Sensitivity to Cd or Zn of host and symbiont of ectomycorrhizal Pinus sylvestris L. (Scots pine) seedlings

Pinus sylvestris seedlings infected with either the ectomycorrhizal (ECM) fungus Paxillus involutus or Suillus variegatus were exposed to a range of Cd or Zn concentrations. This was done to investigate the relationship between the sensitivity of ECM fungi and their host plants over a wide range of concentrations. P. involutus ameliorated the toxicity of Cd and Zn to P. sylvestris with respect to root length, despite significant inhibition of ECM infection levels by Cd (Cd EC₅₀ [effective concentration which inhibits ECM infection by 50%] values were: P. involutus 3.7 μg g^-1 Cd; S. variegatus 2.3 μg g^-1 Cd). ECM infection by P. involutus also decreased Cd and Zn transport to the plant shoots at potentially toxic concentrations and also influenced the proportion of Zn transported to the roots and shoots, with a higher proportion retained in the roots of the seedlings. ECM infection did increase host biomass production, but this was not affected by the presence of Cd or Zn. Root and shoot biomass production by P. sylvestris, in both the presence and absence of ECM fungi, was unaffected by Cd and Zn at all concentrations tested. This revised version was published online in August 2006 with corrections to the Cover Date.     

The effect of ectomycorrhizal fungi on Zn uptake and distribution in seedlings of Pinus sylvestris L

Seedlings of pine, infected with two different ectomycorrhizal fungi, Suillus bovinus (Fr.) O. Kuntze and an unidentified isolate (BP), were exposed to various external Zn concentrations. An additional strain of S. bovinus, cultured on a Zn-enriched medium, was also used. The effect of mycorrhizal associations on Zn uptake and distribution within the plant was determined by AAS.The results show that nonmycorrhizal seedlings have the capability to control the uptake and translocation of Zn to the shoot until the external Zn concentration reaches a threshold level, where no limitation of uptake is possible. Excess Zn is accumulated in the root system to protect the shoot against toxic tissue concentrations. The effect of an ectomycorrhizal infection on Zn uptake and distribution depends on (1) the fungal species (2) the external concentration and (3) the Zn content of the fungal culture medium. Under conditions of low external Zn supply, especially a mycorrhizal infection with S. bovinus led to an increased Zn uptake in root and needles of Pinus sylvestris. Under high external conditions the mycobionts varied considerably in their capability to reduce the transport of Zn to the shoot. Only by an infection with S. bovinus the plant was able to maintain the shoot tissue concentration on a low level. This effect can be enhanced by pretreatment of S. bovinus with high Zn concentrations.     

Effects of soil cadmium on Pinus sylvestris L. seedlings

The effects of Cd on the growth and distribution of Cd and mineral nutrients within plant tissues were investigated for Pinus sylvestris L. seedlings grown in mineral forest soil with increasing levels of Cd addition (0–100 mg kg^–1). Approximately 20% of added Cd was found to be extractable from sandy loam forest soil. Root growth was less affected by Cd than shoot growth, which showed a significant reduction in the 100 mg Cd kg^–1 treatment. Cadmium accumulated in roots up to 325 mg kg^–1. Decreased concentrations of K in needles and Ca in stems with increasing Cd levels suggest a disturbance of mineral nutrition as a result of Cd addition.     

Colonization routes of Pinus sylvestris inferred from distribution of mitochondrial DNA variation

Understanding the present-day distribution of molecular variation requires knowledge about the history of the species. Past colonization routes and locations of refugia of Scots pine (Pinus sylvestris) were inferred from variation in mitochondrial DNA in material collected from 37 populations located in countries within, and immediately adjacent to the continent of Europe. Two mitochondrial regions, nad1 intron (exon B/C) and nad7 intron 1, were included in the study. Differentiation in maternally inherited mitochondria was high (G _ST′ = 0.824). Two new haplotypes were found at the nad7 intron 1. The occurrence of a 5-bp indel variant was restricted to the Turkish Kalabak population and a 32 bp only found in Central, Eastern, and Northern Europe. The complete absence of the 32-bp indel from the Mediterranean peninsulas supports the view that coniferous forests existed outside these areas during the last glacial maximum, and these populations contributed to the subsequent colonization of the northern parts of Europe. P. sylvestris shares features of its glacial and postglacial history with two other northern, cold-tolerant tree species, Picea abies and Betula sp. These three species differ from many other European trees for which pollen core and molecular evidence indicate colonization from southern refugia after the last glacial period.     

Lignosulfonate promotes the interaction between Scots pine and an ectomycorrhizal fungus Pisolithus tinctorius in vitro

Lignosulfonate (LS) is a lignin-based polymer obtained as a by-product from paper industry, which may have potential as an amendment with macronutrients. We studied effects of LS on the interaction between Scots pine (Pinus sylvestris L.) seedlings and hypocotyl cuttings and the ectomycorrhizal (ECM) fungusPisolithus tinctorius (Pers.) Coker and Couch. The experiments were performed in vitroon the MMN agar medium containing Fe–LS chelate at the concentrations of 0, 5, 10 and 25 mg/L. Inoculation with P. tinctoriusincreased root growth of the seedlings. Fe–LS enhanced P. tinctorius induced formation of lateral roots and had a dose-dependent positive effect on the establishment of mycorrhizas on the seedlings. The growth of the fungal mycelium was improved by Fe–LS, which might cause faster and more intensive contact with the roots and, thus, better root growth and mycorrhiza formation. P.tinctorius enhanced also adventitious root formation and subsequent root growth of the hypocotyl cuttings but without any synergistic effect with Fe–LS. Our study with P. tinctorius and Scots pine in vitro indicates that a low-cost by-product Fe–LS, obtained from paper industry, may be a potential tool to improve the efficiency of fungal inoculations, thus, facilitating the early interaction between an ECM fungus and host seedling.     

Food reserves of scots pine (Pinus sylvestris L.)

Summary Starch, soluble sugars, triacylglycerols, diacylglycerols and free fatty acids were measured in 30-year-old Scots pine (Pinus sylvestris L.) trees during an annual cycle in the sapwood (youngest ten xylem rings). The radial distribution of carbohydrates and lipids was studied in the trunkwood of 90 -to 150-year-old Scots pine trees collected at the end of the growing season. Determination of the compounds was performed using specific enzymatic assays, capillary gas chromatography and thin layer chromatography. The amounts of glucose, fructose, sucrose, and galactose/arabinose in the sapwood were slightly higher in winter than in summer. Raffinose/stachyose increased up to 5-fold during the cold period. At the beginning of the growing season starch amounts rose, and then decreased in summer and autumn. No concentration changes were observed in the total amounts of diacylglycerols and fatty acids throughout the year. Triacylglycerol levels were slightly higher in February than in summer and autumn. Relative frequencies of individual fatty acids were similar in all lipid fractions. Glucose, fructose, sucrose, starch and triacylglycerols disappeared almost entirely at the transition zone from sapwood to heartwood. In contrast, free fatty acids and galactose/arabinose rose in centripetal direction, and diacylglycerols remained constant across trunk cross-sections. The relative amounts of individual fatty acids changed markedly in the free fatty acid fraction and in the triacylglycerols when crossing the sapwood-heartwood boundary. Concentration changes of reserve materials are discussed in relation to season, mobilization and translocation processes, dormancy, frost resistance, and heartwood formation. The results are compared to those found in needles.     

Food reserves of Scots pine (Pinus sylvestris L.)

Summary The amounts of starch, soluble sugars, triacylglycerols, diacylglycerols and free fatty acids were studied in Scots pine (Pinus sylvestris L.) during an annual cycle in current-year needles and in 1-, 2- and 3-year-old needles collected shortly after bud break. Determination of the compounds was performed using specific enzymatic assays, capillary gas chromatography and thin layer chromatography. Newly emerging needles contained relatively large amounts of starch, but only trace amounts of fat. During autumn and winter, fat content rose, while starch content decreased; amounts of both these reserve materials were very high the next spring shortly before bud break and decreased again during shoot elongation. Concentration of intermediates in triacylglycerol biosynthesis (diacylglycerols and free fatty acids), were low in summer and high in winter. The same pattern was observed for fructose and glucose (the predominant soluble sugars), galactose/arabinose and raffinose/melibiose. In contrast, sucrose concentrations were highest in spring and in autumn. Mature needles of different ages collected in May showed significant differences only in their triacylglycerol and starch content. Concentration changes of reserve materials are discussed in relation to season, mobilization and translocation processes, dormancy, frost resistance and the possibility of carbohydrate-fat interconversions.     

Soluble carbohydrates of Pinus sylvestris L. sapwood and heartwood

Summary The amounts of glucose, fructose, sucrose, arabinose/galactose, raffinose/stachyose and starch were investigated in the outer sapwood, innermost sapwood, transition zone and heartwood of four stems of Pinus sylvestris L. The samples were taken in October and the determination of the compounds was done enzymatically. It was not possible to distinguish arabinose from galactose and raffinose from stachyose. The amounts of glucose, fructose and sucrose were greatest in the outer sapwood and decreased gradually towards the innermost sapwood and the heartwood. In the outermost heartwood glucose, fructose and sucrose were only present in trace amounts. Raffinose/stachyose showed highest concentrations in the outer sapwood and decreased towards the heartwood. In contrast, the concentrations of arabinose/galactose increased towards the heartwood and the greatest amount was found in the inner heartwood. When identified by thin-layer chromatography (TLC), arabinose was found to be present in greater amounts than galactose. The amount of starch decreased markedly towards heartwood. However, the amounts of sugars in all the studied stems was very variable. The changes in the amounts of carbohydrates in the different zones of the stems and the possible relationships of these phenomena with heartwood formation are discussed.     

Plastids and glycolipids in the stemwood of Pinus sylvestris L.

Summary The ultrastructure of plastids in xylem ray parenchyma cells of Pinus sylvestris L. was studied and compared with the glycolipid composition of the stemwood. Seasonal changes of the ultrastructure were studied by taking samples regularly throughout the year. The plastids resemble amyloplasts. They usually have one large starch grain, and considerable variation in structure and starch content was observed, especially in the innermost sapwood and in the sapwood-heartwood transition zone. Electron-dense deposits were observed attached to the plastid membranes and envelopes, especially in the transition zone, from April to November. The plastids were aggregated near the nucleus and the starch disappeared during the winter (January–March). The glycolipids, monogalactosyldiacylglycerol (MGDG) and di-galactosyldiacylglycerol (DGDG), were present only in the sapwood, in trace amounts. The glycolipid content was slightly greater in the outer sapwood than in the sapwood-heartwood transition zone. DGDG was the dominant lipid of the two.     

The effects of flooding on the formation of ectomycorrhizae in Pinus sylvestris seedlings

Pinus sylvestris seedlings, grown in a vertical petri dish system, were inoculated with five different mycorrhizal fungi. Half of the root system in the petri dish was subjected to periodic flooding, and mycorrhizal colonization was studied. Thelephora terrestris, Laccaria laccata, and Hebeloma crustuliniforme were not sensitive to flooding, whereas Suillus flavidus and S. bovinus were highly sensitive. The latter failed to colonize the root even when flooded for only 2 min per day four times a week.     

Long-term needle litterfall of a Scots pine Pinus sylvestris stand: relation to temperature factors

Summary Needle litterfall of a Scots pine was caught over 24 years (1962–1986) with litter-traps in a Scots pine stand in southeastern Finland. The age of the trees averaged 111 years in 1962. The stand was naturally recruited and only minor silvicultural treatments occurred during its history. Litterfall showed great year-to-year variation, the minimum being 18 g/m² (in 1968) and maximum 213 g/m² (in 1973). There was no overall trend in the amount of litterfall, and the age of the stand was thus not important in determining the needle fall. We used time domain time series analysis (ARIMA) and standard climatic data (temperature, precipitation) to investigate the relationship of litterfall to climatic factors. Mean July temperature was clearly correlated with needle litterfall. High temperature in July coincided with enhanced litterfall in the same and the next year. Litterfall enhanced litterfall in the same and the next year. Litterfall increased also after high temperatures during March–April, but only in the same year. In addition to these the litterfall had a 4-year self-dependency. This is approximately the same as the mean longevity of needles in the study area. Altogether the time series model we propose covers about 90% of the variance of the original time series.     

Lipids and sterols of Pinus sylvestris L. sapwood and heartwood

Summary The lipid and sterol content and composition of three lipid fractions (free fatty acids/ sterols, triacylglycerols and sterol/triterpenoid esters) extracted from three stem discs of Pinus sylvestris were assessed to investigate metabolic changes related to heartwood formation. The wood was separated into (1) cambial zone, (2) outer sapwood, (3) inner sapwood, (4) transition zone, (5) outer heartwood and 6) inner heart-wood. The fractions were separated by thin-layer chromatography (TLC) and analysed by gas-liquid chromatography (GLC). The amount of fatty acids of sapwood triacylglycerols was about 1.5% (dry wt.) but a large reduction occurred in the transition zone. In contrast, noticeable amounts of free fatty acids were present only in the heart-wood. The most important fatty acids in the sapwood fractions were 16:0, 18:0, 18:1, 18:2 (the dominant fatty acid in all fractions), 18:3 and 20:3. Together 18:1 and 18:2 formed about 70% of the total triacylglycerol fatty acids. Of the sterol/ triterpenoid esters, 18:2 and 18:3 were predominant. The fatty acid composition of all fractions changed in the transition zone. The sterols found were sitosterol, stigmastanol, campesterol and campestanol. The amount of sterol esters increased towards the heartwood, and the amount of free sterols was lowest in the inner sapwood. Sitosterol was the dominant sterol in both free sterols and sterol esters.     

Molecular characterization of a cDNA clone encoding glutamine synthetase from a gymnosperm,Pinus sylvestris

A full-length cDNA clone (pGSP114) encoding glutamine synthetase was isolated from a gt11 library of the gymnosperm Pinus sylvestris. Nucleotide sequence analysis showed that pGSP114 contains an open reading frame encoding a protein of 357 amino acid residues with a calculated molecular mass of 39.5 kDa. The derived amino acid sequence was more homologous to cytosolic (GS1) (78–82%) than to chloroplastic (GS2) (71–75%) glutamine synthetase in angiosperms. The lack of N-terminal presequence and C-terminal extension which define the primary structure of GS2, also supports that the isolated cDNA encodes cytosolic GS. Southern blot analysis of genomic DNA from P. sylvestris and P. pinaster suggests that GS may be encoded by a small gene family in pine. GS mRNA was more abundant in cotyledons and stems than in roots of both Scots and maritime pines. Western blot analysis in P. sylvestris seedlings showed that only one GS polypeptide, similar in size to GS1 in P. pinaster, could be detected in several different tissues. Our results suggest that cytosolic GS is mainly responsible for glutamine biosynthesis in pine seedlings.This paper is dedicated to the memory of Dr. Jesús S. Olavarría.     

Seasonal variation of neutral lipids in Pinus sylvestris L. sapwood and heartwood

Summary The lipid levels and fatty acid composition of three fractions (free fatty acids, triacylglycerols and sterol/triterpenoid esters) extracted from the sapwood and heartwood of three stems of Pinus sylvestris were determined to investigate both seasonal changes in sapwood and possible metabolic changes related to heartwood formation. Seasonal changes were observed only in the amount of the free fatty acids in the sapwood: the level of free fatty acids was greatest at the beginning and end of the growing season. In the January and March samples the amount of the free fatty acid fraction in the sapwood was very small. The amount of the other fractions remained at the same level throughout the study. Marked seasonal changes in the fatty acid composition occurred only in the free fatty acid fraction of the sapwood: the saturation grade increased during the winter.     

Regulation of photoassimilate allocation in Pinus sylvestris seedlings by the nutritional status of the mycorrhizal fungus Suillus variegatus

Summary Seedlings of Pinus sylvestris L. were grown on defined nutrient solutions on carbon filters, either sterile or infected with the basidiomycete Suillus variegatus O. Kuntze. After mycorrhizas were established, the shoot of the seedling was subjected to ¹⁴CO₂ photosynthesis. ¹⁴C-labelled photoassimilates were translocated to both mycorrhizas and non-infected root tips. Microautoradiographs of mycorrhizas indicated that omission of external sugars did not affect the formation of mycorrhizas; ¹⁴C-photoassimilates were supplied to cortex, Hartig net and the mantle of hyphae surrounding the rootlet. Nutrient solution containing sugars (malt extract, glucose) enhanced the growth of the fungus. As a consequence, ¹⁴C-photoassimilates from the seedling were accumulated in the mantle, but defence mechanisms of the host cannot be excluded. When soluble nitrogen was omitted from the nutrient solution and replaced by chitin precipitated on the filter-bearing mycorrhizas, the fungus appeared strongly labelled in the mantle, where the fungal chitinase provided soluble nitrogen compounds, necessary for the growth of the seedling.     

Individual-based modelling of Pinus sylvestris invasion after grazing abandonment in the French Massif Central

In the Chaîne des Puys, a mid-elevation volcanic mountain of the French Massif Central, Scots pine proves to be an invasive species colonizing abandoned lawns or heathlands, and forms in a few years monospecific natural forests. Most of the abandonment occurred 30 to 40 years ago and this process has now stopped. Thus, we lack data on the very first phase of tree colonization. We anticipate that a simulation tool could bring an appreciable help in (i) rebuilding the entire colonization process – including the initial phase – of pine settlement and (ii) answering questions about the origin of the narrow and unimodal distributions of age of pine stands we observed. In addition, such a simulator could help managers to forecast extension of Scots pine and to predict growth and evolution of present secondary forests. A spatially explicit individual-based model is presented. The model takes into account both space and time and includes growth of trees, seed production and seed dispersal, death and competition between individuals. The influence of the initial parameters are analyzed and elements of validation given. The model was then used to predict tree settlement and stand establishment using the initial conditions from a natural stand studied in the field whose characteristics before abandonment were known (number and age-distribution of trees, location of mother trees, time of abandonment). Three simulations were achieved by using the same initial conditions but following different scenarios for the recruitment process. The scenario of a fluctuating resistance of the resident vegetation (that controls the susceptibility of the environment to tree establishment) seemed as one of the most probable to explain the actual stand characteristics. We thus concluded that dynamic models could be improved by taking into account the resistance of the vegetation to colonization as a fluctuating parameter instead of a static and permanent attribute.     

Occurrence of carpophores of ectomycorrhizal fungi in selected stands ofPinus sylvestris in the Netherlands in relation to stand vitality and air pollution

The relation between the occurrence of carpophores of mycorrhizal fungi, tree vitality, and air pollution in ‘young’ (5–10 years) and ‘old’ (50–80 years) stands ofPinus sylvestris L. was investigated. In the Netherlands 21 homogeneous plots of 1000 m² were selected on dry, sandy soils. Tree vitality was assessed in the summer of 1985 and during the autumn the plots were searched for carpophores of mycorrhizal fungi. In the young plots 3 times more species of ectomycorrhizal fungi and 20 times more carpophores per plot were found than in the old plots. In the old plots, the number of carpophores and the number of species showed significant positive correlations with the average number of needleyears of the trees. The number of carpophores and the number of species were significantly negative correlated with the concentrations of SO₂ and O₃ in the air and the amount of NH₃ emission. The number of needleyears, the crown density and the overall tree vitality were found to be significantly negative correlated with the amount of NH₃ emission, and the number of needleyears also with the concentration of O₃ in the air. Such correlations were not found for the young plots. No differences were found in soil chemical properties between the plots. Evidence was obtained that the fructification of mycorrhizal fungi of old stands ofPinus sylvestris is influenced by air pollution either directly, or indirectly by way of tree vitality. It is hypothesized that the young plots offer more advantageous circumstances for fructification of the fungi because of the disturbance of the upper soil-layer at the time of planting.     

Competition for nitrogen between Pinus sylvestris and ectomycorrhizal fungi generates potential for negative feedback under elevated CO2

We investigated fungal species-specific responses of ectomycorrhizal (ECM) Scots pine (Pinus sylvestris) seedlings on growth and nutrient acquisition together with mycelial development under ambient and elevated CO₂. Each seedling was associated with one of the following ECM species: Hebeloma cylindrosporum, Laccaria bicolor, Suillus bovinus, S. luteus, Piloderma croceum, Paxillus involutus, Boletus badius, or non-mycorrhizal, under ambient, and elevated CO₂ (350 or 700 μl l⁻¹ CO₂); each treatment contained six replicates. The trial lasted 156 days. During the final 28 days, the seedlings were labeled with ¹⁴CO₂. We measured hyphal length, plant biomass, ¹⁴C allocation, and plant nitrogen and phosphorus concentration. Almost all parameters were significantly affected by fungal species and/or CO₂. There were very few significant interactions. Elevated CO₂ decreased shoot-to-root ratio, most strongly so in species with the largest extraradical mycelium. Under elevated CO₂, ECM root growth increased significantly more than hyphal growth. Extraradical hyphal length was significantly negatively correlated with shoot biomass, shoot N content, and total plant N uptake. Root dry weight was significantly negatively correlated with root N and P concentration. Fungal sink strength for N strongly affected plant growth through N immobilization. Mycorrhizal fungal-induced progressive nitrogen limitation (PNL) has the potential to generate negative feedback with plant growth under elevated CO₂. Responsible Editor: Herbert Johannes Kronzucker     

The effect of ozone and season on the pool sizes of cyclitols in Scots pine (Pinus sylvestris)

Summary The effect of 100 and 200 g · m^-3 (50 and 100 ppb) ozone and ambient air on 4-year-old grafts of a Scots pine clone was tested in closedtop fumigation chambers. Ozone decreased the myo-inositol and inccreased the pinitol levels in the 1-year-old needles, whereas the effect in the current-year needles was less distinct. In neither case did ambient air lead to any detectable effects on these parameters compared with controls. However, there was a pronounced chamber effect in the levels of myo-inositol and pinitol compared with trees standing in the open field, where inositol was higher and pinitol lower. There is some evidence, based on visible symptoms as well as biochemical changes, that season is an important modifier of the effect of ozone on Scots pine.     

Identification of Naemacyclus minor hyphae within needle tissues of Pinus sylvestris by immunoelectron microscopy

Cultural investigations revealed that Naemacyclus minor, Lophodermium seditiosum and Cenangium ferruginosum were the most frequent colonizers of asymptomatic and symptomatic Pinus sylvestris needles. Since ultrastructural observations showed that morphological features were not suitable to differentiate hyphae of N. minor from hyphae of other isolates, the on-section immunogold labelling technique was applied in combination with an anti-N. minor specific immunoserum. The specificity of this serum was tested against culture hyphae of all isolates. Anti-N. minor specific immunoserum was then used to identify N. minor hyphae in thin sections of green P. sylvestris needles. The infection loci identified were restricted to small tissue areas located in the vicinity of stomata. In the hypodermis, hyphae and endocell-containing hyphae were located within the lumina of host cells but outside the protoplast. The growth of hyphae from cell to cell occurred through pits. The hyphae spreat into the mesophyll intercellularly and continued with the intracellular colonization of moribund and dead mesophyll cells in a later stage of infection. The observed host-parasite interactions at cellular and ultrastructural level are discussed in connection with the still controversial interpretation of the pathogenicity of N. minor.