Soil
Soil samples were collected from a perennially flooded paddy field located in Leshan, Sichuan Province, China (29.2593 N, 103.9403 E). Surface soil was collected at a depth of 0 to 20 cm through a “five points” sampling strategy in a 25 m × 25 m field. All soil samples were transported immediately to the laboratory on ice and stored at 4 °C. Plant residues, roots, and stones were removed, and the soil was drained well enough to pass through a 2 mm sieve. These soils were used in greenhouse batch experiments; they were chosen as they contain no native compatible rhizobia that can nodulate with Glycine max variety C08. The basic properties of the soil were: pH 5.3 (soil:water = 1:2.5); total carbon (TC), total nitrogen (TN), H and S contents, 1.95%, 0.16%, 1.01% and 0.05%, respectively; cation exchange capacity (CEC) 16.55 cmol kg−1; dissolved organic carbon (DOC) and dissolved organic nitrogen (DON), 37.58 mg kg−1 and 2.93 mg kg−1, respectively; exchangeable sodium (Na), potassium (K), calcium (Ca) and magnesium (Mg), 0.2497, 0.7898, 4.323 and 1.72 mg kg−1, respectively. Cultivated soybean (Glycine max) variety C08 was used in this study.
Greenhouse experiment and symbiotic phenotype testing
The greenhouse experiment was of a complete factorial randomized block design (Fig. 1a) that consisted of two rhizobial genotype treatments and two planting patterns. The rhizobial genotype treatments included: 1) B. diazoefficiens USDA 110 wild type, isolated from soybean [52]; and 2) B. diazoefficiens USDA 110 noeI mutant, obtained from our previous study [50]. The two planting patterns were 1) planted with cultivated soybean (C08) and 2) intact soil without plants (unplanted). Planted and unplanted soils that were not inoculated with rhizobia were instead inoculated with sterile 0.8% NaCl (w/v) solution as negative control treatments. As such, the negative control of unplanted soil is also referred to as bulk soil.
Soybean seeds were selected for fullness and uniformity before being surface-sterilized in 95% ethanol for 30 seconds and then further sterilized with 2.5% (w/v) sodium hypochlorite (NaClO) solution for 3-5 minutes, after which they were rinsed seven times with sterilized deionized water. The sterilized seeds were germinated on 0.8% water-agar (w/v) plates in the dark at 28 °C for 36-48 h. Uniform germinated seedlings were selected and transferred into pots (10 by 12 cm height by diameter) containing 500 g of soil. Each treatment was inoculated with 1 mL of rhizobial culture (optical density at 600 nm [OD600] concentration of 0.2, diluted with 0.8% NaCl solution), as described in our previous study [50]. Plants were grown in the greenhouse (day/night cycle 16/8 h, 25/16 °C and a relative humidity of 60%) and were harvested 45 days post-inoculation (dpi). Several symbiotic phenotypes were recorded for plants inoculated with the wild type and the mutant. Leaf chlorophyll concentrations were determined using a SPAD-502 meter (Konica Minolta, Osaka, Japan) [53]. Plant height, weight of fresh nodules and the number of nodules were measured after sampling and shoot and root weights were determined after being dried at 65 °C for 5 days. Nodule nitrogenase activity was measured using the acetylene reduction method as described in Buendiaclaveria et al. [54].
Sampling of unplanted soil, rhizosphere, rhizoplane, endosphere, and nodule
The method for sampling unplanted soil, rhizosphere, rhizoplane, endosphere and nodules followed the protocol described Edwards et al. [55] with the following modifications. Briefly, the plants were removed from each pot and the loosely attached soil on the roots was removed with gentle shaking, leaving the root-adhering soil layer (approximately 1 mm of soil). The soil collection steps were performed on ice. Firstly, the roots were placed in a sterile 50 mL falcon tube containing 30 mL of sterile pre-cooled PBS (phosphate-buffered saline) buffer (with pH 7.3-7.5) and vortexed for 15 s, and the turbid solution was filtered through a 100-μm aseptic nylon mesh strainer into a new 50-mL tube to remove root fragments and large sediments, followed by centrifuging for 5 min at 12,000 × g at 4 °C. The supernatant was discarded, and the soil washed from the roots was defined as rhizosphere soil, which was then frozen with liquid nitrogen and stored at -80 °C. For rhizoplane samples, the washed roots were transferred to a falcon tube with 30 mL PBS and sonicated for 30 s at 50-60 Hz twice. The roots were then removed, and the rhizoplane samples was collected by centrifugation at 12,000×g for 5 min at 4 °C and stored at -80 °C until DNA extraction. The washed roots were cleaned and sonicated again as described before to ensure that all microbes were removed from the root surface. Two more sonication procedures using clean PBS solution were performed, and the sonicated roots were surface-sterilized in 70% (v/v) ethanol for 2 min and then in 2.5% (w/v) NaClO solution for 5 min, followed by washing with PBS solution for seven times. The root nodules were collected by separating them from roots using sterile blades. The roots were defined as endosphere samples and stored at -80 °C alongside the nodules. Unplanted soil samples were collected from unplanted pots approximately 2 cm below the soil surface and stored at -80 °C until DNA extraction.
DNA extraction, 16S rRNA gene sequencing, and analysis
Genomic DNA of each sample was extracted using the FastDNA Spin Kit for Soil (MP Biomedicals, LLC., Solon, OH, USA) following the manufacturer's protocol. DNA concentration and purity were evaluated photometrically using a NanoDrop ND-1000 UV-Vis spectrophotometer (NanoDrop Technologies, Wilmington, DE, United States). The extracted DNA was stored at -80 °C until further analysis. Primers 515F (5’-GTGCCAGCMGCCGCGGTAA-3’) and 806R (5’-GGACTACHVGGGTWTCTAAT-3’) were used to amplify the variable V4 region of the bacterial 16S rRNA gene. PCR conditions as follows: 94 °C, 5 min, 94 °C, 30 s, 52 °C, 30 s, 72 °C, 30 s, 72 °C, 10 min, 30 cycles. Sequencing libraries were generated using NEBNext® Ultra™ DNA Library Prep Kit for Illumina® (New England Biolabs, MA, USA) following the manufacturer's recommendations and index codes were added. The library quality was assessed on the Qubit@ 2.0 Fluorometer (Thermo Fisher Scientific, MA, USA) and Agilent Bioanalyzer 2100 systems (Agilent Technologies, Waldbronn, Germany). Finally, the library was sequenced on an Illumina_Hiseq2500 platform and paired-end reads of length 250 bp were generated (Guangdong Magigene Biotechnology Co., Ltd. Guangzhou, China). The resulting paired sequence reads were then merged, trimmed, filtered, aligned, and clustered to define the operational taxonomic unit (OTU) using USEARCH v.11.06 [56]. Briefly, sequences with ≥ 97% similarity were assigned to the same OTU by the UPARSE-OTU algorithm in USEARCH; and chimera detection was performed with VSEARCH 2.11 [57]. Putative chimeric sequences and singletons were discarded.
Root exudate collection and UPLC-MS/MS analysis
Full and uniform soybean seeds were surface sterilized and germinated as described above. To enhance root growth, germinated seedlings were transferred to sterile pots containing sterile vermiculite and grown in the greenhouse for 7 days under the same conditions as described above. At harvest, the soybean plants were pulled from their pots and washed to remove the vermiculite, then four plants were transferred to a 9-well sponge lattice placed in a glass jar (12.6 cm in height and 8.5 cm in diameter) containing 100 mL 25% (v/v) of sterile nitrogen-free Rigaud–Puppo solution [58]. The plant roots grew through the holes of the lattice into the nutrient solution. These hydroponics systems were inoculated with 4 mL of USDA 110 WT and noeI mutant cultures as described above with 4 mL 0.8% NaCl added to the control samples. To provide an aerobic environment for rhizobia, oxygen was pumped into the nutrient solution; each treatment contained three replicate hydroponics systems. The systems were incubated for 7 days in a climate-controlled growth chamber (day/night cycle 14/10 h, 28/16 °C and relative humidity of 60%). To check the sterility of the hydroponics systems, aliquot of 500 µL from each system was spread and cultured on tryptone-yeast (TY) medium plates. Soybean root exudates were collected by centrifugation at 10,000 rpm for 20 min (5 °C), filtered using a 0.25-µm cellulose nitrate filter and then stored at -20 °C until further analysis.
Eleven standard flavonoids (supplied by J&K or ANPEL) were determined during experiment: naringenin, hesperetin, genistein, daidzein, 7, 4′-dihydroxyflavone, apigenin, chrysin, luteolin, isoliquiritigenin, morin, coumestrol; deuterated genistein was used as the internal standard. The calibration curve was prepared by the serial dilution of a mixture of eleven standards by methanol with concentrations as follows: 50, 25, 10, 5, 1, 0.5, 0.1 μg/L. The internal standard was also added to all samples to achieve a final concentration of 10 μg/L. The calibration curve was obtained by plotting the peak area ratio (y) of the standard to the internal standard versus the ratio of their concentrations (x). The curve was fitted to a linear function with a weight of 1/nx (R2 > 0.99), with “n” being the calibration level. The concentrations of the compounds in the sample were determined by their peak area ratio with the internal standard and were determined using the calibration curve. All standards and samples were filtered through a PTFE syringe filter (0.22 μm) and stored at -80 °C until further analysis.
The internal standard was added to each hydroponics culture (100 mL) to give a concentration of 10 μg/L after which the solution was passed through a Resprep C18 solid-phase extraction cartridge [Sep-Pak Vac 6cc (500 mg), Waters, USA]. Flavonoids were eluted by 10 mL methanol and then freeze-dried with liquid nitrogen. For quantification, samples were resuspended in 1 mL of 50% (v/v) methanol solution and 10 µL aliquots were injected into a Waters ACQUITY I-class UPLC coupled with Xevo TQ-XS Triple Quadrupole Mass Spectrometer in the electrospray ionization negative mode (Waters, USA). Liquid chromatography was performed on a 100 mm × 2.1 mm BEH C18 column with a particle size of 1.7 µm. The mobile phase consisted of solvent A (water) and solvent B (100% acetonitrile) and the flow rate was 0.3 mL/min. The optimized linear gradient system was as follows: 0–1 min, 5% B; 1–10 min, 35% B; 10–12 min, 95% B; 12–15.5 min, re-equilibrium to 5% B. The parameters of the mass spectrometer were as follows: capillary voltage 2.5 kV, cone voltage 80 V, desolvation temperature 600 °C, desolvation gas flow 1100 L/h, cone gas flow 250 L/h, nebulizer gas flow 7 bar, and collision gas flow 0.15 mL/min of argon. A multiple reaction monitoring (MRM) mode was employed for quantitative analysis. Mass spectral parameters were optimized for each analyte and are shown in Supplementary Table S1.
Impacts of the mixture of flavonoids on soil microbiome
To determine the effect of flavonoids on the structure of the soil microbiota, watery solutions were prepared containing a mixture of the eleven flavonoid standards according to the quantitative analysis of flavonoids secreted by soybean. The final concentration of daidzein was 1 µg/g, and the other ten flavonoids were added following their ratios to daidzein. From the soil described above, 100 g were placed into pots and pre-incubated under the greenhouse conditions described above for one week to activate the soil microbiomes. 1 mL of the mixture solution was added into each pot twice a week for 4 weeks. The control treatment had the same volume of sterile water added; each treatment consisted of three replicates. All pots were watered twice a week during the incubation period. The soil samples were collected after incubation, with DNA extracted and the 16S rRNA gene sequenced and analyzed as described above.
Physicochemical characterization of soil
The soil physicochemical characteristics of each treatment were measured following the methods described by Bao [59]. Soil pH was measured using a suspension of soil and deionized water at a ratio of 1:2.5 (w/v). Soil total C, N, H and S contents were determined separately using an elemental analyzer (Flash EA 1112, Thermo Finnigan). DOC and DON were measured using a TOC analyzer (Multi N/C 3100, Analytik Jena AG). Soil exchangeable Na, K, Ca and Mg were extracted with 1 M ammonium acetate and measured by atomic absorption spectrophotometry (NovAA300, Analytik Jena AG). CEC was measured in a continuous colorimetric flow system (Skalar SAN++ System, Netherlands).
Statistical analysis
The resulting OTU table was normalized by the negative binomial model using the package phyloseq [60] in R (version 3.6.0). Weighted UniFrac [61] distances were calculated from the normalized OTU tables using the R package vegan, Principal coordinate analyses (PCoA) utilizing the weighted UniFrac distances to assess the differences in microbial communities between treatments. To measure the β-diversity significance, permutational multivariate analyses of variance (PERMANOVA) was conducted using the function adonis in vegan [62]. Shannon, Chao 1 and Fisher indices and the number of observed species were calculated using the function diversity in R package vegan. Kruskal-Wallis tests followed by Dunn’s multiple-comparison test were performed to assess differences between treatments. The statistical analysis of taxonomic and functional profiles (STAMP) was applied to identify different species associated with rhizobial treatments [63]. To explore the correlation between microbial communities and environmental properties, weighted UniFrac distance-based RDA (db-RDA) and Variation partitioning analysis (VPA) were performed using the function capscale and varpart in the package vegan, respectively. To determine OTU enrichment in each treatment, a generalized linear model (GLM) approach using edgeR [55] was conducted. Microbial co-occurrence networks were constructed based on Spearman correlations among 300 dominant OTUs. The nodes in this network represent OTUs and links indicate potential microbial interactions. We adjusted all P-values of the correlation matrix using the Benjamini and Hochberg FDR controlling procedure. The indirect correlation dependencies were distinguished using the network deconvolution method [64]. The subnetworks for various compartments were induced based on OTU-presenting in corresponding samples. The cutoff for correlation value was determined through random matrix theory (RMT)-based methods [65]. Network properties were calculated with the igraph [66] package in R and visualized in Gephi 0.8.2 [67]. Fisher’s Least Significant Difference (LSD) test (p < 0.05) and Duncan multiple-comparison test (p < 0.05) using R package agricolae [68] were employed to analyze the difference of soybean symbiotic phenotypes and relative abundance of bacterial taxa, respectively. All figures in this study were generated using ggplot2 [69] in R and OriginPro 2017.