Chemoraces and Habitat Specialization of Claviceps purpurea Populations

We studied genetic variability of 100 isolates of Claviceps purpurea by using randomly amplified polymorphic DNA (RAPD), an EcoRI restriction site polymorphism in the 5.8S ribosomal DNA (rDNA), the alkaloids produced, and conidial morphology. We identified three groups: (i) group G1 from fields and open meadows (57 isolates), (ii) group G2 from shady or wet habitats (41 isolates), and (iii) group G3 from Spartina anglica from salt marshes (2 isolates). The sclerotia of G1 isolates contained ergotamines and ergotoxines; G2 isolates produced ergosine and ergocristine along with small amounts of ergocryptine; and G3 isolates produced ergocristine and ergocryptine. The conidia of G1 isolates were 5 to 8 μm long, the conidia of G2 isolates were 7 to 10 μm long, and the conidia of G3 isolates were 10 to 12 μm long. Sclerotia of the G2 and G3 isolates floated on water. In the 5.8S rDNA analysis, an EcoRI site was found in G1 and G3 isolates but not in G2 isolates. The host preferences of the groups were not absolute, and there were host genera that were common to both G1 and G2; the presence of members of different groups in the same locality was rare. Without the use of RAPD or rDNA polymorphism, it was not possible to distinguish the three groups solely on the basis of phenotype, host, or habitat. In general, populations of C. purpurea are not host specialized, as previously assumed, but they are habitat specialized, and collecting strategies and toxin risk assessments should be changed to reflect this paradigm shift.     

Semicontinuous cultivation of immobilized Claviceps purpurea

Summary Mycelia of Claviceps purpurea CBS 164.59 were immobilized in 2%, 4%, and 8% calcium alginate. Alkaloid production by free cells declined after 60 days, while immobilized cells retained their activity for 200 days. The cumulative alkaloid production for all fermentation cycles using 8% calcium alginate immobilized mycelia was 25 times higher than that from free cells. The best yields of the ergopeptide ergometrine were reached with 4% gel immobilized mycelia, while higher gel concentrations caused a shift in the alkaloid biosynthesis towards high clavine alkaloid production.Beginning with the third cycle of reincubation the immobilized mycelia showed a marked tendency to fragmentize into vacuolated arthrosporoid-like structures and produced violet-black pigments so that the beads recalled sclerotial structures of parasitically living Claviceps.Dedicated to Prof. Dr. K. Esser to his 60th birthday     

N-acetyl-L-tryptophan in Claviceps purpurea PRL 1980

Claviceps purpurpea PRL 1980 converts L-tryptophan to N-acetyl-L-trytophan. There is little acetylation of D-tryptophan. Added N-acetyl-L -trypotophan. has no effect on alkaloid production. L-Tyrosine addition results in production of a compound which is probably N-acetyltyrosine and also causes accumulation of 4-γ,γ-dimethylally-tryptophan.     

Heterokaryosis and Alkaloid Production in Claviceps purpurea

Strain 275 FI of Claviceps purpurea, which produces large amounts of peptide alkaloids in submerged culture, is a heterokaryon; after several generations on agar media, it segregates the single components. These components (by us labeled V, C, and W) are stable and produce almost no alkaloids under described conditions of submerged culture. The mycelium of strain 275 FI consists of hyphae with multinucleate cells and does not produce conidia. Strains V, C, and W form numerous anastomoses when grown together on agar. By combining strains V and C, a heterokaryon similar to 275 FI in appearance has been obtained. This new strain produces amounts of alkaloids much larger than those produced by V and C separately or in associated submerged culture. We conclude, therefore, that in strain 275 FI the heterokaryotic condition is favorable to the production of alkaloids. Several conidiaproducing cultures of C. purpurea of various origins, as well as sclerotia of the same species, have been examined. The results demonstrated that the heterokaryotic condition is rare in the cultures, but it is frequent in the mycelium from sclerotia. Since it is known that the production of alkaloids is typical of the sclerotial phase in C. purpurea, it is suggested that this capacity is related to the heterokaryosis of the producing strains.     

High-production mutant Claviceps purpurea 59 accumulating secoclavines

Abstract A new cloning vehicle, pEAP37 was constructed to develop the excretion system of Escherichia coli . This plasmid, derived from pEAP1, carried the penicillinase gene from an alkalophilic Bacillus sp. and the chloramphenicol acetyltransferase gene from pBR329 as selective markers. The Bacillus N-4 cellulase gene, Bacillus 1139 cellulase gene and Aeromonas sp. 212 xylanase L gene was inserted into pEAP37, and the distribution of the plasmid-encoded enzymes was analyzed. Most of these enzyme activities were found in the periplasmic space of E. coli when these extracellular enzyme genes were inserted into pBR322. On the other hand, most of these activities were observed in the culture medium when inserted into pEAP37.     

Influence of inorganic phosphate and immobilization on Claviceps purpurea

Summary The influence of inorganic phosphate and immobilization on cells of Claviceps purpurea strain 1029/N5 producing ergot peptides in shake culture was examined. Immobilization in Ca-alginate beads resulted in a marked reduction of some metabolic activities, i.e. the periods of alkaloid formation and cell growth were prolonged. High concentrations of inorganic phosphate (1 g/l KH₂PO₄) could reduce or stop alkaloid formation both by free and immobilized cells at any time during fermentation. The optimum phosphate concentration for alkaloid production by immobilized cells (about 0.5 mM) was a quarter of that required by free cells. This optimum shift was attributed to (i) the diminished phosphate demand of immobilized cells, due to their reduced metabolic activities, and (ii) the phosphate-dependent morphological behaviour of the biocatalyst. The observed decrease in alkaloid concentrations during later periods of the fermentation supported the idea of alkaloid-degradative enzymes, activated by high phosphate concentrations. Immobilization showed an advantageous influence on this undesirable effect. Offprint requests to: H.J. Rehm     

Application of oxygen vectors to Claviceps purpurea cultivation

The application of a two-phase fermentation system for the production of ergot peptide alkaloids by Claviceps purpurea is described. Perfluorocarbons (PFC) are used as oxygen vectors in Claviceps fermentation for the first time. In shake-flask cultivations, the inclusion of PFC in the medium brings about a five-fold increase in the total alkaloid production and a six-fold increase in the pharmaceutically important component, ergotamine. This rise cannot be correlated with the concentration of the added PFC and it is thought that the enhancement is due to a combination of factors, including the influence of PFC. Other oxygen vectors, such as several hydrocarbons, prove to be poor oxygen carriers in our study. Cultivations with PFC in a bioreactor are reproducible, the maximum total alkaloid and ergotamine production being attained on the 11th and 9th days, respectively. The relatively lower increase in the total alkaloid production in the bioreactor as compared to the shake-flasks is attributed to the unequal oxygen availability in the reactor. Processes with PFC offer the operational advantage of a five-fold reduction in aeration rate.     

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.     

Ergotamine Production in Submerged Culture and Physiology of Claviceps purpurea

Strain 275 FI of Claviceps purpurea, which produces large amounts of peptide alkaloids in submerged culture, and strains V, C, and W, spontaneously obtained from 275 FI and practically unable to produce alkaloids, were compared. Strain 275 FI differs from the other strains in its capacity to accumulate lipids and sterols, as well as in its capacity to produce alkaloids. Strain 275 FI utilizes large quantities of sucrose and citric acid simultaneously; strain V utilizes large amounts of sucrose but little citric acid; strain C utilizes large quantities of citric acid but only small amounts of sucrose; strain W consumes only small amounts of both substances. We conclude that the production of large quantities of alkaloids, as well as the accumulation of lipids and sterols, is correlated with the simultaneous utilization of large amounts of sucrose and citric acid.     

Metabolism of the germinating sclerotium of Claviceps purpurea.

The concentration of free amino acids, particularly alanine, altered as stromata developed on germinating sclerotia. Lipid reserves in the sclerotium provided the principal carbon and energy source, and the nutrients required for stomatal growth appeared to be provided by the sclerotial tissues immediately beneath the point of attachment of the stoma. Alkaloid did not disappear from the sclerotia during germination. Very high levels of polyol, particularly mannitol, were attained in the stromata as germination progressed; the synthesis of polyol was usually accompanied by that of soluble sugars, notably glucose. In an attempt to ascertain the role of mannitol in germination, the relationship between polyol and soluble sugars was investigated in excised stromata after their submergence in buffered media containing a variety of soluble carbohydrates. Mannitol was synthesized under these conditions by way of phosphorylated intermediates, and chiefly in the capitulum, after rapid, and probably passive, absorption of the exogenously supplied sugar. The tissues equilibrated rapidly with the supplied carbohydrate, whose concentration was maintained by further uptake whilst mannitol was being synthesized. The concentrations of assimilated glucose and synthesized mannitol tended toward the same value during incubation and the results suggested that the synthesis was compartmentalized within the stromata. Very little exogenously supplied mannitol was assimilated.     

Transformation of Claviceps purpurea using a bleomycin resistance gene

Summary To develop a DNA-mediated transformation system for Claviceps purpurea a vector was constructed using a bleomycin-resistance gene (bleo ^R) fused in frame to the Aspergillus nidulans trp C promoter as a dominant selection marker. The construct was shown to be functional in Aspergillus nidulans and Aspergillus niger and used to transform a wild strain of Claviceps purpurea. Transformats were obtained at low frequencies; they were shown to contain transforming DNA integrated into the chromosomal DNA, probably in multimeric copies and at multiple sites. Combined Southern, Northern and resistance level analysis indicate that the A. nidulans promoter is functional in C. purpurea.Dedicated to Professor Dr. Dr. h. c. K. Esser on the occasion of his 65^th birthday     

Model of growth and ergot alkaloid production by Claviceps purpurea

A growth model for Claviceps purpurea in submerged batch culture is presented. In developing the model, the basic principles of the growth and the morphological properties of C. purpurea are considered. The growth of C. purpurea is assumed to occur in a three-step manner; the first step involves the assimilation and the growth of cells; the second one involves cell division, and the third one involves transformation of the mature cells to a state where they have no ability to divide but do have the ability to produce ergot alkaloids and then they gradually die. Inorganic phosphate is assumed to be the limiting substrate for the first and the second steps in conditions of carbon source being in excess. The model constants are determined by model simulation and graphical searching techniques to find the minimum value of the absolute difference between the experimental and the simulated curves for biomass, alkaloids, and sucrose.     

Distribution and habitat specialization of species affect local extinction in dragonfly Odonata populations

The object of our study was to determine the effect of distribution and habitat specialization of odonate species on local extinction in streams in central Finland. We studied the local extinction of the 20 most abundant dragonfly (Odonata) species in 34 small creeks and brooks in central Finland. The historical presence of each studied species in our research area was confirmed using existing records gathered between 1930 and 1975. A minimum of five records was available for each species. During the summers of 1995 and 1996, we investigated the current persistence of 219 separate populations with historical presence. In total, 98 historical populations were vanished. As predicted, we found that species with a narrow distribution were less persistent than species with a broad distribution. Therefore, the extinction risk of a species was inversely related to the width of its regional distribution. Using reference works, species were categorized into two main breeding habitat types: lotic species or lentic species. The species main habitat type was a significant predictor of local extinction risk after statistical removal of the effect of regional distribution on extinction risk. The lotic species had lower local extinction risk than other species. Altogether, the highest extinction risk was found in habitat-specialist species associated with peatlands, probably due to loss of natural breeding habitat. On the other hand, extinction risk was lower in widely distributed habitat generalist species than true lotic species. The local extinction within species was more common in small dynamic upstream than in larger stable downstream habitats. The results of this study are consistent with meta-population theory.