Symptomatology and morphology of Claviceps cyperi on yellow nut sedge in South Africa.

Symptoms of ergot on yellow nut sedge, germination of sclerotia of the causal organism, Claviceps cyperi, and morphology of fresh specimens of the pathogen are described for the first time. The initial symptom of infection was a black sooty layer on inflorescences of infected plants due to colonization of the ergot honeydew by Cladosporium cladosporioides. Sclerotia of C. cyperi started to develop in March and April and could be discerned as small protuberances on inflorescences in the place of seed. Mature sclerotia were purplish-black. They generally remained viable for less than a year and germinated without prior cold treatment, although exposure for 21 d to 5 C before incubation significantly increased the germination rate. Under moist conditions at 24 C in the laboratory, germination commenced within 4-8 wk. Stromata took about 12 d to mature. Mature capitula were distinctly lobulate with a perithecium embedded in each lobe and a collar-like appendage around the base. Although dimensions of sclerotia, stipes, capitula, asci and ascospores were larger than in the original description, the general morphology supports treatment of C. cyperi as a distinct species.

Cultural characteristics, morphology, and variation within Claviceps africana and C. sorghi from India.

Sorghum ergot in India is caused by Claviceps africana and C. sorghi. The distributions of these two species in India is not known. Eighty-nine sorghum ergot isolates were cultured from young sphacelia obtained from male sterile sorghum plants artificially inoculated using inoculum collected in the field. Based on cultural characteristics, the isolates were separated into two groups which differed distinctly in the morphology of their sphacelia, conidia, and sclerotia. Marked differences also were observed in rates of secondary conidial production and disease spread between the groups. In combination with molecular evidence, our results confirm that the isolates placed in Group I represent C. africana and Group II isolates represent C. sorghi. C. africana was found to be widely distributed in all sorghum growing areas of India. The species first described as occuring in India, C. sorghi, appears to be restricted to a few locations in the states of Maharashtra, Andhra Pradesh, and Karnataka.

Secretion of acid phosphatase in Claviceps purpurea--an ultracytochemical study.

The lead phosphate precipitation method showed the reaction product of acid phosphatase (which reflects the presence of the enzyme glycoprotein) in peripheral cytoplasmic vesicles in the ascomycetous fungus Claviceps purpurea. The product appeared to diffuse from these vesicles (diameter 100-200 nm) towards the cell wall, usually to its sites covered by the capsular fibres exhibiting also acid phosphatase activity. This observation of diffusion of secretory glycoprotein in the cytoplasmic matrix and its orientation to the plasmalemma and capsular fibrils suggests an alternative to the well-described secretory mechanism of transport and exocytosis of glycoproteins via membrane-bound transport conveyors fusing with the cell membrane. It confirms and enlarges our previous finding of the reaction product of acid phosphatase performed by ultrastructural cytochemistry in vacuoles (lysosomes), in the growing cell septum, in cytoplasmic vesicles and in the fibres of the external capsule.

Developmental studies of Claviceps paspali seed cultures for the submerged production of lysergic acid derivatives.

Metabolic pattern of mycelial Claviceps paspali seed cultures during the submerged cultivation was established. By comparing it with conidial and mycelial Claviceps purpurea strains it was found that the biosynthesis of RNA, DNA, and proteins followed a similar course in all Claviceps strains, so the fall of RNA content in mycelium may be considered a general biochemical indicator for optimally developed inoculum. But, two different patterns of carbohydrate and lipid metabolism were observed one for conidial and one for mycelial strains.

Changes in cytoplasmic ultrastructure during submerged cultivation of a peptide alkaloids-producing strain of Claviceps purpurea (Fr.) Tul.

A strain of Claviceps purpurea, designated Pepty 695/S produces ergotoxine alkaloids under particular conditions of fermentation. The onset of alkaloid synthesis occurs around the second day of cultivation. Alkaloid formation is connected with morphological and ultrastructural changes. In the first 3-5 days of cultivation short thickened, septated hyphae, organized in plectenchymatic pellets as well as large single cells are formed. The hyphae are ultrastructurally characterized by increasing number of lipid droplets, deposits of glycogen and by extended ER membranes, which apparently may form numerous vesicles. The correlations between lipid and alkaloid synthesis are discussed.

Electron-cytochemical localization of phospho(enol)pyruvate carboxylase (EC 4.1.1.31) in fungal cells.

New cytochemical method, based on biochemical experiments, was elaborated for the ultrastructural localization of phospho(enol)pyruvate carboxylase (EC 4.1.1.31). The procedure was used to study the saprophytic submerged mycelium of the ascomycetous fungus Claviceps purpurea Tul. producing clavine alkaloids. The pelleted mycelium was fixed in ice cold 3% glutaraldehyde in 50 mM cacodylate buffer pH 7.2 and washed repeatedly in the same cold buffer The reaction mixture contained 100 mM Tris-HCl buffer pH 9.0, 10 mM phospho(enol)pyruvate, 30 mM sodium potassium tartrate, 3 mM Pb(NO3)2, 60 mM MgCl2 and 30 mM NaHCO3. Enzyme activity was localized in vacuoles, particularly inside lipid globules (spherosomes) and less frequently in membranous vesicles. Acetyl-CoA activated PEP-carboxylase both in cell free extracts and in the cytochemical staining. Aspartate inhibited the enzyme in the biochemical assay with coupled malate dehydrogenase system; the cytochemical reaction was not influenced, probably due to the interference of asparagine synthase (EC 6.3.1.1).

Fine structural localization of alkaloid synthesis in endoplasmic reticulum of submerged Claviceps purpurea.

Acetyl coenzyme A (CoA) carboxylase (EC 6.4.1.2), an enzyme catalyzing the synthesis of malonyl-CoA, was cytochemically localized in endoplasmic reticulum (ER) of sclerotia-like cells of submerged Claviceps purpurea Tul. producing clavine alkaloids. The enzymic activity was structurally bound in unit membranes of ER strands which, later on, evolved into vacuoles containing lipoprotein material. The reaction product was absent from ER in nonvacuolized filamentous hyphae and ovoid asexual spores containing numerous lipid globules; it was also absent from ER in the mycelium of submerged C. purpurea strain producing no alkaloids. In view of our previous morphogenetic observations and the available biochemical evidence, the observed localization of acetyl-CoA carboxylase was assumed not to coincide with fatty acid biosynthesis but to represent sites of alkaloid synthesis.

Fine structure of imbibed sclerotial cells of Claviceps purpurea (Fr.) Tul revealed by freeze-etching.

The fine structure of the cortex of the natural sclerotium of Claviceps purpurea was studied. The cell wall of sclerotial cells is thickened due to overproduction of the fibrillar component of the wall. The intracellular spaces of the cortex tissue form a continuous system which is apparently instrumental in mediating communication between the growing sclerotium and the external milieu. The cytoplasmic membrane of imbibed sclerotial cells carriers abundant signs of secretory activity. Secretion vesicles, the content of which is discharged into external space, apparently contain exo 1,3-glucanase. Cytoplasmic vesicles migrating toward the plugged pores of the thickened septa apparently involve hydrolase secretion too. Spherosomes with lipid content are a predominant component of the cytoplasm of sclerotial cells. The activity of the membrane systems of imbibed cells indicates that the mobilization of lipids sets in only after activation of the hydrolases. Findings of phagocytosis of lipid granules by vacuoles are relatively frequent so that lipolysis might proceed in the vacuoles. Alkaloids could not be detected with the aid of freeze-etching.

A simple method for preparing specimens of Claviceps purpurea for scanning electron microscopy.

The method is a modification of freeze-drying and includes also a simultaneous coating during rotation. It utilizes a common vacuum coating apparatus with a modified miniature electric motor. The cells of C. purpurea in the resulting specimen retain their original shape.

Electron-cytochemical demonstration of acid phosphatase in saprophytic Claviceps purpurea.

p-Nitrophenylphosphate in combination with lead salt technique was used for the cytochemical localization of acid phosphatase (EC 3.1.3.2) in saprophytic submerged culture of Claviceps purpurea Tul. The lead reaction product was found in capsular fibrils, in the newly formed parts of the cell wall and in the vacuoles of aged cells (autolysosomes). Phosphatase activity was present also in particulate intra-cytoplasmatic organelles. The concentric layering of lead deposits in these organelles indicates their relationship to endoplasmic reticulum membranes.

Origin of sclerotia-like cells in submerged Claviceps purpurea producing clavine alkaloids.

Ultrathin sectioning of submerged mycelium of Claviceps purpurea Tul. producing clavine alkaloids revealed yeast-like budding resulting in asexual spores-blastospores. These deciduous spores were born by extended hyphal cells and retained the same ultrastructure of cell organelles. Both the extended hyphae and the blastospores resembled the cells of ergot sclerotial tissue. A surface culture of C. purpurea Tul. producing no alkaloids was used as a reference.

[Morphological differentiation of cells in submerged cultures of Claviceps purpurea (Fr.) Tul., producing alkaloids]. / Morfologicheskaia differentsiatsiia kletok v pogruzhennykh kul'turakh Claviceps purpurea (Fr.) Tul., obrazuiushchikh alkaloidy

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.

Cytochemical detection of polysaccharides on the surface of the cell membrane complex in fungi.

Cytochemical staining in toto (periodic acid, thiosemicarbazide, OSO4) revealed the presence of polysaccharide lamellae on the surface of the cell membrane complex of fungi. The membraneous clusters in the vacuolar bodies of Claviceps purpurea were covered with these lamellae at both surfaces, as it was also the case with the endoplasmic reticulum membranes, the tonoplast and the cytoplasmic membrane. In Saccharomyces cerevisiae, the polysaccharide lamellae were visible on the surface of the endoplasmic reticulum membranes and the plasmalemma; the strain revealed polysaccharide deposits also on the tonoplasts of small vacuoles and in glucanase vesicles. We assume that these observations give precision to the localization of the enzymes synthetizing the glycoprotein components of the fungal cell wall.

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.