Optimizing Initial Nitrogen Application Rates to Improve Peanut (Arachis hypogaea L.) Biological Nitrogen Fixation

Abstract

The application of nitrogen fertilizer is crucial to the growth and biological nitrogen fixation of peanut, especially in the seedling stage where nodules have not yet formed. However, it is still uncertain how much initial nitrogen fertilizer should be applied to promote peanut root growth, nodule formation, and biological nitrogen fixation (BNF). There, a 2-year pot experiment was conducted using Huayu 22 (HY22, large-grain cultivar) and Huayu 39 (HY39, small-grain cultivar) as experimental materials to research the effects of different initial nitrogen fertilizer application rates on peanut root growth (root weight, root length, root mean diameter, root activity) and biological nitrogen fixation capacity (nodule number, nodule weight, biological nitrogen fixation, and nitrogen fixation potential per plant). N₀, as control, four initial nitrogen fertilizer application rates were established: 15 kg·hm⁻² (N₁₅), 30 kg·hm⁻² (N₃₀), 45 kg·hm⁻² (N₄₅), and 60 kg·hm⁻² (N₆₀). The present results showed that the nodule number, nodule dry weight, nitrogenase activity, and biological nitrogen fixation of the HY22 cultivar under the N₁₅ treatment were higher compared to those under other treatments over the two growing seasons. In addition, the cultivar of HY39 treated with the N₁₅ treatment also increased the nitrogen fixation potential per plant and BNF relative to other treatments. Although the application of 60 kg·hm⁻² nitrogen increased the root surface area and root volume, it decreased the nitrogenase activity, nodule dry weight, and nitrogen fixation potential per plant of HY22 and HY39 varieties in both growing seasons. Above all, an initial nitrogen application of 15 kg·hm⁻² may be the optimal treatment for promoting peanut nodule formation and biological nitrogen fixation.

1. Introduction

Nitrogen is a key element in the crop growth process, with sufficient nitrogen to ensure high yields often being provided in the form of industrial nitrogen fertilizer in agricultural settings [1,2]. In contrast to this industrial-grade synthetic nitrogen fertilizer, the symbiotic relationship between leguminous plants and rhizobia offers an alternative mechanism through which these crops can obtain N from atmospheric N₂ through biological nitrogen fixation (BNF) [3]. Peanut (Arachis hypogaea L.) is an economically key oil-producing crop in China, and as they are legumes they can also engage in biological nitrogen fixation. However, this BNF activity can only provide an estimated 40–60% of the nitrogen required for optimal plant growth such that exogenous nitrogen is necessary to achieve maximal yields [4,5]. The addition of excessively high nitrogen levels can suppress rhizosphere nitrogen fixation, completely inhibiting both acetylene reduction activity (ARA) and nodulation when these soil nitrogen levels are above a given threshold [6,7]. This inhibition tends to become more severe as soil nitrogen levels rise. Mousumi-Mondal’s study showed that the application of higher nitrogen fertilizers inversely impacted root nodulation and nitrogen fertilizer application efficiency and concluded that 25 kg hm⁻² as a base application in combination with Rh inoculant would be very beneficial for irrigated peanut in India [8]. There was also research indicating that 72 kg hm⁻² nitrogen fertilizer in combination with Bradyrhizobium sp. increased symbiotic nitrogen fixation in Egypt [9]. In the context of peanut cultivation in China, large amounts of nitrogen are applied as a basal fertilizer that can limit BNF, contributing to lower nitrogen utilization rates that ultimately compromise crop yields and quality while also contributing to the ecological deterioration of the soil. Studies have shown that higher yields and lower environmental pollution can be achieved by optimizing the balance between the application of nitrogen fertilizer and the natural process of biological nitrogen fixation in legumes [10].

Low soil nitrogen concentrations are known to promote soil nodule development, whereas BNF in these nodules is suppressed in response to higher nitrogen concentrations [11,12]. As this symbiotic BNF process requires substantial energy input, leguminous plants will preferentially utilize available soil nitrogen under low-nitrate conditions prior to establishing N₂-fixing symbiosis, thereby reducing nitrate source competition between nodules and host plants [13]. These nodules develop high nitrate levels, but excessive nitrate accumulation within the nodules can suppress symbiotic rhizobia-host interactions [14]. Applying appropriate levels of nitrogen in growth can support root nodule formation and development in leguminous crops, with some evidence suggesting that the early stages of growth are the most important period during which to promote increases in root nodule numbers [15]. Given the importance of the early stage of growth as a determinant of root nodule development, appropriate nitrogen supplementation provides an opportunity to improve the uptake of soil and fertilizer-derived nitrogen by host plant root systems while effectively initiating root nodule formation, mainly through an abundant supply of photo-assimilate to the nodules, thus supporting root growth and activity [16].

While the initial nitrogen concentrations applied to peanut seedlings is a key factor in the formation of nodule development and crop yields, studies also proved that the inhibitory effect of one-time application of basal nitrogen on nodulation is greater in the early stage of peanuts. However, split application of controlled release fertilization can slow down the inhibitory effect of nitrogen. It is not clear how much initial nitrogen fertilizer is optimal to apply, making it difficult to accurately match peanut’s nitrogen needs to controlled release fertilization. Some reports have demonstrated that nitrogen can influence the length, surface area, and roots during early phases of growth, in turn influencing BNF activity [17]. Root systems provide a nutrient-rich niche conducive to nodule development, promoting BNF [18]. The close physical, genetic, and biochemical interactions between rhizomes and roots are well documented, particularly in the context of nodule development, symbiotic nitrogen fixation activity, and associated transport activity. However, there have been few efforts to date to simultaneously examine the associations and relationships linking root architecture and BNF. Accordingly, the purpose of the present research was to optimize the initial nitrogen application of two peanut varieties commonly used in production, improve root growth (root weight, root length, root mean diameter, root activity) and biological nitrogen fixation (nodule number, nodule weight, nodule nitrogenase activity, biological nitrogen fixation, and nitrogen fixation potential per plant), and to provide support for realizing the input of nitrogen fertilizer according to the need and breaking through the bottleneck of efficient use of N in peanut.

2. Materials and Methods

2.1. Experimental Design

This research was conducted in 2020 and 2021 under the experimental pot conditions at Shandong Academy of Agricultural Sciences Station (36°43′ N, 117°05′ É) in Jinan, China. Pots (diameter: 30 cm, height: 28 cm, volume: 3.2 dm³) were packed with 10 kg of a quartz and vermiculite mixture (3:1, v/v). The total N content was 2.21 g·kg⁻¹, Experimental crops included the Huayu 22 and Huayu 39 peanut cultivars, which are, respectively, large-grain and small-grain varieties, using seeds that were kindly provided by Shandong Peanut Research Institute, Qingdao, China. Four different nitrogen treatment conditions were established based on the concentration of urea applied, including the N₀ (0 kg·hm⁻²), N₁₅ (15 kg·hm⁻²), N₃₀ (30 kg·hm⁻²), N₄₅ (45 kg·hm⁻²), N₆₀ (60 kg·hm⁻²) treatments. The arrangements for the 2-year experiment were consistent. Three replicates were established per treatment in a randomized design, with 10 pots per replicate. Three of the pots in each replicate utilized ¹⁵N-labeled urea with 10.3% abundance, while the remaining seven pots utilized regular urea. P and K fertilizers were provided in the form of 180 kg·hm⁻² potassium phosphate monobasic (KH₂PO₄), while Ca fertilizer was applied in the form of 150 kg·hm⁻² Calcium sulfate (CaSO₄). Bradyrhizobium yuanmingense, a rhizobium CCBAU21353 isolated from peanut root nodules.

All fertilizer treatments were applied using the same approach. Briefly, quartz and vermiculite (3:1, v/v) were thoroughly mixed with N, P, and K fertilizers at the defined concentrations on 20 May 2020 and 14 May 2021. Peanut seeds were disinfected for 2 min with 70% ethanol, disinfected for 8 min with 0.1% (w/v) HgCl₂, rinsed with sterile water 7–8 times, dried of surface water, inoculated for 2 h with rhizobia, and then sown in 2 holes per pot, with 2 seeds being added per hole. After emergence, 1 plant was cultivated per hole for 2 total peanut plants per pot. The peanuts were watered every 7 days, and irrigation and other management were consistent.

2.2. Morphological and Physiological Characteristic Measurements

Peanut plants grown under various initial nitrogen concentrations were sampled at random 30 days after planting. Measured parameters included stem height, total branch number, number of stem nods, number of stem green leaves, root dry weight (DW), and aboveground DW. A ruler was used when measuring stem height and lateral branch length. Biomass measurements were made by separating roots and aboveground biomass, heating samples for 30 min at 105 °C in an oven, and then drying these samples for 48 h at 75 °C, after which root and aboveground DW was measured and the root/shoot ratio was determined.

2.3. Root System Measurement

The peanuts were randomly sampled 30 days after sowing; 6 pots per treatment were selected at random to assess root activity and architecture. the triphenyl tetrazolium chloride (TTC) method was applied to calculate the root activity. In brief, root tip samples of 0.5 g were placed in a beaker, and an equal mixture of 0.4% TTC and phosphate-buffered solution was added in 10 mL; the roots were fully submerged in the solution and kept at 37 °C for 1~3 h without light. Thereafter, 1 mol L⁻¹ sulfuric acid was added in 2 mL in order to stop the reaction. The sample was dried, then extracted via ethyl acetate. The absorbance was detected at 485 nm. Root morphological parameters were evaluated with a scanner (Epson V800 Photo, SEIKO EPSON CORP, Jakarta, Indonesia), and WinRHIZO was applied to determine the root length, and volume. The root activity was calculated with reference to the following formula:

Root activity = amount of TTC reduction (µg)/fresh root weight (g) × time (h)

(1)

2.4. Biological Nitrogen Fixation Measurement

The nitrogen fixation number of peanut nodules was calculated by detecting the ¹⁵N abundance of plant samples using the ¹⁵N labeling tracer method. Peanut plant samples were ground by a plant tissue grinder, then finely ground by a ball mill (MM400, Retsch Crop, Haan, Germany) and passed through a 0.15 mm sieve. Total N levels were measured with an automatic nitrogen analyzer (XY-2 SAMPLER, SEAL, Germany), and ¹⁵N abundance was calculated via mass spectrometry (MAT-271, Thermo Finnigan, Germany). The nitrogen derived from nitrogen fertilizer (Ndff) and BNF was calculated with reference to the following formula:

Ndff = (¹⁵N atom% excess in plant sample/¹⁵N atom% excess in fertilizer) × 100

(2)

Total N accumulation (mg/plant) = Plant dry weight × Plant N content

(3)

Plant ¹⁵N fertilizer absorption(mg/plant) = Plant total N accumulation × Ndff

(4)

BNF (mg/plant) = total N accumulation − ¹⁵N fertilizer absorption

(5)

2.5. Nitrogenase Activity Measurement

Acetylene (C₂H₂) reduction to ethylene (C₂H₄) was used to measure nitrogenase activity. Briefly, washed peanut root nodules were added to a 50 mL closed glass bottle. Then, 5 mL of air was removed from each bottle and replaced with 5 mL of C₂H₂, after which bottles were sealed for 2 h. Ethylene levels were then measured via gas chromatography, and nodule numbers and weight were then assessed after drying, with the nitrogen fixation potential per plant being calculated as follows:

nitrogen fixation potential per plant (mmol·h⁻¹)                                                    
= nitrogenase activity (μmol·g⁻¹·h⁻¹) × Root nodule dry weight per plant (g)

(6)

2.6. Statistical Analysis

The effects of year, cultivar, N treatment, and their interaction on peanut root development and biological nitrogen fixation characteristic were tested using a two-way ANOVA with SPSS 25.0. The repeated factors “year”, “cultivar” and “N treatment” were combined with the repeated factor “peanut trait.” Using the LSD approach, multiple comparisons of each indicator under various treatments were carried out at a significant probability level of 0.05. We applied DPS (p < 0.05) significance analysis to all the data and processed and plotted results using Origin 2019.

3. Results

3.1. Peanut Root Architecture

Root architecture is a term that is used to refer to root morphology and spatial distributions, directly reflecting the growth of roots in a manner that is responsive to environmental nitrogen levels (

Table 1. Peanut root architecture in the 2020 and 2021 growing seasons.

Year	Cultivar	Nitrogen Application	Root Length (cm)	Root Surface Area (cm2)	Root Average Diameter (mm)	Root Volume (cm3)
2020	HY22	N0	5516 ± 160 c	712 ± 9 c	0.416 ± 0.01 a	7.24 ± 0.12 c
		N15	5738 ± 115 bc	728 ± 11 bc	0.414 ± 0.008 a	7.4 ± 0.23 b
		N30	5956 ± 139 ab	748 ± 3 ab	0.413 ± 0.002 a	7.57 ± 0.09 ab
		N45	6027 ± 119 a	751 ± 13 a	0.396 ± 0.01 b	7.72 ± 0.1 ab
		N60	6195 ± 106 a	768 ± 16 a	0.409 ± 0.005 ab	7.75 ± 0.14 a
	HY39	N0	4182 ± 37 b	565 ± 7 c	0.415 ± 0.016 a	5.17 ± 0.15 b
		N15	4334 ± 130 b	611 ± 5.2 b	0.408 ± 0.006 a	5.37 ± 0.08 ab
		N30	4576 ± 102 a	619 ± 5.43 b	0.405 ± 0.007 a	5.45 ± 0.09 ab
		N45	4664 ± 88 a	633 ± 6.46 a	0.403 ± 0.013 a	5.54 ± 0.06 ab
		N60	4643 ± 167 a	640 ± 8.55 a	0.406 ± 0.013 a	5.57 ± 0.41 a
2021	HY22	N0	4293 ± 374 c	551 ± 63 c	0.443 ± 0.048 a	5.66 ± 0.94 c
		N15	4978 ± 200 b	612 ± 49 bc	0.404 ± 0.04 a	6.05 ± 0.24 bc
		N30	5236 ± 273 ab	707 ± 55 ab	0.404 ± 0.032 a	7.08 ± 0.7 abc
		N45	5662 ± 459 ab	733 ± 29 a	0.393 ± 0.011 a	7.56 ± 0.89 ab
		N60	5848 ± 483 a	743 ± 73 a	0.389 ± 0.035 a	8.24 ± 1.1 a
	HY39	N0	3101 ± 100 c	410 ± 13 c	0.419 ± 0.025 a	4.33 ± 0.34 b
		N15	3518 ± 187 bc	440 ± 3 bc	0.406 ± 0.029 a	4.39 ± 0.29 b
		N30	3746 ± 247 b	479 ± 50 ab	0.4 ± 0.026 a	4.89 ± 0.8 ab
		N45	3924 ± 362 ab	489 ± 29 ab	0.398 ± 0.019 a	4.86 ± 0.25 ab
		N60	4259 ± 173 a	515 ± 12 a	0.397 ± 0.013 a	5.28 ± 0.19 a

Note: Values represent the means ± standard deviation (SD, n = 3). The letters (a, b, c) within a column and in the same year indicate significant differences among treatments by Duncan’s range test (p < 0.05).

). Analysis of variance (ANOVA) showed that year, cultivar and N treatment had significant effects on peanut root length, volume, and surface area, and the interaction of year and N treatment had a strong influence on root length as well as surface area, whereas the interaction among the three had nonsignificant effects (

Table 2. Analysis of variance for the effects of year, cultivar and nitrogen on root architecture.

Source of Variation		Root Length (cm)	Root Surface Area (cm2)	Root Average Diameter (mm)	Root Volume (cm3)
ANOVA	Year(Y)	**	**	NS	**
	Cultivar(C)	**	**	NS	**
	Nitrogen(N)	**	**	NS	**
	Y×C	NS	**	NS	NS
	Y×N	*	*	NS	NS
	C×N	NS	NS	NS	NS
	Y×C×N	NS	NS	NS	NS

Note: * and ** are significant level of 0.05 and 0.01; NS means not significant.

). According to the above result, nitrogen application obviously improved root length and surface area, but not root diameter. In 2020 and 2021, the HY22 exhibited a longer root length under N₆₀ conditions relative to the other treatments. We found that the root length and root volume were significantly increased by 12.31% and 7.04% of HY22 in 2020, respectively, compared to N₀. In two years, the HY39 in the N₄₅ and N₆₀ group exhibits obviously higher root length, surface area, and volume than those in the other treatments. it can be included that the root length and surface area under N₆₀ were significantly increased by 37.34%, and 25.61% of HY39 in 2021, respectively, compared to N₀. These findings indicated that root length and volume for the HY22 and HY39 cultivars can be significantly enhanced under 60 kg·hm⁻², thereby improving root absorption area in a manner conducive to peanut root absorption efficiency.

3.2. Peanut Root Activity

As shown in Figure 1, the cultivar and N treatment had an obvious influence on peanut root activity, and the interaction of year and cultivar and cultivar and nitrogen treatment can significantly impact the peanut root activity. The growth and metabolic level of roots, that is, root activity, is one of the important physiological indicators of plant growth. Plants with strong root activity can better absorb water and nutrients in the soil and provide sufficient nutrient supply. Nitrogen fertilizer application improved root activity over the two years. For the HY22 in 2020 and 2021, higher root activity levels were evident under N₁₅ treatment conditions (31.43% and 30.39% above N₀), with no increases as N concentrations were further increased. There were no obvious differences in root activity for the HY39 cultivar among the N₁₅, N₃₀, and N₄₅ treatment conditions over the two years, with increases of 11.72%, 8.69%, and 10.4% in 2020, respectively, and 15.03%, 16.19, and 17.85% in 2021, respectively, compared with the N₀. These findings suggested that under N₁₅ treatment conditions, the peanut seedling root system exhibits more vigorous activity and exhibits a higher capacity to engage in nutrient absorption.

3.3. Nodulation and BNF Activity

As shown in Figure 2, Analysis of variance (ANOVA) showed that year, cultivar, N treatment, and the interaction of cultivar and N treatment obviously impacted the nodule number and dry weight, whereas the interaction among the year and cultivar had nonsignificant effects. Root nodule numbers and dry weight showed a continuous decreasing trend with increasing N input in both years. From 2020 to 2021, N₃₀, N₄₅, and N₆₀ treatments reduced the nodule number and dry weight by 28.91–68.42% and 29.2–62.07%, respectively, among the 2 cultivars compared with N₀. Compared with other nitrogen treatments, N₁₅ treatment significantly increased the nodule number and dry weight, but it was not significantly different from N₀. Nitrogen application higher than N₁₅ had a significant inhibitory effect on the peanut nodulation ability, and the higher the nitrogen level, the stronger the inhibitory effect. So N₁₅ treatment had stronger nodulation ability in both varieties.

The result of nitrogenase activity and BNF potential per plant in various nitrogen levels for the two years can be seen from

Table 3. Peanut nitrogenase activity in the 2020 and 2021 growing seasons.

Year	Cultivar	Nitrogen Application	Nitrogenase Activity (μmol·g−1·h−1)	Nitrogen Fixation Potential (μmol·plant−1 h−1)
2020	HY22	N0	44.86 ± 2.39 a	4.51 ± 0.59 a
		N15	56.47 ± 1.96 a	5.36 ± 0.25 a
		N30	49.32 ± 6.07 a	3.5 ± 0.59 b
		N45	33.40 ± 8.70 b	1.64 ± 0.57 c
		N60	27.29 ± 8.11 b	1.25 ± 0.36 c
	HY39	N0	41.68 ± 9.79 a	2.05 ± 0.54 a
		N15	46.64 ± 5.14 a	1.98 ± 0.11 a
		N30	35.82 ± 10.34 ab	1.26 ± 0.45 b
		N45	33.2 ± 12.25 ab	0.93 ± 0.35 bc
		N60	19.82 ± 5.78 b	0.37 ± 0.13 c
2021	HY22	N0	24.87 ± 3.24 a	2.02 ± 0.19 a
		N15	25.21 ± 1.00 a	2.30 ± 0.13 a
		N30	21.3 ± 1.51 ab	1.36 ± 0.34 b
		N45	16.32 ± 4.19 bc	0.64 ± 0.36 c
		N60	15.68 ± 2.51 c	0.52 ± 0.22 c
	HY39	N0	25.35 ± 2.66 ab	1.19 ± 0.32 a
		N15	29.09 ± 3.10 a	1.53 ± 0.21 a
		N30	25.65 ± 0.75 ab	0.36 ± 0.12 b
		N45	23.21 ± 2.12 b	0.30 ± 0.13 b
		N60	21.90 ± 1.93 b	0.15 ± 0.07 b

Note: Values represent the means ± standard deviation (SD, n = 3). The letters (a, b, c) within a column and in the same year indicate significant differences among treatments by Duncan’s range test (p < 0.05).

. Analysis of variance (ANOVA) showed that year, N treatment and the interaction of year and cultivar had a significant influence on nitrogenase activity, whereas the interaction among the cultivar and N treatment had nonsignificant effects. Analysis of variance (ANOVA) showed that nitrogen fixation potential per plant was affected by the interaction among the three (

Table 4. Analysis of variance for the effects of year, cultivar and nitrogen on nitrogenase activity.

Source of Variation		Nitrogenase Activity (μmol·g−1·h−1)	Nitrogen Fixation Potential (μmol·plant−1 h−1)
ANOVA	Year (Y)	**	**
	Cultivar (C)	NS	**
	Nitrogen application (N)	**	**
	Y × C	**	**
	Y × N	*	**
	C × N	NS	**
	Y × C × N	NS	*

Note: * and ** are significant at the 0.05 and 0.01 levels; NS means not significant.

). Both the HY22 and HY39 exhibited peak nitrogenase activity under N₁₅ treatment conditions over the two years, with 25.88% and 11.9% above N₀ levels, respectively, in 2020. The BNF potential per plant is obviously higher in N₁₅ compared to that in other N treatments. The single-plant nitrogen fixation potential of the HY22 under N₁₅ conditions was increased by 18.85% and 13.86% during 2020 and 2021, respectively, relative to the N₀ condition, whereas for the HY39, this potential increased by 28.57% relative to N₀ condition in 2021. Accordingly, the initial nitrogen concentrations for the small-grain HY22 and the large-grain HY39 should, respectively, be 15 kg·hm⁻², which exhibited a more notably increased single-plant nitrogen fixation potential than the other treatments, thereby enhanced the nodulation capacity of peanut.

3.4. Peanut Seedling Growth

Analysis of variance (ANOVA) showed that cultivars and N treatment had obvious influence on the main stem height, total branch number, main stem nod number, and main stem green leaves, whereas the interaction among the year and N treatment had nonsignificant effects. For both experimented peanut cultivars, initial nitrogen application strongly accelerated the growth of the peanut (Figure 3). Nitrogen inputs to both varieties significantly increased the main stem height, total branch number, main stem nods number, and main stem green leaves. The HY22 cultivar with the N₄₅ treatment increased the main stem height, main stem nod number, and main stem green leaves compared with other treatments during 2020 to 2021. For HY39, nitrogen fertilizer inputs did not significantly increase the main stem nod number and main stem green leaves. However, the lateral branch length under N₃₀, N₄₅, and N₆₀ was significantly higher than in the other treatments during both years.

Analysis of variance (ANOVA) showed that year, cultivars, and N treatment had obvious effects on root dry weight, while interaction among the year and N treatment had significant effects. It was clear that the maximum root dry weight in the large-grain HY22 peanut cultivar was N₄₅ conditions over both years and in the order of N₄₅ > N₆₀ > N₃₀ > N₁₅ > N₀ in 2020 (Figure 4). The root dry weight of the small-grain HY39 cultivar was significantly decreased under the N₀ treatment relative to other treatments in 2020. In the 2021 growing season, the root dry weight under the N₃₀ and N₄₅ treatments was obviously higher compared to the other treatments. Root growth inevitably influences aboveground plant growth. As shown in Figure 4, the aboveground dry weight of these two peanut cultivars rose continuously with increasing nitrogen application to a peak value under the N₄₅ treatment of the HY22 cultivar and under the N₆₀ treatment of the HY39 cultivar.

3.5. Peanut Root Shoot Ratio

Analysis of variance (ANOVA) showed that year, cultivar, and N treatment had an obvious influence on root shoot ratio, and the interaction of year and cultivar and N treatment had remarkable effects, whereas the interaction among the cultivar and N treatment had nonsignificant effects. In our study, the root shoot ratio of HY22 and HY39 was higher in N₁₅ than in other treatments (Figure 5). This may be due to the root-foraging response such that it increased dry matter distribution to the belowground root system.

3.6. Peanut Nitrogen Content and Ndff

Analysis of variance (ANOVA) showed that the year, cultivar, and N treatment had strong effects on the N content and Ndff of various organs, except for no interaction between year and cultivar; the interaction between the other two significantly affected Ndff. Nitrogen fertilizer inputs significantly increased nitrogen content and Ndff in all organs of peanut compared with the N₀ treatment, reaching the maximum at the N₆₀ (

Table 5. Peanut nitrogen content and Ndff in different organs in the 2020 and 2021 growing seasons.

Year	Cultivar	N Level	N Content of Root (g·kg−1)	N Content of Stems (g·kg−1)	N Content of Leaves (g·kg−1)	Ndff in Roots (%)	Ndff in Stems (%)	Ndff in Leaves (%)
2020	HY22	N0	10.09 ± 0.2 d	14.15 ± 0.08 e	16.69 ± 0.21 e	1.41 ± 0.03 e	1.57 ± 0.02 e	1.63 ± 0.03 e
		N15	10.72 ± 0.17 c	14.42 ± 0.08 d	17.22 ± 0.21 d	10.9 ± 0.26 d	10.95 ± 0.18 d	11.32 ± 0.1 d
		N30	10.93 ± 0.2 c	14.7 ± 0.13 c	17.95 ± 0.1 c	17.94 ± 0.42 c	18.08 ± 0.29 c	18.19 ± 0.19 c
		N45	11.67 ± 0.04 b	15.3 ± 0.05 b	18.47 ± 0.2 b	36.34 ± 0.48 b	36.89 ± 0.1 b	36.18 ± 0.29 b
		N60	12.2 ± 0.14 a	15.93 ± 0.16 a	18.85 ± 0.12 a	46.22 ± 0.33 a	45.99 ± 0.23 a	46.65 ± 0.34 a
	HY39	N0	8.93 ± 0.22 d	13.01 ± 0.03 d	16.62 ± 0.44 d	4.14 ± 0.04 e	3.87 ± 0.09 e	3.98 ± 0.1 e
		N15	9.17 ± 0.01 d	13.86 ± 0.14 c	16.88 ± 0.13 d	14.34 ± 0.16 d	14.18 ± 0.11 d	14.31 ± 0.06 d
		N30	9.63 ± 0.18 c	13.95 ± 0.11 c	17.75 ± 0.34 c	24.08 ± 0.37 c	24.16 ± 0.22 c	24.16 ± 0.37 c
		N45	11.39 ± 0.09 b	14.67 ± 0.11 b	19.47 ± 0.2 b	42.11 ± 0.34 b	41.92 ± 0.36 b	41.76 ± 0.48 b
		N60	11.79 ± 0.05 a	15.42 ± 0.22 a	21.14 ± 0.42 a	56.13 ± 0.59 a	55.74 ± 0.74 a	55.64 ± 0.74 a
2021	HY22	N0	18.12 ± 0.57 b	14.31 ± 0.39 c	24.92 ± 1.33 c	1.67 ± 0.41 e	1.19 ± 0.17 e	1.48 ± 0.46 e
		N15	18.84 ± 0.68 b	14.95 ± 0.19 bc	26.53 ± 1.39 bc	16.59 ± 0.26 d	16.76 ± 0.07 d	16.74 ± 0.12 d
		N30	19.53 ± 0.00 b	14.64 ± 0.23 c	27.09 ± 0.33 b	30.55 ± 0.20 c	30.52 ± 0.35 c	30.41 ± 0.54 c
		N45	20.06 ± 0.50 b	15.49 ± 0.40 b	27.56 ± 0.34 b	40.01 ± 0.50 b	40.32 ± 0.32 b	39.98 ± 0.36 b
		N60	21.93 ± 2.00 a	16.47 ± 0.58 a	29.85 ± 0.56 a	48.19 ± 0.38 a	49.69 ± 0.76 a	48.84 ± 0.59 a
	HY39	N0	16.04 ± 0.24 c	11.61 ± 0.76 c	20.9 ± 1.74 d	1.23 ± 0.08 e	1.27 ± 0.24 e	1.33 ± 0.09 e
		N15	18.67 ± 0.56 b	12.56 ± 0.48 bc	23.03 ± 0.83 c	20.3 ± 0.29 d	20.43 ± 0.17 d	20.03 ± 0.47 d
		N30	19.16 ± 0.76 b	13.4 ± 0.49 ab	23.79 ± 0.29 bc	35.36 ± 0.31 c	35.72 ± 0.32 c	35.78 ± 0.29 c
		N45	19.83 ± 0.23 ab	13.74 ± 0.78 a	25.53 ± 0.9 ab	43.34 ± 0.36 b	43.67 ± 0.15 b	42.29 ± 0.36 b
		N60	20.97 ± 1.7 a	13.61 ± 0.43 ab	26.19 ± 0.52 a	56.13 ± 0.59 a	55.74 ± 0.74 a	55.64 ± 0.74 a
ANOVA	Year(Y)		**	**	**	**	**	**
	Cultivar(C)		**	**	**	**	**	**
	Nitrogen(N)		**	**	**	**	**	**
	Y × C		NS	**	**	NS	NS	NS
	Y × N		*	NS	*	**	**	**
	C × N		NS	*	*	**	**	**
	Y × C × N		NS	*	*	**	**	**

Error bars denote the SD of the mean (SD, n = 6). The letters (a, b, c, d, e) within a column and in the same year indicate significant differences among treatments by Duncan’s range test (p < 0.05). * and ** are significant at the 0.05 and 0.01 levels; NS means not significant.

). Compared with N₀, the N₁₅, N₃₀, N_45, and N₆₀ treatments of HY22 and HY39 in 2020 showed an increase of 6.2%, 8.34%, 15.61%, 20.87% and 2.72%, 7.82%, 27.56%, 32.1% in root nitrogen content, which was consistent with the results of 2021. Ndff represents the percentage of plant nitrogen from 15N-labeled fertilizer and can be used to reflect the competitive ability of plant organs to absorb fertilizer nitrogen. The Ndff of the root, stem, and leaves receiving the different N conditions differed in the following order: N₆₀ > N₄₅ > N₃₀ > N₁₅> N₀. The results of 2 years were consistent.

To confirm the above results, the impact of nitrogen levels on nitrogen supply characteristics for the two sources of nitrogen was also assessed in 2020 and 2021 (Figure 6), Analysis of variance (ANOVA) showed that the year, cultivars, and N treatment strongly impacted the BNF, and the interaction between the two and three also had a significant effect. The BNF of HY39 was obviously higher in the N₁₅ treatment, and in the order of N₁₅ > N₃₀ > N₄₅ > N₀ > N₆₀. We found that BNF was significantly increased by 10.07%, 16.77%, 24.99% and 42.55% under N₁₅ treatments in 2020, respectively, compared to N₃₀, N₄₅, N₀ and N₆₀. And it was increased by 8.86%, 13.6%, 15.69% and 43.36% in 2021, respectively. The HY22 showed a significantly higher BNF under N₁₅ and N₃₀ in 2020, with an observed BNF of 82.58 and 81.66 mg·plant⁻¹, respectively. But the maximum BNF was observed under the N₁₅ treatment in 2021. Accordingly, these results suggest that smaller (N₁₅) initial nitrogen concentrations can help increase the proportion of nitrogen fixed by peanut root nodules.

3.7. Pearson Correlations for Key Indices of Root Growth Strategy and BNF

Figure 7 showed that the increase in nodule dry weight was obviously positively associated with both the root dry weight and root activity. The increase in BNF was also significantly positively associated with the root dry weight, root length, root activity, nodule number, nodule dry weight and total nitrogen accumulation. This suggests that variation in the peanut root system may impact nodulation and biological nitrogen fixation. The root system provides both the space and nutrient supply for nodules, thus increasing BNF.

4. Discussion

4.1. Peanut Root Growth

Nutrient availability is particularly important for root morphological characteristics [19]. Nitrogen is mainly available to plants in the form of NO₃^--N and NH₄⁺-N, which can influence root surface area, length, and volume in a manner that shapes root morphological development during early growth phases, in turn shaping the efficiency with which roots take up nutrients [20]. In our study, the application of exogenous nitrogen obviously enhanced the root dry weight and volume of the peanut. Studies have shown that the change in root architecture mainly depends on the growth of lateral roots, which can develop in the direction of relative nutrient enrichment in nutrient-poor soils [21]. The peanut root system is not only an important nutrient absorption organ but also a carrier of nodule attachment, which affects peanut nodulation and nitrogen fixation. Meier [22] found that local ammonium supply promotes lateral root emergence. The point to emphasize here is that the key regulatory components of lateral root formation are utilized by legume plants to form nodules [23]. In our study, during the early stage of peanut root growth, 45 kg·hm⁻² initial nitrogen application significantly enhanced root dry weight, and volume for the HY22 and HY39. However, 45 kg·hm⁻² initial nitrogen application significantly decreased nodule formation. This indicates that when the nitrogen concentration exceeds 15 kg·hm⁻², the nitrogen absorbed by peanuts is preferentially allocated to root growth rather than forming nodules. Consequently, 15 kg·hm⁻² exogenous nitrogen significantly promoted lateral root elongation, increased root dry matter accumulation, and had a promoting effect on nodule formation.

Our research shows that the root/shoot ratio declined under high levels of nitrogen application, and the root activity, and root/shoot ratios, under the N₁₅ were obviously higher compared to other treatments over the two years. This is generally in line with the findings of others [24]. In general, under nitrogen stress, crops will increase the distribution of assimilates to enhance water and nutrient uptake, thus increasing the relative dry weight of the root system and the root/shoot ratios. On the contrary, nitrogen sufficiency conditions lead to a decrease in the root/shoot ratios [25]. Although the initial nitrogen dose of 45 kg·hm⁻² can promote root development, it has already had an inhibitory effect on root nodule formation, but it is obvious that low nitrogen(N₁₅) significantly increases root activity, promotes root nutrient absorption, increases root to shoot ratio, and influences below-ground partitioning to improve root system plasticity. In addition, the nodulation process of legumes is very sensitive to the available nitrogen in the environment. Legumes can adjust their nitrogen fixation efficacy in a timely manner according to the availability of carbon sources in their bodies, thus maintaining the carbon and nitrogen balance in the plant and adapting to changes in the surrounding environment [26]. Root growth inevitably influences aboveground plant growth. Nitrogen application increased the N content and Ndff of stems and leaves, which in turn increased main stem height, total branch number, main stem nods number, and main stem green leaves, and increased the accumulation of aboveground dry matter. This is consistent with previous research [27,28].

4.2. Biological Nitrogen Fixation

Large amounts of nitrogen fertilizer inputs at one time in China can inhibit nitrogen fixation in peanuts, and slow and controlled addition of nitrogen fertilizer can reduce the inhibitory effect of inorganic nitrogen fertilizer on root nodule infection. Analysis of nitrogen supply parameters of different nitrogen sources further showed that BNF was significantly higher in both peanut varieties under N₁₅ treatment. We found that BNF was significantly increased by 10.07–42.55% under N₁₅ treatments in 2020, respectively, compared to other N treatments. Our research shows that the number of root nodules, root dry weight, ARA, and BNF potential per plant under the N₁₅ was obviously higher compared to other N treatments over the two years. The nitrogenase activity under N₁₅ treatment conditions was increased by 11.9–25.88% above N₀ levels, and the single-plant nitrogen fixation potential was increased by 13.86–18.85%. These findings suggest that small (N₁₅) doses of nitrogen can improve the proportion of nitrogen fixed by root nodules, thus providing key source of nitrogen for peanut growth. A few research efforts to date have explored the stimulating effects of small nitrogen doses on soybean seedling growth and early nodulation. For example, Gan [29] found that high mineral N concentrations (10 mM) in the form of nitrate, ammonium, or ammonium nitrate resulted in the significant suppression of root nodule numbers and dry weight values, whereas lower concentrations (1 or 3.75 mM) obviously improved nodule numbers, total N₂ fixed per plant. Kato [30] additionally determined that lotus root nodule nitrogenase activity were markedly inhibited at 27 h after nitrate addition, with both nitrogenase activity and nodule growth being suppressed by nitrate nitrogen in a manner related to the timing and location of the nitrogen application.

Peanuts grow rapidly during the seedling stage and exhibit some degree of nitrogen requirement during this period despite the fact that associated rhizobia lack the ability to fix nitrogen. Initial nitrogen application above this value of N₁₅ at the seedling stage inhibits nitrogen fixation. In our study, numbers of root nodules and associated dry weight continuously declined as the exogenous initial nitrogen concentration increased, demonstrating that excessive nitrogen can significantly inhibit the BNF capacity of peanut plants in line with prior evidence [31,32]. In conclusion, the present results suggest that the initial nitrogen dose applied for the large-grain HY22 cultivar and small-grain HY39 cultivar should be approximately 15 kg·hm⁻². Above these levels, increased nitrogen fertilizer application will adversely impact peanut plant BNF activity.

4.3. The Association between Biological Nitrogen Fixation and Root Growth Strategy

Roots and rhizomes are closely linked physically, the root systems of legumes form symbiotic nodules with rhizobacteria, and the root system provides a nutrient-rich ecological niche for nodules development that promotes biological nitrogen fixation. With both environmental nitrogen and microbial signals regulating the optimal generation of lateral roots and nodules in leguminous crops [33]. Our studies showed that the increase in BNF was obviously positively associated with the root dry weight, root surface area, nodule number, and total nitrogen accumulation. This suggests that changes in the peanut root system may impact nodulation and biological nitrogen fixation. Recent work has shown that nodules also influence changes in root architecture. Root nodules alter root system responses to changes in external nitrate levels [34]. These nodules can both directly influence plant phenotypes through the regulation of plant hormones and other signals that shape changes in plant root systems, while also indirectly influencing them through changes in stress responses as in the case of the shortening of root length due to nitrogen fixation in low nitrogen environments [35].

5. Conclusions

A two-year pot optimization experiment showed that initial nitrogen application of 15 kg·hm⁻² could improve the nitrogenase activity, nitrogen fixation potential per plant, and BNF. Therefore, based on the results of this experiment, the N15 treatment may be the most appropriate N concentration to promote the establishment of a symbiotic nitrogen fixation system, especially at the seedling stage of the plant.