Hyphal Mass Research Articles

The bacteriumPaenibacillus validusstimulates growth of the arbuscular mycorrhizal fungusGlomus intraradicesup to the formation of fertile spores

Two isolates of Paenibacillus validus (DSM ID617 and ID618) stimulated growth of the arbuscular mycorrhizal fungus Glomus intraradices Sy167 up to the formation of fertile spores, which recolonize carrot roots. Thus, the fungus was capable of completing its life cycle in the absence of plant roots, but relied instead on the simultaneous growth of bacteria. The supernatant of a mixed batch culture of the two P. validus isolates contained raffinose and another, unidentified trisaccharide. Among the oligosaccharides tested, raffinose was most effective in stimulating hyphal mass formation on plates but could not promote growth to produce fertile spores. A suppressive subtractive hybridization library followed by reverse Northern analyses indicated that several genes with products involved in signal transduction are differentially expressed in G. intraradices SY 167 when grown in coculture with P. validus (DSM 3037). The present investigation, while likely representing a significant step forward in understanding the arbuscular mycorrhizal fungus symbioses, also confirms that its optimal establishing and functioning might rely on many, as yet unidentified factors.

Structural characterization of (1→3)-β- d-glucans isolated from blastospore and hyphal forms of Candida albicans

Glucans are (1→3)-β-linked linear and branched polymers containing anhydroglucose repeat units. They comprise a major portion of the cell wall of saprophytic and pathogenic fungi. Glucans activate a wide range of innate immune responses. They are also released from the fungal cell wall as exopolymers into the blood of patients with fungal infections. Extensive studies have been done on glucans isolated from saprophytic fungi, such as Saccharomyces cerevisiae; however, much less is known about the glucans produced by the polymorphic fungal pathogen Candida albicans. We have undertaken an extensive structural characterization and comparison of glucans isolated from C. albicans blastospores and hyphae using high-resolution, solution-state proton nuclear magnetic resonance spectroscopy (NMR). In addition, we developed a simple and straightforward method for the production of Candida hyphae that resulted in gram quantities of hyphal mass. Also, we compared and contrasted the Candida glucans isolated by two different protocols with those isolated from S. cerevisiae. Isolation protocols provide high purity glucans with source-based structural differences. Structural details provided by this NMR analysis included the degree of polymerization, molecular weight, degree and type of branching, and structural composition. We observed that Candida glucans, derived from blastospores or hyphae, are different compared to those isolated from S. cerevisiae with regard to side-chain branching along the backbone and at the reducing terminus. These structural details are an important prerequisite for biomedical studies on the interaction of isolated fungal cell wall glucans with the innate immune system.

Helicobasidium mompa isolates from sweet potato in continuous monoculture fields

Isolates of the violet root rot fungus Helicobasidium mompa were collected from herbaceous and tree plants. Their host preference was studied by inoculation experiments using carrots, sweet potatoes, and apple stocks. It was found that sweet potato isolates from Kyushu produced infection cushions on carrots and sweet potatoes but not on apple stocks. Other isolates did not show host preference. Sweet potato isolates were also characterized by ready hyphal mass (sclerotium) production. They were thought to have adapted to the habitat with high disturbance by annual tillage.

Identification and characterization of anaerobic gut fungi using molecular methodologies based on ribosomal ITS1 and 185 rRNA.

The gut fungi are an unusual group of zoosporic fungi occupying a unique ecological niche, the anaerobic environment of the rumen. They exhibit two basic forms, with nuclear migration throughout the hyphal mass for polycentric species and with concentration of nuclear material in a zoosporangium for monocentric species. Differentiation between isolates of these fungi is difficult using conventional techniques. In this study, DNA-based methodologies were used to examine the relationships within and between two genera of monocentric gut fungi gathered from various geographical locations and host animals. The ribosomal ITS1 sequence from 16 mono- and 4 polycentric isolates was PCR-amplified and sequenced; the sequences obtained were aligned with published sequences and phylogenetic analyses were performed. These analyses clearly differentiate between the two genera and reflect the previously published physiological conclusions that Neocallimastix spp. constitute a more closely related genus than the relatively divergent genus Piromyces. The analyses place two type species N. frontalis and N. hurleyensis together but, contrary to a recent suggestion in the literature, place them apart from the other agreed species N. patriciarum. In situ hybridization and slot-blotting were investigated as potential methods for detection of and differentiation between monocentric gut fungi. DNA slot-blot analysis using ribosomal sequences is able to differentiate between gut fungal genera and thus has considerable potential for use in ecological studies of these organisms.

Viability and wall permeability of the extramatrical hyphae of the ectomycorrhizal fungus Hebeloma cylindrosporum

The extramatrical hyphae of ectomycorrhizae are known to absorb ions from the soil for the symbiotic association, but the fraction of hyphae involved in this activity has not been studied. For hyphae to potentially absorb ions, they must have permeable walls and be viable. In the present study, the proportion of living, extramatrical hyphae of Hebeloma cylindrosporum (Romagnesi) was measured by a grid-intersect method after staining with fluorescein diacetate, disodium fluorescein, or ethidium bromide. Results with ethidium bromide indicated that 89% of the hyphae were alive when the association was 2 weeks old and that this value declined to 76% at 26 weeks. In contrast, both fluorescein treatments resulted in staining of only 25% of the hyphae at 2 weeks and 7% at 26 weeks. The difference in measured viability was ascribed to wall permeability. Ethidium bromide was applied following an ethanol pretreatment, which apparently increased the permeability of the hyphal wall as well as that of its membranes. Our results indicate that only a fraction of hyphae are likely involved in ion uptake (as indicated by the fluorescein tests), a fraction that declines as the association ages. Further, parts of the hyphal mass are alive but encased in impermeable walls; these parts may act as conduits for the ions that were absorbed by the younger regions.Key words: Hebeloma cylindrosporum, ectomycorrhizae, ion uptake, permeability, viability.

Suppression of bioremediation by Phanerochaete chrysosporium by soil factors

The timing of onset of mineralization of pyrene by Phanerochaete chrysosporium correlated with the production of ligninase activity as monitored by the decolorization of the polymeric dye Poly R-478. A layer of native soil decreased the growth of P. chrysosporium by visual assessment of hyphal mass and measurement of ergosterol, a fungal membrane sterol. The native soil layer slowed the production of ligninase as determined by Poly R-478 decolorization and the rate of mineralization of pyrene was decreased. Inhibition of pyrene mineralization was also observed with layers of sterilized silt and clay but not by sand. Mineralization, fungal growth and rate of decolorization of the dye were improved when the native soil was sterilized by autoclaving. Suspensions of the native soil contained a consortium of bacteria and fungi. Several of the bacteria were antagonistic to the growth of P. chrysosporium on solid medium. The onset of dye decolorization by P. chrysosporium on a nitrogen-limited medium was delayed by certain bacteria. These findings suggest that in certain soils, bioremediation by P. chrysosporium could be suppressed by indigenous soil microbes as well as by the abiotic features of the soil composition.

Quantification of fungal morphology, gaseous transport and microbial dynamics in soil: an integrated framework utilising fractal geometry

The consequences of heterogeneous structure for nutrient acquisition by soil fungi, microbial dynamics and transport in soil are studied. Fractal geometry provides the unifying theme and forms the basis of a theoretical framework for studying dynamics in heterogenous media. The interpretation of foraging strategies of soil fungi are presented which suggest that the processes governing branching and hyphal mass distribution are independent. Classical diffusion is shown to be inappropriate for the study of diffusion in heterogeneous soil and a new theory is proposed which incorporates heterogeneity and pore tortuosity. The consequences of structure for microbial spatial and temporal dynamics are examined and it is found that an understanding of these and related processes such as nutrient cycling must include the role of soil structure. While stressing the need to appreciate the relevance of the theory to any particular application, it is shown that quantitative fractal geometry can yield insights into the mechanism whereby spatial organisation influences the interaction between structure and biotic processes in the soil.

Extramatrical hyphae as sinks for carbon in ectomycorrhizal pine seedlings

Allocation of carbon to extramatrical (EM) hyphae has heretofore not been based on accurate estimates of EM hyphal mass. To test the demand of EM hyphae for carbon, pine seedlings that had been inoculated by two different techniques (pouch inoculated, P+, and commercial vermiculite inoculated, V+) were labeled with 14C in root mycocosms, and the amount and distribution of the label quantified at 4 and 9 days. Carbon use in host, mycorrhizae and EM hyphae depended on spatial and temporal relationships between host and mycobiont. Isotope translocation to seedling components also differed among the inoculation treatments. Based on gravimetric estimates of hyphal mass, more 14C remained in hyphae from the P+ seedlings than in hyphae from the V+ seedlings after 9 days. Overall, 14C allocation in V+ seedlings was similar to that in the uninoculated control seedlings. It is hypothesized that EM hyphae of P+ seedlings emanated directly from host roots and may have been more closely linked with the host than were V+ hyphae, allowing P+ hyphae to derive carbon directly from the host, while V+ hyphae existed semi-saprophytically and were less of a sink for carbon.

Temporal allocation of 14C to extramatrical hyphae of ectomycorrhizal ponderosa pine seedlings

Ponderosa pine seedlings were inoculated with Hebeloma crustuliniforme either in growth pouches before they were transplanted to root-mycocosms (P seedlings), or at the time of transfer to root-mycocosms (V seedlings). Uninoculated seedlings served as controls (U seedlings). The use of root-mycocosms allowed examination of portions of hyphae separate from roots and rooting substrate but still in symbiosis with the host. The results thus provided a quantitative basis for estimating hyphal mass and carbon allocation to extramatrical hyphae. The amount of (14)CO(2) fixed after a 2-h exposure was greatest for P seedlings and least for uninoculated seedlings. Four and nine days after exposure, (14)C content was greatest in uninoculated seedlings and least in inoculated seedlings. In isotope distribution and dry mass accumulation, V seedlings were more similar to U than to P seedlings. Calculated on a dry weight basis, the allocation of isotope to mycelium suggested that extramatrical hyphae of P seedlings were a stronger sink for carbon than extramatrical hyphae of V seedlings. Differences in inoculation methods resulted in differences in carbon allocation and physiology of extramatrical hyphae that could affect seedling establishment and survival. Seedlings inoculated by one method cannot serve as surrogates for mycorrhizal seedlings produced by other inoculation techniques.

A root-mycocosm for growing ectomycorrhizal hyphae apart from host roots while maintaining symbiotic integrity

A culture system is described to grow mycorrhizal plants which allows experimental measurements to be made on mycorrhizae, and a portion of intact ectomycorrhizal fungi while in symbiosis, but growing apart from the rooting medium and host roots. A portion of the extramatrical hyphae is kept apart from the rooting medium by a restrictive passageway between the root and fungal chambers. The passageway is small enough to restrict coniferous fine roots to the root chamber, but large enough to allow fungal hyphae to grow out of the root chamber onto pre-weighed glass fiber filter paper situated in the fungal chambers. The pre-weighed filter paper allows for gravimetric estimation of the hyphal mass in the fungal chamber. The pieces of the modular Root-Mycocosm can be assembled in various configurations with one or two seedling chambers and one, two or three fungal chambers. Ponderosa pine (Pinus ponderosa Laws.) seedlings were inoculated withHebeloma crustuliniforme (Bull.: St. Amans) Quel in either commercial-vermiculite inoculum or in plastic growth-pouches and grown in the Root-Mycocosm. Hyphae were allowed to grow into the fungal chambers and after four weeks, amounted to 5.5±0.81 SE and 6.4±1.5 SE mg for pouch and commercial inoculum techniques, respectively.

Preparation of fungal starter culture in liquid fluidized bed reactor

Pregerminated Trichoderma reesei (Rut C-30) spores were grown on corncob particles in a liquid fluidized bed reactor (LFBR). Hyphal mass covered particles were recovered from the top of the reactor in 24 h and used as starters for solid substrate fermentation. The starter from LFBR gave better biomass production than spore or mycelial inoculum.

A Comparison of Glucosamine and Biovolume Conversion Factors for Estimating Fungal Biomass

Glucosamine content or biovolume of fungi cannot be used to estimate fungal biomass unless the ratio of these quantities to fungal mass is known. We measured the interspecific and temporal variation of seven attributes of three salt-marsh fungi grown on damp Spartina alterniflora. Glucosamine (jig) and biovolume (mm3 fresh) per mg dry weight, mg carbon, and mg nitrogen were calculated. There were significant differences (p < 0.01) among species (Buergenerula spartina, Fusarium sp., Phaeosphaeria typharum) and among ages (2, 4, 12 wk) for glucosamine. Glucosamine and biovolume per unit mass increased with age. There were significant correlations (p < 0.01) between glucosamine and biovolume conversion factors (r = 0.89) and between residual hyphal mass after amino sugar analysis and hyphal nitrogen content (r = 0.85). Glucosamine did not correlate as well with hyphal surface area (p < 0.05). Considering all species and ages, glucosamine content varied from 8.5-92.8 jig GlcN mg-' dry weight (23-299 [Lg GlcN mg-' C; 170-3094 jig GlcN mg1 N). It is necessary to know species-specific and age-specific biomass conversion factors to accurately estimate fungal biomasses using biochemical or direct-count quantification techniques.

Perithecial formation inGelasinospora reticulispora V. Microscopically versus photobiologically recognizable differentiation of hyphae

Morphological changes in the early development of photobiologically induced perithecium weve microscopically observed with hyphae in apically growing mycellum ofGelasinospora reticulispora. No morphological differentiation was recognized in any hyphae during the inductive darkness and the following lag period. Semispherical protrusion was first noticed on slender hyphae 7 to 9 hr after the photoinductive treatment, and it developed into a coil-shaped mass within 3 to 6 hr thereafter. This hyphal mass grew to a young perithecium having 50 to 100 μm in diameter ca. 24 hr after the photoinduction.The density of induced pertithecium was dependent upon spot size of irradiation; the smaller the exposing area of mycelium, the higher the density. The result indicated that most slender hyphae can be responsive to photobiological induction of perithecium.

The Parasexual Cycle in Aspergillus Flavus

(1973). The Parasexual Cycle in Aspergillus Flavus. Mycologia: Vol. 65, No. 5, pp. 1201-1205.

The Morphology and Cytology of the Aecidium Cup

1. The essential features in the development of the cup are similar to those found in the development of the caeoma. The initial hyphal mass, or primordium, is formed by hyphae growing radially toward the center of the future cup. 2. The cup is more deeply seated and produces a greater number of sterile cells and gametes to each gametophoric hypha. The gametes form a fertile layer two or more cells in thickness. The sterile cells that form the pseudoparenchyma of the cup are homologous with the "buffer" cells of the caeoma. 3. Sexual cell fusions, by the breaking down of the cell walls between two equal gametes, were found in 6 additional species of cupulate aecidia, namely Ur. Caladii, P. Claytoniata, P. Violae, P. Hydrocotyles, P. Eatoniae, and P. angustata. Although the actual fusion stages were not seen in the last named species, the presence of two-legged basal cells is evidence that the fusions are of the same type as those found in the other species. No central organs ("fertile hyphae") or multinucleated cells were found. The organization of the cup, therefore, is merely that of a remarkably unified colony of gametophores. 4. Triple cell fusions were observed in P. Claytoniata and P. Violae, and trinucleated aecidiospores were frequently found in both of these species and in Ur. Caladii. Several quadrinucleated aecidiospores and a chain of quadrinucleated cells were found in P. Claytoniata. 5. The first fusion cells are formed at the center of the gametic tissue, and the subsequent ones are formed on all sides of this center in centrifugal order, until the lateral borders of the aecidium are reached. 6. The fusing cells may have their long axes in general in the long axis of the cup, for example P. Claytoniata, P. Violae, P. Hydrocotyles, and Ur. Caladii, or tangential to its curved basal surface, for example P. Eatoniae. 7. The presence or absence of a peridium is a natural but not very fundamental distinction between the aecidium cup and the caeoma. The production of a peridium is correlated with the deep location of the cup and the extensive formation of sterile cells. 8. As has long been known, the peridial cells are metamorphosed aecidiospores and aecidiospore initial cells. The central arch of the peridium is formed from the apical aecidiospores of the interior spore chains and the lateral walls from entire peripheral spore chains. The first peridial cells are produced at the center of the arch and the peridium enlarges from this point centrifugally until the bases of the lateral walls are reached. Its subsequent enlargement is by the basipetal growth and sterilization of the peripheral spore chains.