Monitoring of filamentous fungal growth by in situ microspectrophotometry, fragmented mycelium absorbance density, and 14C incorporation: alternatives to mycelial dry weight.

Monitoring of filamentous fungal growth by spectrophotometry is generally considered not feasible. This report describes the monitoring of growth of the filamentous fungi Trichophyton mentagrophytes, Rhizopus oryzae, and Sporothrix schenckii in broth by two new spectrophotometric methods and by 14C incorporation from [U-14C]glucose. Microcultures (200 microliter) were prepared in 96-well, flat-bottom microtiter trays, and macrocultures (4 ml) were prepared in glass vials proportionally scaled up from microcultures. Mycelium accumulation in microcultures was measured without terminating the cultures by in situ microspectrophotometry. Accumulation in macrocultures was monitored by uniformly fragmenting the mycelium with a Broeck tissue grinder and by measuring absorbance density in plastic cuvettes with a dual-beam spectrophotometer. Absorbance measurements were found to increase linearly with mycelial weight. In situ absorbance correlated with absorbance density of fragmented mycelium, indicating that both methods monitored growth equivalently. Both defined lag-, exponential-, and stationary-growth phases. Increases in 14C incorporation, absorbance, and mycelial dry weight were kinetically identical for macrocultures and microcultures of T. mentagrophytes. For R. oryzae and S. schenckii, with the exception of R. oryzae growing in microcultures, incorporation of 14C also defined lag, exponential, and stationary growth after selection of the appropriate isotope-specific activity. This incorporation correlated directly with absorbance. We conclude that in situ microspectrophotometry, fragmented mycelium absorbance density, and, to a lesser extent, 14C incorporation can be used to effectively monitor filamentous fungal growth.     

Filamentous fungal growth assay: correlation between [U-14C] glucose uptake and dry weight determinations

The filamentous fungus Trichophyton mentagrophytes was grown in [U-14C] glucose supplemented nutrient broth. Growth was monitored in 200 microliter microcultures and 5 ml macrocultures by measuring the incorporation of 14C into trichloroacetic acid-insoluble macromolecules and in macrocultures by dry weight determination. Adjustment of the specific activity of the [U-14C] glucose medium supplement from 1080 muCi/mmole to 108 muCi/mmole decreased the rate of isotope uptake and prolonged availability so that growth beyond 36 h could be monitored radiometrically. The dry mycelial weight of the fungus was directly proportional (r = 0.995) to the amount of isotope incorporated. Isotope uptake in microcultures and macrocultures was kinetically identical. These data clearly indicate that the uptake of 14C by T. mentagrophytes can be used to monitor fungal growth accurately in both macrocultures and microcultures.     

Defining individual size in the model filamentous fungus Neurospora crassa

It is challenging to apply the tenets of individuality to filamentous fungi: a fungal mycelium can contain millions of genetically diverse but totipotent nuclei, each capable of founding new mycelia. Moreover, a single mycelium can potentially stretch over kilometres, and it is unlikely that its distant parts share resources or have the same fitness. Here, we directly measure how a single mycelium of the model ascomycete Neurospora crassa is patterned into reproductive units (RUs), meaning subpopulations of nuclei that propagate together as spores, and function as reproductive individuals. The density of RUs is sensitive to the geometry of growth; we detected 50-fold smaller RUs when mycelia had expanding frontiers than when they were constrained to grow in one direction only. RUs fragmented further when the mycelial network was perturbed. In mycelia with expanding frontiers, RU composition was strongly influenced by the distribution of genotypes early in development. Our results provide a concept of fungal individuality that is directly connected to reproductive potential, and therefore to theories of how fungal individuals adapt and evolve over time. Our data show that the size of reproductive individuals is a dynamic and environment-dependent property, even within apparently totally connected fungal mycelia.     

CTBT (7-chlorotetrazolo[5,1-c]benzo[1,2,4]triazine) producing ROS affects growth and viability of filamentous fungi

CTBT (7-chlorotetrazolo[5,1-c]benzo[1,2,4]triazine) causes intracellular superoxide production and oxidative stress and enhances the susceptibility of Saccharomyces cerevisiae, Candida albicans, and C.?glabrata cells to cycloheximide, 5-fluorocytosine, and azole antimycotic drugs. Here, we demonstrate the antifungal activity of CTBT against 14 tested filamentous fungi. CTBT prevented spore germination and mycelial proliferation of Aspergillus niger and the pathogenic Aspergillus fumigatus. The action of CTBT is fungicidal. CTBT increased the formation of reactive oxygen species in fungal mycelium as detected by 2',7'-dichlorodihydrofluorescein diacetate and reduced the radial growth of colonies in a dose-dependent manner. Co-application of CTBT and itraconazole led to complete inhibition of fungal growth at dosages lower than the chemicals alone. Antifungal and chemosensitizing activities of CTBT in filamentous fungi may be useful in combination treatments of infections caused by drug-resistant fungal pathogens.     

Effect of cadmium on growth of potentially pathogenic soil fungi

The effect of Cd on the mycelial growth of some potentially pathogenic soil fungi was investigated. Sixty-four strains from twenty-five fungal species were tested for their susceptibility to Cd. Final colony diameter, radial growth rate and final dry mass of mycelium (for Cd: 1–200 ppm) were measured. EcD₅₀ values were calculated from these parameters. The intra- and interspecific variability in the results obtained is presented. Among the keratinolytic fungal group, the genera Arthrographis, Trichophyton and Chrysosporium (including the Chrysosporium anamorph of Aphanoascus reticulisporus) were the most resistant to Cd. T. mentagrophytes was the most sensitive of all Trichophyton species tested. Among the nonkeratinolytic fungal group, the genera Pseudallescheria, Absida and Rhizopus were highly resistant to Cd. Strains of Aspergillus were more resistant to Cd than strains of Penicillium. Zygomycetes were more resistant to Cd than Ascomycetes with Fungi Imperfecti. Nonkeratinolytic fungi showed higher resistance to Cd than keratinolytic fungi. The two last differences resulted from the extremely high EcD₅₀ values for R. oryzae and A. corymbifera. This revised version was published online in June 2006 with corrections to the Cover Date.     

Ultrastructural Studies on the Yeastlike and Mycelial Phases of Sporotrichum schenckii

Fine details of the internal and external morphology of the yeastlike and mycelial phases of the dimorphic fungal pathogen Sporotrichum schenckii as seen in ultrathin sections are described and illustrated by electronphotomicrography. Comparisons of yeastlike phase ultrastructure were made using two different methods of fixation and embedding. The internal morphology of the two forms of yeastlike S. schenckii was in many ways similar to that of similarly dimorphic fungi and yeasts studied by other authors. However, the use of the glutaraldehyde-osmium in agar fixation technique suggests the presence of an electron transparent capsular or slime layer with associated electron dense microfibrils to be present external to the cell wall of the yeastlike phase but not the mycelial phase. Mycelial phase S. schenckii was found to contain many of the internal microstructures reported for other filamentous dimorphic fungi. Conidia production in agitated liquid culture was found to be restricted since only rare sessile conidia were observed.     

Use of the tetrazolium salt MTT to measure cell viability effects of the bacterial antagonist Lysobacter enzymogenes on the filamentous fungus Cryphonectria parasitica

Despite substantial interest investigating bacterial mechanisms of fungal growth inhibition, there are few methods available that quantify fungal cell death during direct interactions with bacteria. Here we describe an in vitro cell suspension assay using the tetrazolium salt MTT as a viability stain to assess direct effects of the bacterial antagonist Lysobacter enzymogenes on hyphal cells of the filamentous fungus Cryphonectria parasitica. The effects of bacterial cell density, fungal age and the physiological state of fungal mycelia on fungal cell viability were evaluated. As expected, increased bacterial cell density correlated with reduced fungal cell viability over time. Bacterial effects on fungal cell viability were influenced by both age and physiological state of the fungal mycelium. Cells obtained from 1-week-old mycelia lost viability faster compared with those from 2-week-old mycelia. Likewise, hyphal cells obtained from the lower layer of the mycelial pellicle lost viability more quickly compared with cells from the upper layer of the mycelial pellicle. Fungal cell viability was compared between interactions with L. enzymogenes wildtype strain C3 and a mutant strain, DCA, which was previously demonstrated to lack in vitro antifungal activity. Addition of antibiotics eliminated contributions to MTT-formazan production by bacterial cells, but not by fungal cells, demonstrating that mutant strain DCA had lost complete capacity to reduce fungal cell viability. These results indicate this cell suspension assay can be used to quantify bacterial effects on fungal cells, thus providing a reliable method to differentiate strains during bacterial/fungal interactions.     

申克孢子丝菌菌丝相至酵母相转化的实验研究

目的观察申克孢子丝菌菌丝相向酵母相转化的形态学变化并初步研究连续传代后菌株转化为酵母相的百分率。方法将95株申克孢子丝菌临床株于脑心浸液琼脂培养基上连续传代至酵母相,利用显微镜及血细胞计数板计数菌丝和孢子比例并记录显微镜下形态。结果 95株申克孢子丝菌中,经1次传代即成功转化有23株,经2次传代有14株,经3次传代有10株,经4次传代有6株,4次传代后总计55.8%实验菌株转化为酵母相。结论部分申克孢子丝菌由菌丝相向酵母相转化需经过连续多次传代,连续传代增加了菌丝相至酵母相的转化率。     

Polymorphism of Sporothrix schenckii surface polysaccharides as a function of morphological differentiation.

The alkali-extractable polysaccharides from different morphological types of two Sporothrix schenckii strains (1099.12 and 1099.18) were investigated. Dissociation of morphological phase transition and temperature effects was possible in a synthetic medium which produced cultures with 100% yeast forms either at 25 or at 37 degrees C. Only rhamnomannans with single-unit alpha-L-rhamnopyranosyl side chains were formed by the yeast forms irrespective of the incubation temperature. The higher temperature inhibited formation of 4-O- and 2,4-di-O-substituted alpha-D-mannopyranose units in the rhamnomannan. An apparently unsporulated mycelium culture of one S. schenckii strain (1099.12) synthesized a galactomannan whose structure was partially determined by methylation analysis and by proton and 13C nuclear magnetic resonance spectroscopy. In another strain (1099.18), a mannan was excreted in the medium of an apparently conidia-less mycelial form at 25 degrees C with short incubation. Its structure was also partially determined. An apparent mixture of this mannan and a rhamnomannan rich in alpha-L-rhamnopyranosyl-(1 leads to 2)-alpha-L-rhamnopyranose side chains formed in these cultures on prolonged incubation. The proportion of the excreted rhamnomannan increased as the mycelium sporulated and conidia were more numerous. Mannans or galactomannans may be transient polysaccharides in the young mycelium of S. schenckii. As the culture develops, rhamnomannans are formed in amounts usually masking the presence of other mannose-containing polysaccharides. It is suggested that in S. schenckii different polysaccharides are formed with side chains containing different proportions of rhamnose, mannose, or galactose, as a function of morphological differentiation.     

Control of zoopathogenic fungi in vitro by polyamine biosynthesis inhibitors.

Effect of inhibitors of polyamine (PA) biosynthesis, alpha-difluoromethylornithine (DFMO), methylglyoxal bis (guanylhydrazone)--MGBG and bis (cyclohexylammonium) sulphate (BCHA) on mycelial growth of three clinically important fungi-Trichophyton mentagrophytes, Microsporum gypseum and Aspergillus flavus was examined in vitro. All inhibitors at concentrations 1 to 50 mM produced greater inhibition of mycelial growth in all fungi tested in a dose-dependent manner. MGBG was the most effective inhibitor, and T. mentagrophytes was the most sensitive fungus to all inhibitors followed by M. gypseum and A. flavus. The results suggested that control of fungal diseases in animals and human beings with specific inhibitors of PA biosynthesis is possible.     

Microcycle Conidiation and Spore-Carrying Capacity of Colletotrichum gloeosporioides on Solid Media

The effect of spore inoculum density, medium concentration, and temperature on slime-spot formation, spore yield, and mycelium production by Colletotrichum gloeosporioides on agar media were studied with a simple microplate assay. A steady-state spore yield (spore-carrying capacity) independent of inoculum density was reached only on media that supported good fungal growth and sporulation. The spore-carrying capacity was reached earlier, the denser the inoculum. On standard mycological media a high inoculum density (2.5 × 10⁶ spores per ml) resulted in a slimy mass of conidia forming a slime spot, a phenomenon associated with greatly reduced mycelium formation and indicative of microcycle conidiation. In contrast, for a similar inoculum density, enhanced mycelial growth preceded sporulation and overrode slime-spot formation on highly concentrated media; a very low medium concentration resulted in much less mycelium, but spore production was also decreased. Exposure to suboptimal growth temperatures of 36 to 48°C for up to 8 days did not induce microcycle conidiation from inocula that did not form a slime spot at 28°C.     

Aspergillus oryzae in solid-state and submerged fermentations. Progress report on a multi-disciplinary project

We report the progress of a multi-disciplinary research project on solid-state fermentation (SSF) of the filamentous fungus Aspergillus oryzae. The molecular and physiological aspects of the fungus in submerged fermentation (SmF) and SSF are compared and we observe a number of differences correlated with the different growth conditions. First, the aerial hyphae which occur only in SSFs are mainly responsible for oxygen uptake. Second, SSF is characterised by gradients in temperature, water activity and nutrient concentration, and inside the hyphae different polyols are accumulating. Third, pelleted growth in SmF and mycelial growth in SSF show different gene expression and protein secretion patterns. With this approach we aim to expand our knowledge of mechanisms of fungal growth on solid substrates and to exploit the biotechnological applications.     

Humoral immune response against soluble and fractionate antigens in experimental sporotrichosis

Sporotrichosis is a chronic granulomatous mycosis caused by the dimorphic fungus Sporothrix schenckii, which is widely distributed in nature, and presents a saprophytic mycelial form on plant debris and soil. The immunological mechanisms involved in the prevention and control of sporotrichosis are not yet fully understood. In this study, mice were studied after infection with Sporothrix schenckii. In the first week after infection, fungal loading increased and thence decreased drastically 14 days after infection. Analysis by immunoblotting showed that the sera of all mice tested had antibodies reacting only with a 70 kDa antigen, with predominance of IgG1 and IgG3. Taken together, our results show that antigens from S. schenckii induced a specific humoral response in infected mice.     

Nocardia-like mutants of a soil coryneform bacterium

Summary A rod-shaped, morphologically stable coryneform soil isolate, strain 9b, gives rise to filamentous and nocardioform mycelial mutants when submitted to mutagenic treatment. Stepwise selection for increasing average length of the rods appears to be necessary to obtain the mycelial form. The mycelial mutant reproduces by fragmentation of the mycelium in the stationary phase of growth and the rough variant exhibits aerial hyphae.     

Copper uptake inAspergillus niger during batch growth and in nongrowing mycelial suspensions

Copper accumulation by the filamentous fungusAspergillus niger from a glucose mineral salts medium containing copper in the concentration range 16 to 157 μM was maximal in the lag phase of growth. In the subsequent linear growth phase, the mycelial copper contents were dramatically reduced on a per gram dry weight basis. The fungal mycelium exhibited pelleted morphology and exponential growth was not apparent. The medium pH was reduced during growth in flask cultures, but this was not responsible for the reduction in copper uptake as indicated by the similar effect in cultures grown in a stirred-tank fermenter with electronic maintenance of pH at 5.5. Voltammetric analysis of medium which had supported growth of the fungus showed that copper added at a final concentration of 40 μM was complexed. Energy-dependent copper uptake from 2-(N-morpholino)ethanesulfonic acid buffer at pH 5.5 containing 40 μM copper could not be demonstrated in nongrowing mycelium. Incubation at 4°C reduced copper uptake while the presence of 10 mM glucose or preincubation of the mycelium in 1 mM sodium azide had no effect on copper uptake.     

Inositol-Limited Growth, Repair, and Translocation in an Inositol-Requiring Mutant of Neurospora crassa

The biochemical consequences of inositol limitation in an inositol auxotroph of Neurospora crassa have been examined as a means of disclosing the cellular role of inositol. The cellular levels of inositol in the inl mutant were proportional to the concentration of inositol in the growth medium whereas inositol phosphate levels remained relatively constant at about 0.1 mumol/g (dry weight). After 72 h of growth, about 57-fold more protein per milligram (dry weight) was released by the mutant grown on limiting inositol than by the inositol-supplemented control. When the inositol-limited growth medium was osmotically buffered with 1% NaCl, 3% NaCl, or 6% sorbitol, there was about 33, 74, or 54%, respectively, less protein released by the mutant. These results are consistent with cell lysis occurring in the mutant grown on limiting inositol because of a structurally weakened cell wall and membrane deterioration. When sufficient inositol for normal mycelial growth was supplied to an inositol-deficient mycelium, there was within 2 h a rapid incorporation of inositol to 85% of control levels. This incorporation occurred without significant growth by any area of the mycelium. About 10 to 15% of the total cell inositol was translocated forward from the older mycelial areas to the growing tips; only 2 to 5% of the total cell inositol was translocated backward toward the older mycelial areas. Possible mechanisms of translocation are discussed.     

Growth of chitinolytic dune soil beta-subclass Proteobacteria in response to invading fungal hyphae

It has frequently been reported that chitinolytic soil bacteria, in particular biocontrol strains, can lyse living fungal hyphae, thereby releasing potential growth substrate. However, the conditions used in such assays (high bacterial density, rich media, fragmented hyphae) make it difficult to determine whether mycolytic activity is actually of importance for the growth and survival of chitinolytic bacteria in soils. An unidentified group of beta-subclass Proteobacteria (CbetaPs) was most dominant among the culturable nonfilamentous chitinolytic bacteria isolated from Dutch sand dune soils. Here we demonstrate that the CbetaPs grew at the expense of extending fungal mycelium of three dune soil fungi (Chaetomium globosum, Fusarium culmorum, and Mucor hiemalis) under nutrient-limiting, soil-like conditions. Aggregates of CbetaPs were also often found attached to fungal hyphae. The growth of a control group of dominant nonchitinolytic dune soil bacteria (beta- and gamma-subclass Proteobacteria) was not stimulated in the mycelial zone, indicating that growth-supporting materials were not independently released in appreciable amounts by the extending hyphae. Therefore, mycolytic activities of CbetaPs have apparently been involved in allowing them to grow after exposure to living hyphae. The chitinase inhibitor allosamidin did not, in the case of Mucor, or only partially, in the cases of Chaetomium and Fusarium, repress mycolytic growth of the CbetaPs, indicating that chitinase activity alone could not explain the extent of bacterial proliferation. Chitinolytic Stenotrophomonas-like and Cytophaga-like bacteria, isolated from the same dune soils, were only slightly stimulated by exposure to fungal hyphae.     

Cellular automata simulations of fungal growth on solid substrates

Growth of filamentous fungi on the surface of cereal grains is a critical aspect of solid substrate fermentation (SSF). Numerous mathematical models have been developed to describe various aspects of fungal growth in SSF. These models consider hyphal geometry and nutrient availability as determinants of colony morphology and fungal physiological state. This work describes the use of cellular automata (CA) as an alternative method of modeling fungal growth. CA models reliant on a very limited set of rules or "knowledge base" display a rich array of behaviors that mimic fungal growth. By incorporating probablistic growth rules into CA models, colony characteristics such as biomass accumulation rate, colony radial growth rate, mycelial density and fungal differentiation are readily generated.     

Lipid metabolism of Pleurotus sajor caju

The biosynthesis of lipids in the mycelium and sporophore of Pleurotus sajor caju was studied. Whereas in the mycelium the biosynthesis of lipids was directly primarily towards storage (e.g. tri-acylglycerols), in the sporophore it was directed towards structural components (e.g. sterols). The incorporation of ¹⁴C precursors into non-polar and polar lipid fractions was generally similar for ¹⁴C acetate, ¹⁴C palmitate, ¹⁴C oleate and ¹⁴C linoleate in the case of mycelium and sporophore. It appears that linoleic acid was utilised as a source of acetate for lipid biosynthesis in the sporophore. A significantly higher incorporation of label was seen in sporophore sterol than in mycelial sterol. Malate dehydrogenase activity increased in the mycelium grown in the presence of lipids. Lipase of P. sajor caju was inducive. The growth of P. sajor caju was enhanced by increased lipid utilisation. The implications of these results on commercial cultivation of this fungus are discussed.     

Evaluation of the in vitro and in vivo dimorphism of Sporothrix schenckii, Blastomyces dermatitidis, and Paracoccidioides brasiliensis isolates after preservation in mineral oil

Morphological differentiation has commanded attention for its putative impact on the pathogenesis of invasive fungal infections. We evaluated in vitro and in vivo the dimorphism from mycelial to yeast-phase of Sporothrix schenckii, Blastomyces dermatitidis and Paracoccidioides brasiliensis isolates, two strains for each species, preserved in mineral oil. S. schenckii strains showed typical micromorphology at 25 degrees C but one strain was unable to complete the dimorphic process in vitro. After in vivo passage through mice the strains had the ability to turn into yeast-like cells and to form colonies on brain-heart infusion medium at 36 degrees C. B. dermatitidis strains grew as dirty white to brownish membranous colonies at 25 degrees C and their micromorphology showed thin filaments with single hyaline conidia. At 36 degrees C the colonies did not differ from those grown at 25 degrees C, but produced a transitional micromorphology. P. brasiliensis strains grew as cream-colored cerebriform colonies at 25 degrees C showing a transitional morphology. B. dermatitidis and P. brasiliensis strains did not turn into yeast-like cells in vivo. The present results demonstrate that B. dermatitidis and P. brasiliensis strains were unable to complete the dimorphic process even after in vivo passage, in contrast to the S. schenckii strain.