The effect of some amino acids on the growth and biosynthesis of ergot alkaloids and quinocitrinins in the fungus Penicillium citrinum Thom 1910

The effect of some amino acids, added to the medium either during inoculation or in the stationary growth phase, on the growth and biosynthesis of ergot alkaloids and quinocitrinins in the fungus Penicillium citrinum VKM FW-800 has been studied. Exogenously added amino acids were mostly utilized in primary metabolism. When added during inoculation, tryptophan and leucine virtually did not influence fungal growth and synthesis of the alkaloids, whereas the addition of isoleucine enhanced the biomass accumulation. When added in the stationary growth phase, tryptophan stimulated the synthesis of both ergot alkaloids and quinocitrinins. Leucine added in the stationary growth phase did not influence the synthesis of ergot alkaloids but inhibited the synthesis of quinocitrinins. Isoleucine inhibited the synthesis of both ergot alkaloids and quinocitrinins irrespective of the time of its addition to the medium.     

The effect of some amino acids on the growth and biosynthesis of ergot alkaloids and quinocitrinins in the fungus Penicillium citrinum Thom 1910

The effect of some amino acids, added to the medium either during inoculation or in the stationary growth phase, on the growth and biosynthesis of ergot alkaloids and quinocitrinins in the fungus Penicillium citrinum VKM FW-800 has been studied. Exogenously added amino acids were mostly utilized in primary metabolism. When added during inoculation, tryptophan and leucine virtually did not influence fungal growth and synthesis of the alkaloids, whereas the addition of isoleucine enhanced the biomass accumulation. When added in the stationary growth phase, tryptophan stimulated the synthesis of both ergot alkaloids and quinocitrinins. Leucine added in the stationary growth phase did not influence the synthesis of ergot alkaloids but inhibited the synthesis of quinocitrinins. Isoleucine inhibited the synthesis of both ergot alkaloids and quinocitrinins irrespective of the time of its addition to the medium.     

Effect on microelements on biosynthes of secondary metabolites in the fungus Penicillium citrinum Thom VKM F-1079

Penicillium citrinum VKM F-1079 was found to produce clavine ergot alkaloids and citrinin, a secondary O-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 affected but little fungal growth and alkaloid production. The effect of microelements on the main kinetic parameters of growth and alkaloid production was studied.     

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.     

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.     

Transport of ergot alkaloids and quinocitrinins in the producing fungus Penicillium citrinum

The decrease in the concentration of alkaloids in the culture liquid of Penicillium citrinum grown to the early stationary phase was found to be due to the uptake of quinocitrinins and ergot alkaloids by fungal cells. The ability of the fungal mycelium to absorb autogenous quinocitrinins does not depend on the mycelium age, whereas its ability to absorb ergot alkaloids is higher in the young than in the 12-day-old mycelium. The uptake of exogenously added ergot alkaloids by the fungal mycelium is accompanied by excretion of intracellular quinocitrinins. The addition of quinocitrinins to the medium was found to exert different effects in different growth stages. Namely, the uptake of exogenously added quinocitrinins by the actively growing young mycelium inhibits the excretion of ergot alkaloids, but the excretion of ergot alkaloids by the 12- day-old mycelium occurs throughout the cultivation period. The excretion of both ergot alkaloids and quinocitrinins does not require energy.     

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.     

Transport of ergot alkaloids and quinocitrinins in the producing fungus <Emphasis Type="Italic">Penicillium citrinum</Emphasis>

The decrease in the concentration of alkaloids in the culture liquid of Penicillium citrinum grown to the early stationary phase was found to be due to the uptake of quinocitrinins and ergot alkaloids by fungal cells. The ability of the fungal mycelium to take up autogenous quinocitrinins does not depend on the mycelium age, whereas its ability to take up ergot alkaloids is higher in the young than in the 12-day-old mycelium. The uptake of exogenously added ergot alkaloids by the fungal mycelium is accompanied by excretion of intracellular quinocitrinins. The addition of quinocitrinins to the medium was found to exert different effects in different growth stages. Namely, the uptake of exogenously added quinocitrinins by the actively growing young mycelium inhibits the excretion of ergot alkaloids, but the excretion of ergot alkaloids by the 12-day-old mycelium occurs throughout the cultivation period. The excretion of both ergot alkaloids and quinocitrinins does not require energy.     

Effects of different levels of ergot in concentrate on the health and performance of male calves

A number of studies dealing with the effects of ergot and ergot alkaloids on the health and performance of poultry and pigs were reported in the past, but only a few studies and field reports are available for ruminants. Therefore, a dose-response study was carried out with calves since young animals are considered to be especially sensitive to ergot. A total of 35 male Holstein calves were randomly assigned to three feeding groups after one month of feeding milk replacer. The mean initial live weight of the calves was 49.4±5.7 kg. One control group was fed an ergot-free concentrate (n=12), one group an ergot proportion of 1000 mg/kg in the concentrate (n=ll), and another group was fed a concentrate containing 5000 mg/kg ergot (n=12). Hay, grass silage and water were available forad libitum consumption, whereas the daily concentrate portion was restricted to 2 kg. Live weight, health parameters and feed intake were monitored over the experimental period of 84 days. In addition, blood samples were taken from theVena jugularis at the beginning and at the end of the experiment and analysed for ergot alkaloids and liver parameters. Total dry matter intake, live weight gain and feed-to-gain ratio were not significantly influenced by increasing ergot proportions when the whole experimental period was considered, although there was a trend for an ergot-related decrease in concentrate intake during the first 6 weeks of the experiment. After this period of time, it seemed that calves got used to the presence of ergot in the concentrate and were able to adjust their intake to the level of the control group. Moreover, health and liver parameters, such as total bilirubin, aspartate aminotransferase, glutamate dehydrogenase, gamma-glutamyl transpeptidase and creatine kinase in the serum were not significantly influenced by dietary treatments. Concentrations of the individual ergot alkaloids in serum were lower than the detection limits of the applied HPLC-method. In conclusion, it can be assumed that an ergot contamination of the concentrate up to 5000 mg/kg resulted in a transient depression of concentrate intake by the calves. However, no significant effects on health and performance could be detected when the entire test period of 84 days was considered. Presented at the 28^th Mykotoxin-Workshop, Bydgoszcz, Poland, May 29–31, 2006     

Association of ergot alkaloids with conidiation in Aspergillus fumigatus

Ergot alkaloids are mycotoxins that affect the nervous and reproductive systems of exposed individuals through interactions with monoamine receptors. They have been studied more widely in ergot fungi and grass endophytes but also are found in Aspergillus fumigatus, an opportunistic human pathogen that reproduces and disseminates exclusively through conidia. The ergot alkaloids festucla-vine and fumigaclavines A, B and C are present in or on conidia of A. fumigatus. Cultures of the fungus that are free of conidia are difficult to obtain, obscuring comparisons of conidia versus vegetative hyphae as sources of the ergot alkaloids. To create conidiation-deficient strains of A. fumigatus we manipulated the bristle A gene (brlA), which controls vesicle formation or budding growth necessary for conidiation in Aspergillus spp. Disruption of brlA in A. fumigatus, via homologous recombination, resulted in a nonconidiating mutant that produced bristle-like structures instead of conidiophores and conidia. Moreover the disrupted strain failed to produce ergot alkaloids as verified by HPLC analyses. Complementation with a wild-type allele restored conidiation and ergot alkaloid production. These results suggest that ergot alkaloids are not produced within the vegetative mycelium of the fungus and are associated directly with conidiation.     

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.     

Comparative studies on the effect of ergot contaminated feed on performance and health of piglets and chickens

Two dose response trials were conducted with piglets and chickens to study the effects of increasing amounts of ergot (Claviceps purpurea) with a defined alkaloid content and pattern on performance, biochemical serum characteristics and organ weights (of chickens). The ergot was mixed into the cereal-soybean meal based diets at levels of 0, 0.5, 1, 2 and 4 g/kg. The total alkaloid content of the ergot was analysed to be 2775 mg/kg and showed the following composition: ergometrine 8.1%, ergotamine 5.4%, ergocomine 3.2%, alpha-ergocryptine 1.9%, ergocristine 14.9% and residue 66.5%. Each treatment was tested with eight castrated male and eight female piglets over a period of 35 days (8 kg initial live weight) and 28 male chickens for 21 days (43 g initial live weight). Cumulative daily dry matter intake and live weight gain [g/d] were 595, 535, 560, 577 and 490 and 413, 399, 420, 443 and 347 for the piglets fed the unsupplemented control diet and the diets containing 0.5, 1, 2 and 4 g ergot per kg, respectively. Feed intake and live weight gain of the piglets fed the highest ergot supplemented diet were significantly decreased. Serum aspartate aminotransferase activity of the 4 g ergot treatment was significantly increased. Also serum albumin concentrations showed significant linear alterations. Serum activities of glutamate dehydrogenase, gamma-glutamyltransferase, total protein and porcine growth hormone were not significantly influenced by dietary treatment. The experiment with chickens demonstrated no significant effects on performance due to dietary ergot exposure. The serum activities of glutamate dehydrogenase and alanine aminotransferase were not significantly influenced by dietary treatment while serum activities of gamma-glutamyltransferase and aspartate aminotransferase and the concentrations of albumin and total bilirubin were significantly affected. Heart weights showed a significant linear decrease due to ergot feeding. According to these results, piglets seemed to react more sensitively on the occurrence of ergot in the diet as compared to chickens. The critical level of total ergot alkaloids for piglets seemed to be in the range from 5.6 mg to 11.1 mg/kg diet for the present study. Ergot effects on signs of inflammation in the proximal duodenum occurred in chickens fed diets containing 2.8 mg and 11.1 mg total ergot alkaloids/kg although live performance remained unaffected. Further studies are necessary to define the critical level of ergot alkaloids in dependence on alkaloid pattern.     

Comparative studies on the effect of ergot contaminated feed on performance and health of piglets and chickens

Two dose response trials were conducted with piglets and chickens to study the effects of increasing amounts of ergot (Claviceps purpurea) with a defined alkaloid content and pattern on performance, biochemical serum characteristics and organ weights (of chickens). The ergot was mixed into the cereal-soybean meal based diets at levels of 0, 0.5, 1, 2 and 4?g/kg. The total alkaloid content of the ergot was analysed to be 2775?mg/kg and showed the following composition: ergometrine 8.1%, ergotamine 5.4%, ergocornine 3.2%, α-ergocryptine 1.9%, ergocristine 14.9% and residue 66.5%. Each treatment was tested with eight castrated male and eight female piglets over a period of 35 days (8?kg initial live weight) and 28 male chickens for 21 days (43?g initial live weight). Cumulative daily dry matter intake and live weight gain [g/d] were 595, 535, 560, 577 and 490 and 413, 399, 420, 443 and 347 for the piglets fed the unsupplemented control diet and the diets containing 0.5, 1, 2 and 4?g ergot per kg, respectively. Feed intake and live weight gain of the piglets fed the highest ergot supplemented diet were significantly decreased. Serum aspartate aminotransferase activity of the 4?g ergot treatment was significantly increased. Also serum albumin concentrations showed significant linear alterations. Serum activities of glutamate dehydrogenase, γ-glutamyltransferase, total protein and porcine growth hormone were not significantly influenced by dietary treatment. The experiment with chickens demonstrated no significant effects on performance due to dietary ergot exposure. The serum activities of glutamate dehydrogenase and alanine aminotransferase were not significantly influenced by dietary treatment while serum activities of γ-glutamyltransferase and aspartate aminotransferase and the concentrations of albumin and total bilirubin were significantly affected. Heart weights showed a significant linear decrease due to ergot feeding. According to these results, piglets seemed to react more sensitively on the occurrence of ergot in the diet as compared to chickens. The critical level of total ergot alkaloids for piglets seemed to be in the range from 5.6?mg to 11.1?mg/kg diet for the present study. Ergot effects on signs of inflammation in the proximal duodenum occurred in chickens fed diets containing 2.8?mg and 11.1?mg total ergot alkaloids/kg although live performance remained unaffected. Further studies are necessary to define the critical level of ergot alkaloids in dependence on alkaloid pattern.     

Ergot: from witchcraft to biotechnology

The ergot diseases of grasses, caused by members of the genus Claviceps , have had a severe impact on human history and agriculture, causing devastating epidemics. However, ergot alkaloids, the toxic components of Claviceps sclerotia, have been used intensively (and misused) as pharmaceutical drugs, and efficient biotechnological processes have been developed for their in vitro production. Molecular genetics has provided detailed insight into the genetic basis of ergot alkaloid biosynthesis and opened up perspectives for the design of new alkaloids and the improvement of production strains; it has also revealed the refined infection strategy of this biotrophic pathogen, opening up the way for better control. Nevertheless, Claviceps remains an important pathogen worldwide, and a source for potential new drugs for central nervous system diseases.     

Abundant respirable ergot alkaloids from the common airborne fungus Aspergillus fumigatus

Ergot alkaloids are mycotoxins that interact with several monoamine receptors, negatively affecting cardiovascular, nervous, reproductive, and immune systems of exposed humans and animals. Aspergillus fumigatus, a common airborne fungus and opportunistic human pathogen, can produce ergot alkaloids in broth culture. The objectives of this study were to determine if A. fumigatus accumulates ergot alkaloids in a respirable form in or on its conidia, to quantify ergot alkaloids associated with conidia produced on several different substrates, and to measure relevant physical properties of the conidia. We found at least four ergot alkaloids, fumigaclavine C, festuclavine, fumigaclavine A, and fumigaclavine B (in order of abundance), associated with conidia of A. fumigatus. Under environmentally relevant conditions, the total mass of ergot alkaloids often constituted >1% of the mass of the conidium. Ergot alkaloids were extracted from conidia produced on all media tested, and the greatest quantities were observed when the fungus was cultured on latex paint or cultured maize seedlings. The values for physical properties of conidia likely to affect their respirability (i.e., diameter, mass, and specific gravity) were significantly lower for A. fumigatus than for Aspergillus nidulans, Aspergillus niger, and Stachybotrys chartarum. The demonstration of relatively high concentrations of ergot alkaloids associated with conidia of A. fumigatus presents opportunities for investigations of potential contributions of the toxins to adverse health effects associated with the fungus and to aspects of the biology of the fungus that contribute to its success.     

The Fungus <Emphasis Type="Italic">Penicillium citrinum</Emphasis>, Isolated from Permafrost Sediments,as a Producer of Ergot Alkaloids and New Quinoline Alkaloids Quinocitrinines

Quinocitrinines and ergot alkaloids were synthesized by the strain Penicillium citrinum VKM FW-800 as the culture grews. The major part of these secondary metabolites was secreted into the medium. In the phase of growth deceleration, these metabolites were partly absorbed by the producer cells. Zinc ions stimulated both the primary and secondary metabolic processes. Addition of this microelement into the culture medium stimulated biomass accumulation and the synthesis of clavine alkaloids and quinocitrinines.     

Derivation of the multiply-branched ergot alkaloid pathway of fungi

Ergot alkaloids are a large family of fungal specialized metabolites that are important as toxins in agriculture and as the foundation of powerful pharmaceuticals. Fungi from several lineages and diverse ecological niches produce ergot alkaloids from at least one of several branches of the ergot alkaloid pathway. The biochemical and genetic bases for the different branches have been established and are summarized briefly herein. Several pathway branches overlap among fungal lineages and ecological niches, indicating activities of ergot alkaloids benefit fungi in different environments and conditions. Understanding the functions of the multiple genes in each branch of the pathway allows researchers to parse the abundant genomic sequence data available in public databases in order to assess the ergot alkaloid biosynthesis capacity of previously unexplored fungi. Moreover, the characterization of the genes involved in the various branches provides opportunities and resources for the biotechnological manipulation of ergot alkaloids for experimentation and pharmaceutical development.     

Selection of ergot alkaloid producers by induced mutagenesis

Using the induced mutagenesis technique, A series of genetically modified Claviceps sp. 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.     

Fungus Penicillium citrinum, isolated from permafrost sediments, as a producer of ergot alkaloids and new quinoline alkaloids quinocitrinines

Quinocitrinines and ergot alkaloids are synthesized by the strain Penicillium citrinum VKM FW-800 as the culture grows. The major part of these secondary metabolites are secreted into the medium. In the phase of growth deceleration, these metabolites were partly absorbed by the producer cells. Zinc ions stimulated both the primary and secondary metabolic processes. Addition of this microelement into the culture medium stimulated biomass accumulation and the synthesis of clavine alkaloids and quinocitrinines.     

Abundant Respirable Ergot Alkaloids from the Common Airborne Fungus Aspergillus fumigatus

Ergot alkaloids are mycotoxins that interact with several monoamine receptors, negatively affecting cardiovascular, nervous, reproductive, and immune systems of exposed humans and animals. Aspergillus fumigatus, a common airborne fungus and opportunistic human pathogen, can produce ergot alkaloids in broth culture. The objectives of this study were to determine if A. fumigatus accumulates ergot alkaloids in a respirable form in or on its conidia, to quantify ergot alkaloids associated with conidia produced on several different substrates, and to measure relevant physical properties of the conidia. We found at least four ergot alkaloids, fumigaclavine C, festuclavine, fumigaclavine A, and fumigaclavine B (in order of abundance), associated with conidia of A. fumigatus. Under environmentally relevant conditions, the total mass of ergot alkaloids often constituted >1% of the mass of the conidium. Ergot alkaloids were extracted from conidia produced on all media tested, and the greatest quantities were observed when the fungus was cultured on latex paint or cultured maize seedlings. The values for physical properties of conidia likely to affect their respirability (i.e., diameter, mass, and specific gravity) were significantly lower for A. fumigatus than for Aspergillus nidulans, Aspergillus niger, and Stachybotrys chartarum. The demonstration of relatively high concentrations of ergot alkaloids associated with conidia of A. fumigatus presents opportunities for investigations of potential contributions of the toxins to adverse health effects associated with the fungus and to aspects of the biology of the fungus that contribute to its success.