Claviceps purpurea

Biosynthesis of the dihydrogenated forms of ergot alkaloids is of interest because many of the ergot alkaloids used as pharmaceuticals may be derived from dihydrolysergic acid (DHLA) or its precursor dihydrolysergol. The maize (Zea mays) ergot pathogen Claviceps gigantea has been reported to produce dihydrolysergol, a hydroxylated derivative of the common ergot alkaloid festuclavine. We hypothesized expression of C. gigantea cloA in a festuclavine-accumulating mutant of the fungus Neosartorya fumigata would yield dihydrolysergol because the P450 monooxygenase CloA from other fungi performs similar oxidation reactions. We engineered such a strain, and high performance liquid chromatography and liquid chromatography-mass spectrometry analyses demonstrated the modified strain produced DHLA, the fully oxidized product of dihydrolysergol. Accumulation of high concentrations of DHLA in field-collected C. gigantea sclerotia and discovery of a mutation in the gene lpsA, downstream from DHLA formation, supported our finding that DHLA rather than dihydrolysergol is the end product of the C. gigantea pathway.

Ergot alkaloids are bioactive indole derivatives produced by a broad range of fungi. They are divided into three groups: ergopeptines, clavines, and lysergic acid. Claviceps purpurea has a wide host range and infects approximately 400 plant species. They can have a major impact on the central nervous system of mammals due to their structural similarities to neurotransmitters. In medieval history, there were two prominent types of symptoms: first was convulsive ergotism, which caused spasms, paranoia, and hallucinations while the second was gangrenous ergotism, which caused impaired peripheral sensibility, oedema, and limb loss. Clavicipitaceous endophytes can grow endobiotically or epibiotically. Ergot

Ergotism is a toxic condition resulting from overexposure to the ergot compounds produced by various fungi of the genus Claviceps. Traditionally, such exposure was due to ingestion of infected grains, but long-term or excessive use of medications containing ergot derivatives or drug-drug interactions between these medications can result in ergotism. Ergotamine, typically used to treat migraine, has less than 5% bioavailability due to extensive first-pass metabolism by cytochrome P450 3A4 (CYP3A4). Concurrent intake of ergotamine and strong CYP3A4 inhibitors, such as the HIV protease inhibitors (PIs), can lead to clinical ergotism. A total of 13 cases of clinical ergotism in HIV-infected patients has been published since 1997 (most recently reviewed by Frohlich et al).

Background: Ergopeptines are a predominant class of ergot alkaloids produced by tall fescue grass endophyte Neotyphodium coenophialum or cereal pathogen Claviceps purpurea. The vasoconstrictive activity of ergopeptines makes them toxic for mammals, and they can be a problem in animal husbandry. Results: We isolated an ergopeptine degrading bacterial strain, MTHt3, and classified it, based on its 16S rDNA sequence, as a strain of Rhodococcus erythropolis (Nocardiaceae, Actinobacteria). For strain isolation, mixed microbial cultures were obtained from artificially ergot alkaloid-enriched soil, and provided with the ergopeptine ergotamine in mineral medium for enrichment. Individual colonies derived from such mixed cultures were screened for ergotamine degradation by high performance liquid chromatography and fluorescence detection. R. erythropolis MTHt3 converted ergotamine to ergine (lysergic acid amide) and further to lysergic acid, which accumulated as an end product. No other tested R. erythropolis strain degraded ergotamine. R. erythropolis MTHt3 degraded all ergopeptines found in an ergot extract, namely ergotamine, ergovaline, ergocristine, ergocryptine, ergocornine, and ergosine, but the simpler lysergic acid derivatives agroclavine, chanoclavine, and ergometrine were not degraded. Temperature and pH dependence of ergotamine and ergine bioconversion activity was different for the two reactions.

to the line of travel on either side of the horse. Video footage was recorded into the Equi-neTecTM (Monroe, GA, USA) software program for analysis. Blood via jugular puncture was taken the morning following video recording. Hematocrit values were analyzed immediately. Whole blood and plasma was frozen for later analysis. Thiobarbituric acid reactive substances (TBARS) analysis was conducted on plasma according to procedures established by C. Jo and D.U. Ahn [3]. Data was analyzed using SAS version 9.3 Proc GLM with repeated measures over time to determine the effects of treatment, time, and treatment by time interactions. P values of P<0.05 were considered significant. After 90 days, glucosamine horses showed greater front leg retraction distance (P¼.01) as compared to earlier time points. No significant difference in this parameter was seen in control horses. Additionally, a trend toward greater front fetlock flexion was observed in glucosamine supplemented horses as compared to controls. No differences between control and treatment were observed in TBARS analysis. In conclusion, oral supplementation of glucosamine sulfate resulted in modest changes in biomechanical parameters measured. Differences in blood oxidative levels between treatments were not observed.

Feedingstuffs are frequently contaminated with sclerotia (ergot) of the phytopathogenic fungus Claviceps purpurea. Ergot contains ergot alkaloids (EA), but the amount and composition of these substances may be highly variable hampering the extrapolation of the amount of EA from the ergot content. 600 different feed samples from the harvest years 2011–2014 (rye, triticale, wheat, mixed cereal grains or compound feed), preferably those with visible ergot infestation, were analyzed for both parameters ergot and EA content in a German monitoring program. The analytical results were employed for statistical evaluation using the software JMP® 13. The data show that the correlation between the ergot and the EA content was rather poor, especially for rye, the grain most frequently contaminated with ergot and EA. Consequently, effects on animal health correlated much better with EA than with ergot content. Furthermore, it shows that the current European law for undesirable substances (Direct...

Claviceps purpurea infects the seed heads of cereal grains and grasses and produces ergopeptine alkaloids that cause hyperthermia and agalactia in cattle during periods of heat stress. A field experiment was undertaken to examine the effects of ergopeptine alkaloids found in barley on thermal status of dairy cattle during periods of heat stress. Production end points were also measured to identify the effect of the change in thermal status. Contaminated barley screenings containing known levels of ergopeptine alkaloids were fed to lactating Holstein cattle (10 g total ergopeptine alkaloids/kg BW/day) for 10 days during summer heat stress. Air temperature increased 14.4 C during the first 8 days of treatment and then declined the same during the last 2 days. Extreme daily values for rectal temperature and respiration rate, using averages of all animals, showed maximum increases of 2.3 C and 56.8 breaths/minute, respectively, during this period. Group afternoon milk production decreased 2 kg/day during the heat stress period, with no measurable change in feed intake. A greater level of hyperthermia occurred in cattle consuming the diet with ergopeptine alkaloids, with only marginal symptoms of ergot toxicosis reflected in feed intake and milk production. Therefore, the ergopeptine alkaloid dose used in this study represents a level for minimal induction of the ergot toxicity response.

A competitive inhibition enzyme-linked immunosorbent assay (CI-ELISA) was developed which was able to detect ergot alkaloids in seed and flour a t the picograms per gram level. A monoclonal antibody (MAb) directed against ergonovine was found to be sensitive to the secondary metabolites of Claviceps spp. having an intact ergoline moiety in wheat, bahiagrass, bluegrass, and tall fescue seeds. The assay could detect the alkaloids of Claviceps purpurea when sclerotia were diluted 10-5 by weight in whole wheat flour, or approximately one sclerotium in 20 kg of wheat. Ergonovine added to ergot-free wheat flour was detected at 10 ng/g. Total elapsed time for the assay is 1.5 h for 96 samples in the microplate format.

hBsTR~cT.-Dihydrolysergic acid is converted t o dihydroergotamine in cultures of Dihydrolysergyl coenzyme h Dihydrolysergyl coenzyme A was converted in a cell-free system an ergotamine-producing st rain of Claviceps purpurea. was synthesized. t o an unidentified product.

A holistic approach based on high resolution and multiple stage mass spectrometry was developed for identification of less studied or novel ergot alkaloid derivatives. Initially, the fragmentation of nine known ergot alkaloids was studied to establish a strategy for the identification of novel ergot alkaloids. Ions with m/z 223 and m/z 251 were found to be common for all ergopeptines, ergoamides and ergopeptams. Subsequently, parent scan experiments using these ions were performed to screen grain samples for the presence of possible ergot alkaloid derivatives. Besides the six most common ergot alkaloids and their corresponding epimers (for which reference standards were available), eleven other ergot alkaloid derivatives were identified following the proposed strategy.

A holistic approach based on high resolution and multiple stage mass spectrometry was developed for identification of less studied or novel ergot alkaloid derivatives. Initially, the fragmentation of nine known ergot alkaloids was studied to establish a strategy for the identification of novel ergot alkaloids. Ions with m/z 223 and m/z 251 were found to be common for all ergopeptines, ergoamides and ergopeptams. Subsequently, parent scan experiments using these ions were performed to screen grain samples for the presence of possible ergot alkaloid derivatives. Besides the six most common ergot alkaloids and their corresponding epimers (for which reference standards were available), eleven other ergot alkaloid derivatives were identified following the proposed strategy.

Descrevem-se três surtos de síndrome distérmica (hipertermia) associada à intoxicação por Claviceps purpurea, em bovinos de leite durante o verão de 1999-2000, em três estabelecimentos do Rio Grande do Sul. De um total de 66 bovinos que ingeriram a ração contaminada com o fungo, 37 (56%) adoeceram até 3 meses após a introdução da ração contaminada. Os principais sinais clínicos foram temperatura retal elevada, pêlos compridos, longos e sem brilho, salivação intensa, respiração ofegante, com a boca aberta e, em alguns casos, com a língua para fora da cavidade oral. Os animais acometidos procuravam sombra ou permaneciam dentro d&#39;água. Houve diminuição de 10 a 30% no consumo de alimentos e perda de peso. A redução na produção de leite foi de 30 a 50%. Os sinais clínicos se intensificavam durante o dia e eram diretamente proporcionais à elevação da temperatura ambiental. Os achados de necropsia em um bovino que foi eutanasiado, incluíram leve enfisema pulmonar, principalmente na reg...

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Following publication of the original article [1], the author has found two mistakes in the title made by the BMC production office. The title of the original article and this erratum have been changed from 'fIdentification of B. napus small RNAs responsive to infection by a necrotrophic pathogen' to 'Identification of Brassica napus small RNAs responsive to infection by a necrotrophic pathogen. The original article has been corrected.

A laboratory experiment was conducted to determine the feasibility of density separation for reducing ergot contamination in barley. Four 1.1-m3 (30-bu) lots of contaminated barley were conditioned with an air-screen cleaner followed by density separator. Samples were collected before and after conditioning with each machine. The amount of ergot in collected samples was determined separating and weighing the ergot and calculating the percentage by weight. The ergot contamination level was significantly reduced from 0.52%, 0.16%, 0.10%, and 0.12% to 0.02%, 0.01%, 0.02%, and 0.01%, respectively, for the four contaminated lots near the conditioning operations. An economic analysis indicated that it was advantageous to clean barley and use it as an animal feed rather than to sell it at salvage value.

Background: Ergotism is caused by the fungus Claviceps purpurea, which parasitizes cereal grains and is ingested by man through flour milled from contaminated cereals. An outbreak of ergotism in Ethiopia in 1978 resulted from exposure to ergot alkaloids from C. puruprea sclerotia. Objectives: The objective of this study was to investigate consumption of cereal grains grown locally as the most likely cause of the outbreak of gangrenous ergotism so that control measures could be applied. Methods : During June to August, 2001, there were reports of a large number of cases of gangrene in Arsi Zone, Ethiopia. A multidisciplinary team assessed the outbreak of the decease. Non-structured in-depth interviews were conducted with heads of households of the affected, and each of the patients was also interviewed. Grain samples were then collected from the interviewed households and analyzed for ergot alkaloids. Acute toxicity studies were also conducted by feeding male, non-pregnant and pregnant Swiss albino mice with the collected grain samples. Results: Mycological cultures of grain samples yielded ergot alkaloids. All the grain samples contained ergot alkaloids, but with varying concentration. The highest concentration of ergotamine was observed in grain samples No. 4 (2.51 mg/100 g) and No. 6 (2.66 mg/100 g). Grain samples No. 2 and 7 had similar concentration of ergotamine, but more than four-fold higher than in grain sample No. 3. In contrast, the concentration of ergometrine in grain samples No. 4 (1.15mg/100 g) and No.6 (1.21mg/100 g) were twofold lower than ergotamine. The highest death (55%) of mice was observed in those test groups fed on grain samples No. 4 and No. 6. Cases of abortion were noted after 3 days of feeding in all pregnant mice with the exception of those allocated to grain sample No. 3. Conclusion: We conclude on the basis of these results that the outbreak of gangrene in Arsi Zone, Ethiopia, is attributed to the ingestion of barley containing ergotized wild oats.

Common reed Phragmites australis (Cav.) Trin. ex Steud. is one of the most widely distributed angiosperms with important ecological functions. In recent decades, it has been affected by a severe decline known as reed die-back syndrome (RDBS), the causal factors of which are still under investigation. Among the biotic factors that influence the dynamic of the reed population, the role of microorganisms is still poorly understood. During surveys carried out on P. australis populations in Central Italy, Claviceps-like sclerotia were detected: is Claviceps infection related to P. australis sexual reproduction and seed production? Could Claviceps infection be involved in the RDBS? These are the questions that we address. We characterized the sclerotia at the morphological, molecular, and chemical level and we demonstrated that they belong to Claviceps arundinis Pažoutová & M. Kolařík. To our knowledge, this is the first report of C. arundinis on P. australis in Italy. Furthermore, the association of C. arundinis with RDBS was evaluated considering a set of macromorphological traits generally related to RDBS, such as P. australis clumping habit, culm height and diameter. No correlation was seen between the occurrence of C. arundinis and the declining status of reed populations.

Because the occurrence of Claviceps in European pastures may have been overlooked to cause serious health problem for grazing animals, we documented the degree of Claviceps contamination in two horse pastures and estimated whether the horses could have ingested a critical quantity of alkaloids. We counted the Claviceps sclerotia and determined alkaloid levels using high performance liquid chromatography with fluorescence detection. Depending on the location, the number of sclerotia varied from 0.09 to 0.19 per square meter (central area) and from 0.23 to 55.8 per square meter (border strips). Alkaloid levels in individual sclerotia also varied in different genera of grasses, ranging from 0.98 ± 0.17 μg/kg in Agrostis sp. to 25.82 ± 9.73 μg/kg in Dactylis sp., equivalent to 0.98 μg/kg and 7.26 mg/kg. Sclerotia from Dactylis contained high levels of ergosine (0.209 % ± 0.100 %) and ergocristine (0.374 % ± 0.070 %). Depending on the localization in pastures, alkaloid levels in forage (dry matter, DM) ranged from 16.1 to 45.4 μg/kg in central areas and from 23.9 to 722 μg/kg in border strips. The amount of alkaloids that a horse could have ingested depended on its daily DM uptake, which was higher in the central areas (5.85 kg/day) than in the border strips (2.73 or 0.78 kg/day). In the central areas, this amount of alkaloids ranged from 94.2 to 265.9 μg/ day; and in the border strips, from 65.3 (in 2.73 kg DM/day) to as much as 563.8 μg/day (in 0.78 kg DM/day). All these amounts are higher than the European averages for alkaloids ingested by horses via feedstuffs.

Cows were fed ration for 9 wk containing 5, 48, 201, and 822 μg kg−1 ergot alkaloids. The objective was to evaluate the impact of ergot consumption in beef cow–calf operations. Ergot alkaloids up to 822 μg kg−1 did not alter the weight of peripartum and postpartum beef cows (P = 0.93) or nursing calves (P = 0.08), rectal temperature (P = 0.16), or plasma prolactin concentrations (P = 0.30) at moderate ambient temperatures. Ergot did not influence the time (&gt;1 ng mL−1; P = 0.79) or the progesterone concentration (P = 0.38) at the time of first postpartum rise or the size of the first (14 ± 0.6 mm; P = 0.40) and second (13 ± 0.5 mm; P = 0.41) follicles to ovulate. The maximum size of the first postpartum corpus luteum (CL) was 4 mm larger in the 822 μg kg−1 ergot group compared with the control (P = 0.03) for the first ovulation post partum, but not for the second (P = 0.11). There was no effect of ergot exposure on the number of days until the appearance of the first (43 ± 4 d; P ...

Dihydroergotamine is a semisynthetic natural product derived from ergotamine, an ergot alkaloid. It is used to treat migraines, a neurological disease characterized by recurrent moderate to severe headaches. In this work, the in vitro metabolism of dihydroergotamine was evaluated in a biomimetic phase I reaction, aiming to verify all possible formed metabolites. Dihydroergotamine was submitted to an in vitro metabolism assay using rat liver microsomes, and the metabolites were analyzed by HPLC-MS/MS. The biomimetic reactions were performed with Jacobsen catalyst for scaling up production of oxidized metabolites. Two hydroxylated metabolites were isolated and characterized by MS/MS and (1)H NMR analysis.

Ergot alkaloids (EAs) are mycotoxins produced by several species of fungi of the genus Claviceps, among which Claviceps purpurea is the most widespread in Europe. This species has been found in many economically important cereal grains, such as rye, wheat, triticale, barley, millet and oats. The distribution of EAs contamination has a sporadic incidence with many factors involved in its occurrence, greatly varying between fungal strains, geographic regions, host plants and regional/local weather conditions, at the same time emphasizing that cool, damp weather favours ergot by enhancing the germination of sclerotia. The aim of this study was to investigate the occurrence of ergot sclerotia and EAs in wheat and rye grain samples (n = 64) taken during the year 2021 from Croatian cereal producers located in the Central and Eastern parts of Croatia. In two of the rye samples the presence of ergot sclerotia was detected in the amount of 259 mg/kg and 536 mg/kg, whereas none of the wheat s...

Previous research of colonies formed by yeast Saccharomyces cerevisiae growing on glycerol agar medium revealed two major cell types of U and L cells that are formed within these colonies. This colonial cell differentiation seem to be caused by communication among yeast cells as well as whole colonies and affected by changes in the environment (for example changes in nutrients). Studies of U and L cells showed that U cells are more resistant against biological, chemical and physical stresses than L cells. The aim of this thesis was to isolate U and L cell types and investigate their resistance against selected low molecular weight chemical substances produced in Ehrlich pathway. Ehrlich pathway was discovered in 1907 and was classified as amino acid catabolic pathway in yeast S. cerevisiae. The low molecular intermediates are formed in Ehrlich pathway which are called fusel (original name from German) alcohols and acids. These chemical substances are widely used in food industry and...

Kořeninová paprika patří mezi jedno z nejvyužívanějších koření v mnoha částech světa. Přítomnost nežádoucích patogenů může být závažným problémem pro konzumenty. Zvláště kontaminace mikroskopickými houbami, z nichž někteří zástupci produkují mykotoxiny, představuje pro člověka velkou hrozbu. Dnešní konvenční metody pro určování kontaminací mikroorganismy jsou náročné na čas a některé méně časté druhy patogenů může být obtížné určit. V rámci bakalářské práce byly aplikovány metody molekulární biologie pro detekci kontaminace ve vzorcích kořeninové papriky. Pro sekvenaci byly vybrány ITS oblasti, které vykazují vysokou variabilitu u jednotlivých druhů organismů a jsou vhodným nástrojem pro jejich taxonomii. Díky sekvenační analýze ITS oblastí, byly identifikovány sekvence o vysoké podobnosti s ITS oblastmi mnoha zástupců mikroskopických hub zejména ze třídy Ascomycotina, Deuteromycotina a několika druhů kvasinek. V práci se jednalo o kvalitativní nikoli o kvantitativní detekci, která nevylučuje zdravotní nezávadnost vzorků, která je prokázaná protokoly o zdravotní nezávadnosti.

Background: Methylergometrine is an ergot alkaloid of ergonovine used to treat post-partum hemorrhage secondary to uterine atony. Mistaking methylergometrine for vitamin K with accidental administration to the neonate is a rare iatrogenic illness occurring almost exclusively in the delivery room setting. Complications of ergot alkaloids in neonates include respiratory depression, cyanosis, seizures, and death. Case Report: A term male infant was inadvertently given 0.04 mg of methylergometrine intramuscularly. The error was only noted when the baby developed central cyanosis, after administration, identifying it as methylergometrine rather than vitamin K. The local poison center was notified, and the infant was transferred to the neonatal intensive care unit level III for observation. Few minutes after cyanosis, the infant was noted to have oxygen desaturations and prolonged apnea required oxygen via nasal cannula and ventilation as a respiratory support to maintain his oxygen. ECG and ECHO were done and ECG was normal and ECHO showed Large ASD secondum. Feeding was started by 48 hours of life, and the infant was discharged home in good condition after a 96-hour stay without further complications. Disscussion: Because of the potential for serious adverse events, Look-alike Sound-alike policy and precaution is required to be applied to prevent accidental administration of methylergometrine to the neonate as a result of possible confusion with vitamin K in the early post-partum period.

Background: Methylergometrine is an ergot alkaloid of ergonovine used to treat post-partum hemorrhage secondary to uterine atony. Mistaking methylergometrine for vitamin K with accidental administration to the neonate is a rare iatrogenic illness occurring almost exclusively in the delivery room setting. Complications of ergot alkaloids in neonates include respiratory depression, cyanosis, seizures, and death. Case Report: A term male infant was inadvertently given 0.04 mg of methylergometrine intramuscularly. The error was only noted when the baby developed central cyanosis, after administration, identifying it as methylergometrine rather than vitamin K. The local poison center was notified, and the infant was transferred to the neonatal intensive care unit level III for observation. Few minutes after cyanosis, the infant was noted to have oxygen desaturations and prolonged apnea required oxygen via nasal cannula and ventilation as a respiratory support to maintain his oxygen. ECG and ECHO were done and ECG was normal and ECHO showed Large ASD secondum. Feeding was started by 48 hours of life, and the infant was discharged home in good condition after a 96-hour stay without further complications. Disscussion: Because of the potential for serious adverse events, Look-alike Sound-alike policy and precaution is required to be applied to prevent accidental administration of methylergometrine to the neonate as a result of possible confusion with vitamin K in the early post-partum period.

Common reed Phragmites australis (Cav.) Trin. ex Steud. is one of the most widely distributed angiosperms with important ecological functions. In recent decades, it has been affected by a severe decline known as reed die-back syndrome (RDBS), the causal factors of which are still under investigation. Among the biotic factors that influence the dynamic of the reed population, the role of microorganisms is still poorly understood. During surveys carried out on P. australis populations in Central Italy, Claviceps-like sclerotia were detected: is Claviceps infection related to P. australis sexual reproduction and seed production? Could Claviceps infection be involved in the RDBS? These are the questions that we address. We characterized the sclerotia at the morphological, molecular, and chemical level and we demonstrated that they belong to Claviceps arundinis Pažoutová & M. Kolařík. To our knowledge, this is the first report of C. arundinis on P. australis in Italy. Furthermore, the association of C. arundinis with RDBS was evaluated considering a set of macromorphological traits generally related to RDBS, such as P. australis clumping habit, culm height and diameter. No correlation was seen between the occurrence of C. arundinis and the declining status of reed populations.

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.

Genus Claviceps is a plant pathogen able to produce a group of toxins, ergot alkaloids (EAs), whose effects have been known since the Middle Ages (ergotism). Claviceps purpurea is the most important representative specie, known to infect more than 400 monocotyledonous plants including economically important cereal grains (e.g., rye, wheat, triticale). EAs are not regulated as such. Maximum limits are in the pipeline of the EU Commission while at present ergot sclerotia content is set by the Regulation (EC) No. 1881/2006 in unprocessed cereals (0.05% as a maximum). This study aimed to investigate the presence of the six principal EAs (ergometrine, ergosine, ergocornine, α-ergocryptine, ergotamine and ergocristine) and their relative epimers (-inine forms) in rye- and wheat-based products. Of the samples, 85% resulted positive for at least one of the EAs. Wheat bread was the product with the highest number of positivity (56%), followed by wheat flour (26%). Rye and wheat bread samples...

Claviceps purpurea, the fungal pathogen that causes the cereal disease ergot, produces glycerides that contain high levels of ricinoleic acid [(R)-12-hydroxyoctadec-cis-9-enoic acid] in its sclerotia. Recently, a fatty acid hydroxylase (C. purpurea FAH [CpFAH]) involved in the biosynthesis of ricinoleic acid was identified from this fungus (D. Meesapyodsuk and X. Qiu, Plant Physiol. 147:1325-1333, 2008). Here, we describe the cloning and biochemical characterization of a C. purpurea type II diacylglycerol acyltransferase (CpDGAT2) involved in the assembly of ricinoleic acid into triglycerides. The CpDGAT2 gene was cloned by degenerate RT-PCR (reverse transcription-PCR). The expression of this gene restored the in vivo synthesis of triacylglycerol (TAG) in the quadruple mutant strain Saccharomyces cerevisiae H1246, in which all four TAG biosynthesis genes (DGA1, LRO1, ARE1, and ARE2) are disrupted. In vitro enzymatic assays using microsomal preparations from the transformed yeast strain indicated that CpDGAT2 prefers ricinoleic acid as an acyl donor over linoleic acid, oleic acid, or linolenic acid, and it prefers 1,2-dioleoyl-sn-glycerol over 1,2-dipalmitoyl-sn-glycerol as an acyl acceptor. The coexpression of CpFAH with CpDGAT2 in yeast resulted in an increased accumulation of ricinoleic acid compared to the coexpression of CpFAH with the native yeast DGAT2 (S. cerevisiae DGA1 [ScDGA1]) or the expression of CpFAH alone. Northern blot analysis indicated that CpFAH is expressed solely in sclerotium cells, with no transcripts of this gene being detected in mycelium or conidial cells. CpDGAT2 was more widely expressed among the cell types examined, although expression was low in conidiospores. The high expression of CpDGAT2 and CpFAH in sclerotium cells, where high levels of ricinoleate glycerides accumulate, provided further evidence supporting the roles of CpDGAT2 and CpFAH as key enzymes for the synthesis and assembly of ricinoleic acid in C. purpurea.

The retention of six alkaloids in hard red spring wheat infected by ergot (Claviceps purpurea) was determined in mill streams and in processed Oriental noodles, pasta, and pan bread. Individual and total alkaloids (ergonovine, ergosine, ergotamine, ergocornine, α-ergokryptine, and ergocristine) were determined by a modification of a previously reported reversed-phase high-performance liquid chromatography procedure. Generally, ergocristine was the predominant alkaloid present, followed by ergotamine. Ergot is more plastic than hard wheat endosperm, hence it flattened during smooth-roll reduction grinding. After milling, ergot alkaloids were concentrated in the late reduction streams and in the shorts derived from the reduction system. Low concentrations of ergot alkaloids were retained in high quality patent flour. Ergot alkaloids were quite stable during end-use processing. Processing of flour into pasta and Oriental noodles had little effect on levels of ergot alkaloids, and a sub...

A semicontinuous fermentation with immobilized mycelium of Claviceps paspali, producing mainly methylcarbinolamide of lysergic acid, ergometrine and the amide of lysergic acid, was carried out. It was found that immobilization itself improved ergometrine production. The type and concentration of alginate used dit not influence the alkaloid spectrum. The age and quantity of inoculum as well as the length of a particular cycle affected total alkaloid production but did not seem to affect the changes in relative amounts of particular alkaloids. The best results were obtained with 1000 mg dry mycelium weight of 6-day-old fungus from the vegetative phase in 100 ml production medium immobilized in 4% alginate of medium or low viscosity and by recycling every 10 days. When no fungus was found outside the gel beads it was possible to improve the yield of ergometrine from 52% in the first cycle to 85% in the fourth cycle of the semicontinuous procedure.

Citrinin (CIT) is a mycotoxin responsible for the contamination of many agricultural products, like wheat, barley, corn, rice and their products, as also other foodstuffs and feedstuffs used in human and animal nutrition. It is essentially produced by Penicillium citrinum, although it can also be biosynthesised from Penicillium expansum and Penicillium verrucosum and some species of Aspergillus and Monascus. However, several studies have shown that CIT is known for its genotoxic, hepatotoxic, fetotoxic and teratogenic properties. The aim of this study is to investigate the occurrence of CIT in wheat grain cultivated in Kosovo andAlbania. Given the fact that wheat flour is the most consumed product in Kosovo and Albania, it is necessary to analyse the CIT in wheat in these two countries. In total, 60 wheat samples were tested from Fusha e Kosovës (Kosovo), Myzeqeja (Albania) and Fusha e Maliqit (Albania), as places withthe highest wheat production. The enzyme- linked immunosorbent a...

The toxicity of sorghum ergot (Claviceps africana) was assessed in young pigs over 28 days. Forty-eight pigs of both sexes and 2 breeds (Large White and Duroc) were allocated across 6 grower diets, balanced for fibre and predicted digestible energy, and containing 0, 0.3, 0.6, 1.3, 2.5, or 5% ergot sclerotia [the 5% sclerotia diet contained 70 mg alkaloids/kg (&gt;90% dihydroergosine)]. Blood samples taken on Days 0 and 28 were analysed for prolactin and clinical, biochemical, and haematological indices of health. Feed consumption and liveweight were individually monitored. There were no clinical signs of illness attributable to ergotism in the pigs. Blood prolactin concentrations were significantly depressed in pigs receiving 9 mg alkaloids/kg (0.6% sclerotia) and by &gt;80% in pigs receiving 35 and 70 mg alkaloids/kg, clearly indicating a potential to interfere with lactation in sows. Reductions in feed intake and poor feed conversion were observed over the first 7 days with &gt;9...

Ergot alkaloids are vasoconstrictors frequently detected in low concentrations in livestock feed. The Canadian Food Inspection Agency permits up to 3,000 µg ergot alkaloids per kg cattle feed. The objective of this study was to examine the effects of feeding low concentrations of ergot alkaloids over 9-weeks on vascular dynamics in the caudal and internal iliac arteries of beef cows. A relationship between ergot alkaloid concentration in feed and hemodynamic changes in the caudal and internal iliac arteries was hypothesized. Periparturient beef cows were randomized into four groups and group fed mixed rations containing <15 µg ergot alkaloids per kg of dry matter intake (Control, n = 9), 48 µg/kg (Low, n = 9), 201 µg/kg (Medium, n = 8), and 822 µg/kg (High, n = 6). Three experimental periods comprised the study: pre-treatment (2 weeks), treatment (9 weeks), and post-treatment (3 weeks). B-mode and Doppler ultrasonography was performed weekly to measure hemodynamic endpoints. Plasma prolactin concentrations and rectal temperatures were measured weekly. Caudal artery diameter decreased (Treatment * Experimental Period i.e., Tx * EP, p < 0.001) by 14% in the High group during the treatment period. Reductions (Tx * EP, p < 0.001) in caudal artery blood flow (37%, 29%) and blood volume per pulse (29%, 11%) were recorded during the treatment period in the High and Medium groups. Internal iliac artery diameter and blood flow decreased (Tx * EP, p ≤ 0.004) by 13% and 40% during the treatment period in the Medium group. Moderate reductions (Tx * EP, p ≤ 0.042; 12-25%) in the mean blood velocity during the treatment and post-treatment periods and decreases (Tx * EP, p ≤ 0.01; 12-17%) in the peak systolic velocity of both arteries during the post-treatment period were also detected. Prolactin did not change in any group during the treatment period (p = 0.462). Rectal temperatures were within the normal physiological range for beef cows. In conclusion, we documented moderate vasoconstriction in the caudal artery and the internal iliac artery in cows fed 201-822 µg ergot alkaloids per kg of dry matter intake for 9-week period near parturition. The pattern of alterations was similar between the caudal and internal iliac arteries. Results of this study suggest that feeding up to 822 µg/kg produce reversible pharmacological changes in beef cow vasculature and warrant reconsideration of current regulations for cattle.

Cieľom predkladanej práce bolo vyvinúť účinný protokol polyakrylamidovej gélovej elektroforézy na báze dodecylsulfátu sodného (SDS-PAGE), ako aj želatínovej zymografie, na detekciu proteínov. Na štúdium elektroforetického profilovania proteínov bolo použitých 100 vzoriek od pacientov s kolorektálnym karcinómom (CRC) v rôznych štádiách. Bol navrhnutý presný protokol na optimalizáciu podmienok, ako je pH používaných roztokov, koncentrácie jednotlivých zložiek gélov, množstvo vzorky nanášanej do gélu, časové rozmedzie farbenia, odfarbovania a iné. Optimalizáciou týchto faktorov boli metódou zymografie detegované matrixové metaloproteinázy v svojej latentnej aj aktívnej forme.

The retention of six alkaloids in hard red spring wheat infected by ergot (Claviceps purpurea) was determined in mill streams and in processed Oriental noodles, pasta, and pan bread. Individual and total alkaloids (ergonovine, ergosine, ergotamine, ergocornine, α-ergokryptine, and ergocristine) were determined by a modification of a previously reported reversed-phase high-performance liquid chromatography procedure. Generally, ergocristine was the predominant alkaloid present, followed by ergotamine. Ergot is more plastic than hard wheat endosperm, hence it flattened during smooth-roll reduction grinding. After milling, ergot alkaloids were concentrated in the late reduction streams and in the shorts derived from the reduction system. Low concentrations of ergot alkaloids were retained in high quality patent flour. Ergot alkaloids were quite stable during end-use processing. Processing of flour into pasta and Oriental noodles had little effect on levels of ergot alkaloids, and a sub...

Background: Ergotism is caused by the fungus Claviceps purpurea, which parasitizes cereal grains and is ingested by man through flour milled from contaminated cereals. An outbreak of ergotism in Ethiopia in 1978 resulted from exposure to ergot alkaloids from C. puruprea sclerotia. Objectives: The objective of this study was to investigate consumption of cereal grains grown locally as the most likely cause of the outbreak of gangrenous ergotism so that control measures could be applied. Methods : During June to August, 2001, there were reports of a large number of cases of gangrene in Arsi Zone, Ethiopia. A multidisciplinary team assessed the outbreak of the decease. Non-structured in-depth interviews were conducted with heads of households of the affected, and each of the patients was also interviewed. Grain samples were then collected from the interviewed households and analyzed for ergot alkaloids. Acute toxicity studies were also conducted by feeding male, non-pregnant and pregnant Swiss albino mice with the collected grain samples. Results: Mycological cultures of grain samples yielded ergot alkaloids. All the grain samples contained ergot alkaloids, but with varying concentration. The highest concentration of ergotamine was observed in grain samples No. 4 (2.51 mg/100 g) and No. 6 (2.66 mg/100 g). Grain samples No. 2 and 7 had similar concentration of ergotamine, but more than four-fold higher than in grain sample No. 3. In contrast, the concentration of ergometrine in grain samples No. 4 (1.15mg/100 g) and No.6 (1.21mg/100 g) were twofold lower than ergotamine. The highest death (55%) of mice was observed in those test groups fed on grain samples No. 4 and No. 6. Cases of abortion were noted after 3 days of feeding in all pregnant mice with the exception of those allocated to grain sample No. 3. Conclusion: We conclude on the basis of these results that the outbreak of gangrene in Arsi Zone, Ethiopia, is attributed to the ingestion of barley containing ergotized wild oats.

Claviceps purpurea, the causal agent of ergot of perennial ryegrass seed crops, overwinters as sclerotia in the soil and releases airborne ascospores in the spring that infect flower ovaries and replace seed with sclerotia. Burkard spore traps were used to quantify the dispersal phenology and concentration of ascospores in perennial ryegrass seed fields in the Columbia Basin of Oregon. Weather factors were measured concurrently with spore trapping. Nonparametric regression, box-and-whisker plots, and univariate analysis were used to visualize and identify trends between ascospore concentrations and weather variables. Most ascospores (75.4%) were trapped when minimum soil temperatures were between 16.2 and 20.4°C. Over 67% of the total ascospores trapped were observed when minimum air temperatures were between 6.8 and 12.4°C and 64% of ascospores were trapped when daily mean dew point was between 3.7 and 8.2°C. Environmental favorability index (EFI) models were developed and validate...

Citrinin (CIT) is a mycotoxin responsible for the contamination of many agricultural products, like wheat, barley, corn, rice and their products, as also other foodstuffs and feedstuffs used in human and animal nutrition. It is essentially produced by Penicillium citrinum, although it can also be biosynthesised from Penicillium expansum and Penicillium verrucosum and some species of Aspergillus and Monascus. However, several studies have shown that CIT is known for its genotoxic, hepatotoxic, fetotoxic and teratogenic properties. The aim of this study is to investigate the occurrence of CIT in wheat grain cultivated in Kosovo andAlbania. Given the fact that wheat flour is the most consumed product in Kosovo and Albania, it is necessary to analyse the CIT in wheat in these two countries. In total, 60 wheat samples were tested from Fusha e Kosovës (Kosovo), Myzeqeja (Albania) and Fusha e Maliqit (Albania), as places withthe highest wheat production. The enzyme- linked immunosorbent a...

Ergot of cereals, caused by Claviceps purpurea, results in yield loss and downgrading of infested grain because of toxic alkaloids in the sclerotia. Resistant wheat genotypes are known, but their effectiveness against different C. purpurea isolates over geographic regions has not been studied. The objective was to examine the pathogenic variability among isolates of C. purpurea on wheat lines differing in resistance. Under controlled environmental conditions, fourty one single spore isolates of C. purpurea were developed from Canadian and United Kingdom collections and inoculated onto a set of wheat genotypes composed of durum wheat lines 'Melita', 'Kyle', and 9260B-173A, and hexaploid spring wheat lines 'Cadillac', 'Vista', 'Kenya Farmer', 'Lee' and HY630. Honeydew production and weight of sclerotia produced per spike were assessed. There were significant differences among the wheat genotypes for overall reactions to the pathogen isolates, and among pathogen isolates for geographic origin and host origin. Twenty virulence phenotypes were identified using the honeydew production data, and 23 virulence phenotypes identified using the sclerotial weight data from the 41 isolates. The existence of different virulence phenotypes indicates that variability in virulence exists in populations of C. purpurea, and knowledge of virulence phenotypes is necessary to effectively breed for resistant commercial lines.

The grass parasitic fungus Claviceps purpurea sensu lato produces sclerotia with toxic indole alkaloids. It constitutes several genetic groups with divergent habitat preferences that recently were delimited into separate proposed species. We aimed to 1) analyze genetic variation of C. purpurea sensu lato in Norway, 2) characterize the associated indole alkaloid profiles, and 3) explore relationships between genetics, alkaloid chemistry and ecology. Approximately 600 sclerotia from 14 different grass species were subjected to various analyses including DNA sequencing and HPLC-MS. Molecular results, supported by chemical and ecological data, revealed one new genetic group (G4) in addition to two of the three known; G1 (C. purpurea sensu stricto) and G2 (C. humidiphila). G3 (C. spartinae) was not found. G4, which was apparently con-specific with the recently described C. arundinis sp. nov, was predominantly found in very wet habitats on Molinia caerulea and infrequently in saline habitats on Leymus arenarius. Its indole-diterpene profile resembled G2, while its ergot alkaloid profile differed from G2 in high amounts of ergosedmam. In contrast to G1, indole-diterpenes were consistently present in G2 and G4. Our study

RATIONALE: The biological activities most commonly associated with indole-diterpenoids are tremorgenicity in mammals and toxicity in insects through modulation of ion channels. The neurotoxic effects of some analogues are the cause of syndromes such as 'ryegrass staggers' and 'Paspalum staggers' in cattle and sheep. Our purpose was to obtain and interpret mass spectra of some pure Claviceps-related indole-diterpenoids (paspaline, paspalinine, paxilline, paspalitrems A and B) to facilitate identification of related compounds for which standards were not available. METHODS: C. paspali-infected Paspalum dilatatum as well as C. purpurea sclerotia obtained from infected Phalaris arundinacea were extracted and the extracts separated via liquid chromatography. Low-and high-resolution mass spectra were then obtained of known and potentially unknown indole-diterpenoids. RESULTS: At least 20 different indole-diterpenoids were detected in the C. paspali extract with molecular masses ranging from 405 Da (C 28 H 40 NO) to 517 Da (C 32 H 40 NO 5). The C. purpurea sclerotia were shown to contain several indole-diterpenoids with molecular masses ranging from 405 Da (C 28 H 40 NO) to 419 Da (C 28 H 38 NO 2). CONCLUSIONS: This study demonstrates for the first time that C. purpurea may also produce indole-diterpenoids. This might explain why grazing of Phalaris spp. is occasionally connected with a tremorgenic syndrome in cattle, called 'phalaris staggers'.

Ergot alkaloids (EAs) are mycotoxins produced by several species of fungi of the genus Claviceps, among which Claviceps purpurea is the most widespread in Europe. This species has been found in many economically important cereal grains, such as rye, wheat, triticale, barley, millet and oats. The distribution of EAs contamination has a sporadic incidence with many factors involved in its occurrence, greatly varying between fungal strains, geographic regions, host plants and regional/local weather conditions, at the same time emphasizing that cool, damp weather favours ergot by enhancing the germination of sclerotia. The aim of this study was to investigate the occurrence of ergot sclerotia and EAs in wheat and rye grain samples (n = 64) taken during the year 2021 from Croatian cereal producers located in the Central and Eastern parts of Croatia. In two of the rye samples the presence of ergot sclerotia was detected in the amount of 259 mg/kg and 536 mg/kg, whereas none of the wheat s...

Key message Four QTL for ergot resistance (causal pathogen Claviceps purpurea) have been identified in the durum wheat cultivar Greenshank. Abstract Claviceps purpurea is a pathogen of grasses that infects flowers, replacing the seed with an ergot sclerotium. Ergot presents a significant problem to rye, barley and wheat, in particular hybrid seed production systems. In addition, there is evidence that the highly toxic alkaloids that accumulate within sclerotia can cross-contaminate otherwise healthy grain. Host resistance to C. purpurea is rare, few resistance loci having been identified. In this study, four ergot resistance loci are located on chromosomes 1B, 2A, 5A and 5B in the durum wheat cv. Greenshank. Ergot resistance was assessed through analysis of phenotypes associated with C. purpurea infection, namely the number of inoculated flowers that produced sclerotia, or resulted in ovary death but no sclerotia, the levels of honeydew produced, total sclerotia weight and average s...