Effect of raw humus under two adult Scots pine stands

Tree Physiology Advance Access published December 19, 2011

Tree Physiology 00, 1–13 doi:10.1093/treephys/tpr125

Research paper

Effect of raw humus under two adult Scots pine stands on ectomycorrhization, nutritional status, nitrogen uptake, phosphorus uptake and growth of Pinus sylvestris seedlings

1Department of Soil Ecology, UFZ-Helmholtz Centre for Environmental Research, Theodor-Lieser-Strasse 4, 6120 Halle (Saale), Germany; 2University of Leipzig, Chair of Soil Ecology, Johannisallee 21-23, 03104 Leipzig, Germany; 3Martin Luther University Halle-Wittenberg, Biocenter of the University, Weinbergweg 22, 06120 Halle (Saale), Germany; 4Corresponding author : ([email protected])

Received May 10, 2011; accepted November 2, 2011; handling Editor Torgny Näsholm

Ectomycorrhiza (EM) formation improves tree growth and nutrient acquisition, particularly that of nitrogen (N). Few studies have coupled the effects of naturally occurring EM morphotypes to the nutrition of host trees. To investigate this, pine seedlings were grown on raw humus substrates collected at two forest sites, R2 and R3. Ectomycorrhiza morphotypes were identified, and their respective N uptake rates from organic (2-13C, 15N-glycine) and inorganic (15NH4Cl, Na15NO3, 15NH4NO3, NH415NO3) sources as well as their phosphate uptake rates were determined. Subsequently, the growth and nutritional status of the seedlings were analyzed. Two dominant EM morphotypes displayed significantly different mycorrhization rates in the two substrates. Rhizopogon luteolus Fr. (RL) was dominant in R2 and Suillus bovinus (Pers.) Kuntze (SB) was dominant in R3. 15N uptake of RL EM was at all times higher than that of SB EM. Phosphate uptake rates by the EM morphotypes did not differ significantly. The number of RL EM correlated negatively and the number of SB EM correlated positively with pine growth rate. Increased arginine concentrations and critical P/N ratios in needles indicated nutrient imbalances of pine seedlings from humus R2, predominantly mycorrhizal with RL. We conclude that different N supply in raw humus under Scots pine stands can induce shifts in the EM frequency of pine seedlings, and this may lead to EM formation by fungal strains with different ability to support tree growth. Keywords: arginine; growth; mycorrhization; N uptake; P uptake; Pinus sylvestris; raw humus; Rhizopogon luteolus; Suillus bovinus

Introduction Boreal and temperate forests are typically considered as nitrogen (N) limited, and the role of associated ectomycorrhizal (EM) fungi is to help their host trees compensate for this limitation by mobilizing nutrients from soil organic matter (Finlay 2004). From this point of view, the diversity of EM fungi present at a site is crucial as the performance in N and phosphorus (P) acquisition from mineral and organic sources may vary among EM fungi. This is particularly evident in EM fungi colonizing boreal forest ecosystems in which a large

­ roportion of N and P is sequestered in organic forms not p readily available to the dominant plant species (Read 1991). However, as a consequence of human activity, atmospheric depositions of ammonium and nitrate in soils have increased since the nineteenth century, which profoundly altered the nutrient status of forest trees and the structure and function of associated EM communities (Lilleskov et al. 2002). Plant responses to such changes of the nutritional status can involve enhanced sulfur (S) or N needle contents and reduced or stimulated tree growth.

© The Author 2011. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected]

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Horst Schulz1,4, Tina Schäfer1,2, Veronika Storbeck1,2, Sigrid Härtling1, Renate Rudloff1, Margret Köck3 and François Buscot1,2

2  Schulz et al.

Materials and methods Sampling site and humus characteristics Two 79-year-old Scots pine stands (P. sylvestris) in Dübener Heide nature park, a large forested area in central Germany (Saxony-Anhalt and Saxony), were selected for sampling humus

Tree Physiology Volume 00, 2011

material. The pine stands Rösa2 (R2) and Rösa3 (R3) are located near Rösa (12°29′E, 51°37′N), and are both classified as Calamagrostio Cultopinetum sylvestris ecosystem type (Hofmann 1994) with grasses and nitrophilous herbaceous species (Amarell 1997). According to Schulz and Härtling (2001, 2003), the two plots represent pine stands with different degrees of needle damage symptoms: in R2, higher N contents and degrees of needle tip necroses were found on needles of the second age class compared with R3. Bulk deposition of ammonium and nitrate in both pine stands was measured as throughfall by means of pine bark (Schulz et al. 1997). Cumulative data from 1991 to 2007 (R2: 144.4 kg NH4+N; R3: 157.1 kg NH4+N; and R2: 158.0 kg NO3−N; R3: 159.2 kg NO3−N) showed that both pine stands received nearly identical deposits. The soils are spodi-dystric cambisols that developed from glacial sediments. Humus material (15 subsamples per plot) was collected in March 2008 from the OF and OH layers from plots R2 and R3 (100 × 100 m) and combined to produce one composite material from each plot. The fresh material was transported to our experimental station in Bad Lauchstädt, sieved at 6 mm, and stored for pot experiments and chemical characterization. The humus form of both plots was classified according to AG Boden (2005) as raw humus moder with mean pH values of 4.0 (R2) and 4.3  (R3) in 0.1 mol l−1 KCl extracts. The water-holding capacities were determined as 139% (R2) and 134% (R3). The total contents of macronutrients in dry material of the humus samples were determined after digestion with HNO3 and analysis using ICP-OES (Spectro A.I. Ciros, Kleve, Germany): for R2, K (0.55%), Mg (0.12%), Ca (0.44%) and P (0.063%); and for R3, K (0.45%), Mg (0.11%), Ca (0.51%) and P (0.078%). The total contents of carbon (31.3%) and N (1.49%) for R2 and those (29.7% and 1.42%, respectively) for R3 in humus were analyzed using an elemental analyzer (Vario EL, Elementar, Hanau, Germany). The net N mineralization in homogenized humus substrates was determined using the incubation method described by Zöttl (1958) at 15 °C and a water content of 50% of the water-holding capacity. After a time period of 3 days, 11.3 µg N g−1 DW nitrate in humus from R2, and 13.7 µg N g−1 DW nitrate in humus from R3 were mineralized. Ammonium accumulation in humus from both stands was not detected within an incubation time of 21 days.

Tree seedlings and growing conditions The experiment started on 18 March 2008 with the planting of tree seedlings of P. sylvestris in potted soils. Prior to planting, each 10 l pot was filled with 1500 g of quartz sand to ensure drainage. Three 2-year-old seedlings (bare-rooted) obtained from the ‘Fürst Pückler’ seed orchard (Zeischa, Saxony, Germany) were planted in each pot with 1570 g of DW humus. The height of the tree seedlings varied between 15 and 20 cm. The pots were placed in a greenhouse under controlled ­conditions. The temperature was 22 °C during day and 18 °C at

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As part of a long-term field study on the vitality of Pinus sylvestris in the Dübener Heide nature park (north-eastern Germany), Schulz et al. (1998, 1999) observed that both the growth of Scots pines and the nutritional status of their needles vary significantly, particularly between stands with adult trees. These variations coincided with imbalances in S and N nutrition of the adult trees that, however, could not be related to variations in the N supply of trees in the neighborhood of the seedlings (Schulz and Härtling 2003). The needles supplied with surplus N (>18 mg N per g dry weight (DW)) can contain high amounts of soluble amino acids, particularly arginine (Huhn and Schulz 1996). Such arginine accumulations in Scots pine needles may be due to atmospheric NH3 loads (Perez-Soba et al. 1994), N depositions (van Dijk and Roelofs 1998) or high amounts of plant-available N in organic layers of the soil (Nohrstedt et al. 1996). Nutrient deficiencies, in particular insufficient P supply to the needles, have been considered as further causes of surplus N (Ericsson et al. 1993). The symptoms abate when N supply decreases and disappear entirely upon P fertilization (Quist et al. 1999). Thus, it can be concluded that the accumulation of arginine in needles is primarily the result of an imbalance between N and P availability in conifer stands (Ericsson et al. 1993). However, it must also be assumed that there are additional causes for high arginine contents in conifer needles, as Scots pine stands in the Dübener Heide nature park accumulate arginine to very different extents, although the stands show no significant differences in the input and availability of N and P in their organic soil layers (Huhn and Schulz 1996). The aim of our current investigations is therefore to further clarify the causes for high arginine accumulations in pine needles. The present work was based on the hypothesis that significant differences in abundance and/or diversity of EM fungi in the soil organic layers of different stands lead to unequal N uptake by the Scots pine roots and thus to imbalances in N and P nutrition and to variable growth. To test this hypothesis, pot experiments with P. sylvestris seedlings on raw humus from two adult Scots pine stands of the Dübener Heide nature park with different supplies of N and P were set up. In the context of this project, the following questions were addressed: (i) Do cultivated seedlings show differences in the diversity of EM they trapped from their respective substrates and in their mycorrhization rates? (ii) If so, are there EM-type specific differences in the uptake of phosphate and in the N forms accumulated? (iii) Does the influence of raw humus from the two adult Scots pine stands on the nutritional status and growth of the Scots pine seedlings differ?

Effect of raw humus on P. sylvestris seedlings  3 night. Extra lights were not used. Watering was carried out each day during the vegetation period by pot-weighting and adjusting the soil moisture to 50% of the water-holding capacity.

Measurements of growth parameters and sample processing

Morphotyping, species identification and determination of mycorrhization rates of selected morphotypes One month prior to the main sampling (6 July 2009), three saplings from one randomly selected pot each of R2 and R3 were screened for associated EM morphotypes. Morphotypes were classified on the basis of ramification, shape, color, outer mantle characteristics, and presence or absence of emanating hyphae and rhizomorphs according to the method described by Agerer (1991). Several root tips of each morphotype were individually stored in 50 µl extraction buffer (pH 8) consisting of 2% (w/v) cetyltrimethylammonium bromide (STAB), 100 mM Tris, 1.4 M NaCl and 20 mM EDTA. Root tips were ground with sterile micropestles before extraction buffer volumes were increased to 300 µl. Following 1 h incubation at 65 °C, samples were mixed with 300 µl chloroform and centrifuged (15 min, 13,000 g). Supernatants were transferred to fresh tubes, precipitated with 400 µl of isopropanol for 1 h at −20 °C and centrifuged (10 min,

32P

uptake experiments

A modified version of the ‘teabag’ method of Epstein et al. (1963) was used to determine the phosphate uptake kinetics. Fine root mixed samples (~6–7 g) (wet weight) were enclosed in nylon bags (10 cm × 10 cm, 2 mm mesh) and stored in 0.5 mM CaCl2 at 4 °C for 18 h to remove any unlabeled nutrients in the free space of the roots. The nylon bags were incubated in 900 ml of uptake solution (0.5 mM CaCl2, pH 6.0), which Tree Physiology Online at http://www.treephys.oxfordjournals.org

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On 6 July 2009, i.e., 15 months after tree planting, five pots of each humus variant (R2, R3) were harvested. Plant harvesting was conducted by cutting shoots and then immediately retrieving the entire humus substrate containing the roots of the tree seedlings from each pot. Shoots were separated into twigs and needles (first and second age class), dried at 80 °C and weighed. Before drying, the length of the apical twigs (first age class) was measured. The roots of each tree seedling were rinsed under tap water to remove soil particles and solutes from the root surfaces. Samples were then stored in plastic bags filled with tap water at 3 °C and maximally for 6 days until EM morphotypes and mycorrhization rates were determined. Previous experience had shown that this was a good way of preserving fine roots and mycorrhizal tips. Five pots of each humus variant (R2, R3) were used for phosphate uptake experiments. Again, the seedlings were separated into shoots and roots. The roots were thoroughly washed with water and subsequently rinsed in 0.5 mM CaCl2 to remove soil particles. Three mixed samples of fine roots were prepared by cutting from each rootstock (per pot and humus variant) using a pair of scissors. Mixed samples of needles (first age class) from each seedling per pot and humus variant (n = 5) were frozen in liquid N and stored in plastic bags for analysis of soluble amino acids and total nutrients. The fresh material was pulverized in liquid N using a microdismembrator (Braun, Melsungen, Germany) or dried at 60 °C to a constant weight and ground once more to a fine powder using an MM2 ball mill (Retsch, Haan, Germany).

13,000 g). Supernatants were discarded, pellets were washed with 70% ethanol and centrifuged (5 min, 13,000 g). Air-dried pellets were dissolved in 50 µl of deionized water. The ITS region of the rDNA was amplified using the primer pair ITS-1f (5′-CTTGGTCATTTAGAGGAAGTAA-3′) and ITS-4 (5′-TCCTCC GCTTATTGATATGC-3′). DNA was amplified in 40 µl reaction volumes containing 20 µl GoTaq® Green Mastermix (Promega, Mannheim, Germany), 17 µl deionized water, 0.5 µl of each primer (25 pmol) and 2 µl of DNA template. Polymerase chain reactions (PCRs) were carried out in an Eppendorf 5333 Mastercycler (Hamburg, Germany); an initial denaturation of 5 min at 95 °C was followed by 39 cycles of 30 s at 95 °C, 30 s at 55 °C and 1 min at 72 °C, followed by a final extension period of 7 min at 72 °C. To verify the correct sorting of morphotypes and to reduce the sequencing effort, 2–5 µl of PCR products were digested with restriction enzymes HinfI and MboI (Fermentas, St Leon-Rot, Germany). Restriction fragment length polymorphism (RFLP) patterns were typed by determining fragment lengths (Gene Tools software by SynGene, Cambridge, UK). Using the Good Enough RFLP Matcher (GERM) program by Dickie et al. (2003), we compared the obtained patterns. If available, several representatives of each RFLP pattern were selected; PCR products were purified with ExoSAP (USB, Cleveland, OH, USA) and used as templates in cycle sequencing with ITS-1f or ITS-4 as sequencing primers using an ABI PRISM Big DyeTerminator Cycle Sequencing Kit v.3.1 (Applied Biosystems, Darmstadt, Germany) according to the manufacturer’s protocol. Sequences were edited (BioEdit, Carlsbad, CA, USA) and compared with sequences in the databases UNITE (http://unite.ut.ee/ analysis.php) and NCBI (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Sequences were deposited at the NCBI Genbank under accession numbers HQ259630–HQ259660. As two species, Rhizopogon luteolus Fr. (RL) (Figure 1a) and Suillus bovinus (Pers.) Kuntze (SB) (Figure 1b), dominated as EM partners of the examined root tips, we decided to focus on these two morphotypes in the main sampling in July 2009. In this main sampling, for each of the 30 examined seedlings (15 saplings per humus variant R2 and R3 from five pots), 5 × 100 root tips were examined under a dissecting microscope. For each sample, the numbers of non-mycorrhizal, nonvital mycorrhizal and vital mycorrhizal root tips were counted. Vital mycorrhizal root tips were sorted into three categories: RL, SB and other morphotypes.

4  Schulz et al. where C is the total P concentration (µmol l−1) in the incubation medium, V is the volume (0.9 l) of the incubation medium, DPMsample (disintegrations per h per g DW) is the radioactivity in the sample and DPMref (disintegrations) is the radioactivity in the incubation medium. Analysis of the incubation conditions and phosphate concentrations revealed that (i) the linear increase uptake period applies and (ii) phosphate uptake is not saturated at the phosphate concentrations investigated. 15N

c­ ontained 5  µCi (0.2 MBq) of radioactive carrier-free phosphoric acid [32P]H3PO4 (Hartmann Analytic GmbH, Braunschweig, Germany; specific activity 7000 Ci mmol−1) at room temperature. To determine the P uptake rates of total phosphate over a range of concentrations, uptake solutions were supplemented with increasing phosphate concentrations as indicated (2, 10 and 40 µM KH2PO4). Five replicate teabags were used for each concentration. The samples were subjected to permanent stirring of the uptake solution in order to ensure uniform distribution of radioactive phosphate. The root bags were harvested after 20 min by immediately transferring them into ice-cold unlabeled 0.5 mM KH2PO4 solution and washing them for 1 min in order to remove unspecific adsorbed radioactive phosphate from root surfaces. The root samples were dried overnight at 100 °C, cut and homogenized. Four aliquots of 0.15 g DW of root per sample were taken for determination of the 32P content by Cerenkov counting in a liquid scintillation counter (Tri-Carb 2100 TR, PerkinElmer LAS GmbH, Rodgau, Germany). The phosphate uptake rate was calculated according to the equation



µmol P h−1 g−1 DW = (C ´ V ´ DPMsample ) / DPMref )

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The second period of the pot experiment started on 13 July 2009 in the greenhouse. Five pots per humus variant (R2, R3) and different tracer treatments (a total of 60 pots with 180 saplings) were chosen for 15N humus labeling in a randomized block design. For each treatment, i.e., water control, 15NH4Cl, Na15NO3, 15NH4NO3, NH415NO3 and (2-13C,15N) glycine, 17.61 mg 15N was added to 1572 g of humus DW per pot, corresponding to 0.587 mmol l−1 15N in 2000 ml of total soil solution and assuming full mixing of the injected 15N with the available soil NH4+ or NO3− pool. All compounds had an isotopic enrichment of 98 atom% of both 15N and 13C. Before tracer injection, the soil water volume per pot was adjusted to 1900 ml using deionized water. The labeling solutions (100 ml) were injected into the soil using a needle syringe in five 20-ml doses, as described by Schulz et al. (2011). All tracer experiments were started on Monday morning (13 July 2009) and were stopped when the whole plants were harvested (22 July 2009). We did not observe any flow out of the soil solution from the pots during the short-time (10 days) tracer experiment. Isotope tracing was used for assessment of the individual tree seedling species and date 15N partitioning on a dry mass basis (µg 15N day−1 g−1 DW).

Root sample preparation and analysis of roots and needles

15N

in EM fine

The roots of all saplings per tracer treatment and humus variant were harvested 10 days after tracer application. Plant harvesting was conducted as described above. The root samples were retrieved from the soil core within 6 h of harvesting. For 15N analysis, only fine roots mycorrhizal with RL and SB were used. Under a dissecting microscope, the mycorrhizal fine roots of each EM type and humus variant were separated into EM root tips and non-mycorrhizal proximal root fragments. Due to the rather low amounts of EM root material retrieved during harvest, equal numbers of individual samples of both humus variants were mixed for each 15N tracer treatment. In addition, mixed samples of fine roots and needles (first age class) from each seedling per pot, humus variant and tracer treatment were frozen in liquid N and stored in plastic bags. The fresh material was then pulverized in liquid N using a microdismembrator (Braun, Melsungen, Germany) for ­chemical and

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Figure 1. ​Typical mycorrhizal morphotypes of RL (a) and SB (b) found on P. sylvestris seedlings grown in raw humus ­substrates of both R2 and R3.

tracing and experimental design

Effect of raw humus on P. sylvestris seedlings  5 biochemical analysis or dried at 60 °C to a constant weight and ground once more to a fine powder using an MM2 ball mill (Retsch, Haan, Germany) for stable isotope analyses. Fine powder (2–3 mg) was weighed into tin capsules and analyzed with three replicates for total N, total C, 15N and 13C, applying the method of Gehre et al. (1994) and using an elemental analyzer (Dumas Combustion, EuroVector, Milan, Italy) directly coupled via a ConFlo III interface (Thermo Electron, Oberhausen, Germany) to an isotope ratio mass spectrometer MAT 253 (Thermo Electron, Oberhausen, Germany). The specific 15N uptake rate for each tracer in the fine powder was calculated as follows: µg 15N day −1 g−1 DW = (((a − ao )N %) / t ) ´ 100

where a and ao are the atom percentage concentrations of 15N in roots and needles treated with 15N-enriched soil solution and in the control samples, respectively, N% is the total N percentage concentration and t is the time in days from 15N application until tree harvest (10 days).

Measurements of total nutrients in fine roots and needles The total contents of K, Mg, Ca and P in the dry material of fine roots and needles were determined after digestion with HNO3 and analyzed using ICP-OES (Spectro A.I. Ciros, Kleve, Germany). The total contents of carbon and N were analyzed using an elemental analyzer (Vario EL Elementar, Hanau, Germany).

Measurements of soluble nutrients in humus, fine roots and needles For humus analysis, 0.5 g of fresh material was extracted with 25 ml of deionized water on a shaker for 40 min. After centrifugation, the supernatant was membrane-filtered and analyzed by ion chromatography as described below. For fine root and needle analysis, 500 mg of pulverized fresh material was extracted (Ultra Turrax T 25, IKA-Labortechnik, Staufen, Germany) with cooling for 1 min in 12.5 ml of deionized water. After centrifugation, the supernatant was twice membrane-­ filtered (45 µm). The analysis of nitrate and phosphate was carried out using an ICS-2000 Ion Chromatography System (Dionex Corporation, Sunnyvale, USA). For the separations, we used an analytical AS17-C column (2 × 250 mm) with an column (2 × 50 mm) with a flow rate of AG17-C guard ­ 0.5 ml min−1 (Dionex Corporation, Sunnyvale, USA). A sample volume of 10 µl of membrane-filtered soil or foliar extract was injected and run in a linear KOH gradient of 1 mmol l−1 from 0 to 1.5 min, of 15 mmol l−1 to 5 min, of 40 mmol l−1 to 9 min, of 60 mmol l−1 to 13 min, of 1 mmol l−1 to 14 min and equilibrated at this concentration for a further 5 min before the start of the next analysis. Ammonium in humus, fine root and needle samples was measured after extraction with 1% K2SO4 (6.25 g/25 ml) using

Measurements of amino acids in humus, fine roots and needles Pulverized fresh fine root/needle material (0.7 g) was extracted in 7.4 ml of 4% sulfosalicylic acid. After centrifugation, 4 ml of the supernatant were added to a 5-ml volumetric vessel, the pH was adjusted to ~3.0 using 0.1 ml of 30% NaOH, and the contents were then topped up with deionized water. Subsequently, the crude extract was used for derivatization and high-performance liquid chromatography (HPLC) separation. The derivatization procedure for amino acids with 9-­fluorenylmethyl chloroformate and the HPLC conditions were as described by Huhn and Schulz (1996). For the determination of α-amino N in humus samples, 1 g fresh material was extracted with 5 ml of deionized water and subsequently filtered through a fluted filter and membrane filter (<45 µm). The concentrations of asparagine, glutamine, glutamate, arginine and alanine were measured as described above and summed up on a dry mass basis (µg α-amino N g−1 DW).

Measurements of nitrate reductase activity in fine roots For the determination of nitrate reductase (NR) activity in fine root fresh material, a modified version of the method of Genenger et al. (2003) was used. Approximately 250 mg of powdered frozen plant material was mixed with 4 ml of 0.1 mol l−1 potassium phosphate buffer (pH 7.5) containing 12 mM l−1 KNO3 and 1% isopropanol. The mixture was incubated for 3 h at 25 °C in the dark. After incubation, the sample buffer was centrifuged for 20 min at 4 °C and at 13,800 g. The supernatant was used for the determination of formed nitrite. The supernatant (550 µL) was mixed with 1000 µl of 1% ­sulfanilamide in 1.5 N HCl and 1000 µl of 0.02% N-naphthylethylene diamine. In the control experiment, the N-naphthylethylene diamine was replaced with water. After 15 min of incubation, the absorbance was measured at 540 nm against water. The NR activity is quoted in units of nmol NO2− min−1 g DW−1.

Data analysis The experiment was based on a randomized pot design. Five pots for each treatment and humus variant were used as replicates. We conducted a non-parametric Mann–Whitney U test using Statistica (V.8.0, StatSoft (Europe) GmbH, Hamburg, Germany) with humus variant (R2, R3) as the main effect to examine growth differences in length of apical twig, needle biomass, root biomass, shoot/root ratio and total nutrients in needles. Single analyses of variance (ANOVA) with the Tukey honestly significant difference (HSD) test were used to examine significant differences in availability of plant-available nutrients in humus, specific 15N uptake rates of EM root tips, non-­mycorrhizal proximal root fragments and needles between

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a gas-sensitive electrode (Type NH 500/2, WTW, Weilheim, Germany), as described by Schulz et al. (1999).

6  Schulz et al. the fungi, tracer treatments and humus variants. To evaluate the ­relationships between phosphate uptake rates and total phosphate concentration in the uptake solutions, arginine concentrations and P/N ratios in needles, growth parameters and the number of EM root tips per type, we carried out correlation analyses, taking the r2 values into account.

Table 2. ​Mean values ± SD (n = 5) of growth parameters (apical twig first age class, total needle biomass of first age class, total root biomass, shoot/root ratio) of P. sylvestris seedlings grown in raw humus substrates from Rösa2 (R2) and Rösa3 (R3).The different superscript letters represent statistically significant differences between the humus variants (Mann–Whitney U test) at P < 0.05. Humus variant

Results Availability of N and P in humus at the time of tree seedling planting

Seedling growth The elongation of apical twigs (i.e., first age class produced in 2009) of P. sylvestris seedlings was significantly different between both humus variants (Table 2). Overall, 15 months after planting (March 2008), the mean length of apical twigs was 52% higher in raw humus from R3 than in R2 (26 vs. 17 cm). The needle biomass of first age class was also significantly increased by raw humus from R3. In contrast, the total root biomass and the shoot/root ratio both tended to be higher in R3 in comparison with R2, but without significance (Table 2).

Nutritional status of pine seedlings To investigate whether the humus variant affected the nutritional status of pine seedlings, macronutrient and amino acid analyses were conducted in fine roots and needles (first age class) after harvesting in July 2009, i.e., 15 months after tree planting (see the results summarized in Tables 3 and 4). Table 1. ​Mean values ± SD (n = 5) of plant-available nutrients in raw humus substrates from Rösa2 (R2) and Rösa3 (R3) at the time of tree seedling planting. The different superscript letters represent statistically significant differences between the humus variants (Mann– Whitney U test) at P < 0.05. Humus variant Ammonium-N Nitrate-N

α-Amino-N Phosphate-P

(µg g−1 DW) R2 R3

9.48 ± 2a 12.73 ± 3a

21.94 ± 2a 2.9 ± 0.02a 7.57 ± 2b 2.1 ± 0.02a

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0.39 ± 0.1a 0.66 ± 0.3a

R2 R3

Total needle biomass (first class)

Total root biomass

(cm)

(gram dry weight)

(gram dry weight)

17.17 ± 4a 26.13 ± 5b

10.87 ± 5a 14.90 ± 6b

9.45 ± 2a 12.20 ± 4a

Shoot/root ratio

3.57 ± 0.2a 3.92 ± 0.5a

Significant differences between the humus variants were only found for total N and P (Table 3). The concentrations of arginine in solution within fine roots and needles varied accordingly, while glutamine, glutamate, glycine and alanine showed no significant differences between the humus variants (Table 4).

Identification of EM fungi Prior to the main sampling in July 2009, we screened three saplings from each of R2 and R3. Together, 10 morphotypes were found (see Table S1 available as Supplementary Data at Tree Physiology Online). Restriction fragment length polymorphism digestion with HinfI and MboI was employed for verifying the correct sorting of morphotypes and rationalizing the sequencing effort. None of the morphotypes yielded identical patterns to other ones, but the PCR products from DNA of the Tomentella ellisii (Sacc.) Jülich & Stalpers morphotype resulted in two different patterns. According to the data obtained from cycle sequencing and subsequent BLAST search, 6 out of 10 detected morphotypes could be identified to species level and two morphotypes were identified to genus level (see Table S1 available as Supplementary Data at Tree Physiology Online). Two ­morphotypes—one per variant—remained unidentified. In both R2 and R3 substrates we identified RL, SB, Thelefora terrestris Ehrh. and Wilcoxina sp. In R3, we additionally detected Rhizopogon roseolus (Corda) Th. Fr., T. ellisii and Tuber sp., while Phialocephala fortinii Wang & Wilcox was specifically detected in R2. As we were interested in those EM species that might play a major role in sapling nutrition in pot cultures rather than in an exhaustive assessment of all occurring morphotypes, we decided to focus on RL and SB, which appeared to be the dominant morphotypes in both humus types in the preliminary screening.

Mycorrhizal colonization of fine roots With 90% in humus variant R2 and >88% in R3, the total mycorrhization rates were nearly identical for both treatments. However, the respective percentages of EM formed by RL and SB differed significantly between both humus variants (Table  5). In R2,

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The concentrations of plant-available inorganic N (NH4+, NO3−), organic N (α-amino N) and water-extractable phosphate (PO43–) in both humus substrates at the time of tree seedling planting are given in Table 1. Significant differences between the humus substrates were only found for nitrate. The NH4+/ NO3− ratio tended to be higher in R3 in comparison with R2, while the total concentrations of soluble amino acids (α-amino N) were comparable between the humus substrates. Very small soluble PO43– concentrations with mean values of 0.39 (R2) and 0.66 (R3) µg P g−1 DW were measured in both raw humus substrates, in comparison with total P concentrations of 0.63 (R2) and 0.78 ± 0.02 (R3) mg P g−1 DW.

Apical twig length (first class)

Effect of raw humus on P. sylvestris seedlings  7 Table 3. ​Mean values ± SD (n = 5) of total N, phosphorus, potassium, magnesium and calcium contents in fine roots and needles (first age class) of P. sylvestris seedlings grown in raw humus from Rösa2 (R2) and Rösa3 (R3). The different superscript letters represent statistically significant differences between the humus variants (Mann–Whitney U test) at P < 0.05. Humus variant

N

P

K

Mg

Ca

12.21 ± 0.6a 10.99 ± 0.5b

0.81 ± 0.03a 0.88 ± 0.03b

1.59 ± 0.2a 1.49 ± 0.3a

0.77 ± 0.06a 0.85 ± 0.04a

4.00 ± 0.3a 4.62 ± 0.3a

12.49 ± 0.6a 11.43 ± 0.5b

0.70 ± 0.03a 0.85 ± 0.03b

1.98 ± 0.2a 1.86 ± 0.3a

0.57 ± 0.03a 0.61 ± 0.03a

2.13 ± 0.2a 1.98 ± 0.2a

(mg g−1 DW) Fine roots  ​ ​R2  ​ ​R3 Needles  ​ ​R2  ​ ​R3

Humus variant

Arginine

Glutamine

Glutamate

Alanine

Glycine

(µg g−1 DW) Fine roots  ​ ​R2  ​ ​R3 Needles  ​ ​R2  ​ ​R3

1455.83 ± 763a 454.21 ± 203b

744.86 ± 147a 822.71 ± 227a

319.11 ± 28a 275.96 ± 37a

235.39 ± 68a 254.16 ± 46a

27.42 ± 4a 28.00 ± 2a

1338.08 ± 809a 258.43 ± 326b

96.05 ± 21a 95.20 ± 13a

253.25 ± 57a 270.49 ± 47a

46.54 ± 5a 51.64 ± 6a

6.46 ± 1a 5.74 ± 1a

Table 5. ​Mean values ± SD (n = 5) of EM root tip numbers per type as well as number of dead EM root tips and total EM root tips of P. sylvestris seedlings grown in raw humus from Rösa2 (R2) and Rösa3 (R3). The different superscript letters represent statistically significant differences between the humus variants (Mann–Whitney U test) at P < 0.05. Humus variant

R2 R3

Number of ectomycorrhizal root tips (%) RL

SB

EM (unclassified)

EM (dead)

EM (total)

32.65 ± 12a 7.23 ± 6b

4.95 ± 6a 26.14 ± 8b

24.29 ± 7a 27.35 ± 3a

28.56 ± 5a 27.84 ± 5a

90.45 ± 2a 88.56 ± 3a

32.65% of the root tips were mycorrhizal with RL against only 4.95% by SB. In R3, the situation was almost inverted with 7.23% of RL but 26.14% for SB EM. 32P

uptake in fine roots

To clarify whether the measured differences in seedling P nutritional status of needles were linked to the observed differences in EM colonizations, we performed [32P]-phosphate uptake studies with excised fine roots in a concentration range of 2–40 µM total phosphate-P in the uptake solution. The total phosphate uptake in the fine roots of pine seedlings grown in humus variants R2 and R3 increased significantly with respect to the phosphate concentration in the uptake solution (Figure 2). The P uptake in fine roots from humus variant R2 in the concentration range 10–40 µM PO4 -P tended to be higher than in fine roots from raw humus substrate R3; however, the mean difference of 11.8 µg P h−1 g−1 DW between the humus variants at 40 µM phosphate P con-

centration in the uptake solution was not statistically significant, even though the mean phosphate concentrations of 91.0 ± 10 µg P g−1 DW (R2) and 125.0 ± 10 µg P g−1 DW (R3) in fine roots after 32P measurements between the humus variants were significantly different as also measured before 32P measurements in fine roots (R2: 66.0 ± 10 µg P g −1 DW; R3: 106.0  ± 10 µg P  g−1 DW) and needles (146.0  ± 10 µg P g−1 DW; 162.0 ± 12 µg P g−1 DW).

Effects of 15N tracer additions on availability of inorganic N and P in raw humus We measured the concentrations of inorganic N and P in both humus substrates before and after tracer addition. In July 2009, 15 months after tree seedling planting, the concentrations of NH4+ -N between the controls (before and after tracer addition) were significantly increased in both humus substrates R2 and R3, while the concentrations of NO3− -N and PO43– -P were unchanged (Table 6). However, the effects of 15N tracer

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Table 4. ​Mean values ± SD (n = 5) of soluble amino acids in fine roots and needles (first age class) of P. sylvestris seedlings grown in raw humus from Rösa2 (R2) and Rösa3 (R3). The different superscript letters represent statistically significant differences between the humus variants (Mann–Whitney U test) at P < 0.05.

8  Schulz et al. a­ ddition on the availability of NH4+ -N, NO3− -N and PO43– -P in both humus substrates were not observed. The concentrations of plant-available nutrients were not significantly different between the treatments and both raw humus substrates. The higher extractable ammonium concentrations were a general response to altered soil moisture in the humus substrates after tracer addition. 15N

uptake in EM fine roots

15N

Figure 2. ​Rates of phosphate uptake in relation to phosphate concentrations in the uptake solution by excised fine roots of tree seedlings (P. sylvestris) grown in raw humus from two old Scots pine stands (R2, R3). The non-linear relationships were fitted using the model function y = (Pmax × x)/(Km + x), Michaelis–Menten plot.

Table 6. ​Mean values ± SD (n = 5) of plant-available N and P concentrations in raw humus before and 10 days after tracer addition (harvesting). Different lowercase superscript letters within the rows indicate significant differences between the humus variants. Different uppercase superscript letters within the columns indicate significant differences between the treatments (ANOVA, Tukey HSD test) at P < 0.05. Reference: control, before tracer addition. Treatment

Control (before tracer addition) Control (after tracer addition) 15NH Cl 4 Na15NO3 15NH NO 4 3 NH415NO3 (2-13C, 15N) Glycine

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Humus substrate R2

Humus substrate R3

NH4+ -N (µg g−1 DW)

NO3− -N (µg  g−1 DW)

PO43− -P (µg g−1 DW)

NH4+ -N (µg g−1 DW)

NO3− -N (µg g−1 DW)

PO43− -P (µg g−1 DW)

13.70 ± 3a,A 34.02 ± 5a,B 36.54 ± 2a,B 24.08 ± 3a,B 29.18 ± 3a,B 36.51 ± 2a,B 35.12 ± 5a,B

6.27 ± 3a,A 5.27 ± 2a,A 6.79 ± 5a,A 3.90 ± 2a,A 5.63 ± 3a,A 7.52 ± 3a,A 5.13 ± 2a,A

0.48 ± 0.2a,A 0.45 ± 0.2a,A 0.23 ± 0.1a,A 0.36 ± 0.2a,A 0.37 ± 0.1a,A 0.42 ± 0.1a,A 0.25 ± 0.1a,A

16.20 ± 3a,A 32.73 ± 3a,B 30.75 ± 3b,B 31.06 ± 3b,B 34.04 ± 4a,B 35.32 ± 1a,B 35.08 ± 2a,B

3.33 ± 2a,A 2.97 ± 1a,A 2.35 ± 1a,A 4.26 ± 2a,A 2.47 ± 1a,A 3.20 ± 1a,A 2.58 ± 1b,A

0.55 ± 0.2a,A 0.61 ± 0.3a,A 0.63 ± 0.1b,A 0.54 ± 0.1a,A 0.56 ± 0.1a,A 0.46 ± 0.1a,A 0.48 ± 0.2a,A

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measurements indicated that EM root tips and the nonmycorrhizal proximal root fragments of colonized fine roots acquired N from all added N sources, but in different amounts and in a manner dependent on the fungus species (Table 7). Overall, the two EM types exhibited significant differences in their 15N uptake from the distinct N sources in the EM root tips (F = 277.23, P = <0.001); no significant differences were noted in the proximal non-mycorrhizal root fragments

(F = 2.27, P = 0.1480). RL EM root tips always took up a higher 15N amount than SB EM root tips, i.e., 3.6 times more 15N from 15NH Cl, 1.9 times more from Na15NO , 1.8 times 4 3 more from 15NH4NO3, 4.1 times more from NH415NO3 and 2.2 times more from 2-13C,15N-glycine addition. The percentages of total inorganic 15N uptake in the root tips of RL and SB were 84.0% and 86.3%, respectively, while the percentages of organic 15N uptake in the root tips of RL and SB were 15.3% and 13.6%, respectively. Comparisons of 15N uptake in EM root tips revealed a different uptake of ammonium and nitrate by both species. As shown in Table 7, the uptake of nitrate in both EM types was significantly higher than that of ammonium when the two N forms were given individually. In contrast, when both forms were given simultaneously in the form of NH4NO3, ammonium uptake was higher even if not significantly for RL. Similar N-uptake patterns according to the given 15N form were found in the non-mycorrhizal proximal fine root parts of the EM formed by SB and RL. Again, nitrate was preferred over ammonium in the case of individual providing, but ammonium uptake was higher than that of nitrate by simultaneous providing as NH4NO3 (Table 7). The two EM species also exhibited significant differences in 13C uptake from 2-13C, 15N glycine by EM root tips (RL: 99.95 ± 18 µg 13C g−1 DW; SB: 31.02 ± 1 µg 13C g−1 DW). In contrast, no significant differences were found in 13C levels accumulated in the non-mycorrhizal proximal root fragments (RL: 46.35 ± 9 µg 13C g−1 DW; SB: 57.85 ± 2 µg 13C g−1 DW). Consequently, plotting 13C contents against 15N contents of EM root tips (Table 4) resulted in a statistically significant regression curve (y = −0.0003 + 0.2137x; R2 = 0.99; n = 9; P = <0.001; see Figure S1 available as Supplementary Data at Tree Physiology Online). The slope of the regression curve (0.21) indicates the relative amounts of 13C and 15N in EM root tips and can be compared with the 13C/15N ratio (0.88) of the added glycine tracer. This suggests that at least 24% of accumulated 15N in EM root tips had been taken up in organic form by both species.

Effect of raw humus on P. sylvestris seedlings  9 Table 7. ​Mean values ± SD (n = 3) of 15N uptake rates in EM fine roots of P. sylvestris harvested 10 days after tracer addition. Different lowercase superscript letters within the rows indicate significant differences between the fungi. Different uppercase superscript letters within the columns indicate significant differences between specific 15N uptake rates after individual and simultaneous tracer treatment (ANOVA, Tukey HSD test) at P < 0.05. Tracer treatment

N uptake rate (µg 15N day−1 g−1 DW) RL

15NH Cl 4 Na15NO3 15NH NO 4 3 NH415NO3 (2-13C, 15N)

EM root tips

Non-mycorrhizal proximal root fragments

EM root tips

Non-mycorrhizal proximal root fragments

602.53 ± 41a,A 915.40 ± 7a,B 621.02 ± 147a,A 490.19 ± 64a,A 474.32 ± 76a

190.07 ± 20a,A 290.35 ± 34a,B 273.38 ± 49a,A 157.48 ± 8a,B 185.09 ± 12a

168.07 ± 10b,A 485.88 ± 38b,B 338.01 ± 24b,A 119.09 ± 14b,B 175.16 ± 3b

153.12 ± 4a,A 237.91 ± 5a,B 285.17 ± 24a,A 116.20 ± 8a,B 242.57 ± 7a

uptake in needles

The results of the 15N measurements are summarized in Table 8. The specific 15N acquisition from all N sources in needles of the first age class was significantly higher in pine seedlings grown in humus substrate R2 compared with R3. When 15N acquisition in needles from different N sources was compared after individual (15NH4Cl vs. Na15NO3) or simultaneous tracer addition (15NH4NO3 vs. NH415NO3), there were no significant differences.

Relationship between NR activity in fine roots and ammonium accumulation in needles In fine roots of pine seedlings grown in humus substrate R2, significantly higher NR activities were measured. The enzyme activities were closely correlated with the increased enrichment of ammonium in the needles (Figure 3).

Relationships between arginine concentrations and uptake rates and P/N ratios in needles

15N

In order to find out whether arginine contents in needles of P. sylvestris seedlings were influenced by the uptake of 15N from various N forms or by critical P/N ratios as responses to nutrient imbalances, we carried out correlation analyses. The various 15N treatments showed no marked differences in the tendency for increased needle arginine contents with increasing 15N uptake rates (Figure 4a–d). However, under 15NH4NO3 supply, a lower arginine accumulation appeared to be related to 15NH4+ uptake, whereas the opposite was true under NH415NO3 supply when arginine accumulation was related to 15NO3− uptake (Figure 4c). In contrast, no relationships between glutamine contents and 15N uptake rates were observed (data not shown). The overall means of the glutamine contents (µg g−1 DW) in needles of pine seedlings grown in humus substrates R2 (109.84 ± 41) and R3 (112.31 ± 44) showed no significant ­differences. On the other hand, the trend of decreasing P/N ratios indicated increasing nutrient imbalances in needles of pine seedlings grown in humus substrate R2 (Figure 5).

Figure 3. ​Relationships between NR activity in fine roots and ammonium concentrations in needles (first age class) of P. sylvestris seedlings. Open symbols, seedlings grown in raw humus substrate R2; filled symbols, seedlings grown in raw humus substrate R3.

Relationships between elongation of apical twigs and number of colonized root tips Finally, we carried out correlation analyses in order to find out whether the elongation of apical twigs of P. sylvestris seedlings was influenced by the number of colonized root tips. The relationships showed a positive correlation (r2 = 0.27) between the elongation of apical twigs and EM colonization by SB (Figure 6a) and a negative correlation (r2 = −0.32) with EM colonization by RL (Figure 6b).

Discussion The results of this study confirm the hypothesis that the accumulation of arginine in P. sylvestris seedlings grown in raw humus substrates with different N supply from two old Scots pine stands indeed corresponded to a P/N imbalance in the needles. Increased concentrations of arginine in conifer tissues after N fertilization have already been described (Durzan and Steward 1967, Barnes and Bengston 1968, Ebell and McMullan 1970).

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15N

Glycine

SB

10  Schulz et al.

Figure 5. ​ Relationships between arginine concentrations and P/N ratios in needles (first age class). Open symbols, seedlings grown in raw humus substrate R2; filled symbols, seedlings grown in raw humus substrate R3.

Rabe and Lovatt (1986) also described increased de novo arginine biosynthesis in citrus rootstock cultivars under conditions of P deficiency. More recently, Näsholm and Ericsson (1990),

Tree Physiology Volume 00, 2011

Ericsson et al. (1993) and Edfast et al. (1990) reported on arginine accumulation in Norway spruce and Scots pine needles, which they primarily ascribed as metabolic response to an imbalance between N and P availability. However, it has not been shown so far that mycorrhiza fungi with increased N uptake capacity may also be responsible for enhanced arginine contents in Scots pine needles. Furthermore, our results demonstrate (a) that low availability of P in raw humus is not the sole reason for increased arginine contents in pine needles but (b) that arginine accumulation in needles can also be triggered by high nitrate availability and EM fungi with high N uptake rates. In total, the increased arginine concentrations in needles of P. sylvestris seedlings grown in the raw humus substrate R2 can also be explained as follows: (a) high availability of ammonium and/or nitrate in raw humus substrate, (b) high conversion of nitrate to ammonium in roots, and (c) increased uptake and accumulation of N in roots and needles with stimulation of de novo arginine biosynthesis. This interpretation is supported by the findings of this study. We showed that the availability of mineral P in both raw humus substrates was relatively low, especially with regard to the high ammonium and nitrate availability in both raw humus

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Figure 4. ​Relationships between arginine contents and 15N uptake rates of different N forms in needles (first age class) of P. sylvestris seedlings. Open symbols, seedlings grown in raw humus substrate R2; filled symbols, seedlings grown in raw humus substrate R3.

Effect of raw humus on P. sylvestris seedlings  11

substrates (Table 1). However, the measured plant-available P ­concentrations were not significantly different and were not influenced by 15N tracer additions (Table 6). It may therefore be argued that pine seedlings grown in both raw humus substrates accumulate arginine due to an inadequate P supply (see Ericsson et al. 1993). This hypothesis of a nutrient imbalance is especially reinforced by the trend of decreasing P/N ratios found in the needles (Figure 5). Except for pine seedlings grown in substrate R3, the P/N ratios of needles were lower than or equal to the critical limit of 0.055 suggested by Ericsson et al. (1993) for Picea abies. In contrast, the relationships between other nutrition elements (K, Mg, Ca) and N in needles did not differ between the two humus variants (data not shown). Thus, we can conclude at first that the accumulation of arginine in potted pine seedlings is primarily the result of an imbalance between N and P availability in the raw humus substrates. However, it remains to be shown why the P/N ratios, arginine accumulation and growth of pine seedlings grown in substrates R2 and R3 were significantly different in the case of comparable P availability.

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Figure 6. ​Relationships between elongation of apical twigs and the number of colonized root tips of P. sylvestris seedlings: (a) RL; (b) SB.

We did not register any significant difference between the uptake capacity of phosphate in the fine roots of pine ­seedlings grown in both substrates (Figure 2). In contrast, the N uptake capacity in EM fine roots of pine seedlings grown in the two humus substrates was significantly different due to variations in the individual numbers of colonized root tips. The study showed that P. sylvestris formed more EM with RL on R2 than on R3 while just the opposite pattern was found with SB. This dominance of EM with RL over EM with SB corresponded to depressed tree growth (Figure 6a and b). Therefore, seedling growth appeared to be influenced by a shift in the dominant EM fungal partner as the total mycorrhization rates were similar on both substrates (Table 5). These results correspond to those of Repácˇ (2007), who found correlations with substrate-type treatments to correspond to variations in the EM-type frequencies. A similar relationship between plant growth and mycorrhization by distinct mycobionts was also observed in several other studies (Schoenberger and Perry 1982, Wallander et al. 1994, Vonderwell and Enebak 2000). In addition, our 15N labeling experiments showed that the correlation between dominant EM species and plant growth probably corresponds to different and specific abilities of the mycobionts to assimilate different N forms from the substrate. This is supported by the significantly different uptake rates of 15N from three mineral and one organic (glycine) N form by root tips mycorrhizal with RL and SB. These differences were, however, only detectable in EM root tips themselves because for all N forms no significant differences in 15N uptake rates were found on non-mycorrhizal proximal root fragments, which is in agreement with the fact that EM form on the fine root part where most of the nutrient uptake occurs (Chalot and Brun 1998, Smith and Read 2008). The uptake of 15N from all N sources in EM root tips of RL was at all times higher than in the case of EM root tips with SB (Table 7). We also measured higher NR activities in the fine roots of pine seedlings grown in humus substrate R2, which correlated closely with the increased enrichment of ammonium in the needles (Figure 3). These results show that the conversion of nitrate to ammonium was higher in the fine roots of pine seedlings grown in R2 and that mineral N was preferentially transported in the form of ammonium in the shoots. Consequently, we also measured increased 15N uptake rates in the needles of pine seedlings grown in R2 (Table 8, Figure 4a–d), and the remaining N, which was not stored into protein, was accumulated in intermediary storage pools as arginine (see also Flaig and Mohr 1992). It is known that arginine might play a role in stabilizing polyphosphate (Finlay et al. 1992). However, Rabe and Lovatt (1986) favor the accumulation of arginine during P deficiencies as the mechanism for detoxifying needle tissue of excess ammonia. Rabe and Lovatt (1986) also suggested that the de novo arginine biosynthetic pathway is expensive in terms of ATP and carbon, which probably causes ­additional

12  Schulz et al. Table 8. ​Mean values ± SD (n = 5) of 15N uptake in needles (first age class) of P. sylvestris seedlings harvested 10 days after tracer addition. Different lowercase superscript letters within the rows indicate significant differences of specific 15N uptake rates between the humus variants. Different uppercase superscript letters within the columns indicate significant differences between specific 15N uptake rates after individual or simultaneous tracer treatment (ANOVA, Tukey HSD test) at P < 0.05. Tracer treatment

15NH Cl 4 Na15NO3 15NH NO 4 3 NH415NO3 (2-13C, 15N)

N uptake rate (µg 15N day−1 g−1 DW) Humus substrate R2

Humus substrate R3

4.68 ± 2a,A

2.76 ± 1b,A 3.20 ± 1b,A 3.11 ± 1b,A 2.89 ± 1b,A 1.82 ± 1b,B

4.84 ± 1a,A

Glycine

4.78 ± 3a,A 3.88 ± 1a,A 2.84 ± 1a,A

Acknowledgments We thank Drs M. Gehre and Falk Bratfisch (Department of Isotope Biogeochemistry) for 15N and 13C measurements and S. Mothes and Jürgen Steffen (Department of Analytical Chemistry) for conducting element measurements. We also thank the service team at our experimental station in Bad Lauchstädt, whose technical assistance made this work possible. Furthermore, we thank two anonymous reviewers for valuable comments on the former version of the manuscript.

Supplementary data Supplementary data for this article are available at Tree Physiology online.

Tree Physiology Volume 00, 2011

AG Boden. 2005. Bodenkundliche Kartieranleitung. Bundesanstalt für Geowissenschaften und Rohstoffe in Zusammenarbeit mit den Staatlichen Geologischen Diensten, Hannover, 438 p. Agerer, R. 1991. Characterization of ectomycorrhizas. In Methods in Microbiology, Vol. 23. Eds. J.R. Norris, D.J. Read and A.K. Varma. Academic Press, London, pp 489–500. Amarell, U. 1997. Anthropogene vegetationsveränderungen in den Kiefernforsten der Dübener Heide. In Regeneration und Nachhaltige Landnutzung-Konzepte für Belastete Regionen. Eds. R. Feldmann, K. Henle, H. Auge, J. Flachowsky, S. Klotz and R. Krönert. Springer, Berlin, pp 110–117. Barnes, R.L. and G.W. Bengston. 1968. Effects of fertilization, irrigation, and cover cropping on flowering and on nitrogen and soluble sugar composition of slash pine. For. Sci. 14:172–180. Chalot, M.D. and A. Brun. 1998. Physiology of organic nitrogen acquisition by ectomycorrhizal fungi and ectomycorrhizas. FEMS Microbiol. Rev. 22:21–44. Dickie, A.I., P.G. Avis, D.J. McLaughlin and P.B. Reich. 2003. GoodEnough RFLP Matcher (GERM) program. Mycorrhiza 3:171–172. Durzan, D.J. and F.C. Steward. 1967. The nitrogen metabolism of Picea glauca (Moench) Voss and Pinus banksiana Lamb. as influenced by mineral nutrition. Can. J. Bot. 45:695–710. Ebell, L.F. and E.E. McMullan. 1970. Nitrogenous substances associated with differential cone production responses of Douglas-fir to ammonium and nitrate fertilization. Can. J. Bot. 48:2169–2177. Edfast, A.B., T. Näsholm and A. Ericsson. 1990. Free amino acid concentrations in needles of Norway spruce and Scots pine trees on different sites in areas with two levels of nitrogen deposition. Can. J. For. Res. 28:1132–1136. Epstein, E., W.E. Schmid and D.W. Rains. 1963. Significance and technique of short-term experiments on solute absorption by plant ­tissue. Plant Cell Physiol. 4:79–84. Ericsson, A., L.G. Norden, T. Näsholm and M. Walheim. 1993. Mineral nutrient imbalances and arginine concentrations in needles of Picea abies (L.) Karst. From areas with different levels of airborne deposition. Trees 8:67–74. Finlay, R.D. 1992. Uptake and translocation of nutrients by ectomycorrhizal fungal mycelia. In Mycorrhizas in Ecosystems. Eds. D.J. Read, D.H. Lewis, A.H. Fitter and I.J. Alexander. CAB International, Wallingford, pp 91–97. Finlay, R.D. 2004. Mycorrhizal fungi and their multifunctional roles. Mycologist 18:91–96. Flaig, H. and H. Mohr. 1992. Assimilation of nitrate and ammonium by Scots pine (Pinus sylvestris) seedling under conditions of high nitrogen supply. Physiol. Plant. 84:568–576. Gehre, M., Hofmann, D. and P. Weigel. 1994. Methodische Untersuchungen zum 15N-ConFlo-IRMS system. Isotopenpraxis Isot. Environ. Health Stud. 30:239–245. Genenger, M., Zimmermann, S., Frossard, E. and I. Brunner. 2003. The effects of fertiliser or wood ash on nitrate reductase activity in Norway spruce fine roots. For. Ecol. Manag. 175:413–423. Hofmann, G. 1994. Der Wald. Sonderheft Waldökosystemkatalog. Deutscher Landwirtschafts, Berlin. Huhn, G. and Schulz, H. 1996. Contents of free amino acids in Scots pine needles from field sites with different levels of nitrogen deposition. New Phytol. 134:95–101. Lilleskov, E.A., T.J. Fahely, T.R. Horton and G.M. Lovett. 2002. Belowground ectomycorrhizal fungal community change over a nitrogen deposition gradient in Alaska. Ecology 83:104–115. Näsholm, T. and A. Ericsson. 1990. Seasonal changes in amino acids, protein and total nitrogen in needles of Scots pine trees. Tree Physiol. 6:267–281.

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stress to the plant. Thus, this pathway would seem prone to growth ­inhibition during P deprivation. It was apparently also the case for pine seedlings grown in raw humus substrate R2, the growth of which was significantly decreased. In summary, the results of our pot experiments with seedlings of P. sylvestris cultivated in raw humus from two old Scots pine stands show that arginine accumulation in needles occurs in the case of an excess of N in relation to P in the substrate. There was low natural availability of P in both raw humus substrates, but it seems that the reason for a higher arginine accumulation in seedlings grown on R2 was probably an enhanced N uptake as well as N accumulation in pine seedlings (roots and needles), which resulted from both a higher N supply in raw humus of R2 and a shift in the dominant fungal partner on EM root tips. Our analyses did not enable us to find the origin of the different mycobiont dominance between both sites R2 and R3. Therefore, we can only speculate that this difference results from a random distribution of the EM fungi in soils or from differences in the nutrient supply at the two sites.

References

Effect of raw humus on P. sylvestris seedlings  13 Schulz, H., G. Huhn and S. Härtling. 1998. Responses of sulphur and nitrogen-containing compounds in Scots pine needles along a deposition gradient in eastern Germany. In Changes of Atmospheric Chemistry and Effects on Forest Ecosystems. A Roof Experiment without a Roof, Vol. 3. Eds. R.F. Hüttl and K. Bellmann. Nutrients in Ecosystems. Kluwer Academic Publishers, Dordrecht, pp 37–63. Schulz, H., S. Härtling, G. Huhn and G. Schüürmann. 1999. Nachweis ökotoxikologischer Wirkungen in Kiefernökosystemen— Ergebnisse einer langfristig angelegten Freilandstudie. In Ökotoxikologie-Ökosystemare Ansätze und Methoden. Eds. J. Oehlmann and B. Markert. Ecomed Verlagsgesellschaft, Landsberg, pp 248–267. Schulz, H., S. Härtling and C.F. Stange. 2011. Species-specific differences in nitrogen uptake and utilization by six European tree species. J. Plant Nutr. Soil Sci. 174:28–37. Smith, S.E. and D.J. Read. 2008. Mycorrhizal symbiosis. Academic Press, New York. Van Dijk, H.F.G. and J.G.M. Roelofs. 1988. Effect of excessive ammonium deposition on the nutritional status and condition of pine needles. Physiol. Plant. 73:494–501. Vonderwell, J.D. and S.A. Enebak. 2000. Differential effects of rhizobacterial strain and dose on the ectomycorrhizal colonization of loblolly pine seedlings. For. Sci. 46:437–441. Wallander, H., J.E. Nylund and B. Sundberg. 1994. The influence of IAA, carbohydrate and mineral concentration in host tissue on ectomycorrhizal development on Pinus sylvestris L. in relation to nutrient supply. New Phytol. 127:521–528. Zöttl, H. 1958. Die Bestimmung der Stickstoffmineralisation im Waldhumus durch den Brutversuch. Z. Pflanzenern. Bodenkd. 81:35–49.

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Nohrstedt, H.Ö., U. Sikström, E. Ring, T. Näsholm, P. Högberg and T. Persson. 1996. Nitrate in soil water in three Norway spruce stands in southwest Sweden as related to N depositions and soil, stand, and foliage properties. Can. J. For. Res. 26:836–848. Perez-Soba, M., I. Stulen and L.J.M. van der Eerden. 1994. Effect of atmospheric ammonia on the nitrogen metabolism of Scots pine (Pinus sylvestris) needles. Physiol. Plant. 90:629–636. Quist, M.E., T. Näsholm, J. Lindeberg, C. Johannison, L. Högbom and P. Högberg. 1999. Responses of a nitrogen-saturated forest to a sharp decrease in nitrogen input. J. Environ. Qual. 28:970–1977. Rabe, E. and C.J. Lovatt. 1986. Increased arginine biosynthesis during phosphorus deficiency. Plant Physiol. 81:774–779. Read, D.J. 1991. Mycorrhizas in ecosystems. Experientia 47:376–391. Repácˇ , I. 2007. Ectomycorrhiza formation and growth of Picea abies seedlings inoculated with alginated-bead fungal inoculum in peat and bark compost substrates. Forestry 80:517–530. Schoenberger, M.M. and D.A. Perry. 1982. The effect of soil disturbance on growth and ectomycorrhizae of Douglas-fir and western hemlock seedlings: a greenhouse bioassay. Can. J. For. Res. 12:343–353. Schulz, H. and S. Härtling. 2001. Biochemical parameters as biomarkers for the early recognition of environmental pollution on Scots pine trees. II. The antioxidative metabolites ascorbic acid, glutathione, α-tocopherol and the enzymes superoxide dismutase and glutathione reductase. Z. Naturforsch. 56c:767–780. Schulz, H. and S. Härtling. 2003. Vitality analysis of Scots pines on basis of a multivariate approach. For. Ecol. Manag. 186:73–84. Schulz, H., G. Huhn, B. Niehus, G. Liebergeld and G. Schüürmann. 1997. Determination of throughfall rates on the basis of pine bark loads: results of a field study. Air Waste Manage. Assoc. 47:510–516.