2.1.2 Plant-soil interaction experiment
We conducted the plant-soil interaction experiment in a greenhouse
located at the Botanical Garden Leipzig, Germany, in July 2017. We
recorded an average temperature of 23.5°C and an average relative
humidity of 58.6% for the time of the experiment in the greenhouse. We
used PVC tube microcosms (height 20 cm, diameter 10 cm, bottom closed
with 250 µm mesh) filled with 1.6 L inoculated substrate and watered
each microcosm twice. We prepared the inoculated substrate by mixing
autoclaved (twice at 134°C for 20 min) 50:50 sand-peat (Floradur B Pot
Clay Medium, Floragard, Oldenburg, Germany) background substrate with
liquid field soil inoculum 3 weeks prior to the establishment of the
experiment. In June 2017 (i.e., ~ 7 years after
the establishment of the experiment), we collected field soil from plant
communities established in 2010 as part of the Trait-Based Experiment
(Ebeling et al., 2014). We collected and pooled six soil cores (2 cm x
10 cm) from each plant community accounting for within-plot
heterogeneity. We sieved each field soil through a 4 mm mesh and
subsequently dissolved 100 g field soil in 1 L demineralized water. We
then added the liquid soil inoculum to our autoclaved background
substrate (10 mL liquid inoculum per 1 kg background substrate) and
stored each mixture in closed-lid plastic boxes at room temperature for
3 weeks. Each substrate-inoculum mixture was thoroughly mixed three
times per week and stored with an open lid for 1 h once per week. We
cleaned all used instruments, i.e., sieves, boxes, beakers,
mixer, before and after each step with distilled water and 70% ethanol
to minimize cross contamination.
We established the following inoculated substrates (hereafter, soil
legacy levels): (1) monocultures of each plant species, (2) the three
possible two-species mixtures, and (3) the three-species mixture
(Appendix Table A2 ). Each soil legacy level represents the plot
from the Trait-Based Experiment, we sampled the soil from. We
transplanted four similarly developed seedlings per microcosm. Seedlings
of plant species were only planted into soil legacy levels that also
contained the respective species in the field experiment. This set-up
resulted in twelve unique soil legacy level-planted species
combinations. Each soil legacy level-planted species combination was
replicated ten times (total number of microcosms: 120). All microcosms
were randomly placed on tables in the greenhouse and covered with net
cages to prevent unwanted herbivory. We watered all microcosms three
times per week and randomized the position on the tables every 7 days.
We fertilized all microcosms with 250 mL Hoagland solution after 5 weeks
to counteract any loss of nutrients and ensure optimal growth. After 7
weeks of growth, we harvested five microcosms per soil legacy
level-planted species combination (see below). The next day, we infested
two randomly selected plants per microcosms of the remaining microcosms
with three 2nd instar Spodoptera exigua larvae
each (see above). We covered and closed each plant just above the soil
with an organza net to ensure that the larvae could not escape. After 7
days of herbivory, we harvested the remaining microcosms (see below).