Influence of autoclaved saprotrophic fungal mycelia on proteolytic activity in ectomycorrhizal fungi

The production of proteolytic enzymes by several strains of ectomycorrhizal fungi i.e., Amanita muscaria (16-3), Laccaria laccata (9-12), L. laccata (9-1), Suillus bovinus (15-4), Suillus bovinus (15-3), Suillus luteus (14-7) on mycelia of Trichoderma harzianum, Trichoderma virens and Mucor hiemalis and sodium caseinate, yeast extract was evaluated. The strains of A. muscaria (16-3) and L. laccata (9-12) were characterized by the highest activity of the acidic and neutral proteases. Taking the mycelia of saprotrophic fungi into consideration, the mycelium of M. hiemalis was the best inductor for proteolytic activity. The examined ectomycorrhizal fungi exhibited higher activity of acidic proteases than neutral ones on the mycelia of saprotrophic fungi, which may imply the participation of acidic proteases in nutrition.     

Cytoskeleton and mitochondrial morphology of saprotrophs and the pathogen Heterobasidion annosum in the presence of Suillus bovinus metabolites

Ectomycorrhizal fungi are known to synthesize antifungal compounds both in vitro and in symbiosis with the host-plants. Culture filtrates of the ectomycorrhizal fungus Suillus bovinus (at pHs of 2.5–6) showed antifungal activity towards saprotrophs Trichoderma harzianum, and Trichoderma virens and the pathogen Heterobasidion annosum, by significantly suppressing their growth relative to sterile liquid medium at the same pHs. In the presence of the culture filtrates, hyphae of the saprotrophs and the pathogen were characterized by distensions, irregular and frequent branching, tip damage and cytoplasm coagulation. Since hyphal abnormalities may be evoked by disruptions in the cytoskeleton and mitochondria, their structural changes were also examined. Depolymerization of microtubules was confirmed for all of the fungi. Serious damage to mitochondria morphology may cause significant functional impairment. Growth of mycelia was inhibited in the lower pH S. bovinus culture filtrate, and the mitochondrial morphology was altered. This suggests that the activity of antifungal compounds synthesized by ectomycorrhizal fungus is significantly affected by pH.     

Ni2+ induces changes in the morphology of vacuoles, mitochondria and microtubules in Paxillus involutus cells

Organelles of ectomycorrhizal fungi are known to respond to changes in the extracellular environment. The response of vacuoles, mitochondria and microtubules to short-term nickel (Ni2+) exposure were investigated in hyphal tip cells of a Paxillus involutus from a heavy metal-rich soil. Vacuoles, mitochondria and microtubules were labelled with Oregon Green 488 carboxylic acid diacetate, 3,3'-dihexyloxacarbocyanine iodide (DiOC6(3)) and anti-alpha-tubulin antibodies, respectively; hyphae were treated with NiSO4 in the range of 0-1 mmol l(-1) and examined microscopically. Untreated hyphal tip cells contained tubular vacuole and mitochondrial networks. Ni2+ caused loss of organelle tubularity and severe microtubule disruption that were exposure-time and concentration dependent. Fine tubular vacuoles thickened and eventually became spherical in some hyphae, tubular mitochondria fragmented and microtubules shortened and aggregated into patches in most hyphae. Tubular vacuoles reformed on NiSO4 removal and tubular mitochondria in the presence of NiSO4 suggesting cellular detoxification. These results demonstrate that Ni2+ induces changes in organelle and microtubule morphology. Recovery of tubular organelles to pretreatment morphology after Ni2+ exposure suggests cellular detoxification of the metal ion.     

Changes in hyphal morphology and activity of phenoloxidases during interactions between selected ectomycorrhizal fungi and two species of <Emphasis Type="Italic">Trichoderma</Emphasis>

Patterns of phenoloxidase activity can be used to characterize fungi of different life styles, and changes in phenoloxidase synthesis were suspected to play a role in the interaction between ectomycorrhizal and two species of Trichoderma. Confrontation between the ectomycorrhizal fungi Amanita muscaria and Laccaria laccata with species of Trichoderma resulted in induction of laccase synthesis, and the laccase enzyme was bound to mycelia of ectomycorrhizal fungi. Tyrosinase release was noted only during interaction of L. laccata strains with Trichoderma harzianum and T. virens. Ectomycorrhizal fungi, especially strains of Suillus bovinus and S. luteus, inhibited growth of Trichoderma species and caused morphological changes in its colonies in the zone of interaction. In contrast, hyphal changes occurred less often in the ectomycorrhizal fungi tested. Species of Suillus are suggested to present a different mechanism in their interaction with other fungi than A. muscaria and L. laccata.     

Conidia of Trichoderma virens as a phosphorus source for mycorrhizal Pinus sylvestris seedlings

In this study, the mobilization and further translocation of phosphorus from conidia of saprotrophic fungus Trichoderma virens into Pinus sylvestris seedlings by nondestructive measuring of ³²P was assessed. The radioactive phosphorus flux from the conidia to the Scots pine seedlings forming mycorrhiza with Laccaria laccata and Suillus bovinus amounted up to 27.82% and 7.42%, respectively, on the 28th day of the experiment, while at the same time in nonmycorrhizal pine seedlings, the detected radioactivity reached only 0.56%. Our studies revealed that both ectomycorrhizal fungi: L. laccata and S. bovinus, mobilized the phosphorus from radioactive conidia of T. virens. On this basis, we conclude that activities of the mycosymbionts may facilitate absorption and further translocation of phosphorus from organic matter into the host plants.     

Ultrastructural neuronal pathology in transgenic mice expressing mutant (P301L) human tau

Transgenic mice expressing mutant (P301L) human tau develop neurofibrillary tangles, amyotrophy and progressive motor disturbance. We present ultrastructural features of neuronal degeneration in this model that suggests involvement of both neurofibrillary and autophagic processes in neurodegeneration. Neurons undergoing neurofibrillary degeneration contain tau-immunoreactive, 15–20 nm-wide straight or wavy filaments with no periodic twists. Tau filaments were found in two types of affected neurons. One type resembled neurons with neurofibrillary tangles (NFT) that were filled with numerous filaments that displaced sparse cytoplasmic organelles to the periphery. Microtubules were almost completely absent. The nucleus remained centrally located, but showed lobulations due to deep infoldings. The other type resembled ballooned neurons seen in some human tauopathies. The nucleus was peripherally placed, but normal appearing. The cytoplasmic organelles were dispersed throughout the swollen perikarya, the Golgi complex was fragmented and duplicated, while mitochondria and other organelles appeared normal. Tau filaments similar to those in NFT were sparse and not tightly packed. Microtubules were also sparse. Many autophagic vacuoles were present in these cells. Heterogeneous appearing axonal swellings resembling spheroids in human tauopathies were present in gray and white matter. Unlike normal appearing axons, axonal spheroids were filled with tau-immunoreactive filaments and autophagic vacuoles, in addition to normal appearing neurofilaments and microtubules. These P301L transgenic mice exhibit many features common to human tauopathies, making them a valuable model to study the pathogenesis of these uncommon disorders.     

PROTONEMAL ORGANIZATION AND GROWTH IN THE MOSS DAWSONIA SUPERBA: ULTRASTRUCTURAL CHARACTERISTICS

The elongating, tip cell of the protonema of Dawsonia superba is characterized by a variety of organelles distributed uniformly over the length of the cell. Chloroplasts, vacuoles, mitochondria and dictyosomes are present in the terminal portion of the cell. Dictyosomes are abundant throughout the cell and their production of vesicles is pronounced. Stratification of the cytoplasm and exclusion of organelles from the tip are not observed. The orientation of microtubules and microfibrils follows the pattern observed in filamentous fern prothallia. Microtubules are not observed at the tip of the protonema, but microfibrils are abundant and randomly oriented. While microtubules and microfibrils in lower regions of the apical dome are arranged randomly, more basally they assume an axial arrangement.     

The dynamic behaviour of mitochondria in living zygotes during maturation and meiosis in Chlamydomonas reinhardtii

To understand fully the function of mitochondria during the development of cells and organs, it is important to elucidate the dynamics of their morphology. However, the detailed morphology of mitochondria during meiosis has not yet been studied in algae. We examined the mitochondrial morphology of Chlamydomonas reinhardtii and classified zygotes into seven types by mitochondrial morphology in order to analyse the morphological change in mature and meiotic zygotes. We also investigated the oxygen consumption of living zygotes and the effects of tubulin and actin polymerization inhibitors on mitochondria, using fluorescence microscopy and oxygen electrodes. During zygote maturation, mitochondria fragmented into small particles, with a large decrease in oxygen consumption. When mature zygotes were exposed to light, mitochondria became tubular and formed a network, and oxygen consumption gradually recovered. At the same time, particle-like mitochondrial nucleoids became stringy and produced new nucleoid particles. Tubular mitochondria accumulated around the cell nucleus and then spread throughout the cell. Cell division followed (first and second rounds), and the resultant daughter cells had tubular mitochondria in a mesh-like arrangement. An inhibitor of tubulin polymerization, demecolcine, inhibited the assembly of mitochondria around the cell nucleus, whereas an inhibitor of actin polymerization, latrunculin B, inhibited the formation of tubular mitochondria. These results suggest that microtubules are probably involved in mitochondrial accumulation around the cell nucleus, whereas microfilaments may maintain the tubular network of mitochondria.     

The effect of soil pH on the ability of ectomycorrhizal fungi to increase the growth of Eucalyptus globulus Labill.

We examined the effect of two levels of soil pH (5 and 6) on the ability (effectiveness) of ectomycorrhizal fungi to increase the growth of Eucalyptus globulus Labill. at a deficient supply of P. Plants were inoculated with one of six fungal isolates [Laccaria laccata (Scop. ex Fr.) Berk. and Br. (isolates A and B), Pisolithus tinctorius (Pers.) Coker and Couch (isolates A and B), Descolea maculata Bough. and Mal. and Setchelliogaster sp. nov.] and were grown in a P-deficient sand, in pots, in a temperature-controlled glasshouse. Seedlings were harvested 89 days after planting and were assessed for dry matter production, tissue P concentrations, ectomycorrhizal colonization of roots and hyphal development in soil.Uninoculated plants had less than 5% of their fine root length colonized by ectomycorrhizal fungi. In contrast, inoculated plants had 30% or greater of their fine root length ectomycorrhizal. Inoculation increased the uptake of P and growth of plants for all isolates and at both levels of soil pH, although growth responses to inoculation were greater at pH 6, particularly for the two L. laccata isolates. Isolates which colonized roots most extensively increased plant growth to the greatest extent. D. maculata was the most effective fungal isolate at pH 5, and both D. maculata and L. laccata A were most effective at pH 6. The effects of soil pH on plant growth were also related to some extent to the effects of soil pH on colonized root length. Growth responses to inoculation were related less well to hyphal development in soil. The L. laccata isolates formed more hyphae in soil (on a per pot, per m of fine root, and per m of colonized fine root basis) than other fungal isolates, but were not always more effective in increasing plant grown.     

Oribatid mite (Acari, Oribatida) feeding on ectomycorrhizal fungi

The coexistence of a large number of soil animals without extensive niche differentiation is one of the great riddles in soil biology. The main aim of this study was to explore the importance of partitioning of food resources for the high diversity of micro-arthropods in soil. In addition, we investigated if ectomycorrhizal fungi are preferentially consumed compared to saprotrophic fungi. Until today, ectomycorrhizal fungi have never been tested as potential food resource for oribatid mites. We offered six ectomycorrhizal fungi [Amanita muscaria (L.) Hook., Boletus badius (Fr.) Fr., Cenococcum geophilum Fr., Laccaria laccata (Scop.) Fr., Paxillus involutus (Batsch) Fr. and Piloderma croceum J. Erikss. & Hjortstam], one ericoid mycorrhizal fungus [Hymenoscyphus ericae (D.J. Read) Korf & Kernan] and three saprotrophic fungi [Agrocybe gibberosa (Fr.) Fayod, Alternaria alternata (Fr.) Keissl. and Mortierella ramanniana (A. Møller) Linnem.] simultaneously to each of the mainly mycophagous oribatid mite species Carabodes femoralis (Nicolet), Nothrus silvestris Nicolet and Oribatula tibialis Nicolet. The ericoid mycorrhizal fungus H. ericae and the ectomycorrhizal fungus B. badius were preferentially consumed by each oribatid mite species. However, feeding preferences differed significantly between the three species, with O. tibialis being most selective. This study for the first time documented that oribatid mites feed on certain ectomycorrhizal fungi.     

Behaviour of the hyphae of<Emphasis Type="Italic"> Laccaria laccata</Emphasis> in the presence of<Emphasis Type="Italic"> Trichoderma harzianum</Emphasis> in vitro

The growth rate and the behaviour of Laccaria laccata and Trichoderma harzianum hyphae in co-culture and in the rhizosphere of 3-month-old Pinus sylvestris seedlings grown in vitro were investigated. In the interaction zone, hyphae of L. laccata became more pigmented and formed short branches growing towards the hyphae of the saprobic fungus, coiled around them and penetrated sporadically. Vacuolated hyphae of T. harzianum showed protoplasm granulation and breaks in walls followed by release of protoplasts. In the rhizosphere, the mantle hyphae of L. laccata showed a tendency to surround conidia of T. harzianum. No obvious penetration of the conidial walls by the hyphae of the mycorrhizal fungus was observed by scanning electron microscopy. Instead, in rare cases, the hyphae of L. laccata showed marked wrinkles, and a partial degradation of a mucilaginous material covering the mantle appeared to occur.     

Coinoculation efficacy of ectomycorrhizal fungi on Pinus patula seedlings in a nursery

 The coinoculation efficacy of the ectomycorrhizal fungi Laccaria laccata and Thelephora terrestris on the growth and mycorrhizal development of Pinus patula seedlings was studied and compared to individual inoculation of these fungi in a nursery. The total number of mycorrhizas was higher in seedlings inoculated with the combined inoculum than with the individual inocula. The colonization by T. terrestris was higher than L. laccata when the seedlings were inoculated with the two fungi simultaneously. Coinoculation significantly increased the height and dry weight of the seedlings compared with individual inoculation, both in steam-sterilized and unsterilized soil. Accepted: 12 May 1997     

The time of urea treatment and its effects on the succession of ammonia fungi in two warm temperate forests in Japan

We studied the effects of the timing of urea treatment on the succession of ammonia fungi. In two evergreen Castanopsis cuspidata forests and in one deciduous Quercus serrata forest, we applied 343g urea to 25 and 15 plots of 0.5m², respectively, at three different times of the year. Ten of the early-phase (EP) species, considered to be saprotrophic, and 6 of the late-phase (LP) ones, considered ectomycorrhizal, fruited. In both phases, the commencement, peak, and cessation of fruiting took place simultaneously among all the plots treated at the same time. The fruiting occurred in summer and autumn. Quantity and size of the fruit bodies was larger in the LP than in the EP species. Fruiting of EP species was affected by the treatment time and that of LP species by interaction of the treatment time and vegetation type. EP was short and occurred as one period, whereas LP was long and occurred as two or more fruiting seasons. We found that species composition, dominant species, and degree of its dominance in fruiting of the ammonia fungi are predictable for different treatment times of the year and different vegetation types.     

Dynamics of lead accumulation in mycorrhizal and non-mycorrhizal Norway spruce (Picea abies (L.) Karst.)

Twelve-week-old seedlings of Norway spruce (Picea abies (L.) Karst), non-mycorrhizal or mycorrhizal with Laccaria laccata, Paxillus involutus or Pisolithus tinctorius were exposed to 5 M Pb for either 32 or 42 days in a quartz sand-nutrient solution system. Ultrathin sections of mycorrhizal and non-mycorrhizal short roots were examined by X-ray microanalysis. After 42 days Pb treatment, the Pb content of the cortex cell walls was lower in the non-mycorrhizal short roots and in the P. involutus mycorrhizae than in the mycorrhizae of L. laccata or P. tinctorius. The Pb content of the cell walls of the hyphal mantle was higher in P. involutus than in L. laccata or P. tinctorius. The short term experiment over 32 days showed that the Pb content of the cortex cell walls strongly increased during the first 16 days in the non-mycorrhizal roots and the L. laccata mycorrhizae, whereas it increased more slowly in the P. involutus mycorrhizae. After 32 days Pb treatment, the Pb content in the cortex cell walls in the P. involutus mycorrhizae was similar to that in the non-mycorrhizal roots. P. involutus also decreased Pb translocation from the roots to the stems. Mycorrhizal infection was not affected by Pb but with P. involutus, the amount of extramatrical mycelium was reduced by 50% on day 32 compared to day 16. The extramatrical mycelium of L. laccata was not reduced by Pb. It is concluded that ectomycorrhizal fungi differ in their effect on Pb accumulation in the roots of Norway spruce. The binding capacity of the extramatrical mycelium seems to be an important factor.     

Effects of different ectomycorrhizal fungi on somatic embryogenesis of <Emphasis Type="Italic">Abies cephalonica</Emphasis> Loud

The effects of ectomycorrhizal (ECM) fungi, including Laccaria bicolor (Maire) Orton, Laccaria laccata (Scop., Fr.) Berk. and Br., along with two strains of Pisolithus tinctorius (Pers.) Coker and Couch, on the proliferation and subsequent maturation of two embryogenic cell lines of Abies cephalonica Loud., designated lines 6 and 8, were investigated. In the presence of these ECM fungi, the proliferation of both embryogenic cell lines was inhibited. L. bicolor and P. tinctorius strain 2 resulted in the highest inhibition rates. On the other hand, cultivation of embryogenic cultures along with ECM fungi, termed a dual culture, increased radial growth of both P. tinctorius strains; whereas, L. bicolor and L. laccata did not grow as well in the presence of embryogenic cell masses. The dual culture during the proliferation period of embryogenic cells, however, enhanced the subsequent embryo formation and maturation of A. cephalonica; i.e. the capability of embryogenic cell lines to form somatic embryos as well as increasing the mean number of somatic embryos per 1 g fresh weight of embryogenic cell mass. However, levels of responses were highly dependent on the interaction between the specific embryogenic cell line and fungal strain.     

Brassinosteroids, microtubules and cell elongation in Arabidopsis thaliana. II. Effects of brassinosteroids on microtubules and cell elongation in the bul1 mutant

In order to elucidate the involvement of brassinosteroids in the cell elongation process leading to normal plant morphology, indirect immunofluorescence and molecular techniques were use to study the expression of tubulin genes in the bul1-1 dwarf mutant of Arabidopsis thaliana (L.) Heynh., the characteristics of which are reported in this issue (M. Catterou et al., 2001). Microtubules were studied specifically in the regions of the mutant plant where the elongation zone is suppressed (hypocotyls and petioles), making the reduction in cell elongation evident. Indirect immunofluorescence of α-tubulin revealed that very few microtubules were present in mutant cells, resulting in the total lack of the parallel microtubule organization that is typical of elongating cells in the wild type. After brassinosteroid treatment, microtubules reorganized and became correctly oriented, suggesting the involvement of brassinosteroids in microtubule organization. Molecular analyses showed that the microtubule reorganization observed in brassinosteroid-treated bul1-1 plants did not result either from an activation of tubulin gene expression, or from an increase in tubulin content, suggesting that a brassinosteroid-responsive pathway exists which allows microtubule nucleation/organization and cell elongation without activation of tubulin gene expression. Received: 28 April 2000 / Accepted: 6 October 2000     

The fungus-specificity of mycorrhization helper bacteria (MHBs) used as an alternative to soil fumigation for ectomycorrhizal inoculation of bare-root Douglas-fir planting stocks with Laccaria laccata

Mycorrhization helper bacteria (MHBs) isolated and selected from the Douglas fir-Laccaria laccata symbiotic system have previously been shown to be fungus-specific: they promote ectomycorrhizal establishment of Laccaria laccata but inhibit mycorrhiza formation by other fungi. In this paper, two experiments in a nursery producing two years-old bare-root Douglas-fir planting stocks confirm the specificity of MHBs under field conditions. They also show that, by selectively helping the introduced L. laccata against the resident symbionts, MHBs are an interesting alternative (safer and easier) to soil fumigation for the success of routine controlled mycorrhization of planting stocks in forest nurseries.     

Spatial congruence between exine pattern,microtubules and endomembranes in Vigna pollen

The role of microtubules and endomembranes in pollen wall pattern formation in Vigna vexillata L. was examined using fluorescence laser scanning confocal microscopy. Indirect immunofluorescence using anti--tubulin antibodies revealed that the arrangement of the cortical microtubular cytoskeleton in microspores resembled the reciprocal of the reticulate ektexine ornamentations of mature V. vexillata pollen. Patches of microtubules in cortical cytoplasm corresponded in location with the lumina of the exine reticulum and with apertural sites. Microtubules were absent from cytoplasm under muri (ridges) of the exine reticulum. Labeling of microspores during the mid-tetrad stage with the endomembrane-specific fluorochrome DiOC₆ produced a pattern similar to that of the microtubules; i.e., DiOC₆ staining was localized in cytoplasm underlying lumina and absent from cortical cytoplasm underlying sites of muri. This report represents the first observation of congruence of the pattern of occurrence of any subcellular organelles with exine pattern and, in particular, the congruence of both microtubules and endomembranes in cortical cytoplasm with the lumina of the reticulate exine.     

Concomitant changes in mitochondria and intermediate filaments during heat shock and recovery of chicken embryo fibroblasts

Utilizing video-enhanced differential interference contrast microscopy of chicken embryo fibroblasts, we observed dramatic changes in the localization and morphology of mitochondria shortly after cells were subjected to a mild heat shock. At normal temperatures mitochondria were distributed in the cell cytoplasm as elongated, tubular, and dynamic organelles but upon heat shock they moved to the perinuclear region and formed a tight ring of short swollen and—in some cases—fused vesicles. Vital dye staining of mitochondria with rhodamine 123 and indirect immunofluorescence staining with antibodies against the mitochondrial-matrix protein, HSP 60, confirmed these results. Using cells double labeled with antibodies to vimentin and the HSP 60 protein, we found that the changes in mitochondria were accompanied by perturbations of the intermediate filament network that we and others have reported previously for heat shocked cells. Microtubules remained largely unaltered by our heat shock treatment and the redistribution of intermediate filaments and mitochondria occurred even in the presence of taxol, a microtubule stabilizing drug. The effects of heat shock on mitochondria were reversed when cells were returned to normal temperatures and their recovery to their normal state coincided with return of normal intermediate filament morphology. This recovery was blocked in cells treated with actinomycin D during heat shock, a result indicating that a heat shock protein may be required for recovery. These data are consistent with previously published observations that mitochondria are associated with the intermediate filament network but they extend this interaction to a cell system responding to a physiological stress normally experienced by the intact organism.     

Influence of the cytoskeleton,energy supply,and protein synthesis on the structure of the endoplasmic reticulum

Summary FITC-Con A fluorescence was used to visualize rER arrangement of endothelial cells derived fromXenopus laevis tadpole hearts. In particular determinants of rER organization, intracellular localization and the interrelationships with other organelles were analysed. rER occurs in association with nucleus, mitochondria and microtubules.The structure of rER is strongly affected by energy metabolism and by microtubules. In order to elucidate the interdependence of structure and function we examined the influence of cellular respiration, net lactate production and protein synthesis on rER morphology, as well as the relationship between energy metabolism and protein synthesis. ER morphology is determined primarily by energy consuming intracellular transport mechanisms. Energy needed for protein synthesis is supplied by the respiratory chain while ATP from aerobic glycolysis only compensates when respiration is disturbed.Abbreviations ACM amphibian culture medium - ATP adenosine triphosphate - FITC-Con A fluoresceine-isothiocyanate-coupled con-canavalin A - MT microtubule - rER rough endoplasmic reticulum - sER smooth endoplasmic reticulum - TRITC-phalloidin tetramethyl-rhodaminyl-isothiocyanate-coupled phalloidin - pXTH primary cells fromXenopus laevis tadpole hearts - XTH-2 endothelial cell line derived fromXenopus laevis tadpole hearts