Microscopic observations of germination and septum formation in pycnidiospores of Botryodiplodia theobromae

A population of aseptate pycnidiospores of the fungus Botryodiplodia theobromae can be induced to germinate or to form septa delimiting two cells; this developmental process is dependent upon nutritional and environmental factors. Transmission electron microscope investigations indicate that during germination of the aseptate spore, a new inner wall layer is synthesized de novo at the site of germ tube emergence. Formation of the septum also involves the de novo synthesis of an inner wall layer which comprises the majority of the septum and completely surrounds the spore. The wall of the germ tube emerging from the septate spore is a direct extension of this inner layer deposited during the formation of the septum. Although the early stages of spore germination may involve localized enzymatic degradation of the internal layers of the spore wall, transmission and scanning electron micrographs of germinating spores show that the outer wall layers are physically fractured by the emerging germ tube. It is suggested that spore germination and septum formation are initially similar processes regarding cell wall genesis but that some mechanism responsive to environmental and nutritional conditions determines the course of development.     

Scanning electron and light microscopy of appressorium development in Piptocephalis unispora (Mucorales)

Appressorium development in the mycoparasite Piptocephalis unispora was studied by means of scanning electron microscopy using the techniques of critical point drying, sputter coating and light microscopy. The germ tube which contacts both the young host hypha or a germinating spore swells at the tip to form an appressorium closely adpressed to the surface of the host. Lateral proliferation of hyphae may occur from the mature appressorium. Factors affecting the sites of appressorium development are suggested and their significance discussed.     

Wall fibrils ingerminating sporangiospores of Gilbertella persicaria (Mucorales)

Summary In germinated sporangiospores of Gilbertella persicaria, negatively contrasted fibrils, 20–70 Å diam, are seen in thin sections of the inner vegetative wall that is continuous with the germ tube wall. The fibrils are randomly oriented in a loose network in this wall and in the germ tube wall. Germ tubes have an additional surface layer of fine, positively contrasted fibrils which appear as a nap-like coating on the hyphae. Patterns of wall fibril orientation are not revealed by transverse sections of spore and germ tube walls, whereas oblique and tangential sections are favorable for examining cell wall architecture in situ. Staining patterns show textural and compositional differences among various wall layers.     

Ultrastructure of the Wall of the Germinating Sporangiospore of Linderina pennispora (Mucorales)

Carbon replicas of germinating sporangiospores of Linderinapennispora show the outer wall complex to break open basally,during the phase of swelling, and the surface of the germ tubeto be smooth. Chemical treatment reveal the microfibrillar wallof the germ tube to be continuous with the microfibrillar innerwall complex of the spore. Microfibrils of the germ tube arerandornly arranged and appear to be finer than those of thespore wall. Ultra-thin sections reveal the wall of the germtube to consist of an outer electron-dense layer and an innermicrofibrillar electron-transparent layer and both layers originatein the basal region of the spore between the plasmalemma andthe inner wall complex of the spore.     

Fine structure of germinatingPenicillium megasporum conidia

Summary Penicillium megasporum conidia have spore walls consisting of several layers. There is no visible change in the outer wall layers during spore germination, but the inner layers increases in thickness on only one side of the spore, resulting in a rupture of the outer wall layers and subsequently in germ tube formation. Invaginations in the plasma membrane disappear as the germ tube forms and emerges, and the nucleus migrates into the developing germ tube. Mitochondria gather at the base of the germ tube during its formation. During germination, the amount of lipid in the spore decreases and portions migrate into the germ tube. Membrane-bound, electron dense bodies are present in resting spores. These bodies decrease in size as germination proceeds, and the cytoplasm in the developing germ tube appears much more electron dense than the cytoplasm within the spore.     

Ultrastructure of spore development in <Emphasis Type="Italic">Scutellospora heterogama</Emphasis>

The ultrastructural detail of spore development in Scutellospora heterogama is described. Although the main ontogenetic events are similar to those described from light microscopy, the complexity of wall layering is greater when examined at an ultrastructural level. The basic concept of a rigid spore wall enclosing two inner, flexible walls still holds true, but there are additional zones within these three walls distinguishable using electron microscopy, including an inner layer that is involved in the formation of the germination shield. The spore wall has three layers rather than the two reported previously. An outer, thin ornamented layer and an inner, thicker layer are both derived from the hyphal wall and present at all stages of development. These layers differentiate into the outer spore layer visible at the light microscope level. A third inner layer unique to the spore develops during spore swelling and rapidly expands before contracting back to form the second wall layer visible by light microscopy. The two inner flexible walls also are more complex than light microscopy suggests. The close association with the inner flexible walls with germination shield formation consolidates the preferred use of the term ‘germinal walls’ for these structures. A thin electron-dense layer separates the two germinal walls and is the region in which the germination shield forms. The inner germinal wall develops at least two sub-layers, one of which has an appearance similar to that of the expanding layer of the outer spore wall. An electron-dense layer is formed on the inner surface of the inner germinal wall as the germination shield develops, and this forms the wall surrounding the germination shield as well as the germination tube. At maturity, the outer germinal wall develops a thin, striate layer within its substructure.     

Electron microscopy of spore germination and cell wall formation in Mucor rouxii

Summary The fine structure of ungerminated and aerobically germinated sporangiospores of Mucor rouxii was compared. The germination process may be divided into two stages: I, spherical growth; II, emergence of a germ tube. In both stages, germination is growth in its strictest sense with overall increases in cell organelles; e.g., the increase in mitochondria is commensurate with the overall increase in protoplasmic mass. Noticeable changes occurring during germination are the disappearance of electron-dense lipoid bodies, formation of a large central vacuole and, most strikingly, formation of a new cell wall. Unlike many other fungi, M. rouxii does not germinate by converting the spore wall into a vegetative wall. Instead, as in other Mucorales, a vegetative wall is formed de novo under the spore wall during germination stage I. This new wall grows out, rupturing the spore wall, to become the germ tube wall. Associated with the apical wall of the germ tube is an apical corpuscle previously described. The vegetative wall exhibits a nonlayered, uniformly microfibrillar appearance in marked distinction to the spore wall which is triple-layered, with two thin electron dense outer layers, and a thick transparent inner stratum. The lack of continuity between the spore and vegetative walls is correlated with marked differences in wall chemistry previously reported. A separate new wall is also formed under the spore wall during anaerobic germination leading to yeast cell formation. On the other hand, in the development of one vegetative cell from another, such as in the formation of hyphae from yeast cells, the cell wall is structurally continuous. This continuity is correlated with a similarity in chemical composition of the cell wall reported earlier.     

A monoclonal antibody specific for conidia and mycelium wall layer of Penicillium and Aspergillus.

A monoclonal antibody was obtained from BALB/c mice immunized with Penicillium frequentans mycelium. The specificity of the antibody was evaluated by enzyme-linked immunosorbent and indirect immunofluorescence assays against the same mycelium. This IgM antibody cross-reacted with various strains of the Penicillium and Aspergillus genera. By indirect immunofluorescence assays, the antibody was able to stain about 10% of Penicillium and Aspergillus conidia, but major part of conidia did not absorb the fluorescence-labeled antibody before swelling. During germination of P. frequentans conidia, the germ tube wall which constitutes a continuation of an inner wall layer was also stained. During germination of P. griseofulvum, the protrusion of the germ tube wall was not always recognized by the antibody because the germ tube wall was constituted by a continuation of an outer spore wall layer. The study of the staining patterns of the spores and the protrusions suggests that the antibody specifically recognizes an antigen of the inner spore wall layer. The monoclonal antibody reacts with extracellular galactomannans produced by genera Aspergillus and Penicillium but is not directed against beta-(1,5)-linked galactofuranose units.     

Ultrastructure of hyphae and ascospores in the genus Eremascus eidam

Hyphae and ascospores of Eremascus fertilis and E. albus were studied in ultrathin sections. The lateral wall of the hyphae had a thick electron-light inner layer and a thin dark outer layer. The septa had a simple central pore with or without a plug, and there were Woronin bodies in the vicinity. The wall of the ascospores of E. fertilis showed a thick light inner layer and a thin dark outer layer. In the wall of the spores of E. albus a dark fibrillar layer was present between the light inner layer and the dark outer layer. The spores of this species germinated with a tube the wall of which was continuous with a newly formed layer inside the spore wall.This investigation was supported by the Netherlands Organization for the Advancement of Pure Research (Z. W. O.)     

Ultrastructure of germination and mucilage production inHalosphaeria appendiculata (Halosphaeriaceae)

The germination of ascospores of the marine fungusHalosphaeria appendiculata was investigated with transmission electron microscopy. Prior to germination, settled ascospores became surrounded by a fibro-granular layer. Small, membrane-bounded vesicles and larger electron-dense membrane-bounded vesicles aggregated at the site of germ tube formation where the plasmalemma adjacent to the aggregation was convoluted. The vesicles appeared to fuse with the plasmalemma, releasing their contents. Enzymatic digestion of the spore wall probably occurred at the time of germ tube emergence. After the nucleus had migrated into the newly formed germ tube, a septum was formed to delimit the germ tube from the ascospore. The growing germ tube can be divided into 3 morphological regions, namely the apical, sub-apical and vacuolated regions, and is typical of other fungi. A mucilaginous sheath was associated with the older mycelium. The germ tube displaced the polar appendage, and the ascospore, germ tube and appendage were enclosed in a mucilaginous sheath. In ascospores which subtended old germ tubes, the nucleus and lipid body became irregular in shape and the cytoplasm was more vacuolated. Microbody-like structures remained associated with the lipid throughout development, and were present in old ascospores.     

Ultrastructural study of spore wall morphogenesis inOphioglossum thermale kom. var.nipponicum (Miyabe et Kudo) nishida

Spore wall morphogenesis ofOphioglossum thermale var.nipponicum was examined by transmission electron microscopy. The spore wall of this species consists of three layers: endospore, exospore, and perispore. The spore wall development begins at the tetrad stage. At first, the outer undulating lamellar layer of the exospore (Lo) is formed on the spore plasma membrane in advance of the inner accumulating lamellar layer (Li) of the exospore. Next, the homogeneous layer of the exospore (H) is deposited on the outer lamellar layer. Both lamellar layers may be derived from spore cytoplasm; and the homogeneous layer, from the tapetum. Then the endospore (EN) is formed. It may be derived from spore cytoplasm. The membranous perispore (PE), derived from the tapetum, covers the exospore surface as the final layer. Though the ornamentation of this species differs distinctly from that ofO. vulgatum, the results mentioned above are fundamentally in accordance with the data obtained fromO. vulgatum (Lugardon, 1971). Therefore, the pattern of spore wall morphogenesis appears to be very stable in the genusOphioglossum.     

The tapetum inSchizaea pectinata (Schizaeaceae) and a comparison with the tapetum inPsilotum nudum (Psilotaceae)

A combination tapetum consisting of a cellular, parietal component and a plasmodial component occurs inSchizaea pectinata. A single, tapetal initial layer divides to form an outer parietal layer which maintains its cellular integrity until late in spore wall development. The inner tapetal layer differentiates into a plasmodium which disappears after the outer exospore has developed. In the final stages of spore wall development, granular material occurs in large masses and is dispersed as small granules throughout the sporangial loculus. No tapetal membrane develops. Comparisons are drawn with the combination tapetum found inPsilotum nudum.     

Untersuchungen über den chemischen Aufbau von Sporen- und Keimschlauchwänden der Uredosporen des Weizenrostes (Puccinia graminis var. tritici)

Zusammenfassung Zellwände und Keimschläuche von Uredosporen des Weizenrostes (Puccinia graminis var. tritici) wurden isoliert, und ihre chemische Zusammensetzung wurde quantitativ untersucht. Als gemeinsame Bausteine enthalten Sporenwände und Keimschlauchwände Proteine, Lipide und die Neutralzucker Galaktose, Glucose und Mannose. Die einzelnen Komponenten liegen in unterschiedlicher Menge vor. Auch qualitativ unterscheiden sich die Sporenwände und die Keimschlauchwände: Melanin ist nur in den Sporenwänden vorhanden, in den Keimschlauchwänden dagegen nicht. Der polymer gebundene Aminozucker der Keimschlauchwände ist N-Acetylglucosamin, das mit großer Wahrscheinlichkeit als Chitin vorliegt. Die Sporenwände enthalten dagegen polymeres Glucosamin (vermutlich Chitosan).Sporenwände sind in 3% iger NaOH löslich. Aus diesem Extrakt läßt sich mit Fehlingscher Lösung ein Galaktoglucomannan fällen, das überwiegend aus Mannose besteht. Aus der entsprechenden Fraktion der Keimschlauchwände, in der ebenfalls Mannose überwiegt, kann mit Fehlingscher Lösung kein Mannan gewonnen werden. Der in NaOH unlösliche Satz der Keimschlauchwände ist zum größten Teil aus Glucose und N-Acetylglucosamin aufgebaut. Es gibt keine identischen Polysaccharidfraktionen von Sporen- und Keimschlauchwänden. Sie sind heteropolymer und setzen sich jeweils aus Galaktose, Glucose und Mannose zusammen.

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Investigations on the chemical composition of spore walls and germ tube walls of wheat rust (Puccinia graminis var. tritici) uredospores

Summary Spore walls and germ tube walls from uredospores of wheat stem rust (Puccinia graminis var. tritici) were isolated and their chemical compositions determined quantitatively. The spore and germ tube walls are commonly composed of proteins, lipids, and the neutral sugars mannose, glucose and galactose. Carbon and nitrogen content, total lipids, composition of bound amino acids, total glucosamine and chitin content, and neutral sugars of spore and germ tube walls were compared. While the carbon content of the germ tube walls is only slightly higher than that of the spore walls, the germ tube walls contain twice as much nitrogen and lipids as the spore walls. The higher nitrogen content of the germ tube walls is due to higher amounts of bound amino acids and hexosamine. The polymeric germ tube wall hexosamine is insoluble in 3% NaOH, while the bulk of the polymeric spore wall hexosamine will go into solution when treated with 3% NaOH. The polymeric amino sugar of the germ tube wall is N-acetylglucosamine, which in all probability is present as chitin. In comparison, spore walls contain polymeric glucosamine (probably chitosan).The predominant neutral sugar of the spore walls is polymeric mannose (90%) while the germ tube walls contain polymeric glucose and mannose in nearly equal amounts. Galactose occurs in both wall types as a minor constituent.From spore walls completely dissolved in 3% NaOH we were able to precipitate a galactoglucomannan with fehling's solution. This galactoglucomannan was composed mainly of mannose. The corresponding fraction of the germ tube wall gave no precipitate with Fehling's solution. An alkali insoluble fraction of the germ tube wall consists mainly of glucose and N-acetylglucosamine. There are no identical polysaccharide fractions in spore walls and germ tube walls. They are always heteropolymers. Melanine is found in spore walls but not in germ tube walls.

     

Electron microscopy of germinating ascospores ofSaccharomyces cerevisiae

The wall of mature ascospores ofSaccharomyces cerevisiae showed in sections under the electron microscope a dark outer layer and a lighter inner layer. The latter was composed of a greyish inner part and a light outer part. During germination, the spore grew out at one side and the dark outer layer was broken. Of the light inner layer, the inner greyish part became the wall of the vegetative cell, but the extented part of the cell had a new wall.     

ULTRASTRUCTURE OF GERMINATING CARPOSPORES OF PORPHYRA VARIEGATA (KJELLM.) HUS (BANGIALES,RHODOPHYTA)1

The fine structure of released, attached, and germinating carpospores of Porphyra variegata (Kjellm.) Hus is described. Adhesive vesicles, formed during sporogenesis and discharged upon settling of the spore, produced a layer of adhesive mucilage around the spore and filled a deep imagination on the spore's ventral side. The mucilage layer was punctured by the emergence of a germ tube. Both spore and germ tube were lined by newly deposited cell wall. Germination was accompanied by vacuolation and starch mobilization. The morphological development of the sporeling was not noticeably influenced by the great variability of the timing, location, and orientation of septum formation. The attached carpospore possessed a plastid like that of gametophyte cells: stellate with one large central pyrenoid and no peripheral encircling thylakoids. Cells of mature vegetative cells of the conchocelis had plastids that were elongate and parietal and had multiple pyrenoids and encircling thylakoids. Most stages in the transition between the two forms of plastids occurred during carpospore germination.     

A structural comparison of cyst and germ tube wall inPhytophthora palmivora

Summary An electron microscopic analysis of germinating cysts ofPhytophthora palmivora involving freeze-etching, thin sectioning, and replica techniques reveals that both cyst and hyphal wall comprise a two-phase system with a fibrillar and an amorphous component. The cyst wall is fibrillar throughout with the fibrils tightly interwoven and embedded in an amorphous matrix on the internal side of the wall. The hyphal wall consists of a fibrillar inner layer with the fibrils lightly covered by some amorphous material and an amorphous outer layer devoid of any fibrillar material. Both cyst and germ tube walls are wholly or partially covered by a fluffy coat of variable thickness. In the zone of germ tube emergence cyst wall and germ tube wall overlap and are tightly apposed. Thus, the germ tube wall is not a simple extension of the cyst wall but a new structural entity separated from the cyst wall by a thin line of demarcation.     

COMPARATIVE DEVELOPMENT OF THE RED ALGAL PARASITES BOSTRYCHIOCOLAX AUSTRALIS GEN. ET SP. NOV. AND DAWSONIOCOLAX BOSTRYCHIAE (CHOREOCOLACACEAE,RHODOPHYTA)1

The development of two red algal parasites was examined in laboratory culture. The red algal parasite Bostrychiocolax australis gen. et sp. nov., from Australia, originally misidentified as Dawsoniocolax bostrychiae (Joly et Yamaguishi-Tomita) Joly et Yamaguishi-Tomita, completes its life history in 6 weeks on its host Bostrychia radicans (Montagne) Montagne. Initially the spores divide to form a small lenticular cell, and then a germ tube grows from the opposite pole. Upon contact with the host cuticle, the germ tube penetrates the host cell wall. The tip of the germ tube expands, and the spore cytoplasm moves into this expanded tip. The expanded germ tube tip becomes the first endophytic cell from which a parasite cell is cut off that fuses with a host tier cell. The nuclei of this infected host cell enlarge. As parasite development continues, other host-parasite cell fusions are formed, transferring more parasite nuclei into host cells. The erumpent colorless multicellular parasite develops externally on the host, and reproductive structures are visible within 2 weeks. Tetrasporangia are superficial and cruciately or tetra-hedrally divided. Spermatia are formed in clusters. The carpogonial branches are four-celled, and the carpogonium fuses directly with the auxiliary (support) cell. The mature carposporophyte has a large central fusion cell and sympodially branched gonimoblast filaments. Early stages of development differ markedly in Dawsoniocolax bostrychiae from Brazil. Upon contact with the host, the spore undergoes a nearly equal division, and a germ tube elongates from the more basal of the two spore cells, penetrates the host cell wall, and fuses with a host tier cell. Subsequent development involves enlargement of the original spore body and division to form a multicellular cushion, from which descending rhizoidal filaments form that fuse with underlying host cells. This radically different development is in marked contrast to the final reproductive morphology, which is similar to B. australis and has lead to taxonomic confusion between these two entities. The different spore germination patterns and early germ-ling development of B. australis and D. bostrychiae warrant the formation of a new genus for the Australian parasite.     

Ultrastructure of developing basidiospores in <Emphasis Type="Italic">Rhizopogon roseolus</Emphasis> (= <Emphasis Type="Italic">R. rubescens</Emphasis>)

The ultrastructure of developing basidiospores in Rhizopogon roseolus is described. When viewed in the fruiting body chamber using scanning electron microscopy, basidiospores appear narrowly ellipsoid and have smooth walls. Eight basidiospores are usually produced on the apex of each sterigma on the basidium. Transmission electron micrographs showed that basidiospores formed by movement of cytoplasm (including the nuclei) via the sterigmata, and then each basidiospore eventually became separated from its sterigma by an electron-lucent septum. The sterigma and basidium subsequently collapsed, resulting in spore release. Freshly released spores retained the sterigmal appendage connected to the collapsed basidium. After spore release, the major ultrastructural changes in the spore concerned the lipid bodies and the spore wall. During maturation, lipid bodies formed and then expanded. Before release, the spore wall was homogeneous and electronlucent, but after release the spore wall comprised two distinct layers with electron-dense depositions at the inner wall, and the dense depositions formed an electron-dense third layer. The mature spore wall complex comprised at least four distinct layers: the outer electron-lucent thin double layers, the mottled electron-dense third layer, and the electron-lucent fourth layer in which electron-lucent granular substances were dispersed.     

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.     

Ultrastructure of the nematode pathogen, Bacillus penetrans

The ultrastructure and development of Bacillus penetrans in root-knot nematodes, Meloidogyne spp., was studied with a transmission electron microscope. Host infection was by a germ tube from the cup-shaped sporangium containing the endospore. The prokaryotic vegetative cells contained septa formed by an ingrowth of the inner layer of the trilaminate cell wall and were associated with mesosomes. Structure of the endospore was similar to other bacteria with a spore protoplast enclosed within two cortical layers and three spore coats. An exosporium which may function in attachment and host specificity surrounded the endospore. Ultrastructural changes accompanying sporulation were similar to those reported for other endospore-forming bacteria but with some parasite specialization. The filamentous vegetative growth was characteristic of some Actinomycetales. Endospore development at the apices of dichotomously branched filaments of the thallus resembled the genus Actinobifida.