Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 415
Filtrar
1.
Molecules ; 27(21)2022 Oct 28.
Artigo em Inglês | MEDLINE | ID: mdl-36364148

RESUMO

Ergot is the spore form of the fungus Claviceps purpurea. Ergot alkaloids are indole compounds that are biosynthetically derived from L-tryptophan and represent the largest group of fungal nitrogen metabolites found in nature. The common part of ergot alkaloids is lysergic acid. This review shows the importance of lysergic acid as a representative of ergot alkaloids. The subject of ergot and its alkaloids is presented, with a particular focus on lysergic acid. All methods of total lysergic acid synthesis-through Woodward, Hendrickson, and Szantay intermediates and Heck coupling methods-are presented. The topic of biosynthesis is also discussed.


Assuntos
Claviceps , Alcaloides de Claviceps , Ácido Lisérgico , Ácido Lisérgico/metabolismo , Claviceps/metabolismo
2.
Toxins (Basel) ; 14(9)2022 08 24.
Artigo em Inglês | MEDLINE | ID: mdl-36136518

RESUMO

As the contamination of cereal grains with ergot has been increasing in Western Canada, studies were undertaken to evaluate the impacts of heating (60, 80, 120, or 190 °C) alone or in combination with pelleting on concentrations of ergot alkaloids. Fifteen samples of ergot-contaminated grain from Alberta and Saskatchewan were assayed for R and S epimers of six alkaloids (ergocryptine, ergocristine, ergocornine, ergometrine, ergosine, and ergotamine) using HPLC MS/MS. Five samples with distinct alkaloid profiles were then selected for heating and pelleting studies. Heating resulted in a linear increase (p < 0.05) of total R and total S epimers with increasing temperature, although some individual R epimers were stable (ergometrine, ergosine, ergotamine). Pelleting also increased (p < 0.05) concentrations of total R and total S epimers detected, although ergometrine concentration decreased (p < 0.05) after pelleting. A feeding study arranged in a 2 × 2 factorial structure used 48 backgrounding Angus-cross steers fed four different diets: (1) Control Mash (CM, no added ergot), (2) Control Pellet (CP), (3) Ergot Mash (EM), or (4) Ergot Pellet (EP). Pelleting heated the ergot to 90-100 °C under 4 bars pressure, but the ergot used in the feeding study was not otherwise heated. Alkaloid concentrations of EM and EP varied by up to 1.1 mg/kg depending on the feed matrix assayed. No differences among treatments were noted for growth performance, feed intake, feed conversion, concentrations of serum prolactin and haptoglobin, hair cortisol, or in temperatures of extremities measured by infrared thermography. The only negative impacts of ergot alkaloids were on blood parameters indicative of reduced immune function or chronic inflammation. Pelleting did not heighten the negative clinical outcomes of ergot, although alkaloid concentrations of pelleted feed increased depending on the matrix assayed. It was hypothesized that the heat and pressure associated with pelleting may enhance the recovery of alkaloids from pelleted feed.


Assuntos
Claviceps , Alcaloides de Claviceps , Alberta , Ração Animal/análise , Animais , Bovinos , Claviceps/química , Grão Comestível/química , Ergonovina/análise , Alcaloides de Claviceps/análise , Ergotamina/análise , Ergotaminas/análise , Haptoglobinas/análise , Calefação , Hidrocortisona , Prolactina , Espectrometria de Massas em Tandem/métodos
3.
J Agric Food Chem ; 70(29): 8931-8941, 2022 Jul 27.
Artigo em Inglês | MEDLINE | ID: mdl-35830571

RESUMO

Detoxification of ergot-contaminated feed by ammonia would be a practical application, given that ammonia is routinely used in the agriculture industry. To assess the effects of ammonia on ergot alkaloids, natural ergot-contaminated wheat was ammoniated. The total concentration of ergot alkaloids (R- and S-epimers) decreased after exposure to ammonia (8-29%). Separately, the total R-epimers decreased in concentration (40-66%), whereas the total S-epimers increased (21-81%). Specific ergot alkaloids demonstrated degradation and/or epimerization after exposure to ammonia, potentially associated with structural differences, and influenced the total concentrations observed. Ammonization of ergot standards resulted in potential degradation products and epimerization, supporting the above results. The use of ultrahigh-performance liquid chromatography-tandem mass spectrometry provides an updated assessment of the detoxification potential of ammonia for ergot alkaloids and the quantification of the S-epimers. Ammonia alters the R- and S-epimers of ergot alkaloids, which may lead to a potential practical detoxification process of ergot-contaminated feed.


Assuntos
Claviceps , Alcaloides de Claviceps , Amônia , Cromatografia Líquida de Alta Pressão/métodos , Alcaloides de Claviceps/análise , Contaminação de Alimentos/análise , Compostos Heterocíclicos de 4 ou mais Anéis , Triticum/química
4.
PLoS One ; 17(2): e0263496, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35143550

RESUMO

Pangenome analyses are increasingly being utilized to study the evolution of eukaryotic organisms. While pangenomes can provide insight into polymorphic gene content, inferences about the ecological and adaptive potential of such organisms also need to be accompanied by additional supportive genomic analyses. In this study we constructed a pangenome of Claviceps purpurea from 24 genomes and examined the positive selection and recombination landscape of an economically important fungal organism for pharmacology and agricultural research. Together, these analyses revealed that C. purpurea has a relatively large accessory genome (~ 38%), high recombination rates (ρ = 0.044), and transposon mediated gene duplication. However, due to observations of relatively low transposable element (TE) content (8.8%) and a lack of variability in genome sizes, prolific TE expansion may be controlled by frequent recombination. We additionally identified that within the ergoline biosynthetic cluster the lpsA1 and lpsA2 were the result of a recombination event. However, the high recombination rates observed in C. purpurea may be influencing an overall trend of purifying selection across the genome. These results showcase the use of selection and recombination landscapes to identify mechanisms contributing to pangenome structure and primary factors influencing the evolution of an organism.


Assuntos
Claviceps/genética , Genoma Fúngico , Recombinação Genética , Elementos de DNA Transponíveis , Especificidade de Hospedeiro , Polimorfismo de Nucleotídeo Único , Seleção Genética
5.
Phytopathology ; 112(4): 842-851, 2022 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-34698539

RESUMO

Partial resistance to the biotrophic fungal pathogen Claviceps purpurea, causal agent of ergot, has been found that colocates with mutant alleles of the wheat Reduced height (Rht) loci on chromosomes 4B and 4D. These Rht loci represent the wheat orthologs of the Arabidopsis Della genes. To investigate the role of the Rht mutant DELLA proteins in ergot resistance, we assessed C. purpurea infection in wheat near-isogenic lines (NILs) carrying the gibberellic acid (GA)-insensitive semidwarf alleles Rht-B1b and Rht-D1b and the severe dwarf alleles Rht-B1c and Rht-D1c. NILs of the GA-sensitive alleles Rht8 (chromosome 2D) and Rht12 (chromosome 5A) were also included. A general trend toward increased resistance to C. purpurea, with smaller and lighter sclerotia, was observed on the NILs Rht-B1b, Rht-D1b, Rht-B1c, and Rht-D1c, and also on Rht8. Levels of the bioactive GA4 and the auxin indole-3-acetic acid increased after inoculation with C. purpurea, following similar patterns and implicating a potential auxin-mediated induction of GA biosynthesis. In contrast, jasmonic acid (JA) levels fell in the parental lines 'Mercia' and 'Maris Huntsman' after inoculation with C. purpurea, but increased in all the Rht-mutant NILs. Inoculation with C. purpurea did not show any informative changes in the levels of salicylic acid. Our results suggest that GA-mediated degradation of the DELLA proteins and down-regulation of JA-signaling pathways supports infection of wheat by C. purpurea. As these responses are generally associated with necrotrophic fungal pathogens, we propose that the biotroph C. purpurea may have a necrotrophic growth stage.


Assuntos
Claviceps , Triticum , Claviceps/genética , Hormônios/metabolismo , Ácidos Indolacéticos/metabolismo , Doenças das Plantas/microbiologia , Triticum/genética , Triticum/metabolismo
6.
Metab Eng ; 69: 198-208, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34902590

RESUMO

Privileged ergot alkaloids (EAs) produced by the fungal genus Claviceps are used to treat a wide range of diseases. However, their use and research have been hampered by the challenging genetic engineering of Claviceps. Here we systematically refactored and rationally engineered the EA biosynthetic pathway in heterologous host Aspergillus nidulans by using a Fungal-Yeast-Shuttle-Vector protocol. The obtained strains allowed the production of diverse EAs and related intermediates, including prechanoclavine (PCC, 333.8 mg/L), chanoclavine (CC, 241.0 mg/L), agroclavine (AC, 78.7 mg/L), and festuclavine (FC, 99.2 mg/L), etc. This fungal platform also enabled the access to the methyl-oxidized EAs (MOEAs), including elymoclavine (EC), lysergic acid (LA), dihydroelysergol (DHLG), and dihydrolysergic acid (DHLA), by overexpressing a P450 enzyme CloA. Furthermore, by optimizing the P450 electron transfer (ET) pathway and using multi-copy of cloA, the titers of EC and DHLG have been improved by 17.3- and 9.4-fold, respectively. Beyond our demonstration of A. nidulans as a robust platform for EA overproduction, our study offers a proof of concept for engineering the eukaryotic P450s-contained biosynthetic pathways in a filamentous fungal host.


Assuntos
Claviceps , Alcaloides de Claviceps , Vias Biossintéticas/genética , Claviceps/genética , Claviceps/metabolismo , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Alcaloides de Claviceps/genética , Alcaloides de Claviceps/metabolismo , Saccharomyces cerevisiae/metabolismo
7.
Clin Dermatol ; 39(6): 1088-1094, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34920829

RESUMO

It has been widely accepted that ergot is a fungal disease caused by infection with the parasitic Claviceps purpurea leading to the development of typical black kernels n the plant. Ingestion of infected rye grains containing ergot alkaloids-usually in the form of contaminated rye bread-causes poisoning, also known as ergotism. The negative impacts of ergot contamination of grain on the health of humans and animals were first documented in ancient times. The history of ergotism shows the influence of food on human health. Although ergot has been known for ages, until the 18th century, its nature was not recognized. It was a part of the rye plant and it was used in traditional medicine. The diet was based was mostly on rye that led to neurologic disorders and gangrene. In the Middle Ages, in regions where rye was a dietary staple, many cases of a peculiar epidemic were recorded. Two names are usually used to describe it: "Saint Anthony's fire" and "holy fire," although there are many more appellations. The history of ergotism is a very important part of history of dermatology. The saint who people prayed to for protection against the disease was Anthony the Great (251-356). Monks of the Order of Saint Anthony played a particular role in treating ergotism by natural methods and specialized in treating skin diseases. Ergot alkaloids still pose a risk to human and animal safety if they appear in food.


Assuntos
Ergotismo , Animais , Claviceps , Ergotismo/epidemiologia , Alimentos , Humanos
8.
Toxins (Basel) ; 13(12)2021 12 02.
Artigo em Inglês | MEDLINE | ID: mdl-34941699

RESUMO

Research into ergot alkaloid production in major cereal cash crops is crucial for furthering our understanding of the potential toxicological impacts of Claviceps purpurea upon Canadian agriculture and to ensure consumer safety. An untargeted metabolomics approach profiling extracts of C. purpurea sclerotia from four different grain crops separated the C. purpurea strains into two distinct metabolomic classes based on ergot alkaloid content. Variances in C. purpurea alkaloid profiles were correlated to genetic differences within the lpsA gene of the ergot alkaloid biosynthetic gene cluster from previously published genomes and from newly sequenced, long-read genome assemblies of Canadian strains. Based on gene cluster composition and unique polymorphisms, we hypothesize that the alkaloid content of C. purpurea sclerotia is currently undergoing adaptation. The patterns of lpsA gene diversity described in this small subset of Canadian strains provides a remarkable framework for understanding accelerated evolution of ergot alkaloid production in Claviceps purpurea.


Assuntos
Claviceps/genética , Alcaloides de Claviceps/biossíntese , Micotoxinas/química , Canadá , Claviceps/metabolismo , Grão Comestível/microbiologia , Alcaloides de Claviceps/genética , Variação Genética , Micotoxinas/genética , Secale/microbiologia , Triticale/microbiologia , Triticum/microbiologia
9.
Toxins (Basel) ; 13(11)2021 11 13.
Artigo em Inglês | MEDLINE | ID: mdl-34822583

RESUMO

Ergot fungi (Claviceps spp.) are infamous for producing sclerotia containing a wide spectrum of ergot alkaloids (EA) toxic to humans and animals, making them nefarious villains in the agricultural and food industries, but also treasures for pharmaceuticals. In addition to three classes of EAs, several species also produce paspaline-derived indole diterpenes (IDT) that cause ataxia and staggers in livestock. Furthermore, two other types of alkaloids, i.e., loline (LOL) and peramine (PER), found in Epichloë spp., close relatives of Claviceps, have shown beneficial effects on host plants without evidence of toxicity to mammals. The gene clusters associated with the production of these alkaloids are known. We examined genomes of 53 strains of 19 Claviceps spp. to screen for these genes, aiming to understand the evolutionary patterns of these genes across the genus through phylogenetic and DNA polymorphism analyses. Our results showed (1) varied numbers of eas genes in C. sect. Claviceps and sect. Pusillae, none in sect. Citrinae, six idt/ltm genes in sect. Claviceps (except four in C. cyperi), zero to one partial (idtG) in sect. Pusillae, and four in sect. Citrinae, (2) two to three copies of dmaW, easE, easF, idt/ltmB, itd/ltmQ in sect. Claviceps, (3) frequent gene gains and losses, and (4) an evolutionary hourglass pattern in the intra-specific eas gene diversity and divergence in C. purpurea.


Assuntos
Claviceps/genética , Alcaloides de Claviceps/biossíntese , Genes Fúngicos/genética , Alcaloides Indólicos/isolamento & purificação , Claviceps/metabolismo , Evolução Molecular , Família Multigênica , Filogenia
10.
BMC Genomics ; 22(1): 766, 2021 Oct 26.
Artigo em Inglês | MEDLINE | ID: mdl-34702162

RESUMO

BACKGROUND: The phytopatogen Claviceps paspali is the causal agent of Ergot disease in Paspalum spp., which includes highly productive forage grasses such as P. dilatatum. This disease impacts dairy and beef production by affecting seed quality and producing mycotoxins that can affect performance in feeding animals. The molecular basis of pathogenicity of C. paspali remains unknown, which makes it more difficult to find solutions for this problem. Secreted proteins are related to fungi virulence and can manipulate plant immunity acting on different subcellular localizations. Therefore, identifying and characterizing secreted proteins in phytopathogenic fungi will provide a better understanding of how they overcome host defense and cause disease. The aim of this work is to analyze the whole genome sequences of three C. paspali isolates to obtain a comparative genome characterization based on possible secreted proteins and pathogenicity factors present in their genome. In planta RNA-seq analysis at an early stage of the interaction of C. paspali with P. dilatatum stigmas was also conducted in order to determine possible secreted proteins expressed in the infection process. RESULTS: C. paspali isolates had compact genomes and secretome which accounted for 4.6-4.9% of the predicted proteomes. More than 50% of the predicted secretome had no homology to known proteins. RNA-Seq revealed that three protein-coding genes predicted as secreted have mayor expression changes during 1 dpi vs 4 dpi. Also, three of the first 10 highly expressed genes in both time points were predicted as effector-like. CAZyme-like proteins were found in the predicted secretome and the most abundant family could be associated to pectine degradation. Based on this, pectine could be a main component affected by the cell wall degrading enzymes of C. paspali. CONCLUSIONS: Based on predictions from DNA sequence and RNA-seq, unique probable secreted proteins and probable pathogenicity factors were identified in C. paspali isolates. This information opens new avenues in the study of the biology of this fungus and how it modulates the interaction with its host. Knowledge of the diversity of the secretome and putative pathogenicity genes should facilitate future research in disease management of Claviceps spp.


Assuntos
Claviceps , Micotoxinas , Paspalum , Animais , Bovinos , Claviceps/genética , Virulência
11.
J Nat Prod ; 84(10): 2630-2643, 2021 10 22.
Artigo em Inglês | MEDLINE | ID: mdl-34553942

RESUMO

Claviceps purpurea is an ergot fungus known for its neurotropic alkaloids, which have been identified as the main cause of ergotism, a livestock and human disease triggered by ergot consumption. Tetrahydroxanthone dimers, the so-called ergopigments, presumably also contribute to this toxic effect. Overexpression of the cluster-specific transcription factor responsible for the formation of these pigments in C. purpurea led to the isolation of three new metabolites (8-10). The new pigments were characterized utilizing HRMS, NMR techniques, and CD spectroscopy and shown to be xanthone dimers. Secalonic acid A and its 2,4'- and 4,4'-linked isomers were also isolated, and their absolute configuration was investigated. The contribution of secalonic acid A, its isomers, and new metabolites to the toxicity of C. purpurea was investigated in HepG2 and CCF-STTG1 cells. Along with cytotoxic properties, secalonic acid A was found to inhibit topoisomerase I and II activity.


Assuntos
Claviceps/química , Xantenos/química , Células Hep G2 , Humanos , Estrutura Molecular , Inibidores da Topoisomerase , Xantonas
12.
Toxins (Basel) ; 13(7)2021 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-34357964

RESUMO

In the past centuries consumption of bread made of ergot-infected flour resulted in mass poisonings and miscarriages. The reason was the sclerotia of Claviceps purpurea (Fr.) Tul.-a source of noxious ergot alkaloids (ergotamine and ergovaline). The authors have searched the 19th century medical literature in order to find information on the following topics: dosage forms of drugs based on ergot and their application in official gynecology and obstetrics. The authors also briefly address the relevant data from the previous periods as well as the 20th century research on ergot. The research resulted in a conclusion that applications of ergot in gynecology and obstetrics in the 19th century were limited to controlling excessive uterine bleeding and irregular spasms, treatment of fibrous tumors of the uterus, and prevention of miscarriage, abortion, and amenorrhoea. The most common dosage forms mentioned in the works included in our review were the following: tinctures, water extracts (Wernich's and Squibb's watery extract of ergot), pills, and powders. The information documented in this paper will be helpful for further research and helpful in broadening the understanding of the historical application of the described controversial crude drugs. Ergot alkaloids were widely used in obstetrics, but in modern times they are not used in developed countries anymore. They may, however, play a significant role in developing countries where, in some cases, they can be used as an anti-hemorrhage agent during labor.


Assuntos
Alcaloides de Claviceps/uso terapêutico , Claviceps , Ergotamina , Ergotaminas , Ginecologia , Dietilamida do Ácido Lisérgico/análogos & derivados , Obstetrícia
13.
BMC Plant Biol ; 21(1): 316, 2021 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-34215204

RESUMO

BACKGROUND: Ergot, caused by the fungal pathogen Claviceps purpurea, infects the female flowers of a range of cereal crops, including wheat. To understand the interaction between C. purpurea and hexaploid wheat we undertook an extensive examination of the reprogramming of the wheat transcriptome in response to C. purpurea infection through floral tissues (i.e. the stigma, transmitting and base ovule tissues of the ovary) and over time. RESULTS: C. purpurea hyphae were observed to have grown into and down the stigma at 24 h (H) after inoculation. By 48H hyphae had grown through the transmitting tissue into the base, while by 72H hyphae had surrounded the ovule. By 5 days (D) the ovule had been replaced by fungal tissue. Differential gene expression was first observed at 1H in the stigma tissue. Many of the wheat genes differentially transcribed in response to C. purpurea infection were associated with plant hormones and included the ethylene (ET), auxin, cytokinin, gibberellic acid (GA), salicylic acid and jasmonic acid (JA) biosynthetic and signaling pathways. Hormone-associated genes were first detected in the stigma and base tissues at 24H, but not in the transmitting tissue. Genes associated with GA and JA pathways were seen in the stigma at 24H, while JA and ET-associated genes were identified in the base at 24H. In addition, several defence-related genes were differential expressed in response to C. purpurea infection, including antifungal proteins, endocytosis/exocytosis-related proteins, NBS-LRR class proteins, genes involved in programmed cell death, receptor protein kinases and transcription factors. Of particular interest was the identification of differential expression of wheat genes in the base tissue well before the appearance of fungal hyphae, suggesting that a mobile signal, either pathogen or plant-derived, is delivered to the base prior to colonisation. CONCLUSIONS: Multiple host hormone biosynthesis and signalling pathways were significantly perturbed from an early stage in the wheat - C. purpurea interaction. Differential gene expression at the base of the ovary, ahead of arrival of the pathogen, indicated the potential presence of a long-distance signal modifying host gene expression.


Assuntos
Claviceps/fisiologia , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , Transcriptoma/genética , Triticum/genética , Triticum/microbiologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Genes de Plantas , Reguladores de Crescimento de Plantas/farmacologia , RNA-Seq , Fatores de Tempo , Triticum/efeitos dos fármacos
14.
J Agric Food Chem ; 69(25): 7137-7148, 2021 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-34148344

RESUMO

The plant parasitic fungus Claviceps purpurea sensu lato produces sclerotia containing toxic ergot alkaloids and uncharacterized indole diterpenoids in grasses including cereals. The aim of this study was to detect as many peptide ergot alkaloids and indole diterpenoids in ergot sclerotia as possible by using a liquid chromatography-high-resolution mass spectrometry (LC-HRMS/MS) approach and applying filtering of diagnostic fragment ions for data extraction. The sample set consisted of 66 Claviceps sclerotia from four different geographic locations in southeastern Norway as well as Saskatchewan, Canada. The host plants included both wild grasses and important cereal grains such as rye. DNA sequencing showed that the sclerotia were from three Claviceps species, i.e., Claviceps purpurea sensu stricto (s.s.), Claviceps humidiphila, and Claviceps arundinis (former C. purpurea genotypes G1, G2, and G2a, respectively). All sclerotia from cereal grains were from C. purpurea s.s. Diagnostic fragment filtering was based on detecting specific product ions in MS/MS data sets that are well-conserved across the different ergot alkaloid subgroups and indole diterpenoids of the paspaline/paxilline type. The approach extracted mass spectra from 67 peptide ergot alkaloids (including C-8 epimers and lactam variants) and five indole diterpenoids. In addition, three clavines were detected by using targeted analysis. The sum of the peak areas for ergot alkaloids, which have been assigned as "major" analogues by the European Food Safety Authority (ergometrine, ergosine, ergotamine, α-ergocryptine, ergocornine, ergocristine, and their 8-S epimers), accounted for at least 50% of the extracted total ergot alkaloid metabolome. Univariate and multivariate statistical analyses showed that several of the alkaloids were specific for certain species within the C. purpurea species complex and could be used as chemotaxonomic markers for species assignment.


Assuntos
Claviceps , Diterpenos , Alcaloides de Claviceps , Canadá , Cromatografia Líquida , Claviceps/genética , Indóis , Metaboloma , Noruega , Espectrometria de Massas em Tandem
15.
Toxins (Basel) ; 13(5)2021 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-34064772

RESUMO

Ergot alkaloids are mycotoxins formed by fungi of the Claviceps genus, which are some of the most common contaminants of food and feed worldwide. These toxins are a structurally heterogeneous group of compounds, sharing an ergoline backbone. Six structures and their corresponding stereoisomers are typically quantified by either HPLC-FLD or HPLC-MS/MS and the values subsequently summed up to determine the total ergot alkaloid content. For the development of a screening method targeting all ergot alkaloids simultaneously, the alkaloids need to be transferred to one homogeneous structure: a lysergic acid derivative. In this study, two promising cleaving methods-acidic esterification and hydrazinolysis-are compared, using dihydroergocristine as a model compound. While the acidic esterification proved to be unsuitable, due to long reaction times and oxidation sensitivity, hydrazinolysis reached a quantitative yield in 40‒60 min. Parallel workup of several samples is possible. An increasing effect on the reaction rate by the addition of ammonium iodide was demonstrated. Application of hydrazinolysis to a major ergot alkaloid mix solution showed that all ergopeptines were cleaved, but ergometrine/-inine was barely affected. Still, hydrazinolysis is a suitable tool for the development of a sum parameter screening method for ergot alkaloids in food and feed.


Assuntos
Claviceps/metabolismo , Alcaloides de Claviceps/análise , Micotoxinas/análise , Cromatografia Líquida de Alta Pressão , Alcaloides de Claviceps/química , Hidrazinas/química , Micotoxinas/química , Estereoisomerismo , Espectrometria de Massas em Tandem
16.
J AOAC Int ; 104(3): 546-554, 2021 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-33394021

RESUMO

BACKGROUND: Ergot alkaloids are mycotoxins produced by the fungus Claviceps, which can contaminate grains and pose a health risk to humans and animals. Validation of an ergot alkaloid method in collaborative projects can be challenging due to instability of analytes, a lack of reliable reference materials, and a fully validated reference method. OBJECTIVE: To extensively evaluate performance of a quantitative UHPLC-MS/MS method to detect ten ergot alkaloids at concentrations between 16 and 500 ng/g in grains. METHOD: The method performance was evaluated in the Blinded Method Test (BMT) exercise, which allowed organizers to successfully address the challenges. Forty completely blinded test samples were prepared in an independent laboratory and shipped to a participating laboratory to analyze on two separate days. RESULTS: Precision, accuracy, and HorRatr values met or exceeded the U.S. Food and Drug Administration recommendations. The design of the BMT exercise provided a high degree of confidence in data and conclusions drawn. CONCLUSIONS: The method performed in a manner as expected, and the method can be used by the laboratory for routine testing of wheat and rye grains. HIGHLIGHTS: BMT of laboratory methods facilitate validation of tests by evaluating performance in an unbiased manner.


Assuntos
Claviceps , Alcaloides de Claviceps , Animais , Cromatografia Líquida de Alta Pressão , Humanos , Secale , Espectrometria de Massas em Tandem , Triticum
17.
Genome Biol Evol ; 13(2)2021 02 03.
Artigo em Inglês | MEDLINE | ID: mdl-33512490

RESUMO

The genus Claviceps has been known for centuries as an economically important fungal genus for pharmacology and agricultural research. Only recently have researchers begun to unravel the evolutionary history of the genus, with origins in South America and classification of four distinct sections through ecological, morphological, and metabolic features (Claviceps sects. Citrinae, Paspalorum, Pusillae, and Claviceps). The first three sections are additionally characterized by narrow host range, whereas section Claviceps is considered evolutionarily more successful and adaptable as it has the largest host range and biogeographical distribution. However, the reasons for this success and adaptability remain unclear. Our study elucidates factors influencing adaptability by sequencing and annotating 50 Claviceps genomes, representing 21 species, for a comprehensive comparison of genome architecture and plasticity in relation to host range potential. Our results show the trajectory from specialized genomes (sects. Citrinae and Paspalorum) toward adaptive genomes (sects. Pusillae and Claviceps) through colocalization of transposable elements around predicted effectors and a putative loss of repeat-induced point mutation resulting in unconstrained tandem gene duplication coinciding with increased host range potential and speciation. Alterations of genomic architecture and plasticity can substantially influence and shape the evolutionary trajectory of fungal pathogens and their adaptability. Furthermore, our study provides a large increase in available genomic resources to propel future studies of Claviceps in pharmacology and agricultural research, as well as, research into deeper understanding of the evolution of adaptable plant pathogens.


Assuntos
Claviceps/genética , Evolução Molecular , Genoma Fúngico , Claviceps/classificação , Genes Fúngicos , Genômica , Especificidade de Hospedeiro , Sequências Repetitivas Dispersas , Anotação de Sequência Molecular , Filogenia
18.
Phytopathology ; 111(5): 831-841, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33141647

RESUMO

Ergot, caused by Claviceps purpurea sensu lato, is an economically important seed replacement disease of Kentucky bluegrass (Poa pratensis) and perennial ryegrass (Lolium perenne) seed crops. C. purpurea sensu stricto is considered the primary Claviceps species responsible, but genetic diversity and cryptic species within C. purpurea sensu lato have previously been reported. Fifty-six C. purpurea sensu lato isolates collected from P. pratensis (n = 21) and L. perenne (n = 35) in Oregon and Washington between 2010 and 2014 were characterized via random amplified polymorphic DNA (RAPD), partial internal transcribed spacer (ITS), ß-tubulin and elongation factor-1α (EF-1α) sequences, conidial size, and ergot alkaloid chemotype. Based on RAPD analysis, seven isolates from P. pratensis and 33 isolates from L. perenne collected in Oregon corresponded to C. purpurea sensu stricto, and 13 isolates collected from P. pratensis in Washington and Oregon were identified as C. humidiphila. Partial ITS, ß-tubulin, and EF-1α sequences identified 10 isolates from P. pratensis as C. humidiphila, and seven isolates from P. pratensis and 33 isolates from L. perenne were identified as C. purpurea sensu stricto. Several isolates generated ambiguous RAPD bands or sequences that prevented identification. Ergot alkaloid chemotype profiling found that ergocornine and its epimer were predominant in sclerotia from P. pratensis, whereas ergotamine and its epimer were most abundant in sclerotia from L. perenne. This study confirms the presence of the C. purpurea sensu lato species complex in the U.S. Pacific Northwest and suggests that more research is needed to characterize and mitigate Claviceps spp. infection of grass seed crops in North America.


Assuntos
Claviceps , Alcaloides de Claviceps , Claviceps/genética , Doenças das Plantas , Poaceae , Técnica de Amplificação ao Acaso de DNA Polimórfico , Sementes , Washington
19.
J Biotechnol ; 325: 341-354, 2021 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-33053363

RESUMO

Claviceps purpurea is a filamentous fungus well known as a widespread plant pathogen, but it is also an important ergot alkaloid producer exploited by the pharmaceutic industry. In this work, we demonstrated that CRISPR/Cas9 can be a tool for directed mutagenesis in C. purpurea targeting pyr4 and TrpE genes encoding the orotidine 5'-phosphate decarboxylase involved in pyrimidine biosynthesis and the α-subunit of the anthranilate synthase involved in tryptophan biosynthesis, respectively. After protoplast transformation and single spore isolation, homokaryotic mutants showing uridine or tryptophan auxotrophy were selected. In all cases, insertions or insertions combined with deletions were found mostly 3 bp upstream of the PAM sequence. However, transformation efficiencies of CRISPR/Cas9 and CRISPR/Cas9 mediated homology-directed repair only slightly improved in comparison to homologous recombination-mediated knocking-out of the TrpE gene. Moreover, Trp auxotrophs were non-infectious towards rye plants likely due to a decreased production of the plant hormones auxins, which are synthesized by C. purpurea from indole-3-glycerolphosphate in Trp-dependent and Trp-independent biosynthetic pathways, and help the fungus to colonize the plant host. It was demonstrated that the CRISPR/Cas9 vector containing autonomous replicative sequence AMA1 can be fully removed by further culturing of C. purpurea on non-selective media. This method enables introducing multiple mutations in Claviceps and makes feasible metabolic engineering of industrial strains.


Assuntos
Claviceps , Sistemas CRISPR-Cas/genética , Claviceps/genética , Edição de Genes , Mutagênese , Protoplastos
20.
J Sci Food Agric ; 101(3): 1047-1052, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-32761809

RESUMO

BACKGROUND: Harmful botanical impurities may contaminate feed and feed materials and be a potential danger to animal or human health, or to the environment. The aim of this study was to establish rapid and sensitive methods that can be used in routine official controls to determine botanical impurities such as Datura stramonium, Ricinus communis, Crotaliaria spp., and Ambrosia spp. in animal feed and raw materials. Claviceps sclerotia were also detected in cereals, due to the similarities of the targets and the analytical procedure. Regulation (EU) 625/2017, which replaces Reg. 2004/882/EC, states that EU member states should conduct official controls in assessed and accredited laboratories and that the analytical methods must be validated before use by considering parameters such as specificity, precision, recovery, and measurement uncertainly. RESULTS AND CONCLUSION: The results demonstrate that all of the methods tested are suitable for the official quantitative analyses required by EU official legislation. © 2020 Society of Chemical Industry.


Assuntos
Ração Animal/análise , Análise de Alimentos/métodos , Contaminação de Alimentos/análise , Plantas Tóxicas/química , Ambrosia/química , Animais , Claviceps/química , Crotalaria/química , Datura stramonium/química , Grão Comestível/química , União Europeia , Humanos , Ricinus/química
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...