2.3 Experimental design
In mid-October 2017, we collected seeds from mature individuals of X. strumarium near Qipanshan, Shenyang. Seeds of G. max were bought from Dafeng Seed Industry Development Co., Ltd, Shenyang China. In late April 2018, both invasive and native plant seeds were disinfected with 0.5% potassium permanganate solution, washed with distilled water and sown in pots (25 cm × 16 cm × 14 cm) filled with 4 kg soil. The initial soil physical and chemical characters were as follows: soil pH, 7.34; organic matter content, 44 g kg− 1; total N content, 0.22 g kg− 1; total phosphorus content, 0.06 g kg− 1; total potassium content, 1.48 g kg− 1; available N content, 125.67 mg kg− 1; available phosphorus content, 3.10 mg kg− 1; available potassium content, 55 mg kg− 1.
In mid-May, seedlings were thinned to one individual plant per pot for the monoculture planation, and one G. max individual and one X. strumarium individual per pot for the mixture. There were five replicates for each planting types. For each pot, root physiological traits (acid phosphatase), mycorrhizal colonization rate and ‘activity’ of biological N-fixation were measured. We set additional five replicates for each monoculture and mixture planting to measure plant biomass and leaf morphological traits.
2.4 Plant sampling and trait measurements
Plants were harvested in late July 2018. Shoot biomass was measured after the samples were dried at 60 ℃ for 48 hours. Root biomass was also measured separately for coarse (diameter > 2mm) and fine (diameter < 2mm) roots.
Total leaf area of an individual plant was measured by Li-3000C (Li-COR, Lincoln, NE, USA), and then dried in an oven at 60 ℃ for 48 hours, and weighed. In addition, randomly selected sub-samples of fine roots were scanned, and the images were used to calculate fine root length and diameter using WinRhizo Pro (2016 software, Regent Instruments, Canada), then weighed. Surface area of the fine root sub-sample was calculated as: π × root diameter × root length. We then calculated total surface area of fine roots of an individual plant according to the portion of the fine root sub-sample to total fine root biomass of the individual plant.
Root acid phosphatase activity (Apase) was measured following Png et al. (2017), using para-nitrophenyl phosphate (pNPP) as the substrate (Kavka et al. 2021; Png et al. 2017). Briefly, two fresh root samples, approximately 0.2 g, were prepared, each added with 9 mL of sodium acetate-acetic acid buffer (pH = 5.0) and then ground and shaken (100 rpm) in water-bath at 20°C for 5 min. One ground root sample was then added with 1 mL of pNPP substrate (5 mM) and incubated for 30 min. The other ground root sample using as a control was added with sodium acetate-acetic acid buffer (pH = 5.0) and incubated for 30 min. The reaction solution with pNPP was then centrifuged (2500 rpm for 5 min and then 4000 rpm for 5 min), and supernatant (0.5 mL) was added with 4.5 mL of sodium hydroxide (0.11 M) to terminate the reaction. The concentration of para-nitrophenol (pNP) in the final solution was calculated using absorbance value at 405 nm by UV-VIS spectrophotometer (UV-Vis Spectrophotometer, Shimadzu, Japan). Root acid phosphatase activity (Apase) was then calculated as: Apase = pNP/ (reaction time × root weight).
Mycorrhizal colonization rate was measured following previous studies (Bi et al. 2023; Chen et al. 2022; Trouvelot et al. 1986). Specifically, 50 root segments (~ 1 cm in length) were randomly selected including terminal two root branch orders, which usually have the highest mycorrhizal colonization rate in the root system. These root segments were put into 5% KOH solution at 70°for 10 min and cleaned with distilled water. The roots were then consecutively soaked in 5% acetic acid solution and 5% acetic acid ink for root dyeing. The extent of mycorrhizal colonization was examined using a microscope (Nikon MODEL ECLIPSE Ni-U, Japan). Mycorrhizal colonization rate was divided into five categories, each with a weight of 0.95, 0.70, 0.30, 0.05 and 0.01, respectively. Root mycorrhizal colonization rate was calculated as follows:
n1-n5 is the number of root fragments examined in each above mycorrhizal colonization category.
The ‘quantity’ aspect of the N-fixing strategy for the soybean was assessed by the nodule number and nodule biomass per plant as well as the biomass of a single nodule. The ‘activity’ aspect of the N-fixing strategy was indicated by the number of nifH gene copies in the nodule (Libourel et al. 2023; Yang, Xiang, et al. 2023; Yang et al. 2020). The nifH gene copies were determined by the following three steps: 1) Nodule DNA extraction. Total DNA was extracted from 0.2 g fresh nodule using the FAST DNA spin kit for Soil (Omega Bio-tek, Norcross, GA, U.S.) following the manufacturer’s instructions. The extracted DNA was quantified using a NanoDropND-2000 spectrophotometer (NanoDrop2000, Thermo Fisher Scientific, USA). 2) Quantitative real-time PCR assay. The abundance of nifH genes in nodule was measured using a StepOne™ Real-Time PCR instrument (Bioer Technology Co., Ltd, Hangzhou). The degenerate oligonucleotide primers used to amplify the nifH gene were primer nifH-F (5´-AAAGGYGGWATCGGYAARTCCACCAC-3´) and nifH-R (5´-TTGTTSGCSGCRTACATSGCCATCAT-3´). The reaction mixture (20 µL) contained 2 µL of template DNA, 10 µL of ChamQ SYBR COLOR qPCR Master Mix (2×) (Vazyme Biotech Co., Ltd, Nanjing), 0.4 µL of each primer (5 µM), and nuclease-free water. Initial denaturation step was at 95°C for 5 min, followed by 40 cycles of 95°C for 30 s, 56°C for 30 s, and 72°C for 40 s. Melting curve analysis was performed as follows: 95°C for 15 s, 60°C for 30 s, and 95°C for 15 s. Melting curve calculation and determination of Tm values were performed using the polynomial algorithm function of LightCycler Software v.1 (Roche Applied Science). 3) To quantify the nifH gene present in the samples, a standard was prepared by performing end-point Polymerase Chain Reaction (PCR) with the nifH-F and nifH-R primer set. Plasmid copy number calculation was as follows: (copies µL− 1) = concentrations × 10− 9 × 6.02 × 1023 / (molecular weight × 660). Selected 10− 3 ~ 10− 8 diluent of the standard product to prepare the standard curve. Standard product amplification efficiency was calculated from standard curves according to the equation E = 10(−1/slope). The samples for each treatment were analyzed. Results in g µL− 1 were converted in number of nifH copies using the following formula (assuming an average of 660 g mol− 1 per base pair): number of copies= [DNA (g) × Avogadro’s number (molecules mol− 1)] / [number of DNA base pairs in nifH fragment × 660 (g mol− 1)]. The resulting numbers were expressed as per g of nodule (fresh weight).
We also collected bulk soil and rhizosphere soil in each pot to measure soil total C and N content as well as 15N content. Specifically, the pots were destroyed to expose plant roots. The soil easily shaken off the roots was treated as bulk soil and the soil adhering to roots and not easily shaken off was carefully collected as rhizosphere soil. The soil samples were then ground with GT200 (Crinoer, Beijing Greidman Instrument and Equipment Co., LTD.) to determine soil C and N content using Elementary analytical instrument (Elementary analytical instrument, Germany). Soil 15N content was measured using SerCon Integra 2 Integrated EA-IRMS isotope mass spectrometer (SeCron, Cheshire Crewe, UK). The C, N and 15N contents for leaves and roots were also measured in a similar procedure. the contribution of total leaf N by the N-fixation (%Ndfa) was calculated following previous studies (Balboa and Ciampitti 2020; Cox et al. 2022):
X. strumarium was used as the reference plant. Leaf δ15N of the reference plant (δ15N (X. strumarium)) is calculated as the average leaf δ15N of this plant across pots containing this plant. δ15N (G. max) is the leaf 15N in each pot with G. max.
B is the leaf δ15N value of legume plants depending solely on atmospheric N for their N source. We used an average B value of -1.97‰ across legume species (Balboa and Ciampitti 2020). δ15N (X. strumarium) is the average leaf δ15N of X. strumarium. The absolute amount of N in G. max leaves contributed by N-fixation of nodules (i.e., Ndfa amount) was calculated as: