystem | Molecular basis of fungicide resistance | Scoop.it
The spread of antimicrobial resistance and global change in air temperature represent two major phenomena that are exerting a disastrous impact on natural and social issues but investigation of the interaction between these phenomena in an evolutionary context is limited. In this study, a statistical genetic approach was used to investigate the evolution of antimicrobial resistance in agricultural ecosystem and its association with local air temperature, precipitation, and UV radiation. We found no resistance to mancozeb, a nonspecific fungicide widely used in agriculture for more than half a century, in 215 Alternaria alternata isolates sampled from geographic locations along a climatic gradient and cropping system representing diverse ecotypes in China, consistent with low resistance risk in many nonspecific fungicides. Genetic variance accounts for ~35% of phenotypic variation, while genotype–environment interaction is negligible, suggesting that heritability plays a more important role in the evolution of resistance to mancozeb in plant pathogens than phenotypic plasticity. We also found that tolerance to mancozeb in agricultural ecosystem is under constraining selection and significantly associated with local air temperature, possibly resulting from a pleiotropic effect of resistance with thermal and other ecological adaptations. The implication of these results for fungicide and other antimicrobial management in the context of global warming is discussed.
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The Y137H mutation in the FgCYP51B protein confers reduced sensitivity to tebuconazole in Fusarium graminearum

The Y137H mutation in the FgCYP51B protein confers reduced sensitivity to tebuconazole in Fusarium graminearum | Molecular basis of fungicide resistance | Scoop.it
Fusarium graminearum is the main pathogen of Fusarium Head Blight (FHB), a worldwide plant disease and one of the most significant wheat diseases in China. Demethylation inhibitor (DMI) fungicides, such as tebuconazole (TEC), are widely used to control FHB, but long-term use leads to low efficacy against FHB. Earlier studies showed that DMI resistance is associated with the fungal sterol 14α-demethylase (CYP51) gene, and that point mutations in the CYP51 gene are the primary mechanism of resistance to DMI fungicides. The aims of this study were to clarify the molecular mechanisms of resistance to TEC and identify the binding sites on the FgCYP51B protein.

RESULTS
Site-directed mutagenesis was used to change the FgCYP51B gene of wide-type strain PH-1 from tyrosine to histidine at residue 137 (Y137H) to generate mutant transformant, which was confirmed to be resistant to TEC compared to the parental strains. A three-dimensional FgCYP51B model was constructed, and molecular docking simulation studies were conducted to identify the optimum binding mode with TEC. The wild-type FgCYP51B protein displayed stronger affinity to TEC than that of the mutational FgCYP51B in the molecular docking analysis.

CONCLUSION
These results indicate that a Tyr137 amino acid mutation in the FgCYP51B gene could lead to resistance to TEC and that Y137 forms part of the tebuconazole binding pocket.
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Erg4A and Erg4B Are Required for Conidiation and Azole Resistance via Regulation of Ergosterol Biosynthesis in Aspergillus fumigatus

Ergosterol, a fungus-specific sterol enriched in cell plasma membranes, is an effective antifungal drug target. However, current knowledge of the ergosterol biosynthesis process in the saprophytic human fungal pathogen Aspergillus fumigatus remains limited. In this study, we found that two endoplasmic reticulum-localized sterol C-24 reductases encoded by both erg4A and erg4B homologs are required to catalyze the reaction during the final step of ergosterol biosynthesis. Loss of one homolog of Erg4 induces the overexpression of the other one, accompanied by almost normal ergosterol synthesis and wild-type colony growth. However, double deletions of erg4A and erg4B completely block the last step of ergosterol synthesis, resulting in the accumulation of ergosta-5,7,22,24(28)-tetraenol, a precursor compound of ergosterol. Further studies indicate that erg4A and erg4B are required for conidiation but not for hyphal growth. Importantly, the Δerg4A Δerg4B mutant still demonstrates wild-type virulence in a compromised mouse model but displays remarkable increased susceptibility to antifungal azoles. Our data suggest that inhibitors of Erg4A and Erg4B may serve as effective candidates for adjunct antifungal agents with azoles.
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Plasticity of the MFS1 Promoter Leads to Multidrug Resistance in the Wheat Pathogen Zymoseptoria tritici

Plasticity of the MFS1 Promoter Leads to Multidrug Resistance in the Wheat Pathogen Zymoseptoria tritici | Molecular basis of fungicide resistance | Scoop.it
The ascomycete Zymoseptoria tritici is the causal agent of Septoria leaf blotch on wheat. Disease control relies mainly on resistant wheat cultivars and on fungicide applications. The fungus displays a high potential to circumvent both methods. Resistance against all unisite fungicides has been observed over decades. A different type of resistance has emerged among wild populations with multidrug-resistant (MDR) strains. Active fungicide efflux through overexpression of the major facilitator gene MFS1 explains this emerging resistance mechanism. Applying a bulk-progeny sequencing approach, we identified in this study a 519-bp long terminal repeat (LTR) insert in the MFS1 promoter, a relic of a retrotransposon cosegregating with the MDR phenotype. Through gene replacement, we show the insert as a mutation responsible for MFS1 overexpression and the MDR phenotype. Besides this type I insert, we found two different types of promoter inserts in more recent MDR strains. Type I and type II inserts harbor potential transcription factor binding sites, but not the type III insert. Interestingly, all three inserts correspond to repeated elements present at different genomic locations in either IPO323 or other Z. tritici strains. These results underline the plasticity of repeated elements leading to fungicide resistance in Z. tritici and which contribute to its adaptive potential.
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Screening and Characterization of a Non-cyp51A Mutation in an Aspergillus fumigatus cox10 Strain Conferring Azole Resistance

Screening and Characterization of a Non-cyp51A Mutation in an Aspergillus fumigatus cox10 Strain Conferring Azole Resistance | Molecular basis of fungicide resistance | Scoop.it
The rapid and global emergence of azole resistance in the human pathogen Aspergillus fumigatus has drawn attention. Thus, a thorough understanding of its mechanisms of drug resistance requires extensive exploration. In this study, we found that the loss of the putative calcium-dependent protein-encoding gene algA causes an increased frequency of azole-resistant A. fumigatus isolates. In contrast to previously identified azole-resistant isolates related to cyp51A mutations, only one isolate carries a point mutation in cyp51A (F219L mutation) among 105 independent stable azole-resistant isolates. Through next-generation sequencing (NGS), we successfully identified a new mutation (R243Q substitution) conferring azole resistance in the putative A. fumigatus farnesyltransferase Cox10 (AfCox10) (AFUB_065450). High-performance liquid chromatography (HPLC) analysis verified that the decreased absorption of itraconazole in related Afcox10 mutants is the primary reason for itraconazole resistance. Moreover, a complementation experiment by reengineering the mutation in a parental wild-type background strain demonstrated that both the F219L and R243Q mutations contribute to itraconazole resistance in an algA-independent manner. These data collectively suggest that the loss of algA results in an increased frequency of azole-resistant isolates with a non-cyp51A mutation. Our findings indicate that there are many unexplored non-cyp51A mutations conferring azole resistance in A. fumigatus and that algA defects make it possible to isolate drug-resistant alleles. In addition, our study suggests that genome-wide sequencing combined with alignment comparison analysis is an efficient approach to identify the contribution of single nucleotide polymorphism (SNP) diversity to drug resistance.
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A Major Facilitator Superfamily Transporter-Mediated Resistance to Oxidative Stress and Fungicides Requires Yap1, Skn7, and MAP Kinases in the Citrus Fungal Pathogen Alternaria alternata

A Major Facilitator Superfamily Transporter-Mediated Resistance to Oxidative Stress and Fungicides Requires Yap1, Skn7, and MAP Kinases in the Citrus Fungal Pathogen Alternaria alternata | Molecular basis of fungicide resistance | Scoop.it
Major Facilitator Superfamily (MFS) transporters play an important role in multidrug resistance in fungi. We report an AaMFS19 gene encoding a MFS transporter required for cellular resistance to oxidative stress and fungicides in the phytopathogenic fungus Alternaria alternata. AaMFS19, containing 12 transmembrane domains, displays activity toward a broad range of substrates. Fungal mutants lacking AaMFS19 display profound hypersensitivities to cumyl hydroperoxide, potassium superoxide, many singlet oxygen-generating compounds (eosin Y, rose Bengal, hematoporphyrin, methylene blue, and cercosporin), and the cell wall biosynthesis inhibitor, Congo red. AaMFS19 mutants also increase sensitivity to copper ions, clotrimazole, fludioxonil, and kocide fungicides, 2-chloro-5-hydroxypyridine (CHP), and 2,3,5-triiodobenzoic acid (TIBA). AaMFS19 mutants induce smaller necrotic lesions on leaves of a susceptible citrus cultivar. All observed phenotypes in the mutant are restored by introducing and expressing a wild-type copy of AaMFS19. The wild-type strain of A. alternata treated with either CHP or TIBA reduces radial growth and formation and germination of conidia, increases hyphal branching, and results in decreased expression of the AaMFS19 gene. The expression of AaMFS19 is regulated by the Yap1 transcription activator, the Hog1 and Fus3 mitogen-activated protein (MAP) kinases, the ‘two component’ histidine kinase, and the Skn7 response regulator. Our results demonstrate that A. alternata confers resistance to different chemicals via a membrane-bound MFS transporter.
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The binding mechanism between azoles and FgCYP51B, sterol 14α‐demethylase of Fusarium graminearum

The binding mechanism between azoles and FgCYP51B, sterol 14α‐demethylase of Fusarium graminearum | Molecular basis of fungicide resistance | Scoop.it
BACKGROUND

Fusarium graminearum is the main pathogen of Fusarium Head Blight (FHB), a worldwide plant disease and one of the major wheat diseases in China. The control of the FHB is mainly dependent on the application of DMIs fungicides. Fungal sterol 14α-demethylase enzymes (CYP51) are the main target for DMIs fungicides. In order to investigate the binding mechanism between azoles and CYP51B in F.graminearum, the molecular modeling study and biological evaluation were performed.
RESULTS

Firstly, the homology model based on the crystal structure of Aspergillus fumigatus was built. Secondly, molecular docking and molecular dynamics (MD) simulation were used to identify the optimum binding mode between Propiconazole (PRP), Diniconazole (DIN), Triadimenol (TRL), Tebuconazole (TEC), and Triadimefon (TRN) with FgCYP51B. Furthermore, binding free energy of the five protein-inhibitor complexes was calculated by Molecular Mechanics Generalized Born Surface Area and Poisson–Boltzmann Surface Area (MM-GB/PBSA) methods. The key residues to the selective binding of azoles to FgCYP51B were recognized by per-residue free energy decomposition analysis. The five ligands have similar binding mode in the active pocket. Inhibitors PRP and TEC have more beneficial binding free energy to the enzyme than TRN, TRL and DIN. Furthermore, the Amino acid residues Phe511, Val136, Ile374, Ala308, Ser312 and Try137 of FgCYP51B are the key residues interacting with azoles fungicides. The EC50 value of PRP, TEC, DIN, TRL and TRN is 0.024, 0.047, 0.148, 0.154 and 0.474 mg/L according to the experimental evaluation, respectively. These five molecules exhibited potential inhibitory activity against CYP51B protein from the F. graminearum.
CONCLUSION

The results suggest that azoles fungicides for FgCYP51B should possess more hydrophobic groups interacting with residues Phe511, Val136, Ile374, Ala308, Ser312 and Tyr137. PRP and TEC should be preferred to control Fusarium Head Blight disease than DIN, TRL and TRN.
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Steve Marek's curator insight, July 21, 2017 7:40 PM
Protein-fungicide structure models predict sensitivity/resistance. Very cool!
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Molecular and biochemical characterization of dimethachlone resistant isolates of Sclerotinia sclerotiorum

Molecular and biochemical characterization of dimethachlone resistant isolates of Sclerotinia sclerotiorum | Molecular basis of fungicide resistance | Scoop.it
Sclerotinia sclerotiorum is a necrotrophic fungal plant pathogen with a broad host range. The dicarboximide fungicide dimethachlone has been used to control this pathogen for more than a decade and resistance to dimethachlone has recently been reported in China. Compared with sensitive isolates, the three dimethachlone resistant isolates with resistance ratios of 78.3, 85.5, and 94.8 exhibited significantly (P < 0.05) higher cell membrane permeability and peroxidase and polyphenol oxidase activities. Dimethachlone at 0.25 μg/mL significantly increased cell membrane permeability and enhanced activity of the two enzymes in both resistant and sensitive isolates. There were no significant differences in glycerol or oxalate content between the resistant and sensitive isolates. Dimethachlone treatment increased glycerol content in the resistant isolates and reduced in the sensitive isolates (P < 0.01). Sequencing of three genes involved in two-component signal pathway and of three genes in mitogen-activated protein (MAP) kinase cascade demonstrated that the dimethachlone resistant isolates HLJ4 and HLJ6 harbored point mutations of I232T and G1087D, respectively, in the deduce amino acid sequence of the histidine kinase (HK) gene Sshk. HLJ4 had a point mutation of P96L in the deduced amino acid sequence of the MAP kinase-kinase gene SsPbs. The expression levels of the Sshk gene were higher in HLJ4 and HLJ6 than in HLJ3 and the sensitive isolate HLJMG2, and transcription of the Sshk gene was up-regulated by dimethachlone for the three resistant isolates.
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Mutations in sdh genes in field isolates of Zymoseptoria tritici and impact on the sensitivity to various succinate dehydrogenase inhibitors

Mutations in sdh genes in field isolates of Zymoseptoria tritici and impact on the sensitivity to various succinate dehydrogenase inhibitors | Molecular basis of fungicide resistance | Scoop.it
Zymoseptoria tritici is the causal agent of septoria tritici blotch (STB), a foliar wheat disease important worldwide. Succinate dehydrogenase inhibitors (SDHIs) have been used in cereals for effective control of STB for several years, but resistance towards SDHIs has been reported in several phytopathogenic fungi. Resistance mechanisms are target-site mutations in the genes coding for subunits B, C and D of the succinate dehydrogenase (SDH) enzyme. Previous monitoring data in Europe indicated the presence of single isolates of Z. tritici with reduced SDHI sensitivity. These isolates carried mutations leading to amino acid exchanges: C-T79N, C-W80S in 2012; C-N86S in 2013; B-N225T and C-T79N in 2014; and C-V166M, B-T268I, C-N86S, C-T79N and C-H152R in 2015. The current study provides results from microtitre and greenhouse experiments to give an insight into the impact of different mutations in field isolates on various SDHIs. In microtitre tests, the highest EC50 values for all tested SDHIs were obtained with mutants carrying C-H152R. Curative greenhouse tests with various SDHIs confirmed the findings of microtitre tests that isolates with C-H152R are, in general, controlled with lower efficacy than isolates carrying B-T268I, C-T79N and C-N86S. SDHI-resistant isolates of Z. tritici found in the field were shown to have cross-resistance towards all SDHIs tested. So far, SDHI-resistant isolates of Z. tritici have been found in low frequencies in Europe. Therefore, FRAC recommendations for resistance management in cereals, including a limited number of applications, alternation and combination with other MOAs, should be followed to prolong SDHI field efficacy.
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Bioactivity and structure-activity relationship of cinnamic acid esters and their derivatives as potential antifungal agents for plant protection

Bioactivity and structure-activity relationship of cinnamic acid esters and their derivatives as potential antifungal agents for plant protection | Molecular basis of fungicide resistance | Scoop.it
A series of cinnamic acid esters and their derivatives were synthesized and evaluated for antifungal activities in vitro against four plant pathogenic fungi by using the mycelium growth rate method. Structure−activity relationship was derived also. Almost all of the compounds showed some inhibition activity on each of the fungi at 0.5 mM. Eight compounds showed the higher average activity with average EC50 values of 17.4–28.6 μg/mL for the fungi than kresoxim-methyl, a commercial fungicide standard, and ten compounds were much more active than commercial fungicide standards carbendazim against P. grisea or kresoxim-methyl against both P. grisea and Valsa mali. Compounds C1 and C2 showed the higher activity with average EC50 values of 17.4 and 18.5 μg/mL and great potential for development of new plant antifungal agents. The structure−activity relationship analysis showed that both the substitution pattern of the phenyl ring and the alkyl group in the alcohol moiety significantly influences the activity. There exists complexly comprehensive effect between the substituents on the phenyl ring and the alkyl group in the alcohol moiety on the activity. Thus, cinnamic acid esters showed great potential the development of new antifungal agents for plant protection due to high activity, natural compounds or natural compound framework, simple structure, easy preparation, low-cost and environmentally friendly.
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A novel major facilitator superfamily transporter in Penicillium digitatum (PdMFS2) is required for prochloraz resistance, conidiation and full virulence

A novel major facilitator superfamily transporter in Penicillium digitatum (PdMFS2) is required for prochloraz resistance, conidiation and full virulence | Molecular basis of fungicide resistance | Scoop.it
A novel MFS transporter gene, Pdmfs2, was isolated from P. digitatum. The full-length DNA of Pdmfs2 had a 1590 bp ORF encoding a full-size MFS transporter with 529 amino acids. In a prochloraz-resistant strain (PdHS-F6), Pdmfs2 transcript level was up-regulated compared with the prochloraz-sensitive strain (PdHS-E3) and could be induced by 7 μg prochloraz/ml. The deletion of Pdmfs2 (ΔPdmfs2) in PdHS-F6 led to increased susceptibility to prochloraz and lower EC50 value (the concentration of prochloraz producing 50 % growth inhibition) compared with the PdHS-F6 or complementation strain (COPdmfs2). The ΔPdmfs2 strain was defective in conidia yield and virulence towards citrus fruits, while the complementation of Pdmfs2 could restore the phenotypic features to a large extent.
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Regulatory roles of introns in fungicide sensitivity of Fusarium graminearum - Li - 2017 - Environmental Microbiology - Wiley Online Library

Regulatory roles of introns in fungicide sensitivity of Fusarium graminearum - Li - 2017 - Environmental Microbiology - Wiley Online Library | Molecular basis of fungicide resistance | Scoop.it
Although the roles of introns have been much debated in eukaryotic organisms, none of them have been functionally characterized in Fusarium graminearum. In this study, we characterized the roles of introns in regulation of fungicide-sensitivity of F. graminearum. β2tub, cyp51A and myosin-5 are important target genes of benzimidazoles, triazoles and cyanoacrylates respectively. To explore the sensitivity regulation functions of introns in target genes, several detailed deletion studies were completed on the intronic regions of β2tub, cyp51A and myosin-5. Phenotypic analyses showed that deletion of the fourth intron from β2tub gene (designated β2Δi4), the sole intron from cyp51A gene (cyp51A-Δi) and the second intron from myosin-5 gene (myo5-Δi2) exhibited an increased sensitivity to corresponding fungicides. In contrast, deletion of the first or second intron from β2tub gene exhibited a decreased sensitivity to carbendazim. qRT-PCR showed that the mRNA transcript levels of target genes were significantly downregulated in β2Δi4, cyp51A-Δi and myo5-Δi2 respectively. Meanwhile, Western blot assays revealed that the protein expression levels of β2tub was also dramatically reduced in β2Δi4, but accumulated in β2Δi1 and β2Δi2. Overall, our results indicate that introns in target genes significantly regulate the fungicide-sensitivity by influencing expression of the corresponding resident genes in F. graminearum.
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Glutathione transferase-mediated benzimidazole-resistance in Fusarium graminearum - ScienceDirect

Glutathione transferase-mediated benzimidazole-resistance in Fusarium graminearum - ScienceDirect | Molecular basis of fungicide resistance | Scoop.it
Fusarium graminearum laboratory mutants moderately (MR) and highly (HR) benzimidazole-resistant, carrying or not target-site mutations at the β2-tubulin gene were utilized in an attempt to elucidate the biochemical mechanism(s) underlying the unique BZM-resistance paradigm of this fungal plant pathogen. Relative expression analysis in the presence or absence of carbendazim (methyl-2-benzimidazole carbamate) using a quantitative Real Time qPCR (RT-qPCR) revealed differences between resistant and the wild-type parental strain although no differences in expression levels of either β1- or β2-tubulin homologue genes were able to fully account for two of the highly resistant phenotypes. Glutathione transferase (GST)-mediated detoxification was shown to be -at least partly- responsible for the elevated resistance levels of a HR isolate bearing the β2-tubulin Phe200Tyr resistance mutation compared with another MR isolate carrying the same mutation. This benzimidazole-resistance mechanism is reported for the first time in F. graminearum. No indications of detoxification involved in benzimidazole resistance were found for the rest of the isolates as revealed by GST and glutathione peroxidase (GPx) activities and bioassays using monoxygenase and hydrolase detoxification enzyme inhibiting synergists. Interestingly, besides the Phe200Tyr mutation-carrying HR isolate, the remaining highly-carbendazim resistant phenotypes could not be associated with any of the target site modification/overproduction, detoxification or reduced uptake-increased efflux mechanisms.
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Nucleic adaptability of heterokaryons to fungicides in a multinucleate fungus, Sclerotinia homoeocarpa

Nucleic adaptability of heterokaryons to fungicides in a multinucleate fungus, Sclerotinia homoeocarpa | Molecular basis of fungicide resistance | Scoop.it
Sclerotinia homoeocarpa is the causal organism of dollar spot in turfgrasses and is a multinucleate fungus with a history of resistance to multiple fungicide classes. Heterokaryosis gives rise to the coexistence of genetically distinct nuclei within a cell, which contributes to genotypic and phenotypic plasticity in multinucleate fungi. We demonstrate that field isolates, resistant to either a demethylation inhibitor or methyl benzimidazole carbamate fungicide, can form heterokaryons with resistance to each fungicide and adaptability to serial combinations of different fungicide concentrations. Field isolates and putative heterokaryons were assayed on fungicide-amended media for in vitro sensitivity. Shifts in fungicide sensitivity and microsatellite genotypes indicated that heterokaryons could adapt to changes in fungicide pressure. Presence of both nuclei in heterokaryons was confirmed by detection of a single nucleotide polymorphism in the β-tubulin gene, the presence of microsatellite alleles of both field isolates, and the live-cell imaging of two different fluorescently tagged nuclei using laser scanning confocal microscopy. Nucleic adaptability of heterokaryons to fungicides was strongly supported by the visualization of changes in fluorescently labeled nuclei to fungicide pressure. Results from this study suggest that heterokaryosis is a mechanism by which the pathogen adapts to multiple fungicide pressures in the field.
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Jonathan Lapleau's curator insight, January 17, 9:29 AM
More we understand pests, better we can fight them back !
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Characterization of boscalid-resistance conferring mutations in the SdhB subunit of respiratory complex II and impact on fitness and mycotoxin production in Penicillium expansum laboratory strains ...

Characterization of boscalid-resistance conferring mutations in the SdhB subunit of respiratory complex II and impact on fitness and mycotoxin production in Penicillium expansum laboratory strains ... | Molecular basis of fungicide resistance | Scoop.it
Laboratory mutants of Penicillium expansum highly resistant (Rfs: 90 to > 500, based on EC50s) to Succinate Dehydrogenase Inhibitors (SDHIs) were isolated after UV-mutagenesis and selection on media containing boscalid. A positive correlation was found between sensitivity of isolates to boscalid and other SDHIs such as isopyrazam and carboxin but not to fungicides affecting other cellular pathways or processes, such as the triazole flusilazole, the phenylpyrrole fludioxonil, the anilinopyrimidine cyprodinil and the benzimidazole benomyl. Most of the boscalid-resistant strains were more sensitive to the SDHI fluopyram and the QoI pyraclostrobin. In order to investigate the mechanism responsible for the observed resistance profiles, part of the SdhB subunit isolated the wild type and boscalid-resistant isolates, was genetically characterized. Comparison of the deduced amino-acid sequence between resistant and wild-type isolates revealed two point mutations at a position corresponding to codon 272 of the respective SdhB protein in Botrytis cinerea. The substitution of histidine by arginine was found in boscalid-resistant isolates which were equally sensitive to fluopyram compared with the wild-type whereas the replacement of histidine by tyrosine was found in strains with increased sensitivity to fluopyram. No adverse effects of resistance mutations were observed on fitness determining parameters such as osmotic sensitivity, sporulation and pathogenicity, while mycelial growth rate and spore germination was negatively affected in some of the mutants studied. P. expansum mutant strains displayed significantly perturbed patulin and citrinin levels as compared to the wild-type parent strain both in vitro and in vivo as revealed by thin layer (TLC) and high performance liquid chromatography (HPLC).
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A new mechanism for reduced sensitivity to demethylation‐inhibitor fungicides in the fungal banana black Sigatoka pathogen Pseudocercospora fijiensis

A new mechanism for reduced sensitivity to demethylation‐inhibitor fungicides in the fungal banana black Sigatoka pathogen Pseudocercospora fijiensis | Molecular basis of fungicide resistance | Scoop.it
The Dothideomycete Pseudocercospora fijiensis, previously Mycosphaerella fijiensis, is the causal agent of black Sigatoka, one of the most destructive diseases of bananas and plantains. Disease management depends on fungicide applications with a major share for sterol demethylation-inhibitors (DMIs). The continued use of DMIs puts a considerable selection pressure on natural P. fijiensis populations enabling the selection of novel genotypes with reduced sensitivity. The hitherto explanatory mechanism for this reduced sensitivity was the presence of non-synonymous point mutations in the target gene Pfcyp51, encoding the sterol 14α-demethylase enzyme. Here, we demonstrate a second mechanism involved in DMI sensitivity of P. fijiensis. We identified a 19bp element in the wild type (wt) Pfcyp51 promoter that concatenates in strains with reduced DMI sensitivity. A PCR assay identified up to six Pfcyp51 promoter repeats in four field populations of P. fijiensis in Costa Rica. We used transformation experiments to swap the wild type promoter of a sensitive field isolate with a promoter from a strain with reduced DMI sensitivity that comprised multiple insertions. Comparative in vivo phenotyping showed a functional and proportional upregulation of Pfcyp51, which consequently decreased DMI sensitivity. Our data demonstrate that point mutations in the Pfcyp51 coding domain as well as promoter inserts contribute to reduced DMI sensitivity of P. fijiensis. These results bring new insights into the importance of the appropriate use of DMIs and the need for the discovery of new molecules for black Sigatoka management.
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Identifying when it is financially beneficial to increase or decrease fungicide dose as resistance develops

Identifying when it is financially beneficial to increase or decrease fungicide dose as resistance develops | Molecular basis of fungicide resistance | Scoop.it
When fungicide efficacy declines due to the development of resistance in the pathogen population, growers have to either change to an alternative mode of action or adjust their treatment program. Adjustments may include to either decrease (or stop) use of the mode of action, or to increase the total dose applied (by increasing number of applications and/or dose per application, where permitted) to try to maintain effective disease control. We explore the circumstances under which increasing/decreasing total applied fungicide is financially optimal. We use a model, based on field data, and show how to optimise the dose of fungicide applied when fungicide resistance develops in a pathogen population. We use the model to explore contrasting patho-systems and fungicide classes. When qualitative fungicide resistance develops, the shape of the disease-yield loss relationship determines whether the optimal total dose increases or decreases with increasing frequency of resistance in the pathogen population. When quantitative fungicide resistance develops, such that effective control can still be obtained with doses close to the maximum permitted dose, the optimal dose increases with increasing frequency of resistance in the pathogen population.
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Peter Ojo Uwukhor's curator insight, October 4, 2017 6:56 AM
What happens when fungi resist fungicides. This is borne out of a thesis on the antagonistic and biological measures of fungi Trichoderma against fusarium oxysporium affecting tomato plants in the presence of fungicides such as mancozeb. Research shows that some high levels of chemical such as mancozeb influence and help in the sporulation of fusarium. and thus slows down the inhibitoon process of the antagonism by the trichoderma antagonist. this work is on going and suggestions are needed to buttress the points for fungi resistance and adaptation to fungicides
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Changes in field dose‐response curves for DMI and QoI fungicides against Zymoseptoria tritici, related to laboratory sensitivity phenotyping and genotyping assays

Changes in field dose‐response curves for DMI and QoI fungicides against Zymoseptoria tritici, related to laboratory sensitivity phenotyping and genotyping assays | Molecular basis of fungicide resistance | Scoop.it
BACKGROUND

Insensitivity of Z. tritici to de-methylation inhibitor (DMI) and quinone outside inhibitor (QoI) fungicides has been widely reported from laboratory studies, but the relationships between laboratory sensitivity phenotype or target site genotype and field efficacy remain uncertain. This article reports field experiments quantifying dose response curves, and investigates the relationships between field performance and in vitro EC50 values for DMIs, and frequency of the G143A substitution conferring QoI resistance.
RESULTS

Data were analysed from 83 field experiments over 21 years. Response curves were fitted, expressed as percentage control, rising towards an asymptote with increasing dose. Decline in DMI efficacy over years was associated with a decrease in the asymptote, and reduced curvature. Field ED50 values were positively related to in vitro EC50 values for isolates of Z. tritici collected over a 14 year period. Loss of QoI efficacy was expressed through a change in asymptote. Increasing frequency of G143A was associated with changes in field dose response asymptotes.
CONCLUSION

New resistant strains are often detected by resistance monitoring and laboratory phenotyped/genotyped before changes in field performance are detected. The relationships demonstrated here between laboratory tests and field performance could aid translation between laboratory and field for other fungicide groups.
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Regulatory roles of introns in fungicide sensitivity of Fusarium graminearum

Regulatory roles of introns in fungicide sensitivity of Fusarium graminearum | Molecular basis of fungicide resistance | Scoop.it
Although the roles of introns have been much debated in eukaryotic organisms, none of them have been functionally characterized in Fusarium graminearum. In this study, we characterized the roles of introns in regulation of fungicide-sensitivity of F. graminearum. β2tub, cyp51A and myosin-5 are important target genes of benzimidazoles, triazoles and cyanoacrylates, respectively. To explore the sensitivity regulation functions of introns in target genes, several detailed deletion studies were completed on the intronic regions of β2tub, cyp51A, myosin-5. Phenotypic analyses showed that deletion of the 4th intron from β2tub gene (designated β2Δi4), the sole intron from cyp51A gene (cyp51A-Δi) and the 2nd intron from myosin-5 gene (myo5-Δi2) exhibited an increased sensitivity to corresponding fungicides. In contrast, deletion of the 1st or 2nd intron from β2tub gene exhibited a decreased sensitivity to carbendazim. qRT-PCR showed that the mRNA transcript levels of target genes were significantly down-regulated in β2Δi4, cyp51A-Δi and myo5-Δi2, respectively. Meanwhile, western blot assays revealed that the protein expression levels of β2tub was also dramatically inhibited in β2Δi4, but accumulated in β2Δi1 and β2Δi2. Overall, our results indicate that introns in target genes significantly regulate the fungicide-sensitivity by influencing expression of the corresponding resident genes in F. graminearum.
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Targeting Fungicide Inputs According to Need.   Annual Review of Phytopathology, 2017

Empty deFungicides should be used to the extent required to minimize economic costs of disease in a given field in a given season. The maximum number of treatments and maximum dose per treatment are set by fungicide manufacturers and regulators at a level that provides effective control under high disease pressure. Lower doses are economically optimal under low or moderate disease pressure, or where other control measures such as resistant cultivars constrain epidemics. Farmers in many countries often apply reduced doses, although they may still apply higher doses than the optimum to insure against losses in high disease seasons. Evidence supports reducing the number of treatments and reducing the applied dose to slow the evolution of fungicide resistance. The continuing research challenge is to improve prediction of future disease damage and account for the combined effect of integrated control measures to estimate the optimum number of treatments and the optimum dose needed to minimize economic costs. The theory for optimizing dose is well developed but requires translation into decision tools because the current basis for farmers’ dose decisions is unclear.scription
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Damage resistance protein (Dap) contributes to azole resistance in a sterol-regulatory-element-binding protein SrbA-dependent way

Damage resistance protein (Dap) contributes to azole resistance in a sterol-regulatory-element-binding protein SrbA-dependent way | Molecular basis of fungicide resistance | Scoop.it
The targeting of stress-response regulators has emerged as a powerful strategy to enhance azole drug efficacy and to abrogate azole drug resistance. Previously, we reported that a damage resistance protein (Dap) family, composed of DapA, DapB, and DapC, could respond to azole stress stimuli in Aspergillus fumigatus, although the exact response mechanisms remain unknown. In this study, RNA-seq analysis found that a total of 180 genes are induced by azole in a dapA-dependent manner. These genes are involved in oxidation-reduction, metabolic processes, and transmembrane transport. Following azole stress stimuli, DapA and DapC consistently show a stable endoplasmic reticulum (ER)-localization pattern. In comparison, the sterol-regulatory element-binding protein SrbA is capable of nuclear translocation from the ER after azole-stress stimuli, suggesting that SrbA, but not Daps, can directly sense azole stress. Moreover, we found that SrbA is required for the normal expression of DapA and DapC but not of DapB. In addition, in the absence of SrbA, the enhanced expression of DapA induced by azole-itraconazole is blocked, indicating that SrbA is required for the DapA response to azole stress. Double mutants together with overexpression experiments suggest that DapA might act downstream of SrbA to respond to azole stress stimuli. Compared with the ΔsrbA strain, no additional increase in sensitivity was observed in the double mutants ΔsrbAΔdapB and ΔsrbAΔdapC, indicating that DapA might be of central importance in the response to azole drugs. Thus, our findings demonstrate that Dap proteins indirectly sense azole stress and link the function of the azole stress-regulator SrbA with the role of Daps in azole susceptibility.
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Strobilurins as growth‐promoting compounds: How Stroby regulates Arabidopsis leaf growth

Strobilurins as growth‐promoting compounds: How Stroby regulates Arabidopsis leaf growth | Molecular basis of fungicide resistance | Scoop.it
Strobilurins are an important class of agrochemical fungicides used throughout the world on a wide variety of crops as protection against fungal pathogens. In addition to this protective role, they are reported to also positively influence plant physiology. In this study, we analyzed the effect of Stroby® WG, a commercially available fungicide consisting of 50% (w/w) kresoxim-methyl (KM) as active strobilurin compound, on Arabidopsis leaf growth. Treatment of seedlings with Stroby resulted in larger leaves due to an increase in cell number. Transcriptome analysis of Stroby-treated rosettes demonstrated an increased expression of genes involved in redox homeostasis, iron metabolism and sugar transport. Stroby treatment strongly induced the expression of the subgroup Ib basic helix-loop-helix (bHLH) transcription factors, which have a role in iron homeostasis under iron-limiting conditions. Single loss-of-function mutants of three bHLHs and their triple bhlh039 bhlh100 bhlh101 mutant did not respond to Stroby treatment. Although iron and sucrose content was not affected, nitric oxide (NO) levels and nitrate reductase (NR) activity were significantly increased in Stroby-treated rosettes as compared with control plants. In conclusion, we suggest that the Stroby-mediated effects on growth depend on the increased expression of the subgroup Ib bHLHs and higher NO levels.
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The Fungicidal Activity of Thymol against Fusarium graminearum via Inducing Lipid Peroxidation and Disrupting Ergosterol Biosynthesis

The Fungicidal Activity of Thymol against Fusarium graminearum via Inducing Lipid Peroxidation and Disrupting Ergosterol Biosynthesis | Molecular basis of fungicide resistance | Scoop.it
Thymol is a natural plant-derived compound that has been widely used in pharmaceutical and food preservation applications. However, the antifungal mechanism for thymol against phytopathogens remains unclear. In this study, we identified the antifungal action of thymol against Fusarium graminearum, an economically important phytopathogen showing severe resistance to traditional chemical fungicides. The sensitivity of thymol on different F. graminearum isolates was screened. The hyphal growth, as well as conidial production and germination, were quantified under thymol treatment. Histochemical, microscopic, and biochemical approaches were applied to investigate thymol-induced cell membrane damage. The average EC50 value of thymol for 59 F. graminearum isolates was 26.3 μg·mL−1. Thymol strongly inhibited conidial production and hyphal growth. Thymol-induced cell membrane damage was indicated by propidium iodide (PI) staining, morphological observation, relative conductivity, and glycerol measurement. Thymol induced a significant increase in malondialdehyde (MDA) concentration and a remarkable decrease in ergosterol content. Taken together, thymol showed potential antifungal activity against F. graminearum due to the cell membrane damage originating from lipid peroxidation and the disturbance of ergosterol biosynthesis. These results not only shed new light on the antifungal mechanism of thymol, but also imply a promising alternative for the control of Fusarium head blight (FHB) disease caused by F. graminearum.
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Steve Marek's curator insight, April 20, 2017 11:40 AM
An active ingredient in mouthwash e.g. Listerine