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summary Ten Armillaria isolates, collected from various host plants and widespread geographical origins in tropical Africa, were cultivated on orange fragments in the presence of water and ran different culture media in order to optimize enzyme and mycelial cord production. Seven enzymes involved in the primary metabolism of nitrogen and carbon (glutamate dehydrogenases, aspartate aminotransferase, malate dehydrogenase, glucose n-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase and isocitrate dehydrogenase) were extracted from the mycelial cords and analyzed by polyacry lamide gel electrophoresis. Cluster analysis based on calculated similarity values derived from isozyme banding patterns separated the isolates into five groups. Two isolates considered as belonging to A. mellea ssp. africana (an African species closely related to European A. mellea) were present in a clearly Separated cluster when compared to the other isolate groups. Two Kenyan isolates, belonging to an as yet unnamed biological species, which were characterized by the production of few slow growing mycelial cords, were also found in a separate cluster with slightly greater similarity coefficients to the other isolates. The six other isolates, referred to as isolates of A. heimii (a highly variable species with different sexual systems) fell into three sub-clusters of variable homology. The two homothallic heimii isolates from Tanzania and Malawi, which were very closely related, displaying 100% isozyme similarity, exhibited a higher degree of similarity with the two other homothallic heimii isolates from Zimbabwe and Congo, than with the two heterothallic unifactorial heimii isolates from Cameroon and Gabon. The value of isozymes in the classification of African Armillaria spp. is discussed.

Mycorrhizas of Pinus sylvestris, collected from zinc wastes in Poland and France were investigated using transmission electron microscope (TEM) and scanning electron microscope (SEM) equipped with energy dispersion spectroscopy (EDS) and electron energy loss spectroscopy (EELS). At both sites, mycorrhizas of Hebeloma were the most frequent, however, they were often characterised by a sparse or only locally developed fungal mantle. Mycorrhizas formed by suilloid fungi were much less frequent, and usually produced a clearly defined fungal mantle characterised by abundant formation of pigments and crystals covering the hyphae of the outer mantle. These two groups of mycorrhizas differed in their heavy metal binding properties. A biofiltering effect of Pb and Zn by the fungal mantle was observed only in the case of suilloid mycorrhizas, which represented up to 10% of the total number of mycorrhizas. No statistical differences between the mantle, the cortical cell walls and the vascular tissue were demonstrated in mycorrhizas formed by other fungi dominating on industrial wastes. In the case of Hebeloma and Inocybe, however, elements such as Cu and Cd were present in higher amounts in the extra-matrical mycelium, whereas no or only low amounts of these elements were detected within fungal mantles, mainly in mycorrhizas from the French waste. Analysis of the root systems has shown relatively high percentage of nonmycorrhizal short roots, suggesting the inhibition of mycorrhiza formation or a decreased number of mycorrhizal propagules. The role of dead roots and mycorrhizas in biosorption and immobilization of heavy metals was discussed.

External hyphae, which play a key role in nitrogen nutrition of trees, are considered as the absorbing structures of the ectomycorrhizal symbiosis. Here, we have cloned and characterized Hebeloma cylindrosporum AMT1, GLNA and GDHA genes, which encode a third ammonium transporter, a glutamine synthetase and an NADP-dependent glutamate dehydrogenase respectively. Amt1 can fully restore the pseudohyphal growth defect of a Saccharomyces cerevisiae mep2 mutant, and this is the first evidence that a heterologous member of the Mep/Amt family complements this dimorphic change defect. Dixon plots of the inhibition of methylamine uptake by ammonium indicate that Amt1 has a much higher affinity than the two previously characterized members (Amt2 and Amt3) of the Amt/Mep family in H. cylindrosporum. We also identified the intracellular nitrogen pool(s) responsible for the modulation of expression of AMT1, AMT2, AMT3, GDHA and GLNA. In response to exogenously supplied ammonium or glutamine, AMT1, AMT2 and GDHA were downregulated and, therefore, these genes are subjected to nitrogen repression in H. cylindrosporum. Exogenously supplied nitrate failed to induce a downregulation of the five mRNAs after transfer of mycelia from a N-starved condition. Our results demonstrate that glutamine is the main effector for AMT1 and AMT2 repression, whereas GDHA repression is controlled by intracellular ammonium, independently of the intracellular glutamine or glutamate concentration. Ammonium transport activity may be controlled by intracellular NH4+. AMT3 and GLNA are highly expressed but not highly regulated. A model for ammonium assimilation in H. cylindrosporum is presented.

The gene encoding a superoxide dismutase (PiSOD) was cloned by suppressive subtractive hybridization from cDNA library of the ectomycorrhizal fungus, Paxillus involutus, grown under cadmium-stress conditions. The encoded protein was presumed to be localized in the peroxisomes because it contained a C-terminal peroxisomal localization peptide (SKL) and lacked an N-terminal mitochondrial transit peptide. Complementation of an Escherichia coli SOD null strain that is unable to grow in the presence of paraquat or cadmium indicated that cloned Pisod encoded a functional superoxide dismutase. Sensitivity of PiSOD activity to H2O2 but not KCN, and sequence homologies to other SODs strongly suggest that it is a manganese-containing superoxide dismutase. Monitoring PiSOD transcript, immunoreactive polypeptide and superoxide dismutase activity following cadmium stress suggests that the principal level of control is post-translational. This is, to our knowledge, the first insight in the characterization of molecular events that take place in an ectomycorrhizal fungus during exposure to heavy metals.

The impact of inoculation with Paxillus involutus on the utilization of organic carbon compounds by birch roots was studied by feeding [14C]Glu or [14C]malate to the partners of the symbiosis, separately or in association, and by monitoring the subsequent distribution of 14C. Inoculation increased [14C]Glu and [14C]malate absorption capacities by up to eight and 17 times, respectively. Six- and 15-d-old mycorrhizal roots showed about four-fold higher [14C]Glu and [14C]malate absorption capacities compared with 60-d-old mycorrhizal roots, suggesting that the early stages of mycorrhiza formation induced higher requirements for C skeletons. Moreover, the results demonstrated that inoculation strongly modified the fate of [14C]Glu and [14C]malate. It was demonstrated that exogenously supplied Glu and malate might serve as C skeletons for amino acid synthesis in mycorrhizal birch roots and in the free-living fungus. Gln was the major 14C-sink in mycorrhizal roots and in the free-living P. involutus. In contrast, citrulline and insoluble compounds were the major 14C sinks in non-mycorrhizal roots, whatever the 14C source. It was concluded that mycorrhiza formation leads to a profound alteration of the metabolic fate of exogenously supplied C compounds. The ecological significance of amino acid and organic acid utilization by mycorrhizal plants is further discussed.

Degradation of beech leaves by the white rot fungus Sporotrichum pulverulentum was investigated for 4 weeks under laboratory conditions. Observations by transmission electron microscopy revealed that the degradation patterns depended on the nature of the foliar tissues and on the stage of decay. Fibres, parenchyma cells and specific zones of the sclerenchyma tissue, corresponding to the cells located between vessels, were strongly degraded, while vessels and epidermal cells were more resistant to degradation. During the early stages of degradation, a selective removal of components from the lignin and hemicellulose-rich layers was observed in the cell walls of sclerenchyma fibres and parenchyma cells. At a more advanced stage of decay, a simultaneous disappearance of all cell wall layers (primary wall, secondary wall, middle lamella and cell corners) occurred, irrespective of cell type. In parenchyma cells, removal of the intracellular brown pigments occurred prior to degradation of the cell walls, while in epidermis, the cell walls were altered first. In sclerenchyma cells surrounding the vessels, hyphae were found to be closely associated with decayed areas, while in all the other tissues, there was no contact of the fungus with lignocellulosic compounds and polyphenols, suggesting a diffusion of the fungal degradative enzymes. A fibrillar mucilagenous matrix, often detected between hyphae and cell walls, might make this diffusion easier. Cellulase activity was detected in the decayed tissues by the release of reducing sugars localized in the vicinity of the leaf cell walls and often somewhat distant from the hyphae. The role of the fungal enzymes involved in degradation of cell wall components and brown pigments in beech leaves is discussed.

The effects of the heavy metals Cu, Cd, Ni, Pb and Zn on [(14)C]methylamine and [(14)C]aminoisobutyric acid uptake were studied in the free-living fungus Paxillus involutus and in mycorrhizal and non-mycorrhizal birch roots. The uptake of both N sources by P. involutus was inhibited by the five metals tested. However, Cu(2+) and Pb(2+) had a greater inhibitory effect. Non-competitive inhibitions were determined between heavy metals and [(14)C]methylamine uptake. [(14)C]Methylamine uptake was reduced by one third by 2 µM Cd(2+) and Cu(2+) in non-mycorrhizal roots, whereas that of mycorrhizal roots was not affected. However, it was reduced by 30 to 80% by 200 µM Cd(2+) and Cu(2+) irrespective of the mycorrhizal status. [(14)C]Aminoisobutyric acid uptake in mycorrhizal roots was not significantly affected by Cd(2+) and Cu(2+), whereas that of non-mycorrhizal roots was decreased by 77% at 200 µM Cu(2+). [(14)C]Aminoisobutyric acid uptake was 4.5 to 6 fold higher in mycorrhizal roots, compared with non-mycorrhizal roots, even under metal exposure. The high efficiency of N acquisition by mycorrhizal birch seedlings under metal exposure might be regarded as a mechanism of stress avoidance.

Cadmium uptake and subcellular compartmentation in the ectomycorrhizal fungus Paxillus involutus were investigated using radiotracer flux analyses. Concentration-dependent Cd2+-uptake kinetics were characterized by a smooth, non-saturating curve that could be dissected into linear and saturable components. The linear-uptake kinetic component was interpreted as representing binding of Cd to apoplastic components, whereas the remaining saturable component was the result of carrier-mediated transport across the plasma membrane. Cell-wall-bound Cd was almost completely removed during desorption from cell-wall preparations. Cd2+ desorption from intact mycelium was found to be a function of time involving three compartments corresponding in series to cell wall (50%), cytoplasm (30%) and vacuole (20%), when mycelia were exposed to a 0.05 microM Cd concentration. At 4 degrees C, most of the Cd recovered was due to the cell-wall-bound fraction, suggesting that transport across the plasma membrane is a metabolically mediated process. Carbonyl cyanide m-chlorophenylhydrazone (CCCP) inhibited Cd accumulation in P. involutus mycelia by up to 28%, which indicates that transport of Cd2+ was partially dependent on the membrane potential. Cd2+ uptake into symplasm is linked to Ca2+ transport, as revealed by the inhibition of Cd accumulation by the Ca2+ ionophore A23187. The present work demonstrates the ability of the ectomycorrhizal fungus P. involutus to take up and further accumulate Cd in different compartments. Binding of Cd onto cell walls and accumulation of Cd in the vacuolar compartment may be regarded as two essential metal-detoxification mechanisms. These data represent a first step towards the understanding of metal-tolerance mechanisms in mycorrhizal fungi.

Using [(14)C]methylamine as an analogue of ammonium, the kinetics and the energetics of NH(4)(+) transport were studied in the ectomycorrhizal fungus, Paxillus involutus (Batsch) Fr. The apparent half-saturation constant (K(m)) and the maximum uptake rate (V(max)) for the carrier-mediated transport derived from the Eadie-Hofstee transformation were 180 µM and 380 nmol (mg dry wt)(-1) min(-1,) respectively. Both pH dependence and inhibition by protonophores indicate that methylamine transport in P. involutus was dependent on the electrochemical H(+) gradient. Both long-term and short-term uptake experiments were consistent with regulation of ammonium/methylamine transport processes by the presence of an organic nitrogen source. Analysis of methylamine uptake by different P. involutus isolates revealed no obvious trend in the uptake capacities in relation to N deposition at the collection site. Kinetic parameters were determined in P. involutus/Betula pendula (Roth.) axenic association and in detached mycorrhizal roots isolated from forest sites. Enhanced methylamine uptake in the presence of the fungal symbiont was demonstrated. Homogeneous V(max) values were found for axenic and detached mycorrhizas, whereas K(m) values showed greater variations.

The growth characteristics and uptake capacities for NO3− and NH4+, as well as activities of nitrogen assimilating-enzymes have been determined in Scleroderma verrucosum. Biomass, grown on agar or liquid MMN media, was higher on nitrate. When S. verrucosum was grown in the presence of equal amounts of ammonium and nitrate, uptake rate for NH4+ was considerably higher than that of NO3−. In addition, it was found that a limitation in ammonium concentration was a prerequisite for nitrate uptake. Nitrate reductase was stimulated when the fungus was transferred from ammonium to nitrate containing media, indicating that the enzyme was inducible by nitrate and repressed by ammonium. Glutamine synthetase and NAD-glutamate synthase activities were clearly detected in S. verrucosum, while the NADP-glutamate dehydrogenase was almost undetectable. This is consistent with the view that ammonium assimilation occurs through the GS/GOGAT cycle in S. verrucosum.

The effect of 700 μmol CO 2 mol - 1 , 200 nmol ozone mol -1 1 and a combination of the two on carbon allocation was examined in Pinus halepensis co-cultured with Betula pendula in symbiosis with the ectomycorrhizal fungus Paxillus involutus. The results show that under low nutrient and ozone levels, elevated CO 2 has no effect on the growth of B. pendula or P. halepensis seedlings nor on net carbon partitioning between plant parts. Elevated CO 2 did not enhance the growth of the fungus in symbiosis with the birch. On the other hand, ozone had a strong negative effect on the growth of the birch, which corresponded with the significantly reduced growth rates of the fungus. Exposure to elevated CO 2 did not ameliorate the negative effects of ozone on birch; in contrast, it acted as an additional stress factor. Neither ozone nor CO 2 had significant effects on biomass accumulation in the pine seedlings. Ozone stimulated the spread of mycorrhizal infection from the birch seedlings to neighbouring pines and had no statistically significant effects on phosphoenolpyruvate carboxylase (PEPC) or ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activity in the pine needles or on PEPC activity in pine roots.

The Paxillus involutus (Fries) Karsten-Betula pendula Roth association was studied during the early stages of formation. Cytological studies revealed fungal colonization behind the root cap and gradually around the entire root apex. Ultrastructural investigations were carried out and insoluble polysaccharide distribution was followed. The density of starch grains increased in plant cells especially after 4 d of contact between the two partners, but later on decreased strongly in the root cap. Large amounts of glycogen were revealed in the hyphae in certain mycorrhizal regions after 6 d of contact: in the Hartig net, in the inner sheath but only near the net, and all along the outer sheath surrounding the mycorrhiza. Thickenings of the epidermal cell walls were detected as early as 2 d after contact and then varied according to the distance from the root tip. Such polysaccharide distributions are assumed to show a transfer of carbohydrates from the root to the fungus and are discussed in terms of carbon requirements for both partners.

Exposure of Rhizopogon roseolus mycelia to 15 mmol·dm−3 cadmium for 24 h induces a different pattern of Mn-SOD on polyacrylamide gels, probably being a changed form of an originally existing one. The presence of cadmium affects the chromatographic properties of this enzyme and its mobility through the acrylamide gel. This new isoform was purified using DEAE Trisacryl chromatography. Cadmium induced isoform adsorbed stronger to the ligands and was eluated with a Tris-HCl buffer containing 0.1 mol·dm−3 NaCl. SOD from control samples (not treated with cadmium) was eluated with the same buffer without NaCl.

Variation within the internal transcribed spacer (ITS) and the intergenic spacer (IGS) of the ribosomal RNA gene of isolates representing seven European species of Armillaria was examined by PCR, coupled with RFLP analysis and partial sequencing of the ITS region.

The ectomycorrhizal Laccaria bicolor , cultivated axenically in modified Pachlewski's medium, can use a broad spectrum of nitrogen and carbon sources. The fungus exhibited greater growth in the presence of ammonium or nitrate than with ammonium nitrate. With the latter salt, the drop in pH of the culture medium indicated that ammonium was taken up preferentially to nitrate. L. bicolor grew more poorly on amino acids such as glycine, alanine, aspartate and glutamate, the latter three being very poor carbon sources. By contrast, glycine was used as nitrogen and carbon sources. Glucose and maltose in mixture, with a 20:5 ratio, were the most effective carbohydrates for promoting growth, followed by starch, dextrins, maltose, glucose and sorbitol. L. bicolor failed to grow in the presence of sucrose and galactose.

Seedlings of Betula pendula Roth. were grown in the presence of Paxillus involutus (Batsch) Fr., and metabolic changes during mycorrhiza formation were examined by measuring organic acid and amino acid pools and related enzyme activities, following sequential harvests. Glutamine, aspartate and asparagine pools were always lower in infected roots than in non-infected roots, especially during Hartig net initiation and formation. Glutamate concentration was similar in both tissues. Citrate and malate were the two major organic acids detected and their concentrations were equal in infected and non-infected roots. Aspartate aminotransferase, glutamine synthetase, NAD-dependent malate dehydrogenase and glucose-6-phosphate dehydrogenase activities were higher in infected roots than non-infected roots. For all enzymes revealed on polyacrylamide gels, both root and fungal isoforms were present in infected roots. Quantitative changes in enzyme capacities and metabolite pools indicated that mycorrhiza formation caused a re-arrangement of the main metabolic pathways during the very early stages following contact, which might be related to the structural changes.

The NAD-dependent glutamate dehydrogenase (GDH) (EC 1.4.1.2) from Laccaria bicolor was purified 410-fold to apparent electrophoretic homogeneity with a 40% recovery through a three-step procedure involving ammonium sulfate precipitation, anion-exchange chromatography on DEAE-Trisacryl, and gel filtration. The molecular weight of the native enzyme determined by gel filtration was 470 kDa, whereas sodium dodecyl sulfate-polyacrylamide gel electrophoresis gave rise to a single band of 116 kDa, suggesting that the enzyme is composed of four identical subunits. The enzyme was specific for NAD(H). The pH optima were 7.4 and 8.8 for the amination and deamination reactions, respectively. The enzyme was found to be highly unstable, with virtually no activity after 20 days at -75 degrees C, 4 days at 4 degrees C, and 1 h at 50 degrees C. The addition of ammonium sulfate improved greatly the stability of the enzyme and full activity was still observed after several months at -75 degrees C. NAD-GDH activity was stimulated by Ca2+ and Mg2+ but strongly inhibited by Cu2+ and slightly by the nucleotides AMP, ADP, and ATP. The Michaelis constants for NAD, NADH, 2-oxoglutarate, and ammonium were 282mgr;M, 89mgr;M, 1.35 mM, and 37 mM, respectively. The enzyme had a negative cooperativity for glutamate (Hill number of 0.3), and its Km value increased from 0.24 to 3.6 mM when the glutamate concentration exceeded 1 mM. These affinity constants of the substrates, compared with those of the NADP-GDH of the fungus, suggest that the NAD-GDH is mainly involved in the catabolism of glutamate, while the NADP-GDH is involved in the catalysis of this amino acid. Copyright 1997 Academic Press. Copyright 1997 Academic Press

The kinetics, energetics and specificity of a general amino acid transporter were studied in the ectomycorrhizal fungus Paxillus involutus (Batsch) Fr. The uptake of amino acids showed features characteristic of active transport. After correction for a non-mediated transport component, the kinetics of glutamate, glutamine, alanine and aspartate uptake measured over a wide concentration range followed the simple Michaelis-Menten saturation curves, The apparent K-m derived from the Eadie-Hofstee plots ranged from 7 mu M for alanine to 27 mu M for glutamate, Dinitrophenol, carbonyl cyanide m-chlorophenylhydrazone and NaN3 strongly inhibited amino acid uptake, whereas dicyclohexylcarbodiimide. vanadate and the ionophores monensin and nonactin had no effect on the uptake. Both ph dependence and inhibition by protonophores are consistent with a proton symport mechanism for amino acid uptake by P. involutus, Competition studies indicated a broad substrate recognition by the uptake system, which resembles the general amino acid permease of yeast, Dixon plots of the inhibition of glutamate uptake by alanine, lysine and methionine sulfoximine showed that inhibitions were competitive, Tire physiological importance of this transporter for the exchange of nitrogenous compounds between fungal and host plant cells in ectomycorrhizal associations is discussed. (Less)

summary NADP-glutamate dehydrogenase (NADP-GDH) is a key enzyme of N assimilation in ectomycorrhizal fungi, but little is known about its regulation. In the present study. the effects of N source and concentration cm NADP-GDH specific activity, polypeptide content and biosynthesis have been investigated in the ectomycorrhizal basidiomycete Laccaria bicolor S238N. Mycelium was transferred from NH+4-rich medium to either NO−3-rich or N-free medium. Under both these conditions, intracellular NH.−3 and glutamine content decreased dramatically. The specific activity of NADP-GDH was simultaneously increased 3 4.5-fold. Immunological analysis using anti-GDH antibodies showed that depression of enzyme activity resulted from increased concentration of the GDH polypeptide. In addition, assessment of the steady-state biosynthesis of NADP-GDH by in vivo labelling of proteins demonstrated that the observed changes in enzyme concentration are explained by differences in the relative rate of enzyme biosynthesis. These results suggested that the source of N regulates synthesis of NADP-GDH and that this regulation is under transitional and/or transcriptional control.

Oxalate accumulation in external medium under hyphal mats of two ectomycorrhizal species is strongly stimulated (1.7 to 35 fold) by a simultaneous depletion of available K + and Mg 2+ . Pisolithus tinctorius strain 441 accumulates oxalate both on NH 4 -N and on NO 3 -N whereas Paxillus involutus strain COU only accumulates oxalate on NO 3 -N. On NO 3 -N, under a simultaneous K + and Mg 2+ deficiency, P. involutus COU is a very active oxalate producer compared to P. tinctorius 441. The present results could explain the various mineralogical evolutions of a phlogopite mica previously recorded under P. involutus COU or P. tinctorius 441 and suggest a key role for fungal oxalic acid during mineral weathering in response to nutrient deficiency.

Glutamine synthetase (GS, EC 6.3.1.2) was purified to apparent electrophoretic homogeneity from roots of Pseudotsuga menziesii (Mirb) Franco by a three-step procedure involving diethylaminoethyl (DEAE)-Trisacryl chromatography, affinity chromatography on Matrex Gel Red A, and preparative polyacrylamide gel electrophoresis. The enzyme was purified 40-fold with a 16% recovery. The native enzyme had a molecular mass of 460 ± 5 kDa as estimated by gel filtration, interpolation of the Ferguson plots and non-denaturing gradient-PAGE. It was composed of two different subunits of 54 and 64 kDa. Affinity constants for glutamate (Glu), glutamine (Gln), ATP and ADP were 2.6, 10.5, 0.5 and 0.083 mM, respectively. The enzyme exhibited a negative cooperativity for ammonium (Hill number of 0.7) with two K m values which were 11 and 75 μM in the presence of ammonium concentrations lower and higher than 1.3 mM, respectively. Glycine and ADP appeared as potential inhibitors of the GS activity. The optimum pH values were 7.2 and 7.6 for the transferase and the biosynthetic assays, respectively. The enzyme lost 30% of its activity within 25 days of storage at 4°C. The optimum temperatures of activity were 40°C and 45°C for the transferase and biosynthetic activities, respectively.

A simple and rapid method was developed for the separation and determination of a large number of organic acids by using capillary zone electrophoresis, with hydrostatic injection for 30 s and UV detection at 254 nm. Electropherograms were obtained with 4-hydroxybenzoate buffer (5 m M ) adjusted to pH 4.75 at 25°C. The electroosmotic flow modifier (commercial solution) was added to the buffer at a concentration of 25 ml 1 −1 . Co-migration problems could be solved by modifying the pH of the electrophoresis buffer or by addition of Ca 2+ to the buffer.

Mycelium of Pisolithus arrhizus cultured on agar media containing cadmium dust (collected from industrial electrofilters) and on media without the dust was analysed by electron spectroscopic imaging (ESI) and electron energy loss spectroscopy (EELS) connected with the transmission electron microscope. Complementary investigations of polysaccharide (PATAg) and cysteine rich protein localization were carried out. The analyses revealed abundant deposition of glycogen in the form of net-like material in the cytoplasm of the dust-supplemented mycelium, accompanied by increased levels of Ca. Heavy metals like Cd, Cu and Fe accumulated mainly in electron opaque granules and in the outer pigmented layer of the cell wall, both characterized by the presence of polysaccharides and cysteine-rich proteins.

Purification and study of the antibiotic substances produced byLentinus squarrosulus have been carried out. The substances, excreted in the culture medium, were extracted withn-butanol. The butanolic extract inhibited growth ofRigidopurus lignosus, the agent of white rot ofHevea brasiliensis, and also ofMucor ramannianus, Rhodotorula mucilaginosa, Saccharomyces cerevisiae, andBacillus subtilis. The antibiotic fractions were purified by silicic acid column chromatography, then by reversed-phase (C18) high performance liquid chromatography (HPLC) followed by preparative adsorption thin-layer chromatography on silica gel. Two purified compounds were obtained: Ls1, which was active againstB. subtilis, and Ls2, which in addition was also active againstR. lignosus, M. ramannianus, and yeasts. Only the Ls2 compound is analyzed in this work. It was characterized by chemical reactions, ultraviolet spectroscopy, infrared spectroscopy, and proton nuclear magnetic resonance spectroscopy, which indicated the hydrophilic character of the molecule and the presence of alcoholic functions as well as a glycosidic moiety. The properties differed from those of already known antibiotics produced by different species ofLentinus.

summary Previous studies of nitrogen-assimilating enzymes in ectomycorrhizal associations of the Pinaceae (Norway spruce, Douglas fir) and the Fagaceae (beech) have suggested that the host plant may regulate the expression of some fungal enzymes. To improve our understanding of the regulation of nitrogen-assimilating enzymes in ectomycorrhizal plants, we have extended this work to a third plant family, the Myrtaceae. Glutamate dehydrogenases, specific either for NAD (NAD-GDH) or NADP (NADP-GDH), and aspartate amminotransferase (AAT) were investigated by enzyme assays and electrophoretic patterns on polyacrylamide gels. Non-mycorrhizal roots of four species of Eucalyptus (E. diversicolor, E. globulus, E. nitens and E. regnans) were characterized by a high activity of NAD-GDH, while only NADP-GDH was found in Hebeloma westraliense in pure culture. In associations of these eucalyptus with Hebeloma westratiense, both NAD- and NADP-GDHs were detected. By contrast, NAD- and NADP-GDHs found in the free-living mycelium of Lacrayia lacrata were not detected in the associated ectomycorrhizas. In ectomycorrhizas of five species of eucalypts (E. diversicolor, E. globulus, E. grandis, E. regnans and E. urophylla) associated with either Laccaria laccata, Scleroderma verrucosum or Pisolithus tinctorius, fungal AAT. which was very active in free-living mycelia, was not detected in the symbiotic tissues. In contrast, the two root AAT isoenzymes remained active in the mycorrhizas and were even stimulated. These results suggest that fungal gene expression is moderated by the host plant but also depends on the fungal associate. Factors which may contribute to the observed differences in enzyme activity between Hebeloma and Laccaria are discussed.

Three basidiomycetes, Lentinus squarrosulus, Cerrena meyenii and Gloeophyllum striatum were studied as potential antagonists against Rigidoporus lignosus , the cause of white rot of Hevea brasiliensis . During direct confrontation between colonies of R. lignosus and the three antagonists on malt agar, R. lignosus colonies ceased growth and were overgrown by the opposing fungi. The most effective antagonist was L. squarrosulus . Confrontation of R. lignosus with culture filtrates from the antagonistic fungi led to an inhibition of the parasite, the best result being obtained with L. squarrosulus filtrates. Confrontation on Hevea wood blocks revealed that L. squarrosulus was the only test fungus able to invade the zone previously colonized by R. lignosus and to kill the latter; furthermore, it caused significantly greater decay of the test blocks. Extracts of culture filtrates of L. squarrosulus were more active against R. lignosus at acidic pH values with a maximum at pH 5 but retained a significant activity up to pH 10. The antibiotic compound of L. squarrosulus was thermostable and remained active when heated for 15 min at 100 °C.

The monosodium salt of a synthetic auxin (2,4-D), and a quaternary ammonium salt (BrB), were tested on mitochondria isolated from fresh potato tubers. The two compounds inhibited mitochondrial respiration, the quaternary ammonium salt being the most effective. The uncoupling activity of 2,4-D resulted in the loss of energy production. Moreover, 2,4-D inhibited succinate dehydrogenase, malate dehydrogenase and NAD-dependent malic enzyme activities leading to a strong inhibition of electron flow. The mode of action of BrB was shown to be more complex. It was demonstrated that BrB acts on the membrane structure, its effects being concentration-dependent. Thus, low BrB concentrations inhibiting phosphorylation process by impeding phosphate entry to the matrix. In the presence of high BrB concentrations, mitochondrial membranes were permeable to cytochrome c. In addition, BrB efficiency diminished when the mitochondrial protein concentration was increased. Consequently, BrB probably caused, as a detergent, a large disorganization of the mitochondrial membranes.

summary In order to investigate the relative contribution of glutamine synthetase and NADP-glutamate dehydrogenase to the assimilation of ammonium (NH4+) by spruce ectomycorrhizas, changes in free amino acid content and kinetics of 15N incorporation into free amino acids were measured together with the effect of specific enzyme inhibitors. Exposure of detached ectomycorrhizas to (15NH4) 2SO4 showed that the greatest flow of 15N enters into the amido group of glutamine. Label was also detected in glutamic acid, alanine and γ-aminobutyric acid. Large amounts of alanine and glutamate accumulated in response to the addition of methionine sulfoximine (MSX) together with a decrease in 15N incorporation into both amido- and ammo-nitrogen of glutamine. These results are consistent with a major role of glutamate dehydrogenase and glutamine synthetase in nitrogen assimilation in the symbiosis and do not suggest any significant role for glutamate synthase in the synthesis of glutamate. A large accumulation of unlabelled asparagine in response to MSX and albizziine inhibition suggests the occurrence of an unlabelled NH4+ pool in the host plant. The transfer of nitrogen compounds between the fungal cells and the host tissues is discussed.

Hebeloma cylindrosporum strain h 17 was grown on media containing either glutamate or ammonium as nitrogen source. Growth tests and in vitro activity measurements revealed that both glutamine synthetase (GS. EC 6.3.1.2) and NADP-specific glutamate dehydrogenase (NADP-GDH, EC 1.4.1.4) are fully functional in wild type mycelia grown on glutamate or ammonium as sole nitrogen source. However, NADP-GDH appeared to be more active than GS in stationary growing mycelia. NADP-GDH is also able to sustain adequate ammonium assimilation in methionine sulfoximine (MSX)-treated mycelia since they grew as well as mycelia fed with ammonium alone. The NADP-GDH also appeared to be L-glutamate inducible whereas GS was repressed by ammonium. The NADP-GDH deficient strain, when transferred from a glutamate containing medium to an ammonium containing medium, exhibited a derepressed GS, although this enzyme did not fully substitute for the deficiency of NADP-GDH in ammonium assimilation. The low NADP-GDH activity of the mutant strain exhibited a reduced mobility on a 6% constant polyacrylamide gel. By contrast, the two enzymes had identical molecular weights, estimated to be ca 295 kDa on gradient polyacrylamide gel. The involvement of NADP-GDH and GS enzymes in nitrogen assimilation is discussed.

The composition of the buffer for extraction of nitrate reductase (EC 1.6.6.3) from Sphaerostilbe repens was studied in order to obtain optimal and stable activity. A good extraction buffer consisted of 100 m m K-phosphate buffer, pH 7·5, 1% casein, 1 m m -EDTA, 10 m -FAD, 1 m -sodium molybdate and 5% fresh weight PVPP. In spite of these protectants, nitrate reductase in crude extracts or submitted to gel filtration proved to be very unstable. Thus fungal extracts were stable at − 25 °C for about 3 d but at 0° the activity was reduced by half within 24 h and at 20° enzyme activity was completely lost in less than one hour. Stability was greatly improved when the enzyme was extracted in the presence of 1 m m -PMSF and partially purified by a two-step procedure including ammonium sulphate fractionation and DEAE-Trisacryl chromatography. The optimal pH value for the reduction on nitrate was about 7·5. The enzyme was NADPH-specific and required the addition of flavin adenine dinucleotide for maximum activity. The Michaelis constants for nitrate and NADPH were 1·4 and 0·032 m m respectively. Nitrate reductase activity was induced by nitrate and repressed by ammonium. The induction resulted in an atypical curve characterized by minor increase of activity at the 8th min of incubation on nitrate and a major increase 40 min later.

Seasonal changes in growth, nutrient content, protein amount, amino acid levels and enzyme activities were measured in both L. laccata inoculated or non inoculated Douglas fir tissues. The increased growth of inoculated fir is accompanied by higher nutrient and protein amounts and higher enzyme activity levels in foliar tissues. By contrast only slight changes were observed in root and stem tissues.

Sulphite at a concentration of 1 mM did not strongly affect the growth of mycelium. Higher concentrations of 5–20 mM almost completely inhibited the growth of mycelium and superoxide dismutase (SOD) [EC.1.15.1.1.] activity. The activity of this enzyme was not detectable on polyacrylamide gels. The lack of induction of SOD and the resulting oxidative stress may in part be responsible for the growth inhibition caused by high concentrations of sulphite.

External hyphae, which play a key role in nitrogen nutrition of trees, are considered as the absorbing structures of the ectomycorrhizal symbiosis. Here, we have cloned and characterized Hebeloma cylindrosporum AMT1, GLNA and GDHA genes, which encode a third ammonium transporter, a glutamine synthetase and an NADP-dependent glutamate dehydrogenase respectively. Amt1 can fully restore the pseudohyphal growth defect of a Saccharomyces cerevisiae mep2 mutant, and this is the first evidence that a heterologous member of the Mep/Amt family complements this dimorphic change defect. Dixon plots of the inhibition of methylamine uptake by ammonium indicate that Amt1 has a much higher affinity than the two previously characterized members (Amt2 and Amt3) of the Amt/Mep family in H. cylindrosporum. We also identified the intracellular nitrogen pool(s) responsible for the modulation of expression of AMT1, AMT2, AMT3, GDHA and GLNA. In response to exogenously supplied ammonium or glutamine, AMT1, AMT2 and GDHA were downregulated and, therefore, these genes are subjected to nitrogen repression in H. cylindrosporum. Exogenously supplied nitrate failed to induce a downregulation of the five mRNAs after transfer of mycelia from a N-starved condition. Our results demonstrate that glutamine is the main effector for AMT1 and AMT2 repression, whereas GDHA repression is controlled by intracellular ammonium, independently of the intracellular glutamine or glutamate concentration. Ammonium transport activity may be controlled by intracellular NH4+. AMT3 and GLNA are highly expressed but not highly regulated. A model for ammonium assimilation in H. cylindrosporum is presented.

Nutrient transport, namely absorption from the soil solution as well as nutrient transfer from fungus to plant and carbon movement from plant to fungus are key features of mycorrhizal symbiosis. This review summarizes our current understanding of nutrient transport processes in ectomycorrhizal fungi and ectomycorrhizas. The identification of nutrient uptake mechanisms is a key issue in understanding nutrition of ectomycorrhizal plants. With the ongoing functional analysis of nutrient transporters, identified during sequencing of fungal and tree genomes, a picture of individual transport systems should be soon available, with their molecular functions assessed by functional characterization in, e.g., yeast mutant strains or Xenopus oocytes. Beyond the molecular function, systematic searches for knockout mutants will allow us to obtain a full understanding of the role of the individual transporter genes in the physiology of the symbionts. The mechanisms by which fungal and plant cells obtain, process and integrate information regarding nutrient levels in the external environment and the plant demand will be analyzed.

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Thirty-nine ectomycorrhizal isolates of Paxillus involutus, Pisolithus tinctorius, Suillus bovinus, S. luteus and S. variegatus were tested on cadmium, copper, nickel and zinc amended media to determine their in vitro tolerance, measured as inhibition of biomass production. Twenty-one isolates were from heavy metal polluted sites, whereas the others were from non-contaminated soils. There was a strong interspecific variation in metal tolerance. S. luteus, S. variegatus and P. tinctorius were more tolerant of Cu, Cd and Zn when compared with P. involutus, whereas the reverse was true for Ni. A high intraspecific heterogeneity in metal tolerance was also found. EC 50 values for isolates originating from polluted sites were not statistically different from EC 50 values for isolates originating from non-contaminated sites. The findings are discussed in relation to the potential benefits of ectomycorrhizal fungi in protecting their host plants from metal contamination.

A lectin (HHL) was isolated from the fruiting body of the mushroom Hygrophorus hypothejus by a combination of affinity chromatography on stromas of group B erythrocytes embedded in polyacrylamide gel, and DEAE-trisacryl and gel filtration chromatography. Its molecular mass, as determined by gel filtration, is estimated to be 68 000 kDa and its structure is tetrameric with four identical subunits assembled with non-covalent bonds. HHL agglutinates specifically A and B blood group erythrocytes and in hemagglutination inhibition assays, exhibits sugar-binding specificity toward lactose, the anomeric α form being more effective than the β form.

The internal transcribed spacer (ITS) of rDNA was analysed by PCR/RFLP in 66 strains belonging to 11 putative species of the Morchellaceae and 3 species of the Discinaceae. For certain taxa, in particular Morchella esculenta, Morchella conica, and Morchella elata, isolates from distant geographical origins were available. Nevertheless, no intraspecific variations of ITS length and restriction was noted. All genera were separated and within Morchella; four putative species could be clearly distinguished in the group of the yellow morels (sectio adnatae). In contrast, no distinction could be obtained between the compared taxa in the group of the black morels (sectio distantes). Sequence analyses of the ITS were performed in one strain of seven putative Morchella. The sequence comparison confirmed the distinctions within the yellow morels and allowed us to separate one putative taxon from the others in black morels. Only the 5,8 gene could be aligned in all sequenced samples. The ITS1 and ITS2 sectors could only be aligned within the black and yellow morels, respectively. The genetic and phylogenetic distances between black and yellow morels, respectively revealed by maximum parsimony and neighbour joining analyses, both confirm the great distance between these species groups and address the question of a separation in the genus Morchella.Key words: Morchellaceae, Discinaceae, ITS.

Thirteen Armillaria isolates, collected from various geographical areas in tropical Africa and previously characterized by cultural morphology, pairing tests and isozyme analysis, were evaluated using the polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP). DNA regions corresponding to the intergenic spacer (IGS) and internal transcribed spacer (ITS) were amplified and analyzed by restriction enzyme digestion. The IGS amplification products were about 875 bp long and uniform in length among the isolates. The amplified-ITS region showed two different lengths corresponding to two groups. The first group included the isolates believed to belong to A. mellea ssp. africana and two Kenyan isolates (K11 and K12) belonging to a yet unnamed biological species. The second group included isolates identified as A. heimii and a Tanzanian isolate (T7). Each length variant of the ITS showed distinct RFLP banding patterns. Digestion with EcoRI confirmed the two polymorphic groups while the endonucleases AluI and NdeII discriminated the A. mellea isolates from the Kenyan isolates K11 and K12. In addition, the latter enzyme showed a slight dissimilarity between the A. heimii isolates from Western and Eastern Africa (C1 and Z1). Digestion with HinfI cleaved the isolates of A. heimii into two sub-groups corresponding to the heterothallic and homothallic forms. This endonuclease also indicated that the isolate T7, originating from Tanzania, was clearly similar to the heterothallic species A. heimii. Data presented support the maintenance of three distinct species of Armillaria in tropical Africa with A. heimii as a variable species, the isolates of which were separated in accordance with their sexual system. The results indicate that PCR-RFLP can be used as a simple and speedy taxonomical tool for the ecological studies of Armillaria species.

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A field experiment in the Vosges (a low mountain range in north-eastern France) had shown that a CaMg amendment of an acidic forest soil reduced the total number of fungal propagules per gramme dry soil, while the number of species considerably increased. Some rhizosphere fungi isolated from the amended and/or control plots were grown in three media with ammonium or nitrate as the sole source of nitrogen, and with or without aluminium. Complexing organic acids released by the fungi and likely to interfere with tree nutrition were analyzed in the culture medium after fourteen days incubation. The largest amounts of complexing organic acids are released by fungi isolated from the acid control plot. These rhizospheric fungi take part in soil acidification, which results in the long-term decrease of soil mineral reserves in a forest soil subjected to permanent leaching. Therefore, we conclude from this work that fungi might contribute to the deficient mineral nutrition of the Norway spruce on acidic soils, by impoverishment of the root environment in essential cations. However, among the causes of the Norway spruce yellowing, rhizosphere deleterious fungi act as secondary agents or contributing factors rather than primary causes of decline.

Pireaux, J-C., Hayani-Obeidou, W., Chalot, M., Botton, B., and Dizengremel, P. 1995. Mitochondria in the white-rot fungus Phanerochaete chrysosporium: Purification and evidence for a mitochondrial isoform of aspartate aminotransferase. Experimental Mycology 19: 91-100. A very high specific aspartate aminotransferase activity was found in young Phanerochaete chrysosporium cultures. In order to obtain more information about the localization of aspartate aminotransferase, mitochondria were isolated and purified. The purification gradient showed two mitochondrial fractions. Mitochondria localized near the top of the gradient were severely damaged, whereas mitochondria aggregated at the bottom part were largely intact (91 and 94% intactness for outer and inner membrane, respectively). The highest oxidation rates were obtained with succinate and NADH as substrates. Malate oxidation was improved by exogenous NAD + while NADPH oxidation was partially Ca 2+ dependent. All these oxidations were correctly coupled to the phosphorylation. Experiments using standard respiratory chain inhibitors indicate that, in P. chrysosporium mitochondria, the alternative pathway was present, but not engaged. On nondenaturing uniform polyacrylamide gels, four aspartate aminotransferase isoforms were detected, one being localized in the mitochondrial matrix. On nondenaturing polyacrylamide gradient gels, the nonmitochondrial isoforms had similar electrophoretic mobilities with a molecular mass estimated at 110 kDa, while the mitochondrial isoform had a molecular mass of about 160 kDa.

A hemagglutinating and hemolytic lectin (PSL) has been isolated from carpophores of the parasitic mushroom Laetiporus sulfureus by affinity chromatography on Sepharose. Its molecular weight, as determined both by gel filtration and by electrophoresis in non-denaturing conditions, is about 190,000 and its structure is tetrameric, with two distinct types of subunits (about 60,000 and 36,000). It appeared homogeneous on HPLC gel filtration but exhibited microheterogeneity on isoelectric focusing. Hapten inhibition assay indicated that the Laetiporus lectin is specific for N-acetyllactosamine residues and that hemagglutinating and hemolysis activities are supported by the same site.

Using antibodies raised against glutamine synthetase (GS) and NADP-glutamate dehydrogenase (NADP-GDH) from Laccaria laccata, we examined tissular localization of GS and NADP-GDH in symbiotic tissues of Douglas fir/L. laccata ectomycorrhizas by immunogold labeling. Thin sections of mycorrhizal roots were first treated either with an anti-GS- or antiNADP-GDH-specific antibody and then with a colloidal gold marker. Both enzymes appeared to be cytoplasmic. Our results also indicated the presence of GS in some fungal cells in dense cytoplasmic patches. It also appeared that GS is more abundant than NADP-GDH. The distribution of these nitrogen-assimilating enzymes in the fungal Hartig net and the sheath did not differ significantly. No labelling was observed in host cells.

Summary Pathways of inorganic nitrogen assimilation are very often different in higher plants and fungi and this raises the intriguing problem of their coexistence and interactions in ectomycorrhizas. Results based on amino acid accumulation, enzyme assays, electrophoretic patterns on gels and immunogold labelling have consistently shown that beech ectomycorrhizas are similar to higher plants in that glutamine synthetase (GS) and glutamate synthase (GOGAT) are operative, while fungal glutamate dehydrogenese (NADP-GDH) is strongly repressed. By contrast, in spruce and Douglas-fir ectomycorrhizas, fungal NADP-GDH was found to be involved in ammonium assimilation, jointly with the GS enzyme. Enzyme repression seems to be due to the host plant but is moderated by the structure of the ectomycorrhizas and especially by the degree of association of the fungus with root tissues, as found in Eucalypt ectomycorrhizas. In all the ectomycorrhizas investigated, fungal aspartate aminotransferase (AAT) was found to be r...

Glutamine synthetase (GS) and NADP-dependent glutamate dehydrogenase (NADP-GDH) play a key role in nitrogen assimilation in the ectomycorrhizal fungus Laccaria laccata (Scop. ex Fr. Cke) strain S 238. The two enzymes were purified to apparent electrophoretic homogeneity by a three-step procedure involving diethylaminoethyl (DEAE)-Trisacryl and affinity chromatography, and DEAE-5PW fast protein liquid chromatography. This purification scheme resulted in a 23 and 62% recovery of the initial activity for GS and NADP-GDH, respectively. Purified GS had a specific activity of 713 nanomoles per second per milligram protein and a pH optimum of 7.2. Michaelis constants (millimolar) for the substrates were NH(4) (+) (0.024), glutamate (3.2), glutamine (30), ATP (0.18), and ADP (0.002). The molecular weight (M(r)) of native GS was approximately 380,000; it was composed of eight identical subunits of M(r) 42,000. Purified NADP-GDH had a specific activity of 4130 nanomoles per second per milligram protein and a pH optimum of 7.2 (amination reaction). Michaelis constants (millimolar) for the substrates were NH(4) (+) (5), 2-oxoglutarate (1), glutamate (26), NADPH (0.01), and NADP (0.03). Native NADP-GDH was a hexamer with a M(r) of about 298,000 composed of identical subunits with M(r) 47,000. Polyclonal antibodies were produced against purified GS and NADP-GDH. Immunoprecipitation tests and immunoblot analysis showed the high reactivity and specificity of the immune sera against the purified enzymes.

Publisher Summary This chapter describes some immunological procedures already used in mycorrhiza to study protein synthesis as well as tissue and intracellular locations of a few enzymes. In addition to more generally used techniques such as those for the estimations of the different forms of nitrogen, the chapter covers a large range of enzymological techniques such as, purification, quantification, localization, and the use of enzyme inhibitors that appear to have considerable future potential in the study of nitrogen metabolism in mycorrhiza. Much research is devoted to electrophoretic and immunological procedures, which are very sensitive methods for elucidating metabolic pathways and are used after the protein purification steps which are the key to many areas of biochemical research on mycorrhiza. However, with the recent advances in gene cloning and expression it is not only protein researchers who need to be able to use these techniques, but also biochemists and molecular biologists, working on a wide range of problems. The chapter also discusses extraction and recovery of the nitrogen fractions.