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SEQUENTIAL FORMATION AND ACCUMULATION OF PRIMARY AND SECONDARY SHUNT METABOLIC PRODUCTS IN CLAVICEPS PURPUREA.

The fungus Claviceps purpurea was grown on a rich and a limited nutrient medium such that alkaloid was produced after 8 days on the former medium and after 3 days on the latter medium. Cultures grown on both were assayed for the primary shunt metabolic products, polyols, trehalose, lipids, ribonucleic acid, and polyphosphate, and the secondary metabolic product, ergot alkaloid. Although differing considerably in composition, the two media nevertheless allowed formation of both primary and secondary shunt products. In both instances, however, the secondary product, ergot alkaloid, did not form until formation and accumulation of the primary products had ceased and the mycelial content of these products was actually decreasing. In both instances, alkaloid formation took place after the total dry weight of the mycelium had begun to decrease but while the dry weight of the residual, or structural portion of the mycelium, was either constant or increasing. The dilution of labeling in mannitol isolated from mycelia grown on rich medium containing 1,6-C(14)-labeled mannitol was 2.2. Thus, about half of the mycelial mannitol was actually mannitol which had been taken up directly from the medium.

Ascomycota↗

A CDC42 homologue in Claviceps purpurea is involved in vegetative differentiation and is essential for pathogenicity.

Claviceps purpurea, a biotrophic pathogen of cereals, has developed a unique pathogenic strategy including an extended period of unbranched directed growth in the host's style and ovarian tissue to tap the vascular system. Since the small GTPase Cdc42 has been shown to be involved in cytoskeleton organization and polarity in other fungi, we investigated the role of Cdc42 in the development and pathogenicity of C. purpurea. Expression of heterologous dominant-active (DA) and dominant-negative (DN) alleles of Colletotrichum trifolii in a wild strain of C. purpurea had significant impact on vegetative differentiation: whereas DA Ctcdc42 resulted in loss of conidiation and in aberrant cell shape, expression of DN Ctcdc42 stimulated branching and conidiation. Deletion of the endogenous Cpcdc42 gene was not lethal but led to a phenotype comparable to that of DN Ctcdc42 transformants. DeltaCpcdc42 mutants were nonpathogenic; i.e., they induced no disease symptoms. Cytological analysis (light microscopy and electron microscopy) revealed that the mutants can penetrate and invade the stylar tissue. However, invasive growth was arrested in an early stage, presumably induced by plant defense reactions (necrosis or increased production of reactive oxygen species), which were never observed in wild-type infection. The data show a significant impact of Cpcdc42 on vegetative differentiation and pathogenicity in C. purpurea.

Amino Acid Sequence↗

Cytochemical localization of polysaccharides in Claviceps paspali ultrastructure during submerged fermentation of alkaloids.

Morphological characteristics of two types of elements in the submerged mycelium of Claviceps paspali are described. Distribution of polysaccharides in the cell wall and cytoplasm was cytochemically determined at the ultrastructural level. Polysaccharide deposition into the cell walls was proportional to the increase in the alkaloid yield. In the cytoplasm, on the other hand, the presence of polysaccharide grains indicated an absence of alkaloid synthesis.

Alkaloids↗

Morphogenesis and ultrastructure of Claviceps purpurea during submerged alkaloid formation.

Criteria for morphogenetic and ultrastructural distinction between conidia and chlamydospores of a submerged culture of Claviceps purpurea (Fr.) Tul. are described. Both the hyphae of the sphacelia (asexual) stage and the conidia contained granular cytoplasm. Cytoplasmic invaginations in vacuoles were transformed to electron-opaque bodies and disintegrated prior to germination. The budding of conidia had basipetal succession. The chlamydospores were formed by rounding up the terminal cells of filamentous hyphae. Homogeneous nonvacuolized cytoplasm with lipid droplets and lipid-forming bodies was characteristic of young chlamydospores. Cristate mitochondria did not appear in the chlamydospores before the alkaloid production phase. Simultaneously a specific organelle in the chlamydospores, a dense body, appeared to absorb intracellular lipids and form large deposits of phospholipid material. No germination of chlamydospores was observed. The ultrastructural pattern described for chlamydospores was also observed in hyphae with reduced proliferation during the alkaloid production phase.

Cell Membrane↗

Uptake and efflux of succinic acid by uninduced mycelium of Claviceps purpurea.

Claviceps purpurea PRL 1980 grew on partially dissociated succinic acid (pH 4) but not on fully dissociated succinic acid (pH 7.2). Myeclium suspended in 42 mM solution of partially ionized succinic acid (pH 4; 60.1% nonionized, 39% monoanion, and 0.9% dianion, K+ salt) over a period of 25 min accumulated more succinic acid carbon than mycelium suspended in highly ionized solution (pH 6.8; 0.01% nonionized, 4.8% monoanion, and 95% dianion). The greater accumulation from partially ionized solution was not attributable solely to metabolism of succinic acid nor to the lower external concentration of potassium ion. Rate of uptake by sodium azide and iodoacetate-treated mycelium was proportional to external concentration at least up to 200 mumol/ml. External potassium or sodium ion was not required for uptake by inhibited or uninhibited mycelium and external sodium ion and glucose did not allow concentration of succinic acid. The internal concentrations of succinic acid carbon expressed as succinic acid in cell water were about the same as the external concentrations. Uptake was not appreciably affected by extent of ionization of external succinic acid but accumulation was markedly affected. A plot of accumulated succinic acid carbon against external pH produced a bimodal curve with the two maxima corresponding to the maximal concentrations of nonionized and monoanion succinic acid. The bimodal curve probably results from overlapping of two separate curves; the nonionized form accumulating efficiently because of one interaction with the cell and the monoanion form accumulating efficiently because of another interaction. Uptake from concentrated solution is by diffusion and efflux is rapid but not complete. Efflux is not retarded by presence of phosphate in the external solution.

Azides↗

The cellular role of nitrogen in the biosynthesis of alkaloids by submerged culture of Claviceps purpurea (Fr.) Tul.

Asparagine was a superior nitrogen source for clavine-alkaloid production in Claviceps purpurea. Its transport into the cell excedded the cell's biosynthetic need for this amino acid. Asparagine entered the cell without degradation. This disturbed the relative pool sizes of various amino acids resulting in a change in the genetically determined ratio at which amino acids were utilized for protein synthesis. Overproduction of alkaloids (4500 mug.ml-1) may be associated with increased availability of tryptophan because of the enhanced assimilation of asparagine-derived ammonia via glutamine synthetase (EC 6.3.1.2). However, ammonium salts in the fermentation broth led to a depression of the alkaloid yield. Partial replacement of the ammonium salt by aspartic acid elevated alkaloid production.

Amino Acids↗

The relationship between conidiation and alkaloid production in saprophytic strains of Claviceps purpurea.

A direct relationship between alkaloid synthesis and conidia formation was observed in a series of mutants of the saprophytic fungus Claviceps purpurea 129 which produces high quantities of clavine alkaloids (4500 microgram-ml-1). Sclerotia-like mutants forming mostly arthrospores and only a low number of conidiospores were exceptional in that they were characterized by an inverse relationship between alkaloid synthesis and conidial formation. Conidiospores of the parent strain 129 differed both in their size and ultrastructure. They include oval microconidia (2 X 3.5 micrometer) and prolonged macroconidia (2 X 10.5 micrometer). Both conidial types originated on short lateral sporophores terminated by phialids. Most conidia were characterized by electron-dense cytoplasm with a number of lipidic inclusions; a minority of conidia contained granular cytoplasm and vacuoles with membrane invaginations. Bicellular conidia were only rarely observed.

Claviceps↗

Metabolic changes in a conidia-induced Claviceps paspali strain during submerged fermentation.

Chemical changes in the mycelium of the conidial Claviceps paspali mutant strain, isolated after gamma irradiation, were followed during the course of submerged fermentation and compared with the mycelial parent strain; both strains are capable of producing simple lysergic acid derivatives. The syntheses of lipids, carbohydrates, phosphates, nucleic acids, proteins, and alkaloids, as well as nutrient uptake, were determined. It was found that conidiation induced by mutagenic treatment was accompanied by a set of changes in the metabolic pattern. In the conidial mutant, the primary and secondary metabolic activities were repressed and the protein to nonprotein compound ratio of the cells was changed in favour of protein compounds.

Carbohydrates↗

Ergotism and photosensitization in swine produced by the combined ingestion of Claviceps purpurea sclerotia and Ammi majus seeds.

Poisoning of domestic animals happens frequently in the southeast of Buenos Aires Province (Argentina). Intoxications are produced mainly by the ingestion of plants and mycotoxins, but animals are rarely affected simultaneously by both types of agents. One herd of pigs suffered simultaneous intoxications by ergot alkaloids from Claviceps purpurea sclerotia and furocoumarins from Ammi majus seeds. Pigs were fed a diet composed of wheat (poor quality) or corn and protein and vitamin supplements. This diet was completed with forage sorghum. Nervous signs were first observed 5-7 days after the initiation of feeding the suspect ration. These signs were followed by cutaneous irritation. Snout ulcers, eyelid edema, and conjunctivitis were observed in several piglets. Ten days after the start of feeding the incriminated ration, 8 abortions were observed. Many of the sows that were nursing piglets developed udder edema and teat cracking. Dermal lesions were observed in most of the animals with unpigmented areas in the skin but not in a Duroc-Jersey boar. Removal of the incriminated diet and feeding of another diet prepared with good-quality wheat allowed all the animals to recover in 15 days. The herd experienced normal pregnancies and parturitions, litter sizes, and piglet weights when fed a cleaned portion of the poor-quality wheat. No photosensitization lesions were observed. Examination of impurities in the suspected wheat indicated the presence of 2.2% of A. majus seeds and 0.14% of C. purpurea sclerotia. The quantitative analysis indicated the presence of 3.2 g xanthotoxin and 0.65 g bergaptene/100 g A. majus seeds and 0.73 g ergot alkaloids (expressed as ergonovine) per 100 g of C. purpurea sclerotia. Qualitative analysis demonstrated the presence of ergotamine, ergocristine, and ergonovine. These results indicate that clinical signs and lesions were caused by the ingestion of large quantities of these biologically active compounds.

Animal Feed↗

Association of insects and ergot (Claviceps purpurea) in Kentucky bluegrass seed production fields.

Insects in Kentucky bluegrass seed production fields in Oregon, Idaho, and Washington were sampled just before harvest and their association with ergot conidia of Claviceps purpurea Fr. (Tul.) was evaluated during 1996-1998. A diversity of insects was observed at all three locations. The most abundant beneficial insects collected with sweep nets were Nysium spp., Nabis spp., ichneumonid wasps, and Hippodamia spp. The cranberry girdler, Chrysoteuchia topiaria (Zeller), was the only important pest on grass seed collected by sweep net. Numbers of aphids such as Sitobion avenae (F.), cicadellids and thrips such as Anaphothrips spp. and Aptinothrips spp. that were collected with all aphid sampler were below economic thresholds. Other insect groups occurred in low numbers. Noctuid moths collected in universal blacklight traps included nine species of cutworms and armyworms. Protogrotis obscura (B. & McD.) was the most common cutworm species and was present in all fields. The moth Chortodes rufostrigata (Pack.) previously reported only from wet meadows in northeast and south central Oregon was found in Kentucky bluegrass fields in central Oregon, suggesting that irrigated Kentucky bluegrass seed production fields may simulate a montane meadow habitat. Conidia of C. purpurea were found on a diversity of insects, including moths, flies, leafhoppers, and thrips. Up to 100% of moths and 75% of flies collected from some fields carried conidia of C. purpurea. No correlation between ergot honeydew present in a field and number of insects with conidia of C. purpurea was detected.

Animals↗

[Stimulatory effect of sodium deoxycholate on alpha-ergocryptine productivity of Claviceps purpurea 94002 (EKPN94002)].

The stimulation of sodium deoxycholate (SDC) on alpha-ergocryptine production of Claviceps purpurea strain 94002 (EKPN94002) was investigated. Preliminary results indicated that SDC, as a stimulator and at appropriate concentrations, could obviously increase the extracellular and the total production levels of the ergot alkaloid. Satisfied results were obtained when 6.0 mg.L-1 of SDC was present in the seed and fermentation media. The possible mechanism was discussed.

Claviceps↗

[Morphological differentiation of cells in submerged cultures of Claviceps purpurea (Fr.) Tul., producing alkaloids].

Differentiation of the cells of the submerged culture of Claviceps purpurea (Fr.) Tul. was studied by electron microscopy. Two types of oviform cell were found: (1) the conidia which had one nucleus and vacuolized cytoplasm and were not involved in the production of alkaloids; (2) the chlamydospores with two nuclei, homogeneous cytoplasm, and high content in lipids. The chlamydospores, like the cells of sclerotia, were found to produce alkoloids.

Cell Nucleus↗

[Optimization of conditions for storage and cultivation of the fungus Claviceps sp.--a producer of the ergot alkaloid agroclavine].

Conditions of agroclavine biosynthesis by the mutant Claviceps sp. strain s 106 were studied. The content of agroclavine was maximum (1.5-2 g/l) on days 15-16 of cultivation in the complex medium T25, containing sucrose, citric acid, and yeast extract. Agroclavine was the major component of the alkaloid fraction (90-95%). Storage of the culture at -70 degrees C in T25 supplemented by 7% glycerol provided a stable level of alkaloid formation.

Air↗

Hyperthermic syndrome in dairy cattle associated with consumption of ergots of Claviceps purpurea in southern Brazil.

A hyperthermic syndrome (HS) associated with consumption of ration contaminated with Claviceps purpurea is described in 4 dairy cattle herds from different counties in the state of Rio Grande do Sul, southern Brazil. Outbreaks of this mycotoxicosis occurred during the summer of 1999-2000 and autumn of 2002 when 25% to 70% of cattle from those herds were affected. The disease occurred after the introduction of a new batch of feed contaminated with sclerotia of C. purpurea. First clinical signs were observed up to 3 mo after the ration was introduced and consisted of pyrexia, intense salivation and drooling, open-mouth, difficult breathing, in some cases with the tongue protruded, decrease in milk yield, decrease in food intake and weight loss. Affected cattle compulsively sought shade or water ponds. Signs were more pronounced during daytime. The severity of the clinical picture varied according to changes in environmental temperature during the day. There was complete recovery approximately 60 d after feeding with the contaminated ration was discontinued. Abortions occurred during the outbreak and soon after the remission of the clinical signs associated with HS. In one of the herds, reproductive performance and fertility were seriously impaired even after clinical signs of HS completely disappeared. Diagnosis was based on epidemiology, clinical signs and pathology. The pathogenetic mechanisms involved in the occurrence of HS are briefly discussed.

Animals↗

[Preservation of Claviceps purpurea, the producer of peptide ergot alkaloids, by the method of freeze drying].

A saprotrophic strain of Claviceps purpurea VNIIA 312A, an organism producing peptide +ergot alkaloids with prolactin inhibiting activity was shown to die under lyophilization conditions. To provide long-term storage of strain 312A, L-drying or drying under vacuum from liquid state was used with success. Three protective media were tested. Favourable results were obtained by using 25 per cent maltose solution as a protective medium. Preservation of the culture viability was accompanied by maintenance of the culture capacity for active formation of the biomass and production of +ergot alkaloids.

Claviceps↗

[Effect of various factors on the biosynthesis of alkaloids in a culture of Claviceps CP II].

The effect of a carbohydrate medium component, aeration, tryptophane and Tween additions on the biosynthesis of alkaloids by Claviceps CP II was being studied. The quantity of synthesized alkaloids and the composition of produced alkaloids depended on the nature of the carbohydrate and its concentration. A few alkaloids were accumulated on media containing xylose, lactose and glucose, whereas active production of alkaloids was observed when galactose maltose, sucrose and sorbit were used. Intensified aeration and introduction of Tween-80 and Tween-40 resulted in an increased alkaloid yield. Exogenous tryptophane had slight stimulatory effect on alkaloid production.

Alkaloids↗

Proteinases of various Claviceps purpurea strains.

Extra- and intracellular proteinases from various Claviceps purpurea strains grown in submerged culture have been studied. A maximum level of intracellular proteinases was observed on the 6th day of culture growth, whereas extracellular activity continued to increase throughout the culture growth. Proteinases were purified and characterized. The ergotamine strain secreted one aspartic and two serine proteinases, whereas from the disrupted mycelium only the aspartic proteinase could be isolated. The ergocornine strain secreted the aspartic proteinase in two forms and the ergocristine strain produced an aspartic and a serine proteinase.

Claviceps↗

Mycotoxic tremorgens of Claviceps paspali and Penicillium cyclopium: a comparative study of effects on sheep and cattle in relation to natural staggers syndromes.

Claviceps paspali sclerotia were dosed orally at various levels to sheep and cattle and the neurotoxic effects are described. Evidence is presented which indicates that the tremors are produced by a neutral tremorgen fraction. The ergot alkaloids, principally D-lysergic acid alpha-hydroxyethylamide and its isolysergic acid isomer, are not involved. The mixture of C paspali tremorgens has not been separated and characterised but its components are chemically related to other known tremorgens, such as penitrem A, produced by saprophytic moulds, notably Penicillium cyclopium. P cyclopium mycelium and C paspali sclerotia were dosed to produce comparable effects in sheep and in cattle. Similarly, effects of penitrem A and tremorgen concentrate of C paspali were compared after intravenous dosage to sheep. Observed enurotoxic effects of both moulds were identical in most respects. A low level of tremorgenic activity was detected at the honeydew stage of C paspali. These findings are discussed in relation to staggers syndromes occurring in grazing livestock.

Administration, Oral↗