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PubMed2003

In vitro interaction of the truffle Terfezia terfezioides with Robinia pseudoacacia and Helianthemum ovatum.

The type of the in vitro root interactions of Terfezia terfezioides with the plants Robinia pseudoacacia and Helianthemum ovatum was investigated including detailed anatomical and ultrastructural characterization. No difference in growth was detected at different phosphate concentrations on agar synthetic medium between the inoculated and control plants during a short-time cultivation. The fungal colonization of the roots increased with higher phosphate level in both plant species, but was always lower in R. pseudoacacia roots. Septate hyphae formed frequently intracellular branched coils in dead cortical cells. In H. ovatum intercellular hyphae were observed forming finger-like structures reminiscent of Hartig-net structures in ectomycorrhizae. A loose hyphal envelope covered the root surface of both colonized and noncolonized roots. The features resembled similar structures described earlier during the mycorrhizae of different Terfezia species. Our detailed anatomical and ultrastructural study shows that the in vitro root interactions of the T. terfezioides cannot be considered unambiguously as mycorrhiza.

Ascomycota↗
PubMed1991

The use of some fluorescent stains for studying morphogenesis of micromycetes.

The use of some classical fluorochromes and optical brighteners in the fluorescence microscopy of micromycetes was investigated. Of the 16 compounds tested on slide cultures of Trichoderma viride 3 were too toxic, whereas the other stained primarily hyphae with various intensity. Reproductive structures did not stain or stained only weakly. With respect to vital staining the optical brightener Blankophor RKH exhibited most favorable properties. It did not inhibit either the growth or sporulation and stained intensively hyphae, septa and growth apices in particular. It also induced intensive fluorescence of a growing yeast culture.

Fluorescent Dyes↗
PubMed2002

Primary cutaneous mucormycosis complicating the use of adhesive tape to secure the endotracheal tube.

PURPOSE: To report a rare case of primary cutaneous mucormycosis (PCM), complicating securing of the endotracheal tube with adhesive tape. CLINICAL FEATURES: A 39-yr-old woman with systemic lupus erythematosus (SLE) developed four annular, punched out ulcers with a necrotic centre and elevated border in a linear distribution over the left cheek, under the tape securing the endotracheal tube. A tissue biopsy revealed broad, branching, nonseptate hyphae found in epidermis and dermis consistent with mucormycosis, best demonstrated with silver staining. Cultures were positive for Rhizopus species. Treatment with iv amphotericin B was successful. CONCLUSION: Because of the rarity of the disease and the difficulty of culturing the causative organism, diagnosis of mucormycosis is often elusive. Tissue biopsy and microscopic visualization of nonseptate hyphae with right-angled branching are the only methods for making the diagnosis. Skin biopsy of new ulcerative or plaque-like lesions should be obtained in immunocompromised patients. Early diagnosis and prompt treatment are critical for favourable outcomes in PCM.

Adhesives↗
PubMed1997

Flammulina as a model system for fungal graviresponses.

Gravitropic bending of fruiting bodies of Flammulina velutipes (Curtis) Karst. is based on the differential growth of the transition zone between stem and cap. Reorientation becomes visible as early as 2 h after displacing the fruiting body from the vertical to the horizontal position. It is preceded by a preferential accumulation of microvesicles within the hyphae on the lower side of the transition zone and related to an increase in the vacuolar compartment required for hyphal extension. A model made of a bundle of interconnected balloons is used to demonstrate that a differential volume increase at one flank is sufficient to bend the entire structure in the opposite direction. Gravitropic raising of intact stems or segments derived from the transition zone requires positional information which can be accomplished by three major, coordinated events: (i) gravisensing by the individual hyphae within the transition zone, (ii) unidirectional signalling by means of a soluble growth factor creating a vertical concentration gradient, and (iii) translation of the concentration signal into elongation growth.

Basidiomycota↗
PubMed2002

A novel Streptomyces gene, samR, with different effects on differentiation of Streptomyces ansochromogenes and Streptomyces coelicolor.

A 1.4-kb DNA fragment from Streptomyces ansochromogenes accelerated mycelium formation of S. ansochromogenes when present on a multicopy plasmid. The DNA fragment contains one complete open reading frame, designated samR, encoding a protein with 213 amino acids that contains a likely DNA-binding helix-turn-helix motif close to its N-terminus. The deduced SamR protein resembles the product of the hppR gene, which is involved in the regulation of catabolism of 3-(3-hydroxyphenyl) propionate in Rhodococcus globerulus. A samR disruption mutant was constructed that presented a bald phenotype and failed to form aerial hyphae and spores. We suggest that samR plays an important role in the emergence of aerial hyphae from substrate mycelium. An almost identical gene of Streptomyces coelicolor was also subjected to gene disruption. Surprisingly, the mutant was able to develop an aerial mycelium, but it remained white and deficient in sporulation instead of forming gray spores.

Amino Acid Sequence↗
PubMed2003

Characterization of the Penicillium chrysogenum antifungal protein PAF.

The filamentous fungus Penicillium chrysogenum abundantly secretes the small, highly basic and cysteine-rich protein PAF ( Penicillium antifungal protein). In this study, the antifungal activity of PAF is described. PAF inhibited the growth of a variety of filamentous fungi, including opportunistic human pathogenic and phytopathogenic fungi, whereas bacterial and yeast cells were unaffected. PAF reduced the conidial germination and hyphal extension rates in a dose-dependent manner and induced severe changes in cell morphology that resulted in crippled and distorted hyphae and atypical branching. Growth-affected hyphae suffered from oxidative stress, plasma membrane leakage, and metabolic inactivity, which points to an induction of multifactorial effects in sensitive fungi. In contrast to other known antifungal proteins, the effects of PAF were only partially antagonized by cations.

Antifungal Agents↗
PubMed1998

Heat shock effects on second messenger systems of Neurospora crassa.

Exposure of growing hyphae of Neurospora crassa to heat shock (44 degrees C) or ethanol (2.6 M) for 1 h induced a significant increase in the cAMP level, which reached a maximum approximately 2 min after the beginning of treatment and then decreased to control values despite continued heat or ethanol exposure. A 10-s heat shock or a 5-s ethanol shock also resulted in a transient cAMP increase 2 min after the pulse. Heat shock or ethanol treatment led to an increase in the amount of catalytic subunits of the cAMP-dependent protein kinase A in the nucleus almost synchronously with the increase of cAMP in the cytoplasm. The concentration of cGMP decreased a few seconds after the beginning of heat shock (44 degrees C) or ethanol treatment (2.6 M) to approximately 50% of the control level. Exposure to heat shock (44 degrees C, 1 h) led to an increase in the amount of inositol phosphates 0.5-2 min after the onset of heat shock. Thereafter, inositol phosphate levels dropped to control values despite continued heat exposure. Incubation of growing hyphae with cAMP or 8-Br-cAMP led to a two- to threefold increase of inositol phosphates 10-300 s after the beginning of incubation. Heat treatment furthermore caused a rapid release of calcium from vacuoles as determined by Fura-2 measurement of the calcium content released from isolated vacuoles. These heat-shock-dependent second messenger changes may play a role in the heat-shock-induced phase shifts of the circadian clock and heat-shock-induced conidiation.

Calcium↗
PubMed2004

Filamentous fungi: the indeterminate lifestyle and microbial ecology.

The filamentous fungi have dynamic and variable hyphal structures within which cytoplasm can be moved, synthesized, and degraded, in response to changes in environmental conditions, resource availability, and resource distribution. Their study has gone through several phases. In the first phase, direct observation was emphasized without undue concern for interior structures or in the presence of cytoplasm. By the mid-1970s, single biochemical proxies (ergosterol, marker fatty acids, chitin derivatives, etc.) were being used increasingly. The use of these surrogate single measurements continues, in spite of their inability to provide information on the physical structure of the filamentous fungi. Molecular approaches also are being used, primarily through the use of bulk nucleic acid extraction and cloning. Because the sources of the nucleic acids used in such studies usually are not known, taxonomic and phylogenetic information derived by this approach cannot be linked to specific fungal structures. Recently, a greater emphasis has been placed on assessing physical aspects of indeterminate fungal growth, involving the assessment of cytoplasm-filled and evacuated (empty) hyphae. Both of these parameters are important for describing filamentous fungal growth and function. The use of phase contrast microscopy and varied general stains, as well as fluorogenic substrates with observation by epifluorescence microscopy, has made it possible to provide estimates of cytoplasm-filled hyphal lengths. Using this approach, it has been possible to evaluate the responses of the indeterminate fungal community to changes in environmental conditions, including soil management. It is now possible to obtain molecular information from individual bacteria and fungal structures (hyphae, spores, fruiting bodies) recovered from environments, making it possible to link individual fungal structures with their taxonomic and phylogenetic information. In addition, this information can be considered in the context of the indeterminate filamentous fungal lifestyle, involving the dynamics of resource allocation to hyphal structural development and synthesis of cytoplasm. Use of this approach should make it possible to gain a greater appreciation of the indeterminate filamentous fungal lifestyle, particularly in the context of microbial ecology.

Cytoplasm↗
PubMed2005

Fungal diversity in rock beneath a crustose lichen as revealed by molecular markers.

Lichen-forming fungi have been assumed to be more or less restricted to the surface of the substrate on which they grow, Conclusive identification of hyphae or an assessment of the fungal diversity inside lichen-covered rock has not been possible using methods based on direct observation. We circumvented this problem by using a DNA sequencing approach. Cores were drilled from a Devonian arcosic sandstone rock harboring the crustose lichen Ophioparma ventosa (L.) Norman on the surface. The cores were cut vertically, and DNA was extracted from the pulverized rock slices. A series of polymerase chain reactions using fungal-specific primers as well as Ophioparma ventosa specific primers were employed to amplify the internal transcribed spacer region of the nuclear ribosomal DNA. The results show that hyphae of O. ventosa penetrate approximately 10-12 mm into the rock. Consequently, the hyphal layer formed by the lichen fungus inside the rock could be 7-12 times as thick as the symbiotic thallus at the surface of the rock. In addition, eight non-lichenized fungal taxa and five that could not be identified to species were encountered. One fungal species in the order Helotiales occurs in six of the eight cores. The significance of these results to the colonization and weathering of rock by lichenized fungi is discussed.

Base Sequence↗
PubMed1996

Detection of endogenous beta-glucuronidase activity in Aspergillus niger.

An endogenous beta-glucuronidase that hydrolyses the chromogenic substrate 5-bromo-4-chloro-3-indolyl-beta-D-glucuronide (X-gluc) in Aspergillus niger is reported. The activity was induced when the fungus was grown in media containing xylan, but was either very low, or absent, when grown on glucose. Endogenous beta-glucuronidase was primarily located in newly formed hyphae, and was apparent at pH values between 3 and 6. Hydrolysis of X-gluc was sensitive to the inhibitor D-saccharic acid 1,4-lactone and was irreversibly inactivated by heating. The bacterial uid A beta-glucuronidase reporter gene was strongly expressed in the hyphae of transformed A. niger but, in contrast to the endogenous activity, the enzyme was also active at pH 7-8.5. Histochemical localization of uidA expression in A. niger, without interference from the endogenous beta-glucuronidase activity, was achieved by staining at this pH.

Aspergillus niger↗
PubMed1999

Amylose-like polysaccharide accumulation and hyphal cell-surface structure in relation to citric acid production by Aspergillus niger in shake culture.

When 120 mg glucose/ml was used as a carbon source, in shake culture Aspergillus niger Yang no. 2 maximally produced only 15.4 mg citric acid/ml but accumulated 3.0 mg extracellular polysaccharide/ml. The polysaccharide secreted by mycelia of Yang no. 2 in shake culture was confirmed to be an amylose-like alpha-1,4-glucan by hydrolysis analysis with acid, amylase and glucoamylase. However, in static cultures, such as semisolid and surface cultures free from physical stresses caused by shaking damage, Yang no. 2 produced more citric acid but did not accumulate the polysaccharide. With cultivation time in shake culture, the amount of extracellular polysaccharide and the viscosity of the culture broth increased. The increase of shaking speed caused a remarkable increase in the accumulation of extracellular polysaccharide, e.g. 11.2 mg extracellular polysaccharide/ml was accumulated in the medium at a shaking speed of 200 rpm. The addition of 2.0 mg carboxymethylcellulose (CMC)/ml as a viscous additive to the medium reduced drastically the amount of extracellular polysaccharide accumulated to 1.5 mg/ml, but increased the citric acid produced to 52.0 mg/ml. However, intracellular polysaccharide accumulation kept up a steady rate of 0.26 microgram/mg dried mycelium through the entire period of cultivation. The addition of 3.0 mg polysaccharide/ml purified from the culture broth to the medium at the start of a culture resulted in a decrease of extracellular polysaccharide accumulation but an increase of citric acid accumulation. From electronmicroscopic observation, cell surfaces of hyphae cultivated with CMC were smooth, while hyphae cultivated without CMC had fibrous and granular polysaccharide on the cell surface. These results suggested that Yang no. 2 secreted the polysaccharide on the cell surface as a viscous substance and/or a shock absorber to protect itself from physical stresses caused by shaking damage in shake culture.

Adaptation, Physiological↗
PubMed2000

Uniparental mitochondrial transmission in sexual crosses in Cryptococcus neoformans.

Restriction fragment length polymorphism (RFLP) in the large ribosomal RNA region of the mitochondrial DNA (mtDNA) was developed as a genetic marker for investigating mitochondrial transmission in sexual crosses of the human pathogenic basidiomycetous yeast Cryptococcus neoformans. Strain JEC20 of C. neoformans var. neoformans (mat a) was mated with six strains of C. neoformans var. grubii (mat alpha). Successful mating was indicated by the formation of hyphae and basidiospores. These basidiospores were examined for mtDNA RFLP genotypes. All 570 basidiospores examined from the six crosses showed the mtDNA genotype of strain JEC20. The failure to recover the C. neoformans var. grubii mtDNA in any cross indicates that the C. neoformans var. grubii mtDNA is either selectively eliminated in the newly formed dikaryon or selectively excluded in the immediate dikaryotic hyphae of the newly formed dikaryon.

Crosses, Genetic↗
PubMed1999

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 Spitzenkörper. The cytoskeleton of the hyphal tube has been implicated in the organisation of the Spitzenkörper 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 Spitzenkörper and the initiation of apical branching.

Computer Simulation↗
PubMed2001

Isolation and characterization of four genes encoding pyruvate, phosphate dikinase in the oomycete plant pathogen Phytophthora cinnamomi.

The oomycete genus Phytophthora contains some of the world's most devastating plant pathogens. We report here the existence in P. cinnamomi of four genes encoding the pyrophosphate-utilizing glycolytic/gluconeogenic enzyme pyruvate, phosphate dikinase (PPDK). The coding regions of the four genes are >99% identical. At least three of the genes comprise a small gene cluster, which may have arisen through recent gene duplication and inversion events. Levels of Pdk mRNA are low in vegetative hyphae, but increase rapidly and transiently upon transfer of cultures to nutrient-free media, conditions that trigger asexual sporulation. PPDK protein and enzyme activity levels do not show a similar increase during sporulation. Assays of PPDK activity in P. cinnamomi hyphal extracts suggest that the majority of glycolytic flux in sporulating hyphae probably occurs via PPDK, rather than pyruvate kinase. This finding, combined with the existence of Phytophthora-expressed sequence tags encoding two other pyrophosphate-utilizing enzymes, indicates that pyrophosphate-based metabolism may be important in Phytophthora. The possibility that PPDK and other enzymes of pyrophosphate-based metabolism may provide targets for the development of novel control measures for Phytophthora and other oomycete pathogens is discussed.

Amino Acid Sequence↗
PubMed2003

Morphological development of anthers induced by the dimorphic smut fungus Microbotryum violaceum in female flowers of the dioecious plant Silene latifolia.

When inoculated with the dimorphic smut fungus Microbotryum violaceum (Pers.) G. Deml and Oberwinkler, the female flower of the dioecious plant Silene latifolia (Miller) E.H.L. Krause develops anther-like structures filled with spores instead of pollen grains. Using natural scanning electron microscopy, Nomarski interference microscopy, and fluorescence microscopy, we investigated the morphological modifications of the host plant resulting from this parasitism and the localization of smut hyphae in the flower bud. Flowers of infected plants lasted significantly longer than those of healthy plants, probably because the infection strengthened floral organs, such as the flower base and the anther filaments. Smut hyphae were observed throughout all organs of the young flower buds of infected plants, including sepals, petals, stamens, and pistil primordia. In healthy female flowers, anthers initiated sporogenous cell formation, but lacked parietal cell layers. By contrast, the parietal cell layers of infected female flowers differentiated into tapetal tissue, middle cell layers, and endothecial layers, as in the anthers of healthy male flowers. Smut spore formation in the infected anther was initiated in intercellular regions between the sporogenous cells, resulting in degeneration of premature sporogenous cells, tapetal tissue, and middle cell layers. The development of the endothecial layers and epidermis in the infected anther were morphologically normal.

Basidiomycota↗
PubMed2004

Combined mucormycosis and aspergillosis of the oro-sinonasal region in a patient affected by Castleman disease.

One case of aspergillosis and mucormycosis occurring in a patient with stage-IV Castleman disease was investigated. The patient, who had undergone polychemotherapy and was in otherwise good general condition, without lymphadenopathies or imbalance of the immune system, presented with a palatal ulceration that progressively involved the palatal mucosa and bone, the paranasal sinuses and the orbit. Repeated cultural examinations were always negative. He had undergone multiple cytological smears of the inflammatory infiltration and biopsies of both the oral and nasal mucosa, which resulted in extensive necrotic debris and suppurative inflammation, and, on the very last biopsy, fungal hyphae, spores and conidia were also detected. These were large, branching, mostly non-septate hyphae, associated with conidiophores and conidia, the latter appearing dark brown to black in the histological preparations. Following the diagnosis of combined mucormycosis and aspergillosis, the patient underwent prolonged topic and systemic antibiotic treatment that resulted curative. Mucormycosis usually is a fatal complication of head and neck or systemic disorders, leading to severe immune suppression. Nevertheless, early diagnosis may be achieved using a combination of special stains and may lead to effective antibiotic treatment and cure of the patient, even if associated with other opportunistic infections, such as aspergillosis.

Aged↗
PubMed2001

Aortic lesions in aspergillosis: histopathological study of two autopsy cases.

The involvement of the aorta in aspergillosis is relatively rare, and its histopathological features have not been described in detail. In two autopsy cases of invasive aspergillosis in immunologically compromised patients with hematological disorders, the aspergillotic lesions of the aorta showed different characteristic histopathological features depending on the three layers of aortic wall. Intimal lesions were formations of the mural thrombus composed of aggregates of numerous fungal hyphae arranged perpendicularly to the aortic wall. In the media, smooth muscle cells were completely necrotic (anemic infarct of the media), while the framework of elastic fibers was well preserved, with only a few fungal hyphae detected. In the adventitia, an intense, partly granulomatous chronic inflammatory change with fibrosis was observed in association with mycotic embolic occlusion of the vasa vasorum. These histopathological changes were considered to represent one of the typical tissue reaction patterns in the wall of an elastic artery in aspergillosis.

Aorta, Thoracic↗
PubMed1997

A ras homologue of Neurospora crassa regulates morphology.

In order to study the role of signal transduction pathways in the regulation of morphology in Neurospora crassa, we cloned and characterized a ras homologue, termed NC-ras2. The predicted protein product of this gene is composed of 229 amino acid residues and contains all the consensus sequences shared by the ras protein family. The gene is located in linkage group V. An NC-ras2 disruptant showed morphological characteristics very similar to those of the smco7 mutant, which also maps to linkage group V. Nucleotide sequence analysis revealed that the smco7 mutant harbored a single base deletion in the NC-ras2 gene, which is predicted to result in the truncation of the protein product. Introduction into the smco7 mutant of an NC-ras2 clone yielded stable transformants with a wild-type phenotype. The smco7 mutant exhibited very slow hyphal growth and the rate of conidial formation was approximately one two-hundredth of wild type. The smco7 mutation causes both the changes in the pattern of hyphal growth and the defects in cell wall synthesis. Both the diameter and the length of the apical compartment were shorter in the hyphae of the smco7 mutant. These results suggest that NC-ras2 is identical to smco7, and that the signal transduction pathway mediated by the NC-ras2 protein regulates the apical growth of hyphae, cell wall synthesis, and conidial formation in N. crassa.

Amino Acid Sequence↗