An unexpected role of EasDaf: catalyzing the conversion of chanoclavine aldehyde to chanoclavine acid.

Ergot alkaloids (EAs) are a diverse group of indole alkaloids known for their complex structures, significant pharmacological effects, and toxicity to plants. The biosynthesis of these compounds begins with chanoclavine-I aldehyde (CC aldehyde, 2), an important intermediate produced by the enzyme EasDaf or its counterpart FgaDH from chanoclavine-I (CC, 1). However, how CC aldehyde 2 is converted to chanoclavine-I acid (CC acid, 3), first isolated from Ipomoea violacea several decades ago, is still unclear. In this study, we provide in vitro biochemical evidence showing that EasDaf not only converts CC 1 to CC aldehyde 2 but also directly transforms CC 1 into CC acid 3 through two sequential oxidations. Molecular docking and site-directed mutagenesis experiments confirmed the crucial role of two amino acids, Y166 and S153, within the active site, which suggests that Y166 acts as a general base for hydride transfer, while S153 facilitates proton transfer, thereby increasing the acidity of the reaction. KEY POINTS: • EAs possess complicated skeletons and are widely used in several clinical diseases • EasDaf belongs to the short-chain dehydrogenases/reductases (SDRs) and converted CC or CC aldehyde to CC acid • The catalytic mechanism of EasDaf for dehydrogenation was analyzed by molecular docking and site mutations.

Toxigenic Endophyte-Infected Tall Fescue and Ergot Alkaloids.

"Fescue toxicosis" and reproductive ergotism present identical toxidromes in late-gestational mares and, likely, other equids. Both toxic syndromes are caused by ergopeptine alkaloids (EPAs) of fungal origin, and they are collectively referred to as equine ergopeptine alkaloid toxicosis (EEPAT). EPAs are produced by either a toxigenic endophyte (Epichloë coenophiala) in tall fescue and/or a nonendophytic fungus (Claviceps purpurea), infecting small grains and grasses. EEPAT can cause hypoprolactinemia-induced agalactia/dysgalactia, prolonged gestation, dystocia, and other reproductive abnormalities in mares, as well as failure of passive transfer in their frequently dysmature/overmature/postmature foals. Prevention relies on eliminating exposures and/or reversing hypoprolactinemia.

Two Satellite Gene Clusters Enhance Ergot Alkaloid Biosynthesis Capacity of Aspergillus leporis.

Ergot alkaloids are fungal specialized metabolites that are important in agriculture and serve as sources of several pharmaceuticals. Aspergillus leporis is a soil saprotroph that possesses two ergot alkaloid biosynthetic gene clusters encoding lysergic acid amide production. We identified two additional, partial biosynthetic gene clusters within the A. leporis genome containing some of the ergot alkaloid synthesis (eas) genes required to make two groups of clavine ergot alkaloids, fumigaclavines and rugulovasines. Clavines possess unique biological properties compared to lysergic acid derivatives. Bioinformatic analyses indicated the fumigaclavine cluster contained functional copies of easA, easG, easD, easM, and easN. Genes resembling easQ and easH, which are required for rugulovasine production, were identified in a separate gene cluster. The pathways encoded by these partial, or satellite, clusters would require intermediates from the previously described lysergic acid amide pathway to synthesize a product. Chemical analyses of A. leporis cultures revealed the presence of fumigaclavine A. However, rugulovasine was only detected in a single sample, prompting a heterologous expression approach to confirm functionality of easQ and easH. An easA knockout strain of Metarhizium brunneum, which accumulates the rugulovasine precursor chanoclavine-I aldehyde, was chosen as expression host. Strains of M. brunneum expressing easQ and easH from A. leporis accumulated rugulovasine as demonstrated through mass spectrometry analysis. These data indicate that A. leporis is exceptional among fungi in having the capacity to synthesize products from three branches of the ergot alkaloid pathway and for utilizing an unusual satellite cluster approach to achieve that outcome. IMPORTANCE Ergot alkaloids are chemicals produced by several species of fungi and are notable for their impacts on agriculture and medicine. The ability to make ergot alkaloids is typically encoded by a clustered set of genes that are physically adjacent on a chromosome. Different ergot alkaloid classes are formed via branching of a complex pathway that begins with a core set of the same five genes. Most ergot alkaloid-producing fungi have a single cluster of genes that is complete, or self-sufficient, and produce ergot alkaloids from one or occasionally two branches from that single cluster. Our data show that Aspergillus leporis is exceptional in having the genetic capacity to make products from three pathway branches. Moreover, it uses a satellite cluster approach, in which gene products of partial clusters rely on supplementation with a chemical intermediate produced via another gene cluster, to diversify its biosynthetic potential without duplicating all the steps.

Ergot Alkaloids Contribute to the Pathogenic Potential of the Fungus Aspergillus leporis.

Opportunistically pathogenic fungi have varying potential to cause disease in animals. Factors contributing to their virulence include specialized metabolites, which in some cases evolved in contexts unrelated to pathogenesis. Specialized metabolites that increase fungal virulence in the model insect Galleria mellonella include the ergot alkaloids fumigaclavine C in Aspergillus fumigatus (syn. Neosartorya fumigata) and lysergic acid α-hydroxyethylamide (LAH) in the entomopathogen Metarhizium brunneum. Three species of Aspergillus recently found to accumulate high concentrations of LAH were investigated for their pathogenic potential in G. mellonella. Aspergillus leporis was most virulent, A. hancockii was intermediate, and A. homomorphus had very little pathogenic potential. Aspergillus leporis and A. hancockii emerged from and sporulated on dead insects, thus completing their asexual life cycles. Inoculation by injection resulted in more lethal infections than did topical inoculation, indicating that A. leporis and A. hancockii were preadapted for insect pathogenesis but lacked an effective means to breach the insect's cuticle. All three species accumulated LAH in infected insects, with A. leporis accumulating the most. Concentrations of LAH in A. leporis were similar to those observed in the entomopathogen M. brunneum. LAH was eliminated from A. leporis through a CRISPR/Cas9-based gene knockout, and the resulting strain had reduced virulence to G. mellonella. The data indicate that A. leporis and A. hancockii have considerable pathogenic potential and that LAH increases the virulence of A. leporis. IMPORTANCE Certain environmental fungi infect animals occasionally or conditionally, whereas others do not. Factors that affect the virulence of these opportunistically pathogenic fungi may have originally evolved to fill some other role for the fungus in its primary environmental niche. Among the factors that may improve the virulence of opportunistic fungi are specialized metabolites--chemicals that are not essential for basic life functions but provide producers with an advantage in particular environments or under specific conditions. Ergot alkaloids are a large family of fungal specialized metabolites that contaminate crops in agriculture and serve as the foundations of numerous pharmaceuticals. Our results show that two ergot alkaloid-producing fungi that were not previously known to be opportunistic pathogens can infect a model insect and that, in at least one of the species, an ergot alkaloid increases the virulence of the fungus.

Phylogenetic patterns of bioactive secondary metabolites produced by fungal endosymbionts in morning glories (Ipomoeeae, Convolvulaceae).

Heritable fungal endosymbiosis is underinvestigated in plant biology and documented in only three plant families (Convolvulaceae, Fabaceae, and Poaceae). An estimated 40% of morning glory species in the tribe Ipomoeeae (Convolvulaceae) have associations with one of two distinct heritable, endosymbiotic fungi (Periglandula and Chaetothyriales) that produce the bioactive metabolites ergot alkaloids, indole diterpene alkaloids, and swainsonine, which have been of interest for their toxic effects on animals and potential medical applications. Here, we report the occurrence of ergot alkaloids, indole diterpene alkaloids, and swainsonine in the Convolvulaceae; and the fungi that produce them based on synthesis of previous studies and new indole diterpene alkaloid data from 27 additional species in a phylogenetic, geographic, and life-history context. We find that individual morning glory species host no more than one metabolite-producing fungal endosymbiont (with one possible exception), possibly due to costs to the host and overlapping functions of the alkaloids. The symbiotic morning glory lineages occur in distinct phylogenetic clades, and host species have significantly larger seed size than nonsymbiotic species. The distinct and widely distributed endosymbiotic relationships in the morning glory family and their alkaloids provide an accessible study system for understanding heritable plant-fungal symbiosis evolution and their potential functions for host plants.

Quantification of Ergot Alkaloids via Lysergic Acid Hydrazide-Development and Comparison of a Sum Parameter Screening Method.

Ergot alkaloids are a group of mycotoxins occurring in products derived from various grasses (e.g., rye) and have been regulated in the EU recently. The new maximum levels refer to the sum of the six most common ergot alkaloids in their two stereoisomeric forms in different food matrices. Typically, these twelve compounds are individually quantified via HPLC-MS/MS or -FLD and subsequently summed up to evaluate food safety in a time-consuming process. Since all these structures share the same ergoline backbone, we developed a novel sum parameter method (SPM) targeting all ergot alkaloids simultaneously via lysergic acid hydrazide. After extraction and clean-up, in analogy to the current European standard method EN 17425 (ESM) for ergot alkaloid quantitation, the samples were derivatized by an optimized hydrazinolysis protocol, which allowed quantitative conversion after 20 min at 100 °C. The new SPM was evaluated against another established HPLC-FLD-based method (LFGB) and the HPLC-MS/MS-based ESM using six naturally contaminated rye and wheat matrix reference materials. While the SPM provided comparable values to the ESM, LFGB showed deviating results. Determined recovery rates, limits of detection and quantification of all three employed methods confirm that the new SPM is a promising alternative to the classical approaches for ergot alkaloid screening in food.

Ergot Alkaloids on Cereals and Seeds: Analytical Methods, Occurrence, and Future Perspectives.

Ergot alkaloids are secondary metabolites resulting from fungi of the genus Claviceps that have proven to be highly toxic. These mycotoxins commonly infect cereal crops such as wheat, rye, barley, and oats. Due to the increase worldwide consumption of cereal and cereal-based products, the presence of ergot alkaloids in food presents a concern for human safety. For this reason, it is essential to develop several analytical methods that allow the detection of these toxic compounds. This review compiles and discusses the most relevant studies and methods used in the detection and quantification of ergot alkaloids. Moreover, the decontamination techniques are also addressed, with special attention to sorting, cleaning, frying, baking, peeling, and ammonization methods, as they are the only ones already applied to ergot alkaloids.

Impact of fetal exposure to mycotoxins on longissimus muscle fiber hypertrophy and miRNA profile.

BACKGROUND: Longissimus muscle samples were collected from lambs exposed in utero to mycotoxins [E-, endophyte-free tall fescue seed without ergot alkaloids (negative control) or E + , endophyte-infected tall fescue seed containing ergot alkaloids] during mid-gestation (MID; E + /E-) or late-gestation (LATE; E-/E +) harvested at two developmental stages (FETAL, gestational d133) or (MAT, near maturity, 250 d of age; n = 3/treatment/developmental stage). Muscle samples were examined to determine the impact of in utero mycotoxin exposure on skeletal muscle fiber hypertrophy and the miRNA profile at FETAL and MAT. RESULTS: Longissimus weight was greater (P < 0.05) in E + /E- lambs compared to E-/E + lambs at MAT; however, FETAL longissimus weight did not differ (P > 0.10) between fescue treatments. Type I fiber cross sectional area was larger (P < 0.10) for E + /E- than E-/E + at MAT but did not differ (P > 0.10) between fescue treatments at FETAL. Type II fiber area was larger (P < 0.05) at MAT in E + /E- compared to E-/E + but did not differ (P < 0.05) between fescue treatments at FETAL. Cross-sectional Type I and Type II longissimus muscle fiber area increased (P < 0.05) from FETAL to MAT by 6.86-fold and 10.83-fold, respectively. The ratio of Type II:Type I muscle fibers was lower (P = 0.04) at MAT compared to FETAL. There were 120 miRNA differentially expressed (q < 0.05) between FETAL and MAT. Maternal fescue treatment did not alter (q > 0.05) expression of miRNAs in the longissimus muscle. miR-133, -29a, -22-3p, and -410-3p were identified as highly significant with a log2 fold change > 4. In vitro satellite cell cultures showed that selected miRNAs (miR-22-3p, 29a, 27a, and 133a) are differentially regulated during proliferation and differentiation indicating a role of miRNA in muscle hypertrophy. CONCLUSIONS: Exposure to mycotoxins did not alter fiber type but had long-term impacts on postnatal muscle hypertrophy and cross-sectional area. The miRNA profile of the longissimus was not altered by Maternal mycotoxin exposure at FETAL or MAT. Developmental age altered the miRNA transcriptome and mRNA expression of known genes related to muscle growth. These results indicate that Maternal exposure to E + fescue seed during LATE gestation can alter postnatal muscle hypertrophy in sheep; however, these changes are not regulated by the miRNA transcriptome of the longissimus muscle.

Alkaloid Biosynthetic Enzyme Generates Diastereomeric Pair via Two Distinct Mechanisms.

Ergopeptines constitute one of the representative classes of ergoline alkaloids and carry a tripeptide extension on the lysergic acid core. In the current study, we discovered and structurally characterized newly isolated ergopeptine-like compounds named lentopeptins from a filamentous fungus Aspergillus lentulus, a close relative of A. fumigatus. Interestingly, in lentopeptins, the common lysergic acid moiety of ergopeptines is replaced by a cinnamic acid moiety at the N-terminus of the peptide segment. Moreover, lentopeptins lack the C-terminal proline residue necessary for the spontaneous cyclization of the peptide extension. Herein, we report the atypical lentopeptin biosynthetic pathway identified through targeted deletion of the len cluster biosynthetic genes predicted from the genome sequence. Further in vitro characterizations of the thiolation-terminal condensation-like (T-CT) didomain of the nonribosomal peptide synthetase LenA and its site-specific mutants revealed the mechanism of peptide release via diketopiperazine formation, an activity previously unreported for CT domains. Most intriguingly, in vitro assays of the cytochrome P450 LenC illuminated the unique mechanisms to generate two diastereomeric products. Lentopeptin A forms via a stereospecific hydroxylation, followed by a spontaneous bicyclic lactam core formation, while lentopeptin B is produced through an initial dehydrogenation, followed by a bicyclic lactam core formation and stereospecific hydration. Our results showcase how nature exploits common biosynthetic enzymes to forge new complex natural products effectively (213/250).

Overproduction of medicinal ergot alkaloids based on a fungal platform.

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.

Beyond the cyclopropyl ring formation: fungal Aj_EasH catalyzes asymmetric hydroxylation of ergot alkaloids.

Ergot alkaloids (EAs) are among the most important bioactive natural products. FeII/α-ketoglutarate-dependent dioxygenase Aj_EasH from Aspergillus japonicus is responsible for the formation of the cyclopropyl ring of the ergot alkaloid (EA) cycloclavine (4). Herein we reconstituted the biosynthesis of 4 in vitro from prechanoclavine (1) for the first time. Additionally, an unexpected activity of asymmetric hydroxylation at the C-4 position of EA compound festuclavine (5) for Aj_EasH was revealed. Furthermore, Aj_EasH also catalyzes the hydroxylation of two more EAs 9,10-dihydrolysergol (6) and elymoclavine (7). Thus, our results proved that Aj_EasH is a promiscuous and bimodal dioxygenase that catalyzes both the formation of cyclopropyl ring in 4 and the asymmetric hydroxylation of EAs. Molecular docking (MD) revealed the substrate-binding mode as well as the catalytic mechanism of asymmetric hydroxylation, suggesting more EAs could potentially be recognized and hydroxylated by Aj_EasH. Overall, the newly discovered activity empowered Aj_EasH with great potential for producing more diverse and bioactive EA derivatives. KEY POINTS: • Aj_EasH was revealed to be a promiscuous and bimodal FeII/α-ketoglutarate-dependent dioxygenase. • Aj_EasH converted festuclavine, 9,10-dihydrolysergol, and elymoclavine to their hydroxylated derivatives. • The catalytic mechanism of Aj_EasH for hydroxylation was analyzed by molecular docking.

Why Do Plant-Pathogenic Fungi Produce Mycotoxins? Potential Roles for Mycotoxins in the Plant Ecosystem.

For many plant-pathogenic or endophytic fungi, production of mycotoxins, which are toxic to humans, may present a fitness gain. However, associations between mycotoxin production and plant pathogenicity or virulence is inconsistent and difficult due to the complexity of these host-pathogen interactions and the influences of environmental and insect factors. Aflatoxin receives a lot of attention due to its potent toxicity and carcinogenicity but the connection between aflatoxin production and pathogenicity is complicated by the pathogenic ability and prevalence of nonaflatoxigenic isolates in crops. Other toxins directly aid fungi in planta, trichothecenes are important virulence factors, and ergot alkaloids limit herbivory and fungal consumption due to insect toxicity. We review a panel discussion at the American Phytopathological Society's Plant Health 2021 conference, which gathered diverse experts representing different research sectors, career stages, ethnicities, and genders to discuss the diverse roles of mycotoxins in the lifestyles of filamentous fungi of the families Clavicipitaceae, Trichocomaceae (Eurotiales), and Nectriaceae (Hypocreales).

Methods of Lysergic Acid Synthesis-The Key Ergot Alkaloid.

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.

Molecular and Alkaloid Characterization of Claviceps purpurea Sensu Lato From Grass Seed Production Areas of the U.S. Pacific Northwest.

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.

Acute Treatments for Episodic Migraine in Adults: A Systematic Review and Meta-analysis.

Importance: Migraine is common and can be associated with significant morbidity, and several treatment options exist for acute therapy. Objective: To evaluate the benefits and harms associated with acute treatments for episodic migraine in adults. Data Sources: Multiple databases from database inception to February 24, 2021. Study Selection: Randomized clinical trials and systematic reviews that assessed effectiveness or harms of acute therapy for migraine attacks. Data Extraction and Synthesis: Independent reviewers selected studies and extracted data. Meta-analysis was performed with the DerSimonian-Laird random-effects model with Hartung-Knapp-Sidik-Jonkman variance correction or by using a fixed-effect model based on the Mantel-Haenszel method if the number of studies was small. Main Outcomes and Measures: The main outcomes included pain freedom, pain relief, sustained pain freedom, sustained pain relief, and adverse events. The strength of evidence (SOE) was graded with the Agency for Healthcare Research and Quality Methods Guide for Effectiveness and Comparative Effectiveness Reviews. Findings: Evidence on triptans and nonsteroidal anti-inflammatory drugs was summarized from 15 systematic reviews. For other interventions, 115 randomized clinical trials with 28 803 patients were included. Compared with placebo, triptans and nonsteroidal anti-inflammatory drugs used individually were significantly associated with reduced pain at 2 hours and 1 day (moderate to high SOE) and increased risk of mild and transient adverse events. Compared with placebo, calcitonin gene-related peptide receptor antagonists (low to high SOE), lasmiditan (5-HT1F receptor agonist; high SOE), dihydroergotamine (moderate to high SOE), ergotamine plus caffeine (moderate SOE), acetaminophen (moderate SOE), antiemetics (low SOE), butorphanol (low SOE), and tramadol in combination with acetaminophen (low SOE) were significantly associated with pain reduction and increase in mild adverse events. The findings for opioids were based on low or insufficient SOE. Several nonpharmacologic treatments were significantly associated with improved pain, including remote electrical neuromodulation (moderate SOE), transcranial magnetic stimulation (low SOE), external trigeminal nerve stimulation (low SOE), and noninvasive vagus nerve stimulation (moderate SOE). No significant difference in adverse events was found between nonpharmacologic treatments and sham. Conclusions and Relevance: There are several acute treatments for migraine, with varying strength of supporting evidence. Use of triptans, nonsteroidal anti-inflammatory drugs, acetaminophen, dihydroergotamine, calcitonin gene-related peptide antagonists, lasmiditan, and some nonpharmacologic treatments was associated with improved pain and function. The evidence for many other interventions, including opioids, was limited.

Determination of the Main Ergot Alkaloids and Their Epimers in Oat-Based Functional Foods by Ultra-High Performance Liquid Chromatography Tandem Mass Spectrometry.

An ultra-high performance liquid chromatography coupled to tandem mass spectrometry method is proposed for the determination of the major ergot alkaloids (ergometrine, ergosine, ergotamine, ergocornine, ergokryptine, ergocristine) and their epimers (ergometrinine, ergosinine, ergotaminine, ergocorninine, ergokryptinine, and ergocristinine) in oat-based foods and food supplements. A modified QuEChERS (quick, easy, cheap, effective, rugged, and safe) procedure was applied as sample treatment, reducing the consumption of organic solvent and increasing sensitivity. This method involved an extraction with acetonitrile and ammonium carbonate (85:15, v/v) and a clean-up step based on dispersive solid-phase extraction, employing a mixture of C18/Z-Sep+ as sorbents. Procedural calibration curves were established and limits of quantification were below 3.2 µg/kg for the studied compounds. Repeatability and intermediate precision (expressed as RSD%) were lower than 6.3% and 15%, respectively, with recoveries ranging between 89.7% and 109%. The method was applied to oat-based products (bran, flakes, flour, grass, hydroalcoholic extracts, juices, and tablets), finding a positive sample of oat bran contaminated with ergometrine, ergosine, ergometrinine, and ergosinine (total content of 10.7 µg/kg).

Determination of principal ergot alkaloids in swine feeding.

BACKGROUND: Ergot alkaloids are secondary metabolites produced by fungi in the genus Claviceps. They contaminate a large variety of cereals, such as rye, triticale, wheat and barley. The ingestion of contaminated cereals might cause adverse health effects in humans and animals. In fact, pigs, cattle, sheep, and poultry are involved in sporadic outbreaks and, although there are several studies about occurrence of ergot alkaloids in grain cereals, there are scarce studies focused on compound feed. RESULTS: Twelve ergot alkaloids have been quantified in 228 feed samples intended for swine. The analytes were extracted using QuEChERS with Z-Sep+ as sorbent in the clean-up step, which reduced the matrix effect, allowing limits of quantification between 2.1 and 21.7 µg kg-1 . The analytes were subsequently quantified by ultra-high-performance liquid chromatography with tandem mass spectrometry (UHPLC-MS/MS). A total of 29 samples (12.7%) revealed contamination by at least one ergot alkaloid, and among contaminated samples, 65% were contaminated by more than one. Only 6 of 12 target ergot alkaloids showed concentrations above the limit of quantification. The concentrations for individual ergot alkaloids ranged between 5.9 µg kg-1 for ergosinine to 145.3 µg kg-1 for ergometrine (the predominant ergot alkaloid), while the total ergot alkaloid content ranged from 5.9 to 158.7 µg kg-1 . CONCLUSIONS: The occurrence of ergot alkaloids in feed samples in Spain seems to be lower than in other regions of Europe. All the samples fulfilled current recommendations of the feed industry about practical limits for ergot alkaloids in pig feeds. This suggests that the feeds are safe for pig consumption, regarding the presence of ergot alkaloids. © 2021 Society of Chemical Industry.

Differentially expressed genes in cotyledon of ewes fed mycotoxins.

BACKGROUND: Ergot alkaloids (E+) are mycotoxins produced by the endophytic fungus, Epichloë coenophiala, in tall fescue that are associated with ergotism in animals. Exposure to ergot alkaloids during gestation reduces fetal weight and placental mass in sheep. These reductions are related to vasoconstrictive effects of ergot alkaloids and potential alterations in nutrient transport to the fetus. Cotyledon samples were obtained from eight ewes that were fed E+ (n = 4; E+/E+) or E- (endophyte-free without ergot alkaloids; n = 4; E-/E-) seed during both mid (d 35 to 85) and late (d 85-133) gestation to assess differentially expressed genes associated with ergot alkaloid induced reductions in placental mass and fetal weight, and discover potential adaptive mechanisms to alter nutrient supply to fetus. RESULTS: Ewes fed E+/E+ fescue seed during both mid and late gestation had 20% reduction in fetal body weight and 33% reduction in cotyledon mass compared to controls (E-/E-). Over 13,000 genes were identified with 110 upregulated and 33 downregulated. Four genes had a |log2FC| > 5 for ewes consuming E+/E+ treatment compared to controls: LECT2, SLC22A9, APOC3, and MBL2. REViGO revealed clusters of upregulated genes associated glucose, carbohydrates, lipid, protein, macromolecular and cellular metabolism, regulation of wound healing and response to starvation. For downregulated genes, no clusters were present, but all enriched GO terms were associated with anion and monocarboxylic acid transport. The complement and coagulation cascade and the peroxisome proliferator-activated receptor signaling pathway were found to be enriched for ewes consuming E+/E+ treatment. CONCLUSIONS: Consumption of ergot alkaloids during gestation altered the cotyledonary transcriptome specifically related to macronutrient metabolism, wound healing and starvation. These results show that ergot alkaloid exposure upregulates genes involved in nutrient metabolism to supply the fetus with additional substrates in attempts to rescue fetal growth.

Identification of the polyketide synthase PKS7 responsible for the production of lecanoric acid and ethyl lecanorate in Claviceps purpurea.

Claviceps purpurea is a plant pathogenic fungus which is still highly relevant in modern agriculture as it infects grasses such as rye and wheat. The disease caused by the consumption of contaminated grain or flour has been known since the Middle Ages and is termed ergotism. The main cause for the toxicity of this fungus is attributed to the ergot alkaloids. Apart from these alkaloids and the ergochromes known as ergot pigments, the secondary metabolism of C. purpurea is not well investigated. This study demonstrated the function of the polyketide synthase PKS7 in C. purpurea by determining the effect of its overexpression on metabolite profiles. For the first time, the depsides lecanoric acid, ethyl lecanorate, gerfelin, and C10-deoxy gerfelin were discovered as secondary metabolites of C. purpurea. Additionally, to estimate the contribution of isolated secondary metabolites to the toxic effects of C. purpurea, lecanoric acid, ethyl lecanorate, and orsellinic acid were tested on HepG2 and CCF-STTG1 cell lines. This study provides the first report on the function of C. purpurea PKS7 responsible for the production of depsides, among which lecanoric acid and ethyl lecanorate were identified as main secondary metabolites.

Several Metarhizium Species Produce Ergot Alkaloids in a Condition-Specific Manner.

Genomic sequence data indicate that certain fungi in the genus Metarhizium have the capacity to produce lysergic acid-derived ergot alkaloids, but accumulation of ergot alkaloids in these fungi has not been demonstrated previously. We assayed several Metarhizium species grown under different conditions for accumulation of ergot alkaloids. Isolates of M. brunneum and M. anisopliae accumulated the lysergic acid amides lysergic acid α-hydroxyethyl amide, ergine, and ergonovine on sucrose-yeast extract agar but not on two other tested media. Isolates of six other Metarhizium species did not accumulate ergot alkaloids on sucrose-yeast extract agar. Conidia of M. brunneum lacked detectable ergot alkaloids, and mycelia of this fungus secreted over 80% of their ergot alkaloid yield into the culture medium. Isolates of M. brunneum, M. flavoviride, M. robertsii, M. acridum, and M. anisopliae produced high concentrations of ergot alkaloids in infected larvae of the model insect Galleria mellonella, but larvae infected with M. pingshaense, M. album, M. majus, and M. guizhouense lacked detectable ergot alkaloids. Alkaloid concentrations were significantly higher when insects were alive (as opposed to killed by freezing or gas) at the time of inoculation with M. brunneum Roots of corn and beans were inoculated with M. brunneum or M. flavoviride and global metabolomic analyses indicated that the inoculated roots were colonized, though no ergot alkaloids were detected. The data demonstrate that several Metarhizium species produce ergot alkaloids of the lysergic acid amide class and that production of ergot alkaloids is tightly regulated and associated with insect colonization.IMPORTANCE Our discovery of ergot alkaloids in fungi of the genus Metarhizium has agricultural and pharmaceutical implications. Ergot alkaloids produced by other fungi in the family Clavicipitaceae accumulate in forage grasses or grain crops; in this context they are considered toxins, though their presence also may deter or kill insect pests. Our data report ergot alkaloids in Metarhizium species and indicate a close association of ergot alkaloid accumulation with insect colonization. The lack of accumulation of alkaloids in spores of the fungi and in plants colonized by the fungi affirms the safety of using Metarhizium species as biocontrol agents. Ergot alkaloids produced by other fungi have been exploited to produce powerful pharmaceuticals. The class of ergot alkaloids discovered in Metarhizium species (lysergic acid amides) and their secretion into the growth medium make Metarhizium species a potential platform for future studies on ergot alkaloid synthesis and modification.