Effect of microelements on the biosynthesis of secondary metabolites by the fungusPenicillium citrinum thom VKM F-1079

Penicillium citrinum VKM F-1079 was found to produce clavine ergot alkaloids and citrinin, a secondaryO-heterocyclic metabolite. Citrinin was produced in the idiophase, whereas the production of ergot alkaloids paralleled fungal growth. The addition of manganese ions to the growth medium stimulated the biosynthesis of both citrinin and ergot alkaloids. Zinc ions stimulated only citrinin synthesis. The presence of these microelements in the growth medium influenced the proportion between the ergot alkaloids synthesized. Copper, manganese, and iron ions slightly affected fungal growth and alkaloid production. The effect of microelements on the main kinetic parameters of growth and alkaloid production was studied.     

Role of weed grasses in the etiology of ergot disease in wheat

Forty isolates of the ergot fungus Claviceps purpurea collected from nineteen gramineous host species were used to inoculate male-sterile wheat. The isolates segregated into highly and weakly infective groups. The marked pathogenicity, on wheat, of the fungal strains occurring on certain grass species has been correlated with distinctive patterns of alkaloids within the sclerotia. Analysis of the alkaloid content of 241 samples of naturally occurring ergot sclerotia from twenty gramineous host species has confirmed the existence of host restricted strains characterized also by their particular spectra of alkaloids. Similarity of the alkaloid spectra of ergot sclerotia from blackgrass (Alopecurus myosuroides) and wheat, ease of cross-infection from blackgrass to wheat and an association between blackgrass infestation and the occurrence of ergot sclerotia in surveyed wheat crops have confirmed the hypothesis that the presence of this early flowering weed grass increases the risk of high levels of ergot infection in wheat.     

Screening for Ergot Alkaloid Producers among Microscopic Fungi by Means of the Polymerase Chain Reaction

The potential of the polymerase chain reaction for the detection of ergot alkaloid producers among microscopic fungi of the generaPenicilliumand Clavicepswas evaluated. Twenty-three strains of various species of fungi with a previously studied capacity for alkaloid production were used. The internal fragment of the gene encoding 4-dimethylallyltryptophan synthase, the enzyme catalyzing the first step in the biosynthesis of ergot alkaloids, was amplified using degenerate primers. This approach revealed an about 1.2-kb specific DNA fragment in micromycetes synthesizing ergot alkaloids with complete tetracyclic ergoline system. Microorganisms that produce alkaloids with modified C or D ergoline rings, as well as -cyclopiazonic acid, did not yield the PCR fragment of the expected size. This fragment was also not found in fungi incapable of ergot alkaloid production.     

The ergot alkaloid gene cluster: Functional analyses and evolutionary aspects

Ergot alkaloids and their derivatives have been traditionally used as therapeutic agents in migraine, blood pressure regulation and help in childbirth and abortion. Their production in submerse culture is a long established biotechnological process. Ergot alkaloids are produced mainly by members of the genus Claviceps, with Claviceps purpurea as best investigated species concerning the biochemistry of ergot alkaloid synthesis (EAS). Genes encoding enzymes involved in EAS have been shown to be clustered; functional analyses of EAS cluster genes have allowed to assign specific functions to several gene products. Various Claviceps species differ with respect to their host specificity and their alkaloid content; comparison of the ergot alkaloid clusters in these species (and of clavine alkaloid clusters in other genera) yields interesting insights into the evolution of cluster structure. This review focuses on recently published and also yet unpublished data on the structure and evolution of the EAS gene cluster and on the function and regulation of cluster genes. These analyses have also significant biotechnological implications: the characterization of non-ribosomal peptide synthetases (NRPS) involved in the synthesis of the peptide moiety of ergopeptines opened interesting perspectives for the synthesis of ergot alkaloids; on the other hand, defined mutants could be generated producing interesting intermediates or only single peptide alkaloids (instead of the alkaloid mixtures usually produced by industrial strains).     

Procedure for isolating the endophyte from tall fescue and screening isolates for ergot alkaloids

A procedure was developed to isolate and determine ergot alkaloid production by Acremonium coenophialum, the endophytic fungus of tall fescue. The procedure established that macerated leaf sheath or pith from inflorescence stem placed either in a liquid medium or on a corn meal-malt extract agar medium produced isolated mycelium and characteristic conidia within a 3- to 3.5-week period. Once isolated, each fungus was placed in another liquid medium, M104T, where competent strains produced total ergot alkaloids ranging from 38 to 797 mg/liter. Several isolates were negative for ergot alkaloid synthesis. The production of ergot alkaloids by individual isolates was unstable; isolates rapidly degenerated in their ability to produce ergot alkaloids during subculture. However, the procedure as presented allows the assessment of an isolate for ergot alkaloid synthesis during its initial isolation.     

Procedure for isolating the endophyte from tall fescue and screening isolates for ergot alkaloids.

A procedure was developed to isolate and determine ergot alkaloid production by Acremonium coenophialum, the endophytic fungus of tall fescue. The procedure established that macerated leaf sheath or pith from inflorescence stem placed either in a liquid medium or on a corn meal-malt extract agar medium produced isolated mycelium and characteristic conidia within a 3- to 3.5-week period. Once isolated, each fungus was placed in another liquid medium, M104T, where competent strains produced total ergot alkaloids ranging from 38 to 797 mg/liter. Several isolates were negative for ergot alkaloid synthesis. The production of ergot alkaloids by individual isolates was unstable; isolates rapidly degenerated in their ability to produce ergot alkaloids during subculture. However, the procedure as presented allows the assessment of an isolate for ergot alkaloid synthesis during its initial isolation.     

New producers of biologically active compounds—fungal strains of the genus <Emphasis Type="Italic">Penicillium</Emphasis> isolated from permafrost

Screening of producers of secondary metabolites was carried out among 25 fungal strains of Penicillium genus isolated from permafrost in Arctic and Antarctic regions and Kamchatka. Nearly 50% of the investigated strains synthesize biologically active substances of alkaloid nature: ergot alkaloids, diketopiperazinees, and quinoline derivatives. A large group of the identified metabolites belongs to mycotoxins. A strain of Penicillium waksmanii was found producing epoxyagroclavine-I and quinocitrinines. The main physiological and biochemical characteristics of this producer were investigated.     

Contribution of ergot alkaloids to suppression of a grass-feeding caterpillar assessed with gene knockout endophytes in perennial ryegrass

Neotyphodium and Epichloë species (Ascomycota: Clavicipitaceae) are fungal symbionts (endophytes) of grasses. Many of these endophytes produce alkaloids that enhance their hosts’ resistance to insects or are toxic to grazing mammals. The goals of eliminating from forage grasses factors such as ergot alkaloids that are responsible for livestock disorders, while retaining pasture sustainability, and of developing resistant turf grasses, require better understanding of how particular alkaloids affect insect herbivores. We used perennial ryegrass Lolium perenne L. (Poaceae) symbiotic with Neotyphodium lolii × Epichloë typhina isolate Lp1 (a natural interspecific hybrid), as well as with genetically modified strains of Lp1 with altered ergot alkaloid profiles, to test effects of ergot alkaloids on feeding, growth, and survival of the black cutworm, Agrotis ipsilon (Hufnagel) (Lepidoptera: Noctuidae), a generalist grass‐feeding caterpillar. Neonates or late instars were provided clippings from glasshouse‐grown plants in choice and rearing trials. Wild‐type endophytic grass showed strong antixenosis and antibiosis, especially to neonates. Plant‐endophyte symbiota from which complex ergot alkaloids (ergovaline and lysergic acid amides such as ergine) or all ergot alkaloids were eliminated by endophyte gene knockout retained significant resistance against neonates. However, this activity was reduced compared to that of wild‐type Lp1, providing the first direct genetic evidence that ergot alkaloids contribute to insect resistance of endophytic grasses. Similarity of larval response to the two mutants suggested that ergovaline and/or ergine account for the somewhat greater potency of wild‐type Lp1 compared to the knockouts, whereas simpler ergot alkaloids contribute little to that added resistance. All of the endophyte strains also produced peramine, which was probably their primary resistance component. This study suggests that ergot alkaloids can be eliminated from an endophyte of perennial ryegrass while retaining significant insect resistance.     

An Ergot Alkaloid Biosynthesis Gene and Clustered Hypothetical Genes from Aspergillus fumigatus

The ergot alkaloids are a family of indole-derived mycotoxins with a variety of significant biological activities. Aspergillus fumigatus, a common airborne fungus and opportunistic human pathogen, and several fungi in the relatively distant taxon Clavicipitaceae (clavicipitaceous fungi) produce different sets of ergot alkaloids. The ergot alkaloids of these divergent fungi share a four-member ergoline ring but differ in the number, type, and position of the side chains. Several genes required for ergot alkaloid production are known in the clavicipitaceous fungi, and these genes are clustered in the genome of the ergot fungus Claviceps purpurea. We investigated whether the ergot alkaloids of A. fumigatus have a common biosynthetic and genetic origin with those of the clavicipitaceous fungi. A homolog of dmaW, the gene controlling the determinant step in the ergot alkaloid pathway of clavicipitaceous fungi, was identified in the A. fumigatus genome. Knockout of dmaW eliminated all known ergot alkaloids from A. fumigatus, and complementation of the mutation restored ergot alkaloid production. Clustered with dmaW in the A. fumigatus genome are sequences corresponding to five genes previously proposed to encode steps in the ergot alkaloid pathway of C. purpurea, as well as additional sequences whose deduced protein products are consistent with their involvement in the ergot alkaloid pathway. The corresponding genes have similarities in their nucleotide sequences, but the orientations and positions within the cluster of several of these genes differ. The data indicate that the ergot alkaloid biosynthetic capabilities in A. fumigatus and the clavicipitaceous fungi had a common origin.     

The influence of ergot-contaminated feed on growth and slaughtering performance,nutrient digestibility and carry over of ergot alkaloids in growing-finishing pigs

Abstract

Diets containing 0, 1 and 10 g ergot (Claviceps purpurea) per kg, corresponding to mean total alkaloid contents of 0.05, 0.60 and 4.66 mg/kg (sums of ergometrine, ergotamine, ergocornine, α-ergocryptine, ergocristine, ergosine and their -inine isomers analysed by a HPLC-method), were each fed ad libitum to 12 pigs in the BW range of 30–115 kg to study the effect of ergot-contaminated feed on growth and slaughtering performance and the carry over of ergot alkaloids. Additionally, balance trials were conducted to investigate the digestibility of nutrients. Tendencies towards reduced feed intake and BWG were observed at a feeding level of 4.66 mg total alkaloids per kg diet. Typical symptoms of ergot poisoning were not observed. Heart and spleen weights showed significant linear increases. Differences in carcass quality due to dietary treatment were not detected. No genuine ergot alkaloids were found in physiological samples. The balance trials demonstrated a significantly decreased protein digestibility for the most highly supplemented diet.     

Selection of Ergot Alkaloid Producers by Induced Mutagenesis

Using the induced mutagenesis technique, a series of genetically modified Clavicepssp. VKM F-2609 strains that display high levels of agroclavine and elymoclavine synthesis were selected by induced mutagenesis. Compared to the parent strain, c106 displayed a 40-fold higher level of agroclavine synthesis, and c66 displayed an eightfold higher level of elymoclavine synthesis. The levels of synthesis of other alkaloids were decreased in these strains. The effects of various carbohydrates on the strain growth and ergot alkaloid biosynthesis was then investigated in both the parent strain and c106. The largest amount of agroclavine was synthesized by c106 strain growing on a medium with maltose.     

Investigations into the occurrence of alkaloids in ergot and single sclerotia from the 2007 and 2008 harvests

As a contribution to the occurrence of ergot alkaloids in ergot from German rye and triticale, samples from the 2007 and 2008 harvests were analyzed. Twelve alkaloids—six pairs of main alkaloids and their corresponding epimers—were determined in extracts prepared under alkaline conditions by HPLC with fluorescence detection without preceding purification. The total alkaloid content was found to be 0.03–0.18% in ergot from rye (n = 19) and 0.06–0.22% in ergot from triticale (n = 4), respectively. Furthermore, single sclerotia (n = 40) were investigated in terms of alkaloid content and distributional pattern. The main alkaloids in ergot were ergocristine, ergotamine and ergocornine, although the alkaloid composition was highly variable. Presented in part at the 30th Mykotoxin-Workshop, Utrecht, The Netherlands, April 28–30, 2008     

Effect of Tween 80 on alkaloid-producing cultures ofClaviceps paspali

Addition of Tween 80 to submerged cultures ofClaviceps paspali (Stevens and Hall) growing in a simple defined medium increased biomass formation and caused a temporary change in alkaloid synthesis intensity. The Tween-supplemented culture reached maximal alkaloid yields four days earlier than the control. The shift of alkaloid production was associated with a shift of organic acids and amino acids in the cell-pool. Thus the maximal formation of alkaloids was characterized by a decrease in the amount of citric acid, by the disappearance of succinic, fumaric and oxaloacetic acids and by increased accumulation of methionine, cysteine, alanine and histidine. The slow alkaloid synthesis was accompanied by a relatively high content of citric and succinic acids in the cell-pool and by the absence of methionine. The data are consistent with the hypothesis that ergot alkaloids participate in the regulation of the metabolism of cultures.     

Production of ergot alkaloids by Aspergillus fumigatus (Fresenius)

Summary Nutritional requirements for the production of ergot alkaloids were studied with Aspergillus fumigatus under submerged conditions of fermentation, in a chemically defined medium. Glucose in combination with mannitol and triammonium citrate were found to be the best sources of carbon and nitrogen for the production of alkaloids. Carbon to nitrogen ratio of 4.16 : 1 was found optimum. Phosphate at elevated concentration inhibited alkaloid production.     

Secondary metabolites in taxonomy of the <Emphasis Type="Italic">Penicillium</Emphasis> fungi

A correlation was established between species specificity and synthesis of specific secondary metabolites by the Penicillium fungi. Strains of the subgenus Aspergilloides usually synthesize metabolites of polyketide nature. Most strains of the subgenus Furcatum produce clavine ergot alkaloids and metabolites of diketopiperazine nature. The only clavine ergot alkaloids and diketopiperazine alkaloids produced by strains of the subgenus Biverticillium are rugulovasines and rugulosuvines, respectively. Species designations of the strains of the subgenus Penicillium isolated from permafrost soil, the Mir orbital complex, and sites undergoing anthropogenic load were refined based on the marker secondary metabolites. Changes in the taxonomic position of some strains in the genus Penicillium are suggested.     

Studies on Claviceps purpurea (Fr.) Tul. parasitic on Phragmites communis Trin

Isolates from C. purpurea sclerotia occurring naturally on Phragmites communis usually sporulated vigorously on the culture medium employed, and their failure to produce alkaloid in vitro was associated with a thin white growth form. Such isolates also failed to produce sclerotia on the host plants tested. A variant having a plectenchymatic morphology in vitro and producing a thick pigmented non-sporulating growth form yielded alkaloid (up to approximately 300μg/ml mainly δ8–9 and δ9–10 lysergic acids and chanoclavine) in surface or submerged culture and developed typical ergot sclerotia (containing 0·2-0·4% alkaloid, mainly ergotoxine and ergotamine) in vivo. Improved alkaloid yield in vitro was obtained from a strain reselected after passing through a parasitic phase. Aetiological aspects of the P. communis ergot are discussed.     

Physiological status of submergedClaviceps during enzymic assembly of ergot alkaloids

Ergot alkaloids are formed only for arelatively brief time during the lifespan of the culture and under conditions of reduced proliferation. They cannot be taken for waste products of general metabolism. Ergot strains are capable of carrying out all the simple steps of the anthranilic acid—tryptophan cycles. Alkaloids influence activities of certain enzymes of primary metabolism in the ergot mycelium,e.g. tryptophan synthetase, acetyl-CoA carboxylase, citrate synthase, isocitrate lyase, and malate synthase. Ergot alkaloids do not belong to a group of physiologically inert secondary metabolites. A tentative scheme of the enzymic assembly of the ergoline nucleus is presented. The increased yield of ergoline alkaloids may be attributed to the following points: (1) Unbalnced growth of the culture. (2) Support of competition of fatty acids and alkaloid biosynthesis for acetyl-CoA. (3) Decreased activities of tricarboxylic acid and glyoxylate cycles. (4) Positive association between the rate of protein turnover and alkaloid formation. (5) Stimulation of both tryptophan synthesis and degradation via kynurenine—anthranilate. (6) Regulation of tryptophan-histidine cross-pathway. (7) Continuous control of the alkaloid level during fermentation.     

The Fungus Penicillium citrinum Thom 1910 VKM FW-800 Isolated from Ancient Permafrost Sediments As a Producer of the Ergot Alkaloids Agroclavine-1 and Epoxyagroclavine-1

The study of the secondary metabolites of the relict strain Penicillium citrinum VKM FW-800 isolated from ancient Arctic permafrost sediments showed that this fungus produces agroclavine-1 and epoxyagroclavine-1, which are rare ergot alkaloids with the 5R,10S configuration of the tetracyclic ergoline ring system. The production of the alkaloids by the fungus showed a biphasic behavior, being intense in the phase of active growth and slowing down in the adaptive lag phase and in the stationary growth phase. The addition of zinc ions to the incubation medium led to a fivefold increase in the yield of the alkaloids. The alkaloid-producing Penicillium fungi isolated from different regions exhibited the same tendencies of growth and alkaloid production.     

Evidence for an ergot alkaloid gene cluster in Claviceps purpurea

A gene (cpd1) coding for the dimethylallyltryptophan synthase (DMATS) that catalyzes the first specific step in the biosynthesis of ergot alkaloids, was cloned from a strain of Claviceps purpurea that produces alkaloids in axenic culture. The derived gene product (CPD1) shows only 70% similarity to the corresponding gene previously isolated from Claviceps strain ATCC 26245, which is likely to be an isolate of C. fusiformis. Therefore, the related cpd1 most probably represents the first C. purpurea gene coding for an enzymatic step of the alkaloid biosynthetic pathway to be cloned. Analysis of the 3′-flanking region of cpd1 revealed a second, closely linked ergot alkaloid biosynthetic gene named cpps1, which codes for a 356-kDa polypeptide showing significant similiarity to fungal modular peptide synthetases. The protein contains three amino acid-activating modules, and in the second module a sequence is found which matches that of an internal peptide (17 amino acids in length) obtained from a tryptic digest of lysergyl peptide synthetase 1 (LPS1) of C. purpurea, thus confirming that cpps1 encodes LPS1. LPS1 activates the three amino acids of the peptide portion of ergot peptide alkaloids during D-lysergyl peptide assembly. Chromosome walking revealed the presence of additional genes upstream of cpd1 which are probably also involved in ergot alkaloid biosynthesis: cpox1 probably codes for an FAD-dependent oxidoreductase (which could represent the chanoclavine cyclase), and a second putative oxido-reductase gene, cpox2, is closely linked to it in inverse orientation. RT-PCR experiments confirm that all four genes are expressed under conditions of peptide alkaloid biosynthesis. These results strongly suggest that at least some genes of ergot alkaloid biosynthesis in C. purpurea are clustered, opening the way for a detailed molecular genetic analysis of the pathway. Received: 26 August 1998 / Accepted: 19 October 1998     

Studies onClaviceps parasitic onPanicum species in India

Panicum repens andP. antidotale were found to be infected withClaviceps sp. This is the first report of ergot onP. repens. The pyrenomycete produced abundant sclerotia on the host plants. The sclerotia contained 0.71 and 0.68 % alkaloids, respectively, which predominantly consisted of chanoclavine, festuclavine and agroclavine. The infected grasses were possibly mycotoxic. Submerged cultures ofClaviceps strain isolated fromPanicum spp produced significant amount of chanoclavine, festuclavine and agroclavine. No pharmaceutically important alkaloid was found in sclerotia or in submerged culture.