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STRUCTURE AND FORMATION OF CALCIUM OXALATE CRYSTAL DEPOSITS ON THE HYPHAE OF A WOOD ROT FUNGUS.
Abstract
A basidiomycete fungus was found growing on rotting Pinus ponderosa wood near Pagosa Springs, Colorado. This fungus produces large masses of calcium oxalate crystals in association with the hyphae. The calcium oxalate deposits take the form of druses, multiple crystal aggregates. Examination of the hyphae and druses by transmission electron microscopy shows that a crystal sheath covers the individual crystals. The crystal sheath is continuous with the external part of the hyphal cell wall, and zones of the wall, just below the druses, are substantially thinner than the adjacent cell wall. Results of this research support the hypothesis that crystals are produced by the hyphae and do not support the idea that the crystals are precipitated on the hyphal surface.
... Oxalate minerals are fundamental organic minerals widespread in nature, present in humans, animals and plants [3][4][5][6][7][8][9][10][11][12][13][14], as well as in naturally occurring minerals [15][16]. ...
... Evidence for the existence of primitive life forms like lichens and fungi can be based upon the formation of oxalates. These minerals form as the result of expulsion of heavy metals from fungi, lichens and plants [9][10][11][12], since these organisms can control their heavy metal intake through expulsion of metal salts such as oxalates. The production of simple organic acids as oxalic and citric acids has profound implications for metal speciation in biogeochemical cycles [12]. ...
Natroxalate mineral, Na2C2O4, is a fundamental oxalate mineral widespread in nature, present in humans, animals and plants, as well as in naturally occurring minerals. The characterization of oxalate minerals is extraordinarily important since these organic minerals are indicators of environmental events and of the presence of biological activity, because they are commonly of biological origin. These minerals are currently under study to investigate the possible biological activity on Mars. The identification of these compounds is usually performed by X-ray diffraction and Raman spectroscopy. Theoretical calculations are of great value for the study and interpretation of the results of these experimental techniques. In this work, natroxalate mineral structure and Raman spectrum was studied by first principle calculations based on the density functional theory. The computed structure of natroxalate reproduces the one determined experimentally by X-ray diffraction (monoclinic symmetry, space group P21/c; lattice parameters a = 3.449 Å, b = 5.243 Å; c = 10.375 Å). Lattice parameters, bond lengths, bond angles and X-ray powder pattern were found to be in very good agreement with their experimental counterparts. Raman spectrum was then computed by means of density functional perturbation theory and compared with the experimental spectrum. Since the results were also found in agreement with the experimental data, a normal mode analysis of the theoretical spectra was carried out and used in order to assign the main bands of the Raman spectrum. The band found at about 567 cm⁻¹, described as a single peak in previous experimental works, is shown clearly to have two contributing bands. Finally, two bands of the observed spectrum, located at the wavenumbers 1750 and 1358 cm⁻¹, were not found in the theoretical spectrum. This is because these bands correspond to an overtone, 2ν1 (ν1 = 875 cm⁻¹), and a combination band, ν1 + ν2 (ν1,ν2 = 875, 481 cm⁻¹), respectively. Finally, the fundamental thermodynamic properties of natroxalate mineral were determined. The calculated specific heat at 298.15 K is in excellent agreement with the experimental value, the difference being less than 1%. Since for most of these properties there are not experimental values to compare with, their values were predicted.
... Callot et al. (1985aCallot et al. ( , 1985b have suggested that microcrystalline calcite forms both within and on the external walls of fungal mycelia in calcareous soils. Mycorrhizal fungi living in forest litter and compost can secrete microcrystalline calcium oxalate (Foster and Marks 1967;Malajczuk and Cromack 1982;Arnott 1982;Arnott and Webb 1983). The oxalate crystallites adhere to both the hyphae and the fruiting bodies (Horner et al. 1983;Powell and Arnott 1985), and exhibit a variety of crystal habits, including acicular forms, which have different morphologies on adjacent hyphae. ...
... The oxalate crystallites adhere to both the hyphae and the fruiting bodies (Horner et al. 1983;Powell and Arnott 1985), and exhibit a variety of crystal habits, including acicular forms, which have different morphologies on adjacent hyphae. Arnott and Webb (1983) were able to establish that the hyphae produced the oxalate crystals within the cell wall, although the mechanism is not known. In living fungi, the crystals have been variously regarded as a means of removing excess Ca, as a means of conserving Ca in Cadeficient environments, as excess photosynthate, and as a defence against browsing microfauna. ...
Scanning electron microscope (SEM) studies of calcareous soils and calcretes from South Australia reveal a fossilized community of soil micro-organisms dominated by filamentous structures preserved in fine detail by calcite. In the various calcrete lithological facies, the filaments form dense mats within channels and voids, and also occur within the matrix where they are intimately associated with micrite. The calcite forming the filaments has a variety of crystal habits: the nature of the microcrystals is specific to each filament but varies significantly between adjacent filaments. In the calcareous soils there are various stages between the primary filaments and the calcite encrusted structures characteristic of the calcretes, suggesting that in vivo biochemical processes dominate the mechanisms of calcification. This hypothesis is supported by the specificity of the habit of calcite microcrystals on each filament. It is suggested that the organisms deposit calcite microcrystals within the mucilaginous sheath or in the cell wall (or both) as a detoxification mechanism in response to their highly calcareous environment. Based on the identification of structures resembling fruiting bodies, at least some of the filaments appear to have been fungal hyphae, which are known to be responsible for stabilizing macroaggregates in soils. Calcified filaments may produce permanently stabilized macroaggregates which provide the locus for further carbonate precipitation, leading to eventual induration of the soil.
... Oxalate minerals are widespread in nature and they may be found in animals, plants, and naturally occurring mineral assemblages [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15]. Organic minerals are mostly formed by inorganic natural processes but they may also be produced organically via biomineralization [16]. ...
In this study, the structural properties, main characteristics of the Raman spectrum, and the thermodynamic properties of the ammonium oxalate monohydrate mineral oxammite, were investigated in theoretical solid-state calculations based on the periodic density functional theory using plane waves and pseudopotentials. The optimized structure of oxammite agreed very well with that obtained from low temperature X-ray diffraction data by structure refinement (orthorhombic symmetry, space group P21 21 2; lattice parameters: = 8.017 Å; = 10.309 Å; = 3.735 Å). The calculated structural properties, including the lattice parameters, bond lengths and angles, and X-ray powder pattern, accurately reproduced the experimental information. The Raman spectrum determined theoretically agreed with that obtained experimentally. The assignment of the Raman spectral bands significantly improved their previous empirical assignment. Thus, the assignment of a large series of bands was modified and the origins of several previously unassigned bands were found. Five bands in the experimental spectrum at 2344, 2161, 1933, 1902, and 815 cm-1, were absent from the computed spectrum and they were identified as combination bands. The band located at 2879 cm-1 was confirmed as an overtone. Furthermore, the theoretical calculations clearly showed that some features described as single peaks in previous experimental studies were due to the contributions of several bands. The thermodynamic properties of the oxammite mineral, including the specific heats, entropies, enthalpies, and Gibbs free energies, were determined as functions of temperature. The specific heat calculated at 323 K, Cp= 202.3 J K-1mol-1, was in good agreement with the corresponding experimental heat capacity, = 211.7 J K-1 mol-1, where the values only differed by about 4%. Finally, using the computed thermodynamic data, the thermodynamic properties of the formation of oxammite as well as the free energies and reaction constants of the reaction for its thermal decomposition were determined.
... [37][38][39][40][41] They likely participate in the removal of heavy metals or the adsorption of water, as they do in other species. 42,43 The number and distribution of crystal idioblasts within the plant body vary among taxa, and some investigators have used the distribution of crystal idioblasts in classification. 27 However, according to Hartl et al., 12 the crystal idioblast distributions were not observed to follow a pattern that could be used to classify the Cactaceae; in the Pereskioideae, Maihuenioideae, and Opuntioideae, COM is predominant, whereas in the Cactoideae, COD prevails. ...
To find markers that distinguish the different Cactaceae species, by using near infrared Raman spectroscopy and scanning electron microscopy, we studied the occurrence, in the stem, of solid deposits in five Cactaceae species (Coryphantha clavata, Ferocactus latispinus,
Opuntia ficus-indica, O. robusta, and O. strepthacantha) collected from their natural habitats from a region of México. The deposits in the tissues usually occurred as spheroidal aggregates, druses, or prismatic crystals. From the Raman spectra, the crystals were
identified either as calcium oxalate monohydrate (CaC2O4·H2O) or calcium oxalate dihydrate (CaC2O4·2H2O).
Opuntia species (subfamily Opuntioideae) showed the presence of CaC2O4·H2O,
and the deposition of CaC2O4·2H2O was present in C. clavata and F. latispinus (subfamily Cactoideae, Cacteae tribe). As a punctual technique, Raman spectroscopy seems to be a useful tool to identify crystal composition. In addition to allowing
the analysis of crystal morphology, this spectroscopic technique can be used to identify Cactaceae species and their chemotaxonomy.
... In connection with respect to the origin of calcium oxalate, Whitney & Arnott (1986) mention two theories: (a) According to Graustein et al (1977) crystals formed when oxalic acid is excreted by the fungus and combine with calcium ions in the environment, (b) In agreement with Arnott (1982) and Arnott & Webb (1983), crystals develop inside the hyphae, grow to an intermediate stage in which the ends cause the extension of the hyphal wall; thus, crystals become more and more external and, finally, disassociate from the hyphae and sometimes collect in groups. However, additional studies with more adequately-preserved material and utilizing transmission electron microscopy are needed to answer the question of crystal origin. ...
... The presence of verrucose and large lenticular crystals on Piloderma hyphae is similar to ornaments of other fungi (e.g., Cromack et al. 1979;Arnott and Webb 1983;Whitney and Arnott 1987;Lapeyrie et al. 1990;Jones et al. 1992;Connolly and Jellison 1994). For example, Resinicium bicolor (Abertini and Schwein: Fr.) (Connolly and Jellison 1994) and Agaricus bisporus (Whitney and Arnott 1987) have styloid crystals 5-40 µm in size and elongated rod-like to plate-like crystals. ...
Piloderma is a broad host range ectomycorrhizal fungal genus that may benefit conifer growth through increased soil nutrient availability via enhanced soil mineral weathering. In an in vitro study, we investigated the ability of Piloderma to extract K and/or Mg from three soil minerals commonly found in soils of central British Columbia: biotite, microcline, and chlorite. The growth, hyphal morphology, and chemical composition were compared among Piloderma grown for 110 days in media optimized for fungal growth as well as in media where K and/or Mg were supplied from biotite, microcline, and chlorite. Piloderma grown in treatments with low K showed fibrillar growths, hyphal swellings, and hyphae devoid of ornamentation, possibly indicating nutrient deficiency. Differences were found in growth rates, morphologies, and Mg content in hyphae grown in chlorite and biotite treatments, suggesting that Mg was limiting to the normal growth of Piloderma . Energy dispersive X-ray analysis indicated that Piloderma extracted significantly more K from biotite than from microcline. The high Ca and O content of hyphal ornamentation were mainly composed of Ca-oxalate crystals. The study indicated that K and Mg are essential for vigorous Piloderma growth and that Piloderma may provide more available K to host plant through accelerated weathering of biotite, compared to microcline and chlorite sources. The differences were attributed to the ability of Piloderma to efficiently extract K from the interlayer of biotite. However, the exact mechanism by which Piloderma supplies plant roots with K extracted from biotite is still largely unknown.
... Introduction 1977; Klappa 1979; Phillips et al. 1987; Wright 1989; Monger et al. 1991 Graustein et al. 1977; Cromack et al. 1979; Arnott & Webb 1983; Horner et al. 1983; Verrecchia et al. 1993)Cromack et al. 1979; Verrecchia et al. 1993Graustein et al. 1977; Cromack et al 1977; de la Torre et al. 1993; Johnston & Vestal 1993 ...
Fungal filaments are the most abundant organic features in weathered profiles developed on chalky limestone ("platy calcrete"). Their activity affects the mineral dynamics of the pore/carbonate microsystem. A theoretical biogeochemical model is proposed to describe the Ca-oxalate-carbonate cycle related to fungal activity in dry environments.
The system studied is the pore itself (defined as the reactor) delimited by its wall and its content: solutions, gases (air and CO2), microorganic material, their transformation products and the minerals present (calcite and calcium oxalate). The system exchanges gas and solution with the outside environment, which includes micritic calcite, solutions, a gaseous phase (air and CO2), and the nanoporosity of the pore wall constituted by the micritic matrix. A diagram of pH = f(log |Ca2+|) is constructed to simulate the behaviour of various fungal excretions, whether the system is open or semi-closed. Two steps are studied. In the first step, the fungi is in full metabolic activity, and assumed to secrete i) an organic diacid (BH2) of soluble calcium salt, or ii) oxalic acid, or iii) soluble sodium oxalate, or iv) CO2. In the second step, bacteria transform the oxalates into carbonates. In the first step, the model concurs with petrographic observations, on the condition that the system is semi-closed and the aggressive agent produced by the hypha is mostly oxalic acid (COOH)2. In the second step, the pore solution becomes saturated in calcite, whether the system is open or semi-closed. This explains the calcium carbonate precipitation inside the pore as needles or microsparite and impregnation of the micritic matrix around the pores. In conclusion, the presence of fungi allows a redistribution of calcium carbonate. This secondary cementation is strong in the case of recrystallization of pore walls and weaker when infilling voids with needles.
... When considering the boreal forest with needle and leaf litter combined, a significant and negative relationship was found between Ca concentration and mass loss; indeed at this site leaf litter with the highest Ca concentrations decomposed less than needle litters with lower Ca concentration. It is well known that calcium oxalate is a common component of fungal cell walls in a variety of fungi (Arnott and Webb 1983); besides ectomycorrhizal fungi, associated with a wide variety of coniferous and hardwood species, occurring in decaying wood, in fragmented litter and in acid humus, have calcium-rich hypha encrustations (Arocena et al. 2001). According to Arnott (1986, 1987) calcium oxalate provides a hydrophobic coating preventing hyphae from becoming hydrated and thus reducing microbial attack; the calcium richer leaf litters might support a larger colonization by fungal populations with calcium-rich hyphae encrustations hampering decomposition and this could explain the inverse relationship we have found between calcium concentration and decomposition rates in the boreal forest. ...
We studied late-stages decomposition of four types of coniferous needle and three types of deciduous leaf litter at two sites,
one nutrient-poor boreal and one nutrient-rich temperate. The late stage was identified by that reached by litters at the
onset of net loss of lignin mass, i.e. at about 1year after the incubation when the highest amount of lignin had been detected;
the study extended over the following 2year period. Decomposition rates were significantly lower at the boreal than at the
temperate site and did not differ between needle litter and leaf litter. In the boreal forest: (1) mass-loss was positively
correlated with N and Mn release, (2) Mn concentration at the start of the late stage was positively correlated with lignin
decay, (3) Ca concentration was negatively correlated to litter mass loss and lignin decay. In the temperate forest neither
lignin, N, Mn, and Ca concentration at the start of the late stage, nor their dynamics were related to litter decomposition
rates and lignin decay. In leaf litter mass-loss and lignin decay were positively correlated with N and Ca release and with
Ca concentration. In needle litter mass-loss was positively correlated to Mn release and N concentration negatively with lignin
decay. We concluded that Ca, N and Mn have different roles in controlling lignin decay depending on type of litter and site
conditions.
... The mechanism brown rot fungi employ to withstand their acidic environment is not known. However previous reports of glucan (fungal or hyphal sheath) production which enveloped and then exposed oxalate crystals produced by wood degrading fungi (79) suggest the possibility that the extracellular glucan matrix secreted by wood degrading fungi may not only help to maintain the pH of the fungal environment, but help protect the organism in acidic environments as well. ...
This overview focuses on fungal attack and degradation of wood by the brown rot fungi. These fungi are perhaps the most important agents involved in the degradation of wood products and in the degradation of dead wood in coniferous ecosystems. In the decay process, brown rot fungi actively metabolize the carbohydrate fraction as well as a more minor portion of lignin, leaving behind a chemically modified lignin residue. The wood loses strength very rapidly in the early stages of degradation because of the rapid depolymerization of the cellulosic fraction. More heavily degraded wood typically displays a cubical, crumbly, brown appearance with little residual strength. Mechanisms employed by brown rot fungi in the biodegradation of wood are both enzymatic and nonenzymatic. These fungi produce no lignin degrading enzymes, but they do have a mechanism that results in lignin modification and slow lignin depletion from wood undergoing decay. Recent work by several groups has suggested that nonenzymatic, low molecular agents produced by the brown rot fungi are responsible for early stages of wood cell wall depolymerization through the production of free radical species. Similarities and differences between brown rot fungi, white rot fungi, and molds are discussed with regard to pH contorl in wood, oxalate production, and metal metabolism. Development of several hypotheses for low molecular weight metabolite function and systems postulated for non-enzymatic degradative activities are reviewed.
... It is generally presumed that CaOx crystals form on the surface of fungal hyphae as a result of precipitation when released oxalic acid interacts with calcium cations (23,43). However, the regularity of the CaOx crystals suggests that their formation is regulated and that they may be formed within the fungal hyphae at specific sites of origin (3,5,7). ...
Piloderma fallax is an ectomycorrhizal fungus commonly associated with several conifer and hardwood species. We examined the formation of
calcium oxalate crystals by P. fallax in response to calcium (0.0, 0.1, 0.5, 1, and 5 mM) and phosphorus (0.1 and 6 mM) additions in modified Melin-Norkrans agar
medium. Both calcium and phosphorus supplementation significantly affected the amount of calcium oxalate formed. More calcium
oxalate was formed at high P levels. Concentrations of soluble oxalate in the fungus and medium were higher at low P levels.
There was a strong positive linear relationship between Ca level and calcium oxalate but only under conditions of phosphorus
limitation. Calcium oxalate crystals were identified as the monohydrate form (calcium oxalate monohydrate [COM] whewellite)
by X-ray diffraction analysis. Prismatic, styloid, and raphide forms of the crystals, characteristic COM, were observed on
the surface of fungal hyphae by scanning electron microscopy. P. fallax may be capable of dissolving hyphal calcium oxalate under conditions of limited Ca. The biomineralization of calcium oxalate
by fungi may be an important step in the translocation and cycling of Ca and P in soil.
Basidiocarps of Geastrum saccatum have calcium oxalate crystals associated with the peridium. In mature, opened basidiocarps eroded crystals occur on the outer surface of the endoperidium. Examination of undehisced basidiocarps reveals that bipyramidal calcium oxalate crystals arise from, and remain associated with, the hyphae that form the endoperidial layer. The development of calcium oxalate crystals in the endoperidial layer prior to basidiocarp dehiscence suggests that the formation of these crystals may be related to the process of exo- and endoperidial separation during basidiocarp maturation.
Walls of sporangiophores and sporangia of Gilbertella persicaria contain conspicuous deposits of calcium oxalate. The morphology of these crystalline deposits varies with location on the fruiting structure. Deposits over most of the sporangiophore consist of elongate, flattened plates embedded in the sporangiophore wall, and these plates often bear single or paired, upright appendages. In the area of transition between sporangiophore and sporangium the crystals consist of short, verrucose projections borne on flattened, polygonal basal plates. Crystals on the sporangium proper are morphologically complex, composed of an apical, flattened, polygonal cap, an angular, upright column, and a flattened, polygonal base plate. Scanning electron micrographs and X-ray elemental analysis of these crystals are presented. The morphology of these crystals and their relationship to sporangiophore and sporangium development are discussed, along with speculations on the function of these deposits.
Aerial hyphae of Agaricus bisporus grown in agar culture, or on natural substrates like rye grain, produce abundant calcium oxalate crystals associated with the hyphal wall. These deposits, when first formed, consist of acicular crystals that cover the surface of the hyphae, giving the elements of the aerial mycelium a bottle-brush appearance. SEM examination of the crystal-bearing hyphae reveals that the crystals are arranged more or less tangentially on the hyphal surface. The crystals appear to originate within the wall of the hyphae, and as they increase in length their distal ends protrude through the hyphal wall. While the crystalline deposits of hyphae grown on malt-extract agar or on rye grain are typically elongate crystals, calcium oxalate deposits of hyphae grown on enriched media consist of both elongate forms and large, plate-like crystals. Scanning and transmission electron micrographs, energy-dispersive x-ray elemental analysis, and x-ray powder diffraction analysis of these crystals are presented.
Mycelium formation and oxalate production by two dsRNA-free, virulent strains and three dsRNA-containing, hypo virulent strains of Cryphonectria (Endothia) parasitica were evaluated after 10 days incubation on agar media containing either 0.010 M or 0.025 M calcium acetate, or no calcium acetate. The dsRNA-containing strains EP 31–2P and EP-752 consistently produced the least mycelium while the dsRNA-free strain EP-155 produced the most mycelium on these three media. Addition of 0.010 M calcium acetate did not affect the amount of growth by strain EP-155 or strain EP-752, enhanced growth of the dsRNA-free strain EP-42, and reduced growth of the dsRNA-containing strains EP 17–8B and EP 31–2P. Growth of all strains was reduced on the medium containing 0.025 M calcium acetate. Strain EP-155 produced the greatest total oxalate on these media, strains EP-42 and EP 17–8B produced significantly less, and dsRNA-containing strains, EP 31–2P and EP-752, produced the least. Formation of oxalate by C. parasitica may have a regulatory role in calcium metabolism and/or oxalic acid production.
Crystals of Ca-oxalate are produced on vegetative or reproductive structures by members of each non-flagellate fungal class — the Ascomycetes, Basidiomycetes, and Zygomycetes. These crystals originate within the hyphal wall, distorting the outer wall layers during subsequent crystal growth. Crystals commonly occur on the vegetative mycelium of leaf and wood-rotting Basidiomycetes, both on field-collected and on cultured material. In culture, crystals are typically produced only on the aerial mycelium, not on the substrate mycelium. This suggests that calcium is translocated from the substrate mycelium via the fungal protoplast to the aerial mycelium, where it is then precipitated with oxalic acid. Mucoralean Zygomycetes also produce mineralized deposits on their aerial sporangia and sporangiophores, again suggesting that calcium is transported from the substrate and immobilized on aerial portions of the fungal thallus. Further, in the zygomycete Gilbertella persicaria, the reduction of calcium levels by Ca-oxalate crystallization coincides with an increased rate of mycelial growth. Thus, relatively high levels of calcium in the media appear to inhibit the growth of Gilbertella persicaria. The precipitation of Ca-oxalate may serve as a way for fungi to regulate or reduce the calcium ion concentration in their microenvironment.
Wood decay experiments using red spruce wood resting on moist soil were conducted to discern temporal and spatial patterns of calcium oxalate (CaOx) crystal production by three species of fungi over the course of decay. All three species produced crystals of calcium oxalate dihydrate, but not monohydrate, in and on wood. Over the course of decay, the production of CaOx crystals was shown to be heterogeneous in both space and time. The relative quantity, morphology and longevity of CaOx crystals varied among species. Gloeophyllum (G.) trabeum produced substantial quantities of 'free' crystals; Fomitopsis (F.) pinicola produced encrusting crystals; and Trichaptum (T.) abietinum produced adhering crystals and druses. Paramorphic corrosion of crystals was observed most frequently in the brown rot fungus G. trabeum and not at all in the white-rot fungus T. abietinum. Often associated with crystal precipitations was the production of crystal surface-obscuring extracellular matrix. All the species observed produced CaOx crystals more consistently in or on soil than in wood. The study of crystal production patterns and crystal morphologies could yield important information about the microenvironmental conditions in wood during biodegradation and the mechanisms by which wood decay fungi decompose lignocellulose.
Biomineralization is the process by which living forms influence the precipitation of mineral materials. The process creates heterogeneous accumulations, composites composed of biologic (or organic) and inorganic compounds, with nonhomogeneous distributions that reflect the environment in which they form. It is only with the advent of more sensitive, higher-resolution techniques that we can identify the exact mineral components, and appreciate the precision and control life-form exercise on their mineralized structures, and the potential that their formation and evolution are responses to the environment and climatic or any other pervasive geochemical changes.
The mineralogy and ecology of the crystals that occur at the microbe-mineral interface, and the evolution of minerals around calcified filaments in a calcretized calcarenite from Temara (Rabat south, Morocco) are the focus of this study. From X-ray diffraction (XRD), high-resolution field emission scanning electron microscopy (FESEM) and atomic force microscopy (AFM) studies, it is apparent that complexity of the interface between mineral-microbe can be investigated at the nanometre scale. Previous workers proposed a model for the evolution of the fungal filament biomineralization that describes the episodic modification of weddellite into whewellite, a phase absent from the present study. The common association of carbonate phases with microorganisms suggests that the organisms enhance conditions suitable for the growth of morphologically diverse crystal forms. A nanocrystalline calcium carbonate phase maybe a transient precursor phase of calcite mediated by the lichen Xanthoria parietina. Despite extensive studies on biomineralization, little is known about the causes of polymorph selection during fungal oxalate mineralization in nature.
Identification of mycelia from the field to the species level is often dependent on the presence of reproductive structures or upon the ability to isolate and study cultures of the organism(s). Some wood decay fungi produce elaborate calcium oxalate crystals which could aid in the identification of fungal mycelium within rotting wood. Decomposing red spruce wood from four sites in northern New England was examined by scanning electron microscopy, and the morphology of the observed crystals was compared to the morphology of the crystals produced in pure culture by fungi isolated and identified from the same pieces of wood. The conclusion was that there is insufficient species specificity in crystal morphology for it, alone, to be used to identify fungi resident in red spruce wood in nature.
Hyphae of many species of bird's nest fungi (Nidulariaceae) were examined for the presence of crystals. Scanning electron microscopy revealed crystals typical of calcium oxalate associated with hyphae. Crystal depositions included complete hyphal encrustation, organized druses, randomly arranged groups, single crystals, and relatively "clean" hyphae. The majority of crystals could be morphologically classified as styloid, raphide, or bipyramidal type. Energy-dispersive X-ray microanalyses confirmed that all crystal types were rich in calcium, and X-ray diffraction identified both the dihydrate (weddellite) and monohydrate (whewellite) forms of calcium oxalate. Morphological variation of crystals was species and medium specific, with generally more crystals being produced on relatively calcium-rich V8 juice agar than on PDA. No association was found between crystal types and geographic and habitat distribution of species. Keywords: wood rotting fungus, decomposition, calcium oxalate.
Oxalate secretion by fungi provides many advantages for their growth and colonization of substrates. The role of oxalic acid in pathogenesis is through acidification of host tissues and sequestration of calcium from host cell walls. The formation of calcium oxalate crystals weakens the cell walls, thereby allowing polygalacturonase to effect degradation more rapidly in a synergistic response. There is good correlation between pathogenesis, virulence, and oxalic acid secretion. Solubility of soil nutrients is achieved by soil-living species, when cations freed by oxalate diffusing in clay layers increases the effective solubility of Al and Fe. Oxalate retained in hyphal mats of mycorrhizal species increases phosphate and sulphate availability. The formation of calcium oxalate crystals provides a reservoir of calcium in the ecosystem. The ability of oxalate to bind divalent cations permits detoxification of copper, particularly evident in wood preserved with copper salts. Oxalate plays a unique role in lignocellulose degradation by wood-rotting basidiomycetes, acting as a low molecular mass agent initiating decay. In addition, in white-rot fungi oxalate acts as a potential electron donor for lignin-peroxidase catalysed reduction and chelates manganese, allowing the dissolution of Mn3+ from the manganese–enzyme complex and thus stimulating extracellular manganese peroxidase activity. The biosynthesis and degradation of oxalate are discussed.Key words: oxalic acid, calcium oxalate, pathogenicity, fungi.
The white-rot wood decay fungus Resinicium bicolor (Abertini & Schwein.: Fr.) Parmasto was studied for its ability to solubilize and translocate ions from the naturally occurring mineral strontianite. Resinicium bicolor colonized a soil mixture culture medium containing strontianite sand, solubilized strontium ions from this mineral phase, translocated the ions vertically, and reprecipitated the strontium into strontium-containing calcium oxalate crystals. Storage of the Sr in crystals was highest in mycelial cords and was dynamic in character. These results suggest that non-mycorrhizal saprotrophic fungi should be evaluated for their potential participation in forest nutrient cycling via biologically weathering parent material and translocating the mobilized mineral nutrients vertically within soils.Key words: fungi, strontium, calcium oxalate, translocation, soil, minerals nutrient cycling.
Monotropa uniflora is an achlorophyllous angiosperm that is obligately mycotrophic. The "monotropoid" mycorrhizae it forms resemble ectomycorrhizae but are distinguished by elaborations of the epidermal cell walls that surround intruding fungal hyphae. Monotropoid mycorrhizae collected from blooming plants in late summer contained calcium oxalate crystals between mantle hyphae. The crystals appeared to form in association with hyphal walls and grew into a matrix outside the hyphae. Production of calcium oxalate by M. uniflora's mycobiont seems to be a coordinated metabolic process rather than a random precipitation event. The significance of calcium translocation and isolation as calcium oxalate to this mycorrhizal fungus is unclear, but the presence of extensive crystal deposits during and after flowering of the host plant suggests a possible link with the nutrient transfer occurring at that time. Mycorrhizal regulation of calcium may affect the availability of mineral nutrients to the associated Monotropa plants. Key words: Monotropa uniflora, mycorrhiza, calcium oxalate, ectomycorrhiza.
Mycelial micro- and macromorphology and growth characteristics, laccase activity tests, pigmentation, teleomorph formation, and genetic variability of 31 collections of the edible medicinal mushroom Flammulina velutipes have been investigated. The strains were isolated from fruiting bodies collected in different geographical regions and wood substrates. Two morphological types (A, B) and three subtypes (A1, A2, A-B) of mycelial colonies with different growth parameters were described in F. velutipes collections on different agar media. Correlations between morphological types and growth characteristics of colonies with substrate nature and geographical origination of strains have been revealed. However, in order to evaluate species-specific media responses for the described mycelial morphotypes, further research with more strains is required. Mycelial macromorphology and growth parameters were more variable than the described microstructures (form, shape of clamp cells and crystals, asexual spores). Significant correlation between the mycelial morphotypes and genetic variability of screened F. velutipes strains has not been revealed.
Genes for morphological and cultural variability may be unlinked to genes for the nuclear ribosomal repeat. Collections of putative F. velutipes were confirmed by rDNA-ITS sequencing to be F. velutipes with the exception of four collections as follows: SB71 (Armenia) is a hybrid or unknown biotype; collections SBIV-1 (Armenia) and SB317 (Germany) are identified as F. elastica; and R12 (Russia) as F. rossica. High genetic diversity was observed in Armenian collections of F. velutipes when compared to collections from Eurasia. At least 16 haplotypes were recovered in Armenian collections. This sequence diversity may be a consequence of the survival of an ancient genetic variation in the Caucasus and Armenia during the glaciation period, while in Europe, the genetic variation was extirpated. A subset of Armenian genetic diversity of F. velutipes is found in Europe.
Morphological and growth characteristics of mycelium will assist in further biotechnological cultivation of F. velutipes. High genetic variability of Armenian collections may be used for improvement of strains to obtain novel biotech products and health-enhancing functional food additives from this valuable medicinal mushroom.
Biomineralization is the process by which living forms influence the
precipitation of mineral materials. The process creates heterogeneous
accumulations, composites composed of biologic (or organic) and
inorganic bcompounds, wbth inhom++++ogeneous distributions that reflect
the environment in which they form. The products are, however,
disequilibrium assemblages, created and maintained during life by
dynamic metabolism and which, on death, may retain some of the original
characteristics. The living forms discussed in this chapter produce
carbonate, phosphate, oxalate, silica, iron, or sulfur-containing
minerals illustrating the remarkable range of biomineralization
chemistries and mechanisms. Biomineralization, in the broadest use of
the term, has played a role in Earth cycles since water appeared on the
surface. The general perception that the onset of biomineralization
coincides with the appearance of fossils that left "hard parts" amenable
to analysis is marked geologically as the dawn of the Cambrian. At least
for some of us, the origins of life, and possibly biomineralization, go
back 3.8 Gyr. The startling chemical and biological range encompassed by
the term biomineralization implies that life forms have adapted and
altered geoenvironments from the beginning, and will continue to do so,
an essential ingredient for establishing and maintaining Earth's
environment in the future as in the past (Figure 1).
The thermal decomposition of natural ammonium oxalate known as oxammite has been studied using a combination of high resolution thermogravimetry coupled to an evolved gas mass spectrometer and Raman spectroscopy coupled to a thermal stage. Three mass loss steps were found at 57, 175 and 188°C attributed to dehydration, ammonia evolution and carbon dioxide evolution respectively. Raman spectroscopy shows two bands at 3235 and 3030 cm-1 attributed to the OH stretching vibrations and three bands at 2995, 2900 and 2879 cm-1, attributed to the NH vibrational modes. The thermal degradation of oxammite may be followed by the loss of intensity of these bands. No intensity remains in the OH stretching bands at 100°C and the NH stretching bands show no intensity at 200°C. Multiple CO symmetric stretching bands are observed at 1473, 1454, 1447 and 1431cm-1, suggesting that the mineral oxammite is composed of a mixture of chemicals including ammonium oxalate dihydrate, ammonium oxalate monohydrate and anhydrous ammonium oxalate.
Species in Trechispora with a poroid hymenophore are described. The monotypic genus Echinotrema is regarded as a synonym and the new combination Trechispora clancularis is proposed. Collections determined as Trechispora mollusca can be divided in three groups supported by macromorphology of basidiomata, micromorphology of hyphae and crystals, and ultrastructure of spores. The groups correspond to species and should be named T. candidissima, T. hymenocystis and T. mollusca. Energy dispersive X-ray microanalysis shows that crystals on hyphae in these three species contain calcium, presumably as calcium oxalate. In each species, crystals show a different and constant morphology which seems to be under genetical control. Crystal morphology is the most reliable separating character. A key to some poroid species with ornamented spores in Corticiaceae is provided.
Piloderma species are broad-host-range fungi associated with a wide variety of conifer and hardwood species to form ectomycorrhizae
(ECM). In this study, we investigated the hypha crystals collected from Piloderma– Picea glauca× engelmannii ECM as an initial step in the elucidation of the role of mycorrhizal fungi in mineral weathering and nutrient cycling. We
compared the morphology and composition of hypha encrustation between field and cultured Piloderma samples. For field samples, the morphology of encrustations was dominated by elongated crystals often underlain by verrucose
crystals. Cultured samples had mostly verrucose crystals. Generally, encrustations on field samples had higher calcium contents
than cultured samples. Calcium contents ranged from 3% in the verrucose crystals of cultured samples to 17% in verrucose and
elongated crystals of field samples. Encrustations had infrared absorption bands at 1333 and 781 cm–1 wavenumbers, indicative of the presence of oxalate. High amounts of C and O in verrucose crystals are likely associated with
the crystal sheath around all encrustations. This composition suggests an intracellular origin for the crystals. It is possible
that encrustations start as verrucose crystals and develop into euhedral elongated crystals susceptible to dislod- gement
into the soil environment. These crystals may prevent the desiccation of the hyphae and inhibit the build-up of calcium and
oxalate in fungus cells.
Extensive, uniform, yellow-brown films are observed on many monuments. The origin of these films, composed predominantly of calcium oxalate, has been investigated by several authors. Oxalate film formation may be related, in some cases, to the activity of such microorganisms as fungi, which presumably form oxalic acid via the metabolic transformation of organic substances already present on the stone. The present work provides an overview of the physiological factors affecting oxalate synthesis by fungi and of oxalic acid in fungi metabolism.
The design of environmentally benign methods for preserving wood in service requires an understanding of the precise sequence of the biochemical events that occur as wood is colonized. We hypothesize that in-situ precipitation of existing calcium ions in association with pectin in wood may prevent the cascade of biochemical events involved in fungal colonization. Preliminary experiments showed that pretreatment of wood blocks with the selective water-soluble calcium-precipitating agent N,N-naphthaloylhydroxylamine (NHA) inhibited decay caused by brown-rot and white-rot fungi as well as damage caused by eastern subterranean termites.
In terrestrial environments, fungi constitute one of the most important microbial agents, but also due to their ability to decay organic matter but also because of their role in various biogeochemical cycles (Gadd, 1999; Verrecchia, 2000). Fungal involvement in carbonate rock destruction, dissolution and precipitation has been suspected for over 150 years (Braconnot, 1825) but convincing hypotheses to explain the processes of carbonate dissolution and precipitation were not proposed before the W’s. Nevertheless, their role in the calcium carbonate cycle remains largely underestimated. The aim of this chapter is to emphasize the great potential of fungi in CaCO3 redistribution and sequestration at the surface of continents.
Evidence for the existence of primitive life forms such as lichens and fungi can be based upon the formation of oxalates. Oxalates are most readily detected using Raman spectroscopy. A comparative study of a suite of natural oxalates including weddellite, whewellite, moolooite, humboldtine, glushinskite, natroxalate and oxammite has been undertaken using Raman spectroscopy. The minerals are characterised by the Raman position of the CO stretching vibration which is cation sensitive. The band is observed at 1468 cm-1 for weddellite, 1489 cm-1 for moolooite, 1471 cm-1 for glushinskite and 1456 cm-1 for natroxalate. Except for oxammite, the infrared and Raman spectra are mutually exclusive indicating the minerals are bidentate. Differences are also observed in the water OH stretching bands of the minerals. The significance of this work rests with the ability of Raman spectroscopy to identify oxalates which often occur as a film on a host rock. As such Raman spectroscopy has the potential to identify the existence or pre-existence of life forms on planets such as Mars.
The production of organic acids by fungi has profound implications for metal speciation, physiology and biogeochemical cycles. Biosynthesis of oxalic acid from glucose occurs by hydrolysis of oxaloacetate to oxalate and acetate catalysed by cytosolic oxaloacetase, whereas on citric acid, oxalate production occurs by means of glyoxylate oxidation. Citric acid is an intermediate in the tricarboxylic acid cycle, with metals greatly influencing biosynthesis: growth limiting concentrations of Mn, Fe and Zn are important for high yields. The metal-complexing properties of these organic acids assist both essential metal and anionic (e.g. phosphate) nutrition of fungi, other microbes and plants, and determine metal speciation and mobility in the environment, including transfer between terrestrial and aquatic habitats, biocorrosion and weathering. Metal solubilization processes are also of potential for metal recovery and reclamation from contaminated solid wastes, soils and low-grade ores. Such 'heterotrophic leaching' can occur by several mechanisms but organic acids occupy a central position in the overall process, supplying both protons and a metal-complexing organic acid anion. Most simple metal oxalates [except those of alkali metals, Fe(III) and Al] are sparingly soluble and precipitate as crystalline or amorphous solids. Calcium oxalate is the most important manifestation of this in the environment and, in a variety of crystalline structures, is ubiquitously associated with free-living, plant symbiotic and pathogenic fungi. The main forms are the monohydrate (whewellite) and the dihydrate (weddelite) and their formation is of significance in biomineralization, since they affect nutritional heterogeneity in soil, especially Ca, P, K and Al cycling. The formation of insoluble toxic metal oxalates, e.g. of Cu, may confer tolerance and ensure survival in contaminated environments. In semi-arid environments, calcium oxalate formation is important in the formation and alteration of terrestrial subsurface limestones. Oxalate also plays an important role in lignocellulose degradation and plant pathogenesis, affecting activities of key enzymes and metal oxido-reduction reactions, therefore underpinning one of the most fundamental roles of fungi in carbon cycling in the natural environment. This review discusses the physiology and chemistry of citric and oxalic acid production in fungi, the intimate association of these acids and processes with metal speciation, physiology and mobility, and their importance and involvement in key fungal-mediated processes, including lignocellulose degradation, plant pathogenesis and metal biogeochemistry.
In many patients with sarcoidosis, the granulomas contain inclusion bodies within giant cells. Many giant cells contain crystalline oxalate that chemically coordinates iron on the surface of the crystal. If this iron is incompletely coordinated and capable of redox cycling, then oxalate might contribute to granuloma formation in the lung.
Using human tissues, isolated alveolar macrophages and respiratory epithelial cells, we measured the ability of calcium oxalate to sequester iron, stimulate cytokine release and cause granuloma formation. We then studied the effects of in vivo oxalate instillation on pulmonary granuloma formation over 3 to 6 months in rats.
Calcium oxalate present in human sarcoid granulomas sequesters significant amounts of iron and ferritin. In alveolar macrophage cultures, oxalate accumulates iron and stimulates ferritin production and giant cell formation. In cultured respiratory epithelial cells, calcium oxalate increases the release of two interleukins (IL), IL-8 and IL-6, involved in granuloma formation by 8 to 10 fold within 24 hours. Intratracheal instillation of calcium oxalate crystals into the lungs of rats is associated with pulmonary iron and ferritin accumulation and organic carbonyl formation consistent with sustained oxidative stress. These exposures were accompanied by influx of alveolar macrophages, giant cell formation, and a granulomatous response in the lung.
These results support an association between calcium oxalate deposition in the lung, iron mediated oxidative stress and formation of some of the granulomas of sarcoidosis.
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