Morphological differences between aerial and submerged sporidia of bio-fongicide Pseudozyma flocculosa CBS 16788.

Pseudozyma flocculosa is a fungus very useful and highly efficient as a biocontrol agent against powdery mildew. The reproduction of this fungus occurs exclusively by asexual production of conidia or sporidia that are the most suitable form for agricultural use and seems to be the most resistant to storage conditions. Despite the advantages offered by P. flocculosa in biological control, the use of this fungus use remains largely limited compared to that of chemical fungicides, at least partly due to the difficulty to obtain sporidia resistant to adverse environmental stresses in submerged culture conditions. Under solid-state and submerged-state cultivation, P. flocculosa strain CBS 16788 produced different types of sporidia. The submerged sporidia (SS) appeared relatively uniform in size, which was 15,4 ± 1,6 µm µm long, and 2,8 ± 0.8 µm wide. The aerial sporidia (AS) varied in shape and size, with a mean length of 8,2 ± 3 µm and width of 2,3 ± 0.6 µm. Under scanning and transmission electron microscopy, the cell wall of submerged sporidia was thinner than that of aerial spores, and the surface was smooth in contrast to the aerial sporidia that had a tendency to have verrucous, brittle surface characteristics. The thickness of the aerial sporidia wall is due to the presence of an outer layer rich in melanin. The sporidia germination was compared on YMPD (yeast extract, malt extract, soy peptone, dextrose and agar) coated coverslips. The aerial sporidia did not show germ tubes until 5 h of incubation, while the submerged sporidia showed many germ tubes after the same time. The resistance against the adverse environmental conditions in relation to the type of sporidia of P. flocculosa is discussed.

Metabolism-dependent bioaccumulation of uranium by Rhodosporidium toruloides isolated from the flooding water of a former uranium mine.

Remediation of former uranium mining sites represents one of the biggest challenges worldwide that have to be solved in this century. During the last years, the search of alternative strategies involving environmentally sustainable treatments has started. Bioremediation, the use of microorganisms to clean up polluted sites in the environment, is considered one the best alternative. By means of culture-dependent methods, we isolated an indigenous yeast strain, KS5 (Rhodosporidium toruloides), directly from the flooding water of a former uranium mining site and investigated its interactions with uranium. Our results highlight distinct adaptive mechanisms towards high uranium concentrations on the one hand, and complex interaction mechanisms on the other. The cells of the strain KS5 exhibit high a uranium tolerance, being able to grow at 6 mM, and also a high ability to accumulate this radionuclide (350 mg uranium/g dry biomass, 48 h). The removal of uranium by KS5 displays a temperature- and cell viability-dependent process, indicating that metabolic activity could be involved. By STEM (scanning transmission electron microscopy) investigations, we observed that uranium was removed by two mechanisms, active bioaccumulation and inactive biosorption. This study highlights the potential of KS5 as a representative of indigenous species within the flooding water of a former uranium mine, which may play a key role in bioremediation of uranium contaminated sites.

Cryptic diversity in Tranzscheliella spp. (Ustilaginales) is driven by host switches.

Species of Tranzscheliella have been reported as pathogens of more than 30 genera of grasses (Poaceae). In this study, a combined morphological and molecular phylogenetic approach was used to examine 33 specimens provisionally identified as belonging to the T. hypodytes species complex. The phylogenetic analysis resolved several well-supported clades that corresponded to known and novel species of Tranzscheliella. Four new species are described and illustrated. In addition, a new combination in Tranzscheliella is proposed for Sorosporium reverdattoanum. Cophylogenetic analyses assessed by distance-based and event-cost based methods, indicated host switches are likely the prominent force driving speciation in Tranzscheliella.

Sugarcane smut: shedding light on the development of the whip-shaped sorus.

Background and Aims: Sugarcane smut is caused by the fungus Sporisorium scitamineum (Ustilaginales/Ustilaginomycotina/Basidiomycota), which is responsible for losses in sugarcane production worldwide. Infected plants show a profound metabolic modification resulting in the development of a whip-shaped structure (sorus) composed of a mixture of plant tissues and fungal hyphae. Within this structure, ustilospores develop and disseminate the disease. Despite the importance of this disease, a detailed histopathological analysis of the plant-pathogen interaction is lacking. Methods: The whip-shaped sorus was investigated using light microscopy, scanning and transmission electron microscopy, histochemical tests and epifluorescence microscopy coupled with deconvolution. Key Results: Sorus growth is mediated by intercalary meristem activity at the base of the sorus, where the fungus causes partial host cell wall degradation and formation of intercellular spaces. Sporogenesis in S. scitamineum is thallic, with ustilospore initials in intercalary or terminal positions, and mostly restricted to the base of the sorus. Ustilospore maturation is centrifugal in relation to the ground parenchyma and occurs throughout the sorus median region. At the apex of the sorus, the fungus produces sterile cells and promotes host cell detachment. Hyphae are present throughout the central axis of the sorus (columella). The plant cell produces callose around the intracellular hyphae as well as inside the papillae at the infection site. Conclusions: The ontogeny of the whip-shaped sorus suggests that the fungus can cause the acropetal growth in the intercalary meristem. The sporogenesis of S. scitamineum was described in detail, demonstrating that the spores are formed exclusively at the base of the whip. Light was also shed on the nature of the sterile cells. The presence of the fungus alters the host cell wall composition, promotes its degradation and causes the release of some peripheral cells of the sorus. Finally, callose was observed around fungal hyphae in infected cells, suggesting that deposition of callose by the host may act as a structural response to fungal infection.

Detection of Tilletia controversa using immunofluorescent monoclonal antibodies.

AIMS: Tilletia controversa is an internationally quarantined pathogenic fungus that causes dwarf bunt of wheat and is similar to Tilletia caries in both teliospore morphology and genetic structure. This study developed a rapid and sensitive immunofluorescence method for differentiating the teliospores of T. controversa from T. caries. METHODS AND RESULTS: The method utilizes monoclonal antibody D-1 against teliospores of T. controversa as well as a PE-Cy3-conjugated goat anti-mouse antibody (overlapping light excitation of 495 and 555 nm). The orange cycle fluorescent signal was stronger against T. controversa teliospores in the outer spore wall and net ridge, whereas only the green signal was observed for the protoplasm of T. caries teliospores. The detection limit of this method was 2.0 µg ml(-1) of the D-1 monoclonal antibody. CONCLUSION: This study describes the production and diagnostic application of a novel mouse monoclonal antibody specific to T. controversa teliospores. SIGNIFICANCE AND IMPACT OF THE STUDY: This method could be used for the on-site identification of T. controversa teliospores in the near future and will help in selecting fungicides to control dwarf bunt of wheat as further technical developments are achieved.

Ultrastructure of spined conidia and hyphae of the rice false smut fungus Ustilaginoidea virens.

Spined conidia and hyphae of Ustilaginoidea virens were examined by light and electron microscopy. Bright-field light microscopy showed that conidia were round to elliptical and warty on the surface with diameters approximately ranging from 3 to 5 microm. Scanning electron microscopy revealed the globose to irregularly rounded and ornamented conidia with prominent spines. The spines were pointed at the apex or irregularly curved, and approximately 200-500 nm long. Ultrastructure of spined conidia and hyphae revealed by transmission electron microscopy showed lipid globules and vacuoles in the cytoplasm enclosed by an electron-transparent cell wall. Conspicuous electron-dense spines were evident on the surface of conidia, and had obclavate or irregularly protruding shapes with varying heights along the conidial cell wall. Microfibrillar structures with stretching or branching patterns were evident in the spine matrix. Some conidia were interconnected by spines from the neighboring conidia by their extended outgrowth. Hyphae had concentric bodies that showed an electron-transparent core surrounded by an electron-dense layer. One or more intrahyphal hyphae were found in hyphal cytoplasm. The fungus is thought to form concentric bodies and intrahyphal hyphae as survival mechanisms against the water- and nutrient-deficient environments that may occur in the necrotic regions of host plants.

[Activity assay, antiserum preparation and cellular localization of ustiloxins].

False smut is an increasingly important rice disease in recent years, caused by a pathogen, Ustilaginoidea virens Cooke, Takahashi. The pathogen fungus grew well in PD medium, and could produce toxins, ustiloxins, which strongly inhibited the growth of radicle and embryo of plants. The rough ustiloxins were extracted with 100% methanol from the culture filtrate in the study. The antiserum against Ustiloxin A, the main component of the toxins, was raised by immunizing New Zealand rabbit. Elisa assay showed that the titers of the two antiserums obtained were 1: 20 000 and 1: 6 000, respectively. The specificity of the antiserums was also confirmed by Immuno-gold labeling.

Development and nature of the partition layer in Tilletia caries teliospore walls.

Developing Tilletia caries teliospores were studied with thin sectioning procedures. After the W1 and W2 spore walls are formed, lamellar material begins to form adjacent to the W2 wall layer. The patches of lamellar material become continuous, and additional layers are added. After the W3 wall starts to form, the lamellar material is difficult to see without special staining. The lamellar material makes it difficult to get resins to penetrate the partition layer of teliospore walls.

Surface rodlets of Tilletia indica teliospores.

Tilletia indica teliospores were studied by use of thin sections and freeze-etch replicas. Surfaces of these spores have rodlet patterns which differ from those previously reported for spores of other fungi. The rodlets on T. indica teliospores average 240 nm in length and are not grouped into fascicles.

A scanning electron microscope comparison of basidial structures in Filobasidiella neoformans and Filobasidiella bacillispora.

A comparison of basidial structures in the two species of Filobasidiella by scanning electron microscopy showed similar morphology for the basidia. Globose swellings developed into oval to clavate mature basidia which produced chains of basidiospores by budding at four points on the apical surface, with sterigmata lacking. F. bacillispora sometimes developed a typical basal swelling during enlargement of the clavate basidium. The basidiospores of F. neoformans were larger, with irregularly roughened walls and lemon-shaped to oval in contrast to the smooth, rod-shaped basidiospores of F. bacillispora.

Ultrastructure of lipid bodies in Tilletia caries teliospores.

The ultrastructure of lipid bodies within developing, dormant, and germinating Tilletia caries (DC). Tul. (race T-16) teliospores was studied by freeze-etching and thin-sectioning techniques. When teliospores were prefixed in sodium cacodylate-buffered glutaraldehyde-acrolein for 24 h before further processing, most of the lipid bodies appeared to have a uniformly osmiophilic matrix. Some of these lipid bodies were surrounded by thin electron-dense lines that appeared to be half-unit membranes. Occasionally this membrane seemed to be absent, allowing for a direct interface between lipid and cytosol. Irregular electron-dense patterns were occasionally observed in lipid bodies of developing, dormant, and germinating teliospores. A lamellar substructure with 6- to 10-nm center-to-center spacing was visible in the electron-dense patterns at high magnifications. Irregular fracture patterns were visible in freeze-etch replicas.

Sorosporium consanguineum: relation between variable nuclear condition and dissociation.

Saprophytic development of Sorosporium consanguineum and its nuclear cycle were studied on laboratory media. During vegetative reproduction, the nuclei of the monokaryotic sporidia were shown to first migrate into developing bud cells where division occurs, one nucleus returning to the parent cell prior to completion of cell division. Following fusion of sporidia of opposite mating type and subsequent formation of infection hyphae, dikaryons eventually dissociated, giving rise to sporidia of both sex groups in the process. As a result of dissociation, shown to occur in several ways, "satellite" sporidial colonies characteristically formed in advance of the parent colony and fusions again occurred between sporidia of opposite mating type. Reports of variable nuclear condition of mycelia in other species of smut fungi in culture are discussed in light of these findings.

Fungal fimbriae. II. Their role in conjugation in Ustilago violacea.

During conjugation in the anther smut fungus Ustilago violacea cells of opposite mating type first pair tightly and then develop a conjugation tube or bridge between them. The cells of both mating types are covered in long fine hairs or fimbriae, some of which appear to end in knobs. Experiments involving enzyme treatments of the cell surface indicate that these fimbriae do not play an essential role in cell pairing, instead pairing seems to be initiated when one or both mating types produce amorphous masses of alpha-amylase-sensitive material. Electron micrographs, enzyme and inhibitor studies, and experiments using restrictive temperatures suggest, however, that fimbriae may be essential for the later stages of conjugation i.e. development of the conjugation tube. If so, it is suggested that they may permit the exchange of macromolecules between the conjugating cells, initiating localized wall-softening and wall-breakdown.

Fungal fimbriae. I. Structure, origin, and synthesis.

Fine hair-like appendages on the cell walls of the another smut Ustilago violacea are described. These hairs are termed fimbriae because of their close similarity to the fimbriae (pili) found on certain Gram-negative bacteria. Cells of U. violacea may carry more than 200 fimbriae varying in length from about 0.5 mum to over 10 mum, and having a diameter of about 60-70 A. Some fimbriae produce knobs similar to those found on bacterial sex fimbriae. Log-phase cells are the most densely fimbriated, while stationary phase cells are devoid of fimbriae. The cells can be defimbriated by sonication, high-speed agitation, or centrifugation through a 40% sucrose solution. The fimbriae can regenerate in these defimbriated cells in about 1 h. This regeneration is inhibited by both cycloheximide and rifampin, but not by chloramphenicol and therefore appears to depend on de novo protein synthesis on cytoplasmic ribosomes. Similar long fimbriae are found on U. maydis and Leucosporidium (Candida) scottii. Short fimbriae, about 0.5 mum long, were found on all the other species of yeast-like fungi examined (Rhodotorula, Saccharomyces, Schizosaccharomyces, Hansenula, Lipomyces, Nadsonia, and Torulopsis spp.).