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.     

Ectomycorrhizal mycelia reduce bacterial activity in a sandy soil

Abstract: Bacterial activity was studied in a growth system containing Pinus contorta seedlings inoculated with different mycorrhizal fungi. Nylon nets enabled separation of soil compartments with extramatrical mycorrhizal hyphae from soil compartments with roots and mycelium. In three separate experiments bacterial activity, estimated as thymidine incorporation, was reduced in soils with Paxillus involutus hyphae compared to controls without mycorrhizal hyphae. This effect was found irrespective of compartments with and without roots were compared. Laccaria bicolor only reduced the activity in one of these three experiments. Thelephora terrestris (tested in two experiments), Laccaria proxima, Suillus variegatus and Hebeloma crustuliniforme (one experiment), also reduced the thymidine and leucine incorporation rates of bacteria. The reduction for these fungi varied between 20% and 50% in all experiments. Numbers of viable bacteria appeared to be reduced by T. terrestris, L. proxima, S. variegatus and H. crustuliniforme in one experiment, while no effect was seen in the other experiments.     

Characterization of the surface hydrophobicity of filamentous fungi

A method for the quantitative analysis of the hydrophobicity of the mycelial mat of filamentous fungi based on contact angle measurements is presented. It was tested for a range of fungi belonging to the classes of basidiomycetes, ascomycetes and deuteromycetes. The measured contact angles of the mycelial mats ranged between hydrophilic (<30 degrees) for the deuteromycetes Fusarium oxysporum Fo47 GUS1 and Trichoderma harzianum P1[pZEGA1] and hydrophobic (>60 degrees) for the ascomycete Cladosporium sp. DSE48.1b and the basidiomycetes Paxillus involutus WSL 37.7, Hebeloma crustiliniforme WSL 6.2, Suillus bovinus WSL 48.1 and Laccaria bicolor WSL 73.1. For some fungi, variations in the hydrophobicity of the mycelium depending on the growth medium, the physiological state and the exposure to water were distinguished.     

In situ identification of intracellular bacteria related to Paenibacillus spp. in the mycelium of the ectomycorrhizal fungus Laccaria bicolor S238N

Bacterial proliferations have recurrently been observed for the past 15 years in fermentor cultures of the ectomycorrhizal fungus Laccaria bicolor S238N, suggesting the presence of cryptic bacteria in the collection culture of this fungus. In this study, intracellular bacteria were detected by fluorescence in situ hybridization in combination with confocal laser scanning microscopy in several collection subcultures of L. bicolor S238N. They were small (0.5 micro m in diameter), rare, and heterogeneously distributed in the mycelium and were identified as Paenibacillus spp. by using a 16S rRNA-directed oligonucleotide probe initially designed for bacteria isolated from a fermentor culture of L. bicolor S238N.     

ALLELOPATHIC POTENTIAL OF RASPBERRY FOLIAR LEACHATES ON GROWTH OF ECTOMYCORRHIZAL FUNGI ASSOCIATED WITH BLACK SPRUCE

In vitro bioassays on seven species of ectomycorrhizal fungi associated with black spruce (Picea mariana (Mill.) BSP) were tested under allelopathic action of raspberry (Rubus idaeus L.). Radial growth inhibitions were observed when fungi were confronted with increasing concentrations (0%–0.1%–0.5%–2.5%) of foliar leachates made from May, July, and September material (1986). For the 2.5% treatment, species Paxillus involutus, Laccaria proximo, Laccaria bicolor, Thelephora terrestris, and Cortinarius pseudonapus grew only 6, 8, 32, 42, and 46% of their control, whereas Hebeloma cylindrosporum and Cenococcum geophilum were stimulated. Tree seedlings from raspberry invaded plantation showed mycorrhizal infection rate over 75%, with fine roots mainly colonized by C. geophilum. In combination with efforts of tree improvement and based on the results of this study, it seems important to select a well-adapted my-corrhizae, when interference by raspberry is involved.     

Concentrations of terpenes in mycorrhizal roots of Scots pine (Pinus sylvestris L.) seedlings grown in vitro

The effect of Paxillus involutus, Laccaria laccata, Suillus luteus, S. bovinus, Hebeloma crustuliniforme and a strain of the ectendomycorrhizal fungus Mrg X (Ascomycotina) on the content of volatile organic compounds in roots of Pinus sylvestris seedlings grown in vitro was investigated. Volatile compounds extracted with a supercritical fluid extraction were primarily terpenes and sesquiterpenes and qualitatively were the same in roots of mycorrhizal and nonmycorrhizal plants. The major monoterpenes were α-pinene, Δ³-carene and β-pinene. Inoculation of plants with the fungi resulted in statistically non-significant increases in the total amount of the volatiles. The mycorrhizal fungi showed diversified effect on the concentrations of several terpenoids.     

Content of phenolics in mycorrhizal roots of Pinus sylvestris seedlings grown in vitro

The effect of Paxillus involutus, Laccaria laccata, Suillus luteus, S. bovinus, Hebeloma crustuliniforme and a strain of the ectendomycorrhizal fungus Mrg X (Ascomycotina) on the content of phenolic compounds: ortho-diphenols and total soluble phenols in roots of Pinus sylvestris seedlings grown in vitro was investigated. Mycorrhizal fungi varied significantly in the ability to influence the concentration of phenolics in Scots pine roots. Plants inoculated with S. luteus accumulated the lowest amount of the phenolics, whereas the amount of phenolics in pine roots inoculated with L. laccata was the highest.     

Phylogenetic, genomic organization and expression analysis of hydrophobin genes in the ectomycorrhizal basidiomycete Laccaria bicolor

Hydrophobins are morphogenetic, small secreted hydrophobic fungal proteins produced in response to changing development and environmental conditions. These proteins are important in the interaction between certain fungi and their hosts. In mutualistic ectomycorrhizal fungi several hydrophobins form a subclass of mycorrhizal-induced small secreted proteins that are likely to be critical in the formation of the symbiotic interface with host root cells. In this study, two genomes of the ectomycorrhizal basidiomycete Laccaria bicolor strains S238N-H82 (from North America) and 81306 (from Europe) were surveyed to construct a comprehensive genome-wide inventory of hydrophobins and to explore their characteristics and roles during host colonization. The S238N-H82 L. bicolor hydrophobin gene family is composed of 12 genes while the 81306 strain encodes nine hydrophobins, all corresponding to class I hydrophobins. The three extra hydrophobin genes encoded by the S238N-H82 genome likely arose via gene duplication and are bordered by transposon rich regions. Expression profiles of the hydrophobin genes of L. bicolor varied greatly depending on life stage (e.g. free living mycelium vs. root colonization) and on the host root environment. We conclude from this study that the complex diversity and range of expression profiles of the Laccaria hydrophobin multi-gene family have likely been a selective advantage for this mutualist in colonizing a wide range of host plants.     

Mycorrhizal infection and ageing affect element localization in short roots of Norway spruce [Picea abies (L) Karst.]

Norway spruce [Picea abies (L.) Karst.] seedlings, nonmycorrhizal of mycorrhizal with Laccaria laccata or Paxillus involutus were grown in a quartz sand-nutrient solution system for 6 months and then treated with 5 M Pb for 4 days. Element contents of cortex cell wall of young, medium and old short roots were determined by X-ray microanalysis of longitudinal thin sections. The Pb content was influenced neither by age nor by the distance from the root tip (up to 1.7 mm) but was significantly lower in the P. involutus mycorrhizae than in the L. laccata mycorrhizae or in nonmycorrhizal short roots. In the P. involutus mycorrhizae, the P content of the cortex cell walls was twice as high in young mycorrhizae than in old mycorrhizae. In the nonmycorrhizal short roots and the L. laccata mycorrhizae, P content was influenced neither by age nor by distance from the root tip. The Ca and Fe contents of the cortex cell walls increased with age in the nonmycorrhizal short roots and the mycorrhizae. It is concluded that the element content of the cortex cell walls of short roots is strongly influenced by age, while the distance from the root tip seems to be of minor importance.     

Aluminium polyphosphate complexes in the mycorrhizal basidiomyceteLaccaria bicolor: A27Al-nuclear magnetic resonance study

The techniques of ²⁷Al- and ³¹P-nuclear magnetic resonance (NMR) spectroscopy were used to investigate the interactions of aluminium with intracellular ligands within the mycelium of the ectomycorrhizal basidiomycete Laccaria bicolor (Maire) Orton (S238). The vegetative mycelium was grown on medium containing 0.5 mM AlCl₃ for 0.5 to 3 d. The ²⁷Al-NMR spectra showed that aluminium was rapidly taken up and accumulated into polyphosphate complexes in the vacuole. Comparison with Al-polyphosphate complexes obtained in vitro on model systems indicated that Al forms at least three mixed-solvation complexes with Pi and polyphosphates, that there is more than one complex present under any set of conditions, and that the equilibrium between these complexes shifts dramatically with Al concentration in the medium. The high phosphate concentrations in the growth medium favoured the accumulation of the Al-polyphosphate complexes. When mycelium containing Al-polyphosphate complexes was transferred to Al-free nutrient solution for 9 d, the Alpolyphosphate complexes were not remobilized. The sequestration of Al in the polyphosphate complexes could therefore make a significant contribution to the protection of mycorrhizal plants against aluminium toxicity.Abbreviations NMR nuclear magnetic resonance - PolyP polyphosphate(s) - PP₁ terminal phosphate of PolyP - PP₃ middle phosphate of PolyP We thank Prof. Daniel Canet (Laboratoire de Méthodologie RMN, University of Nancy I, Vandceuvre-lès-Nancy, France) for his constant encouragement and Christine Delaruelle for skilled technical assistance in growing the fungal cultures. This work was supported by a research grant from the Commission of the European Communities (STEP-CT90-0059, Role of Ectomycorrhiza in Stress Tolerance of Forest Trees) to F.M. and a travel grant from the Institut National de la Recherche Agronomique to I.K.; R.C. is a recipient of a Postdoctoral Fellowship from the Natural Sciences and Engineering Research Council of Canada.     

Distribution of lead in mycorrhizal and non-mycorrhizal Norway spruce seedlings

Non-mycorrhizal spruce seedlings (Picea abies Karst.) and spruce seedlings colonized with Lactarius rufus (Scop.) Fr. or two strains of Paxillits involutus (Batsch) Fr. were grown in an axenic silica sand culture system with frequently renewed nutrient solution. After successful mycorrhizal colonization, the seedlings were exposed to 1 μM PbCI₂ for 19 weeks. The degree of infection in all of the mycorrhizal treatments approached 100% during the experiment and was not affected by exposure to Pb. However, the number of root tips per root dry weight and the shoot: root ratio, both in the non-mycorrhizal and the mycorrhizal seedlings, had decreased after the 19 week treatment with PbCl₂ Using X-ray microanalysis, the distribution and concentration of Pb in the tissues of mycorrhizal and non-mycorrhizal root tips were compared. In the mycorrhizae of seedlings exposed to Pb no significant accumulation of Pb in the hyphal mantle or in fungal cell walls of the Hartig net were detected. Lead accumulated primarily in the cortex cell walls both of non-mycorrhizal and mycorrhizal root tips. No significant difference of Pb concentrations in root cortex cell walls of non-mycorrhizal and mycorrhizal seedlings was found; except for seedlings colonized with Paxillus involutus strain 537. However, at the endodermis no effect of mycorrhizal fungal colonization on the Pb tissue concentration was detected. The presence of the fungal sheath did not prevent Pb from reaching the root cortex. The endodermis acted as a barrier to Pb radial transport in both mycorrhizal and non-mycorrhizal seedling roots.     

Structure and dynamics of experimentally introduced and naturally occurring laccaria sp. Discrete genotypes in a douglas fir plantation

Ectomycorrhizal fungi have been introduced in forest nurseries to improve seedling growth. Outplanting of inoculated seedlings to forest plantations raises the questions about inoculant persistence and its effects on indigenous fungal populations. We previously showed (M.-A. Selosse et al. Mol. Ecol. 7:561-573, 1998) that the American strain Laccaria bicolor S238N persisted 10 years after outplanting in a French Douglas fir plantation, without introgression or selfing and without fruiting on uninoculated adjacent plots. In the present study, the relevance of those results to sympatric strains was assessed for another part of the plantation, planted in 1985 with seedlings inoculated with the French strain L. bicolor 81306 or left uninoculated. About 720 Laccaria sp. sporophores, collected from 1994 to 1997, were typed by using randomly amplified polymorphic DNA markers and PCR amplification of the mitochondrial and nuclear ribosomal DNAs. All plots were colonized by small spontaneous discrete genotypes (genets). The inoculant strain 81306 abundantly fruited beneath inoculated trees, with possible introgression in indigenous Laccaria populations but without selfing. In contrast to our previous survey of L. bicolor S238N, L. bicolor 81306 colonized a plot of uninoculated trees. Meiotic segregation analysis verified that the invading genet was strain 81306 (P < 0.00058), implying a vegetative growth of 1.1 m. year-1. This plot was also invaded in 1998 by strain S238N used to inoculate other trees of the plantation. Five other uninoculated plots were free of these inoculant strains. The fate of inoculant strains thus depends less on their geographic origin than on unknown local factors.     

The effects of heavy metals,content of nutrients and inoculation with mycorrhizal fungi on the level of terpenoids in roots of <Emphasis Type="Italic">Pinus sylvestris</Emphasis> seedlings

The effect of two substrates differing in the amount of toxic metals and nutrients on the content of volatile organic compounds in roots of Pinus sylvestris seedlings inoculated with Paxillus involutus, Laccaria laccata, Suillus luteus and a strain of the ectendomycorrhizal fungus Mrg X (Ascomycotina) in pot experiment was investigated. Volatile compounds extracted with a supercritical fluid extraction were primarily terpenes and sesquiterpenes, and they were qualitatively the same in roots of mycorrhizal and nonmycorrhizal plants grown on both substrates. The major monoterpenes were α-pinene, Δ³-carene and β-pinene. On the unpolluted substrate, inoculation of plants with the fungi resulted in a similar amount of the volatiles, while on the polluted substrate the mycorrhizal roots showed significantly lower content of terpenoids than nonmycorrhizal roots. The mycorrhizal fungi showed different effects on the concentrations of several terpenoids on both substrates.     

Apatite as a P source in mycorrhizal and non-mycorrhizal Pinus sylvestris seedlings

The objectives of the study are firstly to test the ability of ectomycorrhizal pine seedlings to use apatite as a P source in comparison with non-mycorrhizal pine seedlings and secondly, to determine if there is a relation between exudation of organic acids and the ability to use apatite as a P source. Non-mycorrhizal Pinus sylvestris (L.) seedlings and seedlings ectomycorrhizal with 4 different isolates of ectomycorrhizal fungi were grown for 220 days in sand/peat filled pots with apatite (Ca₅(F,OH)(PO₄)₃) as the sole P source. In an additional experiment, non-mycorrhizal Pinus sylvestris (L.) seedlings and seedlings ectomycorrhizal with 2 different isolates of ectomycorrhizal fungi were grown without any P source for 250 days. All other nutrients were supplied in a balanced nutrient solution.Ectomycorrhizal seedlings grew less than non-mycorrhizal seedlings but ectomycorrhizal seedlings produced a large external mycelium not included in the biomass estimates. All seedlings in the present study had low shoot:root ratios compared to seedlings growing under optimal conditions. All seedlings grown with apatite as P source had higher foliar P concentrations (0.71–2.11 mg/g) than seedlings growing without any P source (0.57–0.75 mg/g) indicating a significant ability to use apatite as a P source. Seedlings colonized by Suillus variegatus and Paxillus involutus had higher concentrations and total contents of P in shoots compared with non-mycorrhizal seedlings, indicating significant improvement of P uptake by these fungi in comparison with non-mycorrhizal seedlings or seedlings colonized Piloderma croceum.No clear relationship between exudation of organic acids and uptake of P was found. Seedlings colonized by S. variegatus reduced the pH of the soil more than seedlings colonized by P. involutus or non-mycorrhizal seedlings. It is suggested that S. variegatus colonization improves the P uptake by reducing the pH of the soil while P. involutus improves P uptake by having a greater ability to absorb dissolved phosphate than non-mycorrhizal roots or roots colonized by the other fungi used in the study.     

Survival of an introduced ectomycorrhizal Laccaria bicolor strain in a European forest plantation monitored by mitochondrial ribosomal DNA analysis

Mitochondrial and nuclear genes have different inheritance, thus studies of fungal populations should use both mitochondrial and nuclear markers. Using nuclear markers, the S238N strain of the ectomycorrhizal basidiomycete Laccaria bicolor ((Maire) Orton) has been previously shown to persist for at least 10 yr after outplanting in a plantation of Douglas fir ( Pseudotsuga menziesii (Mir.) Franco) inoculated with this strain. In the present study, we have sampled 539 sporophores of Laccaria spp. from this plantation, some of which had the S238N nuclear genotype, to study mitochondrial DNA polymorphism and persistence of the inoculated S238N mitochondrial genome. Length polymorphism in fragments of the large subunit of mitochondrial ribosomal DNA (LrDNA) allowed distinction of the haplotypes present in the plantation at the species level. In addition, heteroduplex analysis and sequencing revealed intraspecific polymorphism of LrDNA among the L. bicolor sporophores and enabled specific identification of S238N LrDNA. This haplotype was only retained in sporophores carrying the S238N nuclear genome, confirming the survival of this introduced strain in a natural population.     

In Situ Identification of Intracellular Bacteria Related to Paenibacillus spp. in the Mycelium of the Ectomycorrhizal Fungus Laccaria bicolor S238N

Bacterial proliferations have recurrently been observed for the past 15 years in fermentor cultures of the ectomycorrhizal fungus Laccaria bicolor S238N, suggesting the presence of cryptic bacteria in the collection culture of this fungus. In this study, intracellular bacteria were detected by fluorescence in situ hybridization in combination with confocal laser scanning microscopy in several collection subcultures of L. bicolor S238N. They were small (0.5 μm in diameter), rare, and heterogeneously distributed in the mycelium and were identified as Paenibacillus spp. by using a 16S rRNA-directed oligonucleotide probe initially designed for bacteria isolated from a fermentor culture of L. bicolor S238N.     

Effects of cadmium on growth and glucose utilisation of ectomycorrhizal fungi in vitro

Effects of Cd on growth and glucose utilisation of Paxillus involutus, Rhizopogon subcaerulescens and Suillus bovinus were investigated in vitro in liquid culture. S. bovinus was the species most sensitive to Cd in terms of dry matter production and P. involutus was less sensitive than R. subcaerulescens. Greater production of hyphae of P. involutus than the other fungi appeared to confer some degree of Cd resistance, possibly by binding Cd onto cell walls. Growth of the three fungi was increased by glucose addition. While Cd significantly reduced dry matter production of the fungi, there were no significant differences in glucose consumption caused by Cd treatment. This suggests that the use of glucose might have been diverted to detoxification and/or repair mechanisms. Further studies on respiration rates and energy metabolites of these fungi under Cd exposure are needed in order to clarify the results of the present study.     

The effect of tree-root exudates on the growth rate of ectomycorrhizal and saprotrophic fungi

Summary The object of this investigation was the promotion by root exudates of the growth rate of ectomycorrhizal fungi, discovered by Elias Melin in 1954. Eight ectomycorrhizal and ten non-mycorrhizal species were used as test fungi in the experiments. Different species often reacted differently: none of the eight isolated strains of Suillus luteus were promoted by pineroot exudate, whereas the growth rates of all seven strains of S. granulatus were increased. Among the other ectomycorrhizal species, S. variegatus, Laccaria bicolor, Pisolithus tinctorius and Thelephora terrestris, each represented by only one, two or three strains, usually reacted to the pine root exudate with an increased growth rate; S. bovinus and Paxillus involutus did not respond at all. Hitherto, studies of root-exudate effects on fungi have been based exclusively on the responses of ectomycorrhizal species; in the present study saprotrophic fungi were also used as test organisms. Seven out of ten saprotrophic species reacted with markedly accelerated growth when exposed to a pineroot exudate. Melin's assumption that a constituent of the root exudate, the M-factor, could replace the exudate growth-promoting activity was verified. By means of TLC fraction it was found that the fatty acid palmitic acid alone caused an increase in growth rate equal to that of the pine-root exudate. In line with previously published data by Gogala (1970), we also showed that certain cytokinins, especially isopentenylaminopurine, could act as substitutes for the total root exudate. Thus both palmitic acid and isopentenylaminopurine are able to function as M-factors equivalent to a root exudate.     

Temporal persistence and spatial distribution of an American inoculant strain of the ectomycorrhizal basidiomycete Laccaria bicolor in a French forest plantation

Selected strains of ectomycorrhizal fungi, such as the basidiomycete Laccaria bicolor , are currently being used as inoculants in nurseries to improve growth of forest trees after outplanting. Information is needed on the survival of these introduced strains in forests and their impact on indigenous biodiversity. Dissemination and persistence of an American strain, L. bicolor S238N, were studied 10 years after outplanting in a Douglas fir plantation located at Saint-Brisson (Morvan, France). About 430 Laccaria spp. sporophores were collected over 3 years. Inheritance of nuclear ribosomal DNA, as well as RAPD markers, was characterized in L. bicolor S238N, using a haploid progeny set of 91 monokaryons. More than 50 markers were identified (19 heterozygous and 33 homozygous or cytoplasmic markers), which unambiguously confirmed that the introduced strain was still present in the inoculated plots. Neither selfing ( P < 0.0008) nor introgression with indigenous strains was detected although in vitro interfertility between the American strain and indigenous L. bicolor was identified. No ingress of the introduced genet into adjacent uninoculated plots colonized by various local Laccaria genets was detected. It is proposed that the spatial distributions identified have developed through mycelial propagation of the introduced strain and intraspecific competition with native genets. Although longer-term data is still lacking, the stability of the inoculant strain and the limited disturbance to indigenous populations described support large-scale nursery production of this host-fungal combination.