Biotite and microcline as potassium sources in ectomycorrhizal and non-mycorrhizal Pinus sylvestris seedlings

 The aim of this study was to investigate the role of plants colonised by two ectomycorrhizal fungi, Paxillus involutus and Suillus variegatus, in mobilising potassium (K) from biotite and microcline, two minerals common in acid to medium-acid bedrock. This was carried out in a 33-week pot study with seedlings of Pinus sylvestris growing in symbiosis with the fungi, where no K was added or where K was added in the form of biotite or microcline. The mineral additions were similar to those found in natural soils. All seedlings, including non-mycorrhizal, were able to access the K in biotite, leading to stimulated growth and K uptake relative to controls. Microcline addition induced growth depression in all seedlings except those colonised by P. involutus, which were stimulated. The soil solution from S. variegatus-colonised seedlings grown with biotite had higher concentrations of citric and oxalic acid. Citric acid concentration was positively correlated to the fungal biomass (ergosterol) in the soil, as well as to the foliar K in S. variegatus-colonised seedlings. Seedlings growing without K addition had low K concentrations in the shoot. Magnesium (Mg) concentrations were enhanced in seedlings with severe K shortage, indicating that Mg can substitute for K, while calcium concentrations did not vary significantly. Accepted: 18 January 1999     

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.     

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.     

外生菌根真菌菌丝对马尾松苗氮素传递的影响

为探究外生菌根真菌菌丝对马尾松Pinus massoniana幼苗氮素的吸收、传递及分配规律的影响,本研究应用¹⁵N示踪技术,采用四室隔网系统进行盆栽试验。研究表明：供体接种外生菌根真菌后,根外菌丝能够穿过粘土层和隔离层到达受体室,在供、受体植株之间形成菌丝桥。接种不同外生菌根真菌均显著增加供体植株苗高、地径及生物量(P<0.05),但对受体植株的影响有差异,其中接种橙黄硬皮马勃Scleroderma citrinum (Sc)对植株苗高、地径及生物量具有显著的促进作用(P<0.05)。接种对供、受体植株全氮含量无显著性的规律(P>0.05),只有接种东方豆马勃Pisolithus orientalis (Po)的植株全氮含量显著高于其他处理。接种能够显著提高供体植株根、茎、叶的吸氮量,且叶>根>茎,受体植株中,接种Po、Sc和褐环乳牛肝菌Suillus luteus (Sl)的植株总吸氮量较不接种(NE)显著增加(P<0.05),而接种混合菌(Sm)的植株与NE无显著差异(P>0.05)。接种Sl的植株¹⁵N传递率最高(75.86%),受体植株对¹⁵N吸收量最大(91.68 μg/pot),Po次之。供体植株中,除接种Sl的植株¹⁵N主要分配在叶外,其他处理的¹⁵N分配中心均在根部,在受体植株中,接种Po、Sl、Sm的植株¹⁵N主要分配在根部,而NE和接种Sc的植株¹⁵N主要分配在茎。综上,在马尾松供、受体苗木间能形成菌丝桥,接种外生菌根真菌能够促进马尾松供体植株苗高、地径的生长及生物量的积累,提高植株吸氮量,对于受体植株不同菌种的作用有差异,其中接种Sc的苗木表现最好。此外,不同的菌种菌丝对植株的全氮含量、¹⁵N吸收量、传递率及分配率的影响也不同,其中,以接种Po和Sl的苗木表现较好。     

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.     

Competition between ectomycorrhizal fungi colonizing Pinus densiflora

 Interactive competition of Pisolithus tinctorius (Pers.) Coker et Couch with an unidentified species Tanashi 01 and Suillus luteus (L.: Fr.) S. F. Gray was investigated using a rhizobox. Pinus densiflora Sieb. et Zucc. was used as the host plant and mycelia were distinguished by hyphal color. The speed of mycelial spread differed between the fungi;P. tinctorius and Tanashi 01 grew faster than S. luteus. A P. tinctorius mycorrhizal seedling and a Tanashi 01 mycorrhizal seedling were transplanted on opposite sides of the rhizobox. The mycelia and mycorrhizae of P. tinctorius were overgrown by Tanashi 01 hyphae and development of P. tinctorius was gradually inhibited. The areas occupied by mycelia and mycorrhiza of P. tinctorius decreased by 52% and 37%, respectively, 154 days after transplantation relative to that at 91 days. In the overlap area of P. tinctorius and Tanashi 01, the latter fungus infected new root tips emerging from P. tinctorius mycorrhiza, which lacked a mantle of P. tinctorius hyphae, and formed a composite mycorrhizal structure. P. tinctorius mycorrhizae were progressively replaced by Tanashi 01 mycorrhizae. Mycelial spread of P. tinctorius and S. luteus were naturally inhibited but there was no interaction in mycorrhizal formation. Accepted: 18 June 1999     

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.     

Coinoculation efficacy of ectomycorrhizal fungi on Pinus patula seedlings in a nursery

 The coinoculation efficacy of the ectomycorrhizal fungi Laccaria laccata and Thelephora terrestris on the growth and mycorrhizal development of Pinus patula seedlings was studied and compared to individual inoculation of these fungi in a nursery. The total number of mycorrhizas was higher in seedlings inoculated with the combined inoculum than with the individual inocula. The colonization by T. terrestris was higher than L. laccata when the seedlings were inoculated with the two fungi simultaneously. Coinoculation significantly increased the height and dry weight of the seedlings compared with individual inoculation, both in steam-sterilized and unsterilized soil. Accepted: 12 May 1997     

Morphological analysis of early contacts between pine roots and two ectomycorrhizal Suillus strains

 Selection of ectomycorrhizal strains for application in forestry is mostly based on the evaluation of symbiotic performance in small-scale experiments. Two Suillus collinitus strains isolated from a Mediterranean and an alpine area were inoculated onto two pine tree species (Pinus pinea and P. nigra ssp. laricio var. corsicana) typical of these two environments. The early events during contact between the cell surfaces of plant and fungal partners were analysed morphologically using ultrastructural and immunocytochemical techniques. All four plant-fungus combinations led to a similar degree of mycorrhizal infection and to a similar colonization pattern. The first contact of fungal hyphae with root cap cells usually involved breakdown of the outermost electron-opaque layer of the plant cell walls. Hyphae further developed between this layer and the underlying wall strata. Ultrastructural observations revealed that S. collinitus strain J3-15-24, isolated from a Mediterranean area, induced a defence reaction in the roots of P. nigra, which grows typically in alpine areas. These observations suggest functional differences between the two fungal strains in their mycorrhizal capabilities. Accepted: 3 April 1998     

Ethanol synthesis, nitrogen, carbohydrates, and growth in tissues from nitrogen fertilized Pseudotsuga menziesii (Mirb.) Franco and Pinus ponderosa Dougl. ex Laws. seedlings

 Seedlings of Douglas-fir, Pseudotsuga menziesii (Mirb.) Franco, and ponderosa pine, Pinus ponderosa Dougl. ex Laws., were grown in a controlled environment and fertilized with nutrient solutions containing 150 ppm (+N), or 0 ppm nitrogen (−N). These treatments greatly altered seedling growth, and the concentrations of N and carbohydrates in their tissues. Metabolically active tissues, such as roots, incubated with a limited supply of O₂ became hypoxic faster and synthesized more ethanol than less active tissues, such as needles. All tissues that were incubated for 4 h in N₂ synthesized ethanol. Needles incubated in N₂ and light had much lower quantities of ethanol than needles in N₂ and dark, suggesting that O₂ from photosynthetsis limited internal anoxia. Most tissues from +N seedlings synthesized greater quantities of ethanol in N₂ anoxia than tissues from −N seedlings, probably because they were able to produce more enzymes with a greater availability of N. However, this increase in ethanol synthesis between N treatments was most pronounced in the phloem. Ethanol and soluble sugar concentrations were negatively related in needles and positively related in roots of N+ seedlings, but not −N seedlings. Starch concentrations had no effect on the amount of ethanol produced by any tissue. Regardless of N treatments, all tissues from ponderosa pine produced more N₂-induced ethanol than Douglas-fir, in part because its tissues contained different concentrations of soluble sugars and N as a consequence of phenological differences between the species. However, ponderosa pine tissues may also maintain greater quantities of anaerobic enzymes, or their isozymes than Douglas-fir. Received: 22 February 1998 / Accepted 23 June 1998     

Tissue density and growth response of ectomycorrhizal fungi to nitrogen source and concentration

 Amanita rubescens Pers., Lactarius affinis Pk., Leccinum aurantiacum (Fr.) S.F. Gray, Tylopilus felleus (Bull. ex Fe.) Karsten, and two isolates of Suillus intermedius (Smith & Thiers) Smith & Thiers collected from an approximately 55-year-old Pinus resinosa Ait. plantation, and Pisolithus tinctorius (Pers.) Coker & Couch obtained from another source, were tested for their abilities to grow with protein as the primary source of nitrogen. Protein plates contained 63 mg l^–1 N as bovine serum albumen and 7 mg l^–1 N as arginine. Control plates contained only 7 mg l^–1 N as arginine. All isolates except Leccinum aurantiacum and one isolate of S. intermedius attained greater dry weight with protein as the primary source of N. Lactarius affinis, Leccinum aurantiacum, P. tinctorius, and both isolates of S. intermedius had higher tissue densities on protein medium. Amanita rubescens had lower tissue density. To determine if increase in tissue density was an effect of total N concentration or an effect of N source (protein versus arginine), we performed a second experiment in which arginine concentration was increased (7 mg l^–1 N versus 70 mg l^–1 N). The second experiment also included Cenococcum geophilum Fr. but excluded T. felleus. Higher tissue densities with increased nutrients were found in C. geophilum, Lactarius affinis, Leccinum aurantiacum, and both isolates of S. intermedius. Only A. rubescens and P. tinctorius did not have increased densities. The results suggest that these ectomycorrhizal fungi alter their growth forms according to N concentration. At low N concentrations, a growth form likely to promote exploitation of a large volume of medium for a given biomass is produced. At high concentrations, a growth form likely to promote exploitation of a rich source of N is produced. Whether ectomycorrhizal fungi growing in association with roots would act in a similar fashion is not known. Accepted: 30 July 1998     

Mycorrhiza formation and growth of Eucalyptus globulus seedlings inoculated with spores of various ectomycorrhizal fungi

 As many eucalypts in commercial plantations are poorly ectomycorrhizal there is a need to develop inoculation programs for forest nurseries. The use of fungal spores as inoculum is a viable proposition for low technology nurseries currently producing eucalypts for outplanting in developing countries. Forty-three collections of ectomycorrhizal fungi from southwestern Australia and two from China, representing 18 genera, were tested for their effectiveness as spore inoculum on Eucalyptus globulus Labill. seedlings. Seven-day-old seedlings were inoculated with 25 mg air-dry spores in a water suspension. Ectomycorrhizal development was assessed in soil cores 65 and 110 days after inoculation. By day 65, about 50% of the treatments had formed ectomycorrhizas. By day 110, inoculated seedlings were generally ectomycorrhizal, but in many cases the percentage of roots colonized was low (<10%). Species of Laccaria, Hydnangium, Descolea, Descomyces, Scleroderma and Pisolithus formed more ectomycorrhizas than the other fungi. Species of Russula, Boletus, Lactarius and Hysterangium did not form ectomycorrhizas. The dry weights of inoculated seedlings ranged from 90% to 225% of the uninoculated seedlings by day 110. Although plants with extensively colonized roots generally had increased seedling growth, the overall mycorrhizal colonization levels were poorly correlated to seedling growth. Species of Laccaria, Descolea, Scleroderma and Pisolithus are proposed as potential candidate fungi for nursery inoculation programs for eucalypts. Accepted: 7 May 1998     

Uptake of 15N-labelled alanine,ammonium and nitrate in Pinus sylvestris L. ectomycorrhiza growing in forest soil treated with nitrogen,sulphur or lime

The uptake of ¹⁵N-labelled alanine, ammonium and nitrate was studied in ectomycorrhizal morphotypes of intact Pinus sylvestris seedlings. PCR-RFLP analysis of the ITS-region of fungal rDNA was used to identify the morphotypes. Seedlings were grown in forest soil collected at an experimental site in southern Sweden. The treatments compared were a control, N fertilisation (600 kg N ha^-1 as urea), sulfur application (1200 kg S ha^-1) and lime application (6000 kg CaCO₃ ha^-1). The forest, which had been dominated by Picea abies, was clear-cut two years before the forest soil was sampled. Soil was also collected from an adjacent standing forest. The aim of the present study was to detect changes in the ectomycorrhizal communities in forest soils and relate these changes to the functional parameter of uptake of nitrogen from organic (alanine and protein) and inorganic (ammonium and nitrate) sources.Liming resulted in the detection of a morphotype not found in other samples, and one morphotype was only found in samples from the standing forest (the fungi in these two morphotypes could not be identified). All mycorrhizal root tips showed a higher ¹⁵N concentration after exposure to different nitrogen forms than non-mycorrhizal long roots. Uptake of¹⁵ N from a labelled solution of alanine or ammonium was higher (about tenfold) than uptake from a ¹⁵N-labelled solution of nitrate. Uptake of ammonium and alanine varied between 0.2 and 0.5 mg N g^-1 h^-1 and between 0.1 and 0.33 mg N g^-1 h^-1, respectively, among the different morphotypes.In seedlings grown in the control soil and in soil from standing forest, alanine and ammonium were taken up to a similar degree from a supply solution by all morphotypes, whereas ammonium uptake was higher than alanine uptake in seedlings grown in lime-treated soil (about twofold) and, to a lesser extent, in the nitrogen- and sulfur-treated soils. The higher ammonium uptake by morphotypes from the limed soil was confirmed in pure culture studies. In cases where ammonium was used as the N source, an isolate of the S. variegatus morphotype collected in the limed soil produced more biomass compared with isolates of S. variegatus collected in nitrogen- or sulphur-treated soil. One isolate of a silvery white morphotype produced about equal amounts of biomass on alanine and ammonium, whereas all S. variegatus isolated performed better with ammonium as their N source. Based on the results it is hypothesised that liming can induce a shift in the ectomycorrhizal community, favouring individuals that mainly utilise inorganic nitrogen over those that primarily utilise organic nitrogen.     

Suppression of common root pathogens by helper bacteria and ectomycorrhizal fungi in vitro

 Root pathogens cause considerable loss of tree seedlings in nurseries and are generally difficult to control using conventional methods. Inoculation with ectomycorrhizal fungi may provide some suppression of pathogens. Bacteria (so-called mycorrhization helper bacteria) have been isolated that stimulate mycorrhiza formation on seedling roots and enhance seedling growth; however, their role in pathogen inhibition has not been explored. Four strains of helper bacteria were inoculated together with the ectomycorrhizal fungal species Laccaria bicolor, L. proxima and Suillus granulatus on culture plates to determine inhibition of the pathogens Fusarium oxysporum and Cylindrocarpon sp. Buffered medium was used to rule out acidification of the medium as a mechanism of inhibition. None of the ectomycorrhizal fungal species alone inhibited the growth of Fusarium but all showed slight inhibition of Cylindrocarpon growth. Helper bacterium strain MB3 (Bacillus subtilis) was effective in inhibiting both pathogens and, when inoculated with either L. proxima or S. granulatus, inhibition of Fusarium growth was enhanced over MB3 alone. With Cylindrocarpon, however, only S. granulatus inoculated along with MB3 showed enhanced inhibition over MB3 alone. The other three bacterial strains had little effect on the growth of Fusarium or Cylindrocarpon. More research is necessary to determine if these inhibitory effects are reproducible in situ. Accepted: 23 October 1996     

In vitro ectomycorrhizal formation in Picea glehnii seedlings

 Twenty isolates of ectomycorrhizal fungi – 3 from Picea glehnii, 12 from other coniferous trees, and 5 from decidous trees – were tested for the ability to form mycorrhizae with P. glehnii, using an in vitro synthesis technique. Macroscopically, mycorrhizal formation was observed 3 months after inoculation, when the lateral roots began to grow. Mycelial growth was observed in all inoculated treatments, generally around and along the roots. Six months after inoculation, seedlings were harvested and the mycorrhizae were observed microscopically. Fourteen of the 20 isolates formed ectomycorrhizae with a dense sheath and a deep Hartig net; 1 formed ectendomycorrhizae with a rudimentary mantle, a well-developed Hartig net and intracellular hyphae; 3 formed pseudomycorrhizae with a mantle but without the Hartig net; and only 2 of the fungi tested, Chalciporus pipeparatus 5/92 and Lyophyllum sp. 61/92, did not form mycorrhizae at all. P. glehnii was a good host species since it had low specificity to ectomycorrhizal fungi isolated from trees other than P. glehnii. Accepted: 6 May 1996