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Inhibitory effect of essential oils on apical growth of Aspergillus fumigatus by vapour contact.

The inhibitory effect of seven essential oils on the apical growth of hyphae of Aspergillus fumigatus was studied using a bio cell tracer by vapour contact in a sealed vessel. Based on the inhibitory pattern, these essential oils were classified into three groups. The first group, composed of citron, lavender and tea tree oils, stopped the apical growth in a loading dose of 63 micrograms ml-1 air, but allowed the regrowth of the hyphae after removal of the vapour, indicating fungistatic action. The second group, consisting of perilla and lemon-grass oils, stopped the apical growth in a loading dose of 6.3 micrograms ml-1 air, and did not allow the regrowth after gaseous contact at 63 micrograms ml-1 air, indicative of fungicidal action. The third group, consisting of cinnamon bark and thyme oils, retarded the growth in a dose of 6.3 micrograms ml-1 air, stopped it in a dose of 63 micrograms ml-1 air, and incompletely suppressed regrowth of the hyphae. Gas chromatographic analysis revealed that vapours of essential oils were absorbed on fungal mycelia and agar medium most abundantly by the first group, followed by the second and third groups, reflecting the volatility of the respective groups. Suppression of the apical growth by vapour contact was ascribed to the direct deposition of essential oils on fungal mycelia, together with an indirect effect via the agar medium absorbed.

Aspergillus fumigatus↗

The root endophytic fungus Piriformospora indica requires host cell death for proliferation during mutualistic symbiosis with barley.

Fungi of the recently defined order Sebacinales (Basidiomycota) are involved in a wide spectrum of mutualistic symbioses (including mycorrhizae) with various plants, thereby exhibiting a unique potential for biocontrol strategies. The axenically cultivable root endophyte Piriformospora indica is a model organism of this fungal order. It is able to increase biomass and grain yield of crop plants. In barley, the endophyte induces local and systemic resistance to fungal diseases and to abiotic stress. To elucidate the lifestyle of P. indica, we analyzed its symbiotic interaction and endophytic development in barley roots. We found that fungal colonization increases with root tissue maturation. The root tip meristem showed no colonization, and the elongation zone showed mainly intercellular colonization. In contrast, the differentiation zone was heavily infested by inter- and intracellular hyphae and intracellular chlamydospores. The majority of hyphae were present in dead rhizodermal and cortical cells that became completely filled with chlamydospores. In some cases, hyphae penetrated cells and built a meshwork around plasmolyzed protoplasts, suggesting that the fungus either actively kills cells or senses cells undergoing endogenous programmed cell death. Seven days after inoculation, expression of barley BAX inhibitor-1 (HvBI-1), a gene capable of inhibiting plant cell death, was attenuated. Consistently, fungal proliferation was strongly inhibited in transgenic barley overexpressing GFP-tagged HvBI-1, which shows that P. indica requires host cell death for proliferation in differentiated barley roots. We suggest that the endophyte interferes with the host cell death program to form a mutualistic interaction with plants.

Apoptosis↗

Electron microscopic findings for micafungin-treated experimental pulmonary aspergillosis in mice.

We performed a scanning and transmission electron microscopic study on the efficacy of micafungin (MCFG) to understand what kind of damage MCFG causes to Aspergillus and to confirm its previously reported in vitro killing activity against Aspergillus in a mouse model of pulmonary aspergillosis. Aspergillus hyphae in MCFG-treated mice displayed hyphal burst, evidenced as either flattened or atrophied appearance and leakage of cellular contents after collapse of the cell wall. Thus, MCFG can induce the destruction of Aspergillus hyphae at the focus of infection. The results of the present study indicate that MCFG improves pulmonary aspergillosis due to lethal damage to Aspergillus hyphae. This action can effectively reduce the invasive ability of Aspergillus even though MCFG does not sterilize the fungal burden.

Animals↗

Reversible and permanent effects of the carbon sources and various antibiotics on the morphology and metabolic properties of Ustilago cynodontis cells.

The effects of various carbon sources and of antibiotics on the morphology of hypha cells of the fungus Ustilago cynodontis is described. Nonfermentable substrates promote readily reversible yeastlike colonies from hypha cells: all the hypha cells spread on these substrates give rise to yeastlike colonies that revert to the mycelial phenotype when transferred to glucose medium. Among the antibiotics tested, chloramphenicol (CAP) is found to promote, under certain circumstances, a long-lasting, even permanent modification on the morphology of the colonies: the colonies developed on CAP-glucose media are yeastlike, and a percentage of them give rise to colonies whose morphology remains yeastlike even on drug-free media: this effect is also obtained with cells cultivated in liquid medium. This permanent morphological modification is accompanied by a change of metabolic properties. Similar permanent effects are obtained with ethidium bromide, suggesting that mitochondrial functioning is involved in these modifications.

Anti-Bacterial Agents↗

Gastrointestinal basidiobolomycosis. Morphologic findings in a cluster of six cases.

We describe the histopathologic features of 6 cases of gastrointestinal basidiobolomycosis examined at 4 Phoenix, AZ, area hospitals during the last 4 years. Resected stomach and intestinal specimens were characterized by marked mural thickening with fibrosis, prominent tissue eosinophil infiltration and palisading granulomatous inflammation around pale fungal hyphae. In 2 cases, there was colonic perforation. Basidiobolus ranarum hyphae (associated with spore-like spherules in 4 cases) were identified within tissue sections; the irregularly branched, thin-walled, occasionally septated hyphae were typically surrounded by a thick eosinophilic cuff (Splendore-Hoeppli phenomenon). Although the histologic features of B ranarum are well described in the skin and subcutaneous tissue, gastrointestinal involvement has presented considerable diagnostic difficulty. Before the occurrence of this cluster of cases, intra-abdominal B ranarum infection has been reported only rarely.

Adult↗

Unusual histopathology of mucormycosis in acute myelogenous leukemia.

Mucormycosis is a mycotic disease caused by certain genera of the class Zygomycetes (order Mucorales). It is usually a rapidly progressive opportunistic infection characterized by vascular invasion, neutrophilic response, and intravascular thromboses. Entomophthoromycosis describes another disease caused by Zycomycetes, but of the order Entophthorales. In contrast to mucormycosis, entomophthoromycosis is not associated with underlying disease. Vascular invasion by the hyphae does not occur, and the hyphae are surrounded by eosinophilic sheathes. These sheathes are regarded as one form of the Splendore-Hoeppli phenomenon. The authors report an unusual case of mucormycosis in a severely pancytopenic patient dying of acute myelogenous leukemia. At autopsy, eosinophilic sheathes with radiating fibrils were seen about intravascular hyphae, simulating entomophthoromycosis. This pseudo-Splendore-Hoeppli phenomenon appears to be caused by adsorption of plasma and fibrin to the hyphal walls in the absence of both neutrophilic response and extensive fibrin clotting.

Aged↗

Inhibition of hyphal development in Trichophyton mentagrophytes arthroconidia by ketoconazole and miconazole.

In the presence of 0.5 mg/l ketoconazole and miconazole, arthroconidia from three isolates of Trichophyton mentagrophytes produced germ tubes, which continued to grow for 12 h before further elongation ceased. Lower concentrations reduced the rate of elongation, but did not halt the process. Higher concentrations had no greater effect than 0.5 mg/l. The inhibitory effect of ketoconazole was independent of time of addition of drug during the first six hours of germination. Ketoconazole inhibition was reversible after 12 h contact, whereas miconazole inhibition was not. Morphological effects of drug treatment were investigated by scanning electron microscopy. In the absence of imidazoles, hyphae were straight and regular; ketoconazole treated hyphae were short, irregular and branching; miconazole treatment produced extensive debris and spores forming within the hyphae were visible.

Ergosterol↗

A novel class of ectomycorrhiza-regulated cell wall polypeptides in Pisolithus tinctorius.

Development of the ectomycorrhizal symbiosis leads to the aggregation of fungal hyphae to form the mantle. To identify cell surface proteins involved in this developmental step, changes in the biosynthesis of fungal cell wall proteins were examined in Eucalyptus globulus-Pisolithus tinctorius ectomycorrhizas by two-dimensional polyacrylamide gel electrophoresis. Enhanced synthesis of several immunologically related fungal 31- and 32-kDa polypeptides, so-called symbiosis-regulated acidic polypeptides (SRAPs), was observed. Peptide sequences of SRAP32d were obtained after trypsin digestion. These peptides were found in the predicted sequence of six closely related fungal cDNAs coding for ectomycorrhiza up-regulated transcripts. The PtSRAP32 cDNAs represented about 10% of the differentially expressed cDNAs in ectomycorrhiza and are predicted to encode alanine-rich proteins of 28.2 kDa. There are no sequence homologies between SRAPs and previously identified proteins, but they contain the Arg-Gly-Asp (RGD) motif found in cell-adhesion proteins. SRAPs were observed on the hyphal surface by immunoelectron microscopy. They were also found in the host cell wall when P. tinctorius attached to the root surface. RNA blot analysis showed that the steady-state level of PtSRAP32 transcripts exhibited a drastic up-regulation when fungal hyphae form the mantle. These results suggest that SRAPs may form part of a cell-cell adhesion system needed for aggregation of hyphae in ectomycorrhizas.

Amino Acid Sequence↗

High level activation of vitamin B1 biosynthesis genes in haustoria of the rust fungus Uromyces fabae.

In the rust fungus Uromyces fabae, the transition from the early stages of host plant invasion toward parasitic growth is accompanied by the activation of many genes (PIGs = in planta induced genes). Two of them, PIG1 (= THI1) and PIG4 (= THI2), were found to be highly transcribed in haustoria, and are homologous to genes involved in thiamine (vitamin B1) biosynthesis in yeast. Their functional identity was confirmed by complementation of Schizosaccharomyces pombe thiamine auxotrophic thi3 (nmt1) and thi2 (nmt2) mutants, respectively. In contrast to thiamine biosynthesis genes of other fungi that are completely suppressed by thiamine, THI1 and THI2 expression was not affected by the addition of thiamine to rust hyphae grown either in vitro or in planta. Immunoblot analysis revealed decreasing amounts of THI1p in extracts from spores, germlings, and in vitro-grown infection structures with increasing time after inoculation. Immunofluorescence microscopy of rust-infected leaves detected high concentrations of THI1p in haustoria, and only low amounts in intercellular hyphae. In the sporulating mycelium, THI1p was found in the basal hyphae of the uredia, but not in the pedicels and only at very low levels in uredospores. These data indicate that the haustorium is an essential structure of the biotrophic rust mycelium not only for nutrient uptake but also for the biosynthesis of metabolites such as thiamine.

Amino Acid Sequence↗

Transhyphal electrical currents in fungi.

Representative mycelial fungi from the phycomycete, ascomycete and basidiomycete groups (Achlya bisexualis, Neurospora crassa, Aspergillus nidulans, Schizophyllum commune and Coprinus cinereus) all generated steady electrical currents around their hyphal tips; the generation of a transhyphal ion current may therefore be a universal characteristic of hyphal growth. As with all other tip growing organisms, positive current always entered apically and left distally; non-growing hyphae did not drive transcellular currents. The current density, measured approximately 30 micron from the membrane surface at the hyphal tips, varied between 0.05 and 0.06 microA cm-2 in different fungi and tended to be larger in wider, rapidly extending hyphae than in thinner, slow growing hyphae. The possibility that these currents serve to localize growth at the apex is discussed.

Electricity↗

Glycogen and trehalose accumulation during colony development in Streptomyces antibioticus.

Streptomyces antibioticus accumulated glycogen and trehalose in a characteristic way during growth on solid medium. Glycogen storage in the substrate mycelium took place during development of the aerial mycelium. The concentration of nitrogen source in the culture medium influenced the time at which accumulation started as well as the maximum levels of polysaccharide stored. Degradation of these glycogen reserves was observed near the beginning of sporulation. The onset of sporogenesis was always accompanied by a new accumulation of glycogen in sporulating hyphae. During spore maturation the accumulated polysaccharide was degraded. No glycogen was observed in aerial non-sporulating hyphae or in mature spores. Trehalose was detected during all phases of colony development. A preferential accumulation was found in aerial hyphae and spores, where it reached levels up to 12% of the cell dry weight. The possible roles of both carbohydrates in the developmental cycle of Streptomyces are discussed.

Disaccharides↗

Localization of growth and secretion of proteins in Aspergillus niger.

Hyphal growth and secretion of proteins in Aspergillus niger were studied using a new method of culturing the fungus between perforated membranes which allows visualization of both parameters. At the colony level the sites of occurrence of growth and general protein secretion were correlated. In 4-d-old colonies both growth and secretion were localized at the periphery of the colony, whereas in a 5-d-old colony growth and secretion also occurred in a more central zone of the colony where conidiophore differentiation was observed. However, in both cases glucoamylase secretion was mainly detected at the periphery of the colonies. At the hyphal level immunogold labelling showed glucoamylase secretion at the tips of leading hyphae only. Microautoradiography after labelling with N-acetylglucosamine showed that these hyphae were probably all growing. Glucoamylase secretion could not be demonstrated immediately after a temperature shock which stopped growth. These results indicate that glucoamylase secretion is located at the tips of growing hyphae only.

Acetylglucosamine↗

In situ localization of the secretion of lignin peroxidases in colonies of Phanerochaete chrysosporium using a sandwiched mode of culture.

Protein secretion and growth were investigated in Phanerochaete chrysosporium by using cultures sandwiched between perforated polycarbonate membranes. Labelling of colonies with radioactive N-acetylglucosamine and L-methionine indicated a close correlation between growth and general protein secretion, even in a central area of the colony secreting the idiophase enzymes lignin peroxidase (LiP) and manganese-dependent lignin peroxidase (MnP). Comparison of the sites of release into the medium of newly synthesized proteins and immuno-detected lignin peroxidases suggested that diffusion of the enzymes from the walls was a limiting step in the release of peroxidases into the medium. Microautoradiography of colonies exposed to N-acetyl[3H]glucosamine revealed the apical growth of thin hyphae and branches (4 to 5 microns diameter on average) in the central secreting area. These secondary hyphae showed peroxidase activity and reacted with lignin peroxidase antibodies. Although it was not possible to directly visualize secretion at hyphal tips, the results suggest that peroxidases (LiP and MnP) are initially secreted at the apex of secondary growing hyphae and later slowly released into the surrounding medium.

Fungi↗

Regulation of chitin synthesis during dimorphic growth of Candida albicans.

Candida albicans has three genes encoding chitin synthase enzymes. In wild-type strains, the expression of CHS2 and CHS3 peaked 1-2 h after the induction of hyphal growth, whilst mRNA levels in a non-germinative strain, CA2, remained low under the same conditions. CHS1 gene expression did not peak during germ tube formation but remained at low levels in both yeast and hyphal growth. The pattern of gene expression did not predict the changes in measured chitin synthase activities or changes in chitin content during dimorphic transition. Chitin synthase activity increased steadily, and did not peak shortly after germ tube induction, and activity profiles were similar in germ-tube-competent and germ-tube-negative strains. The phenotype of a delta chs2 null mutant suggested that CHS2 encoded the major enzyme activity in vitro and was largely responsible for elevated chitin synthase activities in microsomal preparations from hyphal cells compared to yeast cells. However, CaChs3p was responsible for synthesis of most chitin in both yeast and hyphae. Three independent chitin assays gave markedly different estimates of the relative chitin content of yeast and hyphae and wild-type and chs mutants. Only one of the methods gave a significantly higher chitin content for hyphal compared to yeast cell walls and a lower chitin content for hyphae of the delta chs2 null mutant compared to the parental strain.

Candida albicans↗

Association of early sporulation genes with suggested developmental decision points in Streptomyces coelicolor A3(2).

Cytological analysis of a series of Streptomyces coelicolor A3(2) mutants with disruptions of early sporulation (whi, for white aerial mycelium) genes in an isogenic background has provided new information about the role of whiH, and confirmed and extended previous knowledge about whiG, whiA and whiB. The characteristic straight aerial hyphae of whiG mutants contained normally spaced vegetative-like septa, while mutants in whiA or whiB had abnormally long and coiled aerial hyphae almost devoid of septation. whiG, whiA and whiB were all absolutely required for sporulation septation, and for all visible signs of nucleoid condensation and partitioning and other changes associated with later stages of sporulation. On the other hand, whiH appeared to enhance low basal levels of these processes. Thus, whiH mutant aerial hyphae were divided into loosely coiled fragments of variable sizes by what appeared to be a few sporulation septa. These fragments showed some spore-like characteristics and contained condensed and aberrantly partitioned nucleoids. whiG, whiA and whiB were epistatic to whiH on the criterion that they prevented such fragments from forming in double mutants. These spore-like features and the synthesis of clearly detectable levels of the whiE-directed grey spore pigment were not due to any residual activity of previously studied whiH alleles since they were retained by a constructed whiH null mutant. A model is presented that explains the mutant phenotypes by proposing two early developmental decision points involved in commitment to sporulation septation, one requiring whiG and the other requiring whiA and whiB.

Alleles↗

Macromolecular syntheses during the cell cycles of yeast and hyphal phases of Candida albicans.

Synchronous cultures of yeast and hyphal phases of Candida albicans showed exponential increases in RNA content and stepwise exponential increases in DNA content. The periods of DNA synthesis in the two phases coincided with one another and with the budding peaks of the yeast phase. Hyphae grown in synchronous cultures also showed an exponential increase in length. The hyphal phase was therefore normal. Hyphal nuclear division occurred after hyphal DNA synthesis. Germination was a unique event for a hypha and unlike yeast bud formation, preceded the first period of DNA synthesis. The exponential increase in RNA and DNA in asynchronous cultures of hyphae in serum paralleled the exponential increase in the numbers of cells in asynchronous cultures of yeasts in defined media. Thus there are no factors in serum which inhibit the normal exponential growth of C. albicans.

Candida albicans↗

A scanning electronmicroscopy investigation of the structure of colonies of different morphologies produced by phenotypic switching of Candida albicans.

Variant colony formation by Candida albicans has been described and the phenomenon of phenotypic switching has been studied extensively. Whereas the microstructure of non-variant colonies has been investigated by scanning electronmicroscopy (SEM), the relationship between switched variant colonies and microstructure has not been described. The object of this study was to investigate and compared by SEM the microstructure of the normal colony type and five common variant colony types of C. albicans and to determine whether a pattern of dimorphic growth could account for the characteristic colony morphologies. A general relationship between colony type and structure was observed; smooth colonies consisted entirely of blastospores whereas regular, irregular-wrinkled and semi-rough colonies consisted of different proportion of true hyphae and blastospores. Regular extreme-jagged shaped colonies consisted of an almost pure culture of pseudohyphae, and colonies producing aerial hyphae were composed of pseudo- and true hyphae, as well as blastospores. These results show a clear relationship between colony morphology and development of particular cell types.

Candida albicans↗

Enzyme inactivation related to a hyperoxidant state during conidiation of Neurospora crassa.

The conidiation process of Neurospora crassa is characterized by three morphogenetic steps: hyphal adhesion, aerial hyphal formation, and production of conidia. Previous data indicated the occurrence of a hyperoxidant state at the onset of all three morphogenetic steps. Because glutamine synthetase (GS) and the biosynthetic glutamate dehydrogenase [GDH(NADP)] enzymes are susceptible to inactivation by reactive oxygen species, we followed these enzyme activities during conidiation and under different physiological conditions and related them to the hyperoxidant states and morphogenesis. Loss of GS activity occurred prior to all three morphogenetic steps, coinciding with an increase in total protein oxidation. Oxidized GS polypeptides were detected during hyphal adhesion. Loss of GDH(NADP) activity also occurred during hyphal adhesion and before aerial hyphal formation; the enzyme polypeptide and activity decreased in the adhered hyphae to low values and no GDH(NADP) was detected in aerial hyphae. The catabolic GDH [GDH(NAD)] behaved in an opposite manner, increasing its activity during hyphal adhesion and aerial hyphae development. These results are discussed with regard to cell differentiation and the conidiation process in N. crassa.

Glutamate Dehydrogenase↗