Zinc-tolerant Suillus bovinus improves growth of Zn-exposed Pinus sylvestris seedlings

Scots pine (Pinus sylvestris L.) seedlings inoculated or not (NM) by a Zn-sensitive or a Zn-tolerant isolate of the ectomycorrhizal fungus Suillus bovinus (L. Fr.) Roussel were exposed to 0.1 or 150 μM Zn²⁺ for 9 months. We hypothesized that inoculation with a Zn-tolerant S. bovinus isolate should result in added Zn resistance of the host plant. Plant and fungal growth as well as nutrient profiles and photosynthetic pigments in pine needles were quantified. In NM plants and in plants colonized by the Zn-sensitive isolate, plant growth, N, P, Mg and Fe assimilation were strongly inhibited under Zn stress and concurred with significantly reduced chlorophyll concentrations. In contrast, plants colonized by the Zn-tolerant isolate grew much better and remained physiologically healthier when exposed to elevated Zn. These results provide further evidence for the important role metal-adapted mycorrhizal fungi play as an effective biological barrier against metal toxicity in trees.     

Zinc toxicity in ectomycorrhizal Pinus sylvestris

Six strains of ectomycorrhizal fungi were compared for their ability to increase zinc tolerance in Pinus sylvestris L. seedlings. Pioneer and late-stage fungi as well as one strain collected from a Zn-polluted site were included in the study. The accumulation of zinc in the host plants was determined at two different sublethal substrate Zn concentrations. The mycobionts varied considerably in their protection of the autobionts against zinc toxicity. Several fungal species reduced zinc accumulation in the pine seedlings. A Thelephora terrestris (Ehrh.) Fr. Strain, however, increased the Zn concentration in its host plants. Specific zinc-retaining capacity of the mycelium and density of the extramatrical mycelium were important features determining the effectiveness of the zinc retention in the fungal symbiont.The experiments were carried out in a growth chamber where plants were cultivated in root observation chambers. The determination of the extent of substrate colonization showed that an elevated zinc concentration in a substrate might be an important stress factor for the growth of an ectomycorrhizal fungus. The growth of the extramatrical mycelium of some species was inhibited at the highest zinc concentration supplied. A determination of the in vitro zinc tolerance of the fungi could not always predict their tolerance as symbionts, where the latter characteristic did not determine the Zn retention capacity of their mycelium.In this paper we also discuss the possibility that on Zn-polluted soils ectomycorrhizal species and strains are selected that are Zn-tolerant and, in addition, are able to protect their own energy source, the autobiont, from toxicity. These mycorrhizal fungi act as a safety net, that can immobilize large amounts of zinc, thus preventing transport to the host plant.     

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.     

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.     

Ectomycorrhizal development on Scots pine (Pinus sylvestris L.) seedlings in different soils

In the present study ectomycorrhizal development of Laccaria bicolor, Rhizopogon luteolus and Suillus bovinus associated with Scots pine (Pinus sylvestris) seedings was studied as affected by primary stand humus, secondary stand humus, podsolic sandy soil or peat in perspex growth chambers. After 9 weeks, ectomycorrhizal development with S. bovinus was significantly greater in peat and primary stand humus than in secondary stand humus or podsolic sandy soil. Ectomycorrhizal development with R. luteolus in secondary stand humus was higher than in primary stand humus. Degree of ectomycorrhizal development of L. bicolor, R. lutuelus and S. bovinus on Scots pine was related to potassium concentration, organic matter content and pH of the soils suggesting that chemical composition of the soils affects ectomycorrhizal development.     

Utilization of organic nitrogen at two different substrate pH by different ectomycorrhizal fungi growing in symbiosis with Pinus sylvestris seedlings

The aim of this study was to test the potential of four isolates of ectomycorrhizal (EM) fungi to utilize organic nitrogen (N) at two different substrate pHs. The organic N source (¹⁵N labelled lyophilised fungal mycelium) was mixed with either untreated peat/sand mixture (pH 4.9) or peat/sand mixture limed to a pH of 5.9 and put in cylindrical containers added to each pot. The content of the containers was separated from the roots of Pinus sylvestris seedlings by a nylon mesh and a 2 mm air gap to reduce diffusion of labelled N to the roots. The mycorrhizal plants (except those colonized by Suillus variegatus 2) took up significantly more ¹⁵N from the labelled mycelium than uncolonized seedlings. Liming significantly reduced the uptake of ¹⁵N by one of the EM fungi (unidentified) but not the other tested species (Paxillus involutus and two isolates of S. variegatus). The EM fungal isolates differed in their influence on the bacterial activity of the soil. This was reduced with P. involutus at both pH levels and increased with one of the two S. variegatus isolates at the high pH and with the other S. variegatus isolate at the low pH level. Liming the soil generally increased bacterial activity. The influence of liming on the proportion of organic N uptake in relation to inorganic N uptake by ectomycorrhizal trees is discussed.     

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.     

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.     

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.     

Inheritance of autumn frost hardiness in Pinus sylvestris L. seedlings

Summary Inheritance of frost hardiness was analysed making use of a 12×12 incomplete factorial mating design. Owing to space limitations only 59 families could be tested in four experiments. To link the four experiments, some families were common to two or more experiments. The seedlings were grown in climate chambers under conditions inducing autumn hardening. The plants were exposed to a freezing temperature of –10 °C for three hours at night lengths of 11–13 h. A statistical model was developed for analyses of variance of our data. The genetic variation and the variation due to the cultivation regimes during autumn hardening were of the same magnitude. The additive effects were the most important ones for induction of frost damage. No interaction following long-distance crossing was noted. Mixed model equations were used for ranking of the parents. The results obtained support a polygenic inheritance of frost hardiness. The large within-population variation offers good opportunities for hardiness breeding.     

Effects of elevated nickel and cadmium concentrations on growth and nutrient uptake of mycorrhizal and non-mycorrhizal Pinus sylvestris seedlings

The effects of Ni and Cd on growth and nutrient uptake of mycorrhizal and non-mycorrhizal Pinus sylvestris L. seedlings were investigated in a pot experiment. Seedlings were either inoculated with Laccaria bicolor (Maire) Orton or left uninoculated before being planted in pots containing a mixture of sandy soil from the B-horizon of a coniferous forest, small stones and pure quartz sand. The pots were supplied with small amounts of a balanced nutrient solution every 24 h using peristaltic pumps. Nickel or Cd were added as chlorides to the nutrient solution at levels of 85 M Ni (Ni 1), 170 M Ni (Ni 2), or 8.9 M Cd. Mycorrhizal colonisation of the roots was nearly 100% in the mycorrhizal treatments. The mycorrhizal seedlings grew significantly better than the non-mycorrhizal ones. The weight of mycorrhizal seedlings in the Ni 2 treatment was 29% lower than that of the mycorrhizal controls, but still 34% greater than that of the non-mycorrhizal seedlings not exposed to metals. There was an overall, statistically significant, negative effect of metals on plant yield. Mycorrhizal plants had lower root:shoot (R:S) ratios than non-mycorrhizal plants and the R:S ratio was increased by metal exposure, particularly in the non-mycorrhizal seedlings. Plant concentrations of Cd or Ni were not affected by mycorrhizal colonisation, but total uptake of Cd and Ni was higher in bigger mycorrhizal seedlings. Nickel decreased P concentration in all seedlings and Cd decreased P concentration in the non-mycorrhizal seedlings. Generally, the mycorrhizal seedlings grew better than non-mycorrhizal ones and had better P, K, Mg and S status. Root growth was not significantly affected by the metal treatments. The reduction in mean shoot growth of non-mycorrhizal plants, relative to the metal-free control, appeared higher than in mycorrhizal plants but was not statistically significant due to high variation in the non-mycorrhizal plants not exposed to metals. The main mycorrhizal effect was thus increased nutrient uptake and growth of the seedlings.     

Photoreactivation of the UV light effects on growth of Scots pine (Pinus sylvestris L.) seedlings

Summary Ultraviolet-B light (UV-B) and ultraviolet-A light (UV-A) at higher doses exert a strong inhibitory (toxic) effect on axis growth in Scots pine (Pinus sylvestris L.) seedlings. This effect is unrelated to control of growth rate by phytochrome. Rather, after a toxic UV dose growth of the pine seedling no longer responded to phytochrome. Both, the effect of UV-B as well as the inhibiting effect of UV-A could be photoreactivated by blue light (B). The action of UV-A was 2 fold: (i) it exerted a toxic effect which could be photoreactivated by B, and (ii) applied after UV-B it photoreactivated to some extent the toxic UV-B effect. Obviously, the UV-A range causes a toxic effect, and at the same time is capable of photoreactivating the toxic UV effect. At higher doses the toxic effect prevails.     

模拟氮沉降和接种外生菌根真菌对马尾松(Pinus massoniana)幼苗营养元素的影响

量化植物地上部和地下部元素含量对于理解和预测植物养分平衡如何响应大气氮沉降的变化至关重要。通过盆栽试验研究了氮沉降增加背景下外生菌根真菌对马尾松幼苗营养元素的影响。对马尾松幼苗进行了接种两种外生菌根真菌：（彩色豆马勃（Pisolithus tinctorius,Pt）与厚环乳牛肝菌（Suillus grevillei,Sg））以及4种氮素浓度添加：0 kg N hm^-2a^-1（N0）、正常氮沉降30 kg N hm^-2a^-1（N30）、中度氮沉降60 kg N hm^-2a^-1（N60）、重度氮沉降90 kg N hm^-2a^-1（N90）,共12个处理,测定了马尾松地上部和地下部大量元素和微量元素的含量。结果表明：施氮改变了营养元素在马尾松幼苗地上部和地下部的含量,马尾松幼苗磷（P）、钙（Ca）、铁（Fe）、锰（Mn）等元素均在N60时达到临界值,而当输入的量超过了马尾松对氮的需求时,氮沉降会使马尾松营养元素含量较最适浓度时降低,地上部碳（C）随施氮浓度的升高先升高后降低,N随施氮浓度的升高而升高,根系和叶片钾（K）、Ca、镁（Mg）均随施氮浓度的升高而降低,施氮也降低了根系C及微量元素的含量。但在同一施氮浓度下,接种外生菌根真菌（EMF）后能够提高大多数元素的含量,N90时接种厚环乳牛肝菌（Sg）和彩色豆马勃（Pt）的叶片N含量与对照相比分别提高112.6%和138.6%,根系N含量分别提高73.1%、71.6%;N60时接种Sg和Pt的植株叶片P含量比不施氮未接种对照分别提高了166.3%、132.9%,根系P含量分别提高了40.8%、38.5%。EMF能够维持植物养分平衡,从而降低高施氮量对植物的影响效果。这为未来气候变化情景中氮沉降增加下接种EMF可以调节植物元素含量,从而达到更适应环境的元素平衡来促进生长提供理论依据。     

Conidia of Trichoderma virens as a phosphorus source for mycorrhizal Pinus sylvestris seedlings

In this study, the mobilization and further translocation of phosphorus from conidia of saprotrophic fungus Trichoderma virens into Pinus sylvestris seedlings by nondestructive measuring of ³²P was assessed. The radioactive phosphorus flux from the conidia to the Scots pine seedlings forming mycorrhiza with Laccaria laccata and Suillus bovinus amounted up to 27.82% and 7.42%, respectively, on the 28th day of the experiment, while at the same time in nonmycorrhizal pine seedlings, the detected radioactivity reached only 0.56%. Our studies revealed that both ectomycorrhizal fungi: L. laccata and S. bovinus, mobilized the phosphorus from radioactive conidia of T. virens. On this basis, we conclude that activities of the mycosymbionts may facilitate absorption and further translocation of phosphorus from organic matter into the host plants.     

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.     

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.     

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.