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531

Research

Blackwell Publishing, Ltd.

Mycorrhizas improve nitrogen nutrition of

Trifolium repens

after 8 yr of selection under elevated atmospheric

CO

2

partial pressure

Hannes Gamper

1,3

, Ueli A. Hartwig

2

and Adrian Leuchtmann

1

1

Swiss Federal Institute of Technology (ETH Zürich), Geobotanical Institute, Zollikerstrasse 107, CH-8008 Zürich, Switzerland;

2

Academia Engiadina,

Quadratscha 18, CH-7503 Samedan, Switzerland;

3

Present address: Department of Biology, PO Box 373, University of York, Heslington, York, YO10 5YW, UK

Summary

• Altered environmental conditions may change populations of arbuscular mycor-rhizal fungi and thereby affect mycorrhizal functioning. We investigated whether8 yr of free-air CO

2

enrichment has selected fungi that differently influence thenutrition and growth of host plants.• In a controlled pot experiment, two sets of seven randomly picked single sporeisolates, originating from field plots of elevated (60 Pa) or ambient CO

2

partial pres-sure (pCO

2

), were inoculated on nodulated

Trifolium repens

(white clover) plants.Fungal isolates belonged to the

Glomus claroideum

or

Glomus intraradices

speciescomplex, and host plants were clonal micropropagates derived from nine genets.• Total nitrogen (N) concentration was increased in leaves of plants inoculated withfungal isolates from elevated-pCO

2

plots. These isolates took up nearly twice as muchN from the soil as isolates from ambient-pCO

2

plots and showed much greater stim-ulation of biological N

2

fixation. The morpho-species identity of isolates had a morepronounced effect on N

2

fixation and on root length colonized than isolate identity.• We conclude that rising atmospheric pCO

2

may select for fungal strains that willhelp their host plants to meet increased N demands.

Key words:

arbuscular mycorrhizal fungi (AMF), global environmental change,

Glomus claroideum

,

Glomus intraradices

, nitrogen acquisition, selection, symbioticfunctioning,

Trifolium repens

(white clover).

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©

New Phytologist

(2005)

doi

: 10.1111/j.1469-8137.2005.01440.x

Author for correspondence:

Hannes Gamper Tel: +44 (0) 1904 328553 Fax: +44 (0) 1904 328510 Email: hag500@york.ac.uk

Received:

7 January 2005

Accepted:

8 March 2005

Introduction

Arbuscular mycorrhizal fungi (AMF, Glomeromycota) are aubiquitous group of soil fungi closely involved in the cyclingof nutrients between plants and soil ( Jakobsen

et al

., 1992;Smith & Read, 1997). They depend exclusively on organiccarbon (C) sources provided by their host plants and thusare likely to respond to environmentally induced alteration inplant photosynthesis. Responses of mycorrhizas to conditionsof elevated atmospheric CO

2

partial pressure (pCO

2

) arevariable and diverse (Staddon & Fitter, 1998; Rillig & Allen,1999; Fitter

et al

., 2000; Rillig

et al

., 2002; Treseder, 2004).The most notable observed response to elevated pCO

2

is an

enlargement of the extraradical hyphal network and thusthe surfaces for nutrient absorption (Klironomos

et al

., 1998;Sanders

et al

., 1998; Staddon

et al

., 2004).Previous research on the effects of elevated atmospheric

CO

2

on AMF has mainly focused on increased allocation ofphotosynthetates to mycorrhizas. Whether host plants directlychannel the flow of C to their symbiotic root fungi or whetherthe latter just profit from an increased C supply of a leaky rootsystem is not known (Fitter

et al

., 2000). Moreover, plantmineral nutrient deficiency caused by stimulation of photo-synthesis under elevated atmospheric pCO

2

has rarely beenconsidered. Host plants may be able to favour mycorrhizalfungal strains that are beneficial in terms of mineral nutrition.

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A recent meta-analysis of field studies revealed thatmycorrhizal fungal abundance is increased disproportion-ately relative to root length under elevated pCO

2

conditions(Treseder, 2004). In the same study, Treseder put forward theplant investment hypothesis to explain changes in mycor-rhizal fungal colonization rates upon either phosphorus (P)and nitrogen (N) fertilization or atmospheric CO

2

fumiga-tion. Under this hypothesis, plants adjust allocation of C tomycorrhizal fungi according to the degree to which plantgrowth is nutrient limited (Treseder, 2004). If plant controlover C flows exists, as suggested by the term ‘investment’,plants may preferentially stimulate growth of particularsymbionts and thus change the composition of fungal strainassemblages according to their needs. Plant-mediated selec-tion may be hypothesized to have happened as a result ofprolonged exposure to free-air CO

2

enrichment (FACE).Conditions of elevated atmospheric pCO

2

under FACEare coupled with plant nutrient deficiency (Bazzaz, 1990;Fischer

et al

., 1997; Suter

et al

., 2002; Pendall

et al

., 2004) andshould favour mycorrhizal fungal strains that are more effi-cient in nutrient acquisition and thus more beneficial. Con-versely, under mineral nutrient fertilization or anthropogenicnitrogen (N) deposition, an increase in the abundance ofless beneficial AMF strains has been found (Johnson, 1993;Egerton-Warburton & Allen, 2000).

Adaptive changes in AMF assemblages in response toaltered nutritional conditions may take place via preferentialproliferation and competition. There is considerable variabil-ity among AMF strains or taxa with respect to nutrientacquisition and transfer capabilities [for phosphorus (P),e.g. Jakobsen

et al

., 1992; Munkvold

et al

., 2004; Smith

et al

.,2004; for N, e.g. Hawkins

et al

., 2000; Azcon

et al

., 2001],which is a precondition for selection. Nutrition-mediatedselection was observed in a nonrelated plant pathogen inresponse to elevated pCO

2

(Chakraborty & Datta, 2003) andin a natural yeast population in response to C or N limitation(Goddard & Bradford, 2003).

The long-term response of fertile grasslands is thought todepend heavily on legumes and their associated root symbi-oses (Soussana

et al

., 1996; Zanetti

et al

., 1997; Gamper

et al

., 2004). This group of plants usually forms tripartitenutritional symbioses with bacteria, collectively referred to asrhizobia, and AMF. In this super-symbiotic system, the nod-ule symbiosis provides biologically fixed N, whereas AMFenhance mainly the acquisition of poorly available P. Inexchange, the legume hosts supply their bacterial and fungalroot symbionts with photosynthates. Therefore, under nutrient-poor soil conditions, a true reciprocal exchange of limitingresources results in mutual benefit for all three symbiotic part-ners (Olesniewicz & Thomas, 1999; Schulze, 2004).

Maintenance of both the mycorrhizal and nodule symbi-oses is energetically costly. Each root symbiosis can accountfor up to 20% of net photosynthesis, which is diverted fromhost plant metabolism (Bethlenfalvay

et al

., 1985; Murphy,

1986; Jakobsen & Rosendahl, 1990; Schulze, 2004). Therefore,legumes harbouring both symbioses may acquire symbiontsthat are particularly efficient in carbohydrate use, because ofcompetition between the two root symbionts, whereas grassesunder conditions of elevated pCO

2

may preferentially choosesymbionts conferring N nutritional benefit, because of severeN deficiency. In either case, selection for more beneficialfungal strains under exposure to contrasting pCO

2

conditionsshould be strong. In a model grassland under FACE, thefractional root length colonized by AMF and the relativecontribution of biological nitrogen fixation to total plant Nwere found to be significantly higher (Zanetti

et al

., 1997;Gamper

et al

., 2004; M. Richter, ETH, Zürich, Switzerland,pers. comm.), suggesting an increased contribution ofsymbionts to plant nutrition. From plots of this modelgrassland, which has been exposed to either elevated (60 Pa)or ambient atmospheric pCO

2

for 8 yr, we isolated represent-ative AMF strains with the aim of testing them for theirsymbiotic abilities.

The present study focused on whether growth historiesunder different atmospheric pCO

2

conditions may haveselected for AMF strains with altered symbiotic functioning.We specifically tested whether a set of isolates grown in theFACE plots under elevated CO

2

would (i) improve host plantmineral nutrition and (ii) more heavily colonize roots, com-pared with a set of isolates grown under ambient conditions.In a pot experiment,

Trifolium repens

(white clover) plantswere inoculated with the two sets of AMF strains andgrown under identical and strictly controlled conditions. Wehypothesized that severe N and perhaps P limitations underelevated pCO

2

(Fischer

et al

., 1997; Richter

et al

., 2003) haveinduced host plant-mediated selection for nutritionally morebeneficial strains. The abundance of beneficial strains wasexpected to be increased in the FACE plots under elevatedpCO

2

, increasing the likelihood of isolating strains that willimprove the nutrition of experimental host plants. All AMFsingle spore inocula used in the present study were producedon the same host plant species and under identical glasshouseand soil conditions after isolation from trap cultures. Thus,two growth cycles under identical conditions should havelimited potential maternal effects. Seven isolates each weresampled at random to be included in the experimental setsof AMF isolates from the two pools with contrasting CO

2

selection histories. Uniform and functional nodulation wasensured by inoculating the experimental one-to-one fungus–plant pairs with a common rhizobial strain. Harvest took placeafter 72 d of growth under identical, ambient pCO

2

conditions.Because the AMF isolates belonged to two distinct groups of

spore morphotypes, we were able to further assess the effectof fungal taxonomic grouping on effect size and significanceof difference in measured parameters. Thus we investigatedwhether the identity of morpho-species or AMF strain wasmore important for symbiotic measures. To our knowledge,this is the first experiment under controlled conditions and

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using multiple single spore isolates that has assessed thephysiological effects of past selection under experimentallyelevated atmospheric CO

2

on AMF in the field.

Materials and Methods

AMF inocula

AMF inocula were prepared from open-pot trapping cultures(Gilmore, 1968) with soil samples taken from the Swiss-FACEexperiment, located 20 km north-east of Zürich, Switzerland.This experiment consisted of permanent monoculture plots of

Lolium perenne

L. cv. Bastion (perennial ryegrass) and

Trifoliumrepens

L. cv. Milkanova (white clover) and the mixture of bothplant species in ambient and elevated pCO

2

(60 Pa) treatments,and in an additional cross-factorial N-fertilization treatment (14and 56 g m

−

2

yr

−

1

) at the subplot level. At the time of samplingin autumn 2000, half of the plots had been fumigated withCO

2

for 8 yr. The soil at the site of the FACE experimentis a clay loam, classified as fertile, eutric cambisol [Food andAgriculture Organization (FAO) Classification System]. Forfurther details see Zanetti

et al

. (1996) or Suter

et al

. (2002).After 5 months of trap culturing, pure AMF isolates

were established from single spores on

Plantago lanceolata

L.(ribwort plantain), using a potting substrate which consistedof 1 : 2 : 2 volumes of pasteurized field soil from theupper horizon [clay loam, pH (H

2

O) 5.8, available P (Olsen)52 mg P kg

−

1

, re-inoculated with a filtrate of the naturalmicroflora], silica sand (0.7–1.2 mm) and an expandedattapulgite clay (Oil Dry Chem-Sorb WR 24/18, BrenntagMediterranée Export, Vitrolles Cedex, France). All success-fully isolated single spore cultures (approx. 10%) belonged tothe genus

Glomus

and were propagated in bi-species cultures

of

Zea mays

cv. Corso and

P. lanceolata

over a period of7.5 months. Identical soil conditions and host plants duringthe two growth cycles of single spore isolation and inoculumproduction were expected to have minimized potential mater-nal effects caused by host–plant differences in trap cultures(Table 1).

Fourteen fungal isolates were recovered from plots sub-jected to ambient pCO

2

(ambCO

2

isolates), and 44 fromplots subjected to elevated pCO

2

(elevCO

2

isolates). Fromeach of these pools, seven isolates were randomly chosen irre-spective of morpho-species identity, previous field conditionsor trap-culture origin (Table 1). This strategy of randomlychoosing AMF isolates avoided any bias caused by

a priori

expectations. Isolates belonged to the

Glomus claroideum

Schenck & Smith (Gc) and the

Glomus intraradices

(Gi)Schenck & Smith species complexes. Gi isolates were initiallysubclassified as

G. intraradices

and

G. aggregatum

Schenck &Smith, but this became untenable after single spore and inoc-ulum propagation [for a similar finding see Johnson (1993),p. 752]. Certain inocula of Gi isolates contained spore aggre-gates of up to macroscopic size and root cortices of

Z. mays

and

P. lanceolata

that were densely packed with completelyhyaline vesicles. Both AMF species were found in considerableabundance in the root systems of

T. repens

in the originalFACE plots (H. Gamper, U. A. Hartwig & A. Leuchtmann,unpublished).

Plant material

Clonal material was produced from uniform single nodecuttings of different genets of

T. repens

L. cv. Milkanova(Dansk Planteforaedling, Research Division, Store Heddinge,Denmark). Leaf nodes were surface-disinfected and precultured

pCO2 (Pa) Morpho-species Field host(s)

N fertilization (g m−2 yr−1) Trap hosts Isolate number

35 Gc Tr 14 Df & La 412Tr 56 Df & La 52

Gi Tr 56 Df & La 57Tr 56 Df & La 588Lp & Tr 14 Df & La 538Lp & Tr 14 Df & La 539Lp & Tr 14 Df & La 540

60 Gc Lp & Tr 14 Ob & Tp 283Lp & Tr 14 Ob & Tp 284Lp & Tr 56 Lp & Tr 562

Gi Lp & Tr 56 Pl & Zm 484Lp & Tr 56 Pl & Zm 492Lp & Tr 56 Pl & Zm 494Lp & Tr 56 Pl & Zm 498

Fungal isolates belonged to the Glomus claroideum (Gc) or Glomus intraradices (Gi) species complex.Df, Dipsacus fullonum L.; La, Leontodon autumnalis L.; Lp, Lolium perenne L.; Ob, Oenothera biennis L.; Pl, Plantago lanceolata L.; Tp, Tagetes patula L.; Tr, Trifolium repens L.; Zm, Zea mays L. cv. Corso.

Table 1 Identity and histories of host plant species cover, atmospheric CO2 partial pressure (pCO2) and nitrogen (N) fertilization in the field of origin of the 14 single spore isolates of arbuscular mycorrhizal fungi (AMF) used in this experiment.

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on cellulose filter paper on top of 1 × Hoagland (Hoagland& Arnon, 1938) nutrition solution agar (0.7%, weight/volume). After 10 d, micropropagates were transplanted tofinely ground Perlite, and after an additional 14 d plantlets ofthe nine most vigorous and uniform genets (labelled A, B, andF–L) were selected for the current inoculation experiment.

Experimental set-up and growth conditions

The inoculation experiment used nine replicate 1 l pots(14 × 10 cm) for each of the 14 AMF inocula. Replicate potswere planted each with a different micropropagate of the nineT. repens genets, resulting in an orthogonal design with eachplant genet/AMF isolate combination represented once (126pots). As control, six additional sets of pots with the nine plantgenets lacking AMF inoculum were used (54 pots).

Inocula, stored at 4°C for 8 months before use, were thor-oughly mixed into AMF-free potting substrate (compositionas described above) in a volumetric ratio of 2 : 5. The inoculaconsisted of dried pot culture material, which containedspores, colonized root fragments, and extraradical hyphae.Nonmycorrhizal control pots (NM-control) received anAMF-free mock-inoculum, but were otherwise identical to theinoculated pots. Additional NM-control pots, also replicatednine times, were established for five different P-fertilizationtreatments. These pots received cumulated amounts of 50,100, 150, 200 and 250 mg readily available phosphorus(KH2PO4) per kg of dry potting substrate in eight weeklyapplications (50 ml) from the third week onwards. Substratesurfaces of all pots were covered with a layer of coarse silicasand to prevent the formation of adventive roots from leafnodes of stolons which otherwise could have given plantsunequal access to soil nutrients.

To balance the non-AMF microbial community qualita-tively (Koide & Li, 1989), 50 ml of a suspension of a filtrateprepared from the fungal and mock inocula was applied toeach pot. To determine the proportion of biologically fixedN (%bfN2), 10 g Na15NO3 (10% enriched) and 5 g Ca(15NO3)2(5% enriched) were added as soil 15N tracer in the samesuspension. As reference plants, L. perenne seedlings weregrown in two extra pots to which the same amount of 15Nwas added (McAuliffe et al., 1958). To promote uniformnodulation of T. repens plants, dense plate-washes of Rhizobiumleguminosarum bv. trifolii (strain RBL 5020; Leiden, theNetherlands) were added.

Two identical climate chambers (Conviron, PGV-36,Winnipeg, Manitoba, Canada) were required to carry all pots.Sets of four and five pots of each AMF isolate or P fertilizationtreatment were randomly chosen and assigned to one of theclimate chambers, in which they were positioned randomly.Growth conditions consisted of a light phase of 16 h [450–550 µmol m−2 s−1 photosynthetic fluence rate (PPFR) overthe waveband 400–700 nm at plant height], including dawnphases of 30 min twice a day, a day/night temperature profile

of 23/15°C at plant height and 70–80% relative air humidity.Cool white fluorescent lamps and incandescent bulbs (OsramSylvania Inc, St. Marys, PA, USA) provided the high photo-synthetic active irradiance. Automatic drip-irrigation withdeionized water from a tensiometer-type system (TropfBlumat, Weninger GmbH & Co. KG, Telfs, Austria)maintained substrate humidity at about 40%, or 50–60% ofits water-holding capacity, preventing any leaching frompots. Twenty-four, 32 and 43 d after transplantation, 50 mlof 0.1 × Hoagland nutrient solution (Hoagland & Arnon,1938), lacking P, was applied to each pot. On day 43 anadditional 50 ml of a dense suspension of rhizobia was addedto each pot.

Plant growth measurements

Seventy-three days after inoculation, plants of T. repenswere destructively harvested and dry masses determined afterdrying to constant weight for at least 48 h at 65°C. The wholeplant leaf area was determined by passing all leaf laminaethrough a Li-Cor 3000A leaf area meter (Li-Cor Inc., Lincoln,NE, USA).

From each washed root system, a random subsample(≤ 1 g) was preserved in 70% ethanol for root length determi-nation using the grid line intersect method (Tennant, 1975)adapted for computer scanner-assisted estimation. Lengthestimates for the whole root systems were made based on dryweights of the subsamples and the whole root systems. Thedry weight of whole root systems was corrected for the twosubsamples, and the root : shoot dry weight ratio, specificroot length and specific leaf area were calculated using the dryweights of the respective plant fractions.

Fungal growth measurements

Root colonization was determined in the root fragments(∼1.5 cm in length) of a random subsample (≤ 0.3 g) takenfrom the middle zone of root systems. Root fragments werecleared and stained using a modified method of Phillips &Hayman (1970) in which lacto-phenol was substituted bylacto-glycerol and methyl blue was used in addition to trypanblue. Proportions (%) of root length colonized by hyphae,arbuscules and vesicles were determined by enumerating100 intersections of at least 20 root fragments per sample,according to the magnified intersections method at ×200magnification (McGonigle et al., 1990). The total root lengthcolonized by AMF (RLC) was calculated by multiplyingthe absolute root length by the proportion of root lengthcolonized by hyphae.

The density of extraradical hyphal length was estimatedfor duplicate samples of 5 g of fresh material from three cores(diameter 1 cm) of potting substrate, which were takenbetween the plant and the pot edge and thoroughly mixed. Amodified membrane filter technique based on the formula

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of Newman (1966) was used (Jakobsen et al., 1992; Sylvia,1992) to determine hyphal length. Briefly, after an aqueousextraction, intersections of hyphal fragments were enumeratedon 0.45-µm gridded nitrocellulose filters of 25 mm diameter(HAWG, Millipore, Bedford, MA, USA), while calculatedlength estimates were corrected for the dilution of suspen-sions and water contents of each individual sample of pottingsubstrate.

Nutrient concentrations in plant tissue

Chemical analyses of P and N were carried out on subsamplesof the ground leaves (without petioles) and roots. These plantfractions were chosen because they represent the physiol-ogically most active organs.

For measuring P concentrations, samples of approximately500 mg of dry powder (40°C) were incinerated at 500°C for6 h, dissolved in 10% HCl, filtered (0.5 µm) and analysedby the method of Murphy & Riley (1962). Dilutions to therange required were measured in a continuous flow Skalarapparatus (Skalar Analytic GmbH, Erkelenz, Germany) at880 nm, where the absorbance by the ascorbic acid-reducedantimony-phospho-molybdate complex is maximal.

The 15N isotope dilution method (McAuliffe et al., 1958)was used to determine partitioning of N in T. repens betweensymbiotic N2 fixation and uptake from the potting substrate.Total N and percentage 15N were determined in samples of2 ± 0.1 mg dry powder (40°C) of T. repens (and L. perenne asa reference plant) at the Stable Isotope Facility of the Universityof California, Davis, using a continuous flow isotope ratio massspectrometer (cf-IRMS, Europa Integra; PDZ-Europa Scientific,Sandbach, UK). Atom per cent 15N excess (i.e. 15N enrich-ment) was calculated and applied to the formula of McAuliffeet al. (1958) to estimate %bfN2, the remainder being assumedto represent N that was taken up from the substrate.

Statistical analyses

The experimental designs for the separate AMF inoculationand NM-control experiments were each cross-factorialwith respect to the treatment T. repens genet. There was noreplication of the treatment combinations of AMF isolate (14levels) × plant genet (nine levels) and P fertilization (six levels)× plant genet (nine levels) in the two separate experiments.The AMF isolate treatment (for the inoculation experiment)and P-fertilization level (for the NM-control experiment)were replicated by the nine genets of T. repens. This experi-mental design provided control over the variability withinAMF inocula and among genets of T. repens. It allowed detectionof the effects of AMF isolates and genets of T. repens, but notof the AMF isolate × plant genet interaction.

Linear mixed analysis of variance (ANOVA) modelswith nested factors were used to assess the effects of the pre-experimental CO2 history of AMF isolates (two levels), the

morpho-species identity of AMF isolates (two levels), theisolate identity (14 levels), the plant genet identity (nine levels)and the technical factor climate chamber (two levels), in SPSSv.11.0.2 for MacOSX (SPSS Inc., Chicago, IL, USA). Analysesof covariance (ANCOVAs) were applied to the data to assessthe importance of root and hyphal length for mineral nutritionof the plants. Similarly, the potential effects of differences inspore numbers in the inocula on all dependent variables wereassessed. AMF isolate and plant genet were treated as randomfactors, except where inter-isolate variability was examined inFigs 2 and 3 (see later). CO2 history, morpho-species andclimate chamber were defined as fixed factors. Morpho-speciesidentity was nested within CO2 history and the interactionterm (CO2 history × morpho-species) within AMF isolates.NM-control treatments were excluded from ANOVA, becausewe were particularly interested in effects caused by the twosets of AMF isolates with different CO2 histories and not ineffects caused by the colonization status of plants.

The initial statistical model was simplified throughelimination of the interaction term, in cases where it didnot significantly contribute to variance. Schwarz’s BayesianInformation Criterion (BIC) under maximum likelihood(ML) variance estimation was used to control improvementin model goodness. Final significances, reported in the Resultssection, were determined via restricted maximum likelihood(REML) variance estimation.

In two additional analyses of variance, morpho-specieswas replaced by either the pre-experimental N-fertilizationtreatment or the pre-experimental host plant species treat-ment of the FACE plots. These analyses were used to checkfor potential interference of these treatments as selectionfactors, in addition to the CO2 history. All reported statisticalresults are from mixed ANOVA, which included the factormorpho-species identity of AMF isolates, if not statedotherwise. Significance values for the factor CO2 history werealmost identical in the three separate ANOVAs on the samedependent variable.

Phosphorus fertilization and plant genet effects onNM-controls were analysed by two-way ANOVA, followedby multiple pairwise comparisons of the means of factor levelsusing Tukey–Kramer’s honestly significant difference test of v.5.1 (SAS Institute Inc., Cary, NC, USA). Dunnett’s test,as implemented in v.5.1, was used for multiple pairwisecomparisons between the mock-inoculated NM-controltreatment and all the AMF inoculation and P-fertilizationtreatments.

Pearson pairwise correlation coefficients were calculatedand their significance determined in two-tailed t-tests asindicators of the direction of the relationship between mea-sured plant and fungal parameters. Guidelines in Zar (1999)were followed when data had to be transformed to meetassumptions in ANOVA. Weights and concentrations wereln(x + 1), counts were √(x + 3/8) and ratios were arcsin√(x)-transformed.

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Results

Pre-experimental growth of single spore AMF isolates underdifferent atmospheric pCO2 conditions significantly influencedthe effects of the isolates on the mineral nutrition of experi-mental host plants. The foliar N concentration of T. repenshost plants was increased when the plants were inoculatedwith AMF isolates from FACE plots that had been exposed toelevated pCO2, compared with isolates that had been growingunder ambient pCO2 (Fig. 1a). A higher proportion of thetotal foliar N concentration could be ascribed to soil uptakein plants colonized by elevCO2 isolates than in plants colonizedby ambCO2 isolates (Fig. 1b), although the majority of foliarN was biologically fixed N2. Differences in both soil N uptakeand biologically fixed N2 were significant (Fig. 2). Root Nconcentrations did not differ significantly between the twogroups of inoculated plants, but the patterns of partitioning of%bfN2 and soil N uptake in roots were similar to those inleaves and did differ significantly (Fig. 2). Biologically fixedN2 accounted for 98.8 ± 0.2% [mean ± standard error ofthe mean (SEM), n = 14] of foliar N in mycorrhizal plants,compared with 91.8 ± 1.8% (mean ± SEM, n = 6) in nonmy-corrhizal control plants. The two sets of AMF isolates withcontrasting CO2 histories did not produce significant differ-ences in foliar P concentrations.

Fig. 2 Effects of fungal selection history on nitrogen fixation in Trifolium repens (white clover). Effects are shown of fungal selection, under ambient or elevated (60 Pa) atmospheric CO2 partial pressure (pCO2), of 14 single spore isolates of arbuscular mycorrhizal fungi (AMF) on percentages of total foliar (a) and root (b) nitrogen fixed (%bfN2) in the nodule symbiosis of T. repens host plants. Fungal isolates had been growing under ambient and elevated atmospheric pCO2 for 8 yr before isolation and propagation under identical conditions. They belonged to the Glomus claroideum (Gc) or Glomus intraradices (Gi) species complexes. Bars represent the mean with the standard error of the mean for nontransformed raw data (not corrected for the effect of fungal morpho-species identity; n = 9). For F-values and corresponding significance levels, refer to Table 3. Means not sharing letters differ significantly according to Tukey–Kramer’s honestly significant difference (HSD) test (P < 0.05).

Fig. 1 Effects of fungal selection history on host plant nitrogen (N) nutrition. (a) Foliar N concentration and (b) proportion of total foliar N taken up from the soil of nodulated Trifolium repens (white clover) plants, calculated as 100% minus the proportion of biologically fixed nitrogen (%bfN2). Plants were inoculated with two sets of seven single spore isolates of arbuscular mycorrhizal fungi that had been grown under either ambient (open bars) or elevated (60 Pa) (closed bars) atmospheric CO2 partial pressure (pCO2) for 8 yr before isolation and propagation under identical conditions. Bars represent mean with standard error of the mean for nontransformed raw data (n = 7). F-values were calculated from mixed ANOVA for the pCO2 history effects on (a) foliar N concentration (F1,9 = 7.21; P = 0.026) and (b) the proportion of foliar N derived from the potting substrate (F1,11 = 6.92; P = 0.024). *, significant difference at P ≤ 0.05.

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The CO2 histories of the two sets of AMF isolates affectedthe percentage of root length colonized by hyphae and vesiclesand the total root length colonized by hyphae (RLC, Fig. 3).Other morphometric plant parameters, including total plantdry weight, shoot : root dry weight ratio, total and specific leafareas, and total and specific root lengths did not differ betweenthe plants colonized by AMF isolates with different CO2histories. Similarly, there was no difference between the extra-radical hyphal length densities of the two sets of AMF isolates[7.40 ± 0.65 m g−1 (mean ± SEM, n = 14)].

Pre-experimental N fertilization and host plant, two otherpotential selecting factors, had no effect on the plant and fungalparameters studied, with two exceptions: there was a signifi-cant main effect of the host plant on percentage of root lengthcolonized by hyphae (F1,11 = 10.76, P = 0.007) and vesicles(F1,11 = 8.18, P = 0.016); this effect did not, however, interactwith the effect of CO2 history.

Extraradical hyphal length density and root length werenot significant covariates for foliar N and P concentrations. Theproportions of root length colonized by the different AMF

Fig. 3 Effects of fungal selection history on root colonization in Trifolium repens (white clover). Effects are shown of fungal selection, under ambient and elevated (60 Pa) atmospheric CO2 partial pressure (pCO2), of 14 single spore isolates of arbuscular mycorrhizal fungi (AMF) on the percentage of root length colonized by hyphae (a), arbuscules (b) and vesicles (c) and on the total colonized root length (d) of experimental T. repens host plants. Fungal isolates had been growing under ambient and elevated atmospheric pCO2 for 8 yr before isolation and propagation under identical conditions. They belonged to the Glomus claroideum (Gc) or Glomus intraradices (Gi) species complexes. Bars represent the mean with the standard error of the mean for nontransformed raw data (not corrected for the effect of fungal morpho-species identity; n = 9). For F-values and corresponding significance levels, refer to Table 3. Means not sharing letters differ significantly according to Tukey–Kramer’s HSD test (P < 0.05).

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structures or the total colonized root length did not explainfoliar N concentration either. The lack of a contribution toplant nutrition by surrogates of root and hyphal surfaces isalso evident from the significant negative pairwise correlationslisted in Table 2, and from negative correlations (P < 0.01)between the root length colonized by hyphae and %bfN2 ofleaves (r2 = −0.264) and roots (r2 = −0.564).

The 14 AMF single spore isolates did not significantlycontribute to either foliar [2.00 ± 0.05 mg g−1 (mean ± SEM,n = 14)] or root [1.66 ± 0.03 mg g−1 (SEM, n = 14)] Pconcentrations of the T. repens host plants, when comparedwith the mock-inoculated NM-control [2.02 ± 0.18 and 1.75 ±0.15 mg g−1 (SEM, n = 9), respectively], although increasingthe P fertilization of the NM-controls significantly increasedboth these values. P concentrations differed significantly (Tukey’sHSD test at P < 0.05: Q = 3.61) among the NM-control plantsreceiving cumulated amounts of 50 and 150, 100 and 200, and150 and 250 mg readily available P (data not shown). FoliarN concentrations did not significantly differ between the mock-inoculated NM-control and the AMF inoculation treatments[35.99 ± 2.24 (mean ± SEM, n = 9) and 39.14 ± 0.91 mg g−1

(mean ± SEM, n = 14), respectively; Dunnett’s test: d = 2.93,

P > 0.241]. Proportions of symbiotically fixed nitrogen in theleaves and roots did not differ between the mock-inoculatedNM-control and the AMF inoculated plants either, except forthe fungal inoculation treatments 52 and 57, which showedsignificantly lower values (Dunnett’s test: d = 2.93,P < 0.05). These values were comparable to those of the fiveNM-controls with addition of P fertilizer (data not shown).Moreover, only two AMF inoculation treatments (one Gc iso-late from each of the CO2 fumigation histories) reduced totalhost plant biomass [Dunnett’s test: d = 2.93; isolate 283:8.27 ± 1.08 g (mean ± SEM, n = 9), P = 0.027; isolate 412:8.65 ± 1.40 g (mean ± SEM, n = 9), P = 0.032; mock-inocu-lation control: 16.33 ± 1.63 g (mean ± SEM, n = 9)], mainlyas a result of reductions in shoot dry weight. None of theNM-control plants was colonized by AMF. Differences inspore numbers of inocula did not contribute to any of the dif-ferences in the measured parameters, as evidenced byANCOVA (data not shown).

Effect sizes explained by morpho-species identity of AMFisolates for the parameters presented in Figs 2 and 3 were largerthan those explained by isolate identity, although both effectswere significant (Table 3). Indeed, the values of F-ratios

P concentration N concentration

Root Foliar Root Foliar

Root length −0.502*** −0.469*** −0.569*** −0.593***Extraradical hyphal length −0.292*** −0.246** −0.298*** −0.293***RLC −0.391*** −0.387*** −0.438*** −0.353***

Fungal isolates belonged to the Glomus claroideum (Gc) or Glomus intraradices (Gi) species complexes.Significance levels derived from two-tailed t-tests: **, P < 0.01; ***, P < 0.001.

Table 2 Pairwise Pearson correlations for root and foliar phosphorus (P) and nitrogen (N) concentrations, and root, extraradical hyphal and total colonized root length (RLC) in Trifolium repens plants colonized by 14 different single spore isolates of arbuscular mycorrhizal fungi (n = 126).

Table 3 Summary table for mixed ANOVA testing the parameters foliar and root nitrogen fixed in the nodule symbiosis (%bfN2), percentage of root length colonized by hyphae, arbuscules, and vesicles, and total colonized root length for effects of factors CO2 history, arbuscular mycorrhizal fungi (AMF) morpho-species identity (Glomus claroideum or G. intraradices), AMF isolate identity, Trifolium repens genet identity and climate chamber.

Parameter

Factor

CO2 history (morpho-species) (df = 2)

Morpho-species identity (df = 1)

Isolate identity (CO2 history × morpho-species) (df = 10)

Plant genet identity (Z-value)

Climate chamber(df = 1)

Foliar %bfN2 21.31*** 0.59ns 6.71*** 0.28ns 0.06ns

Root %bfN2 48.57*** 58.77*** 6.48*** 1.29ns 0.98ns

Hyphae (%) 32.21*** 65.79*** 14.03*** 1.29ns 0.26ns

Arbuscules (%) 1.36ns 16.48*** 3.30** 0.30ns 0.22ns

Vesicles (%) 49.18*** 306.15*** 14.45*** 1.10ns 0.64ns

Total colonization (m) 14.72*** 53.82*** 6.50*** 1.53ns 0.93ns

F-values and corresponding significance levels are given for all fixed factors, and the Wald Z statistic for the random factor plant genet identity.Significance levels: ns, P > 0.05; **, P < 0.01; ***, P < 0.001.Denominator degrees of freedom (df) were 103 for all factors, except for the factor climate chamber for which 80 < df < 105.

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from ANOVA for the factor morpho-species identity wereconsistently higher than those for the factor isolate identityfor all dependent variables, foliar %bfN2, root %bfN2, hyphal,arbuscular, and vesicular root colonization, and RLC (Table 3).In general, %bfN2 and percentage of root length colonizedby arbuscules were higher in T. repens host plants colonized byAMF isolates of G. claroideum than in those colonized byisolates belonging to the G. intraradices species complex. Theopposite was found for hyphal and vesicular root colonizationrates and RLC.

Discussion

Effects of fungal selection history

In this pot experiment, we showed that 8 yr of FACE selectedfor more plant-beneficial AMF strains in a permanent,agricultural model grassland. Experimental T. repens plantshad 10% higher foliar N concentrations when inoculatedwith AMF isolates from field plots with elevated pCO2conditions (elevCO2 isolates), compared with plantsassociated with isolates from plots with ambient pCO2conditions (ambCO2 isolates, Fig. 1a). ElevCO2 isolatesstimulated both biological N2 fixation in the nodulesymbiosis and nitrate uptake from the growth substrate,as measured by the 15N dilution method. Nitrogen uptakefrom soil was nearly twice as high in plants colonized byelevCO2 isolates as in plants colonized by ambCO2 isolates(Fig. 1b). Synergism between the arbuscular mycorrhizaland nodule symbioses (e.g. Olesniewicz & Thomas, 1999)and the contribution of AMF to plant N nutrition (Tobaret al., 1994; Hawkins et al., 2000; Azcon et al., 2001) havebeen previously observed. Recently, Bago et al. (2004) reportedon the ability of an G. intraradices isolate to take up nitrateefficiently through a change in extraradical hyphal growth underin vitro conditions. The new finding of our study is that only8 yr of CO2 fumigation had favoured AMF strains with improvedN nutritional capabilities, which is particularly noteworthyfor asexual, slow-growing organisms where adaptation toenvironmental change is thought to be slow (Sanders, 2002).

The observed selective change in the AMF field assemblagewas probably plant-mediated. In this FACE experiment,mineral nutrient deficiency and growth limitation of hostplants occurred under elevated atmospheric pCO2 (Fischeret al., 1997; Suter et al., 2002). Moreover, accumulation ofsoil organic matter under elevated pCO2 may have increasedmicrobial immobilization of mineral nutrients (Zak et al.,2000; Schneider et al., 2004). Nutrient retention in soils andincreased plant demand under elevated pCO2 both makenutritional symbioses more important. An immediate uptakeof nutrients released from decaying organic material bymycorrhizal fungi may be crucial under elevated pCO2. Infact, acquisition of N from organic sources by plants in theFACE experiment became more important as the experiment

went on (Schneider et al., 2004). It is possible that theglutamine synthetase activities involved in N acquisitiondiffered in plants inoculated with the two sets of AMF, ashas been observed by Smith et al. (1985) and Breuninger et al.(2004). The shift in mycorrhizal fungal properties towardsmore beneficial strains under long-term atmospheric CO2fumigation is consistent with the concept of a mutualism–parasitism continuum (Johnson et al., 1997) and concurswith the evolutionary predictions deduced from the plantinvestment hypothesis (Treseder, 2004).

Another, although less likely, explanation for the observeddifferences in N concentration of plant tissues is unequalenergetic costs (C costs) of AMF isolates. Increased availabilityof C under elevated atmospheric pCO2 could have favouredmore C-demanding AMF strains, leaving fewer C resources toexperimental host plants for building up biomass. This mayhave increased N concentration in hosts of the elevCO2 isolates(Jarrell & Beverly, 1981). Significantly smaller biomasses oftwo of the 14 AMF inoculation treatments and no growthresponse in the NM-controls to P fertilization (data notshown) suggest that P was not limiting and thus C costs mightindeed have been present. Moreover, more intense hyphal andvesicular root colonization observed in plants with elevCO2isolates may indicate that C investments into biomass ofAMF were enhanced (Fig. 3). In addition, limited energeticresources may have shifted the partitioning of N acquisitionfrom the energetically more expensive symbiotic N2 fixationto nitrate uptake from soil.

The past N-fertilization regime and field hosts in FACEplots did not interfere with the observed effects of the CO2history on the plant nutritional parameters. There were nostatistically significant main or interaction effects of hostplant, N fertilization, and CO2 history. Therefore, the observeddifferences in the symbiotic traits affecting plant nutritionwere the result of selection resulting from differences inatmospheric pCO2. Only hyphal and vesicular root colonizationwas affected by the field-host history, suggesting that thesetraits may have been influenced by pre-experimental CO2conditions and/or host plant.

The observed effect of field hosts on hyphal and vesicularroot colonization rates suggests a selective effect of hostplant species in FACE plots. In the pot experiment, we foundhigher hyphal and vesicular root colonization rates for AMFstrains originating from mixed T. repens and L. perenne plots,compared with those originating from monocultures ofT. repens. Differences in C and N metabolism of grasses andlegumes may have affected the AMF assemblages of the twohosts with respect to the relative abundance of strains withdifferent colonization abilities. On the one hand, L. perennecould have selected for more C-demanding AMF strains,because as a non-N2-fixing plant it has been shown to sufferparticularly from N deficiency and C-sink limitation underelevated atmospheric pCO2 (Fischer et al., 1997; Suter et al.,2002). On the other hand, the assemblage of AMF strains

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from T. repens monocultures may have adapted to their hosts,such that fungal strains that require less intense root coloniza-tion for the same symbiotic benefit became more abundant.

The tripartite symbiosis among legume host plants, AMF,and rhizobial strains involves a close interdependence of theplant C and mineral nutrient metabolisms. Regulation of theN status in legumes is complex and cannot yet be explainedby a unifying concept (Schulze, 2004). Feedback mechanismsvia hormones as well as C costs, N status and O2 permeabilityin nodules were invoked to explain patterns in plant Nnutrition (Bethlenfalvay et al., 1985; Murphy, 1986; Hartwiget al., 1994; Hartwig, 1998; Catford et al., 2003). Thus, giventhe regulatory complexity of legume physiology, furtherstudies would be needed to uncover the mechanisms that haveled to the observed N nutritional patterns in the experimentalT. repens plants. Nevertheless, it is fairly clear from the resultsof ANCOVA that the improved plant N nutrition conferredby the elevCO2 isolates was not a result of better soil exploi-tation by AMF hyphae or their host plant roots. Enlarging thepotential surfaces for absorption would not have been aneconomic scavenging strategy for nitrate, an element that ishighly mobile and evenly distributed in soils. The negativecorrelations between the plant tissue P and N concentrationsand the plant and fungal organs involved in nutrient uptake(Table 2) indicate that plants appear to regulate their invest-ment into the root system and mycorrhizal partners accordingto their actual requirements. Conversely, from a fungal point ofview, extraradical hyphal growth of AMF seems to be diminishedwhen host plants are better supplied with mineral nutrients,perhaps because of limitation in available C resources.

Effects of mycorrhizal inoculation and P fertilization

In general, dry weights of plants did not differ among theAMF inoculation treatments and the mock-inoculation control,except for the dry weights of host plants inoculated with twoGc isolates, which were significantly lower (see Results andDiscussion sections above). This may reflect the high photo-synthetic compensation ability of T. repens towards mycorrhizalC costs (Wright et al., 1998). Moreover, mycorrhizal plants,compared to the unfertilized mock-inoculated plants, did notdiffer in their foliar and root P and N concentrations, indicatingthat the AMF isolates did not contribute, or only marginallycontributed, to plant mineral nutrition under the presentexperimental conditions. An explanation for this limitednutritional function of AMF might be the nutrient-rich fieldsoil contained in our potting substrate. Therefore, it is evenmore striking that, in plants inoculated with the two sets ofAMF isolates, significant differences in N nutrition were found.

P fertilization applied to NM-control plants of T. repensneither increased biomass of plants nor decreased root : shootdry weight ratio and specific root length, although it increasedfoliar and root P concentrations. The lack of symptoms ofmineral nutrient deficiency and the absence of a growth

response to P fertilization clearly indicate that P was availablein sufficient quantity. In fact, nonlimiting soil P shouldexclude significant, additional plant P nutritional benefitsfrom the symbiosis, and thus C costs imposed by AMF shouldbe particularly manifested (Peng et al., 1993; Graham et al.,1996). Our experimental fungal isolates may have been selectedby the original soil conditions, which were rich in available P,and thus should be adapted to such growth conditions. Theseconditions may have enhanced fungal C costs and made Nmore limiting, and thus the present type of potting substratewas suitable for detecting N-nutritional AMF effects. Moreover,the experimental outcome may have been influenced by thechoice of the rhizobial strain, as strong interactions betweenthe bacterial and fungal root symbioses are well established(Ianson & Linderman, 1993; Xavier & Germida, 2002).

Effects of morpho-species and isolate identity of AMF

Both morpho-species and AMF isolate identity contributedsignificantly to the observed patterns in AMF root colon-ization and proportion of N fixed in the nodule symbiosis(Figs 2 and 3). Yet, effect sizes expressed in the form ofF-values were consistently higher for morpho-species(G. claroideum or G. intraradices) than for isolate identity(Table 3). However, our finding that morpho-species identityat the species level is relevant to functional differences is newfor arbuscular mycorrhizal research and might challengethe report by Munkvold et al. (2004), which showed thatfunctional variability among AMF isolates may override thevariability found among morpho-species and phylo-species.Moreover, Hart & Klironomos (2002) compared thecoefficients of variance of the mycorrhizal plant growthresponse at several levels of biological integration of AMF andfound that sizes in the coefficients of variance were comparableat the morpho-species and isolate levels. By contrast, variationsat the isolate level in our data are consistent with the geneticvariability found among isolates of G. intraradices also from asingle field site (Koch et al., 2004).

Based on the hierarchy of biological integration, largereffect sizes at higher taxonomic levels should generally beexpected. Our data provide evidence for hierarchical structur-ing of effect sizes at the species level, and supplement know-ledge of even more pronounced variation in fungal growthpatterns at the level of AMF families (Hart & Reader, 2002).Consequently, future experiments should include adequatereplication at each level of taxonomic integration to morecomprehensively resolve the sources of variance of measuredparameters caused by experimental treatments or environ-mental selection.

Conclusions

Knowledge about the impact of rising atmospheric pCO2on mycorrhizal symbioses is crucial for predicting potential

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changes in ecosystems. We have shown that elevatedatmospheric pCO2 for only 8 yr in model grasslands selectedAMF strains that can improve the N nutrition of T. repenshost plants, a common species of temperate grasslands.Improved N nutrition was found to result from bothstimulation of biological N2 fixation in the nodule symbiosisand soil N uptake. Alleviation of mineral nutrient deficiencyby AMF strains could be particularly relevant for nonlegumes,which are known to experience severe N-growth limitationunder elevated atmospheric pCO2.

Intraspecific variability observed among isolates of theG. claroideum and G. intraradices species complexes, as well asinterspecific variability between these two taxonomic groups,suggests that future inoculation experiments should considerboth intra- and interspecific variation. Further experimentswould be needed to scale up our findings to the ecosystemlevel. In particular, an experiment with a nonlegume hostplant lacking the buffering effect of biological N2 fixationwould be of interest, as in such a system the N-nutritionaleffects of AMF might be even more pronounced. This is thefirst experiment conducted under controlled conditions thatdemonstrates that altered atmospheric pCO2 may induceshifts in AMF composition with consequences for the mycor-rhizal symbiosis.

Acknowledgements

We would like to thank Sonja Müller, Lucinda Palma,Yolanda Callau and Ionut Popa for technical assistanceand Prof. Josef Nösberger, Prof. Nina Buchmann and Prof.Emmanuel Frossard from the Institute of Plant Sciences(ETH) for kind permission to use their research facilities.Dr Hans-Rudolf Roth and Mr Bruno Tona from the statisticalconsulting service at ETH Zürich assisted in designing theexperiment, and Dr Dieter Ramseier, PD Dr Andreas Lüscherand Dr Matthias Suter helped with statistical analyses.Comments by Prof. Ian R. Sanders, Prof. Alistair H. Fitter, theEditor, and Dr Jan Jansa helped to improve the presentationof our findings and are gratefully acknowledged. Financialsupport for this research was provided by a research grantfrom the Federal Institute of Technology (ETH) Zürich andby funds supporting the Swiss FACE experiment.

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