Coelomomyces psorophorae vartasmaniensis Couch+Laird (1988) (Coelomomycetaeceae: Blastocladiales), a fungal pathogen of the mosquitoAedes australis

The germination of sporangia inCoelomomyces psorophorae vartasmaniensis (C. p. tas.) is uncoordinated and thus there is a mixture of developmental stages in any given population. Continuous urografin gradients separated out the critical stages of germinating sporangia giving four bands, each band representing a consecutive stage of germination. These stages were investigated for changes in the sporangial wall using Transmission Electron Microscopy (TEM). The sporangia have a typical two-layered wall, an electron dense outer layer which can be divided into three distinct sub-layers D1, D2, and D3 and an inner electron transparent secondary wall. Stage 3 sporangia have an intact D1 layer on their outer wall. In the subsequent stages (4 & 4b) there is a progressive breakdown of this layer.     

Release of Sporangiospores by a Strain of Actinoplanes

Dehiscence of Actinoplanes sp. 7-10 sporangia is triggered by wetting of the spores. This process requires time because of the hydrophobic nature of the sporangial envelope; it can be speeded up and enhanced by a wetting agent. Once wetted, the spores swell, usually ripping the sporangial wall, and escape as motile elements when functional flagella are synthesized. Flagellation and motility are separate phenomena, both of which lose intensity with age. Spores from old sporangia can regain motility when supplied with an exogenous carbon source, but, when provided only with water, phosphate buffer, or amino acids, flagellation takes place without motility. Deflagellation-reflagellation experiments indicated that functional flagella can be reformed only in presence of both amino acids and glucose which must be added within 180 min of deflagellation. Inoperative flagella were formed in the presence of inhibitors of nucleic acid synthesis, such as 6-azauracil, but inhibitors of protein synthesis, such as chloramphenicol, did not interfere with reflagellation. Flagellated spores remained so after germination.     

A putative DEAD-box RNA-helicase is required for normal zoospore development in the late blight pathogen Phytophthora infestans

The asexual multinucleated sporangia of Phytophthora infestans can germinate directly through a germ tube or indirectly by releasing zoospores. The molecular mechanisms controlling sporangial cytokinesis or sporangial cleavage, and zoospore release are largely unknown. Sporangial cleavage is initiated by a cold shock that eventually compartmentalizes single nuclei within each zoospore. Comparison of EST representation in different cDNA libraries revealed a putative ATP-dependent DEAD-box RNA-helicase gene in P. infestans, Pi-RNH1, which has a 140-fold increased expression level in young zoospores compared to uncleaved sporangia. RNA interference was employed to determine the role of Pi-RNH1 in zoospore development. Silencing efficiencies of up to 99% were achieved in some transiently-silenced lines. These Pi-RNH1-silenced lines produced large aberrant zoospores that had undergone partial cleavage and often had multiple flagella on their surface. Transmission electron microscopy revealed that cytoplasmic vesicles fused in the silenced lines, resulting in the formation of large vesicles. The Pi-RNH1-silenced zoospores were also sensitive to osmotic pressure and often ruptured upon release from the sporangia. These findings indicate that Pi-RNH1 has a major function in zoospore development and its potential role in cytokinesis is discussed.     

THE DEVELOPMENT AND CYTOLOGY OF THE EPIBIOTIC PHASE OF PHYSODERMA PULPOSUM

Lingappa , Yamuna . (U. Michigan, Ann Arbor.) The development and cytology of the epibiotic phase of Physoderma pulposum. Amer. Jour. Bot. 46(3) : 145-150. Illus. 1959.—Physoderma pulposum, a chytrid parasite on Chenopodium album L. and Atriplex patula L., has a zoosporangial epibiotic phase. The latter consists of extramatrical sporangia and intramatrical bushy rhizoids, both enclosed in large protruding galls. The sporangia are subspherical, up to 350μ in diameter, and may produce hundreds of planospores. If planospores settle on the host surface, they develop narrow germ tubes which penetrate the epidermal cells and develop into rhizoids. The planospore body, however, remains on the host surface and develops into a mature epibiotic sporangium in about 20-25 days at 16°C., 12-15 days at 20-25°C., or 6-8 days at 30°C. During development, its nucleus and daughter nuclei divide mitotically with intranuclear spindles until the sporangium contains several hundred nuclei. This is followed by progressive cleavage which delimits the planospore rudiments. When mature sporangia are placed in fresh water, the planospores are quickly formed within 1 hr. at 25°C. and begin to swarm within the sporangia. They escape in large numbers through an opening formed by the deliquescence of a papillum in the sporangial wall. The planospores are subspherical or elongate, 3-5 × 4-6 μ, and each has an eccentric orange-yellow refractive globule and a flagellum 18-22 μ in length. The electron micrographs of the flagella indicate that the flagella are absorbed from tip backward during encystment of the planospores. By periodic inoculation of the host plants with planospores from epibiotic sporangia, as well as from germinating resting sporangia, generation after generation of epibiotic sporangia have been obtained for 4 years. This proves the existence of a eucarpic, epibiotic, ephemeral zoosporangial phase in P. pulposum. Field observations on the duration and sequence of development of the fungus indicate that the endobiotic resting sporangial phase always follows the epibiotic phase. The results of infection experiments also indicate that the epi- and endobiotic phases belong to one and the same fungus, P. pulposum.     

Germination of the resting sporangia ofPhysoderma maydis,the causal agent ofPhysoderma disease of maize

Summary The structural and developmental characteristics of the resting sporangium in uniflagellate phycomycetes, together with the type of zoospore, are of high taxonomic value. Among these fungi, however, only a few electron microscopic investigations have been published on this topic, mainly due to technical problems. In the present study ofPhysoderma maydis (Blastocladiales) these problems were overcome as the resting sporangia in this species are formed synchronously, in large numbers, the germination is readily induced and the impermeability of the resting sporangium wall can be circumvented by shaking the prefixed sporangia with glass beads.The germination of the resting sporangia ofP. maydis is described by correlative light and electron microscopic studies and discussed in relation to related investigations on sporogenesis: The germination process starts by a breakdown of large electron-dense accretions found in the resting stage. Simultaneously, the peripheral location of the lipid bodies is lost. The large operculum is pushed open by a protrusion of the inner sporangial wall; an additional wall layer is formed during this process. Synaptonemal complexes are found in the nuclei at this stage, as are nuclear division figures which suggests anEuallomyces type of life cycle for this fungus. Cleavage vesicles, formed from dictyosomes or endoplasmic reticulum, ultimately separate the sporangial content into meiospores. The sequential assembly of organelles into the side body complex is described. Sequestering of the ribosomes into a nuclear cap is interpreted as taking place immediately prior to zoospore discharge.     

Ultrastructure of Wall Swellings of Germinating Sporangia of Phytophthora palmivora (Butl.) Butl.

Germ tubes of directly germinating sporangia of P. palmivoraincubated in yeast extract solution at 30 ?C usually developedinto prominent swellings from which hyphae later emerged. Thegerm tubes arose as an extension of a new germination wall formedinternal to the sporangial wall prior to germination. The germtube swellings contained typical hyphal organelles. The germtube swelling possessed a thicker wall than both hyphae growingout of it and germ tubes that did not form swellings.     

Maullinia ectocarpii gen. et sp. nov. (Plasmodiophorea), an intracellular parasite in Ectocarpus siliculosus (Ectocarpales, Phaeophyceae) and other filamentous brown algae

An obligate intracellular parasite infecting Ectocarpus spp. and other filamentous marine brown algae is described. The pathogen forms an unwalled multinucleate syncytium (plasmodium) within the host cell cytoplasm and causes hypertrophy. Cruciform nuclear divisions occur during early development. Mature plasmodia become transformed into single sporangia, filling the host cell completely, and then cleave into several hundred spores. The spores are motile with two unequal, whiplash-type flagella inserted subapically and also show amoeboid movement. Upon settlement, cysts with chitinous walls are formed. Infection of host cells is accomplished by means of an adhesorium and a stachel apparatus penetrating the host cell wall, and injection of the cyst content into the host cell cytoplasm. The parasite is characterized by features specific for the plasmodiophorids and is described as a new genus and species, Maullinia ectocarpii.     

Study of the release of cell wall degrading enzymes during adhesion of Chlamydomonas gametes

A quantitative assay for cell wall release in Chlamydomonas has been used to study the timing of release of cell wall degrading enzyme (lysin) during adhesion. Lysin activity, which shows a broad pH range and requires divalent cations, is released as a pulse within 1–2 min after mixing of mt^? and mt⁺ gametes. Thereafter, there is no further lysin release. Gametes of both mating types release the activity during aggregation with isolated gametic flagella of the opposite mating type, although mt⁺ gametes appear to release more lysin activity than mt^? gametes. Electrophoretic analysis of cell wall proteins before and after lysin degradation indicate that the major wall proteins are unchanged after wall breakdown.     

Maullinia ectocarpii gen. et sp. nov. (Plasmodiophorea), an Intracellular Parasite in Ectocarpus siliculosus (Ectocarpales, Phaeophyceae) and other Filamentous Brown Algae

An obligate intracellular parasite infecting Ectocarpus spp. and other filamentous marine brown algae is described. The pathogen forms an unwalled multinucleate syncytium (plasmodium) within the host cell cytoplasm and causes hypertrophy. Cruciform nuclear divisions occur during early development. Mature plasmodia become transformed into single sporangia, filling the host cell completely, and then cleave into several hundred spores. The spores are motile with two unequal, whiplash-type flagella inserted subapically and also show amoeboid movement. Upon settlement, cysts with chitinous walls are formed. Infection of host cells is accomplished by means of an adhesorium and a stachel apparatus penetrating the host cell wall, and injection of the cyst content into the host cell cytoplasm. The parasite is characterized by features specific for the plasmodiophorids and is described as a new genus and species, Maullinia ectocarpii.     

Ultrastructural Changes Associated with Spore Formation in Sporangia and Sporangiola of Thamnidium elegans Link

Fully formed pre-cleavage sporangia and sporangiola of Thamnidiumelegans Link were bounded by a primary wall plus a thick, internalsecondary wall layer. In sporangia in late pre-cleavage, Golgi-likecisternae were associated with groups of cytoplasmic vesiclesof characteristic size and appearance which were not found insporangia containing large cleavage vesicles. In both sporangia and sporangiola, protoplast cleavage was effectedby enlargement of endogenous cleavage vesicles each containinga lining layer of variable appearance, mutual fusion of cleavagevesicle membranes and fusion of cleavage vesicle membranes withthe plasmalemma. Golgi-like cisternae and small vesicular profileswere present in sporangium protoplasts at all stages of cleavagevesicle enlargement. In sporangia, the columella zone was delimitedby cleavage vesicles and separated from the sporogenous zoneby a fibrillar wall. A similar wall, which sometimes protrudedto form a small columella, was formed in sporangiola. Recently delimited spore protoplasts were bounded by plasmalemmamembrane derived from cleavage vesicle bounding membrane andsporangium or sporangiolum plasmalemma and surrounded by aninvesting layer derived from cleavage vesicle lining material.The investing layer at first appeared single, but later twoelectron opaque profiles were discernible. The spore wall wasformed between the investing layer and the plasmalemma. Wallsof sporangia and sporangiola which contained fully formed sporesconsisted of the primary layers only.     

Allomyces neo-moniliformis gametogenesis

Summary InAllomyces neo-moniliformis meiosis takes place during resting sporangium germination. The meiospores are characteristically binucleate and biflagellate as described byEmerson (1938) andTeter (1944). A variation in the number of nuclei and flagella per meiospore from two is correlated with germination of the resting sporangia under reduced oxygen tension. The meiospores are extremely poor swimmers and are typically amoeboid. At encystment the gamma bodies of the cell are mobilized and appear involved in cyst wall synthesis. A single mitotic division of each nucleus gives rise to four nuclei. Gamete cleavage is as described for spore cleavage inBlastocladiella (Lessie andLovett 1968). The assembly of the nuclear cap and side body complex of the spore are extremely late processes in gametogenesis. The gametes are released when the single papilla dissolves. The gametes fuse in pairs and after zygote formation the cell is uninucleate with two flagella. The biflagellate zygote is an active swimming cell. The presence of homothallism or hetero-thallism inA. neo-moniliformis is discussed.     

Electron microscopy of primary zoosporogenesis inPlasmodiophora brassicae

Primary zoosporogenesis in resting sporangia ofPlasmodiophora brassicae that had been incubated for 14 d in culture solution containing turnip seedlings was examined by transmission electron microscopy. A single zoospore differentiated within each sporangium, the differentiation being initiated by the emergence, of two flagella in the tight space formed by invagination of the plasma membrane within the sporangium. The differentiazing zoospore was similar in intracellular aspects to sporangia within clubroot galls. Then a deep groove formed on the zoospore cell body by further invagination of the plasma membrane. Two flagella appeared to coil around the zoospore cell body in parallel along this groove. Thereafter, the cell body lost the groove and became rounded following the protoplasmic condensation (contraction of cell body) during late development, and assumed an irregular shape at the stage of maturation. Intracellular features in, developing and mature zoospores were complicated, being characterized by electron-dense nuclei and mitochondria, microbodies, cored vesicles and various unidentified cytoplasmic vesicles and granules. A nucleolus-like region was observed only in the nucleus of the mature zoospore. A partially opened germ, pore was also seem in the sporangium containing the mature zoospore.     

STRUCTURE AND COMPOSITION OF THE RESISTANT SPORANGIAL WALL IN THE FUNGUS ALLOMYCES

The fine structure and chemical composition of the wall of resistant sporangia of Allomyces neo-moniliformis were investigated. Studies with the electron microscope showed that the wall is approximately 1.3 μ in thickness and is of complex construction. It consists essentially of three parts: a five-layered outer wall, two layers of “cementing substances,” and a single-layered inner wall. The presence of a highly convoluted cell membrane was also demonstrated. Six structural components were found to make up the walls of the resistant sporangia: glucose, glucosamine, chitin, melanin, protein, and lipids. Comparison of the structure and composition of the walls of resistant sporangia with the walls of hyphae and zoospores of Allomyces as reported by other investigators showed that, while the structure is very different, the composition is quite similar with only melanin and lipids apparently being absent from the zoospore and hyphal walls.     

Alteration of the Cell Surface during the Vegetative Cell Cycle of the Unicellular Green Alga Chlamydomonas reinhardtii

Alterations of the cell surface during the vegetative cell cycleof the unicellular green alga Chlamydomonas reinhardtii wereinvestigated using polyclonal antibodies against the purifiedand subsequently deglycosylated insoluble cell wall componentand against a 100 kDa polypeptide of the deglycosylated, chaotrope-solublewall fraction, respectively. Both antibodies recognized epitopeswithin the non-glycosylated domains of a ‘150 kDa’chaotrope-soluble glycoprotein (=GP3B) localized in the outerlayers of the C. reinhardtii cell wall. Immunofluorescence studiesindicated that both antibodies reacted with the surface of ‘late’sporangia (harvested 1 h before liberation of the zoospores),but not with the cell surfaces of released zoospores, growingcells and young sporangia, respectively. After pretreatmentwith aqueous LiCl, however, the cell surfaces of zoospores,growing cells and young sporangia became accessible to theseparticular antibodies. Highly purified preparations of the insolublewall fraction revealed strong immunofluorescence with both antibodiesbut not with the corresponding preimmune sera. Based on thesedata, we concluded that the antigenic sites of the insolubleglycoprotein framework of the C. reinhardtii wall are maskedby LiCl-soluble glycoproteins in single cell stages and youngsporangia, but not or to a lesser extent in the case of themother walls of ‘late’ sporangia. The conclusionwas supported by findings that (I) the multilayered structureof the mother-cell wall was disturbed in ‘late’,but not in young sporangia and that (II) the amounts of chaotropesolublecell wall glycoproteins present in the LiCl-extracts from intactsporangia decreased during ripening of the sporangia. (Received January 10, 1996; Accepted May 27, 1996)     

Anatomical Study on the Pedicels of Two Ferns

Pedicel is a stalk connecting sporangia with frond in ferns, and its structure and function are not clear. In this paper, we studied the pedicels of Dryopteris zhuweimingii and Pentarhizidium orientale. We found that the pedicels consist of three lines of cells, and the cell wall can be separated into two layers. The inner layer is secondary cell wall (S1) that spiral and cling to the outer layer. The outer layer is primary cell wall. And when we broke the pedicel into two sections, the inner layer of the cell wall could be pulled out like a spiral belt. This structure may be important to support and protect the sporangia.     

STRUCTURE AND GERMINATION OF BLASTOCLADIELLA EMERSONII RESISTANT SPORANGIA

Resistant sporangia of Blastocladiella emersonii were induced by the addition of bicarbonate, potassium chloride, sodium chloride, or ammonium chloride to the medium and by the exposure of the zoospores to ultraviolet irradiation. Mature resistant sporangia induced by all of these conditions exhibit similar areolate wall pitting. Under suitable conditions resistant sporangia in all cases examined germinated with the cracking of the outer sporangial wall, with the formation of exit tubes by the inner sporangial wall, and with the cleavage and release of zoospores through discharge papillae formed in the tips of the exit tubes.     

The specific identity and the life history of JapaneseSyringoderma (Syringodermatales,phaeophyceae)

Observations on JapaneseSyringoderma from Rishiri Island, which had been previously identified asS. australe, were made based on newly collected fertile material. These results and comparisons with the type specimen ofSyringoderma abyssicola (=Chlanidophora abyssicola) revealed that the Rishiri Island plants should be identified asSyringoderma abyssicola. Syringoderma abyssicola from Rishiri Island formed unilocular sporangia among the paraphyses on the fan-shaped blades in winter. The first products (uni-spores) in the unilocular sporangia form flagella, and soon after form cell walls before release. Then these reduced gametophytes divide into tetrads and form swarmers, each of which contains a chloroplast with a stigma. These swarmers germinate into branched filaments, from which thicker erect filaments of apical growth tissue. At 5–10 C these erect filaments formed fan-shaped blades under long-day conditions, and unilocular sporangia under short-day conditions corresponding respectively to spring and winter at Rishiri Island. Accordingly, the seasonal growth pattern of the species is considered to be controlled by responses to photoregime and temperature. Dedicated to the memory of the late Professor Muneao Kurogi.     

The arrangement of microfibrils in sporangial walls of Allomyces

Summary The microfibrillar component of the walls of zoosporangia and resistant sporangia of the phycomycete Allomyces arbusculus has been studied in the electron microscope, after chemical removal of the amorphous wall materials. In the zoosporangium wall the microfibrils are randomly arranged, as in the outer layer of the hyphal walls, and the sporangial wall is of even thickness. In the resistant sporangia the microfibrillar layer of the wall is perforated by numerous pores 0.25 in diameter. The microfibrils are randomly arranged over much of the wall but tend to be concentrically arranged in the vicinity of the pores. On the inside of the wall the microfibrils form a thickened rim around the pore.     

Oligosaccharide side chains of wall molecules are essential for cell-wall lysis in Chlamydomonas reinhardtii

The glycoproteins of the cell walls of Chlamydomonas are lysed during the reproductive cycle by proteases (autolysins) which are specific for their substrates. The autolysin which digests the wall of sporangia to liberate the zoospore daughter cells in the vegetative life cycle is a collagenase-like enzyme which attacks only selected domains in its wall substrates containing (hydroxy)-proline clusters. Cell-wall fractions obtained by salt-extraction (NaClO₄) and oxidizing agents (NaClO₂) and the insoluble residue were tested as substrates. The most-crosslinked insoluble inner part of the wall is the best substrate for the sporangia autolysin. Oligosaccharides obtained from the insoluble cell-wall fraction of sporangia by hydrolysis with Ba(OH)₂ inhibit autolysin action. We conclude that the oligosaccharide side chains of wall substrates are essential for forming the reactive enzyme-substrate complex.Abbreviations CSW chlorite-soluble cell-wall fraction - ICW insoluble cell-wall fraction - PSW salt-soluble fraction - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis