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.

Evaluation of Ergot Resistance in Hard Red Spring Wheat in North Dakota.

Higher levels of ergot (Claviceps purpurea [Fr.] Tul.) were reported in North Dakota hard red spring wheat (HRSW) in 2018, leading to questions pertaining to management and cultivar resistance. To better understand pathogen and HRSW cultivar responses, greenhouse experiments were conducted from 2020 to 2021 to evaluate the aggressiveness of nine C. purpurea isolates and ergot resistance in 21 HRSW cultivars. Results from the aggressiveness assay indicated significant cultivar-by-isolate interactions for the total weight of sclerotia produced and ergot incidence. Mean data across all cultivars by isolate combinations suggested isolates CC-3 and IA-Tim were the most aggressive, and these were subsequently used in ergot resistance experiments. Results from ergot resistance screening indicated that none of the HRSW cultivars were immune to C. purpurea because all cultivars produced sclerotia. However, differences in ergot incidence, kernel incidence, aborted kernel incidence, total sclerotia weight, sclerotia length, and sclerotia width occurred among cultivars. Both 'ND-Frohberg' and 'TCG-Spitfire' had the lowest ergot incidence values and were among the lowest in total sclerotia weight. 'Waldron' and 'LCS-Trigger' had the highest ergot incidence and the highest total sclerotia weight. Given that most concerns with ergot occur postharvest, we suggest two categories to describe ergot resistance: host resistance (fate of inoculation for a stigma) and logistical resistance (size characteristics of a sclerotium that influence its ability to remain with a seed lot after harvest and cleaning). This research provides a strong foundation for our understanding of HRSW resistance to ergot that will influence variety decisions in ergot-prone areas in North Dakota.

The C-8-S-isomers of ergot alkaloids - a review of biological and analytical aspects.

Ergot alkaloids are secondary metabolites that are produced by fungi and contaminate cereal crops and grasses. The ergot alkaloids produced by Claviceps purpurea are the most abundant worldwide. The metabolites exist in two configurations, the C-8-R-isomer (R-epimer) and the C-8-S-isomer (S-epimer). These two configurations can interconvert to one another. Ergot alkaloids cause toxic effects after consumption of ergot-contaminated food and feed at various concentrations. For bioactivity reasons, the C-8-R-isomers have been studied to a greater extent than the C-8-S-isomer since the C-8-S-isomers were considered biologically inactive. However, recent studies suggest the contrary. Analytical assessment of ergot alkaloids now includes the C-8-S-isomers and high concentrations of specific C-8-S-isomers have been identified. The inclusion of the C-8-S-isomer in regulatory standards is reviewed. This review has identified that further research into the C-8-S-isomers of ergot alkaloids is warranted. In addition, the inclusion of the C-8-S-isomers into regulatory recommendations worldwide for food and feed should be implemented. The objectives of this review are to provide an overview of historic and current studies that have assessed the C-8-S-isomers. Specifically, this review will compare the C-8-R-isomers to the C-8-S-isomers with an emphasis on the biological activity and analytical assessment.

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.

Effect of Field-applied Fungicides on Claviceps purpurea Sclerotia and Associated Toxins in Wheat.

Claviceps purpurea (Fr.) Tul is the causal organism for ergot impacting grass hosts, including wheat. The pathogen produces ergot alkaloids (EAs) during the development of mature sclerotia leading to potential wheat quality discounts or rejection at the point of sale. Cultural practices are recommended for the management of ergot in wheat, but there is limited information pertaining to the use of in-season fungicides to help reduce ergot. The objective of this research was to evaluate the efficacy of four fungicides (prothioconazole + metconazole, pydiflumetofen + propiconazole, azoxystrobin + propiconazole, and fluxapyroxad + pyraclostrobin) on sclerotia characteristics, and EAs associated with C. purpurea. A field experiment was established using a male-sterile hard red spring line with fungicide applications occurring at complete full head emergence (Feekes Growth Stage 10.5). Individual plots were harvested and cleaned, and ergot sclerotia were collected. Physical characteristics and toxin production were examined. Fungicides had a significant (p < .05) impact on total ergot body weight (EBW), with all fungicides having lower EBW than the nontreated control. The fungicide premixture of pydiflumetofen + propiconazole had the lowest EBW among all treatments. Fluxapyroxad + pyraclostrobin had the lowest levels of EAs among fungicides. Results suggest that fungicide premixtures can potentially reduce EBW and influence EA production in wheat.

Ergot alkaloids in sclerotia collected in Japan: synthetic profiles and induction of apoptosis by Clavine-type compounds.

The genus Claviceps (Clavicipitaceae) is famous for producing ergot alkaloids (EAs) in sclerotia. EAs can cause ergotism, resulting in convulsions and necrosis when ingested, making these compounds a serious concern for food safety. Agroclavine (2), a typical Clavine-type EA, is a causative agent of ergotism and is listed as a compound to be monitored by the European Food Safety Authority. Clavine-type EAs are known to cause cytotoxicity, but the mechanism has not been elucidated. We performed annexin V and PI double-staining followed by flow cytometric analysis to detect apoptosis in HepG2 and PANC-1 cells after exposure to Clavine-type EAs. Clavine-type EAs reduced cell viability and induced apoptosis in both cell lines. We then performed LC-MS analysis of EAs from 41 sclerotia samples of Claviceps collected in Japan. 24 out of 41 sclerotia extracts include peptide-type EAs (ergosine/inine: 4/4', ergotamine: 5, ergocornine/inine: 6/6', α-ergocryptine/inine: 8/8', and ergocristine/inine: 9/9') and 19 sclerotia extracts among 24 sclerotia detected peptide type EAs include Clavine-type EAs (pyroclavine: 1, agroclavine: 2, festuclavine: 3) by LC-MS. We then performed a metabolomic analysis of the EAs in the sclerotia using principal component analysis (PCA). The PCA score plots calculated for EAs suggested the existence of four groups with different EA production patterns. One of the groups was formed by the contribution of Clavine-type EAs. These results suggest that Clavine-type EAs are a family of compounds requiring attention in food safety and livestock production in Japan.

Wheat ergot fungus-derived and modified drug for inhibition of intracranial aneurysm rupture due to dysfunction of TLR-4 receptor in Alzheimer's disease.

BACKGROUND: Alzheimer's disease (AD) is a form of dementia that strikes elderly people more frequently than it does younger people. The cognitive skills and memory of Alzheimer's sufferers continue to deteriorate over time. Recent studies have shown that patients with AD have greater amounts of inflammatory markers in their bodies, which suggests that inflammation occurs early on in the progression of the disease. There is a possibility that Aß oligomers and fibrils can be recognised by TLRs, in addition to the microglial receptors CD14, CD36, and CD47. When Aß binds to either CD36 or TLR4, it sets off a chain reaction of inflammatory chemokines and cytokines that ultimately results in neurodegeneration. Diabetes and Alzheimer's disease have both been recently related to TLR4. The activation of TLR4 has been connected to a variety of clinical difficulties that are associated with diabetes, in addition to the internal environment of the body and the microenvironment of the brain. TLR4 inhibitors have been shown in clinical investigations to not only lessen the likelihood of getting sick but also to increase the average longevity. RESULT: In this work we used molecular docking and molecular dynamics modelling to investigate the effectiveness of FDA-approved antidiabetic plant derived drugs in combating the TLR4 receptor. Molecular docking experiments were used to make a prediction regarding the most important interactions involving 2-Bromoergocryptine Mesylate. With a binding affinity of -8.26 kcal/mol, it stood out from the other candidates as the one with the greatest potential. To verify the interaction pattern that takes place between 2-Bromoergocryptine Mesylate and the TLR4 receptor, a molecular dynamic simulation was run at a time scale of 150 nanoseconds. Because of this, 2-Bromoergocryptine Mesylate was able to make substantial contact with the active site, which led to increased structural stability during the process of the complex's dynamic development. CONCLUSION: As a result of this, the results of our research may be relevant for future research into the efficacy of 2-bromoergocryptine mesylate as a potential lead treatment for TLR4 receptors in intracranial aneurysm rupture in AD.

Dopamine agonists for the treatment of pituitary tumours: From ergot extracts to next generation therapies.

Dopamine agonists are a key tool in the therapeutic arsenal of endocrinologists worldwide. They exert their effects by binding to dopamine-2 (D2) receptors expressed by pituitary tumour cells to modulate hormonal secretion and tumour size. They are the established first-line treatment for prolactinomas which express high levels of D2 receptors. Growing data support their use as an adjuvant treatment option for other pituitary tumours including growth hormone, adrenocorticotrophic hormones, thyroid hormone secreting adenomas and nonfunctional pituitary tumours, all of which have been shown to express D2 receptors as well, albeit to varying extents. For those pituitary tumours inadequately treated by dopamine agonist alone, combined agonism of D2 and somatostatin receptors represent a new frontier in clinical development. Here we review the development and role of dopamine agonist for the treatment of prolactinomas, the literature supporting their adjuvant use for the treatment of all other pituitary tumours, and recent progress in the development of the next generation of chimeric compounds that target D2 and other receptor subtypes highly expressed on pituitary tumour cells.

Pre-invasion assessment on African invasive grasses revealed five new species of ergot fungi, Claviceps section Pusillae.

Ergot, the genus Claviceps comprises several deeply diverged lineages, recently classified as sections. Among them, the section Pusillae, is the most speciose, with a centre of distribution in Africa but occurring worldwide, often as a consequence of its invasive potential. This section includes the most severe plant pathogens such as Claviceps africana and C. gigantea, responsible for toxicoses and a significant reduction in the seed yields of Sorghum and Zea. In this study we surveyed ergot diversity in South Africa, focusing on grasses native to this region, but known for their high potential of invasiveness. The revision based on molecular and phenotypic markers revealed 16 species, with a high proportion of undescribed diversity, confirming Africa as a hot spot for this section. Five new species, Claviceps tulasnei, Claviceps eulaliae, Claviceps hypertheliae, Claviceps fredericksoniae and Claviceps arundinellae were described from Setaria, Eulalia, Hyperthelia, Miscanthus and Arundinella respectively. Claviceps texensis infecting Cenchrus, previously only identified from the same host in Texas, USA, was confirmed to be present in Africa, which is assumed to be its primary area of distribution. In addition, the host grass genus Anthephora is newly reported as a host of Claviceps digitariae. The most of the taxa were negligible concerning alkaloid production, with the exception of C. fredericksoniae, which is a sister of potent alkaloid producer C. africana, and produces mainly DH-ergosine, together with traces of DH-ergocornine. The host/parasite associations within Pusillae section is very narrow, suggesting that co-speciation is the major speciation driver in this group. Host grasses of the described species are already recognised invasive species and their ovarial parasites need to be monitored. This is highlighted by the fact that all Pusillae produced air-borne secondary conidia, which is autapomorphy of this section and considered to be important for their invasive abilities.

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.

Effects of Heating, Pelleting, and Feed Matrix on Apparent Concentrations of Cereal Ergot Alkaloids in Relation to Growth Performance and Welfare Parameters of Backgrounding Beef Steers.

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

Ammonization of the R- and S-Epimers of Ergot Alkaloids to Assess Detoxification Potential.

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

A large accessory genome and high recombination rates may influence global distribution and broad host range of the fungal plant pathogen Claviceps purpurea.

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

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.

The Role of the Wheat Reduced height (Rht)-DELLA Mutants and Associated Hormones in Infection by Claviceps purpurea, the Causal Agent of Ergot.

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

Evolution of the Ergot Alkaloid Biosynthetic Gene Cluster Results in Divergent Mycotoxin Profiles in Claviceps purpurea Sclerotia.

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

Ergotism and Saint Anthony's fire.

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

Mining Indole Alkaloid Synthesis Gene Clusters from Genomes of 53 Claviceps Strains Revealed Redundant Gene Copies and an Approximate Evolutionary Hourglass Model.

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

Genome-wide analysis of Claviceps paspali: insights into the secretome of the main species causing ergot disease in Paspalum spp.

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

Insights into Ergochromes of the Plant Pathogen Claviceps purpurea.

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