Regulatory mechanism of phosphorus supply intensity on the plant nitrogen acquisition in soybean: Insight from the differences of rhizobia diversity between high-oil soybean and non-high-oil soybean

Regulatory mechanism of phosphorus supply intensity on the plant nitrogen acquisition in soybean: Insight from the differences of rhizobia diversity between high-oil soybean and non-high-oil soybean | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Regulatory mechanism of phosphorus supply intensity on the plant nitrogen acquisition in soybean: Insight from the differences of rhizobia diversity between high-oil soybean and non-high-oil soybean HanShuo Zhang, MingCong Zhang, Yang Hu, Anni Bai, Wei Zhou This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8195203/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 28 Mar, 2026 Read the published version in BMC Plant Biology → Version 1 posted 14 You are reading this latest preprint version Abstract Phosphorus supply plays a critical role in regulating symbiotic nitrogen acquisition in soybeans, yet the mechanisms underlying varietal differences between high-oil and non-high-oil varieties remain poorly understood. This study investigated the varietal-specific mechanisms of phosphorus supply intensity on plant nitrogen acquisition via rhizobial community restructuring using two high-oil (Kenong 18, Kenong 39) and two non-high-oil varieties (Heihe 43, Longken 310) under five phosphorus levels (0, 35, 70, 105, 140 kg·hm − 2 ). The results showed that high-oil varieties exhibited superior growth performance and nitrogen acquisition efficiency at 105 kg·hm − 2 phosphorus supply, with increases of 19.99% in plant height, 4.13% in shoot dry weight and 17.96% in root dry weight versus controls. Nodule number, dry weight and haemoglobin content increased by 83%, 30% and 33.02%, respectively, in high-oil genotypes. Enhanced nitrogen metabolism was evidenced by significantly elevated GOGAT/GS activities (9.32–17.13%) and leaf total nitrogen content. Crucially, under optimal phosphorus conditions, high-oil varieties enriched specific nitrogen-fixing rhizobia, such as Bradyrhizobium sp. 173_3_ module and Rhizobium sp ., and exhibited stronger correlations between community structure and soil available phosphorus (RS-AP), along with a predicted greater potential for nitrogen acquisition and aerobic chemoheterotrophy. This study demonstrates that optimal phosphorus supply enhances symbiotic nitrogen acquisition efficiency in high-oil soybeans by driving the assembly of more specialized rhizobial communities, providing microbial mechanistic insights for varietal-specific phosphorus management in soybean cultivation. Phosphorus supply Soybean Nitrogen acquisition High-oil soybean Rhizobial community Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction Soybean (Glycine max) , serves as a critical global source of both edible protein and vegetable oil [ 1 ] , playing an indispensable role in food systems and industrial applications [ 2 , 3 ] . In China, the Northeast region dominates national production, accounting for approximately 41% of domestic output [ 4 ] . Nonetheless, the persistent gap between domestic supply and demand has resulted in substantial reliance on imports, highlighting an urgent need to enhance both the yield and quality of Chinese soybean [ 5 ] . Historically, breeding efforts have often faced a trade-off between selecting for higher seed yield and higher oil content, presenting a key scientific and practical challenge [ 6 , 7 ] . Therefore, achieving synergistic improvement in soybean yield and oil production represents a crucial objective for ensuring national food security and promoting sustainable agricultural development [ 8 ] . The symbiotic relationship with nitrogen-fixing rhizobia (predominantly Bradyrhizobium and Sinorhizobium spp.) is a cornerstone of soybean productivity [ 9 , 10 ] , capable of fulfilling up to 70% of the plant's nitrogen demand through biological nitrogen fixation (BNF) within root nodules [ 11 ] . Beyond nitrogen provision, rhizobia enhance soybean growth, yield, and quality by modulating seed composition [ 12 ] . Critically, soybean varieties bred for distinct metabolic priorities-such as high-oil varieties prioritizing lipid biosynthesis versus conventional varieties prioritizing protein accumulation-may establish distinct interactions with rhizobia. These varieties differences in rhizobial partnerships and nitrogen acquisition efficiency could directly influence carbon allocation patterns, thereby ultimately determining seed oil-to-protein ratios [ 13 ] . Phosphorus, as a key macronutrient and fundamental component of ATP, nucleic acids, and phospholipids, critically regulates soybean growth and physiological processes [ 14 ] ). In the context of the soybean-rhizobia symbiosis, P supply regulates nitrogen acquisition through multiple interconnected mechanisms [ 15 , 16 ] . First, the inherent energy demand of biological nitrogen acquisition means that phosphorus availability directly limits nitrogenase activity by governing ATP supply [ 17 ] , with deficiency severely curtailing this process irrespective of nodule development [ 18 , 19 ] . Second, P deficiency can antagonize nodulation signaling pathways, potentially altering flavonoid exudation and impairing early infection events such as infection thread formation [ 20 ] . Furthermore, an emerging perspective suggests that P supply acts as a powerful environmental filter [ 21 ] , restructuring the rhizobial community in the rhizosphere and nodules to potentially select for more adaptive strains with synergistic traits like phosphorus solubilization [ 22 ] . However, how these P-driven changes in rhizobial communities and nitrogen acquisition efficiency differentially affect nitrogen acquisition and carbon allocation in soybean varieties with contrasting metabolic sinks-specifically [ 23 ] , high-oil varieties optimized for lipid biosynthesis versus conventional non-high-oil varieties-remains poorly understood and constitutes a key knowledge gap [ 24 ] . Furthermore, phosphorus availability critically shapes the rhizosphere microbial community [ 25 ] , which in turn influences nutrient cycling and plant health. An optimal phosphorus supply stabilizes microbial dynamics and selectively promotes strains with beneficial traits, such as phosphorus solubilization and the production of plant growth-promoting substances [ 26 ] .. These microbial activities enhance phosphorus use efficiency and stimulate root development and nodulation, thereby indirectly supporting the soybean-rhizobia symbiotic partnership [ 27 ] . Consequently, phosphorus serves a dual role: it directly sustains plant physiological processes and indirectly modulates the microbial partners that are essential for sustainable soybean cultivation [ 21 ] . Despite its importance, the genotype-specific responses of the soybean–rhizobia symbiosis to phosphorus supply, particularly the underlying microbial mechanisms, remain poorly understood. While appropriate phosphorus application enhances yield and quality, excessive use can reduce phosphorus use efficiency and even depress yield [ 28 , 29 ] .. Furthermore, how rhizobial community structures vary among soybean varieties and respond to phosphorus levels is not yet clear. This study investigates the regulatory role of phosphorus supply intensity on nitrogen acquisition in high-oil and non-high-oil soybeans. We hypothesize that high-oil varieties host a more responsive rhizobial community under optimal phosphorus supply, thereby enhancing nitrogen assimilation efficiency. To test this, we will: (1) compare plant growth and root architecture traits; (2) evaluate nodulation and the activities of key nitrogen assimilation enzymes (GS/GOGAT); and (3) characterize the composition and functional potential of the nodule rhizobial communities. Our findings aim to establish a theoretical basis for precision phosphorus management strategies tailored to different soybean varieties. 2. Materials and methods 2.1 Plant and soil materials The experimental soil was collected from the top layer (0–20 cm) of fields at the Jiu San Branch of the Beidahuang Group and the Heshan Farm Science and Technology Park, located in Heihe, Heilongjiang Province, China (48°43′N, 124°56′E). The soil is classified as a typical black soil (Mollisol). The air-dried soil was ground and passed through a 2-mm sieve for subsequent use. Seeds of soybean ( Glycine max (L.) Merr.), including two high-oil varieties, Kenong18 (KN18) and Kenong39 (KN39), and two non-high-oil varieties, Heihe43 (HR43) and Longken310 (LK310), all bred in Heilongjiang Province, were used in this study. No artificial rhizobial inoculation was performed, relying solely on the native rhizobia present in the field soil. 2.2 Experimental design and cultivation This study established five phosphorus (P) application levels: 0, 0.007, 0.014, 0.021, and 0.028 g P₂O₅ per kg of soil (simulating field application rates of 0, 35, 70, 105, and 140 kg P₂O₅·hm − 2 , respectively). Phosphorus was applied as superphosphate and thoroughly mixed with the substrate before potting. Germinated soybean seeds were planted in individual pots, each containing 500 g of the prepared substrate (composed of soil and vermiculite at a ratio of 3:1 by weight). The plants were cultivated in a greenhouse at the College of Agriculture, Heilongjiang Bayi Agricultural University, under controlled conditions: 23°C, with a 16/8 h light/dark photoperiod. Sterilized deionized water was supplied every two days to maintain soil moisture at approximately 60% of the water holding capacity until the plants reached the V6 stage. All treatments were arranged in a completely randomized design with three independent replications. At the V6 stage, soybean plants were harvested. The root systems were carefully excavated. Rhizosphere soil (RS) was collected by gently shaking the roots to remove the loosely adhered soil. Bulk soil (BS) was collected from areas in the pot with no visible root presence. Plant shoots, roots, and nodules were separated. Shoots and roots were used for measurements of plant height, root length, and dry weight. Root systems were scanned for morphological analysis. Nodules were counted and weighed, with a subset used for leghemoglobin (Lb) content determination and another stored at -80°C for subsequent DNA extraction and analysis of the rhizobial community structure. Leaf samples were collected, immediately frozen in liquid nitrogen, and stored at -80°C for subsequent analysis of nitrogen assimilation enzymes and nutrient content. Soil samples were air-dried for physicochemical analysis. 2.3 Assessment of plant physiological indices Plant height and root length were measured on fresh samples using vernier calipers. Shoot dry weight (SDW) and root dry weight (RDW) were determined after oven-drying the samples to a constant mass [ 30 ] . Root systems were scanned using an Epson TWAIN Pro scanner, and the images were analyzed with WinRHIZO Pro software (Regent Instruments, Inc., Quebec, Canada) to determine total root length (TRL), average root diameter (AvgDiam), root surface area (RSA), root volume (RV), and the root apical number (RAN) [ 31 ] . Nodules were counted and weighed to determine nodule number and dry weight. Leghemoglobin (Lb) content in nodules was quantified fluorometrically with excitation and emission wavelengths set at 405 nm and 650 nm, respectively [ 32 ] . For leaf analyses, total nitrogen (TN) content was assessed using the Kjeldahl method. The activities of glutamate synthase (GOGAT) and glutamine synthetase (GS) were determined according to the methods described by Ullah et al [ 33 ] . Soluble protein (SP) content was measured using the Coomassie brilliant blue G-250 staining method [ 34 ] . 2.4 Assessment of soil physicochemical properties Air-dried rhizosphere soil (RS) and bulk soil (BS) samples were passed through 1-mm and 0.25-mm sieves for subsequent analyses [ 35 ] . Soil pH and electrical conductivity (EC) were measured in a soil-water suspension (1:2.5, w/v) using a digital pH meter and a conductivity meter, respectively [ 36 ] . Soil total nitrogen (TN) content was determined using a semi-automatic nitrogen analyzer [ 37 ] . Total phosphorus (TP) and available phosphorus (AP) were measured by the molybdenum-antimony colorimetric method [ 38 ] . Ammonium nitrogen (NH₄⁺-N) and nitrate nitrogen (NO₃⁻-N) concentrations were determined colorimetrically using the indophenol blue method and the hydrazine sulfate reduction method, respectively [ 39 ] . 2.5 Molecular analyses Total DNA was extracted from surface-sterilized nodules using a commercial kit. The concentration and purity of the extracted DNA were verified, and the DNA was stored at -20°C prior to further analysis. The rpoB gene, selected for its high phylogenetic resolution in distinguishing closely related rhizobial species, was amplified by polymerase chain reaction (PCR) using the primers rpoB1479-F (5′-GAT CGA RAC GCC GGA AGG-3′) and rpoB1831-R (5′-TGC ATG TTC GAR CCC AT-3′) [ 40 ] . The PCR amplification was performed in duplicate for each sample. The 20 µL reaction mixture contained 4 µL of 5× FastPfu Buffer, 2 µL of 2.5 mM dNTPs, 0.8 µL of each primer (5 µM), 0.4 µL of FastPfu Polymerase, and 10 ng of template DNA. The thermal cycling conditions were as follows: initial denaturation at 95°C for 2 min; 25 cycles of denaturation at 95°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 30 s; with a final extension at 72°C for 5 min. The PCR products were confirmed by electrophoresis on a 1% agarose gel and purified using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, U.S.A.). Equimolar amounts of purified amplicons from the same sample were pooled together. The pooled libraries were subjected to paired-end sequencing on the Illumina MiSeq platform (Majorbio Bio-pharm Technology Co., Ltd., Shanghai, China). Subsequent bioinformatic analysis, including taxonomic classification and diversity assessments, was performed following the pipeline established by Jiang et al. (2024). 2.6 Statistical analyses All data were subjected to a one-way analysis of variance (ANOVA) using the SPSS 25.0 software package. When the ANOVA revealed significant effects, treatment means were compared using Duncan's multiple range test at a significance level of P < 0.05. All results, obtained from three independent replicates, are presented as the mean ± standard deviation (SD). Redundancy analysis (RDA) was performed to explore the relationships between rhizobial community composition and environmental variables using the vegan package in R 4.5.1. Figures were generated using Origin Pro 2024 and the ggplot2 package in R. 3. Results 3.1 Plant growth and phenotypic visualization of roots Visual observation at the optimal phosphorus (P) supply of 105 kg·hm -2 clearly demonstrated more robust growth in high-oil soybean varieties compared to non-high-oil varieties (Fig. 1 a)Quantification of growth parameters across the P gradient confirmed this varietal difference (Fig. 1 b-e). Plant height, shoot dry weight, and root dry weight of high-oil varieties were generally superior to those of non-high-oil varieties across all P levels, with the most pronounced differences observed at 105 kg·hm -2 P supply (Fig. 1 b, d, e). At this optimum, the plant height of high-oil varieties was 19.99% greater than that of non-high-oil varieties. Similarly, shoot and root dry weights of high-oil varieties peaked, showing significant increases of 4.13% and 17.96%, respectively, compared to non-high-oil varieties under the same P supply. The response of root length to increasing P supply was distinct from that of other growth parameters (Fig. 1 c). Root length of high-oil varieties increased from 0 to 70 kg·hm -2 but then declined at 105 kg·hm -2 . In contrast, non-high-oil varieties exhibited a continuous increase. Despite this, the root length of high-oil varieties at 105 kg·hm -2 remained 31.05% longer than that of non-high-oil varieties. Comprehensive root system architecture analysis revealed that a P supply of 70 kg·hm -2 was most effective in promoting root expansion for both variety types (Table 1 ). At this level, high-oil varieties significantly outperformed non-high-oil varieties in key morphological traits. The total root length (TRL) of high-oil varieties was 7.0% greater than that of non-high-oil varieties. More notably, high-oil varieties exhibited substantially larger root surface area (RSA, 21.8% greater), root volume (RV, 22.9% greater), and number of root tips (26.6% greater). In contrast, the average root diameter (AvgDiam) was less responsive to P supply and showed no consistent varietal difference. Table 1 Root architecture traits of high-oil and non-high-oil soybean varieties in response to phosphorus supply. Data are presented as mean ± standard deviation (n = 3). Different uppercase letters indicate significant differences among P levels, and lowercase letters indicate significant differences between varieties at the same P level ( P < 0.05, Duncan's test). Treatment Phosphate fertilizer application rate (kg·hm − 2 ) Length (cm) AvgDiam (mm) RootVolume (cm 3 ) SurfArea (cm 2 ) Tips KN18 0 1134.07 ± 53.88Cb 0.89 ± 0.19Aa 162.43 ± 8.81Cab 285.52 ± 18.18Ca 814.00 ± 37.04Da 35 1186.38 ± 26.71Cab 0.88 ± 0.09Aa 149.89 ± 11.29Cb 272.10 ± 14.49Ba 786.00 ± 70.87Da 70 1219.89 ± 42.64Ca 0.87 ± 0.20Aa 125.76 ± 5.75Bc 218.97 ± 18.71Bb 673.33 ± 59.53Cb 105 1246.82 ± 42.49Ea 0.88 ± 0.26Aa 173.95 ± 4.54Bab 249.90 ± 17.85Dab 711.67 ± 45.98Dab 140 1356.40 ± 51.22Ba 0.95 ± 0.10Aa 194.36 ± 11.69Ba 347.60 ± 20.28ABa 1074.00 ± 63.00Ca KN39 0 1404.67 ± 62.28Ba 0.93 ± 0.17Aa 168.60 ± 11.45Bb 318.35 ± 18.13ABa 989.00 ± 33.05Cab 35 1476.00 ± 94.91Ba 0.74 ± 0.06Aa 127.77 ± 5.85Bc 243.03 ± 10.98Bb 843.00 ± 48.14Bc 70 1479.57 ± 56.54Ca 0.90 ± 0.35Aa 176.34 ± 5.32Bb 275.01 ± 19.86BCb 899.00 ± 45.18Cbc 105 1549.22 ± 41.13Ab 0.98 ± 0.10Aa 232.50 ± 20.78Aa 386.00 ± 21.15Aa 1474 ± 113.78Aa 140 1620.88 ± 39.62Aab 0.96 ± 0.21Aa 205.40 ± 11.71Ab 353.70 ± 27.60Aab 1335.67 ± 69.01Aa BR43 0 1682.72 ± 34.71Aa 1.00 ± 0.17Aa 156.43 ± 6.84Ac 291.14 ± 25.53Ac 1043.00 ± 70.06Ab 35 1707.99 ± 66.96Aa 0.92 ± 0.10Aa 200.00 ± 12.97Ab 316.12 ± 24.15Abc 1175.67 ± 49.37Ab 70 1584.34 ± 44.39Aa 1.03 ± 0.16Aa 216.87 ± 11.11ABa 363.79 ± 19.62Aa 1259.33 ± 60.09Ba 105 1584.18 ± 55.78Aa 0.94 ± 0.11Aa 186.63 ± 3.28Bb 348.35 ± 22.05Aa 1183.00 ± 58.64Ba 140 1608.52 ± 44.86Aa 0.92 ± 0.20Aa 148.01 ± 5.58Ac 279.83 ± 20.95Ab 983.67 ± 58.11Ab LK310 0 1613.83 ± 30.16Ba 0.92 ± 0.01Aa 183.85 ± 5.28Bb 293.70 ± 21.80ABb 1005.33 ± 60.80Bb 35 1329.59 ± 42.90Bb 0.97 ± 0.17Aa 203.54 ± 6.42Ba 314.20 ± 28.90BCa 1230.67 ± 108.56Ba 70 1381.93 ± 69.32Bab 0.98 ± 0.22Aa 175.27 ± 7.06Bb 297.06 ± 19.82Ba 1087.67 ± 28.59BCb 105 1455.88 ± 27.99Ba 0.88 ± 0.26Aa 136.45 ± 6.08Bc 221.83 ± 16.47Bb 778.33 ± 52.60BCc 140 1371.58 ± 33.08Dab 0.91 ± 0.27Aa 177.10 ± 9.30Bb 249.29 ± 15.32CDb 757.33 ± 67.69Dc 3.2 Nodule formation and leghemoglobin content Nodulation characteristics, key indicators of symbiotic efficiency, were significantly influenced by phosphorus (P) supply and exhibited clear varietal differences (Fig. 2 ). The number of nodules formed on high-oil varieties was significantly higher than on non-high-oil varieties across most P levels (Fig. 2 a). This difference was particularly pronounced at 35 kg·hm -2 and 105 kg·hm -2 . At the optimal P supply of 105 kg·hm -2 , the nodule number of high-oil varieties reached its peak, being 83% greater than that of non-high-oil varieties. However, a further increase in P supply to 140 kg·hm -2 led to a decline in nodulation in both variety types. A similar trend was observed for nodule dry weight (Fig. 2 b). Overall, high-oil varieties developed heavier nodules than non-high-oil varieties under all P supply intensities. The maximum nodule dry weight for high-oil varieties was also recorded at 105 kg·hm -2 , which was 30% greater than that of non-high-oil varieties at the same P level. The leghemoglobin (Lb) content in nodules, a direct indicator of nodule activity and nitrogen-fixing potential, was also markedly affected by P supply (Fig. 2 c). The Lb content in high-oil varieties increased with P supply up to 105 kg·hm -2 , reaching a peak value of 49.84 mg·g -1 FW. Across the P gradient, the total Lb content in nodules of high-oil varieties was consistently higher, showing an increase ranging from 11.34% to 33.02% compared to non-high-oil varieties. 3.3 Nitrogen assimilation and accumulation The activity of key nitrogen assimilation enzymes and nitrogen accumulation in leaves were significantly influenced by phosphorus (P) supply, with high-oil varieties demonstrating a superior capacity for nitrogen metabolism (Fig. 3 ). The activity of glutamate synthase (GOGAT) in leaves of both varietal types increased with rising P supply from 0 to 105 kg·hm -2 , peaking at the 105 kg·hm -2 level (Fig. 3 a). At this optimum P supply, the GOGAT activity in high-oil varieties was 9.32% higher than in non-high-oil varieties. Across the P gradient, the GOGAT activity in high-oil varieties was consistently and significantly elevated compared to non-high-oil varieties. Glutamine synthetase (GS) activity exhibited a similar pattern, increasing initially with P supply before declining at the highest level (140 kg·hm -2 ) (Fig. 3 b). The GS activity in high-oil varieties was markedly higher than in non-high-oil varieties under all P regimes. The most striking difference was observed at 105 kg·hm -2 , where the GS activity in high-oil varieties was 17.13% higher than that in non-high-oil varieties. Consistent with the enhanced enzyme activities, the leaf total nitrogen (TN) content of high-oil varieties showed a strong positive response to P application from 35 to 105 kg·hm -2 (Fig. 3 c). At 105 kg·hm -2 , the leaf TN content in high-oil varieties was 30.52% higher than in non-high-oil varieties, underscoring the role of optimal P in promoting nitrogen accumulation. In contrast to TN, the soluble protein (SP) content was generally higher in the leaves of non-high-oil varieties across the P gradient (Fig. 3 d). The SP content in both types increased with P application, reaching maximum values at 105 kg·hm -2 (44.58 mg·g -1 FW in non-high-oil varieties and 39.21 mg·g -1 FW in high-oil varieties). 3.4 Rhizobial community structure and composition To elucidate the microbial mechanism underlying the pronounced differences in plant growth and nodulation observed at the optimal phosphorus (P) supply of 105 kg·hm -2 (Fig. 1 – 3 ), a comparative analysis of the rhizobial communities in nodules from the 0 and 105 kg·hm -2 P treatments was conducted, which represented the two phenotypic extremes. Principal coordinates analysis (PCoA) revealed a clear separation of rhizobial communities driven primarily by P supply and varietal type (Fig. 4 a). The first principal coordinate (PC1), explaining 88.34% of the total variation, distinctly segregated nodules from the P-supplied treatments (35–140 kg·hm -2 ) from the no-P control. Notably, the communities in high-oil varieties under 105 kg·hm -2 P were markedly separated from their no-P controls along PC1, whereas this separation was less evident in non-high-oil varieties. Functional divergence in rhizobial genes between high-oil and non-high-oil soybean varieties, as evidenced by relative abundance variations, is demonstrated (Fig. 4 b). Both varieties exhibited high relative abundance in aerobic_chemoheterotrophy and chemoheterotrophy, indicating these metabolic functions dominate their rhizobial communities. However, high-oil soybeans exhibited more concentrated and elevated distributions in these functions in comparison to non-high-oil varieties, suggesting that their rhizobial communities utilise organic carbon sources more efficiently, thereby providing a stable energy foundation for symbiotic nitrogen fixation. It is noteworthy that high-oil soybeans exhibited a substantially elevated levels of activity for key enzymes central to the nitrogen assimilation process, which is consistent with their augmented nitrogen accumulation capacity. This finding serves to emphasise their superior nitrogen-fixing potential. Conversely, non-high-oil varieties exhibited a reduced abundance of nitrogen fixation-related genes, indicative of diminished symbiotic efficiency. Furthermore, high-oil soybeans exhibited distinctive distributions in ureolysis gene abundance, emphasising their rhizobia's diversified nitrogen source utilisation. Non-high-oil varieties demonstrated weaker performance in this function, indicating limited metabolic flexibility in their rhizobial communities. The relative abundance of key rhizobial genera was significantly altered by P supply in a varietal-dependent manner (Fig. 4 c). The relative abundance of eight key rhizobial genera was significantly reshaped by P supply in a varietal-dependent manner (Fig. 4 c). In high-oil varieties (KN18, KN39), optimal P supply (105 kg·hm -2 ) led to a substantial enrichment of several nitrogen-fixing bacteria, including Bradyrhizobium_sp_173_3_module , Bradyrhizobium_sp_112_module , Bradyrhizobium_diazoefficiens , Rhizobium_sp, Bradyrhizobium_diazotrophicus and Novosphingobium_kaempferiae in high-oil varieties. Conversely, non-high-oil varieties (BR43, LK310) displayed a contrasting response. The abundance of Bradyrhizobium_diazotrophicus (Fig. 4 ) decreased under optimal P in non-high-oil varieties, and Bradyrhizobium_sp_OR_306 showed only a marginal increase compared to the pronounced enrichment in high-oil varieties. Furthermore, the abundance of Tardiphaga_sp was significantly elevated by P supply in non-high-oil varieties but remained largely unchanged in high-oil varieties. 3.5 Correlations between environmental variables and microorganisms in nodules Redundancy analysis (RDA) revealed that the first two axes, RDA1 and RDA2, cumulatively explained 82.75% of the variation in rhizobial OTU structure (70.52% and 12.23%, respectively). RDA1 was identified as the primary axis of variation, driven mainly by phosphorus supply intensity (TP), which showed a significant negative correlation with RDA1 (arrow pointing left). High-oil soybeans exhibited distinct varietal-specific responses to phosphorus: at 0 kg·hm − 2 P supply, KN18 and KN39 clustered closely on the right side of RDA1, indicating strong consistency in rhizobial community structure under phosphorus-limited conditions, with a weak association with TP. At 105 kg·hm − 2 P supply, the rhizobial communities of high-oil varieties displayed a clear phosphorus-responsive gradient. The community of B showed a moderate association with TP, suggesting the initiation of directional restructuring. Notably, D was the only sample among all that exhibited a strong association with TP (minimal arrow angle) and was positioned on the left side of RDA1. Its community structure was fully adapted to the high-phosphorus environment and formed a tight coupling with nitrogen-related factors such as NH 4 + -N, NO 3 − -N, and TN, highlighting a targeted adaptation of the rhizobial community to nitrogen acquisition processes driven by phosphorus. In stark contrast, regardless of phosphorus supply intensity (0 or 105 kg·hm − 2 ), all non-high-oil soybean samples clustered on the right side of RDA1, showing a weak association with phosphorus factors. These samples exhibited high within-group dispersion and no significant differentiation between groups, indicating that their rhizobial communities lacked both phosphorus-dependent directional restructuring and effective coupling with nitrogen factors. 3.6 Correlation analysis between physiological indices and rhizobial abundance The correlative relationships between rhizobial communities and edaphic factors in high-oil and non-high-oil soybean varieties under a phosphorus supply of 105 kg·hm -2 are visualized in the heatmap of Fig. 6 . The richness of the rhizobial community in high-oil soybeans exhibited a significantly stronger correlation with key soil environmental factors compared to conventional soybeans, demonstrating a notable advantage, particularly in response to phosphorus supply. Specifically, at the phosphorus response level, the rhizobial community of high-oil soybeans showed a highly significant positive correlation with soil available phosphorus (AP) (Mantel's *r* ≈ 0.5,*p*< 0.001), a correlation strength substantially higher than that observed in conventional soybeans (Mantel's *r* < 0.25, with *p*-values mostly in the 0.01–0.05 range). This statistical finding was strongly corroborated at both physiological and community levels: under the 105 kg·hm -2 phosphorus level, high-oil soybeans exhibited higher measured AP values in the rhizosphere, coupled with a significant enrichment of Bradyrhizobium species within their nodules. This suggests that high-oil soybeans may enhance the perception and response of their rhizobial community to AP signals, thereby driving the directional assembly of the community towards a more beneficial symbiotic function. Furthermore, regarding nitrogen-related factors, the rhizobial community of high-oil soybeans also showed highly significant positive correlations with soil total nitrogen (TN) and ammonium nitrogen (NH 4 + -N) (*** *p* < 0.001), in the same direction as the correlation with AP, preliminarily revealing a synergistic effect of phosphorus and nitrogen factors in regulating the structure of its rhizobial community. This synergistic regulatory pattern corresponded to a concurrent improvement in host physiological performance: under optimal phosphorus supply, nitrogenase activity in high-oil soybeans increased significantly by 33.02%, and leaf total nitrogen content increased by 17.96%. This implies that phosphorus supply not only directly optimizes the rhizobial community structure but may also, through interaction with nitrogen factors, systematically enhance the overall nitrogen metabolism efficiency in the rhizobia-host interaction. In contrast, the correlations between the rhizobial community of conventional soybeans and AP, TN, and NH 4 + -N were all weaker (Mantel's *r* < 0.3) and exhibited lower significance levels (mostly * *p* < 0.05), failing to form a similar phosphorus-nitrogen synergistic regulatory network. This stark contrast, from a community ecology perspective, provides further evidence that high-oil soybean, as a specific genotype, possesses unique and efficient regulatory mechanisms in phosphorus signal perception, rhizobial community assembly, and host-microbe interaction. 4. Discussion 4.1 Effects of phosphorus supply intensity on growth indicators of different soybean varieties Consistent with established knowledge, our results confirm that phosphorus (P) is a key macronutrient critically regulating soybean growth and development [ 41 ] . The observed general increase in plant height and biomass with increasing P supply, up to an optimum of 105 kg·hm − 2 , aligns with findings from prior studies [ 42 ] . However, the present study further reveals a significant varietal difference in P responsiveness [ 43 ] . The superior plant height, shoot dry weight, and root dry weight of high-oil varieties, particularly at 105 kg·hm − 2 , suggest a more efficient utilization of P resources for biomass accumulation, which may be attributed to their distinct metabolic priorities centered on oil biosynthesis [ 44 ] . The root system, as the primary organ for P acquisition, exhibited high plasticity [ 45 ] . The enhanced total root length, surface area, and volume in high-oil varieties under 70 kg·hm − 2 P supply (Table 1 ) are indicative of a superior foraging strategy under sub-optimal P conditions. This aligns with the classic concept that root architectural traits are crucial for plant phosphorus acquisition [ 46 ] and show high plasticity in response to varying P supply intensities [ 47 ] . The remarkable increase in root surface area (RSA) and root volume (RV) in high-oil varieties likely facilitated greater soil exploration and P uptake, providing a physiological basis for their robust growth. The insensitivity of the average root diameter to P treatment is consistent with some reports [ 48 ] and may be related to the specific developmental stage (V6) at which measurements were taken. Furthermore, optimal P supply significantly promoted nodulation, a cornerstone of soybean nitrogen nutrition [ 49 ] . The peak in nodule number and dry weight at 105 kg·hm − 2 in high-oil varieties (Fig. 2 a, b) is in agreement with the established positive influence of phosphorus application on these symbiotic parameters [ 50 ] . Our findings reinforce that proper P nutrition is essential for building and sustaining an effective nitrogen-fixing machinery in soybean, with high-oil varieties demonstrating a heightened symbiotic investment under optimal conditions [ 51 ] . 4.2 Effects of phosphorus supply intensity on physiological and biochemical characteristics of different soybean varieties Nitrogen metabolism lies at the heart of plant productivity and seed quality [ 52 ] . Our findings demonstrate that phosphorus supply intensity exerts a profound regulatory influence on this process, with high-oil varieties exhibiting a more responsive nitrogen assimilation system [ 53 ] . The significantly higher activities of glutamate synthase (GOGAT) and glutamine synthetase (GS) in high-oil varieties under optimal P supply (105 kg·hm⁻²) (Fig. 3 a, b) indicate a P-enhanced capacity for ammonium assimilation. The GS/GOGAT pathway is recognized as the primary route of nitrogen assimilation in higher plants [ 54 ] , and its activation here underscores the role of P in facilitating efficient nitrogen metabolism [ 55 ] . This coordinated upregulation of key enzymes provides a mechanistic explanation for the concurrent surge in leaf total nitrogen (TN) content in high-oil varieties (Fig. 3 c), which was 30.52% higher than in non-high-oil varieties. This result strongly supports the premise that optimal P nutrition enhances the nitrogen fixation capacity of nodules, thereby increasing nitrogen delivery to the host plant [ 56 , 57 ] . The differential response in soluble protein (SP) content further illuminates the distinct metabolic fates of nitrogen in the two varietal types [ 58 ] . The generally higher SP content in non-high-oil varieties (Fig. 3 d) is consistent with their metabolic prioritization of protein synthesis. The observation that SP content increased with P application, peaking at 105 kg·hm − 2 in both types, aligns with previous reports that P availability affects soluble protein contents in plants [ 59 ] . However, the fact that non-high-oil varieties maintained higher SP levels underscores their inherent sink strength for protein accumulation. This varietal divergence in nitrogen partitioning-towards oil-associated precursors in high-oil types and towards storage proteins in non-high-oil types-must be considered when developing P management strategies aimed at optimizing quality traits [ 60 ] . 4.3 Effects of phosphorus supply intensity on rhizobial communities and functions in different soybean varieties Our study provides compelling evidence that the host plant genotype, modulated by phosphorus (P) supply, serves as a powerful filter in shaping the assembly and function of the nodule rhizobial community. Coupled with the distinct clustering of their communities in the PCoA (Fig. 4 a), demonstrate a more dynamic and responsive symbiotic partnership. This finding aligns with the emerging perspective that nutrient availability can act as an environmental filter to restructure rhizobial communities [ 61 ] . The stronger correlation between the rhizobial communities of high-oil varieties and rhizosphere available P (RS-AP), as revealed by RDA (Fig. 5 a), further reflects their superior phosphorus utilization efficiency, which in turn enhances nutrient acquisition and symbiotic performance. The genus-specific responses underscore a sophisticated, varietal-dependent selection for microbial partners. The signifiant enrichment of key nitrogen-fixing genera such as Bradyrhizobium_sp_173_3_module and Rhizobium_sp in high-oil varieties under 105 kg·hm − 2 P (Fig. 4 c) suggests that these varieties actively recruit a more effective microbiome under favorable nutrient conditions. This selective enrichment likely contributes to their enhanced nodulation and nitrogen acquisition. In contrast, the decline in Bradyrhizobium_diazotrophicus abundance in non-high-oil varieties under the same conditions (Fig. 4 c) highlights that the impact of P is not universal but is finely tuned by the host's physiological and metabolic status [ 62 ] . It can be postulated that in high-oil soybeans, P is primarily allocated to support symbiotic nitrogen fixation that fuels lipid biosynthesis, whereas in non-high-oil varieties, P might be directed towards other metabolic pathways prioritizing protein accumulation. The predicted functional potential of the nodule microbiomes offers a genetic-level corroboration of the observed physiological advantages. The significantly increased abundance of genes related to Nitrogen_Fixation and Aerobic_Chemoheterotrophy in high-oil varieties (Fig. 5 ) correlates well with their superior nodulation capacity (Fig. 2 ), heightened activities of nitrogen assimilation enzymes (Fig. 3 a, b), and consequently, greater nitrogen accumulation (Fig. 3 c). This metabolic efficiency likely stems from an optimized carbon allocation to the rhizobial symbionts, fueling the energy-intensive process of biological nitrogen fixation [ 14 ] . The distinct distribution in Ureolysis gene abundance further emphasizes the diversified nitrogen source utilization strategies of the rhizobial communities in high-oil varieties, potentially offering an additional metabolic flexibility. Collectively, these data support a model where optimal P supply promotes the assembly of a more proficient and metabolically versatile rhizobial consortium in high-oil soybean varieties, which synergistically enhances the overall symbiotic efficiency and plant performance. 5. Conclusion In conclusion, this study demonstrates a pronounced varietal differentiation in the response of soybean to phosphorus supply, underpinned by distinct rhizobial community dynamics. High-oil soybean varieties exhibited greater sensitivity and responsiveness to phosphorus fertilization, with optimal growth, nodulation, and nitrogen assimilation achieved at a supply intensity of 105 kg·hm − 2 . The enhanced performance was mechanistically linked to a phosphorus-driven restructuring of the nodule rhizobial community, characterized by increased diversity and the selective enrichment of key nitrogen-fixing genera. This tailored microbial consortium demonstrated a higher predicted genetic potential for nitrogen acquisition and energy metabolism, which synergistically supported more efficient host nitrogen acquisition and assimilation. These findings provide a scientific basis for precision phosphorus management strategies tailored to specific soybean varieties, with the potential to enhance both productivity and resource use efficiency in agricultural systems. Declarations Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Availability of data and materials All data generated or analysed during this study are included in this published article and its supplementary information files. The raw 16S rRNA gene sequencing data have been deposited in the NCBI database under BioProject Accession: PRJNA1377704 (ID: 1377704). Competing interests The authors declare that they have no competing interests. Funding This work was supported by Major Project of Agricultural Biological Breeding (2023ZD0403106), the Low-carbon Green Agriculture of Grain Crops Project (LJGXCG2022-107), the Postdoctoral Scientific Research Startup Fund Project of Heilongjiang Province (LBH-Q21162), Guiding Science and Technology Plan Project of Daqing City (zd-2025-030, zd-2025-031). Competing Interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. 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16:49:22","extension":"jpeg","order_by":9,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":3915234,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8195203/v1/d8ee6fa29f521e842acd15eb.jpeg"},{"id":98338580,"identity":"8fd89975-cacc-4a90-92ee-2bdbdb7bec11","added_by":"auto","created_at":"2025-12-16 16:49:22","extension":"jpeg","order_by":10,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":3449346,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8195203/v1/c9c2d9e48377eaa4debc2c43.jpeg"},{"id":98437374,"identity":"f3511f0a-8fa2-4447-a5f1-fd1166807e37","added_by":"auto","created_at":"2025-12-17 16:57:15","extension":"jpeg","order_by":11,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1986954,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8195203/v1/3b1af651cf15f423f269646a.jpeg"},{"id":98438608,"identity":"226f15c9-6ad7-480a-a05b-4697049c42fc","added_by":"auto","created_at":"2025-12-17 16:59:38","extension":"jpeg","order_by":12,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":4090080,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8195203/v1/34e93a8adca36ab4987b441a.jpeg"},{"id":98438633,"identity":"ee023593-4d52-446d-a44c-7b10824c9b4e","added_by":"auto","created_at":"2025-12-17 16:59:41","extension":"xml","order_by":19,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":139869,"visible":true,"origin":"","legend":"","description":"","filename":"6a9185dd5ae44dfea21a2f2d76a58b7e1structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8195203/v1/aaebfbb73f50f43e1b309f51.xml"},{"id":98437860,"identity":"820bc500-24a7-460f-a9e0-6363ddfe9c0a","added_by":"auto","created_at":"2025-12-17 16:58:11","extension":"html","order_by":20,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":151184,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8195203/v1/8d3f1575995f71976593590f.html"},{"id":98338559,"identity":"45c89d87-6844-4904-9681-e7d57f91f78b","added_by":"auto","created_at":"2025-12-16 16:49:21","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":164991,"visible":true,"origin":"","legend":"\u003cp\u003eGrowth responses of high-oil and non-high-oil soybean varieties to phosphorus supply. (a) Representative plant phenotypes at 105 kg P₂O₅ hm\u003csup\u003e-2\u003c/sup\u003e. (b) Plant height, (c) root length, (d) shoot dry weight, and (e) root dry weight under different P supply intensities. Different uppercase letters indicate significant differences among P levels, and lowercase letters indicate significant differences between varieties at the same P level (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, Duncan's test).\u003c/p\u003e","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8195203/v1/1e0a27e170fcfa60a2ef2077.png"},{"id":98439264,"identity":"ed404c74-c03e-4da6-8e3d-d51c95368b01","added_by":"auto","created_at":"2025-12-17 17:01:30","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":22968,"visible":true,"origin":"","legend":"\u003cp\u003eNodulation and nodule activity in high-oil and non-high-oil soybean varieties under different phosphorus supply intensities. (a) Nodule number, (b) Nodule dry weight, and (c) Leghemoglobin content in nodules. Different uppercase letters indicate significant differences among P levels, and lowercase letters indicate significant differences between varieties at the same P level (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, Duncan's test).\u003c/p\u003e","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8195203/v1/910e7123fd39f0b1b0f6d7f3.png"},{"id":98438369,"identity":"b3a8ebbc-ebd2-4918-b79a-d9471b2101d6","added_by":"auto","created_at":"2025-12-17 16:59:07","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":57905,"visible":true,"origin":"","legend":"\u003cp\u003eNitrogen assimilation and accumulation in leaves of high-oil and non-high-oil soybean varieties under different phosphorus supply intensities. (a) Glutamate synthase (GOGAT) activity, (b) Glutamine synthetase (GS) activity, (c) Leaf total nitrogen content, and (d) Leaf soluble protein content. Different uppercase letters indicate significant differences among P levels, and lowercase letters indicate significant differences between varieties at the same P level (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, Duncan's test).\u003c/p\u003e","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8195203/v1/dced45c8c95dd18da0b00879.png"},{"id":98438272,"identity":"2c36bc20-1c39-4f35-a40f-ce27c2ff2c30","added_by":"auto","created_at":"2025-12-17 16:58:54","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":42639,"visible":true,"origin":"","legend":"\u003cp\u003eComparative analysis of rhizobial communities in soybean nodules under phosphorus 0 kg·hm\u003csup\u003e-2\u003c/sup\u003e and 105 kg·hm\u003csup\u003e-2\u003c/sup\u003e. (a) Most abundant functional categories obtained using PICRUSt for root nodule samples of high-oil and non-high-oil soybean varieties (KEGG database at level 2) . (b) Principal coordinates analysis (PCoA) based on Bray-Curtis distance. (c-j) Relative abundance of key rhizobial taxa showing significant responses.Treatments are coded as: A: KN18 (0 kg·hm\u003csup\u003e-2\u003c/sup\u003e), B: KN18 (105 kg·hm\u003csup\u003e-2\u003c/sup\u003e), C: KN39 (0 kg·hm\u003csup\u003e-2\u003c/sup\u003e), D: KN39 (105 kg·hm\u003csup\u003e-2\u003c/sup\u003e), E: BR43 (0 kg·hm\u003csup\u003e-2\u003c/sup\u003e), F: BR43 (105 kg·hm\u003csup\u003e-2\u003c/sup\u003e), G: LK310 (0 kg·hm\u003csup\u003e-2\u003c/sup\u003e), H: LK310 (105 kg·hm\u003csup\u003e-2\u003c/sup\u003e).\u003c/p\u003e","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8195203/v1/3ca8aa59682eadf7b2f684c8.png"},{"id":98338567,"identity":"74650ce7-b2f7-4fbc-a8fe-14b4b9d0f5c9","added_by":"auto","created_at":"2025-12-16 16:49:22","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":16920,"visible":true,"origin":"","legend":"\u003cp\u003eRedundancy analysis of rhizosphere and bulk soil environmental factors on rhizobia species abundance in soybean nodules . Treatments are coded as: A: KN18 (0 kg·hm\u003csup\u003e-2\u003c/sup\u003e), B: KN18 (105 kg·hm\u003csup\u003e-2\u003c/sup\u003e), C: KN39 (0 kg·hm\u003csup\u003e-2\u003c/sup\u003e), D: KN39 (105 kg·hm\u003csup\u003e-2\u003c/sup\u003e), E: BR43 (0 kg·hm\u003csup\u003e-2\u003c/sup\u003e), F: BR43 (105 kg·hm\u003csup\u003e-2\u003c/sup\u003e), G: LK310 (0 kg·hm\u003csup\u003e-2\u003c/sup\u003e), H: LK310 (105 kg·hm\u003csup\u003e-2\u003c/sup\u003e).\u003c/p\u003e","description":"","filename":"Onlinefloatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8195203/v1/814cc69d7d9bff4dfa297e4f.png"},{"id":98439371,"identity":"05046c01-92d3-46d8-9c8e-83844da6e37e","added_by":"auto","created_at":"2025-12-17 17:01:43","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":32187,"visible":true,"origin":"","legend":"\u003cp\u003eCorrelation analysis heat map between rhizobia communities and soil environmental factors in rhizosphere and non-rhizosphere soils of high-oil and non-high-oil soybean varieties under 105 kg·hm\u003csup\u003e-2\u003c/sup\u003e phosphorus supply. Note: Line width represents Mantel's r values (correlation strength between rhizobia community composition and environmental factors) and color indicates \u003cem\u003ep\u003c/em\u003e values. Spearman's correlation coefficients between soil environmental parameters are shown through color gradients. Statistical significance is denoted as \u003csup\u003e*\u003c/sup\u003e \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e**\u003c/sup\u003e \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, \u003csup\u003e***\u003c/sup\u003e \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Onlinefloatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-8195203/v1/3842772e3e3c01abd9faeff8.png"},{"id":105755805,"identity":"fe2c879d-a08b-48ef-975d-21776cfc9b25","added_by":"auto","created_at":"2026-03-30 16:30:49","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1710753,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8195203/v1/12a484c8-4efa-4773-8903-4cf28e082ea0.pdf"},{"id":98438573,"identity":"4236b63f-4431-4b76-8e68-6c122cced0ee","added_by":"auto","created_at":"2025-12-17 16:59:33","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":17672,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterial1.docx","url":"https://assets-eu.researchsquare.com/files/rs-8195203/v1/b7c442dea521fcc5482395d5.docx"},{"id":98437881,"identity":"14ed67f7-fdf8-426f-8707-aff245f55f22","added_by":"auto","created_at":"2025-12-17 16:58:12","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":12237,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterial2.docx","url":"https://assets-eu.researchsquare.com/files/rs-8195203/v1/d0686fa5efa05103e7d2befb.docx"},{"id":98338571,"identity":"d02516b3-84fd-42d1-a461-741059b43a9c","added_by":"auto","created_at":"2025-12-16 16:49:22","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":585251,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterial3.docx","url":"https://assets-eu.researchsquare.com/files/rs-8195203/v1/fdcec41edc0f3e46bd5ebdc3.docx"},{"id":98338572,"identity":"844b90b8-52a5-4954-aab2-e6fcb679e035","added_by":"auto","created_at":"2025-12-16 16:49:22","extension":"xlsx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":22987,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterial4.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8195203/v1/36867bf8e1f7e1282d2f1529.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Regulatory mechanism of phosphorus supply intensity on the plant nitrogen acquisition in soybean: Insight from the differences of rhizobia diversity between high-oil soybean and non-high-oil soybean","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eSoybean \u003cem\u003e(Glycine max)\u003c/em\u003e, serves as a critical global source of both edible protein and vegetable oil\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e, playing an indispensable role in food systems and industrial applications \u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e. In China, the Northeast region dominates national production, accounting for approximately 41% of domestic output \u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e. Nonetheless, the persistent gap between domestic supply and demand has resulted in substantial reliance on imports, highlighting an urgent need to enhance both the yield and quality of Chinese soybean \u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e. Historically, breeding efforts have often faced a trade-off between selecting for higher seed yield and higher oil content, presenting a key scientific and practical challenge\u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e. Therefore, achieving synergistic improvement in soybean yield and oil production represents a crucial objective for ensuring national food security and promoting sustainable agricultural development \u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe symbiotic relationship with nitrogen-fixing rhizobia (predominantly \u003cem\u003eBradyrhizobium\u003c/em\u003e and \u003cem\u003eSinorhizobium\u003c/em\u003e spp.) is a cornerstone of soybean productivity\u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e, capable of fulfilling up to 70% of the plant's nitrogen demand through biological nitrogen fixation (BNF) within root nodules \u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e. Beyond nitrogen provision, rhizobia enhance soybean growth, yield, and quality by modulating seed composition\u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e. Critically, soybean varieties bred for distinct metabolic priorities-such as high-oil varieties prioritizing lipid biosynthesis versus conventional varieties prioritizing protein accumulation-may establish distinct interactions with rhizobia. These varieties differences in rhizobial partnerships and nitrogen acquisition efficiency could directly influence carbon allocation patterns, thereby ultimately determining seed oil-to-protein ratios \u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003ePhosphorus, as a key macronutrient and fundamental component of ATP, nucleic acids, and phospholipids, critically regulates soybean growth and physiological processes \u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e). In the context of the soybean-rhizobia symbiosis, P supply regulates nitrogen acquisition through multiple interconnected mechanisms \u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e. First, the inherent energy demand of biological nitrogen acquisition means that phosphorus availability directly limits nitrogenase activity by governing ATP supply \u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e, with deficiency severely curtailing this process irrespective of nodule development \u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e. Second, P deficiency can antagonize nodulation signaling pathways, potentially altering flavonoid exudation and impairing early infection events such as infection thread formation \u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e. Furthermore, an emerging perspective suggests that P supply acts as a powerful environmental filter\u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e, restructuring the rhizobial community in the rhizosphere and nodules to potentially select for more adaptive strains with synergistic traits like phosphorus solubilization \u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e. However, how these P-driven changes in rhizobial communities and nitrogen acquisition efficiency differentially affect nitrogen acquisition and carbon allocation in soybean varieties with contrasting metabolic sinks-specifically\u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e, high-oil varieties optimized for lipid biosynthesis versus conventional non-high-oil varieties-remains poorly understood and constitutes a key knowledge gap\u003csup\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eFurthermore, phosphorus availability critically shapes the rhizosphere microbial community\u003csup\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e, which in turn influences nutrient cycling and plant health. An optimal phosphorus supply stabilizes microbial dynamics and selectively promotes strains with beneficial traits, such as phosphorus solubilization and the production of plant growth-promoting substances\u003csup\u003e[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e.. These microbial activities enhance phosphorus use efficiency and stimulate root development and nodulation, thereby indirectly supporting the soybean-rhizobia symbiotic partnership \u003csup\u003e[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e. Consequently, phosphorus serves a dual role: it directly sustains plant physiological processes and indirectly modulates the microbial partners that are essential for sustainable soybean cultivation\u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eDespite its importance, the genotype-specific responses of the soybean\u0026ndash;rhizobia symbiosis to phosphorus supply, particularly the underlying microbial mechanisms, remain poorly understood. While appropriate phosphorus application enhances yield and quality, excessive use can reduce phosphorus use efficiency and even depress yield\u003csup\u003e[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e.. Furthermore, how rhizobial community structures vary among soybean varieties and respond to phosphorus levels is not yet clear. This study investigates the regulatory role of phosphorus supply intensity on nitrogen acquisition in high-oil and non-high-oil soybeans. We hypothesize that high-oil varieties host a more responsive rhizobial community under optimal phosphorus supply, thereby enhancing nitrogen assimilation efficiency. To test this, we will: (1) compare plant growth and root architecture traits; (2) evaluate nodulation and the activities of key nitrogen assimilation enzymes (GS/GOGAT); and (3) characterize the composition and functional potential of the nodule rhizobial communities. Our findings aim to establish a theoretical basis for precision phosphorus management strategies tailored to different soybean varieties.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003e2.1 Plant and soil materials\u003c/h2\u003e\n \u003cp\u003eThe experimental soil was collected from the top layer (0\u0026ndash;20 cm) of fields at the Jiu San Branch of the Beidahuang Group and the Heshan Farm Science and Technology Park, located in Heihe, Heilongjiang Province, China (48\u0026deg;43\u0026prime;N, 124\u0026deg;56\u0026prime;E). The soil is classified as a typical black soil (Mollisol). The air-dried soil was ground and passed through a 2-mm sieve for subsequent use. Seeds of soybean (\u003cem\u003eGlycine max\u003c/em\u003e (L.) Merr.), including two high-oil varieties, Kenong18 (KN18) and Kenong39 (KN39), and two non-high-oil varieties, Heihe43 (HR43) and Longken310 (LK310), all bred in Heilongjiang Province, were used in this study. No artificial rhizobial inoculation was performed, relying solely on the native rhizobia present in the field soil.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\n \u003ch2\u003e2.2 Experimental design and cultivation\u003c/h2\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n \u003cp\u003eThis study established five phosphorus (P) application levels: 0, 0.007, 0.014, 0.021, and 0.028 g P₂O₅ per kg of soil (simulating field application rates of 0, 35, 70, 105, and 140 kg P₂O₅\u0026middot;hm\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e, respectively). Phosphorus was applied as superphosphate and thoroughly mixed with the substrate before potting. Germinated soybean seeds were planted in individual pots, each containing 500 g of the prepared substrate (composed of soil and vermiculite at a ratio of 3:1 by weight). The plants were cultivated in a greenhouse at the College of Agriculture, Heilongjiang Bayi Agricultural University, under controlled conditions: 23\u0026deg;C, with a 16/8 h light/dark photoperiod. Sterilized deionized water was supplied every two days to maintain soil moisture at approximately 60% of the water holding capacity until the plants reached the V6 stage. All treatments were arranged in a completely randomized design with three independent replications.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\n \u003cp\u003eAt the V6 stage, soybean plants were harvested. The root systems were carefully excavated. Rhizosphere soil (RS) was collected by gently shaking the roots to remove the loosely adhered soil. Bulk soil (BS) was collected from areas in the pot with no visible root presence. Plant shoots, roots, and nodules were separated. Shoots and roots were used for measurements of plant height, root length, and dry weight. Root systems were scanned for morphological analysis. Nodules were counted and weighed, with a subset used for leghemoglobin (Lb) content determination and another stored at -80\u0026deg;C for subsequent DNA extraction and analysis of the rhizobial community structure. Leaf samples were collected, immediately frozen in liquid nitrogen, and stored at -80\u0026deg;C for subsequent analysis of nitrogen assimilation enzymes and nutrient content. Soil samples were air-dried for physicochemical analysis.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\n \u003ch2\u003e2.3 Assessment of plant physiological indices\u003c/h2\u003e\n \u003cp\u003ePlant height and root length were measured on fresh samples using vernier calipers. Shoot dry weight (SDW) and root dry weight (RDW) were determined after oven-drying the samples to a constant mass \u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/sup\u003e. Root systems were scanned using an Epson TWAIN Pro scanner, and the images were analyzed with WinRHIZO Pro software (Regent Instruments, Inc., Quebec, Canada) to determine total root length (TRL), average root diameter (AvgDiam), root surface area (RSA), root volume (RV), and the root apical number (RAN) \u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e31\u003c/span\u003e]\u003c/sup\u003e. Nodules were counted and weighed to determine nodule number and dry weight. Leghemoglobin (Lb) content in nodules was quantified fluorometrically with excitation and emission wavelengths set at 405 nm and 650 nm, respectively \u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e]\u003c/sup\u003e. For leaf analyses, total nitrogen (TN) content was assessed using the Kjeldahl method. The activities of glutamate synthase (GOGAT) and glutamine synthetase (GS) were determined according to the methods described by Ullah et al\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/sup\u003e. Soluble protein (SP) content was measured using the Coomassie brilliant blue G-250 staining method \u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e34\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n \u003ch2\u003e2.4 Assessment of soil physicochemical properties\u003c/h2\u003e\n \u003cp\u003eAir-dried rhizosphere soil (RS) and bulk soil (BS) samples were passed through 1-mm and 0.25-mm sieves for subsequent analyses \u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e35\u003c/span\u003e]\u003c/sup\u003e. Soil pH and electrical conductivity (EC) were measured in a soil-water suspension (1:2.5, w/v) using a digital pH meter and a conductivity meter, respectively \u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e36\u003c/span\u003e]\u003c/sup\u003e. Soil total nitrogen (TN) content was determined using a semi-automatic nitrogen analyzer \u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e37\u003c/span\u003e]\u003c/sup\u003e. Total phosphorus (TP) and available phosphorus (AP) were measured by the molybdenum-antimony colorimetric method \u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e38\u003c/span\u003e]\u003c/sup\u003e. Ammonium nitrogen (NH₄⁺-N) and nitrate nitrogen (NO₃⁻-N) concentrations were determined colorimetrically using the indophenol blue method and the hydrazine sulfate reduction method, respectively \u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e39\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\n \u003ch2\u003e2.5 Molecular analyses\u003c/h2\u003e\n \u003cp\u003eTotal DNA was extracted from surface-sterilized nodules using a commercial kit. The concentration and purity of the extracted DNA were verified, and the DNA was stored at -20\u0026deg;C prior to further analysis. The \u003cem\u003erpoB\u003c/em\u003e gene, selected for its high phylogenetic resolution in distinguishing closely related rhizobial species, was amplified by polymerase chain reaction (PCR) using the primers \u003cem\u003erpoB1479-F\u003c/em\u003e (5\u0026prime;-GAT CGA RAC GCC GGA AGG-3\u0026prime;) and \u003cem\u003erpoB1831-R\u003c/em\u003e (5\u0026prime;-TGC ATG TTC GAR CCC AT-3\u0026prime;)\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e40\u003c/span\u003e]\u003c/sup\u003e. The PCR amplification was performed in duplicate for each sample. The 20 \u0026micro;L reaction mixture contained 4 \u0026micro;L of 5\u0026times; FastPfu Buffer, 2 \u0026micro;L of 2.5 mM dNTPs, 0.8 \u0026micro;L of each primer (5 \u0026micro;M), 0.4 \u0026micro;L of FastPfu Polymerase, and 10 ng of template DNA. The thermal cycling conditions were as follows: initial denaturation at 95\u0026deg;C for 2 min; 25 cycles of denaturation at 95\u0026deg;C for 30 s, annealing at 55\u0026deg;C for 30 s, and extension at 72\u0026deg;C for 30 s; with a final extension at 72\u0026deg;C for 5 min. The PCR products were confirmed by electrophoresis on a 1% agarose gel and purified using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, U.S.A.). Equimolar amounts of purified amplicons from the same sample were pooled together. The pooled libraries were subjected to paired-end sequencing on the Illumina MiSeq platform (Majorbio Bio-pharm Technology Co., Ltd., Shanghai, China). Subsequent bioinformatic analysis, including taxonomic classification and diversity assessments, was performed following the pipeline established by Jiang et al. (2024).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\n \u003ch2\u003e2.6 Statistical analyses\u003c/h2\u003e\n \u003cp\u003eAll data were subjected to a one-way analysis of variance (ANOVA) using the SPSS 25.0 software package. When the ANOVA revealed significant effects, treatment means were compared using Duncan\u0026apos;s multiple range test at a significance level of P\u0026thinsp;\u0026lt;\u0026thinsp;0.05. All results, obtained from three independent replicates, are presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). Redundancy analysis (RDA) was performed to explore the relationships between rhizobial community composition and environmental variables using the vegan package in R 4.5.1. Figures were generated using Origin Pro 2024 and the ggplot2 package in R.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Plant growth and phenotypic visualization of roots\u003c/h2\u003e \u003cp\u003eVisual observation at the optimal phosphorus (P) supply of 105 kg\u0026middot;hm\u003csup\u003e-2\u003c/sup\u003e clearly demonstrated more robust growth in high-oil soybean varieties compared to non-high-oil varieties (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea)Quantification of growth parameters across the P gradient confirmed this varietal difference (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb-e). Plant height, shoot dry weight, and root dry weight of high-oil varieties were generally superior to those of non-high-oil varieties across all P levels, with the most pronounced differences observed at 105 kg\u0026middot;hm\u003csup\u003e-2\u003c/sup\u003e P supply (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb, d, e). At this optimum, the plant height of high-oil varieties was 19.99% greater than that of non-high-oil varieties. Similarly, shoot and root dry weights of high-oil varieties peaked, showing significant increases of 4.13% and 17.96%, respectively, compared to non-high-oil varieties under the same P supply. The response of root length to increasing P supply was distinct from that of other growth parameters (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). Root length of high-oil varieties increased from 0 to 70 kg\u0026middot;hm\u003csup\u003e-2\u003c/sup\u003e but then declined at 105 kg\u0026middot;hm\u003csup\u003e-2\u003c/sup\u003e. In contrast, non-high-oil varieties exhibited a continuous increase. Despite this, the root length of high-oil varieties at 105 kg\u0026middot;hm\u003csup\u003e-2\u003c/sup\u003e remained 31.05% longer than that of non-high-oil varieties.\u003c/p\u003e \u003cp\u003eComprehensive root system architecture analysis revealed that a P supply of 70 kg\u0026middot;hm\u003csup\u003e-2\u003c/sup\u003e was most effective in promoting root expansion for both variety types (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). At this level, high-oil varieties significantly outperformed non-high-oil varieties in key morphological traits. The total root length (TRL) of high-oil varieties was 7.0% greater than that of non-high-oil varieties. More notably, high-oil varieties exhibited substantially larger root surface area (RSA, 21.8% greater), root volume (RV, 22.9% greater), and number of root tips (26.6% greater). In contrast, the average root diameter (AvgDiam) was less responsive to P supply and showed no consistent varietal difference.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eRoot architecture traits of high-oil and non-high-oil soybean varieties in response to phosphorus supply. Data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (n\u0026thinsp;=\u0026thinsp;3). Different uppercase letters indicate significant differences among P levels, and lowercase letters indicate significant differences between varieties at the same P level (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Duncan's test).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTreatment\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePhosphate fertilizer application rate\u003c/p\u003e \u003cp\u003e(kg\u0026middot;hm\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLength\u003c/p\u003e \u003cp\u003e(cm)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAvgDiam (mm)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eRootVolume\u003c/p\u003e \u003cp\u003e(cm\u003csup\u003e3\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eSurfArea\u003c/p\u003e \u003cp\u003e(cm\u003csup\u003e2\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eTips\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"4\" rowspan=\"5\"\u003e \u003cp\u003eKN18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1134.07\u0026thinsp;\u0026plusmn;\u0026thinsp;53.88Cb\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.89\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19Aa\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e162.43\u0026thinsp;\u0026plusmn;\u0026thinsp;8.81Cab\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e285.52\u0026thinsp;\u0026plusmn;\u0026thinsp;18.18Ca\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e 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colname=\"c2\"\u003e \u003cp\u003e70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1219.89\u0026thinsp;\u0026plusmn;\u0026thinsp;42.64Ca\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.87\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20Aa\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e125.76\u0026thinsp;\u0026plusmn;\u0026thinsp;5.75Bc\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e218.97\u0026thinsp;\u0026plusmn;\u0026thinsp;18.71Bb\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e673.33\u0026thinsp;\u0026plusmn;\u0026thinsp;59.53Cb\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e105\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1246.82\u0026thinsp;\u0026plusmn;\u0026thinsp;42.49Ea\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.88\u0026thinsp;\u0026plusmn;\u0026thinsp;0.26Aa\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e173.95\u0026thinsp;\u0026plusmn;\u0026thinsp;4.54Bab\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e249.90\u0026thinsp;\u0026plusmn;\u0026thinsp;17.85Dab\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e711.67\u0026thinsp;\u0026plusmn;\u0026thinsp;45.98Dab\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e140\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1356.40\u0026thinsp;\u0026plusmn;\u0026thinsp;51.22Ba\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e 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\u003cp\u003e0.93\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17Aa\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e168.60\u0026thinsp;\u0026plusmn;\u0026thinsp;11.45Bb\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e318.35\u0026thinsp;\u0026plusmn;\u0026thinsp;18.13ABa\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e989.00\u0026thinsp;\u0026plusmn;\u0026thinsp;33.05Cab\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1476.00\u0026thinsp;\u0026plusmn;\u0026thinsp;94.91Ba\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.74\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06Aa\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e127.77\u0026thinsp;\u0026plusmn;\u0026thinsp;5.85Bc\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e243.03\u0026thinsp;\u0026plusmn;\u0026thinsp;10.98Bb\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e843.00\u0026thinsp;\u0026plusmn;\u0026thinsp;48.14Bc\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1479.57\u0026thinsp;\u0026plusmn;\u0026thinsp;56.54Ca\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.90\u0026thinsp;\u0026plusmn;\u0026thinsp;0.35Aa\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e176.34\u0026thinsp;\u0026plusmn;\u0026thinsp;5.32Bb\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e275.01\u0026thinsp;\u0026plusmn;\u0026thinsp;19.86BCb\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e899.00\u0026thinsp;\u0026plusmn;\u0026thinsp;45.18Cbc\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e105\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1549.22\u0026thinsp;\u0026plusmn;\u0026thinsp;41.13Ab\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.98\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10Aa\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e232.50\u0026thinsp;\u0026plusmn;\u0026thinsp;20.78Aa\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e386.00\u0026thinsp;\u0026plusmn;\u0026thinsp;21.15Aa\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1474\u0026thinsp;\u0026plusmn;\u0026thinsp;113.78Aa\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e140\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1620.88\u0026thinsp;\u0026plusmn;\u0026thinsp;39.62Aab\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.96\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21Aa\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e205.40\u0026thinsp;\u0026plusmn;\u0026thinsp;11.71Ab\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e353.70\u0026thinsp;\u0026plusmn;\u0026thinsp;27.60Aab\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1335.67\u0026thinsp;\u0026plusmn;\u0026thinsp;69.01Aa\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"4\" rowspan=\"5\"\u003e \u003cp\u003eBR43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1682.72\u0026thinsp;\u0026plusmn;\u0026thinsp;34.71Aa\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17Aa\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e156.43\u0026thinsp;\u0026plusmn;\u0026thinsp;6.84Ac\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e291.14\u0026thinsp;\u0026plusmn;\u0026thinsp;25.53Ac\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1043.00\u0026thinsp;\u0026plusmn;\u0026thinsp;70.06Ab\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1707.99\u0026thinsp;\u0026plusmn;\u0026thinsp;66.96Aa\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.92\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10Aa\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e200.00\u0026thinsp;\u0026plusmn;\u0026thinsp;12.97Ab\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e316.12\u0026thinsp;\u0026plusmn;\u0026thinsp;24.15Abc\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1175.67\u0026thinsp;\u0026plusmn;\u0026thinsp;49.37Ab\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1584.34\u0026thinsp;\u0026plusmn;\u0026thinsp;44.39Aa\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16Aa\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e216.87\u0026thinsp;\u0026plusmn;\u0026thinsp;11.11ABa\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e363.79\u0026thinsp;\u0026plusmn;\u0026thinsp;19.62Aa\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1259.33\u0026thinsp;\u0026plusmn;\u0026thinsp;60.09Ba\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e105\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1584.18\u0026thinsp;\u0026plusmn;\u0026thinsp;55.78Aa\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.94\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11Aa\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e186.63\u0026thinsp;\u0026plusmn;\u0026thinsp;3.28Bb\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e348.35\u0026thinsp;\u0026plusmn;\u0026thinsp;22.05Aa\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1183.00\u0026thinsp;\u0026plusmn;\u0026thinsp;58.64Ba\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e140\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1608.52\u0026thinsp;\u0026plusmn;\u0026thinsp;44.86Aa\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.92\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20Aa\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e148.01\u0026thinsp;\u0026plusmn;\u0026thinsp;5.58Ac\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e279.83\u0026thinsp;\u0026plusmn;\u0026thinsp;20.95Ab\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e983.67\u0026thinsp;\u0026plusmn;\u0026thinsp;58.11Ab\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"4\" rowspan=\"5\"\u003e \u003cp\u003eLK310\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1613.83\u0026thinsp;\u0026plusmn;\u0026thinsp;30.16Ba\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.92\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01Aa\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e183.85\u0026thinsp;\u0026plusmn;\u0026thinsp;5.28Bb\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e293.70\u0026thinsp;\u0026plusmn;\u0026thinsp;21.80ABb\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1005.33\u0026thinsp;\u0026plusmn;\u0026thinsp;60.80Bb\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1329.59\u0026thinsp;\u0026plusmn;\u0026thinsp;42.90Bb\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.97\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17Aa\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e203.54\u0026thinsp;\u0026plusmn;\u0026thinsp;6.42Ba\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e314.20\u0026thinsp;\u0026plusmn;\u0026thinsp;28.90BCa\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1230.67\u0026thinsp;\u0026plusmn;\u0026thinsp;108.56Ba\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1381.93\u0026thinsp;\u0026plusmn;\u0026thinsp;69.32Bab\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.98\u0026thinsp;\u0026plusmn;\u0026thinsp;0.22Aa\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e175.27\u0026thinsp;\u0026plusmn;\u0026thinsp;7.06Bb\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e297.06\u0026thinsp;\u0026plusmn;\u0026thinsp;19.82Ba\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1087.67\u0026thinsp;\u0026plusmn;\u0026thinsp;28.59BCb\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e105\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1455.88\u0026thinsp;\u0026plusmn;\u0026thinsp;27.99Ba\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.88\u0026thinsp;\u0026plusmn;\u0026thinsp;0.26Aa\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e136.45\u0026thinsp;\u0026plusmn;\u0026thinsp;6.08Bc\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e221.83\u0026thinsp;\u0026plusmn;\u0026thinsp;16.47Bb\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e778.33\u0026thinsp;\u0026plusmn;\u0026thinsp;52.60BCc\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e140\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1371.58\u0026thinsp;\u0026plusmn;\u0026thinsp;33.08Dab\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.91\u0026thinsp;\u0026plusmn;\u0026thinsp;0.27Aa\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e177.10\u0026thinsp;\u0026plusmn;\u0026thinsp;9.30Bb\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e249.29\u0026thinsp;\u0026plusmn;\u0026thinsp;15.32CDb\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e757.33\u0026thinsp;\u0026plusmn;\u0026thinsp;67.69Dc\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Nodule formation and leghemoglobin content\u003c/h2\u003e \u003cp\u003eNodulation characteristics, key indicators of symbiotic efficiency, were significantly influenced by phosphorus (P) supply and exhibited clear varietal differences (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The number of nodules formed on high-oil varieties was significantly higher than on non-high-oil varieties across most P levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). This difference was particularly pronounced at 35 kg\u0026middot;hm\u003csup\u003e-2\u003c/sup\u003e and 105 kg\u0026middot;hm\u003csup\u003e-2\u003c/sup\u003e. At the optimal P supply of 105 kg\u0026middot;hm\u003csup\u003e-2\u003c/sup\u003e, the nodule number of high-oil varieties reached its peak, being 83% greater than that of non-high-oil varieties. However, a further increase in P supply to 140 kg\u0026middot;hm\u003csup\u003e-2\u003c/sup\u003e led to a decline in nodulation in both variety types. A similar trend was observed for nodule dry weight (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). Overall, high-oil varieties developed heavier nodules than non-high-oil varieties under all P supply intensities. The maximum nodule dry weight for high-oil varieties was also recorded at 105 kg\u0026middot;hm\u003csup\u003e-2\u003c/sup\u003e, which was 30% greater than that of non-high-oil varieties at the same P level. The leghemoglobin (Lb) content in nodules, a direct indicator of nodule activity and nitrogen-fixing potential, was also markedly affected by P supply (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). The Lb content in high-oil varieties increased with P supply up to 105 kg\u0026middot;hm\u003csup\u003e-2\u003c/sup\u003e, reaching a peak value of 49.84 mg\u0026middot;g\u003csup\u003e-1\u003c/sup\u003e FW. Across the P gradient, the total Lb content in nodules of high-oil varieties was consistently higher, showing an increase ranging from 11.34% to 33.02% compared to non-high-oil varieties.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Nitrogen assimilation and accumulation\u003c/h2\u003e \u003cp\u003eThe activity of key nitrogen assimilation enzymes and nitrogen accumulation in leaves were significantly influenced by phosphorus (P) supply, with high-oil varieties demonstrating a superior capacity for nitrogen metabolism (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The activity of glutamate synthase (GOGAT) in leaves of both varietal types increased with rising P supply from 0 to 105 kg\u0026middot;hm\u003csup\u003e-2\u003c/sup\u003e, peaking at the 105 kg\u0026middot;hm\u003csup\u003e-2\u003c/sup\u003e level (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). At this optimum P supply, the GOGAT activity in high-oil varieties was 9.32% higher than in non-high-oil varieties. Across the P gradient, the GOGAT activity in high-oil varieties was consistently and significantly elevated compared to non-high-oil varieties. Glutamine synthetase (GS) activity exhibited a similar pattern, increasing initially with P supply before declining at the highest level (140 kg\u0026middot;hm\u003csup\u003e-2\u003c/sup\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). The GS activity in high-oil varieties was markedly higher than in non-high-oil varieties under all P regimes. The most striking difference was observed at 105 kg\u0026middot;hm\u003csup\u003e-2\u003c/sup\u003e, where the GS activity in high-oil varieties was 17.13% higher than that in non-high-oil varieties. Consistent with the enhanced enzyme activities, the leaf total nitrogen (TN) content of high-oil varieties showed a strong positive response to P application from 35 to 105 kg\u0026middot;hm\u003csup\u003e-2\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). At 105 kg\u0026middot;hm\u003csup\u003e-2\u003c/sup\u003e, the leaf TN content in high-oil varieties was 30.52% higher than in non-high-oil varieties, underscoring the role of optimal P in promoting nitrogen accumulation. In contrast to TN, the soluble protein (SP) content was generally higher in the leaves of non-high-oil varieties across the P gradient (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed). The SP content in both types increased with P application, reaching maximum values at 105 kg\u0026middot;hm\u003csup\u003e-2\u003c/sup\u003e (44.58 mg\u0026middot;g\u003csup\u003e-1\u003c/sup\u003e FW in non-high-oil varieties and 39.21 mg\u0026middot;g\u003csup\u003e-1\u003c/sup\u003e FW in high-oil varieties).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Rhizobial community structure and composition\u003c/h2\u003e \u003cp\u003eTo elucidate the microbial mechanism underlying the pronounced differences in plant growth and nodulation observed at the optimal phosphorus (P) supply of 105 kg\u0026middot;hm\u003csup\u003e-2\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), a comparative analysis of the rhizobial communities in nodules from the 0 and 105 kg\u0026middot;hm\u003csup\u003e-2\u003c/sup\u003e P treatments was conducted, which represented the two phenotypic extremes.\u003c/p\u003e \u003cp\u003ePrincipal coordinates analysis (PCoA) revealed a clear separation of rhizobial communities driven primarily by P supply and varietal type (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). The first principal coordinate (PC1), explaining 88.34% of the total variation, distinctly segregated nodules from the P-supplied treatments (35\u0026ndash;140 kg\u0026middot;hm\u003csup\u003e-2\u003c/sup\u003e) from the no-P control. Notably, the communities in high-oil varieties under 105 kg\u0026middot;hm\u003csup\u003e-2\u003c/sup\u003e P were markedly separated from their no-P controls along PC1, whereas this separation was less evident in non-high-oil varieties.\u003c/p\u003e \u003cp\u003eFunctional divergence in rhizobial genes between high-oil and non-high-oil soybean varieties, as evidenced by relative abundance variations, is demonstrated (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). Both varieties exhibited high relative abundance in aerobic_chemoheterotrophy and chemoheterotrophy, indicating these metabolic functions dominate their rhizobial communities. However, high-oil soybeans exhibited more concentrated and elevated distributions in these functions in comparison to non-high-oil varieties, suggesting that their rhizobial communities utilise organic carbon sources more efficiently, thereby providing a stable energy foundation for symbiotic nitrogen fixation. It is noteworthy that high-oil soybeans exhibited a substantially elevated levels of activity for key enzymes central to the nitrogen assimilation process, which is consistent with their augmented nitrogen accumulation capacity. This finding serves to emphasise their superior nitrogen-fixing potential. Conversely, non-high-oil varieties exhibited a reduced abundance of nitrogen fixation-related genes, indicative of diminished symbiotic efficiency. Furthermore, high-oil soybeans exhibited distinctive distributions in ureolysis gene abundance, emphasising their rhizobia's diversified nitrogen source utilisation. Non-high-oil varieties demonstrated weaker performance in this function, indicating limited metabolic flexibility in their rhizobial communities.\u003c/p\u003e \u003cp\u003eThe relative abundance of key rhizobial genera was significantly altered by P supply in a varietal-dependent manner (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). The relative abundance of eight key rhizobial genera was significantly reshaped by P supply in a varietal-dependent manner (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). In high-oil varieties (KN18, KN39), optimal P supply (105 kg\u0026middot;hm\u003csup\u003e-2\u003c/sup\u003e) led to a substantial enrichment of several nitrogen-fixing bacteria, including \u003cem\u003eBradyrhizobium_sp_173_3_module\u003c/em\u003e, \u003cem\u003eBradyrhizobium_sp_112_module\u003c/em\u003e, \u003cem\u003eBradyrhizobium_diazoefficiens\u003c/em\u003e, Rhizobium_sp, \u003cem\u003eBradyrhizobium_diazotrophicus\u003c/em\u003e and \u003cem\u003eNovosphingobium_kaempferiae\u003c/em\u003e in high-oil varieties. Conversely, non-high-oil varieties (BR43, LK310) displayed a contrasting response. The abundance of \u003cem\u003eBradyrhizobium_diazotrophicus\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e) decreased under optimal P in non-high-oil varieties, and \u003cem\u003eBradyrhizobium_sp_OR_306\u003c/em\u003e showed only a marginal increase compared to the pronounced enrichment in high-oil varieties. Furthermore, the abundance of \u003cem\u003eTardiphaga_sp\u003c/em\u003e was significantly elevated by P supply in non-high-oil varieties but remained largely unchanged in high-oil varieties.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Correlations between environmental variables and microorganisms in nodules\u003c/h2\u003e \u003cp\u003eRedundancy analysis (RDA) revealed that the first two axes, RDA1 and RDA2, cumulatively explained 82.75% of the variation in rhizobial OTU structure (70.52% and 12.23%, respectively). RDA1 was identified as the primary axis of variation, driven mainly by phosphorus supply intensity (TP), which showed a significant negative correlation with RDA1 (arrow pointing left). High-oil soybeans exhibited distinct varietal-specific responses to phosphorus: at 0 kg\u0026middot;hm\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e P supply, KN18 and KN39 clustered closely on the right side of RDA1, indicating strong consistency in rhizobial community structure under phosphorus-limited conditions, with a weak association with TP. At 105 kg\u0026middot;hm\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e P supply, the rhizobial communities of high-oil varieties displayed a clear phosphorus-responsive gradient. The community of B showed a moderate association with TP, suggesting the initiation of directional restructuring. Notably, D was the only sample among all that exhibited a strong association with TP (minimal arrow angle) and was positioned on the left side of RDA1. Its community structure was fully adapted to the high-phosphorus environment and formed a tight coupling with nitrogen-related factors such as NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N, NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N, and TN, highlighting a targeted adaptation of the rhizobial community to nitrogen acquisition processes driven by phosphorus. In stark contrast, regardless of phosphorus supply intensity (0 or 105 kg\u0026middot;hm\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e), all non-high-oil soybean samples clustered on the right side of RDA1, showing a weak association with phosphorus factors. These samples exhibited high within-group dispersion and no significant differentiation between groups, indicating that their rhizobial communities lacked both phosphorus-dependent directional restructuring and effective coupling with nitrogen factors.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.6 Correlation analysis between physiological indices and rhizobial abundance\u003c/h2\u003e \u003cp\u003eThe correlative relationships between rhizobial communities and edaphic factors in high-oil and non-high-oil soybean varieties under a phosphorus supply of 105 kg\u0026middot;hm\u003csup\u003e-2\u003c/sup\u003e are visualized in the heatmap of Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e. The richness of the rhizobial community in high-oil soybeans exhibited a significantly stronger correlation with key soil environmental factors compared to conventional soybeans, demonstrating a notable advantage, particularly in response to phosphorus supply.\u003c/p\u003e \u003cp\u003eSpecifically, at the phosphorus response level, the rhizobial community of high-oil soybeans showed a highly significant positive correlation with soil available phosphorus (AP) (Mantel's *r* \u0026asymp; 0.5,*p*\u0026lt; 0.001), a correlation strength substantially higher than that observed in conventional soybeans (Mantel's *r* \u0026lt; 0.25, with *p*-values mostly in the 0.01\u0026ndash;0.05 range). This statistical finding was strongly corroborated at both physiological and community levels: under the 105 kg\u0026middot;hm\u003csup\u003e-2\u003c/sup\u003e phosphorus level, high-oil soybeans exhibited higher measured AP values in the rhizosphere, coupled with a significant enrichment of Bradyrhizobium species within their nodules. This suggests that high-oil soybeans may enhance the perception and response of their rhizobial community to AP signals, thereby driving the directional assembly of the community towards a more beneficial symbiotic function.\u003c/p\u003e \u003cp\u003eFurthermore, regarding nitrogen-related factors, the rhizobial community of high-oil soybeans also showed highly significant positive correlations with soil total nitrogen (TN) and ammonium nitrogen (NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N) (*** *p* \u0026lt; 0.001), in the same direction as the correlation with AP, preliminarily revealing a synergistic effect of phosphorus and nitrogen factors in regulating the structure of its rhizobial community. This synergistic regulatory pattern corresponded to a concurrent improvement in host physiological performance: under optimal phosphorus supply, nitrogenase activity in high-oil soybeans increased significantly by 33.02%, and leaf total nitrogen content increased by 17.96%. This implies that phosphorus supply not only directly optimizes the rhizobial community structure but may also, through interaction with nitrogen factors, systematically enhance the overall nitrogen metabolism efficiency in the rhizobia-host interaction.\u003c/p\u003e \u003cp\u003eIn contrast, the correlations between the rhizobial community of conventional soybeans and AP, TN, and NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N were all weaker (Mantel's *r* \u0026lt; 0.3) and exhibited lower significance levels (mostly * *p* \u0026lt; 0.05), failing to form a similar phosphorus-nitrogen synergistic regulatory network. This stark contrast, from a community ecology perspective, provides further evidence that high-oil soybean, as a specific genotype, possesses unique and efficient regulatory mechanisms in phosphorus signal perception, rhizobial community assembly, and host-microbe interaction.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e4.1 Effects of phosphorus supply intensity on growth indicators of different soybean varieties\u003c/h2\u003e \u003cp\u003eConsistent with established knowledge, our results confirm that phosphorus (P) is a key macronutrient critically regulating soybean growth and development \u003csup\u003e[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]\u003c/sup\u003e. The observed general increase in plant height and biomass with increasing P supply, up to an optimum of 105 kg\u0026middot;hm\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e, aligns with findings from prior studies \u003csup\u003e[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]\u003c/sup\u003e. However, the present study further reveals a significant varietal difference in P responsiveness\u003csup\u003e[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]\u003c/sup\u003e. The superior plant height, shoot dry weight, and root dry weight of high-oil varieties, particularly at 105 kg\u0026middot;hm\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e, suggest a more efficient utilization of P resources for biomass accumulation, which may be attributed to their distinct metabolic priorities centered on oil biosynthesis\u003csup\u003e[\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe root system, as the primary organ for P acquisition, exhibited high plasticity\u003csup\u003e[\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]\u003c/sup\u003e. The enhanced total root length, surface area, and volume in high-oil varieties under 70 kg\u0026middot;hm\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e P supply (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) are indicative of a superior foraging strategy under sub-optimal P conditions. This aligns with the classic concept that root architectural traits are crucial for plant phosphorus acquisition \u003csup\u003e[\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]\u003c/sup\u003e and show high plasticity in response to varying P supply intensities \u003csup\u003e[\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]\u003c/sup\u003e. The remarkable increase in root surface area (RSA) and root volume (RV) in high-oil varieties likely facilitated greater soil exploration and P uptake, providing a physiological basis for their robust growth. The insensitivity of the average root diameter to P treatment is consistent with some reports \u003csup\u003e[\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]\u003c/sup\u003e and may be related to the specific developmental stage (V6) at which measurements were taken.\u003c/p\u003e \u003cp\u003eFurthermore, optimal P supply significantly promoted nodulation, a cornerstone of soybean nitrogen nutrition\u003csup\u003e[\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]\u003c/sup\u003e. The peak in nodule number and dry weight at 105 kg\u0026middot;hm\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e in high-oil varieties (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea, b) is in agreement with the established positive influence of phosphorus application on these symbiotic parameters \u003csup\u003e[\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]\u003c/sup\u003e. Our findings reinforce that proper P nutrition is essential for building and sustaining an effective nitrogen-fixing machinery in soybean, with high-oil varieties demonstrating a heightened symbiotic investment under optimal conditions \u003csup\u003e[\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e4.2 Effects of phosphorus supply intensity on physiological and biochemical characteristics of different soybean varieties\u003c/h2\u003e \u003cp\u003eNitrogen metabolism lies at the heart of plant productivity and seed quality\u003csup\u003e[\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]\u003c/sup\u003e. Our findings demonstrate that phosphorus supply intensity exerts a profound regulatory influence on this process, with high-oil varieties exhibiting a more responsive nitrogen assimilation system\u003csup\u003e[\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]\u003c/sup\u003e. The significantly higher activities of glutamate synthase (GOGAT) and glutamine synthetase (GS) in high-oil varieties under optimal P supply (105 kg\u0026middot;hm⁻\u0026sup2;) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea, b) indicate a P-enhanced capacity for ammonium assimilation. The GS/GOGAT pathway is recognized as the primary route of nitrogen assimilation in higher plants \u003csup\u003e[\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]\u003c/sup\u003e, and its activation here underscores the role of P in facilitating efficient nitrogen metabolism \u003csup\u003e[\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]\u003c/sup\u003e. This coordinated upregulation of key enzymes provides a mechanistic explanation for the concurrent surge in leaf total nitrogen (TN) content in high-oil varieties (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec), which was 30.52% higher than in non-high-oil varieties. This result strongly supports the premise that optimal P nutrition enhances the nitrogen fixation capacity of nodules, thereby increasing nitrogen delivery to the host plant \u003csup\u003e[\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe differential response in soluble protein (SP) content further illuminates the distinct metabolic fates of nitrogen in the two varietal types \u003csup\u003e[\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]\u003c/sup\u003e. The generally higher SP content in non-high-oil varieties (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed) is consistent with their metabolic prioritization of protein synthesis. The observation that SP content increased with P application, peaking at 105 kg\u0026middot;hm\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e in both types, aligns with previous reports that P availability affects soluble protein contents in plants \u003csup\u003e[\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]\u003c/sup\u003e. However, the fact that non-high-oil varieties maintained higher SP levels underscores their inherent sink strength for protein accumulation. This varietal divergence in nitrogen partitioning-towards oil-associated precursors in high-oil types and towards storage proteins in non-high-oil types-must be considered when developing P management strategies aimed at optimizing quality traits \u003csup\u003e[\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e4.3 Effects of phosphorus supply intensity on rhizobial communities and functions in different soybean varieties\u003c/h2\u003e \u003cp\u003eOur study provides compelling evidence that the host plant genotype, modulated by phosphorus (P) supply, serves as a powerful filter in shaping the assembly and function of the nodule rhizobial community. Coupled with the distinct clustering of their communities in the PCoA (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea), demonstrate a more dynamic and responsive symbiotic partnership. This finding aligns with the emerging perspective that nutrient availability can act as an environmental filter to restructure rhizobial communities \u003csup\u003e[\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]\u003c/sup\u003e. The stronger correlation between the rhizobial communities of high-oil varieties and rhizosphere available P (RS-AP), as revealed by RDA (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea), further reflects their superior phosphorus utilization efficiency, which in turn enhances nutrient acquisition and symbiotic performance.\u003c/p\u003e \u003cp\u003eThe genus-specific responses underscore a sophisticated, varietal-dependent selection for microbial partners. The signifiant enrichment of key nitrogen-fixing genera such as \u003cem\u003eBradyrhizobium_sp_173_3_module\u003c/em\u003e and \u003cem\u003eRhizobium_sp\u003c/em\u003e in high-oil varieties under 105 kg\u0026middot;hm\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e P (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec) suggests that these varieties actively recruit a more effective microbiome under favorable nutrient conditions. This selective enrichment likely contributes to their enhanced nodulation and nitrogen acquisition. In contrast, the decline in \u003cem\u003eBradyrhizobium_diazotrophicus\u003c/em\u003e abundance in non-high-oil varieties under the same conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec) highlights that the impact of P is not universal but is finely tuned by the host's physiological and metabolic status \u003csup\u003e[\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e]\u003c/sup\u003e. It can be postulated that in high-oil soybeans, P is primarily allocated to support symbiotic nitrogen fixation that fuels lipid biosynthesis, whereas in non-high-oil varieties, P might be directed towards other metabolic pathways prioritizing protein accumulation.\u003c/p\u003e \u003cp\u003eThe predicted functional potential of the nodule microbiomes offers a genetic-level corroboration of the observed physiological advantages. The significantly increased abundance of genes related to Nitrogen_Fixation and Aerobic_Chemoheterotrophy in high-oil varieties (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e) correlates well with their superior nodulation capacity (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), heightened activities of nitrogen assimilation enzymes (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea, b), and consequently, greater nitrogen accumulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). This metabolic efficiency likely stems from an optimized carbon allocation to the rhizobial symbionts, fueling the energy-intensive process of biological nitrogen fixation \u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e. The distinct distribution in Ureolysis gene abundance further emphasizes the diversified nitrogen source utilization strategies of the rhizobial communities in high-oil varieties, potentially offering an additional metabolic flexibility. Collectively, these data support a model where optimal P supply promotes the assembly of a more proficient and metabolically versatile rhizobial consortium in high-oil soybean varieties, which synergistically enhances the overall symbiotic efficiency and plant performance.\u003c/p\u003e \u003c/div\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eIn conclusion, this study demonstrates a pronounced varietal differentiation in the response of soybean to phosphorus supply, underpinned by distinct rhizobial community dynamics. High-oil soybean varieties exhibited greater sensitivity and responsiveness to phosphorus fertilization, with optimal growth, nodulation, and nitrogen assimilation achieved at a supply intensity of 105 kg\u0026middot;hm\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e. The enhanced performance was mechanistically linked to a phosphorus-driven restructuring of the nodule rhizobial community, characterized by increased diversity and the selective enrichment of key nitrogen-fixing genera. This tailored microbial consortium demonstrated a higher predicted genetic potential for nitrogen acquisition and energy metabolism, which synergistically supported more efficient host nitrogen acquisition and assimilation. These findings provide a scientific basis for precision phosphorus management strategies tailored to specific soybean varieties, with the potential to enhance both productivity and resource use efficiency in agricultural systems.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analysed during this study are included in this published article and its supplementary information files. The raw 16S rRNA gene sequencing data have been deposited in the NCBI database under BioProject Accession: PRJNA1377704 (ID: 1377704).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by Major Project of Agricultural Biological Breeding (2023ZD0403106), the Low-carbon Green Agriculture of Grain Crops Project (LJGXCG2022-107), the Postdoctoral Scientific Research Startup Fund Project of Heilongjiang Province (LBH-Q21162), Guiding Science and Technology Plan Project of Daqing City (zd-2025-030, zd-2025-031).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHanshuo Zhang: Writing \u0026ndash; original draft, Writing \u0026ndash; review \u0026amp; editing. Anni Bai: Data curation. Yang Hu: Supervision, review \u0026amp; editing. Mingcong Zhang: Project administration, Resources, Supervision, review \u0026amp; editing. Wei Zhou: Data curation.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eLIU Y, ZHANG S, LI J, et al. An R2R3-type MYB transcription factor, GmMYB77, negatively regulates isoflavone accumulation in soybean [Glycine max (L.) Merr.] [J]. Plant Biotechnol J. 2025;23(3):824\u0026ndash;38.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZHAO Q, XU Y, LIU Y. Soybean oil bodies: A review on composition, properties, food applications, and future research aspects [J]. Food Hydrocolloids, 2022, 124(107296.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHAMZA M, BASIT A W, SHEHZADI I, et al. Global impact of soybean production: A review [J]. 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Plants. 2023;12(15):2861.\u003c/span\u003e\u003c/li\u003e \u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-plant-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pbio","sideBox":"Learn more about [BMC Plant Biology](http://bmcplantbiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pbio/default.aspx","title":"BMC Plant Biology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Phosphorus supply, Soybean, Nitrogen acquisition, High-oil soybean, Rhizobial community","lastPublishedDoi":"10.21203/rs.3.rs-8195203/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8195203/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePhosphorus supply plays a critical role in regulating symbiotic nitrogen acquisition in soybeans, yet the mechanisms underlying varietal differences between high-oil and non-high-oil varieties remain poorly understood. This study investigated the varietal-specific mechanisms of phosphorus supply intensity on plant nitrogen acquisition via rhizobial community restructuring using two high-oil (Kenong 18, Kenong 39) and two non-high-oil varieties (Heihe 43, Longken 310) under five phosphorus levels (0, 35, 70, 105, 140 kg\u0026middot;hm\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e). The results showed that high-oil varieties exhibited superior growth performance and nitrogen acquisition efficiency at 105 kg\u0026middot;hm\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e phosphorus supply, with increases of 19.99% in plant height, 4.13% in shoot dry weight and 17.96% in root dry weight versus controls. Nodule number, dry weight and haemoglobin content increased by 83%, 30% and 33.02%, respectively, in high-oil genotypes. Enhanced nitrogen metabolism was evidenced by significantly elevated GOGAT/GS activities (9.32\u0026ndash;17.13%) and leaf total nitrogen content. Crucially, under optimal phosphorus conditions, high-oil varieties enriched specific nitrogen-fixing rhizobia, such as \u003cem\u003eBradyrhizobium sp. 173_3_\u003c/em\u003emodule and \u003cem\u003eRhizobium sp\u003c/em\u003e., and exhibited stronger correlations between community structure and soil available phosphorus (RS-AP), along with a predicted greater potential for nitrogen acquisition and aerobic chemoheterotrophy. This study demonstrates that optimal phosphorus supply enhances symbiotic nitrogen acquisition efficiency in high-oil soybeans by driving the assembly of more specialized rhizobial communities, providing microbial mechanistic insights for varietal-specific phosphorus management in soybean cultivation.\u003c/p\u003e","manuscriptTitle":"Regulatory mechanism of phosphorus supply intensity on the plant nitrogen acquisition in soybean: Insight from the differences of rhizobia diversity between high-oil soybean and non-high-oil soybean","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-16 16:49:17","doi":"10.21203/rs.3.rs-8195203/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-12-25T08:49:22+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-24T09:40:33+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-21T10:58:42+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-18T15:24:23+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-16T04:13:57+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"204796057500967517907967513762440448114","date":"2025-12-14T10:41:58+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"42280612961969569301987014753274282954","date":"2025-12-13T11:29:49+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"185699740867666769596305023550235885147","date":"2025-12-11T09:17:36+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"118835378674011202218043202289656945945","date":"2025-12-11T08:08:31+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-12-11T07:56:07+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-12-11T07:48:08+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-12-10T07:43:19+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-12-09T14:03:55+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Plant Biology","date":"2025-12-09T13:48:39+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-plant-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pbio","sideBox":"Learn more about [BMC Plant Biology](http://bmcplantbiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pbio/default.aspx","title":"BMC Plant Biology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"71851f0a-63e5-4546-9b89-b40dc20b0049","owner":[],"postedDate":"December 16th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-03-30T16:26:21+00:00","versionOfRecord":{"articleIdentity":"rs-8195203","link":"https://doi.org/10.1186/s12870-026-08459-0","journal":{"identity":"bmc-plant-biology","isVorOnly":false,"title":"BMC Plant Biology"},"publishedOn":"2026-03-28 16:12:41","publishedOnDateReadable":"March 28th, 2026"},"versionCreatedAt":"2025-12-16 16:49:17","video":"","vorDoi":"10.1186/s12870-026-08459-0","vorDoiUrl":"https://doi.org/10.1186/s12870-026-08459-0","workflowStages":[]},"version":"v1","identity":"rs-8195203","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8195203","identity":"rs-8195203","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}