Experimental design
The experiment was conducted in a glasshouse at the Northeast Institute of Geography and Agroecology of the Chinese Academy of Sciences (125°24’30”E, 43°59’49”N), from June to August 2023. The glasshouse maintained a controlled temperature range of 24 to 30°C, with natural light conditions. The soil used was collected from the Jilin Songnen grassland ecosystem national observation and research station, Northeast Normal University, China (123°37’2”E, 44°34’47”N). The soil was sterilized using 25 kGy of 60Coγirradiation at the CNNC Tongfu Radiation Technology Co., Ltd (Changchun, China). PVC pots (internal diameter 10 cm, depth 20cm) were used for the experiment. The experiment employed a fully factorial design with eight GCF treatments and six AM fungal treatments with five replicates of each combination for a total of 240 pots.
The six AM fungal treatments were: no AMF inoculation (control), inoculation one (low diversity) of the AMF species: Gigaspora rosea (Nicolson and Schenck, 1979), Glomus etunicatum (Becker and Gerdemann, 1977), Rhizophagus irregularis (Tisserant et al., 2013, formerly named Glomus intraradices ), andFunneliformis mosseae (Nicolson and Gerdemann, 1968). These AM fungi are common in the farming–pastoral ecotone of northern China (Xiang et al., 2014). The high AMF diversity treatment was a mix of all four of the above AMF fungi. This totals six AM fungal treatments: no-AMF control, four single AMF species, and one mixture of all four AMF species.
AM fungal inoculants were prepared by culturing the AMF species on the host plant Trifolium repens for 4 months. Inoculation was done by first filling the pots with sterilized soil three quarters full and then added 20g of AM fungi inoculum (about 100 spores) to each tube. The mixed-inoculum treatment was created by mixing each of the four AM inoculants together in equal mass (5g) to total the 20g. A 3 cm layer of sterile soil was placed on top to avoid cross contamination and ensure the soil weight of all pots were consistent (all contained 2 kg of substrate).
Six seeds of Triticum aestivum (germination rate: 98%) were evenly planted in each pot. Seeds were sterilized with 1.25% sodium hypochlorite for 10 minutes and thoroughly washed with distilled water (dH2O). All seeds successfully germinated and survived throughout the duration of the experiment. Plants were watered three times a week to maintain soil water content at 60% of soil water holding capacity (WHC). Pots were randomly rearranged every two weeks to minimize spatial variability. Each tube received a single application of 20 ml of Hoagland nutrient solution 50 days after seed germination. After 80 days, plant and soil samples were harvested.
The eight GCF treatments included: 1) a control, 2) nitrogen addition (NH4NO3, 10 g N m-2year-1), 3) antibiotic pollution (ciprofloxacin, 100 μg kg-1 soil), 4) microplastic pollution (4 g polyethylene kg-1 soil ), 5) pesticide pollution (triadimefon wettable powder, 50 g ha-1), 6) soil saline-alkali (NaCl: Na2SO4: NaHCO3: Na2CO3 = 4:1:6:4, pH 8.80 ± 0.05), 7) drought (30% of WHC) and 8) a treatment of all six GCFs together. The global change treatments 3 – 6 were applied once to the soil just prior to planting. For the nitrogen addition treatment, we added NH4NO3 twice during the experiment on the 20th and 40thday after sowing separately. The drought was imposed throughout the experiment by maintaining a 30% WHC, with other treatments maintaining 60% WHC. The details on global changes treatment can be found in the supporting information.