pH-Dependent accumulation of anticancer compound on mycelia in fermentation of marine fungus.

The real-time distribution of anticancer 1403C in fermentation broth of marine fungus Halorosellinia sp. was investigated. It was closely related with pH variations, which was, 1403C in the supernatant decreased while that in the mycelia increased with pH rising. There was only 0.5 % of the total 1403C left in the supernatant when pH reached 7.0. The scanning electron microscope then provided information that compounds precipitated on the mycelia when pH rose. Then, the pH-regulation experiments proved that 1403C mainly secreted extracellular and easily dissolved in acidic condition but precipitated and absorbed on the mycelia with the increase of broth pH. Thereby, a pH-regulation strategy was proposed and applied to accumulate 1403C on the mycelia before draw-off of fermentation broth. It significantly simplified purification process and is critical for 1403C preparation of industrial scale.

Potentiation of mycovirus transmission by zinc compounds via attenuation of heterogenic incompatibility in Rosellinia necatrix.

Heterogenic incompatibility is considered a defense mechanism against deleterious intruders such as mycovirus. Rosellinia necatrix shows strong heterogenic incompatibility. In the heterogenic incompatibility reaction, the approaching hyphae hardly anastomosed, a distinctive barrage line formed, and green fluorescent protein (GFP)-labeled hyphae quickly lost their fluorescence when encountering incompatible hyphae. In this study, transmission of a hypovirulence-conferring mycovirus to strains with different genetic backgrounds was attempted. Various chemical reagents considered to affect the programmed cell death pathway or cell wall modification were examined. Treatment with zinc compounds was shown to aid in transmission of mycoviruses to strains with different genetic backgrounds. In incompatible pairings, treatment with zinc compounds accelerated hyphal anastomosis; moreover, cytosolic GFP was transmitted to the newly joined hyphae. These results suggest that zinc compounds not only increase hyphal anastomosis but also attenuate heterogenic incompatibility.

A new species and a new record of Diatrypaceae from Panama.

Based on opportunistic collections of fungi in Panama, two species of Diatrypaceae (Xylariales) are described and illustrated. One of them, Eutypella semicircularis, found twice on branches probably belonging to Alnus acuminata, is new to science. It differs from known species of Eutypella and Peroneutypa with strongly allantoid or semicircular ascospores, such as E. crustata and P. curvispora, in having larger ascospores that sometimes almost form a circle and broader stromata, among other differences. Molecular data of the ITS rDNA region show that the new species is a sister taxon of Eutypella cerviculata, the type species of the genus Eutypella. Diatrype bermudensis on stems of Guadua angustifolia is reported from Panama for the first time, being known up to now apparently only from Bermuda.

New Annulohypoxylon species from Brazil.

Four new Annulohypoxylon species are described from northeastern Brazil: A. austrobahiense, A. macrodiscum, A. hemicarpum and A. purpureopigmentum. A key is provided.

Diatrypasimilis australiensis, a novel xylarialean fungus from mangrove.

A marine xylarialean fungus, isolated from roots of Rhizophora (mangrove) in Australia, displays morphology of eight ellipsoidal dark brown ascospores in a cylindrical ascus having a refractive apical apparatus. Each ascospore has a longitudinal germ slit. The fungus grew very slowly and produced dark brown water-soluble pigment(s) on various media. It developed unique, column-shaped, indeterminate synnemata on which needle-shaped conidia were produced. The sexual stage of this fungus was not observed under the laboratory conditions tested. Molecular phylogenetic analyses of the combined sequences of nuclear ribosomal RNA genes and their internal transcribed spacers placed it at a basal position in the clade of Diatrypaceae of the Xylariales with comparatively high statistical support. However the morphological features and phylogenetic position of this organism do not closely resemble any known fungal taxa. Therefore this fungus is proposed to be a representative of a novel taxon and described as Diatrypasimilis australiensis gen. et sp. nov.

Diversity of diatrypaceous fungi associated with grapevine canker diseases in California.

Eutypa lata is the most common diatrypaceous fungus in grapevine cankers. This study was undertaken to determine the presence and diversity of other members of the Diatrypaceae in grapevines in California. We isolated into pure culture 11 species of Diatrypaceae from the wood of diseased grapevines including Cryptosphaeria pullmanensis, Cryptovalsa ampelina, Diatrype oregonensis, D. stigma, D. whitmanensis, Diatrype sp., Diatrypella verrucaeformis and four putative species of Eutypella. Species were identified or grouped morphologically and by phylogenetic analyses of the complete sequence of the internal transcribed spacer (ITS) of the rDNA and beta-tubulin gene. Diatrypaceous fungi also occurred frequently on the native flora surrounding vineyards, thus serving as possible inoculum sources for these putative pathogens.

Taxonomic and nomenclatural aspects of Hypoxylon taxa from southern South America proposed by Spegazzini.

The holotypes and isotypes of 20 Hypoxylon taxa described by Spegazzini have been examined and their taxonomic positions and nomenclatural problems are discussed. Two new combinations, Annulohypoxylon apiahynum comb. nov. and A. subeffusum comb. nov., are proposed. H. goliath is considered a synonym of Rosellinia bunodes. H. albostigmatosum and H. guarapiense are synonyms of H. anthochroum, H. anthracoderma of H. monticulosum, H. mbaiense of H. notatum, H. paulistanum of H. diatrypeoides, H. plumbeum and H. rubiginosum var. microcarpum of H. perforatum. H. porteri and H. intermedium belong in Biscogniauxia capnodes, H. puiggarii in Annulophypoxylon subeffusum, H. subvinosum. in H. lenormandii, H. turbinatum var. guaraniticum in Phylacia turbinata and H. valsarioides in Creosphaeria sassafras. H. leptascum is transferred to A. leptascum, H. circostomum to Nemania circostoma and H. latissimum to N. latissima. The holotype of H. albostigmatosum has been recovered, thus the lectotypification by Shear no longer is needed. H. subnigricans and H. umbilicatum are confirmed as good taxa. H. anthochroum and H. lenormandii are reported as first records from Argentina (Tucumán).

Microdochium triticicola sp. nov. from roots of Triticum aestivum in the United Kingdom.

Morphological and molecular studies led to the description of Microdochium triticicola as new species isolated from roots of wheat in the United Kingdom. The morphological features of all known Microdochium species are tabulated.

Theissenia rogersii sp. nov. and phylogenetic position of Theissenia.

Theissenia rogersii deviates from known Theissenia species primarily in having large ascospores with a thick wall layer and a unique configuration of two stromatal tissue types, one carbonaceous and the other fibrous. The carbonaceous tissue forms palisades on and beneath the perithecial layer as well as encasing individual perithecia, whereas the fibrous tissue fills the spaces between columns of the palisades as well as between encased perithecia. Phylogenetic analyses based on DNA sequences of beta-tubulin and alpha-actin genes placed Theissenia in the subfamily Hypoxyloideae among the genera that are characterized by having bipartite stromata (i.e. with the stromata differentiated into an outer dehiscing layer and an inner perithecium-bearing layer).

The genus Pyrenomyxa and its affinities to other cleistocarpous Hypoxyloideae as inferred from morphological and chemical traits.

Types and authentic specimens of Hypoxylon piceum, Pulveria porrecta, and Pyrenomyxa invocans were studied for morphological traits and extrolite (= secondary metabolite) profiles generated by analytical HPLC with UV-visual and mass spectrometric detection. The orange stromatal pigments of P. invocans are rubiginosin A and mitorubrinol. It lacks three different types of extrolites (BNT, macrocarpone and hypomiltin) that are known from Hypoxylon taxa and occur in H. piceum and P. porrecta. In agreement with morphological traits, the latter two names are regarded as synonymous and transferred to Pyrenomyxa. Another species from Eastern Russia, Pyrenomyxa morganii sp. nov., is recognized. It contains yet unidentified azaphilones besides BNT and orsellinic acid, and its culture produces 5-methylmellein and a virgariella-like anamorph. These findings suggest a close relationship of Pyrenomyxa to Hypoxylon and emphasize the utility of chemotaxonomic traits for fungal taxonomy in general. Pyrenomyxa is accepted ad interim until the phylogenetic relationships among Hypoxylon have been further evaluated by means of chemotaxonomic, morphological and molecular methods.

Cytological analysis of mycelial incompatibility in Rosellinia necatrix.

When the mycelia of Rosellinia necatrix encounter mycelia with a different genetic background, distinct barrage lines form. In this study, we observed hyphal interactions between compatible and incompatible R. necatrix pairs by means of light and electron microscopy. Although we observed perfect hyphal anastomosis in compatible pairs of isolates, the hyphae never anastomosed in incompatible pairs (i.e., the hyphae remained parallel or crossed over without merging). These behaviours appeared to result from the detection of or failure to detect one or more diffusible factors. The attraction to other hyphae in pairs of incompatible isolates was increased by supplementation of the growing medium with activated charcoal, although no anastomosis was observed and ultrastructural observation confirmed a lack of hyphal anastomosis. Programmed cell death (PCD) started with one of the two approaching hyphae. Heterochromatin condensation and genomic DNA fragmentation were not observed. Moreover, cell damage began with the tonoplast and continued with the plasma and nuclear membranes, suggesting that the PCD observed in heterogenic incompatibility of R. necatrix was a vacuole-mediated process.

[Leptosphaeria (?) senegalensis, Segrétain, Baylet, Darasse et Camain. Perithecia and asci. II. Asci and ascospores]. / Les périthèces et les asques du Leptosphaeria (? Senegalensis Segretain, Baylet, Darasse et Camain. II. Les asques et les ascospores.

The ascal wall of Leptosphaeria senegalensis typically belongs to the bitunicate type; in its fin al state it comprises an exoascus separated from the endoascus by a clear space. The three-layered endoascus is formed of fibrillae whose most internal are accordion-like folded. The ascospores have a complex wall; from the interior to the exterior it comprises: an endospore, an irregularity thickened epispore, a perispore, and an ectospore thickened in a cap at the posterior end of the spore. The comparison of ascospores of two "real" Leptosphaeria (L. acuta et L. maculans) with those of L. senegalensis shows that the latter species must no longer be included in the genus Leptosphaeria.

[Electron microscopy of mitochondrial DNA in Podospora anserina and the presence of a multimeric range of circular DNA molecules from senescent cultures]. / Etude au microscope électronique du DNA mitochondrial de Podospora anserina et présence d'une série multimérique de molécules circulaires de DNA dans des cultures sénescentes.

Mitochondrial DNA from young cultures of race s of Podospora anserina was isolated. Its density in Cesium chloride density equilibrium gradients was 1.694 g/cc. Examination by the electron microscope revealed that ca 1% of this DNA consisted of circles, 31 micrometer in contour length; the remaining DNA was composed of linear molecules ranging in length from 2 to 33 micrometer. In DNA of similar density obtained from senescent cultures of the same race s, about 11% of the molecules consisted of a multimeric set of circles ranging in size from 0.9 to 15 micrometer, with most being in the 1.8 and 2.7 micrometer classes. The similarity of these DNA molecules with the mitochondrial DNA from rho(-) yeast mutants is discussed.

Cryptic species related to Daldinia concentrica and D. eschscholzii, with notes on D. bakeri.

Daldinia macaronesica (from the Canary Islands and Madeira), D. palmensis (from the Canary Islands), D. martinii and D. raimundi (from Sicily), and D. vanderguchtiae (from Jersey, Channel Islands) spp. nov., are described, based on new combinations of teleomorphic and anamorphic characters. They all resemble the pantropical D. eschscholzii and/or the European D. concentrica with regard to teleomorphic characters and secondary metabolite profiles generated by analytical HPLC. The status of the newly described taxa was established by SEM of ascospores and microscopic studies of their anamorphs in comparison with various materials of the aforementioned known species. HPLC and SEM studies on the holotype of D. bakeri confirmed its relationships to D. fissa and D. loculata. Yellowish pigments contained in the type specimen of D. bakeri are probably artificial.

Successful crosses and molecular tetrad and progeny analyses demonstrate heterothallism in Mycosphaerella graminicola.

Monospore isolates of Mycosphaerella graminicola considered to originate from one ascus were analysed by the polymerase chain reaction (PCR) with 32 RAPD primers. Eighteen of these revealed three classes of polymorphisms, which enabled a RAPD-based tetrad analysis. Four pairs of isolates resulting from a single diploid nucleus were determined. A procedure to cross these isolates was developed to investigate the mating system. Three of six crosses were successful, and the segregation of mating types in accordance with the tetrad analysis strongly points to a bipolar heterothallic mating system in M. graminicola. Random ascospore progenies from the successful crosses, each comprising 54 isolates, were studied with three primers to determine the mode of inheritance of the RAPD markers. Mendelian segregation and recombination of RAPD markers was observed in all progenies.

Ultrastructure of developing ascospores in Sordaria brevicollis.

The ultrastructure of ascospore wall formation in the pyrenomycete Sordaria brevicollis was studied in developing asci at progressive time intervals. From early spore delimitation through final stage of maturation, the wall of the ascospore differentiated into four composite layers, the periascosporium the delineation ascosporium, the subascosproium, and the endoascosproium, While ascospores were at the hyaline stage of development,they possessed only the periascosporium and delineation ascosporium as their wall components. At about 7 to 8 days from the initiation of the cross, the spores developed a yellow color, and this coloration was always associated with the elaboration of the subascorsporium just internal to the ascosporium. Asthe spores continued to progressively darken in color, the subascosporium was seen to increase in complexity, electron density, and thickness. Soon after the formation of the subascosporium, the endoascosporium began to develop de novo and was, therefore, the last wall layer formed as the spore approached maturity.

Isolation and characterization of plasma membranes from the fungus Podospora anserina.

This paper describes a method for separating and isolating plasma membranes from the septated fungus Podospora anserina. Plasma membranes were isolated from protoplasts (young cell plasma membranes) and mycelia (both young and aged cell plasma membranes). The procedure of fractionation consisted of a combination of differential and isopycnic centrifugations. Characterization of cellular membranes and enrichment of the fractions with plasmalemma were carried out by assays on enzymatic activities. A plasma membrane fraction was isolated in a buoyant density peak of 1.087 g/cm3, where three enzymatic activities bound to plasma membrane, adenylate cyclase, chitin synthase, and beta-glucan synthase at low affinity for UDP-Glc, peaked together. Good purity of this fraction was determined by the absence or the very low level of other enzymatic activities used as markers for intracellular membranes, i.e., succinate dehydrogenase, alpha-mannosidase, NADPH cytochrome c reductase, and beta-glucan synthase at high affinity for UDP-Glc activities.

Incompatibility in Podospora anserina: comparative properties of the antagonistic cytoplasmic factors of a nonallelic system.

In the fungus Podospora anserina, the interaction between the nonallelic incompatible R and V genes has two consequences: a lytic reaction due to the synthesis of specific proteolytic enzymes, and a quenching in protein and ribonucleic acid synthesis. The incompatibility reaction when vegetative or sexual R and V cells fuse is asymmetric: it is induced only in the R protoplasm. The cessation in ribonucleic acid and protein synthesis was investigated in heterokaryotic strains carrying the antagonistic R and V genes and their "neutral" r and v alleles. Asymmetry between R and V genes lies in the fact that the strains homozygous for the R genes are the only strains that cannot grow. From these results it is postulated that the V-gene product is a diffusible cytoplasmic factor and that the R-gene product, which is nonautonomous, is a ribosomal component.

A nonmammalian homolog of the PAF1 gene (Zellweger syndrome) discovered as a gene involved in caryogamy in the fungus Podospora anserina.

The car1 gene of the filamentous fungus Podospora anserina was cloned by complementation of a mutant defective for caryogamy (nuclear fusion), a process required for sexual sporulation. This gene encodes a protein that shows similarity to the mammalian PAF1 protein (Zellweger syndrome). Besides sequence similarity, the two proteins share a transmembrane domain and the same type of zinc finger motif. A combination of molecular, physiological, genetical, and ultrastructural approaches gave evidence that the P. anserina car1 protein is actually a peroxisomal protein. This study shows that peroxisomes are required at a specific stage of sexual development, at least in P. anserina, and that a functional homolog of the PAF1 gene is present in a lower eucaryote.