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Soil Biology & Biochemistry 81 (2015) 323e328

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Soil Biology & Biochemistry

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Arbuscular mycorrhizal fungi reduce decomposition of woody plantlitter while increasing soil aggregation

E.F. Leifheit a, b, E. Verbruggen a, b, M.C. Rillig a, b, *

a Institut für Biologie, Plant Ecology, Freie Universit€at Berlin, Altensteinstr. 6, 14195, Berlin, Germanyb Berlin-Brandenburg Institute of Advanced Biodiversity Research (BBIB), 14195, Berlin, Germany

a r t i c l e i n f o

Article history:Received 2 July 2014Received in revised form7 December 2014Accepted 9 December 2014Available online 19 December 2014

Keywords:Arbuscular mycorrhizal fungiMicroorganismsDecompositionSoil aggregationWood

* Corresponding author. Institut für Biologie, PlanBerlin, Altensteinstr. 6, 14195 Berlin, Germany. Tel.: þ4(0)30 838 53886.

E-mail address: matthias.rillig@fu-berlin.de (M.C.

http://dx.doi.org/10.1016/j.soilbio.2014.12.0030038-0717/© 2014 Elsevier Ltd. All rights reserved.

a b s t r a c t

The decomposition of plant organic matter and the stability of soil aggregates are important componentsof soil carbon cycling, and the relationship between decomposition rate and arbuscular mycorrhizal fungi(AMF) has recently received considerable attention. The interaction of AMF with their associated mi-croorganisms and the consequences for litter decomposition and soil aggregation still remain fairlyunclear. In a laboratory pot experiment we simultaneously tested the single and combined effects of oneAMF species (Rhizophagus irregularis) and a natural non-AMF microbial community on the decomposi-tion of small wooden sticks and on soil aggregation. To disentangle effects of hyphae and roots we placedmesh bags as root exclusion compartments in the soil. The decomposition of the wooden sticks in thiscompartment was significantly reduced in the presence of AMF, but not with the non-AMF microbialcommunity only, compared to the control, while aggregation was increased in all treatments comparedto the control. We suggest that AMF directly (via localized nutrient removal or altered moisture condi-tions) or indirectly (by providing an alternative carbon source) inhibited the activity of decomposers,leading to different levels of plant litter degradation under our experimental settings. Reduceddecomposition of woody litter in presence of AMF can be important for nutrient cycling in AMF-dominated forests and in the case of woody plants and perennials that develop lignified roots ingrasslands.

© 2014 Elsevier Ltd. All rights reserved.

1. Introduction

The decomposition of plant litter in soil is a key ecologicalprocess and can be an important factor determining soil carbon (C)storage. Since two thirds of the earth's carbon are stored interrestrial ecosystems (Jobbagy and Jackson, 2000; Amundson,2001) the storage of soil C is a key component of the global car-bon cycle, and because arbuscular mycorrhizal fungi (AMF) havebeen shown to affect litter decomposition they could have animportant influence on this process (e.g. Cheng et al., 2012; Hermanet al., 2012; Drigo et al., 2013). Until recently AMF were thought tocontribute to soil C storage mainly through channeling plant pho-tosynthates into soil and contributing to stabilization of C withinsoil aggregates (Six et al., 2004; Talbot et al., 2008). However, this

t Ecology, Freie Universit€at9 (0)30 838 53165; fax: þ49

Rillig).

potentially positive effect on soil C levels can be offset if AMFsimultaneously promote decomposition of plant litter: even thoughAMF do not have saprotrophic capabilities, they can enhancedecomposition of organic matter (OM) (Hodge et al., 2001; Kolleret al., 2013). Indeed, Cheng et al. (2012) showed that plant litterdecomposed faster in the presence of AMF, especially under con-ditions of elevated CO2 and nitrogen (N) concentrations. Likewise,Hodge et al. (2001) have shown an increased plant capture of Nfrom a patch containing leaf litter, simultaneously with a reductionof C in the patch, in the presence of AMF, and hypothesized thatAMF promoted decomposition by stimulating the activity ofhyphosphere bacteria.

Meanwhile, there are also studies that indicate that soil carbonlevels do not necessarily decrease in response to AMF: a long-termfield study (17 and 6 years) found carbon stocks to positivelycorrelate with AMF extraradical hyphae (Wilson et al., 2009), and amesocosm experiment found AMF root colonization to positivelyassociate with the amount of stable soil C (Manning et al., 2006).One potential reason for the observation that AMF do not decreasesoil C levels could be that they differentially affect plant litter

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depending on litter quality: litter can be slowly or fast decomposingdepending on its chemical constitution (Milcu et al., 2011; Cotrufoet al., 2013). Slowly decomposing litter such as lignified plant ma-terial is a major contributor to soil OM and its decomposition de-pends on a number of factors including humidity, size/shape ofwood particles and the organisms involved (Boddy and Watkinson,1995). In the soil environment fungi are the main decomposers ofwoody litter and the relationship between numerous saprotrophicfungi and wood decomposition has been studied intensively (e.g.Boddy and Watkinson, 1995; Worrall et al., 1997; Clinton et al.,2009). However, to our knowledge the effect of AMF on decom-position of woody material has not yet been studied, even thoughlignified plant roots, stems and leaves can represent a considerateproportion of litter inmany natural ecosystems (Heim and Schmidt,2007).

As mentioned before, another way bywhich AMFmay affect soilC cycling is through their effects on soil structure, i.e. the size anddistribution of soil aggregates and pores, which determinesnutrient and water availability, oxygen diffusion and relations ofpredator and prey (Rillig and Mummey, 2006). OM can be furtherphysically protected within soil aggregates and thus a higher soilaggregate stability can contribute to OM stabilization in the soil(reviewed in Six et al., 2004). The role of AMF in soil aggregateformation and stabilization is well documented (see review of Rilligand Mummey, 2006) and we recently showed in a meta-analysis,using a wide range of studies, that AMF generally increase soilaggregation (Leifheit et al., 2014).

While it is clear that AM fungi are important for soil aggregation,they are usually part of a wider natural microbial community withnumerous organism interactions, i.e. bacteria and non-AM fungican also play an important role in soil aggregation (Tisdall, 1994)and litter decomposition (Hodge et al., 2001). Rillig et al. (2005)showed that various AMF species differentially affect microbialcommunity composition and that these differences are importantfor soil aggregation. Furthermore, the combination of fungal spe-cies and host plant, the characteristics of the fungal species and thesoil microbial community composition can strongly influence theeffects of AMF on soil aggregation (Schreiner et al., 1997; Piotrowskiet al., 2004). Given that AMF affect litter decomposition indirectlythrough their effects on decomposers, e.g. by imposing N limitationor altering rhizodeposition and thereby altering the C supply toother microorganisms (Cheng et al., 2012; Nuccio et al., 2013), thecomposition of the microbial community present may also be ex-pected to determine the effect of AMF on litter decomposition rate.

In this study we aim to assess how AMF and other soil micro-organisms interact to affect 1) woody litter decomposition and 2)soil aggregation. Therefore we tested whether the decompositionof small wood pieces would be influenced by the presence of AMF,while excluding effects of plant roots, and if potential effects wouldbe enhanced or reduced by the presence of a more natural micro-bial assemblage. For soil aggregation we expected a positive, ad-ditive effect of AMF and associated microorganisms as both groupsare capable of forming and stabilizing soil aggregates.

2. Materials and methods

2.1. Experimental design and setup

In a 2� 2 factorial experiment we tested for the effects ofpresence/absence of AMF and a natural microbial community andthe interaction between these two factors. Each treatment combi-nationwas replicated 10 times for a total of 40 pots. The experimentwas located in a climate chamber with an average day/night tem-perature of 20/16 �C. The soil was a loamy sand collected from anexperimental field of Freie Universit€at Berlin, which had the

following properties: pH 7.1 (CaCl2), 6.9 mg P/100 g soil (calcium-acetate-lactate), 0.12% N (total) and 1.87% C (total) (for analyticalmethods see Rillig et al. (2010)). The vegetation at the field site isdominated by grassland species (Trifolium repens L., Arrhenatherumelatius (L.) P. Beauv. ex J. Presl & C. Presl, Bellis perennis L., Plantagolanceolata L., Elymus repens L. Gould, Medicago sativa L.) with a fewcherry trees (Prunus spec.) at the borders. The soil was autoclavedtwice on two consecutive days with at least 24 h in between inorder to ensure absence of viable microbial propagules. The soilwas dried in the autoclaving bags at 60 �C and subsequently brokenup with a rubber mallet to reduce soil aggregation. The soil wasthen sieved using a 2 mm sterilized sieve and simultaneouslymixed with 20% autoclaved sand in order to partially compensatefor nutrient release during sterilization, resulting in a sand contentof 79%. One and a half liters of the soil-sand-mixture were trans-ferred into 3 L round plastic pots with a 2 cm layer of sterilized sandon the bottom and on the top. Close to the center of the pot a meshbag (120 ml volume, 38 mm pore size) was installed to allow thepenetration of hyphae and the passage of bacteria while excludingroots (hereafter referred to as root exclusion compartment). Thetop of the mesh bags was left open and was positioned above thesoil surface (see Fig. S1, Supplementary Material). Pots were re-randomized regularly throughout the experiment.

Seeds of P. lanceolata (a perennial mycorrhizal rosette formingherb of grasslands, ruderal areas and farmland) were sterilized in10% bleach for 10 min and in 70% ethanol for 30 s, rinsed indeionized water after each step, and sown directly into the soil.After seedling emergence plants were thinned to one plant per pot(further referred to as root compartment). During the growingperiod plant leaves were cut twice to a height of approximately15 cm to prevent excessive growth of leaves (to avoid contamina-tion from neighboring pots) and roots (to avoid pot bound roots).Plants were watered as needed three times per week. We usedP. lanceolata as plant species because it is common in the grasslandwhere the soil for the experiment was collected.

Control pots were left non-inoculated. For the AMF treatmentwe inoculated approximately 1000 spores per pot of Rhizophagusirregularis, a member of the Glomeraceae family and frequentlyreferred to as “the model AMF” (Stockinger et al., 2009) and a verycommonly encountered AMF species in temperate Europe andcommonly used in mycorrhizal research (€Opik et al., 2010; Mooraet al., 2011) (Schenk & Smith, isolate DAOM197198, SymplantaGmbH & Co. KG). The sterile spores were contained (pre-mixed) ina rock flour material (attapulgit clay based powder) that was mixedwith deionized water and pipetted into a cut-off pipette tip thatwas positioned under the plant towards the roots. The controlsreceived sterilized carrier material. Fresh field soil from the up-permost 30 cm, collected at the same field site as above, was used toproduce a microbial wash from a soil filtrate (200 g of soil in 2 L oftap water) where the smallest sieve had a size of 20 mm, thusexcluding larger spores such as those of AMF. This microbial washrepresents the non-AMF microbial community. We inoculated themicrobial wash (‘MW’) to the seedlings of the treatments andautoclaved microbial wash to controls. Inoculation with both AMFand MW will later be referred to as the ‘AMF þ MW’ treatment.

To measure microbially mediated decomposition we usedwooden sticks that were inserted in the soil and determined theirmass loss rate at the end of the experiment (Sinsabaugh et al., 1993;Arroita et al., 2012). The wooden sticks (4 � 2 mmwidth, Meyer &Weigand GmbH, Germany) were cut into pieces of ca. 30 mmlength, autoclaved and weighed (154.43 mg ± 0.35, N ¼ 30). Weused a Tilia species, which is a common tree in the surrounding ofthe field site where the soil was collected. One stick was introducedinto the soil of each compartment (root exclusion and rootcompartment) using a spatula. Each stick contained approximately

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76 mg C and 1.5 mg N. We used wood as OM because the rela-tionship between woody litter decomposition and AMF has not yetbeen studied but might nevertheless be important in AMF domi-nated systems such as tropical forests or grasslands, where woodymaterial is present in the form of woody stem litter and/or lignifiedroots. Because our primary focus was on direct decomposition ofthe OM (but not necessarily nutrient transport by AMFmycelia) andthe wood sticks can be readily recovered, it was not necessary touse isotopically labeled plant materials.

2.2. Harvest and sample storage

The root exclusion compartment was harvested 5 months afterinoculation. The newly formed holes were filled with a plastic tubeto prevent the surrounding soil from collapsing. In order to extendthe plant growth period for as long as possible we continued wa-tering the plants until they would have needed another cut (seeabove), which was 6 weeks after the harvest of the root exclusioncompartment (6.5 months after inoculation). All materials weredried at 40 �C, weighed and stored at room temperature untillaboratory analysis. Soil subsamples were immediately frozen inliquid N and stored at �80 �C.

2.3. Lab analyses

The dried soil was sieved through a 4 mm sieve and the woodensticks were recovered from the soil of both the root exclusion androot compartments, cleanedwith scalpel and brush and dry-weightwas determined. One half of each wooden stick was ball milled andanalyzed for percentages of total C and N content (EuroEA, Heka-Tech, Germany).

All soil analyses were performed for the root exclusioncompartment only. Because our primary focus was on the effects ofAMF and MW on soil aggregation and decomposition in absence ofroots, nutrient and physic-chemical parameters were only analyzedin this compartment, while decomposition in the root compart-ment was only measured as an additional estimate for comparisonand external validity. Additionally, the two compartments are notdirectly comparable due to the 6 week gap in harvesting.

Ball milled soil was analyzed for percentages of total C and Nwith an Elemental Analyzer (EuroEA, HekaTech, Germany). Hyphaewere extracted from 4.0 g of soil (Jakobsen et al., 1992), stainedwithink and vinegar and hyphal length in m $ g�1 soil was measuredaccording to Rillig et al. (1999). On six randomly selected sampleswe additionally determined the amount of blue-stained and ‘lightbrown’ colored non-AM fungal hyphae as a rough indication ofpossible differences in fungal communities. Briefly, all hyphae thatdid not meet the criteria for AMF hyphae as described in Mosse(1959) (dark-to light-blue stained aseptate hyphae with charac-teristic unilateral angular projections) but that were stained bluewere counted as ‘blue-stained’ non-AMF and all aseptate hyphaewith a characteristic golden color were counted as ‘light brown’non-AMF.

From each treatment water stable soil aggregates were assessedfor eight subsamples by wet sieving with a series of stacked sievesof the size 2 mm, 1 mm, 0.5 mm and 0.212 mm with the smallestsieve at the bottom (modified fromKemper and Rosenau,1986).Weimmersed the stack of sieves in a bucket of water (40 cm high,30 cm diameter). 50.0 g of soil were rewetted by capillary action,placed on top of the uppermost sieve and moved up and down for4 min (30 strokes per minute), while the soil on the uppermostsieve was completely immersed in water the entire time. Weseparated the coarse matter (mainly sand particles) by crushing theaggregates and pushing the soil through the respective sieve.Coarsematter and soil were collected and dried at 80 �C for 36 h. All

calculations were corrected for coarse matter. The fraction of waterstable aggregates (WSA) in each size class was calculated asdescribed in Barto et al. (2010). Here, we only report the data for thetotal relative amount of water stable aggregates. Detailed data onthe different size classes are given in the Supplementary Material(Table S4, Supplementary Material).

For a subsample of five replicates the following parameters wereanalyzed. Plant available phosphate in 5.0 g of dry soil wasextracted with 100 ml of a 0.05 M calcium-acetate-lactate-solutionwhile shaking for two hours. The supernatant was filtered througha Phosphorus-free filter and phosphate concentration was deter-mined photometrically (Analytical Continuous Flow Analyzer SANPlus, Skalar, The Netherlands). Ammonium was extracted from a5.0 g sample of frozen soil with a 0.01 M CaCl2 solution andmeasured photometrically (ISO 14255, Analytical Continuous FlowAnalyzer SAN Plus, Skalar, The Netherlands). Soil pH and electricalconductivity were analyzed in 0.01 M CaCl2 and deionized water,respectively, according to ISO 10390:2005 and ISO 11265:1997.Roots were washed, dried at 40 �C and weighed. To demonstratethe success of inoculation we determined the percent root coloni-zation in each treatment: roots were stained with ink and vinegar(as described in Vierheilig et al., 1998) and percentage of AMFstructures was determined microscopically at 200� magnificationwith the gridline intersect method (100 intersects per sample)(McGonigle et al., 1990; Rillig et al., 1999).

2.4. Statistics

All statistical analyses were conducted in the statistical softwareR version 2.15.2 (R Core Team, 2013). For the analysis of single andcombined effects of inoculation with AMF and MW we used two-way ANOVAs with AMF and MW as factors. Data were log trans-formed if necessary to meet the assumptions of normality of re-siduals. If conditions for homoscedasticity were not met we used ageneralized least squares model (gls) using the package ‘nlme’(Pinheiro et al., 2013). AMF hyphal length residuals were not nor-mally distributed and treatment effects were therefore analyzedusing a KruskaleWallis rank sum test. For the C and N concentra-tions of the wood sticks at harvest we additionally applied a TukeyHonest Significant Difference post-hoc test to specifically comparethe treatments among each other. To test whether C and N con-centrations of the wood changed during the experiment, concen-trations at the start of the experiment and at harvest werecompared using the ‘lm’ function of the ‘stats’ package in R(Chambers, 1992). The weight loss of the wood sticks of the rootexclusion compartment was analyzed with an ANOVAwith the fullnumber of replicates (N ¼ 10) and we repeated the analysisexcluding three samples for which the recovery of the whole stickwas uncertain. The results of these two analyses were the same andwe report the results excluding those three samples as outliersfrom our statistics. Due to the larger soil volume the recovery ofwood sticks from the root compartment was more difficultcompared to the root exclusion compartment and we thereforereport statistics for eight replicates, for which we could fullyrecover the sticks.

3. Results

The weight loss (%) of the wooden sticks in the root exclusioncompartment was significantly reduced in the presence of AMF(p ¼ 0.0001) (see Fig. 1A). Overall weight loss in the root exclusioncompartment was approximately 47% in the control and the MWtreatment. The presence of AMF reduced the weight loss to anaverage of 31%. Overall weight loss in the root compartment wasapproximately 25% in the control and MW treatment, while the

Fig. 1. Average (±se) gravimetric weight loss (%) of the wood sticks in the root exclusion compartment (A) and the root (B) compartment after the experiment. C: Average (±se)hyphal length (m g�1 soil) of AMF hyphae in the root exclusion compartment. D: Average (±se) hyphal length (m g�1 soil) of ‘light brown’ and blue-stained non-AMF hyphae (N ¼ 6)and the total amount of non-AMF hyphae (N ¼ 10) in the root exclusion compartment. E: Average (±se) percentage of total water stable aggregates (%) in the root exclusioncompartment. F: Mean (±se) C: N ratio in the wood sticks at the end of the experiment (C: N ratio at the start of the experiment was 473 (40)). Control ¼ non-inoculated treatment,AMF ¼ treatment with Rh. irregularis, MW ¼ treatment with microbial wash, AMF þ MW ¼ treatment with combined inoculation of AMF and MW.

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presence of AMF reduced the weight loss to an average of 13%(p ¼ 0.003, see Fig. 1B).

The C: N ratio of the wood sticks in the AMF þ MW treatmentwas significantly higher compared to the AMF and the MW treat-ment (p ¼ 0.02, p ¼ 0.005, respectively) (see Fig. 1F). This increasewas caused by a significant reduction in N concentration in theAMFþMWtreatment compared to the AMFand theMW treatment(p ¼ 0.02, p ¼ 0.004, respectively) (see Table S1, SupplementaryMaterial). The total carbon concentration was not different be-tween the treatments (p � 0.07 for all treatments), and did notchange compared to the start concentration (see Table S1,Supplementary Material). Concentrations of N in the wood sticksall increased during the experiment (p � 0.03 for all treatments),while C: N ratios decreased during the experiment (p < 0.02 for alltreatments).

Soil nutrient concentrations (ammonium, phosphate, C, N) andelectrical conductivity were not significantly different between thetreatments (see Table S1, SupplementaryMaterial). For pH therewasa slight increase in all inoculated treatments compared to the con-trol (p<0.02 for each treatment), but all valueswere in the range of aneutral soil pH: 6.80e7.02 (Table S2, Supplementary Material).

R. irregularis successfully colonized the host plant roots toaround 59 (±11) % in the AMF treatment and 75 (±4) % in theAMF þ MW treatment, while AMF root colonization was absent inthe control. AMF soil hyphal length was significantly higher in theAMF-inoculated treatments (p < 0.0001) (see Fig. 1C). Non-AMFhyphae were significantly higher in the MW treatment and the

AMF þ MW treatment (p � 0.03 for both treatments) (see Fig. 1D).The length of non-AMF ‘light brown’ hyphae in the soil wassignificantly influenced by the AMF and the MW treatment(p < 0.01 for both treatments). Non-AMF blue-stained hyphaeincreased in the presence of AMF (p < 0.05) (see Fig. 1D).

The formation of water stable aggregates in the root exclusioncompartment was significantly increased in all inoculated treat-ments compared to the control (AMF: p < 0.001, MW: p < 0.01,AMF þ MW: p < 0.05), but there were no differences between theinoculated treatments (see Fig. 1E). Root dry weight in the rootcompartment and water content in the root exclusion compart-ment were not significantly different between the treatments (seeTable S3, Supplementary Material).

4. Discussion

Our results show a decreased decomposition of wood sticks intreatments inoculated with AMF, independent of whether plantroots or a natural soilmicrobial communitywere present or not. Thissuggests that the presence of AMF has directly or indirectly inhibitedthe activity of microbiota capable of plant litter decomposition. Sucha reduction is commonly found in ectomycorrhizal fungi, where itwas first described by Gadgil and Gadgil (1971). The so-called‘Gadgil-effect’ has been proposed to be caused by indirect effects ofmycorrhizae on decomposers through altering soil nutrient avail-ability, moisture, and/or direct inhibition of saprotrophic fungi(Bending, 2003). Other than competitive inhibition of decomposing

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fungi or bacteria, alternative potential causes for their reduced ac-tivity may lie in root and fungal C deposits: if plants lower rhizode-posits that are used to scavenge for nutrients in response to AMFcolonization, thismight also lead to reduced activity of decomposers,e.g. wood decay fungi, that would otherwise be stimulated by thesedeposits (Jones et al., 2004). However, given that the root exclusioncompartment did not contain roots, for this effect to have caused ourobservations those exudates would have needed to be highly watersoluble andmove into the compartment. For fungal exudates to havecaused reduced decomposition in the root exclusion compartmentthe effect of hyphal exudation would need to be opposite to theplant's: it should have served as an alternative C source to the de-composers and thus reduced decomposition of the wood stick.

In our experiment nutrient concentrations, pH and EC of theroot exclusion compartment were not different in AMF inoculatedtreatments, which would have indicated changed chemical condi-tions in the soil that could have altered microbial activity or pro-liferation (Baath and Anderson, 2003; Marschner et al., 2005).Another potentially relevant parameter for decomposer activity isthe water content of the soil, which was also equal in all treatmentsat harvest. However, even though nutrients and other parametersdid not change significantly, they may have been responsible in amore localized fashionwhere AMF scavenge nutrients exactly frompatches where they are released or removed water on a microporescale, without strong effects on overall nutrient or water levels.Therefore, indirect effects of AMF through changing nutrient orwater levels cannot be excluded.

Contrary to nutrient levels in the soil, nutrient concentrations inthe wood sticks at harvest differed between the treatments. Acommon effect in wood decomposition studies is that carboncontent in the decayed wood varies substantially depending on thetype of wood or the fungi present, whereas it is generally observedthat nutrients, especially N as we find here as well, increase duringtime of decomposition (Boddy and Watkinson, 1995; Clinton et al.,2009; Preston et al., 2012). In this case, growth of microorganismscauses nutrients to be translocated into the wood, where they areretained in the wood-mycelium-biomass, while carbon is miner-alized or respired (Boddy and Watkinson, 1995). Final N concen-tration increased in all wood sticks compared to the starting wood,but differed markedly between the treatments: in the AMF þ MWtreatment the concentration was on average only half of that in allother treatments. A potential reason for this could be a highermicrobial turnover promoting N removal from localized OMpatches by AMF in the presence of a more complex microbial foodweb, which was recently demonstrated in the presence of protozoa(Koller et al., 2013).

The results of our study contrast with other studies that foundenhanced decomposition of OM in the presence of AMF (Hodgeet al., 2001; Cheng et al., 2012). However, most studies thattested effects of AMF on litter decomposition used relatively short-term experiments (4e10 weeks) and a comparatively large amountof (ground) leaf litter as organic substrate (0.42e4.44 % C in theorganic patch/compartment) (e.g. Hodge et al., 2001; Cheng et al.,2012; Koller et al., 2013), while we employed a small amount ofintact woody plant material (0.04% wood C in the soil of the rootexclusion compartment) and measured decomposition afterapproximately 23 weeks. Some of the studies that found increaseddecomposition in presence of AMF added N to their treatments (e.g.Cheng et al., 2012), which can favor bacterial dominance in the soil,leading to increased turnover rates of high quality OM (Manning,2013). Berg (2000) proposed that N availability is the factor thatis simultaneously responsible for increased initial decompositionand decreased subsequent decomposition of litter. Analogously, thepresence of AMF may have a stimulating effect in early stages ofdecomposition and an inhibitory effect on decomposition in later

stages of decomposition (or on slowly decomposing OM moregenerally). This could explain opposing accounts in the literaturebut requires further research and understanding of the underlyingmechanisms. Reduced decomposition in presence of AMF ispotentially important inwoody systems and/or in grasslands whereslowly decomposing material - such as lignified roots e is present.Wider implications of this potential effect such as increased soil Csequestration would be especially interesting to study.

Although the control treatment was not inoculated, thedecomposition rate in the control was among the highest of ourtreatments and approximately the same as in the MW treatment.The presence of decomposing microorganisms in the control isunsurprising, as experiments in greenhouses or climate chambersgenerally do not remain sterile when airborne propagules of bac-teria or fungi can enter the soil. However, absolute decay ratesfound in our experiment cannot be categorized as comparativelyhigh or low: mass loss rates of wood can be highly variable asdecomposition depends heavily on the presence of particular de-composers and the type of wood. Worrall et al. (1997) observedweight loss of two different wood types between 20 and 30% onaverage for typical decay fungi (Aphyllophorales) after 12 weeks ofincubation, but other fungi caused mass losses up to >80%.

By using a root exclusion compartmentwe could show that thereare direct positive effects of AMF hyphae, a natural soil microbialcommunity excluding AMF, and the combination of both on soilaggregate formation. However, the single effects of AMF and mi-crobial wash were non-additive, contrary to our hypothesis, as thesoil aggregation level was not higher when these two treatmentswere combined compared to either treatment alone. It is possiblethat the relatively high sand content of the soil-sand-mixture (79%)did not allowmore soil aggregation because of low amounts of clayor organic matter, which are soil components that usually enhanceaggregation (Tisdall and Oades, 1982). The combined inoculation ofa single AMF species and a whole soil microbial community in ourexperiment might have induced strong interspecific competitionprocesses (Smith and Read, 2008). In AMF inoculated treatmentsthis competition might have suppressed non-AMF species with ahigh soil aggregating ability such as other filamentous fungi orbacteria that excrete “sticky” polysaccharides (Tisdall, 1994). Inabsence of AMF, i.e. without the competition, these species couldproliferate more freely and thus contribute to soil aggregation.

5. Conclusions

To our knowledge this is the first study that shows reduceddecomposition of woody plant litter in presence of AMF. Moreover,even when a natural soil microbial community (excluding AMF)was present, the reduction of decomposition entirely depended onwhether AMFwere present as well. We suggest that future researchshould test plant litter types of differing qualities for theirdecomposition rates and fate of C in terms of loss or incorporationin microbial biomass or soil OM e.g. through using litter labeledwith stable isotopes. The use of different AMF species, a natural soilmicrobial community, different soil types, aggregation levels andfertilizer levels would be important additions to those experiments.

The findings of increased soil aggregation in the presence ofAMF and AMF teamed with a natural soil microbial communitysupport the role of AMF in the physical protection of OM in soilaggregates. The lack of an additive effect of AMF in combinationwith associated microbes on soil aggregation still needs to bevalidated, given the context dependency of AMF effects. To eluci-date this question further experimental investigationwith differentsoil textures and different AM fungal species involved in the pres-ence and absence of a non-AM microbial community will benecessary.

E.F. Leifheit et al. / Soil Biology & Biochemistry 81 (2015) 323e328328

Acknowledgments

This work was funded by a grant (RI 1815/1-1) from the Deut-sche Forschungsgemeinschaft (German Research Foundation). Wethank Pete Manning and an anonymous reviewer for helpfulcomments.

Appendix A. Supplementary data

Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.soilbio.2014.12.003.

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