2.1 Growth substrate and compartment
The soil was collected from Shangzhuang experimental station, Beijing, China (40°140′ N, 116°190′ E) with the following properties: soil pH 8.2 (soil: water, 1:5), 11.5 g kg− 1 organic carbon, 2.6 mg kg− 1 Olsen-P, 0.72 g kg− 1 total N, 8.5 mg kg− 1 available N (NH4+ + NO3−) and 32.3 mg kg− 1 exchangeable K (Wang et al. 2021a). Texture was a silt loam. The soil was air-dried and sieved through a 2 mm sieve and then the soil was sterilized by γ-radiation (> 25 kGy) to eliminate indigenous microorganisms. The growth substrate was prepared by mixing sterilized soil and sand (w/w, 2:1).
A three-compartment growth microcosm (length × width × height = 20 × 13 × 10 cm) was used in the experiment (Fig. S1A). The microcosm was made of polyvinyl chloride sheet sandwiched by 30-µm nylon mesh between the three compartments (two root compartments; RCs, and one hyphal compartment; HC) to allow fungal hyphae but not plant roots to enter the other compartments. Each RC contained 700 g growth substrate with another 20 g inoculum, while the HC contained 1600 g of growth substrate.
For ensuring the initial growth of maize and soybean, nutrient solutions were added in RCs and HC with the following concentration (mg kg− 1 soil): 100 P as KH2PO4; 113 K as K2SO4; 43 Mg as MgSO4·7H2O; 5.9 Fe as Fe-EDTA; 6.7 Mn as MnSO4·H2O; 10 Zn as ZnSO4·7H2O; 2 Cu as CuSO4·5H2O; 0.67 B as H3BO3; 0.17 Mo as Na2MoO4·5H2O. In addition, 100 mg kg− 1 N as Ca(NO3)2·4H2O was supplied only in the RCs. In other words, 70 mg P and 70 mg N were added in the RCs with maize and soybean, respectively, while 160 mg P was added in the HC.
2.2 Experimental design
The experiment was conducted from November 15th, 2021 to February 21st, 2022 (13 weeks) in a greenhouse at China Agricultural University (40°1'29"N,116°16'33"E) where both temperature and light are controlled. Light intensity was 400 ~ 1000 µmol photons m− 2 s− 1 with a time period from 8 am to 6 pm every day. The RCs were irrigated by deionized water every second day by weighting the microcosm to maintain soil moisture content at about 75% of field capacity. The HC was irrigated once a week.
Maize (Zea mays L. cv. Zhengdan 958) and soybean (Glycine max (L.) Merrill cv. Jidou 12) were used to establish microcosms with monoculture (maize or soybean was grown in the two chambers of RC) and mixture (maize was grown in one chamber of RC and soybean in the other). Seeds were surface-sterilized with 10% H2O2 for 30 min, rinsed thoroughly in sterile distilled water and pre-germinated on filter paper. Then two pre-germinated seeds of uniform size were placed in each RC. Maize seedlings were thinned to one seedling in each RC 5 days after sowing, while soybean seedlings were thinned to one seedling 7 days after sowing.
One of the RCs was inoculated with the AM fungus Rhizophagus irregularis (R. irregularis, BGC JX04B; the model AM fungal strain (Stockinger et al. 2009)), or sterilized inoculum, yielding seven treatments (Fig. 1). R. irregularis was provided by the Beijing Academy of Agriculture and Forestry Sciences and further propagated by using maize as host for four months to obtain enough AMF inoculum for our study. The seedings in the RC with AMF inoculation were inoculated with 25 g AMF inoculum (350 spores kg− 1 soil). The seedlings in the RC without AMF inoculation were inoculated with 25 g inoculum sterilized at 121°C for 30 min and received 20 mL of filtrate without AM fungal propagules filtered through a 30-µm nylon mesh sieve to balance the initial microbial communities (Singh et al. 2019; Liu et al. 2021). All treatments with soybean were inoculated with rhizobia (Sinorhizobium fredii; strain CCBAU 45436). The germinated soybean seeds were immersed in the rhizobium suspension (OD value = 1) for 30 min before sowing and 5 mL rhizobium suspension was added after sowing to each soybean compartment (Li et al. 2022a). The seedlings inoculated with AMF (+ AMF) were defined as donor plant and the neighbor seedlings with sterilized inoculum (-AMF) as receiver. The treatments with two RCs supplied with sterilized inoculum were non-mycorrhizal treatments. Each treatment was replicated eight times and a total of 56 microcosms were established and randomly arranged (Fig. 1).
2.3 Harvest and sample analysis
Maize (tasseling period) and soybean (in pod-forming stage) were harvested from all RCs in the eight replicates of each treatment. Each plant was separated into shoot and root. Roots in the four of eight replicates were washed with deionized water, and 50 root segments with a length of 1 cm were randomly selected for the determination of AMF colonization. Subsequently, the soybean root nodules were removed by cutting and stored in a 10 mL centrifuge tube for counting. After counting, the root nodules, remaining roots and shoots were dried at 105℃ for 30 min, and then at 70℃ for two days to constant weight before weighing.
Dry ashing method was used to determine shoot P concentration (Thomas et al. 1967). Briefly, 0.2 g of well-ground maize or soybean shoot samples were placed in a 25 mL porcelain crucible and placed in a muffle furnace for dry ashing. During dry ashing, the samples were heated to 180°C, kept for half an hour to facilitate carbonization of the sample, then kept for 4 hours in a muffle furnace at 450°C. Two mol L− 1 nitric acid were added to the ash for digestion first, then 18 mL deionized water were added to dilute the solution to a total volume of 20 mL. After the above procedures, the solutions were filtered with P-free filter paper, then 1 mL filtrate was taken from the solution of each sample and diluted 10 times and vanadium molybdenum yellow colorimetric method was used for determination.
We assessed AMF colonization by using the method from McGonigle et al. (1990) and Wang et al. (2022). Briefly, root clippings were immersed in 10% KOH, kept in 90℃ water bath for 30 min, then acidified in 2% HCl for 10 min and finally soaked in a plastic box containing 0.05% trypan blue in lacto-glycerol (lactic acid: glycerol: deionized water = 1:1:1) for 30 min in a 90℃ water bath. We then destained with lacto-glycerol at room temperature for 2 days and cut the root into 1 cm pieces. Fifteen pieces were placed on each microscope slide, and 10 visual fields were observed in each root piece, a total of 150 visual fields were scored for mycorrhizal colonization.
The mycorrhizal growth response (MGR) of plant shoot biomass, N and P content were calculated as:
$$\text{MGR = ln(}\frac{\text{AM}}{\text{NM}}\text{) }$$
where AM is plant shoot biomass, N content and P content in the mycorrhizal treatments and NM is plant shoot biomass, N content and P content in the corresponding non-mycorrhizal treatment. Here we calculated the MGR of maize and soybean in each RC separately instead of taking the average of two RCs with AMF because we distinguished donor and receiver plant in the experiment i.e., we calculated the MGR of receiver or donor maize and receiver or donor soybean (3 or 5 vs 1; 9 or 11 vs 7; 4 or 6 vs 2; 10 or 12 vs 8).
For the other four of eight replicates of each treatment, N concentration and atom% 15N natural abundance were measured with an elemental analyzer connected to a stable-isotope mass spectrometer (DELTAplus XP, Thermo Finnigan Electron Corporation, Germany) (Li et al. 2022a). The precision of isotopic measurements was ± 0.1‰. The isotopic abundance was expressed as (Weremijewicz et al. 2016):
$${\text{δ}}^{\text{15}}\text{N(}\text{‰}\text{) }\text{= }\frac{{\text{R}}_{\text{sample }}\text{–}{\text{ R}}_{\text{standard}}}{{\text{R}}_{\text{standard}}}\text{× 1000}$$
Where R represents the 15N: 14N ratio of a sample or of the standard which is atmospheric N.
The fraction of N2 fixed by soybean was calculated as:
$$\text{%Ndfa = }\frac{{\text{δ}}^{\text{15}}{\text{N}}_{\text{maize}}\text{-}{\text{ δ}}^{\text{15}}{\text{N}}_{\text{soybean}}}{{\text{δ}}^{\text{15}}{\text{N}}_{\text{maize}}\text{- B}}\text{× 100}$$
Where δ15Nmaize and δ15Nsoybean are the δ15N value of maize and soybean, respectively. The B value used in our calculation was − 1.37‰ (Balboa and Ciampitti 2020). Sole maize without AMF inoculation was used as reference plant to calculate the fraction of N derived from atmosphere (%Ndfa).
The amount of N derived from atmosphere (Ndfa) for soybean was calculated as follow:
$$\text{Ndfa = %Ndfa × }{\text{N}}_{\text{soybean}}$$
To assess the overyielding, the expected biomass and N, P content of maize/soybean mixture were calculated. With maize as donor, the expected value was calculated by maize as donor in monoculture + soybean as receiver in monoculture; When soybean as donor, the expected value was calculated by soybean as donor in monoculture + maize as receiver in monoculture.
2.4 Statistical analysis
We first tested, through two-way analysis of variance (ANOVA), for general effects of the mycorrhizal treatment (df = 2 for mycorrhiza, three levels of AMF inoculation: -AMF, donor, receiver; two levels of cropping system: monoculture and mixture). A second ANOVA tested for specific effects of being a donor or receiver of CMNs (df = 1, two levels of AMF inoculation: donor, receiver; two levels of cropping system: monoculture and mixture). The first ANOVA was conducted for shoot biomass, N and P concentration, N and P content, δ15N value, Ndfa, nodule number and nodule weight, while the second ANOVA was conducted for the same parameters and additionally for AMF colonization. Before ANOVAs, the data were checked for homogeneity of variances with Levene’s test and normality with Shapiro-Wilk test.
After ANOVAs, significant differences among treatments were tested by Tukey’s honestly significant difference post-hoc test (Tukey HSD test). All statistical analyses were performed with SPSS 20.0 (IBM SPSS software), and figures were made with SigmaPlot 12.5 (Systat).