Genetic diversity of the ectomycorrhizal fungus Pisolithus tinctorius based on RAPD-PCR analysis

 Twenty Pisolithus tinctorius isolates from different geographic locations and different hosts were characterized by the random amplified polymorphic DNA technique. Thirteen arbitrary primers generated 87 DNA fragments, all of them polymorphic. These data were used to calculate genetic distances among the isolates. The pairwise genetic distances ranged from 1 to 100%, with an average of 58.7%. Cluster analysis based on the amplified fragments grouped the isolates according to their host and geographical origins. Group I contained isolates collected in Brazil and group II those collected in the Northern Hemisphere. In addition to the diversity seen at the molecular level, the isolates also showed host specificity. Greenhouse experiments demonstrated that isolates from the Northern Hemisphere colonized mainly Pinus whereas isolates from Brazil colonized only Eucalyptus. The molecular data suggest that the Pisolithus tinctorius isolates analyzed belong to two distinct groups. The data also suggest new guidelines for future investigations on the taxonomy and systematic of this important fungus species. Furthermore, these results support future experiments aimed at the selection and development of improved isolates of P. tinctorius. Accepted: 3 October 1997     

A 31P nuclear magnetic resonance study of phosphate levels in roots of ectomycorrhizal and nonmycorrhizal plants of Castanea sativa Mill.

 ³¹P-Nuclear Magnetic Resonance (NMR) was used to assess phosphate distribution in ectomycorrhizal and nonmycorrhizal roots of Castanea sativa Mill. as well as in the mycorrhizal fungus Pisolithus tinctorius in order to gain insight into phosphate trafficking in these systems. The fungus P. tinctorius accumulated high levels of polyphosphates during the rapid phase of growth. Mycorrhizal and nonmycorrhizal roots accumulate orthophosphate. Only mycorrhizal roots presented polyphosphates. The content in polyphosphates increased along the 3 months of mycorrhiza formation. In mycorrhizal roots of plants cultured under axenic conditions, the orthophosphate pool decreased along the culture time. In nonmycorrhizal roots the decrease in the orthophosphate content was less pronounced. The level of orthophosphate in mycorrhizal roots was significantly lower than in nonmycorrhizal ones, which indicates that this system relies upon the fungal polyphosphates as a major source of phosphate. Received: 28 July 1998 / Accepted: 21 October 1998     

Subcellular compartmentation of elements in non-mycorrhizal and mycorrhizal roots of Pinus sylvestris: an X-ray microanalytical study. II. The distribution of calcium, potassium and sodium

The inter- and intracellular distribution of the elements calcium, potassium and sodium in non-mycorrhizal and mycorrhizal roots of Pinus sylvestris dependent on different external nutrient supply conditions was detected by energy-dispersive X-ray microanalysis after cryofixation, freeze-drying and pressure infiltration of the material. In non-mycorrhizal and mycorrhizal roots, calcium was mainly detectable in the apoplastic regions. The levels in vacuoles and cytoplasm were near the limits of detection by X-ray microanalysis. Incubation with high concentrations of potassium and sodium, or mycorrhizal infection with Suillus bovinus and Pisolithus tinctorius reduced the amounts of calcium detectable in the roots, especially in the apoplast of cortical cells. The studies revealed that: potassium is mainly localized in cytoplasm and cell walls; the cytoplasmic content is regulated over a wide range of external potassium concentrations; potassium levels in the inner parts of roots are higher than in the outer parts. Mycorrhizal infection with Suillus bovinus had no effect on the inter- and intracellular distribution of potassium in roots but, if the external supply was low, the potassium content in shoots was reduced. In non-mycorrhizal pine roots and those infected with Paxillus involutus an increase in the sodium content of all cell compartments was observed after treatment with high external concentrations of NaH₂PO₄. However, an increase in sodium content in mycorrhizas of S. bovinus was not detected. The X-ray microanalytical results are discussed in relation to the apoplastic movement of nutrients in non-mycorrhizal and mycorrhizal fine roots of pine and to the demand for these nutrients in different intracellular compartments.     

Endogenous polyamine content and metabolism in the ectomycorrhizal fungus Paxillus involutus

Endogenous polyamine content of the ectomycorrhizal fungus Paxillus involutus , as well as the activity of its biosynthetic enzymes in relation to mycelia ageing were investigated in this work. Polyamines in free, PCA-soluble and insoluble conjugated forms, are present in Paxillus involutus mycelia in relatively high amounts and the ratio of putrescine to spermidine is age-dependent. Both arginine- and ornithine-decarboxylases are present, but putrescine biosynthesis proceeds mostly via ornithine decarboxylase and decreases with the age of mycelia. There was a large release of free polyamines from mycelia which showed age-dependent features. Clear polyamine uptake was observed in 2-wk-old mycelia and no competition between putrescine and cadaverine was detected. Putrescine uptake seems to reduce ornithine decarboxylase activity, but does not affect arginine decarboxylase.     

Screening for rapidly evolving genes in the ectomycorrhizal fungus Paxillus involutus using cDNA microarrays

We have examined the variations in gene content and sequence divergence that could be associated with symbiotic adaptations in the ectomycorrhizal fungus Paxillus involutus and the closely related species Paxillus filamentosus. Strains with various abilities to form mycorrhizae were analysed by comparative genomic hybridizations using a cDNA microarray containing 1076 putative unique genes of P. involutus. To screen for genes diverging at an enhanced and presumably non-neutral rate, we implemented a simple rate test using information from both the variations in hybridizations signal and data on sequence divergence of the arrayed genes relative to the genome of Coprinus cinereus. C. cinereus is a free-living saprophyte and is the closest evolutionary relative to P. involutus that has been fully sequenced. Approximately 17% of the genes investigated were detected as rapidly diverging within Paxillus. Furthermore, 6% of the genes varied in copy numbers between the analysed strains. Genome rearrangements associated with this variation including duplications and deletions may also play a role in adaptive evolution. The cohort of divergent and duplicated genes showed an over-representation of either orphans, genes whose products are located at membranes, or genes encoding for components of stress/defence reactions. Some of the identified genomic changes may be associated with the variation in host specificity of ectomycorrhizal fungi. The proposed procedure could be generally applicable to screen for rapidly evolving genes in closely related strains or species where at least one has been sequenced or characterized by expressed sequence tag analysis.     

Interactions between Pisolithus tinctorius and its hosts: a review of current knowledge

 Pisolithus tinctorius (Pers.) Coker and Couch [Syn. = P. arhizus (Scop.: Pers.) Rauschert] (Pt) is a widespread ectomycorrhizal basidiomycete forming mycorrhizas with a variety of hosts. Developmental and functional aspects of the symbiosis are well documented and thus Pt has been adopted as a model organism for investigations of the molecular basis of ectomycorrhizal interactions. In this review of the current state of knowledge of interactions between Pt and its hosts we demonstrate that Pt displays much intraspecific heterogeneity of host specificity, physiology and the benefits the fungus can impart upon the host plant. It is not clear at present how far such heterogeneity reflects systematic segregation within Pt. Accepted: 20 May 1997     

Nitrogen and phosphorus acquisition by the mycelium of the ectomycorrhizal fungus Paxillus involutus and its effect on host nutrition

The contribution of the extramatrical mycelium to N and P nutrition of mycorrhizal Norway spruce ( Picea abies (L.) Karst.) was investigated. Seedlings either inoculated with Paxillus involutus (Batsch) Fr. or non-mycorrhizal were grown in a two compartment sand culture system where hyphae were separated from roots by a 45 μm nylon net. Nutrient solution of the hyphal compartment contained either 1.8 m m NH₄⁺ and 0.18 m m H₂PO₄⁻ or no N and P. Aluminium added to the hyphal compartment as a tracer of mass flow was not detected in the plant compartment, indicating that measurements of N and P transfer by the mycelium were not biased by solute movement across the nylon net.
The addition of N and P to the hyphal compartment markedly increased dry weight, N and P concentration and N and P content of mycorrhizal plants. Calculating uptake from the difference in input and output of nutrient in solution confirmed a hyphal contribution of 73% and 76% to total N and P uptake, respectively. Hyphal growth was increased at the site of nutrient solution input.     

Evolution of nucleotide sequences and expression patterns of hydrophobin genes in the ectomycorrhizal fungus Paxillus involutus

Hydrophobins are small, secreted proteins that play important roles in the development of pathogenic and symbiotic fungi. Evolutionary mechanisms generating sequence and expression divergence among members in hydrophobin gene families are largely unknown. Seven hydrophobin (hyd) genes and one hyd pseudogene were isolated from strains of the ectomycorrhizal fungus Paxillus involutus. Sequences were analysed using phylogenetic methods. Expression profiles were inferred from microarray experiments. The hyd genes included both young (recently diverged) and old duplicates. Some young hyd genes exhibited an initial phase of enhanced sequence evolution owing to relaxed or positive selection. There was no significant association between sequence divergence and variation in expression levels. However, three hyd genes displayed a shift in the expression levels or an altered tissue specificity following duplication. The Paxillus hyd genes evolve according to the so-called birth-and-death model in which some duplicates are maintained for a long time, whereas others are inactivated through mutations. The role of subfunctionalization and/or neofunctionalization for preserving the hyd duplicates in the genome is discussed.     

Influence of mycorrhization on physiological parameters of micropropagated Castanea sativa Mill. plants

 Mycorrhizal micropropagated Castanea sativa plants were studied in terms of growth and physiological parameters following in vitro mycorrhization with Pisolithus tinctorius. Mycorrhization enhanced growth of micropropagated chestnut plants, increased their protein content and photosynthetic rates, decreased the respiratory rates and CO₂ compensation point. RuBisCO activity was not significantly different in mycorrhizal and control plants, although there was an increase in the amount of RuBisCO in the former. Mycorrhization increased plant biomass and improved plants physiological status, thus enhancing the acclimatization process. Accepted: 21 May 1997     

Cytological and physiological behaviour of Euglena gracillis cells entrapped in a calcium alginate gel

The immobilisation of Euglena gracilis Z cells in a calcium alginate matrix maintained respiratory and photosynthetic activities and ultrastructural integrity. Moreover, immobilization did not prevent Euglena cells from greening inside the gel beads. Electron microscopy demonstrated that the immobilized cells were fixed in the same cellular state as they were when the immobilization occurred. This can he explained by simultaneous reaction of both Ca2⁺ and the alginate with the cells. Some hypotheses about the role of C2⁺ are discussed. In addition, long term storage (2 years) in calcium alginate has been performed permitting applications in algal storage.     

Translocation of 32P between interacting mycelia of a wood-decomposing fungus and ectomycorrhizal fungi in microcosm systems

Interactions between saprotrophic and ectomycorrhizal fungi have been largely ignored, although their mycelia often share the same microsites. The mycelial systems show general similarity to each other and, although the enzymatic potential of the saprotrophic fungi is generally considered to be higher, the importance of organic nutrient sources to ectomycorrhizal fungi is now widely accepted. In the experiments described here, nutritional interactions involving transfer of elements from one mycelium to the other have been monitored dynamically using radioactive tracers and a non-destructive electronic autoradiography system. Microcosms were used in which mycelial systems of the ectomycorrhizal fungi Suillus variegatus and Paxillus involutus , extending from Pinus sylvestris host plants, were confronted with mycelia of the saprotroph Hypholoma fasciculare extending from wood blocks. The fungi showed a clear morphological confrontation response. The mycorrhizal mycelium often formed dense patches over the Hypholoma mycelia. Up to 25% of the ³²P present in the Hypholoma mycelium was captured by the mycorrhizal fungi and translocated to the plant host within 30 d. The transfer of ³²P to the saprotroph from labelled mycorrhizal mycelium was one to two orders of magnitude lower. The significance of this transfer as a 'short cut' in nutrient cycling is discussed.     

In vitro synthesis of Pisolithus-Eucalyptus ectomycorrhizae: synchronization of lateral tip emergence and ectomycorrhizal development

 A simple and reproducible in vitro system is described for the synthesis of Pisolithus-Eucalyptus grandis ectomycorrhizae. Hyphal discs from actively growing colonies were placed in large petri dishes containing minimum nutrient agar overlaid with cellophane and allowed to grow for 7 days. Seeds were then surface sterilized and placed above the expanding fungal colonies and the plates slanted. Seedlings that germinated and grew in the presence of fungal hyphae had twice as many lateral root tips as seedlings that germinated before they were transferred onto hyphal mats. In addition, the lateral root tips of inoculated seedlings had a faster maturation rate and emerged closer to the primary root apex than non-inoculated seedlings. All lateral tips emerged in contact with fungal hyphae and the differentiation of ectomycorrhizae was followed by examining lateral tips basipetally along a single primary root. Typical ectomycorrhizae had formed on 4-day-old lateral tips, i.e. a mantle, radially elongated epidermal cells and a Hartig net commencing about 0.3 mm behind the lateral root apex. Thereafter, the mantle continued to thicken and the apical meristem diminished. The Hartig net often surrounded the apex of 11- to 12-day-old lateral root tips. This model system will facilitate detailed studies on synchronized ectomycorrhizal development and associated molecular and biochemical changes. Accepted: 12 January 1996     

Spatial patterns of gene expression in the extramatrical mycelium and mycorrhizal root tips formed by the ectomycorrhizal fungus Paxillus involutus in association with birch (Betula pendula) seedlings in soil microcosms

Functional compartmentation of the extramatrical mycelium of ectomycorrhizal (ECM) fungi is considered important for the operation of ECM associations, although the molecular basis is poorly characterized. Global gene expression profiles of mycelium colonizing an ammonium sulphate ((NH4)2SO4) nutrient patch, rhizomorphs and ECM root tips of the Betula pendula-Paxillus involutus association were compared by cDNA microarray analysis. The expression profiles of rhizomorphs and nutrient patch mycelium were similar to each other but distinctly different from that of mycorrhizal tips. Statistical analyses revealed 337 of 1075 fungal genes differentially regulated among these three tissues. Clusters of genes exhibiting distinct expression patterns within specific tissues were identified. Genes implicated in the glutamine synthetase/glutamate synthase (GS/GOGAT) and urea cycles, and the provision of carbon skeletons for ammonium assimilation via beta-oxidation and the glyoxylate cycle, were highly expressed in rhizomorph and nutrient patch mycelium. Genes implicated in vesicular transport, cytoskeleton organization and morphogenesis and protein degradation were also differentially expressed. Differential expression of genes among the extramatrical mycelium and mycorrhizal tips indicates functional specialization of tissues forming ECM associations.     

Relationships between fungal uptake of ammonium, fungal growth and nitrogen availability in ectomycorrhizal Pinus sylvestris seedlings

 Nitrogen deposition and intentional forest fertilisation with nitrogen are known to affect the species composition of ectomycorrhizal fungal communities. To learn more about the mechanisms responsible for these effects, the relations between fungal growth, nitrogen uptake and nitrogen availability were studied in ectomycorrhizal fungi in axenic cultures and in symbiosis with pine seedlings. Effects of different levels of inorganic nitrogen (NH₄) on the mycelial growth of four isolates of Paxillus involutus and two isolates of Suillus bovinus were assessed. With pine seedlings, fungal uptake of ¹⁵N-labelled NH₄ was studied in short-term incubation experiments (72 h) in microcosms and in long-term incubation experiments (3 months) in pot cultures. For P. involutus growing in symbiosis with pine seedlings, isolates with higher NH₄ uptake were affected more negatively at high levels of nitrogen availability than isolates with lower uptake. More NH₄ was allocated to shoots of seedlings colonised by a high-uptake isolate, indicating transfer of a larger fraction of assimilated NH₄ to the host than with isolates showing lower NH₄ uptake rates. Thus low rates of N uptake and N transfer to the host may enable EM fungi avoid stress induced by elevated levels of nitrogen. Seedlings colonised by S. bovinus transferred a larger fraction of the ¹⁵N label to the shoots than seedlings colonised by P. involutus. Seedling shoot growth probably constituted a greater carbon sink in pot cultures than in microcosms, since the mycelial growth of P. involutus was more sensitive to high NH₄ in pots. There was no homology in mycelial growth rate between pure culture and growth in symbiosis, but N uptake in pure culture corresponded to that during growth in symbiosis. No relationship was found between deposition of antropogenic nitrogen at the sites of origin of the P. involutus isolates and their mycelial growth or uptake of inorganic nitrogen. Accepted: 18 September 1998     

Cross-colonization of Scots pine (Pinus sylvestris) seedlings by the ectomycorrhizal fungus Paxillus involutus in the presence of inhibitory levels of Cd and Zn

The effects of Cd and Zn on cross-colonization by Paxillus involutus of Scots pine seedlings was examined by using pairs of ectomycorrhizal (ECM) and non-mycorrhizal (NM) seedlings grown in the same vessel. This was done to assess, first, the ability of P. involutus to colonize NM Scots pine seedlings by growth from colonized roots of other Scots pine seedlings in the presence of Cd or Zn, and, second whether ECM colonization of Scots pine by P. involutus provided a competitive advantage over NM seedlings. Ectomycorrhizal colonization of Scots pine was shown to be more sensitive than Scots pine itself to Cd and Zn, but prior colonization did provide a competitive advantage with respect to biomass production. This beneficial effect over NM seedlings was, however, equal in the control, Cd and Zn treatments, and was due simply to growth stimulation in the presence of ECM colonization. Cross-colonization from an ECM to a NM seedling was reduced but not prevented by Cd and Zn. Cd had a more negative effect on cross-colonization than on initial colonization of seedlings, whereas Zn had an equally inhibitory effect on both parameters. These results have important implications for plant establishment on metal-contaminated sites. If cross-colonization between plants is reduced by toxic metals, plant establishment on contaminated sites might be retarded.