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.     

The NADPH oxidase Cpnox1 is required for full pathogenicity of the ergot fungus Claviceps purpurea

The role of reactive oxygen species (ROS) in interactions between phytopathogenic fungi and their hosts is well established. An oxidative burst mainly caused by superoxide formation by membrane-associated NADPH oxidases is an essential element of plant defence reactions. Apart from primary effects, ROS play a major role as a second messenger in host response. Recently, NADPH oxidase (nox)-encoding genes have been identified in filamentous fungi. Functional analyses have shown that these fungal enzymes are involved in sexual differentiation, and there is growing evidence that they also affect developmental programmes involved in fungus-plant interactions. Here we show that in the biotrophic plant pathogen Claviceps purpurea deletion of the cpnox1 gene, probably encoding an NADPH oxidase, has impact on germination of conidia and pathogenicity: Deltacpnox1 mutants can penetrate the host epidermis, but they are impaired in colonization of the plant ovarian tissue. In the few cases where macroscopic signs of infection (honeydew) appear, they are extremely delayed and fully developed sclerotia have never been observed. C. purpurea Nox1 is important for the interaction with its host, probably by directly affecting pathogenic differentiation of the fungus.     

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.     

Correlation between growth and ergot alkaloid biosynthesis in Claviceps purpurea batch fermentation

Summary Following the growth kinetics of a C. purpurea ergotoxine-producing strain it was observed that alkaloid synthesis and alkaloid distribution depend on fungal growth velocity during idiophase. Formation of extracellular alkaloids is favoured by higher fungal growth velocity, while intracellular alkaloids begin to accumulate at a lower rate of growth. Simple lysergic acid derivatives prevali among extracellular alkaloids, whereas ergotoxines predominate among intracellular alkaloids. By varying cultivation conditions, by feeding the culture, or by varying the inoculum size, alkaloid composition can be influenced within the limits of strain capabilities.     

Epidemiology of ergot disease (Claviceps purpurea) in open-flowering male-sterile cereals

Ergot sclerotia collected from one source germinated over a period of about 6 wk, but those from many sources, over a period of about 5 months. The duration of ascospore discharge followed a similar pattern.
Individual ovaries of male-sterile barley remained susceptible to infection for about 3 wk. After infection, 7 to 10 days elapsed before honeydew production started and this secondary source of inoculum (conidia) continued to be produced for about 2 wk in individual ovaries before ergot sclerotia started to develop.
A field trial demonstrated that the duration of ovary susceptibility is lengthened in practice because of unevenness in flowering and the production of successive flushes of tillers. The potential for increase of the disease from a small primary focus of infection was large with the majority of the infections occurring in the back- or late-tillers.     

De novo biosynthesis of cytokinins in the biotrophic fungus Claviceps purpurea

Disease symptoms of some phytopathogenic fungi are associated with changes in cytokinin (CK) levels. Here, we show that the CK profile of ergot‐infected rye plants is also altered, although no pronounced changes occur in the expression of the host plant's CK biosynthesis genes. Instead, we demonstrate a clearly different mechanism: we report on the first fungal de novo CK biosynthesis genes, prove their functions and constitute a biosynthetic pathway. The ergot fungus Claviceps purpurea produces substantial quantities of CKs in culture and, like plants, expresses enzymes containing the isopentenyltransferase and lonely guy domains necessary for de novo isopentenyladenine production. Uniquely, two of these domains are combined in one bifunctional enzyme, CpIPT‐LOG, depicting a novel and potent mechanism for CK production. The fungus also forms trans‐zeatin, a reaction catalysed by a CK‐specific cytochrome P450 monooxygenase, which is encoded by cpp450 forming a small cluster with cpipt‐log. Deletion of cpipt‐log and cpp450 did not affect virulence of the fungus, but Δcpp450 mutants exhibit a hyper‐sporulating phenotype, implying that CKs are environmental factors influencing fungal development.     

Biochemical characterization of the inoculum of Claviceps purpurea for submerged production of ergot alkaloids

Summary Biochemical and morphological changes during the growth and development of Claviceps purpurea seed cultures under submerged conditions were established. The fall in RNA content at about the middle of the exponential phase of growth has been found to be associated with the transition from sphacelial to sclerotial growth and may be considered the major indicator of changes in the metabolism of the fungus. It may also serve as a biochemical indicator for the optimal activity of Claviceps purpurea seed cultures.     

Activation of ergot alkaloid biosynthesis in prototrophic isolates by Claviceps purpurea protoplast fusion.

Intraspecific protoplast fusions were carried out with active ergocornine-ergokryptine and inactive ergocristine Claviceps purpurea strains and vice versa. The isolated prototrophic strains from both types of crosings produced all three alkaloid types, showing that biosynthesis of distinct alkaloid was activated in an inactive partner strain. The prototrophic isolates were stable on minimal medium but they segregated by subculturing on complete medium. In comparison with the original partner strains, differences in morphological and cytological characteristics were also established.     

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 well wall and in the vacuoles of aged cells (autolysosomes). Phosphatase activity was present also in particulate intracytoplasmatic organelles. The concentric layering of lead deposits in these organelles indicates their relationship to endoplasmic reticulum membranes.Abbreviation Used pNPP p-nitrophenylphosphate     

Effect of the composition of a nutrient medium on growth and synthesis of ergot alkaloids by Claviceps purpurea in the saprotrophic culture]

The impact of changes in the ratio of the medium-668 components on growth and synthesis of ergot alkaloids in saprotrophic cultures was studied with mathematical design of the experiment. Medium-668 used for cultivation of strain VNIIA-312A-producing peptide +ergot alkaloids was shown to be balanced with respect to the ratio of all the medium components. An important role of phosphate in control of culture growth and alkaloids synthesis was elucidated. It was demonstrated that by changing the ratio of the medium components it was possible to control accumulation and excretion of the alkaloids which permitted development of the conditions required for product isolation.     

Association of insects and ergot (Claviceps purpurea) in Kentucky bluegrass seed production fields.

Insects in Kentucky bluegrass seed production fields in Oregon, Idaho, and Washington were sampled just before harvest and their association with ergot conidia of Claviceps purpurea Fr. (Tul.) was evaluated during 1996-1998. A diversity of insects was observed at all three locations. The most abundant beneficial insects collected with sweep nets were Nysium spp., Nabis spp., ichneumonid wasps, and Hippodamia spp. The cranberry girdler, Chrysoteuchia topiaria (Zeller), was the only important pest on grass seed collected by sweep net. Numbers of aphids such as Sitobion avenae (F.), cicadellids and thrips such as Anaphothrips spp. and Aptinothrips spp. that were collected with all aphid sampler were below economic thresholds. Other insect groups occurred in low numbers. Noctuid moths collected in universal blacklight traps included nine species of cutworms and armyworms. Protogrotis obscura (B. & McD.) was the most common cutworm species and was present in all fields. The moth Chortodes rufostrigata (Pack.) previously reported only from wet meadows in northeast and south central Oregon was found in Kentucky bluegrass fields in central Oregon, suggesting that irrigated Kentucky bluegrass seed production fields may simulate a montane meadow habitat. Conidia of C. purpurea were found on a diversity of insects, including moths, flies, leafhoppers, and thrips. Up to 100% of moths and 75% of flies collected from some fields carried conidia of C. purpurea. No correlation between ergot honeydew present in a field and number of insects with conidia of C. purpurea was detected.     

An oleate hydroxylase from the fungus Claviceps purpurea: cloning, functional analysis, and expression in Arabidopsis

Claviceps purpurea, a fungal pathogen responsible for ergot diseases in many agriculturally important cereal crops, produces high levels of ricinoleic acid (12-hydroxyoctadec-cis-9-enoic acid) in its sclerotia. It has been believed for many years that the biosynthesis of this fatty acid in C. purpurea involves a hydration process with linoleic acid as the substrate. Using degenerate polymerase chain reaction, we cloned a gene from the sclerotia encoding an enzyme (CpFAH) that has high sequence similarity to the C. purpurea oleate desaturase, but only low similarity to plant oleate hydroxylases. Functional analysis of CpFAH in yeast (Saccharomyces cerevisiae) indicated it acted predominantly as a hydroxylase, introducing hydroxyl groups at the 12-position of oleic acid and palmitoleic acid. As well, it showed Delta(12) desaturase activities on 16C and 18C monounsaturated fatty acids and, to a much lesser extent, omega(3) desaturase activities on ricinoleic acid. Heterologous expression of CpFAH under the guidance of a seed-specific promoter in Arabidopsis (Arabidopsis thaliana) wild-type and mutant (fad2/fae1) plants resulted in the accumulation of relatively higher levels of hydroxyl fatty acids in seeds. These data indicate that the biosynthesis of ricinoleic acid in C. purpurea is catalyzed by the fungal desaturase-like hydroxylase, and CpFAH, the first Delta(12) oleate hydroxylase of nonplant origin, is a good candidate for the transgenic production of hydroxyl fatty acids in oilseed crops.     

The ergot alkaloid gene cluster in Claviceps purpurea: extension of the cluster sequence and intra species evolution

The genomic region of Claviceps purpurea strain P1 containing the ergot alkaloid gene cluster [Tudzynski, P., H?lter, K., Correia, T., Arntz, C., Grammel, N., Keller, U., 1999. Evidence for an ergot alkaloid gene cluster in Claviceps purpurea. Mol. Gen. Genet. 261, 133-141] was explored by chromosome walking, and additional genes probably involved in the ergot alkaloid biosynthesis have been identified. The putative cluster sequence (extending over 68.5kb) contains 4 different nonribosomal peptide synthetase (NRPS) genes and several putative oxidases. Northern analysis showed that most of the genes were co-regulated (repressed by high phosphate), and identified probable flanking genes by lack of co-regulation. Comparison of the cluster sequences of strain P1, an ergotamine producer, with that of strain ECC93, an ergocristine producer, showed high conservation of most of the cluster genes, but significant variation in the NRPS modules, strongly suggesting that evolution of these chemical races of C. purpurea is determined by evolution of NRPS module specificity.     

Preservation of Claviceps purpurea, the producer of peptide ergot alkaloids, by the method of freeze drying

A saprotrophic strain of Claviceps purpurea VNIIA 312A, an organism producing peptide +ergot alkaloids with prolactin inhibiting activity was shown to die under lyophilization conditions. To provide long-term storage of strain 312A, L-drying or drying under vacuum from liquid state was used with success. Three protective media were tested. Favourable results were obtained by using 25 per cent maltose solution as a protective medium. Preservation of the culture viability was accompanied by maintenance of the culture capacity for active formation of the biomass and production of +ergot alkaloids.     

Isolation and Characterization of Vacuoles from the Ergot Fungus Claviceps purpurea

Protoplasts of Claviceps purpurea were prepared by treatment of mycelium with a lytic mixture of snail gut enzyme and cellulase from Trichoderma viride. Such protoplasts could be efficiently lysed by Triton X-100 treatment at high osmotic pressure without Ca²⁺ or Mg²⁺, allowing the release of intact vacuoles in high yields. Vacuoles obtained from cells grown in modified Vogel medium (vegetative-type cells not producing alkaloids) were isolated and purified by centrifugation from a 5% Ficoll 400 (wt/vol) phase into the interphase between two layers, one containing 0.25 M each of mannitol and sucrose, and one containing 0.5 M mannitol. Vacuoles derived from cells grown in a medium favoring ergot alkaloid synthesis (sclerotia-like cells) were isolated by gentle centrifugation of filtered protoplast lysates without addition of Ficoll 400. Biochemical analyses of the vacuole fraction isolated from either kind of cell revealed their function as compartments harboring several hydrolytic enzymes. However, the enrichment of free amino acids in vacuoles of sclerotia-like cells was less pronounced than that in vacuoles of vegetative-type cells, indicating a difference in metabolic compartmentation in the two types of cells.