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Keywords:

  • 454 sequencing;
  • chronosequence;
  • ectomycorrhiza;
  • ergosterol;
  • external mycelia

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information
  • Here, species composition and biomass production of actively growing ectomycorrhizal (EM) mycelia were studied over the rotation period of managed Norway spruce (Picea abies) stands in south-western Sweden.
  • The EM mycelia were collected using ingrowth mesh bags incubated in the forest soil during one growing season. Fungal biomass was estimated by ergosterol analysis and the EM species were identified by 454 sequencing of internal transcribed spacer (ITS) amplicons. Nutrient availability and the fungal biomass in soil samples were also estimated.
  • Biomass production peaked in young stands (10–30 yr old) before the first thinning phase. Tylospora fibrillosa dominated the EM community, especially in these young stands, where it constituted 80% of the EM amplicons derived from the mesh bags. Species richness increased in older stands.
  • The establishment of EM mycelial networks in young Norway spruce stands requires large amounts of carbon, while much less is needed to sustain the EM community in older stands. The variation in EM biomass production over the rotation period has implications for carbon sequestration rates in forest soils.

Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Nutrient uptake of boreal forest trees is mainly mediated through symbiotic ectomycorrhizal (EM) fungi, which colonize the tree roots producing an extensive network of external mycelium in the soil (Smith & Read, 2008). In addition to its importance for nutrient uptake, EM mycelium serves as an important food source for soil animals and saprotrophic microorganisms, and in this way fuels the soil ecosystem with energy derived from the photosynthetic activity of the trees. These EM mycelia can also contribute significantly to soil respiration; up to 50% of soil respiration has been attributed to roots and their associated EM fungi (Höberg et al., 2001). The large proportion of photosynthetic activity that is allocated to EM fungi (10–50%, reviewed by Simard et al., 2002) indicates a large potential for EM fungi to influence carbon (C) flux below ground and consequently the sequestration of carbon in the soil.

The allocation of C to fine roots in managed forest soils has been shown to vary over the rotation period, with a distinct maximum in young stands (King et al., 2007). The amount of C allocated to EM fungi over the same period may follow a similar pattern, as the growth of fine roots is positively correlated with that of EM mycelium (Majdi et al., 2008). Maximum belowground C allocation appears to coincide with intense nutrient demand at canopy closure, when the leaf area index is at a maximum (Simard et al., 2002), which is reached between 25 yr and 40 yr in Norway spruce (Picea abies) forests in south-central Sweden (Schmalholz & Hylander, 2009). However, studies on the production of EM fruit bodies in forests of different ages have provided inconclusive results. Some studies report a peak in production in young forest (Chisilov & Demidova, 1998; Hintikka, 1988), while others report no variation with forest age (Bonet et al., 2004).

Ectomycorrhizal fungal communities in boreal forests are highly diverse (Dahlberg, 2001), and successions of EM communities over the rotation period of different types of forest ecosystems are well documented from studies of sporocarps or root tips (Kranabetter et al., 2005; Palfner et al., 2005; Twieg et al., 2007). Successional change in species composition have been explained by changes in soil chemistry such as pH and nitrogen (N) availability, and the chemical characteristics of the organic material, which usually becomes more recalcitrant as the forest ages (Deacon & Flemming, 1992; Jumpponen et al., 1999). In addition, a variation in C delivery by the host may influence EM succession, as indicated by the observed changes in EM community composition in response to elevated CO2 concentrations (Fransson et al., 2001; Parrent & Vilgalys, 2007). In earlier research, EM fungi have been described in terms of early- and late-stage fungi based on their occurrence with forest age (Last et al., 1987; Deacon & Flemming, 1992), but these concepts have been criticized in more recent studies for being insufficient to describe fungal successions in forest ecosystems (Kranabetter et al., 2005; Palfner et al., 2005; Twieg et al., 2007).

In the present study, the aim was to estimate production of EM biomass, as extraradical mycelium, over a Norway spruce chronosequence ranging from 0 to 130 yr of age in south-western Sweden. In addition, we analysed how the EM fungal community changed over this period, using novel high-throughput 454 sequencing (Margulies et al., 2005; Buée et al., 2009) of internal transcribed spacer (ITS) amplicons. We used sand-filled ingrowth mesh bags to estimate EM production and to identify the active, extraradical EM community in the soil (Wallander et al., 2001; Hedh et al., 2008). This approach has been used in a number of studies for both quantifying EM growth in soil and for analysing changes in EM communities in response to nutrient amendments or forest management practices (Kjöller, 2006; Korkama et al., 2007; Parrent & Vilgalys, 2007; Hedh et al., 2008; Majdi et al., 2008). A peak in EM growth was expected during canopy closure. We also expected changes in EM community composition with stand age, owing to shifting host C allocation patterns, soil chemical parameters and organic matter quality.

Materials and Methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Study sites

The sites studied are located in the Tönnersjöheden Experimental Forest (lat. 56°42′N, long. 13°06′E, alt. 60–140 m above sea level) in south-western Sweden (Malmström, 1937), representing maritime climate conditions (yearly mean air temperature +6.7°C, yearly mean precipitation 1064 mm, mean length of growing season 215 d). Forty Norway spruce (Picea abies (L.) H. Karst) stands were selected with the ages evenly distributed between 0 and 130 yr (see the Supporting Information Table S1). The different stands represented the following forest development classes: fresh and recently cultivated clearcuts (= 10), young stands before first thinning (= 10), stands in the active thinning phase (= 10) and old stands after the active thinning phase (= 10). Each class was further divided into one younger and one older subclass, resulting in eight different classes with five stands in each. All sites were located on mineral soils representing the soil types till or gravel. The type of former land use at the sites was varied, representing first and second rotation of pure Norway spruce stands following coniferous-dominated stands, broadleaf-dominated stands and former open Calluna heathland (Table S1).

Experimental design

To estimate the growth of EM fungi we used fungal ingrowth bags made of nylon mesh (50 μm mesh size, 8 × 8 × 1 cm). This mesh size allows the ingrowth of fungal hyphae, but not roots (Wallander et al., 2001). The mesh bags were filled with 30 g acid-washed quartz sand (0.36–2.0 mm, 99.6% SiO2; Ahlsell AB, Malmö, Sweden). Five replicate mesh bags were buried at the interface between the organic horizon and the mineral soil in each spruce stand (a total of 5 × 40 = 200 bags). The mesh bags were placed in a row, c. 1 m apart, on 1 June 2005. The mesh bags were harvested on 15 November 2005, after an incubation period of 22 wk. Soil samples were collected from the humus layer (c. 3–10 cm) and from the upper region (0–5 cm) of the mineral soil using a soil corer (4 cm diameter). Five cores, taken at random locations (c. 1–2 m away from the location of the mesh bags) within each site were pooled to give one composite sample.

The mesh bags were stored at +8°C for up to 6 h before being taken to the laboratory. The sand from all five mesh bags from each site was combined and mixed, and a subsample of 10 g was taken for the analysis of ergosterol as an indicator of fungal biomass (see later). Another subsample of 10 g was gently shaken with water in a glass flask to loosen the sand from the fungal mycelium. The procedure was repeated until the water was clear and no apparent mycelium was visible in the water. The mycelium was collected on a nylon mesh, and it was assumed that insignificant amounts of hyphae were retained on the sand particles. The mycelium was collected in Eppendorf tubes and the approximate volume was recorded by comparing with an Eppendorf tube with following markings: 20, 60, 150, 300 and 500 μl. This sample was then used for DNA extraction and identification of the EM community. The samples were stored at −20°C and freeze-dried until analysed.

DNA extraction, PCR and 454 sequencing

The mycelium samples were mixed with Al2O3 powder to a total volume of c. 200 μl and ground together with glass beads in a Fast Prep shaker (FP120; MP Biomedicals, Irvine, CA, USA). Between 50 mg and 100 mg of this mixture was then used for DNA extraction with 1 ml extraction buffer (3% cetyl trimethylammonium bromide (CTAB), 2 mM EDTA, 150 mM Tris-HCl and 2.6 M NaCl, pH 8) at 65°C for 1 h, followed by precipitation with 1.5 volumes of isopropanol. After centrifugation (15 min, 16 200 g ) the pellet was resuspended in 50 μl Milli-Q water.

Polymerase chain reaction was carried out in two steps. First, the fungi-specific primer combination ITS1-F (Gardes & Bruns, 1993) and ITS4 (White et al., 1990) were used at an annealing temperature of 57°C for 35 cycles. In the second step, the same ITS primers were used, but elongated with the adaptors required for 454 sequencing (ITS1-F/A adaptor and ITS4/B adaptor) (Margulies et al., 2005). The primers also contained sample-specific tags consisting of five bases (Acosta-Martinez et al., 2008). For the ITS1-F/A adaptor primer, a single tag was used for all samples (5′-GCCTCCCTCGCGCCATCAGACCTGCTTGGTCATTTAGAGGAAGTAA-3′), while for the ITS4/B adaptor primer, unique base combinations were used for each individual sample (5′-GCCTTGCCAGCCCGCTCAGXXXXXTCCTCCGCTTATTGATATGC-3′). The same annealing temperature as above was used in this second step, but for only five cycles. Between and after the two PCR steps, products were purified with the Agencourt AMPure kit (Agencourt Bioscience Corporation, Beverly, MA, USA), in order to remove residual primers and primer dimers.

The concentrations of the purified PCR products were measured with the PicoGreen ds DNA Quantification Kit (Molecular Probes, Eugene, OR, USA) on a luminescence spectrometer (model LS50B; Perkin Elmer, Waltham, MA, USA). Equal amounts of DNA from each sample were pooled to provide a single sample for the entire study. The 454 sequencing was performed on a Genome Sequencer FLX 454 (Roche Applied Biosystems) at the Department of Biotechnology, the Royal Institute of Technology, Stockholm, Sweden. A half-plate was sequenced, starting from the ITS4/B adapter, providing partial coverage (200–300 base pairs) of the fungal ITS regions.

Bioinformatic analysis of sequence data was conducted using the SCATA software (http://scata.mykopat.slu.se). Sequences were filtered for quality, removing short sequences (< 200 bases), sequences with low quality (average read quality < 20 or > 5 bases with base quality < 10) as well as primer dimers and polymers. Primer and sample tag sequences were then removed, but the sample association was stored as meta-data associated with each sequence. All sequences were then searched against each other, using blastn from the NCBI blast package (Altschul et al., 1997) requiring a minimum match length of 190 bp. Sequences were brought together in clusters when the sequence similarity, excluding gaps, exceeded 98.5% (single linkage clustering), resulting in a matrix of cluster relative abundances vs sample identities. Within each cluster, sequences were realigned, using the global alignment program muscle (Edgar, 2004), and consensus sequences were inferred. Clusters were taxonomically identified manually by aligning consensus sequences together with selected reference sequences from NCBI Genbank or the UNITE database (Köljalg et al., 2005), using the CLUSTALW algorithm of megalign (DNAStar Inc., Madison, WI, USA).

Ergosterol analysis

Freeze-dried sand from the mesh bags was analysed for ergosterol to provide an estimate of the EM fungal biomass (Nylund & Wallander, 1992). Ergosterol was also used as an estimate of fungal biomass in soil samples. Total ergosterol (including esterified forms) is most commonly used for fungal biomass estimates in soil, but recently it has been suggested that free ergosterol is a better proxy for living fungi (Yuan et al., 2008). We used one subsample (10 g sand or 1 g soil) for estimates of free ergosterol, and one subsample for total ergosterol. The free ergosterol was extracted in 5 ml methanol, while the total ergosterol was extracted with 5 ml 10% KOH in methanol. After this step, the two methods followed the same protocol. The samples were sonicated for 15 min, extracted overnight and then refluxed at 70°C for 90 min. After cooling, 1 ml H2O and 2 ml cyclohexane were added. The samples were mixed in a vortex apparatus for 20 s, centrifuged for 5 min at 900 g and the cyclohexane phase was then transferred to another test tube. The methanol was extracted with a further 1.5 ml cyclohexane. The cyclohexane was evaporated under N2 and the samples were dissolved in methanol. Before the quantification of ergosterol, the samples were filtered through a 0.5 μm Teflon syringe filter (Millex LCR-4; Millipore). The chromatographic system consisted of a high-performance liquid chromatograph (Hitachi model L2130, Japan), a UV detector (Hitachi model L2400, Japan) and a C18 reversed-phase column (Chromolith, Merck) preceded by a C18 reversed-phase guard column (Elite LaChrome; Hitachi). Extracts were eluted with methanol at a flow rate of 1 ml min−1 and absorbance measured at 282 nm.

Soil chemical analysis

The most available parts of NH4, calcium (Ca), potassium (K) and magnesium (Mg) in the soil samples from the humus layer were estimated by BaCl2 extraction. Subsamples of 25 g of soil were extracted with 100 ml 0.1 M BaCl2 for 1 h. The pH of the solutions was measured with a pH meter and the concentrations of the elements were analysed using Inductively Coupled Plasma - Emission Spectroscopy.

The most available proportion of phosphorus (P) of the same samples was estimated by sulphate fluoride extraction. Subsamples of 15 g were extracted for 1 h in 70 ml 0.05 M Na2SO4 and 0.02 M NaF. The solutions were filtered through a 25 mm RC filter (Lida Manufacturing Corp., Windsor, UK) and were stored at −20°C until analysed. The phosphate concentration in the filtrate was determined using SnCl2 as a reducing agent (Murphy & Riley, 1962).

The C : N ratio was measured with a vario Max CN analyser (Elementar Analysensysteme GmbH, Germany).

Statistics

The forest stands were organized into age classes: 0–1, 5–10, 11–20, 21–30, 31–40, 41–50, 51–90 and 91–130 yr (= 5), and variations in production of extramatrical hyphae in mesh bags, soil fungal biomass and nutrient availabilities between age classes were analysed with one-way ANOVA and Fishers LSD to separate the means. For each stand, Shannon’s diversity index was calculated for EM communities in the ingrowth bags as H ′ = − Σpi logepi, where pi is the proportion of EM amplicons accounted for by cluster i. Relationships between EM diversity and stand age as well as C : N ratio and stand age were tested for significance by linear regression. The relationship between stand age and over-all distribution of EM amplicon between clusters was tested for statistical significance by canonical correspondence analysis (CCA), with stand age as a continuous explaining variable. The analysis was repeated with clusters merged within fungal genera. Relationships between stand age and the relative abundance of amplicons of individual EM clusters and genera were tested by Spearman’s rank correlation. The CCA was conducted using canoco (Microcomputer Power, Ithaca, NY, USA), and all other statistical analyses were performed using the software statistica (StatSoft Inc., Tulsa, OK, USA). A species accumulation curve with stands as replicate samples was produced, using the software estimates (Colwell, RK, University of Connecticut, Storrs, CT, USA), and the asymptote was estimated by the Chao2 method.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Most of the mesh bags contained visible fungal mycelium, except those collected from stands aged 0–1 yr (Fig. 1a). The values of free and total ergosterol in the mesh bags were similar in all age classes except for 30–40 yr, where the total ergosterol was higher than the free ergosterol (Fig. 1b). The age of the stand had a strong influence on hyphal growth in the mesh bags, showing a peak in young stands before the first thinning phase, when the trees were < 30 yr old (ANOVAs for free and total ergosterol, and for EM mycelial volume: < 0.001, Fig. 1)

image

Figure 1.  Amount of ectomycorrhizal (EM) mycelium in sand from mesh bags incubated in forests of different ages. (a) Visual estimates of EM volume collected from mesh bags. (b) Free ergosterol (closed bars) and total ergosterol (open bars). Error bars represent SE. Different letters represents statistically significant differences between treatments, = 5 for each age group, (abc were used to distinguish volume of mycelium in (a), as well as free ergosterol in (b); xyz were used to distinguish total ergosterol in (b)).

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Useful PCR products were obtained from 28 of the 40 forest stands, with most of the unsuccessful samples originating from recent clearcuts or very old stands and containing little mycelium. We only obtained PCR products from a single clearcut stand (0–1 yr) and from two old-growth stands (90–130 yr). The numbers of replicates from the other age classes are reported in Fig. 2. Sequencing yielded a little over 18 000 ITS sequences; the number of sequences per sample ranged from 300 to 1100. These values do not reflect the full potential of the 454 method, as a great deal of the information consisted of nonITS sequences, mainly primer dimers. The 18 000 sequences were arranged in 248 clusters containing 99% of the total number of sequences. Each of the remaining 184 sequences occurred only once in the data set and were therefore dismissed as unreliable.

image

Figure 2.  Species distribution of internal transcribed spacer (ITS) amplicons from ectomycorrhizal (EM) fungi colonizing ingrowth mesh bags. The mesh bags were incubated in the humus layer of Norway spruce (Picea abies) stands of different ages.

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In most samples, EM fungi dominated the communities. Averaged across all samples, 71% of the 18 000 amplicons could be attributed to known EM taxa. By contrast, EM fungi accounted for only 53 (21%) of the 248 clusters, indicating a long tail of rare nonmycorrhizal fungi. Four samples from stands 0, 6, 110 and 124 yr of age deviated in that the proportions of amplicons from known EM fungi were low (0–30%). These samples also had a low content of ergosterol. As the youngest and oldest age classes were represented by only one and two successfully amplifying samples, respectively, and the proportion of EM amplicons in these samples was low, these samples were excluded from further analyses. After exclusion of these samples, the average proportion of amplicons from EM taxa was 78% (median 86%). Among the amplicons from EM taxa, members of the family Atheliaceae dominated. The most common cluster was attributed to Tylospora fibrillosa, which was the most common species in all age classes between 10 yr and 90 yr, and almost completely dominated among the EM amplicons in 10- to 30-yr-old stands (Fig. 2). Amplicons attributed T. asterophora, an as yet unidentified atheloid species and various Piloderma species were also common. In the CCA, stand age explained 7.1% of the eigenvalue of the data, and the relationship was marginally insignificant (= 0.063) according to a Monte Carlo test with 1000 permutations. When clusters were merged within fungal genera, 10.3% of the inertia was explained by stand age and the relationship was significant (= 0.028). The amplicon abundances of the genera Xerocomus (= 0.0006) and Russula (= 0.008), as well as the species Tylopilus felleus (= 0.015) and Byssocorticium pulchrum (= 0.046), were significantly and positively correlated with stand age. Among the nonEM or nonassigned fungi, Sordariomycetes and Leotiomycetes predominated, accounting for on average 8% and 6%, respectively, of the amplicons.

Both the number of EM clusters (= 0.01) and Shannon’s index based on EM taxa (= 0.002) increased significantly with stand age (Fig. 3). The species accumulation curve, based on the stands as sampling units, levelled off with increasing number of stands sampled, indicating that the number of samples collected was sufficient to describe most of the EM species colonizing the ingrowth bags in the area (Fig. 4). The Chao2 estimate of the total EM species richness was 65 (57–89 at the 95% confidence interval).

image

Figure 3.  Diversity of ectomycorrhizal (EM) fungi in relation to age of Norway spruce (Picea abies) stands: (a) number of EM clusters; (b) Shannon diversity index.

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image

Figure 4.  Species accumulation curve (solid line) with 95% confidence interval (fine broken lines). The uppermost broken line indicates Chao2 estimated total species richness of ectomycorrhizal (EM) fungi in Norway spruce (Picea abies) stands.

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When the composite soil samples were analysed, neither the total ergosterol nor the free ergosterol concentration varied significantly with tree age, in the humus layer or the mineral soil. The concentration of free ergosterol was significantly lower (< 0.001) than that of total ergosterol in both the humus (41% ± 2) and the upper mineral soil (20 ± 2%, Fig. 5).

image

Figure 5.  Free (closed) and total (open) ergosterol concentrations in soil samples collected from Norway spruce (Picea abies) stands of different ages: (a) humus layer, (b) mineral soil.

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Nutrient availability in the soil varied to some extent with stand age. Ammonium levels were significantly higher in clearcuts compared with standing forests (Table 1). A negative relationship was found between EM mycelial production and NH4 availability in young forests (0–36 yr, Fig. 6) but not in older forests. The available PO4 peaked in clearcuts and both PO4 and K peaked in the 30–40 yr old forests. The availability of Ca did not vary significantly with stand age (Table 1). The average C : N ratio in the humus was 26.9 ± 0.4, and was positively related to stand age (= 0.025, Fig. 7). pH values showed maxima in clearcuts (Table 1).

Table 1.   Effects of stand age on extractable nutrients and pH in the soil
Parameter0–1 yr5–10 yr10–20 yr 20–30 yr30–40 yr40–50 yr50–90 yr90–130 yr
  1. Values are given as mean ± SE, all values are mg g−1 DW, except pH. Different letters within each row indicate statistically different values (P < 0.05). ns, not significant [correction added on 21 June 2010, after first online publication: the table formatting has been changed to aid clarity].

NH40.53 ± 0.09a0.24 ± 0.07b0.17 ± 0.02b0.20 ± 0.04b0.27 ± 0.07b0.21 ± 0.06b0.21 ± 0.06b0.13 ± 0.02b
PO40.085 ± 0.009ab0.061 ± 0.006bc0.047 ± 0.007c0.053 ± 0.011c0.095 ± 0.02a0.047 ± 0.004c0.042 ± 0.015c0.031 ± 0.004c
K0.27 ± 0.02b0.22 ± 0.04b0.29 ± 0.04b0.31 ± 0.03b0.42 ± 0.06a0.27 ± 0.02b0.27 ± 0.02b0.25 ± 0.03b
Ca (ns)1.3 ± 0.31.6 ± 0.21.1 ± 0.21.0 ± 0.21.6 ± 0.51.3 ± 0.060.7 ± 0.20.8 ± 0.1
pH4.3 ± 0.1a4.1 ± 01ab3.8 ± 0.1c3.7 ± 0.1c4.0 ± 0.1bc3.9 ± 0.2bc3.7 ± 0.1c3.7 ± 0.1c
image

Figure 6.  Relationship between ectomycorrhizal (EM) growth and NH4 concentration in young Norway spruce (Picea abies) forests (0–36 yr), = −0.026–0.07log(x), r2 = 0.43.

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image

Figure 7.  Carbon (C) : nitrogen (N) ratio in the humus layer in relation to Norway spruce (Picea abies) stand age. = 25.8 + 0.25x, r2 = 0.12, = 0.025.

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Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

The majority of the amplicons from fungi colonizing the mesh bags were of EM origin. Although many saprotrophic species were detected, they constituted a minor portion of the amplicons.

In accordance with our expectations, the results indicated a maximal EM mycelial production in young forests (10–20 yr), coinciding with canopy closure when tree growth is rapid and leaf area maximal (Simard et al., 2004). Our observations support the results of Palfner et al. (2005) and Twieg et al. (2007), who found that the ratio of active to senescent mycorrhizal root tips was higher in young than in old forests. Palfner et al. (2005) estimated that 4 × 105 EM root tips were active per m2 in a 12-yr-old Sitka spruce forest, and that this decreased to 1 × 105 m−2 in a 40-yr-old forest, although the total number of root tips was similar.

Our findings suggest that much less C is required to support EM hyphal growth in very young and very old Norway spruce forests, than during the period before the first thinning, when C requirements appear to peak. A better description of this variation in C allocation to root growth and growth of EM hyphae over the rotation period is necessary in order to improve C allocation models of forest ecosystems, which are usually not well developed in this respect (Litton & Giardina, 2008).

The extent to which the peak in belowground C flux in young forests is associated with a peak in C sequestration in the soil is not known. It is difficult to quantify C sequestration over the rotation period because of the large pool of soil C and the small changes that occur during a single forest generation. King et al. (2007) reasoned that the much higher root to shoot C ratio found in young stands of Pinus resinosa (0.80 in 8-yr-old and 0.29 in 55-yr-old stands) should have considerable implications for belowground C accumulation. However, Vesterdal et al. (1995) found no increase in total C accumulation after afforestation of former arable land, although soil C was redistributed from the mineral soil to the forest floor over a period of 29 yr of Norway spruce growth. The contribution of EM fungi to this process was not investigated. The growth of external EM mycelia did not influence the amount of C remaining in litter over a 2-yr period in a study by Langley et al. (2006). However, EM roots were degraded more slowly than nonmycorrhizal roots, suggesting that the mycorrhizal status of roots can have a strong influence on soil C processing rates (Langley & Hungate, 2003).

Although the total species richness may be high, EM communities associated with root tips are usually dominated by a few species (Dahlberg, 2001; Horton & Bruns, 2001). In the present study, the EM community of external mycelia was dominated by fast-growing T. fibrillosa, which constituted 80% of the EM amplicons in the mesh bags collected from 10- to 30-yr-old stands, but declined to an average of 43% in mesh bags collected from 30- to 90-yr-old stands. As the dominance of T. fibrillosa decreased, the diversity of EM fungi in the mesh bags increased (Figs 2, 3). The same pattern was observed by Palfner et al. (2005) in a Sitka spruce forest in northern England, where T. fibrillosa colonized over 90% of the root tips in young forests (1–6 yr), but only 40% of the root tips in 40-yr-old forest. In Canada, Twieg et al. (2007) found increasing diversity of EM fungi on root tips in Douglas-fir but not in paper birch forests. The EM diversity stabilized after c. 65 yr in the Douglas fir forest.

In line with the theories of Grime (1979), T. fibrillosa might be described as a C strategist, being adapted to high population densities. Carbon strategists are characterized by efficient conversion of resources to biomass, leading to rapid growth and ecosystem dominance when resources are abundant. This is in agreement with the observed increase in dominance of Tylospora species in ingrowth bags in response to elevated atmospheric CO2 concentrations (Parrent & Vilgalys, 2007), which presumably increases belowground allocation of photosynthates. It has been shown in microcosm experiments that species with ample production of extraradical mycelium may exclude other EM species from the root systems of the host plants (Wu et al., 1999), decreasing diversity. The competitive advantage of T. fibrillosa may have declined in the maturing forest, leaving room for other species, such as X. badius and B. pulchrum to enter the community. We also found increases in russuloid species (e.g. Russula nigricans) with stand age, which confirms the results of other studies (Visser, 1995; Smith et al., 2002; Kranabetter et al., 2005; Twieg et al., 2007).

The mesh bag method may underestimate EM production (Hendricks et al., 2006), presumably as a result of discrimination against EM species belonging to the contact or short distance exploration types according to the classification of Agerer (2001). Furthermore, it has been shown that species belonging to the genus Cortinarius are under-represented in ingrowth bags, while boletoid species can be over-represented compared with the EM community on root tips (Kjöller, 2006). It should, however, be noted that we detected several species of Cortinarius in the mesh bags in the present study, although always in low amounts. On the other hand, the use of ingrowth mesh bags made it possible to sample the active portion of the EM community from 40 Norway spruce stands with a limited amount of effort. Studies of EM communities on root tips may include inactive EM fungi and such studies are particularly laborious because of the large number of root tips present in the soil. The spatial variation of EM fungi was accounted for by placing several mesh bags in each stand and pooling the samples before analysis. According to the species accumulation curve, most of the variation in the community of EM fungi that colonized the mesh bags was reflected in our measurements (Fig. 4).

Ammonium availability in the soil was negatively related to hyphal growth in mesh bags in young forests, probably as a result of efficient nutrient uptake by EM mycelium during the period of most active growth. If this nutrient-absorbing capacity is impaired, the risk of nutrient losses from the system will increase. Large amounts of nitrate were lost from Douglas fir stands in France (Marques et al., 1997; Jussy et al., 2000) and Sitka spruce forests in Wales (Harrison et al., 1995), probably as a result of impaired growth of extramatrical hyphae owing to elevated N input. Nilsson et al. (2007) found a negative relationship between hyphal production in mesh bags and nitrate loss in oak stands along a N deposition gradient in southern Sweden.

The observed progressive increase in C : N ratio with stand age is in agreement with results from an 8000 yr long chronosequence (Wallander et al., 2009) as well as with the observed increasing C : N ratios in humus layers of increasing age and depth (Lindahl et al., 2007). The increase may be explained by selective uptake of N by mycorrhizal fungi, with subsequent translocation to host tissue.

The trend towards a reduction in the proportion of free ergosterol in older soil organic matter (SOM) is interesting, and may have consequences for the way in which the accuracy of fungal biomass estimates in soil is evaluated. In newly formed young mycelia almost all the ergosterol was in free form. By contrast, in the oldest SOM from the mineral horizon, only 20% of the ergosterol was in free form. This indicates that a portion of ergosterol becomes bound in older SOM, and that this portion is unlikely to be associated with active fungi. Another interesting finding was that the concentration of free ergosterol (but not bound ergosterol) in soil samples tended to be lower in clearcuts than in young plantations. This may be the result of degradation of EM mycelia from the previous forest generation after cutting the forest. de Ridder-Duine et al. (2006) found that free ergosterol constituted a small portion of the total ergosterol in a forest soil with a high SOM content, while most ergosterol was in free form in agricultural soils with a lower SOM content. Ergosterol from inactive fungi may thus be conserved in soils rich in SOM. The amount of fungal biomass produced in the mesh bags was much smaller than the fungal biomass estimated in the soil, which would suggest an unrealistically long turnover time, as discussed in Wallander et al. (2004). This is probably a result of underestimation of the EM production in the mesh bags, as suggested by Hendricks et al. (2006) but is should be noted that the calculated turnover time will be reduced by using the free ergosterol rather than total ergosterol.

In conclusion, the hyphal growth in mesh bags incubated in Norway spruce forests in southern Sweden shows a distinctive peak in young spruce stands that has not entered the first thinning phase (< 30 yr old). This coincides with the period of most active growth of fine roots. A single species, T. fibrillosa, accounted for 80% of the amplicons in the mesh bags during this period. The growth of T. fibrillosa declined after 25 yr when other species such as Xerocomus spp., Russula spp. and B. pulchrum became more active. This massive growth of EM fungi in young forests suggests that this period may be important for soil C sequestration, although more research is required to clarify the residence time of EM-derived C sources in the soil.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Funding was provided by The Swedish Research Council for Environment, Agricultural Sciences and Spatial Planning (Formas), the Swedish Energy Agency and Södra’s Foundation for Research, Development and Education.

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  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information
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Supporting Information

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Table S1 Descriptions of the sites.

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NPH_3324_sm_TableS1.doc90KSupporting info item