Microscopical evidences for the presence of a nucleus inCandida albicans chlamydoconidia

By using the fluorescent, DNA specific stain DAPI (4,6-diamidino-2-phenylindole) some microscopic observations ofCandida albicans pseudomycelium and chlamydoconidia were performed. In this manner blue fluorescent dots were noted both in yeasts, psudomycelium and chlamydoconidia, so evidencing the presence of a nucleus inC. albicans chlamydoconidia.     

Nitrogen Starvation-Induced Changes in Amino Acid and Free Ammonium Pools in <Emphasis Type="Italic">Schizophyllum commune</Emphasis> Colonies

Wood-decay fungi depend upon recycling of nitrogen-containing molecules to maintain growth in nitrogen-deficient environments. One of the pools that can support growth in these organisms is the pool of free amino acids. The free amino acid (AA) composition of Schizophyllum commune mycelium grown on the surface of nitrogen-rich (M = 6.6 mM L-asparagine) and nitrogen-poor medium (M01 = 0.06 mM L-asparagine) has been examined: When mycelium is grown on M, alanine, glutamate, and asparagine account for almost 2/3 of the amino acid pool. The free amino acid concentration is reduced by 75% for mycelium grown on the M01 medium, with alanine and glutamate predominating. In addition, free NH₄⁺ increases by 60% in nitrogen-deprived mycelia. Except for asparagine, which is absorbed by the apices, the concentration of all free amino acids is higher in the centers of M-grown, 4-day-old mycelia than in the apices. Hyphae grown to exponential growth on M and transferred to M01 for 12 h show greater free amino acid and NH₄⁺ concentrations in the apices, most likely indicating increased translocation to the apices.     

New milk medium for germ tube and chlamydoconidia production byCandida albicans

A new medium consisting of UHT milk, tween 80 and agar is described for the development of both germ tube and chlamydoconidia byCandida albicans. In total 172 isolates from clinical specimens, includingC. albicans (112),C. guilliermondii (4),C. krusei (3),C. parasilopsis (16).C. tropicalis (28),Torulopsis glabrata (6) andTrichosporon beigellii (3), were examined in this medium by using the standard method. A higher percentage (98.2%) of germ tube production byC. albicans was found in this medium than in undiluted serum (90.2%). In addition, onlyC. albicans was found to be able to produce a high percentage of chlamydoconidia (95.5%) after 48 hours' incubation. In comparison with the conventional medium, corn meal tween 80 agar (21.4%), this new medium gives a significantly higher percentage and abundance of chlamydoconidia production. Being simple, cheap and easy to prepare, the new milk medium is proposed as very practical in the clinical mycology laboratory.     

Kinetics of growth and chemical composition ofFusarium moniliforme cultivated on carob aqueous extract for microbial protein production

Summary The kinetics of growth and the chemical composition ofFusarium moniliforme cultivated on aqueous carob pod extract were investigated. The extract was adjusted to provide 0.5, 1.0, 2.0 and 4.0% carob sugars supplemented with inorganic salts at the ratio: carob sugar: NH₄H₂PO₄: MgSO₄.7H₂O=1:0.6:0.012. The extract contained 16 mg tannic acid (Folin-Dennis) per g of carob sugar.The phase of vigorous growth was exponential. Tannins were not observed to depress growth. The maximum value of 0.22 h^–1 for a specific growth rate corresponding to a generation time of 3.15 h was obtained when the fungus was cultivated on a 4% carob sugar medium. The dry mycelium produced per g of consumed carob sugar was then 0.515 g.The protein and purine content was affected by the composition of the growth medium. Protein values up to 37.7% true (Lowry) and 53.1% crude (NX6.25) of dry mycelium were recorded. Mean purine contents were 89 and 116 mol/g, corresponding to nucleic acid levels of 5.7 and 7.5% for mycelium grown on 0.5 and 4.0% carob sugar respectively.These findings linked with those previously reported regarding the good appearance and nutritional quality ofF. moniliforme (Drouliscos et al., 1976) make this fungus worthy of consideration for the production of protein.     

A Preparation Method of Aspergillus spp. for Scanning Electron Microscopy

For scaning electron microscopy (SEM) of Aspergillus flavus Link und A. petraku Vörös. Simultaneous fixation and drying in osmium tetroxide (O₅O₄ vapours was superior to the conventional preparation methods. Which involve critical point drying. Mycelium exposed to vapours from a solution of 2% O₅O₄ for 24 h at ca. 20°C retained its turger and natural structures. Best results were achived when young mycelium was used. whereas old mycelium always collapased, irrespective of the fixation method used. Use of this method for fixation of Aspergillus spp. is therefore preferable to convennonal methods which are laborious. time-consuming and often interfere with the natural habit of structures. It may also prove usefull with other fungal structures.     

Physiological studies on Phymatotrichum omnivorum X. Influence of nitrogen sources on free and bound amino acid pools

The effect of nitrogen source on the free and bound amino acids of mycelium of Phymatotrichum omnivorum (Shear) Dugg was investigated. The largest free amino acid pool was present in the natural medium and the smallest in the synthetic medium. Phymatotrichum omnivorum was able to utilize different nitrogen sources with the best growth occurring with NH₄NO₃. The ratio of glycine to alanine and aspartic to glutamic was around 0.25 in the free amino acid pool and around 1 in the bound amino acid pool. The free pool of glutamic acid ranged from 5.6 % to 27.2 % depending upon the nitrogen source in the media. The free pool of alanine ranged from 35.7 % to 17.2 % in relation to the nitrogen source. Most other amino acid ratios did not vary significantly between the free amino acids and the bound amino acids.     

Production of gluconic acid by immobilized in pumice stones mycelium of Aspergillus niger using unconventional oxygenation of culture

Gluconic acid was produced in repeated batch processes with Aspergillus niger AM-11, immobilized in pumice stone particles using an unconventional oxygenation of culture media based on the addition of H₂O₂, decomposed by catalase to O₂ and water. The highest gluconic acid productivity of 8.2 g l^–1 h^–1 was reached with 30 g immobilized mycelium per 150 ml, 10% (w/v) glucose, at 24 °C and pH 6.5, with O₂ at 100% saturation. The immobilized mycelium was successfully reused up to 8 times in 1-h batches with only a slight loss (11%) of gluconic acid productivity.     

Fungi on the hair of large mammals in Egypt

Several isolates of Candida albicans were tested for production of chlamydoconidia and metabolic changes when grown on several different solid and liquid media. A liquid medium, consisting solely of sterilized skimmed milk and a solid medium containing processed cheese stimulated more rapid and greater production of chlamydoconidia than the corn meal agar and the other media tested.     

A novel gene——samfR involved in early stage of Streptomyces ansochromogenes differentiation

A 4.6 kb DNA fragment was cloned from the DNA library ofStreptomyces ansochromogenes using a partial DNA fragment located in the downstream of promoter-P_TH4 as probe. The experiments revealed that this DNA fragment consists ofsaw D gene and a 1.4 kbPvu II fragment which can accelerate mycelium formation ofS. ansochromogenes. The nucleotide sequence of 1.4 kb DNA fragment was determined and analysed; the result indicated that the fragment contains one complete open reading frame (ORF) which encodes a protein with 213 amino acids, and this gene was designated assamfR. The deduced protein has 36% amino acid identities and 52% amino acid similarities in comparison with that encoded byhppR gene, which is involved in the regulation of catabolism for 3-(3-hydroxyphenyl) propionate (3HPP) inRhodococcus globerulus. The function ofsamfR gene was studied using strategy of gene disruption, and the resultingsamfR mutant failed to form aerial hyphae and spores, its development and differentiation stopped at the stage of substrate mycelium in contrast with wild type strain. The results showed that thesamfR gene is closely related toS. ansochromogenes differentiation. Project supported by the National Natural Science Foundation of China (Grant No. 39830010).     

Viability of ectomycorrhizal fungus mycelium entrapped in calcium alginate gel

 We studied the viability of fragmented mycelium of Pisolithus tinctorius and Paxillus involutus entrapped in calcium alginate gel to determine the efficacy of this method of producing ectomycorrhizal fungus inoculum. Fungi were grown in MMN solution at 28  °C before being fragmented in a blender and subsequently entrapped in calcium alginate. We tested different ratios of alginate and mycelium suspension to 0.7 M CaCl₂. The ratio 8 : 10 resulted in well-formed beads of the highest viability for Paxillus involutus (99%) and for Pisolithus tinctorius (75%). Paxillus involutus mycelium was more than 90% viable when entrapped mycelium was 10 to 50 days old, and Pisolithus tinctorius attained its highest viability (55%) for 20- to 40-day-old mycelium. Gel entrapped Paxillus involutus mycelium grew well at all temperatures after 30 days of storage, but viability significantly decreased after 60 days storage at 6  °C on dry filter paper. For gel-entrapped Pisolithus tinctorius mycelium, viability was highest when stored at 25  °C in 0.7 M CaCl₂. Entrapment of Paxillus involutus fragmented mycelium in calcium alginate beads under the conditions that we propose can be used successfully to produce inoculum. Accepted: 11 October 1998     

Changes in protein secretion of Aspergillus niger caused by the reduction of the water activity by potassium chloride

The effect of the rapid reduction of the water activity (a_w) on the extracellular protein and amylolytic activity of Aspergillus niger was studied. An a_w value gradient from 0.90 to 0.99 in KCl solutions was applied for the mycelium treatment. It was found that the a_w reduction considerably influenced the protein secretion. This phenomenon was dependent on the age of the treated mycelium and the range of the a_w gradient. The highest protein and enzyme secretion yields were obtained at a_w = 0.98 using a 72-h old mycelium. In comparison with the non-treated mycelium, the increase in the secretion amounted to about 60% for the amylolytic activity and 37% for the soluble protein, respectively. It was shown that the mycelium incubated in KCl solutions of an a_w value from 0.90 to 0.99 had the ability for regeneration in fresh CZAPEK-DOX medium. The effect of the osmotic shock on the protein secretion was limited only for the treated cell population and declined in the mycelium which was regenerated after the transfer into the culture medium.     

Abortiporus biennis tolerance to insoluble metal oxides: oxalate secretion, oxalate oxidase activity, and mycelial morphology

The ability of Abortiporus biennis to tolerate and solubilize toxic metal oxides (Cu₂O, Al₂O₃, ZnO, CuFe₂O₄Zn, CdO, and MnO₂) incorporated into agar media was investigated and the growth rate, oxalic acid secretion, and mycelial morphology were monitored. Among the tested metal oxides, formation of clear zones underneath the mycelium growing on Cu₂O- and ZnO-amended plates was observed. ZnO, CdO and Cu₂O caused the highest rate of fungal growth inhibition. An increased level of oxalic acid concentration was detected as a response of A. biennis to the presence of Cu₂O, MnO₂, ZnO and CuFe₂O₄Zn in growth medium. The oxalate oxidase (OXO) was found to be responsible for oxalic acid degradation in A. biennis cultivated in metal-amended media. An increased level of OXO was observed in media amended with Cu₂O, ZnO and MnO₂. Confocal microscopy used in this study revealed changes in mycelial morphology which appeared as increased hyphal branching, increased septation and increased spore number.     

Continuous production of gibberellic acid in a fixed-bed reactor by immobilized mycelia of Gibberella fujikuroi in calcium alginate beads

Summary The continuous production of gibberellic acid with immobilized mycelia of Gibberella fujikuroi was maintained over a hundred days in a tubular fixed-bed reactor. Free mycelium at the beginning of the storage phase was harvested from G. fujikuroi shake-flask culture and was immobilized by ionotropic gelation in calcium alginate beads.The continuous recycle production system consisted of a fixed-bed reactor, a container in which the culture medium was heated, stirred and aerated, and valves for sample withdrawal or reactant addition during the first 1320 h (55 days). A two-phase continuous extractor was then added for the last 960 hours (40 days). Free and immobilized mycelium shake-flask cultures with the same strain used in the continuous culture system were also realized to compare growth, maintenance and production parameters. The results show about the same gibberellic acid productivity in both free and immobilized mycelium shakeflask cultures: 0.384 and 0.408 mgGA₃·gBiomass^-1 ·day^-1, respectively, whereas in the continuous system the gibberellic acid production is about twice as large for a similar biomass: 0.768 mgGA₃·gBiomass^-1·day^-1. Several factors affecting the overall productivity of the immobilized systems were found to be: the quality and the quantity of mycelia in the biocatalyst beads and the immobilization conditions.     

Ethylene Production and Mycelium Growth of the Tulip Strain of Fusarium oxysporum as Influenced by Shaking of and Oxygen Supply to the Culture Medium

The fungus Fusarium oxysporum Schlecht f. sp. tulipae Apt. can produce ethylene abundantly in vitro when grown in Pratt's liquid medium with glucose as the only organic substrate. This production starts after a lag phase of about 4 days, and peak production occurs when mycelium weight has reached its maximum value. For several days the rate of production is more or less linearly dependent on pO₂. The total production is also dependent on the oxygen concentration, but pure oxygen inhibits the total production by about 50% as compared with 21% oxygen. The high production in shake cultures, as compared with the low production in stagnant cultures, is probably the result of a better oxygen supply in the culture medium. The mycelium weight proved not to be a valid referential basis for the production of ethylene.     

Effect of phosphate on the toxicity of phosphite in Phytophthora palmivora

The concentration of P_i in the growth medium had a marked effect on the amount of the fungicide phosphite accumulated by Phytophthora palmivora. The mass of mycelium, produced after 7 days growth in a medium containing excess P_i, was not markedly inhibited until phosphite concentrations of 10 mM or greater were supplied. In contrast, in a P_i-deficient medium, growth was inhibited by 0.1 mM phosphite. In the latter medium, the fungus took up significantly more phosphite once P_i had been depleted from the medium (from day 4 to 6). When 7 day old mycelium was presented with either 1 mM P_i or phosphite, the P_i-deficient mycelium transported either anion at almost eight times the rate found in the P_i-excess mycelium. The level of alkaline phosphatase present in the P_i-deficient mycelium was also significantly elevated.     

Utilization of Carbohydrates by Rhynchosporium secalis II. Absorption and Metabolism of Glucose, Galactose and Galacturonic acid

Glucose, galactose and galacturonic acid were taken up at different rates by the fungus Rhynchosporium secalis and were intracellularly converted to other forms of carbohydrate at different rates. These differences explain why, when there is only a single source of nutrient carbon in the growth medium, development of the fungus is greatest when glucose is present and least when galactose is present. Glucose and galactose were taken up by the same mechanism for their uptake showed a reciprocal competitive inhibition. Uptake mechanisms had a high affinity for glucose (apparent K_m 2.76 mM) and galacturonic acid (apparent K_m 3.10 mM) and a low affinity for galactose (apparent K_m 29.67 mM). After uptake, galactose accumulated in the mycelium, whereas glucose and galacturonic acid were rapidly converted to other soluble carbohydrates, principally trehalose and mannitol. The insoluble carbohydrates within the mycelium were little affected by the type of carbohydrate that was supplied to the fungus.     

<Emphasis Type="Italic">Nocardiopsis terrae</Emphasis> sp. nov., a halophilic actinomycete isolated from saline soil

A Gram-positive, moderately halophilic, facultatively alkaliphilic, catalase- and oxidase-positive, obligately aerobic, filamentous actinomycete strain, designated YIM 90022^T, was isolated from saline soil collected from the Qaidam Basin, north-west China. Phylogenetic analysis based on 16S rRNA gene sequences indicated that the new isolate was a member of the genus Nocardiopsis and the sequence similarities between the isolate and the type strains of members of the genus Nocardiopsis were in the range of 95.1–98.7%. Phenotypic and chemotaxonomic properties of this organism also indicated that strain YIM 90022^T was a member of the genus Nocardiopsis. The strain grew well on most of the media tested, producing yellow-white to deep brown substrate mycelium and white aerial mycelium. Light gray to deep brown diffusible pigments were produced. The substrate mycelium was well developed and fragmented with age; the aerial mycelium produced long, straight to flexuous spore chains with non-motile, smooth-surfaced, rod-shaped spores on them. The strain grew in the presence of 1–15% (w/v) total salts (optimum, 3–5%) and at pH 6.0–10.5 (optimum, pH 8.5) and 10–45°C (optimum, 30°C). Whole-cell hydrolysates of strain YIM 90022^T contained meso-diaminopimelic acid and no diagnostic sugars. The predominant menaquinones were MK-10(H₄), MK-9(H₈), MK-10(H₆) and MK-10(H₈). Polar lipids comprised diphosphatidylglycerol, phosphatidylcholine, phosphatidylglycerol and phosphatidylmethylethanolamine. The major cellular fatty acids were iso-C_16:0, anteiso-C_17:0, 10-methyl-C_18:0 and 10-methyl-C_17:0. The DNA G + C content of strain YIM 90022^T was 71.5 mol%. The combination of phylogenetic analysis, DNA–DNA relatedness data, phenotypic characteristics and chemotaxonomic data supported the suggestion that strain YIM 90022^T represents a new species of the genus Nocardiopsis, for which the name Nocardiopsis terrae sp. nov. is proposed. The type strain is YIM 90022^T (=CCTCC AA 208011^T =KCTC 19431^T).     

Distribution of protein and nucleic acids in hyphae and microconidia of Fusarium

Summary Extracts obtained by manual grinding of the mycelium and microconidia of Fusarium oxysporum were fractionated into four particulate portions—designated P₁, P₂, P₃ and P₄—and into a soluble portion by differential centrifugation. Approximately 60% of the protein was recovered in the supernatant fraction whereas the ribonucleic acid content of the P₃ and P₄ particles was particularly high. The P₄ particle fraction was composed of approximately 40% ribonucleic acid and 60% protein and contained a particle with a sedimentation constant of about 85 S. More than 70% of the deoxyribonucleic acid was recovered among the various particle types, but the deoxyribonucleic acid: protein ratio was highest in the P₁ granules. Polysaccharides were particularly concentrated in the P₃ fraction. On the basis of the distribution of bound hexosamine, the extracts appeared to be free of cell wall fragments. Several methods of disruption of the fungus hyphae were examined.This investigation was supported in part by a grant from the United Fruit Company. Present address of senior author: Department of Agricultural Chemistry, Faculty of Agriculture, University of Tokyo.     

Involvement of the glutamine synthetase/glutamate synthase pathway in ammonia assimilation by the wood-rotting fungusPleurotus ostreatus

The mycelium of the wood-rotting fungus,P. ostreatus, contains NAD-dependent glutamate synthase inhibited by azaserine.l-Glutamine andl-glutamate are the most important free amino acids in the mycelium. Feeding of the mycelium with nitrogenous substrates showed thatl-glutamate,l-aspartate andl-alanine are interconnected by way of transaminases. After the inhibition of glutamine synthetase by methionine-S-sulfoximine the synthesis ofl-glutamate was inhibited and the level of all free amino acids decreased. The¹⁵N-NMR spectra of mycelia after the addition of¹⁵NH₄Cl confirmed that the GS/GOGAT is the only pathway of ammonia assimilation inP. ostreatus and NAD-glutamate dehydrogenase should be the deaminating enzyme.     

Metabolism of aromatic acids in the antibiotic-producing basidiomyceteOudemansiella mucida

Aromatic acids were determined in the mycelium and fermentation medium ofOudemansiella mucida. Coumaric acids (bothm- andp-),p-hydroxybenzoic acid (salicylic acid) and benzoic acid were found to predominate in the mycelium. Phenylacetic acid represents the main component in the medium. Phenylalanine ammonia-lyase catalyzing conversion of phenylalanine to cinnamie acid which is further metabolized to benzoic acid was detected in the mycelium. The results are discussed with respect to the synthesis of the antibiotic mucidin.