Trifolium

Over a 2‐year period, from 1 April 2011 to 31 March 2013, the beef cattle and sheep systems operated under the same management guidelines on all three farmlets to enable productivity to be measured on the existing permanent grassland. From 1 April 2013, two of the farmlets entered a ‘transition’ phase, where they moved progressively towards the following treatments:

Blue farmlet, legumes. Sward was improved by reseeding with long‐term grass (perennial ryegrass; Lolium perenne L.) and legume (white clover; Trifolium repens L.) mixtures. Clover‐based systems can replace (Elgersma & Hassink, 1997) up to 150 kg nitrogen (N) ha⁻¹ of industrially‐produced N, which is a major cost for any grassland farm. An opportunity to reduce costs might lie in farmers' becoming more reliant on N that is biologically fixed by legumes. On this farmlet, we enhanced the current small proportion of clover by reseeding, but we do not rely on clover alone to supply the N. In addition, a maximum of 40 kg N ha⁻¹ of fertilizer is permitted in spring in a particularly cold, slow growing season, and organic manures are also used. However, no inorganic N fertilizer was required in the period 2013–2015 on the reseeded fields.

Red farmlet, planned reseeding. Sward improvement through planned and regular reseeding about every 4 years. Currently, new varieties developed by plant breeders for traits associated with improved animal performance (e.g. grasses with enhanced sugar content; polymeric‐oligo‐fructans) or environmental resilience (e.g. deep‐rooting grasses) have been sown on this farmlet. In the future, this treatment will provide opportunities to introduce other new and innovative plant variety traits (e.g. grasses with large lipid content, clover with large polyphenol oxidase content, clover with small protein content) developed to improve the efficiency of nutrient use by the animals and greater environmental resilience that can be incorporated easily.

The progressive reseeding was achieved over 3 years and was intended to allow the systems to continue to run and feed the cattle and sheep. The Blue and Red treatments will be compared with the following Green control.

Green farmlet, permanent pasture. Sward improvement of the existing grassland through the use of artificial fertilizers, and with monitoring of the proportion of original sown species (predominantly perennial ryegrass). Both the Red and Green farmlets are fertilized with nitrogenous fertilizer (see below).

Individual catchments within the Red and Blue farmlets were sprayed with glyphosate to kill the existing grass, followed by ploughing and cultivation, and then reseeded in July to August 2013, 2014 and 2015 (0.40, 0.34 and 0.26 of the farmlets were reseeded in each year, respectively). (See Figure S3 in Supporting Information for a map of the re‐seeding schedule).

White clover flowers at different stages of maturity as well as leaves from Lotus corniculatus plants were decolorized in absolute ethanol for 3 h and stained for the presence of proanthocyanidins and flavan-3-ols using 0.01% (w/v) 4-dimethylaminocinnamaldehyde (DMACA) in absolute ethanol containing 0.8% w/v hydrochloric acid [5 (link)]. Flowers were stained for 20 min and the remaining organs were stained for 2 h before being transferred to 100% ethanol. After DMACA staining, samples were vacuum-infiltrated for 1 min with fixative (6% w/v glutaraldehyde, 4% w/v paraformaldehyde in 50 mM sodium phosphate buffer, pH 7.4) in 1.5 mL microcentrifuge tubes and were incubated for 2 h at 4°C. Samples were then washed three times for 5 min in 50 mM sodium phosphate buffer, pH 7.4.

Heavy metal content in the Bolesław, Bukowno, and Saturn upper layer of metalliferous and reference soil as well as in plant tissues were measured in five sample replicates. To determine total Zn, Pb, and Cd concentrations in soil samples, 0.2 g of soil matrix, previously passed through a 1 mm sieve, was extracted for 15 min with 70% nitric acid (4.5 mL) and hydrofluoric acid (1.5 mL) at 180 °C (Method 3052, US EPA 1996) in a Mars 6 microwave oven (CEM Corporation, Matthews, NC, USA), whereas to measure the metal concentrations in white clover roots and leaves, tissues were treated with concentrated nitric acid (2.5 mL) and 250 µL hydrogen peroxide solution (30% w/w in H₂O) diluted in 2.250 mL double deionized water at 150 °C (Method Plant Material, US EPA 1996) in the above mentioned microwave oven [47 (link)]. Metal analysis of these solutions was carried out by electrothermal atomic absorption spectrometry (AAS), using the Thermo iCE 3400 instrument with Zeeman correction (Thermo Electron Manufacturing Ltd., Cambridge, UK). Quality assurance procedures including the analysis of reagent blanks and standard reference material, i.e., Montana II soil (NIST^® 2711a, Sigma-Aldrich) and tomato leaves (NIST^® 1573a, Sigma-Aldrich), for soil and plant samples, respectively, were performed in parallel. The recovery of Zn, Pb, and Cd was 90%–95%. Data were expressed as mean ± SD. They were analyzed by one-way analysis of variance (ANOVA) followed by the Duncan’s multiple range test. Differences at p < 0.05 were considered as statistically significant.

We obtained land cover data for our study areas from the Cropland Data Layer for 2006–2017 [26 ]. The Cropland Data Layer combines National Land Cover Database [31 ] classifications with information on specific crop types at a 30-m spatial resolution and is ground-truthed with overall accuracy ranging from 76.5% - 92.4% for agricultural and 78% - 86.4% for nonagricultural classes [26 , 31 ]. We defined “grassland” as classes comprised of clover/wildflowers, switchgrass, grass/pasture, alfalfa, or other hay/non alfalfa in the Cropland Data Layer. We calculated the percent grassland cover at two scales around survey locations (local scale: 250-m buffer around survey points [~20 ha], landscape scale: 2,500-m buffer around survey routes [~21563 ha]). These scales represent local- and landscape-scale habitat features with respect to our focal species’ home ranges [29 ] and are similar to scales used in other multi-scale habitat analyses [32 (link)–34 ]. Because these data were not available for the period 2001–2005, we used linear regression to develop a predictive model of grassland cover at both scales around points from existing data and extrapolated values for years with missing data.
Among all years, focal area sites had a mean of 54.0% grassland cover at the local (250-m buffer) scale and a mean of 51.9% grassland cover at the landscape (2,500-m buffer) scale (95% of all stops/routes between 5.4% - 92.2% and 32.7% - 73.7%, respectively). Among all years, paired area sites had a mean of 50.1% grassland cover at the local scale and a mean of 50.0% grassland cover at the landscape scale (95% of all stops/routes between 3.6% - 92.5% and 29.4% - 72.0%, respectively). Percent grassland was generally similar among focal and paired site pairings (S1 Fig).

The seeds of walnut variety Liaohe No. 1 were provided by the Walnut Technology Extension Center of Baokang (Hubei, China). The seeds were surface disinfected with 75% ethanol for 8 min, washed with distilled water, soaked in distilled water for a week, and then sown in autoclaved sands for the germination in an incubator at 28°C/20°C (day and night temperature) and 80% relative humidity. The seedlings with four leaves were transplanted into plastic pots (2.4 L) pre-filled with hydrochloric acid-washing sand to reduce the interference of P in the substrate.
Based on the results of Huang et al. (2020) (link), we selected the D. spurca strain as the fungal material because it showed relatively good effects on improving walnut growth. The D. spurca strain was isolated from the rhizosphere of tomato in Shouguang (Shandong, China). After the morphological identification, the strain of D. spurca was trapped by white clover under potted conditions. After approximately 11 weeks, the D. spurca-colonized roots and potted substrates were collected as the mycorrhizal fungal inoculums and stored at 4°C after natural air-drying. Before use, the spore density was 15 spores/g. The inoculation of D. spurca was carried out at the time of transplanting. A total of 120 g of mycorrhizal fungal inoculums was applied to the designed pot as the inoculation treatment. The equal amount of autoclaved mycorrhizal inoculums was applied to the uninoculated pot as the uninoculation treatment, followed by 2 mL of filtered (25 μm) solution with equal amount of mycorrhizal inoculums added to maintain the consistency of the microbiota except for the target strain.
Seven days after the inoculation, P treatments were applied. P concentrations in the potted substrate were achieved by controlling the KH₂PO₄ level in Hoagland nutrient solutions (pH 7.0), where 1 μmol/L and 100 μmol/L KH₂PO₄ was defined as the low P and moderate P level (Li et al., 2010 ). To reduce the difference in K levels of nutrient solutions among treatments, additional KNO₃ was added to the P-deficient treatment to ensure the consistent K level. The nutrient solution was used at an intensity of 150 mL per pot at the three-day intervals.
All treated seedlings were placed in a greenhouse from May 4, 2020 to August 4, 2020, where environmental conditions were described in detail by Zou et al. (2021) (link)