Microcycle conidiation in Penicillium urticae: an ultrastructural investigation of conidiogenesis.

A cultivation system has been developed for Penicillium urticae which yields 'microcycle' conidiation in submerged culture. Spherical growth of spores was initiated by incubation at 37 degrees C in a growth-favoring medium. Transfer of these enlarged spores to a nitrogen-poor medium at 35 degrees C results in synchronous germination and limited outgrowth followed by roughly synchronous conidiation. A study of the conidiation stage showed that a phialide and an immature conidium began to form at the tip of all germ tubes 18 h after the temperature shift. By 24 h additional phialides commonly appeared as a branch near the tip of the germ tube and the more mature conidia exhibited increasing refractility. The earliest ultrastructural signs of conidiation were various round invaginations in the plasma membrane and a thickening and rounding of the new spore wall which appeared as an inner extension of the phialide cell wall. Upon segregation of the conidium from the phialide cell by conidial wall formation, 'trench-like' invaginations gradually appeared in the plasma membrane and a disorganized rodlet pattern was formed on the outer surface of the maturing conidial wall. Continued maturation involved the formation of chains of conidia and phialide senescence which was characterized by a general degradation of intracellular structure. A comparison with standard surface and submerged culture conidiation indicated that 'microcycle' conidiation, while less prolific, was essentially identical.     

Fine Structure of Germinating Botrytis fabae Sardina Conidia

The fine structure of germinating Botrytis fabae conidia wasstudied using both chemically stained sections and freeze-etchedreplicas. Germinating conidia have fewer organelles than restingconidia, glycogen is absent, and prevacuoles have disappeared.Endoplasmic reticulum which occurs as small strands close tothe cell wall of resting conidia becomes, on germination, multiplesheets surrounding the nuclei. A cross wall is formed at thebase of the germ tube soon after germination commences. Thenew wall material which appears to be continuous with this septalwall is produced, at least partly, from a new wall layer laiddown in the centre of the old conidial wall. An apical corpuscleis present at the apex of young germ tubes. Freeze-etched preparationsshow the formation of lomasomes by the passage of vesicles throughthe plasmalemma of conidia and germ tubes. In young hyphae lomasomescontain a complex arrangement of branching tubules. Some ofthe particles on the outer plasmalemma of young hyphae are arrangedin a geometrical pattern.     

Description of conidia from submerged cultivation of Thermomyces lanuginosus for use as a uniform inoculum.

Conidia produced by submerged cultivation of the thermophilic fungus Thermomyces lanuginosus were superior to conidia from agar plates when used as inoculum, due to a faster and more synchronous germination. With conidia derived from submerged liquid culture at 40-45 degrees C more than 90% germination was achieved at 50 degrees C within 3 h whereas the same percentage germination was only achieved after 5 h incubation of conidia produced on agar plates. The temperature during conidial formation, and conidial age at the time of harvesting, were factors influencing germination of the conidia.     

Electron microscopic observation of cell wall structure during appressorium formation in Colletotrichum lagenarium

Summary The formation of cell walls during the appressorium formation inColletotrichum lagenarium was observed by electron microscope on the materials prepared by replicas and sectioning. The outer layer of conidia cell walls ruptured at the time of germination and the inner layer bulged out to form a germ tube. The germ tubes and primordia of appressoria had smooth surface and were consisted of one-layered cell wall. However, as the appressorium matured, the electron dense materials appeared on the outer surface of the cell wall which grew into granules. These granules are believed to form the outer layer of appressoria. The under side of the appressorium in contact with the glass surface showed a round pore.Contribution No. 191.     

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.     

Effects of caffeine,cyclic 3′, 5′ adenosine monophosphate and cyclic 3′, 5′ guanosine monophosphate in the development of the mycelial form of Sporothrix schenckii

The kinetics of the development of the mycelial form of Sporothrix schenckii from yeast cells and conidia in a minimal basal medium with glucose at pH 4.0 and 25 °C were established. Germ tube formation was used as the index of germination for both yeast cells and conidia. Yeast cells were first observed to develop germ tubes after 3 h of incubation, reaching 92±5%, after 12 h of incubation. Germ tubes were first detected in conidia after 9 h of incubation, and 12 h after inoculation 92±6% of the conidia had germ tubes. After 24 h of incubation, fully developed, sporulating mycelia were observed from both yeast cells and conidia. A delay in germ tube formation from yeast cells was observed when But₂cAMP(10 mM) and But₂cGMP (10 mM) were added to the medium. Also the addition of caffeine, a cyclic nucleotide phosphodiesterase inhibitor, inhibited the yeast to mycelial transition. Conidial germination into the mycelial form was also inhibited when cAMP, But₂cAMP and caffeine were added to the medium. These results suggest the possible involvement of cyclic nucleotides in the control of dimorphism in S. schenckii.     

Fine structure of resting and germinatingPenicillium chrysogenum conidiospores

Summary The fine structure of resting and germinating conidia ofPenicillium chrysogenum has been examined by electron microscopy. In addition to enlargement of the cells, a number of changes in ultrastructure become evident as morphogenesis proceeds. The newly synthesized germ tube is continuous with the corresponding layers of the conidial wall. Some conidial wall layers, however, do not extend into the hyphal wall. Several sections showing initial septum synthesis suggest that a septal pore is not a necessary structural entity. A characteristic orientation of the initial septum formed after germination is described. Aside from numerical considerations, no significant changes occur in nuclei, mitochondria, or ribosomes. The electron micrographs illustrate the presence of spherosomes, lomasomes, and nucleoli; the possible significance of these structures is discussed.     

Cell wall α1-3glucans induce the aggregation of germinating conidia of Aspergillus fumigatus

The germination of Aspergillus fumigatus conidia can be divided into four stages: breaking of dormancy, isotropic swelling, establishment of cell polarity, and formation of a germ tube. Swelling of conidia is associated in liquid medium with a multi-cellular aggregation that produced large clumps of conidia. Conidial aggregation can be specifically prevented by the addition of α1-3glucanase. Swollen conidia specifically adhere to insoluble α1-3glucan chains. Electron microscopy studies showed that cell wall α1-3glucan chains became exposed at the cell surface during the swelling. These results demonstrate that cell wall α1-3glucans play an essential role in the aggregation between swollen conidia. Experiments with α1-3glucan coated latex beads show that α1-3glucan chains interacted between them without the requirement of any other cell wall component suggesting that biophysical properties of α1-3glucans are solely responsible for conidial aggregation.     

Microcycle conidiation in Penicillium urticae: an ultrastructural investigation of spherical spore growth.

A cultivation system has been developed for Penicillium urticae (NRRL 2159A) which yields 'microcycle' conidiation in submerged culture. Spherical growth of spores was initiated by incubation at 37 degrees C in a growth-favoring medium. Transfer of these enlarged spores to a nitrogen-poor medium at 35 degrees C resulted in synchronous germination and limited outgrowth followed by roughly synchronous conidiation. An ultrastructural study of the spherical growth stage indicates a significant loss of cell envelope fine structure and a general increase in the number of cellular organelles. Loss of the complex pattern of rodlets on the spore surface, and the trench-like invaginations of the plasma membrane were most prominent. From an ultrastructural point of view these large spores (about 6 mum in diameter) appeared to be perfectly normal.     

Fine structure, physiology and biochemistry of arthrospore germination in Streptomyces antibioticus.

During germination, Streptomyces antibioticus arthrospores passed through stages: darkening, swelling and germ tube emergence. The first stage, darkening, whose main features were a decrease in absorbance and a loss of refractility, only required exogenous divalent cations (Ca2+, Mg2+ or Fe2+) and energy that can be obtained from the spore reserves. This stage was blocked by agents that inhibit ATP formation but not by antibiotics that inhibit macromolecular synthesis. The second stage, swelling, needed an exogenous carbon source and was not blocked by mitomycin C. In this stage, the spores exhibited the highest cytochrome oxidase and catalase activities and respiratory quotient. The last stage, germ tube emergence, required additional carbon and nitrogen sources. Ammonium compounds were superior to nitrate. Dry weight remained constant during the stages of darkening and swelling, with a rapid increase from the moment of germ tube emergence. Optimum pH and temperature for germination were 8.0 and 45 degrees C, respectively. Heat treatment (55 degrees C for 10 min) had no effect on germination. The fine structure of the spore underwent important changes during germination. The wall of the swollen spore became stratified and the inner layer was continuous with the germ tube wall. Macromolecular synthesis occurred in the sequence RNA, protein and then DNA. Rifampicin, streptomycin and mitomycin C prevented synthesis when added at the start of incubation. The same effect was obtained if the addition was made during germination, except with mitomycin C which inhibited DNA, but not RNA and protein synthesis.     

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.     

Fine Structure of Quiescent and Germinating Aeciospores of Cronartium fusiforme

Aeciospores of the long-cycle heteroecious rust fungus, Cronartium fusiforme, were found to have an extremely thick cell wall with striking spicules protruding from it. The wall was readily degraded by commercial chitinase, but spicules were unaffected. Quiescent spores contained two nuclei with distinct nuclear membranes possessing many pores. Numerous membrane-bounded lipid bodies were found both in wild-type orange and in white mutant aeciospores. An abundance of irregularly ovoid mitochondria was present in quiescent spores. After glutaraldehydeosmium fixation, the surface of the mitochondria appeared to be covered with ribosomes or microtubules in a paracrystalline array, whereas after permanganate fixation only smooth outer mitochondrial membranes were noted. The latter fixative revealed abundant vesicular endoplasmic reticulum in the spore. Spores incubated at 20 C on agar produced one to five distinct germ tubes within 65 to 180 min. These thin-walled tubes exhibited varying degrees of branching, and reached a total hyphal length of 300 to 500 mu prior to rupturing. Emergence of germ tubes took place through a pore in the spore wall and appeared to be mainly a physical flowing of cytoplasm from the spore into the germ tube without division of nuclei or other cell organelles. On completion of germination, the protoplasm of the germ tube contained both nuclei and nearly all of the other spore contents. Mitochondria had smooth outer membranes, were greatly elongated, and possessed distinct longitudinal cristae. A limited amount of rough endoplasmic reticulum was arranged parallel to the germ tube wall. Other organelles seen in germ tubes were lipid bodies, concentric membrane figures, and numerous ribosomes. Lipid bodies appeared smaller and fewer in number than in quiescent spores.     

Effects of long-term storage at different temperatures on conidia of Botrytis cinerea Pers.: Fr.

Survival of Botrytis cinerea conidia was studied after storage without pretreatments at different temperatures (-80 degrees C, -20 degrees C, 4 degrees C and 21 degrees C). Germination tests performed during 3 years showed that viability at 21 degrees C was completely lost after 1 month. Conidia stored for 30 months at -80 degrees C, -20 degrees C and 4 degrees C were able to germinate, respectively, at 79%, 8% and 0.2%. Changes in adenylate level, energy charge and respiration (O(2) consumption) made on each set of conidia were correlated to the germination rate. The 30-month-old stored conidia showed differences in pathogenicity tests on apples. While the pathogenic aggressiveness of conidia stored at -80 degrees C was almost the same as for fresh conidia, it decreased with increasing temperature of storage. An ultrastructural study made on conidia stored for 30 months at -80 degrees C has shown the emergence of a new wall layer in a retraction zone of the cytoplasm by comparison to fresh conidia. However, the integrity of the cytoplasmic content was maintained. The effects of low temperature storage, maintenance of cell integrity and pathogenicity of conidia of B. cinerea are discussed.     

Role of the conidium in dimorphism of Blastomyces dermatitidis

Fine details of yeastlike cell development of Blastomyces dermatitidis from its conidium are described and illustrated by electron micrographs. When cultured in an enriched medium at 37 °C, conidia of two strains of B. dermatitidis readily underwent ultrastructural changes consistent with mycelial to yeast dimorphism. Although hyphal cells contained in the conversion cultures were observed consistently to undergo profound degenerative changes, the conidia rapidly germinated to give rise to short germ tubes which subsequently enlarged to form intermediate yeast mother cells (YMC). The wall of the germ tube arose from the innermost layer of the wall of the germinant. During the transition globoid osmiophilic inclusions of unknown origin and function were observed in vacuolated areas of the germ tube and YMC cytoplasm. Yeastlike daughter cells then budded from the intermediate YMC. Since transformation was readily accomplished under in vitro conditions favoring mycelial to yeast dimorphism, it is suggested that the conidium of B. dermatitidis represents the primary infective unit of this pathogenic fungus.     

Morphology and lipid body and vacuole dynamics during secondary conidia formation in Colletotrichum acutatum: laser scanning confocal analysis

Colletotrichum acutatum may develop one or more secondary conidia after conidial germination and before mycelial growth. Secondary conidia formation and germination were influenced by conidia concentration. Concentrations greater than 1x105 conidia/mL were associated with germination decrease and with secondary conidia emergence. Secondary conidia can form either alone or simultaneously with germ tubes and appressoria. Confocal analysis showed numerous lipid bodies stored inside ungerminated conidia, which diminished during germ tube and appressoria formation, with or without secondary conidia formation. They were also reduced during secondary conidia formation alone. While there was a decrease inside germinated conidia, lipid bodies appeared inside secondary conidia since the initial stages. Intense vacuolization inside primary germinated conidia occurred at the same time as the decrease in lipid bodies, which were internalized and digested by vacuoles. During these events, small acidic vesicles inside secondary conidia were formed. Considering that the conidia were maintained in distilled water, with no exogenous nutrients, it is clear that ungerminated conidia contain enough stored lipids to form germ tubes, appressoria, and the additional secondary conidia replete with lipid reserves. These results suggested a very complex and well-balanced regulation that makes possible the catabolic and anabolic pathways of these lipid bodies.     

Molecular and cellular events during the germination of conidia of Sporothrix schenckii

Hyaline, non pigmented microconidia of Sporothrix schenckii were harvested and allowed to form germ tubes in a basal medium with glucose at pH 4.0 and 25 °C. These conditions supported only the development of the mycelial form of Sporothrix schenckii in a reproducible, synchronized manner which allowed further analysis of the early cellular events ocurring during the germination of the conidia. The relationship between macromolecular synthesis (DNA, RNA and protein synthesis) and nuclear division, hyphal growth and septum formation were established. Following inoculation, protein synthesis was observed after 10 minutes followed by RNA synthesis, after 1 h and DNA synthesis after 2 h. The first nuclear division was observed during the 9 to 12 h interval after inoculation. Germ tube formation slightly preceeded nuclear division and was first evidenced 9 h after the induction of germination but was not completed until 12 h after inoculation. Septation was first observed in the germ tubes 0.25 m from the mother cell-germ tube function 9 h after induction of germination.     

Changes in glucosamine and galactosamine levels during conidial germination in Neurospora crassa.

The levels of glucosamine and galactosamine were determined in conidia, germinating conidia, and vegetative mycelia of Neurospora crassa. In the vegetative mycelia about 90% of the amino sugars were shown to be components of the cell wall. The remaining 10% of the amino sugars were tentatively identified as the nucleotide sugars uridine diphospho-2-acetamido-2-deoxy-D-glucose and uridine diphospho-2-acetamido-2-deoxy-D-galactose. Conidia and vegetative mycelia contained about the same levels of glucosamine. During the first 9 h after the initiation of germination, the total glucosamine content had increased 3.1-fold, whereas the residual dry weight of the culture had increased 7.7-fold. This led to a drop in the glucosamine concentration from 100 mumol/g of residual dry weight to 42 mumol/g. During this time, all of the conidia had germinated and the surface area of the new germ tubes had increased to 10 times that of the conidia. Either germ tubes were initially produced without glucosamine-containing polymers, or these polymers (probably chitin) were deposited only at low densities in the germ tube cell walls. The chitin precursor uridine diphospho-2-acetamido-2-deoxy-D-glucose was present at all times during conidial germination. Conida contained very low levels of galactosamine. During germination, galactosamine could not be detected until the culture had reached a cell density of about 0.6 mg of residual dry weight per ml of growth medium. This was observed regardless of the time required to reach this cell density or the fold increase in dry weight. The accumulation of galactosamine-containing polymers does not appear to be necessary for germ tube formation. The levels of soluble galactosamine (uridine diphospho-2-actamido-2-deoxy-D-galatose) were very low in conidia and increased during germination at the same time that galactosamine appeared in the cellular polymers. In addition, under certain culture conditions, the appearance of galactosamine and the increase in the glucosamine concentration occurred simultaneously.     

Formation and Germination of Septoria tritici Secondary Conidia as Affected by Environmental Factors

The effects of medium, isolate, temperature, light and pH on the formation and germination of Septoria tritici secondary conidia were tested. Of the six media tested, the malt–yeast extract agar was the best and generated 1.82 × 10⁹ conidia/plate. The ten isolates tested showed different ability of conidia production. Darkness significantly reduced conidial formation and enhanced the transition of intermediates. The conidial germination and germ tube growth was strongly inhibited at 30°C. The suitable pH for conidial budding in malt–yeast broth (MYB) was between 5 and 9. At pH 2, 10 and 11, almost no new conidia were formed. The number of conidia reached 1.27 × 10⁸ conidia/ml after 7 days in MYB, significantly more than that in potato dextrose broth, wheat leaf extract and H₂O.     

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.     

Changes in cyclic nucleotide levels and dimorphic transition in Candida albicans.

The relationship between the levels of cyclic nucleotides and dimorphic transition in Candida albicans was examined. The results showed that cells of this pathogenic fungus contained both cyclic adenosine 3',5'-monophosphate (cAMP) and cyclic guanosine 3',5'-monophosphate (cGMP), the concentration of the latter being about one-tenth that of the former in stationary-phase cells of the yeast form. Our results further indicated that germ tube formation induced by incubation at 40 degrees C followed a rise in cAMP concentration in the cell with no accompanying change in cGMP content. Cysteine, which suppressed germination, also reversed the increase in intracellular cAMP concentration. Dibutyryl cAMP (1 MM) significantly promoted germination in proline medium at temperatures of 32 to 34 degrees C. These results suggested that cAMP was one of the controlling factors in the morphological transition in Candida albicans.