Fungal Sheath Research Articles

Characterization of carbohydrate-binding cytochrome b562 from the white-rot fungus Phanerochaete chrysosporium.

cDNA encoding a hemoprotein similar to the cytochrome domain of extracellular flavocytochrome cellobiose dehydrogenase (CDH) was cloned from the white-rot fungus Phanerochaete chrysosporium. The deduced amino acid sequence implies that there is a two-domain structure consisting of an N-terminal cytochrome domain and a C-terminal family 1 carbohydrate-binding module (CBM1) but that the flavin-containing domain of CDH is not present. The gene transcripts were observed in cultures in cellulose medium but not in cultures in glucose medium, suggesting that there is regulation by carbon catabolite repression. The gene was successfully overexpressed in Pichia pastoris, and the recombinant protein was designated carbohydrate-binding cytochrome b562 (CBCyt. b562). The resonance Raman spectrum suggested that the heme of CBCyt. b562 is 6-coordinated in both the ferric and ferrous states. Moreover, the redox potential measured by cyclic voltammetry was similar to that of the cytochrome domain of CDH. These results suggest that the redox characteristics may be similar to those of the cytochrome domain of CDH, and so CBCyt. b562 may have an electron transfer function. In a binding study with various carbohydrates, CBCyt. b562 was adsorbed with high affinity on both cellulose and chitin. As far as we know, this is the first example of a CBM1 connected to a domain without apparent catalytic activity for carbohydrate; this CBM1 may play a role in localization of the redox protein on the surface of cellulose or on the fungal sheath in vivo.

The fungal sheath of ectomycorrhizal pine roots: an apoplastic barrier for the entry of calcium, magnesium, and potassium into the root cortex?

The apoplastic permeability of the fungal sheath of two different ectomycorrhizal associations of Pinus sylvestris L. was analysed by laser microprobe mass analysis (LAMMA) and energy-dispersive X-ray spectroscopy (EDXS) after stable isotope labelling with 25Mg, 41K and 44Ca. Entry of 25Mg and 44Ca into the outer cortical apoplast of non-mycorrhizal roots was detected after 4 min of labelling. After a longer exposure time the endodermis with its Casparian band acted as an efficient apoplastic diffusion barrier for the radial movement of 25Mg and 44Ca into the stele. A fraction of approximately one-third of the apoplastic cations of the root cortex could not be exchanged against the external label even after longer exposure times. The ectomycorrhizal sheath of the two fungal species used, Pisolithus tinctorius (Pers.) Coker & Couch and Suillus bovinus (L. ex Fr.) Kuntze, does not completely inhibit the apoplastic movement of ions into the mycorrhizal root cortex, but retarded the penetration of isotopes into the cortical apoplast. In roots inoculated with S. bovinus, a clear labelling of the cortical apoplast could first be detected after 24 h of exposure to the stable isotope solution. At this time the labelling of the cortical apoplast in these mycorrhizal roots was higher than those of non-mycorrhizal roots and, with EDXS, changes in the element composition of the apoplast were detected. The results indicated that possibly hydrophobins localized in the fungal cell wall might be involved in the increased hydrophobicity of mycorrhizal roots and the lower permeability of the ectomycorrhizal sheath.

Colonization with Hebeloma crustuliniforme increases water conductance and limits shoot sodium uptake in white spruce ( Picea glauca ) seedlings

White spruce [Picea glauca (Moench) Voss] seedlings were inoculated with Hebeloma crustuliniforme and treated with 25 mM NaCl to examine the effects of salinized soil and mycorrhizae on root hydraulic conductance and growth. Mycorrhizal seedlings had significantly greater shoot and root dry weights, number of lateral branches and chlorophyll content than non-mycorrhizal seedlings. Salt treatment reduced seedling growth in both non-mycorrhizal and mycorrhizal seedlings. However, needles of salt-treated mycorrhizal seedlings had several-fold higher needle chlorophyll content than that in non-mycorrhizal seedlings treated with salt. Mycorrhizae increased N and P concentrations in seedlings. Na levels in shoots and roots of salt-treated mycorrhizal seedlings were significantly lower and root hydraulic conductance was several-fold higher than in non-mycorrhizal seedlings. A reduction of about 50% in root hydraulic conductance of mycorrhizal seedlings was observed after removal of the fungal hyphal sheath. Transpiration and root respiration rates were reduced by salt treatments in both groups of seedlings compared with the controls, however, both transpiration and respiration rates of salt-treated mycorrhizal seedlings were as high as those in the non-mycorrhizal seedlings that had not been subjected to salt treatment. The reduction of shoot Na uptake while increasing N and P absorption and maintaining high transpiration rates and root hydraulic conductance may be important resistance mechanisms in ectomycorrhizal plants growing in salinized soil.

Excretion of two proteases by the ectomycorrhizal fungus Amanita muscaria

AbstractIn the soils of mature forests, nitrogen availability is mainly the result of litter decomposition. Thus protein degradation is of major interest for nutrition. Two aspartic proteases were excreted in a pH‐dependent manner by the ectomycorrhizal fungus Amanita muscaria grown in liquid culture. AmProt1 with a molecular mass of approximately 45 kDa was mainly present at pH values up to 5·4, whereas the excretion of AmProt2 with a molecular mass of about 90 kDa was only detectable at pH values between pH 5·4 and 6·3. In addition, the pH optima of both enzymes differed significantly. AmProt1 had a narrow pH optimum around 3, whereas AmProt2 had a broad pH optimum between 3 and 5·5 and a higher affinity to the substrate methylcasein. One cDNA‐clone (AmProt1*) that presumably encodes AmProt1 was identified. Like AmProt1, this cDNA was expressed in a pH‐dependent manner. In addition, carbohydrate and to a lesser extent nitrogen depletion significantly enhanced AmProt1* expression. In fully developed Populus hyb./A. muscaria ectomycorrhizas the expression of AmProt1* was significantly higher in hyphae of the Hartig net compared with those of the fungal sheath. The role of AmProt1 and AmProt2 for fungal physiology and competitiveness is discussed.

Differential expression of the hexose‐regulated fungal genes AmPAL and AmMst1 within Amanita/Populus ectomycorrhizas

Summary At least three fungal ‘networks’ are formed in ectomycorrhizal symbiosis: the Hartig net, the sheath and extramatrical hyphae. Since these structures have different functions regarding uptake, storage and partitioning of carbohydrates and nutrients, regulation and coordination of metabolic fluxes are of special interest for ectomycorrhizal functioning. To get some insight into structure‐specific functions, the Amanita fungal sheath was separated by microdissection from the remaining Populus roots that still contained the Hartig net. Expression of the genes coding for phenylalanine ammonium lyase (AmPAL) and hexose transporter (AmMst1) was investigated separately for both structures by RT‐PCR. When constitutively expressed fungal genes were used as a control, the expression of AmMst1 was enhanced about six‐fold in hyphae of the Hartig net compared with those of the fungal sheath. By contrast, AmPAL was only barely detectable in the Hartig net, but highly expressed in the fungal sheath. Since in pure fungal culture both genes are regulated by monosaccharides, it is supposed that an apoplastic hexose gradient, between hyphae of the Hartig net and those of the fungal sheath, might regulate fungal gene expression and thus hyphal physiology within ectomycorrhizas.

Microautoradiographic localization of phosphate and carbohydrates in mycorrhizal roots of Populus tremula x Populus alba and the implications for transfer processes in ectomycorrhizal associations.

Microautoradiographic studies were carried out to examine the distribution and exchange of phosphate and labeled carbohydrates in mycorrhizal roots of Populus tremula x Populus alba L. following application of 33P-orthophosphate (Pi) and 14CO2. Labeled Pi was not homogeneously distributed along the mycorrhizal longitudinal axis. The fungal sheath and the Hartig net contained more 33Pi in the median parts of the root than in the apical or basal root zones, indicating that uptake and transfer of Pi to the host plant was localized mainly in this area. The Pi was translocated by the Hartig net and the interfacial apoplast to the host plant. It was distributed by way of the stele within the plant. Young leaves and meristematic tissue in the shoot tip were the main sinks for Pi. In plants that were left in the dark for 5 days before 33Pi application, the reduced carbohydrate supply caused a decrease in Pi absorption by mycorrhizal roots. Microautoradiography of mycorrhizal roots after assimilation of 14CO2 revealed that: (1) the fungal partner had a high capacity to attract photosynthates; (2) the main transfer of carbohydrates was localized in the median zone of a mycorrhizal root; (3) carbohydrates that were absorbed by the mycorrhizal fungus were translocated to the fungal sheath and were homogeneously distributed; and (4) in the main exchange zone, cortical cell nuclei showed a high sink capacity, indicating increased metabolic activity in these cells. We postulate that (1) the phosphate demand of the host plant regulates absorption of Pi by the fungus, and (2) a bidirectional transfer of carbohydrates and Pi occurs across the same interface structure in ectomycorrhizal roots of Populus.

Identification of symbiosis‐regulated genes in Eucalyptus globulus–Pisolithus tinctorius ectomycorrhiza by differential hybridization of arrayed cDNAs

SummaryEctomycorrhiza development alters gene expression in the fungal and plant symbionts. The identification of a large number of genes expressed exclusively or predominantly in the symbiosis will contribute greatly to the understanding of the development of the ectomycorrhizal symbiosis. We have constructed a cDNA library of 4‐day‐old Eucalyptus globulus–Pisolithus tinctorius ectomycorrhiza and sequenced 850 cDNAs cloned randomly or obtained through suppression subtractive hybridization (SSH). Based on the absence of a database match, 43% of the ectomycorrhiza ESTs are coding for novel genes. At the developmental stage analysed (fungal sheath formation), the majority of the identified sequences represented ‘housekeeping’ proteins, i.e. proteins involved in gene/protein expression, cell‐wall proteins, metabolic enzymes, and components of signalling systems. We screened arrayed cDNAs to identify symbiosis‐regulated genes by using differential hybridization. Comparisons of signals from free‐living partners and symbiotic tissues revealed significant differences in expression levels (differential expression ratio >2.5) for 17% of the genes analysed. No ectomycorrhiza‐specific gene was detected. The results successfully demonstrate the use of the cDNA array and SSH systems as general approaches for dissecting symbiosis development, and provide the first global picture of the cellular functions operating in ectomycorrhiza.

Identification of symbiosis-regulated genes in Eucalyptus globulus-Pisolithus tinctorius ectomycorrhiza by differential hybridization of arrayed cDNAs.

Ectomycorrhiza development alters gene expression in the fungal and plant symbionts. The identification of a large number of genes expressed exclusively or predominantly in the symbiosis will contribute greatly to the understanding of the development of the ectomycorrhizal symbiosis. We have constructed a cDNA library of 4-day-old Eucalyptus globulus-Pisolithus tinctorius ectomycorrhiza and sequenced 850 cDNAs cloned randomly or obtained through suppression subtractive hybridization (SSH). Based on the absence of a database match, 43% of the ectomycorrhiza ESTs are coding for novel genes. At the developmental stage analysed (fungal sheath formation), the majority of the identified sequences represented 'housekeeping' proteins, i.e. proteins involved in gene/protein expression, cell-wall proteins, metabolic enzymes, and components of signalling systems. We screened arrayed cDNAs to identify symbiosis-regulated genes by using differential hybridization. Comparisons of signals from free-living partners and symbiotic tissues revealed significant differences in expression levels (differential expression ratio >2.5) for 17% of the genes analysed. No ectomycorrhiza-specific gene was detected. The results successfully demonstrate the use of the cDNA array and SSH systems as general approaches for dissecting symbiosis development, and provide the first global picture of the cellular functions operating in ectomycorrhiza.

Ectomycorrhiza formation of Tricholoma matsutake on Pinus densiflora

Mycorrhizal association ofTricholoma matsutake withPinus densiflora was studied. A naturally establishedP. densiflora stand (age: ca. 45 yr) where occurrences ofT. matsutake sporocarps had been confirmed was studied in lbaraki Prefecture, Japan. Pine root systems connected withT. matsutake sporocarps via the fungal white mycelia were sampled in October 1997. The sampled pine roots were covered overall with mycelia. Under a dissecting microscope, the mycelia were confirmed to form fungal sheaths on the lateral roots. Under a light microscope, transverse and longitudinal sections of these roots showed the presence of both fungal sheaths and Hartig nets, which are typical of ectomycorrhizas. The fungal sheath was ca. 1.5–20 μm. in thickness, and felt prosenchymatous in texture. Hartig nets developed continuously at the cortex and extended to the boundary between cortical cells and endodermal cells. The same ectomycorrhizal morphotype on the pine was also recovered from inside the same mycelial colony (i.e., “shiro”) ofT. matsutake from winter to summer. These results suggest thatT. matsutake has a perennial ectomycorrhizal association withP. densiflora.

Chemical and mineral composition of ectomycorrhizosphere soils of subalpine fir (Abieslasiocarpa (Hook.) Nutt.) in the Ae horizon of a Luvisol

Differences in the properties of bulk forest and rhizosphere soils are often attributed to ectomycorrhizal association, or the symbiosis characterized by a fungal sheath surrounding the root (mantle) and intercellular root colonization (Hartig net). We compared the soil pH, total C, N, cation exchange capacity, and the contents of mica, chlorite, kaolinite, 2:1 expandable clays, feldspars and amorphous materials between two ectomycorrhizosphere soils (or soil environment in the vicinity of ectomycorrhizae (ECM)) and non-ectomycorrhizosphere soils to study the influence of ectomycorrhizae on chemical and mineralogical properties of soils. The two ectomycorrhizosphere soils were characterized by ectomycorrhizal colonization dominated by (1) Piloderma spp., and (2) Mycelium radicis atrovirensand cottony yellow-brown (MRA-CYB) types or where Piloderma spp. colonization was <2%. Our results showed that total C and N were higher in ectomycorrhizosphere than non-ectomycorrhizosphere soils, and the ectomycorrhizosphere soils dominated by Piloderma spp. had almost twice the total C and N as ectomycorrhizosphere soils with MRA-CYB. Soil pH was lower by half a pH unit in ectomycorrhizosphere soils compared to non-ectomycorrhizosphere soils. Cation exchange capacity as well as exchangeable Ca2+, Mg2+ and K+ were lower in non-ectomycorrhizosphere soil compared to ectomycorrhizosphere soils. We also found that cation exchange capacity, exchangeable Ca2+, Mg2+ and K+ values in soils dominated by Piloderma spp. were higher compared to ectomycorrhizosphere soils with insignificant Piloderma spp. Our results suggest that transformation rate of mica and chlorite to 2:1 expandable clays was predominant in ectomycorrhizosphere compared to non-ectomycorrhizosphere soils, likely as a result of high production of organic acids and direct extraction of K+ and Mg2+ by fungal hyphae. In ectomycorrhizoplane samples, it is suggested that K+ and possibly amorphous Al2O3 and Fe2O3 could reconstitute the degraded mica and chlorite through the formation of hydroxy-interlayered 2:1 clays. Key words: Ectomycorrhizosphere soils, subalpine fir, Piloderma spp.

Ultrastructure and cytochemistry of early stages of colonization by Gremmeniella abietina in Pinus resinosa seedlings

This paper provides details on the infection processes at the ultrastructural level in Pinus resinosa Ait. seedlings during early stages of colonization by Gremmeniella abietina (Lagerb.) Morelot. Different gold-conjugated enzymes and antibodies were used to cytochemically localize cellulose, pectin, fungal laccase, and the pathogen cells in host tissues. Gremmeniella abietina penetrated into the host through stomata of the short shoot bracts and sparsely colonized both intercellular and intracellular areas of the bract tissues. The colonizing hyphae usually had a thick wall surrounded by an extracellular sheath composed of fibrillar material. Microhyphaelike cells were observed as having penetrated host cell walls. The fungal cells (except the extracellular sheath), even when embedded in cellulosic or pectic material of host tissues, did not appear to contain cellulose or pectin. We suggest that G. abietina is able to degrade cellulose and pectin and that phenoloxidases secreted by the pathogen could be involved in host cell wall degradation. The results indicate that the extracellular sheath of G. abietina is implicated in host–pathogen interactions such as attachment of hyphae to the host surface and cell wall degradation during colonization of host tissues. Key words: Gremmeniella, Pinus, infection processes, cell wall degradation, extracellular fungal sheath, gold labelling.

15N abundance of surface soils, roots and mycorrhizas in profiles of European forest soils.

15N natural abundances of soil total N, roots and mycorrhizas were studied in surface soil profiles in coniferous and broadleaved forests along a transect from central to northern Europe. Under conditions of N limitation in Sweden, there was an increase in δ15N of soil total N of up to 9% from the uppermost horizon of the organic mor layer down to the upper 0-5 cm of the mineral soil. The δ15N of roots was only slightly lower than that of soil total N in the upper organic horizon, but further down roots were up to 5% depleted under such conditions. In experimentally N-enriched forest in Sweden, i.e. in plots which have received an average of c. 100 kg N ha-1 year-1 for 20 years and which retain less than 50% of this added N in the stand and the soil down to 20 cm depth, and in some forests in central Europe, the increase in δ15N with depth in soil total N was smaller. An increase in δ15N of the surface soil was even observed on experimentally N-enriched plots, although other data suggest that the N fertilizer added was depleted in15N. In such cases roots could be enriched in15N relative to soil total N, suggesting that labelling of the surface soil is via the pathway: - available pools of N-plant N-litter N. Under N-limiting conditions roots of different species sampled from the same soil horizon showed similar δ15N. By contrast, in experimentally N-enriched forest δ15N of roots increased in the sequence: ericaceous dwarf shrubs<Scots pine<grass, suggesting increasing use of inorganic N along the sequence. Complementary studies at the major transect sites had shown that 90-99% of fine tree roots had ectomycorrhizas (ECMs). ECMs were 2% more enriched than corresponding non-mycorrhizal fine roots. Fungal sheaths stripped off ECMs were 2.4-6.4 enriched relative to the remaining root core. It is suggested that a flux of N through ECMs to aboveground parts in N-limited forests would leave 15N enriched compounds in fungal material, which could contribute to explain the observed δ15N profiles if fungal material is enriched, because it is a precursor of stable organic matter and recalcitrant N. This could act in addition to the previous explanation of the isotopically lighter soil surface in forests: plant uptake of 15N-depleted N and its redeposition onto the soil surface by litter-fall.

Morphological classification of ectomycorrhizas of Pinus densiflora

Morphological classification of ectomycorrhizas ofPinus densiflora was conducted. Fifty soil samples containing pine ectomycorrhizas, and 40 pine seedlings were collected randomly in two separate reforested stands ofP. densiflora (45 yr old) from May 1992 to October 1994. Fifty-six types of ectomycorrhizas could be classified based upon microscopically observable morphological characteristics. Fifty percent of the types showed cystidia or other specific characteristics such as laticiferous hyphae in the fungal sheaths, verrucose emanating hyphae and a positive hyphal reaction to UV irradiation. Four mycorrhizal types were confirmed to be formed by the fungiRussula delica, R. mariae, R. nigricans, andCenococcum geophilum, respectively. Although the other 52 types were unidentified mycobionts at species level, it was inferred that they were formed by the fungiHebeloma, Lactarius, Russula andTuber. There was a slight difference in the observed mycorrhizal types between the tree ages.

Localization of chitinolytic activities in Fagus sylvatica mycorrhizas

Localization of chitinolytic activities in Fagus sylvatica (beech) mycorrhizas was examined using a range of fluorogenic 4-methylumbelliferyl [4-MU-(GlcNAc)1–4] substrates in order to distinguish between exochitinase, endochitinase and β-N–acetylglucosaminidase activities. The validity of the technique was confirmed using onion epidermis cells. In the beech mycorrhiza, endochitinase activity was not detectable above background fluorescence. Exochitinase activity was detected in the fungal sheath and the Hartig net. β-N–Acetylglucosaminidase activity was also mainly associated with the fungal sheath and Hartig net. Individual fungal hyphae extending from these structures also showed substantial β-N–acetylglucosaminidase activity. The cortical cell walls of the host in the Hartig net region also fluoresced brightly. The localization of β-N–acetylglucosaminidase activity was confirmed using a chromogenic histochemical reagent, 5-bromo-4-chloro-3-indolyl-N–acetyl-β-d-glucosaminide (X-GlcNAc).

Mycorrhizal association of isolates from sporocarps and ectomycorrhizas with Pinus densiflora seedlings

Thirty-two isolates from sporocarps of 27 species of macromycetes, 43 isolates from ectomycorrhizas of Pinus densiflora (Japanese red pine) and 1 isolate from an ectomycorrhiza of Quercus myrsinaefolia were tested for the ability to form mycorrhizas with P. densiflora seedlings in glass tubes. Ten isolates from sporocarps of Hebeloma sp., Laccaria bicolor, Lactarius chrysorrheus, Suillus granulatus, Scleroderma areolatum, Russula mariae and R. nigricans had formed ectomycorrhizas by 8 months after transplantation. Twenty isolates taken from mycorrhizas including of Cenococcum geophilum, R. mariae and R. nigricans formed ectomycorrhizas. The synthesized mycorrhizas were classified based on morphological characteristics such as hyphal arrangement of their fungal sheath, and appearance of cystidia and emanating hyphae. Twenty-one mycorrhizal types were recognized.

Wood degradation by Phellinus noxius: ultrastructure and cytochemistry

An ultrastructural and cytochemical investigation of the development of Phellinus noxius, a white-rot fungus, in wood chips of Betula papyrifera was done to gain insight into the cellular mechanisms of wood cell wall degradation. Extracellular sheaths and microhyphae were seen to be involved in wood colonization. Close association was observed between these fungal structures and wood cell walls at both early and advanced stages of wood alteration. Fungal sheaths were often seen deep inside host cell walls, sometimes enclosing residual wood fragments. Investigations using gold probes indicated the occurrence of β-1,3-glucans within the fungal sheaths, while β-1,4-glucans were detected only within the fungal septa. The positive reaction with the PATAg test revealed that polysaccharides such as β-1,6-glucans were important components of the sheath. Chitin, pectin, β-glucosides, galactosamine, mannose, sialic acid, fucose, and fimbrial proteins were not found to be present in the sheath. Our data suggest that extracellular sheaths and microphyphae produced by P. noxius during wood cell wall colonization play an important role in wood degradation.Key words: cellulose, Phellinus, sheath, wood degradation.

Adhesion of germlings of Botrytis cinerea.

Adhesion of conidia and germlings of the facultative plant parasite Botrytis cinerea occurs in two distinct stages. The first stage, which occurs immediately upon hydration of conidia and is characterized by relatively weak adhesive forces, appears to involve hydrophobic interactions (R. P. Doss, S. W. Potter, G. A. Chastagner, and J. K. Christian, Appl. Environ. Microbiol. 59:1786-1791, 1993). The second stage of adhesion, delayed adhesion, occurs after viable conidia have been incubated for several hours under conditions that promote germination. At this time, the germlings attach strongly to either hydrophobic or hydrophilic substrata. Delayed adhesion involves secretion of an ensheating film that remains attached to the substratum upon physical removal of the germlings. This fungal sheath, which can be visualized by using interference-contrast light microscopy, scanning electron microscopy, or atomic force microscopy, is 25 to 60 nm thick in the region immediately adjacent to the germ tubes. Germlings are resistant to removal by boiling or by treatment with a number of hydrolytic enzymes, 2.0 M periodic acid, or 1.0 M sulfuric acid. They are readily removed by brief exposure to 1.25 N NaOH. A base-soluble material that adheres to culture flask walls in short-term liquid cultures of B. cinerea is composed of glucose (about 30%), galactosamine (about 3%), and protein (30 to 44%).

Root soluble carbohydrates of Afzelia africana Sm. seedlings and modifications of mycorrhiza establishment in response to the excision of cotyledons

Stem length, number of secondary lateral roots, shoot dry weight and reducing sugar concentrations of root were significantly reduced when translocation of reserves from cotyledons to the roots of Afzelia africana seedlings was interrupted by complete or partial cotyledon excision. The sucrose but not the glucose concentration of lateral roots also decreased significantly after complete cotyledon excision. Hartig net development rather than fungal sheath formation was affected after inoculation with the early fungal isolate E1 and by both late-stage fungal isolates L1 and L2 after partial or complete cotyledon excision. However, mycorrhizal colonization by the early fungal isolate E2 was not affected by cotyledonary reserves, suggesting that this fungal isolate has a lower carbohydrate requirement than fungal isolates E1, L1 and L2. The late-stage fungal isolates L1 and L2 induced a hypersensitivity reaction by epidermal cell walls of the host plant after complete cotyledon excision, suggesting they are more dependent than the early fungal isolate E1 on available root carbohydrate substrates for ectomycorrhizal colonization. These results are discussed in the light of the hypothesis that early and late-stage fungi were different carbohydrate requirements, and that the time sequence of colonization was related to the root carbohydrate status, which increased with time.

The influence of culture conditions on mycorrhiza formation between the ectomycorrhizal fungus Pisolithus sp. and Afzelia africana Sm. seedlings

Interactions between an isolate of the ectomycorrhizal fungus Pisolithus sp. and Afzelia africana Sm. seedlings were studied at the structural and ultrastructural levels. Several different conditions were tested with or without sugar and in a sterile or nonsterile medium. In the growth cabinet, the A. africana/Pisolithus sp. interactions did not produce ectomycorrhizas. A fungal sheath was formed but no Hartig net, and an unusual host epidermal cell wall was observed. Hyphae of Pisolithus sp. induced modifications of epidermal cells of 15-day-old A. africana seedlings indicative of non-mycorrhizal interactions, such as wall thickening, wall ingrowth, papillae formation, degraded host wall material and the presence of intracellular hyphae. Wall ingrowth consisted of depositions of host cell wall materials giving a positive reaction for polysaccharides; however, wall thickenings and papillae showed no homogeneous reactions for polysaccharides. In glasshouse conditions, inocula of Pisolithus sp. in the form of spores or mycelia entrapped in peat-vermiculite added to sterilized soil produced typical ectomycorrhizae only with 6-month-old A. africana seedlings. Under these conditions, no conspicuous cell wall reactions occurred on A. africana roots. The results demonstrate that the establishment of an association between an ectomycorrhizal fungus and a potential host plant is strongly influenced by seedling age and/or environmental conditions. Therefore, in vitro synthesis is not a conclusive demonstration of a symbiotic relationship.

Needle-fiber Calcite: A Critical Review and a Proposed Classification

ABSTRACT From a review of the recent literature and our own observations, the different morphologies of needle-fiber calcite are classified. They can be characterized as monocrystalline rods (with or without serrated edges) or as polycrystalline chains. The simplest monocrystalline habit of needle-fiber calcite is the individual micro-rod (M). Larger monocrystalline rods (MA) exist in multiples of two. More complex forms are found as paired monocrystalline rods with serrated plates along their length (MB). This group includes three habits, which can be distinguished by the number of plates and their orientation relative to the rod. Finally, there is a monocrystalline form (MAB), intermediate between the categories MA and MB, which shows the transition from the simple coupled rods to rods with errated plates. Although polycrystalline needles (P) have rarely been reported in the literature, three types of morphologies have been identified. Each type of crystal has a specific origin. The M micro-rods are physicochemical nuclei or calcified bacteria. They do not seem to evolve towards larger or more complex morphologies. The MA rods are related to biomineralization inside the fungal sheaths. The MB rods postdate liberation of MA rods from mycelian bundles. The serrated plates are secondary syntactic crystallizations of physicochemical origin, whose growth can be inhibited for various reasons. The origin of P chains is less well understood, but they seem to be physicochemical crystallizations related to evaporation and desiccation. Therefore, these three groups--M, MA-MB, and P--have similar morphologies but do not seem to be related genetically. The only known link is between the MA and MB rods. There is no evolutionary s quence of needle-fiber calcite habits but a convergence of forms between crystals whose origins are different and independent. Structures described in the literature as convoluted, random, cutane, and nest-like are the result of either evolution of the mycelian bundles or movement of material and organisms in the soil. In the case of M micro-rods, the microfabric is related to the random precipitation around nuclei in pores filled with a highly supersaturated solution. Initially, the P chains also precipitate without preferential organization, and later on, the growth seems to preferentially constitute chains, although the mechanisms are still not understood. Moreover, needle-fiber calcites do not indicate a particular climatic environment.