Hyphal walls of isolated lichen fungi

The hyphal walls of three mycobionts, isolated from the lichens Xanthoria parietina, Tornabenia intricata and Sarcogyne sp. were investigated by two techniques: microautoradiography of fungal colonies exposed to radioactive carbohydrate precursors; and binding, in vivo, of fluorescein conjugated lectins to hyphal walls of such colonies.N-[³H] acetylglucosamine was readily incorporated into tips, young hyphal walls and septa of the three mycobionts and the free-living fungus Trichoderma viride, but not into Phytophthora citrophthora, indicating that chitin is a major component of the mycobionts' hyphal walls. All three mycobionts, but neither of the free-living fungi, incorporated [³H] mannose and [³H] mannitol into their hyphal walls.Fluorescein-conjugated wheat germ agglutinin was bound to the hyphal walls of the three mycobionts and T. viride, but not to the walls of P. citrophthora; the binding pattern was similar to the grain pattern obtained in autoradiographs after short N-[³H] acetylglucosamine labelling. As wheat germ agglutinin binds specifically to chitin oligomers, the lectin binding tests further confirmed that chitin is a mycobiont hyphal wall component.Binding characteristics of several fluorescein-conjugated lectins to the three mycobionts indicated that this technique can yield useful information concerning the chemical composition of hyphal wall surfaces.List of abbreviations FITC fluorescein isothiocyanate - WGA wheat germ agglutinin - TCA trichloroacetic acid - PNA peanut agglutinin - LA lotus agglutinin - Glc NAc N-acetylglucosamine - ConA concanavalin A - SBA soybean agglutinin - WBA waxbean agglutinin Part of an M.Sc. thesis submitted by A. Braun to the Department of Botany, Tel Aviv University.     

Paracoccidioidomycosis in nude mice: Presence of filamentous forms of the fungus

Congenitally athymic nude mice (nu/nu) and their phenotypically normal littermates (nu/+) were intraperitoneally infected with yeast cells of a strain of Paracoccidioides brasiliensis. The nude mice developed a severe and generalized infection with an intense parasitism of several organs, accompanied by a low-grade of tissue reaction. The lesions were characterized by abundant yeast-like cells of the fungus, and in some animals, numerous hyphal forms could be well visualized. In control animals, infection was moderate, almost exclusively restricted to the area of inoculation, and the lesions presented few parasites surrounded by an inflammatory response. Filamentous forms of the fungus were never encountered in these animals.     

Abundance of Collembola (Insecta) inhabiting the hyphal mat of an ectomycorrhizal fungus,Sarcodon scabrosus,in a Pinus densiflora forest

The total number of collembolans collected from the hyphal mat of the ectomycorrhizal fungus Sarcodon scabrosus was less than half of that collected from the adjacent non-mat soil. The same was true of all families of collembolans except one, although not all differences were significant. The exception was the Hypogastruridae, with more individuals in the hyphal mat on the sampling day in the fruiting season; these were also the most abundant collembolans on the fruiting bodies. These results indicate that most collembolans avoid the hyphal mat of S. scabrosus in a Japanese red pine forest.     

Cerrena unicolor isolated from the mycangia of a horntail,Tremex longicollis,in Kochi Prefecture,Japan

A fungus was found to be stored in the mycangia of a horntail,Tremex longicollis, as hyphal fragments. All fungal isolates from the mycangia of 31 adult females of the horntail produced the same colonies on PDA. Basidiocarps ofCerrena unicolor occurred near the emergence hole of the horntail on a dead hackberry tree (Celtis sinensis). The cultures of this fungus were similar to those from the mycangia of the horntail in cultural characteristics. Mating between single-basidiospore mycelia ofC. unicolor and single-arthrospore mycelia from the mycangia of the horntail showed that they were compatible. These results revealed that the fungus isolated from the mycangia ofT. longicollis wasC. unicolor.     

Endomycorrhizal fungi in nitrogen transfer from soybean to maize

Using ¹⁵N as a tracer, interspecific N-transfer was studied during the course of plant development. The use of barriers of differing permeabilities between donor and receiver plants allowed separation of the effect of mycorrhizal colonization, root or hyphal contact and interplant hyphal bridging, on ¹⁵N-transfer from soybean (Glycine max (L.) Merrill) to maize (Zea mays L.). More transfer was measured between mycorrhizal plants, but transport of ¹⁵N from the labelled host plant to Glomus versiforme (Karsten) Berch did not seem to occur at the symbiotic interface, suggesting that the fungus is independent of its host for its N-nutrition, and that the role of hyphal bridges in N-transfer between plants, is not significant. Uptake by the receiver plant of the N excreted by the donor plant root system appears to be the mechanism of N-transfer between plants. The factor most affecting ¹⁵N-transfer between plants was found to be the extent of the contact between plant root systems. The presence of the endomycorrhizal fungus in plant roots reduced ¹⁵N-loss from soybean, but at the same time, its extensive hyphal network improved the efficiency of the maize root system for the recovery of the ¹⁵N excreted by soybeans. The net result was a better conservation of the N resource within the plant system. The transfer of N between mycorrhizal plants was particularly enhanced by the death of the soybean.     

Cytochemical evidence of a fungal cell wall alteration during infection of Eucalyptus roots by the ectomycorrhizal fungus Cenococcum geophilum

When the ectomycorrhizal fungus Cenococcum geophilum changes from a saprophytic to a symbiotic stage, its cell wall structure becomes simplified. The external hyphal wall layer which, in the saprophytic stage, is highly reactive to the Gomori-Swift test becomes poorly reactive and can no longer be distinguished from the internal wall layer in the Hartig net hyphae. The intensely stained external wall layer was also absent from pure cultures of Cenococcum geophilum grown on a medium with a low sugar content. This cell wall alteration could be due to a decrease in the amount of melanin or of melanin plus cystine-containing proteins. This change may be necessary for increased nutrient exchange between symbionts through hyphal walls.     

Studies on growth inhibition by lectins of penicillia and aspergilli

It has previously been shown in our laboratory that wheat germ agglutinin (WGA) binds to Trichoderma viride and inhibits growth of this fungus. Here we report on the effect of WGA, soybean agglutinin (SBA) and peanut agglutinin (PNA) on Penicillia and Aspergilli. Binding of the lectins to the fungi was examined with the aid of their fluorescein isothiocyanate (FITC) conjugated derivatives. FITC-WGA bound to young hyphal walls of all species, in particular to the hyphal tips and septa, in agreement with the chitinous composition of the cell walls of the two genera. Hyphae of all species examined were labelled, though in different patterns, by FITC-SBA and FITC-PNA, suggesting the presence of galactose residues on their surfaces. Young conidiophores, metulae (of the Penicillia), vesicles (of the Aspergilli), sterigmata and young spores, were also labelled. The three lectins inhibited incorporation of [³H]acetate, N-acetyl-D-[³H]glucosamine and D-[¹⁴C]galactose into young hyphae of Aspergillus ochraceus, indicating interference with fungal growth. Inhibition of spore germination by the three lectins was also observed. Preincubation of the lectins with their specific saccharide inhibitors prevented binding and the inhibitory effects. We conclude that lectins are useful tools for the study of fungal cell surfaces, and may also serve as an important aid in fungal classification. The present findings also support the suggestion that one role of lectins in plants is protection against fungal pathogens.Abbreviations Con A concanavalin A - PNA peanut agglutinin - SBA soybean agglutinin - WGA wheat germ agglutinin - FITC fluorescein isothiocyanate - GlcNAc N-acetyl-D-glucosamine - GalNAc N-acetyl-D-galactosamine     

<Emphasis Type="Italic">Septobasidium parviflorae</Emphasis> sp. nov. on <Emphasis Type="Italic">Pinus parviflora</Emphasis> from Japan

Septobasidium parviflorae sp. nov. on Pinus parviflora is described and illustrated. This species is characterized by its whitish-gray, gray to dark gray-colored fungus body with an indeterminate margin, hyphal strands, and cylindrical basidia with long sterigmata. This is the first report of Septobasidium occurring on a member of the genus Pinus in Japan.     

Isolationin vitro ofStrongwellsea castrans [Fungi: Entomophthorales] a pathogen of adult cabbage root flies,Delia radicum [Dipt.: Anthomyiidae]

The entomogenous fungusStrongwellsea castrans was isolatedin vitro for the first time, by incubating conidia projected from infected cabbage root flies (Delia radicum) in a simple, semi-defined liquid medium comprising dextrose, yeast extract and lactalbumin hydrolysate buffered to pH 7. The fungus grew as long unitunicate hyphae. After transfer to a solid nutrient medium, multinucleate hyphal bodies were formed which developed a thick, laminated wall. Neither conidia nor resting spores developed in liquid or on solid media and the fungus survived successive sub-culturing only in liquid media. Using the API-ZYM system, tests on extracts on hyphae ofS. castrans were positive for 11 enzymes but there were no consistent differences in enzyme profiles betweenS. castrans and fungi of the related genusErynia.      

Studies on initiation and development of the partner association inGeosiphon pyriforme (Kütz.) v. Wettstein,a unique endocytobiotic system of a fungus (Glomales) and the cyanobacteriumNostoc punctiforme (Kütz.) Hariot

Summary Phagocytosis ofNostoc filaments byGeosiphon, a fungus closely related to AM forming Glomales, was observed under light microscopes. Incorporation can only be performed ifNostoc primordia come into contact with growing hyphal tips of the fungus. The fungal wall just below the apex softens, and fungal cytoplasm is bulged out repeatedly covering the vegetativeNostoc cells but not the heterocytes. New heterocytes are differentiated by the internalised filament whose cells can increase up to ten times in volume after recovering from incorporation strain. TheNostoc cells are coated stepwise by short finger-shaped protuberances of the fungal hypha. These hernia-like outgrowths first remain separated, after 1 to 2 days they merge. Adjacent hyphal walls inside the complex covering disintegrate. Periphal fungal wall portions are united to form a smooth strong outer envelope. Internalisation is categorised as phagocytosis. The partnership is partly specific,Nostoc strains capable of living endocytobiotically are often partners in other symbioses besidesGeosiphon.Abbreviations AM arbuscular mycorrhiza (formerly VAM vesicular arbuscular mycorrhiza) - DIC differential interference contrast - LD light/dark Dedicated to Prof. Dr. Dr. h.c. Eberhard Schnepf on the occasion of his retirement     

Possible role of ionic gradients in the apical growth of <Emphasis Type="Italic">Neurospora crassa</Emphasis>

The effects of the Ca²⁺/H⁺ exchanger A23187 and the K⁺/H⁺ exchanger nigericin on the growth of Neurospora crassa were analyzed. Both ionophores had the same effects on the fungus. They both inhibited growth in liquid media, apical extension being more affected than protein synthesis. A sudden challenge to either ionophore on solid media rapidly stopped hyphal extension. Additionally, both ionophores induced profuse mycelium branching and upward hyphal growth. Hyphae growing on nigericin-containing media also burst at the apex. Both ionophores caused a rapid inhibition in the apically-occurring synthesis of structural wall polysaccharides, but they did not affect mitochondrial energy conservation. With the use of DiBAC, a membrane-potential sensitive fluorophore, it was excluded that their effects were due to depletion of the plasma membrane potential. Considering that both ionophores exchange H⁺ for different metallic ions, we concluded that their effect was due to dissipation of a proton gradient, which is directly or indirectly involved in the apical growth of the fungus. This revised version was published online in June 2006 with corrections to the Cover Date.     

Lanceispora amphibia gen. et sp. nov., a new amphisphaeriaceous ascomycete inhabiting senescent and fallen leaves of mangrove

Lanceispora amphibia gen. et sp. nov. in the Amphisphaeriaceae is described from senescent and fallen leaves ofBruguiera gymnorrhiza in mangrove forests in the Southwest Islands, Japan. The fungus produces immersed ascomata in leaf tissue, cylindrical asci with an apical ring staining blue with iodine, and oblanceolate ascospores with a septum above the middle. Studies on the fungal succession on the mangrove leaves revealed thatL. amphibia infects senescent leaves on the tree and inhabits intertidal fallen leaves, showing the highest frequency of occurrence at the late stage of decomposition. In culture the optimal conditions for hyphal growth were 20 ppt salinity and 30°C, and those for sexual reproduction were 10 ppt salinity and 25°C. Growth at 0 ppt (fresh water) was depressed. The fungus has amphibious habits, growing on the tree and in intertidal water; and it is adapted to the high osmotic conditions in leaf tissues of the mangrove tree and to the subtropical, brackish water environment of mangrove forests.     

Ramicandelaber, a new genus of the Kickxellales, Zygomycetes

Ramicandelaber, a new genus of Zygomycetes is erected to accommodateRamicandelaber longisporus sp. nov. The fungus has hyphal septa with median plugs and forms homologous structures to sporocladia and pseudophialides. These characteristics suggest that it belongs to the Kickxellales, Zygomycetes.     

Mycorrhiza of the host-specific Lactarius deterrimus on the roots of Picea abies and Arctostaphylos uva-ursi

The ectomycorrhizal basidiomycete species Lactarius deterrimus Gröger is considered to be a strictly host-specific mycobiont of Picea abies (L.) Karst. However, we identified arbutoid mycorrhiza formed by this fungus on the roots of Arctostaphylos uva-ursi (L.) Spreng. in a mixed stand at the alpine timberline; typical ectomycorrhiza of P. abies were found in close relation. A. uva-ursi is known as an extremely unspecific phytobiont. The mycorrhizae of both associations are described and compared morphologically. The mycorrhiza formed by L. deterrimus on both A. uva-ursi and P. abies show typical ectomycorrhizal features such as a hyphal mantle and a Hartig net. The main difference between the mycorrhizal symbioses with the different phytobionts is the occurrence of intracellular hyphae in the epidermal cells of A. uva-ursi. This emphasizes the importance of the plant partner for mycorrhizal anatomy. This is the first report of a previously considered host-specific ectomycorrhizal fungus in association with A. uva-ursi under natural conditions. The advantages of this loose specificity between the fungus and plant species is discussed.     

In vitro evaluation of combination of Trichoderma harzianum and chitosan for the control of sapstain fungi

In vitro assays were undertaken to evaluate the control of two sapstain fungi, Leptographium procerum and Sphaeropsis sapinea by a combination of chitosan or chitosan oligomer and an albino strain of Trichoderma harzianum. Spore germination and hyphal growth of the test fungi were assessed on media amended with chitosan or chitosan oligomer with and without T. harzianum using either simultaneous inoculation with test fungus or inoculation 1, 2, or 3 days after pre-infection with test fungus.There was no mycelial growth of the test fungi regardless of chitosan concentrations used when either L. procerum or S. sapinea was simultaneously inoculated with T. harzianum. However, the dose–response of chitosan or chitosan oligomer on the test fungi was apparent when T. harzianum was not simultaneously inoculated with test fungus but introduced later. There was a greater growth reduction at higher concentrations (0.075–0.1% v/v) of chitosan, and overall chitosan oligomer was more effective than chitosan aqueous solution.Chitosan alone was able to restrict or delay the germination of spores but the combination of chitosan and T. harzianum inhibited spore germination and hence colony formation of test fungi regardless of time delay.     

Study on the hyphal responses of Aspergillus fumigatus

The hyphal responses of an A. fumigatus isolate to a trizolederivative-fluconazole (FCZ) were studied with a Bio-Cell Tracer system. The numerical data were recorded as the original growth rate (Pre-GR), the time needed for FCZ reaching to its target in hypha (τ_on), the growth rate under the FCZ effect (Exp-GR) and the growth rate after FCZ was removed (Post-GR). Based on above numerical data, the inhibitory rates in the exposure and post exposure periods were calculated as the Exp-I% and Post-I% values. It was found there were variable inhibitory rate values (I%) in individual hyphae corresponding to different FCZ concentrations. It was shown by correlation analysis of the numerical data that the Pre-GR values were negatively correlated with the τ_on values and positively correlated with both the Exp-I% and Post-I% values. Additionally, the τ_on values are negatively correlated with the Exp-I% and Post-I% values. Those results suggested that the hyphal growth rate and the susceptibility of the FCZ target be the important factors to determine the hyphal responses to the FCZ effect. Serial morphological alternations were captured while the hyphal growth curves were changing under the FCZ effects. Of the morphological data, the interesting alternations were visualized when the hyphae were affected by 16 μg/ml FCZ. As shifting of the hyphal growth curves, the hyphae were repeatedly seen as swollen tips and germination from the swollen sites. It is indicated that the hyphal tips are the most sensitive parts of this mycelia fungus to the FCZ affects. Additionally, because the hyphal regrowth was observed as germination from the swollen tips before FCZ was removed, an adaptation phenomenon could be proposed. This revised version was published online in June 2006 with corrections to the Cover Date.     

Amelioration of Pb and Mn Toxicity to Arbuscular Mycorrhizal Fungus Glomus intraradices by Maize Root Exudates

The effect of maize root exudates on the toxicity of lead and manganese to arbuscular mycorrhizal fungus (AMF) Glomus intraradices was studied in vitro by observation of intraradical hyphae regrowth from colonised root segments. Higher heavy metal (HM) concentrations strongly reduced the hyphal growth, however, the inhibitory effect was to a large extent eliminated by the addition of maize root exudates to the media. However, the capacity of exudates to ameliorate HM toxicity was limited and did not operate when a threshold HM concentration was reached. This revised version was published online in July 2006 with corrections to the Cover Date.     

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.     

Formation of structures resembling ericoid mycorrhizas by the root endophytic fungus Heteroconium chaetospira within roots of Rhododendron obtusum var. kaempferi

A resynthesis study was conducted to clarify the relationship between the root endophyte, Heteroconium chaetospira and the ericaceous plant, Rhododendron obtusum var. kaempferi. The host plant roots were recovered 2 months after inoculation, and the infection process and colonization pattern of the fungus were observed under a microscope. The hyphae of H. chaetospira developed structures resembling ericoid mycorrhizas, such as hyphal coils within the host epidermal cells. These structures were morphologically the same as previously reported ericoid mycorrhizal structures. The frequencies of hyphal coils within the epidermal cells of host roots ranged from 13 to 20%. H. chaetospira did not promote or reduce host plant growth. This is the first reported study that H. chaetospira is able to form structures resembling mycorrhizas within the roots of ericaceous plants.     

C-NMR analysis of Aspergillus mutants disturbed in pyruvate metabolism

The metabolic consequences of two defects in pyruvate metabolism of the hyphal fungus Aspergillus nidulans have been investigated by natural abundance ¹³C-NMR spectroscopy. A pyruvate dehydrogenase complex (pdh) mutant, grown on acetate, accumulates alanine upon starvation which is derived from mannitol reserves. The -alanine level increases further upon incubation with the non-permissive substrate -glucose. -Glutamate is absent from these spectra as it is required both for the transamination of pyruvate and as a reaction on an impaired energy metabolism in such a pdh-deficient strain. A pyruvate carboxylase (pyc) mutant, grown upon acetate, only starts to accumulate alanine after a long incubation period with -glucose, due to the long-lasting presence of phosphoenolpyruvate carboxykinase and malic enzyme, which are both induced by growth on acetate. When this strain is grown on -fructose and -glutamate, alanine also accumulates within 3 h upon transfer to -glucose.