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.