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3. Brodey RS, Reid CF, Sauer RM: 1966, Metastatic bone neoplasms in the dog. J Am Vet Med Assoc 148:29–43. 4. Chin RP, Barr BC: 1990, Squamous cell carcinoma of the pharyngeal cavity in a Jersey black giant rooster. Avian Dis 34: 775–778. 5. Head KW: 1990, Tumors of the alimentary tract. In: Tumors in domestic animals, ed. Moulton JE, 3rd ed., pp. 347–435. University of California Press, Berkeley, CA. 6. Kotwall C, Sako K, Razak MS, et al.: 1987, Metastatic patterns in squamous cell cancer of the head and neck. Am J Surg 154: 439–442. 7. Latimer KS: 1994, Oncology. In: Avian medicine: principles and application, ed. Ritchie BW, Harrison GJ, Harrison LR, pp. 640–672. Wingers, Lake Worth, FL. 8. Pulley T, Stannard AA: 1990, Tumors of the skin and soft tissues. In: Tumors in domestic animals, ed. Moulton JE, 3rd ed., pp. 23–87. University of California Press, Berkeley, CA. 9. Rose ME: 1981, Lymphatic system. In: Form and function in birds, ed. King AS, McLelland J, vol. 2, pp. 341–384. Academic Press, London, England. 10. Shingaki S, Suzuki I, Kobayashi T, Nakajima T: 1996, Predicting factors for distant metastases in head and neck carcinomas: an analysis of 103 patients with locoregional control. J Oral Maxillofac Surg 54:853–857. 11. Tagaki M, Kayano T, Yamamoto H, et al.: 1992, Causes of oral tongue cancer treatment failures. Analysis of autopsy cases. Cancer 69:1081–1087. 12. Yager JA, Scott DW: 1993, The skin and appendages. In: Pathology of domestic animals, ed. Jubb KVF, Kennedy PC, Palmer N, 4th ed., vol. 1, pp. 531–738. Academic Press, San Diego, CA.

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.

The ultrastructure of zoospores of several zoosporic fungi was examined using a modified cryofixation technique. An atomizer was used to spray a zoospore suspension into the cold propane reservoir of a conventional plunge freeze-substitution apparatus. Spray-freeze fixation and freeze-substitution of zoospores porvided better fixation of vacuolar structures, membranes and the extracellular coat than that obtained with chemical fixation. The overall shape of cryofixed spores was closer to that seen in living zoospores. Two types of vacuoles were seen in cryofixed zoospores ofMonoblepharella andChytridium. One type of vacuole contained electron-opaque material within the lumen while the other type had no visible internal material in the lumen and appeared to be part of the water expulsion vacuole complex. Coated pits and coated vesicles were observed associated with both the water expulsion vacuoles and the plasma membrane inMonoblepharella andPhytophthora, suggesting that endocytosis of the plasma membrane and expulsion vacuoles is part of membrane recycling during osmoregulatory events. An extracellular coat was seen on the outer surface of cryofixed zoospores ofMonoblepharella sp.,Chytridium confervae andPhytophthora palmivora without the use of carbohydrate-specific stains. The spray-freeze method gave good and reproducible fixation of the wall-less spores in quantities greater than those obtained in previously described zoospore cryofixation studies. The technique is potentially useful for cell suspensions in that freeze damage from excess water is limited.

Ungerminated conidia of Blumeria graminis f.sp. hordei were prepared for transmission electron microscopy using cryofixation and freeze-substitution. Conidia were uninucleate, with the nucleus located in the central portion of the conidium, typically off to one side. The nucleus was spherical and contained a distinct nucleolus with an associated satellite. Conidia contained numerous large, spherical vacuoles that occupied much of the spore volume. Vacuolar contents were mostly homogeneous with occasional electron-opaque inclusions. Much of the cytoplasm consisted of lightly staining aggregations of glycoprotein that could be labelled with Concanavalin A – gold and that stained darkly using a modified Thiéry's reaction specific for carbohydrates containing vicinal hydroxyl groups. Mitochondria, strands of endoplasmic reticulum, Golgi body equivalents, multivesicular bodies, microbodies, Woronin bodies, and microtubules were present in the conidium. Cuboidal, stacked, electron-translucent structures also were present in the cytoplasm. The conidium was surrounded by a two-layered wall that labelled with gold-conjugated wheat germ agglutinin-ovomucoid, indicating the presence of chitin. The wall between attached conidia contained areas that did not label with gold-conjugated wheat germ agglutinin-ovomucoid. Keywords: Erysiphe, ultrastructure, electron microscopy, freeze substitution.

GerminatingCochliobolus 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 Spitzenkorper 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.

Gonapodya prolifera was isolated from a pond in Athens, Georgia, and its development in pure culture is reported for the first time. While the fungus grew well on a diversity of media, the morpholo...

Cytochemical methods that employed observation of lectin- or enzyme-gold complexes with an electron microscope were used to study the occurrence and distribution of sugars and polysaccharides in the cell walls of Rhizidiomyces apophysatus and Hyphochytrium catenoides , two fungi belonging to the class Hyphochytriomycetes. Both fungi have the polysaccharides cellulose and chitin in their cell walls and a minor component that contains n -acetyl- d -galactosamine. The discharge tubes emanating from sporangia of R. apophysatus stained positively for chitin but were negative for cellulose and the N -acetyl- d -galactosamine containing compound. Whereas the cell walls of developing sporangia of H. catenoides labelled strongly for cellulose, the results indicated they contained only trace amounts of chitin. The rhizomycelial walls of H. catenoides had a composition similar to that of the sporangia except for the septa that appeared to contain more sites indicative of chitin. The significance of the results relative to earlier studies of these fungi and generalizations with respect to cell wall chemistry and systematic relationships is discussed.

Three monocentric and two polycentric obligately anaerobic zoosporic fungi were isolated from the rumen, freshly voided feces, and dry and moist feces. Rumen isolates were morphologically similar to fecal isolates from the same animal. The monocentric isolates were Piromyces communis, Neocallimastix frontalis, and Caecomyces communis. The polycentric isolates were Orpinomyces bovis and Orpinomyces sp. type PC3. Piromyces communis and N. frontalis were obtained from feces stored for 3 and 6 months, respectively. Orpinomyces bovis and Orpinomyces sp. type PC3 were isolated from dry feces, but not moist feces, stored for 3 and 6 months, respectively. Caecomyces communis was isolated only from feces stored for 6 weeks. All the isolates, except C. communis, formed melanized sporangia in pure culture. Structures that were morphologically similar to the melanized sporangia of rumen fungi were observed in fecal smears.The fecal isolates grew under conditions similar to the rumen, but not under aerobic conditions. Key words: rumen fungi, Chytridiomycetes, fecal fungi.

The development of Caecomyces communis was studied with light and fluorescence microscopy. Caecomyces communis is a monocentric fungus which develops to form either a thallus consisting of one or more sporangia attached to a vegetative cell or a mycelioid thallus. During the formation of a vegetative cell with attached sporangia, the posteriorly uniflagellate zoospore encysts and produces a broad germ tube which develops into an incipient vegetative cell. The vegetative cell enlarges faster than the zoospore cyst which develops into an incipient sporangium. A unisporangiate thallus consists of one sporangium attached to a large, vacuolate, vegetative cell. Thallus development may terminate at this unisporangiate stage or further development into a multisporangiate thallus may occur. During multisporangiate thallus formation, the vegetative cell forms one or more outgrowths that become sporangial stalks. Nuclei occur within the vegetative cell and, at maturity, sporangia are formed at the tips of the outgrowths. Often, instead of forming sporangia at the tips of outgrowths produced by the vegetative cell, the outgrowths enlarge into thick filaments. These filaments branch extensively and nuclei occur within the branches. Sporangia are formed at the tips of such filaments which results in a mycelioid thallus. The development and morphology of C. communis and C equi are compared.

The development from zoospore to a mature thallus in Neocallimastix sp. isolated from a Georgia cow was studied at the light microscope level. The zoospore had 9–14 posteriorly directed flagella, and its shape varied from amoeboid in agar to ovoid in broth. Encysted zoospores developed endogenously into extramatrical ovoid or spherical incipient zoosporangia with extensively branched intramatrical rhizoids that often had constrictions. Sessile mature zoosporangia varied in shape, and zoospores were fully formed within zoosporangia before release through an apical pore. In agar, zoospores encysted close to the parent zoosporangium and developed endogenously into second generation zoosporangia or exogenously into elongate thalli. At maturity, an elongate thallus was made up of a sporangium, a sporangial stalk, a cyst, and branched rhizoids. Elongate thalli were sometimes formed in broth. Melanized resting sporangia were formed on branched thalli in old (> 36 h) cultures. Two isolates of Neocallimastix frontalis from a cow and sheep and Neocallimastix patriciarum were grown under the same conditions as our isolate, and the morphology of zoospore, zoosporangium, and melanized sporangium of the four isolates were compared. In broth, the isolates developed in the same manner and formed elongate thalli and melanized sporangia as described for our isolate. There is insufficient justification, based on morphology alone, for separating the four isolates. The importance of basic light microscopy is discussed. Key words: Neocallimastix, development, morphology.

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.

The mycoparasitism of teliospores of the smut fungus Ustilago bullata was discovered by transmission electron microscopy (TEM). Large, multinucleate chlamydospores germinated, producing hyphae that directly penetrated the walls of mature teliospores after forming an appressorium-like structure. Invagination of the exosporium at the point of penetration suggested mechanical penetration of this outer spore wall layer. The inner endosporium layer was fibrillar in appearance, with irregular electron-transparent regions suggesting enzymatic degradation. The cytoplasm and endosporium of parasitized teliospores were completely disintegrated, leaving only the spiny exosporium layer distributed throughout the sorus. Hyphae of the mycoparasite emerged from the teliospore shell, and their tips penetrated surrounding teliospores. TEM characteristics of the parasite confirmed oomycetous affinities. Pythium vexans was isolated from smutted tissue collected at the same time as tissue used for TEM observations. Pythium vexans was able to parasitize U. bullata teliospores in culture. Key words: hyperparasitism, ultrastructure, smut, Pythium, host–parasite relationships, infection.

Hyphae of Sclerotium rolfsii were grown on cyproconazole-amended agar at fungistatic concentrations of 0.1, 0.75, and 1.0 μg/ml, which respectively produced approximately 50, 80, and 95% inhibition of hyphal extension. Hyphal tip cells were prepared for transmission electron microscopy by cryofixation and freeze substitution methods. Cell walls of control and fungicide-treated hyphal tip cells were compared cytochemically using periodic acid-thiocarbohydrazide-silver protein, gold-conjugated lectins, and immunocytochemical staining. Cell walls of both control and fungicide-treated cells reacted positively to periodic acid-thiocarbohydrazide-silver protein staining although the patterns of staining were different. Apical walls of control cells were generally thinner than those in fungicide-treated hyphae. Two distinct cell wall layers were identified in the subapical walls of control hyphae while distinct wall layers were typically not observed in fungicide-treated hyphae. Staining characteristics of abnormal cell wall deposits were variable after periodic acid-thiocarbohydrazide-silver protein stain. In regions of mature cell wall of control hyphal tip cells, wheat germ agglutinin-binding sites were localized along the inner layer of the cell wall. In fungicide-treated hyphae, binding sites for wheat germ agglutinin occurred throughout the cell wall except in electron opaque wall inclusions. Concanavalin A-binding sites were localized along the plasma membranes of both control and cyproconazole-treated hyphae in subapical cell regions. Electron opaque inclusions in abnormal cell wall deposits had affinities for concanavalin A. Immunocytochemical localization of β-1,3 glucans occurred in mature cell walls of control and fungicide-treated cells. No β-1,3 glucans were detected in electron opaque wall inclusions of hyphae exposed to cyproconazole. Alterations in the cell wall are discussed in light of the mode of action of the fungicide and aspects of cell wall deposition.

The Hyphochytriomycota are a small group of zoospore-producing organisms that, although they live like fungi, i.e., they are nonphotosynthetic organisms that usually possess cell walls during their growth phases and acquire nutrients by absorption, are more closely allied with algal groups. The diagnostic feature of the group is the presence of a single, mastigoneme-bearing flagellum that most often is at the anterior end of the zoospore as it swims. The Hyphochytriomycota have been classified with the Protista (Whittaker 1969) and Protoctists (Margulis et al. 1990), but more recent molecular data place them in a clade with the Oomycetes and heterokont algae. This clade has been called Kingdom Cromista (Cavalier-Smith 1993, and Chap. 1, this Vol., and Kingdom Stramenopila (Alexopoulos et al. 1996); others simply refer to the group as stramenopiles (Leipe et al. 1994). Molecular studies (Van der Auwera et al. 1995) of ribosomal RNA sequences of Hyphochytrium catenoides and inverted repeat sequences of mitochondrial DNA in H. catenoides (Mc Nabb et al. 1988) indicate that the Hyphochytriomycota are close relatives of the Oomycota (see Dick, Chap. 2, this Vol.). Our own molecular studies (S. Lee and M.S. Fuller, unpubl.) of the group support inclusion of the Hyphochytriomycota in a clade with the Oomycota and heterokont algae, but suggest that the Hyphochytriomycota are more closely related to some groups of the heterokont algae than to the Oomycota.

The water expulsion vacuole ofPhytophthora palmivora was studied with the light microscope after the motility of the spores had been slowed by using a low Ca2 + medium. For transmission electron microscopy, the zoospores were prepared using freeze substitution. The water expulsion vacuole consisted of a central vacuole that collapsed and emptied its contents to the external medium. The central vacuole was surrounded by a series of interconnected vacuoles that are called the surrounding vacuole. The surrounding vacuole and the central vacuole were always associated with coated pits. The vesicles found in the region between the central and surrounding vacuole were exclusively coated vesicles. Consistent changes in the organization of the water expulsion vacuole led us to conclude that, as the central vacuole collapses, the surrounding vacuole is transformed and becomes the new central vacuole. No evidence was obtained for contraction of the central vacuole. The processes involved in membrane recycling during the functioning of the water expulsion vacuole are discussed.

The hyphal tip ofSclerotium rolfsii was examined after fixation by freeze substitution. The Spitzenkorper consisted of a dense mass of apical vesicles and microvesicles surrounding a vesicle-free zone. Linear arrangements of microvesicles were occasionally observed within the Spitzenkorper. Abundant microfilaments were seen within the Spitzenkorper region, often in close association with apical vesicles and microvesicles. Microtubules passed through the Spitzenkorper 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.

Two triazoles, cyproconazole and propiconazole, and the imidazole, prochloraz, all commercially available fungicides that inhibit 14a-demethylation during sterol synthesis, were applied to a cholesterolproducing fungus, Allomyces macrogynus, during growth phase. Concentrations of the above inhibitors 1/100-1/500 of the EC50 concentrations needed for inhibition of vegetative growth, caused failure of sporangia to cleave normally and produce uniformly sized, uniflagellate zoospores. Instead, dependent upon the concentration of fungicide used, motile cells were uniflagellate, biflagellate, triflagellate or multiflagellate with the number of nuclei corresponding to the number of flagella. The fungicides were only effective if they were present during the growth phase and impairment of cleavage could not be overcome by adding cholesterol to the discharge medium. Observations were based on light and electron microscope observations. Multinucleate cells with more than one flagellum germinated to produce normal thalli when plated on agar lacking the fungicides. The basis of the interference with cleavage is discussed.

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.

The fine structure of the Harpochytrium hedinii Wille zoospore is reexamined using serial sections. All of the major organelles are located in the posterior two-thirds of the zoospore; the anterior region contains only a few vacuoles and a network of filaments. The basal body complex is located at the center of the posterior end of the zoospore. The nucleus and "nuclear cap" are located directly anterior to the basal body complex on one side of the zoospore. All of the microbodies, lipid globules, and mitochondria are arranged into a complex associated with the rumposome along the opposite side of the zoospore. A spore-wide system of cisternae, the vesicular system, is continuous throughout the posterior region of the zoospore. The overall arrangement of organelles in the H. hedinii zoospore most closely resembles the internal morphologies of certain chytridialean zoospores. This suggests that Harpochytrium may be more closely related to the chytrids than has been previously assumed.

Nonfilamentous, lower, aquatic fungi are potential systems for developmental study. Chytridium confervae , a monocentric, eucarpic chytrid, exemplifies the morphological and developmental simplicity of these fungi. It is easily isolated and maintained in axenic culture, and will grow and reproduce asexually in liquid culture under a variety of conditions. The degree of synchrony of growth, differentiation, and zoospore release of large populations of cells of C. confervae in axenic liquid cultures is directly related to culturing conditions. Under a specific set of conditions, called standard conditions, large populations of cells undergo rapid and consistent exponential growth as measured by increases in cell diameter. If cells are induced to sporulate just prior to the end of the exponential growth phase, a sequence of morphological events associated with zoosporangial differentiation and zoospore release occurs in a predictable pattern and with a high degree of synchrony. Between 2.5 and 4.0 h after induction, a period of time that constitutes 5% of the asexual generation time, over 95% of the cells develop opercula. During this same period, a relatively high percentage of cells undergoes mitosis. Between 6 and 7 h after induction, whole populations of cells release zoospores. The synchronization index is over 0.90 in both petri dish cultures and spinner flask cultures under standard conditions. This degree of synchrony is comparable to that reported in cultures of Blastocladiella emersonii . A variety of midicycles are possible with C. confervae and it can be grown on a defined medium, Crasemann's 1B. The high degree of synchrony and control of large populations of C. confervae in axenic culture makes it adaptable to morphological and molecular studies of development.

The ultrastructure of the zoospores of Oedogoniomyces lymnaeae and Monoblepharella sp. are reexamined using serial sections. The relative molecular weights of the ribosomal RNA of various chytridiomycetous fungi are also determined and compared. The fine structure of each of these zoospores is very similar. The nucleus and "nuclear cap" are centrally located in the zoospores. The anterior region contains lipid globules, a few large, empty vacuoles and numerous, small vesicles. The posterior region is filled with an electron-translucent material throughout which are scattered spherical mitochondria and large vacuoles with electron-opaque inclusions. The basal body complex is located at the center of the posterior end, and the rumposomal complex is adjacent to the plasmalemma in the posterior region of these zoospores. A system of cisternae that are associated with microbodies appears to connect the lipid globules in the anterior region with the rumposomal complex in the posterior region of these zoospores. Small differences in the structure and distribution of certain organelles are considered minor compared with the overall similarities between these two zoospores. It is suggested that Oedogoniomyces be transferred to the Monoblepharidales. The fine structure of the Harpochytrium hedinii zoospore is not only similar to that of the zoospores studied herein, but also resembles the zoospores of chytrids. The possible relationship of Harpochytrium to the Chytridiales is supported by zoospore ultrastructure and the relative molecular weights of ribosomal RNA. The data available at the present time suggest that the order Harpochytriales be abandoned and that Harpochytrium be considered either a genus of nematosporangial, operculate, eucarpic chytrids or a monoblepharidalean genus with Oedogoniomyces. It is recommended that speculation concerning the taxonimic relationship between Harpochytrium and other Chytridiomycetes be delayed until more data concerning other species of Harpochytrium and monoblepharidalean fungi are available.

Development of hyphal septa (pseudosepta) in Allomyces macrogynus begins with the formation of five or more discontinuous pieces of wall material that project inward from the hyphal wall. Lateral fusion of these projections leaves a central pore in the septum that is later filled in by centripetal deposition of wall material. However, lateral fusion of the projections is not complete; peripheral pores remain in the rim of the mature septum. The position of cytoplasmic microtubules corresponds to the position of actively moving cellular particles and organelles. Allomyces reticulatus and A. arbuscula have similar septa. PREVIOUS STUDIES on the physiology and cytology of Allomyces have been concerned primarily with the reproductive aspects of this fungus, such as zoospore ultrastructure, sporangial and gametangial development, pheromones, and life cycles (Emerson, 1941; Kochert, 1978; Lange and Olson, 1979; Griffien, 1981; Olson, Eden and Lange, 1981). The structure ofthe vegetative portion ofthe fungus has received relatively little attention. Since the ecological success of Allomyces, as with any other fungus, generally depends in part upon the success of its vegetative phase, a better understanding of the structural and functional aspects of the thallus itself would be helpful in understanding its biology. Little is known about septa in the zoosporic fungi, and all studies to date have been concerned with the septa at the base of sporangia orgametangia(Powell, 1974;Morrison, 1977; Taylor and Fuller, 1980). The septa of Allomyces are taxonomically important within the Blastocladiales. Allomyces is separated from other members of the Blastocladiales by the presence of septa in the hyphae (Sparrow,1960, 1973; Emerson and Robertson, 1974). These septations are commonly referred to as pseudosepta because they incompletely separate adjacent cells in the vegetative hyphae. Despite their taxonomic significance, published illustrations of hyphal septa in Allomyces are lim1 Received for publication 1 November 1984revision accepted 8 April 1985. We thank R. L. Blanton, C. Haigler, and J. Taylor for their helpful suggestions. We also thank R. Berman and J. Taylor for providing the cultures of Allomyces reticulatus. This paper represents a portion of a dissertation submitted by R. J. Meyer to the Department of Botany of the University of Georgia, Athens. 2 Present address: Department of Botany, University of Toronto, Erindale Campus, Mississauga, Ontario L5L 1C6, Canada. ited to the drawings of Barrett (1912) and the micrograph of Adrian Couch (in Coker, 1930). In a detailed consideration of septa, Barrett ( 19 12) stated that "in older parts of the plant they reach their most perfect development, and then only incompletely separate the protoplasm of the adjacent segments.... They are first seen as separate processes or thickenings protruding inwardly from the wall at the constrictions. These processes increase in length, probably by accretion, until on meeting at the center of the cell, a fusion takes place and a definite central plate results." This is the generally accepted view of how septum development proceeds and what the mature septum looks like in Allomyces. However, there has been some suggestion that variation does exist in the structure of the septum within the genus Allomyces. Emerson and Robertson (1974) reported that they never observed septa with central plates and radiating strands in A. reticulatus Emerson et Robertson. Rather, they observed that A. reticulatus had septa that were "simply ring-like thickenings." At times these rings were incompletely formed and extended "only part way around the hyphal wall." Since there seemed to be variation in the structure or interpretation of septa in diffierent species of Allomyces and since septal development has never been examined ultrastructurally, a reexamination of the hyphal septa in Allomyces was undertaken. MATERIALS AND METHODS-Light microscopy-Living material of A. macrogynus Emerson et Wilson, A. arbuscula E. J. Butler, and A. reticulatus Emerson et Robertson (Emerson cultures Burma 3-35, PI1, and Cal70, respectively) were examined with both bright field and diffierential interference con

This study was undertaken to examine the ultrastructural organization of encysting zoospores, cysts, and germinating cysts of Phytophthora palmivora using the method of freeze substitution. During encystment, changes in the outer surface of the plasma membrane were accompanied by changes in organization of peripheral cisternae. Electron-opaque, vesicular structures and membrane-bound patches of cytoplasm were observed outside the plasma membrane at early stages of encystment. Dictyosomes and tubular vesicles, the latter believed to be derived from peripheral cisternae, were associated with many coated pits and coated vesicles throughout encystment. During germination, variously shaped apical vesicles were observed at the tip of the germ tube. These vesicles appeared to be derived from dictyosomes and it is suggested that their processing involves multivesicular bodies as an intermediate stage.

The gametes and the process of fertilization were examined by light and electron microscopy in the lower eukaryote Allomyces macrogynus. Differences in gamete morphology included the overall larger size and the presence of a larger nuclear apparatus, along with the association of a side-body complex and many more mitochondria in the female gamete. In this species of Allomyces, fertilization was initiated by contact and fusion of specialized regions of the gamete plasma membranes resulting in a binucleate fusion cell surrounded by plasma membrane contributed by both partners. Following plasmogamy, nuclear fusion was initiated by multiple nuclear membrane contacts between adjacent outer membranes. Following inner membrane fusion, small nucleoplasmic bridges were observed which presumably fused with one another and resulted in a single bridge which widened, forming the mature diploid nucleus. After karyogamy, fusion of the nuclear caps did not always occur and zygotes with and without fused caps were observed. Coalescence of the nucleoli completed the events of fertilization, forming a zygote with a single nuclear apparatus (sometimes with two caps) and two flagella. These observations are discussed in relation to fertilization mechanisms and compared to fertilization in other organisms.

The ultrastructural organization of the zoospores of Phytophthora palmivora was examined using freeze substitution as a potential means of improving upon the images obtained with zoospores preserved by conventional chemical fixation. Despite die technical difficulties encountered, freeze substitution was effective in preserving the overall shape of the zoospores and membranous structures, including the plasma membrane, peripheral cisternae, and the water-expulsion vacuole. Coated pits, not previously reported on the plasma membrane of zoospores of fungi, were found in P. palmivora. The nucleus and cytoskeleton of zoospores were not as well preserved as with conventional chemical fixation. Unique features of freeze-substituted zoospores were compared with structures observed in previous studies of zoospores.

The effects of cyproconazole on hyphal tip cells of Sclerotium rolfsii were examined in living cells using Nomarski optics and in fixed cells using epifluorescence staining. Hyphae grown on cyproconazole were often undulated in appearance, generally smaller in diameter, had shorter septal distances, and exhibited a higher degree of branching than did control cells. Swelling of the tip region and disruption of the spitzenkorper were frequently observed within 10 min after exposure to the fungicide at concentrations of 1.0 μg/ml. Staining with 3,3′-dihexyloxacarbocyanine iodine revealed that fewer organized Spitzenkorper were present in cells grown on agar amended with 0.1 and 0.75 μg/ml fungicide. Staining with calcofluor white, after 60 min exposure to 0.75 μg/ml of cyproconazole, showed that apical wall deposition had decreased and patchy subapical deposition appeared. Immunofluorescence staining of the microtubule cytoskeleton revealed altered patterns in microtubule arrays after 30 min exposure to cyproconazole (0.75 μg/ml). Nuclear staining with DAPI demonstrated that positioning of nuclei within the cell was not altered as a result of the fungicide. Studies using [14C]cyproconazole indicated that the maximum levels of fungicide in hyphal cells were obtained within 60 min of exposure to 0.75 μg/ml. The amount of fungicide associated with hyphal cells decreased to a constant level by 240 min.

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.

Chytridium confervae is a eucarpic, monocentric chytrid. We have used light and electron microscopy to study the relationship between the nutrient absorbing rhizoids and the asexually reproductive sporangium during growth. We have also examined the induction of zoosporogenesis by starvation, and subsequent differentiation until zoospore release. During growth the cytoplasm of the rhizoids and the developing sporangium was continuous and similar. At the start of starvation a bundle of fibers that were visible with light microscopy appeared at the junction between the rhizoids and the sporangium. Two hours after initiation of starvation a wall, that was also visible with light microscopy, formed to separate the rhizoids from the sporangium. Electron microscopy revealed a large, ordered array of microtubules in the thallus at the same time that the fibers appeared, and a sharp difference in the density of ribosomes in the cytoplasm of the sporangium and that of the rhizoids that was apparent immediately after starvation. This cytoplasmic difference was preserved by the formation of a cross-wall that was penetrated by plasmodesmata. After the wall was formed the cytoplasm of the rhizoids senesced. Comparison ofC. confervae with other organisms that use arrays of microtubules to move organelles is made and speculation on the role of the microtubules in organelle movement and wall formation inC. confervae is offered.

SUMMARYThe ultrastructure of mitosis in the zoosporangia of the water mold Catenaria anguillulae has been studied. The nuclear divisions prior to the formation of zoospores are mitotic. Centrioles ...

The morphological similarities between the kinetosome and the second centriole of the zoospores of Phlyctochytrium kniepii and P. punctatum (Chytridiomycetes) suggest that the second centriole in the chytrid zoospore is a vestigial flagellum base. It is suggested that the term vestigial kinetosome may also be used when referring to the structure which is presently termed the second centriole of the chytrid zoospore. Morphological similarities between the chytrid zoospores of P. kniepii and P. punctatum and the zoospores of Rhizidiomyces apophysatus (Hyphochytridiomycetes) are noted. The possible biflagellate origin of fungi with uniflagellate zoospores is discussed. The third fiber (C fiber) of the kinetosome triplet is shown to form as an outgrowth of the B fiber of the kinetosome doublet.

The cell walls of selected oomycetous fungi are composed primarily of glucans, and cellulose constitutes a relatively small proportion of the total glucan. The noncellulosic constituents consist of acid-soluble glucan or glucans and insoluble glucan or glucans. These noncellulosic glucan fractions contain beta-(1--3) glucosidic linkages and apparently beta-(1--6) linkages also.

Cell walls of the marine Oomycete, Atkinsiella dubia were prepared and an analysis of the wall constituents was made. The walls contained approximately 80% polysaccharides and 14% proteins along with small quantities of lipid and ash. The carbohydrate fraction was composed primarily of glucan along with 1.8% glucosamine and a trace of galactosamine. An analysis of the amino acid composition of the protein fraction showed the presence of 18 identified amino acids including a surprisingly high (20% of total amino acids) hydroxyproline content. The polysaccharide fractions of the wall were mostly glucans with solubility properties similar to those reported for other Oomycetes. As anticipated, the glucans of mechanically isolated walls were virtually identical to those prepared from chemically isolated walls. The minor glucan component, cellulose, was found to occur in the form of poorly crystalline cellulose I As expected, electron microscopy of wall specimens showed microfibrillar and amorphous regions. It was stressed that Atkinsiella walls, like those of other Oomycetes, contain large quantities of β-1→3 and β-1→6 linked glucan along with a smaller amount of cellulose.

The 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.

Zoospores of Monoblepharella sp. a representative of the order Monoblepharidales, no members of which had ever been studied with the electron microscope, were fixed with glutaraldehyde, followed by OsO4, and their ultrastructure is described. The kinetosome, to which the flagellum is attached, has the appearance of a centriole in cross section. Radiating laterally from the kinetosome is a striated disc which is anchored in a pair of annular lamellae. Microtubules run forward in the cell from a ring in the striated disc. The nucleus is surrounded by a concentration of ribosomes, the latter being partially surrounded and traversed by membranous elements. The zoospores of this Monoblepharella sp. do not have a nuclear cap of the type found in the Blastocladiales. The nature and phylogenetic distribution of nuclear caps in the aquatic fungi is discussed. A highly organized tubular structure at the posterior end of the zoospores is described and its possible function as a photosensitive structure discussed. At encystment the flagellum is withdrawn and cross sections of the flagellum are shown within cysts.

The amoebae of Acrasis rosea grown on a white mutant of Rhodotorula mucilaginosa contained three colored carotenoids. Two of these were xanthophylls, one being torulene with absorption maxima in hexane at 458, 484, and 518 mμ. The second xanthophyll, the most abundant pigment of the amoebae, was an unknown carotenoid with absorption maxima in hexane at 453, 477, and 508 mμ. A carotene with absorption maxima in hexane at 442, 468, and 500 mμ was also present in small amounts but not identified with any known carotene.It was demonstrated, contrary to a previous report, that the amoebae of A. rosea multiplied and became pigmented on carotenoid-lacking yeasts such as Saccharomyces cereviseae. On such colorless yeasts, the pigments were qualitatively the same as those isolated when A. rosea was grown on the colored wild-type and white mutant of R. mucilaginosa. The synthesis of torulene must then be accomplished, at least in its final stages, by the amoebae of A. rosea.