Solubilisation and colonisation of wood ash by ectomycorrhizal fungi isolated from a wood ash fertilised spruce forest

Solubilisation and colonisation of wood ash by ectomycorrhizalfungi isolated from a wood ash fertilised spruce forest

Shahid Mahmood a;*, Roger D. Finlay b, Susanne Erland a, Ha®kan 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

Received 16 August 2000; received in revised form 11 December 2000; accepted 21 December 2000

Abstract

In Sweden application of granulated wood ash has been suggested as a method to supplement nutrient loss resulting from harvesting offorest residues for bioenergy production. Mycelia of two ectomycorrhizal fungi Piloderma sp. 1 and Ha-96-3, were commonly found tocolonise ash granules in a wood ash fertilised spruce forest. Thirty-eight fungal isolates were selected from 10 taxa to investigate the possiblerole of different ectomycorrhizal fungi in nutrient mobilisation from ash. The taxa were Cenococcum geophilum Fr., Piloderma croceumErikss. and Hjortst., Piloderma sp. 1, Thelephora terrestris (Ehrenb.) Fr., Tylospora fibrillosa Donk, and five unidentified species, alloriginating from a wood ash fertilised spruce forest. The isolates were tested for their ability to solubilise tricalcium phosphate (TCP) orhardened wood ash (HWA) in vitro. Ha-96-3, P. croceum and Piloderma sp. 1 were the only taxa which solubilised TCP. Abundant calciumoxalate crystals were formed in TCP and HWA plates with Piloderma sp. 1. Ha-96-3 and two isolates of P. croceum produced intermediateamounts of crystals. Ha-96-1 and T. fibrillosa produced low amounts of crystal but no crystal formation was observed by any of the otherisolates. Piloderma sp. 1 from HWA plates had significantly higher concentrations of P, compared to P. croceum or Ha-96-3. Piloderma sp. 1and P. croceum were further tested for their ability to colonise wood ash in microcosms containing intact mycorrhizal associations. After7 months Piloderma sp. 1 colonised ash amended patches with a dense, mat like mycelium, whereas P. croceum mycelia avoided the ashpatches. Possible differences between these fungi in patterns of carbon allocation were investigated by labelling seedlings with 14CO2.Piloderma sp. 1 mycelia allocated significantly more 14C to ash patches than P. croceum. P. croceum allocated relatively more 14C to controlpatches than to the ash patches. The possible role of ectomycorrhizal fungi in mobilisation of nutrients from wood ash is discussed. ß 2001Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.

Keywords: Ectomycorrhizal fungi ; PCR-RFLP; Tricalcium phosphate; Wood ash; Solubilisation; Calcium oxalate; Spruce forest

1. Introduction

Ectomycorrhizal fungi form symbiotic associations withtree roots and improve uptake of nutrients and water fromsoils with low fertility [1]. Mycorrhizal trees allocate car-bon to mycorrhizal mycelia which take up mineral nu-trients and can also play an important role in makingorganically bound N available to their host plants [1].There is also convincing evidence that ectomycorrhizalfungi are able to weather primary minerals to mobilisenutrients [2^9].

Anthropogenic deposition of pollutants and increasedharvesting of forest residues are known to cause acidi¢ca-tion in coniferous forests [10^12]. Application of granu-

lated wood ash to such forest soils has been recommendedto increase the pH and soil productivity through a slowsupplementation of lost base cations and other nutrientsessential for tree growth [13^15]. The e¡ects of ash fertil-isation on the ectomycorrhizal fungi, especially the mech-anisms by which certain species can capture nutrients fromwood ash, have not been thoroughly investigated. In anearlier investigation [16] of the e¡ects of granulated woodash in a spruce forest, although no signi¢cant e¡ects werereported on individual species, two mycorrhizal species(Piloderma sp. 1 and Ha-96-3) showed a tendency to in-crease in abundance in plots treated with ash. Moreover,Piloderma sp. 1 and Ha-96-3 mycelia were also found tocolonise the ash granules. We hypothesise that these my-corrhizal fungi may play a role in mobilisation of nutrientsfrom the wood ash. Most of the elements in wood ash arereadily leachable except P which is bound in apatite likecompounds with low solubilities [17]. Several investiga-

0168-6496 / 01 / $20.00 ß 2001 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 0 ) 0 0 1 2 4 - 0

* Corresponding author. Tel. : +46 (46) 2223756;Fax.: +46 (46) 2224158; E-mail : [email protected]

FEMSEC 1213 29-3-01 Cyaan Magenta Geel Zwart

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tions document the ability of soil fungi to solubilise other-wise insoluble P compounds both in vitro and in vivo [18^23]. Recently, Wallander et al. [6] and Wallander [8] dem-onstrated the superior ability of ectomycorrhizal pineseedlings (colonised by Suillus variegatus or Paxillus invo-lutus) to use apatite as P source compared to non-mycor-rhizal seedlings. However, there is no information avail-able on the role of ectomycorrhizal fungi which coloniseash granules and tend to increase in abundance on theroots following wood ash applications.

The aims of the present study were: (a) to isolate andidentify ectomycorrhizal fungi colonising tree roots in agranulated wood ash fertilised spruce forest ; (b) to testtheir ability to solubilise tricalcium phosphate (TCP) orhardened wood ash (HWA) in vitro; and (c) to studypatterns of colonisation, carbon allocation and P trans-port by intact mycorrhizal mycelia colonising wood ashamended substrates in microcosms.

2. Materials and methods

2.1. Sampling of ectomycorrhizal roots

Ectomycorrhizal roots were collected from a Norwayspruce (Picea abies (L.) Karst) forest planted in 1957and located at Torup in the southwest of Sweden(56³55PN, 13³05PE). The experimental forest was treatedin 1990 with 0 ton ha31 (control, C), 3 ton ha31 (lowash, LA) and 6 ton ha31 (high ash, HA) of granulatedwood ash. Ectomycorrhizal roots were sampled in May,1997. Cores from three replicates of all treatments weresampled with the aim of ¢nding representatives of all my-corrhizal taxa in the community on the roots, previouslydescribed by Mahmood [16].

2.2. Isolation of fungi from root tips

Mycorrhizas were picked from the root materials ofeach treatment. After surface sterilisation with 30%H2O2 for 20^30 s and rinsing with sterile water, the rootswere plated on Petri dishes ¢lled with 25 ml of 1/2 strengthmodi¢ed Melin^Norkrans (MMN) medium containing 30mg l31 chlorotetracycline and 1 mg l31 Benomyl [24]. Onthe basis of root tip morphology, attempts were made toplate similar looking mycorrhizas on the same plate (notmore than 10 per plate). The resultant axenic cultures weresubsequently maintained on 1/2 MMN medium.

2.3. Molecular identi¢cation of ectomycorrhizal fungi

Ninety-seven axenic cultures resembling basidiomycetesor ascomycetes were selected for molecular identi¢cationof the isolated fungi. Mycelium covering an area about1U1/2 cm on the Petri dish was carefully removed, avoid-ing the agar medium. DNA was extracted using enzymatic

and mechanical lysis of cell walls, SDS extraction bu¡erand phenol/chloroform steps, following the procedures de-scribed by Mahmood et al. [25].

The primers used for PCR were, ITS1, 5P-TCCGTA-GGTGAACCTGCGG-3P and ITS4, 5P-TCCTCCGCTT-ATTGATATGC-3P designed by White et al. [26]. The 50Wl reaction mixture for PCR contained the following ¢nalconcentrations or total amounts: 0.1^10 ng total DNA,500 pmol of each primer, 20 mM Tris (pH 8.3), 200 WMdNTP, 1.5 mM MgCl2, 50 mM KCl and 2.5 units of Taqpolymerase (Perkin Elmer). The thermocycler (Perkin El-mer, GenAmp PCR System 9600) used for ampli¢cationswas programmed for one cycle of 10 min at 94³C, fol-lowed by 35 cycles of 30 s at 94³C, 30 s at 53³C, 1.5min at 72³C and one cycle of 10 min at 72³C. Negativecontrols were used with each batch of samples to makesure that none of the reactants was contaminated. PCRampli¢ed products were analysed by electrophoresis on 2%agarose gels and detected by staining with ethidium bro-mide [27].

RFLP analysis of the PCR products was carried outwith the restriction endonucleases HinfI, MboI and TaqIaccording to manufacturers' recommendations (Boehr-inger Mannheim). The uncut ampli¢cation products andtheir restriction fragments were size fractionated using 1%agarose+1% NuSieve (FMC) gels stained with ethidiumbromide [27]. The gels were documented using a GelDoc 2000 System (Bio-Rad) and Quantity One0 software(Bio-Rad) for imaging and band analysis. Restriction frag-ments of di¡erent fungi were compared to a referencelibrary consisting of RFLPs of identi¢ed fruit-bodies andectomycorrhizal fungi found in our previous communitystructure studies on tree roots at Torup and other forestslocated in southern Sweden [16,25,28,29].

2.4. Preparation and inoculation of tricalcium phosphate(TCP) and hardened wood ash (HWA) plates

A modi¢ed Melin^Norkrans medium was preparedwith the following composition (amount l31 dH2O):MgSO4W7H2O, 0.15 g; NaCl, 0.025 g; MES sodium salt,4.3 g; FeCl3W6H2O, 0.012 g; thiamine^HCl, 100 Wg; D-glucose, 10 g; agar, 15 g. L-alanine, ammonium or nitratewere initially tested as N sources. All isolates were able togrow on all of the tested N sources (data not shown) butL-alanine (0.34 g l31 with KCl 0.26 g l31) was chosen forsubsequent experiments in order to avoid unwanted pHe¡ects. The pH of the medium was adjusted to 5.5 byaddition of HCl or KOH and then autoclaved at 0.7atm for 30 min. Alanine was added to the autoclavedmedium through a Millipore ¢lter. To each Petri dish 25ml of the medium was added with a dispenser and aftersolidi¢cation a surface layer of 5 ml agar (1.5%, w/v) con-taining 0.25% (w/v) TCP (particle size6 0.050 mm), or0.1% (w/v) HWA (particle size6 0.050 mm) (Ljungbyver-ket, Sydkraft va«rme AB, Sweden) was also added.


S. Mahmood et al. / FEMS Microbiology Ecology 35 (2001) 151^161152

The agar surface was covered with cellophane to pre-vent growth of mycelium into the medium. Inoculationswere made with discs of fungal inoculum cut with a corkborer (1.0 cm diameter) from the edge of actively growingcolonies on MMN (1/2 strength) plates. There were sixreplicate Petri dishes for each fungal isolate. The disheswere incubated at 20³C in sealed plastic bags. The fungiwere allowed to grow for 120 days.

2.5. Experiment I

2.5.1. Solubilisation of TCPOnly those fungi with RFLP patterns matching previ-

ously described ectomycorrhizas from Torup [16] or otherforests in the region [25,28,29] were selected for this assay.Thirty-eight isolates from 10 taxa were inoculated on TCPplates containing alanine as an N source and incubated for120 days at 20³C. Visual assessment of solubilisation after120 days was made by observing the clear zones in theagar around and under the colonies after removing thecellophane.

2.6. Experiment II

2.6.1. Crystal formation in TCP and HWA platesSince the TCP solubilisation was consistent within taxa,

a subset of 22 isolates from all 10 taxa was used to mon-itor crystal formation in TCP and HWA plates containingalanine as the sole nitrogen source. Isolates were inoculat-ed onto the cellophane covered agar medium and incu-bated as in Experiment I. Crystal formation was moni-tored using an inverted microscope (Nikon, Japan) fordirect and non-destructive visualisation of the medium inthe Petri dishes. Di¡erences between isolates in crystalproduction were recorded using a rough quantitativescale : abundant crystal formation, few crystals, rare crys-tal formation and no crystal formation.

2.6.2. SEM and X-ray microprobe analysis of puri¢edcrystals

The agar from the clear zone around or under the col-ony was collected, after removing the cellophane mem-brane, and dissolved in 5 ml of ddH2O by gentle macer-ation. The puri¢ed crystals either from TCP or HWAweathering plates were mounted on a glass plate (25U60mm) using a conducting tape and dried overnight in afreeze-drier. The crystals and glass plate were carboncoated for SEM. The crystals were examined for theirmorphology and X-ray micronutrient analysis by using aJEOL JSM 6400 electron microscope equipped with aback-scattered electron detector and a LINK EDS analysisunit.

2.6.3. Analysis for P content in the myceliumPiloderma sp. 1 and Ha-96-3, which colonised wood ash

granules in the ¢eld site at Torup, and Piloderma croceum

were selected for analyses of mycelial P content. The fungiwere grown on cellophane membranes covering HWAplates and mycelia were carefully collected, excluding thecentral MMN containing agar plug and freeze-dried.Dried mycelia were digested in concentrated HNO3 priorto analysis of P by Inductively Coupled Plasma-AtomicEmission Spectroscopy (ICP-AES) (Perkin Elmer, Con-necticut, USA) [30].

2.7. Experiment III

2.7.1. Mycorrhizal synthesis and preparation of plantgrowth chambers

Mycorrhizal associations were successfully synthesisedbetween spruce (P. abies (L.) Karst.) seedlings and ¢veof the 10 previously reported ectomycorrhizal species (Ta-ble 1). Mycorrhizas were synthesised by the method de-scribed by Duddridge [31] as modi¢ed by Finlay et al. [32].Piloderma sp. 1 (isolate no. Tor-35), present on ash gran-ules, and P. croceum (isolate no. Tor-03), which was notpresent on ash granules were selected for further experi-ments in microcosms. Seedlings with well establishedmycorrhizal roots were transferred to microcosms(24.5U24.5U2.5 cm, Nunc1, Denmark) containing a 5^6 mm thick layer of non-sterile peat as a substrate. After2 months, when the mycelia started growing actively fromthe mycorrhizas and colonised peat, small peat ¢lled plas-tic cups (1.5U0.6 cm) were embedded carefully around theroot system, close to the margin of the extending mycor-rhizal mycelium (Fig. 3a). Within each chamber ¢ve con-trol cups contained only peat, while the remaining ¢vecups also contained a surface layer of wood ash (20 mgper cup) (Fig. 3b). Five replicate microcosms were madefor each mycorrhizal fungus and they were randomisedwithin the phytotron (programmed for 300 Wmol m32

s31 PAR, 80% relative humidity and a 18 h/6 h and18³C/16³C day/night cycle) and incubated for further5 months before examining the mycelial colonisation ofthe cups.

2.7.2. 14C labelling procedureTo trace the £ow of carbon from the seedlings to the

symbionts and allocation to the wood ash nutrientpatches, the seedlings were labelled with 14C. Shoots ofseedlings were carefully sealed in plastic boxes(12.2U8.2U3.2 cm, Hofsta«tter and Ebbesen a/s, Den-mark) and 14CO2 (1.5 MBq) was released by adding 500Wl of 15% lactic acid to 40 Wl of 20 mM NaH14CO3. Theseedlings were exposed to 14CO2 for 3 h and the plantswere allowed to translocate 14C for 72 h.

2.7.3. Harvesting and analysis of radioactivityAfter the feeding period, the wood ash and control nu-

trient patches were carefully removed from the peat bycutting the connected mycelia at the edge of plastic cupswith a ¢ne scalpel. Roots were washed gently under run-


S. Mahmood et al. / FEMS Microbiology Ecology 35 (2001) 151^161 153

ning tap water to remove peat. Nutrient cups, shoots androots were dried in an oven at 80³C for 24 h before weightdeterminations. 14C in shoots, roots, and ash and controlpatches was analysed by sample oxidation and liquid scin-tillation counting. The samples were combusted in a sam-ple oxidiser (Model 304, Packard), the 14CO2 evolved wastrapped in Carbosorb and counted on a Tri-Carb 2100TR,Packard scintillation counter.

2.7.4. Analysis for P content in shoot and rootShoots and roots were milled and digested in 15 ml

concentrated HNO3 prior to P content analysis by ICP-AES (see Section 2.6.3).

3. Results

3.1. Isolation and identi¢cation of ectomycorrhizal fungi

Ninety-seven fungal isolates showed characteristics ofbasidiomycetes or ascomycetes. PCR-RFLP analysis ofthese fungi revealed that 57 isolates representing nine

Table 1In vitro solubilisation of tricalcium phosphate (TCP) and calcium oxalate crystal formation in TCP or hardened wood ash (HWA) plates by di¡erentectomycorrhizal fungi

Species/ITS types Identi¢cation references Isolate no. Treatment origin TCP solubilisation Crystal formation in Mycorrhizasynthesis

TCP HWA

C. geophilum [16,25,28,29] Tor-109 HA 3 3 3 +Ha-96-1 [16,29] Tor-52 C 3 3 + +Ha-96-1 [16,29] Tor-55 C 3 3 + +Ha-96-1 [16,29] Tor-77 C 3 nd nd ndHa-96-3a [16,29] Tor-78 LA + ++ ++ +P. croceum [16,25,28] Tor-1 C + 3 + +P. croceum [16,25,28] Tor-2 C + ++ + +P. croceum [16,25,28] Tor-3 LA + + + +P. croceum [16,25,28] Tor-4 LA + nd nd +P. croceum [16,25,28] Tor-5 LA 3 nd nd ndP. croceum [16,25,28] Tor-6 LA + nd nd +P. croceum [16,25,28] Tor-21 HA + nd nd ndP. croceum [16,25,28] Tor-23 HA + 3 + +P. croceum [16,25,28] Tor-24 HA 3 3 + +Piloderma sp. 1a [16] Tor-32 LA ++ +++ +++ +Piloderma sp. 1a [16] Tor-35 LA ++ +++ +++ +Piloderma sp. 1a [16] Tor-37 LA ++ nd nd ndPiloderma sp. 1a [16] Tor-40 HA ++ nd nd ndPiloderma sp. 1a [16] Tor-42 HA ++ +++ +++ +Piloderma sp. 1a [16] Tor-67 HA ++ +++ +++ +T. terrestris [16,25,28,29] Tor-103 HA 3 3 3 3T. ¢brillosa [16,25,28,29] Tor-43 C 3 3 + 3T. ¢brillosa [16,25,28,29] Tor-44 C 3 nd nd ndT. ¢brillosa [16,25,28,29] Tor-50 C 3 nd nd ndT. ¢brillosa [16,25,28,29] Tor-63 LA 3 3 + 3T. ¢brillosa [16,25,28,29] Tor-64 LA 3 nd nd ndT. ¢brillosa [16,25,28,29] Tor-65 LA 3 nd nd ndT. ¢brillosa [16,25,28,29] Tor-68 HA 3 3 + 3T. ¢brillosa [16,25,28,29] Tor-75 HA 3 nd nd ndT. ¢brillosa [16,25,28,29] Tor-76 HA 3 nd nd ndTo«-95-3 [16,25] Tor-31 C 3 3 3 3Ve-95-1 [16,28,29] Tor-90 HA 3 3 3 3Ve-95-3 [28,29] Tor-45 C 3 nd nd ndVe-95-3 [28,29] Tor-47 C 3 3 3 3Ve-95-3 [28,29] Tor-66 LA 3 nd nd ndVe-95-3 [28,29] Tor-85 LA 3 nd nd ndVe-95-3 [28,29] Tor-69 HA 3 3 3 3Ve-95-3 [28,29] Tor-89 HA 3 3 3 3

Fungi were isolated from root tips collected from a spruce forest (treated with two levels of granulated wood ash, LA = 3 ton ha31, HA = 6 ton ha31 orC = untreated) located at Torup in southern Sweden. The fungi were grown for 120 days on TCP or HWA weathering plates. Key (TCP solubilisation) :++, clearance around and under colony; +, clearance under colony; 3, no clearance. Key (crystal formation in TCP/HWA plates): +++, crystals inabundance; ++, a few; +, rare; 3, no crystals. Key (Mycorrhiza synthesis): +, mycorrhiza formation; 3, no mycorrhiza formation; nd, not deter-mined.aMycelia commonly colonised wood ash granules in the ¢eld site at Torup.



taxa had perfect matches with the mycorrhizas previouslyreported at Torup [16]. Twenty-nine isolates (representingthree taxa) showed RFLP patterns identical to ectomycor-rhizal fungi reported in two other spruce forests in south-ern Sweden [28]. Only 11 isolates (representing eight taxa)remained unmatched to the available reference material.The identi¢ed species were Cenococcum geophilum Fr.,P. croceum Erikss. and Hjortst., Thelephora terrestris (Eh-

renb.) Fr., and Tylospora ¢brillosa Donk. In addition,there were eight unidenti¢ed species designated as Piloder-ma sp. 1, Ha-96-1, Ha-96-3, To«-95-3, Ve-95-1, Ve-95-2,Ve-95-3 and Ve-95-7.

3.2. TCP solubilisation and crystal formation

The ability of 38 isolates belonging to 10 taxa to solu-

Fig. 1. Production of calcium oxalate crystals by Piloderma sp. 1 in tricalcium phosphate (TCP) and hardened wood ash (HWA) weathering plates.(a) Crystals under the colony or in the clear zone (inset, SEM of a tetragonal dipyramid crystal) in TCP medium; (b) unsolubilised TCP medium fur-ther from the colony; (c) crystals under or in the periphery of the colony in HWA medium; (d) unsolubilised HWA particles further from the colony.



bilise TCP is summarised in Table 1. Ha-96-3, P. croceumand Piloderma sp. 1 were the only isolates which solubi-lised TCP (solubilisation declined in the order Pilodermasp. 1sHa-96-3sP. croceum), and none of the other iso-lates showed any sign of TCP solubilisation (Table 1). Allisolates of P. croceum or T. ¢brillosa solubilised TCP tothe same extent regardless of whether they were isolated inash treated or control plots in the forest (Table 1).

Only three ectomycorrhizal taxa, Ha-96-3 (single iso-late), P. croceum (two out of ¢ve isolates) and Pilodermasp. 1 (all of four isolates), produced crystals in the medium(Table 1). A distinct clear zone (0.5^1.5 cm) around colo-nies was only observed for Piloderma sp. 1 isolates. More-over, Piloderma sp. 1 produced abundant crystals underthe colonies and in the clear zone around the colonies(Fig. 1a). Ha-96-3 and P. croceum produced a few crystalsand only under the colonies. No crystals were observed inthe unsolubilised TCP medium close to the edge of thePetri dishes or in uninoculated controls (Fig. 1b).

3.3. Crystal formation in HWA weathering plates

All taxa except for C. geophilum, T. terrestris, To«-95-3,Ve-95-1 and Ve-95-3 produced some crystals in the me-dium but all isolates of Piloderma sp. 1 produced muchlarger amounts (Fig. 1c) (Table 1). In all cases, there wererelatively more crystals under or close to the periphery ofthe colonies than further from the actively growing myce-lium. No solubilisation of ash was noticed close to theedge of the plates or in uninoculated controls (Fig. 1d).

3.4. Identi¢cation of crystals

Examination of the puri¢ed crystals from TCP mediumby light and scanning electron microscopy revealed thatthey were predominantly tetragonal dipyramids (Fig. 1a,inset). The other common crystal types (not shown) wereplate like, rods or needles. The puri¢ed crystals fromHWA medium were only of one type and morphologicallyidentical to the tetragonal dipyramids found in the TCPmedium and identi¢ed as calcium oxalate dihydrate crys-tals. The presence of calcium in these crystals was con-¢rmed by X-ray micronutrient analysis and they wereidenti¢ed as di¡erent forms of calcium oxalate.

3.5. P content in the mycelium

Mycelia of Piloderma sp. 1 collected from HWA plateshad a signi¢cantly higher (ANOVA, P6 0.02) P con-centration (5.0 þ 0.3 mg g31) compared to P. croceum(3.4 þ 0.3 mg g31) or Ha-96-3 (3.2 þ 0.2 mg g31) (Fig. 2).

3.6. Colonisation of ash and control patches in microcosms

The mean substrate pH of ash colonised and controlpatches was 6.6 and 3.3 respectively. Piloderma sp. 1 my-

celia growing in symbiotic association with spruce seed-lings colonised ash patches completely with a thick, matlike mycelium, whereas control patches were colonisedmore slowly and incompletely by a less dense mycelium(Fig. 3a,b). P. croceum mycelia avoided ash patches anddistinct hollow zones appeared in the mycelium at the sitesof the ash patches, whereas control patches were colonised(Fig. 4a,b).

3.7. Carbon allocation patterns

Ash patches colonised by Piloderma sp. 1 myceliashowed markedly higher 14C allocation compared toP. croceum or allocation to the controls (Table 2). Ashpatches colonised by Piloderma sp. 1 mycelia received64% of the total 14C allocated to nutrient patches, whereascontrol patches received only 36% (Fig. 5). In contrast toPiloderma sp. 1, P. croceum allocated 38% of the total 14Cto the ash patches and 62% to the control patches (Fig. 5).The pattern of 14C allocation between shoots and roots 72h after feeding 14CO2 was the same for NM and Pilodermasp. 1 seedlings (90^91% in the shoots and 9^10% in theroots). In P. croceum colonised seedlings a larger propor-tion of the 14C was in the roots despite their lower root^shoot ratio: 82% of the activity was present in the shootsand 18% in the roots (Table 2).

3.8. Plant biomass and P content

Total plant biomass of Piloderma sp. 1 seedlings(517 þ 8 mg) was signi¢cantly higher (P6 0.01) than that

Fig. 2. Concentration of P (mg g31) in the mycelia of Piloderma sp. 1and P. croceum or Ha-96-3 when grown on cellophane membranes cov-ering hardened wood ash (HWA) agar medium. The fungal colonieswere grown for 120 days before harvest. Vertical bars represent S.E.M.Di¡erent letters indicate statistically di¡erent values (ANOVA,P = 0.020).



of NM controls (271 þ 61 mg) (Table 2). The total biomassof P. croceum colonised plants was intermediate (430 þ 98mg). Concentrations of P in shoots and roots did not varysigni¢cantly between the treatments (Table 2).

4. Discussion

We successfully managed to isolate nine out of 20 earlierreported ectomycorrhizal species on tree roots at Torupincluding Piloderma sp. 1 and Ha-96-3 which coloniseash granules in the wood ash treated plots (see [16]). Pi-

loderma sp. 1 mycelia were also able to colonise ash inlaboratory microcosms when inoculated on spruce seed-lings. Most of the ectomycorrhizal species selected forthe present study were abundant on tree roots in the ¢eldsite at Torup (see [16]). The ectomycorrhizal taxa Ve-95-2,Ve-95-3 and Ve-95-7 were not found in the previous com-munity study at Torup [16], but they were common on theroots in two other Norway spruce forests in southern Swe-den [28].

Calcium oxalate crystals were formed in great abun-dance by isolates that were e¤cient in solubilising TCP,showing that these isolates produce varying amounts ofoxalic acid. Inter- and intra-speci¢c di¡erences in oxalicacid production by ectomycorrhizal fungi have been dem-onstrated by Wallander et al. [6] and Ahonen-Jonnarth etal. [33]. In the present study there were no indications ofintra-speci¢c variation in ability to solubilise TCP in rela-tion to the treatments the isolates came from (Table 1).Clearing of TCP plates and formation of calcium oxalatecrystals indicate release of P in a soluble form, since nocalcium or phosphate were included in the basal incuba-tion medium and no crystals were observed in dishes with-out TCP or HWA. However in the case of HWA it isdi¤cult to interpret which complexes of minerals weresolubilised. P is mostly bound in apatite like compoundswith low solubility, and elemental analysis showed thatash particles often contained P in combination with Ca,K and O [17]. In the present study, mycelia of Pilodermasp. 1 from HWA plates showed signi¢cantly higher con-centration of P compared to P. croceum or Ha-96-3. Itprovided evidence that Piloderma sp. 1 isolates were ableto solubilise P from its otherwise insoluble complexes bycalcium oxalate formation. However much of the calciumin these crystals may have originated from the largeamounts of calcium carbonate in the ash. A number ofmycorrhizal and non-mycorrhizal soil fungi have been re-

Table 214C content in non-mycorrhizal (NM) spruce seedlings and seedlings growing in symbiosis with Piloderma sp. 1, P. croceum

Parameters Treatments

Piloderma sp. 1a P. croceum NM

14C allocated toash patches (dpm) 7U103 þ 2U103 3U103 þ 1U103 ^blank patches (dpm) 5U103 þ 2U103 4U103 þ 2U103 ^shoot (dpm) 7U106 þ 0.4U106 5U106 þ 0.9U106 6U106 þ 0.7U106

root (dpm) 0.7U106 þ 0.3U106 1U106 þ 0.4U106 0.6U106 þ 0.5U106

Plant growth datashoot wt (mg) 259 þ 12 240 þ 56 154 þ 39root wt (mg) 258 þ 14 190 þ 44 117 þ 23plant biomass (mg) 517 þ 8 430 þ 98 271 þ 61root:shoot 1.01 þ 0.1 0.8 þ 0.08 0.8 þ 0.1P concentration (mg g31) inshoot 0.58 þ 0.29 0.73 þ 0.33 0.87 þ 0.50root 0.83 þ 0.42 0.98 þ 0.44 0.99 þ 0.57

Plants in microcosms were exposed to 14CO2 for 3 h and allowed to assimilate 14C for 72 h before harvest. 14C allocated to nutrient patches (with ashor blank), shoot and root is given in dpm. The table also shows data on shoot, root and plant biomass, root shoot ratios and P concentration in shootsand roots. Values are means þ S.E.M. of ¢ve replicates.aMycelia commonly colonised wood ash granules in the ¢eld site at Torup.

Fig. 5. Percentage 14C allocation to wood ash and control cups colon-ised by Piloderma sp. 1 and P. croceum mycelia. Vertical bars representS.E.M.



ported to solubilise inorganic phosphate by formation ofcalcium oxalate [19,34] and this mechanism may be moreimportant in high pH environments with high levels ofcalcium.

In the present study, Piloderma sp. 1 and Ha-96-3 were

the most e¤cient fungi in weathering/solubilising HWAand TCP. These two species have been found to coloniseash granules in the treated plots at Torup and they alsotended to increase in abundance on roots in ash treatedplots [16]. The in vitro experiments in the present study

Fig. 3. (a) A microcosm with spruce seedling colonised by Piloderma sp. 1: patches/plastic cups containing wood ash amended and unamended peat areembedded in the peat around the root system; (b) colonisation of wood ash and control cups by Piloderma sp. 1 mycelia.



suggest that these fungi have the capability to mobilisenutrients from wood ash.

Our microcosm experiments, however, provided no evi-dence of P translocation from the ash substrate to the hostplant despite the intensive colonisation of the ash by Pi-

loderma sp. 1. P concentrations in the shoots were belowoptimal levels [30]. The lack of P transport from the ash tothe plant by Piloderma sp. 1 may have a number of pos-sible explanations: (1) P may have been solubilised fromthe ash and taken up by the mycorrhizal mycelium but not

Fig. 4. (a) A microcosm with spruce seedling colonised by P. croceum : patches/plastic cups containing wood ash amended and unamended peat are em-bedded in the peat around the root system; (b) colonisation of wood ash and control cups by P. croceum mycelia.



in su¤cient amounts to allow translocation to the hostplant, (2) insu¤cient time may have been allowed for sol-ubilisation of P from the ash, (3) P may have been solu-bilised by the mycorrhizal fungus but not taken up by themycelium.

In the forest site in Torup, spruce needles in the ashfertilised plots showed signi¢cantly higher levels of P com-pared with untreated controls in the ¢rst year followingash application [15]. It is possible that in a ¢eld situationwith a high diversity of ectomycorrhizal fungi, alternativeectomycorrhizal species may take up and translocate Pwhich is solubilised by fungi like Piloderma sp. 1.

Gri¤ths et al. [21] investigated the role of mycorrhizalmats in changing the soil solution chemistry and reportedsigni¢cantly higher concentrations of PO33

4 in mycorrhizalmat soil solutions than in non-mat soil solutions. Produc-tion of oxalic acid was reported to in£uence the weath-ering and solubility of PO33

4 in the mat soils. A recentelemental analysis of the ash granules collected from thesite at Torup showed presence of P in substantial amountseven 7 years after their ¢eld application, whereas most ofthe other elements had been leached out in the soil overthis period (J. Bergholm, personal communication). Thissuggests that solubilisation of P containing minerals byectomycorrhizal weathering is not as fast as in the presentin vitro study. Other environmental di¡erences betweenour laboratory experiment and ¢eld conditions such asthe higher temperature and supply of carbon at high levelsmay in£uence the degree of P solubilisation.

The intensive colonisation of ash substrate by Pilodermasp. 1 may be a response to the higher pH of the ash.Erland [35] reported more extensive external mycelialgrowth of an unidenti¢ed pink ectomycorrhizal isolate(Pink LMT 85:2) at pH 7 than at pH 3.8. The failure ofP. croceum mycelia to colonise ash patches may be due tothe sensitivity of this fungus to the high pH (6.6) in the ashpatches. In studies conducted by Erland and So«derstro«m[24] P. croceum had a narrow pH tolerance interval com-pared with other ectomycorrhizal fungi used. These au-thors grew pine seedlings in limed humus (pH 4^7.5)and noticed that P. croceum did not colonise at pH valuesabove 6.2, and that the optimal pH for root colonisationwas around 5. In the present pure culture studies, P. cro-ceum isolates showed little ability to solubilise TCP orHWA which may be another explanation to why P. cro-ceum did not colonise ash patches.

Bending and Read [36] used a similar experimental ap-proach to study the nutrient mobilising activities of Suillusbovinus and T. terrestris in root observation chamberswith added patches of fermentation horizon organic mat-ter (FHOM) collected from a pine forest soil. In theirstudy, colonisation of FHOM by S. bovinus reduced con-centrations of N, P and K, whereas colonisation byT. terrestris led to a decrease in N and K but did notchange P concentration. Analysis of the inorganic Npool of uncolonised FHOM incubated separately sug-

gested that mineralisation rates were inadequate to explainthe loss of N from the colonised material. Recently, Perez-Moreno and Read [37] used the same observation chambersystem to demonstrate ability of P. involutus to mobilise Nand P from patches of beech, birch and pine litter and totransfer the nutrients to colonised birch seedlings. In ourinvestigation, we did not quantify the nutrients in colon-ised or uncolonised ash patches, because the overall pro-portion of nutrients which could be mobilised from theash during the incubation period was expected to be small.Furthermore all of the material was required for analysesof 14C allocation.

The visual colonisation patterns strongly suggest thatthere were species speci¢c di¡erences in growth and car-bon allocation to the di¡erently treated substrates. Neithermycelial biomass nor branching ratios were measured inthe cups but branching responses to localised nutrient con-centrations are well documented [38,39] and may explainthe apparently thick and mat like mycelium.

The ectomycorrhizal fungi Piloderma sp. 1 and Ha-96-3have the potential to mobilise nutrients in HWA. Suchfungi may play an important role in forests where woodash has been applied to supplement nutrient losses causedby acidi¢cation or intensive biomass harvesting. Furtherstudies are needed to investigate the mechanisms by whichthese fungi mobilise nutrients in ash under ¢eld condi-tions.

Acknowledgements

This work was mainly supported by the Swedish Na-tional Energy Administration (STEM) and partly by theSwedish Council for Forestry and Agricultural Research(SJFR). We thank Ms. Anna Fossum for her technicalassistance in experiment I and Dr. Leif Johansson forhelp with SEM and X-ray microprobe analyses. We alsothank Dr. H-Oë Nohrstedt and Skog-Forsk for access tothe Torup experimental plots.

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Solubilisation and colonisation of wood ash by ectomycorrhizal fungi isolated from a wood ash fertilised spruce forest