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Blackwell Publishing LtdAre there benefits of simultaneous root colonization by different arbuscular mycorrhizal fungi?

Jan Jansa1,2, F. Andrew Smith1 and Sally E. Smith1

1Soil and Land Systems, School of Earth and Environmental Sciences, Waite Campus, The University of Adelaide, Adelaide, 5005, Australia; 2Present address: ETH Zurich, Institute of Plant Sciences, Eschikon 33, CH –8315 Lindau, Switzerland

Summary

• Arbuscular mycorrhizal fungal (A MF) communities were established in pots usingfungal isolates from a single field in Switzerland. It was tested whether multispeciesmixtures provided more phosphorus and supported greater plant growth than singleA MF species.• Two host plants, medic (Medicago truncatula) and leek (Allium porrum), wereinoculated with three AMF species (Glomus mosseae, G. claroideum and G. intraradices),either separately or in mixtures. The composition of the A MF communities in theroots was assessed using real-time PCR to determine the copy number of largeribosomal subunit genes.• Fungal communities in the roots were usually dominated by one A MF species(G. mosseae). The composition of the communities depended on both plant identity andthe time of harvest. Leek colonized by a mixture of G . claroideum and G . intraradicesacquired more P than with either of the two A MF separately.• Direct evidence is provided for functional complementarity among species withinthe A MF community colonizing a single root system. Competition among the speciesposes a major challenge in interpreting experiments with mixed inoculations, but thisis greatly facilitated by use of real-time PCR.

Key words: arbuscular mycorrhizal fungi (A MF), Glomus sp., large ribosomalsubunit (LSU), leek (Allium porrum), medic (Medicago truncatula), multispeciescommunity of A M fungi, plant phosphorus nutrition, real-time PCR.

New Phytologist (2007) doi: 10.1111/j.1469-8137.2007.02294.x

© The Authors (2007). Journal compilation © New Phytologist (2007)

Author for correspondence:Jan JansaTel: 041 52 3549216Fax: 041 52 3549119Email: jan.jansa@ipw.agrl.ethz.ch

Received: 6 July 2007Accepted: 18 September 2007

Introduction

Arbuscular mycorrhizal fungi (AMF) form symbioticassociations with plant roots, and are involved in plantnutrient uptake, growth and tolerance to environmentalstresses (Fitter & Moyersoen, 1996; Smith & Read, 1997).The responses of plants to colonization by AMF vary fromnegative to positive depending on plant and AMF species, aswell as on environmental conditions such as soil nutrientavailability, light intensity and temperature (Smith & Smith,1996; Johnson et al., 1997). This variation has been observedamong AMF isolates belonging to different species, as well asamong isolates of the same species (van der Heijden et al.,

1998; Klironomos, 2003; Munkvold et al., 2004; Smith et al.,2004). (The term ‘isolate’ is used here for a laboratory cultureestablished from one or several spores.) Almost all the dataon variability of AMF functions have been obtained fromexperiments in which the plants have been inoculated withsingle AMF isolates, and plant growth or total phosphorusuptake have been measured. Such experiments are not fullyrelevant to field situations, where more than one AMF speciesis usually present in a single root system (Daft, 1983;Merryweather & Fitter, 1998; Jansa et al., 2003b). Theexperiments have shown variations in P acquisition strategiesby different fungi ( Jakobsen et al., 1992; Smith et al., 2000;Jansa et al., 2005). Therefore the current challenge is to

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establish mixed communities using different AMF specieswithin the roots of host plants to substantiate the theory offunctional complementarity suggested by Koide (2000), whicharose from the work of Smith et al. (2000).

The consequences of simultaneous colonization of a plantby functionally different AMF have been little explored untilvery recently (Lekberg et al., 2007; Maherali & Klironomos,2007). Previously, it has been suggested that if a plant iscolonized by AMF species that are complementary in theirfunctions (e.g. uptake of nutrients from different soil pools),they may prove to be more beneficial for the plant as a mixturethan any of the species separately (Koide, 2000; Alkan et al.,2006; Gustafson & Casper, 2006). Indeed, the diversity ofAMF communities in the roots has been shown to correlatepositively with P and nitrogen concentrations in the shoots ofPlantago lanceolata ( Johnson et al., 2004). However, otherdata do not support the idea of functional complementarity.Rather, some previous studies indicated that maximumbenefits to plants might be achieved with a single, mostefficient AMF species, and that increasing mycorrhizal diversitywould not bring further benefits (Daft & Hogarth, 1983;Edathil et al., 1996).

Direct experimental evidence for functional complementarityin an AMF community has been difficult to obtain, for severalreasons. It requires the availability of inocula of more than oneAMF species of comparable infectivity, preferably isolatedfrom the same ecosystem (to ensure the relevance of the resultsto that ecosystem and to avoid potentially unrealistic com-petition among AMF of different origin). These conditionsare not easy to fulfil. The AMF used for such experimentsshould be identified clearly and tested for their functionsseparately, in advance. Detection of different species in an AMFcommunity inside the roots has been very difficult until recentapplications of molecular methods (van Tuinen et al., 1998;Husband et al., 2002; Redecker et al., 2003), and the quan-tification of different AMF species in the roots is still poorlydeveloped. Ordinary PCR-based approaches are all potentiallybiased because of the differential amplification and cloningefficiencies of the DNA stretches from different AMF species,which provide only limited precision, and they are also laboriousand costly (Clapp et al., 2002; Jansa et al., 2003b; Sanders, 2004).Quantitative competitive and real-time PCR approaches haveonly recently been applied for dissecting the composition ofan AMF community composed of two AMF species (Edwardset al., 1997; Alkan et al., 2004; Isayenkov et al., 2004).

In this study, synthetic communities (assemblages) were estab-lished of three AMF species (Glomus claroideum, G. intraradices,and G. mosseae) isolated from a single field site in Switzerland(Jansa et al., 2002a). These fungi had previously been shownto vary in their strategy to obtain soil P (Jansa et al., 2005).They differed both in the distance from the roots, from whichthey could take up P (labelled with 33P isotope) and in theirefficiency of P uptake from the soil, estimated by measuring33P uptake of maize via a mycorrhizal pathway from labelled

P solution injected into an established mycelial network. Themycelium of G. claroideum took up P from distances less than6 cm from the roots, whereas G. mosseae and G. intraradicesgrew 15 cm away from the roots (Jansa et al., 2003a, 2005).Additionally, the efficiency of P uptake from the soil (P takenup per unit hyphal length) was lower for G. mosseae than forG. intraradices ( Jansa et al., 2005). We aimed to confirm theseeffects on plant nutrition in two different host plants withthe fungi inoculated separately. We also quantified the AMFcommunity composition in roots using a novel real-time PCRassay that permitted determination of the contribution of thedifferent AMF to overall root colonization. Using this methodwe sought to establish whether the time course of colonizationdiffered between AMF, and whether different AMF differedin their abilities to colonize roots when inoculated singly orin mixtures. We also aimed to determine whether an AMFcommunity composed of two or three functionally differentAMF promoted greater growth and total P uptake of the twoplants used (Medicago truncatula and Allium porrum) thansingle AMF species.

Materials and Methods

Real-time PCR for quantification of A MF colonization

PCR primers (for sequences see Table S1 in SupplementaryMaterial) were designed for specific amplification of largeribosomal subunit (LSU) genes from four different AMFspecies, based on sequencing data published previously (Jansaet al., 2003b). These primers (synthesized and purified usingHPLC at GeneWorks, Hindmarsh, SA, Australia) were eachconfirmed not to cross-amplify sequences from the other AMFspecies used in this study by performing cross-amplificationtests with spore DNA, extracted and amplified according toestablished protocols (Jansa et al., 2003b; data not shown).We also established a real-time PCR quantification protocolfor LSU copy number, as follows. First, DNA was extractedfrom single spores of Glomus claroideum Schenck & SmithBEG 155 (BEG, International Bank for the Glomeromycota;www.kent.ac.uk/bio/beg); Glomus intraradices Schenck &Smith BEG 158; and Glomus mosseae (Nicol. & Gerd.) Gerd.& Trappe BEG 161, as described previously (Jansa et al.,2003b). Second, these DNA extracts were used as templatesfor PCR with LR1 and FLR2 primers (Turnau et al., 2001;Jansa et al., 2003b), using cycling conditions identical to thosedescribed previously (Jansa et al., 2002a). Third, the con-centration of LSU copy numbers (NC, copies per l) in eachof the PCR products was calculated by knowing the DNAproduct fragment length, L (759, 761, and 767 bp for G. claroi-deum, G. intraradices and G. mosseae, respectively); concentrationof DNA in the sample (K, g l–1, determined by UV spectro-photometry at 260 nm using salmon sperm DNA asreference); and molecular weight of DNA (660 Da bp–1) usingequation 1, where Na is Avogadro’s constant (6.023 ⋅ 1023).

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NC _ (K ⋅ Na)/(660 ⋅ L) Eqn 1

Fourth, the PCR products were serially diluted with sterilewater so as to obtain billions to thousands of copies per µl.Additionally, we used a pGEM-T Easy plasmid (Promega,Annandale, NSW, Australia) carrying an LSU fragment ofScutellospora pellucida (Nicol. & Schenck) Walker & SandersBEG 163, delimited by LR1 and FLR2 primers, as an internalstandard for quantification of AMF in root DNA extracts (seebelow). These samples were used as templates for a real-timePCR assay, employing the LightCycler 1.0 System (Roche,Castle Hill, NSW, Australia) and Qiagen chemistry (QuantiTectSYBR Green PCR Kit, Qiagen, Doncaster, VIC, Australia).Cycling conditions were as follows: initial denaturation at95φC for 15 min, then 45 cycles with denaturation at 95φCfor 15 s, annealing at 58φC for 60 s (for S. pellucida, G. mosseaeand G. claroideum) or 90 s (for S. pellucida and G. intraradices),followed by elongation at 72φC for 1 min. The cycling wasfinalized by elongation at 72φC for 10 min. Melting curveswere then analysed in the LightCycler to confirm the lengthof amplified DNA fragments. The cross points were recorded,where real-time PCR curves reached their second derivationmaxima. The numbers of cycles at which these points werereached were used as the DNA quantity estimates correlatingwith the DNA copy numbers. This way, quantification oftarget LSU copies in the samples was possible and highlyreproducible within the entire range tested (Fig. S1).

To ensure the robustness of the real-time PCR procedurefor identification of a specific AMF species in the presenceof another AMF species, we performed a dilution assay. TheDNA extracted from roots of M. truncatula colonized by atarget AMF species (e.g. G. mosseae) was serially diluted withDNA from roots colonized by different AMF species, orwith DNA from nonmycorrhizal roots. We confirmed thatthe presence of DNA from a plant or from nontarget AMFspecies did not interfere with the real-time PCR assay(Fig. S2).

Plant growth substrate

The substrate used in the pot experiment consisted ofautoclaved soil (from Mallala, South Australia) and heat-sterilized sand, mixed in the ratio 1 : 9. It had the followingproperties: total P (HNO3 : HClO4 digest) 32 mg kg–1; availableP (Colwell, 1963) 0.83 mg kg–1; immediately available P (E1min)assessed by isotope exchange kinetics approach (Frossard& Sinaj, 1997) 0.045 mg kg–1; P available within 8 wkextrapolated from a short-term (60 min) isotope-exchangekinetics 0.31 mg kg–1, pH(CaCl2) 7.88.

Plants and A MF

Seeds of Medicago truncatula Gaertn. (medic) cv. Jemalongand Allium porrum L. (leek) cv. Vertina were sterilized in 1%

active chlorine solution (diluted commercial bleach) for5 min, then washed with sterile water and germinated for3 or 9 d (for medic and leek, respectively) on moist sand at25φC in darkness. Single seedlings were planted in each potat the start of the experiment (here referred to as sowingtime).

Three AMF isolates belonging to different species of thegenus Glomus, all isolated from a single field site in Switzerland(Jansa et al., 2002a), were included. These were G. mosseaeBEG 161, G. claroideum BEG 155 and G. intraradices BEG158. The inoculum was produced in 1.4-kg pots for 6 monthsbetween April and September 2003, and consisted of colonizedMallala soil and sand (1 : 9) planted with medic and leekmixtures. Colonization of roots in the inoculum pots was98–100% of root length for all three AMF species. The potswith G. intraradices inoculum contained many vesicles insidethe roots, as well as an extensive mycelial network in thesubstrate, on which no spores were observed. Glomus mosseaeinoculum contained 18 spores g–1 and much mycelium.Inoculum of G. claroideum contained 114 spores g–1, as wellas colonized roots and loose mycelium fragments. Inoculumwas harvested just before the start of the pot experiment; theroots were cut to 1-cm fragments and mixed into the substrateof the pot culture, and the moist inoculum was used forestablishment of the pot experiment.

Experimental design

The pot experiment was carried out in pots filled with 400 gsubstrate. There were two host plants (medic and leek), twoharvest times (4 and 8 wk after sowing), and eight inoculationtreatments. Four replicate pots were established for eachtreatment combination. The inoculation treatments werethree single AMF species treatments, three double speciesmixtures, one triple species mixture, and one nonmycorrhizalcontrol. The amount of inoculum for single species inoculationwas 18 g per pot, the amounts of each AMF species for doublespecies treatments were 9 g per pot, and the amounts for triplespecies treatment were 6 g per pot. Inoculum was mixed intothe whole volume of the pot before sowing. Nonmycorrhizalpots were inoculated with 5 ml per pot of aqueous filtrate ofthe triple species inoculum mixture (20% suspension, w/v)filtered twice through Whatman no. 1 paper. Extra potsinoculated with single AMF species or with inoculum filtrate(already described) were established for destructive samplingat weekly intervals to determine the progress of AM colonization(one pot for each sampling time and each inoculationtreatment). Pots were watered daily to weight with deionizedwater to maintain 75% water-holding capacity of the substrate.Each pot received 10 ml Long Ashton nutrient solutionwithout P, weekly (Cavagnaro et al., 2001). Pots were completelyrandomized in the glasshouse and grown at 25 : 22φC(day : night) under mixed natural and supplemental fluorescentlight (16 h photoperiod).

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Harvest and measurements

Four replicate pots for each treatment were harvested 4 and8 wk after sowing. The extra pots were harvested at weeklyintervals. Plant shoots were dried at 105φC for 48 h andweighed. Roots were washed from the substrate withdeionized water and split into three parts. One part (approx.100 mg) was frozen in liquid N and stored at –20φC for DNAextraction; one part was dried at 105φC for 48 h and weighed;and one part was stained according to a protocol basedon Phillips & Hayman (1970) and Brundrett et al. (1984).Briefly, roots were digested in 10% KOH at 80φC for 30 min,rinsed with water, incubated in 3% HCl at room temperaturefor 30 min, then transferred (with no further rinsing) to0.05% Trypan blue in lactic acid : glycerol : water (1 : 1 : 1,v/v/v) and stained at 80φC for 4 h in a water bath. Finally, theroots were incubated overnight in water at room temperature.The extent of root length colonized by hyphae, arbuscules andvesicles was determined on stained root samples according tothe method of McGonigle et al. (1990), recording 100 rootintersects per sample. Subsamples of dry biomass (100–300 mg) of both shoots and roots were digested with 7 mlHNO3 : HClO4 (6 : 1, v/v) at 150φC for 10 h, evaporated todryness, and made up to 20 ml with 1% HCl. Phosphorusconcentration was measured in these extracts by the malachitegreen method (Ohno & Zibilske, 1991). Five grams ofsubstrate were used for estimation of hyphal length density asdescribed previously (Jansa et al., 2003a). The composition ofAMF communities in the roots was estimated by real-timePCR as described above. DNA was extracted by DNeasy PlantMini Kit (Qiagen) from the frozen root samples afterhomogenization in liquid N, following the manufacturer’srecommendations. All samples were spiked with a knownnumber (5 ⋅ 106) of gene copies of S. pellucida LSU clone(internal standard) before homogenization. Specific amplifica-tion of that LSU clone from the purified DNA extract allowedfor correction for both (1) DNA lost during extraction and (2)variable PCR amplification efficiency across the samples (e.g.Weiss et al., 2004; Bustin et al., 2005). Real-time PCR wasperformed with specific primers using crude DNA extract astemplate.

Calculations and statistics

The percentage of root length colonized by AMF hyphae,arbuscules and vesicles is given as the ratio of intersects withthese structures to all root intersects per sample ⋅ 100.Phosphorus concentrations in plant digests were used forcalculation of plant P content. Plant P uptake from thesubstrate was determined by subtraction of P contained in theseed (11.0 and 19.5 µg P per seed for leek and medic,respectively) from total plant P content at harvest. The LSUcopy numbers of each of the AMF species in each sample werecalculated using the regressions given in Fig. S1. anova andcorrelation analyses were performed in statgraphics plus forwindows ver. 3.1 (Manugistics, Inc., Rockville, MD, USA).Regression analyses (linear and sigmoid models) were performedin SigmaPlot 2002 ver. 8.01 (SPSS, Inc., Chicago, IL, USA).Data for percentage root length colonized by the AMF werearcsin- and square root-transformed (Linder & Berchtold, 1976)for statistical analysis so as not to violate anova assumptions.

Results

Inocula of all three fungi were highly infective. Data fromthe weekly harvests of single pots showed that { 50% of rootlength of both medic and leek became colonized withinthe first 4 wk by the individual AMF species (Trypan bluestaining, Fig. 1; Table 1). Glomus mosseae colonized roots ofboth plant species very rapidly, resulting in approx. 80% ofroot length colonized 2 wk after sowing (Fig. 1). Valuesremained high for the remainder of the experiment (8 wk).The development of G. intraradices was initially slower, butthe values for percentage root length colonized were similarto or higher than for G. mosseae by 4 wk, and againremained high up to 8 wk (Fig. 1; Table 1). The developmentof G. claroideum was similar to that of G. intraradices, butthe percentage of root length colonized saturated at a lowervalue than for the other two AMF (Fig. 1; Table 1). Thepercentage of root length containing arbuscules (Table 1)was high for all fungi at all harvests, and similar to thepercentage of root length containing hyphae. Glomus mosseaedid not produce any vesicles in the roots of either host plant,

Fig. 1 Development of colonization of medic and leek roots by three arbuscular mycorrhizal fungal (A MF) species as revealed by Trypan blue staining of roots from sequentially harvested pots. Each point represents one pot. Three-parameter sigmoid regression curves for each of the A MF species are shown. , solid lines, plants inoculated with Glomus

mosseae; , dotted lines, plants inoculated with G . intraradices; , dashed lines, plants inoculated with G . claroideum; , dot-dashed lines, nonmycorrhizal plants.

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and G. intraradices produced substantially more vesicles thanG. claroideum (Table 1). Hyphal length density (HLD) inthe growth substrate was close to zero in noninoculatedpots and ranged between 3.5 and 14 m g–1 substrate in thedifferent inoculated treatments (Fig. 2). The HLDs weresignificantly higher for medic inoculated with G. mosseaethan with other AMF species or their mixtures at 4 wk aftersowing (Fig. 2). At 8 wk after sowing, HLDs were higherfor medic or leek inoculated with G. mosseae or G. intraradicescompared with G. claroideum. The HLDs in the mixedinoculation treatments at 8 wk after sowing were mostly

intermediate between the highest and lowest values observedfor the single AMF species (Fig. 2).

Only the AMF species introduced to the pots by inoculationwere detected by the real-time PCR assay in the pots afterharvest, indicating that no cross-contamination occurred andno PCR cross-amplification was encountered (Table 2). Thenumber of G. mosseae LSU copies per unit weight of rootsin the single inoculation treatment was very high at 4 wkcompared with the other fungi, and decreased substantiallybetween 4 and 8 wk. By contrast, values were much lower forG. intraradices and G. claroideum, and were similar at both 4

Fig. 2 Hyphal length density in the growth substrate of pots planted with medic or leek 4 and 8 wk after sowing. Mean values of four replicates 01 SE of means are shown. O pen bars, nonmycorrhizal plants (N M); closed bars, mycorrhizal plants. Plants colonized by M , Glomus mosseae; I, Glomus intraradices; C , Glomus claroideum. Mixed inoculation treatments are labelled with the respective letter combination in the treatment description. Different letters indicate significantly different means according to least significant difference (LSD) multiple range test following significant AN O VA (P ] 0.05).

Table 1 Colonization of plant roots by arbuscular mycorrhizal fungi (A MF) assessed by the magnified intersection method following Trypan blue staining

Plant age PlantColonization parameter (%)

A MF treatment (M , G . mosseae; C , G . claroideum; I, G . intraradices)

F AN O VA ¶M C I M C MI CI M CI

4 wk Medic H† 85.5 ab 58.5 c 89.0 a 87.0 ab 91.5 a 78.0 b 87.5 ab 6.49***A‡ 84.0 ab 55.0 c 80.0 ab 80.5 ab 88.5 a 73.5 b 82.5 ab 4.33**V§ 0.0 d 4.5 bc 40.0 a 3.0 cd 11.0 b 32.5 a 7.5 bc 22.1***

Leek H 70.5 bc 55.0 d 79.5 ab 75.5 abc 86.0 a 61.0 cd 75.0 ab 5.38**A 65.0 bc 51.5 c 71.0 ab 65.5 bc 82.5 a 57.5 bc 70.0 ab 4.38**V 0.0 d 7.0 bc 33.0 a 3.0 c 13.0 b 24.5 a 9.5 b 17.1***

8 wk Medic H 87.0 b 55.5 c 99.0 a 82.5 b 87.0 b 98.5 a 88.0 b 11.9***A 80.5 b 52.5 c 97.0 a 73.0 b 79.0 b 97.5 a 81.0 b 10.3***V 0.0 d 4.0 c 52.5 a 0.50 cd 22.5 b 48.0 a 19.5 b 30.1***

Leek H 92.5 bc 64.5 d 94.5 ab 81.0 cd 94.0 ab 99.5 a 91.5 bc 8.42***A 88.0 bc 62.5 d 92.0 ab 77.5 cd 87.5 bc 96.5 a 85.5 bc 5.60**V 0.0 d 8.0 c 55.5 a 3.5 cd 40.5 ab 53.5 a 35.0 b 28.1***

**, 0.001 η P ] 0.01; ***, P ] 0.001; different letters following treatment means in each row indicate significant differences (P ] 0.05).Percentage root length colonized by †, hyphae, with or without other structures; ‡, arbuscules; §, vesicles.¶Percentage values (H , A , V %) were arcsin and square-root transformed for AN O VA.

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and 8 wk (Table 2). When G. mosseae was present in theinoculum mixture, it was always a large component of theAMF community in the roots of both medic and leek (Fig. 3),but was relatively more abundant in the former (Table 3). Thedecrease in abundance of G. mosseae LSU copies between thetwo harvests was more marked in leek than in medic (seeinteraction between host plant and harvest time in Table 3).Inoculation with a mixture of G. intraradices and G. claroideumresulted in almost equal colonization of roots by the two AMFspecies 4 wk after sowing, but G. intraradices became dominant

by 8 wk (Fig. 3; Table 3). When summing all the LSU copynumbers per unit of root biomass across all AMF species inthe mixtures, no increased total colonization by AMF mixturescould be substantiated as compared with the single speciestreatments (analysis not shown). Further, no significantcorrelations were found between traditional root colonizationestimates and the real-time PCR assays (checked for each AMFspecies at each harvest).

Mycorrhizal plants were consistently larger than nonmy-corrhizal plants, with the exception of leek inoculated onlywith G. claroideum 4 wk after sowing (Fig. 4). The differencesamong inoculation treatments for both host plants at bothharvest times were highly significant (P ] 0.001). Glomus mos-seae inoculated singly promoted greatest biomass production inboth medic and leek at both harvests. When inoculated singly,G. intraradices promoted greater biomass of both host plantsthan G. claroideum, at 8 wk after sowing only. The biomass ofplants in mixed inoculation treatments never exceeded therange of growth promotion by the respective single-speciesinoculations (Fig. 4). The biomass of medic plants 8 wk aftersowing was similar, and high in all treatments that includedinoculation with G. mosseae, either alone or in a mixture,whereas there was significant variation among the sametreatments in the case of leek (Fig. 4).

Uptake of P showed a similar pattern to growth. Mycorrhizalplants always took up more P from the substrate than nonmy-corrhizal plants, with the single exception of leek inoculatedwith G. claroideum 4 wk after sowing (Fig. 5). Glomus mosseaeinoculated singly promoted greater total P uptake by medicthan the other two AMF species 4 wk after sowing. However,by 8 wk values for plants inoculated with G. intraradices hadreached the same total P content as those inoculated withG. mosseae. Medic inoculated with G. claroideum took up lessP during 8 wk of growth than those inoculated with either

Table 2 Copy numbers of large ribosomal subunit (LSU , thousands mg–1 FW roots) of three arbuscular mycorrhizal fungal (A MF) species in roots of medic and leek

Plant age PlantLSU of A MF species

A MF treatment (M , G . mosseae; C , G . claroideum; I, G . intraradices)

F AN O VAM C I M C MI CI M CI

4 wk Medic G . mosseae 1250 a 0 b 0 b 953 a 867 ab 0 b 1447 a 4.15**G . intraradices 0 c 0 c 146 a 0 c 37 bc 59 b 79 b 8.96***G . claroideum 0 b 121 a 0 b 25 b 0 b 34 b 26 b 8.16***

Leek G . mosseae 1898 a 0 c 0 c 809 bc 2512 a 0.0 c 1762 ab 9.02***G . intraradices 0 c 0 c 284 a 0 c 99 b 130 b 76 bc 9.30***G . claroideum 0 b 359 a 0 b 28 b 0 b 141 b 47 b 5.26**

8 wk Medic G . mosseae 340 a 0 c 0 c 220 ab 110 bc 0 c 94 bc 7.24***G . intraradices 0 b 0 b 258 a 0 b 8 b 293 a 8 b 45.9***G . claroideum 0 c 177 a 0 c 23 bc 0 c 46 b 11 bc 23.3***

Leek G . mosseae 346 a 0 c 0 c 224 ab 157 b 0 c 184 b 9.22***G . intraradices 0 c 0 c 244 a 0 c 44 c 201 ab 81 bc 4.70**G . claroideum 0 b 320 a 0 b 41 b 0 b 31 b 7 b 31.4***

**, 0.001 η P ] 0.01; ***, P ] 0.001; different letters following treatment means in each row indicate significant differences (P ] 0.05).

Fig. 3 Relative proportions of large ribosomal subunit copies of the three arbuscular mycorrhizal fungal (A MF) species in roots of medic and leek inoculated with the A MF mixtures 4 and 8 wk after sowing. Respective composition of A MF inoculum shown in left-hand column. Black sections, Glomus mosseae; hatched sections, Glomus claroideum; cross-hatched sections, Glomus intraradices. Each slice represents a mean of four replicates.

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G. intraradices or G. mosseae. As with biomass production,total P uptake by medic inoculated with AMF species mixturesnever exceeded the range of P uptake by plants inoculatedwith the respective single AMF species (Fig. 5). Colonizationby G. claroideum was associated with lower P uptake by leek fromthe substrate compared with G. mosseae, but not G. intraradicesat both harvests (Fig. 5). Phosphorus uptake by leek inoculatedwith AMF species mixtures did not exceed the range of P uptakeby plants inoculated with the respective single AMF species,with the notable exception of a mixed inoculation withG. intraradices and G. claroideum. This combination sup-ported greater P uptake by leek than either of the two AMFspecies separately 8 wk after sowing (Fig. 5).

Discussion

Root colonization assessed by traditional (non-vital) Trypanblue staining revealed typical sigmoid colonization patterns

by all three AMF species (Fig. 1). When inoculated singly,the three AMF varied in development, including length oflag, slope of the rapid growth phase, and plateau values ofpercentage colonization (Fig. 1; Table 1). Glomus mosseae wasthe fastest colonizer and G. claroideum the slowest, withG. intraradices intermediate, but eventually reaching thesame plateau value as G. mosseae. Importantly, all three AMFhad essentially reached plateau values of colonization by4 wk when the first destructive harvest took place. Real-timePCR assay at 4 and 8 wk, however, gave a different picture.Glomus mosseae showed values an order of magnitude higherfor LSU copy numbers in the roots at 4 wk, compared withthe other fungi. This declined dramatically (to about 1/4)between 4 and 8 wk, so that at 8 wk all the AMF inoculatedsingly showed similar values in roots of both plant species(Table 2). The other fungi showed LSU copy numbers atboth harvests that more closely mirrored the percentagecolonization. If LSU copy number is to be regarded as a proxy

Fig. 4 Combined shoot and root biomass of medic and leek 4 and 8 wk after sowing. Mean values of four replicates 01 SE of means are shown. O pen bars, nonmycorrhizal plants (N M); closed bars, mycorrhizal plants. Plants colonized by M , Glomus mosseae; I, Glomus intraradices; C , Glomus claroideum. Mixed inoculation treatments are labelled with the respective letter combination in the treatment description. Different letters indicate significantly different means according to least significant difference (LSD) multiple range test following significant AN O VA (P ] 0.05).

Table 3 Three-way AN O VA for relative proportions of large ribosomal subunit copy numbers of the three arbuscular mycorrhizal fungal (A MF) species in roots of medic and leek inoculated with A MF mixtures 4 and 8 wk after sowing

Factor df Glomus mosseae Glomus claroideum Glomus intraradices

Host plant (Pl) 1 4.86†* 1.19 ns 0.31 nsInoculation treatment (I) 3 807.9*** 43.5*** 187.6***Harvest (H) 1 25.68*** 3.69(*) 24.8***Pl ⋅ I 3 0.98 ns 1.67 ns 2.54(*)Pl ⋅ H 1 8.45** 0.79 ns 7.18*I ⋅ H 3 2.95* 20.2*** 7.14***I ⋅ Pl ⋅ H 3 1.41 ns 1.04 ns 1.08 nsError 48

ns, Not significant; (*), 0.05 η P ] 0.1; *, 0.01 η P ] 0.05; **, 0.001 η P ] 0.01; ***, P ] 0.001.†F values.

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for living fungal tissue, then these results require carefulconsideration. High initial values and subsequent declinein LSU copy number for G. mosseae could be explained by arapid spread of active mycelium, followed by a decrease invitality of the colonization. Similar decrease in AMF activitywith time has been documented previously using vitalstains and measurement of arbuscular development (Smith &Dickson, 1991). What is not clear is why G. mosseae behaveddifferently from the other two AMF. It could be that thisfungus both developed very rapidly in roots and thendeclined in activity very rapidly. We can speculate thatmultiplication of nuclei within the hyphae of (some) AMFmay occur at times of peak metabolic activities, leading toa discrepancy between DNA quantification and stainingas measures of fungal biomass. Formation of syncytial(multinucleated) cells, which would lead to an increase inLSU copy number, has been described in many differentorganisms (Beer & Arber, 1919; Comoglio et al., 1969; Lamb& Laird, 1976; Cantalejo et al., 2004) and is usually accompaniedby intense metabolic activity. However, it is not knownwhether similar processes occur in the AMF. Furthermore,traditional estimates of the extent of root colonizationinclude both living and dead fungus, and are related to rootlength rather than root weight. They also provide onlysemiquantitative measures of fungal development, becausecolonization intensities (numbers of hyphae, arbuscules andvesicles per colonized intersection) are normally not recorded.These factors, together with the fact that only a narrow rangeof colonization percentage for each AMF species at each harvestwas obtained, resulted in nonsignificant correlation betweenroot colonization estimates by staining and microscopy andreal-time PCR.

Consistent with the fast rate of root colonization and earlypeak in colonization vitality, G. mosseae was the most successfulcompetitor, as shown by the relatively high LSU copynumbers of this species in roots containing mixtures of fungi.Although conclusions at 4 wk might have been biased by thevery high LSU values for G. mosseae, this fungus also dominatedat 8 wk, providing some confidence in the finding. Inoculationwith a mixture of G. intraradices and G. claroideum resulted inalmost equal colonization of roots by the two AMF species at4 wk, but G. intraradices became dominant by 8 wk (Fig. 3;Table 3). When the three fungi were inoculated together,G. mosseae again predominated. Glomus mosseae was alsofaster in colonizing the substrate than the other AMF species,but by 8 wk G. intraradices had similar HLDs. Glomus claroi-deum mycelium developed poorly by comparison (Fig. 2). Allthe data are consistent with G. mosseae being the most effectivecompetitor and G. claroideum the weakest, with G. intraradicesintermediate. This observation is in accordance with previousresults showing that other isolates of G. mosseae and G. caledoniumwere faster colonizers of leek or clover roots compared withother Glomus species (Wilson & Trinick, 1983; Wilson, 1984;Hepper et al., 1988). However, Alkan et al. (2006) showedthat G. intraradices BEG141 occupied a higher proportion ofthe roots of medic at 4 wk than G. mosseae BEG 12, despitethe fact that the infectivity of G. mosseae inoculum was higher.These inconsistencies are probably caused by differences betweenfungal isolates, as well as in growth conditions, P availability,and many other factors shown to influence colonization(Daft, 1983; Boddington & Dodd, 2000; Alkan et al., 2006).

The mechanisms of competition among AMF within acommunity are not clear and need further elucidation withrespect to competition for carbon supply, exchange of signals

Fig. 5 Phosphorus uptake from substrate by medic and leek 4 and 8 wk after sowing. Phosphorus contents in shoots and roots were combined and mean seed P content was then subtracted. Mean values of four replicates 01 SE of means are shown. O pen bars, nonmycorrhizal plants (N M); closed bars, mycorrhizal plants. Plants colonized by M , Glomus mosseae; I, Glomus intraradices; C , Glomus claroideum. Mixed inoculation treatments are labelled with the respective letter combination in the treatment description. Different letters indicate significantly different means according to least significant difference (LSD) multiple range test following significant AN O VA (P ] 0.05).

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and other factors. Nevertheless, a competitive advantage ofearly root colonization has been proposed (Hepper et al.,1988), and this is also suggested by some of our results.However, it is also clear that the composition of (active) AMFcommunities can change with time because of a drop in activityof the very fast colonizers such as G. mosseae and steadyincrease in colonization and activity of G. intraradices over alonger time period (Table 2). This is in agreement withsuggestions of some previous studies (e.g. Wilson & Trinick,1983).

The identity of the host plant also appeared to be animportant determinant of AMF community composition inour experiment (Table 3). This is consistent with accumulatingevidence for selectivity of AMF symbionts by the host plants(Bever et al., 1996; Helgason et al., 2002) and, in some cases,complete failure of particular AMF to colonize one hostspecies, although others did become colonized (Alkan et al.,2006). However, the most common situation appears to bethe one we describe here, which is that colonization by theindividual AMF in a community varies quantitatively (not aspresence or absence), and it is crucial to develop methods thatwill allow the variations to be measured.

All in all, real-time PCR proved a very useful tool for dis-secting AMF community composition in our study andextends the research of Alkan et al. (2006), who employed asimilar approach. Use of ribosomal RNA genes is the onlycurrently viable molecular approach to targeting differentAMF species, as too little is known about other parts of AMFgenomes. Single-copy genes should be targeted in future,overcoming problems likely to be associated with the substantialsequence polymorphism within ribosomal RNA genes sharingthe same cytoplasm ( Jansa et al., 2002b; Sanders, 2004).Preliminary work also indicates that real-time PCR could beused to analyse AMF communities in soil (J.J., unpublisheddata), possibly overcoming both the PCR and cloning biasesassociated with the current methods (Hempel et al., 2007).Our approach may also be useful in assessing vitality of theAMF colonization, although assessment of fungal RNAwould be preferable when this becomes realistic.

We observed strong positive effects of all inoculation treat-ments on biomass production of, and P uptake by, both leekand medic. When inoculated singly, G. claroideum was con-sistently least effective in increasing P uptake and growthcompared with G. mosseae and G. intraradices. These differenceswere related to differences in the rate and extent of colonization,and particularly to development of extraradical mycelium.The HLDs of the fungi were significantly positively correlatedwith plant P uptake (F1,126 _ 175.1, P ] 0.001, R2 _ 0.58). Thisfinding extends previous results showing that the same isolatesof G. mosseae and G. intraradices were able to take up P morequickly and from greater distances from the roots than G. claroi-deum (Jansa et al., 2005). Interestingly, plant P uptake was notalways mirrored in plant biomass. For example, medicbiomass was greater when inoculated with G. mosseae than

with G. intraradices (Fig. 4), but total P uptake was greaterwith G. intraradices (Fig. 5). It is possible that G. intraradicesutilized more carbon from the medic plants, resulting in lowergrowth but higher tissue P concentrations (results not shown)in the short term. This variation is in accord with many pre-vious studies showing that mycorrhizal benefits are stronglydependent on the plant–fungus combination (Smith et al.,2004). Interestingly, the mycorrhizal plants took several timesmore P from the soil than predicted by Colwell (1963) andisotopic exchange kinetic approaches (see above), meaningthat these P-availability indicators worked particularly poorlyfor our substrate, where possibly organic P might present asignificant reserve of slowly plant-available P (Oberson et al.,2001).

The effects of AMF mixtures on plant growth and P uptakewere mostly within the range of the effects exerted by therespective single AMF species. In the main, there was littleevidence for increased P uptake and/or growth of plantscolonized by several AMF species compared with a singlespecies. When G. mosseae was included, the community inthe roots was dominated by this species (Fig. 3), which washighly effective when inoculated singly, and clearly had a majorinfluence on symbiotic performance of plants colonized bymixtures.

In one case we did observe a synergistic effect of dualinoculation. Phosphorus uptake by leek inoculated with amixture of G. intraradices and G. claroideum was greater at8 wk than when inoculated with either of the two AMF speciesseparately. This is the first direct evidence for functionalcomplementarity with respect to P acquisition between AMFspecies colonizing roots, as suggested by Koide (2000). Thesetwo fungi showed similar progress of colonization wheninoculated singly; when inoculated together, this was one ofthe few AMF communities that was not dominated by asingle fungal species at some time point during development(Fig. 3). Synergy between the fungi can therefore probably beexplained on the basis of differences in P uptake by the fungi,which in this experiment had different HLDs in the soil.Previous work has also shown that G. intraradices could bridgeP-poor soil volumes close to the roots without extensivebranching in them (Jansa et al., 2003a). These featureswould predispose G. intraradices for successful cooperationwith G. claroideum over the same period of development, aswe have shown. Previously, the benefits of colonization ofplants by several AMF species were suggested to be in temporalpartitioning of activities (different AMF active at differentperiods of time); as a buffer against change (different AMFadapted to different environmental conditions); and as a meansof minimizing growth depressions that can arise if single,inefficient fungi are inoculated singly (Daft & Hogarth, 1983;Abbott & Gazey, 1994; Pringle & Bever, 2002; Sanders,2003). The question remains why such effects of multipleinoculations are not observed more often. Early experimentsgenerally lacked the technology to estimate precisely the

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composition of AMF communities developing in roots. Inconsequence, lack of synergistic effects among AMF specieswith respect to plant growth (Daft, 1983; Daft & Hogarth,1983; Pearson et al., 1994) and/or plant P uptake may havebeen caused by the fact that one AMF species became dominant,as shown here with G. mosseae. The quantitative moleculartools now available will enable links between the compositionand function of AMF communities to be unravelled. It will beimportant to link the community studies to direct assessmentof nutrient fluxes via hyphae and roots by using radio- andstable isotopes and compartmented cultivation systems.

Acknowledgements

We are grateful to Stephen Rogers (then at CSIRO Land andWater, Adelaide, Australia) for sharing real-time PCR equip-ment and for valuable advice and discussions. We would liketo express our gratitude to Colin Rivers and Debbie Miller fortheir excellent technical assistance, and Sandy Dickson for herhelp with maintaining the pot experiments. Financial supportof the Swiss National Science Foundation for a postdoctoralfellowship to J.J. and the Australian Research Council forproject support is gratefully acknowledged.

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Supplementary Material

The following supplementary material is available for thisarticle online:

Table S1 PCR primers used in this study for specificamplification of large ribosomal subunit genes from differentAMF species

Fig. S1 Relationships between copy numbers of target largeribosomal subunit DNA and the cross points of the real-timePCR curve.

Fig. S2 Real-time PCR amplification of large ribosomalsubunit of Glomus mosseae from root DNA extract of Medicagotruncatula inoculated with G. mosseae BEG161.

This material is available as part of the online article from:http://www.blackwell-synergy.com/doi/abs/10.1111/j.1469-8137.2007.02294.x(This link will take you to the article abstract).

Please note: Blackwell Publishing are not responsible forthe content or functionality of any supplementary materialssupplied by the authors. Any queries (other than missingmaterial) should be directed to the New Phytologist CentralOffice.