A gene regulating ergot alkaloid biosynthesis in Metarhizium brunneum.

Ergot alkaloid synthesis (eas) gene clusters found in several fungi encode biosynthesis of agriculturally and pharmaceutically important ergot alkaloids. Although the biosynthetic genes of the ergot alkaloid pathway have been well characterized, regulation of those genes is unknown. We characterized a gene with sequence similarity to a putative transcription factor and that was found adjacent to the eas cluster of Metarhizium brunneum, a plant symbiont and insect pathogen. Function of the novel gene, easR, was explored by CRISPR-Cas9-derived gene knockouts. To maximize potential for ergot alkaloid accumulation, strains of M. brunneum were injected into larvae of the insect Galleria mellonella. Larvae infected with the wild type contained abundant ergot alkaloids, but those infected with easR knockouts lacked detectable ergot alkaloids. The easR knockout strains had significantly reduced or no detectable mRNA from eas cluster genes in RNAseq and qualitative RT-PCR analyses, whereas the wild-type strain contained abundant mRNA from all eas genes. These data demonstrate that the product of easR is required for ergot alkaloid accumulation and provide evidence that it has a role in the expression of ergot alkaloid biosynthesis genes. Larvae infected with an easR knockout survived significantly longer than those infected with the wild type (P < 0.0001), indicating a role for EasR, and indirectly confirming a role for ergot alkaloids, in the virulence of M. brunneum to insects. Homologs of easR were found associated with eas clusters of at least 15 other ergot alkaloid-producing fungi, indicating that EasR homologs may contribute to regulation of ergot alkaloid synthesis in additional fungi. IMPORTANCE: Ergot alkaloids produced by several species of fungi are important as contaminants of food and feed in agriculture and also as the foundation of numerous pharmaceuticals prescribed for dementia, migraines, hyperprolactinemia, and several other disorders. Information on control of the ergot alkaloid pathway may contribute to strategies to limit their production in agricultural settings or increase their yield for pharmaceutical production. Our results demonstrate that a previously uncharacterized gene clustered with the ergot alkaloid synthesis genes is required for the sufficient transcription of the ergot alkaloid biosynthesis genes. This observation suggests the gene encodes a factor regulating transcription of those biosynthetic genes.

Ergot alkaloid consumption alters serotonin receptor-induced vasoactivity in ovine umbilical vasculature.

Consumption of ergot alkaloids during the second half of gestation has been shown to decrease umbilical artery vasoactivity resulting in decreased birth weights. Negative vascular effects of ergot alkaloids are mediated predominantly through serotonergic and adrenergic receptors in other tissues. Vasoactivity of serotonin (5-HT) receptors 5-HT2A and 5-HT1B/1D in umbilical artery and vein from ewes receiving endophyte-infected seed (E + 1.77 mg ergovaline/hd/d) or a control total mixed ration (CON; 0 mg ergovaline/hd/d) tall fescue seed at d-110 and d-133 of gestation was evaluated. Gravid reproduction tracts were collected from ewes. Two-mm sections of umbilical artery and vein were exposed to increasing concentrations of a 5-HT1B/1D agonist and 5-HT2A agonist. The 5-HT1B/1D agonist did not stimulate a contractile response in artery or vein or either gestation time point. 5-HT2A agonist caused large responses in artery with greatest occurring at d-110 and decreasing in magnitude as days of gestation increased (p < 0.05). On d-110 and 133 of gestation, arteries from CON ewes had greater contractile response than arteries collected from E+ ewes (p < 0.05). Veins responded to increasing concentrations of the 5-HT2A agonist. Maximal d-110 vein response was greater than d-133 when exposed to 5-HT2A agonist (p < 0.05). Unlike the artery, veins from E+ ewes had greater d-133 contractile response than CON (p < 0.05). Vascular contractions of umbilical artery and vein are induced by 5-HT2A receptor activity and not 5-HT1B/1D. Umbilical artery 5-HT2A receptor activity was more sensitive to seed treatment and could be responsible for ergot alkaloid-induced intra-uterine growth restriction.

A Major Facilitator Superfamily Transporter Contributes to Ergot Alkaloid Accumulation but Not Secretion in Aspergillus leporis.

Ergot alkaloids are fungal natural products with important roles in agriculture and medicine. We used heterologous expression and gene knockout approaches to investigate potential roles for the product of a major facilitator superfamily transporter gene (easT) recently found in an ergot alkaloid biosynthetic gene cluster in Aspergillus leporis. A strain of Aspergillus fumigatus previously engineered to accumulate lysergic acid, but which did not convert the precursor agroclavine to lysergic acid efficiently or secrete lysergic acid well, was chosen as an expression host for easT. Expression of easT in this strain resulted in accumulation of significantly more pathway intermediates but no detectable lysergic acid. Secretion of ergot alkaloids was reduced in the easT-expressing strain. EasT localized to discrete vesicle-like structures in the cytosol of A. fumigatus, with no localization detected in the plasma membrane. When easT was knocked out in A. leporis, accumulation of lysergic acid amides was reduced relative to the wild type. There was no negative effect on secretion of ergot alkaloids in the knockout mutant. The data indicate that easT encodes a product that contributes to accumulation of ergot alkaloids, perhaps by transporting intermediates between cellular compartments, but does not have a significant role in secreting ergot alkaloids.

Examining the long-term impacts of ergot alkaloids on fetal heifer development and subsequent growth and reproductive performance.

Consumption of toxic endophyte-infected tall fescue (EI) results in poor reproductive performance in domestic livestock. In this study, the objective was to evaluate the effects of ergovaline exposure during mid-gestation (days 93 through 188 of gestation) on dam performance, the growing female fetus, and the subsequent growth and reproductive performance of the gestationally exposed heifer calves. Pregnant Angus and Simmental-Angus cows were blocked by age (2 to 3, to 7, and >7 y), body weight (BW), and breed; and then randomly assigned to graze either novel endophyte-infected tall fescue (EN; <5% infection rate; n = 27 year 1, n = 16 year 2) or toxic EI (99% infection rate; n = 27 year 1, n = 17 year 2). Weekly BW, body condition scores (BCS), hair coat scores, hair shedding scores (HSS), and blood samples for progesterone (P4) analysis were collected from mid-April through July of 2017 (year 1) and 2018 (year 2). Gestation length, birth weight, placental characteristics, heifer calf growth, onset of puberty, ovarian characteristics, and artificial insemination pregnancy rates were measured. Data were analyzed using the MIXED procedure of SAS. Cows grazing EI pastures had reduced average daily gain, reduced BCS, greater HSS, and decreased P4 concentrations compared to cows on EN pasture (P < 0.01). Birth weights were decreased for heifers whose dams were exposed to EI pastures during their second trimester (P < 0.01). Heifer pregnancy rates were not impacted by EI pasture exposure during gestation for either year of the study. However, a treatment-by-year effect was seen for the pregnancy rate for EI-exposed heifers in year 2; EI-exposed heifers in year 2 had increased pregnancy rates at two of the inseminations. Combined, these data reinforce that consumption of toxic EI during gestation can negatively impact both dam and offspring performance. More studies are needed to evaluate more parameters in an effort to elucidate the possible life-long impacts of ergovaline exposure during gestation.

The U.S. livestock industry incurs over one billion dollars of economic loss every year due to fescue toxicosis, caused by consuming ergot alkaloids produced by an endophytic fungus in some grass species. Identifying means to mitigate the negative effects of fescue toxicosis is needed for U.S. beef producers. Effective treatment for this toxicosis is still needed. The objective of this study was to evaluate the effects of ergovaline exposure during mid-gestation on dam performance, the growing female fetus, and the subsequent growth and reproductive performance of the gestationally exposed heifer calves. We identified specific phenotype traits that undergo developmental programming in utero in response to fescue toxicosis. However, measurements of growth and reproductive performance were not altered by ergot exposure.

Climate Effects on Ergot and Ergot Alkaloids Occurrence in Italian Wheat.

In recent years, there has been an intensification of weather variability worldwide as a result of climate change. Some regions have been affected by drought, while others have experienced more intense rainfall. The incidence and severity of moldy grain and mycotoxin contamination during the growing and harvesting seasons have increased as a result of these weather conditions. Additionally, torrential rains and wet conditions may cause delays in grain drying, leading to mold growth in the field. In July 2023, a wheat field in Lecco (Lombardy, Italy) was affected by torrential rains that led to the development of the Claviceps fungi. In the field, dark sclerotia were identified on some ears. Wheat ears, kernels, and sclerotia were collected and analyzed by LC-MS/MS at IZSLER, Food Chemical Department, in Bologna. The wheat ears, kernels, and sclerotia were analyzed for 12 ergot alkaloids (EAs) according to (EU) Regulation 2023/915 (ergocornine/ergocorninine; ergocristine/ergocristinine; ergocryptine/ergocryptinine; ergometrine/ergometrinine; ergosine/ergosinine; ergotamine/ergotaminine), after QuEChERS (Z-Sep/C18) purification. The analyzed sclerotia showed significant differences in total alkaloid content that vary between 0.01 and 0.5% (w/w), according to the results of the 2017 EFSA scientific report. EAs detected in sclerotia were up to 4951 mg/kg, in wheat ears up to 33 mg/kg, and in kernels were 1 mg/kg. Additional mycotoxins, including ochratoxin A, deoxynivalenol, zearalenone, fumonisins, T2-HT2 toxins, and aflatoxins, were investigated in wheat kernels after purification with immunoaffinity columns (IAC). The analysis revealed the presence of deoxynivalenol in wheat kernels at a concentration of 2251 µg/kg. It is expected that climate change will increase the frequency of extreme weather events. In order to mitigate the potential risks associated with mycotoxin-producing fungi and to ensure the protection of human health, it is suggested that official controls be implemented in the field.

Mycotoxicoses in Veterinary Medicine: Fusarium Toxins, Grass Staggers, and Neothyphodium Toxins.

Fungi are a large group of eukaryotic microorganisms that can readily adapt to diverse environments and occur in almost all climatic zones and continents. Although some fungi are inevitable in the environment for the decay and recycling of organic material, many species are known to produce secondary metabolites, and these mycotoxins, when ingested with food or feed materials, can adversely affect animal and human health. Among the toxigenic fungi, Fusarium species are recognized as so-called field fungi, invading crops and producing mycotoxins predominantly before harvest. Fusarium produces a wide array of mycotoxins, causing different plant diseases. Fusariosis causes significant economic losses in a wide range of crops. Fusarium secondary metabolites, particularly trichothecenes, are potent toxins in mammalian species and cause diverse adverse effects in humans and animals. Other prominent Fusarium toxins with entirely different chemical structures are zearalenone and its derivatives and fumonisins. With an entirely different life cycle, toxins of endophytes belonging to the genus Epichloë and Neothyphodium coenophialum and Neothyphodium lolii comprise an animal health risk, particularly for grazing animals. This review aimed to summarize the adverse effects of selected Fusarium and Epichloë toxins, with a special emphasis on their occurrence in roughages and their mechanisms of action, and describe their effect on animal health and welfare and the potentially related public health risks.

High deoxynivalenol and ergot alkaloid levels in wheat grain: effects on growth performance, carcass traits, rumen fermentation, and blood parameters of feedlot cattle.

This study was designed to assess the impacts of a mixture of deoxynivalenol (DON) and ergot alkaloids (EAs) on growth performance, rumen function, blood parameters, and carcass traits of feedlot cattle. Forty steers (450 ± 6.0 kg) were stratified by weight and randomly allocated to 1 of 4 treatments; control-low (CON-L), control-high (CON-H) which contained low or high wheat screenings that lacked mycotoxins at the same level as the mycotoxin-low (MYC-L; 5.0 mg/kg DON, 2.1 mg/kg EA), and mycotoxin-high (MYC-H: 10 mg/kg DON, 4.2 mg/kg EA) diets that included wheat screening with mycotoxins. Steers were housed in individual pens for a 112-day finishing trial. Intake was 24.8% lower (P < 0.001) for MYC steers compared to CON steers. As a result, average daily gains of MYC steers were 42.1% lower (P < 0.001) than CON steers. Gain to feed ratio was also lower (P < 0.001) for MYC steers compared to CON steers. Platelets, alanine aminotransferase, globulins, and blood urea nitrogen were lower (P ≤ 0.008), and lymphocytes, glutathione peroxidase activity (GPx), and interleukin-10 (IL-10) were elevated (P ≤ 0.002) in MYC steers compared to CON steers. Hot carcass weights and backfat thickness were reduced (P < 0.001) in MYC steers, resulting in leaner (P < 0.001) carcasses and higher (P < 0.007) meat yield compared to CON steers. Results suggest that a mixture of DON and EAs negatively impacted health, performance, and carcass traits of feedlot steers, with the majority of this response likely attributable to EAs. However, more research is needed to distinguish the relative contribution of each mycotoxin to the specific responses observed.

All-in-one 2LabsToGo system for analysis of ergot alkaloids in whole rye.

Ergot alkaloids, naturally occurring mycotoxins of Claviceps fungi, pose health risks. This necessitates accurate analysis methods to ensure food safety. This study explored the open-source miniaturized all-in-one 2LabsToGo system to analyze ergot alkaloids in whole rye samples. It is suited for sustainable atline analysis as it combines all planar chromatography tasks, allowing low-cost quality control in milling plants. The LOD and LOQ of ergocristine were determined to be 0.4 and 1.2 ng/zone, respectively. Detectability of ergot alkaloids was proven to be below the current maximum limit of 500 µg/kg for rye milling products. The repeatability (%RSD) was 4.1 % and the coefficient of determination of the analytical response (R2) was 0.9918 for ergocristine. The mean recovery rate of ergot alkaloids in spiked whole rye grain was close to 100 %. Results of screening whole rye for ergot alkaloids were successfully verified by comparison with those obtained by conventional status quo HPTLC instrumentation.

First Synthesis of Ergotamine-13CD3 and Ergotaminine-13CD3 from Unlabeled Ergotamine.

Ergot alkaloids (EAs) formed by Claviceps fungi are one of the most common food contaminants worldwide, affecting cereals such as rye, wheat, and barley. To accurately determine the level of contamination and to monitor EAs maximum levels set by the European Union, the six most common EAs (so-called priority EAs) and their corresponding epimers are quantified using high-performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS). The quantification of EAs in complex food matrices without appropriate internal standards is challenging but currently carried out in the standard method EN 17425:2021 due to their commercial unavailability. To address the need for isotopically labeled EAs, we focus on two semi-synthetic approaches for the synthesis of these reference standards. Therefore, we investigate the feasibility of the N6-demethylation of native ergotamine to yield norergotamine, which can subsequently be remethylated with an isotopically labeled methylating reagent, such as iodomethane (13CD3-I), to yield isotopically labeled ergotamine and its C8-epimer ergotaminine. Testing the isotopically labeled ergotamine/-inine against native ergotamine/-inine with HPLC coupled to high-resolution HR-MS/MS proved the structure of ergotamine-13CD3 and ergotaminine-13CD3. Thus, for the first time, we can describe their synthesis from unlabeled, native ergotamine. Furthermore, this approach is promising as a universal way to synthesize other isotopically labeled EAs.

Simultaneous determination of total ergot alkaloids in wheat flour by Orbitrap mass spectrometry.

The optimization screening methods for total ergot alkaloids in wheat extracts involve transforming them into a single compound, which is then analyzed via high-resolution Orbitrap mass spectrometry (Orbitrap MS). Orbitrap MS provides highly sensitive and accurate mass measurements, enhancing the selectivity and sensitivity of the analysis. Various hydrolysis and reduction methods have been investigated, and the use of superhydrides has emerged as the most effective method for transforming ergopeptine alkaloids. This study also focused on the epimerization of ergot alkaloids, particularly the differences between R- and S-epimers and their impact on the mass spectra. We validated our method by assessing the linearity, sensitivity, recovery, matrix effects, repeatability, and stability. The limits of detection and quantitation were set at 0.43 and 1.30 µg LSA/kg wheat, respectively. The proposed method offers a robust analytical approach for screening and quantifying total ergot alkaloids in wheat samples, addressing important concerns about their presence in food and feed.

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.

Biological studies of clavine alkaloids targeting CNS receptors.

In contrast to well established psychedelics such as lysergic acid diethylamide (LSD) and psilocybin, ergot alkaloids of the clavine subclass have not been thoroughly investigated, in spite of their broad occurrence in nature and their well-established potent physiological effects. This study presents the current knowledge on the biological properties of clavine alkaloids, draws comparisons to the pharmacology of ergolines and related psychedelics, and demonstrates opportunities to develop novel structure-activity relationship (SAR) profiles. The latter could usher in a new stage of medicinal chemistry studies that enable an expansion of the currently structurally limited portfolio of psychedelic therapeutics.

Development, in-house validation and application of a method using high-performance liquid chromatography with fluorescence detection (HPLC-FLD) for the quantification of 12 ergot alkaloids in compound feeds.

Introduction: Ergot alkaloids (EAs) are toxic substances naturally produced by Claviceps fungi. These fungi infest a wide range of cereals and grasses. When domestic animals are exposed to EAs through contaminated feeds, it is detrimental to them and leads to significant economic losses. For that reason, it is important to monitor feed for the presence of EAs, especially with methods enabling their determination in processed materials. Material and Methods: Ergot alkaloids were extracted with acetonitrile, and dispersive solid phase extraction (d-SPE) was used for clean-up of the extracts. After evaporation, the extracts were reconstituted in ammonium carbonate and acetonitrile and subjected to instrumental analysis using high-performance liquid chromatography with fluorescence detection. The developed method was validated in terms of linearity, selectivity, repeatability, reproducibility, robustness, matrix effect, limits of quantification and detection and uncertainty. The EA content of 40 compound feeds was determined. Results: All the assessed validation parameters fulfilled the requirements of Regulation (EU) 2021/808. At least one of the monitored alkaloids was determined in 40% of the samples. The EAs with the highest incidence rate were ergocryptine, ergometrinine and ergocornine. The total concentrations of EAs ranged from under the limit of quantification to 62.3 µg kg-1. Conclusion: The results demonstrated that the developed method was suitable for simultaneously determining twelve EAs in compound feed and could be used for routine analysis.

Development and validation of an analytical method for the simultaneous determination of 12 ergot, 2 tropane, and 28 pyrrolizidine alkaloids in cereal-based food by LC-MS/MS.

Alkaloids are naturally occurring compounds containing basic nitrogen atoms. They are biosynthesized mainly by plants but also by some fungi species. Many alkaloids are toxic to humans and animals, and they have been classified as food contaminants. Among them, ergot, tropane, and pyrrolizidine alkaloids have maximum levels in foods, established by the Commission Regulation (EU) 2023/915. In this study, an analytical method was successfully developed and validated for the simultaneous determination of 42 ergot, tropane, and pyrrolizidine alkaloids and their N-oxides in cereal-based food. The method includes QuEChERS-based extraction followed by liquid chromatography coupled with tandem mass spectrometry. The proposed method was validated providing recoveries ranging from 71 to 119 %, intra- and inter-day precision lower than 19 %, and limits of quantification between 0.5 and 1.0 µg kg-1. Finally, the analysis of reference materials coming from FAPAS proficiency tests demonstrated the suitability for purpose of the methodology (z-scores < 2). Nine cereal-based products samples were analyzed of which ergot alkaloids were detected in two of them, while one sample showed the presence of three pyrrolizidine alkaloids.

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.

Occurrence of Ergot Alkaloids in Major and Minor Cereals from Northern Italy: A Three Harvesting Years Scenario.

Ergot alkaloids (EAs), mycotoxins produced mainly by fungi of the Claviceps genus, have been frequently reported in rye, while their increasingly frequent occurrence in other cereals is likely related to weather conditions, with the incidence of ergot sclerotia in winter grains being related to heavy rainfall and moist soils at critical periods. However, compared to other regulated mycotoxins, data about the prevalence and occurrence of EAs in major and minor cereals harvested in the Mediterranean growing areas are still scant. In this regard, the current study reported the occurrence of EAs in 18 genotypes of winter cereals harvested over 3 years from an experimental field located in North Italy which were analyzed by HPLC-MS/MS. Results indicate a widespread occurrence of all the major EAs in all the considered cereal crops, especially under supportive meteorological conditions. EA contamination was dependent on the harvest year (p < 0.0001) which was particularly high in 2020 for all the considered species. The results also demonstrated a large co-occurrence of EAs with 98 cereal samples out of 162 contaminated with at least one of the 12 EAs (60% positive samples) in the range LOD: 15,389 µg/kg (median value: 2.32 µg/kg), expressed as the sum of the EAs. Rye was confirmed to be the crop more susceptible to the fungal infection (EAs content up to 4,302 µg/kg). To the best of our knowledge, we have reported the accumulation of EAs in tritordeum (LOD: 15,389 µg/kg) and in emmer (LOD: 1.9 µg/kg) for the first time.

Photoredox catalysis enabling decarboxylative radical cyclization of γ,γ-dimethylallyltryptophan (DMAT) derivatives: formal synthesis of 6,7-secoagroclavine.

An unusual photoredox-catalyzed radical decarboxylative cyclization cascade reaction of γ,γ-dimethylallyltryptophan (DMAT) derivatives containing unactivated alkene moieties has been developed, providing green and efficient access to various six-, seven-, and eight-membered ring 3,4-fused tricyclic indoles. This type of cyclization, which was hitherto very difficult to comprehend in ergot biosynthesis and to accomplish by more conventional procedures, enables the synthesis of ergot alkaloid precursors. In addition, this work describes a mild, environmentally friendly method to activate, reductively and oxidatively, natural carboxylic acids for decarboxylative C-C bond formation by exploiting the same photocatalyst.

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.

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.