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1.
World J Microbiol Biotechnol ; 35(7): 106, 2019 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-31267229

RESUMO

Xenorhabdus nematophila HB310 secreted the insecticidal protein toxin complex. Two chitinase genes, chi60 and chi70, were found in X. nematophila toxin complex locus. In order to clarify the function of two chitinases, chi60 and chi70 genes were cloned and expressed in Escherichia coli Transetta (DE3). As a result, we found that the Chi60 and Chi70 belonged to glycoside hydrolases (GH) family 18 with a molecular mass of 65 kDa and 78 kDa, respectively. When colloidal chitin was treated as the substrate, Chi60 and Chi70 were proved to have the highest enzymatic activity at pH 6.0 and 50 °C. Chi60 and Chi70 had obvious growth inhibition effect against the second larvae of Helicoverpa armigera with growth inhibiting rate of 81.99% and 90.51%. Chi70 had synergistic effect with the insecticidal toxicity of Bt Cry 1Ac, but the Chi60 had no synergistic effect with Bt Cry 1Ac. Chi60 and Chi70 showed antifungal activity against Alternaria brassicicola, Verticillium dahliae and Coniothyrium diplodiella. The results increased our understanding of the chitinases produced by X. nematophila and laid a foundation for further studies on the mechanism of the chitinases.


Assuntos
Antifúngicos/farmacologia , Quitinases/antagonistas & inibidores , Quitinases/genética , Quitinases/metabolismo , Xenorhabdus/metabolismo , Alternaria/efeitos dos fármacos , Animais , Ascomicetos/efeitos dos fármacos , Quitina/metabolismo , Quitinases/classificação , Clonagem Molecular , Sinergismo Farmacológico , Ensaios Enzimáticos , Estabilidade Enzimática , Escherichia coli/genética , Expressão Gênica , Glicosídeo Hidrolases/genética , Concentração de Íons de Hidrogênio , Inseticidas/metabolismo , Inseticidas/farmacologia , Larva/efeitos dos fármacos , Larva/crescimento & desenvolvimento , Peso Molecular , Mariposas/efeitos dos fármacos , Mariposas/crescimento & desenvolvimento , Micotoxinas/genética , Micotoxinas/metabolismo , Filogenia , Domínios Proteicos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Temperatura Ambiente , Verticillium/efeitos dos fármacos , Xenorhabdus/genética
2.
J Agric Food Chem ; 67(31): 8536-8547, 2019 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-31310520

RESUMO

Watermelon Fusarium wilt is a common soil-borne disease that has significantly affected its yield. In this study, fusaric acid-deficient mutant designated as ΔFUBT (mutated from Fusarium oxysporum f. sp. niveum, FON) was obtained. The ΔFUBT mutant showed significant decrease in fusaric acid production but maintained wild-type characteristics, such as in vitro colony morphology, size, and conidiation. A field pot experiment demonstrated that ΔFUBT could successfully colonize the rhizosphere and the roots of watermelon, leading to significant reduction in FON colonization in the watermelon plant. In addition, ΔFUBT inoculation significantly improved the rhizosphere microenvironment and effectively increased the resistance in watermelon. This study demonstrated that a nonpathogenic Fusarium mutant (ΔFUBT) could be developed as an effective microbial control agent to alleviate Fusarium wilt disease in watermelon and increase its yield.


Assuntos
Citrullus/microbiologia , Fusarium/genética , Micotoxinas/genética , Doenças das Plantas/microbiologia , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Ácido Fusárico/metabolismo , Fusarium/crescimento & desenvolvimento , Fusarium/fisiologia , Mutação , Micotoxinas/metabolismo , Raízes de Plantas/microbiologia , Rizosfera
3.
Comput Biol Chem ; 80: 270-277, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31054539

RESUMO

Panomycocin is a naturally produced potent antimycotic/antifungal protein secreted by the yeast Wickerhamomyces anomalus NCYC 434 with an exo-ß-1,3-glucanase activity. In this study the three dimensional structure of panomycocin was predicted and the computational site-directed mutagenesis was performed to enhance its thermal stability in liquid formulations over the body temperature for topical therapeutic applications. Homology modeling was performed with MODELLER and I-TASSER. Among the generated models, the model with the lowest energy and DOPE score was selected for further loop modeling. The loop model was optimized and the reliability of the model was confirmed with ERRAT, Verify 3D and Ramachandran plot values. Enhancement of the thermal stability of the model was done using contemporary servers and programs such as SPDBViewer, CNA, I-Mutant2.0, Eris, AUTO-MUTE and MUpro. In the region outside the binding site of the model Leu52 Arg, Phe223Arg and Gly254Arg were found to be the best thermostabilizing mutations with 6.26 K, 6.26 K and 8.27 K increases, respectively. In the binding site Glu186Arg was found to be the best thermostabilizer mutation with a 9.58 K temperature increase. The results obtained in this study led us to design a mutant panomycocin that can be used as a novel antimycotic/antifungal drug in a liquid formulation for topical applications over the normal body temperature.


Assuntos
Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/genética , Micotoxinas/química , Micotoxinas/genética , Pichia/química , Sequência de Aminoácidos , Substituição de Aminoácidos , Sítios de Ligação/genética , Modelos Moleculares , Mutagênese Sítio-Dirigida/métodos , Mutação , Estabilidade Proteica , Estrutura Terciária de Proteína , Temperatura Ambiente
4.
Food Microbiol ; 82: 240-248, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31027779

RESUMO

The Aspergillus niger aggregate contains 15 morphologically indistinguishable species which presence is related to ochratoxin A (OTA) and fumonisin B2 (FB2) contamination of foodstuffs. The taxonomy of this group was recently reevaluated and there is a need of new studies regarding the risk that these species might pose to food security. 258 isolates of A. niger aggregate obtained from a variety of products from Spain were classified by molecular methods being A. tubingensis the most frequently occurring (67.5%) followed by A. welwitschiae (19.4%) and A. niger (11.7%). Their potential ability to produce mycotoxins was evaluated by PCR protocols which allow a rapid detection of OTA and FB2 biosynthetic genes in their genomes. OTA production is not widespread in A. niger aggregate since only 17% of A. niger and 6% of A. welwitschiae isolates presented the complete biosynthetic cluster whereas the lack of the cluster was confirmed in all A. tubingensis isolates. On the other hand, A. niger and A. welwitschiae seem to be important FB2 producers with 97% and 29% of the isolates, respectively, presenting the complete cluster. The genes involved in OTA and FB2 were overexpressed in producing isolates and their expression was related to mycotoxin synthesis.


Assuntos
Aspergillus/isolamento & purificação , Aspergillus/metabolismo , Microbiologia de Alimentos , Micotoxinas/metabolismo , Aspergillus/classificação , Aspergillus/genética , Aspergillus niger/classificação , Aspergillus niger/genética , Aspergillus niger/isolamento & purificação , Aspergillus niger/metabolismo , DNA Fúngico/genética , Contaminação de Alimentos/análise , Fumonisinas/metabolismo , Expressão Gênica , Genoma Fúngico/genética , Família Multigênica , Micotoxinas/genética , Ocratoxinas/metabolismo , Filogenia , Análise de Sequência de DNA , Espanha
5.
Food Microbiol ; 78: 62-72, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30497609

RESUMO

Cave cheese is a surface mold-ripened variety of cheese produced also in South of Italy, exploiting fungal population naturally occurring on cave walls, as part of secondary microbiota for ripening. In this study, 148 fungal strains were isolated from 22 independent cave cheese samples, collected in 13 Italian geographical locations, mostly in Apulian area. DNA-based identification showed the presence of twenty-four fungal species in the outer part of the cheese ripened in caves. Aspergillus westerdijkiae and Penicillium biforme resulted the most frequently isolated species, followed by Penicillium roqueforti and Penicillium solitum. The 86% of cheese sample presented at least one toxigenic species and the 45% revealed the presence of ochratoxigenic species, A. westerdijkiae and A. steynii, suggesting possible mycotoxin risk during ripening stage in caves, confirmed by the presence of ochratoxin A (OTA) in the rind of 36% of samples. In conclusion, cave cheese is a susceptible product for toxigenic mold growth and in particular OTA contamination, therefore adeguate scientific tools for matching organolectic consumer expectations and complete safety of food should be developed, as well as spontaneously molded and not monitored cheeses should not be consumed to avoid mycotoxin risk.


Assuntos
Cavernas/microbiologia , Queijo/microbiologia , Fungos/crescimento & desenvolvimento , Microbiota/genética , Micotoxinas/isolamento & purificação , Aspergillus/genética , Aspergillus/crescimento & desenvolvimento , Aspergillus/isolamento & purificação , Aspergillus/fisiologia , Microbiologia de Alimentos , Inocuidade dos Alimentos/métodos , Fungos/genética , Fungos/isolamento & purificação , Fungos/fisiologia , Humanos , Itália , Micotoxinas/genética , Ocratoxinas/análise , Penicillium/genética , Penicillium/crescimento & desenvolvimento , Penicillium/isolamento & purificação , Penicillium/fisiologia
6.
J Microbiol ; 56(9): 634-647, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30054815

RESUMO

Corynespora cassiicola is a species of fungus that is a plant pathogen of many agricultural crop plants, including severe target spot disease on cucumber. Cassiicolin is an important effector of pathogenicity of this fungus. In this study, we collected 141 Corynespora isolates from eighteen hosts, and the casscolin gene was detected in 82 C. cassiicola strains. The deduced protein sequences revealed that 72 isolates contained the Cas2 gene, two strains from Gynura bicolor harboured the Cas2.2 gene, and 59 isolates without a cassiicolin gene were classified as Cas0. Phylogenetic analyses was performed for the 141 isolates using four loci (ITS, ga4, caa5, and act1) and revealed two genetic clusters. Cluster A is composed of four subclades: subcluster A1 includes all Cas2 isolates plus 18 Cas0 strains, subcluster A2 includes the eight Cas5 isolates and one Cas0 isolate, and subclusters A3 and A4 contain Cas0 strains. Cluster B consists of 21 Cas0 isolates. Twenty-two C. cassiicola strains from different toxin classes showed varying degrees of virulence against cucumber. Cas0 or Cas2 strains induced diverse responses on cucumber, from no symptoms to symptoms of moderate or severe infection, but all Cas5 isolates exhibited avirulence on cucumber.


Assuntos
Ascomicetos/genética , Proteínas Fúngicas/genética , Variação Genética , Micotoxinas/genética , Sequência de Aminoácidos , Ascomicetos/isolamento & purificação , Ascomicetos/patogenicidade , China , Cucumis sativus/microbiologia , DNA Fúngico/genética , Proteínas Fúngicas/classificação , Micotoxinas/classificação , Filogenia , Doenças das Plantas/microbiologia , Alinhamento de Sequência , Análise de Sequência de DNA , Virulência/genética , Fatores de Virulência/genética
7.
Cornea ; 37(8): 1042-1046, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-29757853

RESUMO

PURPOSE: To identify mycotoxin genes among clinical ocular isolates of Fusarium species and to correlate these with clinical outcomes of Fusarium keratitis. METHODS: Fifty-four clinical isolates of Fusarium were retrieved from the Bascom Palmer Eye Institute Ocular Microbiology Laboratory data bank. Multiplex polymerase chain reactions were run to confirm the identification of Fusarium species [internal transcribed spacer sequence, translation elongation factor 1-α (TEF) and ß-tubulin] and to detect the presence of genes encoding production of fumonisin B mycotoxins (FUM1 and FUM8) and trichothecene mycotoxins (deoxynivalenol and nivalenol). The presence or absence of mycotoxins was compared with patient outcomes. RESULTS: Forty-three (79%) of the 54 isolates were confirmed as Fusarium species, by an internal transcribed spacer sequence in 3 (5.6%) and by TEF in 43 (79.6%) of the 54 isolates. Fumonisin biosynthetic gene 1 (FUM1) was detected in 57.4% (n = 31/54) of the Fusarium isolates. No FUM8, deoxynivalenol genes, and nivalenol genes were detected among these in the clinical isolates group. Initial best-corrected visual acuity ranged from 20/25 to 20/80 in the FUM1 gene-negative group and from 20/20 to light perception in the FUM1 gene-positive group. There was no difference in the time to cure between both groups. The presence of FUM1 genes in 5 fungal isolates seemed to be associated with progression to penetrating keratoplasty in the 5 patients from whom the fungi were isolated. Fusarium solani was recovered from all patients requiring penetrating keratoplasty. CONCLUSIONS: Fumonisin B biosynthetic gene 1 may be common among clinical Fusarium isolates and contribute to worse initial visual acuity and high-risk progression to penetrating keratoplasty.


Assuntos
DNA Fúngico/genética , Infecções Oculares Fúngicas/microbiologia , Fusariose/microbiologia , Fusarium/genética , Ceratite/microbiologia , Reação em Cadeia da Polimerase Multiplex/métodos , Micotoxinas/genética , Adulto , Córnea/microbiologia , Infecções Oculares Fúngicas/genética , Feminino , Fusariose/genética , Fusarium/isolamento & purificação , Humanos , Ceratite/genética , Masculino , Pessoa de Meia-Idade
8.
PLoS Pathog ; 14(1): e1006827, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-29357387

RESUMO

Myosin-I molecular motors are proposed to function as linkers between membranes and the actin cytoskeleton in several cellular processes, but their role in the biosynthesis of fungal secondary metabolites remain elusive. Here, we found that the myosin I of Fusarium graminearum (FgMyo1), the causal agent of Fusarium head blight, plays critical roles in mycotoxin biosynthesis. Inhibition of myosin I by the small molecule phenamacril leads to marked reduction in deoxynivalenol (DON) biosynthesis. FgMyo1 also governs translation of the DON biosynthetic enzyme Tri1 by interacting with the ribosome-associated protein FgAsc1. Disruption of the ATPase activity of FgMyo1 either by the mutation E420K, down-regulation of FgMyo1 expression or deletion of FgAsc1 results in reduced Tri1 translation. The DON biosynthetic enzymes Tri1 and Tri4 are mainly localized to subcellular structures known as toxisomes in response to mycotoxin induction and the FgMyo1-interacting protein, actin, participates in toxisome formation. The actin polymerization disruptor latrunculin A inhibits toxisome assembly. Consistent with this observation, deletion of the actin-associated proteins FgPrk1 and FgEnd3 also results in reduced toxisome formation. Unexpectedly, the FgMyo1-actin cytoskeleton is not involved in biosynthesis of another secondary metabolite tested. Taken together, this study uncovers a novel function of myosin I in regulating mycotoxin biosynthesis in filamentous fungi.


Assuntos
Fusarium , Micotoxinas/biossíntese , Miosina Tipo I/fisiologia , Proteínas Fúngicas/biossíntese , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Fusarium/genética , Fusarium/metabolismo , Regulação Fúngica da Expressão Gênica , Micotoxinas/genética , Micotoxinas/metabolismo , Organismos Geneticamente Modificados , Metabolismo Secundário/genética
9.
Mycotoxin Res ; 34(2): 107-116, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-29299825

RESUMO

Fungi have a crucial role in the correct maturation of salami, but special attention should be addressed to the production of the nephrotoxic, immunotoxic, and carcinogenic mycotoxin ochratoxin A (OTA). In a monitoring study conducted in Northern Italy, OTA was detected by liquid chromatography coupled with mass spectrometry in 13 out 133 samples of traditional salami (9.8% of the total count). Mycological analysis of these samples yielded 247 fungal isolates which were identified to species level. The most frequent species were Penicillium nalgiovense, P. solitum, and P. chrysogenum. P. nordicum, an OTA-producing species commonly found in proteinaceous food, was not found in these samples. Three isolates were found to be Aspergillus westerdijkiae, an OTA-producing species. In order to check the results of the microbiological identification, 19 different strains of Aspergillus and 94 of Penicillium were tested for the presence of a sequence common to OTA-producing fungi by real-time PCR. None of the studied isolates, including the three A. westerdijkiae, possessed the otanpsPN target which is common to OTA-producing strains. Two out of three isolates of the A. westerdijkiae were also PCR-negative for the otanpsPN gene and did not produce OTA in culture. Conversely, this target sequence was amplified from the DNA purified from 14 salami casings including three casings harboring A. westerdijkiae. The amplification of sequences specific for OTA-producing strains performed on total genomic DNA extracted directly from salami casings provided a more suitable approach than PCR analysis of isolates from salami for the OTA-related otanpsPN gene to evaluate the risk of OTA contamination.


Assuntos
Análise de Alimentos , Contaminação de Alimentos , Microbiologia de Alimentos , Fungos/metabolismo , Ocratoxinas/análise , Cromatografia Líquida , DNA Espaçador Ribossômico , Análise de Alimentos/métodos , Fungos/classificação , Fungos/genética , Fungos/isolamento & purificação , Itália , Microbiota , Micotoxinas/análise , Micotoxinas/biossíntese , Micotoxinas/genética , Ocratoxinas/biossíntese , Penicillium/classificação , Penicillium/genética , Penicillium/isolamento & purificação , Penicillium/metabolismo , Reação em Cadeia da Polimerase , Espectrometria de Massas em Tandem
10.
Int J Food Microbiol ; 268: 35-43, 2018 Mar 02.
Artigo em Inglês | MEDLINE | ID: mdl-29324288

RESUMO

Ochratoxin A (OTA) is one of the most important mycotoxins due to its toxic properties and worldwide distribution which is produced by several Aspergillus and Penicillium species. The knowledge of OTA biosynthetic genes and understanding of the mechanisms involved in their regulation are essential. In this work, we obtained a clear picture of biosynthetic genes organization in the main OTA-producing Aspergillus and Penicillium species (A. steynii, A. westerdijkiae, A. niger, A. carbonarius and P. nordicum) using complete genome sequences obtained in this work or previously available on databases. The results revealed a region containing five ORFs which predicted five proteins: halogenase, bZIP transcription factor, cytochrome P450 monooxygenase, non-ribosomal peptide synthetase and polyketide synthase in all the five species. Genetic synteny was conserved in both Penicillium and Aspergillus species although genomic location seemed to be different since the clusters presented different flanking regions (except for A. steynii and A. westerdijkiae); these observations support the hypothesis of the orthology of this genomic region and that it might have been acquired by horizontal transfer. New real-time RT-PCR assays for quantification of the expression of these OTA biosynthetic genes were developed. In all species, the five genes were consistently expressed in OTA-producing strains in permissive conditions. These protocols might favour futures studies on the regulation of biosynthetic genes in order to develop new efficient control methods to avoid OTA entering the food chain.


Assuntos
Aspergillus/genética , Micotoxinas/genética , Ocratoxinas/biossíntese , Penicillium/genética , Aspergillus/metabolismo , Fatores de Transcrição de Zíper de Leucina Básica/genética , Sistema Enzimático do Citocromo P-450/genética , Genoma Fúngico/genética , Família Multigênica/genética , Micotoxinas/metabolismo , Penicillium/metabolismo , Peptídeo Sintases/genética , Policetídeo Sintases/genética
11.
New Phytol ; 217(1): 320-331, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-28895153

RESUMO

The fungus Zymoseptoria tritici is the causal agent of Septoria Tritici Blotch (STB) disease of wheat leaves. Zymoseptoria tritici secretes many functionally uncharacterized effector proteins during infection. Here, we characterized a secreted ribonuclease (Zt6) with an unusual biphasic expression pattern. Transient expression systems were used to characterize Zt6, and mutants thereof, in both host and non-host plants. Cell-free protein expression systems monitored the impact of Zt6 protein on functional ribosomes, and in vitro assays of cells treated with recombinant Zt6 determined toxicity against bacteria, yeasts and filamentous fungi. We demonstrated that Zt6 is a functional ribonuclease and that phytotoxicity is dependent on both the presence of a 22-amino-acid N-terminal 'loop' region and its catalytic activity. Zt6 selectively cleaves both plant and animal rRNA species, and is toxic to wheat, tobacco, bacterial and yeast cells, but not to Z. tritici itself. Zt6 is the first Z. tritici effector demonstrated to have a likely dual functionality. The expression pattern of Zt6 and potent toxicity towards microorganisms suggest that, although it may contribute to the execution of wheat cell death, it is also likely to have an important secondary function in antimicrobial competition and niche protection.


Assuntos
Anti-Infecciosos/isolamento & purificação , Ascomicetos/enzimologia , Doenças das Plantas/microbiologia , Ribonucleases/isolamento & purificação , Triticum/microbiologia , Anti-Infecciosos/metabolismo , Ascomicetos/patogenicidade , Morte Celular/efeitos dos fármacos , Proteínas Fúngicas/genética , Proteínas Fúngicas/isolamento & purificação , Proteínas Fúngicas/metabolismo , Microbiota/efeitos dos fármacos , Micotoxinas/genética , Micotoxinas/isolamento & purificação , Micotoxinas/metabolismo , Folhas de Planta/microbiologia , Ribonucleases/genética , Ribonucleases/metabolismo
12.
J Basic Microbiol ; 57(11): 899-909, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-28902962

RESUMO

Aspergillus flavus is a filamentous fungus which is widespread on agricultural products and also able to cause various human diseases. This species is frequently isolated from indoor air as well, furthermore, it is known as a common causal agent of keratomycosis, particularly in subtropical and tropical areas. It is also able to produce aflatoxins, one of the most carcinogenic mycotoxins which are harmful to animals and humans. In this study, 59 A. flavus isolates from four different habitats and 1 A. minisclerotigenes isolate were investigated. The isolates were identified and confirmed at the species level by the sequence analysis of a part of their calmodulin gene. Applying a combined analysis of UP-PCR, microsatellite, and calmodulin sequence data, the four group of isolates formed separate clusters on the phylogenetic tree. Examining the distribution of mating type genes MAT1-1 and MAT1-2, a ratio of approximately 3:1 was determined, and no correlation was found between the carried mating type gene and the aflatoxin production capability. HPLC analysis revealed that none of the examined isolates collected from indoor air or maize in Central Europe were able to produce aflatoxins, while about half of the isolates from India produced these mycotoxins under the test conditions.


Assuntos
Aspergillus flavus/classificação , Aspergillus flavus/isolamento & purificação , Genótipo , Aflatoxinas/genética , Aflatoxinas/metabolismo , Microbiologia do Ar , Animais , Aspergillus flavus/genética , Calmodulina/genética , DNA Fúngico , Ecossistema , Genes Fúngicos/genética , Humanos , Índia , Micotoxinas/genética , Filogenia , Análise de Sequência , Especificidade da Espécie , Zea mays/microbiologia
13.
Pestic Biochem Physiol ; 138: 97-103, 2017 May.
Artigo em Inglês | MEDLINE | ID: mdl-28456312

RESUMO

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).


Assuntos
Compostos de Bifenilo/farmacologia , Complexo II de Transporte de Elétrons/metabolismo , Proteínas Fúngicas/metabolismo , Micotoxinas/metabolismo , Niacinamida/análogos & derivados , Penicillium/efeitos dos fármacos , Farmacorresistência Fúngica , Proteínas Fúngicas/genética , Fungicidas Industriais/farmacologia , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Mutação , Micotoxinas/genética , Niacinamida/farmacologia , Subunidades Proteicas
14.
Fungal Genet Biol ; 103: 34-41, 2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-28392426

RESUMO

Surveys for crown rot (FCR) and head blight (FHB) of Algerian wheat conducted during 2014 and 2015 revealed that Fusarium culmorum strains producing 3-acetyl-deoxynivalenol (3ADON) or nivalenol (NIV) were the causal agents of these important diseases. Morphological identification of the isolates (n FCR=110, n FHB=30) was confirmed by sequencing a portion of TEF1. To assess mating type idiomorph, trichothecene chemotype potential and global population structure, the Algerian strains were compared with preliminary sample of F. culmorum from Italy (n=27), Australia (n=30) and the United States (n=28). A PCR assay for MAT idiomorph revealed that MAT1-1 and MAT1-2 strains were segregating in nearly equal proportions, except within Algeria where two-thirds of the strains were MAT1-2. An allele-specific PCR assay indicated that the 3ADON trichothecene genotype was predominant globally (83.8% 3ADON) and in each of the four countries sampled. In vitro toxin analyses confirmed trichothecene genotype PCR data and demonstrated that most of the strains tested (77%) produced culmorin. Global population genetic structure of 191 strains was assessed using nine microsatellite markers (SSRs). AMOVA of the clone corrected data indicated that 89% of the variation was within populations. Bayesian analysis of the SSR data identified two globally distributed, sympatric populations within which both trichothecene chemotypes and mating types were represented.


Assuntos
Fusarium/genética , Genética Populacional , Micotoxinas/genética , Argélia , Fusarium/patogenicidade , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , Triticum/microbiologia
15.
Phytopathology ; 107(5): 504-518, 2017 May.
Artigo em Inglês | MEDLINE | ID: mdl-28168931

RESUMO

Ergot alkaloids are highly diverse in structure, exhibit diverse effects on animals, and are produced by diverse fungi in the phylum Ascomycota, including pathogens and mutualistic symbionts of plants. These mycotoxins are best known from the fungal family Clavicipitaceae and are named for the ergot fungi that, through millennia, have contaminated grains and caused mass poisonings, with effects ranging from dry gangrene to convulsions and death. However, they are also useful sources of pharmaceuticals for a variety of medical purposes. More than a half-century of research has brought us extensive knowledge of ergot-alkaloid biosynthetic pathways from common early steps to several taxon-specific branches. Furthermore, a recent flurry of genome sequencing has revealed the genomic processes underlying ergot-alkaloid diversification. In this review, we discuss the evolution of ergot-alkaloid biosynthesis genes and gene clusters, including roles of gene recruitment, duplication and neofunctionalization, as well as gene loss, in diversifying structures of clavines, lysergic acid amides, and complex ergopeptines. Also reviewed are prospects for manipulating ergot-alkaloid profiles to enhance suitability of endophytes for forage grasses.


Assuntos
Claviceps/genética , Alcaloides de Claviceps/genética , Evolução Molecular , Hypocreales/genética , Doenças das Plantas/microbiologia , Poaceae/microbiologia , Vias Biossintéticas , Claviceps/química , Claviceps/metabolismo , Endófitos , Alcaloides de Claviceps/química , Alcaloides de Claviceps/metabolismo , Genômica , Hypocreales/química , Hypocreales/metabolismo , Família Multigênica , Micotoxinas/química , Micotoxinas/genética , Micotoxinas/metabolismo , Simbiose
16.
Toxins (Basel) ; 9(1)2017 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-28054977

RESUMO

The presence of mycotoxins in food represents a severe threat for public health and welfare, and poses relevant research challenges in the food toxicology field. Nowadays, food toxicologists have to provide answers to food-related toxicological issues, but at the same time they should provide the appropriate knowledge in background to effectively support the evidence-based decision-making in food safety. Therefore, keeping in mind that regulatory actions should be based on sound scientific findings, the present opinion addresses the main challenges in providing reliable data for supporting the risk assessment of foodborne mycotoxins.


Assuntos
Microbiologia de Alimentos/métodos , Genômica/métodos , Metabolômica/métodos , Micotoxinas/toxicidade , Toxicologia/métodos , Animais , Qualidade de Produtos para o Consumidor , Difusão de Inovações , Microbiologia de Alimentos/tendências , Previsões , Genômica/tendências , Humanos , Metabolômica/tendências , Micotoxinas/genética , Micotoxinas/metabolismo , Medição de Risco , Toxicologia/tendências
17.
Appl Microbiol Biotechnol ; 101(5): 2043-2056, 2017 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-27921136

RESUMO

PR toxin is a well-known isoprenoid mycotoxin almost solely produced by Penicillium roqueforti after growth on food or animal feed. This mycotoxin has been described as the most toxic produced by this species. In this study, an in silico analysis allowed identifying for the first time a 22.4-kb biosynthetic gene cluster involved in PR toxin biosynthesis in P. roqueforti. The pathway contains 11 open reading frames encoding for ten putative proteins including the major fungal terpene cyclase, aristolochene synthase, involved in the first farnesyl-diphosphate cyclization step as well as an oxidoreductase, an oxidase, two P450 monooxygenases, a transferase, and two dehydrogenase enzymes. Gene silencing was used to study three genes (ORF5, ORF6, and ORF8 encoding for an acetyltransferase and two P450 monooxygenases, respectively) and resulted in 20 to 40% PR toxin production reductions in all transformants proving the involvement of these genes and the corresponding enzyme activities in PR toxin biosynthesis. According to the considered silenced gene target, eremofortin A and B productions were also affected suggesting their involvement as biosynthetic intermediates in this pathway. A PR toxin biosynthesis pathway is proposed based on the most recent and available data.


Assuntos
Vias Biossintéticas/genética , Família Multigênica/genética , Micotoxinas/genética , Micotoxinas/metabolismo , Naftóis/metabolismo , Penicillium/genética , Penicillium/patogenicidade , Acetiltransferases/genética , Inativação Gênica , Oxigenases de Função Mista/genética , Fases de Leitura Aberta/genética , Sesquiterpenos/metabolismo
18.
Methods Mol Biol ; 1542: 13-32, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-27924529

RESUMO

The genus Alternaria includes more than 250 species. The traditional methods for identification of Alternaria species are based on morphological characteristics of the reproductive structures and sporulation patterns under controlled culture conditions. Cladistics analyses of "housekeeping genes" commonly used for other genera, failed to discriminate among the small-spored Alternaria species. The development of molecular methods achieving a better agreement with morphological differences is still needed. The production of secondary metabolites has also been used as a means of classification and identification. Alternaria spp. can produce a wide variety of toxic metabolites. These metabolites belong principally to three different structural groups: (1) the dibenzopyrone derivatives, alternariol (AOH), alternariol monomethyl ether (AME), and altenuene (ALT); (2) the perylene derivative altertoxins (ATX-I, ATX-II, and ATX II); and (3) the tetramic acid derivative, tenuazonic acid (TeA). TeA, AOH, AME, ALT, and ATX-I are the main. Certain species in the genus Alternaria produce host-specific toxins (HSTs) that contribute to their pathogenicity and virulence. Alternaria species are plant pathogens that cause spoilage of agricultural commodities with consequent mycotoxin accumulation and economic losses. Vegetable foods infected by Alternaria rot could introduce high amounts of these toxins to the human diet. More investigations on the toxic potential of these toxins and their hazard for human consumption are needed to make a reliable risk assessment of dietary exposure.


Assuntos
Alternaria/classificação , Micotoxinas/análise , Alternaria/genética , Alternaria/metabolismo , Ração Animal/microbiologia , Animais , Contaminação de Alimentos , Inocuidade dos Alimentos , Interações Hospedeiro-Patógeno , Humanos , Metaboloma , Metabolômica/métodos , Micotoxinas/genética , Micotoxinas/metabolismo , Plantas/microbiologia , Metabolismo Secundário
19.
Methods Mol Biol ; 1542: 107-119, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-27924532

RESUMO

Penicillium are very diverse and cosmopolite fungi, about 350 species are recognized within this genus. It is subdivided in four subgenera Aspergilloides, Penicillium, Furcatum, and Biverticillium; recently the first three has been included in Penicillium genus, and Biverticillium under Talaromyces. They occur worldwide and play important roles as decomposers of organic materials, cause destructive rots in the food industry where produces a wide range of mycotoxins; they are considered enzyme factories, and common indoor air irritants. In terms of human health are rarely associated as human pathogen because they hardly growth at 37°, while the main risk is related to ingestion of food contaminated by mycotoxins produced by several species of Penicillium. Various mycotoxins can occur in foods and feeds contaminated by Penicillium species, the most important are ochratoxin A and patulin; for which regulation are imposed in a number of countries, and at a less extent cyclopiazonic acid. In this chapter we summarize the main aspect of the morphology, ecology and toxigenicity of Penicillium foodborne mycotoxigenic species which belong mainly in subgenus Penicillium sections Brevicompacta, Chrysogena, Fasciculata, Penicillium, and Roquefortorum.


Assuntos
Micotoxinas/metabolismo , Penicillium/classificação , Penicillium/metabolismo , Animais , Produtos Agrícolas/microbiologia , Código de Barras de DNA Taxonômico , Contaminação de Alimentos , Inocuidade dos Alimentos , Humanos , Micotoxinas/química , Micotoxinas/classificação , Micotoxinas/genética , Penicillium/citologia , Penicillium/genética , Fenótipo
20.
Methods Mol Biol ; 1542: 293-309, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-27924546

RESUMO

Multiplex PCR-based methods for simultaneous detection and quantification of different mycotoxin-producing Penicillia are useful tools to be used in food safety programs. These rapid and sensitive techniques allow taking corrective actions during food processing or storage for avoiding accumulation of mycotoxins in them. In this chapter, three multiplex PCR-based methods to detect at least patulin- and ochratoxin A-producing Penicillia are detailed. Two of them are different multiplex real-time PCR suitable for monitoring and quantifying toxigenic Penicillium using the nonspecific dye SYBR Green and specific hydrolysis probes (TaqMan). All of them successfully use the same target genes involved in the biosynthesis of such mycotoxins for designing primers and/or probes.


Assuntos
Reação em Cadeia da Polimerase Multiplex , Micotoxinas/genética , Penicillium/classificação , Penicillium/genética , Reação em Cadeia da Polimerase em Tempo Real , Micotoxinas/biossíntese , Ocratoxinas/biossíntese , Patulina/biossíntese , Patulina/genética , Penicillium/metabolismo
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