Additive and interactive effects of functionally dissimilar soil organisms on a grassland plant community

Soil Biology & Biochemistry 42 (2010) 2266e2275

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Additive and interactive effects of functionally dissimilar soil organisms on a grassland plant community Natalia Ladygina a, *, Frederic Henry b, Merijn R. Kant c, Robert Koller d, Stefan Reidinger e, Alia Rodriguez f, Stephane Saj g, Ilja Sonnemann h, Christina Witt g, Susanne Wurst i a

Department of Ecology, Lund University, Sölvegatan 37, 223 62 Lund, Sweden Department of Terrestrial Ecology, Copenhagen University, Universitetsparken 15, 2100 Copenhagen, Denmark Max Planck Institute for Chemical Ecology, Hans-Knöll-Strasse 8, 07745 Jena, Germany d UMR INRA-INPL “Agronomie et Environnement”, ENSAIA, BP 172, 54505 Vandoeuvre les Nancy, France e School of Biological Sciences, Royal Holloway University of London, Egham, SurreyTW20 OEX, UK f Department of Soil Microbiology, CSIC, Profesor Albareda 1, 18008 Granada, Spain g Department of Ecological and Environmental Sciences, University of Helsinki, Niemenkatu 73, 15140 Lahti, Finland h Centre for Agri-Environmental Research, University of Reading, P.O. Box 237, Earley Gate, Reading RG6 6AR, UK i Center for Terrestrial Ecology, P.O. Box 40, 6666 ZG Heteren, The Netherlands b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 17 March 2010 Received in revised form 20 August 2010 Accepted 24 August 2010 Available online 8 September 2010

The productivity and diversity of plant communities are affected by soil organisms such as arbuscular mycorrhizal fungi (AMF), root herbivores and decomposers. However, it is unknown how interactions between such functionally dissimilar soil organisms affect plant communities and whether the combined effects are additive or interactive. In a greenhouse experiment we investigated the individual and combined effects of AMF (five Glomus species), root herbivores (wireworms and nematodes) and decomposers (collembolans and enchytraeids) on the productivity and nutrient content of a model grassland plant community as well as on soil microbial biomass and community structure. The effects of the soil organisms on productivity (total plant biomass), total root biomass, grass and forb biomass, and nutrient uptake of the plant community were additive. AMF decreased, decomposers increased and root herbivores had no effect on productivity, but in combination the additive effects canceled each other out. AMF reduced total root biomass by 18%, but decomposers increased it by 25%, leading to no net effect on total root biomass in the combined treatments. Total shoot biomass was reduced by 14% by root herbivores and affected by an interaction between AMF and decomposers where decomposers had a positive impact on shoot growth only in presence of AMF. AMF increased the shoot biomass of forbs, but reduced the shoot biomass of grasses, while root herbivores only reduced the shoot biomass of grasses. Interactive effects of the soil organisms were detected on the shoot biomasses of Lotus corniculatus, Plantago lanceolata, and Agrostis capillaris. The C/N ratio of the plant community was affected by AMF. In soil, AMF promoted abundances of bacterial, actinomycete, saprophytic and AMF fatty acid markers. Decomposers alone decreased bacterial and actinomycete fatty acids abundances but when decomposers were interacting with herbivores those abundances were increased. Our results suggests that at higher resolutions, i.e. on the levels of individual plant species and the microbial community, interactive effects are common but do not affect the overall productivity and nutrient uptake of a grassland plant community, which is mainly affected by additive effects of functionally dissimilar soil organisms. Ó 2010 Elsevier Ltd. All rights reserved.

Keywords: AMF Wireworms Nematodes Collembolans Plant biomass Functional groups C/N content Microbial community PLFA

1. Introduction

* Corresponding author. Tel.: þ46 46 222 44 50; fax: þ46 46 222 47 16. E-mail address: [email protected] (N. Ladygina). 0038-0717/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.soilbio.2010.08.027

Plant communities are influenced by abiotic (Ellenberg, 1974: Tilman, 1982) as well as by biotic soil factors (Brown and Gange, 1992; Van der Putten et al., 1993, 2001; De Deyn et al., 2003). Biotic soil factors consist of a wide array of soil organisms that interact with plants in a functionally dissimilar manner. Functionally dissimilar soil

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organism groups such as mycorrhizal fungi (Gange et al., 1993; Van der Heijden et al., 1998a), root herbivores (Brown and Gange, 1992; Schädler et al., 2004) and decomposers (Partsch et al., 2006) influence plant communities in a variety of ways, and their individual impact on productivity ranges from being beneficial to detrimental (Wardle et al., 2004). Besides the multiple biotic interactions in terrestrial ecosystems, interactions between functionally dissimilar soil organism groups and their impact on plant communities have been very rarely explored. With the exception of Gange and Brown (2002), studies on impacts of soil organism groups on plant communities rather assembled soil organisms according to body size (Bradford et al., 2002), abundance (Wurst et al., 2008a) or origin (De Deyn et al., 2003; Kardol et al., 2006) than functional groups. Gange and Brown (2002) manipulated arbuscular mycorrhizal fungi (AMF) and insect root herbivores by applying soil fungicide and insecticide in the field. They observed that plant cover and plant species richness during early succession were increased when AMF were present, but reduced in the presence of insect root herbivores. However, when both AMF and root herbivores were present together in soil the two opposite effects were additive and thus canceled each other out. Knowledge on effects of functionally dissimilar soil organism groups on plant communities is crucial to understand and predict the general impact of soil organisms on plant communities, since their individual effects might change in presence of other functionally dissimilar soil organisms that co-occur in nature. For instance, interactions between functionally dissimilar root herbivores and AMF were shown to reduce root damage by the root herbivores (Hol and Cook, 2005; Currie et al., 2006; De la Peña et al., 2006); however, other studies did not find evidence for significant interactions (Wurst et al., 2004; Eisenhauer et al., 2009). Since most of the studies were carried out in pots with only one plant species present or in Petri dishes (e.g. feeding assays), it is still unclear whether these interactions are strong enough to change individual impacts of functionally dissimilar soil organism groups on the productivity and structure of a plant community. First objective of our study was to examine the individual and combined effects of three functionally dissimilar groups, i.e. AMF (five Glomus species), root herbivores (wireworms and nematodes) and decomposers (collembolans and enchytraeids) on key functional properties of a grassland plant community, such as productivity and nutrient content of the plant community. Our main goal was to see whether the combined effects of the functionally dissimilar soil organism groups can be predicted by knowing their individual effects. This is the case when effects of the functionally dissimilar soil organism groups are rather independent leading to additive and thus predictable effects when combined. Our null hypothesis was that functionally dissimilar soil organisms cause independent effects and thus the combined effects of the soil organisms are additive under the assumption that in the absence of significant interactions any observed effect in combined treatments can be decomposed as the sum of the individual treatment effects (Sokal and Rohlf, 1981). For the plant community structure we further expected changes according to the type of functional group of soil organisms present: AMF may promote AMF-depending plant species (Van der Heijden et al., 1998a); root herbivores (nematodes and wireworms) may preferentially feed on some of the plant species (Hemerik et al., 2003) and therefore suppress their competitive ability; decomposers (collembolans and enchytraeids) may increase nutrient availability and promote plants that are strong competitors for nutrients (Partsch et al., 2006). Second objective of our study was to determine effects of functionally dissimilar soil organisms on soil microbial biomass and community structure using phospholipid fatty acids (PLFA) as

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microbial markers (Zelles, 1999). We hypothesized that microbial biomass and microbial community structure will change in the presence of AMF since AMF may enhance growth and activity of microorganisms by improving, for example, soil aggregation (Van der Heijden et al., 2006). Root herbivores may positively affect microbial communities by grazing on roots, thus releasing nutrients available for microbial growth (Grayston et al., 2001). Microbial biomass and community structure may be changed through grazing activity of the decomposers (enchytraeids and collembolans) that may selectively feed on fungal hyphae (Cole et al., 2002; Jorgensen, 2002). As null hypothesis we expected to observe additive effects of the functionally dissimilar soil organisms on soil microbial biomass and community structure. 2. Material and methods 2.1. Experimental setup We established 80 microcosms in pots of 17.7 cm diameter and 19.5 cm height with soil from a grassland in the Netherlands (Van der Putten et al., 2000). The soil was a sandy loam and had on average 21.3  0.5 g/kg of carbon, 1.3  0.2 g/kg of nitrogen, 0.3  0.02 g/kg of phosphorus, and a pH of 6.3 (Hedlund et al., 2003). All pots were filled with 4.94 kg (dry wt) of soil that had been sieved through a 1 cm mesh and autoclaved at 121  C for 20 min. Then all microcosms were amended with a non-sterile soil solution of the native grassland communities to restore the microbial community of the soil, excluding AMF (Brundrett et al., 1996). This non-sterile soil solution was prepared by suspending 1000 g of the non-autoclaved soil in 4 l of distilled water and filtering it through a 20 mm sieve. This solution contained no mesoor microfauna, which was checked under a stereoscopic microscope (Leica MZ 8). We selected eight plant species that are common in grasslands of the Netherlands (Leps et al., 2001). Seeds (obtained from Appels Wilde Samen GmbH, Darmstadt, Germany) of five grasses (Agrostis capillaris, Anthoxanthum odoratum, Poa pratensis, Festuca rubra, Holcus lanatus), two forbs (Plantago lanceolata, Hypochaeris radicata), and one legume (Lotus corniculatus) were surface sterilized with 5% CaCl2O2 for 15 min before sowing. About 10e15 seeds of every plant species were sown in each microcosm according to a planting scheme (day 1 of the experiment). The indicated numbers of seeds were used due to the unknown germination rate of the plant species. The planting scheme was related to the position of the plants to assure that each plant species had the same position across the pots. Plants were allowed to establish in the microcosms for 7 weeks, at which time transparent perforated plastic bags, reaching up to 26 cm above the rims of the pots and open at the top, were placed around the pots to prevent potential cross contamination of the soil organism treatments. We set up a full-factorial design experiment, resulting in 8 treatments [Control (no soil organisms added), AMF (arbuscular mycorrhizal fungi), Dec (decomposers), Herb (herbivores), AMF þ Dec, AMF þ Herb, Dec þ Herb, AMF þ Dec þ Herb] with 10 replicates per treatment. First we established the AMF treatment in half of the microcosms (N ¼ 40) by adding an AMF inoculum (provided by PlantWorks Ltd., Sittingbourne Research Center, Kent, UK) on day 1 of the experiment. This inoculum contained five species of AMF (class Glomeromycota): Glomus mosseae, G. claroideum, Glomus intraradices, G. etunicatum and G. microaggregatum. AMF inoculation of the microcosms was carried out according to Brundrett et al. (1996) using 90 g of the inoculum, while the nonAMF microcosms were inoculated with 90 g of autoclaved (1 h at 120  C) AMF inoculum. Additionally, the non-AMF microcosms were supplemented with 1 ml of a microbial wash of the AMF

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inoculum to correct for possible differences in microbial communities (Johnson et al., 1997). This microbial wash was prepared by suspending 100 g of the non-autoclaved inoculum in 170 ml of tap water and filtering it through a 25 mm sieve. The Herb treatment consisted of root-feeding nematodes and wireworms. One thousand individuals of the generalist endoparasitic nematode Pratylenchus penetrans (mixed life stages) (provided by HZPC Research B.V., NL) were added to each Herb microcosm in week 3 of the experiment. Wireworms (larvae of Agriotes spp., Elateridae, Coleoptera) were collected from a meadow close to the Royal Holloway campus (Egham, UK) in March 2007 and five individuals were introduced to each Herb microcosm in week 7 of the experiment. If the wireworms would have been assembled with plant species from the same site in the UK the herbivores effects in terms of plant damage might have been different. The Dec treatment was obtained via inoculation with enchytraeids and collembolans. Approximately 650 individuals from the pure culture (0.8 g fresh worms) of enchytraeids Enchytraeus albidus (provided by Karl-Heinz Büchner Zierfisch Futter, Jena, Germany) were added to each Dec microcosm in week 8 of the experiment when the plants were well-established. Ten individuals of each collembolan species Folsomia fimetaria and Protaphorura armata (provided by Paul Henning Krogh, National Environmental Research Institute, Silkeborg, Denmark) were added to each Dec microcosm in week 14 of the experiment. The densities of all added soil organisms were chosen to be close to real meadow/field densities and being well below the treshold levels for plant damage: for AMF (Kula et al., 2005), nematodes (De la Peña et al., 2006), wireworms (Sonnemann, pers. comm.), collembolans (Steinaker and Wilson, 2008) and enchytraeids (Plum and Filser, 2005). The microcosms were kept in a greenhouse with a 15/9 h light/ dark cycle and average temperatures 23  C at day and 19  C at night. The plants were regularly watered and each time the pots were adjusted to equal soil water content (20%) by weighing. The temperature and moisture regimes were chosen close to natural to create optimal growth of plants and habitat conditions for soil organism communities. 2.2. Sampling and analytical procedure We defoliated plants twice: the first time in week 20 (referred to as harvest 1) at 1 cm above the soil surface, and the second time in week 26 (referred to as harvest 2) at ground level, when we also harvested the roots. The harvest 1 served to maintain stressful conditions that also occur in the field under which single or combined effects of the applied soil organisms can become more pronounced (Sørensen et al., 2008). The plant material was weighed after drying at 70  C for 96 h. The aboveground parts of the plant community were harvested separately for each plant species. The roots were washed from the whole pots as a mixed sample of all plant species, since a separation of roots from the individual plant species was not possible. Only sub samples of P. lanceolata roots were harvested separately to check the efficiency of the AMF treatment. These sub samples were cleared in 10% KOH for 3 days, followed by acidification in 1% HCl and staining in 1% HCl containing few drops of Parker QuinkÒ ink dark blue for 1 h (Vierheilig et al., 1998). Slide preparations were made and the percentage root length colonized (% RLC) by AMF was recorded at 800 magnification by the cross-hair eye piece method of McGonigle et al. (1990). Before the roots were washed, soil cores (4.5 cm diameter and 5 cm depth) were taken to extract nematodes, enchytraeids and collembolans. Nematodes were extracted from approximately 100 g of freshly collected soil by a modified Oostenbrink elutriator (Oostenbrink, 1960) and identified according to Bongers (1988). The

collembolans were extracted from 100 g freshly collected soil for three days by a modified Macfadyen extraction (Macfadyen, 1961) into 50% ethylene glycol as preservative. Enchytraeids were extracted from the soil with a Baermann (1917) funnel technique by heating at 37  C for 3 h. All pots were checked for the presence of wireworms and their numbers recorded. Carbon and nitrogen contents of the plant material under the different soil organism treatments were measured using direct combustion on a CN analyzer (Carlo Erba, Milano, Italy). Shoots of all plant species were pooled together, excluding the shoots of the most abundant species H. lanatus and P. lanceolata that were analyzed separately for C and N. The total roots mixture that was also analyzed for C and N included all plant species used in the experiment. Phosphorus analyses could not be carried out because of lack of plant material. Soil for microbial PLFA analysis was sampled from the mixed soil of the whole pot. The soil was sieved (2 mm mesh) and sub samples mixed to avoid heterogeneity within the pots thus yielding a sample of about 10 g of soil. Roots visible in the soil were taken away as they otherwise can interfere with the PLFA analyses. The collected samples were kept frozen at 20  C until further analyses. 2.3. PLFA analysis Phospholipid fatty acids (PLFAs) were extracted from 3 g of fresh soil according to Frostegård et al. (1993). The lipids were fractionated into neutral lipids, glycolipids, and phospholipids on prepacked silica columns (100 mg of sorbent mass; Varian Medical Systems, Palo Alto, CA) by elution with 1.5 ml of chloroform, 6 ml of acetone, and 1.5 ml of methanol, respectively. The fatty acid residues in the neutral lipids and the phospholipids were transformed into free fatty acid methyl esters that were analyzed by gas chromatography (GC) using a 30 m  0.25 mm fused silica capillary column (HewlettePackard, Palo Alto, CA, USA) with H2 as the carrier gas (for details, see Hedlund, 2002). Twenty-three fatty acids were identified from their relative retention times compared to those of the internal standard fatty acid methyl ester 19:0. The following PLFA were chosen to represent bacterial biomass (Frostegård and Bååth, 1996): 15:0, i15:0, a15:0, i16:0, 16:1u9, i17:0, a17:0, cy17:0, 18:1u7, and cy19:0. Fungal biomass was represented by the PLFA markers 18:1u9 and 18:2u6,9 (Frostegård and Bååth, 1996), and AMF by the fungal marker NLFA 16:1u5 (Hedlund, 2002). Actinomycete biomass was represented by the PLFA marker 10Me18:0 (Kroppenstedt, 1985). The nomenclature of the PLFAs follows that used by Tunlid and White (1992). 2.4. Statistical analyses Biomasses of the individual plant species within the community were analyzed with multivariate analysis of variance (MANOVA). The categorical factors were AMF, Herb, Dec and ‘sampling time’. Significant effects of the MANOVA were followed up by separate factorial ANOVAs to determine which dependent variables were affected. Since all categorical factors were involved in the significant MANOVA results, repeated measures analyses of variance (RANOVAs) with a full-factorial design were performed (Quinn and Keough, 2004). To test effects of the three factors (AMF, Herb and Dec) on aboveground total shoot biomass, biomass of the plant functional groups (forbs, grasses, legumes) of the first and second harvest a three-way RANOVAs were also applied. Total plant biomass (shoot biomass harvest 1 þ shoot biomass harvest 2 þ root biomass), root biomass, C/N ratios, N and C content data and abundances of microbial PLFA (nmol/g soil) were analyzed with a three-way factorial ANOVA. Two-way ANOVA was used to test effects of soil organism abundances on each other. Prior to the

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analyses, the data were tested for normality and homogeneity of variance and log-transformed when necessary. Where mentioned, we performed a two-tailed Pearson’s correlation test as well as linear regression analyses on subsets of our data. A Principal Component Analysis (PCA) was used to detect differences in microbial communities from the soil organism treatments. The microbial community composition data was expressed as mol percentage of PLFA. A three-way ANOVA was used to test the significance of PCA scores obtained for microbial communities (Quinn and Keough, 2004). The two PCs were used as dependent variables and soil organism treatments as fixed orthogonal factor. All data are presented as mean values  SE of variables. All statistical tests were carried out with SPSS 17.0 (Chicago, Illinois, USA), PCA with MVSP 3.0 (Anglesey, Wales, UK).

3. Results 3.1. Plant performance Independent main effects of the functionally dissimilar soil organisms were detected for total plant biomass, root biomass, grass and forb biomass, while significant interaction effects were detected for total shoot biomass and individual plant species. AMF decreased (ANOVA: F(1,72) ¼ 9.67, P ¼ 0.003), decomposers increased (ANOVA: F(1,72) ¼ 7.07, P ¼ 0.01), root herbivores had no effect on total plant biomass, while the combined effects were additive (no significant interactions between the treatments were detected). Total root biomass was reduced by 18% in the AMF treatment (ANOVA: F(1,72) ¼ 10.82, P ¼ 0.01), increased by the decomposers by 25% (ANOVA: F(1,72) ¼ 7.37, P ¼ 0.01; Fig. 1); root herbivores had no effect. No soil organism interactions were detected. Total shoot biomass was reduced by 14% by root herbivores and a significant interaction between decomposers and AMF was detected (Fig. 1, Table 1). Alone decomposers tended to have a negative effect, but in presence of AMF they showed a positive effect on total shoot biomass (Fig. 1, Table 1). Shoot biomass of the grasses (3.2  0.1 g (harvest 1) and 2.5  0.1 g (harvest 2) in the control treatment) was reduced in the AMF treatment by 16% and 39%, respectively (RANOVA: F(1,72) ¼ 24.31, P < 0.001). Shoot biomass of the grasses was also reduced by 14% and 25% by root herbivores for the harvest 1 and harvest 2, respectively (RANOVA: F(1,72) ¼ 6.00, P < 0.05); no effects of decomposers or interactions were detected. Shoot biomass of the forbs (0.7  0.1 g (harvest 1) and 0.7  0.1 g (harvest 2) in the

Fig. 1. Effect of functionally dissimilar soil organisms on total root and shoot biomass of the plant community (mean þ SE, n ¼ 10). Shoot biomass for harvest 1 in week 20 and harvest 2 in week 26 are shown separately.

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control treatment) was enhanced in the AMF treatment by 42% and 46%, respectively (RANOVA: F(1,72) ¼ 44.83, P < 0.001), while no effects of the other treatments were detected (Fig. 2). Overall effects on biomasses of individual plant species of both harvests were detected for AMF (MANOVA: F(8,93) ¼ 11.01, P < 0.001, Wilk’s Lambda ¼ 0.51), root herbivores (MANOVA: F(8,93) ¼ 4.15, P < 0.001, Wilk’s Lambda ¼ 0.74) and the interaction between decomposers and root herbivores (MANOVA: F(8,93) ¼ 3.17, P < 0.01, Wilk’s Lambda ¼ 0.79) as well as sampling time (MANOVA: F(8,93) ¼ 11.06, P < 0.001, Wilk’s Lambda ¼ 0.51). Individual repeated measures ANOVAs were applied to detect the effects of the soil organism treatments on the plant species (Table 1). Shoot biomass of the forbs P. lanceolata and H. radicata was twice as high when AMF were present, while the shoot biomass of the grasses A. odoratum, A. capillaris and F. rubra was lower in the AMF treatment. The shoot biomass of the grass H. lanatus was lowest in the AMF treatment, but only at harvest 2 (Fig. 2, Table 1), while decomposers greatly enhanced its shoot biomass (Fig. 2, Table 1). L. corniculatus showed poor regrowth after the defoliation and could not be evaluated in a repeated measures analysis, while a three-way ANOVA on its shoot biomass from harvest 1 showed an interaction between root herbivores and decomposers: root herbivores and decomposers alone tended to increase its biomass, while they had the opposite effect when they were added together (Fig. 2, Table 1). The biomass of P. lanceolata was also reduced when both herbivores and decomposer were added together, while they had no effect when added alone (Fig. 2, Table 1). A. capillaris shoot biomass tended to be positively affected by the decomposers when AMF were present, but negatively in the absence of AMF (Fig. 2, Table 1). In general, the aboveground biomass of the plant community was lower at harvest 2 compared to harvest 1, mainly due to poor growth of H. lanatus and A. capillaris after the defoliation (Fig. 2, Table 1). The shoot biomass of P. pratensis showed no response to treatment with any of the soil organisms (Table 1). The effects of combined functionally dissimilar soil organisms on the nutrient content of the plants could be explained through additivity since no significant interactions between the soil organisms were detected. The shoot N contents (% g dry wt) of P. lanceolata and H. lanatus were enhanced by AMF from 1.50  0.07 to 2.04  0.12 (ANOVA: F(1,72) ¼ 38.73, P < 0.001) and from 1.55  0.05 to 1.73  0.07 (ANOVA: F(1,72) ¼ 10.53, P < 0.01), respectively. When root herbivores were present the shoot N contents of P. lanceolata and H. lanatus were enhanced from 1.50  0.07 to 1.69  0.05 (ANOVA: F(1,72) ¼ 4.67, P < 0.05) and from 1.55  0.05 to 1.71  0.09 (ANOVA: F(1,72) ¼ 10.65, P < 0.01), respectively. The N contents of the shoots, with P. lanceolata and H. lanatus excluded, (in all treatments, 1.87  0.03) and roots mixtures (in all treatments, 1.11  0.03) were unaffected. Both the root herbivores and the decomposer treatments enhanced the C content (% g dry wt) of H. lanatus from 41.53  0.3 to 42.22  0.2 (ANOVA: F(1,72) ¼ 8.71, P < 0.01) and from 41.53  0.3 to 42.77  0.2 (ANOVA: F(1,72) ¼ 5.35, P < 0.05), respectively, while AMF reduced it (ANOVA: F(1,72) ¼ 8.93, P < 0.01) from 41.53  0.3 to 41.31  0.4. The AMF treatment also reduced the C content of the total shoots from 41.74  0.3 to 40.85  0.4 (ANOVA: F(1,72) ¼ 13.89, P < 0.001), while it enhanced the C content of the total root mixture from 32.24  3.7 to 39.68  2.4 (ANOVA: F(1,72) ¼ 6.23, P < 0.05). The C content of P. lanceolata was not affected by the soil organism treatments (in all treatments, 40.91  0.56). When AMF was present C/N ratios increased in total root mixture from 29.56  3.0 to 32.93  2.0 (ANOVA: F(1,72) ¼ 5.40, P < 0.05), but decreased in total shoots mixtures from 22.18  1.0 to 20.92  0.5 (ANOVA: F(1,72) ¼ 5.50, P < 0.05). AMF also decreased the C/N ratio in P. lanceolata and H. lanatus shoots from 26.47  1.0 to 20.76  1.0 (ANOVA: F(1,72) ¼ 20.0, P < 0.001) and from

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Table 1 Treatment effects of arbuscular mycorrhizal fungi (AMF), root herbivores and decomposers on individual and total shoot plant biomass tested in a three-way repeated measures ANOVA. Significant F-values are shown in bold (*P < 0.05, **P < 0.01, ***P < 0.001). Treatment

DF

Shoot biomass Individual plant species

AMF Decomposers Herbivores AMF*D AMF*H D*H AMF*D*H Time (T) T*AMF T*D T*H T*AMF*D T*AMF*H T*D*H T*AMF*D*H Error

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 72

Total

Plantago

Hypochaeris

Holcus

Anthoxantum

Agrostis

Poa

Festuca

Lotus

27.67*** 0.67 1.33 0.06 0.55 6.49* 0.57 5.79* 3.29 1.00 0.001 0.29 0.58 0.59 0.009

15.34*** 0.18 0.12 0.02 1.01 0.17 1.25 0.18 1.41 1.67 3.36 0.90 1.13 0.13 0.96

1.25 3.91* 1.41 0.06 0.15 0.48 0.05 166.57*** 8.27** 1.46 0.07 0.14 0.08 0.18 0.01

8.04** 0.62 0.31 1.10 0.57 0.0001 0.06 2.41 2.01 0.01 0.30 0.16 0.04 0.002 0.54

15.12*** 1.74 2.98 4.26* 0.90 0.83 0.26 51.31*** 2.15 0.09 2.08 1.87 1.72 0.001 0.04

0.55 1.76 0.03 0.19 0.73 1.26 0.40 2.16 0.14 0.24 0.01 2.64 2.58 0.07 0.30

4.75* 0.01 0.65 2.00 0.48 1.01 0.05 0.33 0.01 1.99 0.41 1.94 0.21 2.44 0.19

0.06 0.01 0.04 0.71 0.94 10.03** 1.47 -y e e e e e e e

0.001 0.31 12.25** 4.69* 2.32 0.18 0.16 195.9*** 1.02 0.003 0.12 1.01 0.10 0.01 0.57

y a 3-way ANOVA was used due to lack of data for the 2nd harvest.

26.96  1.0 to 24.25  0.5 (ANOVA: F(1,72) ¼ 15.0, P < 0.001), respectively. When root herbivores were present C/N ratios decreased in H. lanatus shoots from 26.96  1.0 to 25.19  1.0 (ANOVA: F(1,72) ¼ 8.10, P < 0.01). 3.2. Soil organisms At the end of the experiment we determined the AMF root colonization levels of P. lanceolata, and the numbers of root herbivores (wireworms and nematodes) and decomposers (collembolans and enchytraeids) in the microcosms to see whether the groups of functionally dissimilar soil organisms had influenced each other’s abundances. The % RLC by AMF of P. lanceolata in the

AMF treatment was not significantly affected by root herbivores (ANOVA: F(1,32) ¼ 0.29, P ¼ 0.59) or decomposers (ANOVA: F(1,32) ¼ 0.04, P ¼ 0.84) (Table 2). Additionally, neither root herbivores (ANOVA: F(1,36) ¼ 3.26, P ¼ 0.08) nor decomposers (ANOVA: F(1,36) ¼ 0.32, P ¼ 0.57) affected NLFA of AMF in the AMF treatments. No AMF colonization of the P. lanceolata roots was detected in the non-AMF treatment. We found no significant effect of AMF (ANOVA: F(1,71) ¼ 0.03, P ¼ 0.88) or decomposers (ANOVA: F(1,71) ¼ 0.45, P ¼ 0.50) on the abundance of the added nematode P. penetrans (Table 2). Few wireworms and no enchytraeids were found back in the sampled soil therefore no data were available for statistical analyses (Table 2). Higher collembolan abundances were found in the AMF treatment (Table 2). Within the AMF treatment,

Fig. 2. Effect of functionally dissimilar soil organisms on the shoot biomass of the individual plant species at A) harvest 1 in week 20, and B) harvest 2 in week 26 (mean þ SE, n ¼ 10). Forbs: Plantago lanceolata, Hypochaeris radicata, grasses: Holcus lanatus, Anthoxanthum odoratum Agrostis capillaris, Poa pratensis, Festuca rubra, legume: Lotus corniculatus.

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Table 2 Soil organism abundances at the end of the experiment (means  SE, n ¼ 10). Treatment

Wireworms, no/pot

Nematodes Pratylenchus penetrans, no/100 g soil

Arbuscular mycorrhizal fungi (AMF) (% root length colonized)

Collembolans, no/g soil

Control AMF Decomposers Herbivores Dec þ Herb AMF þ Dec AMF þ Herb AMF þ Dec þ Herb

0 0 0 1 1 0 1 1

0 0 0 20.0 26.1 0 24.1 25.4

0 25.60 0 0 0 29.74 26.39 24.12

0.01 3.54 0.04 0.08 0.08 4.73 4.52 3.61

 0.2  0.1  0.4  0.2

 5.5  7.2  9.9  4.1

 4.07

 3.47  4.19  5.09

collembolan abundances were positively correlated with the % RLC by AMF (Pearson correlation ¼ 0.42; P < 0.05). Linear regression analyses between collembolan abundances and each of the measured parameters (e.g. biomass, nutrient uptake etc.) within the AMF treatment were conducted to see whether collembolan abundances changed the AMF effects, but revealed no significant numerical relationships (statistics are not shown).

3.3. Microbial biomass and community structure The greatest overall impact on the soil community had the AMF treatment which enhanced all microbial parameters (Table 3). Even though effects of the soil organism treatments for saprophytic fungal PLFA could be explained through additivity, interactive effects of the soil organisms on microbial biomass and community structure were observed in most of the combined treatments. Such interactive effects were detected for AM fungal NLFA as an interaction of the three soil organism treatments was significant. When testing effects of root herbivores and decomposers on AM fungal NLFA within the non-AMF treatment, where AMF may have been introduced with the added soil organisms, significant enhancing effects were found both for decomposers (ANOVA: F(1,36) ¼ 6.10, P ¼ 0.05) and for an interaction between the decomposers and root herbivores (ANOVA: F(1,36) ¼ 4.82, P ¼ 0.05). Also, the combination of decomposers and root herbivores enhanced bacterial and actinomycete PLFA abundances, while the decomposers alone reduced these abundances. Both main and interactive effects of the three groups of functionally dissimilar soil organisms on the microbial community structure were highly significant. In a principal component analysis

       

0.004 0.88 0.02 0.05 0.03 0.53 0.82 0.60

Enchytraeids, no/100 g soil 0 0 0 0 0 0 0 0

(PCA), when proportions of all PLFAs were compared, the microbial community composition differed among all soil organism treatments on the second axis (ANOVA, AMF F(1,72) ¼ 48.74, P < 0.0001; decomposers F(1,72) ¼ 10.07, P ¼ 0.002; herbivores F(1,72) ¼ 13.32, P ¼ 0.0005; AMF  decomposer interaction F(1,72) ¼ 4.26, P ¼ 0.04; AMF  herbivores interaction F(1,72) ¼ 10.07, P ¼ 0.002) but not on the first axis (statistics are not shown) (Fig. 3). The distribution of PCA loading values showed that differences between soil organism treatments on the second axis were mostly explained by fungal fatty acids such as saprophytic fungi PLFA 18:2u6,9, 18:1u9 and AM fungal NLFA 16:1u5 as well as by 16:0, 18:0 and 18:1u7 fatty acids. Separation along the first axis was mostly due to 10Me16:0, 16:0, 16:1u7, cy17:0, 18:1u7 and cy19:0. 4. Discussion 4.1. Effects of functionally dissimilar soil organisms on plant performance We found that combined treatments of the three major groups of functionally dissimilar soil organisms had additive effects on root biomass, nutrient content and total productivity of the plant community. Interactive effects were detected on the shoot growth of individual plant species and the total shoot biomass of the plant community, indicating that effects of the functionally dissimilar soil organisms on aboveground plant growth are not necessarily additive and thus predictable by knowing their individual effects in absence of other soil organism groups. Among the three groups of functionally dissimilar soil organisms AMF had the largest impact on the plant community. First, it

Table 3 Treatment effects of arbuscular mycorrhizal fungi (AMF), root herbivores and decomposers on abundances of microbial phospholipid fatty acids (PLFA) and neutrallipid fatty acids (NLFA) (mean  SE in nmol/g soil, n ¼ 10). Significant F-values are shown in bold (*P < 0.05, **P < 0.01, ***P < 0.001).

C AMF Herb Dec Dec þ Herb AMF þ Herb AMF þ Dec AMF þ Dec þ Herb AMF Herb Dec Dec*Herb AMF*Herb AMF*Dec AMF*Dec*Herb Error

Control Herbivores Decomposers

DF 1 1 1 1 1 1 1 72

Bacterial PLFA

Actinomycete PLFA

Saprophytic fungi PLFA

Arbuscular mycorrhizal fungi NLFA

118.7  4.3 176.6  4.1 107.6  5.0 106.0  5.5 137.6  8.6 161.2  4.2 175.2  6.7 179.0  7.1 F 177.7*** 0.3 4.1* 13.9*** 3.7 0.002 2.0

3.5  0.2 5.2  0.1 3.3  0.2 3.2  0.2 4.3  0.3 5.1  0.1 5.3  0.2 5.6  0.2 F 161.3*** 3.1 5.4* 9.1** 2.1 0.02 3.2

5.3  0.4 7.1  0.6 4.7  0.3 4.6  0.4 5.7  0.5 6.1  0.3 8.2  1.5 6.9  0.3 F 18.7*** 1.0 1.5 0.6 2.2 0.6 1.2

3.0  0.4 175.2  24.0 2.9  0.2 3.3  0.4 4.7  0.5 159.2  23.8 186.4  25.4 124.2  14.4 F 2229.5*** 0.1 1.7 0.1 7.2** 5.0* 3.9*

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AMF

AMF+Decomposers AMF+Root herbivores AMF+Decomposers+ Root herbivores Control Decomposers

Root herbivores Decomposers +Root herbivores

Fig. 3. Principal component analysis of abundances of microbial phospholipid fatty acids (PLFA) with eight soil organisms treatments, including control, AMF (arbuscular mycorrhizal fungi), root herbivores, decomposers single treatments (filled signs) and combined AMF þ root herbivores, AMF þ decomposers, AMF þ root herbivores þ decomposers, root herbivores þ decomposers treatments (open signs), results are means  SE, n ¼ 10 of principal component scores.

reduced total root biomass of the plant community. This was not unexpected since mycorrhizal plants often produce less root biomass, since the extended hyphal network might compensate for fine roots (Smith and Read, 1997). The higher root biomass in absence of AMF might also be attributed to the higher biomass of grasses, which often have more extensive root systems than forbs or legumes (Sun et al., 1997). Second, plant functional groups (grasses, forbs) were differently affected by AMF. Van der Heijden et al. (1998a) found that mycorrhizal dependency varies between plant species which can lead to the promotion of one, and the suppression of another plant species or plant functional group within a plant community. Accordingly, the addition of AMF promoted the shoot biomass of forbs and reduced the shoot biomass of grasses, probably by changing the relative competitive strength between the two plant functional groups in our study. Consistent with this observation, Gange and Brown (2002) have shown that the proportion of grasses in a plant community can be reduced by AMF. They observed that a reduction of AMF by fungicides caused a switch from forbs to a plant community dominated by perennial grasses. This confirms the important role of AMF in regulating plant productivity and plant species coexistence as suggested by Van der Heijden et al. (1998b). Third, AMF had the greatest effect on individual plant species, supporting the idea that AMF strongly influence the relative abundances of plant species within a community (Klironomos, 2002). The fact that AMF had the largest effect may also be due to the circumstance that they were added first to the treatment pots and were interacting with younger plants than the soil organisms that were added later. AMF were added first because AMF need time to establish and colonize the roots, while root-feeding herbivores were added later to prevent potentially detrimental plant damage by nematodes and wireworms to the younger plants. Decomposers were also added afterwards because an establishing AMF hyphal network could be destroyed by potential grazers such as collembolans. Contrary to AMF, root herbivores did not reduce the root biomass of the plant community but decreased total shoot biomass by reducing the shoot biomass of grasses. Consistent with our observation, root-feeding nematodes and wireworms did not affect the root biomass of a grassland plant community in the study of De Deyn et al. (2007). An explanation for the observed reduction of

grass biomass by root herbivores could be that grasses were the more palatable food resource than forbs. Forbs can contain high levels of secondary metabolites, such as the iridoid glycosides in P. lanceolata (Rønsted et al., 2000), which may act against rootfeeding nematodes (De Deyn et al., 2004). Wireworms can also reduce the growth of grasses (Hemerik et al., 2003), and enhance the abundance of specific root-feeding nematodes (De Deyn et al., 2007), thereby impairing the competitive abilities of their host plants against non-host plant species (Van Ruijven et al., 2003). Decomposers enhanced total root biomass and the total biomass of the plant community. This is in agreement with Partsch et al. (2006) who found that decomposers enhanced the root biomass of a grassland plant community, most likely due to their ability to distribute nutrients and support mineralization leading to an increased nutrient availability for plant growth. As expected, decomposers promoted the growth of a grass species, H. lanatus, probably because this species is a strong competitor for nutrients in soil (Schippers and Kropff, 2001). There were also interactive effects of soil organisms on the shoot biomasses of individual plant species. The shoot biomasses of A. capillaris, L. corniculatus and P. lanceolata were enhanced by decomposers but only when the decomposers were interacting with AMF or root herbivores. The positive interactive effect between decomposers and root herbivores on the shoots of L. corniculatus and P. lanceolata may be due to decomposer activity enhancing root growth through changes in soil nutrient availability and thereby promoting root consumption by root herbivores (Endlweber and Scheu, 2006). The root consumption by herbivores may consequently increase root exudation, leading to enhanced microbial growth and the potential promotion of plant growth promoting rhizobacteria (Grayston et al., 2001). The shoot biomass of A. capillaris and the total shoot biomass of the plant community were affected by an interaction between AMF and decomposers. In the presence of AMF, decomposers had a positive effect on the shoot biomass, while the effect of decomposers tended to be negative in the absence of AMF. One possible explanation is that AMF increase plant P uptake, but the plants can only profit from this (i.e. increase their biomass) if they also get enough additional nutrients, which are provided by decomposer activity (Filser, 2002; Cole et al., 2004; Johnson, 2010). In the absence of AMF, P is likely limiting the plant growth and the nutrients made available by decomposers are immobilized by soil microorganisms, which successfully compete with the plants for nutrients (Dannenmann et al., 2009; Gray and Dighton, 2009). Overall, however, no interactive effects of the functionally dissimilar soil organisms were detected on the nutrient content of the plant community. The higher N content of P. lanceolata and H. lanatus shoots in the presence of either root herbivores or AMF supports the idea that the influence of soil organisms on plant growth is mediated by increased N mobilization. Higher N levels in plant shoots when fed upon by phytophageous nematodes were already observed by Wurst et al. (2006) indicating that root-feeding by nematodes increases microbial activity and mineralization in the rhizosphere (Bardgett et al.,1999; Denton et al.,1999). Moreover, the high levels of shoot N may be explained by the ability of AMF to directly provide plants with organic and inorganic N (Hawkins et al., 2000; Leigh et al., 2008). Higher C levels in shoots of H. lanatus in our study could be due to an induced allocation of C from roots to the shoots in the presence of root herbivores (Poll et al., 2007). However, consistent with previous studies (Wurst and Van der Putten, 2007; Wurst et al., 2008b), P. lanceolata C content was not affected by root herbivores. In presence of AMF, C levels were significantly reduced in the total shoot biomass and in the shoots of H. lanatus, and enhanced in the total root biomass. De Deyn et al. (2009) also observed higher C levels (in terms of glucose) in the roots of

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P. lanceolata inoculated with AMF. Since AMF are completely dependent on C provided by the plant (Smith and Read, 1997) the need of AMF might be reflected in the observed lower shoot and higher root C contents of the plant community. Decreased C/N ratio of shoots in our experiment therefore may be explained either through improved N uptake by AMF or a shift of C to the roots resulting in increased C/N ratio of roots because of AMF demands for assimilated plant C. Decomposers, in turn, enhanced C content of H. lanatus shoots, although in another study neither C nor N contents of a grass were affected by the presence of collembolans (Endlweber and Scheu, 2007). 4.2. Effects of functionally dissimilar soil organisms on each other abundances, microbial community structure and biomass Consistent with previous studies, AMF root colonization was not affected by the presence of insect root herbivores (Gange, 2001; Wurst et al., 2008b) or nematodes (De la Peña et al., 2006). Nematode and wireworms numbers at the end of the experiment were not affected by AMF, albeit AMF have been recently reported to reduce P. penetrans root colonization and reproduction in a dune grass (De la Peña et al., 2006). Collembolans reached high densities in the AMF treatments, including species (Proisotoma minuta) that had not been introduced with the decomposer treatment, probably due to a contamination of the AMF inoculum with collembolans. However, collembolan abundances did not change the effects of AMF on the measured plant parameters. Except for the positive correlation between AMF colonization of P. lanceolata roots and collembolan abundances in soil, the soil organisms had no effect on each other’s abundances at the end of the experiment. Interactive effects of soil organisms were detected at a higher resolution that is for the microbial biomass and microbial community structure. According to our hypothesis, all microbial PLFA abundances were enhanced in the presence of AMF. This effect may be due to a positive AMF contribution to soil aggregation (Van der Heijden et al., 2006) that provides optimal water film microenvironments or habitats for microorganisms (Paradelo and Barral, 2009). Microbial community structure was significantly affected by AMF in interaction with the other soil organism groups. In accordance with our results, it was shown that AMF can modify soil microbial communities (Zhang et al., 2010), probably through the exudation of substances with selective effects on soil microorganisms (Marschner and Baumann, 2003). Root herbivores alone did not affect microbial PLFA abundances but when interacting with decomposers they enhanced the abundances of bacteria and actinomycetes. The interaction effect may be explained, as described above for the shoot biomass, by collembolan-mediated changes in soil nutrient availability, leading to the growth of longer and thinner roots (Endlweber and Scheu, 2006), that become an easier food resource for herbivores. During the consumption of roots by herbivores, nutrients are released into the soil, leading to more C and N available for the microorganisms, therefore enhancing growth of bacteria and actinomycetes (Bardgett et al., 1999; Grayston et al., 2001). Additionally, root herbivores and decomposers can spread microbial propagules, increasing distribution and fitness of microorganisms in soil (Whipps and Budge, 1993; Knox et al., 2003). Also both root herbivores and decomposers may affect soil structure (Vliet and Hendrix, 2003) thus possibly contributing to changes in the bacterial and actinomycete abundances. The fungal PLFA were not affected, likely because the fungi could exploit other C and N resources. According to our hypothesis that decomposers may affect microbial biomass and community structure, bacterial and actinomycete biomasses were significantly reduced, whereas saprophytic

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and AMF biomasses were not affected. Such bacterial biomass reduction might be due to grazing by collembolans, since under certain conditions collembolans preferentially feed on bacteria rather than fungi as was shown in lab experiments (Haubert et al., 2006). Additionally, the negative effects of decomposers on bacteria and actinomycetes may have reduced the competitive strength of the bacteria and actinomycetes. Enchytraeids were not found back in the soil samples at the end of the experiment but could have contributed to the changes in microbial PLFA abundances at an earlier stage of the experiment (Cole et al., 2002). Overall, the detected changes in the structure and biomass of soil microbial communities due to presence of functionally dissimilar soil organisms may have resulted in changes in mineralization processes, and thus nutrient availability for plant growth, indicating complex interrelationships among soil animals, microorganisms and plants. 5. Conclusions We designed a comprehensive experiment to test individual and combined effects of functionally dissimilar soil organisms on soil microbial community as well as on plant community productivity and nutrient content. There were additive and therefore predictable effects of the functionally dissimilar soil organisms on general parameters of the plant community, such as total productivity, total root biomass, grass and forb biomass, and nutrient uptake. There were interactive effects on the shoot biomass of individual plant species, the total shoot biomass, and the soil microbial communities. Our study shows that interactive effects are mainly present at higher resolutions, i.e. on the levels of individual plants and the soil microbial community but do not affect the productivity and nutrient content of the plant community which is affected by additive effects of functionally dissimilar soil organisms. In general, to understand and predict the impact of AMF, root feeders and decomposers on plant communities as well as on soil microbial communities under natural conditions, their relative effects in the presence of other functionally dissimilar soil organisms have to be taken into account. Acknowledgments The study was funded by the EU Marie Curie BIORHIZ Research Network. We thank Ulrike Gloger for C/N analysis, Institute of Zoology, Darmstadt, and all principal investigators of the BIORHIZ project for their support and encouragement. We also thank Richard Bardgett and anonymous reviewers for their useful comments and suggestions. References Baermann, G., 1917. Eine einfache Methode zur Auffindung von Anchylostomum(Nematoden)-Larven in Erdproben. Geneeskundig Tijdschrift voor Ned Indië 57, 131e137. Bardgett, R.D., Denton, S.C., Cook, R., 1999. Below-ground herbivory promotes soil nutrient transfer and root growth in grassland. Ecology Letters 2, 357e360. Bongers, T., 1988. De nematoden van Nederland. Natuurhistorische Bibliotheek van de KNNV, nr 46. Pirola, Schroorl, the Netherlands. Bradford, M.A., Jones, T.H., Bardgett, R.D., Black, H.I.J., Boag, B., Bonkowski, M., Cook, R., Eggers, T., Gange, A.C., Grayston, S.J., Kandeler, E., McCaig, A.E., Newington, J.E., Prosser, J.I., Setala, H., Staddon, P.L., Tordoff, G.M., Tscherko, D., Lawton, J.H., 2002. Impacts of soil fauna community composition on model grassland ecosystems. Science 298, 615e618. Brown, V.K., Gange, A.C., 1992. Secondary plant succession how is it modified by insect herbivory? Vegetation 101, 3e13. Brundrett, N., Bougher, N., Dell, B., Grove, T., Malajczuk, N., 1996. Working with mycorrhiza in forestry and agriculture. ACIAR Monograph 32, 374. Cole, L., Bardgett, R.D., Ineson, P., Hobbs, P.J., 2002. Enchytraeid worm (Oligochaeta) influences on microbial community structure, nutrients dynamics and plant

2274

N. Ladygina et al. / Soil Biology & Biochemistry 42 (2010) 2266e2275

growth in blanket peat subjected to warming. Soil Biology & Biochemistry 34, 83e92. Cole, L., Staddon, P.L., Sleep, D., Bardgett, R.D., 2004. Soil animals influence microbial abundance, but not plantemicrobial competition for soil organic nitrogen. Functional Ecology 18, 631e640. Currie, A.F., Murray, P.J., Gange, A.C., 2006. Root herbivory by Tipula paludosa larvae increases colonization of Agrostis capillaris by arbuscular mycorrhizal fungi. Soil Biology & Biochemistry 38, 1994e1997. Dannenmann, M., Simon, J., Gasche, R., Holst, J., Naumann, P.S., Kögel-Knabner, I., Knicker, H., Mayer, H., Schloter, M., Pena, R., Polle, A., Rennenberg, H., Papen, H., 2009. Tree girdling provides insight on the role of labile carbon in nitrogen partitioning between soil microorganisms and adult European beech. Soil Biology & Biochemistry 41, 1622e1631. De Deyn, G.B., Raaijmakers, C.E., Rik Zoomer, K., Berg, M.P., De Ruiter, P.C., Verfoef, H.A., Bezemer, T.M., van der Putten, W., 2003. Soil invertebrate fauna enhances grassland succession and diversity. Nature 422, 711e713. De Deyn, G.B., Raaijmakers, C.B., van Ruijven, R., Berendse, F., van der Putten, W., 2004. Plant species identity and diversity effects on different trophic levels of nematodes in the soil food web. Oikos 106, 576e586. De Deyn, G.B., Van Ruijven, R., Raaijmakers, C.E., De Ruiter, P.C., Van der Putten, W.H., 2007. Above- and belowground insect herbivore differently affect soil nematode communities in species-rich plant communities. Oikos 116, 923e930. De Deyn, G.B., Biere, A., Van der Putten, W.H., Wagenaar, R., Klironomos, J.N., 2009. Chemical defence, mycorrhyzal colonization and growth responses in Plantago lanceolata L. Oecologia 160, 433e442. De la Peña, E., Echeverria, S.R., Van der Putten, W.H., Freitas, H., Moens, M., 2006. Mechanism of control of root-feeding nematodes by mycorrhizal fungi in the dune grass Ammophila arenaria. New Phytologist 169, 829e840. Denton, C.S., Bardgett, R.D., Cook, R., Hobbs, P.J., 1999. Low amounts of root herbivory positively influence the rhizosphere microbial community in a temperate grassland soil. Soil Biology & Biochemistry 31, 155e165. Eisenhauer, N., Konig, S., Sabais, A.C.W., Renker, C., Buscot, F., Scheu, S., 2009. Impacts of earthworms and arbuscular mycorrhizal fungi (Glomus intraradices) on plant performance are not interrelated. Soil Biology & Biochemistry 41, 561e567. Ellenberg, H., 1974. Zeigerwerte der Gefäßpflanzen Mitteleuropas. Scripta Geobot 9 Goltze. Endlweber, K., Scheu, S., 2006. Effects of Collembola on root properties of two competing ruderal plant species. Soil Biology & Biochemistry 38, 2025e2031. Endlweber, K., Scheu, S., 2007. Interactions between mycorrhizal fungi and Collembola: effects on root structure of competing plant species. Biology and Fertility of Soils 43, 741e749. Filser, J., 2002. The role of Collembola in carbon and nitrogen cycling in soil. Pedobiologia 46, 234e245. Frostegård, Å, Bååth, E., 1996. The use of phospholipid fatty acid analysis to estimate bacterial and fungal biomass in soil. Biology and Fertility of Soils 22, 59e65. Frostegård, Å, Tunlid, A., Bååth, E., 1993. Phospholipid fatty acid composition, biomass and activity of microbial communities from two soil types experimentally exposed to different heavy metals. Applied and Environmental Microbiology 59, 3605e3617. Gange, A.C., Brown, V.K., 2002. Soil food web components affect plant community structure during early succession. Ecological Research 17, 217e227. Gange, A.C., Brown, V.K., Sinclair, G.S., 1993. Vesicular-Arbuscular Mycorrhizal fungi: a determinant of plant community structure in early succession. Functional Ecology 7, 616e622. Gange, A.C., 2001. Species-specific responses of a root- and shoot-feeding insect to arbuscular mycorrhizal colonization of its host plant. New Phytologist 150, 611e618. Gray, D.M., Dighton, J., 2009. Nutrient utilization by pine seedlings and soil microbes in oligotrophic pine barrens forest soils subjected to prescribed fire treatment. Soil Biology & Biochemistry 41, 1957e1965. Grayston, S.J., Dawson, L.A., Treonis, A.M., Murray, P.J., Ross, J., Reid, E.J., MacDougall, R., 2001. Impact of root herbivore by insect larvae on soil microbial communities. Europian Journal of Soil Biology 37, 277e280. Haubert, D., Häggblom, M.M., Langel, R., Scheu, S., Ruess, L., 2006. Trophic shift of stable isotopes and fatty acids in collembola on bacterial diets. Soil Biology & Biochemistry 38, 2004e2007. Hawkins, H.-J., Johansen, A., George, E., 2000. Uptake and transport of organic and inorganic nitrogen by arbuscular mycorrhizal fungi. Plant and Soil 226, 275e285. Hedlund, K., Santa Regina, I., Van der Putten, W.H., Leps, J., Diaz, T., Korthals, G.W., Lavorel, S., Brown, V.K., Gormsen, D., Mortimer, S.R., Rodriquez Barrueco, C., Roy, J., Smilauer, P., Smilauerova, M., Van Dijk, C., 2003. Plant species diversity, plant biomass and responses of the soil community on abandoned land across Europe: idiosyncracy or above-belowground time lags. Oikos 103, 45e58. Hedlund, K., 2002. Soil microbial community structure in relation to vegetation management on former agricultural land. Soil Biology & Biochemistry 34, 1299e1307. Hemerik, L., Gort, G., Brussaard, L., 2003. Food preference of wireworms analyzed with multinomial models. Journal of Insect Behaviour 16, 647e665. Hol, W.H.G., Cook, R., 2005. An overview of arbuscular mycorrhizal fungienematode interactions. Basic and Applied Ecology 6, 489e503. Johnson, N.C., Graham, J.H., Smith, F.A., 1997. Functioning of mycorrhizal associations along the mutualismeparasitism continuum. New Phytologist 135, 575e585.

Johnson, N.C., 2010. Resource stoichiometry elucidates the structure and function of arbuscular mycorrhizas across scales. New Phytologist 185, 631e647. Jorgensen, H.B., 2002. Food Selection and Fitness Optimization in Insects. PhD thesis. Lund University, ISBN: 91-7105-182-1. Kardol, P., Bezemer, T.M., Van der Putten, W.H., 2006. Temporal variation in plantesoil feedback controls succession. Ecology Letters 9, 1080e1088. Klironomos, J., 2002. Feedback with soil biota contributes to plant rarity and invasiveness in communities. Nature 417, 67e70. Knox, O.G.G., Killham, K., Mullins, C.E., Wilson, M.J., 2003. Nematode-enhanced microbial colonization of the wheat rhizosphere. FEMS Microbiology Letters 225, 227e233. Kroppenstedt, R.M., 1985. Fatty acids and menaquinone analysis of actinomycetes and related organisms. In: Goodfellow, M., Minnikin, D.E. (Eds.), Chemical Methods in Bacterial Systematic. Academic Press, London, pp. 173e199. Kula, A.A.R., Hartnett, D.C., Wilson, G.W.T., 2005. Effects of mycorhizal symbiosis on tallgrass prairie planteherbivore interactions. Ecology Letters 8, 61e69. Leigh, J., Hodge, A., Fitter, A.H., 2008. Arbuscular mycorrhizal fungi can transfer substantial amounts of nitrogen to their host plant from organic material. New Phytologist 181, 199e207. Leps, J., Brown, V.K., Diaz, T.A., Gormsen, D., Hedlund, K., Kailova, J., Korthals, G.W., Mortimer, S.R., Rodriguez-Barrueco, C., Roy, J., Regina, S.I., van Dijk, C., Van der Putten, W.H., 2001. Separating the chance effect from diversity per se effect in functioning of plant communities. Oikos 92, 123e134. Macfadyen, A., 1961. Improved funnel-type extractor for soil arthropods. Journal of Animal Ecology 30, 171e184. Marschner, P., Baumann, K., 2003. Changes in bacterial community structure induced by mycorrhizal colonization in split-root maize. Plant Soil 251, 279e289. McGonigle, T.P., Millet, M.H., Evans, D.G., Fairchild, G.L., Swan, J.T., 1990. A new method which gives an objective measure of colonization of roots by vesiculararbuscular mycorrhizal fungi. New Phytologist 115, 495e501. Oostenbrink, M., 1960. Estimating nematode populations by some selected methods. In: Sasser, J.N., Jenkins, W.R. (Eds.), Nematology. Univ. of North Carolina Press, pp. 85e102. Paradelo, R., Barral, M.T., 2009. Effect of moisture and disaggregation on the microbial activity in soil. Soil & Tillage Research 104, 317e319. Partsch, S., Milcu, A., Scheu, S., 2006. The role of decomposer animals (Lumbricidae, Collembola) for plant performance in model grassland systems of different diversity. Ecology 87, 2548e2558. Plum, N.M., Filser, J., 2005. Floods and drought: responce of earthworms and potworms (Oligochaeta: Lumbricidae, Enchytraeidae) to hydrological extremes in wet grasslands. Pedobiologia 49, 443e453. Poll, J., Marhan, S., Haase, S., Hallmann, J., Kandeler, E., Ruess, L., 2007. Low amounts of herbivory by root-knot nematodes affect microbial community dynamics and carbon allocation in the rhizosphere. FEMS Microbiology Ecology 62, 268e279. Quinn, G.P., Keough, M.J., 2004. Experimental Design and Data Analysis for Biologists. Cambridge Univ. Press, Cambridge. Rønsted, N., Gobel, E., Franzyk, H., Rosendal Jensen, S., Olsen, C.E., 2000. Chemotaxonomy of Plantago. Iridoid glycosides and caffeoyl phenylethanoid glycosides. Phytochemistry 55, 337e348. Schädler, M., Jung, G., Brandl, R., Auge, H., 2004. Secondary succession is influenced by belowground insect herbivory on a productive site. Oecologia 138, 242e252. Schippers, P., Kropff, M.J., 2001. Competition for light and nitrogen among grassland species: a simulation analysis. Functional Ecology 15, 155e164. Smith, S.E., Read, D.J., 1997. Mycorrhizal Symbiosis. Academic, London. Sokal, R.R., Rohlf, F.J., 1981. Additivity, in: Biometry: The Principles and Practice of Statistics in Biological Research. Freeman W.H. & Co, New York, pp. 414e417. Sørensen, L.I., Kytoviita, M.-M., Olofsson, J., Mikola, J., 2008. Soil feedback on plant growth in a sub-arctic grassland as a result repeated defoliation. Soil Biology & Biochemistry 40, 2891e2897. Steinaker, D.F., Wilson, S.D., 2008. Scale and density dependent relationships among roots, mycorrhizal fungi and collembola in grassland and forest. Oikos 117, 703e710. Sun, G., Coffin, D.B., Lauenroth, W.K., 1997. Comparison of root distribution of species in North American grasslands using GIS. Journal of Vegetation Science 8, 587e596. Tilman, D., 1982. Resource Competition and Community Structure. Princeton Univ. Press. Tunlid, A., White, D.C., 1992. Biochemical Analysis of Biomass, Community Structure, Nutritional Status, and Metabolic Activity of Microbial Communities in Soil. In: Stotzky, G., Bollag, J.-M. (Eds.), Soil Biochemistry. Marcel Dekker, Inc., New York, pp. 229e262. Van der Heijden, M.G.A., Boller, T., Wiemken, A., Sanders, I.R., 1998a. Different arbuscular mycorrhizal fungal species are potential determinants of plant community structure. Ecology 79, 2082e2091. Van der Heijden, M.G.A., Klironomos, J.N., Ursic, N., Moutoglis, P., StreitwolfEngel, R., Boller, T., Wiemken, A., Sanders, I.R., 1998b. Mycorrhizal fungal diversity determines plant biodiversity, ecosystem variability and productivity. Nature 396, 69e72. Van der Heijden, M.G.A., Streitwolf-Engel, R., Riedl, R., Siegrist, S., Neudecker, A., Ineichen, K., Boller, T., Wiemken, A., Sanders, I.R., 2006. The mycorrhizal contribution to plant productivity, plant nutrition and soil structure in experimental grassland. New Phytologist 172, 739e752. Van der Putten, W.H., Van Dijk, C., Peters, B.A.M., 1993. Plant-specific soil-borne diseases contribute to succession in foredune vegetation. Nature 362, 53e56.

N. Ladygina et al. / Soil Biology & Biochemistry 42 (2010) 2266e2275 Van der Putten, W.H., Mortimer, S., Hedlund, Van Dijk, K.C., Brown, V.K., Leps, J., Rodriguez-Barrueco, C., Roy, J., Diaz Len, T.A., Gormsen, D., Korthals, G.W., Lavorel, S., Santa Regina, I., Smilauer, P., 2000. Plant species diversity as a driver of early succession in abandoned fields: a multi-site approach. Oecologia 124, 91e99. Van der Putten, W.H., Vet, L.E.M., Harvey, J.A., Wackers, F.L., 2001. Linking above e and belowground multitrophic interactions of plants, herbivores, pathogens, and their antagonists. Trends in Ecology and Evolution 16, 547e554. Van Ruijven, J., De Deyn, G.B., Raaijmakers, C.E., Berendse, F., Van der Putten, W.H., 2003. Diversity reduces invasibility in experimental plant communities: the role of plant species. Ecology Letters 6, 910e918. Vierheilig, H., Coughlan, A.P., Wyss, U., Piche, Y., 1998. Ink and vinegar, a simple staining technique for arbuscular-mycorrhizal fungi. Applied and Environmental Microbiology 64, 5004e5007. van Vliet, P.C.J., Hendrix, P.F., 2003. Role of fauna in soil physical processes. In: Abbott, L.K., Murphy, D.V. (Eds.), Soil Biological Fertility: a Key to Sustainable Land Use in Agriculture. Kluwer Academic Publishers, Dordrecht, Netherlands, pp. 61e80. Wardle, D.A., Bardgett, R.D., Klironomos, J.N., Setala, H., Van der Putten, W.H., Wall, D.H., 2004. Ecological linkages between aboveground and belowground biota. Science 304, 1629e1633. Whipps, J.M., Budge, S.P.,1993. Transmission of the mycoparasite Coniothyrium minitans by collembolan Folsomia candida (Collembola: Entomobryidae) and glasshouse sciarid Bradysia sp. (Diptera: Sciaridae). Annals of Applied Biology 123, 165e171.

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Wurst, S., Van der Putten, W.H., 2007. Root herbivore identity matters in plantmediated interactions between root and shoot herbivores. Basic and Applied Ecology 8, 491e499. Wurst, S., Dugassa-Gobena, D., Langel, R., Bonkowski, M., Scheu, S., 2004. Combined effects of earthworms and vesicular-arbuscular mycorrhizas on plant and aphid performance. New Phytologist 163, 169e176. Wurst, S., Langel, R., Rodger, S., Scheu, S., 2006. Effects of belowground biota on primary and secondary metabolites in Brassica oleracea. Chemoecology 16, 69e73. Wurst, S., Allema, B., Duyts, H., Van der Putten, W.H., 2008a. Earthworms counterbalance the negative effect of microorganisms on plant diversity and enhance the tolerance of grasses to nematodes. Oikos 117, 711e718. Wurst, S., Van Dam, M.N., Monroy, F., Biere, A., Van der Putten, W.H., 2008b. Intraspecific variation in plant defense alters effects of root herbivores on leaf chemistry and aboveground herbivore damage. Journal of Chemical Ecology 34, 1360e1367. Zelles, L., 1999. Fatty acid patterns of phospho-lipids and lipopolysaccharides in the characterization of microbial communities in soil: a review. Biology and Fertility of Soils 29, 111e129. Zhang, H., Tang, M., Chen, H., Tian, Z., Xue, Y., Feng, Y., 2010. Communities of arbuscular mycorrhizal fungi and bacteria in the rhizosphere of Caragana korshinkii and Hippophae rhamnoides in Zhifanggou watershed. Plant Soil 326, 415e424.