cAMP promotes hyphal branching in <Emphasis Type="Italic">Mucor globosus</Emphasis>

The growth pattern of Mucor globosus cultured on a medium with or without cyclic adenosine monophosphate (cAMP) was examined. Branching remarkably increased in a mycelium grown on an agar medium containing cAMP. In submerged culture containing cAMP, some sporangiospores grew spherically and formed yeast-like cells, and others showed hyphal growth. These hyphae showed septation and swelling and formed spore-like structures. When these hyphae were transferred to cAMP-free medium, a germ tube emerged from each compartment. These results show that cAMP has two different effects on the development of hyphae: one is the promotion of branching, and the other is the suppression of polarized growth.     

In vitro production of specialized reproductive torulose hyphae byFrankia strain ORS 021001 isolated fromCasuarina junghuhniana root nodules

Summary AFrankia strain (ORS 021001) isolated fromCasuarina junghuhniana root nodules was shown to produce four type of structuresin vitro: vegetative hyphae, sporangiospores within sporangia, N₂-fixing vesicles, and a fourth type of structure which is described in detail in this report. Structures of this latter type which we propose to call reproductive torulose hyphae: (RTH) result from enlargement and multiple segmentation of vegetative hyphae into torulose chaions of spore-like cells. RTH differ from sporangia in three major aspects: morphology, morphogenesis and outgrowth. RTH play an important role in survival and reproduction ofFrankia strain ORS 021001. Adding activated charcoal to the nutrient medium promotes the formation ofFrankia colonies orginating from RTH.     

Production of thermostable and neutral glucoamylase using immobilized <Emphasis Type="Italic">Thermomucor indicae-seudaticae</Emphasis>

Thermomucor indicae-seudaticae was immobilized in alginate, κ-carrageenan, agarose, agar, polyacrylamide and loofah (Luffa cylindrica) sponge (as such or coated with alginate/starch/Emerson YpSs agar), and used for the production of glucoamylase in submerged fermentation. The mycelium developed from alginate-immobilized sporangiospores secreted higher glucoamylase titres (22.7 U ml⁻¹) than those immobilized in other gel matrices and the freely growing mycelial pellets (18.5 U ml⁻¹). Loofah network provided a good support for mycelial growth, but the enzyme production was lower than that attained with alginate beads. Glucoamylase production increased with inoculum density and the optimum levels were achieved when 40 calcium alginate beads (∼5 × 10⁶ immobilized spores) were used to inoculate 50 ml production medium. The alginate bead inoculum displayed high storage stability at 4°C and produced comparable enzyme titres up to 120 days. The glucoamylase production by hyphae emerged from the immobilized sporangiospores was almost stable over eight batches of repeated fermentation. Scanning electron micrographs of alginate beads, after batch fermentation, revealed extensive mycelial growth inside and around the beads.     

Pinnaticoemansia, a new genus of Kickxellales, with a revised key to the genera of Kickxellales

Pinnaticoemansia coronantispora gen. sp. nov. (Kickxellaceae, Kickxellales) is described and illustrated. This species is characterized by imparipinnate sporocladia (sporocladia arranged in pairs), sporangiospores with a three-lobed corona, and the germination pattern of sporangiospores with downward hyphal growth and repeated dichotomy. The key to the genera of Kickxellales by Kurihara et al. (2001) is revised based on the observation of the germination pattern of this fungus in comparison with that of Asellariales.     

Über die Struktur und Teilung der Kerne von Geotrichum candidum und Geotrichum magnusii

Summary The nuclei in germinating spores and growing hyphae ofGeotrichum magnusii andG. candidum have been examined during life and in fixed and stained preparations.The spores and the cells of the hyphae are multinucleate. The nuclei consist of a dense Feulgen-negative nucleolus surrounded by a less dense shell of Feulgen-positive particles. No membrane was seen at the margin of either living or fixed and stained nuclei. The mass of chromatin and the nucleolus divide at the same time by elongation followed by constriction. Chromosomes could not be detected in either resting or dividing nuclei.

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Cyclic adenosine 3′,5′-monophosphate and germination of sporangiospores from the fungus Mucor

Cyclic adenosine 3,5-monophosphate (cAMP) metabolism was examined in germinating sporangiospores of Mucor genevensis and Mucor mucedo. Exogenous cAMP prevented normal hyphal development from sporangiospores. Internal pools of cAMP fluctuated profoundly during development. Spherical growth of the spores was characterized by large pools of cAMP whereas germ tube emergence and hyphal elongation were characterized by small pools of cAMP. These observations suggest a possible role for cAMP in sporangiospore germination. Adenylate cyclase activities fluctuated significantly during germination with maximum values attained during spherical growth. In contrast, cAMP phosphodiesterase activities remained constant throughout germination. Internal cAMP levels may therefore be regulated by adjustment of adenylate cyclase activities. The binding of cAMP by soluble cell proteins was measured. cAMP-binding activity changed greatly during germination. Dormant and spherically growing spores possessed the highest activities. Developing hyphae contained the lowest activities. Use of the photoaffinity label, 8-azido-[^32P]cAMP, in conjunction with sodium dodecyl sulfate-polyacrylamide-gel electrophoresis allowed the identification of a small population of morphogenetic-stage-specific proteins which bind cAMP and may be of regulatory significance to development.     

Bioprospecting for novel oxylipins in fungi: The presence of 3-hydroxy oxylipins in Pilobolus

As previously found in various members of the Mucorales, 3-hydroxy oxylipins in Mucor genevensis are associated with the sporangia, i.e. mainly the columella structure and between aggregating sporangiospores. To determine if this phenomenon is also true in distantly related members, the mucoralean fungus Pilobolus was examined. This fungus is characterized by relatively large sub sporangial-columella structures which actively eject sporangia in a sticky liquid for attachment onto herbage surrounding its growth medium – in this case horse dung. Strikingly, this fungus produced a novel oxylipin i.e. a 3-hydroxy monounsaturated fatty acid, possibly a nonenoic acid, which is mainly associated with the sub sporangial-columella structure and aggregating sporangiospores. The specificity of the antibody against 3-hydroxy oxylipins used in immunofluorescence mapping of the mucoralean fungi, was further confirmed in the yeast, Saccharomycopsis malanga which produces 3-hydroxy palmitate in crystal form. These crystals occur between aggregating yeast cells. On the basis of the available data, we hypothesize that 3-hydroxy oxylipins probably function as adhesives, attaching fungal cells to each other or to other surfaces through entropic based hydrophobic forces and/or hydrogen bonds.     

Wall fibrils ingerminating sporangiospores of Gilbertella persicaria (Mucorales)

Summary In germinated sporangiospores of Gilbertella persicaria, negatively contrasted fibrils, 20–70 Å diam, are seen in thin sections of the inner vegetative wall that is continuous with the germ tube wall. The fibrils are randomly oriented in a loose network in this wall and in the germ tube wall. Germ tubes have an additional surface layer of fine, positively contrasted fibrils which appear as a nap-like coating on the hyphae. Patterns of wall fibril orientation are not revealed by transverse sections of spore and germ tube walls, whereas oblique and tangential sections are favorable for examining cell wall architecture in situ. Staining patterns show textural and compositional differences among various wall layers.     

Behaviour of the hyphae of<Emphasis Type="Italic"> Laccaria laccata</Emphasis> in the presence of<Emphasis Type="Italic"> Trichoderma harzianum</Emphasis> in vitro

The growth rate and the behaviour of Laccaria laccata and Trichoderma harzianum hyphae in co-culture and in the rhizosphere of 3-month-old Pinus sylvestris seedlings grown in vitro were investigated. In the interaction zone, hyphae of L. laccata became more pigmented and formed short branches growing towards the hyphae of the saprobic fungus, coiled around them and penetrated sporadically. Vacuolated hyphae of T. harzianum showed protoplasm granulation and breaks in walls followed by release of protoplasts. In the rhizosphere, the mantle hyphae of L. laccata showed a tendency to surround conidia of T. harzianum. No obvious penetration of the conidial walls by the hyphae of the mycorrhizal fungus was observed by scanning electron microscopy. Instead, in rare cases, the hyphae of L. laccata showed marked wrinkles, and a partial degradation of a mucilaginous material covering the mantle appeared to occur.     

Dormant Cells in the Developmental Cycle of Blakeslea trispora: Distinct Patterns of the Lipid and Carbohydrate Composition

We revealed differences in lipid and carbohydrate composition between cells of mucorous fungi during endogenous and exogenous dormancy. Endogenous dormancy (zygospores) is characterized by high contents of phosphatidylcholine (about 70% of the total phospholipids) and triacylglycerol (over 90% of the total neutral lipids). By contrast, exogenous dormancy (sporangiospores) is accompanied by elevated amounts of sterols, sterol esters, and free fatty acids, which account for over 70% of the total neutral lipids. We established for the first time significant differences in the phospholipid composition between sporangiospores obtained from stylosporangia and sporangioles. Based on the data obtained, we regard the retardation of life-sustaining activities as a biochemical adaptation based on the dormancy state. We also discuss the taxonomical position of Blakeslea trispora.     

Actinoplanes capillaceus sp. nov., a new species of the genus Actinoplanes

Two motile actinomycete strains, K95–5561^T and K95–5562, were isolated from a soil sample collected at Sayama City, Saitama Prefecture, Japan. They produced bell shaped spore vesicles (sporangia) with hairy surfaces on substrate hyphae. When released into water, the sporangiospores became motile by a tuft of polar flagella. The chemotaxonomic and morphological characteristics together with 16S rRNA gene sequence data indicated that the two isolates belonged to the genus Actinoplanes. The two strains were assigned to a single species on the basis of phenotypic, notably cultural, morphological and physiological characteristics, and DNA-DNA pairing data. The two strains were distinguished from representatives of all validly described species of Actinoplanes using a combination of genotypic and phenotypic properties. It is, therefore, proposed that strains K95–5561 and K95–5562 be recognized as a new species of the genus Actinoplanes with the name Actinoplanes capillaceus sp. nov. The type strain of the species is strain K95–5561^T (=JCM 10268^T =IFO 16408^T). The invalidly proposed species `Ampullariella cylindrica', `Ampullariella pekinensis' and `Ampullariella pilifera' were assigned to Actinoplanes capillaceus on the basis of genotypic and phenotypic data.     

The effect of gamma radiation on breaking of dormancy in Phycomyces spores

Dormant sporangiospores of Phycomyces blakesleeanus were activated by Cs-137 gamma rays. After a dose of about 300 krad, between 80 and 90% of the spores germinated normally. However, further development of the mycelium was inhibited. Even with dry spores a partial activation was obtained. Activation by gamma rays was not accompanied by an increase in trehalase activity.     

Staining of Fungi With Saccharated Iron Oxide

A 0.2% aqueous solution of saccharated iron oxide with a pH of 10.8 is shown to be suitable for supravital staining of fungi. Colonies or fresh tissue materials presumed to contain fungi are immersed in the solution for 1-24 hr, fixed in a 10% neutral solution of buffered formalin about 1 hr, washed, and treated with 10% potassium ferrocyanide in 0.5 N, HC1. Cell membranes of hyphae, perithekia, and walls of sporangia appear dark blue; cytoplasm of the hyphae, sporangiospores and spores stain greenish blue or yellowish green.     

Nuclear architecture and DNA location in two VAM fungi

Summary The ultrastructural organization of nuclei in Glomus versiforme and G. mosseae hyphae was examined by sampling at different times during the fungus life cycle. In young and active hyphae, the nucleus is chromocentric with dispersed chromatin and a well-developed nucleolus. On aging, and particularly during arbuscule collapse, the chromatin condenses to give a pyk-notic nucleus with blebs in the envelope and conspicuous spaces between this and the nucleoplasm. DNA location by immunogold or enzyme-gold techniques resulted in uniform labelling over the chromatin. Nuclear degeneration is mirrored by decreased gold labelling and may thus be regarded as an important parameter for evaluation of fungus viability and the ephemeral life of the arbuscule.     

Mycoparsitic behaviour ofFusarium oxysporum Schlechtendahl towardsAspergillus luchuensis Inui

Summary Mycoparasitic behaviour ofFusarium oxysporum Schlechtendahl towardsAspergillus luchuensis Inui was studiedin vitro. Growth of the hyphae ofF. oxysporum inside the hyphae and conidiophores ofA. luchuensis and formation of resting bodies ofF. oxysporum in conidiophores and hyphae ofA. luchuensis were observed. Bursting of the host hypha attacked by the parasitic hyphae was also noticed.     

Ribosomal proteins of the dimorphic fungus, Mucor racemosus

Summary Ribosomal proteins of the dimorphic fungus Mucor racemosus were isolated and characterized by 2-dimensional gel electrophoresis. Proteins from ribosomes of the yeast and mycelial phase were compared, and were found to be qualitatively indistinguishable. The only consistant difference in the patterns of proteins was in a protein of the 40S subunit, S-6. This protein was phosphorylated in yeast and hyphae forms, but not in asexual sporangiospores. Studies on protein S-6 showed that it contained 3 phosphate residues per molecule of protein when maximally phosphorylated. In this form 3 different tryptic peptides were shown to contain a single phosphoserine. The S-6 protein also existed in forms containing 1 or 2 phosphates per molecule, depending on growth conditions.     

Endophyte differentiation inCasuarina actinorhizae

Summary This is an ultrastructural study of development of infected cells in nitrogen fixing root nodules ofCasuarina spp. While several aspects of development are similar to those found in many other actinorhizae, unusual aspects of development of the host cell and differentiation of the endophyte inCasuarina are correlated with unusual changes in the wall of the infected cell. Instead of vesicles the endophyte forms atypical hyphae in mature infected cells. These unusual hyphal forms are termed intracellular hyphae. Intracellular hyphae are nonseptate hyphae which originate and terminate within the same host cell, and have a varying diameter and a multidirectional growth and branching pattern. A laminate surface layer previously undescribed on hyphae ofFrankia is a feature common to mostCasuarina endophytic hyphae and is probably similar chemically to the laminae comprising the multilamellate envelope of endophytic vesicles in other actinorhizae.This paper is Florida Agricultural Experiment Station Journal Series No. 7350.     

Identification of Naemacyclus minor hyphae within needle tissues of Pinus sylvestris by immunoelectron microscopy

Cultural investigations revealed that Naemacyclus minor, Lophodermium seditiosum and Cenangium ferruginosum were the most frequent colonizers of asymptomatic and symptomatic Pinus sylvestris needles. Since ultrastructural observations showed that morphological features were not suitable to differentiate hyphae of N. minor from hyphae of other isolates, the on-section immunogold labelling technique was applied in combination with an anti-N. minor specific immunoserum. The specificity of this serum was tested against culture hyphae of all isolates. Anti-N. minor specific immunoserum was then used to identify N. minor hyphae in thin sections of green P. sylvestris needles. The infection loci identified were restricted to small tissue areas located in the vicinity of stomata. In the hypodermis, hyphae and endocell-containing hyphae were located within the lumina of host cells but outside the protoplast. The growth of hyphae from cell to cell occurred through pits. The hyphae spreat into the mesophyll intercellularly and continued with the intracellular colonization of moribund and dead mesophyll cells in a later stage of infection. The observed host-parasite interactions at cellular and ultrastructural level are discussed in connection with the still controversial interpretation of the pathogenicity of N. minor.     

FTIR spectroscopy as a potential tool to analyse structural modifications during morphogenesis of Candida albicans

Candida albicans is a polymorphic organism that grows under certain conditions as blastospores, hyphae or pseudohyphae. The potentials of FTIR spectroscopy for assessing structural differences in C. albicans blastospores and hyphae were investigated. The main observed differences were localised in the polysaccharide (950–1,185 cm⁻¹), protein (1,480–1,720 cm⁻¹), and the fatty acids (2,840–3,000 cm⁻¹) regions. Quantitative evaluation of differences between hyphae and blastospores by curve-fitting of these regions indicate that these modifications could be due to both changes in structure and content of components of the cell wall such as β-glucans, mannoproteins, and lipids. Furthermore, glycogen consumption could be involved during hyphae elongation. Thus, FTIR spectroscopy can be an interesting tool to investigate differences in structure and in content between blastospores and hyphae. We also demonstrate through this study that differentiation of C. albicans clinical strains using hyphae is feasible, as this has been previously shown with blastospores. This preliminary work on identification of C. albicans using hyphae is a prelude to a larger clinical study for early typing within 7 h from a pure culture.     

Changes in the Lipid Composition of Mucor hiemalis Sporangiospores Related to the Age of the Spore-Forming Culture

Analysis of sporangiospore lipids of the fungus Mucor hiemalis F-1156 showed that alterations occur in the content of fatty acids and individual classes of lipids during long-term cultivation (for about 20 days). The changes in the lipid composition related to the age of the spore-forming mycelium suggest an important role of sporangiospore lipids in spore germination and in further development of the spherical cells formed in this processes. The M. hiemalis F-1156 sporangiospores with a lipid pool exhausted during long-term cultivation can give rise to both mycelial and yeastlike growth.