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Basic and Applied Ecology 13 (2012) 663–672

ffects of arbuscular mycorrhiza on community composition and seedlingecruitment in temperate forest understory

adri Koorema,∗, Ülle Saksa, Virve Sõberb,c, Annika Uibopuua, Maarja Öpika, Martin Zobela,ari Mooraa

Department of Botany, Institute of Ecology and Earth Sciences, University of Tartu, Lai 40, 51005 Tartu, EstoniaDepartment of Zoology, Institute of Ecology and Earth Sciences, University of Tartu, Vanemuise 46, 51014 Tartu, EstoniaDepartment of Biology, Section of Ecology, University of Turku, FIN-20014 Turku, Finland

eceived 3 November 2011; accepted 17 September 2012

bstract

Arbuscular mycorrhizal (AM) fungal communities can influence the species composition of plant communities. This influenceay result from effects of AM on seedling recruitment, although the existing evidence is limited to experimental systems. We

ddressed the impact of AM fungi on the plant community composition and seedling recruitment of two species – Oxaliscetosella and Prunella vulgaris – in a temperate forest understory. We established a field experiment over two years inhich soil fertility (using fertilizer to enhance and sucrose to decrease fertility) and the activity of AM fungi (using fungicide)as manipulated in a factorial design. Species richness, diversity and community composition of understory plants were not

nfluenced by soil fertility or AM fungal activity treatments. However, plant community composition was marginally significantlyffected by the interaction of these treatments as the effect of AM fungal activity became evident under enhanced soil fertility.uppression of AM fungal activity combined with decreased soil fertility increased the number of shoots of herbaceous plants.nchanged activity of AM fungi enhanced the growth of O. acetosella seedlings under decreased soil fertility, but did not

nfluence the growth of P. vulgaris seedlings. We conclude that the role of AM fungi in structuring plant communities dependsn soil fertility. AM fungi can have a strong influence on seedling recruitment, especially for those plants that are characteristicf the habitat.

usammenfassung

Die Gesellschaften der arbuskulären Mycorrhizapilze (AM) können die Artenzusammensetzung von Pflanzengemeinschafteneeinflussen. Dieser Einfluss könnte ein Resultat von Effekten der AM auf die Ansiedlung von Keimlingen sein, obwohlie existierenden Hinweise auf experimentelle Systeme beschränkt sind. Wir untersuchten den Einfluss der AM-Pilze auf die

usammensetzung der Pflanzengesellschaft und die Ansiedlung von Keimlingen von zwei Arten, Oxalis acetosella und Prunellaulgaris, in der Krautschicht eines gemäßigten Waldes. Wir richteten ein Freilandexperiment über zwei Jahre ein, bei dem dieodenfruchtbarkeit (durch den Einsatz von Dünger, um die Fruchtbarkeit zu erhöhen, und Sukrose, um die Fruchtbarkeit zu

enken) und die Aktivität der AM-Pilze (durch den Gebrauch von Fungiziden) in einem faktoriellen Design manipuliert wurde.ie Artenvielfalt, die Diversität und die Gesellschaftszusammensetzung der Pflanzen in der Krautschicht wurden durch dieodenfruchtbarkeits oder die AM-Pilzaktivitätsbehandlung nicht beeinflusst. Die Zusammensetzung der Pflanzengesellschafturde marginal signifikant durch die Interaktion der beiden Varianten beeinflusst, da der Effekt der AM-Pilzaktivität bei

∗Corresponding author. Tel.: +372 737 6221; fax: +372 737 6380.E-mail address: kadri.koorem@ut.ee (K. Koorem).

439-1791/$ – see front matter © 2012 Gesellschaft für Ökologie. Published by Elsevier GmbH. All rights reserved.ttp://dx.doi.org/10.1016/j.baae.2012.09.008

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64 K. Koorem et al. / Basic and Applied Ecology 13 (2012) 663–672

rhöhter Bodenfruchtbarkeit erkennbar wurde. Die Unterdrückung der AM-Pilzaktivität in Kombination mit einer vermindertenodenfruchtbarkeit erhöhte die Anzahl der Keimlinge krautiger Pflanzen. Eine unveränderte Aktivität der AM-Pilze förderteas Wachstum der Oxalis acetosella-Keimlinge bei verminderter Bodenfruchtbarkeit, aber nicht das Wachstum der Prunellaulgaris-Keimlinge. Wir schließen daraus, dass die Rolle der AM-Pilze bei der Strukturierung der Pflanzengesellschaften voner Bodenfruchtbarkeit abhängt. AM-Pilze können einen starken Einfluss auf die Ansiedlung von Keimlingen haben, besondersei den Pflanzen, die für das Habitat charakteristisch sind.

2012 Gesellschaft für Ökologie. Published by Elsevier GmbH. All rights reserved.

rowth;

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eywords: AM fungi; Soil fertility; Soil heterogeneity; Seedling g

ntroduction

Arbuscular mycorrhizal (AM) fungi (phylum Glomeromy-ota) colonize the roots of most terrestrial plants, facilitatingineral nutrient uptake from the soil in exchange for

lant-assimilated carbon (Smith & Read 2008). Theres increasing evidence that mycorrhizal fungi can affectlant growth and modify the strength of biotic interac-ions among plants, but also among plants and heterotrophicrganisms (van der Heijden 2002), indicating that mycor-hiza can greatly influence plant community structure.lthough the potential role of mycorrhiza in structur-

ng plant communities has been discussed for decadesZobel, Moora, & Haukioja 1997), evidence of this rolen different abiotic and biotic environments remains scarceKlironomos et al. 2011).

AM fungi can alter community structure by shifting theompetitive balance in plant communities and giving relativedvantages to species that may otherwise be inferior competi-ors under particular conditions (Moora & Zobel 2010). Moreenerally, AM can modify plant niches and affect plant–soilommunity feedbacks, thereby altering the entire networkf biotic interactions, resulting in a major impact on plantoexistence (Bever et al. 2010). These processes may playcrucial role in determining the fate of plant individuals.

n particular, the effect of AM on plants can be especiallyronounced at the seedling establishment stage (Francis &ead 1995), which is crucial in shaping local species compo-

ition and diversity of plant communities (Dickson & Foster008).

Evidence of the effect of AM fungi on seedlings is scarce.ot experiments have shown that AM facilitates the growthf single seedlings, but not in the presence of competitione.g. Kytöviita, Vestberg, & Tuomi 2003). Further evidencey van der Heijden (2004) in grassland microcosms showshat AM fungi promote seedling establishment by integrat-ng emerged seedlings into extensive hyphal networks andy supplying nutrients to the seedlings. In general, the exist-ng evidence (reviewed by van der Heijden & Horton 2009;

oora & Zobel 2010) shows that the role of AM in seedlingstablishment is species-specific and depends on particu-ar environmental conditions. For instance, AM may be

mportant in unfertile conditions and detrimental in fertileonditions. However, the relevant evidence is limited largelyo artificial systems and information on the effect of AM on

dwi

Benomyl; Vegetation

eedling establishment in natural ecosystems is extremelyparse.

We address the role of AM fungi in shaping the structure oflant communities and seedling establishment in a temperateerb-rich boreonemoral coniferous forest where most under-tory plants are arbuscular mycorrhizal (Uibopuu, Moora,pik, & Zobel 2012). One can therefore speculate that AMlays a role in shaping the understory plant communities inoreonemoral forests. As small-scale soil heterogeneity isypical of natural ecosystems (Farley & Fitter 1999), one canlso assume that the role of AM varies among loci with dif-erent soil fertility. This idea is supported by the re-allocationf resources from belowground to aboveground with increas-ng soil fertility (Bazzaz 2004), which leads to a change fromominant competition for nutrients to competition for lightWilson & Tilman 1993). Therefore we hypothesize that theositive effect of AM fungi is more pronounced in low fertil-ty conditions for both community composition and seedlingstablishment.

aterials and methods

tudy site

The study site is located in Koeru, central Estonia58◦58′N; 26◦05′E) in a flat landscape with a mosaic ofultivated arable areas and forest. It is an herb rich bore-nemoral forest with Picea abies, Corylus avellana andxalis acetosella dominating in the tree layer, shrub layer

nd understory, respectively (see Zobel, Kalamees, Püssa,oosaluste, & Moora 2007; Moora, Öpik, Zobel, & Zobel009 for more details).

xperimental design

A factorial field experiment which manipulated soil fertil-ty (three levels) and activity of AM fungi (two levels) wasstablished in May 2008. The position of each experimen-al unit (round plot with 15 cm diameter) within a uniform0 m × 50 m area was chosen with the help of random coor-

inates (http://www.random.org/). Experimental treatmentsere randomly assigned to 240 experimental units, result-

ng in 40 experimental units per treatment combination. Soil

pplied

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K. Koorem et al. / Basic and A

ertility was enhanced, decreased or unchanged (as a control).oil fertility was enhanced by application of a long-lastingomplex fertilizer Osmocote in an amount of 125 g m−2 yr−1

N – 20 g m−2 yr−1, P – 6 g m−2 yr−1 and K – 11 g m−2 yr−1).ertilizer was added annually at the start of the vegetationeriod in May. Soil fertility was decreased by application ofarbon in the form of sugar (sucrose). Sucrose was addedt the beginning and the end of the vegetation period (Maynd September, respectively), 500 g m−2 for each applica-ion. Carbon addition promotes soil microbial activity, whichesults in increased immobilization of inorganic nitrogennd decreased rates of net N mineralization and nitrificationTörök et al. 2000).

Activity of AM fungi was suppressed by application of theungicide Benomyl (Benlate, 2 g m−2, dissolved in 2686 mlf water), which was applied at 3-week intervals through-ut the growing seasons until the end of the experiment inctober 2009. The treatment in which AM fungal activityas unchanged received an equal amount of tap water as

ungicide treatment to eliminate the effect of water. Benomylas been used successfully to suppress mycorrhizal activ-ty in many studies: it is effective in reducing AM fungalolonization and has the fewest drawbacks of all potentialethods of AM fungal suppression in the field (Hartnett &ilson 2002). However, the addition of Benomyl can also

ffect soil biota (including fungi other than AM fungi) andutrient availability (Smith, Hartnett, & Rice 2000; Welc,avnskov, Kieliszewska-Rokicka, & Larsen 2010).The impact of manipulating soil fertility and AM fun-

al activity on natural regeneration was followed in all 240xperimental units where emergence of the dominant under-tory species, O. acetosella (hereafter Oxalis) was recorded.he number of seedlings of other plant species was insuffi-ient for replicated observation (preliminary observations byhe authors). Therefore, seeds of the second target speciesrunella vulgaris (hereafter Prunella), were sown in 60 ran-omly chosen experimental units (ten replicates per treatmentombination). Seeds of Prunella were collected from thexperimental area in summer 2008 and sown (20 seeds perxperimental unit) in October 2008, four months after thetart of soil fertility and AM fungal activity treatments. Theffect of manipulations on seedling growth was estimatedn half of the experimental units (120): in 60 experimentalnits for Oxalis and 60 experimental units for Prunella (teneplicates per treatment combination for both species).

ollection of samples

Species composition and the number of plant shoots inach experimental unit were recorded twice: before the startf the experimental manipulations (May 2009) and before

he final harvesting (October 2009). The number of emergedeedlings was first recorded in May 2009, a year after thetart of the soil fertility and AM fungal activity treatment.hereafter, the number of emerged seedlings was recorded

Ogp

Ecology 13 (2012) 663–672 665

t 3-week intervals until the end of the experiment, in Octo-er 2009. The cumulative number of Oxalis and Prunellaeedlings which emerged during the vegetation period in009 was used to estimate their emergence success. At thend of June 2009, the three most distant seedlings of each tar-et species were chosen to estimate the effect of treatmentsn their growth. The rest of the emerged seedlings were sub-equently removed from each experimental unit at 3-weekntervals until the end of the experiment to avoid seedlingompetition.

For final harvesting, samples were collected with a spe-ial soil corer (diameter: 15 cm), which enabled removal ofhe soil core with roots and aboveground plant parts fromhe forest floor. Samples were transported to the laboratoryhere the roots and aboveground parts of herbaceous andoody plants were separated from the sample. During thisrocess, the above- and belowground parts of seedlings ofarget species were separated from other herbaceous plantsnd stored individually. The separation of seedling roots fromoil cores was problematic, as the roots were difficult to iden-ify and liable to break. We were able to collect abovegroundiomass of Oxalis from 14 experimental units and below-round biomass from 11 experimental units. We were able toollect both above- and belowground biomass for Prunellaeedlings from 14 experimental units. All biomasses excepthe roots of the seedlings were dried to constant weight at0 ◦C and weighed. The mean weight of the seedlings grownn each experimental unit was used for further biomass analy-es. Roots of the seedlings were dried to a constant weight at0 ◦C, weighed, stored in a dry place at room temperature andsed in order to determine AM fungal colonization (detailselow).

Fifty grams of soil were collected from the upper 10 cm ofhe soil cores for chemical analysis. Soil samples were keptt −80 ◦C until analysis. Soil was processed at the Labora-ory of Agricultural Research Center in Saku, Tallinn wherehe content of soil organic carbon (C), total nitrogen (N),mmonium (NH4), phosphorus (P), and potassium (K) wasetermined (see Appendix A for details).

M analysis

The percentage of AM fungal colonization in roots wasstimated from a seedling individual from each experimen-al unit from which a seedling with a sufficiently largeoot system could be harvested. Roots were stained withrypan blue (Koske & Gemma 1989) and the percentagef root mycorrhizal colonization was assessed using theagnified grid-line intersection method (McGonigle, Miller,vans, Fairchild, & Swan 1990); see Appendix A for details.eedlings from 18 experimental units (five Oxalis and threerunella grown with natural AM fungal activity and seven

xalis and three Prunella grown with suppressed AM fun-al activity) developed sufficiently large root systems for thisrocedure.

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66 K. Koorem et al. / Basic and A

ata processing

The effect of soil fertility manipulation (with three lev-ls: sucrose addition, unchanged, fertilizer addition), andM fungal activity manipulation (with two levels: sup-ressed with fungicide or unchanged) and their interaction onoil nutrient content, seedling root AM fungal colonization%), plant community parameters (above- and belowgroundiomass of vascular plants, species richness, Simpson’sndex, log response ratio of species (see below)) and seedlinghoot (for Oxalis and Prunella) and root dry weight (forrunella) (g) was estimated using 2-way ANOVA. The effectf soil fertility and AM fungal activity on Oxalis seedlingoot biomass was tested with 1-way ANOVA because of aack of data in some treatment combinations. The effect ofxperimental treatments on cumulative number of seedlingsas estimated by Generalized Linear Models (GLZ) (usingoisson distribution and log link function), all with soil fer-

ility and AM fungal activity serving as fixed factors. Theisher LSD post hoc multiple comparison test was appliedith a significance level of 0.05 in order to estimate the differ-

nces between treatments. The above- and/or belowgroundiomass of some seedlings was very low (<0.0001 g, the exacteight was undeterminable). Therefore, we added 0.0001 g

o all above- and belowground biomass measures. Biomassnd percentage of AM fungal colonization data were log- andrcsine transformed, respectively, prior to statistical analysis.ample size varied among response variables and is reflected

n the reported degrees of freedom (Table 1).Changes in the number of plant shoots of the 11 most

bundant (occurring in more than 3% of experimental units)lant species in the experimental units from May 2008 toctober 2009 was assessed by using the log response ratio

LRR). LRR is considered to be the most suitable measure ofelative response due to its statistical properties (Goldberg,ajaniemi, Gurevitch, & Stewart-Oaten 1999). LRR was cal-ulated as a log-ratio of the number of shoots in experimentalnits in October 2009 and May 2008 (LRR = log (no. shoots009/no. shoots 2008)). All analyses were conducted with theindows version of STATISTICA 7 (StatSoft, Inc., Tulsa,K, USA).Species richness (number of species present) in an experi-ental unit and Simpson’s diversity index (calculated using

he number of shoots of species present in an experimentalnit) – except Prunella seedlings when seeds of this speciesere added in both cases – were used as integrative char-

cteristics of vegetation. The Simpson’s index is one of theost meaningful and robust diversity measures available and

rovides a good estimate of diversity even for relatively smallample sizes (Magurran 2004). Simpson’s diversity indexas calculated by EstimateS 7.5 (Version 7.5, R.K. Colwell,npublished). Non-metric multidimensional scaling (NMS)sing Bray–Curtis distance and permutation-based nonpara-

etric MANOVA (PerManova) were used to test whether

lant communities differ under different experimental treat-ents (see Appendix A for details). NMS and PerManova

as

Ecology 13 (2012) 663–672

ere conducted with PC-ORD ver. 5.10 (McCune & Grace002).

esults

mpact of experimental treatments on soilarameters and root AM fungal colonization

Soil C% and N% were the only soil variables measuredhich were not influenced by the soil fertility treatment

Table 1, Appendix A: Table 1, Fig. 1A and B). Soil C/Natio was considerably greater and soil P and K content wasignificantly lower under decreased compared to enhancedoil fertility (Table 1, Appendix A: Table 1, Fig. 1C, E and). Soil NH4 content was greater under enhanced soil fertil-

ty than under untreated soil fertility (Table 1, Appendix A:able 1, Fig. 1D). None of the soil variables measured wasffected by AM fungal activity treatment (Table 1, Appendix: Table 1). Fungicide addition lowered the mean rate of AM

ungal colonization in seedling roots significantly (Table 1,ppendix A: Table 1, Fig. 1G).

ommunity level effects

The number of shoots at the final harvest was unaffectedy the soil fertility treatment, but was greater under sup-ressed AM fungal activity (Table 1, Fig. 1A, Appendix A:able 2). Soil fertility and AM fungal activity treatments hado significant effects on plant species richness or Simpson’siversity index (Table 1, Fig. 1B and C, Appendix A: Table). The NMS and PerManova revealed no clear differencesn understory plant community composition under soil fertil-ty or AM fungal activity treatments (p = 0.69 and p = 0.52,espectively). However, there was a marginally significantnteraction between these treatments (p = 0.07). StratifiedMS showed that under enhanced fertility conditions, thereas a marginally significant difference in community com-osition between treatments with and without suppression ofM fungi (p = 0.07).Aboveground biomass of vascular plants was signifi-

antly affected by the soil fertility treatment (Table 1):lants under enhanced fertility produced significantly moreiomass than plants under untreated and decreased fertil-ty (Fig. 1D, Appendix A: Table 2). Suppressed AM fungalctivity increased significantly the aboveground biomass ofascular plants (Table 1, Appendix A: Table 2). None of thereatments had a significant effect on belowground biomassf vascular plants (Table 1, Fig. 1E, Appendix A: Table 2).

esponses by individual plant species

The shoot log response ratio of only two of the 11 mostbundant plant species reacted significantly to the suppres-ion of AM fungal activity, increasing for Luzula pilosa

K. Koorem et al. / Basic and Applied Ecology 13 (2012) 663–672 667

Table 1. The results of 2-way ANOVA with soil fertility (decreased, unchanged or enhanced), activity of arbuscular mycorrhizal (AM) fungi(suppressed, unchanged) and their interaction serving as fixed factors.

VAR Soil fertility (S) AM activity (AM) S × AM Error DF

DF F p DF F p DF F p

C% 2 0.33 0.72 1 0.89 0.35 2 0.47 0.63 224N% 2 0.36 0.70 1 1.41 0.24 2 3.07 0.05 224C/N 2 4.08 0.02 1 0.48 0.49 2 2.98 0.05 224NH4 2 3.40 0.04 1 0.02 0.89 2 8.46 <0.001 225P 2 31.44 <0.001 1 0.002 0.97 2 0.02 0.98 225K 2 13.61 <0.001 1 0.04 0.83 2 0.14 0.87 225AMcol 2 0.33 0.78 1 19.52 <0.001 2 0.45 0.45 12Shoot 2 1.06 0.35 1 4.06 0.05 2 3.68 0.03 234SPric 2 0.51 0.60 1 0.64 0.42 2 2.45 0.09 231D′ 2 1.19 0.31 1 0.06 0.81 2 0.35 0.71 222ABbm 2 5.45 0.005 1 4.02 0.05 2 1.50 0.23 232BGbm 2 0.52 0.60 1 0.85 0.36 2 0.50 0.61 188LRRluz 2 0.41 0.71 1 32.84 0.03 2 8.00 0.11 2LRRver 2 2.40 0.15 1 26.98 0.001 2 2.83 0.11 9OXAab 2 0.58 0.58 1 2.49 0.15 2 4.31 0.05 8PRUab 2 3.08 0.10 1 0.44 0.53 2 0.26 0.78 8PRUbg 2 0.68 0.53 1 <0.001 0.98 2 0.19 0.83 8

Characteristics given: DF – degrees of freedom; F – F statistic; p – p value. Variables (VAR) given: C% – soil organic C (%); N% – soil total N (%); C/N –ratio of soil organic C and total N; NH4, P, K – soil NH4, P and K content, respectively (mg/kg); AMcol – arbuscular mycrorrhizal colonization in seedlingr n’s divep la piloso nd biom

(fT

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oots (%); shoot – number of shoots; SPric – species richness; D′ – Simpsolants, respectively (g); LRRluz, LRRver – shoot log response ratio of Luzuf Oxalis acetosella seedlings (g); PRUab, PRUbg – above- and belowgrou

Table 1, Fig. 2A, Appendix A: Table 3) and decreasingor Veronica chamaedrys (Table 1, Fig. 2B, Appendix A:able 3).Of 240 experimental units, 147 had naturally emerged

eedlings of Oxalis; 29 of 60 experimental units in whicheeds of Prunella had been sown produced seedlings. Theoil fertility treatment had a marginally significant effect onhe cumulative number of Oxalis seedlings (GLZ: χ2 = 5.85,= 0.05): more seedlings were found under decreased com-ared to enhanced soil fertility (Appendix A: Table 3).uppressed AM fungal activity and the interaction betweenoil fertility and AM fungal activity had no significantffect (GLZ: χ2 = 3.55, p = 0.06 and GLZ: χ2 = 2.11, p = 0.35,espectively; Fig. 2C, Appendix A: Table 3). The cumulativeumber of Prunella seedlings was not influenced by the soilertility treatment (GLZ: χ2 = 0.90, p = 0.64, Appendix A:able 3). Suppression of AM fungal activity decreased theumber of Prunella seedlings (GLZ: χ2 = 12.53, p < 0.001,ppendix A: Table 3), whereas the interaction between

oil fertility and AM fungal activity had no effect (GLZ:2 = 1.52, p = 0.47, Fig. 2D, Appendix A: Table 3).Aboveground biomass of Oxalis seedlings was unaffected

y soil fertility and AM fungal activity treatments (Table 1,ppendix A: Table 3). The interaction between soil fertility

nd AM fungal activity treatments influenced abovegroundiomass of Oxalis seedlings (Table 1): aboveground biomass

f seedlings was enhanced by natural AM fungal activityhen fertility was decreased (Fig. 2E, Appendix A: Table). Soil fertility manipulation (1-way ANOVA, F(2;8) = 0.53,

oS

n

rsity index; ABbm, BGbm – above- and belowground biomass of vasculara and Veronica chamaedrys, respectively; OXAab – aboveground biomassass of Prunella vulgaris seedlings, respectively (g).

= 0.61) and suppression of AM fungal activity (1-wayNOVA, F(1;9) = 2.41, p = 0.16) had no effect on below-round biomass of Oxalis. None of the treatments exhibitedny significant effect on above- or belowground biomass ofrunella seedlings (Table 1, Fig. 2F, Appendix A: Table 3).

iscussion

The results of this experimental study demonstrateuccessful manipulation of soil fertility and AM fungalolonization in the field: the amount of soil nutrients andboveground biomass of vascular plants was increased byertilizer addition, soil C/N ratio was increased by additionf sucrose and AM fungal colonization in seedling roots wasuppressed by addition of fungicide. Although the overallffect on the plant community remained modest, it is in lineith previous findings (Klironomos et al. 2011).Suppression of AM fungal activity increased the number

f shoots slightly, particularly under decreased soil fertility.s the number of naturally recruiting seedlings in the forestnderstory was generally very low, this response is expectedo be due largely to clonal growth, as most of the plant speciesn Koeru forest are able to propagate clonally (Moora et al.009). A positive effect of fungicide treatment on the number

f shoots has also been observed by others (Wilson, Hartnett,mith, & Kobbeman 2001).We observed no significant changes in plant species rich-

ess and diversity due to suppression of AM fungal activity

668 K. Koorem et al. / Basic and Applied Ecology 13 (2012) 663–672

EnhancedUnchangedDecreased14

16

18

20

22

24

Num

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biW1

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espectively) under decreased (sucrose addition), unchanged and enctivity of arbuscular mycorrhizal fungi (solid bars). Different lettisher LSD test.

y fungicide addition. Evidence from other field studies

s mixed; plant richness has increased (Smith, Hartnett, &

ilson 1999), decreased (Zobel, Pilt, Moora, Pärtel, & Liira999) or remained unchanged (O’Connor, Smith, & Smith

OAc

(fertilized) soil fertility and suppressed (open bars) or unchangedand b) denote a significant difference among means according to

002), and plant diversity has increased (Smith et al. 1999;

’Connor et al. 2002) or decreased (Karanika, Mamolos,lifragis, Kalburtji, & Veresoglou 2008) after fungi-

ide addition. Small-scale plant diversity is dependent on

K. Koorem et al. / Basic and Applied Ecology 13 (2012) 663–672 669

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Fig. 2. Mean (±SE) log-response ratio of Luzula pilosa and Veronica chamaedrys (A and B, respectively), Oxalis acetosella and Prunellavulgaris seedling number and shoot biomass (C, D and E, F, respectively) under decreased (sucrose addition), unchanged and enhanced(fertilized) soil fertility and suppressed (open bars) or unchanged activity of arbuscular mycorrhizal fungi (solid bars). Different letters (a andb) denote a significant difference among means according to Fisher LSD test.

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ultiple biotic interactions (Gross et al. 2009), thus mycor-hizal symbiosis alone may not play a crucial role inetermining the number of locally coexisting species.

Suppression of AM fungi led to some differences in plantommunities growing under enhanced soil fertility, whichmplies a loss of the beneficial effect of AM symbiosis forlants in fertile soil (van der Heijden & Horton 2009). Somelant species may suffer from mycorrhizal growth depres-ion in fertile conditions more than others and changes in thebundance of such species may lead to differences in plantommunity composition. This is supported by the observedecrease in the abundance of some plant species under sup-ressed AM fungal activity and an increase in others. Anxpected significant decrease was observed in the abundancef Veronica chamaedrys, a species previously shown to reactositively to AM fungal inoculum collected from old growthtands in Koeru forest (Uibopuu et al. 2012). By contrast,uppressed AM fungal activity increased the abundance of L.ilosa, a species that has been generally recognized as non-ycorrhizal (Harley & Harley 1987). Differential response

f particular plant species to fungicide application has beenhown in previous field experiments (Smith et al. 1999;

ilson et al. 2001; O’Connor et al. 2002; Maron, Marler,lironomus, & Cleveland 2011), and in pot experiments withoeru forest understory plant species (Uibopuu et al. 2012).Seedlings of target species, Oxalis and Prunella, represent-

ng a forest specialist and a generalist species, respectivelyÖpik, Metsis, Daniell, Zobel, & Moora 2009), showed dif-erent responses to experimental treatments. Emergence ofxalis was unaffected by experimental manipulations, but

ubsequent growth was enhanced by natural activity of AMungi when soil fertility was decreased. Under low soil fertil-ty, seedlings experience less shoot competition than in fertileonditions, in which plants allocate more biomass above-round (Bazzaz 2004). At the same time, seedlings growingn low soil fertility conditions may experience more nutri-nt stress and AM symbiosis improves nutrition for manylant species (van der Heijden & Horton 2009). Suppressionf AM fungi under low soil resources may therefore resultn inhibited seedling growth. In contrast to the behavior ofxalis, emergence of Prunella was positively influenced byatural activity of AM fungi, but subsequent growth of theseeedlings showed no significant response to experimentalanipulations. This shows that the growth of seedlings of

his generalist plant species does not depend on the presencef AM fungi even when soil fertility is low.

We hypothesized that both the effect of AM fungi oneedling growth and on plant community composition areore pronounced in locally less fertile conditions. Although

eedlings benefited from AM fungi in less fertile conditions asxpected, this did not manifest in changes to plant communityomposition. By contrast, variation in plant community com-

osition in response to suppression of AM fungi was limitedo the enhanced fertility treatment. This pattern can be inter-reted as indirect evidence that some plant species are unableo discard symbiotic interactions that are no longer beneficial

B

Ecology 13 (2012) 663–672

Johnson, Graham, & Smith 1997), although the presence ofuch a mechanism is subject to future study. Alternatively,t is possible that direct nutrient uptake via plant roots wasower in plants with natural as opposed to suppressed AMungal activity, whilst the mycorrhizal nutrient uptake did notompensate for the difference, resulting in higher biomass oflants with suppressed AM fungal activity. This pattern coin-ides with reported negative growth response of mycorrhizallants with functional fungal nutrient uptake pathway com-ared to non-mycorrhizal plants without it (Grace, Cotsaftis,ester, Smith, & Smith 2009), indicative of the complexity of

nterpreting mycorrhizal roles in natural communities (SmithSmith 2012). The third, not mutually exclusive explanation

s that without potentially suppressed pathogenic fungi in theungicide treatment, the less mycorrhiza-responsive plantsere able to gain greater benefit from increased soil nutri-

nt status having been released from pathogen suppressionMaron et al. 2011).

We conclude that – as in several previous experiments – theffect of suppressed AM fungal activity on the composition oflant communities was not clearly expressed. Research on theole of AM fungi in structuring plant communities is alwaysomplex and the effects may be limited owing to multipleiotic interactions acting in situ (Klironomos et al. 2011),ut also to relative availabilities of soil nutrients (Johnson010). However, we expect that accumulating experimentalvidence will gradually improve the understanding of the rolef AM fungi in plant communities.

cknowledgements

We thank Elise Vanatoa, Mart Kinkar, Lars Götzenberger,ariliis Võsu and John Davison for technical assistance.obert Szava-Kovats kindly commented on the first draftsf the manuscript. This study was supported by grantsrom Estonian Science Foundation (7738, 9050, and 9157),argeted financing (SF0180098s08), and by the Europeanegional Development Fund (Center of Excellence FIBIR).

ppendix A. Supplementary data

Supplementary data associated with this article can beound, in the online version, at http://dx.doi.org/10.1016/.baae.2012.09.008.

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