*2.4. One- and Two-Year Follow-Up Studies on PA-Transfer to Acceptor-Plants on Fields Previously Used for L. squarrosa Cultivation*

These experiments were carried out on 50–400 m2 plots in Zappendorf (Black loess No. 2/clay soil) after harvesting *L. squarrosa* crops.

The experiments were performed growing winter wheat and spring barley as subsequent crops in the first year after cultivation. For the second year, the plots were swapped, and spring barley followed winter wheat and vice versa. Controls using the same crop varieties were grown on a nearby comparable location, with the difference being that no PA-plants (e.g., *L. squarrosa*) were grown on these plots previously. The plots were sampled three times per season: (a) soil right before the start of the cultivation, (b) soil, roots and plantlets at the two-node stage and (c) soil, roots, above ground plant parts and grain/seeds/fruits of the cultivated crop. The subsequent crops were selected according to their importance for local agriculture. A total of three seasons were covered.

In addition, a more diverse range of follow-up crops of common regional varieties from other plant families were also included in these experiments, however, not under the strict procedure of three independent replicates. These other crops included *Brassica napus*, *Pisum sativum* and *Coriandrum sativum*. These experiments were conducted nearby on similar soil and climate conditions (diluvial site, soil value index 54, Halle/Saale, Martin Luther University, Halle-Wittenberg, Germany).

#### *2.5. Investigations of Distance-Related Effects on PA-Transfer*

To investigate the effects of distance on PA-transfer to non-PA-plants, field strips right next to the *L. squarrosa* cultivation were prepared. The location in 2019 was Kühnfeld (Martin Luther University, Halle-Wittenberg, Germany, soil value index 54), and in 2020 it was Zappendorf (soil value index 85, Saxony-Anhalt, Germany). These strips were planted with *Lolium multiflorum*. Plant and soil samples were taken at a distance of 50 cm, 200 cm

and 400 cm away from the field of *L. squarrosa* cutivation. In addition, to monitor and minimize the PA-carry-over via soil, four Kick-Brauckmann vessels with a diameter of 22 cm and a height of 22 cm (0.038 m2 soil) were buried up to the rim in these strips. Two vessels each were placed at a 50 cm and two vessels at 200 cm distance to the *L. squarrosa* cultivation. All four vessels per season also contained *L. multiflorum* as plants. To illustrate this experiment, we added a picture to the Supplementary Materials (Figure S1).

## *2.6. Controls and Pot Experiments*

All experiments described above were accompanied through seasons with control experiments using plots that had no history of PA-plant cultivation (in particular, *L. squarrosa*). On these control patches *Brassica napus*, *Pisum sativum*, *Coriandrum sativum*, *T. aestivum*, *H. vulgare* and *L. multiflorum* were cultivated.

In addition to these field-controls, a one-season model experiment was conducted, using 20 Kick-Brauckmann pots (including replicates) to grow *T. aestivum* and *H. vulgare* separately, under controlled conditions. At the start of the experiment, the pots were individually filled using two kinds of soil: (a) commercial potting soil, and (b) top soil from a harvested *L. squarrosa* field. Shoot samples of both grain crops from both treatments were collected and analyzed for PAs at the two-node stage.

#### *2.7. Sample Collection*

In all experiments, samples of soil at the different growing stages (sowing stage, vegetation stage, two-node stage, and harvesting stage) were taken in the soil layer of the main root horizon (0–30 cm depth) using the boring stick method (8–14 impacts per variant depending on the crop acreage). All soil samples were frozen in plastic zip-lock bags and stored at −20 ◦C, then lyophilized and stored under dry and dark conditions until analysis. On the other hand, whole plants (including roots) at the vegetation stage (two-node stage) and crops at the stage of harvest were manually sampled using a spade (8–10 stakes) to collect about 20–50 plants per variant, depending on the species. In addition, individual plants of weeds growing in the plots were sampled as well. The plant material of the vegetation stage (two-node stage) and weeds were cleaned from adherent soil and separated into roots and shoot material. Harvest-ready plants were separated into roots, straw, and crop product (seeds, fruits); all different plant parts were collected in individual paper bags, air dried and stored at room temperature until analysis. Right before analysis all individual samples of one plot/field were well mixed resulting in an individual cross-section sample per data point, hence each value represents a biological average.
