Ergot alkaloids in sclerotia collected in Japan: synthetic profiles and induction of apoptosis by Clavine-type compounds.

The genus Claviceps (Clavicipitaceae) is famous for producing ergot alkaloids (EAs) in sclerotia. EAs can cause ergotism, resulting in convulsions and necrosis when ingested, making these compounds a serious concern for food safety. Agroclavine (2), a typical Clavine-type EA, is a causative agent of ergotism and is listed as a compound to be monitored by the European Food Safety Authority. Clavine-type EAs are known to cause cytotoxicity, but the mechanism has not been elucidated. We performed annexin V and PI double-staining followed by flow cytometric analysis to detect apoptosis in HepG2 and PANC-1 cells after exposure to Clavine-type EAs. Clavine-type EAs reduced cell viability and induced apoptosis in both cell lines. We then performed LC-MS analysis of EAs from 41 sclerotia samples of Claviceps collected in Japan. 24 out of 41 sclerotia extracts include peptide-type EAs (ergosine/inine: 4/4', ergotamine: 5, ergocornine/inine: 6/6', α-ergocryptine/inine: 8/8', and ergocristine/inine: 9/9') and 19 sclerotia extracts among 24 sclerotia detected peptide type EAs include Clavine-type EAs (pyroclavine: 1, agroclavine: 2, festuclavine: 3) by LC-MS. We then performed a metabolomic analysis of the EAs in the sclerotia using principal component analysis (PCA). The PCA score plots calculated for EAs suggested the existence of four groups with different EA production patterns. One of the groups was formed by the contribution of Clavine-type EAs. These results suggest that Clavine-type EAs are a family of compounds requiring attention in food safety and livestock production in Japan.

Claviceps purpurea expressing polygalacturonases escaping PGIP inhibition fully infects PvPGIP2 wheat transgenic plants but its infection is delayed in wheat transgenic plants with increased level of pectin methyl esterification.

Claviceps purpurea is a biotrophic fungal pathogen of grasses causing the ergot disease. The infection process of C. purpurea on rye flowers is accompanied by pectin degradation and polygalacturonase (PG) activity represents a pathogenicity factor. Wheat is also infected by C. purpurea and we tested whether the presence of polygalacturonase inhibiting protein (PGIP) can affect pathogen infection and ergot disease development. Wheat transgenic plants expressing the bean PvPGIP2 did not show a clear reduction of disease symptoms when infected with C. purpurea. To ascertain the possible cause underlying this lack of improved resistance of PvPGIP2 plants, we expressed both polygalacturonases present in the C. purpurea genome, cppg1 and cppg2 in Pichia pastoris. In vitro assays using the heterologous expressed PGs and PvPGIP2 showed that neither PG is inhibited by this inhibitor. To further investigate the role of PG in the C. purpurea/wheat system, we demonstrated that the activity of both PGs of C. purpurea is reduced on highly methyl esterified pectin. Finally, we showed that this reduction in PG activity is relevant in planta, by inoculating with C. purpurea transgenic wheat plants overexpressing a pectin methyl esterase inhibitor (PMEI) and showing a high degree of pectin methyl esterification. We observed reduced disease symptoms in the transgenic line compared with null controls. Together, these results highlight the importance of pectin degradation for ergot disease development in wheat and sustain the notion that inhibition of pectin degradation may represent a possible route to control of ergot in cereals.

Molecular analysis of ∆6 desaturase and ∆6 elongase from Conidiobolus obscurus in the biosynthesis of eicosatetraenoic acid, a ω3 fatty acid with nutraceutical potentials.

Conidiobolus obscurus, an entomopathogenic fungus able to infect aphids, was previously reported to produce substantial amounts of very long chain polyunsaturated fatty acids (VLCPUFAs) that may mediate the insect infection. However, the genes involved in the biosynthesis of these VLCPUFAs from the order Entomophthorales have yet to be identified. Using degenerate reverse transcriptase-polymerase chain reaction and rapid amplification of the cDNA end methods, we cloned a ∆6 desaturase cDNA (CoD6) and a ∆6 elongase cDNA (CoE6) from C. obscurus. Expression of CoD6 and CoE6 in Saccharomyces cerevisiae revealed CoD6 could introduce a Δ6 double bond into α-linolenic acid (18:3n-3), and CoE6 preferentially elongated 18-carbon Δ6 desaturated fatty acid stearidonic acid (18:4n-3). When the fungus was grown under a temperature shift from 20 °C to 10 °C, the transcript level of CoD6 and CoE6 increased, whereas when the fungal culture was shifted from 20 °C to 30 °C, the transcript level of both genes decreased. The entire eicosatetraenoic acid biosynthetic pathway was reconstituted in yeast using four genes, CoD6 and CoE6 from C. obscurus, CpDes12 (a Δ12 desaturase) and CpDesX (a ω3 desaturase) from Claviceps purpurea. Yeast transformants expressing the four genes produced ten new fatty acids including the final product eicosatetraenoic acid (ETA). This represents the reconstitution of the entire ETA pathway in yeast without supplementation of any exogenous fatty acids.

Deletion of Mid1, a putative stretch-activated calcium channel in Claviceps purpurea, affects vegetative growth, cell wall synthesis and virulence.

The putative Claviceps purpurea homologue of the Saccharomyces cerevisiae stretch-activated calcium ion channel Mid1 was investigated for its role in vegetative growth, differentiation and pathogenicity on rye (Secale cereale). Gene replacement mutants of Cl. purpurea mid1 were not affected in polar growth and branching in axenic culture but showed a significantly reduced growth rate. The growth defect could not be complemented by Ca(2+) supplementation, in contrast to mid1 mutants in yeast, but the altered sensitivity of the mutants to changes in external and internal Ca(2+) concentrations indicates some role of Mid1 in Ca(2+) homeostasis. The major effect of mid1 deletion, however, was the complete loss of virulence: infected rye plants showed no disease symptoms at all. Detailed analyses of in vitro-infected rye ovaries demonstrated that the Deltamid1 mutants had multiple apical branches and were unable to infect the host tissue, suggesting that Mid1 is essential for generating the necessary mechanical force for penetration. This is believed to be the first report of an essential role for a Mid1 homologue in the virulence of a plant-pathogenic fungus.

A novel medium for the enhanced production of cyclosporin A by Tolypocladium inflatum MTCC 557 using solid state fermentation.

Cyclosporin A (CyA) produced by Tolypocladium inflatum is a promising drug owing to its immunosuppressive and antifungal activities. From an industrial point of view, the necessity to obtain a suitable and economic medium for higher production of CyA was the aim of this work. The present study evaluated the effect of different fermentation parameters in solid state fermentation, such as selection of solid substrate, hydrolysis of substrates, initial moisture content, supplementation of salts, additional carbon, and nitrogen sources, as well as the inoculum age and size, on production of CyA by Tolypocladium inflatum MTCC 557. The fermentation was carried out at 25+/-2 degrees for 9 days. A combination of hydrolyzed wheat bran flour and coconut oil cake (1:1) at 70% initial moisture content supported a maximum production of 3,872+/-156 mg CyA/kg substrate as compared with 792+/-33 mg/kg substrate before optimization. Furthermore, supplementation of salts, glycerol (1%w/w), and ammonium sulfate (1%w/w) increased the production of CyA to 5,454+75 mg/kg substrate. Inoculation of 5 g of solid substrate with 6 ml of 72-h-old seed culture resulted in a maximum production of 6,480+95 mg CyA/kg substrate.

Effect of clomiphene on fatty acids, sterols and membrane fluidity in clavine producing Claviceps purpurea strains.

Clomiphene depressed the growth and enhanced clavine production of Claviceps purpurea strains 129,35 and 59. Mycelial content of 18:2 and 16:0 fatty acids decreased, whereas that of 18:1 and 18:0 acids increased. In the mutant strain 59 clomiphene, triadimefon and ergosterol stimulated the impaired function of chanoclavine cyclase. Their effect was counteracted by plant oil. Clomiphene decreased the content of total lipids (44%), triglycerides (32%), sterols (22%) and sterol/phospholipid molar ratio. The PC/PE ratio was 9X increased. Clomiphene and triadimefon enhanced membrane fluidity of protoplasts, ergosterol and oil reverted their effect.

Influence of tryptophan and related compounds on ergot alkaloid formation in Claviceps purpurea (FR.) Tul.

L-Tryptophan did not exert any influence on peptide alkaloid formation in an ergotamine and in an ergosine-accumulating C. purpurea strain. A different picture was observed in a series of related C. purpurea strains. Tryptophan showed a slight stimulatory effect on the ergotoxine producer Pepty 695/S. A blocked mutant of it, designated as Pepty 695/ch which was able to accumulate secoclavines gave similar results. In a high-yielding elymoclavine strain Pepty 695/e, the progeny of the former one, tryptophan up to a concentration of 25 mM stimulated remarkably clavine biosynthesis. Furthermore, tryptophan could overcome the block of synthesis by inorganic phosphate. Increased specific activities of chanoclavine cyclase but not DMAT synthetase were observed in cultures of strain Pepty 695/e supplemented with tryptophan. 5-Methyltryptophan and bioisosteres of tryptophan were ineffective in alkaloid stimulation. These results are compared with those obtained with the grass ergot strain SD 58 and discussed with the relation to other induction phenomena.

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.

Physiological study of ergot: induction of alkaloid synthesis by tryptophan at the enzymatic level.

The enhancement of ergot alkaloid production by tryptophan and its analogues in both normal and high-phosphate cultures is more directly related to increased dimethylallyltryptophan (DMAT) synthetase activity rather than to a lack of regulation of the tryptophan biosynthetic enzymes. Thiotryptophan [beta-(1-benzo-thien-3-yl)-alanine] is rather ineffective in the end product regulation of tryptophan biosynthesis, whereas tryptophan and 5-methyltryptophan are potent effectors. The presence of increased levels of DMAT synthetase in ergot cultures supplemented with tryptophan or thiotryptophan, and to a lesser extent with 5-methyltryptophan, suggests that the induction effect involves de novo synthesis of the enzyme. Thiotryptophan and tryptophan but not 5-methyltryptophan can overcome the block of alkaloid synthesis by inorganic phosphate. The results with thiotryptophan indicate that the phosphate effect cannot be explained merely on the basis of a block of tryptophan synthesis.

Ergot alkaloids. VI. Nitrogen metabolism during the development of sclerotium of Claviceps purpurea.

The developmental transition of ergot from the sphacelial to the sclerotial phase is accompanied by marked changes in morphology, growth and metabolism. The production of alkaloids is paralleled by a concomitant protein synthesis, a decline in the contents of both total N and P, and an attendant rise in the value of the N/P ratio. The total level of free amino acids drops with proceeding development of the sclerotium. The most conspicuous drop, observed at the beginning of the production phase, is due to a sharp decrease in the level of free lysine. This drop attests to profound changes in the metabolism of the parasite in the period of transition from nonproducing sphacelium to producing sclerotium. The gradually decreasing level of free proline reflects the accumulation of the alkaloids. In a parasitic culture, proline is probably supplied in sufficient amounts by the host plant and the main difference between the metabolism of fungal sphacelium and sclerotium is a different utilisation of acetyl-CoA.