Morphogenesis and growth kinetics of <Emphasis Type="Italic">Fusarium verticillioides</Emphasis>

The present study aimed to analyze the growth kinetics and morphogenesis of toxigenic Fusarium verticillioides strains. Growth curves based on mycelial dry weight measured after 24, 48, and 96 h and every 4 days for a period of 60 days were obtained for each strain. The morphogenesis of this fungus was studied during its developmental stages through the fluorescent method Fluorecein Diacetate-FDA and Ethidium Bromide-EB. The growth curves of the strains usually followed a homogeneous pattern comparable to the ideal growth curve. Using the fluorescence method, non-viable cells showed a light bright red coloration and viable cells presented green fluorescence with three fluorescent patterns. The present results showed that the morphogenesis of F. verticillioides is an asynchronous process characterized by the presence of a wide variety of fungal forms until 50 days of culture. The method is very useful to demonstrate the F. verticillioides growth stages as well as the perfect differentiation between viable and non-viable cell.     

Changes in the intracellular concentrations of the adenosine phosphates and nicotinamide adenine dinucleotides ofSaccharomyces cerevisiae during batch fermentation

Significant changes in the intracellular concentrations of adenosine phosphates and nicotinamide adenine dinucleotides were observed during fermentation of grape must by three different strains ofSaccharomyces cerevisiae: S. cerevisiae var.cerevisiae, a typical fermentative yeast strain and two flor-veil-forming strains,S. cerevisiae var.bayanus andS. cerevisiae var.capensis. The intracellular concentration of ATP was always higher inS. cerevisiae var.cerevisiae than in the flor-veil-forming strains. NAD⁺ and NADP⁺ concentrations decreased at faster rates in the flor-veil-forming yeasts than in the other yeast but NADH concentration was the same in all yeasts for the first 10 days of fermentation. NADPH concentration was always lower inS. cerevisiae var.cerevisiae than in the other yeasts and this yeast also showed higher rates of growth and fermentation during the early stages of the fermentation and the presence of non-viable cells at the end of fermentation. In contrast, the flor-veil-forming strains maintained growth and fermentation capabilities for a relatively long time and viable cells were present throughout the entire fermentation process (31 days).The authors are with the Department of Microbiology, Faculty of Sciences, University of Cordoba, Avda. San Alberto Magno s/n, 14004-Córdoba, Spain     

Respiratory capacity, cyclic adenosine 3'',5''-monophosphate, and morphogenesis of Mucor racemosus.

A variety of cultural conditions were examined to determine the relationship between respiratory capacity and the growth of Mucor racemosus in the yeast and mycelial form. The results show that both yeasts and hyphae can develop when the respiratory capacity is low (e.g., in N2). In addition, the yeast form of the fungus could be grown in air in the presence of cyclic adenosine 5'-monophosphate with high respiratory rates characteristic of air-grown mycelia. These results indicate that their is not an obligatory relationship between respiratory capacity and morphogenesis in M. racemosus. Low intracellular levels of cyclic adenosine 5'-monophosphate, however, were correlated with aerobic mycelial development, whereas yeast development under CO2 was characterized by higher cyclic adenosine 5'-monophosphate levels.     

Cellular and humoral immune responses to Candida albicans in subcutaneously infected mice

Pure mycelial and yeast cultures of Candida albicans were produced in a low sulphate medium. Groups of mice were injected subcutaneously with increasing doses of viable or heat-killed mycelial or yeast cells and the kinetics of delayed-type hypersensitivity (DTH), anti-mycelial and anti-yeast antibodies were studied. Both the dose and the morphological phase of C. albicans showed an influence on the development of the DTH, but the viability is the factor which showed the highest influence on this reaction, since on the one hand mice infected with viable yeast or mycelial cells developed higher DTH levels than mice injected with heat killed cells, and on the other hand this factor seems to play an important role in the kinetics of DTH response. The enzyme-linked immunosorbent assay has been adapted to detect antibodies to yeast and mycelial phase cytoplasmic antigens of C. albicans. In contrast with the DTH reactions, neither dose, morphological phase nor viability played an important role on the antibody titer developed. However, the use of mycelial cytoplasmic antigens seems to be better than the yeasts to detect anti -Candida antibodies over the last days studied.     

Growth and macromolecular content of the dimorphic fungus Aureobasidium pullulans and the effect of hydroxyurea and other inhibitors

The growth kinetics and the macromolecular content of the yeast and ethanol-induced hyphal forms of Aureobasidium pullulans were studied. During the morphological transition from yeasts to hyphae, both the protein and RNA content decreased significantly, the mycelial form containing only 76% of the amount of protein in the yeasts, and 38% of the RNA. The DNA was the only component tested whose level increased during the transition. Among several compounds inhibiting macromolecular synthesis, only hydroxyurea showed a remarkable effect on the morphology of A. pullulans, inducing the mycelial morphology. The macromolecular composition of hydroxyurea-treated cultures changed with time in a way similar to that of the ethanol-Tween 80-ammonia medium, and to that of carbon-starved cultures, without ethanol or glucose.     

Evaluation of the biological control by the yeast <Emphasis Type="Italic">Torulaspora globosa</Emphasis> against <Emphasis Type="Italic">Colletotrichum sublineolum</Emphasis> in sorghum

The yeasts are microorganisms with great potential for biotechnological applications in diverse areas. The biological control of phytopathogens by yeasts has showed satisfactory results under laboratory conditions, and it has already produced commercial formulations. With this as focus, this work aims to perform in vitro and in vivo evaluations of the action of a Torulaspora globosa yeast strain (1S112), isolated from sugarcane rhizosphere, against the phytopathogenic mold Colletotrichum sublineolum, the causative agent of anthracnose in sorghum. In vitro experiments included the antagonism test in Petri dishes with morphological hyphal evaluation; yeast killer activity; siderophore, volatile compound and hydrolytic enzyme production. In vivo experiments were conducted in greenhouse conditions with a sorghum variety susceptible to C. sublineolum by evaluating the anthracnose disease for 6 weeks. The results indicated that the yeast strain significantly controlled the fungal growth, either in vitro or in vivo. The strain of T. globosa exhibited killer activity against two sensitive strains, which is a novel capacity for this species. The yeast did not produce siderophores, volatile compounds or hydrolytic enzymes, although it has reduced the mycelial growth, resulting in hyphal deformities but not cell death. The yeast controlled the anthracnose disease in sorghum, either inoculated before or after the fungal spores, suggesting that the competition for space and nutrients to dominate the mold and killer toxin production, altering the hyphal morphology, are mechanisms utilized by the yeast in the biocontrol.     

Effect of oxygen on morphogenesis and polypeptide expression by Mucor racemosus.

The morphology of Mucor racemosus in cultures continuously sparged with nitrogen gas was investigated. When appropriate precautions were taken to prevent oxygen from entering the cultures, the morphology of the cells was uniformly yeastlike irrespective of the N2 flow rate. When small amounts of oxygen entered the cultures the resulting microaerobic conditions evoked mycelial development. Polypeptides synthesized by aerobic mycelia, microaerobic mycelia, anaerobic yeasts, and yeasts grown in a CO2 atmosphere were compared by two-dimensional gel electrophoresis. The results indicated that a large number of differences in polypeptide expression exist when microaerobic mycelia or anaerobic yeasts are compared with aerobic mycelia and that these alterations correlate with a change from an oxidative to a fermentative metabolic mode. Relatively few differences in polypeptide composition exist when microaerobic cells are compared with anaerobic cells, but these changes correlate with a change from the mycelial to the yeast morphology. We hypothesize that oxygen regulates the expression of polypeptides involved in both the metabolic mode and in morphogenesis.     

CYTOLOGICAL INVESTIGATIONS OF KABATIELLA CAULIVORA

Cole , Herbert , Jr ., and Houston B. Couch . (Penn. State U., University Park.) Cytological investigations of Kabatiella caulivora. Amer. Jour. Bot. 46(1) : 12-16. Illus. 1959.—Initial growth of K. caulivora on artificial media is characterized by budding, yeast-like, conidia, exclusive of mycelia. After 14 days growth, at 20°C., mycelial growth becomes macroscopically evident. A study of the germination behavior of a total of 5500 conidia from the initial yeast-like growth stage showed 59 to germinate by the production of mycelia, while the balance germinated by budding. Five thousand conidia of the mycelial growth type were studied in a successive, single-spore transfer series, and, in all cases, conidial germination continued to be by means of germ tubes. Conidia of both growth forms were studied by means of bright field and phase contrast microscopy, and found to be multinucleate—possessing variable nuclear numbers, ranging from 1-8 per cell. Mean nuclear number for both the mycelial and conidial types was 2.8. All nuclei of both growth types appeared to contain the same chromosome complement. The cultural variability exhibited by K. caulivora cannot be reconciled with the concept of dual phenomenon. It is suggested, rather, that the mycelial homotype probably arises as the result of a unidirectional mutation within the conidial growth form.     

Primary cutaneous cryptococcosis

Cottonseed protein agar and a modified Tween-albumin casein hydrolysate (TAC) medium were compared for the yeast phase conversion of Blastomyces dermatitidis strains including fresh isolates as well as strains maintained in long-term storage. It was found that both media converted all the B. dermatitidis (mycelial phase) strains studied to yeast phase in three days. The TAC medium has the added advantage that it is clear and the growth can be recognized earlier than in the opaque cottonseed agar medium. The conversion in most cases was more than 95% and the morphology of the yeast cells was uniformly typical with broad base budding. There was a striking difference between the sensitivity of the yeast and mycelial phases of B. dermatitidis strains. The yeast phase was usually more sensitive to Amphotericin B than the mycelial phase of B. dermatitidis. Similarly, the yeast phases of four out of six strains were more sensitive to ketoconazole than their respective mycelial phases, while two strains showed identical sensitivity in cottonseed agar. The yeast phase organism was more susceptible to Amphotericin B when cottonseed medium was used whereas the yeast phase showed more susceptibility to ketoconazole in TAC medium. Since the sensitivity among the various strains differed, it is necessary to determine the antifungal susceptibility of the pathogenic phase of the organism for initiating proper therapy and monitoring effectiveness.Dr. Rose actively participated in this research; expired February 2, 1984.     

Evaluation of a fluorescent method (fluorescein diacetate and ethidium bromide solution) in the study of the viability Cryptococcus neoformans strains

The effectiveness of the fluorescent viability test (fluorescein diacetate-FDA and ethidium bromide-EB-solution) compared to the plaque counting test (Miles & Misra M & M) was performed on 10 samples of Cryptococcus neoformans cultivated in Sabouraud dextrose agar at 25 °C. The optimum incubation period of 50 minutes was determined. Growth curves of the fungal strains studied based on the mean cell number were drawn for both the FDA & EB and M & M methods. The statistical evaluation (Student's T test) of the average sum of the viable cell counts showed that the FDA-EB method is more sensitive than the M & M test for the studied species. The growth curves of the samples usually followed a homogeneous pattern comparable to other non-dimorphic fungi.     

Cysteine biosynthesis in a fungus, Histoplasma capsulatum.

We have analyzed a step in cysteine biosynthesis in several strains of the pathogenic dimorphic fungus, Histoplasma capsulatum. Mycelial cells of all strains tested are prototrophic. However, the yeast phase cells of most stains do not grow in the absence of -SH-containing compounds due to the apparent lack of an active form of sulfite reductase, a crucial enzyme in the cysteine biosynthetic pathway. In contrast, the yeast phase cells of one strain (Downs) have been found to have an active sulfite reductase and can grow in the absence of cysteine if serine is added. A different metabolic block must thus exist in this strain. Sulfite reductase in the yeast form of Downs strain is completely repressed by growth on cysteine while the mycelial form seems to be constitutive. The yeast and mycelial phase extracts were analyzed on polyacrylamide gels. A distinct protein band appeared in extracts prepared from the yeast cells incubated in minimal or serine-containing media, but not in extracts from mycelia or from cysteine-grown yeast cells.     

Development,persistence and regeneration of foraging ectomycorrhizal mycelial systems in soil microcosms

Development of extraradical mycelia of two strains each of Paxillus involutus and Suillus bovinus in ectomycorrhizal association with Pinus sylvestris seedlings was studied in two dimensions in non-sterile soil microcosms. There were significant inter- and intra-specific differences in extraradical mycelial growth and morphology. The mycelial systems of both strains of P. involutus were diffuse and extended more rapidly than those of S. bovinus. Depending on the strain, P. involutus mycelia were either highly plane filled, with high mass fractal dimension (a measure of space filling) or sparse, low mass fractal dimension systems. Older mycelial systems persisted as linear cords interlinking ectomycorrhizal tips. S. bovinus produced either a mycelium with a mixture of mycelial cords and diffuse fans that rapidly filled explorable area, or a predominately corded mycelium of minimal area cover. In the soil microcosms, mass fractal dimension and mycelial cover tended to increase with time, mycelia encountering litter having significantly greater values. Results are discussed in terms of the ecology of these fungi, their foraging activities and functional importance in forest ecosystems.     

Effect of L-amino acids on Mucor rouxii dimorphism.

Mucor rouxii organisms growing aerobically and exponentially on a well-defined minimal medium are able to differentiate as yeasts or as mycelia, depending on the amino acid as the nitrogen source. When certain amino acids were used as the nitrogen source, spores differentiated only as hyphae, whereas other amino acids gave rise to other morphological forms having different ratios of yeasts to hyphae. In both hyphal and yeast cultures, an aerobic metabolism was predominant, as shown by determining several metabolic parameters such as oxygen tension, glucose consumption, ethanol production, and CO2 release. A complete conversion of yeasts to hyphae was obtained by the appropriate change in the amino acid used as nitrogen source. By preparing spheroplasts from mycelial cultures and transferring them to media with amino acids that induce yeast formation, a 50% yield in the reverse transformation was achieved. A correlation between the change in pH of the medium and cell morphology was observed in different growth conditions. Decrease in the pH of the medium preceded the appearance of hyphae. Also, when the initial pH of the medium was increased, aspartate-containing cultures developed mainly as mycelia, instead of yeasts, with a corresponding decrease in the final pH.     

Antagonistic interactions between garden yeasts and microfungal garden pathogens of leaf-cutting ants

We investigate the diversity of yeasts isolated in gardens of the leafcutter ant Atta texana. Repeated sampling of gardens from four nests over a 1-year time period showed that gardens contain a diverse assemblage of yeasts. The yeast community in gardens consisted mostly of yeasts associated with plants or soil, but community composition changed between sampling periods. In order to understand the potential disease-suppressing roles of the garden yeasts, we screened isolates for antagonistic effects against known microfungal garden contaminants. In vitro assays revealed that yeasts inhibited the mycelial growth of two strains of Escovopsis (a specialized attine garden parasite), Syncephalastrum racemosum (a fungus often growing in gardens of leafcutter lab nests), and the insect pathogen Beauveria bassiana. These garden yeasts add to the growing list of disease-suppressing microbes in attine nests that may contribute synergistically, together with actinomycetes and Burkholderia bacteria, to protect the gardens and the ants against diseases. Additionally, we suggest that garden immunity against problem fungi may therefore derive not only from the presence of disease-suppressing Pseudonocardia actinomycetes, but from an enrichment of multiple disease-suppressing microorganisms in the garden matrix.     

Occurrence and Growth of Yeasts in Yogurts

Yogurts purchased from retail outlets were examined for the presence of yeasts by being plated onto oxytetracycline malt extract agar. Of the 128 samples examined, 45% exhibited yeast counts above 10³ cells per g. A total of 73 yeast strains were isolated and identified as belonging to the genera Torulopsis, Kluyveromyces, Saccharomyces, Candida, Rhodotorula, Pichia, Debaryomyces, and Sporobolomyces. Torulopsis candida and Kluyveromyces fragilis were the most frequently isolated species, followed by Saccharomyces cerevisiae, Rhodotorula rubra, Kluyveromyces lactis, and Torulopsis versatilis. The growth of yeasts in yogurts was related to the ability of the yeasts to grow at refrigeration temperatures, to ferment lactose and sucrose, and to hydrolyze milk casein. Most yeast isolates grew in the presence of 100 μg of sorbate and benzoate preservatives per ml. Higher yeast counts from yogurts were obtained when the yogurts were plated onto oxytetracycline malt extract agar than when they were plated onto acidified malt extract agar.     

Yeast colonizing the intestine of rainbow trout (Salmo gairdneri) and turbot (Scophtalmus maximus)

Yeast were isolated from the intestine of farmed rainbow trout (Salmo gairdneri), turbot (Scophtalmus maximus), and free-living flat-fish (Pleuronectes platessa and P. flesus). The average number of viable yeasts recovered from farmed rainbow trout was 3.0 × 10³ and 0.5 × 10² cells per gram homogenized intestine for white and red-pigmented yeasts, respectively. The dominant species were Debaryomyces hansenii, Saccharomyces cerevisiae, Rhodotorula rubra, and R. glutinis. In 5 of 10 free-lving marine fish, > 100 viable yeast cells per gram intestinal mucus were recovered. Red-pigmented yeasts dominated and composed >90% of the isolates. Colonization experiments were performed by inoculating rainbow trout and turbot with fish-specific, isolated yeast strains and by examining the microbial intestinal colonization at intervals. Inoculation of experimental fish with pure cultures of R. glutinis and D. hansenii HF1 yielded colonization at a level several orders of magnitude higher than before the inoculation. Up to 3.8 × 10⁴, 3.1 × 10⁶, and 2.3 × 10⁹ viable yeast cells per gram intestine or feces were recovered in three separate colonization experiments. The high level of colonizing yeasts persisted for several weeks. The concentrations of yeasts in the tank water never exceeded 10³ viable cells per milliliter. No traces of fish sickness as a result of high yeast colonization were recorded during any of the colonization experiments. For periods of the experiments, the concentration of aerobic bacteria in the fish intestine was lower than the intestinal yeast concentration. Scanning electron microscopy studies demonstrated a close association of the yeasts with the intestinal mucosa. The mucosal colonization was further demonstrated by separating intestinal content, mucus, and tissue. All compartments were colonized by >10³ viable yeast cells per gram. No bacteria were detected on the micrographs, indicating that their affinity for the intestinal mucosa was less than that of the yeasts. Correspondence to: Thomas Andtid     

Influence of carbon and nitrogen sources on glutathione catabolic enzymes inCandida albicans during dimorphism

The effect of carbon sources, glucose and sucrose, and nitrogen sources such as ammonia, glutamate andl-citrulline on the activities of glutathione metabolic enzymes has been studied. Yeast and mycelial cells were used to identify changes in activity levels of glutathione reductase (GSSGR), glutathione transferase (GST), glutathione peroxidase (GPX) and -glutamyl transpeptidase (GGT). Enzyme activities from cells grown in sucrose media were lower than in glucose media regardless of the enzyme tested, morphological form, or the growth interval. In all enzymes except GST, activity was higher in yeast form than in mycelia, regardless of nitrogen source, with lower activity from 24 to 72 h than at 96 h. In citrulline media, yeast form showed the maximum GST, GGT, and GPX activity. In ammonia-amended media, mycelia showed maximum activity in GGT, whereas in glutamate media, mycelia showed the maximum activity in GST. Also, the type of nitrogen source had no effect on GPX activity in the mycelial form. Finally, changing the nitrogen source showed no significant effect on GSSGR activity, either in the yeast or mycelial form.     

Improving dispersed growth of Frankia using Carbopol

In laboratory culture, strains of the actinomycete Frankia grow slowly and form dense mycelial pellets. In this study, we have shown that by adding the anionic polymer Carbopol 941® to liquid growth media, dispersed growth of Frankia is improved. Cell protein measurements indicated higher biomass production in cultures maintained in media supplemented with Carbopol. Fluorescence staining and microscopy were used to compare viability of well-dispersed mycelia and pellets.     

In vitro susceptibility of isolates of Sporothrix schenckii to amphotericin B, itraconazole, and terbinafine: comparison of yeast and mycelial forms

Forty-three clinical isolates of Sporothrix schenckii derived from humans and animals were evaluated in vitro for their susceptibility to amphotericin B, itraconazole, and terbinafine. MICs were determined by the method of micro dilution in liquid media, using protocols M27-A2 for the yeast form and M38-A for the mycelial form, both standardized by the Clinical Laboratory Standards Institute. In general, higher MICs were found for the mycelial form (intervals of up to two dilutions). In the case of amphotericin B, a significant difference in activity was observed, with higher values (p<0.05) found for the mycelial form. MICs for itraconazole and terbinafine were similar for both yeast and mycelial forms but slightly higher for mycelia. Although data presented here indicate different levels of susceptibility when both growth forms were compared, indicating an intrinsic difference between them, it is still difficult to draw a consensus as to which form correlates better with clinical findings. More studies are necessary to determine the criteria for in vitro tests that will lead to efficient therapeutic choices.