Cloning of Pinus sylvestris SCARECROW gene and its expression pattern in the pine root system, mycorrhiza and NPA-treated short roots

The SCARECROW (SCR) gene is central to root radial patterning. Its expression has not been investigated in conifers with morphologically different root types. Additional interest in SCR functions in the Pinus sylvestris root system comes from the effect of ectomycorrhiza formation on the short root apical structure. Here, the P. sylvestris SCR gene (PsySCR) was cloned and its expression investigated by northern blot and in situ hybridization of primary, lateral and short roots and mycorrhiza. Short root dichotomization was induced by auxin transport inhibitor (N-1-naphthylphthalamic acid (NPA)). PsySCR has conserved GRAS family protein motifs at the C-terminus and a variable N-terminus. PsySCR expression occurred in young root tissue and mycorrhiza. In root sections the PsySCR signal runs through the tip in initials for stele and root cap column and becomes upwards-restricted to endodermis in all root types. The PsySCR expression pattern suggests for the first time a regulatory role for SCR in maintaining the endodermal characteristics and radial patterning of roots with open meristem organization. The specific PsySCR localization is also an excellent marker for investigation of the dichotomization process in short roots.     

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.     

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.     

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.     

Succession of mycorrhizal fungi in stands of Pinus sylvestris in the Netherlands

The mycorrhizal mycoflora was investigated in 35 stands of Pinus sylvestris in three types of young (4-13 yr) and three of old (50-80 yr) stands in the Netherlands, differing in number of rotations and soil type. A plot of 1050 m² (30 m x 35 m) within each stand was searched for carpophores during the autumns of 1986 and 1987. 10 soil samples per plot were taken in October 1987 in order to assess the mycorrhizal status of the tree roots. The composition of mycorrhizal mycoflora in the different plots was subjected to TWINSPAN cluster analysis and Detrended Correspondence Analysis. Plot groupings generated by these analyses largely parallelled the stand types, indicating that each stand type has its own mycoflora. Differences in myco-floristic composition between stand types were parallelled by differences in the composition of green vegetation. The young stand types had 3.5–27 x more carpophores and 1.4–6.8 x more species than two of the old stand types One old stand type was intermediate. Considerable differences in species composition between the young stand types were observed. It is concluded that the succession of mycorrhizal fungi is not primarily influenced by ageing of the trees, but rather by changes in the soil. The results were compared with data on changes in the occurrence of fruiting species of mycorrhizal fungi in the Netherlands during this century. It appeared that species which have declined according to these data were more frequent in the young plots than in the old plots. However, these species are reported to be frequent in old stands of P. sylvestris in Estonia and Finland. It is argued that this difference is related to the high nitrogen deposition in the Netherlands.     

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.     

樟子松和油松根叶对春季干旱胁迫的响应

以成年樟子松和油松为对象,分析春季干旱胁迫对叶片和根系形态和生理特性的影响,比较2个树种在干旱胁迫下的响应程度和受伤害大小。结果表明:樟子松叶片含水量、脯氨酸含量、POD和CAT活性均显著高于油松(P0.05),根系活力、含水量、可溶性蛋白和脯氨酸含量及CAT活性、比根长和比表面积均显著高于油松(P0.05)。樟子松叶片丙二醛含量显著低于油松(P0.05),而根系丙二醛含量显著高于油松(P0.05)。可溶性糖和脯氨酸含量及SOD活性在叶片中显著大于根系(P0.05),可溶性蛋白含量及CAT活性在樟子松叶片中显著小于根系(P0.05),POD活性在油松叶片中显著低于根系(P0.05)。上述结果表明,春季干旱胁迫下,樟子松比油松做出了更积极的响应,叶片和根系的响应关系不对应,干旱胁迫对油松叶片伤害更大,而对樟子松根系伤害更大。     

Growth and mycorrhizal community structure of Pinus sylvestris seedlings following the addition of forest litter

We report the effects of pine and oak litter on species composition and diversity of mycorrhizal fungi colonizing 2-year-old Pinus sylvestris L. seedlings grown in a bare-root nursery in Lithuania. A layer of pine or oak litter was placed on the surface of the nursery bed soil to mimic natural litter cover. Oak litter amendment appeared to be most favorable for seedling survival, with a 73% survival rate, in contrast to the untreated mineral bed soil (44%). The concentrations of total N, P, K, Ca, and Mg were higher in oak growth medium than in pine growth medium. Relative to the control (pH 6.1), the pH was lower in pine growth medium (5.8) and higher in oak growth medium (6.3). There were also twofold and threefold increases in the C content of growth medium with the addition of pine and oak litter, respectively. Among seven mycorrhizal morphotypes, eight different mycorrhizal taxa were identified: Suillus luteus, Suillus variegatus, Wilcoxina mikolae, a Tuber sp., a Tomentella sp., Cenococcum geophilum, Amphinema byssoides, and one unidentified ectomycorrhizal symbiont. Forest litter addition affected the relative abundance of mycorrhizal symbionts more than their overall representation. This was more pronounced for pine litter than for oak litter, with 40% and 25% increases in the abundance of suilloid mycorrhizae, respectively. Our findings provide preliminary evidence that changes in the supply of organic matter through litter manipulation may have far-reaching effects on the chemistry of soil, thus influencing the growth and survival of Scots pine seedlings and their mycorrhizal communities.     

Initiation and development of wound tissue and roots on hypocotyl cuttings of Pinus sylvestris in vitro

The rooting of hypocotyl cuttings from 20-day-old seedlings of Pinus sylvestris L. cultured in vitro is discussed. About 40% of the cuttings cultured on medium lacking activated charcoal produced roots during the first two months. When activated charcoal was added to the medium, either root formation (75% formed roots) or wound tissue growth (95% formed large wound tissues) was stimulated in different experiments. These large wound tissues did not develop any roots. The anatomical changes in the basal part of the cuttings were similar during the first two weeks in all the cuttings studied. A vascular cylinder composed of short tracheids with many pores developed. Thereafter the differentiation process became varied. The amount of wound tissue produced and the time for rooting differed among the cuttings. Tracheid nests which were in contact with the vascular system in the hypocotyl via short tracheids were observed after three weeks. Subsequently, roots developed from the tracheid nests. The longer root formation was delayed, the larger the wound tissue became.
Short tracheids were found close to the wound tissue surface. Their ability to adsorb nutrients and water is discussed.     

Nitrogen form, availability, and mycorrhizal colonization affect biomass and nitrogen isotope patterns in Pinus sylvestris

Nitrogen (N) isotope patterns are useful for understanding carbon and nitrogen dynamics in mycorrhizal systems but questions remain about how different N forms, fungal symbionts, and N availabilities influence δ¹⁵N signatures. Here, we studied how biomass allocation and δ¹⁵N patterns in Pinus sylvestris L. cultures were affected by nitrogen supply rate (3% per day or 4% per day relative to the nitrogen already present), nitrogen form (ammonium versus nitrate), and mycorrhizal colonization by fungi with a greater (Laccaria laccata) or lesser (Suillus bovinus) ability to assimilate nitrate. Mycorrhizal (fungal) biomass was greater with ammonium than with nitrate nutrition for Suillus cultures but similar for Laccaria cultures. Total biomass was less with nitrate nutrition than with ammonium nutrition for nonmycorrhizal cultures and was less in mycorrhizal cultures than in nonmycorrhizal cultures. The sequestration of available N by mycorrhizal fungi limited plant N supply. This limitation and the higher energetic cost of nitrate reduction than ammonium assimilation appeared to control plant biomass accumulation. Colonization decreased foliar δ¹⁵N by 0.5 to 2.2‰ (nitrate) or 1.7 to 3.5‰ (ammonium) and increased root tip δ¹⁵N by 0 to 1‰ (nitrate) or 0.6 to 2.3‰ (ammonium). Root tip δ¹⁵N and fungal biomass on root tips were positively correlated in ammonium treatments (r ²?=?0.52) but not in nitrate treatments (r ²?=?0.00). Fungal biomass on root tips was enriched in ¹⁵N an estimated 6–8‰ relative to plant biomass in ammonium treatments. At high nitrate availability, Suillus colonization did not reduce plant δ¹⁵N. We conclude that: (1) transfer of ¹⁵N-depleted N from mycorrhizal fungi to plants produces low plant δ¹⁵N signatures and high root tip and fungal δ¹⁵N signatures; (2) limited nitrate reduction in fungi restricted transfer of ¹⁵N-depleted N to plants when nitrate is supplied and may account for many field observations of high plant δ¹⁵N under such conditions; (3) plants could transfer assimilated nitrogen to fungi at high nitrate supply but such transfer was without ¹⁵N fractionation. These factors probably control plant δ¹⁵N patterns across N availability gradients and were here incorporated into analytical equations for interpreting nitrogen isotope patterns in mycorrhizal fungi and plants.     

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

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

Bacterial endobionts in the big non-mycorrhizal roots of Scots pine (Pinus sylvestris L.)

Subsurface bacterial growth occurred in an N-free medium inoculated with interior tissues of big non-mycorrhizal roots (7 to 8 mm diameter) of 15-20 years-old Scots pine (Pinus sylvestris L.) growing on sand dunes at the Baltic Sea of Poland. The bacteria were not N2 fixers as determined by the acetylene reduction method. Light microscopic and scanning electron microscopic observations revealed massive bacterial clusters residing in the cortical cells underlying epidermis and parenchyma. The bacteria produced yellow-green pigments on King's medium, which fluoresced under ultraviolet (UV) irradiation at 366 nm wavelength, and could be a siderophore-producing Pseudomonas.     

Vegetative Propagation of Pinus sylvestris

Methods for the vegetative propagation of Pinus sylvestris L. from interfascicular shoots are described. Using 5-year-old plants the outgrowth of interfascicular shoots was promoted by removal of terminal and lateral buds; this response was augmented substantially by application of cytokinin, tri-iodobenzoic acid, alar and morphactin alone and especially in combination. The rooting capacity of shoot cuttings from interfascicular shoots appeared to be largely determined by the state of growth of the stock plant. Cuttings from dormant stock plants subjected to short-day treatment followed by a period of low temperature gave the best rooting, especially when the cuttings themselves had been cold-stored prior to planting. Rooting was optimal when such cuttings were treated with a mixture of 25 mg/l of indolebutyric acid and 25 mg/l of napthaleneacetic acid as a 48 h basal soak, and were planted on a heated mistbench under extended illumination; over 90% of such cuttings could be rooted. These results are discussed in relation to bud activity, endogenous hormone levels and promoting tissue extracts also tested.     

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.