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.

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.

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.

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.

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.

Reprogramming of the wheat transcriptome in response to infection with Claviceps purpurea, the causal agent of ergot.

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 By 5 days (D) the ovule had been replaced by fungal tissue.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

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.

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.

Gravimetric quantitative validation of botanic impurities in feed.

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

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.

Genetic and transcriptional dissection of resistance to Claviceps purpurea in the durum wheat cultivar Greenshank.

KEY MESSAGE: Four QTL for ergot resistance (causal pathogen Claviceps purpurea) have been identified in the durum wheat cultivar Greenshank. Claviceps purpurea is a pathogen of grasses that infects flowers, replacing the seed with an ergot sclerotium. Ergot presents a significant problem to rye, barley and wheat, in particular hybrid seed production systems. In addition, there is evidence that the highly toxic alkaloids that accumulate within sclerotia can cross-contaminate otherwise healthy grain. Host resistance to C. purpurea is rare, few resistance loci having been identified. In this study, four ergot resistance loci are located on chromosomes 1B, 2A, 5A and 5B in the durum wheat cv. Greenshank. Ergot resistance was assessed through analysis of phenotypes associated with C. purpurea infection, namely the number of inoculated flowers that produced sclerotia, or resulted in ovary death but no sclerotia, the levels of honeydew produced, total sclerotia weight and average sclerotia weight and size per spike. Ergot testing was undertaken in Canada and the UK. A major effect QTL, QCp.aafc.DH-2A, was detected in both the Canadian and UK experiments and had a significant effect on honeydew production levels. QCp.aafc.DH-5B had the biggest influence on total sclerotia weight per spike. QCp.aafc.DH-1B was only detected in the Canadian experiments and QCp.aafc.DH-5A in the UK experiment. An RNASeq analysis, undertaken to identify wheat differentially expressed genes associated with different combinations of the four ergot resistance QTL, revealed a disproportionate number of DEGs locating to the QCp.aafc.DH-1B, QCp.aafc.DH-2A and QCp.aafc.DH-5B QTL intervals.

Population Genetic Structure of Claviceps purpurea in Cool-Season Grass Seed Crops of Oregon.

Ergot, caused by Claviceps purpurea, is a primary disease concern in irrigated cool-season grass seed production systems of Oregon. In order to better understand the genetic diversity, population structure, and the epidemiology of C. purpurea in grasses grown for seed, 226 isolates were obtained using a hierarchical sampling strategy from two fields each of Kentucky bluegrass (n = 102) and perennial ryegrass (n = 124) and characterized using 12 microsatellite markers. A total of 194 unique multilocus genotypes (MLGs) were identified in this study. There were moderate levels of genotypic diversity (H = 3.43 to 4.23) and gene diversity (Hexp = 0.45 to 0.57) within fields. After clone correction, analysis of molecular variance revealed that 66% of the genetic variation occurred between the two C. purpurea isolates collected from the same seed head of individual plants, indicating that many of the seed heads bearing multiple sclerotia were infected by ascospores rather than conidia. However, the majority of the clonal isolates obtained in this study were collected from the same seed head (i.e., the two isolates were identical MLGs), indicating a role of conidia (honeydew) in secondary infections within seed heads. Genetic differentiation was observed between populations from different hosts (22%) but was confounded by geography. The standardized index of association ranged from 0.007 to 0.122 among the four populations, suggesting potential outcrossing and differences in the relative contribution of ascospores and conidia to ergot among the fields. The results from this study provide insights into the epidemiology of ergot in cool-season grass seed crops of Oregon.

Crystallographic and NMR Investigation of Ergometrine and Methylergometrine, Two Alkaloids from Claviceps purpurea.

Ergometrine and methylergometrine are two alkaloids that are used as maleate salts for the prevention and control of postpartum hemorrhage. Although the two molecules have been known for a long time, few and discordant crystallographic and NMR spectroscopic data are available in the literature. With the aim of providing more conclusive data, we performed a careful NMR study for the complete assignment of the 1H, 13C, and 15N NMR signals of ergometrine, methylergometrine, and their maleate salts. This information allowed for a better definition of their conformational equilibria. In addition, the stereochemistry and the intermolecular interactions in the solid state of the two maleate salts were deeply investigated by means of single-crystal X-ray diffraction, showing the capability of these derivatives to act as both hydrogen-bond donors and acceptors, and evidencing a correlation between the number of intermolecular interactions and their different solubility.

Identification of a crucial amino acid implicated in the hydroxylation/desaturation ratio of CpFAH12 bifunctional hydroxylase.

Claviceps purpurea bifunctional Δ12-hydroxylase/desaturase, CpFAH12, and monofunctional desaturase CpFAD2, share 86% of sequence identity. To identify the underlying determinants of the hydroxylation/desaturation specificity, chimeras of these two enzymes were tested for their fatty acid production in an engineered Yarrowia lipolytica strain. It reveals that transmembrane helices are not involved in the hydroxylation/desaturation specificity whereas all cytosolic domains have an impact on it. Especially, replacing the CpFAH12 cytosolic part near the second histidine-box by the corresponding CpFAD2 part annihilates all hydroxylation activity. Further mutagenesis experiments within this domain identified isoleucine 198 as the crucial element for the hydroxylation activity of CpFAH12. Monofunctional variants performing the only desaturation were obtained when this position was exchanged by the threonine of CpFAD2. Saturation mutagenesis at this position showed modulation in the hydroxylation/desaturation specificity in the different variants. The WT enzyme was demonstrated as the most efficient for ricinoleic acid production and some variants showed a better desaturation activity. A model based on the recently discovered membrane desaturase structures indicate that these changes in specificity are more likely due to modifications in the di-iron center geometry rather than changes in the substrate binding mode.

Epigenetic modification enhances ergot alkaloid production of Claviceps purpurea.

OBJECTIVE: To enhance ergot alkaloid production of Claviceps purpurea Cp-1 strain by epigenetic modification approach. RESULTS: The chemical epigenetic modifiers were screened to promote ergot alkaloid production of the Cp-1 strain. The histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA) was found to significantly enhance the alkaloid productivity of the strain. Particularly, the titers of total ergot alkaloids were gradually increased with the increase of SAHA concentration in the fermentation medium, and the highest production of ergot alkaloids could be achieved at the concentration of 500 µM SAHA. Specially, the titers of ergometrine and total ergot alkaloids were as high as 95.4 mg/L and 179.7 mg/L, respectively, which were twice of those of the control. Furthermore, the mRNA expression levels of the most functional genes in the ergot alkaloid synthesis (EAS) gene cluster were up-regulated under SAHA treatment. It was proposed that SAHA might increase histone acetylation in the EAS gene cluster region in the chromosome, which would loosen the chromosome structure, and subsequently up-regulate the mRNA expression levels of genes involved in the biosynthesis of ergot alkaloids, thereby resulting in the markedly increase in the production of ergot alkaloids. CONCLUSIONS: The ergot alkaloid production by the C. purpurea Cp-1 strain can be effectively increased by the application of histone deacetylase inhibitor. Our work provides a reference for using the chemical epigenetic modifiers to improve SM production in other fungi.

Manipulation of cytokinin level in the ergot fungus Claviceps purpurea emphasizes its contribution to virulence.

Pathogen-derived cytokinins (CKs) have been recognized as important virulence factor in several host-pathogen interactions and it was demonstrated multiple times that phytopathogenic fungi form CKs via the tRNA degradation pathway. In contrast to previous studies, the focus of this study is on the second step of CK formation and CK degradation to improve our understanding of the biosynthesis in fungi on the one hand, and to understand CK contribution to the infection process of Claviceps purpurea on the other hand. The ergot fungus Claviceps purpurea is a biotrophic phytopathogen with a broad host range including economically important crops causing harvest intoxication upon infection. Its infection process is restricted to unfertilized ovaries without causing macroscopic defense symptoms. Thus, sophisticated host manipulation strategies are implicated. The cytokinin (CK) plant hormones are known to regulate diverse plant cell processes, and several plant pathogens alter CK levels during infection. C. purpurea synthesizes CKs via two mechanisms, and fungus-derived CKs influence the host-pathogen interaction but not fungus itself. CK deficiency in fungi with impact on virulence has only been achieved to date by deletion of a tRNA-ipt gene that is also involved in a process of translation regulation. To obtain a better understanding of CK biosynthesis and CKs' contribution to the plant-fungus interaction, we applied multiple approaches to generate strains with altered or depleted CK content. The first approach is based on deletion of the two CK phosphoribohydrolase (LOG)-encoding genes, which are believed to be essential for the release of active CKs. Single and double deletion strains were able to produce all types of CKs. Apparently, log gene products are dispensable for the formation of CKs and so alternative activation pathways must be present. The CK biosynthesis pathway remains unaffected in the second approach, because it is based on heterologous overexpression of CK-degrading enzymes from maize (ZmCKX1). Zmckx1 overexpressing C. purpurea strains shows strong CKX activity and drastically reduced CK levels. The strains are impaired in virulence, which reinforces the assumption that fungal-derived CKs are crucial for full virulence. Taken together, this study comprises the first analysis of a log depletion mutant that proved the presence of alternative cytokinin activation pathways in fungi and showed that heterologous CKX expression is a suitable approach for CK level reduction.

Evolutionary history of ergot with a new infrageneric classification (Hypocreales: Clavicipitaceae: Claviceps).

The ergot, genus Claviceps, comprises approximately 60 species of specialised ovarial grass parasites famous for the production of food toxins and pharmaceutics. Although the ergot has been known for centuries, its evolution have not been resolved yet. Our approach combining multilocus phylogeny, molecular dating and the study of ecological, morphological and metabolic features shows that Claviceps originated in South America in the Palaeocene on a common ancestor of BEP (subfamilies Bambusoideae, Ehrhartoideae, Pooideae) and PACMAD (subfamilies Panicoideae, Aristidoideae, Chloridoideae, Micrairoideae, Arundinoideae, Danthonioideae) grasses. Four clades described here as sections diverged during the Paleocene and Eocene. Since Claviceps are parasitic fungi with a close relationship with their host plants, their evolution is influenced by interactions with the new hosts, either by the spread to a new continent or the radiation of the host plants. Three of the sections possess very narrow host ranges and biogeographical distributions and have relatively low toxicity. On the contrary, the section Claviceps, comprising the rye ergot, C. purpurea, is unique in all aspects. Fungi in this section of North American origin have spread all over the world and infect grasses in all subfamilies as well as sedges, and it is the only section synthesising toxic ergopeptines and secalonic acids. The evolutionary success of the Claviceps section members can be explained by high toxin presence, serving as feeding deterrents and playing a role in their protective mutualism with host plants. Closely related taxa Neoclaviceps monostipa and Cepsiclava phalaridis were combined into the genus Aciculosporium.

Inactivation of the indole-diterpene biosynthetic gene cluster of Claviceps paspali by Agrobacterium-mediated gene replacement.

The hypocrealean fungus Claviceps paspali is a parasite of wild grasses. This fungus is widely utilized in the pharmaceutical industry for the manufacture of ergot alkaloids, but also produces tremorgenic and neurotoxic indole-diterpene (IDT) secondary metabolites such as paspalitrems A and B. IDTs cause significant losses in agriculture and represent health hazards that threaten food security. Conversely, IDTs may also be utilized as lead compounds for pharmaceutical drug discovery. Current protoplast-mediated transformation protocols of C. paspali are inadequate as they suffer from inefficiencies in protoplast regeneration, a low frequency of DNA integration, and a low mitotic stability of the nascent transformants. We adapted and optimized Agrobacterium tumefaciens-mediated transformation (ATMT) for C. paspali and validated this method with the straightforward creation of a mutant strain of this fungus featuring a targeted replacement of key genes in the putative IDT biosynthetic gene cluster. Complete abrogation of IDT production in isolates of the mutant strain proved the predicted involvement of the target genes in the biosynthesis of IDTs. The mutant isolates continued to produce ergot alkaloids undisturbed, indicating that equivalent mutants generated in industrial ergot producers may have a better safety profile as they are devoid of IDT-type mycotoxins. Meanwhile, ATMT optimized for Claviceps spp. may open the door for the facile genetic engineering of these industrially and ecologically important organisms.

Detection and Quantification of Airborne Claviceps purpurea sensu lato Ascospores from Hirst-Type Spore Traps using Real-Time Quantitative PCR.

The U.S. Pacific Northwest states of Oregon and Washington are major producers of cool-season grass seed. Ergot, caused by fungi in the Claviceps purpurea sensu lato group, is an important seed replacement disease of grass worldwide. Microscopic methods that are currently used to quantify airborne Claviceps ascospores captured by spore traps are not currently rapid enough to allow for detecting and reporting of spore numbers in a timely manner, hindering growers from using this information to help manage ergot. We developed a SYBR Green real-time quantitative polymerase chain reaction (qPCR)-based assay for fast and efficient detection and quantification of C. purpurea sensu lato ascospores from Hirst-type spore traps. Species-specificity of the qPCR assay was confirmed against 41 C. purpurea sensu lato isolates collected from six hosts and six other Claviceps spp. Significant relationships were observed between cycle threshold (Ct) values and standard curves of serial dilutions of DNA ranging from 1 pg to 10 ng (R2 = -0.99; P = 0.0002) and DNA extracted from a conidial suspension representing 8 to 80,000 conidia (R2 = -0.99; P = 0.0004). Ct values from qPCR were significantly correlated with results from microscopic examination of spore trap samples from the field (r = -0.68; P < 0.0001) and the procedure was able to detect a single ascospore from spore trap tape samples. The qPCR procedure developed in this study provided a means for quantifying airborne Claviceps ascospores that was highly specific and useful over a wide range of spore densities, and could be performed in a matter of hours instead of days. The qPCR assay developed in this study could be part of an integrated pest management approach to help grass seed growers make risk-based fungicide application decisions for ergot management in grass grown for seed.