Rhizosheath–Mycorrhizal Interactions in Kengyilia hirsuta Enhance Phosphorus Efficiency

TL;DR

This study explores how Kengyilia hirsuta adapts to low phosphorus in desertified grasslands through root and fungal interactions.

Contribution

The study reveals a synergistic 'rhizosheath–mycorrhiza' system that enhances phosphorus efficiency in a desert grass.

Findings

Rhizosheath supports AMF enrichment and phosphatase activity under low phosphorus.

Optimal phosphorus levels promote a synergistic system with high AMF colonization and carbon–phosphorus exchange.

Phosphorus absorption is co-regulated by root morphology and symbiotic interactions.

Abstract

Phosphorus deficiency is a key factor limiting plant growth in desertified grasslands. Elucidating the adaptive strategies of pioneer plants that integrate root morphology and microbial interactions is crucial for understanding the natural restoration of ecosystems. This study investigated the strategies employed by Kengyilia hirsuta, a pioneer grass species in desertified grasslands, to adapt to low-phosphorus environments. By conducting sand culture experiments under varying phosphorus levels (low, optimal, and high), we focused on elucidating the synergistic adaptive mechanisms involving the root–rhizosheath system. The results showed that the rhizosheath serves as a critical micro-ecological niche for enriching arbuscular mycorrhizal fungi (AMF) and enhancing phosphatase activity. Under low-phosphorus stress, the plant strengthened root hair development and its symbiotic association with AMF, which markedly increased acid phosphatase activity and led to the highest phosphorus use efficiency. At the optimal phosphorus level, the plant developed an efficient “rhizosheath–mycorrhiza” synergistic system, characterized by high AMF colonization and spore density, facilitating optimized carbon–phosphorus exchange. Under phosphorus-sufficient conditions, the adaptive strategy transitioned towards root morphological plasticity, exemplified by increased surface area and branching. Multivariate analysis revealed that the phosphorus absorption efficiency of K. hirsuta is co-regulated by both morphological adaptation and symbiotic optimization. This study elucidates the mechanisms of nutrient stress adaptation in desertified grassland plants, providing a theoretical foundation for understanding the natural restoration processes of degraded ecosystems.

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1. Introduction

The Zoige Plateau, located on the southeastern edge of the Qinghai-Tibet Plateau, harbors one of the world’s largest alpine swamp–peat wetlands and serves as a critical water conservation zone for the Yellow River [1,2]. Its unique alpine climate has fostered the evolution of numerous plant species possessing stress-resistant traits. Through long-term natural acclimatization, plants inhabiting desertified grasslands in this region have gradually developed a series of adaptive mechanisms, including drought tolerance, cold resistance, and resistance to wind erosion [3]. Among them, K. hirsuta, a pioneer species in the desertified grasslands of this area, exhibits particularly prominent adaptive characteristics [4]. Research indicates that low phosphorus (P) availability in sandy soils is a key limiting factor constraining vegetation restoration [5]. Phosphorus deficiency can lead to reductions in seed size, fruiting frequency, and survival rate [6], negatively impacting plant growth, development, and yield formation [7]. This phosphorus (P) limitation effect is particularly pronounced in sandy habitats characterized by loose structure and high nutrient leaching, thereby making the elucidation of efficient P acquisition mechanisms in psammophytes crucial for understanding the restoration processes of desertified ecosystems.

Plants typically maintain phosphorus efficiency by enhancing either phosphorus acquisition efficiency (PAE) or phosphorus utilization efficiency (PUE) [8]. Research over the past decades has predominantly focused on the mechanisms underlying PAE improvement [9], which mainly include the following aspects. Firstly, plasticity in root system architecture, such as increasing root density and lateral root branching, expands the absorption interface area for direct P acquisition [10,11]. Secondly, interactions with microorganisms, particularly the establishment of AMF symbiosis, can significantly extend the soil volume explored by plants for P uptake [12]. In many species, the mycorrhizal pathway even plays a dominant role [13]. Thirdly, soil insoluble phosphorus is activated through root exudation of protons, organic anions, and phosphatases [14]. For instance, Sun et al. found that Azospirillum brasilense promoted root hair development, enhanced acid phosphatase activity, and upregulated phosphate transporter expression in Arabidopsis thaliana, thereby improving low-P tolerance [15]. Zhao et al. reported that desert plants under low-P conditions preferentially rely on the mycorrhizal pathway, which can contribute over 70% of total P uptake [16]. Notably, under phosphorus-deficient conditions, maize exhibits a greater reliance on arbuscular mycorrhizal (AM) fungi than on root hairs, indicating a potential antagonistic relationship between morphological adaptation and symbiotic association strategies [17]. This potential strategic trade-off provides an important perspective for understanding how plants optimize resource allocation in variable sandy habitats.

Recent studies have further revealed that the rhizosheath, a specialized root–soil interface structure in sandy land plants, plays a crucial role in phosphorus acquisition. By forming a stable micro-environment through physical encasement, it can enrich acid phosphatase activity to 2–3 times that of ordinary rhizosphere soil [18] and affect the structural stability of the rhizosheath micro-environment [19], constituting a multi-dimensional P capture strategy integrating physical retention, chemical activation, and physiological regulation. As a functional hub, the formation of the rhizosheath involves complex plant–soil interactions. It can enhance P availability through various pathways: secreting organic acids to solubilize Ca-P, releasing protons via the mucilage layer to promote the dissociation of Fe/Al-P, and maintaining specific microbial communities to drive organic P mineralization [10]. Although both the rhizosheath and mycorrhizas have been individually demonstrated to enhance plant P uptake, there remains a lack of systematic understanding regarding how these two systems synergistically operate within the rhizosphere microdomain to cope with P stress in alpine psammophytes such as K. hirsuta. Specifically, to elucidate the adaptive mechanisms by which pioneer plants in sandy environments respond to soil available phosphorus availability through integrated root morphological plasticity, physiological regulation, and microbial interactions, thereby providing a theoretical basis for plant selection in alpine sandy ecosystem restoration and laying the groundwork for identifying and breeding phosphorus-tolerant plant genotypes.

Therefore, this study employed K. hirsuta, a pioneer species in the desertified areas of the Zoige Plateau, as plant material. Through a controlled P gradient experiment combined with root morphological analysis, AMF microscopic observation, and enzyme activity assays, we systematically investigated the synergistic response mechanisms involving root morphological plasticity, AMF symbiotic efficiency, and rhizosheath functional traits. This study proposes the following hypotheses: (1) the strategies employed by K. hirsuta to acquire phosphorus vary under different phosphorus levels; and (2) rhizosheath formation and arbuscular mycorrhizal fungi colonization are regulated by soil phosphorus availability. The aims are to elucidate the key mechanisms by which plants efficiently utilize soil phosphorus in sandy environments, provide a theoretical basis for plant selection in the ecological restoration of alpine sandy lands, and lay the groundwork for mining and breeding P-efficient genotypes of psammophytes.

2. Results

2.1. Analysis of Soil Phosphorus Absorption and Utilization by K. hirsuta Under Different Phosphorus Levels

2.1.1. Relationship Between Root Morphology of K. hirsuta and Soil Phosphorus Absorption and Utilization

The response of plant root morphology to varying phosphorus levels reflects the association between morphological plasticity and soil P availability. As shown in Table 1, changes in soil P levels significantly affected the total root length, total root surface area, total root volume, number of root forks, number of root tips, root hair length, and root hair density of K. hirsuta (p < 0.05). With increasing P concentration, total root length gradually increased, with the P0 treatment being significantly lower than the P1 and P2 treatments (p < 0.05). Furthermore, total root surface area, total root volume, number of root tips, and number of root forks also increased significantly with rising P concentration (p < 0.05). These results indicate that under high-P conditions, plants enhance their capacity for soil phosphorus acquisition by expanding total root surface area and volume, as well as by increasing the number of root tips and forks. Conversely, both root hair length and root hair density significantly decreased with increasing P concentration (p < 0.05). This observation suggests that under low-P conditions, K. hirsuta compensates for the limited root surface area by increasing root hair density, thereby enhancing the efficiency of soil exploration and phosphorus absorption.

Total plant phosphorus content was also significantly influenced by phosphorus supply levels (Figure 1A). Both root and shoot total phosphorus contents exhibited an increasing trend with higher phosphorus supply. Compared with the P0 treatment, the P2 treatment significantly increased the total phosphorus content in both roots and shoots (p < 0.05). Notably, shoot total phosphorus content consistently exceeded root content, showing a highly significant difference (p < 0.001) under both P1 and P2 treatments. This indicates a preferential translocation strategy in K. hirsuta, directing absorbed phosphorus towards aboveground tissues. The response of acid phosphatase (ACP) activity to phosphorus levels displayed distinct tissue specificity (Figure 1B). Root ACP activity decreased significantly with increasing phosphorus supply (p < 0.05). In contrast, leaf ACP activity showed an initial increase followed by a decrease, reaching its maximum under the P1 treatment. This value was significantly higher than those under P0 and P2 treatments (p < 0.05). A cross-tissue comparison revealed that leaf ACP activity was significantly higher than root activity (p < 0.001), suggesting a potentially key role for leaves in phosphorus activation and remobilization.

Biomass accumulation and allocation patterns were also significantly regulated by phosphorus levels (Figure 1C–E). Both fresh and dry weights of roots and shoots increased significantly with rising phosphorus concentration (p < 0.05). Although root fresh weight was significantly greater than shoot fresh weight across all treatments (p < 0.05; Figure 1C), under high phosphorus conditions, a greater proportion of photoassimilates was allocated to shoots. This resulted in shoot dry weight significantly surpassing root dry weight (p < 0.01; Figure 1D), accompanied by a corresponding significant decrease in the root-to-shoot ratio (p < 0.05; Figure 1E). Together, these results demonstrate that sufficient phosphorus supply promotes the preferential allocation of carbon assimilates to shoot growth, thereby optimizing the plant’s overall growth strategy.

2.1.2. Contribution of AMF to Phosphorus Acquisition

The extent of root colonization by AMF was significantly regulated by soil phosphorus levels (Figure 1F–K). The frequency of vesicular colonization exhibited an initial increase followed by a decrease with increasing soil P content (Figure 1F,K). Under the P1 treatment, vesicular colonization reached its peak (p < 0.05). This indicates that a moderate P level is conducive to forming a stable symbiotic structure. In contrast, hyphal colonization rate declined progressively and significantly with increasing P supply (p < 0.05; Figure 1G,J). This suggests a greater reliance on the extension of the AMF hyphal network to expand the P absorption range under low-P conditions. The pattern of arbuscular colonization differed, showing an initial decrease followed by a subsequent recovery (Figure 1H,K). The total colonization rate reached its maximum under the P1 treatment (Figure 1I). These results collectively demonstrate that a moderate P level constitutes the optimal range for maintaining efficient AMF colonization and symbiotic function. This symbiotic pathway can partially compensate for the plant’s inherent limitations in direct P uptake via roots.

2.1.3. Analysis of Phosphorus Absorption and Utilization Efficiency

Phosphorus absorption efficiency (PAE) showed significant organ-specific differences (Figure 2A). Across all treatments, the PAE of shoots was consistently and significantly higher than that of roots (p < 0.05). The disparity was most pronounced under the P2 treatment, where shoot PAE exceeded root PAE by 76.51% (p < 0.01). Overall PAE increased linearly with higher P supply levels (p < 0.05). Shoots contributed predominantly to the overall absorption efficiency, indicating that under high-P conditions, plant phosphorus acquisition relies more heavily on shoot absorption capacity.

Phosphorus utilization efficiency (PUE) exhibited a distinct pattern of variation (Figure 2B). Under both P1 and P2 treatments, root PUE was significantly higher than shoot PUE (p < 0.001). However, compared with the P0 treatment, the phosphorus use efficiency in roots under the P2 treatment significantly decreased (p < 0.05), indicating excessive phosphorus consumption in roots under high phosphorus levels.

2.2. Comparative Analysis of Rhizosheath Characteristics in K. hirsuta Under Different Phosphorus Levels

2.2.1. Rhizosheath Biomass Characteristics

The dry mass of rhizosheath soil per unit root length decreased significantly with increasing phosphorus levels (Figure 2C,D). Compared with the P0 treatment, the P2 treatment significantly decreased (p < 0.05), while no significant difference was observed between the P0 and P1 treatments.

2.2.2. Comparison of Phosphorus Content and Phosphatase Activity Between Rhizosheath and Bulk Soil

Total phosphorus content in the rhizosphere soil exhibited significant spatiotemporal heterogeneity (Figure 2E). The total phosphorus content in the rhizosheath soil was consistently higher than that in the bulk soil across all treatments. This difference reached its peak under the P0 treatment (p < 0.01). As the phosphorus application rate increased, the disparity in total phosphorus content between the rhizosheath and bulk soil gradually diminished, suggesting that low phosphorus levels promote the interception and enrichment of phosphorus by the rhizosheath. Although the total soil phosphorus content increased linearly with higher phosphorus application, the increase was substantially greater in the bulk soil compared to the rhizosheath soil (p < 0.05). This indicates that high phosphorus supply attenuates the relative phosphorus enrichment capacity of the rhizosheath.

The acid phosphatase (ACP) activity in the rhizosheath soil decreased significantly with increasing phosphorus levels (Figure 2F). The activity under the P0 treatment was significantly higher than that under the P2 treatment (p < 0.05), indicating that low-phosphorus stress induces a microbial demand for phosphorus activation within the rhizosheath. Under both P1 and P2 treatments, ACP activity in the rhizosheath soil remained significantly higher than in the bulk soil (p < 0.01), underscoring the functional advantage of the rhizosheath as a “phosphorus activation hotspot.”

Neutral phosphatase (NP) activity in the rhizosheath soil showed an initial decrease followed by an increase (Figure 2G), reaching its lowest value under the P1 treatment and then rebounding significantly under P2 (p < 0.05). Notably, under the P2 treatment, NP activity in the rhizosheath soil was significantly higher than in the bulk soil (p < 0.01).

In contrast, alkaline phosphatase (AKP) activity increased progressively with higher phosphorus application rates (Figure 2H). The AKP activity in rhizosheath soil and rhizosphere soil under the P2 treatment was significantly higher than that under the P0 treatment (p < 0.05). Under both P0 and P2 treatments, AKP activity in the rhizosheath soil was significantly lower than in the bulk soil (p < 0.05).

2.2.3. Comparison of AMF Spore and Hyphal Densities Between Rhizosheath and Bulk SOIL

The density of AMF spores in both rhizosheath and bulk soil displayed an initial increase followed by a decrease, reaching its maximum under the P1 treatment (phosphorus level of 35 mg·kg^−1^) (Figure 3A). The spore density in rhizosheath soil and rhizosphere soil under the P1 treatment was significantly higher than that under both the P0 and P2 treatments (p < 0.05). Across all treatments, spore density in the rhizosheath soil was significantly higher than in the bulk soil (p < 0.001). The difference was most pronounced under the P1 treatment, indicating that the rhizosheath serves as a preferential microsite for AMF propagule colonization and enrichment. Morphological identification (Figure 3C,D) revealed that the dominant species included Diversispora globifera (characterized by yellowish-brown spores, often associated with higher β-glucosidase activity) and Septoglomus constrictum (characterized by reddish-brown spores, linked to organic phosphorus decomposition processes).

The development of the AMF hyphal network was significantly influenced by both phosphorus level and soil spatial position (rhizosheath vs. bulk) (Figure 3B,E). Hyphal density in the rhizosheath soil showed a declining trend with increasing phosphorus application, with the P2 treatment lower than P0 (p < 0.05). In contrast, hyphal density in the bulk soil increased significantly with rising phosphorus levels (p < 0.05). Under the P0 treatment, hyphal density was highest in the rhizosheath soil and was significantly greater than in the bulk soil (p < 0.01). This dense hyphal network effectively expanded the root absorption surface area, thereby directly enhancing phosphorus uptake flux. Both hyphal and spore densities peaked under the P1 treatment, providing further evidence that a soil phosphorus level of 35 mg·kg^−1^ represents the optimal range for maintaining the fully functional symbiotic system between K. hirsuta and AMF.

2.3. Response Characteristics of Root Hormones in K. hirsuta to Phosphorus Levels

Plastic changes in root morphology and the establishment of AMF symbiosis are typically regulated by hormonal signaling networks. To further understand the internal regulatory mechanisms by which K. hirsuta adapts to different phosphorus levels, we analyzed its root hormone content. The analysis revealed distinct response patterns of root hormones to phosphorus supply (Figure 3F–I). Both indole-3-acetic acid (IAA) and abscisic acid (ABA) contents exhibited a U-shaped response pattern, decreasing initially and then rising. The IAA content under the P0 and P2 treatments was significantly higher than that under the P1 treatment (p < 0.05, Figure 3F). The response of ABA was more pronounced, with its content under P0 and P2 levels showing a highly significant increase compared to the P1 treatment (p < 0.01, Figure 3G).

In contrast, the contents of gibberellin (GA) and cytokinin (CTK) followed a unimodal pattern, initially increasing and then decreasing, peaking under the P1 treatment and reaching their lowest levels under P2. GA content in the P2 treatment was significantly lower than in both P0 and P1 treatments (p < 0.05; Figure 3H), and CTK content was also significantly lower than in P1 (p < 0.05; Figure 3I). This indicates that optimal phosphorus supply is conducive to maintaining higher levels of GA and CTK, thereby promoting normal growth and development. Conversely, excessive phosphorus supply (P2) inhibits the biosynthesis of these growth-promoting hormones, leading to reduced growth.

In summary, K. hirsuta fine-tunes the synthesis dynamics of different hormone classes in response to phosphorus availability; IAA and ABA primarily respond to phosphorus stress signals, whereas GA and CTK are associated with optimal growth conditions.

2.4. Response Strategies of Root Morphology and AMF Colonization to Phosphorus Levels Based on Principal Component and Regression Analyses

Principal component analysis (PCA) revealed that the response of K. hirsuta to the soil phosphorus gradient exhibited significant hierarchical adaptive characteristics. The first two principal components (PC1 and PC2) accounted for 85.07% of the cumulative variance, encompassing adaptive mechanisms related to root morphology, mycorrhizal symbiosis, and physiological metabolism (Figure 4A). PC1, which explained 62.63% of the variance, was primarily driven by root morphological indicators. Total root surface area, number of root forks, and phosphorus absorption efficiency exhibited strong positive loadings on PC1. This indicates that under high phosphorus levels, plants adopt a “morphological investment” strategy by expanding their root ecological niche to enhance phosphorus acquisition. Conversely, hyphal density in the rhizosheath and root hair density showed significant negative loadings on PC1. This suggests that carbon allocation to microbial symbiotic structures and fine root architecture is suppressed under high-P conditions, reflecting a trade-off in resource allocation. PC2, explaining 22.43% of the variance, highlighted the synergistic adaptation under moderate phosphorus (P1) levels. The association pattern between AMF spore density in the rhizosheath soil and the total colonization rate, combined with the antagonistic regulatory relationship between IAA and ABA, reveals the contribution of the “rhizosheath–mycorrhiza” interaction system to activating the soil phosphorus pool, mediated by hormonal signaling.

Further spatial analysis elucidated the divergence of ecological strategies under different phosphorus levels. Samples from the P0 treatment clustered in the positive region of the PC2 axis, reflecting a low-carbon phosphorus absorption mode reliant on enhanced AMF symbiosis and rhizosheath function. Samples from the P2 treatment shifted towards the positive region of the PC1 axis, indicating a phosphorus capture strategy dominated by root morphological plasticity. Samples from the P1 treatment were concentrated in the negative region of the PC2 axis, signifying reliance on the localized phosphorus activation advantage conferred by high acid phosphatase activity and a dense hyphal network within the rhizosheath microzone. The reliability of the model was verified by statistical analysis (KMO = 0.78, Bartlett’s sphericity test p < 0.001), providing a quantitative basis for phosphorus management in the ecological restoration of alpine sandy lands.

Based on principal component regression analysis (Appendix A Table A1), a quantitative relationship model was established between the root phosphorus absorption efficiency (Y) of K. hirsuta and the core principal components: Y = 0.305 + 0.020X_1_ + 0.010X_2_ (DW = 2.063, F = 1381.717, p < 0.001, R^2^ = 0.997). The model passed rigorous statistical tests (VIF = 1.000, no residual autocorrelation), indicating that PC1 and PC2 together explained 99.7% of the variation in phosphorus absorption efficiency. PC1, representing root morphological plasticity and biomass accumulation, was the core driving factor (standardized coefficient β = 0.958, p < 0.001). This demonstrates that the “morphological expansion strategy” under high-P conditions significantly enhances phosphorus capture capacity by increasing total root volume and fork number. PC2, reflecting the synergistic effect of mycorrhizal symbiosis and rhizosheath function under moderate P levels, contributed an auxiliary gain effect (β = 0.283, p < 0.001). Its contribution intensity was approximately 35.81% of that from PC1, highlighting the critical role of the rhizosheath as a “carbon–phosphorus exchange hotspot” in phosphorus activation through the enrichment of AMF hyphal networks and phosphatase activity.

This model provides a quantitative decision-making tool for sandy land ecological restoration: maintaining soil phosphorus levels around 35 mg·kg^−1^ can optimally balance the synergistic effects of morphological expansion (PC1) and symbiotic optimization (PC2). Furthermore, screening genotypes with high loadings on PC1 can be applied for the directional breeding of grass species with high phosphorus use efficiency.

2.5. Contribution of Various Traits to Biomass Accumulation Based on Partial Least Squares Regression Analysis

Partial least squares regression (PLSR) analysis, using a variable importance in projection (VIP) threshold > 1, revealed that shoot biomass accumulation in K. hirsuta is co-driven by five functional modules (Figure 4B,C and Appendix A Figure A1). The root morphology-dominated module included core indicators such as total root surface area (VIP = 1.296), root volume (VIP = 1.266), root hair density (VIP = 1.278), and rhizosheath mass (VIP = 1.205). This module directly drives biomass accumulation by expanding the root–soil contact interface and enriching phosphorus-solubilizing microorganisms via the rhizosheath. This finding corroborates the morphological expansion strategy represented by PC1 (β = 0.958) in the principal component regression analysis.

The phosphorus metabolism synergy module was centered on root phosphorus absorption efficiency (VIP = 1.297) and root total phosphorus content (VIP = 1.229). This module involves the synergistic interplay between the efficient translocation of phosphorus to shoots and the extension of the hyphal network, as indicated by hyphal density in the rhizosheath soil (VIP = 0.954). Together, these processes support the low-carbon phosphorus activation pathway reliant on “rhizosheath–mycorrhiza” interactions under moderate phosphorus (P1) conditions.

The rhizosphere enzyme activity module featured key factors including acid phosphatase activity in bulk soil (VIP = 1.171), neutral phosphatase activity in rhizosheath soil (VIP = 0.949), and alkaline phosphatase activity in bulk soil (VIP = 0.955). Here, acid phosphatases primarily drive organic phosphorus mineralization, while neutral and alkaline phosphatases indirectly promote the release of insoluble phosphorus by modulating rhizosphere pH.

The mycorrhizal symbiosis efficiency module had major contributors including hyphal density in bulk soil (VIP = 1.259), total colonization rate (VIP = 0.996), and hyphal density in rhizosheath soil (VIP = 0.954). The extension of the hyphal network into distal rhizosphere regions, combined with spore enrichment in the rhizosheath, forms “hyphal bridges” to overcome phosphorus fixation barriers in sandy soil.

The hormone regulation auxiliary module was primarily centered on gibberellin (GA) content (VIP = 1.173). This module indirectly promotes biomass accumulation through mechanisms such as stimulating cell elongation.

3. Discussion

3.1. Adaptive Response of Root Morphological Plasticity to Soil Phosphorus Gradients

The root system serves as the core interface for plant–soil interactions, profoundly shaping ecological adaptation by regulating key processes such as nutrient mobilization, water uptake, and micro-environmental homeostasis [20,21,22]. This study demonstrates that K. hirsuta dynamically responds to changes in soil phosphorus availability through root morphological plasticity. Although total root length did not show significant differences among P treatments, total root surface area, root volume, number of root tips, and number of root forks increased significantly with rising phosphorus concentration (p < 0.05). This finding aligns with reports that high phosphorus promotes lateral root development in switchgrass [23], barley [24], and cereals [25]. However, unlike species such as Arabidopsis thaliana, which enhance local phosphorus acquisition under low-P stress by significantly increasing root hair length and density [26], K. hirsuta in this study exhibited a significant decrease in both root hair length and density under high-P conditions. This suggests that in the relatively phosphorus-rich sandy habitat, this species may prioritize allocating resources to lateral root extension for broadly exploring heterogeneously distributed nutrient space, rather than investing in the carbon-costly, fine-scale absorption strategy involving root hairs. This trade-off likely represents a crucial evolutionary adaptation to the patchy nutrient distribution characteristic of sandy soils.

3.2. Dual Response of AMF Symbiotic Efficiency to Phosphorus Levels

Arbuscular mycorrhizal fungi (AMF) symbiotic efficiency exhibits a typical threshold-dependent response to soil phosphorus concentration. This study found that the P1 level (35 mg·kg^−1^) represents the optimal range for AMF colonization, where total colonization rate and vesicular colonization frequency reached their maximum. This result is consistent with observations in crops like maize [27] and soybean [28], collectively supporting the “carbon–phosphorus exchange balance” theory. The ability of optimal phosphorus levels to enhance AMF colonization is underpinned by two concurrent selective pressures acting on the plant. First, the soil phosphorus concentration, while not yet sufficient for the plant to rely solely on direct root uptake, still necessitates the acquisition of phosphorus via the mycorrhizal pathway [29]. Second, at this optimal level, the plant possesses adequate photosynthetic products to allocate to belowground symbionts, thereby covering the carbon costs required to maintain mycorrhizal symbiosis [30]. Under low-P conditions, AMF significantly enhance phosphorus acquisition efficiency (PAE) by extending the hyphal network, as seen in apple rootstocks where AMF inoculation markedly increased phosphorus uptake in low-P soil [31]. High-P levels led to reduced hyphal density and spore counts in the rhizosheath, indicating diminished carbon investment by the plant into the AMF symbiosis and consequent suppression of symbiotic efficiency [32]. In tomato experiments, AMF’s growth-promoting effect vanished and mycorrhizal dependency dropped to zero when phosphorus concentration reached 1.3 mM [33], aligning with the conclusions drawn here. From the perspective of carbon allocation trade-offs, the suppression of symbiotic relationships under high phosphorus conditions stems from a resetting of the plant’s “cost–benefit” accounting mechanism. When soil phosphorus is sufficient, plants can directly acquire phosphorus through high-affinity phosphate transporters, rendering the high photosynthetic carbon investment required to maintain mycorrhizal symbiosis economically unjustifiable [34]. Plants systematically downregulate the secretion of branching factors such as strigolactones via systemic signaling pathways and initiate feedback inhibition of mycorrhizal-specific phosphate transporters, thereby actively curtailing the carbon supply to AMF [35,36]. This carbon allocation strategy reflects a “principle of economy” under resource-sufficient conditions, prioritizing the allocation of photosynthetic products to aboveground growth over the maintenance of belowground symbiosis. Notably, AMF spore density in the rhizosheath soil was consistently and significantly higher than in the bulk soil, with the greatest difference observed under the P1 treatment. This highlights the rhizosheath’s critical role as both a “reservoir” for AMF propagules and a “source area” for hyphal network development. This spatial distribution pattern facilitates the extension of the hyphal system into deeper sandy soil, overcoming the direct physical limitations of roots to access fixed phosphorus resources. The underlying mechanism shares similarities with the distribution patterns of AMF in the rhizosphere of Chinese fir [37]. However, the unique “soil–mucilage–microbe” composite structure of the K. hirsuta rhizosheath may further enhance the efficiency of hyphal anchoring and nutrient exchange.

3.3. The Rhizosheath Microzone as a Key Interface for Phosphorus Activation and Enrichment

The rhizosheath is not merely a physical attachment structure but an active biochemical reaction interface. In this study, rhizosheath mass per unit root length decreased with increasing phosphorus application, potentially linked to high-P inhibition of root organic acid secretion, thereby weakening soil particle adhesion [29,38]. When soil phosphorus is sufficient, plants downregulate the expression of organic acid transporters (e.g., MATE family proteins), thereby reducing the efflux of root exudates such as citrate and malate [39,40]. These exudates not only serve as key agents in mobilizing soil phosphorus but also, through their carboxyl functional groups, directly mediate the adhesion of soil particles to the root surface mucilage layer via complexation with ions such as calcium and iron [41,42]. Despite this, the rhizosheath soil consistently maintained significantly higher acid phosphatase activity and harbored a denser AMF hyphal network compared to the bulk soil [36,43]. This indicates that the rhizosheath forms an efficient phosphorus activation microzone by recruiting specific functional microbial communities [44]. Microbes colonizing this niche synergistically promote organic phosphorus mineralization and insoluble inorganic phosphorus dissolution through the secretion of extracellular phosphatases and organic acids [45]. Functionally, this strategy converges with the formation of “proteoid roots” by white lupin for concentrated carboxylate exudation [43]. However, K. hirsuta, via rhizosheath–microbe interactions, achieves a spatially more concentrated and potentially more carbon-efficient phosphorus activation pathway. This is crucial for the efficient utilization of limited nutrient resources in impoverished sandy ecosystems.

This characteristic of the rhizosheath microbiome offers important insights for the exploration and utilization of microbial resources in agricultural ecosystems. First, functional microorganisms enriched in the rhizosheath can serve as candidate strains for biofertilizers; through isolation, cultivation, and functional validation, microbial inoculants tailored for nutrient-poor soils can be developed [46]. Second, based on the assembly principles of the rhizosheath microbial community, synthetic microbial communities (SynComs) can be designed by combining multiple strains with complementary functions to achieve more stable growth-promoting effects. Previous studies have demonstrated that well-designed SynComs can successfully colonize the crop rhizosphere under field conditions and significantly enhance the growth and disease resistance of plants such as maize and tomato [47,48]. In the future, integrating high-throughput isolation and cultivation techniques with SynCom design to thoroughly explore beneficial functional taxa within the “microbial black box” of the rhizosheath, and developing microbial inoculant products suitable for different ecological regions, will represent an important pathway toward achieving green agriculture and sustainable production.

3.4. Hormone Signaling-Mediated Coordinated Regulation of Morphology, Symbiosis, and Metabolism

Plant hormones are central messengers integrating environmental signals with internal developmental programs [49]. This study found that indole-3-acetic acid (IAA) and abscisic acid (ABA) exhibited a U-shaped response pattern to the phosphorus gradient. Low-P-induced IAA accumulation likely promotes lateral root initiation to expand soil exploration, while elevated ABA is associated with stress signaling and adaptive responses such as stomatal regulation. High-P also triggered a similar stress-like hormonal response, indicating that phosphorus excess can also act as a physiological stressor. Xu et al. observed that rhizosheath formation is regulated by auxin under moderate soil drying (MSD) conditions, suggesting that high-P levels may indirectly influence such hormonal signals affecting rhizosheath formation [50]. Concurrently, higher levels of gibberellin (GA) and cytokinin (CTK) under moderate phosphorus (P1) align with a growth-promoting physiological state. Integrating previous findings on IAA positively regulating AMF symbiosis [51] and ABA influencing root hair development [52], we postulate that a sophisticated regulatory system mediated by a hormonal network coordinates carbon allocation and cellular developmental programs. This system synergistically optimizes root morphological plasticity, AMF symbiotic intensity, and rhizosheath microzone function, thereby forming an integrated response mechanism adapted to the spatial heterogeneity of phosphorus in sandy soils. This regulatory network warrants further in-depth exploration at the levels of molecular interactions and ecophysiology in future studies. It should be noted that although this study observed a concordance in trends between hormone levels and AMF colonization parameters, a direct causal relationship between the two has not yet been confirmed through molecular approaches. The current data primarily reveal a synergistic response pattern between hormonal changes and symbiotic efficiency, while the underlying regulatory pathways remain to be further validated through experiments such as those using hormone signaling mutants or exogenous hormone treatments.

4. Materials and Methods

4.1. Plant Material and Growth Substrate

Seeds of K. hirsute were collected in September 2022 in Waqie Town, Hongyuan County, Sichuan Province (33.18° N, 102.62° E, 3490 m a.s.l.) with permission from the local Forestry and Grassland Bureau (Figure 5). The plant species was authoritatively identified by Prof. Yang Junliang of the College of Life Sciences, Sichuan Agricultural University. A voucher specimen (SAUT 201402232) is deposited in the herbarium of the Triticeae Research Institute, Sichuan Agricultural University.

The growth substrate was a sandy loam soil with the following properties: pH 7.69, organic carbon 6.3 g kg^−1^, total phosphorus 0.35 g kg^−1^, nitrate-N 6.68 mg kg^−1^, ammonium-N 6.69 mg kg^−1^, and a maximum field water-holding capacity of 23%. Prior to use, the soil was air-dried and passed through a 4 mm sieve.

4.2. Experimental Design

This study employed a controlled indoor pot experiment. After selection, seeds were stratified at 4 °C for 48 h, surface-sterilized with 75% ethanol for 30 s, and rinsed thoroughly with distilled water. Seeds were then germinated on moist filter paper in Petri dishes within a growth chamber set at 24/18 °C (day/night), a 16/8 h photoperiod, and 60–65% relative humidity. Lighting was provided by LED cold light sources (intensity: 8400 lx, spectral peaks at 450 nm and 660 nm). When seedlings developed leaves approximately 3 cm long, uniform individuals were transplanted into pots (upper diameter 15 cm, bottom diameter 10 cm, height 13 cm) each containing 1.4 kg of the prepared sandy loam soil, with three seedlings per pot.

After transplantation, the plants were housed in an artificial climate greenhouse (Tibetan Plateau Research Institute, Southwest Minzu University), where the environmental control system maintained a temperature of 22 ± 2 °C, a photoperiod of 16/8 h, and relative humidity of 65 ± 5%. Supplemental lighting was provided by high-pressure sodium lamps at an intensity of 10,870 lx. Plants were irrigated regularly with equal volumes of a phosphorus-free nutrient solution. After reaching the three-leaf stage, plants were subjected to three phosphorus (P) level treatments by applying superphosphate (12% P_2_O_5_): P0 (0 mg P kg^−1^ soil), P1 (35 mg P kg^−1^), and P2 (70 mg P kg^−1^). Each treatment consisted of four replicates, with each replicate comprising six pots (totaling 24 pots per treatment) and three plants per pot, which were pooled as composite samples for subsequent measurements. Soil moisture was maintained at 25% of the maximum field water-holding capacity using the weighing method. Sampling and measurements were conducted approximately 30 days after P fertilizer application.

4.3. Measurements and Analyses

4.3.1. Root Morphological Traits

Intact, cleaned root systems were scanned using a root scanner (EPSON Expression 12000XL, Los Alamitos, CA, USA). Parameters including total root length, total root surface area, total root volume, average root diameter, number of root tips, and number of forks were obtained using a root image analysis system (WinRHIZO Pro 2017a). Furthermore, root segments from the root hair zone behind the root cap were observed under a phase-contrast microscope. Root hair length and the number of root hairs per unit root length were measured to calculate average root hair length and root hair density.

4.3.2. Biomass and Root-to-Shoot Ratio

Plants were carefully removed from pots, and roots were washed clean. Shoots and roots were separated, and their fresh weights were recorded. Samples were then placed in paper bags, oven-dried at 110 °C for 30 min to deactivate enzymes, and subsequently dried to constant weight at 75 °C before dry weights were measured. The root-to-shoot ratio was calculated as root dry weight divided by shoot dry weight.

4.3.3. Rhizosheath Weight

Soil loosely attached to roots was gently shaken off. Soil that remained tightly adhering to the root surface was defined as the rhizosheath. Shoots and roots with the attached rhizosheath were separated and their combined fresh weight recorded. Roots were then placed in a beaker with deionized water and subjected to ultrasonication for 40 min to completely detach the rhizosheath soil. Roots were removed, blotted dry with filter paper, and weighed again. Rhizosheath soil weight was calculated as the difference between the two weights. Combined with the measured total root length, the rhizosheath mass per unit root length (mg cm^−1^) was calculated [53].

4.3.4. Root Endogenous Hormone Content

Fresh root samples (0.1 g) were homogenized in 0.9 mL of phosphate-buffered saline (PBS, pH 7.2–7.4) on ice. The homogenate was centrifuged at 4 °C and 3000 rpm for 20 min. The supernatant was collected and stored at −20 °C until analysis. The concentrations of abscisic acid (ABA), indole-3-acetic acid (IAA), gibberellin (GA), and cytokinin (CTK) in the supernatant were determined using enzyme-linked immunosorbent assay (ELISA) kits. Absorbance was measured at 450 nm, and hormone concentrations were calculated based on standard curves.

4.3.5. Plant Acid Phosphatase Activity

Acid phosphatase (ACP) activity in leaf and root tissues was measured using a commercial assay kit (Comin Biotechnology Co., Ltd., Suzhou, China). Approximately 0.1 g of tissue was homogenized in 1 mL of the provided reagent on ice. The homogenate was centrifuged at 4 °C for 10 min, and the supernatant was used as the test solution. Following the manufacturer’s instructions, color development was measured at 405 nm, and ACP activity per unit tissue mass was calculated.

4.3.6. Total Phosphorus Content and Phosphorus Efficiency

Total plant P content was determined using the sulfuric acid-hydrogen peroxide digestion method. A 0.25 g fresh plant sample was digested with 5 mL concentrated H_2_SO_4_ at 200 °C for 30 min, followed by heating at 300 °C until dense white fumes appeared, which was maintained for 15 min. After cooling slightly, 30% H_2_O_2_ was added dropwise, and heating at 300 °C was resumed for 15 min. This step was repeated until the digest became clear. A final 10 min heating step removed residual H_2_O_2_. After complete digestion and dilution, P concentration was determined colorimetrically using the molybdenum blue method with antimony as a reductant.

Plant P content and biomass data were used to calculate phosphorus absorption efficiency (PAE) and phosphorus utilization efficiency (PUE) as follows:

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Soil total P was determined using an acid digestion followed by the molybdenum–antimony blue colorimetric method. A 0.5 g air-dried, sieved soil sample was digested with 5 mL concentrated H_2_SO_4_ and 10 drops of HClO_4_. After complete digestion, dilution, and settling, the P concentration in the supernatant was measured using an AA3 continuous flow analyzer.

4.3.7. Soil Phosphatase Activity

Acid phosphatase (ACP), neutral phosphatase (NP), and alkaline phosphatase (AKP) activities in rhizosheath soil and bulk soil (loose soil not attached to roots) were determined using corresponding assay kits (Comin Biotechnology Co., Ltd.). Approximately 0.1 g of air-dried soil was placed in a 1.5 mL tube, mixed with 50 μL toluene, shaken, and left for 15 min. Samples were then incubated at 37 °C in a water bath for 24 h for catalytic reaction. After incubation and centrifugation at room temperature for 10 min, the supernatant was used for analysis. Following kit instructions, color development was measured at 660 nm using a microplate reader, and phosphatase activity was calculated.

4.3.8. Root Colonization by AMF

The colonization rate of roots by AMF was assessed using the trypan blue staining method. Fresh roots were cut into 1 cm segments and placed in tissue embedding cassettes. The following steps were performed sequentially: clearing in 10% KOH at 90 °C for 80 min; rinsing with distilled water; acidifying in 2% HCl at room temperature for 10 min; rinsing again; and staining with 0.05% trypan blue (0.15 g trypan blue in a 1:1:1 mixture of distilled water, lactic acid, and glycerol) at 90 °C for 30 min. Stained roots were destained in a lactoglycerol solution (1:1:1 distilled water:lactic acid:glycerol) at room temperature for 3 days. For each treatment, four slides were prepared, each containing ten root segments mounted on a slide. For each segment, ten fields of view were observed under a microscope, totaling 400 fields per treatment. Colonization was assessed using the gridline intersect method. A field of view was recorded as colonized (1) if hyphae, arbuscules, or vesicles intersected the grid lines; otherwise, it was recorded as non-colonized (0). Colonization rates were expressed as the percentage of colonized fields relative to the total fields observed, with separate calculations for total colonization, vesicular colonization, arbuscular colonization, and hyphal colonization rates [54].

4.3.9. Soil Spore Density

AMF spore density in soil was determined using the wet-sieving and decanting method followed by sucrose density gradient centrifugation. A 10 g air-dried soil sample was suspended in 100 mL tap water and left to settle for 20–30 min. The supernatant was quickly decanted onto a nested sieve pair consisting of a 20-mesh (top) and a 400-mesh (bottom) sieve. Material retained on the 400-mesh sieve was washed into a 100 mL centrifuge tube with distilled water and centrifuged at 3000 rpm for 3 min. The supernatant and floating debris were discarded. The pellet was resuspended in a 45–50% sucrose solution to two-thirds of the tube volume, thoroughly mixed, and centrifuged at 3000 rpm for 2 min. The supernatant containing spores was quickly filtered onto a 400-mesh sieve and rinsed with distilled water to remove sucrose. Spores were washed from the sieve into a Petri dish and counted under a stereomicroscope. Results were expressed as the number of spores per gram of dry soil.

4.3.10. Soil Hyphal Density

Hyphal density in soil was measured using the gridline intersect method. A 5 g air-dried soil sample (sieved to 1 mm) was suspended in 50 mL tap water, ground, and stirred for 30 s. The suspension was poured over a nested 20-mesh and 400-mesh sieve pair. Material retained on the 400-mesh sieve was washed into a 500 mL beaker with 200 mL tap water. The suspension was stirred with a magnetic stirrer, transferred to a 50 mL centrifuge tube, and centrifuged at 1000 rpm for 30 s, followed by 30 s of settling. A 5 mL sub-sample was drawn from 1 cm below the liquid surface using a pipette and vacuum-filtered through a 0.45 μm pore-size membrane filter.

The filter membrane was placed on a microscope slide, stained with three drops of 0.05% trypan blue, air-dried, mounted with a drop of lactoglycerol, and covered with a coverslip. For each treatment, three slides were prepared. On each slide, 25 random fields of view were observed under a microscope fitted with an eyepiece graticule (1 cm^2^ grid). The number of intersections between hyphae and the grid lines was counted. Hyphal length per gram of dry soil (hyphal density, m g^−1^) was calculated using the standard formula.

4.4. Statistical Analysis

All data are presented as the mean ± standard deviation of four biological replicates. Individual data points for each replicate (n = 4 per treatment) are superimposed on the bars and represented by diamonds. Data were collated and processed initially using Microsoft Excel 2016. Statistical analyses, including one-way analysis of variance (ANOVA) and principal component analysis (PCA), were performed using SPSS 26 software (IBM Corp., Armonk, NY, USA). Duncan’s multiple range test was used for post hoc comparisons where ANOVA indicated significant differences (p < 0.05). Partial least squares regression (PLSR) was employed to analyze the relationships between biomass and various morphological, physiological, mycorrhizal, and phosphorus-related traits. Variable importance in projection (VIP) scores were calculated to assess the contribution of each predictor. Figures were prepared using Origin 2024 software (OriginLab Corporation, Northampton, MA, USA).

5. Conclusions

This study systematically elucidates the adaptive strategies of K. hirsuta in response to varying soil phosphorus availability. Under low-P stress, plants primarily enhance rhizosheath–microbe interactions to activate and enrich insoluble phosphorus pools. At a moderate phosphorus level (35 mg·kg^−1^), an optimal synergistic “rhizosheath–AMF” symbiotic system is established, facilitating efficient carbon–phosphorus exchange. In high-P environments, the strategy shifts towards reliance on expansive root morphological plasticity to enhance phosphorus capture capacity. A hormonal signaling network involving auxin (IAA) and abscisic acid (ABA) plays a key integrative role in this multi-strategy shift by coordinately regulating root architecture, mycorrhizal symbiotic efficiency, and rhizosheath development. This study provides clear insights for phosphorus management in the ecological restoration of sandy grasslands using K. hirsuta: a soil phosphorus level of 35 mg·kg^−1^ is optimal for activating the rhizosheath–AMF synergy, and is therefore recommended as a target value to promote pioneer plant colonization and community establishment in restoration practices. Conversely, a high phosphorus condition (70 mg·kg^−1^) induces root morphological plasticity, leading to disproportionate carbon allocation belowground at the expense of aboveground biomass accumulation, suggesting that excessive phosphorus application should be avoided to prevent strategic shifts and resource inefficiency. The findings elucidate the phosphorus adaptation strategies of pioneer plants in alpine sandy areas from the perspective of synergistic interactions among morphological adaptation, microbial interactions, and physiological responses. This provides a theoretical basis for precise phosphorus nutrient management in ecological restoration and lays a crucial foundation for further research on the molecular regulatory mechanisms of plant phosphorus utilization.