Experimental setup

We conducted a full-factorial mesocosm experiment manipulating the three factors Earthworms (two levels: earthworm addition, +EW vs. no earthworms, −EW), AMF (two levels: AMF inoculation, +AMF vs. no AMF inoculation, −AMF) and Herbicide application (two levels: Roundup application, +RU vs. no Roundup application, −RU; more details on the individual treatments below). The experiment was conducted in December 2011 in a greenhouse of the University of Natural Resources and Life Sciences Vienna (BOKU), Austria. During the course of the experiment mean daytime air temperature inside the greenhouse was 20.1 ± 3.2°C at a mean relative humidity of 55.2 ± 5.4%; mean nighttime air temperature was 15.3 ± 2.6°C at a mean relative humidity of 68.3 ± 6.7%; to ensure optimal light conditions, three 1000-W Radium lamps (type HRI-T100W/D, WE-EF Leuchten, Bispingen, Germany) were installed in 1.5 m distance above the experimental units (14 hours light, 10 hours night).

We used 24 plastic pots (volume: 20 l, diameter: 31 cm, height: 30 cm; further called mesocosms) which were lined out with two layers of garden fleece at the bottom and extended at the upper rim with a 10 cm high barrier of transparent plastic to prevent earthworms from escaping; the fleece and barriers were also installed in mesocosms containing no earthworms to create similar microclimatic conditions among treatments.

Treatments

AMF treatments were prepared in March 2011 by first filling the mesocosms with 12 l steam-sterilized (3 hours at 100°C) field soil (Haplic Chernozem, silt loam) mixed with quartz sand (grain size 1.4–2.2 mm) in a ratio of 40:60 vol/vol. Characteristics of this soil mixture: C org = 24.1 g kg−1, N tot = 0.98 g kg−1, K = 111.2 mg kg−1, P = 58.42 mg kg−1, pH = 7.63. The upper 6 l of the +AMF treatments were filled with the same substrate mixture and amended with 25 g l−1 inoculum of Glomus mosseae (T.H. Nicolson & Gerd.; synonymously Funneliformis mosseae (T.H. Nicolson & Gerd.) C. Walker & A. Schüßler) obtained from a commercial supplier (Symbio-m Ltd., Lanskroun, Czech Republic). The −AMF controls were filled with the same amount of steam-sterilized and thus inactive AMF inoculum. We successfully used this substrate mixture in other experiments involving the same earthworm and AMF taxa45,73,74. Then 400 ml of microbial wash was added to each mesocosm to inoculate the steam-sterilized soil with microorganisms present in field soil75. This microbial wash contained 300 ml soil suspension (3500 g fresh soil dispensed in 7200 ml distilled H 2 O filtered through a sieve-cascade from 2000 μm to 25 μm mesh size) and 100 ml AMF suspension (466 g AMF-inoculum dispensed in 2400 ml distilled H 2 O filtered through the same sieve-cascade).

In April 2011 mesocosms were planted with white clover (Trifolium repens L.). Therefore, T. repens was first propagated from seeds in steam-sterilized potting soil, then 18 seedlings (average height about 10 mm, seedlings consisted of two cotyledons and two real leaves) were transplanted into each mesocosm in a regular hexagonal pattern with an equidistance to each other of 5 cm (240 seedlings m−2). This seed material is commonly used by farmers in mixtures for green manuring and was obtained from the BOKU Department of Crop Sciences. No fertilizers were applied during the course of the experiment.

In December 2011 we added 4 adult individuals of vertically burrowing Lumbricus terrestris L. to the +EW mesocosms (16.6 ± 2.1 g mesocosm−1, equivalent to 220.6 g m−2). Earthworm densities were roughly oriented on the average earthworm biomass in temperate grasslands ranging between 52–305 g m−2 where 50–75% of the biomass consists of anecic species1. Earthworms were purchased from a local fishing bait shop. To acquaint earthworms with experimental conditions, we cultivated them in plastic boxes (climate chamber at 15°C) filled with steam-sterilized field soil and ground oat flakes as food before they were introduced to the mesocosms. Before earthworms were randomly added to the +EW mesocosms, they were washed free of attached soil, dried off on filter paper and weighed. All earthworms buried themselves in the soil within a few minutes. The mesocosms were randomly placed on greenhouse tables and randomly repositioned every second week to avoid treatment interactions with potential microclimatic gradients inside the greenhouse. No additional food was provided for earthworms in the mesocosms as there was abundant dead organic material on the soil surface. An automatic irrigation system added on average 0.5 l tap water day−1 to each mesocosm.

Herbicide was applied five days after earthworm insertion on half of the mesocosms comprising all treatment combinations. We used Roundup Speed (Monsanto Inc., St. Louis, Missouri, USA), a systemic, broad-spectrum herbicide containing 7.2 g l−1 of the active ingredient glyphosate. This herbicide is recommended for use in home and garden areas and was obtained from a garden center in Vienna. Following the instructions for use, we applied the herbicide directly onto the plants from the original bottle with the attached fine mist spray nozzle. We applied the herbicide once on day 5 after earthworm inserting at 4 p.m. without direct sunlight at an air temperature of 25°C. As recommended in the instruction text we sprayed the herbicide so that the plant surface was homogeneously covered and shiny from the herbicide film. This application needed 14 squirts of Roundup Speed with the spray nozzle mesocosm−1 amounting to 177.48 ml m−2.

These treatments were replicated three times in a full-factorial design: two earthworm treatments × two AMF treatments × two RU treatments × three replicates equals totally 24 mesocosms.

Measurements and analyses

Earthworm activity was indirectly assessed during nighttime by 30 toothpicks mesocosm−1 that were vertically inserted (0.5 cm deep) in a consistent pattern. In the following morning the number of toothpicks differing from the original vertical position was considered as a measure of earthworm activity because earthworms crawl over the soil surface when searching for food. Knocked over toothpicks were counted as 1 and inclined toothpicks were counted as 0.5. As another measure of earthworm activity we additionally measured the number of freshly produced casts on the soil surface76. Both activity measurements were done parallel three times before and six times after herbicide application.

Water infiltration and Roundup leaching was measured seven days after the Roundup application by pouring 3 l of distilled water on top of the mesocosms simulating a rain shower of about 40 l m−2 (see also45). The time from pouring the water onto the mesocosms until the last water pool disappeared from the soil surface was recorded and used to calculate the water infiltration rate in l m−2 s−1. We collected 250 ml of the leachate from the saucers at bottom of the mesocosms immediately stored it in a freezer at −20°C before it was analysed for glyphosate and its main metabolite aminomethylphosphonic acid (AMPA) in the laboratories of the BOKU Department of Forest and Soil Sciences using a HPLC-MS/MS method77,78.

Harvest of the mesocosms started 14 days after herbicide application by cutting the remaining or untreated plants at the soil surface to obtain aboveground plant biomass production. Afterwards, soil was removed from the mesocosms in three separate layers 0–5 cm, 5–10 cm and 10–30 cm. Earthworms present in these soil layers were carefully washed free of soil, placed on moist filter paper, counted, weighed and released to the BOKU garden. Roots present in these soil layers were washed free of attached soil particles under a jet of tap water over a 1-mm sieve and sorted out. Dry mass of shoots and roots was determined after 48 hours oven-drying at 55°C. A portion of roots per layer was collected, cleared with boiling KOH for four minutes and stained for one minute with black Sheaffer ink79. Percentage of root length colonized by AMF considering arbuscules and vesicles (i.e. mycorrhization rate) was determined using the grid-line method by counting at least 100 intersections per sample80.

Lipid extraction from pot soil was carried out by extracting 3–4 g of soil (wet weight) with Bligh & Dyer solvent (chloroform: methanol: citrate buffer as 1:2:0.8, pH 4)81. The obtained lipids were fractionated into neutral lipid (NLFAs), glycolipid and phospholipid fatty acids (PLFAs) on a silica column (HF Bond Elut - SI, Varian Inc.) by elution with chloroform, acetone and methanol, respectively. NLFAs and PLFAs were subjected to an alkaline methanolysis in 0.2 M methanolic KOH and the fatty acid methyl esters (FAMEs) were extracted with hexane-chloroform. Samples were dissolved in isooctane and stored at −20°C until analysis. FAMEs were analyzed using an Agilent 7890A gas chromatograph equipped with a flame ionization detector (GC-FID) using a HP Ultra 2 capillary column (25 m × 0.2 mm i.d., film thickness 0.33 μm). The oven temperature program started with 170°C and increased by 28°C min−1 to 288°C, followed by 60°C min−1 to 310°C. FAMEs were identified with the Sherlock Pattern Recognition Software (MIDI®) by comparing retention times to a standard mixture and quantifying based on the internal standard methylnondecanoate (19:0). To verify correct identification (chain length and saturation) a range of samples was additionally analyzed with the Agilent 7890A coupled to a Mass Selective Detector (Agilent 7000 Triplequadrupole) equipped with a HP5MS capillary column (30 m × 0.25 mm i.d., film thickness 0.25 μm), operated in splitless mode with helium as carrier gas. Oven temperature program started with 40°C and increased by 46°C min−1 to 200°C, followed by 5°C min−1 to 238°C, 120°C min−1 to 300°C. A mass range of 40–400 m/z was monitored in Scan mode. The fatty acid 16:1ω5 was applied as general marker for AMF, predominantly Glomales, with the PLFA fraction representing hyphal membranes and the NLFA fraction storage lipids58,59.

Statistical analyses

All variables were tested for homogeneity of variances and normality using the tests after Levene and Kolmogorow-Smirnow, respectively. Data on PLFAs and NLFAs were log-transformed to meet the assumptions for parametric tests. We conducted a three way analysis of variance (ANOVA) to test the effects of Earthworms, AMF and Roundup on PLFAs, NLFAs, water infiltration and Roundup leaching. Here analyses for treatment effects on PLFAs and NLFAs were conducted for each soil layer separately. Earthworm activity (moved toothpicks and surface castings) during the course of the experiment was analyzed conducting a repeated measures ANOVA with Roundup and AMF as factors by only including data from mesocosms containing earthworms. Root AMF colonisation was analyzed for each soil layer using a two-way ANOVA considering the factors Earthworms and Roundup; mesocosms without AMF inoculation were not included. We also performed Pearson correlations between earthworm biomass and earthworm activity (moved toothpicks and surface castings) and between earthworm activity and mean air temperature or mean relative humidity. All statistical tests were performed in PASW Statistics 18 (vers. 18.0.0, IBM Corp., Armonk, New York, USA). Values given throughout the text are means ± SE.