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Article

Different Phosphorus Preferences Among Arbuscular and Ectomycorrhizal Trees with Different Acquisition Strategies in a Subtropical Forest

by
Yaping Zhu
,
Jianhua Lv
,
Pifeng Lei
*,
Miao Chen
and
Jinjuan Xie
College of Life and Environmental Sciences, Central South University of Forestry and Technology, Changsha 410004, China
*
Author to whom correspondence should be addressed.
Forests 2025, 16(8), 1241; https://doi.org/10.3390/f16081241
Submission received: 16 May 2025 / Revised: 20 June 2025 / Accepted: 14 July 2025 / Published: 28 July 2025
(This article belongs to the Section Forest Ecophysiology and Biology)
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Abstract

Phosphorus (P) availability is a major constraint on plant growth in many forest ecosystems, yet the strategies by which different tree species acquire and utilize various forms of soil phosphorus remain poorly understood. This study investigated how coexisting tree species with contrasting mycorrhizal types, specifically arbuscular mycorrhizal (AM) and ectomycorrhizal (ECM) associations, respond to different phosphorus forms under field conditions. An in situ root bag experiment was conducted using four phosphorus treatments (control, inorganic, organic, and mixed phosphorus) across four subtropical tree species. A comprehensive set of fine root traits, including morphological, physiological, and mycorrhizal characteristics, was measured to evaluate species-specific phosphorus foraging strategies. The results showed that AM species were more responsive to phosphorus form variation than ECM species, particularly under inorganic and mixed phosphorus treatments. Significant changes in root diameter (RD), root tissue density (RTD), and acid phosphatase activity (RAP) were observed in AM species, often accompanied by higher phosphorus accumulation in fine roots. For example, RD in AM species significantly decreased under the Na3PO4 treatment (0.94 mm) compared to the control (1.18 mm), while ECM species showed no significant changes in RD across treatments (1.12–1.18 mm, p > 0.05). RTD in AM species significantly increased under Na3PO4 (0.030 g/cm3) and Mixture (0.021 g/cm3) compared to the control (0.012 g/cm3, p < 0.05), whereas ECM species exhibited consistently low RTD values across treatments (0.017–0.020 g/cm3, p > 0.05). RAP in AM species increased significantly under Na3PO4 (1812 nmol/g/h) and Mixture (1596 nmol/g/h) relative to the control (1348 nmol/g/h), while ECM species showed limited variation (1286–1550 nmol/g/h, p > 0.05). In contrast, ECM species displayed limited trait variation across treatments, reflecting a more conservative acquisition strategy. In addition, trait correlation analysis revealed stronger coordination among root traits in AM species. And AM species exhibited high variability across treatments, while ECM species maintained consistent trait distributions with limited plasticity. These findings suggest that AM and ECM species adopt fundamentally different phosphorus acquisition strategies. AM species rely on integrated morphological and physiological responses to variable phosphorus conditions, while ECM species maintain stable trait configurations, potentially supported by fungal symbiosis. Such divergence may contribute to functional complementarity and species coexistence in phosphorus-limited subtropical forests.

1. Introduction

Phosphorus (P) is an indispensable element in plant growth and metabolism, and plays a key role in heredity, energy conversion, signal transmission, and structural material construction [1]. As a vital component of plant cells, phosphorus contributes to the formation of key molecules such as nucleic acids, phospholipids, and adenosine triphosphate (ATP), directly affecting genetic information transmission, energy metabolism, and membrane stability [2]. Although phosphorus constitutes only a small fraction of plant biomass (approximately 0.2% of dry weight), its importance is comparable to that of carbon and nitrogen [3]. In plants, phosphorus mainly exists in the form of phosphate and participates in physiological processes such as photosynthesis, respiration, and energy transfer. In natural ecosystems, plants mainly obtain the phosphorus they need from the soil. However, phosphorus in the soil often exists in the form of insoluble compounds, which makes its bioavailability low and often becomes a key factor limiting plant growth [4]. To overcome this limitation, plants have evolved strategies to enhance phosphorus acquisition from the soil [1,5,6,7].
The strategies by which plants acquire phosphorus can be broadly categorized into three main types: adjustment of root morphology, dependence on mycorrhizal symbiosis, and secretion of root exudates [8]. Morphological modifications, such as changes in root length, root diameter, and root tissue density, represent important adaptations that enhance plant access to phosphorus under conditions of soil phosphorus limitation. By optimizing these root traits, plants can increase the root–soil contact area, thereby improving the efficiency of nutrient uptake, particularly phosphorus acquisition [4,9,10]. In addition to morphological adjustments, mycorrhizal symbiosis is thought to play a crucial role in enhancing phosphorus acquisition. Previous studies revealed that arbuscular mycorrhizal (AM) fungi were generally associated with the uptake of inorganic phosphorus [11,12,13], whereas ectomycorrhizal (ECM) fungi were believed to be more efficient in accessing organic phosphorus sources [8,14,15,16,17]. This functional differentiation is hypothesized to influence plant nutrient acquisition strategies and may contribute to their adaptation to varying soil nutrient conditions. It has been suggested that AM-associated tree species tend to rely on morphological traits such as higher root branching and specific root length (SRL) to enhance soil exploration and phosphorus uptake, while ECM species may benefit from enzymatic mechanisms that facilitate organic phosphorus mobilization. However, direct experimental evidence comparing these strategies under controlled phosphorus treatments is still limited. Understanding how different phosphorus forms (e.g., inorganic, organic, and mixed phosphorus) influence root morphology, root exudation, and mycorrhizal associations in tree species with contrasting mycorrhizal types is therefore essential. Such knowledge could reveal whether these tree species adopt distinct or overlapping strategies to cope with phosphorus limitation and whether these mechanisms contribute to their coexistence in forest ecosystems [18,19,20].
Tree species with different mycorrhizal types are known to exhibit distinct root functional strategies that may enable them to adapt to varying soil nutrient conditions [21]. These strategies are thought to include adjustments in root morphology, enhancement of symbiotic associations, and increased secretion of root exudates, which together facilitate phosphorus acquisition and potentially contribute to niche differentiation and species coexistence within forest ecosystems [22,23]. Previous studies suggest that AM species tend to perform better in soils rich in inorganic phosphorus, possibly relying on traits such as high root branching intensity and specific root length, whereas ECM species are believed to be more efficient in utilizing organic phosphorus through enzymatic mineralization and the development of complex foraging networks [8,24]. In phosphorus-deficient environments, plants may also alter their growth strategies by reallocating carbon to root systems, thereby enhancing nutrient acquisition at the expense of shoot growth [25]. However, most existing evidence on phosphorus-related root trait plasticity comes from studies on individual species or seedlings under controlled conditions, with relatively limited investigation of mature trees in natural forests, particularly in subtropical ecosystems [4,8,24,26,27]. Therefore, this study aims to explore how coexisting tree species with contrasting mycorrhizal associations adjust their phosphorus acquisition strategies in response to different forms of soil phosphorus under field conditions. We hypothesize that (1) AM and ECM tree species exhibit distinct responses to different phosphorus forms, primarily driven by differences in root diameter (RD), tissue density (RTD), length (RL), and surface area (RSA), rather than by variation in mycorrhizal colonization. (2) In AM species, phosphorus availability induces clear trade-offs between morphological traits (such as SRL and SRA) and physiological traits (such as RAP and RTP), while ECM species exhibit weak trait correlations and limited plasticity, reflecting a conservative and decoupled adaptation strategy.

2. Materials and Methods

2.1. Study Area

The experimental site for this study is located in Dashanchong Forest Park in Lukou Town, Changsha County, Changsha City, Hunan Province, China (28°23′58″–28°24′58″ N, 113°17′46″–113°19′08″ E). This area is situated in the typical low mountain and hilly region of central Hunan, with an elevation ranging from 55 to 260 m (Figure 1). The region experiences a central subtropical southeastern monsoon climate, with an annual average temperature of 17.3 °C. The highest monthly mean temperature occurs in July at 30.0 °C, and the lowest in January at 5.5 °C. The annual precipitation ranges from 936.4 to 1954.2 mm, with an average of 1412 to 1559 mm, concentrated mainly between April and August. The soil type is well-drained clay loam lateritic red soil, derived from slate and shale parent rocks, classified as Ferralsols under the World Reference Base for Soil Resources (IUSS Working Group WRB, 2006).
In this study, we selected coexisting tree species with contrasting mycorrhizal associations in Dashanchong Forest Park, including two arbuscular mycorrhizal (AM) species, Choerospondias axillaris and Liquidambar formosana, and two ectomycorrhizal (ECM) species, Cyclobalanopsis glauca and Quercus fabri. Dashanchong Forest Park, located in subtropical China, is characterized by rich biodiversity, containing 1234 vascular plant species across 174 families and 638 genera. These include 29 families, 60 genera, and 120 species of ferns; 5 families, 11 genera, and 15 species of gymnosperms; and 140 families, 567 genera, and 1108 species of angiosperms. The park’s vegetation is diverse, mainly composed of Pinus massoniana and Cyclobalanopsis glauca mixed needle and broadleaf forests, Choerospondias axillaris deciduous broadleaf forests, Cyclobalanopsis glauca evergreen broadleaf forests, and pure Cunninghamia lanceolata plantations. Most of these forest types are secondary forests that have naturally regenerated under forest protection policies following previous logging disturbances. Our research team has established multiple long-term monitoring plots within these secondary forests to investigate the ecological dynamics and successional processes of different forest types.

2.2. Tree Species Selection

In April 2022, a tree species survey was conducted in Dashanchong Forest Park to select tree species with similar growth conditions and environmental settings (i.e., trees of the same species with comparable height and diameter at breast height). The intervals between the trees are about 5 m. Using the root bag method, which effectively isolates roots of different species, different forms of phosphorus supply treatments were applied. This in situ field experiment was designed to investigate the absorption and utilization mechanisms of subtropical forest tree species under various phosphorus forms.

2.3. Phosphorus Addition Treatment

Each tree was subjected to four phosphorus supply treatments: control (no phosphorus application, CK), inorganic phosphorus (Na3PO4), a mixture of inorganic and organic phosphorus (1/3 Na3PO4, 1/3 C10H14N5O7P, 1/3 AMP), and organic phosphorus (1/2 C10H14N5O7P, 1/2 AMP). Two root bags were established for each treatment per tree, resulting in a total of 160 root bags [28,29,30]. During the installation of the root bags, careful excavation of a woody root approximately 5 mm in diameter was conducted along the lateral roots in the direction of the target tree trunk. The depth at which the root bags are buried in the soil is approximately 20 cm. All small lateral roots were removed, and a root segment approximately 20 cm long was placed into nylon mesh root bags containing 1.5 kg of soil, ensuring that all roots that developed later would be new growth. The size of the root bags was 30 cm × 30 cm, with a mesh aperture of 35 (to facilitate nutrient and moisture penetration). After moistening the root bags with the appropriate amount of water, they were covered with the original soil and litter, and each bag was properly labeled. All root bags were installed in April 2022. In order to reduce soil heterogeneity caused by the selection of multiple tree species, soil was collected in Dashanchong Forest Park in April 2022. After clearing the surface litter at the sampling site, approximately 720 kg (0–20 cm) of topsoil was excavated using a shovel. The collected soil was then processed to remove roots, stones, and debris, passed through a 2 mm sieve, and mixed uniformly. After one growing season, the root bags were extracted from the field in November by cutting the woody roots and transported to the laboratory for further analysis.

2.4. Determination of Absorptive Root Traits

Root scanning software WinRHIZO Pro2009 (Regent Instruments Inc., Québec City, QC, Canada) was used to obtain the average diameter, root length. The scanned root segments were stored in the labeled aluminum box and dried in the oven at 60 °C for 48 h to constant weight. Finally, root morphological traits were calculated, including specific root length (SRL), specific root area (SRA), and root tissue density (RTD).

2.5. Determination of Phosphatase Activity in Fine Roots

Fresh root samples were collected and thoroughly cleaned to remove adhering soil. Approximately 30 mg of root samples, measuring about 0.5 cm in length, were weighed and placed into 5 mL centrifuge tubes. To each tube, 3.75 mL of sodium acetate buffer was added. The samples were then subjected to shaking at 200 r/min for 30 min at 25 °C using a centrifuge. Afterward, the supernatant was carefully decanted into a designated slot. Using a micropipette, 200 μL of the root solution was transferred to a 96-well black microplate, followed by the addition of 50 μL of MUB solution and 50 μL of 4-MUB-phosphate. A standard curve for MUB was prepared with the following concentrations: 0, 0.5, 2.5, 5, 10, and 25 μM. After preparing the samples, the microplate was incubated in the dark at 25 °C for 3 h. Fluorescence was measured using a multifunctional microplate reader (BioTek Instruments, Inc., Winooski, VT, USA) at an excitation wavelength of 365 nm and an emission wavelength of 450 nm. The fluorescence values were then converted to MUB concentrations based on the standard curve. The roots were removed from the centrifuge tubes, rinsed with deionized water, and dried at 65 °C until a constant weight was achieved (approximately 72 h), after which they were weighed and recorded. Root acid phosphatase activity was quantified as the amount of substrate (in moles) produced per unit time per gram of root dry weight.

2.6. Determination of Mycorrhizal Traits

Arbuscular mycorrhizal (AM) colonization was assessed using 50 first- to third-order absorptive root segments. The root samples were thoroughly washed, and soil particles were carefully removed. The roots were transferred into centrifuge tubes containing 20% KOH solution and incubated in a 65 °C water bath for 2 h. After digestion, the roots were placed in a beaker and rinsed with clean water for 30 s, then sequentially soaked in 5% acetic acid and a staining solution composed of Parker black ink and 5% acetic acid (1:1) for 1 min. The stained roots were heated in a 60 °C water bath for 30 min and subsequently decolorized in distilled water. All root segments were examined under a light microscope. The presence of structures such as arbuscules, hyphae, or vesicles indicated AM fungal colonization. The colonization intensity of each root segment was recorded on a scale of 0%, 10%, 20%, …, 100%, and a weighted average was calculated. The final AM colonization rate was obtained by dividing the total weighted colonization score by the number of root segments examined. Ectomycorrhizal (ECM) colonization was determined by directly observing 50 thoroughly cleaned first-order root tips under a light microscope. Root tips showing blunt, rod-shaped, or bifurcated structures with abundant fungal hyphae on the surface were considered to be colonized by ECM fungi. The ECM colonization rate was calculated as the number of colonized root tips divided by the total number of root tips observed.

2.7. Determination of Fine Root Phosphorus Content

Dried fine root samples were crushed and placed into 2 mL centrifuge tubes, along with six small steel balls. The samples were then ground into a powder using a fully automated high-speed sample grinder (JXFSTPRP-64, Shanghai Jingxin Industrial Development Co., Ltd., Shanghai, China) for the determination of phosphorus concentration. All fine root samples were subjected to fine grinding in a ball mill to ensure uniformity and particle fineness. The ground samples were stored in pre-labeled ziplock bags in a dry environment to prevent moisture absorption and oxidation. During subsequent analyses, precisely 0.05 g of root sample from each batch was weighed and placed into a conical flask. A sulfuric acid–hydrochloric acid mixed digestion solution was employed to digest the samples, ensuring complete decomposition of organic matter. Upon completion of digestion, total phosphorus content in the samples was determined using the molybdenum antimony colorimetric method, with results expressed as grams of phosphorus per kilogram of root sample (g/kg). This method demonstrates high sensitivity and accuracy, reliably reflecting the total phosphorus content in fine roots.

2.8. Determination of Phosphorus in Soil Resin-P

The determination of soil Resin-P was carried out by using the improved Hedley continuous extraction phosphorus classification method. This method classified the soil phosphorus components into Resin-P, phosphorus sodium bicarbonate (NaHCO3-Po and NaHCO3-Pi), phosphorus sodium hydroxide (NaOH-PO, NaOH-Po, and NaOH-Pi), phosphorus hydrochloride (HCL-P), and residual phosphorus (Residual-P). Extract in sequence according to the different activities of compassion. In this study, only the determination of resin phosphorus in the soil was carried out. The specific operation steps are as follows: (1) Weigh 0.50 g of the air-dried soil that has passed through a 100-mesh sieve and place it in a 50 mL centrifuge tube. (2) Add 30 mL of distilled water to the centrifuge tube, place 1 g of anion exchange resin bag in it, and set the temperature at 180 r/min. After shaking for 2 h, remove the resin package and carefully rinse the adhered soil particles into the centrifuge tube with a small amount of deionized water. Then centrifuge at 4000 rpm for 5 min, discard the centrifuge solution, and the remaining soil sample can be used for the next extraction step. The washed resin package was placed in another clean 50 mL centrifuge tube. A total of 20 mL of 0.5M-HCL solution was added to elute the P adsorbed by the resin package. Oscillation was carried out at 180 r/min for 2 h. A total of 5 mL of the eluent was taken into a 25 mL volumetric flask, and resin-P was determined by molybdenum blue colorimetry (Table 1).

2.9. Statistical Analyses

One-way ANOVA was used to test for treatment effects on each root trait within evolutionary stages and within individual tree species. When significant main effects were detected, Tukey’s HSD post hoc tests were conducted to determine pairwise differences among P treatments (p < 0.05). Trait significance and response direction were visualized using heatmaps based on Pearson correlation coefficients. In addition, principal component analysis (PCA) was conducted to examine multivariate trait variation and to identify major axes of functional differentiation across species and treatments. All statistical analyses were conducted in R 4.3.1.

3. Results

3.1. Comparison of Differences in the Growth and Physiological Indicators of Fine Roots Under Different Forms of Phosphorus Treatment

The biomass of Chax, Lifo, Cygl, and Qufa had no significant effect under different phosphorus morphology treatments (p > 0.05), while RTP had a significant effect under different phosphorus morphology treatments (p < 0.05) (Figure 2). In terms of RTP, Chax and Lifo were significantly higher than CK under the treatment of Na3PO4 and the mixture, while Cygl and Qufa had no significant effect under the treatment of the four phosphorus forms (Figure 2b).
In terms of the physiological indicators of fine roots, MC and RAP showed significant differences among different treatments (p < 0.05). The mycorrhizal infection rates of Chax, Lifo, and Qufa changed little under different treatments, while the mycorrhizal infection rate of Cygl decreased significantly under Na3PO4 treatment (Figure 3a). The activity of root acid phosphatase (RAP) showed significant differences among different phosphorus application treatments and tree species. In Chax, compared with CK and AMP + phytic acid treatments, the mixed treatment significantly reduced RAP activity, suggesting that the combination of inorganic phosphorus and organic phosphorus might have inhibited enzymatic phosphorus mobilization. In Lifo, the RAP activity of the mixed treatment also decreased significantly. The RAP activity of AMP + phytic acid treatment was the highest, but there was no statistically significant difference compared with CK. In Cygl, compared with AMP + phytic acid, Na3PO4 treatment significantly reduced the RAP activity (Figure 3b).

3.2. Comparison of Fine Root Shape Differences Under Different Phosphorus Treatments

Among the four fine root morphological traits, different phosphorus morphological treatments had significant effects on RD and RTD of AM tree species (p < 0.05), but no significant effects on SRL and SRA (p > 0.05). Among the AM tree species, RD was significantly lower under the CK treatment than the Na3PO4 treatment (p < 0.05), and RTD was significantly higher under the Na3PO4 treatment than the other three treatments (p < 0.05). There was no significant difference among ECM tree species in each phosphorus form of treatment (p > 0.05) (Figure 4).
In terms of fine root biomass, AM and ECM tree species had no significant effect on the treatment of phosphorus in the four forms (p > 0.05) (Figure 5a). In terms of RTP in fine roots, the Na3PO4 and Mixture treatments in AM tree species were significantly higher than those in CK and AMP + phytic acid treatments (p < 0.05), while there was no significant effect in ECM tree species (p > 0.05) (Figure 5b).
In both rhizosphere and non-rhizosphere soils, the alkaline phosphatase activity under the CK treatment was significantly higher than that under Na3PO4, Mixture, and AMP + phytic acid treatments in both AM and ECM tree species (p < 0.05) (Figure 6).
In AM species, fine root biomass showed a significant positive correlation with RL and RSA (p < 0.01), and significant negative correlations with SRL, SRA, and RTD (p < 0.001, p < 0.05, and p < 0.01, respectively). In contrast, in ECM species, RL and RSA were not correlated with fine root biomass. Among AM species, RD was highly positively correlated with RSA (p < 0.001) and significantly positively correlated with SRA (p < 0.05), whereas in ECM species, RD tended to be negatively correlated with fine root morphological traits. Additionally, in AM species, RTP showed a highly significant positive correlation with RTD (p < 0.001), while in ECM species, RTD was significantly positively correlated with RTP (p < 0.05). RAP was significantly positively correlated with SRL in AM species. Notably, Rhizo-AP was significantly negatively correlated with RAP and RTD in AM species (p < 0.05 and p < 0.01, respectively), while in ECM species, it showed no significant correlations with most traits. Non Rhizo-AP was significantly negatively correlated with RTD and RTP in AM species (p < 0.01 and p < 0.05, respectively), whereas in ECM species, Non Rhizo-AP tended to show positive or no correlations with most traits. Furthermore, in AM species, Rhizo-resin P displayed generally weak correlations with all traits, while in ECM species, both Rhizo-resin P and Non Rhizo-resin P were significantly positively correlated with Rhizo-AP and Non Rhizo-AP (p < 0.05, p < 0.01, and p < 0.05, respectively) (Figure 7).
Under CK treatment, PC1 (32.9%) and PC2 (24.4%) explained most of the variance. RD, RSA, and RL have a strong positive correlation on PC1, while RAP and RTD are mainly distributed on the PC2 axis. This indicates that these traits exhibit strong changes under CK treatment. Under the treatment of Na3PO4, PCA indicated that the responses of root functional traits among different tree species were relatively consistent. RSA and RL are positively correlated with PC1, while RD and MC are negatively correlated with PC1. While SRA and SRL were positively correlated with PC2, RAP and RTD were negatively correlated with PC2. The concentrated distribution of scoring points indicates that under the supply of Na3PO4, the changes in root functional traits of different tree species are relatively consistent, suggesting that the responses of tree species to Na3PO4 treatment are relatively uniform. Under the Mixture treatment, PCA indicated that the responses of root functional traits among different tree species were relatively consistent. RSA and RL are positively correlated with PC1, while RD and MC are negatively correlated with PC1. SRA and SRL are positively correlated with PC2, while RAP and RTD are negatively correlated with PC2. The aggregated distribution of the score points shows that under the Mixture treatment, the changes in the root functional traits of different tree species are relatively consistent, indicating that the responses of tree species to the Mixture treatment are relatively unified. Under AMP + phytic acid treatment, PCA indicated that the responses of root functional traits among different tree species were relatively consistent. RL, RAP, and RTD are positively correlated with PC1, while RD and RSA are negatively correlated with PC1. MC is positively correlated with PC2, while SRA and SRL are negatively correlated with PC2. The close distribution of the score points indicates that under AMP + phytic acid treatment, the changes in the root functional traits of the tree species are relatively consistent, suggesting that the responses of the tree species to this treatment are relatively unified (Figure 8).
In AM species, the PCA revealed a complex pattern of variation in root functional traits. RL, RSA, RD, RAP, RTD, and MC were positively associated with PC1, while SRA and SRL were positively associated with PC2. The wide dispersion of scores indicates considerable variability in root trait responses among AM species, suggesting pronounced interspecific differences under AM treatment. In ECM species, the PCA showed a more consistent pattern of root trait variation. RL, RSA, and SRA were positively associated with PC1, whereas RD, RAP, RTD, and MC were negatively associated with PC2. The clustering of species scores implies that ECM species exhibited relatively uniform changes in root traits, reflecting a more consistent response among species (Figure 9).

4. Discussion

4.1. Divergent Fine Root Trait Responses of AM and ECM Tree Species to Different Phosphorus Forms

The fine root traits of AM and ECM tree species showed distinct responses to different phosphorus (P) forms, as evidenced by the trait-specific variations observed in Figure 4. In AM species, root diameter (RD) and root tissue density (RTD) were significantly influenced by P treatments, with RD being lower and RTD higher under Na3PO4 compared to the control (CK) and other P sources [1]. This indicates that AM trees actively adjust root morphology in response to inorganic P supply, possibly to enhance foraging efficiency or nutrient uptake. In contrast, ECM species did not exhibit significant morphological variation across treatments, suggesting a more stable root construction strategy regardless of P form [31]. Similarly, RAP varied significantly among AM species, with the Mixture treatment consistently suppressing RAP compared to CK and AMP+Phytic acid treatments. In ECM species, RAP was significantly reduced under Na3PO4 relative to CK and AMP+Phytic acid, implying that excessive inorganic P may inhibit enzymatic activity, especially in ECM-dominated systems [32]. Interestingly, total phosphorus (RTP) in AM fine roots was significantly elevated under Na3PO4 and Mixture treatments, while ECM species showed no significant differences. This suggests that AM species are more responsive to external P supply, particularly inorganic and mixed forms, possibly due to higher physiological plasticity or a stronger dependence on soil-available P [33,34]. In contrast, the relatively stable RTP levels in ECM species indicate their ability to maintain phosphorus homeostasis regardless of external P inputs, likely through their symbiotic associations with ECM fungi [27].
This study supports the conclusion proposed by Liang et al. (2024) [24] and Liu et al. (2015) [29] that arbuscular mycorrhizal (AM) tree species preferentially utilize inorganic phosphorus. Under Na3PO4 and mixed phosphorus treatments, AM species exhibited significantly elevated root total phosphorus (RTP) and acid phosphatase activity (RAP), accompanied by distinct changes in root morphological traits. These findings indicate that AM species possess a stronger capacity and regulatory response for acquiring exogenous, readily available phosphorus. In contrast, we did not observe enhanced enzymatic activity or phosphorus accumulation in ectomycorrhizal (ECM) species under organic phosphorus (AMP + phytic acid) treatment. Nor did ECM species exhibit improved growth performance or trait responses compared to inorganic phosphorus treatments. Although ECM species are often ecologically associated with organic phosphorus mobilization, our experimental results do not provide direct evidence supporting a specific preference for organic P. Rather, ECM species maintained stable trait values across all phosphorus treatments, reflecting a low-plasticity strategy likely buffered by their fungal symbionts. We speculate that this conservative strategy may enable ECM trees to maintain nutrient homeostasis under fluctuating phosphorus conditions, rather than gaining a competitive advantage through preference for a particular P form.

4.2. Phosphorus Form Mediates Root Functional Coordination Through Divergent Morphological and Physiological Pathways

The contrasting responses of arbuscular mycorrhizal (AM) and ectomycorrhizal (ECM) tree species to different phosphorus (P) forms are strongly influenced by their fundamentally distinct nutrient acquisition strategies, shaped by long-term co-evolution with their symbiotic fungi and specific ecological niches [26,35,36]. AM species typically depend on extensive root proliferation and biochemical activity to exploit available inorganic P, while ECM species rely more heavily on fungal-mediated enzymatic mineralization of organic P pools [37]. In AM tree species, traits such as root length (RL), root surface area (RSA), and acid phosphatase activity (RAP) exhibited significant interspecific and treatment-driven variation [1,26]. These traits were consistently correlated with both biomass accumulation and total root phosphorus content (RTP), highlighting a high degree of functional integration in P foraging strategies. Principal component analysis (PCA) further revealed that these traits were tightly clustered along the same axes under specific treatments, indicating functional integration in phosphorus acquisition [38,39]. Notably, the negative correlations between RSA, specific root area (SRA), and biomass suggest a trade-off between morphological proliferation and construction efficiency [40,41], where finer and more expansive root systems enhance phosphorus foraging at the potential cost of reduced biomass gain. The positive relationship between RTD and RTP suggests that denser root tissues may serve as internal phosphorus reservoirs, helping store phosphorus when external availability varies.
In contrast, ECM species displayed a more conserved root trait structure, with PCA indicating closer clustering of species scores and more uniform trait expression. In these species, RL and RSA were positively associated with biomass, whereas RAP and RTD were negatively correlated with nutrient acquisition traits, suggesting a decoupling of morphological and physiological strategies. These patterns imply that ECM trees primarily rely on stable architectural traits and modest enzymatic adjustments, likely leveraging their fungal symbionts’ ability to access organic phosphorus [42,43]. Collectively, these findings demonstrate that phosphorus forms not only shape the magnitude of trait responses but also regulate the mode of trait coordination across mycorrhizal types, reflecting distinct evolutionary adaptations in phosphorus acquisition [36,44]. In this study, mycorrhizal colonization rates ranged from 20% to 40%, lower than previously reported values in similar forests. This may be due to the use of root bags, which restricted hyphal extension and soil contact, as well as interspecific differences in mycorrhizal dependency. Despite this, root trait responses and phosphorus uptake patterns suggest that functional mycorrhizal associations were still active.

4.3. Divergent Enzymatic and Morphological Adjustments Reflect Phosphorus Form Sensitivity

Acid phosphatase activity (RAP), a key physiological trait reflecting organic phosphorus(P) utilization potential, exhibited pronounced treatment- and species-specific differences [43]. Among AM species, the Mixture treatment led to significant reductions in RAP in Lifo and Chax, particularly in the latter, where RAP levels under Mixture were significantly lower than under both CK and AMP+Phytic acid. This suggests that the coexistence of inorganic and organic phosphorus may suppress enzymatic phosphorus mobilization, potentially through feedback inhibition by readily available inorganic P [45]. In contrast, RAP remained stable or slightly elevated under AMP+Phytic acid in AM species, indicating their tendency to activate enzymatic systems in organic P-dominated environments. In ECM species, Cygl showed significantly reduced RAP under Na3PO4, implying a sensitivity to inorganic P inputs and a possible reduction in enzymatic reliance [31,43]. These patterns reveal that RAP is not only indicative of species mineralization capacity but also reflects differential regulatory responses to phosphorus forms across mycorrhizal types [45]. Concurrently, fine root morphological traits exhibited divergent responses, with AM trees showing significant shifts in root diameter (RD) and root tissue density (RTD) under P treatments, while ECM species maintained stable morphology, suggesting a more conservative construction strategy buffered by fungal symbiosis.

5. Conclusions

Our findings show that arbuscular mycorrhizal (AM) tree species exhibit stronger sensitivity to phosphorus form variations compared to ectomycorrhizal (ECM) species. AM trees demonstrated significant shifts in fine root traits, such as root diameter (RD), root tissue density (RTD), and acid phosphatase activity (RAP), under inorganic and mixed phosphorus treatments, accompanied by increased root phosphorus content. In contrast, ECM species maintained stable trait values, reflecting a more conservative response likely buffered by fungal symbiosis. AM species showed strong, consistent correlations among root morphological and physiological traits, indicating a coordinated strategy for phosphorus acquisition. Key traits, including fine root biomass, root length (RL), and surface area (RSA), exhibited trade-offs between resource acquisition and structural investment. Root phosphorus content (RTP) was positively correlated with RTD, while RAP aligned with SRL, reflecting a coordinated mechanism for phosphorus storage and enzymatic mobilization. In contrast, ECM species exhibited weak or inconsistent trait correlations, indicating a more independent regulation of phosphorus acquisition. PCA revealed that AM species displayed high variability and plasticity across treatments, suggesting strong adaptability to different phosphorus forms. Conversely, ECM species showed tightly clustered traits, indicating more stable and conservative root strategies with limited adjustments across phosphorus environments.
This study highlights the distinct response patterns of AM and ECM tree species to phosphorus form variations, providing new insights into plant–fungal symbiosis in phosphorus acquisition. AM species, with their greater adaptability to phosphorus changes, offer significant potential for improving phosphorus acquisition in phosphorus-poor soils. Selecting AM species in agriculture can enhance phosphorus use efficiency, reduce fertilizer dependency, and minimize environmental impact. Future research should investigate performance differences between AM and ECM species under various soil types and phosphorus conditions to optimize phosphorus management, while leveraging AM fungi’s symbiotic properties to develop sustainable agricultural strategies.

Author Contributions

Y.Z. participated in the trial design and manuscript writing; J.L. participated in the management of the trial process; M.C. and J.X. participated in the data analysis and revised the manuscript; P.L. revised the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the National Key R&D Program of China (2023YFE0105100), the Hunan provincial innovation foundation for postgraduate (CX20210859, CX20220717, CX2023CX02053, CX20240706), the Scientific Innovation Fund for Post-graduates of Central South University of Forestry and Technology (CX202101014, CX202201016).

Data Availability Statement

The datasets generated for this study are available on request to the corresponding author.

Acknowledgments

We are grateful to the Central South University of Forestry Science and Technology for providing us with a research environment, as well as financial support from the fund, and the students in the group for their help in this experiment.

Conflicts of Interest

The authors declare that they have no competing interests.

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Figure 1. The location map of Dashanchong Forest Park.
Figure 1. The location map of Dashanchong Forest Park.
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Figure 2. The effects of different phosphorus forms on growth parameters of tree species with different mycorrhizal types. (a) biomass, and (b) fine root phosphorus content. Different letters above boxes indicate significant differences among treatments within the same mycorrhizal type (p < 0.05).
Figure 2. The effects of different phosphorus forms on growth parameters of tree species with different mycorrhizal types. (a) biomass, and (b) fine root phosphorus content. Different letters above boxes indicate significant differences among treatments within the same mycorrhizal type (p < 0.05).
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Figure 3. The effects of different phosphorus forms on the physiological and morphological characteristics of fine roots. (a) mycorrhizal infection rate, and (b) activity of fine root acid phosphatase. Different letters above boxes indicate significant differences among treatments within the same mycorrhizal type (p < 0.05).
Figure 3. The effects of different phosphorus forms on the physiological and morphological characteristics of fine roots. (a) mycorrhizal infection rate, and (b) activity of fine root acid phosphatase. Different letters above boxes indicate significant differences among treatments within the same mycorrhizal type (p < 0.05).
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Figure 4. The effects of different phosphorus forms on root morphological traits in AM and ECM tree species. (a) Average root diameter (RD), (b) specific root length (SRL), (c) specific root area (SRA), and (d) root tissue density (RTD) under four phosphorus treatments: CK (control), Na3PO4, Mixture (Na3PO4 + AMP + Phytic acid), and AMP + Phytic acid. Different letters above boxes indicate significant differences among treatments within the same mycorrhizal type (p < 0.05).
Figure 4. The effects of different phosphorus forms on root morphological traits in AM and ECM tree species. (a) Average root diameter (RD), (b) specific root length (SRL), (c) specific root area (SRA), and (d) root tissue density (RTD) under four phosphorus treatments: CK (control), Na3PO4, Mixture (Na3PO4 + AMP + Phytic acid), and AMP + Phytic acid. Different letters above boxes indicate significant differences among treatments within the same mycorrhizal type (p < 0.05).
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Figure 5. The effects of different phosphorus forms on biomass, phosphorus content, and mycorrhizal traits in AM and ECM tree species. (a) Total biomass, (b) root total phosphorus concentration (RTP), under four phosphorus treatments: CK (control), Na3PO4, Mixture (Na3PO4 + AMP + Phytic acid), and AMP + Phytic acid. Different letters indicate significant differences among treatments within each mycorrhizal type (p < 0.05).
Figure 5. The effects of different phosphorus forms on biomass, phosphorus content, and mycorrhizal traits in AM and ECM tree species. (a) Total biomass, (b) root total phosphorus concentration (RTP), under four phosphorus treatments: CK (control), Na3PO4, Mixture (Na3PO4 + AMP + Phytic acid), and AMP + Phytic acid. Different letters indicate significant differences among treatments within each mycorrhizal type (p < 0.05).
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Figure 6. Effects of different phosphorus sources on alkaline phosphatase (AP) activity in the rhizosphere (a) and non-rhizosphere soil (b) of arbuscular mycorrhizal (AM) and ectomycorrhizal (ECM) plants. Treatments include: CK (control), Na3PO4 (inorganic phosphorus), Mixture (organic-inorganic phosphorus combination), and AMP+Phytic acid (organic phosphorus). Different lowercase letters indicate significant differences among treatments within the same mycorrhizal type (p < 0.05).
Figure 6. Effects of different phosphorus sources on alkaline phosphatase (AP) activity in the rhizosphere (a) and non-rhizosphere soil (b) of arbuscular mycorrhizal (AM) and ectomycorrhizal (ECM) plants. Treatments include: CK (control), Na3PO4 (inorganic phosphorus), Mixture (organic-inorganic phosphorus combination), and AMP+Phytic acid (organic phosphorus). Different lowercase letters indicate significant differences among treatments within the same mycorrhizal type (p < 0.05).
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Figure 7. Pearson correlation matrices for below-ground traits, phosphorus availability, and biomass in (a) arbuscular mycorrhizal (AM) and (b) ectomycorrhizal (ECM) tree species. * p < 0.05; ** p < 0.01; *** p < 0.001.
Figure 7. Pearson correlation matrices for below-ground traits, phosphorus availability, and biomass in (a) arbuscular mycorrhizal (AM) and (b) ectomycorrhizal (ECM) tree species. * p < 0.05; ** p < 0.01; *** p < 0.001.
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Figure 8. Principal component analysis (PCA) of fine root traits under different phosphorus treatments. Panels represent (a) CK, (b) inorganic phosphorus (Na3PO4), (c) mixed phosphorus (Mixture), and (d) organic phosphorus (AMP+Phytic acid). Blue arrows indicate trait loadings; red dots represent species scores; red ellipses show 95% confidence intervals. Percentages on PC1 and PC2 axes denote explained variance. Trait abbreviations: RL, root length; RSA, root surface area; SRL, specific root length; SRA, specific root area; RD, root diameter; RTD, root tissue density; RAP, acid phosphatase activity; MC, mycorrhizal colonization; and Rhizo-AP, rhizospheric phosphatase activity.
Figure 8. Principal component analysis (PCA) of fine root traits under different phosphorus treatments. Panels represent (a) CK, (b) inorganic phosphorus (Na3PO4), (c) mixed phosphorus (Mixture), and (d) organic phosphorus (AMP+Phytic acid). Blue arrows indicate trait loadings; red dots represent species scores; red ellipses show 95% confidence intervals. Percentages on PC1 and PC2 axes denote explained variance. Trait abbreviations: RL, root length; RSA, root surface area; SRL, specific root length; SRA, specific root area; RD, root diameter; RTD, root tissue density; RAP, acid phosphatase activity; MC, mycorrhizal colonization; and Rhizo-AP, rhizospheric phosphatase activity.
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Figure 9. Principal component analysis (PCA) of fine root traits under different phosphorus treatments. Panels represent (a) AM and (b) ECM. Blue arrows indicate trait loadings; red dots represent species scores; red ellipses show 95% confidence intervals. Percentages on PC1 and PC2 axes denote explained variance. Trait abbreviations: RL, root length; RSA, root surface area; SRL, specific root length; SRA, specific root area; RD, root diameter; RTD, root tissue density; RAP, acid phosphatase activity; MC, mycorrhizal colonization; and Rhizo-AP, rhizospheric phosphatase activity.
Figure 9. Principal component analysis (PCA) of fine root traits under different phosphorus treatments. Panels represent (a) AM and (b) ECM. Blue arrows indicate trait loadings; red dots represent species scores; red ellipses show 95% confidence intervals. Percentages on PC1 and PC2 axes denote explained variance. Trait abbreviations: RL, root length; RSA, root surface area; SRL, specific root length; SRA, specific root area; RD, root diameter; RTD, root tissue density; RAP, acid phosphatase activity; MC, mycorrhizal colonization; and Rhizo-AP, rhizospheric phosphatase activity.
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Table 1. Description of belowground foraging traits and definitions of abbreviations.
Table 1. Description of belowground foraging traits and definitions of abbreviations.
Traits AbbreviationUnitsDescription
Morphological traitsRoot average diameterRDmmAverage diameter of absorptive roots
Specific root lengthSRLmg−1Length per unit dry mass of absorptive roots
Specific root areaSRAm2 g−1Root surface area per unit dry mass of absorptive roots
Root tissue densityRTDg cm−3Dry mass per unit root volume of absorptive roots
Root lengthRLcmTotal length of absorptive roots
Root surface areaRSAcm2Total surface area of absorptive roots
Chemical traitsTotal phosphorus content in fine rootsRTPmg g−1Amount of phosphorus per unit dry mass of fine roots
Mycorrhizal fungi traitsMycorrhizal infection rateMC%Percentage of absorptive root length colonized by arbuscular mycorrhizal fungi
Physiological traitsRoot acid phosphatase activityRAPnmol g−1 h−1Number of moles of 4-MUB-phosphate produced per unit time and unit dry mass of absorptive roots
Rhizospheric acid phosphataseRhzio-APμmol g−1 h−1Acid phosphatase activity in rhizosphere soil, indicating the enzymatic potential for organic phosphorus mineralization near root surfaces
Non-rhizospheric acid PhosphataseNon Rhzio-APμmol g−1 h−1Phosphatase acid phosphatase activity in bulk soil outside the rhizosphere, reflecting background microbial phosphorus mineralization
Rhizospheric resin-phosphorusRhzio resin Pmg/kgResin-extractable phosphorus from rhizosphere soil, reflecting plant-available P near the root zone.
Non-rhizospheric resin phosphorusNon Rhzio resin Pmg/kgResin-extractable phosphorus from non-rhizosphere soil, representing background P availability.
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Zhu, Y.; Lv, J.; Lei, P.; Chen, M.; Xie, J. Different Phosphorus Preferences Among Arbuscular and Ectomycorrhizal Trees with Different Acquisition Strategies in a Subtropical Forest. Forests 2025, 16, 1241. https://doi.org/10.3390/f16081241

AMA Style

Zhu Y, Lv J, Lei P, Chen M, Xie J. Different Phosphorus Preferences Among Arbuscular and Ectomycorrhizal Trees with Different Acquisition Strategies in a Subtropical Forest. Forests. 2025; 16(8):1241. https://doi.org/10.3390/f16081241

Chicago/Turabian Style

Zhu, Yaping, Jianhua Lv, Pifeng Lei, Miao Chen, and Jinjuan Xie. 2025. "Different Phosphorus Preferences Among Arbuscular and Ectomycorrhizal Trees with Different Acquisition Strategies in a Subtropical Forest" Forests 16, no. 8: 1241. https://doi.org/10.3390/f16081241

APA Style

Zhu, Y., Lv, J., Lei, P., Chen, M., & Xie, J. (2025). Different Phosphorus Preferences Among Arbuscular and Ectomycorrhizal Trees with Different Acquisition Strategies in a Subtropical Forest. Forests, 16(8), 1241. https://doi.org/10.3390/f16081241

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