Germination of spores of Micromonospora chalcea: physiological and biochemical changes

Germinating spores of Micromonospora chalcea pass through three morphological stages: darkening, swelling and germ tube emergence. The process of germination has pH and temperature optima of 8.0 and 40 degrees C, respectively, and is not affected by activation treatments. Darkening, accompanied by a loss of heat resistance and refractility and a decrease in absorbance of the dormant spores, needs only energy, which can be obtained from endogenous sources, and exogenous cations. Agents that inhibit ATP formation block darkening, but inhibitors of macromolecular synthesis do not affect it. Swelling requires exogenous carbon but not nitrogen sources and is characterized by a 30 to 40% increase in spore diameter. RNA synthesis is necessary for swelling and inhibitors of protein synthesis delay this process. During this stage, maximum respiratory, cytochrome oxidase and catalase activities are reached. DNA synthesis starts at the beginning of germ tube emergence. This final stage requires both exogenous carbon and nitrogen sources and the sequence of macromolecular synthesis is RNA, protein and, finally, DNA. Rifampicin, streptomycin and mitomycin C prevent protein and DNA synthesis regardless of when added during germination. Rifampicin inhibits [3H]uridine incorporation immediately but there is a delay of about 160 min in the case of streptomycin or mitomycin C.     

Differential expression of desaturases and changes in fatty acid composition during sporangiospore germination and development in Mucor rouxii

Polyunsaturated fatty acids (PUFAs), namely, oleic (C18:1), linoleic (C18:2), and gamma-linolenic acid (C18:3), constituted the majority in the total fatty acid content (44%) of sporangiospores of Mucor rouxii. At 30 degrees C, the germination begins within 1h at which time spore swelling occurs, followed by germ tube emergence within 3-4h. Throughout germination, an increase in gamma-linolenic acid (GLA) was observed and its content was highest at germ tube emergence. It took longer for sporangiospores of M. rouxii to germinate at sub-optimal temperatures (15 and 35 degrees C). However, the content of GLA was higher at the germ tube initiation than at the mycelial stage at all temperatures, suggesting the association of GLA and germination of sporangiospores. This finding was substantially confirmed by differential expression of delta9-, delta12-, and delta6-desaturase genes measured during spore germination. The expression of three desaturase genes parallels the pattern of GLA synthesis. By using RT-PCR techniques to follow gene expression, we found that mRNA of delta12- and delta6-desaturase genes were translated as soon as the spores were introduced into a fresh medium while the mRNA of delta9-desaturase gene could not be detected until 2h after introduction. A sharp increase in mRNA of delta6-desaturase genes correlated well with an increase in GLA content at germ tube emergence (4h). These results demonstrated that changes in fatty acid composition of sporangiospore of M. rouxii and differential expression of desaturase genes occurred during germination, and that extensive changes in GLA synthesis associated with some events in germination process.     

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.     

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.     

Reactive products formed by peroxidase catalyzed oxidation of p-phenetidine

The drug 4-nitroquinoline 1-oxide (4NQO) is a potent inhibitor of Dictyostelium discoideum spore germination. This inexpensive, water soluble drug is active at a concentration of 5 micrograms/ml (26 microM) and permeates the spore at all stages in germination. Spores subjected to 4NQO treatment exhibit an irreversible blockage of myxamoebae emergence, but spore activation, post-activation lag, and swelling are not affected. Swollen 4NQO-treated spores lose the outer two spore walls but lack the ability to degrade the innermost wall. The drug does not affect oxygen uptake during post-activation lag or swelling, and only a stage specific depression in O2 uptake is observed when control spores begin to release myxamoebae. When added early in germination, 4NQO blocks the incorporation of [3H] uracil into a cold trichloroacetic acid (TCA) insoluble fraction by 98%. However, when the drug is added midway through germination and followed by a pulse labelling period of 1 h, only 65% inhibition of RNA synthesis is observed. This lack of complete inhibition may occur because the drug requires metabolic activation; thus, new rounds of RNA synthesis may have initiated before the drug became fully activated. 4NQO also blocks the de novo expression of beta-glucosidase activity when added early in germination. Additionally, we observe that vegetative cellular slime mold cells are 100 times more resistant than spores to 4NQO-induced damage. Taken together, our results support the observation that RNA synthesis is only required for the emergence stage of germination and that dormant D. discoideum spores may lack efficient excision repair mechanisms.     

Cell cycle analysis in asynchronous cultures using the BUdR-Hoechst technique.

During synchronized germination of spores of Dictyostelium discoideum, protein synthesis begins almost concomitantly with syntheses of messenger-like RNA (mlRNA) and 4–5S RNA (presumably tRNA) in the swollen spore stage and the initiation of ribosomal RNA (rRNA) synthesis is somewhat delayed. DNA synthesis occurs in the early stages of the amoeba emergence phase. Cycloheximide (200 μg/ml) blocked spore germination as well as total protein synthesis, whereas actinomycin D (60 μg/ml) did not affect either. This concentration of actinomycin D selectively inhibited formation of rRNA but did not influence the synthesis of mlRNA. Examinations of RNA labeled with [¹⁴C]uracil during germination indicated that polysomes initially detectable in the course of the germination process contain ¹⁴C-labeled mlRNA. It was concluded that at least some of mRNA synthesized during germination of D. discoideum spores is involved in protein synthesis required for the germination.     

Germination and outgrowth of Schizosaccharomyces pombe ascospores isolated by Urografin density gradient centrifugation.

A simple method for the isolation of single ascospores of the fission yeast Schizosaccharomyces pombe was examined. Single spores in the 7-day-old sporulating culture of a homothallic strain were separated from remaining vegetative cells by isopycnic centrifugation in the linear gradient from 10 to 60% of Urografin solution at 700 X g for 20 min. Protein content of isolated spores was very low as compared with that of vegetative cells. The isolated spores germinated through the following steps when cultured in a liquid medium at 25--35 degrees C; loss of refractility (darkening) under a phase-contrast microscope, spherical growth (swelling), emergence of germ tubes, elongation of germ tubes, cell plate formation, and cell separation. The absorbance at 650 nm of the spore suspension initially decreased, accompanied by darkening of spores, and then increased with spherical growth. The germination rate of isolated spores reached almost 100%.     

Pattern of protein degradation during germination of Streptomyces antibioticus spores

The pattern of protein degradation during germination of Streptomyces antibioticus spores was studied by the pulse and chase technique. Two different protein fractions were found. First, a fraction of the proteins synthesized during the darkening process (20-30%) was quickly degraded in the 30 min following the labelling period. This rapid protein degradation was partially inhibited by protease inhibitors: p-chloromercuribenzoic acid, phenylmethylsulphonylfluoride, and o-phenanthroline. Second, the remaining 70-80% and the entire protein population formed during spore swelling and germ tube emergence were degraded with a lower and constant rate (3.3-6.0% /h). A stable mRNA fraction of the dormant spores was translated upon incubation of the spores in a minimal synthetic medium (MSM) or in distilled water. However, the degradation of these proteins did not occur unless the spores were then incubated in the MSM. A strong correlation between the degradation pattern of these proteins and that of those quickly degraded at the beginning of germination was observed. Protease activity in cell-free extracts of dormant spores was detected. Inhibition studies suggest the presence of serine, thiol, and metalloproteases. The protease activity, using casein as substrate, remained constant during the darkening process and started to increase progressively from the beginning of spore swelling.     

Fungal spore swelling and germination are restricted by the macrophage phagolysosome

Many species of medically important fungi are prolific in the formation of asexual spores. Spores undergo a process of active swelling and cell wall remodelling before a germ tube is formed and filamentous growth ensues. Highly elongated germ tubes are known to be difficult to phagocytose and pose particular challenges for immune phagocytes. However, the significance of the earliest stages of spore germination during immune cell interactions has not been investigated and yet this is likely to be important for defence against sporogenous fungal pathogens. We show here that macrophages restrict the early phases of the spore germination process of Aspergillus fumigatus and Mucor circinelloides including the initial phase of spore swelling, spore germination and early polarised growth. Macrophages are therefore adept at retarding germination as well as subsequent vegetative growth which is likely to be critical for immune surveillance and protection against sporulating fungi.     

Microcycle conidiation in Penicillium urticae: an ultrastructural investigation of conidial germination and outgrowth.

A cultivation system has been developed for Penicillium urticae (NRRL 2159A) which yields 'microcycle' conidiation in submerged culture. Spherical growth of conidia was initiated by incubation at 37 degrees C in a growth-favoring medium. Transfer of these enlarged conidia to a nitrogen-poor medium at 35 degrees C resulted in synchronous germination and limited outgrowth followed by roughly synchronous conidiogenesis. An ultrastructural study of the germination stage indicated nuclear migration into the emerging germ tube whose new cell wall was an extension of the parent conidium's innermost cell wall layer. Septal formation at the neck of the germ tube followed. The septal pore was filled with particulate material and the septal membranes possessed unusual linear elements in their median hydrophobic zones. The germ tube, which possessed a smooth-surfaced plasma membrane, continued to elongate with periodic septum formation. The parent conidium and later the proximal germ tube showed progressive vacuolation and the cytoplasm became largely occupied by electron-translucent material. In older cells the septal pore was blocked by Woronin bodies. Compared with normal conidial germination this microcycle' germination is far more synchronous and the resultant germling is morphologically simpler. In ultrastructural terms, however, germination appears to be identical with that obtained at 28 degrees C.     

Electrical Sensing Zone Particle Analyzer for Measuring Germination of Fungal Spores in the Presence of Other Particles

Microscopic, respirometric, and electronic sizing methods for measuring germination of fungal spores were compared. With the electronic sizing method, early stages of germination (i.e., spore swelling) were detected long before germ tube emergence or significant changes in respiratory rates were observed. This method, which is rapid, easy, sensitive, and reproducible, also permits measuring the germination of spores when similar-size particles are present in concentrations considerably in excess of the number of spores.     

Conidial germination in the filamentous fungus Fusarium graminearum

The ascomycetous fungus Fusarium graminearum is an important plant pathogen causing Fusarium head blight disease of wheat and barley. To understand early developmental stages of this organism, we followed the germination of macroconidia microscopically to understand the timing of key events. These events, recorded after suspension of spores in liquid germination medium, included spore swelling at 2h, germination tube emergence and elongation from conidia at 8h and hyphal branching at 24h. To understand changes in gene expression during these developmental changes, RNA was isolated from spores and used to interrogate the F. graminearum Affymetrix GeneChip. RNAs corresponding to 5813 genes were detected in fresh spores and 5146, 5249 and 5993, respectively, in spores incubated in germination medium after 2, 8 or 24h (P<0.001). Gene expression data were used to predict the cellular and physiological state of each developmental stage for known processes. Predictions were confirmed microscopically for several previously unreported developmental events such as manifestation of peroxisomes in fresh spores and nuclear division resulting in binuclear cells within macroconidia prior to spore germination. Knowledge of stage-specific gene expression and changes in gene expression levels between developmental stages are an important first step to understanding the molecular mechanisms responsible for spore germination and development.     

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.     

Conidial germination in the filamentous fungus Fusarium graminearum.

The ascomycetous fungus Fusarium graminearum is an important plant pathogen causing Fusarium head blight disease of wheat and barley. To understand early developmental stages of this organism, we followed the germination of macroconidia microscopically to understand the timing of key events. These events, recorded after suspension of spores in liquid germination medium, included spore swelling at 2h, germination tube emergence and elongation from conidia at 8h and hyphal branching at 24h. To understand changes in gene expression during these developmental changes, RNA was isolated from spores and used to interrogate the F. graminearum Affymetrix GeneChip. RNAs corresponding to 5813 genes were detected in fresh spores and 5146, 5249 and 5993, respectively, in spores incubated in germination medium after 2, 8 or 24h (P<0.001). Gene expression data were used to predict the cellular and physiological state of each developmental stage for known processes. Predictions were confirmed microscopically for several previously unreported developmental events such as manifestation of peroxisomes in fresh spores and nuclear division resulting in binuclear cells within macroconidia prior to spore germination. Knowledge of stage-specific gene expression and changes in gene expression levels between developmental stages are an important first step to understanding the molecular mechanisms responsible for spore germination and development.     

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.     

Germination of Rhizopus oligosporus Sporangiospores

The morphology of Rhizopus oligosporus (NRRL 2710) sporangiospores and their physiological requirements for germination were studied. Germination proceeded in two separable phases: phase I (swelling) and phase II (germ tube protrusion). The optimal conditions for germination were 42 degrees C and pH 4.0. Sporangiospores contained insufficient endogenous carbon for swelling or germination to occur in distilled water. Initial swelling during phase I occurred only in the presence of a suitable carbohydrate. Subsequent production of germ tubes during phase II required exogenous sources of both carbon and nitrogen. Spores germinated most rapidly in mixtures of amino acids; l-proline and l-alanine were the most effective. These amino acids, at concentrations as low as 10 M, supported germination when combined with glucose and McIlvaine (citric acid-phosphate) buffer. d-Glucose, d-xylose, and d-mannose were the most effective carbohydrates tested for promotion of germination.     

Physiology and fine structure of sporangiospore germination in Piptocephalis unispora prior to infection

Germination of the sporangiospore of Piptocephalis unispora Benjamin, observed by means of light and electron microscopy, involved the formation of a new inner wall which became continous with the inner layer of the wall of the germ tube. The outer wall layer of the germ tube was continous with the original inner wall layer of the dormant spore. Preliminary details of appressorium structure were noted. Nutritional experiments indicated that sporangiospores required external sources of utilisable nitrogen and carbon compounds for maximal swelling and germ tube production. Limited development occurred when either nutrient was supplied singly. Comparison of germination of the asexual spore with that in other Mucorales, especially the Kickxellaceae, has been made, and the merosporangial status in P. unispora discussed.Non-Standard Abbreviations CH casein hydrolysate - Q spore quotient