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Oligosaccharides produced by submerged cultures of Claviceps africana and Claviceps sorghi.

Oligosaccharides produced by submerged cultures of C. africana and C. sorghi were isolated by semipreparative HPLC. Structure of 6-O-beta-D-fructofuranosyl-D-glucopyranose (blastose), 1,6-bis-O-(beta-D-fructofuranosyl)-alpha-D-glucopyranoside (neokestose) and two sugar alcohols, 1-O-beta-D-fructofuranosyl-D-mannitol (fructosylmannitol) and 1,6-bis-O-(beta-D-fructofuranosyl)-D-mannitol (bisfructosylmannitol) was determined by NMR spectrometry. MALDI TOF MS analysis revealed molecular ions [M+Na]+ that indicate the presence of other tetra- and pentasaccharides (m/z = 689.4 and 851.5, respectively) and corresponding sugar alcohol (m/z = 691.4). Rapid conversion of sucrose into series of oligosaccharides and corresponding sugar alcohols was observed in all tested strains.

Chromatography, High Pressure Liquid↗

A taxonomic study of species of Bothriocephalus Rudolphi, 1808 (Cestoda: Pseudophyllidea) from eels in Japan: morphological and molecular evidence for the occurrence of B. claviceps (Goeze, 1782) and confirmation of the validity of B. Japonicus Yamaguti, 1934.

A taxonomic study of specimens of Bothriocephalus from eels ( Anguilla spp.) in Japan has demonstrated the occurrence of two species, B. claviceps (Goeze, 1782) and B. japonicus Yamaguti, 1934. The former species is a parasite of eels ( A. anguilla and A. rostrata ) in the Holarctic Region and was recently reported from A. marmorata in Japan. The conspecificity of tapeworms newly found in an eel ( A. ? japonica ) from Lake Biwa, central Japan, with B. claviceps has been confirmed by the great similarity of their ITS-2 gene sequences (similarity 95.3% and 95.2%). However, the sequences of worms identified as B. claviceps from A. marmorata differed considerably from those of B. claviceps from two populations of A. anguilla from Europe and the above-mentioned one from Japan (similarity 66.3%, 67.1% and 65.1 %, respectively), thus indicating that the former cestodes may have been misidentified. This assumption was confirmed by morphological evaluation of a voucher specimen from A. marmorata. The morphology of this cestode, as well as those from A. japonica from two localities in Japan (Lakes Biwa and Suwa), indicates their conspecificity with B. japonicus. The validity of this taxon has been confirmed on the basis of a re-examination of the type-specimens. The two taxa, B. japonicus and B. claviceps, differ from each other in the shape and length of the scolex (619-730 microm in B. japonicus versus 1,180-2,100 microm in B. claviceps ), the relative position of the cirro-vaginal and uterine pores (opposite each other in relation to the median line of the body in B. japonicus versus tandem or slightly offset along the median line in the latter species), and the size of the eggs (41-52 x 28-35 microm in B. japonicus versus 50-70 x 31-43 microm in B. claviceps ).

Animals↗

Analysis of Claviceps africana and C. sorghi from India using AFLPs, EF-1alpha gene intron 4, and beta-tubulin gene intron 3.

Isolates of Claviceps causing ergot on sorghum in India were analysed by AFLP analysis, and by analysis of DNA sequences of the EF-1alpha gene intron 4 and beta-tubulin gene intron 3 region. Of 89 isolates assayed from six states in India, four were determined to be C. sorghi, and the rest C. africana. A relatively low level of genetic diversity was observed within the Indian C. africana population. No evidence of genetic exchange between C. africana and C. sorghi was observed in either AFLP or DNA sequence analysis. Phylogenetic analysis was conducted using DNA sequences from 14 different Claviceps species. A multigene phylogeny based on the EF-1alpha gene intron 4, the beta-tubulin gene intron 3 region, and rDNA showed that C. sorghi grouped most closely with C. gigantea and C. africana. Although the Claviceps species we analysed were closely related, they colonize hosts that are taxonomically very distinct suggesting that there is no direct coevolution of Claviceps with its hosts.

Base Sequence↗

Presence of peptide synthetase gene transcripts and accumulation of ergopeptines in Claviceps purpurea and Neotyphodium coenophialum.

The production of toxic ergopeptine alkaloids by the fungi Claviceps purpurea and Neotyphodium coenophialum involves the activity of one or more nonribosomal peptide synthetases. Claviceps purpurea and N. coenophialum each have several different peptide synthetase genes, fragments of which have been cloned previously. An additional Claviceps purpurea peptide synthetase gene was cloned by hydridization with one of the N. coenophialum peptide synthetase gene fragments. We detected the presence of mRNA from the peptide synthetase genes in cultures of different ages grown under conditions favorable or unfavorable for ergopeptine production. All four peptide synthetase genes from Claviceps purpurea were transcribed under at least some of the experimental conditions. Transcripts from three of the four genes were detected under conditions consistent with their potential involvement in ergopeptine biosynthesis. All three peptide synthetase genes previously identified in N. coenophialum were transcribed during symbiotic growth of this fungus with tall fescue, as well as ergopeptine-producing cultures. The data show that all of the peptide synthetase genes are transcribed, that one of the peptide synthetase genes is dissociated from ergopeptine biosynthesis, and, as a result, prioritize the remaining genes for functional analyses by transformation-mediated gene disruption.

Claviceps↗

Claviceps cyperi, a new cause of severe ergotism in dairy cattle consuming maize silage and teff hay contaminated with ergotised Cyperus esculentus (nut sedge) on the Highveld of South Africa.

During December/January 1996/97 typical summer syndrome (hyperthermia and a 30% drop in milk yield) occurred in succession in two Holstein dairy herds (n=240 and n=150 milking cows, respectively) on the South African Highveld. These farms are situated in the midst of the prime maize and dairy farming areas of South Africa where this condition had never been diagnosed before. The individual components of the concentrate on both farms were negative for ergot alkaloids. Endophytic fungi and/or ergot infestation of teff and other grasses fed to the cows were then suspected of being involved, but neither endophytes nor ergot alkaloids could be implicated from these sources. By measuring the serum prolactin levels of groups of sheep (n=5) fed the first farm's total mixed ration (TMR) or its three individual fibre components for a period of 11 days, the source of the ergot alkaloids was identified. A statistically significant decrease in the level of this hormone occurred only in the group on maize silage (which constituted 28% on dry matter base of the TMR). The involvement of the maize silage was further chemically confirmed by the high levels of total ergot alkaloids, predominantly ergocryptine, found by LC-MS in the silage as well as in the TMR (115-975 ppb and 65-300 ppb, respectively). The ergot alkaloid content (mainly ergocryptine) of the maize silage on the second affected farm was 875 ppb. Withdrawal of contaminated silage resulted in gradual recovery of stock on both farms. Nut sedge (Cyperus esculentus and Cyperus rotundus of the family Cyperaceae) has a world-wide distribution and is a common weed in annual crops, and can be parasitized by Claviceps cyperi. Careful examination of the maize silage from both farms revealed that it was heavily contaminated with nut sedge and that it contained minute sclerotia, identified as those of Claviceps cyperi, originating from the latter. Nut sedge was abundant on both farms and it is believed that late seasonal rain had resulted in mature, heavily ergotised nut sedge being cut with the silage. Claviceps cyperi sclerotia, collected on the affected fields in the following autumn contained 3600-4000 ppm ergocryptine. That the dominant alkaloid produced by this particular fungus was indeed ergocryptine, was confirmed by negative ion chemical ionization MS/MS. In one further outbreak in another Holstein herd, teff hay contaminated with ergotised nut sedge and containing 1200 ppb alkaloids, was incriminated as the cause of the condition. This is the first report of bovine ergotism not associated with the Poaceae infected with Claviceps purpureum or endophytes but with the family Cyperaceae and this particular fungal phytopathogen.

Animal Feed↗

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.

Alkaloids↗

Efficient transformation of Claviceps purpurea using pyrimidine auxotrophic mutants: cloning of the OMP decarboxylase gene.

A homologous transformation system was developed for the phytopathogenic fungus Claviceps purpurea. Orotidine-5'-monophosphate decarboxylase (OMPD)-deficient mutants were obtained by UV mutagenesis and selection for resistance against 5-fluoroorotate. These mutants could be complemented well by the corresponding genes of Aspergillus niger (pyrA) and Neurospora crassa (pyr4), yielding significantly higher transformation rates (and lower copy numbers per transformant) than the phleomycin resistance system. The homologous OMPD gene was isolated from a lambda genomic library by heterologous hybridization with the pyr4 gene of N. crassa, identified by complementation of Aspergillus and Claviceps mutants, and used to confirm homologous integration in Claviceps. The pyr transformation system also proved to be very efficient in cotransformation experiments using the bacterial beta-glucuronidase gene (uidA) as a reporter gene, which was also efficiently expressed during the parasitic cycle: honeydew produced by plants infected with pyr/uidA cotransformants was shown to contain significant levels of beta-glucuronidase activity.

Blotting, Southern↗

Induced parasexual processes in Claviceps sp. strain SD58.

A homokaryotic, clavine alkaloid-producing strain of ergot, Claviceps sp. strain SD 58, was used in an attempt to demonstrate parasexuality. Genetically marked auxotrophic strains were produced by mutation with N-methyl-N'-nitro-N-nitrosoguanidine. Protoplast fusion of pairs of unlike doubly auxotrophic strains and isolation of stable prototrophic fusion products were carried out. By growth of the fusion products on complete medium, selective pressure for prototrophy was removed and auxotrophic segregants were allowed to form. Analysis of these and recovery of segregants with nonleaky, non-parent-type combinations of auxotrophic characteristics has provided strong evidence that a parasexual cycle can function in Claviceps sp. strain SD 58. Preliminary work suggests that the genetics of ergot might be studied by mitotic analysis and that protoplast fusion and selection procedures might be useful for the enhancement of favorable characteristics in Claviceps strains.

Claviceps↗

Purification and properties of anthranilate synthetase from the ergot fungus, Claviceps spec., strain SD 58.

A three-enzyme complex containing anthranilate synthetase, phosphoribosyl anthranilate isomerase and indole-3-glycerol phosphate synthetase was partially purified from Claviceps spec., strain SD 58. The anthranilate synthetase activity of the enzyme complex was quite unstable unless glutamine, MgCl2, TRIS and, most importantly, glycerol were included in the extraction buffer. The three-enzyme complex showed a molecular weight of 400,000 when estimated using Sephadex gel filtration, and a molecular weight of 200,000 when using sucrose density gradient centrifugation. At least two bands of anthranilate synthetase were detected on disc gel electrophoresis. An enzyme complex containing phosphoribosyl anthranilate synthetase and indoleglycerol phosphate synthetase, but no anthranilate synthetase, was isolated from Claviceps. This enzyme complex had an apparent molecular weight of 165,000 as estimated by sucrose gradient centrifugation. Anthranilate synthetase is inhibited by L-tryptophan and elymoclavine, the terminal ergot alkaloid produced by this strain of Claviceps. No differences could be detected between the enzyme complexes isolated from 2-day-old growing mycelia and from 6-day-old alkaloid-producing mycelia of the organism.

Age Factors↗

Some characteristics of tryptophan uptake in Claviceps species.

Tryptophan serves as a precursor for the biosynthesis of alkaloids in the ergot fungus, Claviceps purpurea (Fries) Tulasne, and also is believed to act as an inducer of the enzymes necessary for alkaloid production. The characteristics of the transport system responsible for the accumulation of tryptophan in ergot mycelium were investigated, with the goal of clarifying the complex relationships among tryptophan uptake, size of the free intracellular pool of tryptophan, and alkaloid production. The characteristics of tryptophan uptake were studied by pulse feeding radioactively labeled tryptophan to cultures of Claviceps species, strain SD-58, which represented a variety of ages and nutritional states. Tryptophan accumulation in strain SD-58 is mediated by an energy-requiring system which exhibits specificity for neutral aromatic and aliphatic l-amino acids, is pH and temperature dependent, and shows saturation at high substrate concentrations. Tryptophan transport is a function of the intracellular concentration of free tryptophan, the nitrogen deficiency of the mycelium, the rate of growth, and alkaloid production, which were measured in Claviceps strain SD-58 growth in several culture media, some of which promoted alkaloid production and some of which did not. The results indicate that the initial velocity of tryptophan transport is not directly related to alkaloid production.

Alkaloids↗

[Differences in proline metabolism of a peptide alkaloid producing and a nonproducing strain of Claviceps purpurea].

Two strains (pepty 695 and pur 221) of Claviceps purpurea have been used to study the dependence of proline turnover on incubation time (1, 3, 6, and 12 hours) under different culture conditions (synthetic saccharose-citrate medium NL 720 and complex wort medium M 107) by means of tracer technique. In the saprophytically ergotoxine producing strain pepty 695 the proline is utilized in the protein and alkaloid biosyntheses. On the other hand, under non-producing conditions (M 10) the radioactivity of proline-14C is incorporated into many fractions and finally into CO2. In submerged culture (NL 720) the Claviceps strain pur 221 which does not produce ergolines uses proline only to a small extent in the protein biosynthesis, while most of this amino acid is not metabolized. The extent of uptake of proline is not correlated with the alkaloid synthesis. For comparison the turnover of L-glutamate-14C and D,L-ornithine-1-14C by strain pepty 695 cultivated in the NL 720 medium has been studied.

Claviceps↗

Microcycle sporulation in the Claviceps purpurea 244.

The mutant strain Claviceps purpurea 244 forming hyphae composed mainly from sclerotium-like cells was found to sporulate both in liquid and solid media, particularly in the form of terminal chlamydospores (4.0 x 6.5 micrometer). Chlamydospores produced during submerged cultivation germinated, new chlamydospores being formed directly from germ tubes, or, occasionally, conidia (the so-called microcycle) or new vegetative mycelium were formed. The ultrastructures of the chlamydospores and vegetative cells was identical. The cytoplasm was filled with ribosomes and contained lipid inclusions and vacuoles with membrane invaginations. Strain 244 cultivated under submerged conditions produced 150 microgram/ml clavins, with elymoclavin predominating (82%). The parent strain Claviceps pururea 129 only produced chlamydospores on the vegetative mycelium, whereas no microcycle was detected; under submerged conditions it produced mainly agroclavin (85%) at a concentration of 4 mg/ml.

Claviceps↗

Studies on Claviceps parasitic on Panicum species in India.

Panicum repens and P. antidotale were found to be infected with Claviceps sp. This is the first report of ergot on P. 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 of Claviceps strain isolated from Panicum spp. produced significant amount of chanoclavine, festuclavine and agroclavine. No pharmaceutically important alkaloid was found in sclerotia or in submerged culture.

Claviceps↗

Kinetics of soluble glucan production by Claviceps viridis.

Among 18 tested strains of Claviceps spp., 7 produced significant amounts of exocellular polysaccharide (EPS). The maximum production of EPS was found in fermentation broth of Claviceps viridis. The kinetics of growth, substrate consumption, and EPS production in the batch, aerobic, submerged culture of this fungus were investigated in detail. The experimental data were processed by a simple mathematical model describing mass balance of growth, substrate consumption, formation of intermediates, and production of EPS. The parameters of the model were estimated from data obtained in cultivation performed in flasks and two laboratory fermentors of different size. Physiological similarity was obtained during process scale-up in volumetric ratio 1:100. The sugar consumption efficiency (52%) and observed EPS productivity (1.9 kg/m3 per d) were comparable with literature data.

Biomass↗