Molecular basis of fungicide resistance
2.4K views | +1 today
Follow
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
Your new post is loading...
Your new post is loading...
Scooped by Melvin Bolton
Scoop.it!

Characterization of Postharvest Fungicide-Resistant Botrytis cinerea Isolates from Commercially Stored Apple Fruit | Phytopathology

Characterization of Postharvest Fungicide-Resistant Botrytis cinerea Isolates from Commercially Stored Apple Fruit | Phytopathology | Molecular basis of fungicide resistance | Scoop.it
Botrytis cinerea causes gray mold and is an economically important postharvest pathogen of fruits, vegetables and ornamentals. Fludioxonil sensitive B. cinerea isolates were collected in 2011 and 2013 from commercial storage in Pennsylvania. Eight isolates had EC50 values ranging from 0.0004 to 0.0038 µg/ml for fludioxonil and were dual resistant to pyrimethanil and thiabendazole. Resistance was generated in vitro, following exposure to a sub-lethal dose of fludioxonil, in seven of eight dual-resistant B. cinerea isolates. Three vigorously growing B. cinerea isolates with multi-resistance to postharvest fungicides were further characterized and found to be osmosensitive and retained resistance in the absence of selection pressure. A representative multi-resistant B. cinerea strain caused decay on apples treated with postharvest fungicides, which confirmed the in vitro results. The R632I mutation in the Mrr1 gene, associated with fludioxonil resistance in B. cinerea, was not detected in multi-postharvest fungicide resistant B. cinerea isolates, suggesting that the fungus may be using additional mechanism(s) to mediate resistance. Results from this study show for the first time that B. cinerea with dual resistance to pyrimethanil and thiabendazole can also rapidly develop resistance to fludioxonil which may pose control challenges in the packinghouse environment and during long-term storage.
more...
No comment yet.
Scooped by Melvin Bolton
Scoop.it!

Hexokinase plays a critical role in deoxynivalenol (DON) production and fungal development in Fusarium graminearum - Zhang - 2015 - Molecular Plant Pathology - Wiley Online Library

Hexokinase plays a critical role in deoxynivalenol (DON) production and fungal development in Fusarium graminearum - Zhang - 2015 - Molecular Plant Pathology - Wiley Online Library | Molecular basis of fungicide resistance | Scoop.it
Fusarium graminearum, the causal agent of Fusarium head blight, is a common pathogen on small grain cereals worldwide and produces various trichothecenes [deoxynivalenol (DON) is predominant] during infection. A previous study has revealed that DON production is positively correlated with the occurrence of carbendazim (MBC) resistance. Here, we identified and characterized two putative genes encoding hexokinase in F. graminearum (FgHXK1 and FgHXK2), which is a rate-limiting enzyme in DON biosynthesis. The expression level of hexokinase genes and the production of pyruvate, which is the precursor of DON, were up-regulated in the MBC-resistant strain, indicating that hexokinase genes might be involved in increased DON production. Phylogenetic and comparative analyses indicated that FgHXK1 was the predominant hexokinase gene. Gene disruption showed that ΔFgHXK1 severely affected DON production, indicating that FgHXK1 played a role in the regulation of DON biosynthesis. Morphological characterization showed that ΔFgHXK1 led to inhibited vegetative growth and conidiation. Sensitivity tests to MBC and various stresses indicated that both ΔFgHXK1 and ΔFgHXK2 mutants showed no significant difference from parental strains. Pathogencity assays showed that ΔFgHXK1 mutants lost virulence on wheat head and corn stigma; however, they showed no change in sexual reproduction. The FgHXK1-overexpressing transformants were obtained subsequently. Their pyruvate and DON production was confirmed to be increased, indicating that FgHXK1 positively regulated DON biosynthesis. Although additional defects appeared in overexpression mutants, MBC sensitivity showed no change. All of the results indicated that the transcriptional level of FgHXK1 regulated DON biosynthesis, but showed no direct relationship with MBC resistance.
more...
No comment yet.
Scooped by Melvin Bolton
Scoop.it!

Molecular Mechanisms Involved in Qualitative and Quantitative Resistance to the Dicarboximide Fungicide, Iprodione in Sclerotinia homoeocarpa Field Isolates — Phytopathology

Molecular Mechanisms Involved in Qualitative and Quantitative Resistance to the Dicarboximide Fungicide, Iprodione in Sclerotinia homoeocarpa Field Isolates — Phytopathology | Molecular basis of fungicide resistance | Scoop.it
The dicarboximide fungicide class is commonly used to control Sclerotinia homoeocarpa, the causal agent of dollar spot on turfgrass. Despite frequent occurrences of S. homoeocarpa field resistance to iprodione (dicarboximide active ingredient), the genetic mechanisms of iprodione resistance have not been elucidated. In this study, 15 field isolates (seven suspected dicarboximide-resistant, three multi-drug resistance (MDR)-like, and five dicarboximide-sensitive) were used for sequence comparison of a histidine kinase gene Shos1 of S. homoeocarpa. The suspected dicarboximide-resistant isolates displayed non-synonymous polymorphisms in codon 366 (isoleucine to asparagine) in the Shos1, while the MDR-like and sensitive isolates did not. Further elucidation of the Shos1 function using polyethylene glycol-mediated protoplast transformation indicated that S. homoeocarpa mutants (Shos1I366N) from a sensitive isolate gained resistance to dicarboximides but not phenylpyrrole and polyols. The deletion of Shos1 resulted in higher resistance to dicarboximide and phenylpyrrole, and higher sensitivity to polyols than Shos1I366N. Levels of dicarboximide sensitivity in the sensitive isolate, Shos1I366N and Shos1 deletion mutants were negatively correlated to values of iprodione induced-expression of ShHog1, the last kinase in the high-osmolarity glycerol (HOG) pathway. Increased constitutive and induced expression of the ATP-binding cassette (ABC)-multidrug efflux transporter, ShPDR1, was observed in 6 of 7 dicarboximide-resistant isolates. In conclusion, S. homoeocarpa field isolates gained dicarboximide resistance to the fungicide through the polymorphism in the Shos1 and the overexpression of ShPDR1.
more...
No comment yet.
Scooped by Melvin Bolton
Scoop.it!

Demethylase Inhibitor Fungicide Resistance in Pyrenophora teres f. sp. teres Associated with Target Site Modification and Inducible Overexpression of Cyp51 - Mair et al., Frontiers in Microbiol 2016

Demethylase Inhibitor Fungicide Resistance in Pyrenophora teres f. sp. teres Associated with Target Site Modification and Inducible Overexpression of Cyp51 - Mair et al., Frontiers in Microbiol 2016 | Molecular basis of fungicide resistance | Scoop.it
Pyrenophora teres f. sp. teres is the cause of net form of net blotch, an economically important foliar disease in barley (Hordeum vulgare). Net and spot forms of net blotch are widely controlled using site-specific systemic fungicides. Although resistance to succinate dehydrogenase inhibitors and quinone outside inhibitors has been addressed before in net blotches, mechanisms controlling demethylation inhibitor resistance have not yet been reported at the molecular level. Here we report the isolation of strains of net form of net blotch in Australia since 2013 resistant to a range of demethylase inhibitor fungicides. Cyp51A:KO103-A1, an allele with the mutation F489L, corresponding to the archetype F495I in Aspergillus fumigatus, was only present in resistant strains and was correlated with resistance factors to various demethylase inhibitors ranging from 1.1 for epoxiconazole to 31.7 for prochloraz. Structural in silico modelling of the sensitive and resistant CYP51A proteins docked with different demethylase inhibitor fungicides showed how the interaction of F489L within the heme cavity produced a localised constriction of the region adjacent to the docking site that is predicted to result in lower binding affinities. Resistant strains also displayed enhanced induced expression of the two Cyp51A paralogs and of Cyp51B genes. While Cyp51B was found to be constitutively expressed in the absence of fungicide, Cyp51A was only detected at extremely low levels. Under fungicide induction, expression of Cyp51B, Cyp51A2 and Cyp51A1 was shown to be 1.6-, 3- and 5.3-fold higher, respectively in the resistant isolate compared to the wild type. These increased levels of expression were not supported by changes in the promoters of any of the three genes. The implications of these findings on demethylase inhibitor activity will require current net blotch management strategies to be reconsidered in order to avoid the development of further resistance and preserve the lifespan of fungicides in use.
more...
No comment yet.
Scooped by Melvin Bolton
Scoop.it!

Trends and Challenges in Pesticide Resistance Detection

Trends and Challenges in Pesticide Resistance Detection | Molecular basis of fungicide resistance | Scoop.it
Pesticide resistance is a crucial factor to be considered when developing strategies for the minimal use of pesticides while maintaining pesticide efficacy. This goal requires monitoring the emergence and development of resistance to pesticides in crop pests. To this end, various methods for resistance diagnosis have been developed for different groups of pests. This review provides an overview of biological, biochemical, and molecular methods that are currently used to detect and quantify pesticide resistance. The agronomic, technical, and economic advantages and drawbacks of each method are considered. Emerging technologies are also described, with their associated challenges and their potential for the detection of resistance mechanisms likely to be selected by current and future plant protection methods.
more...
No comment yet.
Scooped by Melvin Bolton
Scoop.it!

BMC Genomics

BMC Genomics | Molecular basis of fungicide resistance | Scoop.it
Background
Penicillium digitatum is one of the most destructive postharvest pathogen of citrus fruits, causing fruit decay and economic loss. The emergence of fungicide-resistant strains made the control of P. digitatum more difficult. While the genome of P. digitatum is available, there has been few reports about its resistant mechanism from the transcriptome perspective and there has been no large-scale functional annotation of the genome using expressed genes derived from transcriptomes.

Methods
Total RNA of P. digitatum strain HS-F6 (prochloraz-resistant strain) and HS-E3 (prochloraz-susceptible strain) before and after prochloraz-treatment were extracted and sequenced on an Illumina Hiseq 2000 platform. The transcriptome data of four samples were compared and analyzed using differential expression analysis, novel transcripts prediction and alternative splicing analysis, SNP analysis and quantitative real-time PCR.

Results
We present a large scale analysis about the transcriptome data of P. digitatum. The whole RNA was extracted from a prochloraz-resistant strain (HS-F6) and a prochloraz-susceptible strain (HS-E3) before and after prochloraz-treatment and sequenced by Illumina technology. A total of more than 100 million reads were generated and de novo assembled into 9760 transcripts that contained annotated genes after quality control and sequence assembling. 6625 single nucleotide variations (SNVs) were identified from the sequences aligned against the reference genome. Gene expression profiling analysis was performed upon prochloraz treatment in HS-F6 and HS-E3, and differential expression analysis was used to identify genes related to prochloraz-response and drug-resistance: there are 224 differentially expressed genes in HS-E3 and 1100 differentially expressed genes in HS-F6 after prochloraz-treatment. Moreover, gene expression profile in prochloraz-resistant strain HS-F6 is quite different from that in HS-E3 before prochloraz-treatment, 1520 differential expression genes were identified between the two strains. Gene ontology (GO) term enrichment and KEGG enrichment were then performed to classify the differential expression genes. Among these genes, there are a lot of transporter encoding genes including 14 MFS (Major Facilitator Superfamily) transporters, 8 ABC (ATP-binding cassette transporter) and 3 MATE (multidrug and toxic compound extrusion family) transporters. Meanwhile, the roles of typical MFS, ABC and MATE proteins in prochloraz resistance were investigated using real-time quantitative PCR.
more...
No comment yet.
Scooped by Melvin Bolton
Scoop.it!

Resistance Assessment for Oxathiapiprolin in Phytophthora capsici and the Detection of a Point Mutation (G769W) in PcORP1 that Confers Resistance

Resistance Assessment for Oxathiapiprolin in Phytophthora capsici and the Detection of a Point Mutation (G769W) in PcORP1 that Confers Resistance | Molecular basis of fungicide resistance | Scoop.it

The potential for oxathiapiprolin resistance in Phytophthora capsici was evaluated. The baseline sensitivities of 175 isolates to oxathiapiprolin were initially determinated and found to conform to a unimodal curve with a mean EC50 value of 5.61 × 10-4 μg/ml. Twelve stable oxathiapiprolin-resistant mutants were generated by fungicide adaptation in two sensitive isolates, LP3 and HNJZ10. The fitness of the LP3-mutants was found to be similar to or better than that of the parental isolate LP3, while the HNJZ10-mutants were found to have lost the capacity to produce zoospores. Taken together these results suggest that the risk of P. capsici developing resistance to oxathiapiprolin is moderate. Comparison of the PcORP1 genes in the LP3-mutants and wild-type parental isolate, which encode the target protein of oxathiapiprolin, revealed that a heterozygous mutation caused the amino acid substitution G769W. Transformation and expression of the mutated PcORP1-769W allele in the sensitive wild-type isolate BYA5 confirmed that the mutation in PcORP1 was responsible for the observed oxathiapiprolin resistance. Finally diagnostic tests including As-PCR and CAPs were developed to detect the oxathiapiprolin resistance resulting from the G769W point mutation in field populations of P. capsici.

more...
No comment yet.
Scooped by Melvin Bolton
Scoop.it!

Genome-Wide Association Study Identifies Novel Candidate Genes for Aggressiveness, Deoxynivalenol Production, and Azole Sensitivity in Natural Field Populations of Fusarium graminearum - MPP - Tala...

Genome-Wide Association Study Identifies Novel Candidate Genes for Aggressiveness, Deoxynivalenol Production, and Azole Sensitivity in Natural Field Populations of Fusarium graminearum - MPP - Tala... | Molecular basis of fungicide resistance | Scoop.it
Genome-wide association studies can identify novel genomic regions and genes that affect quantitative traits. Fusarium head blight is a destructive disease caused by Fusarium graminearum that exhibits several quantitative traits, including aggressiveness, mycotoxin production, and fungicide resistance. Restriction site–associated DNA sequencing was performed for 220 isolates of F. graminearum. A total of 119 isolates were phenotyped for aggressiveness and deoxynivalenol (DON) production under natural field conditions across four environments. The effective concentration of propiconazole that inhibits isolate growth in vitro by 50% was calculated for 220 strains. Approximately 29,000 single nucleotide polymorphism markers were associated to each trait, resulting in 50, 29, and 74 quantitative trait nucleotides (QTNs) that were significantly associated to aggressiveness, DON production, and propiconazole sensitivity, respectively. Approximately 41% of these QTNs caused nonsynonymous substitutions in predicted exons, while the remainder were synonymous substitutions or located in intergenic regions. Three QTNs associated with propiconazole sensitivity were significant after Bonferroni correction. These QTNs were located in genes not previously associated with azole sensitivity. The majority of the detected QTNs were located in genes with predicted regulatory functions, suggesting that nucleotide variation in regulatory genes plays a major role in the corresponding quantitative trait variation.
Melvin Bolton's insight:
Share your insight
more...
No comment yet.
Scooped by Melvin Bolton
Scoop.it!

Genetic Analysis Using an Isogenic Mating Pair of Aspergillus fumigatus Identifies Azole Resistance Genes and Lack of MAT Locus’s Role in Virulence

Genetic Analysis Using an Isogenic Mating Pair of  Aspergillus fumigatus  Identifies Azole Resistance Genes and Lack of  MAT  Locus’s Role in Virulence | Molecular basis of fungicide resistance | Scoop.it
Author Summary Invasive aspergillosis (IA) caused by Aspergillus fumigatus is increasing due to medical interventions that suppress the ability of patients’ immune systems to control infections. These invasive lung infections are difficult to diagnose and consequently treatment is frequently not started promptly. Some controversy surrounds the role of mating type in virulence of A . fumigatus and the emergence of azole resistant strains has posed difficult challenges for clinical management of IA. We generated nearly identical A . fumigatus strains with opposite mating types that allowed us to test whether different mating types have different virulence profiles. We found no difference in virulence in three different animal models, which suggests that mating type does not influence virulence. We also took advantage of the essentially identical genomes of both strains to apply classical genetic approaches combined with genomics technologies to identify A . fumigatus genes that contribute to azole resistance. We performed genetic crosses of azole sensitive with azole resistant strains and analyzed the resistance status and genome composition of the offspring. Using this approach we cataloged several genes that were not previously associated with azole resistance. This information will be valuable for finding ways to manage azole resistance in IA patients.
more...
No comment yet.
Scooped by Melvin Bolton
Scoop.it!

Fluconazole induces rapid high-frequency MTL homozygosis with microbiological polymorphism in Candida albicans

Fluconazole induces rapid high-frequency MTL homozygosis with microbiological polymorphism in Candida albicans | Molecular basis of fungicide resistance | Scoop.it
Background

Candida albicans, a common fungal pathogen that can cause opportunistic infections, is regarded as an apparently asexual, diploid fungus. A parasexual cycle was previously found between homozygotes with opposite mating type-like loci (MTLa/α). Fluconazole-resistant strains had a higher proportion of MTL homozygotes, whereas MTL homozygous C. albicans was found in only about 3.2% of clinical strains. MTL heterozygotes had a low frequency (1.4 × 10−4) of white–opaque switching to MTL homozygotes in nature.

Methods

Here, a reference C. albicans strain (SC5314) was used in a fluconazole-induced assay to obtain standard opaque MTL homozygous strains and first-generation daughter strains from the fluconazole inhibition zone. Further separation methods were employed to produce second- and third-generation daughter strains. Polymerase chain reaction analysis based on MTL genes was used to define MTL genotypes, and microscopic observations, a flow-cytometric assay, and an antifungal E-test were used to compare microbiological characteristics.

Results

MTL homozygotes were found at a high frequency (17 of 35; 48.6%) in fluconazole-induced first-generation daughter strains, as were morphological polymorphisms, decreased DNA content, and modified antifungal drug susceptibility. High-frequency MTL homozygosity was identified inside the fluconazole inhibition zone within 24 hours. The DNA content of fluconazole-induced daughter strains was reduced compared with their progenitor SC5314 and standard MTL homozygous strains.

Conclusion

Treatment with fluconazole, commonly used to treat invasive candidiasis, inhibited the growth of C. albicans and altered its microbiological characteristics. Our results suggest that fluconazole treatment induces the high frequency of loss of heterozygosity and microbiological polymorphism in C. albicans.
more...
No comment yet.
Scooped by Melvin Bolton
Scoop.it!

Comparative analyses of fungicide sensitivity and SSR marker variations indicate a low risk of developing azoxystrobin resistance in Phytophthora infestans

Comparative analyses of fungicide sensitivity and SSR marker variations indicate a low risk of developing azoxystrobin resistance in Phytophthora infestans | Molecular basis of fungicide resistance | Scoop.it

Knowledge of the evolution of fungicide resistance is important in securing sustainable disease management in agricultural systems. In this study, we analyzed and compared the spatial distribution of genetic variation in azoxystrobin sensitivity and SSR markers in 140Phytophthora infestans isolates sampled from seven geographic locations in China. Sensitivity to azoxystrobin and its genetic variation in the pathogen populations was measured by the relative growth rate (RGR) at four fungicide concentrations and determination of the effective concentration for 50% inhibition (EC50). We found that all isolates in the current study were sensitive to azoxystrobin and their EC50 was similar to that detected from a European population about 20 years ago, suggesting the risk of developing azoxystrobin resistance inP. infestans populations is low. Further analyses indicate that reduced genetic variation and high fitness cost in resistant mutations are the likely causes for the low evolutionary likelihood of developing azoxystrobin resistance in the pathogen. We also found a negative correlation between azoxystrobin tolerance in P. infestans populations and the mean annual temperature of collection sites, suggesting that global warming may increase the efficiency of using the fungicide to control the late blight.

more...
No comment yet.
Scooped by Melvin Bolton
Scoop.it!

cyp51A-based mechanism of azole resistance in Aspergillus fumigatus: Illustration by a new 3D Structural Model of Aspergillus fumigatus CYP51A protein. Liu et al. 2016 Medical Mycology

cyp51A-based mechanism of azole resistance in Aspergillus fumigatus: Illustration by a new 3D Structural Model of Aspergillus fumigatus CYP51A protein. Liu et al. 2016 Medical Mycology | Molecular basis of fungicide resistance | Scoop.it

Mutations of CYP51A protein (Cytochrome P450 14-α Sterol demethylase) play a central role in the azole resistance of Aspergillus fumigatus. The available structural models of CYP51A protein of A. fumigatus are built based on that of Homo sapiens and that of Mycobacterium tuberculosis, of which the amino acid homology is only 38% and 29% compared with CYP51A protein of A. fumigatus, respectively. In the present study, we constructed a new 3D structural model of A. fumigatus CYP51A protein based on a recently resolved crystal structure of the homologous protein in the fungus S. cerevisiae, which shares 50% amino acid homology with A. fumigatus CYP51A protein. Three azole molecules, itraconazole, voriconazole, and posaconazole, were docked to the wild-type and the mutant A. fumigatus CYP51A protein models, respectively, to illustrate the impact of cyp51A mutations to azole-resistance. We found the mutations that occurred at L98, M220, and Y431 positions would decrease the binding affinity of azoles to the CYP51A protein and therefore would reduce their inhibitory effects. Additionally, the mutations of L98 and G432 would reduce the stability of the protein, which might lead to conformational change of its binding pocket and eventually the resistance to azoles.

more...
No comment yet.
Scooped by Melvin Bolton
Scoop.it!

Augmenting the Activity of Monoterpenoid Phenols against Fungal Pathogens Using 2-Hydroxy-4-methoxybenzaldehyde that Target Cell Wall Integrity - Kim et al - Int J Mol Sci

Augmenting the Activity of Monoterpenoid Phenols against Fungal Pathogens Using 2-Hydroxy-4-methoxybenzaldehyde that Target Cell Wall Integrity - Kim et al - Int J Mol Sci | Molecular basis of fungicide resistance | Scoop.it
Disruption of cell wall integrity system should be an effective strategy for control of fungal pathogens. To augment the cell wall disruption efficacy of monoterpenoid phenols (carvacrol, thymol), antimycotic potency of benzaldehyde derivatives that can ...
Melvin Bolton's insight:

Disruption of cell wall integrity system should be an effective strategy for control of fungal pathogens. To augment the cell wall disruption efficacy of monoterpenoid phenols (carvacrol, thymol), antimycotic potency of benzaldehyde derivatives that can serve as chemosensitizing agents were evaluated against strains ofSaccharomyces cerevisiae wild type (WT), slt2Δ and bck1Δ (mutants of the mitogen-activated protein kinase (MAPK) and MAPK kinase kinase, respectively, in the cell wall integrity pathway). Among fourteen compounds investigated, slt2Δ and bck1Δ showed higher susceptibility to nine benzaldehydes, compared to WT. Differential antimycotic activity of screened compounds indicated “structure-activity relationship” for targeting the cell wall integrity, where 2-hydroxy-4-methoxybenzaldehyde (2H4M) exhibited the highest antimycotic potency. The efficacy of 2H4M as an effective chemosensitizer to monoterpenoid phenols (viz., 2H4M + carvacrol or thymol) was assessed in yeasts or filamentous fungi (Aspergillus, Penicillium) according to European Committee on Antimicrobial Susceptibility Testing or Clinical Laboratory Standards Institute M38-A protocols, respectively. Synergistic chemosensitization greatly lowers minimum inhibitory or fungicidal concentrations of the co-administered compounds. 2H4M also overcame the tolerance of two MAPK mutants (sakAΔ, mpkCΔ) of Aspergillus fumigatus to fludioxonil (phenylpyrrole fungicide). Collectively, 2H4M possesses chemosensitizing capability to magnify the efficacy of monoterpenoid phenols, which improves target-based (viz., cell wall disruption) antifungal intervention.

more...
No comment yet.
Scooped by Melvin Bolton
Scoop.it!

Multi-locus resistance evolution to azole fungicides in fungal plant pathogen populations - Mohd-Assaad - 2016 - Molecular Ecology - Wiley Online Library

Multi-locus resistance evolution to azole fungicides in fungal plant pathogen populations - Mohd-Assaad - 2016 - Molecular Ecology - Wiley Online Library | Molecular basis of fungicide resistance | Scoop.it
Evolution of fungicide resistance is a major threat to food production in agricultural ecosystems. Fungal pathogens rapidly evolved resistance to all classes of fungicides applied to the field. Resistance to the commonly used azole fungicides is thought to be driven mainly by mutations in a gene (CYP51) encoding a protein of the ergosterol biosynthesis pathway. However, some fungi gained azole resistance independently of CYP51 mutations and the mechanisms leading to CYP51-independent resistance are poorly understood. We used whole-genome sequencing and genome-wide association studies (GWAS) to perform an unbiased screen of azole resistance loci in Rhynchosporium commune, the causal agent of the barley scald disease. We assayed cyproconazole resistance in 120 isolates collected from 9 populations worldwide. We found that mutations in highly conserved genes encoding the vacuolar cation channel YVC1, a transcription activator, and a saccharopine dehydrogenase made significant contributions to fungicide resistance. These three genes were not previously known to confer resistance in plant pathogens. However, YVC1 is involved in a conserved stress response pathway known to respond to azoles in human pathogenic fungi. We also performed GWAS to identify genetic polymorphism linked to fungal growth rates. We found that loci conferring increased fungicide resistance were negatively impacting growth rates, suggesting that fungicide resistance evolution imposed costs. Analyses of population structure showed that resistance mutations were likely introduced into local populations through gene flow. Multi-locus resistance evolution to fungicides shows how pathogen populations can evolve a complex genetic architecture for an important phenotypic trait within a short time span.
more...
No comment yet.
Scooped by Melvin Bolton
Scoop.it!

M233I Mutation in the β-Tubulin of Botrytis cinerea Confers Resistance to Zoxamide - Cai et al., - Scientific Reports 

M233I Mutation in the β-Tubulin of Botrytis cinerea Confers Resistance to Zoxamide - Cai et al., - Scientific Reports  | Molecular basis of fungicide resistance | Scoop.it
Three phenotypes were detected in 161 Botrytis cinerea field isolates, including ZoxSCarS (sensitive to zoxamide and carbendazim), ZoxSCarR (sensitive to zoxamide and resistant to carbendazim), and ZoxRCarR (resistant to zoxamide and carbendazim), but not ZoxRCarS (resistant to zoxamide and sensitive to carbendazim). The baseline sensitivity to zoxamide was determined with a mean EC50 of 0.76 μg/ml. Two stable ZoxRCarS isolates were obtained with a resistance factor of 13.28 and 20.43; there was a fitness penalty in mycelial growth rate, sporulation, virulence and sclerotium production. The results suggest that the resistance risk of B. cinerea to zoxamide is low where benzimidazoles have not been used. E198V, E198K and M233I, were detected in the β-tubulin of ZoxSCarR, ZoxRCarR, ZoxRCarS, respectively. Molecular docking indicated that position 198 in β-tubulin were targets for both zoxamide and carbendazim. The mutations at 198 prevented formation of hydrogen bonds between β-tubulin and carbendazim (E198V/K), and changed the conformation of the binding pocket of zoxamide (E198K). M233I had no effect on the binding of carbendazim but resulted in loss of a hydrogen bond between zoxamide and F200. M233 is suggested to be a unique target site for zoxamide and be very important in the function of β tubulin.
more...
No comment yet.
Scooped by Melvin Bolton
Scoop.it!

Characterisation of Ramularia collo-cygni laboratory mutants resistant to Succinate Dehydrogenase Inhibitors - Piotrowska et al. - 2016 - Pest Management Science 

Characterisation of Ramularia collo-cygni laboratory mutants resistant to Succinate Dehydrogenase Inhibitors - Piotrowska et al. - 2016 - Pest Management Science  | Molecular basis of fungicide resistance | Scoop.it
BACKGROUND: Ramularia collo-cygni (Rcc) is responsible for Ramularia leaf spot (RLS), a foliar disease of barley contributing to serious economic losses. Protection against the disease has been almost exclusively based on fungicide applications, including Succinate Dehydrogenase Inhibitors (SDHIs). In 2015 the first field isolates of Rcc with reduced sensitivity to SDHIs were recorded in some European countries. In this study we established baseline sensitivity of Rcc to SDHIs in the UK and characterised mutations correlating with resistance to SDHIs in UV-generated mutants. RESULTS: Five SDHI resistant isolates were generated by UV mutagenesis. In four of these mutants a single amino acid change in a target succinate dehydrogenase (Sdh) protein was associated with decrease in sensitivity to SDHIs. Three of these mutations were stably inherited in the absence of SDHI fungicide and resistant isolates did not demonstrate a fitness penalty. There were no detectable declines in sensitivity in field populations in years 2010-2012 in the UK. 
CONCLUSIONS: SDHIs remained effective in controlling Rcc in the UK in years 2010-2012. However given that the first isolates of Rcc with reduced sensitivity appeared in other European countries in 2015, robust anti-resistance strategies need to be continuously implemented to maintain effective disease control.
more...
No comment yet.
Scooped by Melvin Bolton
Scoop.it!

Increasing resistances to chemical classes of fungicides via selection by association in Botrytis cinerea — Phytopathology

Increasing resistances to chemical classes of fungicides via selection by association in Botrytis cinerea — Phytopathology | Molecular basis of fungicide resistance | Scoop.it
Previous research has shown that Botrytis cinerea isolates with resistance to multiple chemical classes of fungicides exist in eastern strawberry fields. In this study the fungicide resistance profiles of 2130 isolates from flowers of commercial strawberry fields located in multiple states was determined over 4 consecutive strawberry production seasons. Producers were asked to alternate single-site fungicides that were considered ‘low risk’ in their specific location based on resistance monitoring results in their fields. This guidance led to an increase of chemical class diversity used in the spray programs. Results indicated that simultaneous resistance in individual isolates to two, three, four, five, six, and seven classes of fungicides increased over time. The increase in resistances within isolates was likely due to a process we coined ‘selection by association’, where fungicide resistance traits were often linked to the trait being selected rather than the selectable trait itself. Data analysis also indicated that the odds were highest for isolates resistant to one chemical class (1CCR) to be resistant to thiophanate-methyl; for 2CCR isolates to be resistant to thiophanate-methyl and pyraclostrobin; and for 3CCR isolates to be resistant to thiophanate-methyl, pyraclostrobin, and either cyprodinil or fenhexamid. We hypothesize that the more chemical classes are used in a spray program, the faster isolates will be selected with increasing numbers of chemical class resistances by virtue of ‘selection by association’ if such isolates preexist in the population.
more...
No comment yet.
Scooped by Melvin Bolton
Scoop.it!

Fluconazole and Voriconazole Resistance in Candida parapsilosis Is Conferred by Gain-of-Function Mutations in MRR1 Transcription Factor Gene

Fluconazole and Voriconazole Resistance in Candida parapsilosis Is Conferred by Gain-of-Function Mutations in MRR1 Transcription Factor Gene | Molecular basis of fungicide resistance | Scoop.it

Candida parapsilosis is the second most prevalent fungal agent causing bloodstream infections. Nevertheless, there is little information about the molecular mechanisms underlying azole resistance in this species. Mutations (G1747A, A2619C, and A3191C) in the MRR1 transcription factor gene were identified in fluconazole- and voriconazole-resistant strains. Independent expression of MRR1 genes harboring these mutations showed that G1747A (G583R) and A2619C (K873N) are gain-of-function mutations responsible for azole resistance, the first described in C. parapsilosis. Antimicrob Agents Chemother. 2015 Oct; 59(10): 6629–6633

more...
No comment yet.
Scooped by Melvin Bolton
Scoop.it!

Reduced Virulence of Azoxystrobin-Resistant Zymoseptoria tritici Populations in Greenhouse Assays — Phytopathology 2016

Reduced Virulence of Azoxystrobin-Resistant Zymoseptoria tritici Populations in Greenhouse Assays — Phytopathology 2016 | Molecular basis of fungicide resistance | Scoop.it
The development of resistance to multiple fungicide classes is currently limiting disease management options for many pathogens, while the discovery of new fungicide classes may become less frequent. In light of this, more research is needed to quantify virulence trade-offs of fungicide resistance in order to more fully understand the implications of fungicide resistance on pathogen fitness. The purpose of this study was to measure the virulence of azoxystrobin-resistant and -sensitive Zymoseptoria tritici populations collected from North and South Willamette Valley, Oregon, in 2012 and 2015. Inoculum mixtures of known fungicide-resistant phenotypes were used to simulate natural field conditions, where multiple genotypes exist and interact in close proximity. Six greenhouse inoculations were conducted over 2 years, and virulence of the isolate mixtures was evaluated in planta. We considered virulence to be “the degree of pathology caused by the organism” and visually estimated the percent area of leaf necrosis as a measure of virulence. In greenhouse conditions, a consistent association of reduced virulence with azoxystrobin-resistant Z. tritici isolate mixtures was observed. North Willamette Valley and South Willamette Valley populations did not differ in virulence.
more...
No comment yet.
Scooped by Melvin Bolton
Scoop.it!

Co-Occurrence of Two Allelic Variants of CYP51 in Erysiphe necator and Their Correlation with Over-Expression for DMI Resistance

Co-Occurrence of Two Allelic Variants of CYP51 in  Erysiphe necator  and Their Correlation with Over-Expression for DMI Resistance | Molecular basis of fungicide resistance | Scoop.it
Demethylation inhibitors (DMIs) have been an important tool in the management of grapevine powdery mildew caused by Erysiphe necator. Long-term, intensive use of DMIs has resulted in reduced sensitivity in field populations. To further characterize DMI resistance and understand resistance mechanisms in this pathogen, we investigated the cyp51 sequence of 24 single-spored isolates from Virginia and surrounding states and analyzed gene expression in isolates representing a wide range of sensitivity. Two cyp51 alleles were found with respect to the 136th codon of the predicted EnCYP51 sequence: the wild-type (TAT) and the mutant (TTT), which results in the known Y136F amino acid change. Some isolates possessed both alleles, demonstrating gene duplication or increased gene copy number and possibly a requirement for at least one mutant copy of CYP51 for resistance. Cyp51 was over-expressed 1.4- to 19-fold in Y136F-mutant isolates. However, the Y136F mutation was absent in one isolate with moderate to high resistance factor. Two additional synonymous mutations were detected as well, one of which, A1119C was present only in isolates with high cyp51 expression. Overall, our results indicate that at least two mechanisms, cyp51 over-expression and the known target-site mutation in CYP51, contribute to resistance in E. necator, and may be working in conjunction with each other.
more...
No comment yet.
Scooped by Melvin Bolton
Scoop.it!

RNA-sequencing of Cercospora beticola DMI-sensitive and -resistant isolates after treatment with tetraconazole identifies common and contrasting pathway induction

RNA-sequencing of Cercospora beticola DMI-sensitive and -resistant isolates after treatment with tetraconazole identifies common and contrasting pathway induction | Molecular basis of fungicide resistance | Scoop.it
Melvin Bolton's insight:
Cercospora beticola causes Cercospora leaf spot of sugar beet. Cercospora leaf spot management measures often include application of the sterol demethylation inhibitor (DMI) class of fungicides. The reliance on DMIs and the consequent selection pressures imposed by their widespread use has led to the emergence of resistance in C. beticola populations. Insight into the molecular basis of tetraconazole resistance may lead to molecular tools to identify DMI-resistant strains for fungicide resistance management programs. Previous work has shown that expression of the gene encoding the DMI target enzyme (CYP51) is generally higher and inducible in DMI-resistant C. beticola field strains. In this study, we extended the molecular basis of DMI resistance in this pathosystem by profiling the transcriptional response of two C. beticola strains contrasting for resistance to tetraconazole. A majority of the genes in the ergosterol biosynthesis pathway were induced to similar levels in both strains with the exception of CbCyp51, which was induced several-fold higher in the DMI-resistant strain. In contrast, a secondary metabolite gene cluster was induced in the resistance strain, but repressed in the sensitive strain. Genes encoding proteins with various cell membrane fortification processes were induced in the resistance strain. Site-directed and ectopic mutants of candidate DMI-resistance genes all resulted in significantly higher EC50 values than the wild-type strain, suggesting that the cell wall and/or membrane modified as a result of the transformation process increased resistance to tetraconazole. Taken together, this study identifies important cell membrane components and provides insight into the molecular events underlying DMI resistance in C. beticola.
more...
No comment yet.
Scooped by Melvin Bolton
Scoop.it!

Mechanism of action of the benzimidazole fungicide on Fusarium graminearum: interfering with polymerization of monomeric tubulin but not polymerized microtubule — Phytopathology

Mechanism of action of the benzimidazole fungicide on Fusarium graminearum: interfering with polymerization of monomeric tubulin but not polymerized microtubule — Phytopathology | Molecular basis of fungicide resistance | Scoop.it
Tubulins are the proposed target of clinically relevant anticancer drug, anthelmintic, and fungicide. β2-tubulin of plant pathogen Fusarium graminearum was considered as the target of benzimidazoles compounds by homology modeling in our previous work. In this study, α1-, α1-, and β2-tubulin of F. graminearum were produced in E. coli. Three benzimidazole compounds (carbendazim, benomyl, and thiabendazole) interacted with the recombinant β2-tubulin and reduced the maximum fluorescence intensity of 2μM β2-tubulin 47, 50, and 25%, respectively, at saturation of compound-tubulin complexes. Furthermore, carbendazim significantly inhibit the polymerization of α1-/β2-tubulins and α2-/β2-tubulins 90.9±0.4% and 93.5±0.05%, respectively in vitro. The similar result appeared with benomyl on the polymerization of α1-/β2- tubulins and α2-/β2-tubulins were 89.9±0.1% and 92.6±1.2% inhibition ratio, respectively. Besides, thiabendazole inhibited 81.6±1% polymerization of α1-/β2-tubulins, whereas it had less effect on α2-/β2-tubulins polymerization with 20.1±1.9% inhibition ratio. However, the three compounds can not destabilize the polymerized microtubule. To illuminates the issue, mapping the carbendazim binding sites and β/α subunit interface on β/α-tubulin complexes by homology modeling shown the two domains were closed to each other. Understanding the nature of the interaction between benzimidazole compounds and F. graminearum tubulin is fundamental for the development of tubulin specific anti-F. graminearum compounds.
more...
No comment yet.
Scooped by Melvin Bolton
Scoop.it!

Function of the genetic element ‘Mona' associated with fungicide resistance in Monilinia fructicola - Chen - Molecular Plant Pathology - Wiley Online Library

Function of the genetic element ‘Mona' associated with fungicide resistance in Monilinia fructicola - Chen - Molecular Plant Pathology - Wiley Online Library | Molecular basis of fungicide resistance | Scoop.it
The genetic element ‘Mona' was previously shown to be associated with the resistance to demethylation inhibitors (DMIs) in Monilinia fructicola. In this study, promoter activity of the ‘Mona' element was demonstrated genetically and the activity was narrowed down to a 20-bp active region through a series of deletions. ‘Mona' knockout transformants (ΔMona) were generated from DMI-resistant isolate Bmpc7 and EC50 values as well as expression of the MfCYP51 gene were found to be reduced in transformants compared to the parental isolate. When the ‘Mona' element was inserted into the upstream region of the MfCYP51 gene of the DMI-sensitive isolate HG3, the EC50 values as well as the expression of the MfCYP51 gene increased in the transformants compared with the parental sensitive isolate. These results indicated that the ‘Mona' element determined the DMI fungicide resistance through up regulating the expression of the downstream MfCYP51 gene. No fitness penalty was observed in knockout and insertion transformants, i.e., transformants showed similar mycelial growth rate, sporulation, and ability to cause lesions on fruit compared with their parental isolates, suggesting that the ‘Mona' element did not affect basal life activities. 
more...
No comment yet.
Scooped by Melvin Bolton
Scoop.it!

A Novel Sterol Regulatory Element-Binding Protein Gene ( sreA ) Identified in Penicillium digitatum Is Required for Prochloraz Resistance, Full Virulence and erg11 ( cyp51 ) Regulation. Liu e...

A Novel Sterol Regulatory Element-Binding Protein Gene ( sreA ) Identified in  Penicillium   digitatum  Is Required for Prochloraz Resistance, Full Virulence and  erg11  ( cyp51 ) Regulation. Liu e... | Molecular basis of fungicide resistance | Scoop.it
Penicillium digitatum is the most destructive postharvest pathogen of citrus fruits, causing fruit decay and economic loss. Additionally, control of the disease is further complicated by the emergence of drug-resistant strains due to the extensive use of triazole antifungal drugs. In this work, an orthologus gene encoding a putative sterol regulatory element-binding protein (SREBP) was identified in the genome of P . digitatum and named sreA . The putative SreA protein contains a conserved domain of unknown function (DUF2014) at its carboxyl terminus and a helix-loop-helix (HLH) leucine zipper DNA binding domain at its amino terminus, domains that are functionally associated with SREBP transcription factors. The deletion of sreA (ΔsreA) in a prochloraz-resistant strain (PdHS-F6) by Agrobacterium tumefaciens -mediated transformation led to increased susceptibility to prochloraz and a significantly lower EC 50 value compared with the HS-F6 wild-type or complementation strain (CO sreA ). A virulence assay showed that the Δ sreA strain was defective in virulence towards citrus fruits, while the complementation of sreA could restore the virulence to a large extent. Further analysis by quantitative real-time PCR demonstrated that prochloraz-induced expression of cyp51A and cyp51B in PdHS-F6 was completely abolished in the Δ sreA strain. These results demonstrate that sreA is a critical transcription factor gene required for prochloraz resistance and full virulence in P . digitatum and is involved in the regulation of cyp51 expression.
more...
No comment yet.
Scooped by Melvin Bolton
Scoop.it!

Novel Substrate Specificity and Temperature-Sensitive Activity of Mycosphaerella graminicola CYP51 Supported by the Native NADPH Cytochrome P450 Reductase

Novel Substrate Specificity and Temperature-Sensitive Activity of Mycosphaerella graminicola CYP51 Supported by the Native NADPH Cytochrome P450 Reductase | Molecular basis of fungicide resistance | Scoop.it

Mycosphaerella graminicola (Zymoseptoria tritici) is an ascomycete filamentous fungus that causes Septoria leaf blotch in wheat crops. In Europe the most widely used fungicides for this major disease are demethylation inhibitors (DMIs). Their target is the essential sterol 14α-demethylase (CYP51), which requires cytochrome P450 reductase (CPR) as its redox partner for functional activity. The M. graminicolaCPR (MgCPR) is able to catalyze the sterol 14α-demethylation of eburicol and lanosterol when partnered with Candida albicans CYP51 (CaCYP51) and that of eburicol only with M. graminicola CYP51 (MgCYP51). The availability of the functional in vivo redox partner enabled the in vitro catalytic activity of MgCYP51 to be demonstrated for the first time. MgCYP51 50% inhibitory concentration (IC50) studies with epoxiconazole, tebuconazole, triadimenol, and prothioconazole-desthio confirmed that MgCYP51 bound these azole inhibitors tightly. The characterization of the MgCPR/MgCYP51 redox pairing has produced a functional method to evaluate the effects of agricultural azole fungicides, has demonstrated eburicol specificity in the activity observed, and supports the conclusion that prothioconazole is a profungicide.

more...
No comment yet.