RESUMO
The downy mildew pathogen, Pseudoperonospora cubensis, which infects plant species in the family Cucurbitaceae, has undergone major changes during the last decade. Disease severity and epidemics are far more destructive than previously reported, and new genotypes, races, pathotypes, and mating types of the pathogen have been discovered in populations from around the globe as a result of the resurgence of the disease. Consequently, disease control through host plant resistance and fungicide applications has become more complex. This resurgence of P. cubensis offers challenges to scientists in many research areas including pathogen biology, epidemiology and dispersal, population structure and population genetics, host preference, host-pathogen interactions and gene expression, genetic host plant resistance, inheritance of host and fungicide resistance, and chemical disease control. This review serves to summarize the current status of this major pathogen and to guide future management and research efforts within this pathosystem.
Assuntos
Cucurbitaceae/microbiologia , Oomicetos/fisiologia , Cucurbitaceae/genética , Resistência Microbiana a Medicamentos , Europa (Continente) , Fungicidas Industriais , Interações Hospedeiro-Patógeno/genética , Controle de Pragas , Doenças das Plantas , Imunidade Vegetal/genética , Estados UnidosRESUMO
The grapevine downy mildew, Plasmopara viticola, is one of the most devastating pathogens in viticulture. Effective control is mainly based on fungicide treatments, although resistance development in this pathogen is reported for a number of fungicides. In this study we describe for the first time the molecular mechanism of resistance to a carboxylic acid amide (CAA) fungicide. We identified a family of four cellulose synthase (CesA) genes containing conserved domains that are found in all processive glycosyltransferases. Phylogenetic analysis revealed their close relationship to the cellulose synthases of Phytophthora sp. Sequencing of the CesA genes in a CAA- resistant and -sensitive field isolate revealed five single nucleotide polymorphisms (SNPs) affecting the amino acid structure of the proteins. SNP inheritance in F(1)-, F(2)- and F(3)-progeny confirmed resistance to be correlated with one single SNP located in PvCesA3. Only if present in both alleles, this SNP led to the substitution of a glycine for a serine residue at position 1105 (G1105S) in the deduced amino acid sequence, thus conferring CAA- resistance. Our data demonstrate that the identified genes are putative cellulose synthases and that one recessive mutation in PvCesA3 causes inheritable resistance to the CAA fungicide mandipropamid.
Assuntos
Amidas/farmacologia , Ácidos Carboxílicos/farmacologia , Glucosiltransferases/química , Glucosiltransferases/genética , Oomicetos/efeitos dos fármacos , Oomicetos/genética , Praguicidas/farmacologia , Mutação Puntual , Polimorfismo de Nucleotídeo Único , Alelos , Motivos de Aminoácidos , Sequência de Aminoácidos , Substituição de Aminoácidos , Resistência Microbiana a Medicamentos/genética , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Oomicetos/isolamento & purificação , Folhas de Planta/microbiologia , Estrutura Terciária de Proteína , Vitis/microbiologiaRESUMO
BACKGROUND: Aspergillus fumigatus, the causal agent of aspergillosis in humans, is commonly present as a saprophyte in various organic substrates, such as spoiled silages. Aspergillosis is generally combated with demethylation inhibitor (DMI) fungicides, but the recent appearance of resistant medical and environmental strains made current treatment strategies less reliable. The goal of this study was to determine the evolution of A. fumigatus populations during the ensiling process of whole-crop corn, high moisture corn and wet grain corn, and to monitor the sensitivity of isolates from treated and untreated fields to one medical and one agricultural DMI fungicide. RESULTS: A. fumigatus was isolated from fresh forage at harvest at rather low concentrations (102 cfu g-1 ). The low frequency lingered during the silage process (at 60 and 160 days), whereas it significantly increased during air exposure (at 7 and 14 days of air exposure). Field treatment of corn with a mixture of prothioconazole and tebuconazole did not affect the sensitivity of A. fumigatus isolates. One of 29 isolates from the untreated plot was resistant to voriconazole. A unique amino acid substitution (E427K) was detected in the cyp51A gene of 10 of 12 sequenced isolates, but was not associated with DMI resistance. CONCLUSION: A. fumigatus significantly increased during aerobic deterioration of ensilaged corn after silo opening, compared with the low presence in fresh corn and during ensiling. Field treatment of corn with DMI fungicides did not affect the sensitivity of A. fumigatus isolates collected from fresh and ensiled corn. © 2019 Society of Chemical Industry.
Assuntos
Silagem , Aspergillus fumigatus , Desmetilação , Fungicidas Industriais , Humanos , Dinâmica Populacional , Zea maysRESUMO
BACKGROUND: Pythium species attack various vegetable crops causing seed, stem and root rot, and 'damping-off' after germination. Pythium diseases are prevalently controlled by two classes of fungicides, QoIs with azoxystrobin and phenlyamides with mefenoxam as representatives. The present study aimed to test the sensitivity of six Pythium species from different vegetable crops to azoxystrobin and mefenoxam and differentiating species based on ITS, cytochrome b and RNA polymerase I gene sequences. RESULTS: The inter- and intra-species sensitivity to azoxystrobin was found to be stable, with the exception of one Pythium paroecandrum isolate, which showed reduced sensitivity and two cytochrome b amino acid changes. For mefenoxam, the inter-species sensitivity was quite variable and many resistant isolates were found in all six Pythium species, but no RNA polymerase I amino acid changes were observed in them. ITS and cytochrome b phylogenetic analyses permitted a clear separation of Pythium species corresponding to globose- and filamentous-sporangia clusters. CONCLUSION: The results document the necessity of well-defined chemical control strategies adapted to different Pythium species. Since the intrinsic activity of azoxystrobin among species was stable and no resistant isolates were found, it may be applied without species differentiation, provided it is used preventatively to also control highly aggressive isolates. For a reliable use of mefenoxam, precise identification and sensitivity tests of Pythium species are crucial because its intrinsic activity is variable and resistant isolates may exist. Appropriate mixtures and/or alternation of products may help to further delay resistance development. © 2018 Society of Chemical Industry.
Assuntos
Alanina/análogos & derivados , Fungicidas Industriais/farmacologia , Doenças das Plantas/classificação , Pirimidinas/farmacologia , Pythium/classificação , Pythium/efeitos dos fármacos , Estrobilurinas/farmacologia , Transportadores de Cassetes de Ligação de ATP/análise , Alanina/farmacologia , Sequência de Aminoácidos , Produtos Agrícolas/microbiologia , Citocromos b/química , Citocromos b/genética , Citocromos b/metabolismo , DNA Espaçador Ribossômico/análise , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Filogenia , Doenças das Plantas/microbiologia , Pythium/fisiologia , Reação em Cadeia da Polimerase em Tempo Real , Alinhamento de Sequência , Verduras/microbiologiaRESUMO
The carboxylic acid amide (CAA) fungicides mandipropamid, dimethomorph, iprovalicarb, and the phenylamide fungicide mefenoxam (MFX, the active enantiomer of metalaxyl) are anti-oomycete fungicides effective against downy mildews and late blight. Resistance against MFX was reported in nature in several oomycetes including Phytophthora infestans and Plasmopara viticola, whereas resistance against CAAs was reported in P. viticola but not in P. infestans. In this study the mutability of P. infestans for resistance against CAAs and MFX (as a control) was explored under laboratory conditions. UV light or chemical mutagens (e.g., ethyl methan sulfonate [EMS]) were applied to sporangia, and the emergence of mutants resistant to CAAs or MFX, or with altered mating type, was followed. Many mutants resistant to CAAs developed at generation 0 after mutagenesis, but all showed erratic, instable resistance in planta, diminishing after 1 to 8 asexual infection cycles, and failed to grow on CAA-amended medium. In contrast, 19 mutants resistant to MFX were obtained: 6 with UV irradiation (in isolates 28 or 96) and 13 with EMS (in isolates 408, 409, and 410). In three experiments, a shift in mating type, from A1 to A2, was detected. To elucidate whether or not resistance to CAAs is recessive and therefore might emerge only after sexual recombination, A1 and A2 mutants were crossed and the F1 and F2 progeny isolates were tested for resistance. Offspring isolates segregated for resistance to MFX, with resistant isolates maintaining stable resistance in vitro and in planta, whereas all progeny isolates failed to show stable resistance to CAAs in planta or in vitro. The data suggest that P. infestans could be artificially mutated for resistance against MFX, but not against CAAs.
RESUMO
Aspergillus fumigatus, the causal agent of human aspergilloses, is known to be non-pathogenic in plants. It is present as saprophyte in different types of organic matter and develops rapidly during the high-temperature phase of the composting process. Aspergilloses are treated with demethylation inhibitor (DMI) fungicides and resistant isolates have been recently reported. The present study aims to estimate the abundance, genetic diversity and DMI sensitivity of A. fumigatus during the composting process of orange fruits. Composting of orange fruits resulted in a 100-fold increase in A. fumigatus frequency already after 1 week, demonstrating that the degradation of orange fruits favoured the growth of A. fumigatus in compost. Most of A. fumigatus isolates belonged to mating type 2, including those initially isolated from the orange peel, whereas mating type 1 evolved towards the end of the composting process. None of the A. fumigatus isolates expressed simultaneously both mating types. The 52 investigated isolates exhibited moderate SSR polymorphisms by formation of one major (47 isolates) and one minor cluster (5 isolates). The latter included mating type 1 isolates from the last sampling and the DMI-resistant reference strains. Only few isolates showed cyp51A polymorphisms but were sensitive to DMIs as all the other isolates. None of the A. fumigatus isolates owned any of the mutations associated with DMI resistance. This study documents a high reproduction rate of A. fumigatus during the composting process of orange fruits, requesting specific safety precautions in compost handling. Furthermore, azole residue concentrations in orange-based compost were not sufficient to select A. fumigatus resistant genotypes.
Assuntos
Aspergillus fumigatus , Citrus sinensis/microbiologia , Compostagem , Inibidores das Enzimas do Citocromo P-450 , Sistema Enzimático do Citocromo P-450 , Proteínas Fúngicas , Fungicidas Industriais , Polimorfismo Genético , Aspergillus fumigatus/enzimologia , Aspergillus fumigatus/genética , Aspergillus fumigatus/isolamento & purificação , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Proteínas Fúngicas/antagonistas & inibidores , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismoRESUMO
[This corrects the article DOI: 10.1371/journal.pone.0200569.].
RESUMO
ABSTRACT Three carboxylic acid amide (CAA) fungicides, mandipropamid (MPD), dimethomorph (DMM) and iprovalicarb (IPRO) were examined for their effects on various asexual developmental stages of Phytophthora infestans in vitro and in planta. Germination of cystospores and direct germination of sporangia were inhibited with nanomole concentrations of MPD (0.005 mug/ml) and micromole concentrations of DMM (0.05 mug/ml) or IPRO (0.5 mug/ml). A temporary exposure of 1 h to CAAs was not detrimental to germination and infectivity of sporangia or cystospores. CAAs applied to cystospores at 1 h after the onset of germination did not prevent the emergence of germ tubes, but inhibited their further growth and deformed their shape. None of the fungicides affected discharge of zoospores from sporangia or the encystment (cell wall formation/assembly) of the zoospores. Mycelium growth in solid or liquid media was inhibited with micromole concentrations. CAAs mixed with sporangia and drop inoculated onto detached leaves strongly suppressed infection. Curative application at 1 day postinoculation (dpi) required higher concentrations of CAAs than preventive application to inhibit infection and lost its effectiveness at 2 dpi. When sprayed on established late blight lesions 4 days after inoculation, CAAs reduced sporangial production in a dose-dependent manner. Trans-laminar protection of potato or tomato leaves, although achieved with higher doses, was more effective with MPD than with DMM or IPRO. Shade house studies demonstrated superior control of late blight epidemics by MPD compared with the other molecules. The data suggest that germ tube formation by cystospores or sporangia is the most sensitive stage in the life cycle of P. infestans to CAAs. Of the three CAAs, MPD had the highest intrinsic activity against spore germination. This property, together with its better trans-laminar activity, makes MPD more effective than DMM or IPRO in controlling epidemics caused by P. infestans.
RESUMO
QoI fungicides, inhibitors of mitochondrial respiration at the Qo site of cytochrome b in the mitochondrial bc(1) enzyme complex, are commonly applied in vineyards against Plasmopara viticola (Berk. & MA Curtis) Berl. & De Toni. Numerous treatments per year with QoI fungicides can lead to the selection of resistant strains in the pathogen population owing to the very specific and efficient mode of action. In order to evaluate the resistance risk and its development, two different methods, biological and molecular, were applied to measure the sensitivity of oospores differentiated in vineyards, both treated and untreated with azoxystrobin, from 2000 to 2004. Assays using oospores have the advantage of analysing the sensitivity of bulked samples randomly collected in vineyards, describing accurately the status of resistance at the end of the grapevine growing season. Both methods correlated well in describing the resistance situation in vineyards. QoI resistance was not observed in one vineyard never treated with QoI fungicides. In the vineyard where azoxystrobin had been used in mixture with folpet, the selection of QoI-resistant strains was lower, compared with using solely QoI. In vineyards where QoI treatments have been stopped, a decrease in resistance was generally observed.
Assuntos
Farmacorresistência Fúngica , Fungicidas Industriais/farmacologia , Metacrilatos/farmacologia , Oomicetos/efeitos dos fármacos , Pirimidinas/farmacologia , Seleção Genética , Bioensaio , DNA Fúngico/química , Relação Dose-Resposta a Droga , Farmacorresistência Fúngica/genética , Complexo III da Cadeia de Transporte de Elétrons/genética , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Testes de Sensibilidade Microbiana , Oomicetos/genética , Oomicetos/crescimento & desenvolvimento , Reação em Cadeia da Polimerase , Estrobilurinas , VitisRESUMO
Resistance to QoI fungicides in Pyrenophora teres (Dreschsler) and P. tritici-repentis (Died.) Dreschsler was detected in 2003 in France and in Sweden and Denmark respectively. Molecular analysis revealed the presence of the F129L mutation in resistant isolates of both pathogens. In 2004, the frequency of the F129L mutation in populations of both pathogens further increased. The G143A mutation was also detected in a few isolates of P. tritici-repentis from Denmark and Germany. In 2005, the F129L mutation in P. teres increased in frequency and geographical distribution in France and the UK but remained below 2% in Germany, Switzerland, Belgium and Ireland. In P. tritici-repentis, both mutations were found in a significant proportion of the isolates from Sweden, Denmark and Germany. The G143A mutation conferred a significantly higher level of resistance (higher EC50 values) to Qo inhibitors (QoIs) than did the F129L mutation. In greenhouse trials, resistant isolates with G143A were not well controlled on plants sprayed with recommended field rates, whereas satisfactory control of isolates with F129L was achieved. For the F129L mutation, three different single nucleotide polymorphisms (SNPs), TTA, TTG and CTC, can code for L (leucine) in P. teres, whereas only the CTC codon was detected in P. tritici-repentis isolates. In two out of 250 isolates of P. tritici-repentis from 2005, a mutation at position 137 (G137R) was detected at very low frequency. This mutation conferred similar resistance levels to F129L. The structure of the cytochrome b gene of P. tritici-repentis is significantly different from that of P. teres: an intron directly after amino acid position 143 was detected in P. teres which is not present in P. tritici-repentis. This gene structure suggests that resistance based on the G143A mutation may not occur in P. teres because it is lethal. No G143A isolates were found in any P. teres populations. Although different mutations may evolve in P. tritici-repentis, the G143A mutation will have the strongest impact on field performance of QoI fungicides.
Assuntos
Ascomicetos/genética , Citocromos b/genética , Farmacorresistência Fúngica/genética , Sequência de Aminoácidos , Antifúngicos/farmacologia , Ascomicetos/efeitos dos fármacos , Ascomicetos/enzimologia , Sítios de Ligação , Citocromos b/química , Complexo III da Cadeia de Transporte de Elétrons/antagonistas & inibidores , Hordeum/metabolismo , Dados de Sequência Molecular , Mutação de Sentido Incorreto , Polimorfismo de Nucleotídeo Único , Alinhamento de Sequência , Triticum/metabolismoRESUMO
BACKGROUND: Aspergillus fumigatus is a widespread fungus that colonizes dead organic substrates but it can also cause fatal human diseases. Aspergilloses are treated with demethylation inhibitor (DMI) fungicides; however, resistant isolates appeared recently in the medical and also environmental area. The present study aims at molecular characterizing and quantifying A. fumigatus in major environmental habitats and determining its sensitivity to medical and agricultural DMI fungicides. RESULTS: A. fumigatus was isolated only rarely from soil and meadow/forest organic matter but high concentrations (103 to 107 cfu/g) were detected in substrates subjected to elevated temperatures, such as compost and silage. High genetic diversity of A. fumigatus from compost was found based on SSR markers, distinguishing among fungal isolates even when coming from the same substrate sample, while subclustering was observed based on mutations in cyp51A gene. Several cyp51A amino acid substitutions were found in 15 isolates, although all isolates were fully sensitive to the tested DMI fungicides, with exception of one isolate in combination with one fungicide. CONCLUSION: This study suggests that the tested A. fumigatus isolates collected in Italy, Spain and Hungary from the fungus' major living habitats (compost) and commercial growing substrates are not potential carriers for DMI resistance in the environment. © 2017 Society of Chemical Industry.
Assuntos
Antifúngicos/farmacologia , Aspergillus fumigatus/efeitos dos fármacos , Aspergillus fumigatus/genética , Microbiologia do Solo , Antifúngicos/química , Aspergillus fumigatus/classificação , Aspergillus fumigatus/isolamento & purificação , Compostagem , Desmetilação , Farmacorresistência Fúngica , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Variação Genética , PradariaRESUMO
The cytochrome b (cyt b) gene structure was characterized for different agronomically important plant pathogens, such as Puccinia recondita f sp tritici (Erikss) CO Johnston, P graminis f sp tritici Erikss and Hennings, P striiformis f sp tritici Erikss, P coronata f sp avenae P Syd & Syd, P hordei GH Otth, P recondita f sp secalis Roberge, P sorghi Schwein, P horiana Henn, Uromyces appendiculatus (Pers) Unger, Phakopsora pachyrhizi Syd & P Syd, Hemileia vastatrix Berk & Broome, Alternaria solani Sorauer, A alternata (Fr) Keissl and Plasmopara viticola (Berk & Curt) Berlese & de Toni. The sequenced fragment included the two hot spot regions in which mutations conferring resistance to QoI fungicides may occur. The cyt b gene structure of these pathogens was compared with that of other species from public databases, including the strobilurin-producing fungus Mycena galopoda (Pers) P Kumm, Saccharomyces cerevisiae Meyer ex Hansen, Venturia inaequalis (Cooke) Winter and Mycosphaerella fijiensis Morelet. In all rust species, as well as in A solani, resistance to QoI fungicides caused by the mutation G143A has never been reported. A type I intron was observed directly after the codon for glycine at position 143 in these species. This intron was absent in pathogens such as A alternata, Blumeria graminis (DC) Speer, Pyricularia grisea Sacc, Mycosphaerella graminicola (Fuckel) J Schröt, M fijiensis, V inaequalis and P viticola, in which resistance to QoI fungicides has occurred and the glycine is replaced by alanine at position 143 in the resistant genotype. The present authors predict that a nucleotide substitution in codon 143 would prevent splicing of the intron, leading to a deficient cytochrome b, which is lethal. As a consequence, the evolution of resistance to QoI fungicides based on G143A is not likely to evolve in pathogens carrying an intron directly after this codon.
Assuntos
Citocromos b/genética , Farmacorresistência Fúngica/genética , Fungos/enzimologia , Fungicidas Industriais/farmacologia , Genes Fúngicos , Plantas/microbiologia , Substituição de Aminoácidos , Ascomicetos/enzimologia , Ascomicetos/patogenicidade , Basidiomycota/enzimologia , Basidiomycota/patogenicidade , Citocromos b/antagonistas & inibidores , Complexo III da Cadeia de Transporte de Elétrons/antagonistas & inibidores , Fungos/patogenicidade , Íntrons , Oomicetos/enzimologia , Oomicetos/patogenicidade , Mutação Puntual , Reação em Cadeia da PolimeraseRESUMO
Fungicides inhibiting the mitochondrial respiration of plant pathogens by binding to the cytochrome bc1 enzyme complex (complex III) at the Qo site (Qo inhibitors, QoIs) were first introduced to the market in 1996. After a short time period, isolates resistant to QoIs were detected in field populations of a range of important plant pathogens including Blumeria graminis Speer f sp tritici, Sphaerotheca fuliginea (Schlecht ex Fr) Poll, Plasmopara viticola (Berk & MA Curtis ex de Bary) Berl & de Toni, Pseudoperonospora cubensis (Berk & MA Curtis) Rost, Mycosphaerella fijiensis Morelet and Venturia inaequalis (Cooke) Wint. In most cases, resistance was conferred by a point mutation in the mitochondrial cytochrome b (cyt b) gene leading to an amino-acid change from glycine to alanine at position 143 (G143A), although additional mutations and mechanisms have been claimed in a number of organisms. Transformation of sensitive protoplasts of M fijiensis with a DNA fragment of a resistant M fijiensis isolate containing the mutation yielded fully resistant transformants, demonstrating that the G143A substitution may be the most powerful transversion in the cyt b gene conferring resistance. The G143A substitution is claimed not to affect the activity of the enzyme, suggesting that resistant individuals may not suffer from a significant fitness penalty, as was demonstrated in B graminis f sp tritici. It is not known whether this observation applies also for other pathogen species expressing the G143A substitution. Since fungal cells contain a large number of mitochondria, early mitotic events in the evolution of resistance to QoIs have to be considered, such as mutation frequency (claimed to be higher in mitochondrial than nuclear DNA), intracellular proliferation of mitochondria in the heteroplasmatic cell stage, and cell to cell donation of mutated mitochondria. Since the cyt b gene is located in the mitochondrial genome, inheritance of resistance in filamentous fungi is expected to be non-Mendelian and, therefore, in most species uniparental. In the isogamous fungus B graminis f sp tritici, crosses of sensitive and resistant parents yielded cleistothecia containing either sensitive or resistant ascospores and the segregation pattern for resistance in the F1 progeny population was 1:1. In the anisogamous fungus V inaequalis, donation of resistance was maternal and the segregation ratio 1:0. In random mating populations, the sex ratio (mating type distribution) is generally assumed to be 1:1. Therefore, the overall proportion of sensitive and resistant individuals in unselected populations is expected to be 1:1. Evolution of resistance to QoIs will depend mainly on early mitotic events; the selection process for resistant mutants in populations exposed to QoI treatments may follow mechanisms similar to those described for resistance controlled by single nuclear genes in other fungicide classes. It will remain important to understand how the mitochondrial nature of QoI resistance and factors such as mutation, recombination, selection and migration might influence the evolution of QoI resistance in different plant pathogens.
Assuntos
Evolução Biológica , Farmacorresistência Fúngica/genética , Fungos/efeitos dos fármacos , Fungos/fisiologia , Fungicidas Industriais/farmacologia , Complexo III da Cadeia de Transporte de Elétrons/genética , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Fungos/enzimologia , Fungos/genética , Plantas/microbiologiaRESUMO
A single nucleotide polymorphism (snp) in the cytochrome b gene confers resistance to strobilurin fungicides in Erysiphe graminis DC fsp tritici Marchal. On the basis of this point mutation three different types of molecular markers have been developed. Cleaved amplified polymorphic sequences and allele-specific PCR were used to score resistant and sensitive isolates from specifically selected regional populations across Europe. The results of molecular tests were in total agreement with the resistance phenotypes revealed by in vivo tests. Serial dilutions of mixed samples (resistant/sensitive) delimited the detection for strobilurin-resistant alleles to a range of 10-50% for both marker classes. Due to these detection limits no mixture of mitochondria within individual isolates was found. Denaturing high performance chromatography was used to increase the detection sensitivity for the mutant allele. Although the detection limit was lowered to 5-10%, there was no evidence for the existence of mixed mitochondrial genotypes.
Assuntos
Farmacorresistência Fúngica/genética , Fungos/efeitos dos fármacos , Fungos/genética , Fungicidas Industriais/farmacologia , Polimorfismo de Nucleotídeo Único/genética , Genes Fúngicos/genética , Fenótipo , Mutação Puntual/genéticaRESUMO
BACKGROUND: An increasing number of publications have claimed that demethylation inhibitor (DMI) fungicides are confronted with resistance development in the fungus Aspergillus fumigatus and that the origin of resistant isolates may also be outside the medical area. For resistance risk assessment and sourcing the origin of DMI resistance, the primary exposure events ofA. fumigatus with DMI treatments have been analysed case by case, resulting in the pathogen exposure risk (PER). RESULTS: The calculated maximum exposure concentrations (MEC) are highest during medical treatments (human and veterinary), certain fruit and seed treatments and wood preservation, and are much lower for crop protection applications. Most agricultural DMIs are intrinsically â¼10-100 times less active than medical DMIs for A. fumigatus control and potential resistance selection. However, imazalil is used in agriculture and veterinary medicine (as enilconazole) expressing strong intrinsic activity against A. fumigatus. The majority of mutations in the target gene, cyp51, of DMI-resistant isolates are different in A. fumigatus(e.g. TR34/L98H) in comparison with plant pathogens (e.g. A379G, I381V). CONCLUSIONS: The assumed selection risk, ASR (MEC × PER) for resistance evolution to DMIs in A. fumigatus is estimated to be highest for human and veterinary applications. However, environmental origin of DMI-resistant spores from certain sites cannot be ruled out.
Assuntos
Antifúngicos/farmacologia , Aspergilose/microbiologia , Aspergillus fumigatus/efeitos dos fármacos , Farmacorresistência Fúngica , Fungicidas Industriais/farmacologia , Animais , Aspergillus fumigatus/genética , Aspergillus fumigatus/metabolismo , Humanos , Metilação/efeitos dos fármacos , Doenças das Plantas/microbiologiaRESUMO
BACKGROUND: Tolerance to the oomycete-specific carboxylic acid amide (CAA) fungicides is a poorly understood mechanism in Pythium species. The root-rot and damping-off causative agent Pythium aphanidermatum and the CAA fungicide mandipropamid (MPD) were used to investigate the molecular basis of CAA tolerance. RESULTS: Five genes putatively involved in carbohydrate synthesis were identified and characterised: one chitin synthase gene, PaChs, and four cellulose synthase genes PaCesA1 to PaCesA4, of which PaCesA3 encodes the MPD target enzyme. These genes were differentially expressed throughout the life cycle of P. aphanidermatum. Mycelium treated with MPD concentrations slightly affecting mycelial growth did not cause a change in PaCesA3 expression nor a strong upregulation of PaCesA homologues. The high tolerance level of P. aphanidermatum and the lack of PaCesA upregulation imply that MPD tolerance is the result of a specific amino acid configuration in the cellulose synthase 3 (CesA3) target enzyme. Indeed, P. aphanidermatum displays the amino acid L1109 which is also associated with MPD resistance in artificial mutants of Phytophthora species. CONCLUSION: It is concluded that MPD tolerance in P. aphanidermatum is not caused by compensatory mechanisms but most likely by an inherent target-site configuration in PaCesA3 that hinders MPD binding to the enzyme pocket.
Assuntos
Amidas/farmacologia , Ácidos Carboxílicos/farmacologia , Farmacorresistência Fúngica , Fungicidas Industriais/farmacologia , Pythium/efeitos dos fármacos , Pythium/genética , Enzimas/genética , Enzimas/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Doenças das Plantas/parasitologia , Pythium/enzimologia , Pythium/fisiologiaRESUMO
Proper disease control is very important to minimize yield losses caused by oomycetes in many crops. Today, oomycete control is partially achieved by breeding for resistance, but mainly by application of single-site mode of action fungicides including the carboxylic acid amides (CAAs). Despite having mostly specific targets, fungicidal activity can differ even in species belonging to the same phylum but the underlying mechanisms are often poorly understood. In an attempt to elucidate the phylogenetic basis and underlying molecular mechanism of sensitivity and tolerance to CAAs, the cellulose synthase 3 (CesA3) gene was isolated and characterized, encoding the target site of this fungicide class. The CesA3 gene was present in all 25 species included in this study representing the orders Albuginales, Leptomitales, Peronosporales, Pythiales, Rhipidiales and Saprolegniales, and based on phylogenetic analyses, enabled good resolution of all the different taxonomic orders. Sensitivity assays using the CAA fungicide mandipropamid (MPD) demonstrated that only species belonging to the Peronosporales were inhibited by the fungicide. Molecular data provided evidence, that the observed difference in sensitivity to CAAs between Peronosporales and CAA tolerant species is most likely caused by an inherent amino acid configuration at position 1109 in CesA3 possibly affecting fungicide binding. The present study not only succeeded in linking CAA sensitivity of various oomycetes to the inherent CesA3 target site configuration, but could also relate it to the broader phylogenetic context.
Assuntos
Amidas/farmacologia , Ácidos Carboxílicos/farmacologia , Fungicidas Industriais/farmacologia , Glucosiltransferases/genética , Glucosiltransferases/metabolismo , Oomicetos/enzimologia , Filogenia , Sequência de Aminoácidos , Análise por Conglomerados , DNA Fúngico/química , DNA Fúngico/genética , Farmacorresistência Fúngica , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutação de Sentido Incorreto , Oomicetos/classificação , Oomicetos/efeitos dos fármacos , Oomicetos/genética , Mutação Puntual , Análise de Sequência de DNA , Homologia de Sequência de AminoácidosRESUMO
BACKGROUND: Pseudoperonospora cubensis, the causal oomycete agent of cucurbit downy mildew, is responsible for enormous crop losses in many species of Cucurbitaceae, particularly in cucumber and melon. Disease control is mainly achieved by combinations of host resistance and fungicide applications. However, since 2004, resistance to downy mildew in cucumber has been overcome by the pathogen, thus driving farmers to rely only on fungicide spray applications, including carboxylic acid amide (CAA) fungicides. Recently, CAA-resistant isolates of P. cubensis were recovered, but the underlying mechanism of resistance was not revealed. The purpose of the present study was to identify the molecular mechanism controlling resistance to CAAs in P. cubensis. RESULTS: The four CesA (cellulose synthase) genes responsible for cellulose biosynthesis in P. cubensis were characterised. Resistant strains showed a mutation in the CesA3 gene, at position 1105, leading to an amino acid exchange from glycine to valine or tryptophan. Cross-resistance tests with different CAAs indicated that these mutations lead to resistance against all tested CAAs. CONCLUSION: Point mutations in the CesA3 gene of P. cubensis lead to CAA resistance. Accurate monitoring of these mutations among P. cubensis populations may improve/facilitate adequate recommendation/deployment of fungicides in the field.