Plant and Soil 252: 279–290, 2003.© 2003 Kluwer Academic Publishers. Printed in the Netherlands.

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The production of ectomycorrhizal mycelium in forests: Relation betweenforest nutrient status and local mineral sources

David Hagerberg1,3, Gunnar Thelin2 & Håkan Wallander1

1Dept. of Microbial Ecology, Lund University, Sweden. 2Dept. of Plant Ecology, Lund University, Sweden.3Corresponding author∗

Received 4 June 2002. Accepted in revised form 28 November 2002

Key words: Apatite, biotite, ectomycorrhizal fungi, forest, nutrient status, Pinus sylvestris

Abstract

Due to acid rain and nitrogen deposition, there is growing concern that other mineral nutrients, primarily potassiumand phosphorus, might limit forest production in boreal forests. Ectomycorrhizal (EcM) fungi are important forthe acquisition of potassium and phosphorus by trees. In a field investigation, the effects of poor potassium andphosphorus status of forest trees on the production of EcM mycelium were examined. The production of EcMmycelium was estimated in mesh bags containing sand, which were buried in the soil of forests of differentpotassium and phosphorus status. Mesh bags with 2% biotite or 1% apatite in sand were also buried to estimate theeffect of local sources of nutrients on the production of EcM mycelium. No clear relation could be found betweenthe production of EcM mycelium and nutrient status of the trees. Apatite stimulated the mycelial production,while biotite had no significant effect. EcM root production at the mesh bag surfaces was stimulated by apatiteamendment in a forest with poor phosphorus status. The contribution of EcM fungi to apatite weathering wasestimated by using rare earth elements (REE) as marker elements. The concentration of REE was 10 times higherin EcM roots, which had grown in contact with the outer surface of apatite-amended mesh bags than in EcM rootsgrown in contact with the biotite amended or sand-filled mesh bags. In a laboratory study, it was confirmed thatREE accumulated in the roots with very low amounts (<1 %) translocated to the shoots. The short-term effect ofEcM mycelium on the elemental composition of biotite and apatite was investigated and compared with biotite-and apatite-amended mesh bags buried in trenched soil plots, which were free from EcM fungi. The mesh bagssubjected to EcM fungi showed no difference in chemical composition after 17 months in the field. This studysuggests that trees respond to phosphorus limitation by increased exploitation of phosphorus-containing mineralsby ectomycorrhiza. However, the potential to ameliorate potassium limitation in a similar way appears to be low.

Abbreviations: ANOVA – analysis of variance; EcM – ectomycorrhizal; FE1 – field experiment 1; FE2 – field exper-iment 2; ICP-ES – inductively coupled plasma emission spectrophotometry; LE – laboratory experiment; ND – notdetermined; REE – rare earth elements; S-N-K – Student-Newman-Kuels procedure

Introduction

Sustainable forest production demands that the re-moval of mineral nutrients by harvest and leachingdoes not exceed the input of mineral nutrients by

∗ FAX No: +46-46-2224158.E-mail:David.Hagerberg@mbioekol.lu.se

weathering and deposition. Boreal temperate forestsare regarded as nitrogen limited (Tamm, 1991), butdue to the increased anthropogenic deposition of nitro-gen together with the decrease in the storage of basecations due to acid rain, other mineral nutrients, e.g.potassium, phosphorus, magnesium and calcium, maybecome limiting in some forest stands (reviewed byThelin, 2000). Modelling of weathering rates and mass

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balance calculations in southern Sweden indicates thatpotassium will be an element in short supply in thefuture (Barkman and Sverdrup, 1996).

The nutrient concentrations of the needles are of-ten used to determine deficiency (Foerst et al., 1987).However, the ratios of nutrients to nitrogen are of-ten used instead of absolute concentrations since theyare naturally less variable and at least as importantfor tree vitality (Linder, 1995; Thelin, 2000). In thisstudy, we employed four forest sites with varying po-tassium and phosphorus status, including sites withpotassium or phosphorus levels below the deficiencyvalues reported by Linder (1995) and Thelin et al.(2002).

Ectomycorrhizal (EcM) fungi live in symbiosiswith trees and colonise the soil forming an externalmycelium which contributes to the absorbing surfaceover which mineral nutrients can be taken up by thetrees (Bowen, 1973; Schack-Kirchner et al., 2000).The mineral nutrients are transported to the root tips,where they are translocated into the plant root in ex-change for photosynthetically derived carbon (Smithand Read, 1997). In a boreal forest, close to 100% ofthe fine roots may be colonised by EcM fungi (e.g.Kåren and Nylund, 1997).

Studying EcM fungi in the field has been problem-atic since it is difficult to discriminate between my-celia of EcM and saprophytic fungi. However recently,ingrowth mesh bags have been shown to provide goodestimates of EcM fungal growth (Wallander et al.,2001). Wallander et al., (2001) and Hagerberg andWallander (2002) found that 85% of the myceliumcontained in such mesh bags could be related to EcMfungi by comparing the mycelial production in meshbags inside and outside trenched plots. Soil trench-ing inhibits the growth of EcM fungi since the rootsof the host trees are excluded. They also investigatedthe δ13C value, which showed that the mycelia insidemesh bags had δ13C values similar to values of EcMfruit bodies and distinctly different from the values offruit bodies of saprophytic fungi

EcM fungi are able to mobilise and take up ni-trogen from organic matter (Read, 1991), and theyhave also been reported to supply the trees with po-tassium and phosphorus (reviewed by Marschner andDell, 1994) and magnesium (Jentschke et al., 2000). Inlaboratory experiments, EcM fungi have been shownto improve the uptake of potassium and phosphorusfrom poorly soluble minerals (Wallander, 2000a, b;Wallander and Wickman, 1999; Wallander et al.,1997) and van Breemen et al., (2000) suggest that EcM

fungi are able to selectively weather soil minerals inorder to release mineral nutrients.

In laboratory studies, Ekblad et al. (1995) andWallander and Wickman (1999) have found that po-tassium limitation decreases the growth of myceliumfrom EcM Pinus sylvestris. In contrast, the growth ofEcM mycelium was found to be stimulated by phos-phorus limitation (Ekblad et al., 1995; Wallander andNylund, 1992). This might reflect reduced carbon al-location to the roots in plants growing under potassiumlimitation and an increased allocation to roots in plantsgrowing under phosphorus limitation, as discussed byEricsson (1995).

By forming a dense mycelium, EcM fungi are ableto respond to local sources of organic matter (Bendingand Read, 1995; Carleton and Read, 1991; Finlay andRead, 1986), apatite (Bidartondo et al., 2001), woodash (Mahmood et al., 2001; Hagerberg and Wallander,2002), inorganic nitrogen and phosphorus (Brandes etal., 1998) and local areas of elevated pH (Erland etal. 1990). The dense mycelium is believed to promotethe uptake of elements (Unestam and Sun, 1995) andthe release of organic acids, which can increase thedegradation of organic matter and mineral weathering(reviewed by Dutton and Evans, 1996; Landeweert etal., 2001). Similarly, fine roots of Norway spruce re-spond to local sources of higher nutrient availabilityby increased root proliferation (George et al., 1997).

Dissolution of minerals is a slow process. It is thusdifficult to estimate the contribution of EcM fungi tothe weathering rate over short time periods. Since min-eral nutrients are continuously transported from theroots to the crown, the measurement of nutrient con-tent in EcM root tips is not a good way to estimate theflux of nutrients from a mineral source to the trees. Inthe present study, we investigated the possibility of us-ing rare earth elements (REE) as markers for mineralweathering. REE content can be very high in certainminerals such as apatite. Lanthanum has been shownto be taken up by EcM fungi and is transported tothe root tips. It accumulates in the fungal tissue androot cortex but does not easily penetrate into the stele(Robards and Robb, 1974; Vesk et al., 2000). Con-sequently, lanthanum is primarily found in the plantroots rather than in the foliage or stems, and the otherREE have the same distribution (Fu et al. 2001), in-dicating that they might also be retained in the rootcortex.

The aims of this study were to investigate (1) howthe production of EcM mycelium was affected by thepotassium and phosphorus status of their host trees in

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Table 1. Description of the forest sites used in this study

Forest site and Plantation Needle K content Needle K:N Needle P content Needle P:N pHKCl in the organic

reference number year (mg g−1) (Thelin et al., (mg g−1) (Thelin et al., horizon 1988

(Anonymous, 1986) (Thelin et al., 2002) (Thelin et al., 2002) (Berggren et al.,

2002) 2002) 1992)

1994 2000 1994 2000 1994 2000 1994 2000

Björstorp (L 3:1) 1941 3.83 4.20 31.2% 36.6% 1.36 1.20 11.1% 10.4% 2.4

Dyneboda (L 6:4) 1936 5.38 4.38 43.5% 49.8% 1.16 0.82 9.4% 9.3% 2.4

Ignaberga (L 7:1) 1951 3.66 3.92 29.6% 32.6% 1.89 1.46 15.3% 12.2% 2.4

Västra Torup (L 8:4) 1946 1.66 4.97 13.6% 41.4% 1.22 1.24 10.1% 10.4% 2.5

Deficiency levels 4.5 35% 1.3 10%

(Linder, 1995; Thelin

et al., 2002)

the field, (2) how the production of EcM myceliumwas affected by local mineral sources (biotite or apat-ite added to the mesh bag substrate), and how thecolonisation was related to the nutrient status of thetrees, (3) if REE originating from the added miner-als could be detected in root tips growing in contactwith the mesh bags and if we could use these elementsas a measure of mineral dissolution induced by theEcM fungi and (4) if the elemental composition ofthe mineral-amended substrates was changed by theactivity of EcM fungi.

Materials and methods

Forest sites

The field studies were conducted during the years1997 – 2000. Four monitoring sites in the countyof Skåne in southern Sweden used previously forinvestigations of forest damage (Anonymous, 1986)were selected for this study. All of them are plantedwith Norway spruce (Picea abies (L.) Karst.) on aspodosol. The sites were selected in order to be able toinvestigate forest stands with varying K and P statusaccording to Linder (1995) and Thelin et al. (2002).Table 1 shows the nutrient status at the different sitesin the years 1994 and 2000. At Björstorp, the K statuswere below the deficiency level for both of the years,although the K:N in the year of 2000 indicated animprovement of the status. The P status of the samesite was slightly above the deficiency level 1994, but

showed a decreasing trend with a P content slightly be-low deficiency level 2000. At Dyneboda, the K statuswas above the deficiency level both years, althoughthe K content had decreased towards the deficiencylevel between 1994 and 2000, whereas the P statuswas below deficiency level both years. At Ignaberga,the potassium status was below and the phosphorusstatus above the deficiency levels both of the years.Västra Torup exhibited a sufficient P status for bothyears, whereas the K status was below the deficiencylevel in 1994, but not in 2000. All sites exhibited a Castatus well above deficiency levels, with Ca contentsranging from 2.37 to 4.01 mg g−1 and Ca:N rangingfrom 19.3% to 33.0% (Thelin et al., 2002).

Experimental design

Two field and one laboratory experiment was per-formed. In field experiment 1 (FE 1), the effect of thenutrient status of the trees on the production of EcMmycelium was investigated. Furthermore, the colon-isation of local mineral sources by EcM myceliumwas investigated and the translocation of REE to fineroots was estimated. In field experiment 2 (FE2), theeffect of EcM mycelium on the elemental compositionin mineral-amended mesh bags was investigated, byincluding trenched plots where EcM mycelium couldnot grow (Wallander et al., 2001). The laboratory ex-periment (LE) was designed to investigate how REEtaken up by an EcM fungus were distributed in the hostplants.

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Table 2. Elemental composition of the minerals used in the studyas revealed by the lithium borate method (ASTM method D3682).FE1 – field experiment 1, FE2 – field experiment 2, LE – laboratoryexperiment, ND – not determined

Element Biotite (Moen) Apatite Apatite (Kemira)

(mg g−1) FE1, FE2 (Madagascar) (mg g−1) FE2

and LE (mg g−1) FE1

Al 68.2 0.21 ND

Ca 1.3 387 312

K 85.5 0.12 0.18

P ND 189 160

La 0.4 1.2 ND

Pr 0.008 0.3 ND

Nd 0.06 1.2 ND

Sm 0.004 0.1 ND

Eu 0.002 0.01 ND

Gd 0.008 0.1 ND

Tb 0.001 0.01 ND

Dy 0.002 0.04 ND

Ho 0.000 0.007 ND

Er 0.001 0.02 ND

Yb 0.002 0.01 ND

Table 3. Mesh bag and harvest years in the field experiments. FE1– field experiment 1, FE2 – field experiment 2, S – sand-filled meshbags, B – biotite-amended mesh bags, A – apatite-amended meshbags

Forest site Field experiment Mesh bag Year of harvest

Björstorp FE1 S and B 1998

Dyneboda FE1 S, B and A 1998 and 1999

Ignaberga FE1 S, B and A 1998 and 1999

FE2 B and A 2000

Västra Torup FE1 S and B 1998 and 1999

FE1

Ingrowth mesh bags (rectangular shape 5 × 10 cm,mesh size 50 µm) were filled with 120 g acid-washedsea sand (average grain size 0.9 mm, 99.6% SiO2, ‘Sil-versand 90’, Askania AB, Sweden). Some mesh bagswere filled with 120 g acid-washed sand containing2.4 g biotite (from a pegmatite in Moen, Norway, grainsize 0.050 – 0.63 mm) or 1.2 g apatite (originatingfrom Madagascar, Krantz GmbH, Bonn, FRG, grainsize 0.050 – 0.63 mm). The elemental compositionof the minerals is presented in Table 2. Sand-filledmesh bags and bags containing biotite were buriedat all sites, while apatite mesh bags where only bur-

ied at the Dyneboda and Ignaberga sites (Table 3).The mesh bags were buried in the organic horizon,where ectomycorrhizal roots and fungal hyphae aremost abundant. In each plot, 12 replicate sets (pairsor triplets) of mesh bags were buried, one containingsand and one (or two) amended with minerals (1 dmseparation). The mesh bags were buried in Septem-ber (autumn) 1997 (in total 156 bags). Four replicatesets of mesh bags were retrieved from each forest inOctober 1998, and the remaining replicate sets wereharvested in October 1999 (Table 3). During the exper-iment, some mesh bags were excavated and destroyedby animals, therefore, the 1999 samples from Björ-storp were omitted from the dataset and the numberof replicates from the other sites varied between 3and 7. In October 1999, also EcM roots growing incontact with the outer surface of the mesh bags weresampled at Dyneboda, Ignaberga and Västra Torupand analysed regarding elemental composition. Thetotal biomass of EcM roots in contact with the outersurface of the mesh bags was also estimated in samplesfrom Dyneboda and Ignaberga.

FE2Mesh bags, in the shape of a right-angled triangle withthe smaller sides being 7 cm, were filled with 30 gacid-washed sand or sand amended with either 0.6g biotite or 0.3 g apatite. The biotite was the sameas in FE1 but a different apatite was used (apatitti,KEMIRA, Finland, Table 2), since no more Madagas-can apatite was available. Five replicate sets of meshbags were buried in triplets in the Ignaberga forest(Table 3). The mesh bags were buried at a depth of3 cm in the 5.5 cm thick organic horizon. In order toobtain mycorrhizal-free controls, soil trenching wascarried out by inserting plastic tubes (25 cm long,diameter 16 cm) into the ground to exclude the EcMroots, preventing the growth of the external mycelium(Wallander et al., 2001). Five tubes were used andtriplets of mesh bags were buried in the organic ho-rizon (1 cm separation) at the same depth as the meshbags subjected to EcM fungi. The experiment startedin May 1999 and the mesh bags were harvested inOctober 2000.

LEMycorrhizal seedlings of Pinus sylvestris L. colonisedby Suillus variegatus were obtained according to themethod of Duddridge (1986) as modified by Finlay(1989). Each seedling (n = 4) was replanted in a bagmade of 100 µm nylon mesh (about 150 cm3 volume)

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filled with a mixture of acid-washed sand and peat (3:1v:v). This bag was placed in a plastic pot (9×9×9 cm)which was filled with the same substrate supplementedwith 1% (w:w) biotite (particle size, 0.05 – 0.25 µm).In this way, the substrate in the outer compartmentwas accessible only to fungal mycelium, whereas bothroots and EcM fungi could grow in the inner com-partment. The plants were grown for seven months inIngestad medium without K (Ingestad and Kähr, 1985;Nylund and Wallander, 1989). The root systems werewashed under tap water and the shoot and roots werefreeze-dried prior to elemental analysis.

Analyses

Fungal biomass in the mesh bags from FE1 was es-timated in two ways. (1) Water was added to themesh bag content to make a slurry, the floating my-celia were collected on a nylon mesh and the mycelialweight was estimated as the loss of ignition of thedried samples when combusted at 600 ◦C overnight.(2) Ergosterol was extracted from 10 g of mesh bagcontent as described by Wallander and Nylund (1992)and Wallander and Wickman (1999). The water con-tent of the mesh bags was estimated by measuring theweight loss of subsamples of the contents after dryingthem for two days at 70 ◦C.

The elemental composition of the biotite and apat-ites used in the experiments was determined usingthe lithium borate method (ASTM method D3682) atthe Department of Plant Ecology, Lund University,Lund, Sweden. The most available fractions of Al,Ca and K in the mesh bags retrieved in 1998 (FE1)were estimated by BaCl2 extraction. Subsamples of25 g from the mesh bags were extracted with 100 ml0.1 M BaC12 for one hour. The pH of the solutionswas measured and the concentrations of the elementswere analysed using inductively coupled plasma emis-sion spectrophotometry (ICP-ES) at the Department ofPlant Ecology, Lund University.

Roots sampled in FE1 and roots and shootssampled in LE, were digested with concentratedHNO3 for 1 week. The concentrations of Ca, K and Pwere analysed using ICP-ES and the concentrations ofLa, Nd, Sm, Eu, Tb and Yb (in FE 1 also Dy, Er, Gd,Ho and Pr) were analysed using inductively coupledplasma mass spectrometry at the Department of PlantEcology, Lund University.

The elemental composition (Al, Ca, K and P) ofthe sand/mineral mixtures outside and inside trenchedplots (FE2) was investigated in subsamples of 1.0 g

from mesh bags amended with biotite or apatite. Thesubsamples were digested with HNO3 for 1 week. Thesolutions were subsequently diluted 1:10 and the con-centrations of Al, Ca, K and P were analysed usingICP-ES.

Statistical evaluations

All the analyses were performed using the softwareSPSS 11.0 (SPSS, Chicago, USA). The effect of forestsite in FE1 on the production of fungal mycelium insand mesh bags was evaluated with a one-way analysisof variance (ANOVA). The effects of forest site andmineral in FE 1 on the fungal biomass in the meshbags were evaluated with two-way ANOVA. Whenconsidering the effects of biotite, all forest sites wereincluded in the two-way ANOVA, whereas only Dyne-boda and Ignaberga were included when the effect ofapatite was evaluated. Separate ANOVAs were madefor each year. Heterogeneity of variance was detectedwhen evaluating the effects on fungal biomass (my-celial weight), water content and BaCl2-extractableAl and K, and pH and the logarithmic values wereused in the ANOVA (Sokal and Rohlf, 1995). In FE2,the effect of treatments were evaluated using a one-way ANOVA. Differences with a probability less then0.05 were regarded as significant and were evaluatedusing the Student-Newman-Keuls procedure (S-N-K,Sokal and Rohlf, 1969) when considering multiplecomparisons.

Results

The fungal biomass in the mesh bags containing onlysand in FE 1, estimated as the mycelial weight,ranged from 0.04 to 0.28 mg g−1 dw and the er-gosterol content ranged from 0.07 to 0.47 µg g−1

dw (Table 4). There was no statistically significantdifference between the sites (Table 4).

Apatite amendment significantly increased thefungal biomass in the mesh bags in FE1, comparedwith sand-filled mesh bags in 1998 when estimatedboth as mycelial weight and ergosterol content (p< 0.01 and p = 0.04 respectively, Table 4), thoughthere was no effect 1999. The fungal biomass wassimilar in biotite-amended mesh bags and sand-filled.No significant interactions between site and mineralamendment were found (Table 4).

The biomass of EcM roots growing in contact withthe outer surface of the mesh bags (FE1, Figure 1) was

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Table 4. Fungal biomass estimated as the combusted weight of extracted mycelia from mesh bagsand ergosterol content of mesh bags in field experiment 1. The values are presented as the meanvalue for the mesh bags ± the standard error, S – sand-filled mesh bags, B – biotite-amendedmesh bags, A – apatite-amended mesh bags, ND – not determined

Forest site and mesh bag Mycelial mass (mg g−1 dw) Ergosterol content (µg g−1 dw)

substrate

1998a 1999b 1998c 1999d

Björstorp

S 0.18 ± 0.06 ND 0.14 ± 0.03 ND

B 0.20 ± 0.05 ND 0.22 ± 0.04 ND

Dyneboda

S 0.12 ± 0.00 0.17 ± 0.08 0.14 ± 0.04 0.09 ± 0.02

B 0.19 ± 0.03 0.20 ± 0.02 0.18 ±0.01 0.08 ± 0.04

A 0.18 ± 0.03 0.26 ± 0.05 0.47 ± 0.16 0.11 ± 0.02

Ignaberga

S 0.16 ± 0.02 0.19 ± 0.07 0.18 ± 0.03 0.12 ± 0.05

B 0.25 ± 0.09 0.18 ± 0.03 0.20 ± 0.02 0.07 ± 0.02

A 0.28 ± 0.02 0.26 ± 0.05 0.29 ± 0.04 0.07 ± 0.02

Västra Torup

S 0.14 ± 0.01 0.04 ± 0.03 0.30 ± 0.09 0.07 ± 0.02

B 0.23 ± 0.06 0.19 ± 0.11 0.30 ± 0.03 0.13 ± 0.04

ANOVA

Effects of site on fungal biomass in sand-filled bags

Site p=0.44 p=0.052 p=0.18 p=0.58

ANOVA

Effects of biotite and site on the fungal biomass in mesh bag

Site p=0.83 p=0.01 p=0.02 p=0.91

Mineral p=0.14 p=0.11 p=0.25 p=0.93

Site ∗ Mineral p=0.52 p=0.41 p=0.82 p=0.29

ANOVA

Effects of apatite and site on the fungal biomass in mesh bags

Site p=0.02 p=0.23 p=0.52 p=0.96

Mineral p<0.01 p=0.06 p=0.04 p=0.71

Site ∗ Mineral p=0.22 p=0.20 p=0.26 p=0.25

an = 4 except for Dyneboda B, Ignaberga S and B and Vastra Torup B (n = 3).bDyneboda n = 7, Ignaberga n = 6 and V Torup n = 4.cn = 4 except for Ignaberga S (n = 3).dn = 6 except for Dyneboda B (n = 7) and V Torup (n = 4).

significantly higher 1999 at the apatite-amended meshbags (0.23 mg dw bag−1) than at the sand-filled meshbags (0.07 mg dw bag−1) at the Dyneboda site, butnot at the Ignaberga site (S-N-K, p < 0.05). Biotiteamendment did not effect EcM root production.

The levels of BaC12-extractable Al, Ca, and Kmeasured in FE1 1998 were higher (p < 0.001) in thebiotite-amended mesh bags (1.2 ± 0.2 µg Al g−1 dw,3.9 ± 0.4 µg Ca g−1 dw, and 35 ± 2 µg K g−1 dw,

average of means of the forest sites) than in the sand-filled mesh bags (0.18 ± 0.04 µg Al g−1 dw, 1.4 ± 0.2µg Ca g−1 dw and 2.3 ± 0.2 µg K g−1 dw, averageof means of the forest sites). Apatite-amended meshbags contained an elevated extractable level of Ca (5.2± 0.1 µg g−1 dw, p < 0.001; average of means ofthe forest sites). The pHBaCI2 in the sand-filled meshbags was 4.56 ± 0.03 higher in the apatite-amendedmesh bags (p = 0.001). The water content in the sand-

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Figure 1. Biomass of FcM roots growing in contact with the outer surface of the mesh bags at the Dyneboda and Ignaberga sites estimatedin FE1, October 1999. The bars represent the standard error of the mean values (Dyneboda n = 7, Ignaberga n = 6). Columns denoted bythe same letter are not significant on the 0.05 level (S-N-K). Designations: S – sand-filled mesh bags, B – biotite-amended mesh bags, A –apatite-amended mesh bags.

filled mesh bags in FE1 was 2.7 ± 0.2% 1998 and 1.4± 0.2% 1999 (average of means of the forest sites).Biotite amendment increased the water content to 4.7± 0.5% and 2.6 ± 0.5%, respectively, for the 2 years(p < 0.001 for both years), while apatite amendmentdid not influence the water content significantly.

In FE1, the P content of the EcM roots in contactwith the outer surface of the sand-filled mesh bags didnot differ between the investigated sites (Figure 2). AtIgnaberga, there was a significantly higher P content inEcM roots at apatite-amended mesh bags compared tothe other mesh bags (S-N-K, p < 0.05). No increase inEcM root P content could be found at apatite-amendedmesh bags at Dyneboda. The content of Ca in theEcM roots ranged from 1 to 1400 µg g−1 dw, whileK ranged from 4 to 8 µg g−1 dw. Both Ca and K con-tent showed a considerable variation with no signficantdifferences between mesh bag types or sites

The REE supplied from biotite in LE accumulatedto a large extent in the roots shown by the 30 – 160times higher concentrations in the roots than in theshoots (p = 0.016), whereas the content of Ca was sim-ilar between the roots and shoots (Table 5). The con-tents of REE in the EcM roots growing in contact withthe outer surface of the apatite-amended mesh bagsat Ignaberga and Dyneboda (FE1) was significantlyhigher (p < 0.001) than in the EcM roots growing incontact with the sand-filled mesh bags (Figure 3). Theeffect was more pronounced for lighter REE (La toGd) than for the heavier ones (Figure 3). EcM roots

Table 5. Content of Ca and REE in roots and shoots ofPinus sylvestris seedlings colonised by Suillus varie-gatus in pots with an outer compartment, which wasonly accesible to the fungus and to which biotite hadbeen added to the growth substrate

Element Roots Shoots

Ca, mg g−1 2.2 ± 0.2 1.8 ± 0.3

La, µg g−1 0.59 ± 0.07 0.014 ± 0.002

Nd, µg g−1 0.81 ± 0.11 0.014 ± 0.001

Sm, µg g−1 0.16 ± 0.02 0.001 ± 0.00006

Eu, µg g−1 0.03 ± 0.005 < 0.001

Tb, µg g−1 0.023 ± 0.003 < 0.001

Yb, µg g−1 0.045 ± 0.007 0.0015 ± 0.0001

growing in contact with the biotite-amended meshbags showed no increase in REE compared to sand-filled mesh bags (data not shown). The total contentof lighter REE in EcM roots growing in contact withsand-filled mesh bags and apatite amended mesh bagswas 2.2 ± 0.9 µg g−1 and 27 ± 6 µg g−1 respectively,at the Dyneboda site. The corresponding figures forIgnaberga were 2.8 ± 1.0 µg g−1 and 45 ± 13 µg g−1.These values were used to calculate the total amount oflighter REE that had been transported from the apatiteand accumulated in the EcM roots during the period of2 years. The values were 6.1 µg bag−1 lighter REE inthe Dyneboda site and 4.3 µg bag−1 at Ignaberga (us-ing the content of lighter REE in EcM roots from the

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Figure 2. The content of P in EcM roots growing in contact with the outer surface of the mesh bags in FE1 (October 1999). The bars representthe standard error of the mean values. Columns denoted by the same letter are not significant on the 0.05 level (S-N-K). S – sand-filled meshbags, B – biotite-amended mesh bags, A – apatite-amended mesh bags.

sand-filled mesh bags as a background value). Thesevalues correspond to 0.18% of the original amount oflighter REE supplied in the apatite-sand mixture, inDyneboda and 0.13% in Ignaberga.

In FE2, the elemental composition of the contentsof the mesh bags could not be used to determine thedissolution of the minerals, since the minerals werenot uniformly distributed throughout the bags. Insteadthe ratios of K to Al (biotite) or Ca to P (apatite) wereused to determine whether plant nutrients in low sup-ply (K, P) were removed to a greater extent than Al orCa from the mesh bags by EcM fungi.

The original value of HNO3-digestible K:Al was0.91 in the biotite-amended mesh bags (Figure 4A).At harvest K:Al in the mesh bags subjected to mycor-rhizas was 0.97 and the non-mycorrhizal was 0.94; themycorrhizal K:Al being significantly higher than theoriginal value (S-N-K, p < 0.05). In apatite-amendedmesh bags, where the original Ca:P was 1.98, theratios of the mycorrhizal and non-mycorrhizal meshbags did not change significantly during the experi-ment; both being 2.08 (Figure 4B).

Discussion

The assessment of EcM biomass showed that the meshbags in FE 1 were well colonised by fungal mycelium(Table 4). The EcM biomass in sand-filled mesh bags

corresponded to a production of 110 kg ha−1 down to5 cm depth in the soil. Wallander et al. (2001) estim-ated the mycelial production in similar mesh bags to be125 kg ha−1 in another Norway spruce forest in south-ern Sweden. The values for EcM biomass estimatedthrough the ergosterol content (FE 1) indicate that themycelium contain on average 1.2 µg ergosterol mg−1

dw, which is at the lower end of the range of valuesestimated for EcM fungi in pure culture (reviewed byOlsson, 1998); 1.3 – 15.1 µg mg−1dw.

In this study, we could not find any clear relationbetween the production of EcM mycelium and the nu-trient status of the forest (Table 4). The K and P statusof the mature trees used in this study is probably notas poor as for small pine seedlings used in laboratorystudies of K and P limitation (Ekblad et al., 1995; Wal-lander and Nylund, 1992; Wallander and Wickman,1999) where such a relation was found. In laboratoryexperiments, nutrient treatments can be applied thatproduce severe nutrient deficiency symptoms whichprobably are unlikely to occur in natural ecosystems.

Apatite amendment seemed to stimulate the pro-duction of EcM mycelium, whereas biotite amend-ment did not (Table 4). Apatite amendment stimulatedthe EcM fungi more when the forest experiencedP deficiency, as indicated by increased amounts ofEcM root biomass around the apatite-amended meshbags under these conditions (Figure 1). This response

287

Figure 3. The content of various REE in the EcM roots growing in contact with the outer surface of the sand-filled and apatite-amendedmesh bags, normalised to the apatite used in FE1, in FE1 (October 1999). S – sand-filled mesh bags, B – biotite-amended mesh bags, A –apatite-amended mesh bags.

to P deficiency is a common feature among plants(Robinson, 1994). P was found to accumulate in theEcM roots surrounding apatite amended mesh bagsat Ignaberga, but not at Dyneboda (Figure 2). P isknown to accumulate in the fungal mantle of mycor-rhizal roots when supplied in excess amounts (Finlayand Read, 1986; Smith and Read, 1997) but this storedP resource will rapidly be transferred to the host underconditions of low P availability (Morrison, 1962). Thisappeared to have happened at Dyneboda, while theelevated P in mycorrhizal roots at Ignaberga indicatesthat the trees had a sufficient P status. Although wecould not estimate the flux of P from the apatite tothe host trees in the present study, it seems likely thatthe increased production of EcM roots at Dyneboda isa response of the trees to increase the transport of Pfrom the apatite resource. Subsequently, it increasesthe possibility for the EcM fungi to access carbon formycelial production.

Wallander et al. (2002, 2003) collected rhizo-morphs from the same 12 mesh bags at Ignaberga(1998 harvest, FE1) and investigated the elementalcomposition of these rhizomorphs with particle in-duced X-ray emission. They found that apatite amend-ment did not increase the P content of the rhizo-morphs, while the Ca content increased considerablyand calcium oxalate crystals were sometimes foundon the surface of the rhizomorphs. This shows thatthe fungi had interacted with the apatite mineral. Theefficiency of uptake and rate of transport of mineral

nutrients varies between EcM fungal species (Bidar-tondo et al., 2001) and a higher transport rate willnot necessarily result in an increased P content. Thefast-growing EcM species Paxillus involutus has beenfound to colonise mesh bags at Ignaberga (Wallanderet al., 2003) and this fungus has been shown to bemore efficient than other EcM fungi in P translocationto host plants (Bidartondo et al., 2001; Colpaert et al.,1999; Cumming, 1996). An investigation of the spe-cies composition inside the mesh bags would revealif apatite-amended mesh bags were colonised by cer-tain fungi known for higher uptake and translocationcapacity.

The high accumulation of REE in EcM roots grow-ing in contact with the outer surface of the apatite-amended mesh bags (Figure 3) shows that these canbe used as a measure of apatite weathering, while theaccumulation of plant nutrients such as P in the roottips is dependent on the P status of the trees, and canthus not be used in the same way (Fig. 3). The LEconfirmed that REE accumulate in the roots, with verylow amounts being allocated to the shoots.

The translocation of 0.18% lighter REE in Dyne-boda and 0.13% in Ignaberga from apatite to EcMroots is probably an underestimate of the true dis-solution rate, since the content of lighter REE in themycelium and in the solution of the mesh bag sub-strates were not estimated. Some EcM root tips mayalso have degenerated during the experiment, sinceonly about 50% of the EcM root tips have a lifetime

288

Figure 4. (A) The K:Al in mesh bags amended with biotite in FE2. (B) The Ca:P in mesh bags amended with apatite in FE2. The bars respresentthe standard error of the mean values. Points denoted by the same letter are not significant on the 0.05 level (S-N-K).

over 2 years (Majdi et al., 2001). Wallander et al.(2002) estimated the dissolution rate of apatite in themesh bags of FE1 to be 0.04% per year based on theCa content of rhizomorphs collected from the apatite-amended bags. These dissolution rates for apatite areslightly higher than those calculated for apatite inforest soil (0.01 – 0.015% annually) using models ofchemical weathering (Sverdrup and Warfvinge 1993).However, freshly ground apatite, used in this study,has a higher dissolution rate.

Compared with apatite, biotite has a somewhatlower content of La and Nd, and the contents ofthe other REE are much lower (Table 2). The con-tents of La in root tips growing in contact with thebiotite-amended mesh bags were not elevated, and thecontents of REE were at the same level as for the EcMroots growing at the sand-filled bags. The differencebetween apatite and biotite might be explained by themuch slower dissolution rate of biotite than apatite.

EcM fungi did not preferentially remove K frombiotite-amended mesh bags or P from apatite-amendedbags during the 17 months of the study (FE2), asrevealed by K:Al and Ca:P values (Figure 4).

The present study showed that a local source ofK did not stimulate the production of external EcMmycelia or mycorrhizal root tips, even when the foresttrees had low foliage K status. This suggests that the

potential to ameliorate K limitation by increased ex-ploitation of K-containing minerals is low. In contrast,a local source of P stimulated fine root productionand, to some extent, EcM mycelial production in theforest with poor foliage P status. This suggests thattree P deficiency may be ameliorated by exploitationby ectomycorrhiza of P-containing minerals such asapatite.

Acknowledgements

This work was supported by the Swedish NationalEnergy Administration (STEM). We would like tothank Ulrika Rosengren for helping us to find suit-able forest sites for the investigation. Tomas Johanssonand Susanne Erland made valuable comments on themanuscript.

References

Anonymous 1986 Fasta Skogsprovytor i Skåne, Rapport 1. CountyAdministrative Board of Kristianstad, Kristianstad, Sweden. 49pp.

Barkman A and Sverdrup H 1996 Critical Loads of Acidity andNutrient Imbalance for Forest Ecosystems in Skåne, Report 1.Department of Chemical technology II, Lund University, Lund,Sweden. 64 pp.

289

Bending G D and Read D J 1995 The structure and function of thevegetative mycelium of ectomycorrhizal plants V. Foraging be-haviour and translocation of nutrients from exploited litter. NewPhytol. 130, 401–409.

Berggren H, Mattiason G and Nihlgàrd B 1992 Fasta skogsprovytori Skåne, Rapport County Administrative Board of KristianstadKristianstad, Sweden. 53 pp.

Bidartondo M I, Ek H, Wallander H and Söderström B 2001 Donutrient additions alter carbon sink strength of ectomycorrhizalfungi? New Phytol. 151, 543–550.

Bowen G D 1973 Mineral nutrition in ectomycorrhizae. In Physiolo-gical Ecology, Ectomycorrhizae. Their Ecology and Physiology.Eds. Marks G C and Kozlowski T T. pp. 151–205. AcademicPress, New York, USA.

Brandes B, Godbold D L, Kuhn A J and Jentschke G 1998 Nitrogenand phosphorus acquisation by the mycelium of the ectomycor-rhizal fungus Paxillus involutus and its effect on host nutrition.New Phytol. 140, 735–743.

Carleton T J and Read D J 1991 Ectomycorrhizas and nutrienttransfer in conifer – feather moss ecosystems. Can. J. Bot. 69,778–785.

Colpaert J V, van Tichelen K K, van Assche J A and van LaereA 1999 Short-term phosphorus uptake rates in mycorrhizal andnon-mycorrhizal roots of intact Pinus sylvestris seedlings. NewPhytol. 143, 589–597.

Cumming J R 1996 Phosphate-limitation physiology in ectomy-corrhizal pitch pine (Pinus rigida) seedlings. Tree-Physiol. 16,977–983.

Duddridge J A 1986 The development and ultrastructure of ectomy-corrhizas III. Compatible and incompatible interactions betweenSuillus grevillei and 11 species of ectomycorrhizal host in vitroin the absence of exogenous carbohydrate. New Phytol. 103,457–464.

Dutton M V and Evans C S 1996 Oxalate production by fungi: itsrole in pathogenicity and ecology in the soil environment. Can.J. Microbiol. 42, 881–895.

Ekblad A, Wallander H, Carisson R and Huss-Danell K 1995 Fungalbiomass in roots and extramatrical mycelium in relation to mac-ronutrients and plant biomass of ectomycorrhizal Pinus sylvestrisand Alnus incana. New Phytol. 131, 443–451.

Ericsson T 1995 Growth and shoot:root ratio of seedlings in relationto nutrient availability. Plant Soil 168–169, 205–214.

Erland S, Söderström B and Andersson 5 1990 Effects of limingon ectomycorrhizal fungi infecting Pinus sylvestris L. II. Growthrates in pure culture at different pH values compared to growthrates in symbiosis with the host plant. New Phytol. 115, 683–688.

Finlay R D 1989 Functional aspects of phosphorus uptake and car-bon translocation in incompatible ectomycorrhizal associoationsbetween Pinus sylvestris and Suillus grevillei and Boletinuscavipes. New Phytol. 112, 185–192.

Finlay RD and Read D J 1986 The structure and function of the ve-getative mycelium of ectomycorrhizal plants II. The uptake anddistribution of phosphorus by mycelial strands interconnectinghost plants. New Phytol. 103, 157–166.

Foerst K, Sauter U and Neuerburg W 1987 Bericht zur Ehr-nahrungssituation der Wälder in Bayern und über die Anlagevon Walddungeversuchen. Bayerische Forstliche Versuchs- undForschungs-anstalt, Munich, FRG.

Fu FF, Akagi T, Yabuki S and Iwaki M 2001 The variation of REE(rare earth elements) patterns in soil-grown plants: a new proxyfor the source of rare earth elements and silicon in plants. PlantSoil 235, 53–64.

George E, Seith B, Shaeffer C and Marschner H 1997 Responsesof Picea, Pinus and Pseudotsuga roots to heterogeneous nutrientdistribution in soil. Tree Physiol. 17, 39–45.

Hagerberg D and Wallander H 2002 The impact of forest residueremoval and wood ash amendment on the growth of the ec-tomycorrhizal external mycelium. FEMS Microbiol. Ecol. 39,139–146.

Ingestad T and Kähr M 1985 Nutrition and growth of coniferousseedlings at varied relative nitrogen addition rate. Physiol. Plant.65, 109–116.

Jentschke G, Brandes B, Kuhn A J, Schröder W H, Becker J Sand Godbold D L 2000 The mycorrhizal fungus Paxillus involu-tus transports magnesium to Norway spruce seedlings. Evidencefrom stable isotope labelling. Plant Soil 220, 243–246.

Kåren O and Nylund J-K 1997 Effects of ammonium sulphate onthe community structure and biomass of ectomycorrhizal fungiin a Norway spruce stand in southwestern Sweden. Can. J. Bot.75, 1628–1642.

Landeweert R, Hoffland F, Finlay R D, Kuyper T W and van Bree-men N 2001 Linking plants to rocks: Ectomycorrhizal fungimobilize nutrients from minerals. Trends in Ecol. Evol. 16,248–254.

Linder S 1995 Foliar analysis for detecting and correcting nutrientimbalances in Norway spruce. Ecol. Bull. 44, 178–196.

Mahmood S, Finlay R D, Erland S and Wallander H 2001 Sol-ubilisation and colonisation of wood ash by ectomycorrhizalfungi isolated from a wood ash fertilised spruce forest. FEMSMicrobiol. Ecol. 35, 151–161.

Majdi H, Damm F and Nylund J-E 2001 Longevity of mycorrhizalroots depend on branching order and nutrient availability. NewPhytol. 150, 195–202.

Marschner H and Dell B 1994 Nutrient uptake in mycorrhizalsymbiosis. Plant Soil 159, 89–102.

Morrison T M 1962 Absorption of phosphorus from soils bymycorrhizal plants. New Phytol. 56, 247–257.

Nylund J-F and Wallander H 1989 Effects of ectomycorrhiza onhost growth and carbon balance in a semi-hydroponic cultivationsystem. New Phytol. 112, 389–398.

Olsson P-A 1998 The External Mycorrhizal Mycelium. Growthand Interactions with Saprophytic Microorganisms. PhD thesis,Dept. Microbial Ecology, Lund University, Lund Sweden. 41 pp.

Read D J 1991 Mycorrhizas in Ecosystems. Experientia 47, 376–391.

Robards A W and Robb M F 1974 The entry of ions and moleculesinto roots. An investigation using electron opaque tracers. Planta120, 1–12.

Robinson D 1994 Tansley review no 73. The responses of plants tonon-uniform supplies of nutrients. New Phytol. 127, 635–674.

Schack-Kirchner H, Wilpert K V and Hildebrand E F 2000 Thespatial distribution of soil hyphae in structured spruce-forest soil.Plant Soil 224, 195–205

Smith S E and Read D J 1997 Mycorrhizal Symbiosis. AcademicPress, San Diego, USA. 605 pp.

Sokal R R and Rohlf F J 1969 Biometry. 1st ed. W H Freeman, SanFrancisco, USA. 605 pp.

Sokal R R and Rohlf F J 1995 Biometry. 3rd ed. W H Freeman andCompany, New York, USA. 887 pp.

Sverdrup H and Warfvinge P 1993 Calculating field weathering ratesusing a mechanistic geochemical model – PROFILE. J. Appl.Geochem. 8, 273–283.

Tamm C-O 1991 Nitrogen in terrestrial ecosystems. Questions ofproductivity. Springer Verlag, Berlin, FRG. 115 pp.

Thelin G 2000 Nutrient Imbalance in Norway Spruce. PhD thesis,Dept. Plant Ecology Lund University, Lund, Sweden. 44 pp.

290

Thelin G, Rosengren U and Nihlgård B 2002 Barrkemi på SkånskaGran- och Tallprovytor, Rapport 20. County administrative boardof Skåne, Malmö, Sweden. 35 pp.

Unestam T and Sun T P 1995 Extramatrical structures of hydro-phobic and hydrophilic ectomycorrhizal fungi. Mycorrhiza 5,301–311.

van Breemen N, Finlay R. Lundström U, Jongmans A G, GieslerR and Olsson M 2000 Mycorrhizal weathering: A true case ofmineral plant nutrition? Biogeochemistry 49, 53–67.

Vesk P A, Ashford A E, Markovina A L and Allaway W G2000 Apoplasmic barriers and their significance in the exo-dermis and sheath of Eucalyptus pilularis-Pisolithus tinctoriusectomycorrhizas. New Phytol. 145, 333–346.

Wallander H 2000a Uptake of P from apatite by Pinus sylvestrisseedlings colonised by different ectomycorrhizal fungi. PlantSoil 218, 249–256.

Wallander H 2000b Use of strontium isotopes and folar K contentto estimate weathering of biotite induced by pine seedlings col-onised by ectomycorrhizal fungi from two different soils. PlantSoil 222, 215–229.

Wallander H, Johansson L and Pallon J 2002 PIXE analysis to estim-ate the elemental composition of ectomycorrhizal rhizomorphs

grown in contact with different minerals in forest soil. FEMSMicrobiol. Ecol. 39, 147–156.

Wallander H, Mahmood S, Hagerberg D, Johansson L and PallonJ 2003 The use of PIXE to estimate the elemental composi-tion of ectomycorrhizal mycelia identified with PCR/RFLP andgrown in contact with apatite or wood ash in forest soil. FEMSMicrobiol. Ecol. (In press).

Wallander H, Nilsson L O, Hagerberg D and Bååth E 2001 Estim-ation of the biomass and production of external mycelium ofectomycorrhizal fungi in the field. New Phytol. 151, 753–760.

Wallander H and Nylund J-E 1992 Effects of excess nitrogen andphosphorus starvation on the extramatrical mycelium of ectomy-corrhizas of Pinus sylvestris L. New Phytol. 120, 495–503.

Wallander H and Wickman T 1999 Biotite and microcline as po-tassium sources in ectomycorrhizal and non-mycorrhizal Pinussylvestris seedlings. Mycorrhiza 9, 25–32.

Wallander H, Wickman T and Jacks G 1997 Apatite as a P sourcein mycorrhizal and non-mycorrhizal Pinus sylvestris seedlings.Plant Soil 196, 123–131.

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