Alan W. Day

Melanin synthesis by Sclerotinia sclerotiorum.

We confirmed that the melanin produced by Sclerotinia sclerotiorum is a dihydroxynaphthalene (DHN). The specific DHN melanogenesis inhibitor test that uses tricyclazole at low levels (typically2–5 ppm) to cause a confirmatory appearance of soluble red-brown inhibition products does not work when analyzing melanin synthesis in the sclerotia of S. sclerotiorum. We demonstrated the presence of scytalone dehydratase, an enzyme specific to DHN melanogenesis, in melanized sclerotia and melanized nonsclerotial mycelia and observed formation of mycelial nonsclerotial melanin when the fungus was grown on the surface of sterilized dialysis membrane or in rich organic media. Nonsclerotial melanized hyphae in wild type and mutant strains showed the typical excretion of pigmented inhibition products of the DHN pathway in the presence of tricyclazole, and one of these products, 2-hydroxyjuglone, was identified by thin layer chromatography and spectroscopy. We report basic conditions for sclerotial melanin degradation by the white rot fungus Phanerochaete chrysosporium.

Serological Studies on the Fimbriae of Yeasts and Yeastlike Species

Antisera prepared against pure fimbrial proteins of Ustilago violacea (antiserum U) and of Rhodotorula rubra (antiserum R) agglutinate cells carrying fimbriae of appropriate cross-reactivity. All except one of 24 species of smut fungi in the Ustilaginales agglutinated strongly with antiserum U and varied from no response to moderate agglutination with antiserum R. The exceptional species, Tilletia caries, has been shown to be afimbriate under tested conditions. A group of 42 strains of basidiomycetous yeasts varied in their response from no agglutination to either antiserum, agglutination with one antiserum, or agglutination with both antisera. Approximately one-quarter were afimbriate under the tested conditions. Fimbriated basidiomycetous yeasts, like the smut fungi, produce long flexuous fimbriae with a maximum length ca. 10-20 μm. Eleven ascomycetous yeasts produced a short fringe of fimbriae, usually ca. 0.5-1.0 μm long. Many of these species were agglutinated by one or both antisera, but none of five algal species responded to either antibody. No strains responded to control sera, and tests on a few species with purified antisera confirmed the results with crude sera. The agglutination response was correlated with visible fimbriation on all occasions, including studies with temperature-sensitive fimbriated strains. These results indicate that a family of relatively conserved proteins is common to many species of smut fungi, basidiomycetous yeasts, and ascomycetous yeasts.

Regulation of Parasitic Development of the Smut Fungus, Ustilago violacea, by Extracts from Host Plants

Ustilago violacea grows as yeastlike cells on laboratory media, and its parasitic mycelial stage has so far been restricted to its host plants, members of the Caryophyllaceae Aqueous or acetone extracts of all tested host species induced the formation and growth of mycelia on laboratory media and also enhanced the growth and development of conjugation pegs during mating. Extracts from most tested nonhost species had no effect on U. violacea. Races of U. violacea isolated from different caryophyllaceous hosts varied in response, but all races responded to all host extracts under some growth conditions; therefore, there was no evidence that each race is induced to form parasitic hyphae by the specific products of its particular host species. Ustilago violacea is thus able to restrict development of its parasitic phase to conditions when it is exposed to a particular product or group of products (termed silenins) present in its hosts. Only cells carrying both mating types (conjugated a1 + a2 cells or diploid a1a2 cells) responded to silenin by developing infection hyphae. Cells of a single mating type were not affected in growth rate or morphology. Silenin appears to be a member of a new class of plant compounds, termed mycoboethins, which are defined as plant products exploited by particular pathogens to induce highly specific morphogenetic changes leading to parasitism.

Fungal fimbriae. IV. Composition and properties of fimbriae from Ustilago violacea

Abstract The fimbriae of Ustilago violacea consist of long protein fibrils of 7-nm diameter. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the fimbriae of this species and of several other species of Ustilago and Rhodotorula demonstrated that they are composed of a protein of 74, 000 Da which can spontaneously assemble into 7-nm fibrils. No carbohydrate moiety was detected. Fimbrial protein retained both its fibrillar structure and antigenicity when exposed to a variety of chemical treatments, and even when autoclaved. Concentrations of cations greater than 10−1M (monovalent cations) or 5 × 10−2M (divalent cations) resulted in a loss of fimbriae, while the effect of chelators suggested that calcium is important to the structural integrity of fimbriae. Ouchterlony tests using antisera prepared against the fimbriae of U. violacea and R. rubra indicated that while there are differences in the antigenic sites, the fimbrial protein of different basidiomycete species is highly conserved. Fimbrial protein did not react with several mammalian antisera directed against cytoskeletal proteins.

Chapter 27 Genetic and Cell Cycle Analysis of a Smut Fungus (Ustilago violacea)

Publisher Summary This chapter describes the genetic, cytological, and biochemical techniques that are applied in the laboratory to the study of the cell cycle and development in the anther smut. The chapter discusses the biology and culture of Ustilago violacea . Chemostat cultures are preferred for synchrony studies using zonal density-gradient centrifugation. Using chemostat cultures, it is possible to achieve an extremely high level of reproducibility in the zonal separation of sporidia. The chapter describes the techniques that are useful in determining the behavior of the nucleolus, chromatin granules, nucleoplasm, and spindle pole body during division. Two alleles of the mating-type locus that regulate assembly of the copulatory organelle have different cell cycle controls; allele a 1 allows mating only in G 1 and allele a 2 allows mating during the entire cell cycle. The cell cycle controls are allele specific and cis is dominant. When both alleles are active simultaneously they “cancel out” and the cell does not mate. Thus, in the heterozygous diploid mating is inducible only during G 2 and S and a 2 is dominant. Spontaneous changes in the heterozygote affecting the cell cycle control system of at least the a 2 allele appear to arise frequently.

Chemical induction of infection structures in rust fungi: I. Sugars and complex media

Abstract Complex media components were found to induce formation of infection structures in germinated urediospores of bean, corn, cowpea, and sunflower rust fungi. The frequency of these structures was much higher in bean rust and sunflower rust than in the other two species. The structures were morphologically identical to those induced by host plants. Analysis of one complex component, yeast extract, using TLC and HPLC suggested that the active substances were likely to include simple sugars. A survey of sugars indicated that sucrose and glucose (and to a much lesser extent trehalose and fructose) were capable of inducing differentiation. The activity of inducers was partially concentration dependent. Complex components, or combinations of inducers, were more effective than single sugars. Mitogenic lectins did not induce differentiation, but inhibited the action of sucrose. Infection structures significantly increased in length between 24 and 48 h, indicating uptake and utilization of exogenous nutrients.

Chemical induction of infection structures in rust fungi. II: Inorganic ions

Abstract Cations and anions incorporated into agar-containing media were screened for their effects on induction of infection structures in germinating urediospores of three rust fungi. K+ and Mg2+ strongly induced differentiation in bean rust and sunflower rust fungi, while Ca2+, Mn2+, and Na+ were only weak inducers. Differentiation frequency increased with increasing concentration of K+, and up to 70% of germinated spores formed infection structurs. Most potassium salts gave similar induction frequencies indicating that anion effects were relatively unimportant. However, iodide and nitrate anions appeared to interfere with the induction process and virtually eliminated infection structure formation even though they permitted good germ tube growth. Induction by salts was sensitive to total buffer concentration and to surface phenomena possibly related to aeration or humidity factors. Combinations of low levels of K+ and Ca2+ or K+ and sucrose showed synergistic effects and in the latter case resulted in differentiation frequencies in excess of 80% for the sunflower rust fungus. Infection hyphae developed from the infection structures and the average length increased slightly between 24 and 48 hr.

The Effect of Host Extracts on Differentiation in the Genus Ustilago

Ustilago violacea grows as yeastlike sporidia on nutritive media. Leaf extracts from all of 38 tested plant species that host a species of U stilago induced hyphae and inhibited sporulation in U. violacea. However, extracts from most (33 of 39) of the tested plant species that do not host species of smut fungi were inactive. Ustilago scabiosae and U. utriculosae responded similarly to U. violacea to the same range of plant extracts. Species of Ustilago from monocotyledonous hosts formed hyphae on nutritive media in the absence of extracts. Plant extracts inhibited sporulation in these species but did not noticeably affect hyphal growth Farysia olivacea, like U. violacea, remained strictly sporidial in the absence of host extracts. This species, however, was induced to form hyphae by different plant extracts from those active on U. violacea.

A Survey of Extracellular Enzymes in the Smut Fungi

The production of extracellular amylases, lipases, proteases, deoxyribonucleases, and ribonucleases by sporidial cultures of 58 isolates of smut fungi representing 23 species is reported.

Fungal fimbriae: V. Protein A-gold immunocytochemical labeling of the fimbriae ofUstilago violacea

Abstract Protein A-gold immunocytochemical labeling of long protein appendages (fimbriae) on the surfaces of cells of Ustilago violacea is described. The specificity of the technique is confirmed by a series of serological and morphological controls. The technique allows specific identification and localization of fimbrial antigens in electron microscopic studies of a variety of fungi in both pathogenic and saprophytic situations.