Ectomycorrhizal syntheses with Picea abies and three fungal species: a case study on the use of an in vitro technique to identify naturally occurring ectomycorrhizae

Ectomycorrhizal syntheses between Picea abies and the fungal associates Scleroderma citrinum, Boletus luridus, and Tricholoma vaccinum were carried out using Melin's Erlenmeyer flask technique. The symbioses of S. citrinum were characterized by a mantle composed of an outer prosenchymatous and an inner synenchymatous layer. The mantles of B. luridus and T. vaccinum were solely prosenchymatous. Rhizomorphs were produced in all treatments, but only in association with S. citrinum were they differentiated with additional, enlarged hyphae. All synthesized ectomycorrhizae were white or whitish to light orange and greyishorange. On large-scale root sampling in two differing Picea abies forests in Switzerland, nine out of a total of 22 morphological types of ectomycorrhizae were white or yellow in colour and were, therefore, comparable with the synthesized ectomycorrhizae. These nine natural types generally had distinct mantle features (irregular synenchyma, gelatinous matrix, cystides, thick-walled hyphae), but mostly lacked clamp connections. Synthesized ectomycorrhizae, on the other hand, lacked distinct mantle characteristics and always had clamp connections. Natural and synthesized white or yellow ectomycorrhizae did not coincide morphologically and thus identification of the fungal partners of natural symbioses by means of in vitro-synthesis with potential ectomycorrhizal fungi was not possible in the present study.     

Comparative studies on the mycorrhization of Larix decidua and Picea abies by Suillus grevillei

Summary Mycorrhization of Picea abies has been achieved, for the first time, with six strains of Suillus grevillei by a new culture method, using activated charcoal paper and liquid medium as a substrate. Mycorrhization of P. abies and Larix decidua was compared, and the process was found to be significantly different in the two tree species. S. grevillei is not incompatible with P. abies, but it forms mycorrhizae more readily with L. decidua. Hyphal growth was clearly stimulated on the surface of roots of Larix but retarded on Picea. A well organized Hartig net was formed with both tree species, but wall protuberances were frequently observed on the outer cell walls of Picea cortex cells when the Hartig net was not fully developed. No conspicuous cell wall reactions occurred in Larix roots. Cell wall protuberances may be comparable to those in transfer cells and are interpreted as an alternative to Hartig net development. Anatomical differences between roots of Larix and Picea, and physiologically active substances such as recognition factors on the root surfaces, are discussed with respect to their responsibility for the different reactions of S. grevillei.     

Characterization of Picea abies (L.) Karst. ectomycorrhizas: discrepancy between classification according to macroscopic versus microscopic features

Summary In a 3-year study of ectomycorrhizal diversity in 2 Norway spruce stands in Switzerland the macroscopically classified ectomycorrhizal types were analyzed microscopically to compare the results of the 2 approaches. A total of 22 ectomycorrhizal types were macroscopically classified in the 2 stands. Microscopic investigations — particularly of mantle structures — resulted in the identification of 7 additional types to give a total of 29. These additional types resulted from separation of macroscopically identical types occurring on both stands (type-pairs) at the microscopic level. The problematic nature of characterization and classification of ectomycorrhizas is discussed.     

Effect of auxins and ectomycorrhizal elicitors on wall-bound proteins and enzymes of spruce [Picea abies (L.) Karst.] cells

Summary Elicitors of the ectomycorrhizal fungus Hebeloma crustuliniforme and auxins (IAA, NAA and 2,4-D) were tested for their effects on apoplastic proteins and enzymes of suspension cultured cells of Picea abies (L.) Karst. The ectomycorrhizal elicitor increased the amount of some ionically wall-bound proteins (36, 28, 24, 21 kDa) and decreased the amount of others (61, 22 kDa). The elicitor triggered an H₂O₂ burst and enhanced the peroxidase (EC 1.11.1.7) activity of the Picea cells by increasing one of the two wall-bound peroxidase isoforms. Auxins significantly suppressed the elicitor induction of peroxidase but did not influence the elicitor-triggered H₂O₂ burst. The elicitors and auxin did not change the amount and the pattern of wall-bound invertase isoforms (EC 3.2.1.26) of spruce cells. However, auxin reduced the uptake of glucose by spruce cells and increased the acidification of the cell culture medium. Since Hebeloma lacks apoplastic invertase as well as a sucrose uptake system, utilization of plant-derived sucrose depends on the apoplastic plant invertase activity. Although the host invertase is constitutive, the fungus might be able to increase this invertase activity within a mycorrhiza by lowering the pH of the interface towards the pH optimum of the enzyme via the action of auxin. This fungus-released hormone could increase the H⁺ extrusion of plant cells by activation of the plant membrane H⁺-ATPases. Additionally, an auxin-dependent suppression of glucose uptake by cortical root cells could improve the glucose supply for the fungus. Furthermore, the fungal auxin might suppress the elicitor induced formation of defense enzymes, such as peroxidase.     

Effects of ozone on starch accumulation in Norway spruce (Picea abies)

Summary Fumigation with 100 g/m³ and 200 g/m³ ozone in closed-top fumigation chambers induced starch accumulation in chloroplasts of Norway spruce. This accumulation was probably due to a partial inhibition of the starch translocation at night. The intensity of the effect was dependent on the season and the age of the needles. The accumulation was reversed in winter. It is therefore unlikely that such an effect has much significance for plant health.     

Effects of soil pH and calcium on mycorrhizas of Picea abies

The effects of lime, increased soil pH and increased soil Ca concentration on the mycorrhizas of Norway spruce. [Picea abies (L.) Karst.] were studied independently of each other to elucidate the different mechanisms through which lime may influence mycorrhizas in acidic soil. In a field experiment (mature Norway spruce in podzol), lime was applied as CaCO₃; increased Ca concentration without an increase in pH was achieved with CaSO₄; and soil pH was increased without calcium by means of Na₂CO₃ and K₂CO₃ (Na+K treatment). Treatments were done in October, and mycorrhizas were counted from samples collected in the following June and September. All treatments increased the percentage of dead short root tips compared to controls in September, and Na+K already in June. Cenococcum geophilum Fr. increased in proportion in plots treated with Na+K.In a sand culture experiment, Norway spruce seedlings were grown from seed and inoculated with Cenococcum geophilum, or root inoculum, or left uninoculated. When mycorrhizas were beginning to form, CaCO₃ and CaSO₄ treatments were applied. Six weeks later, the percent of dead short root tips in both salt treatments was significantly increased from control, but formation of mycorrhizas was not inhibited by treatments.As all the treatments increased the proportion of dead short root tips, it is concluded that lime directly and adversely affected mycorrhizas of Norway spruce in sand culture and in mor humus. Both increased ionic strength and increased pH may be reasons for this rather than Ca²⁺ specifically.     

Effects of liming and boron fertilization on boron uptake of Picea abies

The effects of liming on concentrations of boron and other elements in Norway spruce [Picea abies (L) Karst.] needles and in the mor humus layer were studied in long-term field experiments with and without B fertilizer on podzolic soils in Finland. Liming (2000+4000 kg ha^-1 last applied 12 years before sampling) decreased needle B concentrations in the four youngest needle age classes from 6–10 mg kg^-1 to 5 mg kg^-1. In boron fertilized plots the corresponding concentrations were 23–35 mg kg^-1 in control plots and 21–29 mg kg^-1 in limed plots. Both liming and B fertilizer decreased the Mn concentrations of needles. In the humus layer, total B concentration was increased by both lime and B fertilizer, and Ca and Mg concentrations and pH were still considerably higher in the limed plots than controls. Liming decreased the organic matter concentration in humus layer, whilst B fertilizer increased it.The results about B uptake were confirmed in a pot experiment, in which additionally the roles of increased soil pH and increased soil Ca concentration were separated by means of comparing the effects of CaCO₃ and CaSO₄. Two-year-old bare-rooted Norway spruce seedlings were grown in mor humus during the extension growth of the new shoot. The two doses of lime increased the pH of soil from 4.1 to 5.6 to 6.1, and correspondingly decreased the B concentrations in new needles from 22 to 12 to 9 mg kg^-1. However, CaSO₄ did not affect the pH of the soil or needle B concentrations. Hence the liming effect on boron availability in these soils appeared to be caused by the increased pH rather than increased calcium concentration.     

Comparative investigations on the differentiation of the endodermis and the development of the Hartig net in mycorrhizae of Picea abies and Larix decidua

Summary The differentiation of the endodermis of mycorrhizal roots of Picea abies and Larix decidua was investigated by means of light and transmission electron microscopy and with fluorescence techniques. The initiation and differentiation of the Hartig net were recorded. Differences between the two tree species were found, as were differences between the two tree species and angiosperms. The Casparian band developed immediately after the origin of endodermal cells from the meristem in mycorrhizae of both tree species. In L. decidua only the primary endodermis was present in most mycorrhizal laterals. The secondary structure of the endodermis was restricted to main roots and proximal parts of larch mycorrhizae. In P. abies mycorrhizae, however, the secondary stage of the endodermis developed soon after the primary endodermis and was characterized by regular alternation of short, active passage cells and elongated, rapidly degenerating cells, the inner surface of which was covered by a thick suberin layer. Hartig net development started in P. abies short roots only after the differentiation of endodermis into the secondary stage, whereas in L. decidua, the Hartig net was already initiated at the primary endodermal stage. Differences were specific for tree species.     

Performance of two Picea abies (L.) Karst. stands at different stages of decline

Summary The development of root tips and apparent ectomycorrhizas was compared in the Fichtelgebirge (FRG) over one growing season in two 30-year-old Picea abies stands, both on soils derived from phyllite but showing varying symptoms of decline. Visual symptoms of tree decline reflected a lower relative and absolute mycorrhizal frequency, a lower number of ectomycorrhizas per m² leaf area and an uneven vertical distribution of root tips and ectomycorrhizas. The number of apparent ectomycorrhizas per ground area was correlated with the amount of magnesium, calcium, and ammonium, and the pH in the free-drainage soil solution, and with the molar calcium to aluminium ratio in mineral soil extracts. The foliage concentrations of magnesium and calcium were correlated with the numbers of apparent ectomycorrhizas per m² leaf or ground area. These observations were used to formulate testable hypotheses concerning the role of the root system and the soil environment in forest decline.     

Intraspecific variation in use of different organic nitrogen sources by the ectomycorrhizal fungus Hebeloma cylindrosporum

The ectomycorrhizal (ECM) fungus Hebeloma cylindrosporum is an appropriate model to study the intraspecific functional diversity of ECM fungi in forest ecosystems. Numerous metabolic genes, specifically genes related to nitrogen assimilation, have been characterised for this species and the spatial and temporal structures of its natural populations have been extensively worked out. In this paper, we reveal the extent to which intraspecific variation exists within this fungus for the ability to use organic nitrogen, an important functional characteristic of ECM fungi. In addition to ammonium and nitrate, H. cylindrosporum can use at least 13 different amino acids out of 21 tested as sole nitrogen source, as well as urea and proteins. By screening 22 genetically different wild type haploid strains we identified obvious differences in use of six nitrogen sources: alanine, glycine, phenylalanine, serine, bovine serum albumin and gelatine. Of the 22 haploid strains, 11 could not use at least one of these six nitrogen sources. The inability of some haploid strains to use a nitrogen source was found to be a recessive character. Nevertheless, obvious differences in use of the four amino acids tested were also measured between wild type dikaryons colonising a common Pinus pinaster root system. This study constitutes the basis for future experiments that will address the consequences of the functional diversity of an ECM fungus on the functioning of the ECM symbiosis under natural conditions.     

Performance of two Picea abies (L.) Karst. stands at different stages of decline

Summary A declining Picea abies (L.) Karst. stand produced as much foliage and branches as a healthy stand but less stemwood at a similar leaf area index and climate. Nutrient analyses revealed that most biomass components at the declining site had lower concentrations of calcium and magnesium, but similar nitrogen and potassium (except for lower potassium in younger needles) and higher phosphorus, manganese and aluminum than the respective components at the healthy site. Comparison of these data with the results from studies on the nutrition and growth of P. abies seedlings (Ingestad 1959) led to the conclusion that the healthy stand is in a balanced nutritional state, while trees at the declining stand have only 56% of the foliar magnesium concentration required to permit growth at a rate which could be achieved at their nitrogen status. It appears that acidic deposition, which involves an input of nitrogen and a leaching of cations from the soil, causes an imbalance in the availability of nitrogen and magnesium. Growth is eventually reduced as magnesium becomes limiting.     

ABA consumption in norway spruce (Picea abies) and white spruce (Picea glauca) somatic embryo cultures

Summary We investigated abscisic acid (ABA) metabolism among Norway and white spruce somatic embryo cultures which exhibited differences in maturation response when placed on racemic abscisic acid [(±)-ABA]. Differences in metabolic rate among the spruce genotypes could affect the ABA pool available for the maturation process, and might therefore be responsible for the differences in maturation response. The production of cotyledonary (stage 3) somatic embryos in cultures (genotypes) of Norway spruce (PA86:26A and PA88:25B) and of white spruce (WS1F cryoD and WS46) was compared. In each species pair one of the two genotypes failed to show stage 3 embryo development (respectively, PA88:25B and WS46). The investigation of ABA metabolism of each species pair showed that no substantial differences in ABA consumption or in the production of metabolites occurred. In each case ABA was metabolized to phaseic acid and dihydrophaseic acid over the 42-day culture period, metabolites were recoverable from the agar-solidified medium, and the sum of residual ABA and metabolites were equivalent to the ABA initially supplied. The results indicate that the process of ABA metabolism occurs essentially independently of somatic embryo maturation. NRCC no. 37345.     

Functional expression of the green fluorescent protein in the ectomycorrhizal model fungus Hebeloma cylindrosporum

Hebeloma cylindrosporum is a model fungus for mycorrhizal studies because of its fast growth rate, simple nutritional requirements, and completion of its life cycle in vitro, and because it is amenable to transformation. To advance cell biological research during establishment of symbiosis, a tool that would enable the direct visualisation of fusion proteins in the different symbiotic tissues [namely, the expression of reporter genes such as Green Fluorescent Protein (GFP)] was still a missing tool. In the present study, H. cylindrosporum was transformed using Agrobacterium carrying the binary plasmid pBGgHg containing the Escherichia coli hygromycin B phosphotransferase (hph) and the EGFP genes, both under the control of the Agaricus bisporus glyceraldehyde-3-phosphate dehydrogenase promoter. EGFP expression was successfully detected in transformants. The fluorescence was uniformly distributed in the hyphae, while no significant background signal was detected in control hyphae. The suitability of EGFP for reporter gene studies in Hebeloma cylindrosporum was demonstrated opening up new perspectives in the Hebeloma genetics.Tobias Müller and Mariam Benjdia contributed equally to this work.     

Performance of two Picea abies (L.) Karst. stands at different stages of decline

Summary Photosynthetic rates and nutrient contents of spruce needles were measured in a region with high levels of air pollution in NE Bavaria, Germany (FRG), and compared to spruce grown under clean air conditions at Craigieburn, in the South Island of New Zealand (NZ). The absolute rates of CO₂ uptake, the slope of the CO₂ response curve at 240 l l^–1 internal CO₂ concentration, and the change of photosynthetic rates with needle age at ambient and saturated CO₂ concentrations were virtually identical at both measuring sites. These results confirm an earlier conclusion, that there is no long-term effect of atmospheric pollutants directly on photosynthetic CO₂ uptake rates with persistent exposure at the FRG site to high levels of anthropogenic air pollution. Photosynthetic capacity at saturating CO₂ concentration was three times higher in the NZ spruce. Needles with high photosynthetic capacity in NZ had lower nitrogen and higher calcium concentrations per unit dry weight but higher concentrations of nitrogen, phosphorus, potassium, magnesium and calcium per unit leaf area, and twice the specific leaf weight.     

Performance of two Picea abies (L.) Karst. stands at different stages of decline

Summary The water relations of Picea abies in a healthy stand with green trees only and a declining stand with trees showing different stages of needle yellowing were investigated in northern Bavaria. The present study is based on observations of trees differing in their nutritional status but apparently green on both sites in order to identify changes in the response pattern which might be caused by atmospheric concentrations of air pollutants and could lead to the phenomenon of decline. Transpiration was measured as water flow through the hydroactive xylem using an equilibrium mass-flow measurement system. Total tree transpiration was monitored diurnally, from July 1985 until October 1985 at both sites. The relationship between transpiration and meteorological measurements indicated that transpiration was a linear function of the vapor pressure deficit. No differences in transpiration of green trees were observed between the two sites. Canopy transpiration was 57%–68% of total throughfall and 41%–54% of total rainfall. Due to this positive water balance, soil water potential at 10 and 20 cm depths remained close to-0.02 MPa (max.-0.09 MPa) for most of the summer. Soil water potential was correlated with the difference between the weekly precipitation and transpiration. No differences in the water relations of apparently healthy trees in the two P. abies stands were observed. It is concluded that differences between green trees at the two sites in terms of nutrient relations or growth rate cannot be explained by changes in whole-tree transpiration or soil water status.     

Dendrochronology of Norway spruce (Picea abies (L.) Karst.) from two range centres in lowland Poland

In Europe, spruce grows in two main regions which meet in Poland, one to the north and east, the other to the south and west. The northeastern area ranges from the northern treeline extending from Norway to Siberia, to southern Sweden, north-eastern Poland and the southern Ural mountains. The southwestern spruce region reaches the mountainous areas of the Alps and the Balkans, the mountains and uplands of the Sudety Mountains, the Carpathians and neighbouring lowlands. Opinions about the distribution of Norway spruce have changed over the years, and its scarcity in the centre of Poland has been strongly debated. The favoured current theory is that Norway spruce once had a continuous distribution in Poland. It is assumed that the rare occurrence in the central Polish lowland is due to a combination of unfavourable soil conditions and previous management activity. The main aim of this work was to analyse climate–growth relationships of Norway spruce in eastern Poland and distinguish regions with similar increment patterns with regards to spruce range. Spruce growth in northern Polish sites is positively correlated with rainfall from May to July. Tree-ring widths in southern sites are more correlated with March temperature. Selected homogenous regions are the same as range types. Trees from the so-called “spruceless area” seem to have similar climate–growth relationships to trees from the southern region. This finding does not settle the question of the origin of the trees, but it does indicate that similar environmental conditions exist in these two areas and proves that the climate was not a limiting factor there.

CO2 fixation in stem slices of Picea abies (L.) Karst: microautoradiographic studies

Summary Microautoradiography was used to show that chlorophyllous cells of young Picea abies stem slices are able to fix ¹⁴CO₂, in the dark as well as in the light. The amount of ¹⁴CO₂ fixed in the dark is much lower than that in the light. In the dark the concentration of radioactive label is equally high in all chlorophyllous cells of the stem. In the light, however, a gradient of radioactive assimilates extends from the stem surface to its centre, with the highest concentration being located in the phelloderm and the outer one-third of the cortex. This is in spite of even illumination and CO₂ supply across the whole stem slice. In the dark, stem slices with and without bark show the same amount of radioactive label in the chlorophyllous cells of xylem, perimedullary region and pith. In the light, however, the concentration of radioactive assimilates in these cells is much higher in stem slices with bark than in stem slices without bark. It is assumed therefore that light fixation products of phelloderm and cortex are transported radially into the tissue inside the cambium.     

IAA-overproducer mutants of Hebeloma cylindrosporum Romagnesi mycorrhizal with Pinus pinaster (Ait.) Sol. and P. sylvestris L. in hydroponic culture

Indole-3-acetic acid (IAA) is thought to play a role in the regulation of ectomycorrhiza development, and vigorous mycorrhiza formers such as Pisolithus and Laccaria have previously been shown to accumulate large amounts of IAA in the culture medium in vitro, particularly in the presence of tryptophan. Recently, 5-fluoroindole-resistant and IAA-overproducing mutant strains of Hebeloma cylindrosporum Romagnesi have been developed and described by Durand et al. (1992). We have used some of these and corresponding wild-type strains as mycobionts on seedlings of Pinus pinaster (Ait.) Sol. and P. sylvestris L. in semi-hydroponic culture in an attempt to study IAA effects independent of species-specific differences. However, no significant differences between strains were found in host growth rate, shoot carbohydrate concentration, root morphology, root IAA concentration or mycorrhizal biomass. Since previous work showed a stimulation by these and other mutants and strains on mycorrhiza formation in Petri dish and test tube cultures, we assume that a semi-hydroponic culture system prevents the build up of tryptophan of fungal origin, which is most likely a precondition for enhanced IAA production.     

Effects of liming and boron fertilization on mycorrhizas of Picea abies

Effects of liming and B fertilization on Norway spruce [Picea abies (L.) Karst.] mycorrhizas were studied in factorial field experiments. The lime was applied twice, about 30 years and 12 years before sampling (2000 and 4000 kg ha^-1 dolomite). B was applied at the rate of 1.5 kg B ha^-1 two years before sampling.Boron fertilization doubled the number of root tips in the top 10 mm of the humus layer. The proportion of dead short root tips was increased from 10 % in control plots to 29 % in the limed plots. Numbers of dead root tips were increased when both lime and B were applied. The % of mycorrhizas with external mycelium was slightly increased and the % of Piloderma croceum Erikss. and Hjortst. was decreased by lime. In conclusion, adverse effects of lime on mycorrhizas were found, which were ameliorated by B fertilization, but lime-induced B deficiency alone was not the only reason for the effects of lime on root mortality.     

Genetic neighbourhood structure in a population of Picea abies L.

Summary Natural populations are currently the basic material for studying forest tree breeding, but little is known on the genotypic spatial structures in these stands. The use of gene markers, such as isozymes, leads to the determination of part of the allelic constitution of individuals. A method is presented here to estimate the degree of genetic relationship between any pair of genotypes. A French Picea abies population is analyzed by these means and a slight but significant correlation between estimated genetic relationship and topographic distance is found.