Hyphal Material Research Articles

Mycorrhizal structures in mycoheterotrophic Thismia spp. (Thismiaceae): functional and evolutionary interpretations

Achlorophyllous, mycoheterotrophic plants often have an elaborate mycorrhizal colonization pattern, allowing a sustained benefit from external fungal root penetrations. The present study reveals the root anatomy and mycorrhizal pattern of eight mycoheterotrophic Thismia spp. (Thismiaceae), all of which show separate tissue compartments segregating different hyphal shapes of the mycorrhizal colonization, as there are intact straight, coiled and peculiarly knotted hyphae as well as degenerated clumps of hyphal material. Those tissue compartments in Thismia roots potentially comprise exo-, meso- and endoepidermae, and exo-, meso- and endocortices, although not all species develop all these root layers. Differences in details among species according to anatomy (number of root layers, cell sizes and shapes) and colonization pattern (hyphal shapes within cells) are striking and can be discussed as an evolutionary series towards increasing mycorrhizal complexity which roughly parallels the recently established phylogeny of Thismia. We suggest functional explanations for why the distinct elements of the associations can contribute to the mycorrhizal advantage for the plants and, thus, we emphasize the relevance of structural traits for mycorrhizae.

From three-dimensional morphology to effective diffusivity in filamentous fungal pellets.

Filamentous fungi are exploited as cell factories in biotechnology for the production of proteins, organic acids, and natural products. Hereby, fungal macromorphologies adopted during submerged cultivations in bioreactors strongly impact the productivity. In particular,fungal pellets are known to limit the diffusivity of oxygen, substrates, and products. To investigate the spatial distribution of substances inside fungal pellets, the diffusive mass transport must be locally resolved. In this study, we present a new approach to obtain the effective diffusivity in a fungal pellet based on its three-dimensional morphology. Freeze-dried Aspergillus niger pellets were studied by X-ray microcomputed tomography, and the results were reconstructed to obtain three-dimensional images. After processing these images, representative cubes of the pellets were subjected to diffusion computations. The effective diffusion factor and the tortuosity of each cube were calculated using the software GeoDict. Afterwards, the effective diffusion factor was correlated with the amount of hyphal material inside the cubes (hyphal fraction). The obtained correlation between the effective diffusion factor and hyphal fraction shows a large deviation from the correlations reported in the literature so far, giving new and more accurate insights. This knowledge can be used for morphological optimization of filamentous pellets to increase the yield of biotechnological processes.

An X-ray microtomography-based method for detailed analysis of the three-dimensional morphology of fungal pellets.

Filamentous fungi are widely used in the production of biotechnological compounds. Since their morphology is strongly linked to productivity, it is a key parameter in industrial biotechnology. However, identifying the morphological properties of filamentous fungi is challenging. Owing to a lack of appropriate methods, the detailed three-dimensional morphology of filamentous pellets remains unexplored. In the present study, we used state-of-the-art X-ray microtomography (µCT) to develop a new method for detailed characterization of fungal pellets. µCT measurements were performed using freeze-dried pellets obtained from submerged cultivations. Three-dimensional images were generated and analyzed to locate and quantify hyphal material, tips, and branches. As a result, morphological properties including hyphal length, tip number, branch number, hyphal growth unit, porosity, and hyphal average diameter were ascertained. To validate the potential of the new method, two fungal pellets were studied-one from Aspergillus niger and the other from Penicillium chrysogenum. We show here thatµCT analysis is a promising tool to study the three-dimensional structure of pellet-forming filamentous microorganisms in utmost detail. The knowledge gained can be used to understand and thus optimize pellet structures by means of appropriate process or genetic control in biotechnological applications.

Stone-Eating Fungi: Mechanisms in Bioweathering and the Potential Role of Laccases in Black Slate Degradation With the Basidiomycete Schizophyllum commune.

Many enzymes, such as laccases, are involved in the saprotrophic lifestyle of fungi and the effects of those may be linked to enhanced bioweathering on stone surfaces. To test this hypothesis, we studied the decomposition of kerogen-enriched lithologies, especially with black slate containing up to 20% of Corg. Indeed, a formation of ditches with attached hyphal material could be observed. To address enzymes involved, proteomics was performed and one group of enzymes, the multicopper oxidase family members of laccases, was specifically investigated. A role in bioweathering of rocks containing high contents of organic carbon in the form of kerogen could be shown using the basidiomycete Schizophyllum commune, a white rot fungus that has been used as a model organism to study the role of filamentous basidiomycete fungi in bioweathering of black slate.

Improving mycorrhizal colonisation of cotton in sodic soils

The mycorrhizal colonisation of cotton in two sodic soils was investigated in a series of glasshouse experiments. Standard techniques to inoculate cotton (Gossypium hirsutum L.) plants in sodic soil were unsuccessful until live hyphal cultures were introduced. In the absence of fresh hyphal material, reliance on spore germination to colonise cotton roots in all tested scenarios was unsuccessful. The presence of a fresh mycorrhizal hyphal network within the inoculum source may play an important role in initiating colonisation of cotton roots in moderately and highly sodic soils. Further investigation into mycorrhizal proliferation under sodic soil conditions is warranted.

Predatory fungi, wood decay, and the carbon cycle

Predatory fungi attack nematodes and other microorganisms using a remarkable array of trapping devices to attract, capture, kill, and digest nematodes for food. The novelty of these relationships, however, has deflected attention from a more fundamental role played by these fungi in the ecosystem. The primary function of predatory fungi appears to be that of wood decay and hence they are cellulolytic or ligno-cellulolytic fungi_that attack other organisms as sources of nitrogen to supplement a primarily carbohydrate (woody) diet. The Carbon to Nitrogen ratio (C:N) of wood is extremely high and nitrogen is the limiting factor for growth of predatory fungi. Predation of nematodes or other organisms adds extra protein (nitrogen) to the system and reduces the C:N to manageable proportions. More importantly, nematode predation, although dramatic, is perhaps of less importance than the ability of wood decay fungi to attack bacteria and perhaps other life forms as nutrient supplements. By definition, therefore, many (most?) wood decay fungi are not saprobes (i.e. living on dead organic material) but are facultative parasites (saprobes that can also parasitize living organisms) for part of their life in which the predatory ‘parasitic’ phase runs parallel to the ‘saprophytic’ wood decay phase and both are essential to success. Based on their roles in building up woody material through mycorrhizal associations, and destroying it through biological decay, it is not surprising that the biomass of fungi in forest soils reaches 90% of the total and exceeds all other micro and meso-organisms combined. Based on the respiration of their massive amounts of hyphal material, fungi are the driving force in the biological component of the terrestrial carbon cycle.

The origin of the attine ant-fungus mutualism.

Cultivation of fungus for food originated about 45-65 million years ago in the ancestor of fungus-growing ants (Formicidae, tribe Attini), representing an evolutionary transition from the life of a hunter-gatherer of arthropod prey, nectar, and other plant juices, to the life of a farmer subsisting on cultivated fungi. Seven hypotheses have been suggested for the origin of attine fungiculture, each differing with respect to the substrate used by the ancestral attine ants for fungal cultivation. Phylogenetic information on the cultivated fungi, in conjunction with information on the nesting biology of extant attine ants and their presumed closest relatives, reveal that the attine ancestors probably did not encounter their cultivars-to-be in seed stores (von Ihering 1894), in rotting wood (Forel 1902), as mycorrhizae (Garling 1979), on arthropod corpses (von Ihering 1894) or ant faeces in nest middens (Wheeler 1907). Rather, the attine ant-fungus mutualism probably arose from adventitious interactions with fungi that grew on walls of nests built in leaf litter (Emery 1899), or from a system of fungal myrmecochory in which specialized fungi relied on ants for dispersal (Bailey 1920) and in which the ants fortuitously vectored these fungi from parent to offspring nests prior to a true fungicultural stage. Reliance on fungi as a dominant food source has evolved only twice in ants: first in the attine ants, and second in some ant species in the solenopsidine genus Megalomyrmex that either coexist as trophic parasites in gardens of attine hosts or aggressively usurp gardens from them. All other known ant-fungus associations are either adventitious or have nonnutritional functions (e.g., strengthening of carton-walls in ant nests). There exist no unambiguous reports of facultative mycophagy in ants, but such trophic ant-fungus interactions would most likely occur underground or in leaf litter and thus escape easy observation. Indirect evidence of fungivory can be deduced from contents of the ant alimentary canal and particularly from the contents of the infrabuccal pocket, a pharyngeal device that filters out solids before liquids pass into the intestine. Infrabuccal pocket contents reveal that ants routinely ingest fungal spores and hyphal material. Infrabuccal contents are eventually expelled as a pellet on nest middens or away from the nest by foragers, suggesting that the pellet provides fungi with a means for the dispersal of spores and hyphae. Associations between such "buccophilous" fungi and ants may have originated multiple times and may have become elaborated and externalized in the case of the attine ant-fungus mutualism. Thus, contrary to the traditional model in which attine fungi are viewed as passive symbionts that happened to come under ant control, this alternative model of a myrmecochorous origin of the attine mutualism attributes an important role to evolutionary modifications of the fungi that preceded the ant transition from hunter-gatherer to fungus farmer.

In situ detection of a fungal glycoprotein-elicitor in stem rust-infected susceptible and resistant wheat using immunogold electron microscopy

Immunoelectron microscopy (IEM) was used to analyze the compatible and incompatible host-pathogen interaction between the obligate, biotroph stem rust (Puccinia graminis f.sp. tritici; Pgt) and primary leaves of wheat (Triticum aestivum L.). The investigation was focused on the subcellular localization of a fungal elicitor glycoprotein of stem rust (Pgt-elicitor). Uredospores as well as fungal infection structures of stem rust on wheat leaves were probed with a specific monoclonal antibody, in order to determine the in situ distribution pattern of the antigen. Binding to the anti-elicitor antibody was observed over the cell wall and the germ pore of germinating uredospores. Immunogold staining was found over the infection structures of stem rust within the wheat leaf tissue of both the compatible and incompatible plant-pathogen interaction. Distinct cell wall layers of the intercellular mycelium, of the haustorial mother cells, as well as of the haustoria were clearly labeled. Gold particles were also detected over the intercellular space and the extrahaustorial matrix in between the extrahaustorial membrane and the haustorial cell wall which indicated a release of elicitor molecules from the fungal cell wall. No labeling was observed over the host cell cytoplasm of the compatible and incompatible interaction, respectively. The immunocytochemical detection of elicitor epitopes over the hyphal cell walls of in vitro grown axenic cultures of P. graminis f.sp. tritici confirmed the occurrence of elicitor molecules in young hyphal material. Elicitor molecules were released by the hyphae of axenic cultures of stem rust in vitro.

Nitrogen metabolism of external hyphae of the arbuscular mycorrhizal fungus Glornus intraradices

summaryThe arbuscular mycorrhizal (AM) fungus Glomus intraradices Schenck and Smith was grown in symbiosis with Cucumis sativus L. ev. Aminex (Fl hybrid) in mesh bags surrounded by a sand‐filled hyphal compartment (HC), allowing only the fungal hyphae to protrude into the HC. The hyphae in the HC were supplied with 15N‐labelled NH4+ or NO3− after 60 d (expt 1). Following a 48 h labelling period, the sand was removed from the HC and the hyphae extracted. In another experiment (expt 2), the hyphae were extracted from the sand before being incubated in vitro in a nutrient solution containing 15N‐labelled NH4+ for 15 h. The hyphal material was incubated in a 0 or 2.5 mM solution of the GOGAT‐inhibitor albizzine prior to labelling. In both experiments the hyphal content of free amino acids and fatty acids were measured as well as the ammo acid l5N enrichment.Asparagine was the hyphal amino acid measured in highest concentration followed by glutamine, glutamate, aspartate and alanine. Ornithine, serine and glycine were detected in lower concentrations, but γ‐aminobutyric acid and citrulline were not detectable. This pattern was the same in both experiments and was unaffected by the type of N applied to the hyphae or by preincubation with albizzine, although the amino‐acid concentration decreased considerably in expt 2 compared with expt 1. Both NH4+‐N and NO3−‐N were assimilated into amino acids but the levels of l0N enrichment following application of NO3− were much lower than those following application of NH4+ indicating that the latter was more readily assimilated. Albizzine decreased the hyphal amino acid concentration by c. 30% (without affecting the 15N enrichment of the individual amino acids) indicating that the AM‐fungal hyphae might possess a GS‐GOGAT enzyme system for assimilation of inorganic N. The fatty‐acid profiles (especially phospholipid fatty acids 16: 1ω5 and 20:5) obtained from the hyphae of G. intraradices showed that contamination of the samples by fungi other than G. intraradices and bacteria was insignificant, and confirmed the usefulness of specific fatty‐acid measurement to estimate soil AM‐fungal content.

Cordyceps brunneapunctata sp. nov. infecting beetle larvae in Thailand

Cordyceps brunneapunctata is described from elaterid beetle larvae buried in forest soils in Thailand where it is found associated with a Hirsutella state. Isolations were made from ascus part-spores, conidia and from hyphal material within the host and all grew in an identical manner.

Identification of endophytic hyphae of Lophodermium piceae in tissues of green, symptomless Norway spruce needles by immunoelectron microscopy

Antiserum specific for Lophodermium piceae hyphae was obtained by absorbing a rabbit L. piceae antiserum with hyphal material of different fungal isolates. The specificity of this absorbed antiserum was tested with hyphae of endophytic fungi isolated from green, asymptomatic needles of Norway spruce (Picea abies Karst.) using the on-section immunogold labeling technique. With this specific, absorbed antiserum, a homogenous labeling was obtained with cultured hyphae of L. piceae, whereas all other spruce needle endophytes examined remained practically unlabeled. Insignificant cross-reactivity was also observed with needle tissues. The absorbed antiserum was then applied for the immunoelectron microscopical identification of L. piceae hyphae in situ in infected tissues of green, asymptomatic Norway spruce needles. The function of papillalike structures observed in infected needle mesophyll as well as the role of the hyphal sheath surrounding endophytic L. piceae hyphae are discussed in connection with the interaction between L. piceae and asymptomatic Norway spruce needles.

Isolation of vesicular–arbuscular mycorrhizal spores: a rapid method for the removal of organic detritus from wet-sieved soil samples

A method for recovery of live vesicular–arbuscular mycorrhizal spores from large soil samples is presented. Spores, hyphal material, and other organic debris are isolated by wet sieving (> 63 μm) and further purified by flotation on 50% glycerol. The organic material is then suspended in Ringer's saline solution and the spores separated from other organic material by centrifuging (10 min at 75 × g) the mixture on a two-layer discontinuous solution gradient consisting of 50% glycerol (specific gravity 1.13 g/cm3) overlaid with 30% glycerol (specific gravity 1.08 g/cm3). The efficiency of recovery is 84 ± 3.4% of the spores present after the initial flotation.

Fusarium Oxysporum Endophthalmitis

To the Editor. —The article by Rowsey et al entitled Fusarium Oxysporum Endophthalmitis in the JanuaryArchives(97:103-105, 1979) raises some interesting points that deserve comment. A diagnosis of fungal infection was made initially when macroconidia, typical of Fusarium sp, were identified on a smear of material obtained at vitrectomy. This is a somewhat unusual circumstance, as we are not aware of Fusarium sporulation in mammalian tissue. It raises the possibility that these spores may have been part of the original inoculum. We would, therefore, be very interested to learn if hyphal material were seen on smear, because this would indicate with certainty that fungal proliferation had occurred within the eye. In the absence of hyphal material, the fascinating possibility needs to be considered that the spores inoculated at the time of injury failed to germinate within the eye, although they remained viable. Germination occurred only when they were

Fusarium Oxysporum Endophthalmitis-Reply

In Reply. —We appreciate the excellent comments of Dr O'Day and associates concerning our patient with F oxysporum endophthalmitis. We did not, in fact, observe hyphal material in the original vitrectomy specimen by phase contrast microscopy. The progressive deterioration of vision and inflammation suggested fungal growth prior to vitrectomy. During the original vitrectomy, several mass lesions were removed from the vitreous but the actual mass on the posterior aspect of the lens capsule was not disturbed; we believed that the risk to the lens was unjustified. Several of these mass lesions could certainly have been an inflammatory reaction to viable spores. The clinician should be aware that fungi do not normally sporulate in tissue. We hope that editors will more rapidly publish Dr O'Day's further fungal experimental and clinical data, for it will assist all clinicians in treating these difficult cases.

Experimental cerebral lesions produced by inoculation with Basidiobolus strains.

Two Basidiobolus strains produced progressive chronic granulomatous lesions in the brain following intracerebral inoculation of mice with hyphal material. Large septate hyphal bodies, together with sperical structures and granular material presumed to be of fungal origin, were present in the granuloma tissue surrounding the IIIrd and lateral ventricles. No progressive lesions developed when Basidiobolus mycelium was inoculated at extra-cerebral sites.