SCANNING ELECTRON MICROSCOPE OBSERVATINS ON ZOOSPORANGIAL ABSCISSION IN PHYCOPELTIS EPIPHYTON (CHLOROPHYA)1

Zoosporangial abscissin in asexually reproductive Phycopeltis epiphyon, Millardet, epiphytic on leaces of Osmanhus fragrans Loureiro and Sabal minor (Jacquin) Person, examined with scanning electron micrsscopy is described and compared with the process in related Cephaleuros virescens Kunze. The laeral wall of he zoosporangium-pedicel septum begins to rupure early in he abscission process and is followed by continued learing of the lateral wall and partial spliing of the septum. The zoosporangium remains attached in a hinge-like fashion to the pedicel by a portion of the lateral wall and septum. A centrally located protuberance emerges from wihin the septa of both the zoosporangium and pedicel, and together with a slight bulging of the septa, is involved in the final separation of the zoosporangium. Although there are differences in surface morphlogy of identical in P. the zoosporangia and the location of the exit pores, the process of abscission is identical in P. epiphyton and C. virescens.     

STRUCTURE AND COMPOSITION OF ZOOSPORANGIAL DISCHARGE PAPILLAE IN THE FUNGUS ALLOMYCES

A study was made of the discharge papillae of zoosporangia (ZS), as well as of resistant sporangia and gametangia, of Allomyces (mainly A. × javanicus). It was found that they differ considerably. The discharge papillae of the resistant sporangia apparently arise by a blister-like modification of the inner wall, whereas those of the ZS and the gametangia consist of a pulley-shaped, refractile plug securely placed in a hole in the wall. This plug can be successfully removed from the ZS wall by treatment with sulfuric acid. Solubility and histochemical tests were used to identify the material of the ZS plugs and the results indicate that the material is a pectic substance, most likely protopectin. The plugs of the gametangia presumably have the same composition. A preliminary study was also made of the discharge papillae of representatives of other families of the aquatic Phycomycetes. The results indicate that such pulley-shaped plugs filling a pore in the wall are characteristic only of the ZS and the gametangia of members of the family Blastocladiaceae to which Allomyces belongs.     

Rhinosporidium seeberi: Light,phase contrast,fluorescent and scanning electron microscopic study

Phase contrast microscopic study indicated the multilayered structure of the sporangial wall of R. seeberi while the scanning electronmicroscopic study revealed a trilaminated wall compared to a thick double walled light microscopic structure. The scanning electronmicroscopy revealed the spores of varying sizes which were found either discretely or in groups interconnected and seen attached to the inner aspect of the sporangial wall. Autofluorescence of sporangia and spores was observed under microscope. Acridine orange staining revealed the presence of DNA materials in the spore and sporangia.     

Microscopical observations onAvena coleoptile cell walls after stretching on a tensile-tester

In order to see if there are any structural damages or modifications of the cell wall ofAvena coleoptiles due to the stretching on an Instron tensile-tester when mechanical properties of the cell wall are measured, we made microscopic observations using cell wall specimens which had been stretched. As far as seen by light microscopic and scanning electron microscopic (SEM) observations there are no appreciable changes in the cell wall, such as breakage, tearing or formation of crack on the surface, due to the stretching which produced 10–30 g stress. It is concluded that stretching of this range causes no artifactitious changes in the wall and hence mechanical properties of the cell wall can be measured by stretching the wall specimen on a tensile-tester.     

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.     

SpoIIB localizes to active sites of septal biogenesis and spatially regulates septal thinning during engulfment in bacillus subtilis

A key step in the Bacillus subtilis spore formation pathway is the engulfment of the forespore by the mother cell, a phagocytosis-like process normally accompanied by the loss of peptidoglycan within the sporulation septum. We have reinvestigated the role of SpoIIB in engulfment by using the fluorescent membrane stain FM 4-64 and deconvolution microscopy. We have found that spoIIB mutant sporangia display a transient engulfment defect in which the forespore pushes through the septum and bulges into the mother cell, similar to the situation in spoIID, spoIIM, and spoIIP mutants. However, unlike the sporangia of those three mutants, spoIIB mutant sporangia are able to complete engulfment; indeed, by time-lapse microscopy, sporangia with prominent bulges were found to complete engulfment. Electron micrographs showed that in spoIIB mutant sporangia the dissolution of septal peptidoglycan is delayed and spatially unregulated and that the engulfing membranes migrate around the remaining septal peptidoglycan. These results demonstrate that mother cell membranes will move around septal peptidoglycan that has not been completely degraded and suggest that SpoIIB facilitates the rapid and spatially regulated dissolution of septal peptidoglycan. In keeping with this proposal, a SpoIIB-myc fusion protein localized to the sporulation septum during its biogenesis, discriminating between the site of active septal biogenesis and the unused potential division site within the same cell.     

ULTRASTRUCTURE OF THE METABASIDIUM OF AURICULARIA FUSCOSUCCINEA

Maturation of the metabasidium of Auricularia fuscosuccinea was followed with light microscopy and transmission electron microscopy. The basidium was divided into four compartments by septa which developed centripetally as in hyphae. Each septum formed a septal pore apparatus with imperforate pore caps. A band of electron-dense material was situated in the middle of the septal pore. There was a large increase in the volume of cytoplasm, excluding vacuoles, in each compartment during sterigmal outgrowth. Compartments were evacuated in basipetal sequence and vacuole enlargement began at the base of a compartment only when sterigmal formation was well advanced. The septal pore apparatus was intact until late in maturation of a compartment when septal swellings occluded the pore. The metabasidial wall was differentiated from those of other hymenial and subhymenial cells. The pattern of basidial maturation is compared with that in other phragmobasidiate and holobasidiate fungi. Use of the septal pore apparatus for phylogenetic and taxonomic purposes is discussed, as is the concept of primary and adventitious septa.     

TWO NEW MEMBERS OF THE BLASTOCLADIACEAE. II. MORPHOGENETIC RESPONSES TO O2 AND CO2

The development of Microallomyces dendroideus and Allomyces reticulatus was studied under various mixtures of O₂, CO₂ and N₂. All thalli grown in broth or agar media in air produce abundant resistant sporangia but rarely produce any zoosporangia. In both fungi, the proportion of zoosporangia borne by the thalli increases as the oxygen level decreases. In addition, increased CO₂ strongly stimulates resistant sporangium germination in M. dendroideus. Neither fungus grows in the absence of oxygen; therefore, they seem to be adapted for rapid multiplication in a microaerophilic niche.     

OBSERVATIONS ON THE LIFE HISTORY OF PAPENFUSSIELLA CALLITRICHA (PHAEOPHYCEAE,CHORDARIALES) IN CULTURE1

Papenfussiella callitricha (Rosenv.) Kylin from eastern Canada was studied in culture. Zoids from unilocular sporangia develop into microscopic, filamentous, dioecious gametophytes which produce isogametes in filament cells and few-chambered plurilocular gametangia. Unfused gametes germinate to reproduce the gametophytes. Fusion takes place between a settled (“female”) and a motile (“male”) gamete. The zygote gives rise to a filamentous plethysmothallus that reproduces asexually by zoids formed in thallus cells and in few-chambered plurilocular zoidangia. Erect macrothalli are produced on the plethysmothallus, beginning with the formation of upright filaments. Later on, these filaments become the terminal assimilators of the macrothalli. Further assimilatory filaments, rhizoids, and unilocular sporangia are produced in a branching region at the base of the terminal assimilator. Zoids from unilocular sporangia formed in culture germinate to reestablish the gametophyte phase. Chromosome counts yielded n = 19 ± 3 for the gametophytes, and 32 ± 6 for the sporophyte, both plethysmothallus and macrothallus.     

GROWTH AND DIFFERENTIATION OF AXIAL AND LATERAL FILAMENTS IN BATRACHOSPERMUM SIRODOTII (RHODOPHYTA)1

Development of the vegetative gametophyte of Batrachospermum sirodotii Skuja was examined with light and both transmission and scanning electron microscopy. Patterns of wall growth were followed using the Calcofluor White ST pulse-chase method. Thallus structure was analysed in terms of the pattern of development of the apical, periaxial and pleuridial initials that generate the axial and whorled lateral filaments characteristic of Batrachospermum. Apical cells of axial filaments elongate initially by tip growth with the nucleus maintaining a distal position. Nuclear division is horizontal. One daughter nucleus migrates basipetally and a thin, convoluted annular septum and perforate-occluded pit connection are then formed. Elongating axial cells subsequently extend by wall deposition at the base of the cell. Periaxial cells are initiated laterally and elongate primarily by tip growth while the nucleus remains within the axial cell. The nucleus then migrates to the boundary between the initial and the axial cell, divides, and one daughter nucleus moves into the initial and the other back into the axial cell. A slightly irregular annular septum and simple-occluded pit connection are then formed. Pleuridial cell initials begin as terminal to subterminal protuberances on periaxial or pleuridial cells. They first extend by tip growth and later by bipolar band growth. The nucleus remains within the parent cell as the pleuridial initial expands and a narrow septal ring is formed between the two cells. It then migrates through the septal ring into the initial and divides transversely. One nucleus passes back into the parent cell and a thick, flat septum and perforate-occluded pit connection are formed. It is concluded that the potentially indeterminate axial filaments and the determinate lateral pleuridia represent distinct developmental types in Batrachospermum.     

Morphogenesis of the reproductive shoots of Welwitschia mirabilis and Ephedra distachya (Gnetales), and its evolutionary implications

For decades, Gnetales appeared to be closely related to angiosperms, the two groups together forming the anthophyte clade. At present, molecular studies negate such a relationship and give strong support for a systematic position of Gnetales within or near conifers. However, previous interpretations of the male sporangiophores of Gnetales as pinnate with terminal synangia conflict with a close relationship between Gnetales and conifers. Therefore, we investigated the morphogenesis of the male reproductive structures of Welwitschia mirabilis and Ephedra distachya by SEM and light microscopy. The occurrence of reduced apices to both halves of the antherophores of W. mirabilis gives strong support for the assumption that the male ‘flowers’ of W. mirabilis represent reduced compound cones. We assume that each half of the antherophore represents a lateral male cone that has lost its subtending bract. Although both halves of the antherophores of Ephedra distachya lack apical meristems, the histological pattern of the developing antherophores supports interpreting them as reduced lateral male cones as well. Therefore, the male sporangiophores of Gnetales represent simple organs with terminal synangia. Although extant conifers do not exhibit terminal synangia, similar sporangiophores are reported for some Cordaitales, the hypothetical sister group of conifers. Moreover, several Paleozoic conifers exhibit male cones with terminal sporangia or synangia. Therefore, we propose that conifers, Cordaitales and Gnetales originated from a common ancestor that displayed simple sporangiophores with a terminal cluster of sporangia.     

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.     

The structure and development of septa inNeurospora crassa

Summary Electron microscopy of septa ofNeurospora crassa has shown that microfibrillar material is a major structural component. These microfibrils are arranged in a predominantly tangential orientation and in young (24 hours) mycelium can often be seen in unextracted septal preparations. In older (5 days) mycelium they are overlain with amorphous material, which can be removed by treatment with Pronase, or by chemical extractions. The structure of septa is described and compared with that of the lateral hyphal wall.Developing septa are typically surrounded by vesicles, and sometimes by alignments of cytoplasmic tubules. Electron and light microscopic autoradiography have shown that N-acetylglucosamine is quickly and specifically incorporated into developing septa, and there is no evidence for its prior incorporation into any cytoplasmic site. Glucose is also incorporated into septa, but less specifically. From the autoradiographic results and from experiments with chemical and enzymic extractions it is suggested that chitin is a major component of the septum. A model of the septum is constructed from these data.     

SEPTUM STRUCTURE IN SPINIGER MEINECKELLUS

The structure of the hyphal septum in Spiniger meineckellus is of the dolipore type and is basically like that reported for other Holobasidiomycetes. The septum forms centripetally across the hypha and is accompanied by invagination of the plasmalemma. That portion of the septum surrounding the single central pore enlarges to form a septal swelling. The laminate central region of the septum wall extends through the septal swelling to the edge of the pore. On both sides of the septum there is a convex, laminate, electron-dense, perforate septal cap that covers the septal swelling and pore. The septal cap is sometimes continuous with endoplasmic reticulum that extends along the septum. Two electron-opaque rings surround the septal pore and rest on the shoulders of the septal swelling. Small, fine, dark filaments extend from these rings to narrow electron-dense bands inside the pore.     

Early Devonian fungi: a blastocladalean fungus with sexual reproduction

In this paper we describe a fossil fungus–Paleoblastocladia milleri gen. et sp. nov.–from the 400 million-year-old Early Devonian Rhynie chert that shares numerous features with modern zoosporic fungi placed in the order Blastocladiales. The fungus occurs in tufts that arise from stomata or between the cuticle and epidermis of Aglaophyton major axes. Thallus development begins from an irregular bipolar basal cell that produces a system of intramatrical rhizoids and clavate-shaped extramatrical, nonseptate hyphae. These hyphae develop into two types of mature thalli. Sporothalli are characterized by several orders of dichotomous branching and the production of terminal, globose zoosporangia, as well as thick-walled, pitted resting sporangia. On separate dichotomously branched thalli (gametothalli) are terminal chains of two or three gametangia, in which the terminal one is slightly larger. Despite the fact that all of the reproductive organs contain either zoospores or gametes, none show evidence of discharge papillae. The fossil fungus is compared with extant members of the Blastocladiales, and the presence of sexual reproduction is discussed.     

Gonidiogenesis in Padixonia bispora Subram. (Hyphomycetes)

(Hyphomycetes). Padixonia bispora produces two kinds of conidium arranged as acropetal tandem pairs. The pair consists of a distal enteroblastic conidium connected via a narrow neck to a proximal broad-based holoblastic conidium. The proximal blastospore is derived from a branched conidiphore axis and initially forms as a distal conidiogenous cell from which arises the distal blastospore. The wall between the two conidia is formed from the narrow neck occluded by a layered plug. Conidial maturation is concurrent with conidiogenesis. Secession of the proximal spore is by a rounding-off mechanism with circumscissile rupture of the outer layer of the continuous wall between the conidiogenous cell and the conidium and concurrent schizolysis of the duplex septum. The distal blastospore is released by mechanical transverse tearing of the plug through an undulate abscission zone. The abscission zone has two major concentric rings of thickening. The outer ring is derived from the outer layer of proximal spore cell wall.     

STUDIES ON THE GENETIC CONTROL OF RESISTANT SPORANGIUM FORMATION IN ALLOMYCES

The diploid sporophyte of the phycomycetous fungus Allomyces arbuscula bears two types of sporangia: thin-walled, colorless, ephemeral zoosporangia (ZS) and thick-walled, dark-brown, resistant sporangia (RS). Normal wild-type cultures (strain Portugal IE) under standard conditions produce approximately 90% of their total sporangia as RS. These RS give the cultures a dark-brown color. A mutant was induced with UV irradiation in which the ratio of ZS to RS was shifted so that only 20% of the total sporangia are RS. These cultures are a pale, tan color. Hybrids between the mutants and wild-types produce ca. 65% RS and are also intermediate in the color of the culture. Meiotic segregation in the RS of the hybrid sporophytes gives gametophytes half of which when selfed produce mutant sporophytes and half of which produce wild-type sporophytes. The shift from RS to ZS formation is thus considered to be the result of a one-gene mutation at a locus ‘R.’ The haploid gametophytes of wild-type strains have in addition to male and female gametangia a small number (2-4%) of RS. In mutant gametophytes the percent RS has dropped to 0.1-0.2%. The proposed genotypes at the ‘R’ locus in Allomyces arbuscula are: wild-type sporophytes (RR), hybrid sporophytes (Rr), mutant sporophytes (rr), wild-type gametophytes (R) and mutant gametophytes (r).     

ULTRASTRUCTURE OF DISPERSED AND IN SITU SPECIMENS OF THE DEVONIAN SPORE RHABDOSPORITES LANGII: EVIDENCE FOR THE EVOLUTIONARY RELATIONSHIPS OF PROGYMNOSPERMS

Abstract: The spore Rhabdosporites (Triletes) langii (Eisenack) Richardson, 1960 is abundant and well preserved in Middle Devonian (Eifelian) ‘Middle Old Red Sandstone’ deposits from the Orcadian Basin, Scotland. Here it occurs as dispersed individual spores and in situ in isolated sporangia. This paper reports on a detailed light microscope (LM), scanning electron microscope (SEM) and transmission electron microscope (TEM) analysis of both dispersed and in situ spores. The dispersed spores are pseudosaccate with a thick walled inner body enclosed within an outer layer that was originally attached only over the proximal face. The inner body has lamellate/laminate ultrastructure consisting of fine lamellae that are continuous around the spore and parallel stacked. Towards the outer part of the inner body these group to form thicker laminate structures that are also continuous and parallel stacked. The outer layer has spongy ultrastructure. In situ spores preserved in the isolated sporangia are identical to the dispersed forms in terms of morphology, gross structure and wall ultrastructure. The sporangium wall is two‐layered. A thick coalified outer layer is cellular and represents the main sporangium wall. This layer is readily lost if oxidation is applied during processing. A thin inner layer is interpreted as a peritapetal membrane. This layer survives oxidation as a tightly adherent membranous covering of the spore mass. Ultrastructurally it consists of three layers, with the innermost layer composed of material similar to that comprising the outer layer of the spores. Based on the new LM, SEM and TEM information, consideration is given to spore wall formation. The inner body of the spores is interpreted as developing by centripetal accumulation of lamellae at the plasma membrane. The outer layer is interpreted as forming by accretion of sporopollenin units derived from a tapetum. The inner layer of the sporangium wall is considered to represent a peritapetal membrane formed from the remnants of this tapetum. The spore R. langii derives from aneurophytalean progymnosperms. In light of the new evidence on spore/sporangium characters, and hypotheses of spore wall development based on interpretation of these, the evolutionary relationships of the progymnosperms are considered in terms of their origins and relationship to the seed plants. It is concluded that there is a smooth evolutionary transition between Apiculiretusispora‐type spores of certain basal euphyllophytes, Rhabdosporites‐type spores of aneurophytalean progymnosperms and Geminospora‐/Contagisporites‐type spores of heterosporous archaeopteridalean progymnosperms. Prepollen of basal seed plants (hydrasperman, medullosan and callistophytalean pteridosperms) are easily derived from the spores of either homosporous or heterosporous progymnosperms. The proposed evolutionary transition was sequential with increasing complexity of the spore/pollen wall probably reflecting increasing sophistication of reproductive strategy. The pollen wall of crown group seed plants appears to incorporate a completely new developmental mechanism: tectum and infratectum initiation within a glycocalyx‐like Microspore Surface Coat. It is unclear when this feature evolved, but it appears likely that it was not present in the most basal stem group seed plants.     

Breakdown of middle lamella pectin by ●OH during rapid abscission in Azolla

Azolla, a small water fern, abscises its roots and branches within 30 min upon treatment with various stresses. This study was conducted to test whether, in the rapid abscission that occurs in Azolla, breakdown of wall components of abscission zone cells by ^●OH is involved. Experimentally generated ^●OH caused the rapid separation of abscission zone cells from detached roots and the rapid shedding of roots from whole plants. Electron microscopic observations revealed that ^●OH rapidly and selectively dissolved a well‐developed middle lamella between abscission zone cells and resultantly caused rapid cell separation and shedding. Treatment of abscission zones of Impatiens leaf petiole with ^●OH also accelerated the separation of abscission zone cells. However, compared with that of Azolla roots, accelerative effects in Impatiens were weak. A large amount of ^●OH was cytochemically detected in abscission zone cells both of Azolla roots and of Impatiens leaf petioles. These results suggest that ^●OH is involved in the cell separation process not only in the rapid abscission in Azolla but also in the abscission of Impatiens. However, for rapid abscission to occur, a well‐developed middle lamella, a unique structure, which is sensitive to the attack of ^●OH, might be needed.