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PubMed1984

D-lysergic acid-activating enzyme from the ergot fungus Claviceps purpurea.

A D-lysergic acid-activating enzyme from the ergot fungus Claviceps purpurea was purified about 145-fold. The enzyme was able to catalyse both the D-lysergic acid-dependent ATP-pyrophosphate exchange and the formation of ATP from D-lysergic acid adenylate and pyrophosphate. Both reactions were also catalysed to a decreased but significant extent with respect to dihydrolysergic acid. The molecular mass of the enzyme was estimated to lie between 135 and 140 kDa. The involvement of the enzyme in the biosynthesis of ergot peptide alkaloids is discussed.

Adenosine Triphosphate↗
PubMed2002

CPMK2, an SLT2-homologous mitogen-activated protein (MAP) kinase, is essential for pathogenesis of Claviceps purpurea on rye: evidence for a second conserved pathogenesis-related MAP kinase cascade in phytopathogenic fungi.

Cpmk2, encoding a mitogen-activated protein (MAP) kinase from the ascomycete Claviceps purpurea, is an orthologue of SLT2 from Saccharomyces cerevisiae, the first isolated from a biotrophic, non-appressorium-forming pathogen. Deletion mutants obtained by a gene replacement approach show impaired vegetative properties (no conidiation) and a significantly reduced virulence, although they retain a limited ability to colonize the host tissue. Increased sensitivity to protoplasting enzymes indicates that the cell wall structure of the mutants may be altered. As the phenotypes of these mutants are similar to those observed in strains of the rice pathogen, Magnaporthe grisea, that have been deprived of their MAP kinase gene mps1, the ability of cpmk2 to complement the defects of delta mps1 was investigated. Interestingly, the C. purpurea gene, under the control of its own promoter, was able to complement the M. grisea mutant phenotype: transformants were able to sporulate and form infection hyphae on onion epidermis and were fully pathogenic on barley leaves. This indicates that, despite the differences in infection strategies, which include host and organ specificity, mode of penetration and colonization of host tissue, CPMK2/MPS1 defines a second MAP kinase cascade (after the Fus3p/PMK1 cascade) essential for fungal pathogenicity.

Claviceps↗
PubMed1999

Identification and characterization of a tri-partite hydrophobin from Claviceps fusiformis. A novel type of class II hydrophobin.

A new type of hydrophobin is encoded by an abundant mRNA of Claviceps fusiformis. The predicted amino-acid sequence of the protein, dubbed CFTH1, shows a putative signal sequence for secretion, followed by three class II hydrophobin domains each preceded by glycine/asparagine rich regions. SDS/PAGE analysis of 60% ethanol extractions of C. fusiformis mycelia from shaken cultures showed CFTH1 at the 50-55-kDa position. N-terminal sequencing of both untreated mature CFTH1 and of a fragment obtained by trypsin digestion revealed that CFTH1 is not processed between the hydrophobin domains. Mass spectroscopy showed a mass of about 36 500 Da, which is about 1500 Da higher than the mass predicted from the constituent amino acids, indicating post-translational modification but not glycosylation. Purified CFTH1 self-assembled at hydrophilic/hydrophobic interfaces and, after assembly at a water/air interface, it was found to be highly surface active. Antibodies raised against CFTH1 localized the protein in a mucilageous coat surrounding submerged vegetative hyphae in liquid shaken culture and, as a discrete layer of about 10 nm thickness at the surface of aerial hyphae of standing cultures, suggesting a role in the formation of aerial hyphae.

Amino Acid Sequence↗
PubMed1996

D-Lysergyl peptide synthetase from the ergot fungus Claviceps purpurea.

The ergot fungus Claviceps purpurea produces the medically important ergopeptines, which consist of a cyclol-structured tripeptide and D-lysergic acid linked by an amide bond. An enzyme activity capable of non-ribosomal synthesis of D-lysergyl-L-alanyl-L-phenylalanyl-L-proline lactam, the non-cyclol precursor of the ergopeptine ergotamine, has been purified about 18-fold from the ergotamine-producing C. purpurea strain D1. Analysis of radioactively labeled enzyme-substrate complexes revealed a 370-kDa lysergyl peptide synthetase 1 (LPS 1) carrying the amino acid activation domains for alanine, phenylalanine, and proline. The activation of D-lysergic acid is catalyzed by a 140-kDa peptide synthetase (LPS 2) copurifying with LPS 1. LPS 1 and LPS 2 contain 4'-phosphopantetheine and bind their substrates covalently by thioester linkage. Kinetic analysis of the synthesis reaction revealed a Km of approximately 1.4 microM for both D-lysergic acid and its structural homolog dihydrolysergic acid, which is one to two orders of magnitude lower than the Km values for the other amino acids involved. The Km values for the amino acids reflect their relative concentrations in the cellular pool of C. purpurea. This may indicate that in in vivo conditions D-lysergyl peptide formation is limited by the D-lysergic acid concentration in the cell. In vitro, the multienzyme preparation catalyzes the formation of several different D-lysergyl peptide lactams according to the amino acids supplied. Specific antiserum was used to detect LPS 1 in various C. purpurea strains. In C. purpurea wild type, the enzyme was expressed at all stages of cultivation and in different media, suggesting that it is produced constitutively.

Amino Acids↗
PubMed1997

Cell volume regulation in Claviceps fusiformis. An animal-type Na,K-ATPase operating in a fungus?

Claviceps fusiformis, an ergot pathogen of pearl millet, was equilibrated in media without and with 0.1 mM ouabain (g-strophantin). Highly significant effects of ouabain on the contents of sodium ions and water were found, suggesting that an active transport of sodium ions, similar to that in animal cells, regulates the cell volume of the fungus. The basic role of the sodium pump appears to be to decrease larger cell volumes and to increase the small ones; for this purpose it can even reverse its direction.

Biological Transport, Active↗
PubMed2004

Viability of Claviceps africana spores ingested by adult corn earworm moths, Helicoverpa zea (Boddie) (Lepidoptera: Noctuidae).

A study was conducted in College Station, TX, to determine the viability of Claviceps africana spores in the digestive tract of adult corn earworm moths, Helicoverpa zea (Boddie). Both sexes were exposed to ergot-infected sorghum panicles for 30 min, and spores were recovered from excreta of the moths at 24-, 48-, and 72-h intervals after feeding. Recovered spores were quantified, and viability was determined by the germination rate of macroconidia. Nearly a 100-fold greater concentration of spores was recovered from female excreta at the three time intervals compared with male excreta. Concentration of spores in female and male excreta was greatest at 24 h, with a significant reduction at the later time intervals. Spore germination rates for both sexes were greater at 24 h, with survival being significantly reduced at the 72-h interval. Spores in female excreta survived longer than those from male excreta. Spore survival over time was significantly reduced in male excreta. Spore concentration and survival were greater from female excreta, which is key, because egg-laying activities on sorghum panicles intensify during flowering, and this source of ergot spores could contribute to the spread of the disease. This study demonstrates that corn earworm moths can internally carry viable ergot spores for several days and can act as primary dispersal agents for the fungus. This is important because contaminated moths migrating from areas in Mexico and southern Texas where ergot is endemic could transmit and spread the disease to other sorghum growing regions of the United States.

Animals↗
PubMed1997

Cel1, probably encoding a cellobiohydrolase lacking the substrate binding domain, is expressed in the initial infection phase of Claviceps purpurea on Secale cereale.

At the host-pathogen interface of hyphae penetrating host cell walls in the rye ovary, a lack of cellulase-gold labeling of beta-1, 4-glucan in host cell walls indicates that enzymatic degradation of cellulose might be an important factor during the infection of rye by Claviceps purpurea. Using cbh1 from Trichoderma reesei as a probe, a putative cellulase gene (cel1) was isolated from a genomic library of the C. purpurea strain T5. The coding region of 1,616 bp contains two introns and a putative signal peptidase cleavage site, leaving a coding capacity of 437 amino acids for the mature protein. The derived amino acid sequence shares significant homology with other fungal cellobiohydrolases and lacks the substrate binding domain. Expression analysis using reverse transcriptase-polymerase chain reaction (RT-PCR) shows that cel1 is induced during the first days of infection of rye by C. purpurea. It may be involved in the penetration and degradation of host cell walls by depolymerizing plant beta-1, 4-glucan and, therefore, play a role in the infection process.

Amino Acid Sequence↗
PubMed1998

Cloning, characterization, and targeted disruption of cpcat1, coding for an in planta secreted catalase of Claviceps purpurea.

Claviceps purpurea has been shown to secrete catalases in axenic and parasitic culture. In order to determine the importance of these enzymes in the host-parasite interaction, especially their role in overcoming oxidative stress imposed on the pathogen by the plant's defense system, the catR gene from A. niger was used to isolate a putative catalase gene from a genomic library of C. purpurea, cpcat1 consists of an open reading frame of 2,148 bp that is interrupted by five introns. Its derived gene product shows significant homology to fungal catalases and contains a putative signal peptide of 19 amino acids and three putative N-glycosylation sites, which indicates that CPCAT1 is a secreted catalase. Disruption of the gene by a gene replacement approach resulted in the loss of two catalase isoforms, CATC and CATD, strongly suggesting that they are both encoded by cpcat1. CATD is the major secreted catalase of C. purpurea and is furthermore the only catalase present in the honeydew of infected rye ears. Deletion mutants of cpcat1 were inoculated on rye plants and showed no significant reduction in virulence. Ovarian tissue and honeydew of plants inoculated with the mutants lacked CATD, confirming that this catalase is not essential for colonization of the host tissue by C. purpurea.

Amino Acid Sequence↗
PubMed2002

The biotrophic, non-appressorium-forming grass pathogen Claviceps purpurea needs a Fus3/Pmk1 homologous mitogen-activated protein kinase for colonization of rye ovarian tissue.

Claviceps purpurea is a common pathogen of a wide range of grasses and cereals that is able to establish a stable, balanced interaction with its host plant and is considered a biotroph. It does not form special penetration structures such as appressoria. To study the signaling processes involved in this special host-pathogen interaction, we have cloned a gene, cpmk1, encoding a mitogen-activated protein (MAP) kinase that shows significant homology to Fus3 of Saccharomyces cerevisiae and to pmk1 of Magnaporthe grisea. Using a gene-replacement approach, we isolated a Acpmk1 mutant and characterized it in detail. Loss of CPMK1 has no obvious effect on vegetative properties (such as growth rate, morphology, and conidia formation); however, infection tests on rye show that the mutant is unable to colonize rye tissue, i.e., it appears to be completely nonpathogenic. Complementation of the mutant with a wild-type copy of cpmk1 fully restores its pathogenicity, confirming that this MAP kinase is essential for infection of rye by C. purpurea. Transformation of the delta pmk1 mutant of M. grisea with a complete copy of cpmk1 (including the C. purpurea promoter) fully restored its ability to form appressoria and its pathogenicity on barley. Although both fungi drastically differ in their pathogenic strategies, this result indicates that the signal pathway involving CPMK1 is highly conserved.

Claviceps↗
PubMed2004

CPTF1, a CREB-like transcription factor, is involved in the oxidative stress response in the phytopathogen Claviceps purpurea and modulates ROS level in its host Secale cereale.

CPTF1, a transcription factor with significant homology to ATF/CREB bZIP factors, was identified during an expressed sequence tag (EST) analysis of in planta-expressed genes of the phytopathogen Claviceps purpurea. Using a gene-replacement approach, deletion mutants of cptf1 were created. Expression studies in axenic culture showed that the H2O2-inducible gene cpcat1 (encoding a secreted catalase) had a reduced basal expression level and no longer responded to oxidative stress in the delta cptf1 mutant. Biochemical analyses indicated that CPTF1 is a general regulator of catalase activity. Delta cptf1 mutants showed significantly reduced virulence on rye. Electron microscopical in situ localization revealed significant amounts of H2O2 in delta cptf1-infected rye epidermal tissues, indicating that the plant tissue displayed an oxidative burst-like reaction, an event not detected in wild-type infections. These data indicate that CPTF1 is involved not only in oxidative stress response in the fungus but also in modulation of the plant's defense reactions.

Amino Acid Sequence↗
PubMed1976

Differentiation of Claviceps purpurea in axenic culture.

The growth form of a strain of Claviceps purpurea in axenic culture has been controlled by the amino nitrogen source. Within the pairs asparagine/aspartic acid or glutamine/glutamic acid the amide promoted sphacelial growth of the colony whereas the acid supported differentiation of plectenchymatic sclerotial tissue and synthesis of ergot alkaloids. Sclerotial colonies showed purple pigmentation. The mycelium had a greater lipid content, rich in ricinoleic acid, and sporulation was much less than in sphacelial colonies. Changes in the relative distribution of amino acids between the free and peptidyl components of the cells was most marked with respect to lysine; growth on asparagine resulted in more than half remaining free, whereas less than 10% remained free on aspartic acid. Lysine supplied exogenously as a nitrogen source did not promote sclerotial differentiation. Frequent transfers to fresh medium of colonies grown on dialysis membrane accentuated the extent of sphacelial or sclerotial growth; four transfers during an 18-day growth period yielded mycelia with an alkaloid content (0-4%, w/w) similar to that of parasitic ergot sclerotia. Asparagine and glutamine were taken up more rapidly from liquid media than their corresponding acids, but each acid exerted a dominant effect over the amide, in a mixture providing equivalent nitrogen, resulting in differentiation from sphacelial to sclerotial growth analogous to that occurring during parasitism. The apparently greater (w/w) proportion of total amino acids in sphacelial mycelia than in sclerotial mycelia mainly reflected the lower lipid content of these tissues, but this factor was insufficient to account for the persistence of a significant proportion of the total lysine amongst the free amino acids. The promotion of sclerotial growth by aspartic and glutamic acids was not confined solely to the experimental strain of the fungus. Although some isolates failed to differentiate into plectenchymatic mycelia on these nitrogen sources, the extent of their sphacelial growth, as indicated by the degree of sporulation, was always much reduced with respect to that promoted by asparagine.

Amino Acids↗
PubMed1976

Carbon assimilation by Claviceps purpurea growing as a parasite.

Carbon assimilation by Claviceps purpurea, growing as a parasite on cereals, has been investigated by supplying the host plant with 14CO2 in a closed system. The presence of the pathogen induced the plant to exude photosynthate which contained high levels of sucrose. During the period of 14CO2 supply, 14C was incorporated into the sucrose and so the path of carbon into the parasite could be traced. Hexoses, derived by the action of the fungal sucrase on sucrose, were assimilated by the pathogen and largely converted into polyols - mainly mannitol and, to a lesser extent, trehalose. The rate of carbohydrate metabolism decreased with maturation of the ergot, and also showed qualitative differences between the basal and apical regions of the ergot which were probably a function of nutrient supply.

Carbon↗
PubMed1989

Induction of heat stress in beef cattle by feeding the ergots of Claviceps purpurea.

Two groups of 4 Hereford steers were housed in a controlled environment room and exposed to simulated high summer temperatures. Both groups were fed a barley grain and hay diet ad libitum. The barley in one diet contained 0.5% w/w ergots of Claviceps purpurea. Within one week of feeding the ergot diet mean rectal temperature was significantly elevated (P less than 0.05) each afternoon (up to 41 degrees C) but returned to normal overnight. Elevated rectal temperature was accompanied by other signs of heat stress, reduced feed intake, body weight loss and depression or serum prolactin concentration. Symptoms disappeared within 1 week of ceasing to feed the ergot diet.

Animal Feed↗
PubMed2000

Sorghum ergot (Claviceps africana) associated with agalactia and feed refusal in pigs and dairy cattle.

OBJECTIVE: To establish the aetiology and define the main clinical features of a syndrome characterised by severe feed refusal, death of piglets and reduced milk production in pigs and dairy cattle. DESIGN: Clinical, pathological, toxicological and epidemiological examination of clinical cases in 10 piggeries and 4 dairies, located between 50 and 150 km south-west of Rockhampton. RESULTS: All cases were associated with the feeding of sorghum grain infected with sorghum ergot (Claviceps africana). There was mild to severe feed refusal when the sorghum was first offered. Sows fed ergot before farrowing had shrunken udders, produced no colostrum, and displayed signs of oestrus. All of their piglets died, apparently from starvation: necropsy of a few piglets showed that they were born alive and walked, but had ingested no milk. Sows fed the grain after farrowing had severe reductions in milk production despite aggressive sucking by piglets, leading to very poor growth of piglets. There were no signs of infectious disease. Ergot in sorghum samples ranged from 1 to 31% ergot sclerotes by weight. Total alkaloid concentrations in mixed feeds ranged from 5 to 40 mg/kg, with dihydroergosine accounting for approximately 90%. At the same time, in the same districts, there were reports of feed refusal and reduced milk production from 4 dairy farms. Grain samples from these farms contained up to 17% C africana ergot sclerotia. CONCLUSION: Agalactia and feed refusal are classical signs of poisoning by rye ergot (C purpurea), but this is the first time that sorghum ergot has been associated with a similar syndrome.

Animal Feed↗
PubMed2003

Evidence that enforced sunlight exposure can cause hyperthermia in cattle ingesting low levels of ergot of rye (Claviceps purpurea), when air temperature and humidity conditions are only moderate.

OBJECTIVE: To determine the rectal temperature response of cattle, following the oral administration of ergot of rye (Claviceps purpurea), under pen conditions of enforced sunlight compared with those of enforced shade. DESIGN AND PROCEDURE: Hereford cross steers were divided into two groups of 18. One group was dosed once, on a Monday morning, with finely ground rye grass ergots at a rate of 180 mg/kg body weight and held in the sun for 7 h each day until Friday afternoon. The other group was not dosed but was similarly held in the sun during the same period. Their rectal temperatures were measured early morning and mid afternoon, from Monday to Friday inclusive. The process was repeated for each group, but this time they were held in the shade. The four treatment options were run concurrently by conducting the experiments over 6 weeks and using 3 animals in each treatment group, each week. RESULTS: The thermic response over all weeks, of the ergot treated, sunlight exposed cattle, was deemed greater than for the other groups, based on the following four parameters. The increase in rectal temperature between early morning and mid afternoon, the size of the mid afternoon rectal temperature rise, the difference between the maximum mid afternoon rectal temperature recorded by an animal in the sun compared with that recorded by the same animal in the shade, and finally the number of animals in a treatment group that recorded rectal temperatures > 40.00 degrees C. The difference in the daily increase in body temperature between the ergot treated, sun exposed cattle and the ergot treated, shaded cattle, was greater than that observed between the sun exposed and shade restricted control cattle. Nine of 18 ergot treated and sun exposed cattle developed hyperthermia; no cattle in the other three groups did. CONCLUSIONS: Some sunlight exposed cattle, dosed with a low amount of ergot of rye, can experience a body temperature elevation above the normal range, even under mild ambient temperature and humidity conditions. Sunlight exposure causes an increase in body temperature in normal cattle that is probably attributable to solar radiant heat. Ergot ingestion causes an increase in body temperature in shaded cattle that is probably attributable to ambient temperature and humidity effects. This response can be increased by sun exposure and this increase is attributable to solar radiant heat effects and possibly also solar radiation. A response to radiant heat is consistent with the known physiological effects of ingested ergot alkaloids, but a response to solar radiation is not.

Animal Feed↗
PubMed1983

Effect of biotin on alkaloid production during submerged cultivation of Claviceps sp. strain SD-58.

Addition of biotin to culture medium NL-406 significantly increased alkaloid yield during submerged cultivation of Claviceps sp. strain SD-58. Alkaloid yield was further enhanced by incorporating leucine in biotin-supplemented culture medium. Increased alkaloid production was associated with an increase in the lipid content of cells and in the number of chlamydospores. Biotin deficiency caused a reduction in alkaloid yield and a parallel decrease in lipid content and chlamydospore numbers.

Alkaloids↗
PubMed1984

Extracellular metabolism of sucrose in a submerged culture of Claviceps purpurea: formation of monosaccharides and clavine alkaloids.

Transformation of extracellular sucrose during cultivation of Claviceps purpurea led to the formation of mono- and oligosaccharides. Maltose was a suitable substrate for submerged fermentation of alkaloids. Fermentation in a medium with maltose was characterized by an insignificant formation of glucans, intensive sporulation, suspension growth of mycelium, and a higher formation of elymoclavine. Glucose alone yielded low levels of total alkaloids and high glucan formation; on the other hand, glucose promoted the formation of elymoclavine.

Claviceps↗
PubMed2000

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 microm long, the conidia of G2 isolates were 7 to 10 microm long, and the conidia of G3 isolates were 10 to 12 microm 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.

Alkaloids↗