On-line study of growth kinetics of single hyphae of Aspergillus oryzae in a flow-through cell

Using image analysis the growth kinetics of the single hyphae of the filamentous fungus Aspergillus oryzae has been determined on-line in a flow-through cell at different glucose concentrations in the range from 26 mg L-1 to 20 g L-1. The tip extension rate of the individual hyphae can be described with saturation type kinetics with respect to the length of the hyphae. The maximum tip extension rate is constant for all hyphae measured at the same glucose concentration, whereas the saturation constant for the hyphae varies significantly between the hyphae even within the same hyphal element. When apical branching occurs, it is observed that the tip extension rate decreases temporarily. The number of branches formed on a hypha is proportional to the length of the hypha that exceeds a certain minimum length required to support the growth of a new branch. The observed kinetics has been used to simulate the outgrowth of a hyphal element from a single spore using a Monte Carlo simulation technique. The simulations shows that the observed kinetics for the individual hyphae result in an experimentally verified growth pattern with exponential growth in both total hyphal length and number of tips.     

A model for hyphal growth and branching.

A mathematical model for hyphal growth and branching is described which relates cytological events within hyphae to mycelial growth kinetics. Essentially the model quantifies qualitative theories of hyphal growth in which it is proposed that vesicles containing wall precursors and/or enzymes required for wall synthesis are generated at a constant rate throughout a mycelium and travel to the tips of hyphae where they fuse with the plasma membrane, liberating their contents into the wall and increasing the surface area of the hypha to give elongation. The hypothesis that there is a duplication cycle in hyphae which is equivalent to the cell cycle observed in unicellular micro-organisms is also included in the model. Predictions from the model are compared with experimentally observed growth kinetics of mycelia of Geotrichum candidum and Aspergillus nidulans. The finite difference model which was constructed is capable of predicting changes in hyphal length and in the number and positions of branches and septa on the basis of changes in vesicle and nuclear concentration. Predictions were obtained using the model which were in good agreement with experimentally observed data.     

The ultrastructure of the growing regions of aerial hyphae ofRhizopus sexualis (Smith) callen

Summary The fine structure of the growing region of the aerial hyphae ofRhizopus sexualis is described. The protoplasmic components are organised in zones. At the hyphal tip there are abundant cytoplasmic vesicles and few ribosomes. Mitochondria, ribosomes, and endoplasmic reticulum are present in the sub-apical region. Nuclei occur in the posterior subapical region, some have been observed containing microtubules and bearing long projections. Microtubules have been observed extending for some distance through the cytoplasm.     

Transmission of the eect of an antifungal agent within a single hypha

A micro-compartment culture method was devised in which a single hypha of Rhizopus stolonifer growing on an agar section traversed an antifungal non-diffusible barrier to another agar section; thus the local environment of the distal or proximal part of the hypha could be controlled independently. The responses in terms of hyphal extension of the test fungus to local application of amphotericin B in this culture system were estimated by using an automatic analysing system. After hyphae had traversed the barrier, distal application of amphotericin B caused no appreciable effect on the proximal hyphae. In contrast, proximal application of amphotericin B caused inhibition of the extension of distal hyphae. The reversal of polarized cytoplasmic streaming also occured during the inhibition of distal hyphal extension. The extents of inhibition of the distal hyphal extension and the cytoplasmic streaming were dependent upon the hyphal distance between the amphotericin B application site and the hyphal tip. These results show that the effect of an antifungal agent on a hypha depends on the region of the hypha exposed. Cytoplasmic streaming may play key role in the transmission of antifungal effects within a single hypha.     

A role for endocytic recycling in hyphal growth

Actin plays multiple complex roles in cell growth and cell shape. Recently it was demonstrated that actin patches, which represent sites of endocytosis, are present in a sub-apical collar at growing tips of hyphae and germ tubes of filamentous fungi. It is now clear that this zone of endocytosis is necessary for filamentous growth to proceed. In this review evidence for the role of these endocytic sites in hyphal growth is examined. One possibility if that the role of the sub-apical collar is associated with endocytic recycling of polarized material at the hyphal tip. The 'Apical Recycling Model' accounts for this role and predicts the need for a balance between endocytosis and exocytosis at the hyphal tip to control growth and cell shape. Other cell differentiation events, including appressorium formation and Aspergillus conidiophore development may also be explained by this model.     

Age-dependent differential responses ofSaprolegnia hyphal tips to a helical growth-inducing factor in the agar substitute,gellan

Saprolegnia ferax produces more-or-less straight, subapically branched, hyphae when growing in liquid or agar-solidified media, with abundant aerial mycelium on the latter. In Contrast, the same medium solidified with gellan gum induced helical growth with reduced branching and almost no aerial mycelium. Helical growth induction was gellan concentration-dependent, peaking at 0.4–0.6% (w/v), when about 60% of tips were helical. Gellan-induced helices showed concentration-dependent inhibition by agarose and polyethylene glycol. Colonies on gellan-agarose, where helices were inhibited, reverted to having aerial mycelium, whereas those on gellan-polyethylene glycol did not. Branches on helical hyphae were initially linear, but converted to helical growth after about 2 h of extension. This transition was often marked by a branch, thus branch and helix competency appeared to be related. Germinating cysts took twice as long as hyphal inocula before producing helical hyphae, reinforcing the suggestion that helix competence was age-related.Achlya, but notPhytophthora, also showed gellan-induced helical growth and aerial mycelium suppression. These results showed (a) that morphogenic regulators of hyphal growth responded to gelling agents, probably high-molecular-weight polysaccharides, (b) that all growing hyphal tips were not equivalent, and (c) that hyphal tips underwent age-related changes in their response to the environment. The gellan-related differences in aerial mycelium mimic hydrophobin-based mycelium behavior and may thus indicate environmental regulation of hydrophobin production.     

Fine-structural Correlates of Growth in Hyphae of Ascodesmis sphaerospora

Mycelial mats of Ascodesmis sphaerospora were fixed and embedded for electron microscopy, and thin sections of 1-mm blocks, taken from the 1st to the 7th mm behind the hyphal tips, were cut parallel to the long axis of the hyphae. The hyphal tip region is characterized by an outer zone of electron-transparent vesicles, 500 to 1,000 A in diameter, and is apparently associated with wall elaboration. Immediately behind this region, dense granules become evident along convoluted membrane systems and along the plasma membrane; in the same region are numerous small lomasomes in the lateral wall. As the hypha grows, septa are laid down at 3- to 7-min intervals at a distance of 200 to 250 μ behind the hyphal tip. A cylinder of endoplasmic reticulum is intimately involved in cross-wall deposition from its earliest stages; as the wall grows in, it becomes increasingly constricted in the pore region, finally assuming a torus-like configuration. Woronin bodies are shown to have a crystalline substructure and to originate in pouch-like membrane systems. Cross-walls from a 7- to 13-hr-old mycelium frequently show highly ordered structures in the vicinity of the pore. These structures may appear either as laminar stacks of discs to one side of the pore or as series of stubby concentric rings within the pore area itself. In the latter case, a mass of granular material is frequently seen plugging the pore. Other unusual organelles and inclusions in 7- to 13-hr hyphae are vesicles containing swirls of beaded or dilated membrane, membrane-enclosed rods, and stacks of unit membranes associated with spherical, electron-transparent vesicles.     

Morphology of Trichoderma reesei QM 9414 in submerged cultures

The microscopic morphology of Trichoderma reesei QM 9414, growing in submerged culture, was studied by image analysis. The morphology was characterized by the total hyphal length, the total number of tips, the number of actively growing tips, and the length of the main hypha. To describe the growth of a single mycelium a simple model is set-up. The main features of the model are: (1) saturation type kinetics for the tip extension of the individual branches within the mycelium; and (2) random branching with a frequency function, which is proportional to the total hyphal length. The model is used to simulate a population of mycelia, where spore germination is described with a log-normal distribution. From the simulation of the population, the average properties of the mycelia, e.g., the average total hyphal length, are calculated, and by fitting the model to experimental data the model parameters are estimated. Finally, the distribution function with respect to the mycelia properties, that is, number of tips and total hyphal length, is calculated, and it corresponds well with the experimental determination of the distribution function. (c) 1995 John Wiley & Sons Inc.     

AN ULTRASTRUCTURAL BASIS FOR HYPHAL TIP GROWTH IN PYTHIUM ULTIMUM

Hyphae of the fungus Pythium ultimum extend by tip growth. The use of surface markers demonstrates that cell expansion is limited to the curved portion of the hyphal apex. Growing and non-growing regions are reflected in internal organization as detected by light and electron microscopy. The young hypha consists of three regions: an apical zone, a subapical zone and a zone of vacuolation. The apical zone is characterized by an accumulation of cytoplasmic vesicles, often to the exclusion of other organelles and ribosomes. Vesicle membranes are occasionally continuous with plasma membrane. The subapical zone is non-vacuolate and rich in a variety of protoplasmic components. Dictyosomes are positioned adjacent to endoplasmic reticulum or nuclear envelope, and vesicles occur at the peripheries of dictyosomes. A pattern of secretory vesicle formation by dictyosomes is described which accounts for the formation of hyphal tip vesicles. Farther from the hyphal apex the subapical zone merges into the zone of vacuolation. As hyphae age vacuolation increases, lipid accumulations appear, and the proportional volume of cytoplasm is reduced accordingly. The findings are integrated into a general hypothesis to explain the genesis and participation of cell components involved directly in hyphal tip growth: Membrane material from the endoplasmic reticulum is transferred to dictyosome cisternae by blebbing; cisternal membranes are transformed from ER-like to plasma membrane-like during cisternal maturation; secretory vesicles released from dictyosomes migrate to the hyphal apex, fuse with the plasma membrane, and liberate their contents into the wall region. This allows a plasma membrane increase at the hyphal apex equal to the membrane surface of the incorporated vesicles as well as a contribution of the vesicle contents to surface expansion.     

On-line study of fungal morphology during submerged growth in a small flow-through cell

A flow-through cell is designed to measure the growth kinetics of hyphae of Aspergillus oryzae grown submerged in a well controlled environment. The different stages of the growth process are characterized, from the spore to the fully developed hyphal element with up to 60 branches and a total length lt up to 10,000 micrometer. Spore swelling is found to occur without change in the form of the spore (circularity index constant at about 1.06) and the spore volume probably increases exponentially. The germ tube appears after about 4 h. The branching frequency and the rate of germ tube extension is determined. After about 10 h growth at a glucose concentration of 250 mg L-1, 6-7 branches have been set, and both the total hyphal length lt and the number of tips increase exponentially with time. The specific growth rate of the hyphae is 0. 33 h-1 while the average rate of the extension of the growing tips approaches 55 micrometer h-1. The growth kinetics for all the branches on the main hypha have also been found. The main hypha and all the branches grow at a rate which can be modeled by saturation kinetics with respect to the branch length and with nearly equal final tip speeds (160 micrometer h-1). Branches set near the apical tip of the main hypha attain their final tip speed in the shortest time, i.e., the value of the saturation parameter is small. Finally, the influence of substrate (glucose) concentration cs on the values of the morphological parameters has been determined. It is found that saturation type kinetics can be used to describe the influence of cs on the growth. Experiments with recirculation of effluent from the cell back to the inlet strongly suggest that the fungus secretes an inducer for growth and branching.     

Aggregation and collapse in a mechanical model of fungal tip growth

 Interconnected hyphal tubes form the mycelia of a fungal colony. The growth of the colony results from the elongation and branching of these single hyphae. The material being incorporated into the extending hyphal wall is supplied by vesicles which are formed further back in the hyphal tip. Such wall-destined vesicles appear conspicuously concentrated in the interior of the hypha, just before the hyphal apex, in the form of an apical body or Spitzenk?rper. The cytoskeleton of the hyphal tube has been implicated in the organisation of the Spitzenk?rper and the transport of vesicles, but as yet there is no postulated mechanism for this. We propose a mechanism by which forces generated by the cytoskeleton are responsible for biasing the movement of vesicles. A mathematical model is derived where the cytoskeleton is described as a viscoelastic fluid. Viscoelastic forces are coupled to the conservation equation governing the vesicle dynamics, by weighting the diffusion of vesicles via the strain tensor. The model displays collapse and aggregation patterns in one and two dimensions. These are interpreted in terms of the formation of the Spitzenk?rper and the initiation of apical branching. Received: 16 September 1996 / Revised version: 20 July 1998     

Effect of restrictive conditions on the growth and morphology of a temperature-sensitive mannose-requiring mutant of Aspergillus nidulans

When incubated at 45 degrees C in the absence of added mannose, pregrown hyphae of a temperature-sensitive, mannose-relief mutant (mnrA455) of Aspergillus nidulans grew normally for a short time (4-5 h) before exhibiting an abnormal morphology consisting of the production by hyphae of discrete spherical swellings called balloons. These swellings could be up to 10 microns in diameter and were produced either at or behind the hyphal apex. Often only one swelling was produced in association with each hyphal tip, but in a significant minority of cases (approximately 19.6%) a second balloon was produced in close association with the first. Hyphal tip extension slowed before and during balloon formation, but growth at individual tips did not usually stop when a balloon began to be formed in the same hypha. All tip extension ceased after approximately 8 h in cultures maintained at 45 degrees C. However, normal growth resumed 45-60 min after transfer of such a culture to the permissive temperature of 37 degrees C even after 48 h at 45 degrees C. Electron microscopic examination indicated that balloons consistently had thicker walls than the surrounding hyphae but that no accumulation of cytoplasmic vesicles was apparent within them. This indicates that a modification of wall structure, probably including deposition of new wall material, was caused by a mannose deficiency, but that this altered wall synthesis and attendant hyphal swelling was not due to diversion of the normal vesicle-mediated tip-extension system to the side walls of hyphae.     

The Organization of mitochondria in growing hyphae of Neurospora crassa

Details of mitochondrial system organization and behavior in the tip of growing N. crassa hyphae and hyphal fragments were studied by means of cell-permeable mitochondria-selective probes in glucose- and sorbitol-containing media. It was shown that filamentous mitochondria concentrate in the 30-μm apical zone of growing hyphae and hyphal fragments independently of the carbon source. The mitochondrial assemblies propagate forward with elongation of hypha, split upon apical branching, and are formed de novo when branches are formed away from the growing tip. These activities resemble microtubule behavior. Co-staining with Mitotracker Red and Mitotracker Green revealed possible functional heterogeneity in subpopulations of mitochondria. It was proposed that the observed features are governed by the microtubular network, while the primary function of mitochondria was to sustain the growth rate. The organizing role of electrical gradients on the growing tip and their influence on mitochondrial and microtubular networks are discussed.     

In planta regulation of extension of an endophytic fungus and maintenance of high metabolic rates in its mycelium in the absence of apical extension.

The fungus Neotyphodium lolii is an endophytic symbiont. It grows in the intercellular spaces of the perennial ryegrass Lolium perenne, producing secondary metabolites which enhance the fitness of the association over that of uninfected L. perenne. We report that the average number of hyphal strands in a given section of a leaf remains constant during the life of a leaf, indicating synchrony of leaf and hyphal extension, including cessation of hyphal extension when leaf extension ceases. We used a constitutively expressed reporter gene as an indicator of the mycelium's metabolic activity during and after hyphal extension. Reporter gene activity decreased when the mycelium stopped extending in liquid culture but not in planta. This indicates that in planta endophyte hyphae remain metabolically highly active when extension has ceased and throughout the life of the leaf they are colonizing. The behavior of the fungus in planta indicates the existence of signaling pathways which (i) synchronize the extension of leaf and hypha by regulating hyphal extension, (ii) suppress hyphal branching, and (iii) stop apical extension of fungal hyphae, without reducing the mycelium's metabolic activity. These signals may be crucial for the symbiosis, by allowing the endophyte to switch the focus of its metabolic activity from extension to the production of secondary metabolites.     

Mycelium development and architecture, and spore production of Scutellospora reticulata in monoxenic culture with Ri T-DNA transformed carrot roots

Mycelium development and architecture and spore production were studied in Scutellospora reticulata from single-spore isolates grown with Ri T-DNA transformed carrot root-organ culture in monoxenic system. Culture establishment, anastomosis occurrence and auxiliary cell development also were examined. Seventy percent of the pregerminated disinfected spores colonized the transformed carrot roots. After 8 mo, the average spore production was 56 (24-130) per 30 cm(3) of medium. Of the spores produced, 75% germinated and produced new generations in monoxenic culture. The mycelium network was formed by thick light-brown hyphae, which exhibit two major architecture patterns related to either root colonization or resource exploitation, and lower-order hyphae, bearing auxiliary cells, branched absorbing structures (BAS), hyphal swellings (HS) and forming anastomoses. BAS were formed abundantly in extramatrical mycelium and frequently had HS resembling vesicles, a feature not previously reported in the Gigasporaceae, to the best of our knowledge. Few anastomosis were observed within the mycelium and most often corresponded to a healing mechanism that form hypha bridges to reconnect broken hyphae or overcoming obstructed areas within a hypha. Numerous auxiliary cells were produced during culture development and their role was inferred.     

In Planta Regulation of Extension of an Endophytic Fungus and Maintenance of High Metabolic Rates in Its Mycelium in the Absence of Apical Extension

The fungus Neotyphodium lolii is an endophytic symbiont. It grows in the intercellular spaces of the perennial ryegrass Lolium perenne, producing secondary metabolites which enhance the fitness of the association over that of uninfected L. perenne. We report that the average number of hyphal strands in a given section of a leaf remains constant during the life of a leaf, indicating synchrony of leaf and hyphal extension, including cessation of hyphal extension when leaf extension ceases. We used a constitutively expressed reporter gene as an indicator of the mycelium's metabolic activity during and after hyphal extension. Reporter gene activity decreased when the mycelium stopped extending in liquid culture but not in planta. This indicates that in planta endophyte hyphae remain metabolically highly active when extension has ceased and throughout the life of the leaf they are colonizing. The behavior of the fungus in planta indicates the existence of signaling pathways which (i) synchronize the extension of leaf and hypha by regulating hyphal extension, (ii) suppress hyphal branching, and (iii) stop apical extension of fungal hyphae, without reducing the mycelium's metabolic activity. These signals may be crucial for the symbiosis, by allowing the endophyte to switch the focus of its metabolic activity from extension to the production of secondary metabolites.     

Immunofluorescence microscopy of the microtubule cytoskeleton during conjugate division in the dikaryon Pleurotus ostreatus N001

Fluorescence microscopy was used to describe the distribution of nuclei and the organization of the microtubule network in hyphae of Pleurotus ostreatus. Dikaryotic hyphae of P. ostreatus N001 grow by tip extension with two closely spaced nuclei moving slowly forward with the growing hyphal tip. During vegetative growth of the hyphae, cytoplasmic microtubules are found as long filaments oriented longitudinally within fungal hyphae. When the apical cell reaches a length of approximately 150 μm, the two nuclei divide synchronously. Mitosis occurs in association with clamp connection formation, with one of the nuclei dividing in the hook of the developing clamp connection and the other in the main hypha. After mitosis, two daughter nuclei move forward to approximately the center of the apical cell, while the other two move backward to a central position in the subapical cell. Two septa are formed, one in the clamp and the other across the main axis of the hypha to delimit the apical cell. The use of fluorescence microscopy made it possible to examine the changes in the cytoplasmic microtubules, the configuration of the mitotic apparatus, the site of septation and the post-mitotic nuclear migrations during conjugate division in P. ostreatus dikaryotic hyphae.     

Time-Lapse Microscopy of Streptomyces coelicolor Growth and Sporulation

Bacteria from the genus Streptomyces are among the most complex of all prokaryotes; not only do they grow as a complex mycelium, they also differentiate to form aerial hyphae before developing further to form spore chains. This developmental heterogeneity of streptomycete microcolonies makes studying the dynamic processes that contribute to growth and development a challenging procedure. As a result, in order to study the mechanisms that underpin streptomycete growth, we have developed a system for studying hyphal extension, protein trafficking, and sporulation by time-lapse microscopy. Through the use of time-lapse microscopy we have demonstrated that Streptomyces coelicolor germ tubes undergo a temporary arrest in their growth when in close proximity to sibling extension sites. Following germination, in this system, hyphae extended at a rate of ~20 μm h⁻¹, which was not significantly different from the rate at which the apical ring of the cytokinetic protein FtsZ progressed along extending hyphae through a spiraling movement. Although we were able to generate movies for streptomycete sporulation, we were unable to do so for either the erection of aerial hyphae or the early stages of sporulation. Despite this, it was possible to demonstrate an arrest of aerial hyphal development that we suggest is through the depolymerization of FtsZ-enhanced green fluorescent protein (GFP). Consequently, the imaging system reported here provides a system that allows the dynamic movement of GFP-tagged proteins involved in growth and development of S. coelicolor to be tracked and their role in cytokinesis to be characterized during the streptomycete life cycle.     

Epichlo? endophytes grow by intercalary hyphal extension in elongating grass leaves.

A fundamental hallmark of fungal growth is that vegetative hyphae grow exclusively by extension at the hyphal tip. However, this model of apical growth is incompatible with endophyte colonization of grasses by the symbiotic Neotyphodium and Epichlo? species. These fungi are transmitted through host seed, and colonize aerial tissues that develop from infected shoot apical meristems of the seedling and tillers. We present evidence that vegetative hyphae of Neotyphodium and Epichlo? species infect grass leaves via a novel mechanism of growth, intercalary division and extension. Hyphae are attached to enlarging host cells, and cumulative growth along the length of the filament enables the fungus to extend at the same rate as the host. This is the first evidence of intercalary growth in fungi and directly challenges the centuries-old model that fungi grow exclusively at hyphal tips. A new model describing the colonization of grasses by clavicipitaceous endophytes is described.     

The product of a developmental gene, crgA, that coordinates reproductive growth in Streptomyces belongs to a novel family of small actinomycete-specific proteins

On solid media, the reproductive growth of Streptomyces involves antibiotic biosynthesis coincident with the erection of filamentous aerial hyphae. Following cessation of growth of an aerial hypha, multiple septation occurs at the tip to form a chain of unigenomic spores. A gene, crgA, that coordinates several aspects of this reproductive growth is described. The gene product is representative of a well-conserved family of small actinomycete proteins with two C-terminal hydrophobic-potential membrane-spanning segments. In Streptomyces avermitilis, crgA is required for sporulation, and inactivation of the gene abolished most sporulation septation in aerial hyphae. Disruption of the orthologous gene in Streptomyces coelicolor indicates that whereas CrgA is not essential for sporulation in this species, during growth on glucose-containing media, it influences the timing of the onset of reproductive growth, with precocious erection of aerial hyphae and antibiotic production by the mutant. Moreover, CrgA subsequently acts to inhibit sporulation septation prior to growth arrest of aerial hyphae. Overexpression of CrgA in S. coelicolor, uncoupling any nutritional and growth phase-dependent regulation, results in growth of nonseptated aerial hyphae on all media tested, consistent with a role for the protein in inhibiting sporulation septation.