Independent role of belowground organisms and plant cultivar diversity in legume-grass communities

Applied Soil Ecology 95 (2015) 1–8

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Applied Soil Ecology journal homepage: www.elsevier.com/locate/apsoil

Independent role of belowground organisms and plant cultivar diversity in legume-grass communities Xiaohui Guoa,* , Jana S. Petermanna,b,c , Conrad Schittkoa , Susanne Wursta,b a b c

Institute of Biology, Freie Universität Berlin, Königin-Luise-Str. 1-3, D-14195 Berlin, Germany Berlin-Brandenburg Institute of Advanced Biodiversity Research (BBIB), D-14195 Berlin, Germany Department of Ecology and Evolution, University of Salzburg, Hellbrunnerstr. 34, 5020 Salzburg, Austria

A R T I C L E I N F O

A B S T R A C T

Article history: Received 23 February 2015 Received in revised form 20 May 2015 Accepted 25 May 2015 Available online xxx

Plant diversity and different groups of belowground organisms have been shown to influence the characteristics of plant communities and their associated aboveground organisms; however, little is known about the interactions and combined effects of these biotic factors. Furthermore, while plant species richness has received a lot of attention, intraspecific diversity has only recently come into the focus of community ecology. The objective of this study was to determine whether the addition of beneficial soil organisms (arbuscular mycorrhizal fungi (AMF) and anecic earthworms (Lumbricus terrestris L.) and different levels of plant cultivar diversity interactively influence productivity, weed establishment, leaf damage by herbivores and pathogens, and the diversity of aboveground herbivores in legume-grass communities. Our results indicated that the addition of AMF increased aboveground plant productivity and tended to decrease the diversity of aboveground herbivores. Earthworms attenuated the effect of AMF addition on AMF root colonization, but had no effect on plant productivity or herbivore diversity. Weed biomass was significantly lower in plant communities with high cultivar diversity compared to low cultivar diversity. We did not find interactive effects of the two soil organism groups and plant cultivar diversity. Our results demonstrate the independent roles and additive positive effects of AMF and plant cultivar diversity on functions such as productivity and resistance against weeds in the field. We suggest that AMF and plant cultivar diversity manipulations may be applied more frequently in agriculture management programs that aim for sustainable yield enhancement and biocontrol of herbivores and weeds. ã2015 Elsevier B.V. All rights reserved.

Keywords: Arbuscular mycorrhizal fungi (AMF) Earthworms Cultivar diversity Productivity Ecosystem function Arthropods

1. Introduction An increasing body of work demonstrates that plant diversity and aboveground communities are influenced by belowground organisms (Wardle et al., 2004; van der Heijden et al., 2008). Plant diversity itself has been found to lead to increased plant productivity and to alter herbivore performance (Hughes et al., 2008; Scherber et al., 2010). However, most studies have focused on species diversity (van der Heijden et al., 1998; Scherber et al., 2010; Eisenhauer et al., 2012), while the role of intraspecific diversity for ecosystem functions has only recently drawn interest (Kotowska et al., 2010; Ostfeld and Keesing, 2012). Moreover, little is known about the interactive effects that intraspecific plant

* Corresponding author at: Functional Biodiversity, Dahlem Centre of Plant Sciences, Freie Universität Berlin, Königin-Luise-Straße 1-3, 14195 Berlin, Germany. Tel.: +49 30 838 50947; fax: +49 30 838 54869. E-mail address: [email protected] (X. Guo). http://dx.doi.org/10.1016/j.apsoil.2015.05.010 0929-1393/ ã 2015 Elsevier B.V. All rights reserved.

diversity and soil biota may have on plant and aboveground herbivore communities, and ecosystem functions. Arbuscular mycorrhizal fungi (AMF) and earthworms are vital components of the rhizosphere and soil biota community. Previous studies have shown that AMF and earthworms can positively influence plant nutrition and productivity (Milleret et al., 2008; Kohler-Milleret et al., 2013; Willis et al., 2013). AMF are generally known to improve plant growth by enhancing nutrient uptake such as available phosphorus (P) and inorganic nitrogen (N), by inducing root system modifications, and by influencing soil structure (Rillig and Mummey, 2006; Smith and Read, 2008). Moreover, the plants’ symbiosis with AMF is thought to improve plant resistance against aboveground herbivores (Pineda et al., 2010). Earthworms, as ecosystem engineers which dominate the biomass of soil invertebrates, are also known to alter the soil environment and nutrient availability through their feeding, burrowing, casting, and dispersing activities (Lavelle, 1988). Furthermore, a review article by Wurst (2010) illustrated that aboveground herbivores can be affected by earthworms either

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negatively or positively depending on abiotic and biotic soil conditions and species identity. Previous studies reported conflicting results about AMF and earthworm interactions and their consequences for plant performance (Eisenhauer et al., 2009; Mammitzsch et al., 2012). Earthworms have been documented to stimulate mycorrhizal infection (Ma et al., 2006), increase the AMF root colonization rate and dispersal of AMF spores (Zarea et al., 2009). However, earthworms may also have no effects on the symbiosis between AMF and plants (Wurst et al., 2004; Eisenhauer et al., 2009), or may reduce the arbuscular mycorrhizal symbiosis by damaging or grazing on fungal mycelia (Pattinson et al., 1997; Bonkowski et al., 2000; Lawrence et al., 2003). Earthworms and AMF were found to have mainly independent effects on plant community productivity (Wurst et al., 2011; Wurst and Rillig, 2011), but this may change with the successional stage of the plant community (Mammitzsch et al., 2012). These results and a previous study on the effect of diverse soil organisms on a plant community (Ladygina et al., 2010) suggest that direct interactions between functionally dissimilar soil organisms exist, but are usually not strong enough to change their individual impacts on ecosystem functions such as primary productivity. Plant species diversity was reported to interact with soil biota and to influence multiple trophic levels and alter ecosystem functions, such as community productivity and plant resistance toward herbivores and plant invaders (van der Heijden et al., 1998; Ladygina et al., 2010; Scherber et al., 2010; Eisenhauer et al., 2012). Yet intraspecific plant diversity has only recently received increasing attention. For example, the diversity of different cultivars of single plant species, such as Oryzae sativa L. (Zhu et al., 2000), Gossypium hirsutum L. (Yang et al., 2012) and Arabidopsis thaliana L. (Kotowska et al., 2010), was reported to have direct and indirect impacts on the enhancement of plant productivity, the resistance to herbivory and plant pathogens, as well as on predators of herbivores. Compared to single cultivars, high cultivar diversity of host plants was found to lead to increased resistance against plant pathogens and insect pests (Ostfeld and Keesing, 2012; Tooker and Frank, 2012). Furthermore, plant cultivar diversity has been reported to influence the genetic diversity (Paffetti et al., 1996; Dalmastri et al., 1999) and community composition of soil microorganisms (Schweitzer et al., 2008). However, to date it remains untested whether plant cultivar diversity interacts with belowground organisms to affect aboveground organisms and ecosystem functions. If these factors have combined positive additive or even synergistic effects, their interactive effects could be exploited to promote ecosystem functions in sustainable agriculture. Many studies on plant-mediated above–belowground interactions have been conducted in highly controlled environments such as greenhouses and climate chambers (Poveda et al., 2005; Olson et al., 2008; Johnson et al., 2012; Kostenko et al., 2012; Singh et al., 2014). However, to evaluate the importance of results obtained under controlled environmental conditions for natural environments, field studies are necessary (Soler et al., 2012). The challenge of field studies manipulating soil biota is to establish a meaningful control treatment. In contrast to former field studies on above–belowground interactions, in the present study soil organisms were added to field soil rather than reducing them by the application of pesticides, because of their potentially severe and uncontrollable side effects (Smith et al., 2000). For instance, to demonstrate the impact of fungi in communities, fungicides are often applied as a control treatment to suppress mutualistic or pathogenic soil fungi. However, fungicides have also been shown to affect earthworms (Xu et al., 2011), soil protozoan (Ekelund, 1999), bacterial diversity (Johnsen et al., 2001) and community structure of soil oribatid

mites (Al-Assiuty et al., 2014). The resulting feedback from the modified belowground community can then affect plant growth and productivity in natural and agricultural systems. By adding certain groups of soil organisms to background levels of the soil biotic community, we took an approach that has the advantage to avoid artificial side effects by treating the control treatments with pesticides, but the disadvantage of potentially underestimating the impact of the soil biota, since they are also present in low numbers in the control treatments. We set up a field experiment with grass–legume mixtures of high and low cultivar diversity and investigated the impacts of AMF and earthworm additions on the plant community, associated antagonistic organisms (e.g., pathogens and herbivores), and ecosystem functions (plant community productivity, resistance against weeds and aboveground antagonists). We worked in an agricultural setting with typical agriculturally–relevant mixtures of the legume Trifolium pratense L. and the grass Lolium perenne L. and used a number of different cultivars (one or four per species) to test effects of plant cultivar diversity. Intraspecific diversity can refer to a number of hierarchical levels of diversity such as the richness of genotypes or subspecies; in our study higher cultivar diversity was used as a proxy for higher intraspecific diversity. Furthermore, several investigations about perennial grass and legume cultivars have been reporting consistent differences in traits including canopy structure, nutritional quality or resistance to fungal diseases among different cultivars (Gilliland et al., 2002; Smit et al., 2005; Jacob et al., 2010; Swieter et al., 2014). Overall, we hypothesized that (1) the addition of AMF, the addition of earthworms, and higher plant cultivar diversity increase plant productivity, and enhance plant community resistance against weeds, aboveground herbivores and plant pathogens. Further, we hypothesized that (2) AMF, earthworms and plant cultivar diversity may interact directly, but their interactions will not be strong enough to alter their individual impacts on ecosystem functions leading to additive combined effects. 2. Materials and methods 2.1. Experiment design 2.1.1. Plot set-up On the campus of Freie Universität Berlin, Germany, we chose an 11 m
23 m area of a former cornfield with sandy loamy soil. Soil samples were randomly taken and mixed for nutrient analyses. The experimental site contained 1.5 g total N kg1, 19.3 g organic C kg1, 325 mg available P kg1, 200 mg available K kg1, and pH 7.4 (LUFA, Rostock, Germany; see the methods in Table S1). The area was ploughed before it was divided into 64 1 m
1 m plots. We established the plots with a distance of 1 m from each other. Between the plots we left a path for management purposes and to reduce the interference between them. A fully-factorial field experiment with three different categorical treatments was designed. The treatments were: arbuscular mycorrhizal fungi addition (no/yes), earthworm addition (no/yes) and cultivar diversity of the plant community (low/high) each with 8 replicates. The treatments were randomly assigned to the plots. 2.1.2. Plant material Differing in characteristics (Table S2), T. pratense cultivars ‘Milvus’ ‘Larus’ ‘Diplomat’ and ‘Taifun’ as well as L. perenne cultivars ‘Lipresso’ ‘Lacerta’ ‘Licampo’ and ‘Sures’ were used for the experiment. The two species were chosen because they are widely grown as commercial grassland species and because they are colonized by AMF in the field (Oehl et al., 2004); furthermore, information relating to impacts of earthworms and AMF is

X. Guo et al. / Applied Soil Ecology 95 (2015) 1–8

2.1.3. Mycorrhiza treatment For the mycorrhiza treatment, 365  0.5 g sand with commercial arbuscular mycorrhizal fungi (AMF) inoculum (from INOQ GmbH; Schnega, Germany), which consisted of the common arbuscular mycorrhizal species Claroideoglomus etunicatum W.N. Becker and Gerd., Glomus multisubstensum Mukerji, Bhattacharjee and J.P. Tewari and Rhizophagus irregularis (Błaszk., Wubet, Renker and Buscot) C. Walker and A. Schüßler comb. nov., was weighed into plastic bags and added to half of the plots as our AMF addition treatment (N = 32) evenly on top of the soil before sowing the seeds. The same amount of sand without mycorrhiza inoculum was added to the AMF control plots (N = 32), before sowing the plants. The inoculum had a pH value of 7.0 and contained 220 mycorrhizal units (including AM hyphae and spores) per cm3 substrate. Each plot was superficially raked afterward. 2.1.4. Earthworm treatment We ordered Lumbricus terrestris L., the most common anecic earthworm in Europe, from a commercial supplier (Klages Angelköder, Brieskow-Finkenheerd, Germany). Fifteen similarsized adult earthworms were chosen and cleaned with cold tap water in order to remove soil particles. They were weighed after cleaning and subsequently added to each earthworm treatment plots six weeks after sowing (on average 77.4  4.9 g per plot). Holes of a depth of 15 cm were made to add the earthworms to the plot. In control (no earthworm addition) plots, the same sized holes were made and covered, without adding earthworms. To evaluate the activity of earthworms in the plots, the number of earthworm casts (including casts of L. terrestris and other earthworm species) were counted immediately after the aboveground harvest as an estimate of earthworm activity in the plots. 2.2. Measurements 2.2.1. Plant Biomass During the course of the experiment other plant species (which we refer to as weeds in this study) besides the sown plants colonized the plots. To maintain the dominance of T. pratense and L. perenne populations, all weeds in the plots were removed twice (in week 4 and week 12) after sowing. Weed removal had a minor influence on total plot biomass as the biomass of weeds was a very small percentage of total plot biomass (2.7  0.2%; see also Figs. 1 and 2). To assess the abundance of weeds growing within each plot, they were collected during the second weeding campaign (in week 12), dried at 60  C for 72 h and then weighed to determine their biomass. In order to assess the influence of soil organisms and plant cultivar diversity on community productivity, we harvested aboveground plant biomass in October, 2012, in week 24 of the

Aboveground biomass (g.m-2)

1200

L. perenne T. pratense

1000 800 600 400 200 0 Control

AMF

Fig. 1. The effect of AMF addition on aboveground biomass of T. pratense (filled bars) and L. perenne (open bars). Mean values and standard error (SE) are shown for each treatment. n = 32 per treatment.

35 30 Weed biomass (g.m-2)

available for these species (Wurst et al., 2003; Eisenhauer et al., 2009; Sabais et al., 2012). Based on common agricultural practice (Moorby et al., 2009), T. pratense and L. perenne were grown in twospecies mixtures using a ratio of 2:1 (by seed numbers). The experiment included a high cultivar diversity treatment (N = 32), which contained all eight cultivars of both species, and a low cultivar diversity treatment (N = 32), which included one randomly chosen variety from T. pratense and from L. perenne, respectively, per experimental plot. In each plot, we sowed 3.25 g of T. pratense seeds (appr. 1350 seeds) and 1.14 g of L. perenne seeds (appr. 660 seeds) as a mixture. To produce a defined number of seedlings sowing densities were adjusted using seed germination rates in each variety. The seeds were sown in April, 2012. On the path between the experimental plots, we evenly sowed seeds of Festuca rubra L. The path was regularly mowed with a hand mower, the first time eight weeks after sowing.

3

25 20 15 10 5 0 Low cultivar diversity

High cultivar diversity

Fig. 2. The effect of plant cultivar diversity on the biomass of weeds (dry weight in g m2). Mean values and standard error (SE) are shown for each treatment. n = 32 per treatment.

experiment. We placed 20 cm
40 cm frames in the center of every plot, cut all the aboveground shoots at 5 cm above the ground level and sorted them into L. perenne, T. pratense and weeds. After sorting, the biomass was dried at 60  C for 72 h and weighed. To measure belowground biomass and AMF root colonization rate, roots were collected with soil cores at harvest. Four medium sized soil cores (diameter 5 cm, depth 10 cm) were taken at the same positions of each 1 m
1 m plot, placed on the diagonal line in 25 cm distance to each corner. To determine the effectiveness of the AMF addition treatment, the percentage of root colonization by AMF was measured. Roots were cleaned from soil particles and weighed after drying at 60  C for 72 h. To determine the mycorrhizal status of roots (arbuscules, vesicles and hyphae), 30 random sub-samples per fresh root were taken, cleared in 10% KOH and stained with 0.05% trypan blue in lactoglycerol, and checked for AMF root colonization. AMF root colonization rate was quantified with 250
magnification using the magnified intersections method by counting 100 intersections (McGonigle et al., 1990). 2.2.2. Herbivore abundance and diversity To determine the effect of treatments on aboveground herbivores, arthropods were collected with aspirators (also known as pooters) and forceps for eight minutes per plot by two persons in

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week 16 after sowing. All arthropods were preserved in ethanol and later identified to species level, their numbers was counted and the Shannon diversity index (H0 ) of aboveground herbivore was calculated. 2.2.3. Leaf damage by herbivores and pathogens To assess the degree of damage of the leaves by herbivores and fungal pathogens, 50 randomly chosen leaves of T. pratense per plot were scored as damaged (part of leaf missing or powdery mildew developing on the leaf surface) or undamaged (no part of leaf missing or without powdery mildew on leaf surface) in week 16 after sowing. The leaf damage of L. perenne by insects or fungal pathogens was not monitored as their leaves were mostly intact across all experimental plots. 2.3. Statistical analyses Factorial ANOVAs were used to test the effects of all treatments (AMF, earthworms and plant cultivar diversity) and their interactions on AMF root colonization rate and the leaf damage of T. pratense by aboveground herbivores and fungal pathogens. Homogeneity of variances (Levene’s test) and normal distribution of errors (visual residues analysis, Shapiro–Wilk test) were checked to test the qualification of the data for ANOVAs. To achieve normality and homoscedasticity, data on total aboveground biomass and belowground biomass were log transformed, Shannon diversity of herbivores was square transformed, and weed mass was square-root transformed. Where the data still did not qualify, generalized linear models (GLMs) were used instead (Bolker et al., 2009). GLMs with a gamma error distribution and inverse link function were used on data of herbivore abundance and aboveground biomass of T. pratense and L. perenne as they were gamma distributed with a constant coefficient of variation. Further, for the count data (the number of L. terrestris casts) we used GLMs with poisson errors and log link function. All statistical analyses were performed using the software ‘R 2.15.3’ (R Development Core Team, 2012). The results are presented as means  SE. 3. Results 3.1. Plant biomass AMF addition significantly increased total aboveground biomass compared to the control (Fig. 1, Table 1). This was mainly due to a positive effect of AMF on the aboveground biomass of T. pratense which strongly dominated the mixtures (Fig. 1, Table 2). In contrast, AMF addition did not change the aboveground biomass of L. perenne (Fig. 1, Table 2). Total aboveground biomass was not influenced by plant cultivar diversity (low cultivar diversity:

961.6  40.9 g m2, high cultivar diversity: 953.4  51.4 g m2) or earthworm addition (Earthworm addition: 942.5  38.1 g m2, Control: 972.5  53.4 g m2). None of the three treatments had significant effects on belowground biomass (Table 1). A significantly higher biomass of weeds occurred in plant communities with low cultivar diversity compared to high cultivar diversity (Fig. 2, Table 1). 3.2. AMF colonization and earthworm activity The results showed an interaction effect of the AMF and the earthworm addition treatment on AMF root colonization rate (Table 1). AMF addition only increased AMF root colonization rate in plots without earthworm addition (Fig. 3). In the plots with earthworm addition, AMF root colonization rate was not increased with AMF addition (Fig. 3). AMF root colonization rate was not affected by the cultivar diversity of the plant community (Table 1). Earthworm casts were analyzed to evaluate earthworm activity; the results show that the number of L. terrestris casts was significantly higher in the earthworm addition treatment compared to the control treatment (Earthworm addition: 0.97  0.19, Control: 0.53  0.14; Table 2). 3.3. Leaf damage and herbivore diversity The percentage of leaves of T. pratense with fungal pathogen damage was marginally increased in the high compared to the low plant cultivar diversity treatment (low cultivar diversity: 49.9  2.5%, high cultivar diversity: 55.6  1.8%), but not affected by AMF addition or earthworm addition (Table 1). The leaf damage of T. pratense by herbivores showed a similar increasing trend in plant communities with high cultivar diversity from 44.3  1.9% to 48.8  1.6% (Table 1). A total of 701 individuals of 69 different herbivore species were found in the experimental plots. The most abundant herbivore species were Haltica oleracea L. (Coleoptera, 213 individuals), Acyrthosiphon pisum Harris (Aphididae, 55 individuals), Lygus pratensis L. (Hemiptera, 49 individuals), Gastroidea polygoni L. (Coleoptera, 33 individuals) and Rhyparochromus lynceus Fabr. (Heteroptera, 18 individuals). The abundance of herbivores was not influenced by the treatments (Table 2). Compared to the control, Shannon diversity of aboveground herbivores was marginally reduced by the AMF addition treatment, but unaffected by plant cultivar diversity and by the earthworm treatment (Table 1). 4. Discussion We manipulated beneficial soil organisms, AMF and earthworms, and plant cultivar diversity in the field to investigate their

Table 1 Results of three-way ANOVAs showing the effects of arbuscular mycorrhizal fungi (AMF), earthworms (E) and plant cultivar diversity (CD) on the total aboveground plant biomass, belowground biomass, the biomass of weeds, Shannon diversity of aboveground herbivores, AMF root colonization rate, the leaf damage of T. pratense by herbivores and fungal pathogens in the field experiment. n = 8 per treatment. Significant P-values (P < 0.05) are printed in bold and marginally significant P-values (0.05 < P<0.1) are printed in bold and italics.

AMF CD E AMF:CD AMF:E CD:E AMF:CD:E

Aboveground biomass (log)

Belowground biomass (log)

Weeds biomass Shannon's diversity of (sqrt) aboveground herbivores (square)

AMF root Leaf damage of T. colonization rate pratense by herbivores

Leaf damage of T. pratense by fungal pathogen

d.f.

F

P

F

P

F

P

F

P

F

P

F

P

F

P

1,62 1,62 1,62 1,62 1,62 1,62 1,62

4.350 0.086 0.055 0.144 0.114 1.412 0.098

0.042 0.770 0.816 0.706 0.738 0.240 0.756

1.601 0.506 0.056 2.489 1.978 0.375 0.078

0.211 0.480 0.814 0.120 0.165 0.543 0.781

2.603 4.248 0.017 0.858 0.129 1.114 1.949

0.112 0.044 0.898 0.358 0.720 0.296 0.168

3.322 0.021 0.316 0.451 2.202 1.663 1.344

0.074 0.887 0.576 0.505 0.143 0.203 0.251

0.571 0.076 0.383 0.426 8.936 0.143 0.005

0.453 0.784 0.539 0.517 0.004 0.707 0.945

0.210 2.935 0.364 0.098 1.286 0.001 0.308

0.648 0.092 0.549 0.755 0.262 0.981 0.581

0.006 3.180 0.692 0.190 2.150 1.413 0.057

0.937 0.080 0.409 0.665 0.148 0.240 0.813

X. Guo et al. / Applied Soil Ecology 95 (2015) 1–8

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Table 2 Results of GLMs showing the effects of arbuscular mycorrhizal fungi (AMF), earthworms (E) and plant cultivar diversity (CD) on the number of L. terrestris casts, aboveground herbivore abundance, aboveground biomass of T. pratense and aboveground biomass of L. perenne in the field experiment. n = 8 per treatment. Significant P-values (P < 0.05) are printed in bold and marginally significant P-values (0.05 < P < 0.1) are printed in bold and italics.

AMF CD E AMF:CD AMF:E CD:E AMF:CD:E

Number of L. terrestris casts

Herbivore abundance

Aboveground biomass of T. pratense

Aboveground biomass of L. Perenne

d.f.

Dev.

P

Dev.

P

F

P

Dev.

P

1,62 1,62 1,62 1,62 1,62 1,62 1,62

0.000 1.340 4.143 3.124 2.303 0.035 2.175

1.000 0.247 0.042 0.077 0.129 0.851 0.140

0.287 0.235 0.081 0.016 0.063 0.295 0.150

0.400 0.446 0.654 0.840 0.694 0.394 0.543

3.511 0.122 0.027 0.018 0.050 0.671 0.018

0.066 0.728 0.869 0.895 0.824 0.416 0.893

1.375 0.007 0.985 2.306 0.180 2.713 0.475

0.214 0.932 0.292 0.107 0.653 0.081 0.465

AMF root colonization rate

25% b

20%

ab

15%

ab a

10%

5%

0% Control

AMF

E

E+AMF

Fig. 3. Effects of AMF and earthworm addition on AMF root colonization rate in plots inoculated with earthworms (E), arbuscular mycorrhizal fungi (AMF), both earthworms and arbuscular mycorrhizal fungi (E + AMF), and non-inoculated (control). Different letters indicate significant differences between the treatments (P < 0.05, Tukey HSD post-hoc multiple comparison test). Mean values and SE are shown for each treatment. n = 16 per treatment.

main and interactive effects on plant communities, higher trophic levels, and ecosystem functions. As predicted, we found mainly independent effects of the soil organisms and cultivar diversity on the functions of the plant community. Below we first discuss the effects of AMF, earthworm and plant cultivar diversity on plant performance in detail, and then how they may affect higher trophic levels. Consistent with a number of earlier studies (van der Heijden et al., 2003; Smith and Smith, 2011; Mammitzsch et al., 2012), our study documents that total aboveground biomass of plant communities was increased in response to AMF addition. The effect was relatively moderate and we attribute this to the high P level of the background soil might which have reduced the efficacy of AMF on plant productivity. A recent review by Smith and Smith (2011) summarized the contributions of arbuscular mycorrhizal symbioses to plant nutrient uptake processes. The root colonization of AMF and the AMF extraradical hyphae can not only promote the absorption of P, but also deliver N to the plant. This facilitation of the absorption of soil nutrients affects plant height, root–shoot ratio and reproductive structures including flowers, fruits and seeds (Shumway and Koide, 1995; Koide, 2010). Still, some neutral or negative results of AMF on plant growth have also been found in previous studies (Jankong and Visoottiviseth, 2008), which seems contradictory to our findings. We speculate that these inconsistent effects of AMF on plant growth depend on plant species (Jankong and Visoottiviseth, 2008), AMF species (Uibopuu et al., 2009; Zaller et al., 2011a), and soil characteristics (Johnson et al., 1997). In our study, AMF addition tended to increase the biomass of T. pratense, while we did not detect effects of AMF on L. perenne. This

indicates that AMF promoted the growth of the mycotrophic plants (van der Heijden et al., 2003), since Haynes (1980) suggested that T. pratense may be more mycotrophic than L. perenne. In previous studies AMF were found to have positive effects on T. pratense aboveground biomass (Eisenhauer et al., 2009; Zaller et al., 2011b; Sabais et al., 2012). However, studies reported inconsistent results for L. perenne. Eisenhauer et al. (2009) found that the biomass of T. pratense was increased, whereas the biomass of L. perenne was decreased in the presence of AMF. In our experiment L. perenne thrived until week 15 (personal observation), but at the end of the experiment, T. pratense was the dominant species of the plant communities. The reduced mowing during summer could explain the unbalanced growth of T. pratense and L. perenne since mowing is often more detrimental to legumes than grasses (Cosgrove and Brougham, 1985). The severe competition for limited resources (e.g., light, water, nutrients) between legume and grass or within the grass population, could also be an explanation for the reduced growth and the mortality of the grass in the experiment (Zhang et al., 2010). It is worth to mention that the present study is one of the few studies (Li et al., 2012) that added AMF to a background soil microbial community and led to significantly increased plant productivity. In most field studies fungicides or herbicides were used for the control treatments, which proved to be effective (Brown and Gange, 1989; Hartnett and Wilson, 1999; Maron et al., 2010, 2013). However, pesticides can have strong non-target effects. Mounting evidence suggests that the application of fungicides or herbicides can impact non-target soil organisms (Yang et al., 2011) and alter the community structure of soil organisms (Al-Assiuty et al., 2014). Thus, it seems reasonable to improve yields by adding mycorrhiza to a natural background community, supporting a possible application of plant mutualists in organic agriculture. While a positive AMF treatment effect on total aboveground plant biomass was detected, the interactive effect of the AMF and the earthworm addition treatments on AMF root colonization did not emerge in the final biomass measure. This result is consistent with our second hypothesis, since we expected that interactions between the soil biota are not strong enough to have consequences for ecosystem functions such as plant productivity (Ladygina et al., 2010). AMF root colonization increased by AMF addition alone. However, in combination with earthworms, the AMF treatment was less effective. This is in line with previous studies (Bonkowski et al., 2000), where the reproduction of AMF and the colonization rate was decreased due to the destruction of hyphal connections by added earthworms through their feeding and burrowing activities. The decreased AMF root colonization rate in the AMF and earthworm interaction plots in our study could be explained by a disturbance by earthworms (Bonkowski et al., 2000). On the other hand, the slightly increased AMF root colonization rate in the plots where earthworms, but no AMF were added might be

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attributed to earthworms as dispersal vectors (Gormsen et al., 2004). The contrasting effects might depend on AMF abundance. Earthworms could have negative effects at high AMF abundance (in the addition treatment), but positive effects at low AMF abundance. Moreover, AMF root colonization rate might also depend on AMF species, because these differ in number and size of spores and likely in suitability for dispersal by earthworms (Pattinson et al., 1997; Gormsen et al., 2004). Inconsistent with our first hypothesis, earthworms were found to have no influence on plant productivity. The earthworm species we used is an anecic worm, L. terrestris, which usually constructs vertical burrows, occurs in deeper soil layers (1.5 m or more), and visits the surface to feed and mate (Canti, 2003). We did not build fences around the plots to prevent the escape of earthworms, as fences only prevent their escape for a short time (Eisenhauer et al., 2009; Eisenhauer pers. comm.). The number of casts, measured as a monitoring method to evaluate earthworm numbers, was significantly higher in the earthworm addition treatments compared with the control. However, the effect of earthworms was apparently not strong enough to create significant differences in plant productivity or in the performance of aboveground herbivores. Inconsistent with recent studies about the positive relationship of plant productivity and plant cultivar diversity (Hughes et al., 2008; Kotowska et al., 2010), no significant effect of plant cultivar diversity on plant biomass was detected in our study. van Ruijven and Berendse (2005) found that positive species richness– productivity relationships strengthened over time in perennial plant communities through increased nutrient use efficiency, both spatially and temporally. Such time-related mechanisms may explain the neutral effect of plant cultivar diversity here, since our study lasted only one growing season. However, our data show that the establishment of weeds was significantly higher in the plant communities with low cultivar diversity compared with plant communities with high cultivar diversity. The weeds originated from the soil seed bank and from new invasions via seed rain. Communities consisting of many plant cultivars may construct a higher spatial complexity or use nutrients more efficiently, preemptying recruitment niches for weeds (Tilman et al., 1999). This is also in line with the finding that the resistance of plant communities against plant invaders is enhanced by plant species diversity through impacts on community stability (Eisenhauer et al., 2008; Scherber et al., 2010). As for the effects of plant cultivar diversity on higher trophic levels, we found high plant cultivar diversity tended to lead to more damaged T. pratense plants by both herbivores and fungal pathogens compared to low plant cultivar diversity. This is inconsistent with the traditional view of a positive effect of plant cultivar diversity on disease suppression (Zhu et al., 2000). However, resistance effects can depend on the chosen cultivars, as Wolfe (1985) illustrated. Kotowska et al. (2010) also found that leaf herbivory was more severe in populations with high plant cultivar diversity and argued that this was due to their high productivity. In our study, we did not find a difference in plant productivity between communities of different cultivar diversity, making this mechanism unlikely here. Consistent with a recent review by Joern and Laws on the mechanisms underlying arthropod diversity (Joern and Laws, 2013), which suggested arthropod diversity to be affected by above and belowground interactions, we also observed that the diversity of aboveground herbivores tended to decrease with addition of AMF. This could be due to changes in plant defensive chemicals induced by AMF root colonization (Koricheva et al., 2009). There is accumulating knowledge suggesting AMF as a critical factor for plant resistance to aboveground herbivory (Koricheva et al., 2009; Kempel et al., 2010). Priming of

jasmonate-regulated defense mechanisms of plants could be an explanation for mycorrhiza-induced resistance (Pozo and AzconAguilar, 2007; Kempel et al., 2010). Indirect defenses of plants against aboveground herbivores may also be induced by AMF through altering volatile profiles to attract parasitoids (Guerrieri et al., 2004). Besides, AMF was also reported to influence the abundance of predators through altering the abundance of particular groups of aboveground herbivores (Ueda et al., 2013) and hence shape higher trophic level diversity. 5. Conclusion We found independent effects of AMF and genetic diversity on plant community and herbivore performance. Our results indicate that AMF addition may have a potential for forage yield enhancement and control of herbivores in the field, and that planting more cultivars could have additional beneficial impacts with regard to weed control. Overall, the application of AMF and the use of a higher number of cultivars in legume grass mixtures are management options that could be easily applied in sustainable agriculture. In general, we suggest that future research should pay more attention to plant intraspecific diversity and beneficial soil organisms and their combined impacts on ecosystem functions. Acknowledgements We wish to thank Monika Fünning for help with the mycorrhizal analysis, and Jenny Speier, Dr. Ilja Sonnemann, Dr. Caspar Schöning, Jörg Schmidt and students from the Ecology and Zoology courses of the summer semester, 2012 at Free University of Berlin for help with field work. We thank Dr. Ulf Feuerstein (Euro Grass Breeding GmbH & Co., KG) for providing seeds of T. pratense and L. perenne. We acknowledge the support of Xiaohui Guo by the German Academic Exchange Service (DAAD). This work was partly funded by the Dahlem Center of Plant Sciences (DCPS). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j. apsoil.2015.05.010. References Al-Assiuty, A.-N.I.M., Khalil, M.A., Ismail, A.-W.A., van Straalen, N.M., Ageba, M.F., 2014. Effects of fungicides and biofungicides on population density and community structure of soil oribatid mites. Sci. Total Environ. 466–467, 412–420. Bolker, B.M., Brooks, M.E., Clark, C.J., Geange, S.W., Poulsen, J.R., Stevens, M.H.H., White, J.-S.S., 2009. Generalized linear mixed models, a practical guide for ecology and evolution. Trends Ecol. Evo. 24, 127–135. Bonkowski, M., Griffiths, B.S., Ritz, K., 2000. Food preferences of earthworms for soil fungi. Pedobiologia 44, 666–676. Brown, V.K., Gange, A.C., 1989. Differential effects of above- and below-ground insect herbivory during early plant succession. Oikos 54, 67–76. Canti, M.G., 2003. Earthworm activity and archaeological stratigraphy: a review of products and processes. J. Archaeolog. Sci. 30, 135–148. Cosgrove, G.P., Brougham, R.W., 1985. Grazing management influences on seasonality and performance of ryegrass and red clover in a mixture. Proc. N. Z. Grassland Assoc. 46, 71–76. Dalmastri, C., Chiarini, L., Cantale, C., Bevivino, A., Tabacchioni, S., 1999. Soil type and maize cultivar affect the genetic diversity of maize root-associated Burkholderia cepacia populations. Microb. Ecol. 38, 273–284. 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 Biol. Biochem. 41, 561–567. Eisenhauer, N., Milcu, A., Sabais, A.C.W., Scheu, S., 2008. Animal ecosystem engineers modulate the diversity–invasibility relationship. PLoS One 3. Eisenhauer, N., Reich, P.B., Isbell, F., 2012. Decomposer diversity and identity influence plant diversity effects on ecosystem functioning. Ecology 93, 2227–2240.

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