Arbuscular mycorrhizal fungi colonization facilitates nitrogen uptake in cotton under nitrogen - reduction condition

Abstract

Background

Cotton is an economically important global crop, the yield and quality of which are strongly influenced by soil nitrogen. Low nitrogen use efficiency poses an important challenge to improve cotton yield and quality. The use of arbuscular mycorrhizal fungi (AMF) has been proposed as an effective solution to this challenge. Therefore, we conducted an indoor experiment using a compartmentalized culture system with cotton as the material and established three nitrogen treatments (1 g·kg⁻¹, 0.7 g·kg⁻¹, and 0 g·kg⁻¹) to investigate whether symbiosis between AMF and cotton roots could improve the nitrogen absorption capacity of cotton.

Results

The results showed that under high-nitrogen, low-nitrogen, and nitrogen- free treatments, the contributions of AMF colonization to root NO₃⁻-N and NH₄⁺-N were 5.89%, 10.10%, 19.92% and 24.35%, 12.37%, 13.16% respectively. Furthermore, the symbiosis between AMF and roots promoted the absorption of soil NO₃⁻-N, NH₄⁺ -N, and dissolved organic nitrogen, and was beneficial for increasing the content of soil readily oxidizable carbon. Additionally, AMF colonization was significantly positively correlated with root tissue density, cotton biomass, and soil microbial activity, but significantly negatively correlated with soil total organic carbon.

Conclusions

Therefore, under nitrogen - reduction condition, roots will be more dependent on the contribution of mycelium to NO₃⁻-N, and AMF colonization was significantly positively correlated with root tissue density (P < 0.05), suggesting that mycelium may prolong its functional cycle by improving the root structure, thereby reducing the carbon and nitrogen consumption in host organ reconstruction. However, this mechanism needs to be further verified in combination with the direct measurement of root turnover rate.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12870-025-07204-3.

Background

Cotton (Gossypium hirsutum L.), the world’s dominant fiber crop, has its productivity and fiber quality strongly limited by nitrogen [1, 2]. In China, most cotton is grown on soil that is chronically deficient in plant-available nitrogen. Although high rates of irrigation and synthetic nitrogen fertilizer can increase crop yield, only 25–50% of the applied nitrogen is actually assimilated by the crop. The remainder is lost from the soil–plant system through leaching, denitrification, and volatilization, which together accelerate soil fertility decline, suppress yield potential, and cause off-farm environmental degradation [3–5].

Arbuscular mycorrhizal fungi (AMF) are emerging as a promising strategy to address nitrogen-related challenges in cotton cultivation. Numerous studies have shown that the symbiosis between AMF and roots can reduce nutrient loss, activate soil nutrients, and enhance nitrogen uptake efficiency, particularly in nutrient-deficient soil [6–12]. Specifically, AMF mycelia secrete carbon-containing compounds (such as glucose, organic acids, amino acids, e.g.) into the soil, which can alter soil texture, and enhance microbial diversity. These compounds also serve as a food source for soil bacteria, actinomycetes, and fungi, thereby promoting nitrogen transformation [13, 14]. Furthermore, AMF can directly facilitate plant nitrogen uptake through extraradical mycelial absorption and intraradical mycelial transport, or indirectly influence nitrogen uptake by altering root traits (specific root area, root branching intensity, root tissue density, e.g.) and regulating the expression of nitrogen-related transporters [14–17]. Simultaneously, mycorrhizal fungi can fix nitrogen in the mycelial network and rhizosphere environment, creating a nitrogen reservoir, that reduces nitrogen leaching following fertilization [18–20]. Although substantial evidence has confirmed that AMF colonization can promote plant nitrogen uptake, the individual contributions of roots and mycelia to nitrogen uptake have not been clearly distinguished [21]. Additionally, it remains unclear whether AMF colonization differentially responds to various forms of nitrogen. Therefore, it is necessary to investigate how AMF colonization affects root characteristics and microbial activity, and to quantify the differences in mycelial contributions to plant uptake of various nitrogen forms.

Soil carbon storage is closely related to mycorrhizal symbiosis. Hodge and Fitter [22] demonstrated that the symbiosis between AMF and roots attracts free-living soil microbes, particularly nitrogen-mineralizing bacteria, to the mycorrhizosphere. This interaction promotes the decomposition of soil organic carbon. The primary driver of this process is the secretion of extracellular enzymes by fungi and bacteria. These enzymes increase the mineralization rate of soil organic carbon, thereby releasing plant-available nitrogen [23–25]. This suggests that the symbiosis between AMF and plant roots can consume soil carbon. However, some studies indicate that the mycelial pathway also significantly contributes to soil carbon sequestration, and in some cases, even more than the roots [26–29]. For example, Zhu et al. [28] found that after symbiosis with roots, the contribution of AMF mycelium to soil carbon storage can be as high as 68%, compared to only 32% from the roots. However, it remains unclear whether the carbon input from the symbiosis of mycelium and roots affects readily oxidizable carbon, inert organic carbon, and total organic carbon.

A thorough understanding of the characteristics and mechanisms of nitrogen uptake and utilization by AMF colonization in plants is essential for achieving efficient nutrient utilization and regulation in farmland ecosystems. Therefore, the present study selected cotton as the research object. A compartmental growth system was utilized to physically isolate the growth of roots and mycelium, with the intention of investigating the following questions under varying nitrogen rates: (1) Are there disparities in the uptake of inorganic nitrogen by mycelium? (2) How does AMF colonization influence root traits and microbial activity? (3) How does AMF colonization impact soil carbon content?

Methods

Test materials

The soil used in our experiment was obtained from the Second Company (85°59 ‘42"E, 44°19’19"N) of the Shihezi University Test Site, Xinjiang, China. This region has an average annual precipitation of 210.6 mm, evaporation of 1664.1 mm, a frost-free period of approximately 170 d, an average annual temperature of 7.0 ℃, and an annual sunshine duration of 2861.2 h. The tested cotton variety was “Xinluzao 84” (China Xinjiang Hexin Technology Development Co., LTD). In addition, the soil in this area has been continuously planted with cotton for the past three years, during which manual weeding was carried out. Each year, 375 kg·hm⁻² of nitrogen fertilizer (urea) and 300 kg·hm⁻² of phosphorus and potassium fertilizer (KH₂PO₄) are applied. The water and fertilizer are managed through the method of film - mulched drip irrigation. The initial soil fungi spore density was 17 spores·g⁻¹, with Paraglomus as the dominant genus [30]. The initial soil physicochemical properties are as follows: the soil type is calcisols (the soil type according to WRB for soil resources), with soil bulk density of 1.3 g·cm⁻³, pH 8.30 (soil-water ratio 2.5:1, 25 °C), organic matter content of 7.70 g·kg⁻¹, total nitrogen of 0.97 g·kg⁻¹, available phosphorus of 20.10 g·kg⁻¹, available potassium of 250.65 mg·kg⁻¹, NH₄⁺-N content of 2.11 mg·kg⁻¹, and NO₃⁻-N content of 0.53 mg·kg⁻¹ (Table S1).

Experimental design

We used a two-factor experimental design. Factor 1 was nitrogen application treatment with three rates, i.e., N1: 1 g·kg⁻¹ (the typical application rate used for the drip irrigation of cotton under film in Xinjiang field, 375 kg·hm⁻²); N2: 0.7 g·kg⁻¹ (250 kg·hm⁻²); and N3 (no nitrogen application). Nitrogen fertilizer was applied with water using a syringe. It should be noted that, due to the limited amount of soil in the experimental setup, the nitrogen concentration used in the field was increased by approximately 7 times when converted for the indoor experiment to ensure that nitrogen application did not restrict the healthy growth of cotton. Factor 2 was AMF treatment, which aimed to distinguish between the direct effects of AMF and the joint effects of roots and mycelium. For this, 1.2-mm-thick iron plates were used to fabricate a device (13 × 10 × 13 cm) with the middle portion separated using nylon mesh. One side was the mycorrhizal chamber, and the other side was the mycelial chamber. Nylon mesh with two different pore sizes was used across three rates, i.e., F₀⁻ (soil without AMF, nylon mesh aperture = 1 μm); F₁⁺ (soil containing AMF, nylon mesh aperture = 48 μm, allowed AMF mycelium to enter the mycelial chamber); and F₀⁺ (soil containing AMF, nylon mesh aperture = 1 μm, which neither the roots nor the AMF mycelium could enter the mycelial chamber) [31] (Fig. 1). In addition, the air space between the mycelial chamber and the mycorrhizal chamber is set at 3 mm to reduce the influence of nutrient flow [32]. There are a total of 9 treatments, with each treatment replicated 4 times.

Fig. 1.

Experimental design pattern diagram: (a) F₀⁻, (b) F₁⁺ and (c) F₀⁺⁰. Note: F₀⁻ : soil without AMF; F₁⁺ and F₀⁺ : soil containing AMF

Cotton seeds with good appearance, no mildew, no pests and no damage, were soaked in 5% H₂O₂ for 15 min and then rinsed with running water. The mycorrhizal and mycelial chambers were loaded with 600 and 1200 g of soil, respectively. The F₀⁺ and F₁⁺ treatments included fresh soil with AMF. In the F₀⁻ treatment, fresh soil (unsterilized soil) was autoclaved (120 °C), and then loaded with 10 mL of soil filtrate without AMF to restore the soil microflora. Four seeds were planted per growth chamber. Subsequently, the whole device was cultured in a GXZ-430D intelligent light incubator (Ningbo City Science and Technology Park Xinjiangnan Instrument Co., Ltd.) with a daily light intensity of 24,000 lx for 16 h at 32 °C and darkness for 8 h at 25 °C. An appropriate amount of deionized water was added daily. After four cotton leaves had developed, two plants with the same growth potential and uniform spacing were maintained in the growth chamber. 2 mL corresponding nitrogen rate was then applied to the mycelial chamber for treatments N1 and N2 every 2 days. For N3, the same volume of deionized water was applied. The form of nitrogen used was urea. Simultaneously, 2 mL 0.7 g​·L⁻¹ of modified Hoagland non-nitrogen nutrient solution was added to the growth chambers (Components in Table S2). After 60 d, the plants were harvested.

Index determination

Plant index measurement

First, cotton height was measured using a tape measure (cm). Next, the roots, stems, and leaves were collected. Root length (RL), surface area (RSA), diameter (RD), volume (RV), and branch count (RBC) were measured using an EPSON EXPRESSION 10000XL root scanner (Seiko Epson Corp., Japan) and analyzed with WinRHIZO software (Pro 2013e, Regent Instruments Inc., Canada). Subsequently, a portion of the root samples was stored at 4 ℃ in the refrigerator for the determination of mycorrhizal colonization rate, root NH₄⁺-N and NO₃⁻-N content. The remaining roots, stems, and leaves were put in envelopes and placed in an oven at 105 ℃ for 30 min, and then placed in an oven at 70 ℃ for 48 h for the determination of plant nitrogen content and biomass. Specific root length (SRL), specific root area (SRA), root branching intensity (RBI), root tissue density (RTD) were calculated as follows:

1

2

3

4

Plant NO₃⁻-N, NH₄⁺-N, and total nitrogen (TN) content were determined using salicylic acid–sulfuric acid colorimetry [33], ultraviolet spectrophotometry [34], and colorimetry [35]. The contribution rate of AMF to plant nitrogen was calculated as follows:

5

The AMF colonization rate was determined by Dickson and Smith [36] using the calculation formula.

6

The colonization of roots by AMF is shown in Fig. S1 and Table S3.

Soil index measurement

The experimental soil was passed through a sieve with a 2 mm aperture. Soil microbial biomass carbon (MBC) and microbial biomass nitrogen (MBN) were determined using the chloroform fumigation method [37]. Soil chitinolytic enzyme (β-N-acetyl-glucosaminidase, NAG), lignin-degrading enzyme (peroxidase, PER, and polyphenol oxidase, PPO), and protease were evaluated using ultraviolet spectrophotometry [38], iodine liquid titration, and sodium caseinate colorimetry, respectively [39].

Soil NH₄⁺-N and NO₃⁻-N contents were determined using phenol disulfonic acid and indigophenol blue colorimetry, respectively [40]. The soil free amino acid (FAA) content was determined using ninhydrin colorimetry [41]. The soil nitrogen mineralization rate and the FAA–net amino acid production rate (FAA–NPR) were measured by indoor culturing [42]. The soil TN was determined by the Kjeldahl nitrogen analyzer method [35]. The difference between soil TN and soil inorganic nitrogen is equivalent to the soil dissolved organic nitrogen (DON).

The contents of soil readily oxidizable carbon (ROC) and total organic carbon (TOC) were determined using 333 mmol·L⁻¹ potassium permanganate oxidation and external heating of potassium dichromate, respectively [43]. The inert organic carbon (IOC) content of the soil was subsequently determined as the difference between the TOC and ROC [44]. Detailed measurement methods for soil enzymes and soil nutrients can be found in the supplementary file (pages 4–8).

Data analysis

Excel 2019 (Beijing Kingsoft Office Software Co., Ltd) and SPSS 27.0 (Statistical Product and Service Solutions; IBM company) were used for data processing and analysis. Before the analysis, a variance homogeneity test was performed, but logarithmic conversion was applied when homogeneity was not met. Tukey ‘s HSD multiple comparison test was used to compare the mean values between the groups. The significance level was set at α = 0.05. All plots were produced using Origin 2024 (OriginLab Corporation). Moreover, Mantel test and random forest weight analysis were conducted using Chiplot (https://www.chiplot.online/) and SPSSAU (https://spssau.com/indexs.html) respectively. The PLS-SEM model (SmartPLS 4.0 software) was used to predict the effects of AMF colonization on cotton growth and soil carbon.

Results

Effects of nitrogen application and AMF colonization on nitrogen content in cotton organs

With an increase in nitrogen application, the TN content of roots and shoot parts changed significantly. However, the root TN content was higher than that of the shoot parts, with a difference of 0.02–105.91 mg·g⁻¹ (Fig. 2,a and b). At the same nitrogen rate, the root TN content of F₀⁺ was higher than F₁⁺ and F₁⁻. In addition, the contribution rate of AMF to the TN content of cotton differed under the different nitrogen rates. Specifically, with the reduction nitrogen application, the TN contribution of mycelium to roots first increases and then decreases, but decreases to the shoot parts (Fig. 2,c).

Fig. 2.

Effects of nitrogen application and AMF on nitrogen content in cotton: (a) shoot TN content, (b) root TN content, (c) contribution rate of mycelium to nitrogen in shoots and roots, (d) influence of mycelium on the NO₃⁻-N and NH₄⁺-N content of roots, (e) the contribution rate of mycelium to the relative contents of NO₃⁻-N and NH₄⁺-N in roots, (f) changes in NO₃⁻-N and NH₄⁺-N in roots without AMF Note: different lower case letters of all figures indicate significant differences (P < 0.05)

There exist disparities in the contributions made by mycelium to the root inorganic nitrogen. Under the same nitrogen application rate, root NO₃⁻-N content was significantly higher than NH₄⁺-N (Fig. 2,d). Under the different nitrogen application rates, the contributions of AMF mycelium to NO₃⁻-N and NH₄⁺-N in the roots were significantly different. Specifically, the AMF mycelium showed the highest contribution to root NH₄⁺-N (24.35%) and the lowest contribution to root NO₃⁻-N (5.89%) at N1. The contribution of mycelium to root NO₃⁻-N and NH₄⁺-N at N2 and N3 is opposite to N1 (Fig. 2,e). In addition, with increasing nitrogen application rate, NO₃⁻-N and NH₄⁺-N of F₀⁻-treated roots both increased. The root NO₃⁻-N content was higher than the NH₄⁺-N content, with a difference of 0–16 mg·g⁻¹ (Fig. 2,f).

Effects of nitrogen application and AMF colonization on soil nitrogen

At the same nitrogen application rate, the soil NO₃⁻-N content of F₀⁺ and F₁⁺ decreased compared to F₀⁻, and there was a significant difference between the F₀⁺ and F₁⁺ at N3 and N2 (Fig. 3,a). At the N3 rate, the NO₃⁻-N content of F₀⁺ was significantly higher than F₁⁺. At the N2 rate, the NO₃⁻-N content of F₁⁺ was significantly higher than F₀⁺. At the N1 rate, there was no significant difference in the soil NO₃⁻-N content between F₁⁺ and F₀⁺ (Fig. 3,a). Under the same nitrogen application rate, the NO₃⁻-N mineralization rate of F₁⁺ was the highest, but F₀⁺ treatment was the lowest (Fig. 3,c). In addition, under the same nitrogen application rate, the NH₄⁺-N content of F₁⁺ was significantly lower than that of F₀⁺ and F₀⁻. At the N2 rate, there was no significant difference in the NH₄⁺-N content between the F₀⁻ and F₀⁺. At the N1 rate, the NH₄⁺-N content of F₀⁻ was higher than that of F₀⁺ (Fig. 3,b). Simultaneously, with an increase in nitrogen application, the NH₄⁺-N mineralization rate gradually decreased (Fig. 3,d).

Fig. 3.

Effects of nitrogen application and AMF on soil (a) NO₃⁻-N content, (b) NH₄⁺-N content, (c) NO₃⁻-N mineralization rate, (d) NH₄⁺-N mineralization rate, (e) free amino acid content (FAA), (f) free amino acid–net production rate (FAA–NPR) Note: different lower case letters of all figures indicate significant differences (P < 0.05)

AMF colonization can also affect the content of soil organic nitrogen. At the same nitrogen application rate, the FAA content of F₀⁺ and F₁⁺ was lower than that of F₀⁻ (Fig. 3,e), but the FAA–NPR of F₀⁺ and F₁⁺ was higher than that of F₀⁻, with that of F₁⁺ being the highest (Fig. 3,f). Finally, with an increase in nitrogen application, the soil DON content increased, and under the same application rate, the DON content of F₀⁻ was the highest (Fig. 4,a).

Fig. 4.

Effects of nitrogen application and AMF on soil (a) dissolved organic nitrogen (DON), (b) inert organic carbon (IOC), (c) readily oxidizable carbon (ROC), total organic carbon (TOC) content. Note: different lower case letters of all figures indicate significant differences (P < 0.05)

Effects of nitrogen application and AMF colonization on soil carbon

AMF colonization can impact the soil carbon content. Specifically, at the N2 rate, the total amounts of ROC and IOC were higher than N1 and N3 rates. At the same nitrogen application rate, the F₁⁺ treatment had the highest IOC and ROC content. Specifically, there was a significant difference between the IOC content of F₀⁺ and F₀⁻, whereas there was no significant difference in the ROC content (Fig. 4,b and c). Moreover, Nitrogen application and AMF colonization significantly affected the soil TOC content, with the N2 rate the highest amount. At the same nitrogen rate, the relative effect of AMF on soil TOC content was as follows: F₁⁺ >F₀⁺ >F₀⁻ (Fig. 4,d). Meanwhile, root traits were significantly positively correlated with ROC, IOC and TOC.

Discussion

Relationship between AMF colonization and root traits

How can the nitrogen-absorption strategy be adjusted when cotton growth is limited by nitrogen? Root morphology and configuration are adaptive strategies developed by plants in response to their growth environment [45], and they determine the spatial expansion ability of roots and methods of nutrient acquisition [46]. In this study, although AMF did not promote RD, RBI and SRL growth, they increased RTD (Fig. 5,a). AMF had a large effect on SRA and RTD (Fig. 5,b). That is, mycorrhizal symbiosis increases root longevity and maintains the nitrogen balance between cotton and mycelium [47, 48]. The saved photosynthetic carbon promotes the morphological construction of the mycelium and cotton aerial parts [49, 50]. Additionally, under low-nitrogen conditions, AMF mycelium absorbed more NO₃⁻-N than NH₄⁺-N, which was contrary to previous findings indicating a preference for NH₄⁺-N [51]. This discrepancy may be attributed to the nitrogen preference of cotton within the study area. Specifically, the examined cotton had a higher NO₃⁻-N content than NH₄⁺-N content, while while the soil had a higher NH₄⁺-N content than NO₃⁻-N content. A study conducted by Zhang et al. [52] in this region also supports this conclusion. NO₃⁻-N is more conducive to root growth and nutrient accumulation, which in turn promotes better root morphology. The redundancy analysis also showed that soil NO₃⁻-N had a greater influence on root characteristics than NH₄⁺-N (Fig. 5,a). Overall, under nitrogen reduction conditions, AMF colonization can not only directly promote root growth but also indirectly facilitate it through the roots’ degree of dependence on inorganic nitrogen.

Fig. 5.

(a) Effect of soil nitrogen and root mycorrhizal colonization on root growth (1, 2, 3 and 4 in the first quadrant represent the order of environmental factors on cotton roots, **<0.05, ns > 0.05), (b) using random forest to analyze the weight influence of infection rate on cotton roots. Note: RMC: root mycorrhizal colonization, SRA: specific root area, RTD: root tissue density, RD: root diameter, RBI: root branching intensity, SRL: specific root length

Relationship between AMF colonization and cotton nitrogen uptake

Arbuscular mycorrhizae, as the symbiont of soil fungi and plant roots, play an important role in promoting nitrogen uptake by host plants [53, 54]. Frey and Schuepp [55] found that the nitrogen absorbed and transported by AMF mycelium to the host plant accounted for 30% of the TN in the plant. Tanaka and Yano [56] suggested that up to 75% of the nitrogen in plants is absorbed by AMF mycelium. The results of our study showed that the contribution rates of AMF to root nitrogen were 13.44%, 21.31% and 8.66% under high, low and no nitrogen treatments, respectively. This indicates that the application of exogenous nitrogen affects the contribution rate of AMF to cotton nitrogen, and that the dependence of cotton on AMF is higher under low-nitrogen conditions. This finding is consistent with the results of Zhang et al. [57]. Notably, AMF can still contribute nitrogen to cotton under high-nitrogen condition, but this contribution is relatively low (13.44%). This further suggests that when exogenous nitrogen is abundant, the mycelial primarily function to store nitrogen, with the nitrogen supply to cotton being of secondary importance. In addition, the AMF colonization rate in roots reached 83.33% in the absence of nitrogen (Table S3), whereas the contribution rate of AMF to aboveground TN was only 42.22%. Tanaka and Yano [56] showed that the contribution rate of AMF to aboveground TN in plants could reach as high as 74% under high colonization rates. This indicates that the high AMF colonization did not result in higher nitrogen acquisition but instead required cotton to allocate more carbohydrates. Under this condition, AMF may exhibit “deceptive colonization” behavior. We used the PLS-SEM model to confirm the significant negative effect between AMF colonization and plant TN (Fig. 6). Therefore, appropriate nitrogen reduction measures can enhance the nitrogen contribution of AMF to cotton, thereby improving soil nitrogen uptake and utilization. However, excessive nitrogen reduction may lead to competition between cotton and AMF for nitrogen.

Fig. 6.

PLS-SEM model prediction of the effects of AMF colonization on cotton growth and soil carbon (Plant Biomass refer to Fig. S2 respectively). Note: TOC: soil total organic carbon, TN: total nitrogen, AMF: arbuscular mycorrhizal fungi; *<0.05, **<0.01, ***<0.001

The relationship between AMF colonization and soil carbon

Sinsabaugh et al. [58] showed that carbon input into mycelium by plants accounted for 10–20% of plant photosynthetic carbon, of which 6–14% was input into the soil through mycelial turnover. The remaining carbon was imported into the soil in the form of mycelial secretions, which can be utilized by soil microorganisms and enhance their activity. In turn, this promotes organic carbon decomposition [25, 59]. Our results showed that soil microbial activity had a significant positive correlation with AMF colonization (Fig. 7). This is conducive to enhancing soil microbial activity and organic carbon utilization. However, previous studies have shown that the activation of mycelium at low-nitrogen rates may promote the formation of stable organic carbon. The potential mechanisms of this include the following: (1) low nitrogen accelerates the turnover of microorganisms and increases the amounts of microbial metabolites and mycelial residues; (2) low nitrogen promotes the transfer of plant photosynthetic carbon by AMF from the rhizosphere hot zone to non-rhizosphere soil, where microbial activity is low. This results in weak mycelial excitation; (3) nutrient competition between AMF and soil microorganisms is strong under low-nitrogen conditions, which inhibits the inter-mycelial excitation effect to a certain extent; and (4) low nitrogen stimulates soil microbial activity, improves soil nutrient availability, and promotes host plant growth and rhizosphere deposition. This indirectly increases the input of plant-derived carbon [60–62]. However, under the condition of AMF colonization, the changes in root characteristics are conducive to soil carbon accumulation (Fig. 7). This indicates that, following AMF colonization, there are disparities in the functions induced by carbon inputs from mycelium and roots into the soil, and these inputs ultimately aim to maintain the dynamic equilibrium of soil carbon.

Fig. 7.

Mantel test of microbial activity and root characteristics on AMF colonization, soil nitrogen mineralization rate and soil carbon. Note: N_nit: NO₃⁻-N mineralization rate, N_amo: NO₄⁺-N mineralization rate, IOC: inert organic carbon, ROC: readily oxidizable carbon, TOC: total organic carbon, FAA–NPR: free amino acid–net production rate, MBC: microbial biomass carbon, MBN: microbial biomass nitrogen, NAG: β-N-acetyl-glucosaminidase, PER: peroxidase, PPO: polyphenol oxidase; microbial activity (PER, PPO, NAG, Protease, MBC/MBN; Refer to Fig. S3 respectively), root characteristics (SRA, SRL, RBI, RD, RTD); *<0.05, **<0.01, ***<0.001

Trade-off between AMF colonization on carbon investment and nitrogen gains

Central to the relationship between roots and AMF is the exchange of carbon (lipids and sugars) and nitrogen [28, 63]. When the soil nitrogen supply rate is high and the roots can meet the plant needs, the contribution of the mycelial pathway to plant nitrogen is small [64, 65]. Conversely, when soil nitrogen supply is low, plants preferentially allocate photosynthetic carbon underground to promote nitrogen uptake. Plants are more dependent on the root pathway, the mycelial pathway, or both, depending on the trade-off between carbon investment and nitrogen yield [64, 66, 67]. In our study, the contribution of mycelium to root nitrogen content was 21.31% under low-nitrogen condition. This indicates that nitrogen uptake by cotton is primarily achieved through the root pathway. The reasons for this may include the following: (1) the increase in root longevity simultaneously reduces high inputs of root nitrogen and carbon; (2) in a complex symbiotic environment, root investment is more stable over time due to the unpredictable environment and unstable resource supply [68].

In our study, AMF colonization significantly increased root tissue density and biomass, but reduced soil carbon input (Figs. 5a, and 6). These results indicate that although the contribution rate of AMF mycelium to plant nitrogen was low, AMF may indirectly promote root nitrogen uptake by affecting root and mycelial longevity under low-nitrogen condition. However, owing to the limitations of the test method, we were unable to quantify the indirect role of mycelium. Indeed, the contribution of existing mycelium to plant nitrogen was underestimated. Under low-nitrogen condition, according to the cost–benefit theory of root construction [69], an increase in root tissue density indicates an extension of root longevity. Moreover, Wu [70] also showed that moderate nitrogen reduction (272 kg N ha⁻¹) can significantly improve root longevity and productivity. Our study provides evidence explaining the increase in root longevity under low-nitrogen conditions. Thus, mycorrhizal fungal colonization can improve root absorption efficiency and prolong root longevity under low-nitrogen conditions [71]. This may alter the trade-off relationship between root absorption efficiency and root persistence. Although root biomass and AMF colonization rates decreased at high nitrogen rates, the contribution of AMF to plant TN increased. This may be due to the high nitrogen content in the soil, where nitrogen storage in the mycelium reaches saturation, and surplus nitrogen is transported to the cotton. Secondly, the higher nitrogen rate causes the cotton roots to be in a “comfortable state.” Although the number and elongation range of fine roots were greatly reduced [72], the death and reconstruction of fine roots take some time, at which point the contribution rate of mycelium to cotton TN may increase. Thus, carbon and nitrogen trading between AMF and cotton is closely related to soil nitrogen, root longevity, and root reconstruction time.

Limitations and implications

The study shows that under different nitrogen levels, the root-AMF symbiosis significantly impacts root characteristics, soil carbon and nitrogen content, and cotton biomass. Optimizing the root-AMF relationship and enhancing the function of the symbiosis can improve nutrient use efficiency and crop productivity, and enhance carbon and nitrogen cycling and the overall functioning of agricultural ecosystems. Therefore, we should incorporate the close association between AMF biological functions, root characteristics, and soil nitrogen levels into soil carbon-nitrogen models.

Certainly, this study also has some limitations. For example: (1) this study explored the impact of root characteristics and AMF colonization on soil carbon but did not quantify the contributions of roots and mycelium to soil carbon input; (2) the rhizosphere is a complex micro-ecosystem. In addition to the role of AMF, the impacts of the root-AMF-PGPR (plant growth-promoting rhizobacteria) interactions on soil carbon and nitrogen processes, as well as the underlying mechanisms (such as the role of root exudates), need to be further explored; (3) this study only investigated the effects of the root-AMF symbiosis during the vegetative growth stage of cotton. The impacts on cotton yield and underlying mechanisms still need further exploration; (4) this experiment was conducted under controlled conditions and cannot fully simulate the complex real-world field environment. Therefore, the existing results cannot be directly extrapolated to the field. Future research needs to further explore methods for studying AMF in situ field environments, develop an integrated model of roots-nitrogen-microbes, and develop microbial fertilizers based on efficient nitrogen fertilizer use.

Conclusions

We have shown that the symbiosis between AMF and roots could improve the nitrogen-absorption capacity (Fig. 8), and that this effect varies under different nitrogen application rates. At low nitrogen rate, the symbiosis could enhance the activity of soil microorganisms and increase RTD. In addition, after nitrogen reduction, the contribution rate of mycelium to NO₃⁻-N and TN in cotton increased.

Fig. 8.

Schematic diagram of AMF colonization promoting N uptake in cotton

Supplementary Information

Below is the link to the electronic supplementary material.

Abbreviations

NAG

 β-N-acetylglucosamine glycosidase

PER

Peroxidase

PPO

Polyphenol oxidase

FAA

Free amino acid

FAA–NPR

Free amino acid–net production rate

DON

Dissolved organic nitrogen

TN

Total nitrogen

TOC

Total organic carbon

N_nit

NO₃⁻-N mineralization rate

N_amo

NO₄⁺-N mineralization rate

RD

Root diameter

RBI

Root branching intensity

SRA

Specific root area

SRL

Specific root length

RTD

Root tissue density

RMC

Root mycorrhizal colonization

Author contributions

Hushan Wang: Methodology, Software, Data curation, Formal analysis, Writing - original draft. Yijian Wang: Software, Investigation, Formal analysis, Data curation. Xiaojiao Cheng: Methodology, Formal analysis, Investigation. Yunzhu He: Methodology, Validation, Investigation. Zihui Shen: Data curation, Validation. Wangfeng Zhang: Conceptualization, Methodology, Writing - review & editing. Xiaozhen Pu: Conceptualization, Methodology, Data curation, Project administration, Resources, Writing - review & editing.

Funding

We would like to acknowledge the support from National natural science foundation of China (32460538), Xinjiang production and construction corps science and technology program (2024DB015) and Xinjiang production and construction corps guiding science, technology program (2023ZD049) and youth innovative talents project of Shihezi university (CXBJ202201) for this research.

Data availability

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

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.