E¡ects of hardened wood ash on microbial activity, plant growthand nutrient uptake by ectomycorrhizal spruce seedlings

Shahid Mahmood a;1, Roger D. Finlay b, Ann-Mari Fransson c, Hafikan Wallander a;�

a Department of Microbial Ecology, University of Lund, Ecology Building, S-223 62 Lund, Swedenb Department of Forest Mycology and Pathology, Swedish University of Agricultural Sciences, Box 7026, S-750 07 Uppsala, Sweden

c Department of Plant Ecology, University of Lund, Ecology Building, S-223 62 Lund, Sweden

Received 28 May 2002; received in revised form 22 August 2002; accepted 24 August 2002

First published online 19 September 2002

Abstract

Plant growth, nutrient uptake, microbial biomass and activity were studied in pot systems containing spruce seedlings colonised withdifferent ectomycorrhizal fungi from an ash-fertilised forest. The seedling root systems were enclosed in mesh bags inside an outercompartment containing crushed, hardened wood ash. Three different species of mycorrhizal fungi and a non-mycorrhizal control wereexposed to factorial combinations of ash and N addition. Ash treatment had a highly significant, positive effect on plant growth and onshoot and root concentrations of K, Ca and P, irrespective of mycorrhizal status. Mycorrhizal inoculation had a significant effect on plantgrowth, which was proportionally greater in the absence of ash. N addition had a significant positive effect on plant biomass inmycorrhizal 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 themycorrhizal 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% inmicrobial biomass. Bacterial activity as determined by [3H]-thymidine and [14C]-leucine incorporation was significantly greater in ashtreatments than in controls with no ash addition. Principal component analysis (PCA) of phospholipid fatty acids (PLFAs) showed a cleardifference in bacterial community structure between samples collected from ash-treated pots and controls without ash.? 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.

Keywords: Ectomycorrhizal fungi ; Wood ash; Spruce; Nutrient uptake; Bacterium; Phospholipid fatty acid; [3H]-thymidine incorporation; [14C]-leucineincorporation

1. Introduction

Wood ash application has been proposed as a counter-measure to soil acidi¢cation due to atmospheric depositionof pollutants or intensive harvesting of forests for bioen-ergy production [1^4]. Ectomycorrhizal fungi are impor-tant among soil microorganisms, as they form symbioticassociations with tree roots and assist in the uptake ofnutrients [5]. It is therefore crucial to evaluate the responseof ectomycorrhizal fungi to ash application and their pos-sible ability to mobilise nutrients in the ash. In an earlier

investigation of the ectomycorrhizal community structureon tree roots in a spruce forest fertilised with wood ash,we found no signi¢cant e¡ects on individual species,although two species/ITS-types tended to increase afterwood ash addition. Nucleic acid analysis based on PCR-RFLP of mycelia colonising the ash granules suggestedthat these two ITS-types (Piloderma sp. 1, Ha-96-3) anda third ITS-type (Tor-97-1) were able to colonise the ashgranules [6]. Isolates of Piloderma sp. 1 and Ha-96-3 ex-hibited pronounced abilities to solubilise tricalcium phos-phate and hardened wood ash in vitro [7]. In the samestudy, mycelia of Piloderma sp. 1 collected from ash-amended cultures contained signi¢cantly higher concentra-tions of P compared to Ha-96-3 or Piloderma croceum,indicating their ability to solubilise and take up P fromash. In ash, P is bound in compounds with low solubilitysuch as apatite [8]. In another investigation, using intactsymbiotic associations with spruce seedlings in laboratory

0168-6496 / 02 / $22.00 ? 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.PII: S 0 1 6 8 - 6 4 9 6 ( 0 2 ) 0 0 3 8 0 - X

* Corresponding author. Tel. : +46 (46) 222 3759;Fax: +46 (46) 222 4158.E-mail address: hakan.wallander@mbioekol.lu.se (H. Wallander).

1 Present address: Department of Molecular and Cell Biology, Instituteof Medical Sciences, University of Aberdeen, Aberdeen AB25 2ZD, UK.

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microcosms, mycelia of Piloderma sp. 1 were able to col-onise wood ash, whereas mycelia of P. croceum did notcolonise the ash [7]. Moreover, in a competition experi-ment with Piloderma sp. 1 and P. croceum, colonisation ofspruce roots by Piloderma sp. 1 signi¢cantly increased inthe presence of wood ash, whereas colonisation by P. cro-ceum decreased [9]. All the seedlings grown in the ash-treated substrates had a signi¢cantly higher biomass com-pared to the ones grown in non-ash-treated controls [9].These experiments indicated the ability of some mycorrhi-zal fungi to solubilise P from wood ash and to improveplant growth, but provided no conclusive proof of nutrienttransfer to plants.

The present study was designed to provide quantitativeestimates of the solubilisation and transfer of nutrientsfrom ash to mycorrhizal plants. Larger, three-dimensionalgrowth systems and longer growth periods were used thanin previous studies, in order to allow us to assess e¡ects onplant growth in more realistic systems. In addition, tominimise e¡ects of N limitation on the uptake of othernutrients, the experiment was conducted at two di¡erentN levels. Fungi with contrasting abilities to colonise woodash were chosen. These fungi were isolated from mycor-rhizal roots growing in an ash-fertilised spruce forest.Since microorganisms other than ectomycorrhizal fungimay also play a vital role in mobilisation of nutrients,changes in microbial biomass, activity and communitystructure were also investigated.

2. Materials and methods

2.1. Mycorrhiza synthesis

Mycorrhizal associations were synthesised betweenspruce (Picea abies (L.) Karst.) seedlings and ¢ve fungalisolates: Piloderma croceum Erikss. and Hjortst. (isolatesno. 02, 24), and two isolates of Piloderma sp. 1 (isolatesno. 35, 67) and an unidenti¢ed fungus (Ha-96-3, isolateno. 78). All of these isolates originated from mycorrhizalroots collected from a wood ash-fertilised spruce forest atTorup in southern Sweden [6,7]. Mycorrhizas were synthe-sised by the method described by Duddridge [10] as modi-¢ed by Finlay et al. [11]. All isolates produced abundantmycorrhizal roots within three months. Only well-colon-

ised spruce seedlings (s 75% colonised root tips) wereselected for this experiment.

2.2. Plant growth system

Plastic pots (9U9U10 cm) were ¢lled with a homoge-nised mixture (1:1 v/v) of acid-washed sand (Silversand 90,Askania, Sweden) and peat (Solmull0, Hasselfors Garden,Sweden), amended with two levels of a slow release Nfertiliser (1 g l31, low N or 2 g l31, high N) (methyleneurea, Kemira, Finland (39% N)). The elemental composi-tion of the sand and peat is given in Table 1. The centralportion of each pot (root compartment) containing theroot system of each seedling and sand^peat substratewas enclosed in a nylon mesh bag (mesh size 50 Wm). Inhalf of the pots, hardened wood ash, ground to a particlesize of 0.10^0.25 mm (Ljungbyverket, Sydkraft va«rme,Sweden, see Table 1 for elemental composition) was mixed(6 g l31, +Ash) into the soil in the outer compartment(mycelial compartment). This amount corresponded ap-proximately to 6 tonnes per hectare, which was the highestamount applied in the experimental forest where the ecto-mycorrhizal isolates were collected [6]. The soil in the oth-er pots was left unamended (3Ash). The substrate-treat-ment combinations were thus: low N 3Ash, high N3Ash, low N +Ash and high N +Ash. Non-mycorrhizalspruce seedlings were used as controls. There were ¢vereplicate pots for each treatment. The pots were arrangedin a randomised design in a phytotron programmed for300 Wmol m32 s31 PAR, 80% relative humidity and an18:6 h and 18:16‡C day/night cycle. The plants weregrown for 120 d before harvesting.

2.3. Harvesting and analysis

Upon termination of the experiment, plants were takenout of the mesh bags and the root systems washed on asieve under running tap water to remove soil particles.Shoots were oven-dried at 80‡C for 24 h and roots werefreeze-dried before weight determinations. Soils from themycelial compartments of all pots was centrifuged for 30min at 16270Ug to collect soil solutions, which were im-mediately frozen at 320‡C until further analysis. Sub-sam-ples of soil from the mycelial compartments were alsostored at 320‡C.

Table 1Concentrations (mg g31) of di¡erent elements in hardened wood ash, quartz sand and peat used as growth substrate in the present experiment

Substrate Elemental concentration (mg g31)

Al B Ca Cd Cr Cu Fe K Mg Mn Na Ni P Pb S Zn

Ash 11.5 0.4 260 0.02 0.05 0.13 7.3 94 24 19 6.8 0.03 17 0.1 17 5.1Quartz sand 0.2 nd 0.08 nd 0.002 0.005 0.9 0.04 1.9 0.008 0.02 0.02 0.01 nd 0.1 0.01Peat 0.4 0.04 2.0 ^ 0.001 0.001 1.0 0.2 1.2 0.03 0.2 0.001 0.2 0.01 1.2 0.01

Values are means of two replicates. nd, not determined; ^, not detectable.

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2.4. Elemental analysis of plant material

Elemental analysis was performed on plant materialfrom the high N treatments only. Shoots and roots weremilled, weighed and digested in 15 ml concentrated HNO3

prior to elemental (P, K, Ca, Mg) analysis by inductivelycoupled plasma^atomic emission spectroscopy (ICP^AES)(Perkin Elmer, CT, USA) [12]. The Kjeldahl method wasused to analyse shoot N [12].

2.5. Analysis of soil solution

The concentrations of PO334 and oxalate in the soil so-

lution were determined by ion chromatography using aVarian 5000 HPLC [13]. The pH (H2O) was measured insoil solution, collected from the mycelial compartment, byusing a PHR-146 micro pH electrode (Lazar ResearchLaboratories, USA).

2.6. Thymidine and leucine incorporation in bacteriaextracted from the growth substrate

One gram of soil from the mycelial compartment washomogenised in 40 ml distilled H2O by shaking for 1 h at150 r.p.m. The soil suspension was centrifuged at 750Ugfor 10 min and the supernatant was ¢ltered through glasswool. Bacterial activity in the suspension was determinedby thymidine and leucine incorporation rates using meth-ods described by Bafiafith [14,15]. Methyl [3H]-thymidine(200 nM) and [14C]-leucine (775 nM) were added to 2.0ml of bacterial suspension and incubated for 2 h at 20‡C.To stop the reaction, 1 ml formalin (5%) was added, andafter this the suspension was ¢ltered through a WhatmanGF/F glass-¢bre ¢lter and washed with 3U5 ml ice-coldethanol followed by 3U5 ml ice-cold tricholoroacetic acid.The ¢lter was placed in a scintillation vial and incubatedin 1 ml 0.1 M NaOH at 90‡C for 1.5 h. After cooling toroom temperature, 10 ml scintillation £uid was added andthe vials allowed to stand for 1^2 days before scintillationcounting on a Beckman LS 6500 scintillation counter.

2.7. Analysis of phospholipid fatty acids

Fungal biomass and microbial community structurewere analysed in the soils collected from the mycelial com-partments by phospholipid fatty acid (PLFA) analysis [16^17]. In brief, 5 g of soil was extracted in 10 ml one-phasechloroform:methanol:citrate bu¡er (1:2:0.8 v/v/v) for 2 h.After centrifugation at 5000 r.p.m. for 10 min, the resul-tant pellets were washed with 4.8 ml of one-phase mixtureand the supernatants were combined. The supernatantswere split into two phases by adding 5 ml chloroformand 5 ml citrate bu¡er, and 6 ml of the lower phasewere sampled and used for phospholipid analysis. Theextracted lipids were fractionated into neutral lipids, gly-colipids and phospholipids on silicic acid (100^200 mesh,

Unisil, Clarkson, Williamsport, PA, USA) columns byeluting with chloroform, acetone and methanol. The phos-pholipids were subjected to a mild alkaline methanolysis[18], which transforms the phospholipids into free fattyacid methyl esters. These were analysed on a HewlettPackard 5890 gas chromatograph with a £ame ionisationdetector and a 50-m HP5 capillary column. The content ofPLFA 18:2g6,9 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 consideredas an estimator of bacterial biomass [20].

2.8. Analysis of microbial P

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

2.9. Statistical analysis

The e¡ect of ash on plant growth parameters was ¢rsttested in a one-way ANOVA and then separate two-wayANOVA analyses were performed on data from the +Ashand 3Ash treatments to evaluate the e¡ects of fungal in-oculation and N addition on plant growth parameters.Within the high N treatment a two-way ANOVA wasperformed to investigate the e¡ects of fungal inoculationand ash addition on the elemental concentrations of theseedlings. Subsequently the data on P solubilisation bud-gets, elemental contents of plants, microbial activity andbiomass was subjected to one-way ANOVA analyses toassess the e¡ects of 3Ash and +Ash treatments separately.Least signi¢cant di¡erences (Fisher’s LSD) were used toevaluate di¡erences between treatments. All statisticalanalyses were performed with the program Systat 7.0 forWindows (SPSS). PLFA composition was analysed by us-ing principal component analysis (PCA).

3. Results

3.1. Plant growth data

The application of ash to the mycelial compartment ofthe pots had a strong positive in£uence (P6 0.001) on thegrowth of the seedlings (Fig. 1, Table 2). The mycorrhizaltreatment e¡ect on growth was statistically signi¢cant(P= 0.001) in both +Ash and 3Ash treatments, but theproportional e¡ect of mycorrhizal inoculation was higherin the 3Ash treatments. The e¡ect of N addition was notsigni¢cant in 3Ash but was highly signi¢cant in the +Ashtreatments.

3.2. Elemental analysis of roots and shoots

All the seedlings grown in +Ash pots showed higherconcentrations of P and K in roots and shoots compared

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to the 3Ash controls (Table 3). All seedlings from the3Ash controls had shoot P concentrations below the crit-ical level, which indicates that P is limiting growth (1.1^1.3mg g31, [12]). In these controls mycorrhizal inoculationhad no e¡ect on shoot Ca and Mg concentrations either.Only non-mycorrhizal seedlings in the 3Ash controls hadshoot K concentrations below the critical level, which in-dicates that K is limiting growth (3.5^4.0 mg g31, [12]). In+Ash treatments, no clear trends could be noticed in theelemental concentrations of mycorrhizal or non-mycorrhi-zal seedlings. Shoot N concentrations were higher in seed-lings grown in 3Ash pots compared to those in +Ashtreatments, which is probably an e¡ect of the accumula-tion of excess N in the 3Ash treatment where P or Klimited growth. Roots of mycorrhizal seedlings had higherCa, K, Mg and P concentrations in the 3Ash controlscompared to the non-mycorrhizal seedlings (Table 3). Inthe +Ash treatments the only element that was in£uencedby mycorrhizal inoculation was Ca. Concentrations of thiselement in the roots were higher in all mycorrhizal treat-ments, especially for Piloderma sp. 1.

Total plant contents of K, Ca Mg and P were increasedby ash addition (Fig. 2). In the absence of ash, mycorrhi-

zal inoculation increased the contents of these elements inall cases (Fig. 2). Under ash conditions the mycorrhizale¡ect was less pronounced, but P. croceum increased thecontents of Ca, K and Mg.

3.3. Soil solution properties

At the harvest, soil solution pH values for +Ash and3Ash treatments were 6.4 V 0.1 and 4.5 V 0.1, respectively.The concentration of PO33

4 in solution extracted from thecompartment colonised by mycelia of Piloderma sp. 1 inthe +Ash treatment was higher compared to the all othertreatments (P= 0.009, Fig. 3). PO33

4 concentrations wereextremely low in solution from all 3Ash controls (Fig. 3).Oxalate concentration in the soil solution varied between1^6 Wmol l31, with no di¡erences among treatments.

3.4. P budgets

The data on overall P budgets suggested that only asmall fraction of P (5^6.7%) was solubilised from theash at the time of harvest (Table 4). This amount of solu-bilised P is composed of microbial P, which constituted

Fig. 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 (3Ash). a: Seedling biomass as a¡ected by 3Ash treat-ment under two levels of N. b: Seedling biomass as in£uenced by +Ash treatment under two levels of N. Vertical bars represent S.E.M. of ¢ve repli-cates.

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Table 2Growth parameters of non-mycorrhizal spruce seedlings and seedlings growing in symbiosis with Piloderma sp. 1 (isolates 35 and 67), P. croceum (iso-lates 02 and 24) or Ha-96-3 (isolate 78)

Treatments Mycorrhizal status Shoot wt (mg) Root wt (mg) Root:Shoot

Low N 3Ash Non-mycorrhizal 95 V 20 59 V 17 0.6 V 0.1Piloderma sp. 1 (35) 254 V 29 182 V 30 0.7 V 0.1Piloderma sp. 1 (67) 286 V 30 229 V 14 0.8 V 0.1P. croceum (02) 223 V 49 168 V 44 0.7 V 0.1P. croceum (24) 176 V 20 137 V 14 0.8 V 0.1Ha-96-3 (78) 159 V 36 105 V 27 0.6 V 0.04

High N 3Ash Non-mycorrhizal 105 V 18 56 V 7 0.6 V 0.05Piloderma sp. 1 (35) 383 V 30 232 V 19 0.6 V 0.04Piloderma sp. 1 (67) 327 V 88 218 V 49 0.7 V 0.1P. croceum (02) 199 V 26 118 V 8 0.6 V 0.1P. croceum (24) 233 V 25 110 V 11 0.5 V 0.02Ha-96-3 (78) 260 V 60 150 V 36 0.6 V 0.05ANOVA (P-value)Fungus 6 0.001 6 0.001 0.029N 0.031 n.s. 6 0.001Fungus*N n.s. n.s. n.s.

Low N +Ash Non-mycorrhizal 451 V 54 292 V 22 0.7 V 0.04Piloderma sp. 1 (35) 603 V 44 360 V 10 0.6 V 0.05Piloderma sp. 1 (67) 563 V 47 363 V 24 0.7 V 0.1P. croceum (02) 509 V 49 313 V 24 0.6 V 0.1P. croceum (24) 430 V 15 284 V 10 0.7 V 0.04Ha-96-3 (78) 405 V 57 266 V 32 0.7 V 0.1

High N +Ash Non-mycorrhizal 514 V 25 305 V 18 0.6 V 0.04Piloderma sp. 1 (35) 722 V 66 400 V 32 0.6 V 0.1Piloderma sp. 1 (67) 826 V 70 435 V 18 0.5 V 0.1P. croceum (02) 605 V 58 397 V 9 0.7 V 0.1P. croceum (24) 571 V 48 360 V 25 0.6 V 0.03Ha-96-3 (78) 557 V 57 398 V 50 0.7 V 0.1ANOVA (P-value)Fungus 6 0.001 0.002 n.s.N 6 0.001 s 0.001 n.s.Fungus*N n.s. n.s. n.s.

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 inthe root-free/mycelial compartment (+Ash) or left unamended (3Ash). Values are means V S.E.M. of ¢ve replicates; 3Ash and +Ash treatments havebeen tested (P6 0.05, ANOVA) separately.

Table 3Elemental concentrations (mg g31) 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)

Treatments Mycorrhizal status Elemental concentration mg g31

P shoot P root K shoot K root Ca shoot Ca root Mg shoot Mg root N shoot

High N 3Ash Non-mycorrhizal 0.7 V 0.03 cd 0.4 V 0.01 d 2.3 V 0.3 f 1.1 V 0.06 e 1.9 V 0.2 c 0.6 V 0.06 e 1.1 V 0.1 a 0.4 V 0.02 d 37 V 2.6 aPiloderma sp. 1 (67) 0.5 V 0.07 bc 0.8 V 0.04 c 4.3 V 0.4 d 4.1 V 0.5 d 1.9 V 0.2 c 1.8 V 0.1 d 1.2 V 0.1 a 1.4 V 0.1 b 24 V 2.4 bP. croceum (02) 0.7 V 0.1 cd 0.9 V 0.06 c 3.2 V 0.4 e 4.1 V 0.3 d 2.3 V 0.4 c 1.5 V 0.07 d 1.2 V 0.2 a 1.3 V 0.09 bc 32 V 2.0 aHa-96-3 (78) 0.5 V 0.02 bc 0.9 V 0.03 c 3.9 V 0.4 de 4.0 V 0.5 d 1.7 V 0.3 c 1.6 V 0.1 d 1.2 V 0.1 a 1.4 V 0.2 bc 25 V 3.4 b

High N +Ash Non-mycorrhizal 1.3 V 0.1 a 2.4 V 0.2 a 9.8 V 0.8 b 13.4 V 0.8 a 3.8 V 0.7 ab 2.9 V 0.1 c 0.6 V 0.08 c 1.1 V 0.08 c 7.2 V 0.3 cPiloderma sp. 1 (67) 1.0 V 0.05 a 1.5 V 0.03 b 7.6 V 0.4 c 9.5 V 0.5 c 2.9 V 0.3 bc 5.1 V 0.4 a 0.6 V 0.06 c 1.5 V 0.2 b 6.2 V 0.2 cP. croceum (02) 1.2 V 0.1 a 1.4 V 0.1 b 10.9 V 0.3 b 11.9 V 0.7 b 4.2 V 0.4 a 3.9 V 0.2 b 0.8 V 0.09 b 1.8 V 0.1 a 6.5 V 0.9 cHa-96-3 (78) 1.1 V 0.1 a 1.4 V 0.09 b 12.4 V 0.9 a 11.5 V 0.6 b 3.5 V 0.5 ab 3.4 V 0.05 c 1.1 V 0.07 a 1.4 V 0.06 b 6.7 V 0.4 cANOVA (P-value)Ash 6 0.001 6 0.001 6 0.001 6 0.001 6 0.001 6 0.001 6 0.001 6 0.001 0.02Fungus 0.06 0.02 6 0.001 n.s. n.s. 6 0.001 0.06 6 0.001 6 0.001Ash*Fungus n.s 6 0.001 6 0.001 6 0.001 n.s. 0.001 n.s. 0.01 0.06Critical levelsThelin et al. [12] 1.1^1.3 ^ 3.5^4.0 ^ 0.4^2.0 ^ 0.4^0.7 ^ 12^13

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/mycelialcompartment (+Ash) or left unamended (3Ash). Values are means V S.E.M. of ¢ve replicates. Di¡erent letters denote di¡erences among means(P6 0.05, ANOVA).

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0.5^1.5% of the total pool, the plants, which contained3.6^4.6%, and the soil solution, which contained 0.9^1.5% of the P originally present in the ash.

3.5. Microbial biomass and activity

Bacterial activity as determined by [3H]-thymidine and[14C]-leucine incorporation was higher (P6 0.001 for bothisotopes) in substrates collected from the mycelial com-partments of +Ash pots than in 3Ash pots (Table 5).However, there was no statistically signi¢cant e¡ect ofmycorrhizal inoculation on bacterial activity.

Abundant mycelia resembling the inoculated mycorrhi-zal fungi were observed in all inoculated pots at harvest

but never in the non-mycorrhizal controls. However, nostatistical di¡erences between non-mycorrhizal and mycor-rhizal treatments were found when analysing the concen-tration of PLFAs of the soil. Fungal biomass signi¢cantlyincreased in response to ash addition (P6 0.001, Table 5).

No clear e¡ect of ash or mycorrhizal inoculation treat-ment was evident on total bacterial PLFA concentrations(Table 5). The ratio between fungal and bacterial PLFAsin +Ash and 3Ash pots was 0.06^0.08 and 0.04^0.07,respectively. PCA of the bacterial PLFAs showed a dis-tinct separation between samples collected from +Ash my-celial compartments and those from 3Ash controls (Fig.4). The following PLFAs increased after ash addition:16:1g5, cy17:0 and a15:0, while the following PLFAsdecreased after ash addition: a17:0, i17:0, i17:0, i15:0,10me17:0.

4. Discussion

In the present study, addition of ash enhanced growthof spruce seedlings, irrespective of mycorrhizal status. Thiswas clearly an e¡ect of K and P added with the ash, sinceconcentrations of these elements were below critical levelsin the seedlings in the 3Ash treatment (Table 3, [12]).Improved plant growth in response to ash additions hasbeen reported previously in both ¢eld and laboratory stud-ies [22^24]. Application of wood ash to peatlands nor-mally improves tree growth more than application to min-eral soils [25]. The reason could be that peatlands haverelatively low concentrations of P and K compared tothe mineral soils. Re-fertilisation of peat soils with K isprobably more important than fertilisation with P duringthe post-fertilisation period [26].

Mycorrhizal inoculation had the most pronounced e¡ecton plant biomass in the 3Ash treatment. This was prob-ably an e¡ect of uptake of K from the mycelial compart-ment by the external mycelium of mycorrhizal plants.Non-mycorrhizal seedlings in the same treatment were un-able to capture nutrients from the root free compartmentand consequently had a very low K concentration, indicat-ing that K was the main element limiting their growth.Although plant concentrations of Ca and Mg were appar-ently above the critical de¢ciency levels, there was a pos-itive mycorrhizal e¡ect on the uptake of these elements.These results con¢rm the potential role of ectomycorrhizalmycelia on the uptake of K and Mg [27^28]. In the +Ashtreatment, levels of these elements were above the criticalde¢ciency level in all plants, including the non-mycorrhizal

6

Fig. 2. Elemental content (mg plant31) 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 woodash was added in the root-free/mycelial compartment (+Ash) or left unamended (3Ash). a^d: Plant contents of Ca (a), K (b), Mg (c) and P (d). Verti-cal bars represent S.E.M. of ¢ve replicates. Di¡erent letters denote di¡erences among means (P6 0.05, ANOVA); 3Ash and +Ash treatments havebeen tested separately.

Fig. 3. Phosphate concentration (Wmol l31) in the soil solution collectedfrom the root-free/mycelial compartments of non-mycorrhizal spruceseedlings and seedlings growing in symbiosis with Piloderma sp. 1 (67),P. croceum (02) or Ha-96-3 (78). The seedlings were grown in pots con-taining sand^peat substrate treated with a high level of N; hardenedwood ash was added in the root-free/mycelial compartment (+Ash) orleft unamended (3Ash). Vertical bars represent S.E.M. of ¢ve replicates.Di¡erent letters denote di¡erences among means (P6 0.05, ANOVA);3Ash and +Ash treatments have been tested separately.

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controls; however, shoots of all seedlings had very lowconcentrations of N, indicating that N had become thelimiting 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 withlow solubility [8]. However, the concentration of PO33

4 inthe solution collected from the +Ash treatment was highcompared with published ¢eld estimates [29]. This demon-strates that the release of P from the ash must have beensu⁄cient for plant growth. Higher PO33

4 concentrationswere found in substrates colonised by Piloderma sp. 1mycelia in the +Ash treatment compared to other treat-ments, indicating that Piloderma sp. 1 stimulated P releasefrom the ash. However, there was no indication that thefungus transported more P to the plant than other mycor-rhizal fungi or in non-mycorrhizal controls, con¢rming theresults of Mahmood et al. [7], obtained using smaller mi-crocosm systems.

Arvidsson and Lundkvist [30] estimated nutrient con-

centrations in spruce needles in forests treated with hard-ened wood ash within a range of climate and fertility gra-dients in Sweden and found consistently higher P, K andCa concentrations ¢ve years after the treatment. The ini-tial P concentrations in tree needles from plots treatedwith 6 tonnes ha31 granulated wood ash at Torup (thesite where the mycorrhizal fungi used in the present studywere isolated) were markedly higher compared to the un-treated control. Data on the tree growth after four con-secutive growing seasons (5 yr) also showed better growthincrements in the 6 tonnes ha31 granulated wood ashtreatment compared to the control or 3 tonnes ha31 treat-ments (S. Jacobson, personal communication). E⁄cientmycorrhizal mycelial uptake and transport of P, whichhas already been solubilised by Piloderma sp. 1 or othermicroorganisms, would contribute to this improvedgrowth.

In contrast to the present ¢nding, Wallander et al. [31]and Wallander [32] reported the superior ability of pine

Table 4P 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)

Treatments Mycorrhizal status Amount of P (mg) solubilised Fraction of P (%) in

soil solution microbial biomass plant biomass

High N 3Ash Non-mycorrhizal 0.2 V 0.02 a 0.03 V 0.005 ab 0.3 V 0.02 a 0.3 V 0.1 aPiloderma sp. 1 (67) 0.6 V 0.09 b 0.02 V 0.004 a 0.5 V 0.1 b 1.3 V 0.3 bP. croceum (02) 0.5 V 0.04 b 0.05 V 0.01 b 0.5 V 0.04 b 0.9 V 0.1 bHa-96-3 (78) 0.4 V 0.06 b 0.03 V 0.01 ab 0.5 V 0.04 b 0.8 V 0.2 b

High N +Ash Non-mycorrhizal 2.2 V 0.2 y 0.9 V 0.1 x 1.5 V 0.4 y 4.3 V 0.2 yPiloderma sp. 1 (67) 2.2 V 0.06 y 1.5 V 0.04 y 0.8 V 0.2 x 4.4 V 0.1 yP. croceum (02) 2.1 V 0.1 y 1.1 V 0.03 x 0.7 V 0.2 x 4.6 V 0.3 yHa-96-3 (78) 1.7 V 0.1 x 0.9 V 0.1 x 0.5 V 0.05 x 3.6 V 0.3 x

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/mycelialcompartment (+Ash) or left unamended (3Ash). 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 V S.E.M. of ¢ve replicates. Di¡erent letters denote di¡erences among means(P6 0.05, ANOVA); 3Ash and +Ash treatments have been tested separately.

Table 5Microbial activity and estimates of bacterial and fungal biomass in the root-free/mycelial compartments of pots with non-mycorrhizal spruce seedlingsand seedlings growing in symbiosis with Piloderma sp. 1 (67), P. croceum (02) or Ha-96-3 (78)

Treatments Mycorrhizal status Microbial activitya Bacterial biomassb

(nmol g31)Fungal biomassc

(nmol g31)

[3H]-thymidine d.p.m. [14C]-leucine d.p.m.

High N 3Ash Non-mycorrhizal 4U103 V 0.3U103 3U103 V 0.2U103 5.9 V 0.5 0.4 V 0.05Piloderma sp. 1 (67) 5U103 V 1.0U103 3U103 V 0.8U103 6.0 V 0.7 0.5 V 0.1P. croceum (02) 5U103 V 0.2U103 3U103 V 0.2U103 5.7 V 0.1 0.4 V 0.03Ha-96-3 (78) 4U103 V 0.8U103 3U103 V 0.6U103 5.4 V 0.4 0.3 V 0.03

High N +Ash Non-mycorrhizal 15U103 V 2U103 12U103 V 0.2U103 6.0 V 0.7 1.1 V 0.2Piloderma sp. 1 (67) 25U103 V 2U103 18U103 V 0.2U103 6.8 V 0.4 1.3 V 0.3P. croceum (02) 18U103 V 4U103 13U103 V 0.3U103 4.9 V 0.5 0.6 V 0.1Ha-96-3 (78) 22U103 V 4U103 17U103 V 0.3U103 4.7 V 0.4 0.8 V 0.1

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 com-partment (+Ash) or left unamended (3Ash). Microbial activity and fungal biomass increased signi¢cantly (P6 0.001, ANOVA) in response to +Ash.There was no signi¢cant e¡ect of hardened wood ash or ectomycorrhizal inoculation on bacterial biomass. Values are means V S.E.M. of ¢ve replicates ;3Ash and +Ash treatments have been tested (P6 0.05, ANOVA) separately.aExpressed as incorporation of [3H]-thymidine and [14C]-leucine.bEstimate of sum of PLFAs of bacterial origin.cTotal amount of PLFA 18:2g6,9.

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seedlings colonised by some ectomycorrhizal fungi to useapatite as a P source compared to non-mycorrhizal seed-lings. This discrepancy may be explained by the relativelyhigher amounts of apatite available for solubilisation inWallander’s experiments. The release of P from the ashin our experiments may have been caused by exudationof oxalate by fungi, bacteria or roots. Gri⁄ths et al. [29]reported signi¢cantly higher concentrations of PO33

4 andoxalate in soil solution of soils colonised by mat-formingectomycorrhizal fungi compared to non-mat soils. In aprevious in vitro study [7], Piloderma sp. 1 demonstratedpronounced ability to solubilise hardened wood ash byformation of abundant calcium oxalate crystals whengrown on ash-amended culture medium. In the samestudy, the concentration of P in the mycelium of Piloder-ma sp. 1 was higher compared to P. croceum or Ha-96-3.In the present study we found low oxalate concentrationin the soil solution in all treatments (1^6 Wmol l31 soilsolution), which is probably a result of precipitation of

calcium oxalate in the presence of ash. It is likely thatan instant estimate of oxalate concentration does not re-£ect the dynamics of oxalate exudation into the soil solu-tion.

In base-poor forest ecosystems at the Hubbard Brookexperimental forest, New Hampshire, USA, ectomycorrhi-zal fungi were found to be important in supplying Ca tothe trees from apatite sources in the soil [33]. In thepresent study Piloderma sp. 1 appeared to transport Cafrom the ash to the roots, since the roots colonised by thisfungus had the highest Ca concentrations among thetested plants when grown in the presence of ash. Only aminor portion of this Ca appeared to be transferred to theplants, since the Ca concentrations in the shoots were low-er in plants colonised by Piloderma sp. 1 than in otherplants. This suggests that the colonization of wood ashby Piloderma sp. 1, found both in laboratory [7] and in¢eld [6] studies, is stimulated by Ca rather than P in theash. The ecological implications of ash-colonising fungi

Fig. 4. PCA using frequencies of bacterial PLFAs originating from soils collected from the root-free/mycelial compartments of non-mycorrhizal spruceseedlings and seedlings growing in symbiosis with Piloderma sp. 1 (67), P. croceum (02) or Ha-96-3 (78). The seedlings were grown in pots containingsand^peat substrate treated with a high level of N; hardened wood ash was added in the root-free/mycelial compartment (+Ash) or left unamended(3Ash). Bars represent S.E.M. of ¢ve replicates.

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may be that the fungus selectively removes Ca from theash while P is released into the soil solution. This P willbecome available to other organisms in the soil, includingspecies of ectomycorrhizal fungi that may transport P totheir host trees. Ca taken up by the ash-colonising fungi islikely to precipitate as calcium oxalate on fungal hyphaewhich, apart from being a result of mineral dissolution,has also been suggested to protect the hyphae from micro-bial attack and predation by soil animals [34]. Accordingto Snetselaar and Whitney [35], fungi accumulate calciumoxalate to avoid potential Ca and oxalate toxicity and,furthermore, Jennings [36] has suggested that calcium ox-alate regulates cytoplasmic pH as well as being a structuralmaterial of hyphae.

Analysis of the soil from the Torup experimental forestshows an increase in the concentration of base cations(Ca, Mg, K) in the ash-fertilised plots (S. Jacobson, per-sonal communication). However, the dissolution of P fromash granules seems to be slow, as indicated by very lowconcentrations of PO33

4 in the soil solution (S. Jacobson,personal communication). A recent elemental analysis ofthe ash granules collected from the site at Torup showedpresence of P in substantial amounts as long as sevenyears after their ¢eld application, whereas most of theother elements had been leached out in the soil over thisperiod (J. Bergholm, personal communication). Similarly,in the present study, only a small fraction (5.0^6.7%) of Pwas weathered from the ash and a large pool of P (93% ofthe total P supplied) was still bound in the ash at the timeof harvest.

A signi¢cant increase in microbial activity, measured by[3H]-thymidine and [14C]-leucine incorporation, in re-sponse to ash treatment could have been due to an ash-induced pH increase in the substrate. Using a similarmethod, Bafiafith et al. [20] reported a 1.6-fold increase inbacterial activity in a site polluted with alkaline dust (pH6.6) over that in the control site (pH 4.13). Soil amend-ments with certain primary minerals may also cause anincrease in bacterial activity and the presence of ectomy-corrhizal mycelia may also have a positive or negativein£uence on bacterial activity [16]. However, in our study,presence of ectomycorrhizal mycelia had no statisticallysigni¢cant e¡ect on bacterial activity.

The high background levels of fungal biomass measuredin non-mycorrhizal controls are probably an e¡ect of highamounts of fungal PLFAs in the peat used as growthsubstrate and makes it di⁄cult to draw conclusions con-cerning the e¡ects of ash treatment on mycorrhizal myce-lia. No e¡ects on total bacterial biomass could be estab-lished using bacterial PLFA concentrations either.

Bafiafith et al. [37] reported a reduction in microbial bio-mass (total PLFAs) and a decreased index of fungal:bac-terial PLFAs, indicating a larger reduction of fungi thanbacteria due to the highest rate of wood ash fertilisation.In the present study, +Ash treatment a¡ected neither bio-mass nor the fungal:bacterial ratio. However, in contrast

to the ¢ndings of Fritze et al. [38], who reported nochange in the level of fungal ergosterol in response toash application, we found an increase in fungal biomass(PLFA 18:2g6,9) in +Ash treatments. The change in com-position of bacterial PLFAs following ash treatment in thepresent experiment was similar to the change found byBafiafith et al. [37] after wood ash fertilization of a forestsoil.

High microbial activity, biomass and P in the root-freecompartment of non-mycorrhizal treatments strongly sug-gest that microbes other than ectomycorrhizal fungi mayalso contribute to the mobilisation of nutrients in ash.Clarholm [2] has emphasised the importance of microbialutilisation of P derived from wood ash. Further studies areneeded to investigate the role of di¡erent ectomycorrhizalmycelia in capturing nutrients already mobilised from ashand transporting them to the host plants.

Acknowledgements

This work was ¢nanced by the Swedish National EnergyAdministration (STEM).

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