Effects of hardened wood ash on microbial activity, plant growth and nutrient uptake by ectomycorrhizal spruce seedlings

Authors

  • Shahid Mahmood,

    1. Department of Microbial Ecology, University of Lund, Ecology Building, S-223 62 Lund, Sweden
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    • 1

      Department of Molecular and Cell Biology, Institute of Medical Sciences, University of Aberdeen, Aberdeen AB25 2ZD, UK.

  • Roger D. Finlay,

    1. Department of Forest Mycology and Pathology, Swedish University of Agricultural Sciences, Box 7026, S-750 07 Uppsala, Sweden
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  • Ann-Mari Fransson,

    1. Department of Plant Ecology, University of Lund, Ecology Building, S-223 62 Lund, Sweden
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  • Håkan Wallander

    Corresponding author
    1. Department of Microbial Ecology, University of Lund, Ecology Building, S-223 62 Lund, Sweden
      *Corresponding author. Tel.: +46 (46) 222 3759; Fax: +46 (46) 222 4158. hakan.wallander@mbioekol.lu.se
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*Corresponding author. Tel.: +46 (46) 222 3759; Fax: +46 (46) 222 4158. hakan.wallander@mbioekol.lu.se

Abstract

Plant growth, nutrient uptake, microbial biomass and activity were studied in pot systems containing spruce seedlings colonised with different ectomycorrhizal fungi from an ash-fertilised forest. The seedling root systems were enclosed in mesh bags inside an outer compartment containing crushed, hardened wood ash. Three different species of mycorrhizal fungi and a non-mycorrhizal control were exposed to factorial combinations of ash and N addition. Ash treatment had a highly significant, positive effect on plant growth and on shoot and root concentrations of K, Ca and P, irrespective of mycorrhizal status. Mycorrhizal inoculation had a significant effect on plant growth, which was proportionally greater in the absence of ash. N addition had a significant positive effect on plant biomass in mycorrhizal treatments with ash, but no effect in non-mycorrhizal treatments or most of the mycorrhizal treatments without ash. Piloderma sp. 1, which was earlier found to colonise wood ash granules in field studies, appeared to accumulate Ca from ash in the mycorrhizal roots. 5–6.7% of the total P in the ash was solubilised, with 0.9–1.5% in solution, 3.6–4.6% in the plants and 0.5–1.5% in microbial biomass. Bacterial activity as determined by [3H]-thymidine and [14C]-leucine incorporation was significantly greater in ash treatments than in controls with no ash addition. Principal component analysis (PCA) of phospholipid fatty acids (PLFAs) showed a clear difference in bacterial community structure between samples collected from ash-treated pots and controls without ash.

1Introduction

Wood ash application has been proposed as a countermeasure to soil acidification due to atmospheric deposition of pollutants or intensive harvesting of forests for bioenergy production [1–4]. Ectomycorrhizal fungi are important among soil microorganisms, as they form symbiotic associations with tree roots and assist in the uptake of nutrients [5]. It is therefore crucial to evaluate the response of ectomycorrhizal fungi to ash application and their possible ability to mobilise nutrients in the ash. In an earlier investigation of the ectomycorrhizal community structure on tree roots in a spruce forest fertilised with wood ash, we found no significant effects on individual species, although two species/ITS-types tended to increase after wood ash addition. Nucleic acid analysis based on PCR-RFLP of mycelia colonising the ash granules suggested that these two ITS-types (Piloderma sp. 1, Ha-96–3) and a third ITS-type (Tor-97–1) were able to colonise the ash granules [6]. Isolates of Piloderma sp. 1 and Ha-96–3 exhibited pronounced abilities to solubilise tricalcium phosphate and hardened wood ash in vitro [7]. In the same study, mycelia of Piloderma sp. 1 collected from ash-amended cultures contained significantly higher concentrations of P compared to Ha-96–3 or Piloderma croceum, indicating their ability to solubilise and take up P from ash. In ash, P is bound in compounds with low solubility such as apatite [8]. In another investigation, using intact symbiotic associations with spruce seedlings in laboratory microcosms, mycelia of Piloderma sp. 1 were able to colonise wood ash, whereas mycelia of P. croceum did not colonise the ash [7]. Moreover, in a competition experiment with Piloderma sp. 1 and P. croceum, colonisation of spruce roots by Piloderma sp. 1 significantly increased in the presence of wood ash, whereas colonisation by P. croceum decreased [9]. All the seedlings grown in the ash-treated substrates had a significantly higher biomass compared to the ones grown in non-ash-treated controls [9]. These experiments indicated the ability of some mycorrhizal fungi to solubilise P from wood ash and to improve plant growth, but provided no conclusive proof of nutrient transfer to plants.

The present study was designed to provide quantitative estimates of the solubilisation and transfer of nutrients from ash to mycorrhizal plants. Larger, three-dimensional growth systems and longer growth periods were used than in previous studies, in order to allow us to assess effects on plant growth in more realistic systems. In addition, to minimise effects of N limitation on the uptake of other nutrients, the experiment was conducted at two different N levels. Fungi with contrasting abilities to colonise wood ash were chosen. These fungi were isolated from mycorrhizal roots growing in an ash-fertilised spruce forest. Since microorganisms other than ectomycorrhizal fungi may also play a vital role in mobilisation of nutrients, changes in microbial biomass, activity and community structure were also investigated.

2Materials and methods

2.1Mycorrhiza synthesis

Mycorrhizal associations were synthesised between spruce (Picea abies (L.) Karst.) seedlings and five fungal isolates: Piloderma croceum Erikss. and Hjortst. (isolates no. 02, 24), and two isolates of Piloderma sp. 1 (isolates no. 35, 67) and an unidentified fungus (Ha-96–3, isolate no. 78). All of these isolates originated from mycorrhizal roots collected from a wood ash-fertilised spruce forest at Torup in southern Sweden [6,7]. Mycorrhizas were synthesised by the method described by Duddridge [10] as modified by Finlay et al. [11]. All isolates produced abundant mycorrhizal roots within three months. Only well-colonised spruce seedlings (>75% colonised root tips) were selected for this experiment.

2.2Plant growth system

Plastic pots (9×9×10 cm) were filled with a homogenised mixture (1:1 v/v) of acid-washed sand (Silversand 90, Askania, Sweden) and peat (Solmull®, Hasselfors Garden, Sweden), amended with two levels of a slow release N fertiliser (1 g l−1, low N or 2 g l−1, high N) (methylene urea, Kemira, Finland (39% N)). The elemental composition of the sand and peat is given in Table 1. The central portion of each pot (root compartment) containing the root system of each seedling and sand–peat substrate was enclosed in a nylon mesh bag (mesh size 50 μm). In half of the pots, hardened wood ash, ground to a particle size of 0.10–0.25 mm (Ljungbyverket, Sydkraft värme, Sweden, see Table 1 for elemental composition) was mixed (6 g l−1, +Ash) into the soil in the outer compartment (mycelial compartment). This amount corresponded approximately to 6 tonnes per hectare, which was the highest amount applied in the experimental forest where the ectomycorrhizal isolates were collected [6]. The soil in the other pots was left unamended (−Ash). The substrate-treatment combinations were thus: low N −Ash, high N −Ash, low N +Ash and high N +Ash. Non-mycorrhizal spruce seedlings were used as controls. There were five replicate pots for each treatment. The pots were arranged in a randomised design in a phytotron programmed for 300 μmol m−2 s−1 PAR, 80% relative humidity and an 18:6 h and 18:16°C day/night cycle. The plants were grown for 120 d before harvesting.

Table 1.  Concentrations (mg g−1) of different elements in hardened wood ash, quartz sand and peat used as growth substrate in the present experiment
  1. Values are means of two replicates. nd, not determined; –, not detectable.

SubstrateElemental concentration (mg g−1)
 AlBCaCdCrCuFeKMgMnNaNiPPbSZn
Ash11.50.42600.020.050.137.39424196.80.03170.1175.1
Quartz sand0.2nd0.08nd0.0020.0050.90.041.90.0080.020.020.01nd0.10.01
Peat0.40.042.00.0010.0011.00.21.20.030.20.0010.20.011.20.01

2.3Harvesting and analysis

Upon termination of the experiment, plants were taken out of the mesh bags and the root systems washed on a sieve under running tap water to remove soil particles. Shoots were oven-dried at 80°C for 24 h and roots were freeze-dried before weight determinations. Soils from the mycelial compartments of all pots was centrifuged for 30 min at 16270×g to collect soil solutions, which were immediately frozen at −20°C until further analysis. Sub-samples of soil from the mycelial compartments were also stored at −20°C.

2.4Elemental analysis of plant material

Elemental analysis was performed on plant material from the high N treatments only. Shoots and roots were milled, weighed and digested in 15 ml concentrated HNO3 prior to elemental (P, K, Ca, Mg) analysis by inductively coupled plasma–atomic emission spectroscopy (ICP–AES) (Perkin Elmer, CT, USA) [12]. The Kjeldahl method was used to analyse shoot N [12].

2.5Analysis of soil solution

The concentrations of PO43− and oxalate in the soil solution were determined by ion chromatography using a Varian 5000 HPLC [13]. The pH (H2O) was measured in soil solution, collected from the mycelial compartment, by using a PHR-146 micro pH electrode (Lazar Research Laboratories, USA).

2.6Thymidine and leucine incorporation in bacteria extracted from the growth substrate

One gram of soil from the mycelial compartment was homogenised in 40 ml distilled H2O by shaking for 1 h at 150 r.p.m. The soil suspension was centrifuged at 750×g for 10 min and the supernatant was filtered through glass wool. Bacterial activity in the suspension was determined by thymidine and leucine incorporation rates using methods described by Bååth [14,15]. Methyl [3H]-thymidine (200 nM) and [14C]-leucine (775 nM) were added to 2.0 ml of bacterial suspension and incubated for 2 h at 20°C. To stop the reaction, 1 ml formalin (5%) was added, and after this the suspension was filtered through a Whatman GF/F glass-fibre filter and washed with 3×5 ml ice-cold ethanol followed by 3×5 ml ice-cold tricholoroacetic acid. The filter was placed in a scintillation vial and incubated in 1 ml 0.1 M NaOH at 90°C for 1.5 h. After cooling to room temperature, 10 ml scintillation fluid was added and the vials allowed to stand for 1–2 days before scintillation counting on a Beckman LS 6500 scintillation counter.

2.7Analysis of phospholipid fatty acids

Fungal biomass and microbial community structure were analysed in the soils collected from the mycelial compartments by phospholipid fatty acid (PLFA) analysis [16–17]. In brief, 5 g of soil was extracted in 10 ml one-phase chloroform:methanol:citrate buffer (1:2:0.8 v/v/v) for 2 h. After centrifugation at 5000 r.p.m. for 10 min, the resultant pellets were washed with 4.8 ml of one-phase mixture and the supernatants were combined. The supernatants were split into two phases by adding 5 ml chloroform and 5 ml citrate buffer, and 6 ml of the lower phase were sampled and used for phospholipid analysis. The extracted lipids were fractionated into neutral lipids, glycolipids and phospholipids on silicic acid (100–200 mesh, Unisil, Clarkson, Williamsport, PA, USA) columns by eluting with chloroform, acetone and methanol. The phospholipids were subjected to a mild alkaline methanolysis [18], which transforms the phospholipids into free fatty acid methyl esters. These were analysed on a Hewlett Packard 5890 gas chromatograph with a flame ionisation detector and a 50-m HP5 capillary column. The content of PLFA 18:2ω6,9 was used to estimate fungal biomass [19], while the total content of i15:0, a5:0, i16:0, i17:0, a17:0, cy17:0, 10Me17:0, 10Me18:0 and cy19:0 was considered as an estimator of bacterial biomass [20].

2.8Analysis of microbial P

Microbial P was analysed in the soils from the mycelial compartments by fumigation extraction [21].

2.9Statistical analysis

The effect of ash on plant growth parameters was first tested in a one-way ANOVA and then separate two-way ANOVA analyses were performed on data from the +Ash and −Ash treatments to evaluate the effects of fungal inoculation and N addition on plant growth parameters. Within the high N treatment a two-way ANOVA was performed to investigate the effects of fungal inoculation and ash addition on the elemental concentrations of the seedlings. Subsequently the data on P solubilisation budgets, elemental contents of plants, microbial activity and biomass was subjected to one-way ANOVA analyses to assess the effects of −Ash and +Ash treatments separately. Least significant differences (Fisher's LSD) were used to evaluate differences between treatments. All statistical analyses were performed with the program Systat 7.0 for Windows (SPSS). PLFA composition was analysed by using principal component analysis (PCA).

3Results

3.1Plant growth data

The application of ash to the mycelial compartment of the pots had a strong positive influence (P<0.001) on the growth of the seedlings (Fig. 1, Table 2). The mycorrhizal treatment effect on growth was statistically significant (P=0.001) in both +Ash and −Ash treatments, but the proportional effect of mycorrhizal inoculation was higher in the −Ash treatments. The effect of N addition was not significant in −Ash but was highly significant in the +Ash treatments.

Figure 1.

Plant biomass (mg) of non-mycorrhizal spruce seedlings and seedlings growing in symbiosis with Piloderma sp. 1 (isolates 35 and 67), P. croceum (isolates 02 and 24) or Ha-96–3 (isolate 78). The seedlings were grown in pots containing sand–peat substrate treated with low and high levels of N; hardened wood ash was added in the root-free/mycelial compartment (+Ash) or left unamended (−Ash). a: Seedling biomass as affected by −Ash treatment under two levels of N. b: Seedling biomass as influenced by +Ash treatment under two levels of N. Vertical bars represent S.E.M. of five replicates.

Table 2.  Growth parameters of non-mycorrhizal spruce seedlings and seedlings growing in symbiosis with Piloderma sp. 1 (isolates 35 and 67), P. croceum (isolates 02 and 24) or Ha-96–3 (isolate 78)
  1. The seedlings were grown in pots containing a sand–peat substrate treated with two levels of N (low N or high N); hardened wood ash was added in the root-free/mycelial compartment (+Ash) or left unamended (−Ash). Values are means±S.E.M. of five replicates; −Ash and +Ash treatments have been tested (P<0.05, ANOVA) separately.

TreatmentsMycorrhizal statusShoot wt (mg)Root wt (mg)Root:Shoot
Low N −AshNon-mycorrhizal95±2059±170.6±0.1
 Piloderma sp. 1 (35)254±29182±300.7±0.1
 Piloderma sp. 1 (67)286±30229±140.8±0.1
 P. croceum (02)223±49168±440.7±0.1
 P. croceum (24)176±20137±140.8±0.1
 Ha-96–3 (78)159±36105±270.6±0.04
High N −AshNon-mycorrhizal105±1856±70.6±0.05
 Piloderma sp. 1 (35)383±30232±190.6±0.04
 Piloderma sp. 1 (67)327±88218±490.7±0.1
 P. croceum (02)199±26118±80.6±0.1
 P. croceum (24)233±25110±110.5±0.02
 Ha-96–3 (78)260±60150±360.6±0.05
 ANOVA (P-value)   
 Fungus<0.001<0.0010.029
 N0.031n.s.<0.001
 Fungus*Nn.s.n.s.n.s.
Low N +AshNon-mycorrhizal451±54292±220.7±0.04
 Piloderma sp. 1 (35)603±44360±100.6±0.05
 Piloderma sp. 1 (67)563±47363±240.7±0.1
 P. croceum (02)509±49313±240.6±0.1
 P. croceum (24)430±15284±100.7±0.04
 Ha-96–3 (78)405±57266±320.7±0.1
High N +AshNon-mycorrhizal514±25305±180.6±0.04
 Piloderma sp. 1 (35)722±66400±320.6±0.1
 Piloderma sp. 1 (67)826±70435±180.5±0.1
 P. croceum (02)605±58397±90.7±0.1
 P. croceum (24)571±48360±250.6±0.03
 Ha-96–3 (78)557±57398±500.7±0.1
 ANOVA (P-value)
 Fungus<0.0010.002n.s.
 N<0.001>0.001n.s.
 Fungus*Nn.s.n.s.n.s.

3.2Elemental analysis of roots and shoots

All the seedlings grown in +Ash pots showed higher concentrations of P and K in roots and shoots compared to the −Ash controls (Table 3). All seedlings from the −Ash controls had shoot P concentrations below the critical level, which indicates that P is limiting growth (1.1–1.3 mg g−1, [12]). In these controls mycorrhizal inoculation had no effect on shoot Ca and Mg concentrations either. Only non-mycorrhizal seedlings in the −Ash controls had shoot K concentrations below the critical level, which indicates that K is limiting growth (3.5–4.0 mg g−1, [12]). In +Ash treatments, no clear trends could be noticed in the elemental concentrations of mycorrhizal or non-mycorrhizal seedlings. Shoot N concentrations were higher in seedlings grown in −Ash pots compared to those in +Ash treatments, which is probably an effect of the accumulation of excess N in the −Ash treatment where P or K limited growth. Roots of mycorrhizal seedlings had higher Ca, K, Mg and P concentrations in the −Ash controls compared to the non-mycorrhizal seedlings (Table 3). In the +Ash treatments the only element that was influenced by mycorrhizal inoculation was Ca. Concentrations of this element in the roots were higher in all mycorrhizal treatments, especially for Piloderma sp. 1.

Table 3.  Elemental concentrations (mg g−1) of macro-nutrients in non-mycorrhizal spruce seedlings and seedlings growing in symbiosis with Piloderma sp. 1 (67), P. croceum (02) or Ha-96–3 (78)
  1. The seedlings were grown in pots containing sand–peat substrate treated with high level of N; hardened wood ash was added in the root-free/mycelial compartment (+Ash) or left unamended (−Ash). Values are means±S.E.M. of five replicates. Different letters denote differences among means (P<0.05, ANOVA).

TreatmentsMycorrhizal statusElemental concentration mg g−1
  P shootP rootK shootK rootCa shootCa rootMg shootMg rootN shoot
High N −AshNon-mycorrhizal0.7±0.03 cd0.4±0.01 d2.3±0.3 f1.1±0.06 e1.9±0.2 c0.6±0.06 e1.1±0.1 a0.4±0.02 d37±2.6 a
 Piloderma sp. 1 (67)0.5±0.07 bc0.8±0.04 c4.3±0.4 d4.1±0.5 d1.9±0.2 c1.8±0.1 d1.2±0.1 a1.4±0.1 b24±2.4 b
 P. croceum (02)0.7±0.1 cd0.9±0.06 c3.2±0.4 e4.1±0.3 d2.3±0.4 c1.5±0.07 d1.2±0.2 a1.3±0.09 bc32±2.0 a
 Ha-96–3 (78)0.5±0.02 bc0.9±0.03 c3.9±0.4 de4.0±0.5 d1.7±0.3 c1.6±0.1 d1.2±0.1 a1.4±0.2 bc25±3.4 b
High N +AshNon-mycorrhizal1.3±0.1 a2.4±0.2 a9.8±0.8 b13.4±0.8 a3.8±0.7 ab2.9±0.1 c0.6±0.08 c1.1±0.08 c7.2±0.3 c
 Piloderma sp. 1 (67)1.0±0.05 a1.5±0.03 b7.6±0.4 c9.5±0.5 c2.9±0.3 bc5.1±0.4 a0.6±0.06 c1.5±0.2 b6.2±0.2 c
 P. croceum (02)1.2±0.1 a1.4±0.1 b10.9±0.3 b11.9±0.7 b4.2±0.4 a3.9±0.2 b0.8±0.09 b1.8±0.1 a6.5±0.9 c
 Ha-96–3 (78)1.1±0.1 a1.4±0.09 b12.4±0.9 a11.5±0.6 b3.5±0.5 ab3.4±0.05 c1.1±0.07 a1.4±0.06 b6.7±0.4 c
 ANOVA (P-value)
 Ash<0.001<0.001<0.001<0.001<0.001<0.001<0.001<0.0010.02
 Fungus0.060.02<0.001n.s.n.s.<0.0010.06<0.001<0.001
 Ash*Fungusn.s<0.001<0.001<0.001n.s.0.001n.s.0.010.06
 Critical levels
 Thelin et al. [12]1.1–1.33.5–4.00.4–2.00.4–0.712–13

Total plant contents of K, Ca Mg and P were increased by ash addition (Fig. 2). In the absence of ash, mycorrhizal inoculation increased the contents of these elements in all cases (Fig. 2). Under ash conditions the mycorrhizal effect was less pronounced, but P. croceum increased the contents of Ca, K and Mg.

Figure 2.

Elemental content (mg plant−1) of macro-nutrients in non-mycorrhizal spruce seedlings and seedlings growing in symbiosis with Piloderma sp. 1 (67), P. croceum (02) or Ha-96–3 (78). The seedlings were grown in pots containing sand–peat substrate treated with a high level of N; hardened wood ash was added in the root-free/mycelial compartment (+Ash) or left unamended (−Ash). a–d: Plant contents of Ca (a), K (b), Mg (c) and P (d). Vertical bars represent S.E.M. of five replicates. Different letters denote differences among means (P<0.05, ANOVA); −Ash and +Ash treatments have been tested separately.

3.3Soil solution properties

At the harvest, soil solution pH values for +Ash and −Ash treatments were 6.4±0.1 and 4.5±0.1, respectively. The concentration of PO43− in solution extracted from the compartment colonised by mycelia of Piloderma sp. 1 in the +Ash treatment was higher compared to the all other treatments (P=0.009, Fig. 3). PO43− concentrations were extremely low in solution from all −Ash controls (Fig. 3). Oxalate concentration in the soil solution varied between 1–6 μmol l−1, with no differences among treatments.

Figure 3.

Phosphate concentration (μmol l−1) in the soil solution collected from the root-free/mycelial compartments of non-mycorrhizal spruce seedlings and seedlings growing in symbiosis with Piloderma sp. 1 (67), P. croceum (02) or Ha-96–3 (78). The seedlings were grown in pots containing sand–peat substrate treated with a high level of N; hardened wood ash was added in the root-free/mycelial compartment (+Ash) or left unamended (−Ash). Vertical bars represent S.E.M. of five replicates. Different letters denote differences among means (P<0.05, ANOVA); −Ash and +Ash treatments have been tested separately.

3.4P budgets

The data on overall P budgets suggested that only a small fraction of P (5–6.7%) was solubilised from the ash at the time of harvest (Table 4). This amount of solubilised P is composed of microbial P, which constituted 0.5–1.5% of the total pool, the plants, which contained 3.6–4.6%, and the soil solution, which contained 0.9–1.5% of the P originally present in the ash.

Table 4.  P solubilisation budgets for pots with non-mycorrhizal spruce seedlings and seedlings growing in symbiosis with Piloderma sp. 1 (67), P. croceum (02) or Ha-96–3 (78)
  1. The seedlings were grown in pots containing sand–peat substrate treated with high level of N; hardened wood ash was added in the root-free/mycelial compartment (+Ash) or left unamended (−Ash). The budgets are based on total amount of P (mg) solubilised in each treatment and the fractions (%) found in soil solution, microbial and plant biomass. Values are means±S.E.M. of five replicates. Different letters denote differences among means (P<0.05, ANOVA); −Ash and +Ash treatments have been tested separately.

TreatmentsMycorrhizal statusAmount of P (mg) solubilisedFraction of P (%) in
   soil solutionmicrobial biomassplant biomass
High N −AshNon-mycorrhizal0.2±0.02 a0.03±0.005 ab0.3±0.02 a0.3±0.1 a
 Piloderma sp. 1 (67)0.6±0.09 b0.02±0.004 a0.5±0.1 b1.3±0.3 b
 P. croceum (02)0.5±0.04 b0.05±0.01 b0.5±0.04 b0.9±0.1 b
 Ha-96–3 (78)0.4±0.06 b0.03±0.01 ab0.5±0.04 b0.8±0.2 b
High N +AshNon-mycorrhizal2.2±0.2 y0.9±0.1 x1.5±0.4 y4.3±0.2 y
 Piloderma sp. 1 (67)2.2±0.06 y1.5±0.04 y0.8±0.2 x4.4±0.1 y
 P. croceum (02)2.1±0.1 y1.1±0.03 x0.7±0.2 x4.6±0.3 y
 Ha-96–3 (78)1.7±0.1 x0.9±0.1 x0.5±0.05 x3.6±0.3 x

3.5Microbial biomass and activity

Bacterial activity as determined by [3H]-thymidine and [14C]-leucine incorporation was higher (P<0.001 for both isotopes) in substrates collected from the mycelial compartments of +Ash pots than in −Ash pots (Table 5). However, there was no statistically significant effect of mycorrhizal inoculation on bacterial activity.

Table 5.  Microbial activity and estimates of bacterial and fungal biomass in the root-free/mycelial compartments of pots with non-mycorrhizal spruce seedlings and seedlings growing in symbiosis with Piloderma sp. 1 (67), P. croceum (02) or Ha-96–3 (78)
  1. The seedlings were grown for 120 d in sand–peat substrate treated with high level of N; hardened wood ash was added in the root-free/mycelial compartment (+Ash) or left unamended (−Ash). Microbial activity and fungal biomass increased significantly (P<0.001, ANOVA) in response to +Ash. There was no significant effect of hardened wood ash or ectomycorrhizal inoculation on bacterial biomass. Values are means±S.E.M. of five replicates; −Ash and +Ash treatments have been tested (P<0.05, ANOVA) separately.

  2. aExpressed as incorporation of [3H]-thymidine and [14C]-leucine.

  3. bEstimate of sum of PLFAs of bacterial origin.

  4. cTotal amount of PLFA 18:2ω6,9.

TreatmentsMycorrhizal statusMicrobial activityaBacterial biomassb (nmol g−1)Fungal biomassc (nmol g−1)
  [3H]-thymidine d.p.m.[14C]-leucine d.p.m.  
High N −AshNon-mycorrhizal4×103±0.3×1033×103±0.2×1035.9±0.50.4±0.05
 Piloderma sp. 1 (67)5×103±1.0×1033×103±0.8×1036.0±0.70.5±0.1
 P. croceum (02)5×103±0.2×1033×103±0.2×1035.7±0.10.4±0.03
 Ha-96–3 (78)4×103±0.8×1033×103±0.6×1035.4±0.40.3±0.03
High N +AshNon-mycorrhizal15×103±2×10312×103±0.2×1036.0±0.71.1±0.2
 Piloderma sp. 1 (67)25×103±2×10318×103±0.2×1036.8±0.41.3±0.3
 P. croceum (02)18×103±4×10313×103±0.3×1034.9±0.50.6±0.1
 Ha-96–3 (78)22×103±4×10317×103±0.3×1034.7±0.40.8±0.1

Abundant mycelia resembling the inoculated mycorrhizal fungi were observed in all inoculated pots at harvest but never in the non-mycorrhizal controls. However, no statistical differences between non-mycorrhizal and mycorrhizal treatments were found when analysing the concentration of PLFAs of the soil. Fungal biomass significantly increased in response to ash addition (P<0.001, Table 5).

No clear effect of ash or mycorrhizal inoculation treatment was evident on total bacterial PLFA concentrations (Table 5). The ratio between fungal and bacterial PLFAs in +Ash and −Ash pots was 0.06–0.08 and 0.04–0.07, respectively. PCA of the bacterial PLFAs showed a distinct separation between samples collected from +Ash mycelial compartments and those from −Ash controls (Fig. 4). The following PLFAs increased after ash addition: 16:1ω5, cy17:0 and a15:0, while the following PLFAs decreased after ash addition: a17:0, i17:0, i17:0, i15:0, 10me17:0.

Figure 4.

PCA using frequencies of bacterial PLFAs originating from soils collected from the root-free/mycelial compartments of non-mycorrhizal spruce seedlings and seedlings growing in symbiosis with Piloderma sp. 1 (67), P. croceum (02) or Ha-96–3 (78). The seedlings were grown in pots containing sand–peat substrate treated with a high level of N; hardened wood ash was added in the root-free/mycelial compartment (+Ash) or left unamended (−Ash). Bars represent S.E.M. of five replicates.

4Discussion

In the present study, addition of ash enhanced growth of spruce seedlings, irrespective of mycorrhizal status. This was clearly an effect of K and P added with the ash, since concentrations of these elements were below critical levels in the seedlings in the −Ash treatment (Table 3, [12]). Improved plant growth in response to ash additions has been reported previously in both field and laboratory studies [22–24]. Application of wood ash to peatlands normally improves tree growth more than application to mineral soils [25]. The reason could be that peatlands have relatively low concentrations of P and K compared to the mineral soils. Re-fertilisation of peat soils with K is probably more important than fertilisation with P during the post-fertilisation period [26].

Mycorrhizal inoculation had the most pronounced effect on plant biomass in the −Ash treatment. This was probably an effect of uptake of K from the mycelial compartment by the external mycelium of mycorrhizal plants. Non-mycorrhizal seedlings in the same treatment were unable to capture nutrients from the root free compartment and consequently had a very low K concentration, indicating that K was the main element limiting their growth. Although plant concentrations of Ca and Mg were apparently above the critical deficiency levels, there was a positive mycorrhizal effect on the uptake of these elements. These results confirm the potential role of ectomycorrhizal mycelia on the uptake of K and Mg [27–28]. In the +Ash treatment, levels of these elements were above the critical deficiency level in all plants, including the non-mycorrhizal controls; however, shoots of all seedlings had very low concentrations of N, indicating that N had become the limiting nutrient by the time the plants were harvested.

Most of the elements in wood ash are readily soluble, except P, which is bound in apatite-like compounds with low solubility [8]. However, the concentration of PO43− in the solution collected from the +Ash treatment was high compared with published field estimates [29]. This demonstrates that the release of P from the ash must have been sufficient for plant growth. Higher PO43− concentrations were found in substrates colonised by Piloderma sp. 1 mycelia in the +Ash treatment compared to other treatments, indicating that Piloderma sp. 1 stimulated P release from the ash. However, there was no indication that the fungus transported more P to the plant than other mycorrhizal fungi or in non-mycorrhizal controls, confirming the results of Mahmood et al. [7], obtained using smaller microcosm systems.

Arvidsson and Lundkvist [30] estimated nutrient concentrations in spruce needles in forests treated with hardened wood ash within a range of climate and fertility gradients in Sweden and found consistently higher P, K and Ca concentrations five years after the treatment. The initial P concentrations in tree needles from plots treated with 6 tonnes ha−1 granulated wood ash at Torup (the site where the mycorrhizal fungi used in the present study were isolated) were markedly higher compared to the untreated control. Data on the tree growth after four consecutive growing seasons (5 yr) also showed better growth increments in the 6 tonnes ha−1 granulated wood ash treatment compared to the control or 3 tonnes ha−1 treatments (S. Jacobson, personal communication). Efficient mycorrhizal mycelial uptake and transport of P, which has already been solubilised by Piloderma sp. 1 or other microorganisms, would contribute to this improved growth.

In contrast to the present finding, Wallander et al. [31] and Wallander [32] reported the superior ability of pine seedlings colonised by some ectomycorrhizal fungi to use apatite as a P source compared to non-mycorrhizal seedlings. This discrepancy may be explained by the relatively higher amounts of apatite available for solubilisation in Wallander's experiments. The release of P from the ash in our experiments may have been caused by exudation of oxalate by fungi, bacteria or roots. Griffiths et al. [29] reported significantly higher concentrations of PO43− and oxalate in soil solution of soils colonised by mat-forming ectomycorrhizal fungi compared to non-mat soils. In a previous in vitro study [7], Piloderma sp. 1 demonstrated pronounced ability to solubilise hardened wood ash by formation of abundant calcium oxalate crystals when grown on ash-amended culture medium. In the same study, the concentration of P in the mycelium of Piloderma sp. 1 was higher compared to P. croceum or Ha-96–3. In the present study we found low oxalate concentration in the soil solution in all treatments (1–6 μmol l−1 soil solution), which is probably a result of precipitation of calcium oxalate in the presence of ash. It is likely that an instant estimate of oxalate concentration does not reflect the dynamics of oxalate exudation into the soil solution.

In base-poor forest ecosystems at the Hubbard Brook experimental forest, New Hampshire, USA, ectomycorrhizal fungi were found to be important in supplying Ca to the trees from apatite sources in the soil [33]. In the present study Piloderma sp. 1 appeared to transport Ca from the ash to the roots, since the roots colonised by this fungus had the highest Ca concentrations among the tested plants when grown in the presence of ash. Only a minor portion of this Ca appeared to be transferred to the plants, since the Ca concentrations in the shoots were lower in plants colonised by Piloderma sp. 1 than in other plants. This suggests that the colonization of wood ash by Piloderma sp. 1, found both in laboratory [7] and in field [6] studies, is stimulated by Ca rather than P in the ash. The ecological implications of ash-colonising fungi may be that the fungus selectively removes Ca from the ash while P is released into the soil solution. This P will become available to other organisms in the soil, including species of ectomycorrhizal fungi that may transport P to their host trees. Ca taken up by the ash-colonising fungi is likely to precipitate as calcium oxalate on fungal hyphae which, apart from being a result of mineral dissolution, has also been suggested to protect the hyphae from microbial attack and predation by soil animals [34]. According to Snetselaar and Whitney [35], fungi accumulate calcium oxalate to avoid potential Ca and oxalate toxicity and, furthermore, Jennings [36] has suggested that calcium oxalate regulates cytoplasmic pH as well as being a structural material of hyphae.

Analysis of the soil from the Torup experimental forest shows an increase in the concentration of base cations (Ca, Mg, K) in the ash-fertilised plots (S. Jacobson, personal communication). However, the dissolution of P from ash granules seems to be slow, as indicated by very low concentrations of PO43− in the soil solution (S. Jacobson, personal communication). A recent elemental analysis of the ash granules collected from the site at Torup showed presence of P in substantial amounts as long as seven years after their field application, whereas most of the other elements had been leached out in the soil over this period (J. Bergholm, personal communication). Similarly, in the present study, only a small fraction (5.0–6.7%) of P was weathered from the ash and a large pool of P (93% of the total P supplied) was still bound in the ash at the time of harvest.

A significant increase in microbial activity, measured by [3H]-thymidine and [14C]-leucine incorporation, in response to ash treatment could have been due to an ash-induced pH increase in the substrate. Using a similar method, Bååth et al. [20] reported a 1.6-fold increase in bacterial activity in a site polluted with alkaline dust (pH 6.6) over that in the control site (pH 4.13). Soil amendments with certain primary minerals may also cause an increase in bacterial activity and the presence of ectomycorrhizal mycelia may also have a positive or negative influence on bacterial activity [16]. However, in our study, presence of ectomycorrhizal mycelia had no statistically significant effect on bacterial activity.

The high background levels of fungal biomass measured in non-mycorrhizal controls are probably an effect of high amounts of fungal PLFAs in the peat used as growth substrate and makes it difficult to draw conclusions concerning the effects of ash treatment on mycorrhizal mycelia. No effects on total bacterial biomass could be established using bacterial PLFA concentrations either.

Bååth et al. [37] reported a reduction in microbial biomass (total PLFAs) and a decreased index of fungal:bacterial PLFAs, indicating a larger reduction of fungi than bacteria due to the highest rate of wood ash fertilisation. In the present study, +Ash treatment affected neither biomass nor the fungal:bacterial ratio. However, in contrast to the findings of Fritze et al. [38], who reported no change in the level of fungal ergosterol in response to ash application, we found an increase in fungal biomass (PLFA 18:2ω6,9) in +Ash treatments. The change in composition of bacterial PLFAs following ash treatment in the present experiment was similar to the change found by Bååth et al. [37] after wood ash fertilization of a forest soil.

High microbial activity, biomass and P in the root-free compartment of non-mycorrhizal treatments strongly suggest that microbes other than ectomycorrhizal fungi may also contribute to the mobilisation of nutrients in ash. Clarholm [2] has emphasised the importance of microbial utilisation of P derived from wood ash. Further studies are needed to investigate the role of different ectomycorrhizal mycelia in capturing nutrients already mobilised from ash and transporting them to the host plants.

Acknowledgements

This work was financed by the Swedish National Energy Administration (STEM).

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