Arbuscular mycorrhizal fungi enhance soybean phosphorus uptake and soil fertility under saline-alkaline stress

Abstract

Fungus-fertilizer interactions can enhance agricultural productivity and effective resource utilization, however, the study of the effect of arbuscular mycorrhizal fungi (AMF) and phosphorus on soil fertility and nutrient uptake of soybeans under salinity stress is still unclear. In this study, a mixture of three AMFs (Funneliformis mosseae, Rhizophagus intraradices, and Diversispora epigaea) was inoculated into the salt-sensitive soybean (Glycine max (L.) Merr.) cultivar ‘Wuxing No.2’ in a pot experiment set up for inoculation, no inoculation and five levels of phosphorus (P₂O₅) supply (such as 0, 50, 100, 250, 500 mg P kg⁻¹), bacterial phosphorus interactions totaling 10 treatments, each treatment 7 replications. Soil nutrient content and soybean nutrient uptake and translocation rates were determined at seasons of flowering pods, tympanic period and harvest period, respectively. Under low phosphorus (50 mg kg⁻¹) conditions, the soil available phosphorus content at the seasons of flowering pods increased by 23.11% compared with the uninoculated group. The accumulation of nitrogen, phosphorus, and potassium in the plants increased significantly, with the phosphorus content in leaves reaching 4.72 mg·g⁻¹, which was 98.50% higher than that in the high-phosphorus non-inoculated treatment. Meanwhile, it optimized nutrient partitioning, promoting the transfer of phosphorus to the stalks (with the phosphorus transport rate in stems being 37.27% in the + AMF P50 treatment) to support grain formation. In contrast, the uninoculated group required a higher phosphorus level (250 mg kg⁻¹) to reach the peak of biomass, with the root fresh weight peaking at 13.71 g. The low phosphorus inoculation treatment can improve soil fertility and plant nutrient uptake and utilization, and promote the efficient use of agricultural resources.

Introduction

Recently, a large number of land salinisation problems of varying degrees exist globally, and the area of salinised soil in China has reached 99.133 million hectares, of which the area of saline-alkaline land in Xinjiang accounts for 22.01% of the country^1,2. Under saline stress, soil nutrients are immobilized, causing a significant decrease in their content, and excessive salinity inhibits the activity of soil microorganisms and reduces their ability to decompose and convert organic matter, thus affecting nutrient release and supply³. Salinity-induced membrane peroxidation destroys membrane integrity, leading to the leakage of essential intracellular components, which in turn hinders plant growth⁴, leading to decreased soybean seed germination and inhibition of seedling growth, and a decrease in soybean yield of up to 24–65% when soil conductivity is greater than 5 dS m⁻¹⁵. Secondary salinisation not only reduces crop yield, but also negatively affects soil microbial diversity, destroys the balance of the ecosystem, and reduces the self-regeneration capacity of the soil.

In the face of the dual inhibition of soil nutrient availability and plant growth under saline-alkali stress, arbuscular mycorrhizal fungi (AMF) provide a potential solution to improve this dilemma. AMF is an very widely distributed class of soil fungi in nature, which can form a symbiotic relationship with about 71% of angiosperms (including most of the crops), and a variety of enzymes and organic acids secrete d by the root system of the plant induced by AMF can promote the decomposition of mineral and organic substances in the soil and thus improve the effectiveness of nutrients in the soil and accelerate the rate of soil nutrient cycling^6–8. This symbiotic relationship contains efficient reactive oxygen species (ROS) scavenging mechanism, which enhances plant defences and growth performance in the early stages of salinity stress^9,10. AMF helps to enhance enzymatic and non-enzymatic antioxidant defense systems, induces systemic tolerance, and attenuates lipid peroxidation^11,12. In addition, AMF extends beyond the inter-root zone through its mycelium, closely connecting the root system to the surrounding soil microenvironment, effectively expanding the root uptake area and significantly enhancing the efficiency of nutrient uptake, especially phosphorus¹³. Phosphorus is a key limiting factor under saline-alkali stress, and its synergistic effect with AMF is particularly important. Phosphorus, as an essential mass element for plant growth, is also one of the main fertilizers to ensure high and stable crop yields¹⁴. Due to the soil-forming characteristics of saline soils, phosphorus in the soil is easily immobilized and difficult to move, resulting in low effective phosphorus content in the soil, thus becoming one of the major limiting factors for crop yield¹⁵. Low phosphorus stress affects the normal growth and development of soybean plants, influencing their flower bud differentiation and causing a reduction in their pod number, thus reducing yield¹⁶. Inoculation of AMF can significantly increase the nutrient content of the plant and promote its growth and development, and the rational regulation of phosphorus level is the key to improve the nutrient uptake and utilization of the plant (Fig. 1).

Fig. 1.

Effect of phosphorus and AMF interactions on the growth and development of soybean plants.

Soybean (Glycine max (L.) Merr.), a moderately salt-tolerant crop, typically has a salt tolerance threshold of 5.0 dS m⁻¹ soil electrical conductivity (EC), with plant mortality increasing significantly at EC ≥ 6.7 dS m⁻¹. Previous studies have demonstrated that AMF inoculation under phosphorus-deficient conditions can significantly enhance soybean growth and phosphorus uptake¹⁷. However, under saline-alkali stress conditions, limited attention has been paid to the optimal amount of phosphorus fertilizer application when inoculating AMF to achieve the best benefits in terms of soil fertility and soybean nutrient absorption. Therefore, a pot experiment was designed in this study. Under saline-alkali stress, the soil fertility and the nitrogen, phosphorus, and potassium contents and nutrient translocation in soybean roots, stems, and leaves were measured under different phosphorus supply levels with AMF inoculation. Following optimal allocation theory, plants should increase the root fraction of biomass (or root exudates) in phosphorus deficient environments. Following the nutritional mutualism hypothesis, phosphorus deficiency should drive plants to outsource their phosphorus acquisition to AMF. We hypothesized that less phosphorus application and AMF inoculation could achieve double enhancement of soil fertility and soybean nutrients, and by determining the optimal ratio threshold between AMF inoculation and phosphorus fertilizer application in saline soybean cultivation, we aimed to provide a strong basis for the study of fungus-fertilizer interactions to promote soybean yield increase.

Materials and methods

Test material

The test soybeans for this experiment were salt-sensitive varieties: ‘Wuxing No.2’ (salt tolerance threshold 3.2 dS m⁻¹). The tested AMF species are: Funneliformis mosseae, with the germplasm resource bank number of BGC HLJ02A; Rhizophagus intraradices, with the BGC number of BGC BJ09; Diversispora epigaea, with the BGC number of BGC 504. The culture medium containing corresponding spores, extraradical hyphae and host plant root segments (provided by the Institute of Mycorrhizal Root Biology, Yangtze University) has an average spore density of 34 spores per gram.

This experiment adopted a pot experiment. The pot specifications are 30 cm × 20 cm × 25 cm (pot mouth diameter × pot bottom diameter × height). The pots were disinfected with 75% alcohol before planting and set aside. The test soil was collected from the experimental field of Shihezi University (0–20 cm), air-dried and sieved through 8 mm sieve, large stones and plant residual root fragments were removed, and the basic physical and chemical properties of the soil were determined after 2 h of autoclaving (115 kPa, 121 °C): soil electrical conductivity is 0.24 dS m⁻¹, soil pH is 7.8, soil alkali-hydrolyzable nitrogen is 46.25 mg·kg⁻¹, soil available phosphorus is 2.21 mg kg⁻¹, soil available potassium is 306.22 mg kg⁻¹, and soil organic matter is 17.25 g kg⁻¹.

Experimental design

The research was conducted in the experimental field of Shihezi University on May 2023. Two inoculation levels (inoculated and non-inoculated; +AMF, -AMF) and five phosphorus application (P₂O₅) levels (0, 50, 100, 250, 500 mg P kg⁻¹) were set up, which were sequentially recorded as no phosphorus (P0), low phosphorus (P50), medium phosphorus (P100, P250), and high phosphorus (P500), with a total of 10 treatments. Each treatment was repeated seven times, for a total of 70 pots. Each pot was filled with 3 kg of sterilized soil and about 1000 spores (Funneliformis mosseae, Rhizophagus intraradices, Diversispora epigaea mixed in equal proportions), where the control group was used with equal amount of sterilized soil.

High-quality soybean seeds of uniform size and full grains were selected, and the soybean seeds were disinfected with 10% H₂O₂ for 10 min, and then rinsed repeatedly with distilled water to kill the natural rhizobia and other microorganisms adhering to the surface, and 9 seeds were applied to each pot, and interplanting was done to 3 plants after 7 days of germination. The phosphorus fertilizer applied was calcium superphosphate (12% P₂O₅), the nitrogen fertilizer applied was urea at a dosage of 180 mg N · kg⁻¹, and the potash fertilizer was KCl at a dosage of 120 mg K₂O· kg⁻¹, all of which were dissolved with water and applied to the pots at the seedling (20%), flowering (30%), and podding (50%) stages, respectively. Soybeans were grown for 30 days to start the saline stress treatment, according to the salt composition of saline land in Xinjiang, the saline solution was configured as follows: NaCl, Na₂SO₄, NaHCO₃, Na₂CO₃ = 12:9:8:1, and the stress concentration was 100 mmol L⁻¹, and the method of watering was used in stages, with 1/5 of the total saline concentration being watered in 8 h intervals for a total of 2 days to reach the preset concentration, and in order to keep the constant saline concentration, each pot was watered with 200 ml of saline solution, weighed once in 1–2 weeks, and weighed and watered every evening, a plastic tray was placed under the pots in order to prevent the loss of salinity, and the solution was poured back into the pots promptly after each irrigation to maintain the soil moisture content up to 70% of the soil’s volumetric weight.

Sample collection

Soybean samples were collected at the seasons of flowering pods, tympanic period and maturity stages. Soil samples were collected by inserting a polyethylene tube with an inner diameter of 2 cm vertically into the potting substrate at a depth of 20 cm, slowly extracting the soil from the core of the tube, removing the visible root residues and mixing them well, and storing the soil at −20 °C. For plant samples, the soybean was uprooted and placed on sterile kraft paper, the main roots and lateral roots were separated, the soil attached to the root surface was gently removed with a soft brush, and the surface impurities were removed by rapid rinsing with deionized water. The surface impurities were removed by quick rinsing with deionized water, and the roots were cut off at the root-neck junction after drying with absorbent paper to obtain stem and root samples respectively. The samples were immediately frozen in liquid nitrogen at −80 °C.

Determination indexes and methods

AMF mycorrhizal infestation rate

Two random soybean roots were chosen, washed with water, and cut into 1 cm segments. These segments underwent a 10% KOH treatment for transparency, followed by a 90 °C water bath for 30 min. After washing, they were acidified with 5% lactic acid and then stained with trypan blue. Under a microscope, each root segment was assessed on a scale of 0, 10, 20, 30, up to 100, with 10 as the initial level. The mycorrhizal infection rate was calculated using the following formula¹⁸:

× 100.

Plant growth

The plant height and root length of soybeans (in cm) were measured using a ruler. An electronic balance (model: BSA224S-CW, manufacturer: Sartorius Scientific Instruments (Beijing) Co., Ltd.) was used to measure the fresh weights (in g) of the above-ground part and roots of the plants. The samples were killed at 105 °C for 15 min in an oven and then dried to a constant weight at 75 °C to measure the dry weights (in g).

Soil nutrient content

Soil alkaline nitrogen was determined by alkaline diffusion method, soil quick phosphorus was determined by sodium bicarbonate leaching-molybdenum antimony colorimetric method, soil quick potassium was determined by ammonium acetate leaching-flame photometer method, and soil organic matter was determined by potassium dichromate volumetric method-external heating method¹⁹.

Calculation of nitrogen, phosphorus, and potassium contents and related indicators in plant roots, stems, and leaves

The plant samples were wet-digested with concentrated H₂SO₄-H₂O₂, and the nitrogen, phosphorus, and potassium contents of the plant samples were determined by Nessler’s colorimetric method, molybdenum-antimony anti-colorimetric method, and flame photometer method respectively¹⁹.

Nitrogen, phosphorus, and potassium translocation amount = nitrogen, phosphorus, and potassium accumulation amount during the flowering period-nitrogen, phosphorus, and potassium accumulation amount during the maturity period²⁰.

Nitrogen, phosphorus, and potassium translocation rate (%) = nitrogen, phosphorus, and potassium translocation amount/nitrogen, phosphorus, and potassium accumulation amount during the flowering period × 100²⁰.

Data processing

The experimental data were processed and graphed using Microsoft Excel 2023. Two-factor analysis of variance and significance analysis (Duncan’s method, P < 0.05) were performed using SPSS 26.0 software.

Results

Effect of phosphorus and AMF on the infestation of soybean roots

As shown in Fig. 2, the AMF infestation rate in soybean roots under saline and alkaline stress followed the pattern of grain tympanic period > seasons of flowering pods > maturity stage as the reproductive process progressed. Phosphorus application significantly regulated the efficiency of mycorrhizal symbiosis, in which the infestation rate peaked at all reproductive stages under P50 treatment, and was significantly increased by 36.80% at the tympanic period compared with P0. The infestation rate at the tympanic period was also increased by 14.54% under P100 treatment compared with P0, whereas the infestation rate at the seasons of flowering pods under P500 treatment was reduced by 20.75%, 25.35%, and 17.24% compared with that under P0, P50, and P100, respectively, which indicated that high phosphorus significantly inhibited AMF colonization in the root system. phosphorus significantly inhibited AMF colonization. At the medium phosphorus level (P100), the infestation rates at the seasons of flowering pods, tympanic and maturity stages increased by 11.64%, 16.14% and 5.35%, respectively, compared with P250, while the P500 treatment decreased by 20.75%−25.35% compared with P250 during the same period. The mycorrhizal infestation rate showed a tendency to increase and then decrease with the increase of phosphorus application, and P50 was the optimal threshold.

Fig. 2.

Effects of phosphorus and AMF on soybean root infection. Note: P0, P50, P100, P250, and P500 were the phosphorus application rates of 0, 50, 100, 250, and 500 mg P kg⁻¹, respectively. Different lowercase letters indicate significant differences in different phosphorus treatments under the same inoculation (P < 0.05). (the same below).

Effects of phosphorus and AMF on the growth of soybeans under Saline-alkali stress

Phosphorus supply level and AMF had significant synergistic effects on soybean growth indexes under saline stress. Soybean plant height, root length and fresh weight of rhizomes and leaves showed a tendency of increasing and then decreasing with increasing phosphorus levels (Table 1), but the peak of the + AMF group was advanced to the medium-low phosphorus level (P50-P100), while that of the -AMF group was delayed to the P250 level. The + AMF group had a plant height of 61.27 cm at P100 (13.95% enhancement from P0, P < 0.05), and the root length reached 26.66 cm at P50 (36.65% increase from P0), which was significantly better than that of the -AMF group (root length of 18.59 cm at P100, + 29.34%).The AMF significantly mitigated the low-phosphorus stress: the + AMF group had a better plant height of 61.27 cm at P100 (13.95% enhancement from P0, P < 0.05), and a better plant height of 26.66 cm at P50 (36.65% increase from P0) than the -AMF group (18.59 cm at P100, + 29.34%). and leaf fresh weight increased by 9.59%, 2.72%, and 6.92%, respectively, compared with P0, while the -AMF group needed until medium phosphorus (P250) to reach the peak (root fresh weight 13.71 g, + 15.5%). Under high phosphorus (P500), the growth indexes of the + AMF group decreased by 4.37%−24.83% from the peak value, and the difference was reduced (P > 0.05) with the -AMF group, which showed an abrupt decrease of 35.4% in root length. Two-way ANOVA showed that there was a highly significant interaction between phosphorus and AMF on plant height, root length, and stem and leaf fresh weight, and AMF optimized plant acclimatization by enhancing the stability of phosphorus uptake.

Table 1.

Effects of phosphorus and AMF on soybean growth under salt and alkali stress.

Treatment		Plant height/cm	Root length/cm	Ground fresh weight/g	Dry weight on the ground/g	leaf dry weight/g
+AMF	P0	53.77 ± 1.08Ac	19.51 ± 1.06Ad	13.87 ± 0.47Aab	16.90 ± 0.39Aa	6.36 ± 0.29Ab
	P50	60.29 ± 0.97Aab	26.66 ± 0.85Aa	15.20 ± 0.63Aa	17.38 ± 0.55Aa	6.80 ± 0.14Aa
	P100	61.27 ± 1.33Aa	23.47 ± 0.61Ab	14.74 ± 0.47Aa	16.14 ± 0.92Aab	6.21 ± 0.21Abc
	P250	59.74 ± 1.26Aab	21.76 ± 0.70Ac	13.86 ± 0.73Aab	16.11 ± 0.73Aab	6.10 ± 0.20Abc
	P500	58.59 ± 1.02Ab	18.36 ± 0.49Ad	13.25 ± 1.06Ab	15.13 ± 0.79Ab	5.91 ± 0.21Ac
-AMF	P0	47.53 ± 2.27Bb	14.35 ± 0.48Bc	11.87 ± 0.35Ab	15.54 ± 0.16Bc	5.49 ± 0.22Bab
	P50	53.29 ± 2.06Bab	16.69 ± 0.92Bb	12.25 ± 0.44Bb	16.31 ± 0.05Bbc	5.41 ± 0.30Bab
	P100	55.29 ± 1.15Ba	18.59 ± 0.70Ba	12.93 ± 0.79Bab	16.51 ± 0.38Bab	5.63 ± 0.52Ba
	P250	56.46 ± 0.60Bab	15.34 ± 0.48Bc	13.71 ± 0.59Ba	17.19 ± 0.65Ba	5.45 ± 0.46Aab
	P500	50.45 ± 2.22Bb	12.01 ± 0.73Bd	11.84 ± 0.81Bc	15.73 ± 0.59Bbc	4.80 ± 0.11Bb
P		**	**	**	**	**
AM		**	**	**	**	**
P×AM		*	**	NS	**	*

Note: P0, P50, P100, P250, and P500 were the phosphorus application rates of 0, 50, 100, 250, and 500 mg P·kg⁻¹, respectively. Different lowercase letters indicate the significant difference of different phosphorus supply treatments under the same inoculation condition, and different uppercase letters indicate the significant difference of different inoculation conditions under the same phosphorus supply treatment (P < 0.05). ** mean P<0.01, * mean P<0.05, NS mean not significant. (the same below).

Effects of phosphorus and AMF on the biomass allocation proportion of soybeans under Saline-alkali stress

The pod biomass allocation proportion in the low-phosphorus inoculation treatment was higher than that in other treatments. It increased by 15.83%, 6.11%, 10.32%, and 13.32% compared with P0, P100, P250, and P500 in the inoculated group respectively, and increased by 19.83%, 17.8%, 13.62%, 6.38%, and 20.52% compared with the non-inoculated group respectively (Fig. 3). For soybean plants inoculated with AMF, under the condition of applying 50 mg kg⁻¹ phosphorus fertilizer, the pod dry matter allocation rate was significantly higher than that in the non-inoculated treatment with 250 mg kg⁻¹ phosphorus fertilizer. This indicates that inoculating AMF significantly improved the absorption efficiency of phosphorus by soybean plants, optimized the nutrient allocation proportion among various plant organs, and promoted high-yield of soybeans.

Fig. 3.

Effects of phosphorus and AMF on biomass allocation ratio under saline-alkali stress.

Effects of phosphorus and AMF on soil nutrient content

As shown in Fig. 4a, under saline-alkali stress, the inoculated group increased the soil alkali-hydrolyzable nitrogen content during the soybean seasons of flowering pods tympanic period, and harvest period compared with the non-inoculated group, showing a trend of P50 > P100 > P250 > P500. Under the low-phosphorus inoculation treatment, it increased by 30.26%, 24.77%, and 18.13% compared with the non-inoculated group, and increased by 23.51%, 15.44%, and 19.64% compared with the high-phosphorus level.

Fig. 4.

Effects of phosphorus and AMF on the contents of alkali-hydrolyzed nitrogen (a), available phosphorus (b), available potassium (c) and organic matter (d) in soil. Note: Different lowercase letters indicate significant differences between different phosphorus supply treatments over the same period, different capital letters indicate significant differences in vaccination levels over the same period (P < 0.05).

With the increase of phosphorus supply level, the content of available phosphorus in soil showed a continuous increasing trend (Fig. 4b). Inoculation with AMF increased the content of available phosphorus in soil. Among them, the improvement effect was the best during the seasons of flowering pods with an increase of 77.16%, 23.11%, 11.54%, 18.63% and 26.72% compared with the non-inoculated group, followed by the harvest period and the tympanic period. During the seasons of flowering pods the growth demand and response were strong, the interaction between roots and fungi was strengthened, and root exudates further stimulated the growth and activity of AMF, prompting it to activate the fixed phosphorus in the soil. throughout the tympanic period, soybeans absorbed a large amount of phosphorus from the soil to meet the huge nutrient demand for grain development, resulting in a reduced increase in the content of available phosphorus in the soil. For the duration of the harvest period, the speed of soybean’s need for phosphorus slowed down, and the inoculated AMF continued to play a role, converting the insoluble phosphorus in the soil into available phosphorus, so the increase range was greater than that in the tympanic period.

With the increase of phosphorus supply level, the content of available potassium in soil showed a trend of first increasing and then decreasing (Fig. 4c). Under the inoculation condition, the content of available potassium in soil at low-phosphorus level was higher than that at other phosphorus application levels, with an increase of 8.13%, 9.03% and 6.01% compared with high-phosphorus levels. Under the non-inoculation condition, it was the largest at P100 during the seasons of flowering pods and tympanic periods, and reached the peak at P250 during the harvest period.

With the increase of phosphorus supply level, the content of soil organic matter showed a trend of first increasing and then decreasing (Fig. 4d). The inoculated group increased the content of soil organic matter during the soybean seasons of flowering pods tympanic period and harvest period compared with the non-inoculated group. Among them, the improvement effect was the best during the harvest period, with an increase of 53.58%, 55.31%, 37.91%, 37.44% and 50.34% compared with the non-inoculated group. Under the low-phosphorus inoculation treatment, it increased by 27.87%, 27.87% and 55.31% compared with the non-inoculated group, and increased by 12.37%, 18.06% and 21.62% compared with high-phosphorus levels. As the growth period progressed, the content of soil nutrients continuously decreased.

Effects of phosphorus and AMF on nutrient absorption in soybean roots, stems and leaves under saline-alkali stress

During the three key growth stages of soybean, the absorption and utilization of nutrients are most obvious during the tympanic period. Generally, the nutrient content in the inoculated group is higher than that in the non-inoculated group (Fig. 5). Among the three major nutrient elements of nitrogen, phosphorus, and potassium, the absorption of phosphorus is greater than that of nitrogen and potassium. Among them, the increase in the phosphorus content of the leaves during the tympanic period is the most significant. With the increase in the amount of phosphorus application, the inoculated group increased by 12.6%, 31.6%, 39.3%, 49.6%, and 65.7% compared with the non-inoculated group. This effect can be attributed to the fact that AMF uses its extensive mycelial network to increase the contact interface between the soil and plant roots and activates the insoluble phosphorus in the soil to promote the overall acquisition ability of various nutrients. However, during the harvest period, there is no significant difference in the potassium content between the inoculated group and the non-inoculated group. It is analyzed that as the growth period of soybeans progresses, the nutrient content required by the plants decreases, and the excessive potassium element may instead inhibit some functions of AMF and reduce the absorption of potassium element.

Fig. 5.

Effects of phosphorus and AMF on nitrogen, phosphorus and potassium contents of soybean roots and leaves under salt and alkali stress. Note: Among them, (a), (b) and (c) are the nitrogen contents of the root, stem and leaves of soybean in the bulging stage, and (d) are the nitrogen contents under different inoculation in the three stages. (e), (f) and (j) are the phosphorus contents in the root, stem and leaves of soybean in the bulging stage, and (h) are the phosphorus contents under different inoculation in the three stages. Among them, (i), (j) and (k) are the potassium contents in the root, stem and leaves of soybean at the grain filling stage, and (l) are the potassium contents under different inoculation in the three stages.

With the increase in the amount of phosphorus applied, the nutrient content in the roots, stems and leaves of soybeans during the tympanic period first increased and then decreased. Generally speaking, the nutrient content in the inoculated group was the highest at the P100 level, and the nutrient content in the non-inoculated group reached its peak at P250. Under the inoculation condition, the phosphorus content in the plant roots and stems was the highest at the P100 level and that in the leaves was the highest at the P50 level, with average values of 2.79 ± 0.06, 1.50 ± 0.08, 4.72 ± 0.11 mg g⁻¹ respectively. Compared with the high-phosphorus non-inoculated treatment in the same period, they increased by 42.35%, 61.00% and 98.50% respectively. The nitrogen content and potassium content of the plant were both the highest at the P100 level under the inoculation condition, with median values of 18.22, 13.19, 25.24 mg g⁻¹ and 29.10, 16.52, 28.66 mg g⁻¹ respectively. Inoculation significantly improved the absorption and utilization efficiency of key nutrients such as nitrogen, phosphorus and potassium by the plant, so that the growth demand of the plant could be met under a lower level of nutrient supply, effectively reducing the redundant input of nutrients and resource waste.

The nitrogen nutrient content of each organ of the plant showed the pattern of seasons of flowering pods > tympanic period > harvest period (Fig. 5d). The potassium nutrient content showed the pattern of tympanic period > seasons of flowering pods > harvest period (Fig. 5i). The phosphorus nutrient content in leaves and non-inoculated stems first increased and then decreased, while in roots and inoculated stems, it showed a continuous decreasing trend (Fig. 5h). During the seasons of flowering pods, soybeans require a large amount of nitrogen nutrients for vegetative and reproductive growth. Potassium is mainly used for the transport and accumulation of photosynthetic products, so the demand is the largest during the tympanic period. As an important element for promoting grain development, the distribution and translocation pattern of phosphorus in the plant is as follows: with the progress of the growth process, phosphorus gradually accumulates in the stems and leaves. Especially during the tympanic period, the phosphorus content in the leaves is significantly higher than that in the stems and roots. Therefore, the proportion of phosphorus content in the tympanic period compared with the seasons of flowering pods is leaves > stems > roots.

Changes in nutrient uptake in soybean roots, stems and leaves by phosphorus and AMF

The nutrient transport amounts in the roots, stems and leaves of the low-phosphorus inoculation treatment were higher than those of the zero-phosphorus, medium-and high-phosphorus level treatments, except that the potassium element in the stems had the highest transport amount at P100 (Table 2). The transport amounts of nitrogen and phosphorus in soybeans and the transport rate of phosphorus under inoculation all showed the order of leaves > stems > roots, and the transport rates of nitrogen and potassium showed the order of stems > leaves > roots. Comparing different nutrient transport rates, the transport rate of phosphorus element in the stems and leaves of the plant was higher than that of nitrogen and potassium. The inoculated group had the greatest increase in the phosphorus transport rate of the plant leaves compared with the non-inoculated group, with an increase of 15.46%, 26.72%, 7.77%, 18.59% and 27.75% respectively as the phosphorus application amount increased.

Table 2.

Changes of phosphorus and AMF on nutrient movement of soybean rhizomes and leaves.

Treatment			Transport amount(mg/plant)			Transport rate(%)
			Root	stem	Leaf	Root	stem	Leaf
Nitrogen	+AMF	P0	27.48 ± 1.67b	45.05 ± 0.68bc	68.21 ± 1.98c	10.13 ± 1.63b	25.94 ± 2.13b	20.57 ± 1.93b
		P50	36.40 ± 5.43a	49.94 ± 0.57a	74.75 ± 3.36a	13.10 ± 1.09a	31.32 ± 3.05a	24.96 ± 2.45a
		P100	34.44 ± 1.55a	47.87 ± 3.47ab	71.34 ± 1.84ab	12.64 ± 1.25ab	27.56 ± 3.19ab	21.59 ± 1.73ab
		P250	33.26 ± 3.05ab	44.50 ± 2.23bc	70.49 ± 0.92b	12.91 ± 2.35a	23.21 ± 1.76b	21.28 ± 1.26b
		P500	31.44 ± 1.87ab	42.33 ± 0.65c	64.69 ± 1.89c	11.62 ± 1.46ab	23.69 ± 2.69b	18.34 ± 2.03b
	-AMF	P0	17.31 ± 1.36c	37.52 ± 0.68c	54.86 ± 1.17c	8.52 ± 1.09b	29.27 ± 3.19a	15.89 ± 1.12a
		P50	24.79 ± 0.97a	44.29 ± 3.28a	59.44 ± 0.70a	10.52 ± 1.23a	26.85 ± 3.23ab	16.32 ± 1.30a
		P100	22.95 ± 0.60b	40.71 ± 0.83b	56.53 ± 1.31b	8.84 ± 0.53ab	24.31 ± 1.49bc	14.63 ± 1.09ab
		P250	21.32 ± 0.96b	35.79 ± 1.38d	51.00 ± 0.63d	8.92 ± 0.92ab	20.74 ± 2.19 cd	14.43 ± 1.45ab
		P500	18.68 ± 0.79c	29.58 ± 0.87e	45.23 ± 0.42e	8.95 ± 0.75ab	19.43 ± 1.37d	12.52 ± 1.29b
Phosphorus	+AMF	P0	1.99 ± 0.15c	3.38 ± 0.35c	16.13 ± 0.22c	4.43 ± 0.13b	36.97 ± 3.29a	52.98 ± 2.88d
		P50	2.55 ± 0.16a	4.34 ± 0.08a	30.60 ± 0.98a	5.80 ± 0.80a	37.27 ± 5.20a	96.93 ± 6.22a
		P100	2.21 ± 0.12bc	4.03 ± 0.13ab	30.39 ± 0.48a	4.81 ± 0.33ab	25.79 ± 2.40b	81.72 ± 3.18b
		P250	2.43 ± 0.09ab	3.91 ± 0.15b	24.49 ± 0.58b	5.62 ± 0.66a	24.27 ± 4.60b	64.13 ± 3.9c
		P500	1.73 ± 0.11d	3.55 ± 0.09c	23.52 ± 0.74b	4.08 ± 0.53b	21.51 ± 1.73b	68.72 ± 2.31c
	-AMF	P0	1.75 ± 0.03c	3.14 ± 0.17c	10.16 ± 0.39e	4.61 ± 0.23b	61.73 ± 4.27a	37.52 ± 2.73b
		P50	2.09 ± 0.13ab	4.12 ± 0.05a	21.43 ± 1.27b	5.71 ± 0.30a	62.99 ± 4.55a	70.21 ± 8.84a
		P100	2.15 ± 0.07a	3.63 ± 0.10a	25.46 ± 0.93a	4.58 ± 0.51ab	50.60 ± 3.22b	73.95 ± 1.04a
		P250	2.21 ± 0.02a	3.32 ± 0.16b	15.86 ± 0.87c	4.54 ± 0.29ab	49.13 ± 6.60b	45.54 ± 5.06b
		P500	1.98 ± 0.13b	3.14 ± 0.04b	12.20 ± 0.45d	3.85 ± 0.39a	50.13 ± 0.98b	40.97 ± 4.99b
Potassium	+AMF	P0	19.35 ± 0.85e	37.36 ± 0.88c	35.13 ± 0.93d	4.23 ± 0.32d	25.46 ± 1.22bc	10.94 ± 0.09c
		P50	40.08 ± 0.26a	45.32 ± 0.79b	76.68 ± 1.23a	9.55 ± 0.40a	28.44 ± 1.89a	25.81 ± 1.20a
		P100	34.61 ± 0.96b	62.13 ± 0.89a	62.37 ± 0.72b	7.51 ± 0.43b	35.80 ± 0.37a	18.66 ± 0.74b
		P250	24.19 ± 0.68c	49.84 ± 1.29bc	40.63 ± 0.84c	5.15 ± 0.18c	26.48 ± 1.52b	11.05 ± 0.27c
		P500	21.37 ± 0.82d	45.31 ± 0.86c	30.87 ± 1.67e	4.23 ± 0.17b	24.90 ± 1.99a	7.89 ± 0.86d
	-AMF	P0	16.78 ± 0.87d	25.22 ± 0.21c	24.85 ± 0.62d	4.53 ± 0.15b	20.43 ± 0.31b	7.17 ± 0.32b
		P50	21.15 ± 0.72b	38.05 ± 0.66a	42.25 ± 0.33b	5.10 ± 0.10b	24.96 ± 1.01a	11.72 ± 1.04c
		P100	27.10 ± 0.84a	32.14 ± 1.14b	46.27 ± 1.04a	7.07 ± 0.20a	20.33 ± 0.54b	12.21 ± 0.49a
		P250	18.35 ± 0.50c	39.09 ± 1.25a	27.41 ± 0.61c	4.64 ± 0.63b	24.35 ± 1.03a	7.71 ± 0.68a
		P500	13.77 ± 0.66e	31.65 ± 0.54b	22.67 ± 0.52e	3.73 ± 0.22c	21.47 ± 1.08b	6.07 ± 0.35bc
P			**	**	**	**	**	**
AM			*	**	*	**	**	**
P×AM			*	*	**	*	NS	**

Effects of phosphorus and AMF on the proportion of nutrient allocation in the roots, stems and leaves of mature soybeans

Inoculation with AMF significantly increased the proportion of phosphorus allocation in the stems. With the increase in the amount of phosphorus applied, the inoculated group increased by 32.55%, 68.78%, 110.50%, 72.27% and 188.97% respectively compared with the non-inoculated group (Fig. 6). Inoculation with AMF significantly enhanced the plant’s ability to absorb phosphorus and promoted the effective transfer of phosphorus from the underground part to the above-ground part. As an important component of soybean grains, the phosphorus content in the stems has a positive impact on increasing soybean yield.

Fig. 6.

Effects of phosphorus and AMF on nutrient distribution of nitrogen (a), phosphorus (b) and potassium (c) in roots and leaves of soybean.

Correlation analysis of soil nutrients, soybean growth and nutrients

Through correlation heat map analysis, significant positive correlations were generally found between soybean plant growth indicators, soil nutrients, plant N and plant K contents (Fig. 7). Unlike the rest of the indicators, soil quick-acting phosphorus content was negatively correlated with most of the variables, probably because high phosphorus application levels led to phosphorus enrichment in the soil, triggering antagonistic effects with other nutrients and indirectly inhibiting plant uptake of nitrogen and potassium as well as the efficiency of photosynthate partitioning.

Fig. 7.

Heat map of correlation analysis between soil nutrients, soybean growth and nutrients.

Discussion

The biomass of various organs of soybeans can reflect the accumulation of nutrients in soybeans. Due to the increase in Na⁺ content under stress, the plants are affected by saline-alkali toxicity. The high-alkali environment of the soil greatly reduces the roots absorption of soil nutrients and inhibits the plant’s nutrient metabolism^21,22. The results of this study show that both phosphorus application and AMF inoculation can affect the biomass of soybeans under saline-alkali stress. Under the condition of non-inoculation with AMF, with the increase in phosphorus application amount, the fresh and dry weights of the above-ground and underground parts of soybean plants showed an obvious increasing trend, but decreased at a high-phosphorus level. This indicates that phosphorus application can increase the biomass of various organs of soybeans. Liu²³ research shows that phosphorus application can promote root growth more, which is similar to the results of this study. In comparing the effects of inoculation and non-inoculation with AMF on the biomass of various parts of soybeans, it was found that AMF inoculation can significantly increase the biomass of soybeans, and the biomass reaches the maximum when the phosphorus application amount is 50 mg kg⁻¹. There is little difference in biomass when the phosphorus application amounts are 100 mg kg⁻¹ and 250 mg kg⁻¹. When the phosphorus application amount is 500 mg kg⁻¹, the biomass of each inoculation treatment is the smallest. Therefore, the inoculation of AMF with a phosphorus application amount of 50 mg kg⁻¹ has the best promoting effect on soybean biomass.

During the salinization process of the soil, its nutrients will be out of balance, resulting in severe nutrient loss. In a high-alkali soil environment, mineral elements such as Mg²⁺ and P will precipitate, resulting in soil nutrient deficiency and hindering the absorption of nutrient elements by plant roots²⁴. This study found that under saline-alkali stress conditions, the contents of soil alkali-hydrolyzable nitrogen, available phosphorus, available potassium and organic matter first increased and then decreased with the increase in phosphorus supply level, and with the extension of the growth period, the increase amount continued to decrease. In addition, through AMF inoculation, the content of soil available phosphorus at different phosphorus supply levels can be significantly increased. It was found that the synergistic effect of AMF was characterized by “low phosphorus dependence and high phosphorus inhibition”. At low phosphorus (P50), mycelium secreted phosphatase to decompose organic phosphorus and acidified the inter-root zone through H⁺-ATPase to release fixed phosphorus²⁵; at the same time, mycorrhizal symbiosis up-regulated host phosphorus transporter genes (e.g., GmPTs) to enhance phosphorus uptake efficiency²⁶. High phosphorus (P500), on the other hand, inhibited AMF infestation rate and mycelial expansion, reducing the symbiotic efficiency and leading to biomass decline. Moreover, AMF can promote the transpiration of plants. This transpiration will stimulate plant roots to absorb a large amount of water and enrich a large number of ionic nitrogen in the soil around the rhizosphere, and the content of alkali-hydrolyzable nitrogen in the soil is also correspondingly increased. At the same time, AMF inoculation will also affect the respiration rate of soil organic matter and accelerate its degradation process, thus promoting the release of nutrients²⁷. AMF inoculation in a low-phosphorus environment significantly increased the nutrient content in the soil rhizosphere, indicating that soybeans rely more on AMF to absorb nutrients and water in the soil in a low-phosphorus environment.

AMF inoculation promotes the absorption and utilization of nutrients by soybean roots. After AMF invades soybean roots, it improves the roots’ absorption and utilization of soil nutrients, changes the growth characteristics of the roots, and promotes the transportation of soybean nutrients from the underground part to the above-ground part²⁸. Previous studies have shown that the arbuscular structure formed by AMF and parasitizing on the roots of host plants can form an extraradical mycelial network, expand the absorption area of root nutrition, reach the areas that the plant roots are difficult to touch, thereby increasing the plant’s absorption of N, P and K, and further promoting the growth and development of the plant²⁹. Studies have shown that at the same phosphorus supply level, AMF inoculation can significantly increase the nitrogen accumulation in the above-ground part of wheat, promote the growth of wheat plants, root development and the absorption of heterogeneous nutrients^30,31. The results of this study show that with the increase in phosphorus application amount, the N, P and K accumulations in soybean roots, stems and leaves first increase and then decrease. AMF inoculation significantly increases the nitrogen, phosphorus and potassium accumulations of soybean plants under saline-alkali stress, and the improvement of nutrient accumulation in soybean leaves is the most obvious, with the highest nutrient accumulation. In addition, AMF also promotes the nutrient transport amount and transport rate of various organs of soybeans.

The absorption and distribution of nutrients can directly affect the growth and development of crops and then affect the yield^32–34. AMF optimizes the transport of N, P, and K to the leaves and enhances photosynthetic product accumulation, which is related to the mechanism by which mycorrhizae promote sucrose unloading and phloem transport³⁵. Appropriate phosphorus levels (P50-P100) coordinate nutrient growth and reproductive allocation and promote dry matter transfer to the seed, while AMF further supports yield formation by enhancing the efficiency of nutrient remobilization (e.g., recycling of N and P in senescent leaves)³⁶. Appropriate phosphorus application can promote tillering and root growth of rice at the seedling stage, optimize the population structure, increase the dry matter distribution of tillering stems of rice and the dry matter transport amount of vegetative organs, ensure more N, P and K to be transported to the above-ground part, and thus increase the accumulation of dry matter³⁷, which is consistent with the results of this study.

Conclusions

In this study, we confirmed that the synergistic effect of AMF inoculation with low and medium phosphorus levels (50–100 mg kg⁻¹) significantly enhanced soil nutrient activation efficiency and soybean stress tolerance under saline and alkaline stress. Inoculation of AMF activated inter-root fixed phosphorus through the secretion of organic acids and cystinomycin, which increased soil quick-acting phosphorus content at the seasons of flowering pods by 23.11% under low phosphorus conditions (P50) compared with that of the uninoculated group, and significantly increased the accumulation of nitrogen, phosphorus, and potassium in plants compared with that of the uninoculated group, and at the same time optimized nutrient partitioning, promote phosphorus transfer to stalks to support seed formation; the uninoculated group required higher phosphorus levels (P250) to reach peak biomass. The results of the study supported the hypothesis of “reducing phosphorus and increasing fungi”, and verified the synergistic advantages of AMF inoculation and 50–100 mg kg⁻¹ phosphorus application in saline soils. It is recommended that AMF inoculation and phosphorus application should be preferred in agricultural production to avoid the inhibition of mycorrhizal function by high phosphorus. In the future, the molecular mechanism of AMF in regulating the inter-root Na⁺/K⁺ balance needs to be analyzed in depth to improve the theory of synergism of mycorrhizal fertilizers.

Author contributions

Zi-cheng Peng and Yun-xia Xing participated in the experimental design and wrote the manuscript. Yu-xin Jia and Feng-hua Zhang analyzed all data and assisted in manuscript preparation and revision. Yue-hong Ma and Shuai-hao Li carried out soybean cultivation and experimental material collection. Hai-chang Yang supervised the overall study and designed the experiment.

Funding

The project was funded by the Corps Science and Technology Plan Project (2022ZD055, 2024AB052), International Science and Technology Cooperation of Shihezi University (GJHZ202304), and the Key Research and Development Plan for the Autonomous Region (2022B02020-1), Innovation Cooperation Special Project Between shihezi University and Huyanghe City (QS2025006), Xinjiang Production and Construction Corps graduate Research and Innovation project (BTYJXM-2024-S17), Chinese International Science and Technology Cooperation Base “Oasis’s Crops Efficient Production and Agricultural Environmental Protection”.

Data availability

The datasets used and/or analysed during the current study available from the corresponding author on reasonable request.

Declarations

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.