Melvin S. Fuller

Ultrastructural aspects of the hyphal tip ofSclerotium rolfsii preserved by freeze substitution

SummaryThe hyphal tip ofSclerotium rolfsii was examined after fixation by freeze substitution. The Spitzenkörper consisted of a dense mass of apical vesicles and microvesicles surrounding a vesicle-free zone. Linear arrangements of microvesicles were occasionally observed within the Spitzenkörper. Abundant microfilaments were seen within the Spitzenkörper region, often in close association with apical vesicles and microvesicles. Microtubules passed through the Spitzenkörper and terminated at the plasmalemma at the extreme hyphal apex. Filasomes were mostly observed within the apical region and were in close proximity to the plasmalemma. Rough ER, mitochondria, microtubules, and vacuoles were abundant in the subapical region and were usually oriented parallel to the long axis of the hypha. Ribosomes were aligned on the outer surfaces of mitochondria. Golgi body equivalents were observed throughout the subapical region and appeared as inflated cisternae of varying shapes and electron opacities. Relationships to other basidiomycetous hyphal tip cells are discussed.

Mitosis in Fungi

Publisher Summary This chapter discusses the ultrastructure of vegetative nuclear division, that is, mitosis in fungi and closely related groups. Mitotic divisions occurring in the vegetative phase of the fungi are essentially the same as in higher organisms, although the details are frequently obscured by the small size of the nuclei. Fungi, as commonly understood, are a highly diverse and polyphyletic group of organisms. Some of the organisms commonly grouped with fungi are placed there only because mycologists have traditionally studied them. The interphase nucleus of the fungi is bounded by an envelope composed of two unit membranes and interrupted by nuclear pores. Centriole structure in fungi does not differ in its basic aspects from that found in other eukaryotic organisms; that is, the centrioles are composed of nine triplet sets of microtubules which, at their proximal ends, enclose a cartwheellike structure. The centrioles of fungi occur in pairs (diplosome) and are often, although not consistently, associated with a pocket in the nuclear envelope. Other aspects of mitosis and their possible usefulness in assessing evolutionary relationships have been discussed in the cited studies of Pickett-Heaps.

Cell wall composition and synthesis via Golgi-directed scale formation in the marine eucaryote, Schizochytrium aggregatum, with a note on Thraustochytrium sp.

SummaryCell walls of Schizochytrium aggregatum and Thraustochytrium sp. were mechanically isolated and subjected to chemical analysis. On a dry weight basis the cell walls contain 21–36% carbohydrate and 30–43% protein. The principal sugar (>95%) of the Schizochytrium wall is l-galactose, while the Thraustochytrium cell wall contains l-galactose, d-galactose and xylose with l-galactose predominating. Ultrastructurally the cell walls of both organisms consist of a laminated structure which yields thin, flexible, nearly circular scales (0.5–1.1 μ in diameter) upon sonic disintegration. Structures presumed to be developing wall scales are found within cisternae of the Golgi apparatus in both organisms. The chemical composition and method of formation of the cell wall in these two protists is distinctly different from that found in the Saprolegniales (Oomycetes), the group with which these organisms have hitherto been aligned.

Fatty acids and sterols of selected hyphochytriomycetes and chytridiomycetes

Abstract The fatty acids and sterols of eight Chytridiomycetes and two Hyphochytriomycetes, and fatty acids of the Oomycete Pythium gracile , were analyzed by gas-liquid chromatography. In addition to the fatty acids anticipated for fungi, the two Hyphochytriomycetes ( Hyphochytrium catenoides and Rhizidiomyces apophysatus ) and four of the Chytridiomycetes ( Catenaria anguillulae, Blastocladiella emersonii, Monoblepharella sp., and Allomyces macrogynus ) contained arachidonic acid as a major fatty acid of the polar lipid fraction, and this fatty acid was detected as a minor component of Rhizophlyctis rosea and Spizellomyces punctatum . Eicosapentaenoic acid constituted 4.6% of the polar lipid fatty acids in Monoblepharella sp., and trace amounts were detected in several other species. Both the gamma (ω-6) and alpha (ω-3) isomers of linolenic acid were detected in all of the species analyzed. Cholesterol was the predominant (≥73%) sterol of B. emersonii, R. rosea, A. macrogynus , and Chytridium confervae , and a minor ( C. anguillulae , and H. catenoides . The major sterols of the other species included lanosterol ( C. anguillulae , 45%), stigmasta-5,22-dien-3β-ol ( H. catenoides , 51%), 24-ethyl-cholesterol ( S. punctatum , 38%; H. catenoides , 17%; Monoblepharella sp., 70%; and R. apophysatus , 84%), 24-methyl-cholesterol ( H. catenoides , 23%; R. apophysatus , 14%; S. punctatum , 53%), and 24-methylene cholesterol ( Rhizophydium sphaerotheca , 51%). Neither ergosterol nor fucosterol was detected in any of the species studied.

The Golgi apparatus, zoosporogenesis, and development of the zoospore discharge apparatus ofChytridium confervae

Abstract C. confervae is a eucarpic, monocentric chytrid that has been cultured synchronously ( L. P. Gauriloff and M. S. Fuller, 1979 , Exp. Mycol. 3 : 3–5). In this study we use light and electron microscopy to examine the development of zoospores and the discharge apparatus, focusing on the multiple roles of the Golgi apparatus. The Golgi apparatus produces, in succession, vesicles with electron-opaque cores that may function in cell wall formation, secretory vesicles that form the extracellular lenticular deposit of the discharge apparatus, cleavage vesicles that fuse to form the plasma membranes of the developing zoospores, and vesicles that contain cell coat material for the zoospores. The discharge apparatus consists of the operculum (a circle of sporangial wall delimited from the rest of the wall), the lenticular deposit, and an outer layer found between the lens and the operculum. At discharge, the operculum dehisces, the outer layer ruptures, and the lenticular deposit expands to form a vesicle that constrains the zoospores. The outer layer provides the mechanical connection between the wall and the vesicle. Comparison of discharge apparatus development with other Chytridiomycetes suggests that the order of the developmental steps leading to discharge may be as important to chytrid taxonomy as the steps themselves.

Leucine-lysine synchronization of Allomyces germlings

SummaryThe leucine-lysine synchronization technique of Dill and Fuller (1970) has been further refined and used to study various biosynthetic events of pre-mitotic germlings of Allomyces neo-moniliformis (the time of DNA replication, RNA synthesis, and protein synthesis), and various morphogenetic changes (germling development, nuclear cap breakdown, and the first mitotic nuclear division). The degree of synchrony induced in a population of germlings appears to be determined by the time when the zoospores are induced to encyst and germinate rather than by the duration of the swimming period of the zoospore. DNA replication, nuclear cap breakdown, early protein synthesis, and morphogenetic development appear to occur prior to messenger RNA synthesis in developing thalli and thus would be under the control of pre-existing messenger RNA. The degree of synchrony of particular morphogenetic or biosynthetic developmental changes induced in a population of A. neo-moniliformis germlings must be determined for each aspect of development which is to be studied.

RESISTANT BODY FORMATION IN NEOCALLIMASTIX SP., AN ANAEROBIC FUNGUS FROM THE RUMEN OF A COW

Evidence of several kinds was sought to confirm that structures thought to be resistant sporangia (RS) in Neocallimastix sp. shared characteristics with RS of other zoosporic fungi. In liquid medium, Neocallimastix formed a chytrid-like thallus made up of a system of intramatrical anchoring rhizoids and an extramatrical sessile or stalked zoosporangium on fiber pieces. As the culture aged, there was a shift from the development of zoosporangia to the production of RS. RS were first observed 36 h after zoospores were inoculated into fresh basal anaerobic medium. Encysted zoospores often developed into elongate thalli that branched monopodially near the tip. During RS formation, the tip of each branch enlarged and the nuclei and cytoplasm migrated towards this enlarged region. A septum was then formed to delimit the developing RS which became pigmented as it matured. The mature RS were dark brown and the pigment in their walls was identified as melanin using histochemical tests. Zoospores had the 1C level of DNA with 1C and 2C levels being observed in developing zoosporangia. The DNA content in the mature RS (4C) was found to be twice the highest level of DNA in the zoosporangium (2C) which suggested that there was a diploidization during RS formation. Additional studies needed to characterize the RS are discussed.

Isolation and Characterization of an Endopolygalacturonase from Cochliobolus sativus and a Cytological Study of Fungal Penetration of Barley

ABSTRACT Endopolygalacturonase (EPG) of Cochliobolus sativus was produced in shake culture and purified by high-performance liquid chromatography. The enzyme had a molecular mass of 34,000 Da, an isoelectric point in the range of 9.0 to 9.5, exhibited endo activity, was nongly-cosylated, and was inhibited by polygalacturonase-inhibiting proteins from bean, pear, and tomato. The amino terminus contained a 14 amino acid region homologous to a region at the N terminus of an EPG of C. carbonum. C. sativus EPG-specific monoclonal antibodies (MAbs) were generated. Western blot analysis confirmed the specificity of the antibodies for the EPG and detected the enzyme in an extract from Hordeum vulgare (cv. Golden Promise) leaf segments infected with C. sativus. Using conventional immunogold and enzyme-gold cytochemical methods, homogalacturonan, esterified pectin, and cellulose were localized in healthy and infected barley leaf epidermis at the electron microscope level. Additionally, the leaf cell wall polysaccharides recognized by purified C. sativus EPG were localized at the electron microscope level, using the purified enzyme as a primary cytochemical reagent, followed by a gold-labeled MAb specific for the enzyme. Loss of polygalacturonic acid in the vicinity of the invading pathogen was visualized cytochemically at the electron microscope level. These observations suggest the involvement of EPG during host penetration by the fungus.

Effects of the demethylase inhibitor, cyproconazole, on hyphal tip cells ofSclerotium rolfsii: II. An electron microscope study

Abstract Hyphal tip cells of Sclerotium rolfsii , prepared by freeze substitution, were examined by transmission electron microscopy after continued growth on cyproconazole-amended potato dextrose agar at fungistatic concentrations of 0.1, 0.75, and 1.0 μg/ml which respectively produced 50, 80, and 95% growth inhibition. Alternatively, observations were made of control tip cells that were transferred onto potato dextrose agar amended with 0.75 μg/ml cyproconazole for 30, 60, and 240 min. Ultrastructural abnormalities observed in hyphal tip cells as a result of cyproconazole treatment included (1) Spitzenko¨rper disorganization, (2) proliferation of smooth endoplasmic reticulum with inflated cisternae, (3) increased vacuolar contents, (4) presence of monovesicular bodies, and (5) irregular cell wall characteristics. Alterations of the Spitzenko¨rper, endoplasmic reticulum, and vacuoles were noted 30 min after exposing control hyphae to cyproconazole (0.75 μg/ml). Abnormal wall depositions were observed 60 min after fungicide exposure. After a 140-min exposure to fungicide, tip cell morphology was similar to that of cells grown in the continual presence of 0.75 μg/ml cyproconazole. The changes in hyphal morphology are discussed relative to the mode of action of cyproconazole.

Induction and formation ofCochliobolus sativus appressoria

SummaryGerminatingCochliobolus sativus spores were induced to form appressoria on a variety of artificial surfaces, including replicas of the barley leaf surface. Evidence was obtained for the involvement of chemical and topographic signals during induction of appressorium formation inC. sativus. Germ tube thigmotropism was also observed in vitro. Ultrastructure relevant to appressorium formation was observed, including the germ tube apex, apical swelling of the germ tube apex prior to appressorium formation, the appressorium with associated septation and the penetration peg. Cytochemical probes applied to germlings at the electron microscope level failed to detect α-D-mannan, α-D-glucan, β-D-galactan, D-glcNAc or D-galNAc polymers in the extracellular mucilage associated with the fungal germlings. The ultrastructure of hyphal apices from germlings grown under different nutritional conditions differed with respect to Spitzenkörper morphology, apex shape and in the quantity of associated extracellular mucilage. Experimental findings are discussed relative to current understanding of appressorium induction in more extensively studied systems.