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PubMed1995

Effects of granulocyte colony-stimulating factor and interferon-gamma on antifungal activity of human polymorphonuclear neutrophils against pseudohyphae of different medically important Candida species.

Polymorphonuclear neutrophils (PMNs) are the major host defense against pseudohyphae, the invasive form of Candida species. We studied the effects of granulocyte colony-stimulating factor (G-CSF) and interferon-gamma (IFN-gamma) on the PMN-induced damage of pseudo-hyphae of Candida albicans, Candida tropicalis, and Candida parapsilosis in vitro by using two antifungal assays: a modified limiting dilution assay and a colorimetric metabolic 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. PMNs from healthy volunteers were incubated with either G-CSF (100-10,000 U/ml) or IFN-gamma (10-5000 U/ml) or buffer at 37 degrees C for 90 min and their capacity to damage nonopsonized pseudohyphae was then measured. C. tropicalis appeared to be the most susceptible species, whereas C. parapsilosis showed the highest rate of resistance to PMN damage. G-CSF (500-10,000 U/ml) and IFN-gamma (100-1000 U/ml) enhanced the antifungal activity of PMNs against C. albicans pseudo-hyphae (P < .01 and P < .05). Among the others, G-CSF enhanced PMN-induced damage of C. parapsilosis at concentrations 500-10,000 U/ml (P < .05), whereas it enhanced damage of C. tropicalis only at 10,000 U/ml (P < .01). IFN-gamma (100-1000 U/ml)-primed PMNs also caused augmented damage of C. parapsilosis (P < .05) but not of C. tropicalis at the same concentrations. Species-dependent differences exist in the responses of PMNs to Candida pseudohyphae and G-CSF as well as IFN-gamma are important immunomodulators of phagocytic host defenses against them.

Adult↗
PubMed1986

DNA replication is not restricted to specific regions in young vegetative Streptomyces mycelia.

In order to determine the localization of DNA-synthesis in Streptomyces granaticolor and Streptomyces hygroscopicus, mycelia (growing either on agar or in liquid medium) were pulse-labelled with 3H-thymidine and prepared for autoradiography. The distribution of silver grains showed no regions of preferential incorporation of 3H-thymidine in mycelia up 300 micron in length. Since mycelia grow by apical elongation of hyphae, the frequency of silver grains was quantitatively analysed along individual main hyphase. No significant difference of labelling was found within zones of different age up to a distance of 80 micron from the hyphal tip. Also, the very youngest part of the hyphae enclosing only the most apically situated nucleoid did not show any deviation from the average frequency of silver grains.

Autoradiography↗
PubMed1990

Qualitative and quantitative changes in beta-1,4-glucosidase accompanying growth of Aspergillus nidulans.

A comparative study of the wild type A. nidulans and a mutant strain aco-T69 (lacking conidia and cleistothecia) revealed the better production of beta-1,4-glucosidase in the former. The relative distribution of the enzyme levels in various morphological structures viz. somatic hyphae, spores and cleistothecia also showed a variation. Highest specific activity was found in the cleistothecial extracts. The electrophoretic analysis of the wild type strain demonstrated the presence of three isoenzymes of beta-1,4-glucosidase i.e. beta-GLU I, beta-GLU II and beta-GLU III, the number of which varies from one to three during the growth of the organism. The parallel study of the mutant strain showed complete absence of beta-GLU II. All three forms were present in the somatic hyphae and cleistothecial extracts while spore extracts depicted the absence of beta-GLU III in the wild type.

Aspergillus↗
PubMed1992

Location of branches within the apical hyphal region of Streptomyces granaticolor mycelia.

The location of branches (ramifications) within the apical hyphal region of Streptomyces granaticolor mycelia was studied in dependence on the specific growth rate by using batch and chemostat cultures. The site where a new branch emerged was correlated to the neighboured elongation sites (e-sites) as well as to the septae. In dependence on the growth rate the distance from a newly formed branch (i.e. e-site) to the apical e-site (tip of the main hypha) changed from 12 to 44 microns and to the e-site on the tip of the preceding neighboured branch from 12 to 27 microns. Thus, except for the slowest grown culture, the new branching site in the apical hyphal region did not represent the midst between the two preexisting e-sites. Comparing branching and septation sites, the data indicated a close correlation between them, if the respective mean values of whole populations were determined. But regarding individual hyphae, only 4-39%--depending on the growth rate--of the apical branches were directly neighboured to a septum. The remaining percentage was situated at one of several potential branching sites, the number of which per subapical cell corresponded to the number of nucleoids.

Cell Division↗
PubMed1992

Effect of temperature and medium composition on mycelial growth of Streptomyces tendae in submerged culture.

To investigate the influence of culture conditions on growth of filamentous bacteria in submerged culture, S. tendae was cultivated at various temperatures and in various media. For this purpose, a temperature-controlled growth chamber was constructed, which allows the cultivation of filamentous bacteria and fungi oxygen saturated medium. To observe the development of mycelia emerging from spores, this growth chamber was mounted on a microscope stage and series of images were analyzed by an image processing system. Growth kinetics obtained in liquid culture were identical to those determined on solid media. Specific growth rate and apical extension rates of individual hyphae seemed to be higher than those observed on solid media, due to a better supply of medium in submerged culture. A two-fold increase in specific growth rate, mean apical extension rate and branching rate was observed when the temperature was increased from 27 degrees C to 37 degrees C, therefore, the length of the hyphal growth unit was not changed. Growth in synthetic media used here was considerably slower compared with complex medium. A decrease in glucose concentration from 40.0 g l-1 to 1.0 g l-1 resulted in an increase in branching rate and specific growth rate, while apical extension rate of individual hyphae was unchanged.

Bacteriological Techniques↗
PubMed1982

Changes in cytoplasmic ultrastructure during submerged cultivation of a peptide alkaloids-producing strain of Claviceps purpurea (Fr.) Tul.

A strain of Claviceps purpurea, designated Pepty 695/S produces ergotoxine alkaloids under particular conditions of fermentation. The onset of alkaloid synthesis occurs around the second day of cultivation. Alkaloid formation is connected with morphological and ultrastructural changes. In the first 3-5 days of cultivation short thickened, septated hyphae, organized in plectenchymatic pellets as well as large single cells are formed. The hyphae are ultrastructurally characterized by increasing number of lipid droplets, deposits of glycogen and by extended ER membranes, which apparently may form numerous vesicles. The correlations between lipid and alkaloid synthesis are discussed.

Cell Nucleus↗
PubMed2002

Mode of action of acibenzolar-S-methyl against sheath blight of rice, caused by Rhizoctonia solani Kühn.

The mode of action of acibenzolar-S-methyl (BTH) was investigated against sheath blight of rice and its pathogen, Rhizoctonia solani. BTH exhibited limited fungitoxicity against R solani, in the form of reduced mycelial growth, hyphal browning and sclerotia formation. Parasite fitness of mycelia and sclerotia formed on BTH-amended media was also reduced. When applied as soil drench or foliar spray, BTH inhibited both disease development on inoculated sheaths and its spread to the younger sheaths. The degree of protection against sheath blight increased with increase in duration between BTH application and inoculation. The curative effect of BTH was poor. When applied through roots a protective effect of BTH was visible even with only a 1-h interval between application and inoculation. However, in the case of foliar application, protective effect was recorded only when the gap between application and inoculation was 24 h. BTH reduced the frequency of penetration by R solani, colonization of host tissue and spread of the hyphae from primary lesions to form secondary lesions. BTH induced swelling of hyphal tips on the sheath surface, formation of papillae, browning of penetrated epidermal cells and degeneration of intra-cellular hyphae colonizing epidermal and mesophyll cells. Therefore, the protective effect of BTH against sheath blight was due to combination of its host defence-inducing activity and its adverse effect on growth and vigor (parasite fitness) of the pathogen.

Dose-Response Relationship, Drug↗
PubMed2004

Determination of specific volatile organic compounds synthesised during Tuber borchii fruit body development by solid-phase microextraction and gas chromatography/mass spectrometry.

Fruit body development is a particular phase of the Tuber life cycle, characterised by the aggregation of different types of hyphae, i.e., vegetative hyphal cells and highly specialised reproductive hyphae (asci). In order to identify the volatile organic compounds (VOCs) produced in different stages of the Tuber borchii ripening fruit body, solid-phase microextraction with gas chromatography and mass spectrometry was used. The volatile organic compounds were extracted using a DVB/CAR/PDMS 50/30 microm fiber placed for 10 min at room temperature in the truffle headspace. The results obtained reveal 49 compounds each of which was present only in a particular stage of maturation. 1-octen-3-ol, aromadendrene, alpha-farnesene and other terpenoid compounds were of particular interest, and their possible biological roles are discussed. The production of aromadendrene in the completely unripe fruit body suggests the existence of communication events in the early stage of ascomata formation between the fungus and the host plant. alpha-Farnesene could represent a chemotactic attractant to saprophytic organisms in order to disperse the fungal spores in the environment. The identification of the VOCs produced by truffles during their maturation could give information about the processes underlying this phase of Tuber life cycle.

Ascomycota↗
PubMedDate not supplied

Scanning electron microscopy of some endophytic streptomycetes in snakevine--Kennedia nigricans.

Soils of all types and locations have generally served as the major sources of streptomycetous bacteria. These organisms are the source of nearly 80% of the world's antibiotics. Now, it is realized that Streptomyces spp. (within the group of prokaryotic filamentous bacteria known as actinomycetes) can exist as endophytes within the interstices of some higher plants. While it is sometimes possible to isolate one or two different streptomycetes from certain plants, most plants are free of these organisms. However, the snakevine (Kennedia nigricans) of the Northern Territory of Australia has yielded at least 39 different endophytic actinomycetes (95% of them being Streptomyces spp.) Most of these isolates possessed no detectable antibiotic properties, while at least seven had antibacterial and antifungal activities. Examination of eight selected cultures by scanning electron microscopy (SEM) as well as environmental scanning electron microcopy (FEI ESEM FEG) (FEI Company, Hillsobro, Ore., USA) revealed unusual patterns, structures, and features of the spores and hyphae of these microorganisms. For instance, as revealed by ESEM FEG for the first time, it has become obvious that extremely fine hair-like structures (average 25-49 nm with gold-coated specimens) exist on the spores and hyphae of some endophytic streptomycetes. The biological purpose of these hair-like protrusions is unknown. Both SEM and ESEM FEG can be effectively used as tools in identification and elucidation of the biology of these organisms. In addition, unusual colony morphology, observed with the unaided eye can very easily be used to distinguish some of these isolates since characteristic donut and pseudo-horn shaped colonies appeared in culture.

Fabaceae↗
PubMed2005

Characterization of thiol-specific antioxidant 1 (TSA1) of Candida albicans.

We previously identified several proteins that are differentially expressed in pathogenic hyphae by comparing protein profiles of yeast and hyphae of Candida albicans. One of these, thiol-specific antioxidant 1 (TSA1), attracted our attention because it may play some roles in surviving an unfavourable oxidative environment created by host cells. Two alleles of the C. albicans TSA1 (CaTSA1) gene are located in opposite orientation on the same chromosome. Using PCR-directed disruption cassettes and URA-Blaster, a series of deletion mutants that lack one to four copies were constructed to examine the functions of CaTSA1. Northern and Western analyses showed that both the transcript and protein products of CaTSA1 decreased proportionally to the disrupted copy number and were completely absent in the null mutant, indicating that all four TSA1 copies are equally functional at the transcriptional level. Intracellular H2O2 increased by an order of magnitude in deletion mutants lacking three to four copies, suggesting that CaTsa1p is not a redundant H2O2 scavenger. CaTsa1p was indispensable for yeast-to-hyphal transition when C. albicans was cultured under oxidative stress. The level of its oxidized form increased approximately five-fold in hyphal cells, whereas that of the reduced form increased two-fold compared to yeast cells. The ratio of oxidized to reduced form was increased three-fold in hyphal cells. This overall increase was found to be controlled at the post-transcriptional level. Interestingly, CaTsa1p is translocated to the nucleus of hyphal cells. These findings may be of biological significance for differentiation and pathogenicity.

Candida albicans↗
PubMed1994

Gravitropism of the basidiomycete Flammulina velutipes: morphological and physiological aspects of the graviresponse.

The fruiting body of the basidiomycete Flammulina velutipes shows a distinct negative gravitropic response. Maturing fruiting bodies in the rapid elongation phase become graviresponsive with basidiospore differentiation. Lateral gravistimulation by horizontal arrangement of the fruiting body results in unilateral growth regulation. Elongation in the upper Stipe side decreases to 40% during gravitropic reorientation of the fruiting body. Overshooting of the gravitropic response during reorientation is precisely regulated. The graviresponsiveness is concentrated to the apical area of the stipe, the transition zone between pileus and stipe, which features a prominent elongation capability. The small size and low vacuolization of the transition zone hyphae compared with differentiated basal stipe hyphae correspond with this physiological function on the light and electron microscopical levels. Curvature experiments using intact and explanted fruiting bodies demonstrated the graviperceptive role of the transition zone. The excision of various amounts of pilear tissue, even the disruption of the whole pileus, had no severe effect on gravitropic curvature, until the transition zone was damaged. Removal of the transition zone resulted in a dramatic loss of graviresponse, whereas the decrease of elongation was less drastic.

Basidiomycota↗
PubMed1998

Hydrophobins and repellents: proteins with fundamental roles in fungal morphogenesis.

Fungal hydrophobins are secreted proteins which react to interfaces between fungal cell walls and the air or between fungal cell walls and solid surfaces. They have been shown to be important in many morphogenetic processes, including sporulation, fruit body development, and infection structure formation. Hydrophobins form hydrophobic surface layers by self-assembly of secreted protein monomers in response to the environment. This process results in amphipathic polymers of interwoven rodlets on surfaces of fungal aerial structures and hyphal aggregations. Hydrophobin self-assembly is also involved in attachment of hyphae to hydrophobic surfaces and this may act as a conformational cue for certain developmental processes. Although hydrophobins appear to be ubiquitous among fungal taxa, a second class of fungal protein with very different biochemical characteristics could fulfill a similar role. These proteins, called repellents, have been identified in only one fungal species so far, but clearly help to make aerial hyphae hydrophobic. The functional similarities between hydrophobins and repellents highlight the importance of aerial development to the fungal lifestyle.

Amino Acid Sequence↗
PubMedDate not supplied

An improved method for affinity probe localization in whole cells of filamentous fungi.

The fungal cell wall, though phylogenetically variable, acts universally as a potent barrier to probing intracellular structures. Thus, the use of high-molecular-weight probes such as antibodies and lectins has proven a formidable challenge. We have devised a preparative method for use with various affinity probes that can be applied to a broad spectrum of filamentous fungal species and used for imaging whole cells. In this study, confocal imaging of whole-mount fungal hyphae after freeze substitution, methacrylate embedment/de-embedment, and infiltration with affinity probes has yielded remarkably improved renderings of the three-dimensional distribution of both microtubules (using antibodies against both alpha- and beta-tubulin) and concanavalin A binding sites. Using this protocol we have been able to document: (1) the three-dimensional distribution of microtubules in all regions of hyphae, (2) the presence of apparent foci for cytoplasmic microtubules, (3) persistent cytoplasmic microtubules during mitosis, and (4) a three-dimensional view of many compartments of the endomembrane system including Golgi-equivalent organelles and apical vesicles. The last result represents the first direct confirmation of apical vesicles comprising the Spitzenkörper.

Affinity Labels↗
PubMedDate not supplied

Colletotrichum: A model genus for studies on pathology and fungal-plant interactions.

Species of Colletotrichum use diverse strategies for invading host tissue, ranging from intracellular hemibiotrophy to subcuticular intramural necrotrophy. In addition, these pathogens develop a series of specialized infection structures, including germ tubes, appressoria, intracellular hyphae, and secondary necrotrophic hyphae. Colletotrichum species provide excellent models for studying the molecular basis of infection structure differentiation and fungal-plant interactions. In this review we cover the various stages of the infection processes of Colletotrichum species, including spore adhesion and germination, germ tube and appressorium differentiation and functions, and biotrophic and necrotrophic development. The contribution of molecular, biochemical, and immunological approaches to the identification of genes and proteins relevant to each stage of fungal development will be considered. As well as reviewing results from several groups, we also describe our own work on the hemibiotrophic pathogen, C. lindemuthianum.

Colletotrichum↗
PubMed2000

A putative spectrin-containing membrane skeleton in hyphal tips of Neurospora crassa.

The apical plasma membrane (PM) is important in hyphal tip growth, where it may regulate tip extensibility via its association with an appropriate membrane skeleton (MS). By cell fractionation and immunocytochemistry we show that proteins with characteristics of actin, spectrin, and integrin are associated in a MS-like manner with the PM of Neurospora crassa hyphae. The spectrin-like protein in particular is highly concentrated at the PM in the region of maximum apical expansion. This protein shares with other spectrins immunoreactivity, molecular weight, PM association, and actin binding capacity. Its distribution in hyphae suggests that it is a dominant component of the MS in true fungi and is critical to hyphal tip growth.

Cell Fractionation↗
PubMed2001

Monoclonal antibodies incorporated into Neotyphodium coenophialum fungal cultures: inhibition of fungal growth and stability of antibodies.

Three monoclonal antibodies (mAbs) produced against proteins from the tall fescue (Festuca arundinacea Schreb.) fungal endophyte Neotyphodium coenophialum hybridize exclusively to a fungal protein under denaturing conditions. The protein is approximately 88 kDa in size. These mAbs were individually incorporated into liquid medium to determine their effects on fungal growth in culture. Neotyphodium-specific mAbs inhibited fungal growth for the duration of the study. Fungal cultures grown in the presence of Neotyphodium-naive mAbs or in the absence of all mAbs grew unimpeded. Bright-field microscopy and immunohistochemical studies of cultures containing Neotyphodium-specific mAbs revealed a change in mycelia morphology with clumps exhibiting a gelatinous matrix containing sparse hyphae, while cultures receiving Neotyphodium-naive mAbs in medium demonstrated unrestricted growth with overlapping and branched hyphae. In liquid culture devoid of fungal isolates, mAbs were stable and detected throughout the experiment, but were below threshold detection levels within 15 min following inclusion in liquid cultures containing Neotyphodium spp., indicating rapid binding to fungal mycelia. Monoclonal antibodies may provide a new method to help control plant pathogenic fungi where chemical or genetic means are not feasible.

Antibodies, Fungal↗
PubMed1999

Interaction of catenaria anguillulae with romanomermis culicivorax

The development of Catenaria anguillulae, at 27 degrees C, in live and heat-immobilized Romanomermis culicivorax was studied at the ultrastructural level. C. anguillulae zoospores infected eggs, preparasites, and postparasites. The zoospores attached predominately to the head of preparasites but in postparasites attachment was primarily on the attenuated tail region. Zoospore attachment usually occurred within 4 h but at high zoospore densities attachment occurred within 30 min. Infection hyphae penetrated the cuticle and developed sporangial swellings within the nematode tissues and pseudocoelom. Sporangia produced exit tubes within 48 h. Those regions of the J3 and J4 nematodes infected by C. anguillulae became immobile whereas uninfected regions remained mobile. The J2 infected nematodes usually died within 1-2 days. Tissues adjacent to C. anguillulae hyphae or developing sporangia appeared to be relatively unaffected and ultrastructurally normal. The lack of an apparent host response in the nematode suggested either a lack of hypersensitivity or a long evolutionary host-parasite relationship. Copyright 1999 Academic Press.

Journal Article↗
PubMed1995

Cell wall synthesis in cotton roots after infection with Fusarium oxysporum. The deposition of callose, arabinogalactans, xyloglucans, and pectic components into walls, wall appositions, cell plates and plasmodesmata.

Fusarium oxysporum f. sp. vasinfectum penetration hyphae infect living cells in the meristematic zone of cotton (Gossypium barbadense L.) roots. We characterized wall modifications induced by the fungus during infection of the protodermis using antibodies against callose, arabinogalactan-proteins, xyloglucan, pectin, polygalacturonic acid and rhamnogalacturonan I in high-pressure frozen, freeze-substituted root tissue. Using quantitative immunogold labelling we compared the cell walls before and after hyphal contact, cell plates with plasmodesmata during cytokinesis, and wall appositions induced by fungal contact. In the already-existing wall, fungal contact induced only minor modifications such as an increase of xyloglucan epitopes. Wall appositions mostly exhibited epitopes similar to the cell plate except that wall appositions had a much higher callose content. This study shows that wall appositions induced by Fusarium oxysporum hyphae are the result of normal cell wall synthesis and the addition of large amounts of callose. The appositions do not stop fungal growth.

Cell Wall↗