Effects of Growth Stages of Pugionium Gaertn. on Soil Microbial Biomass C:N:P Stoichiometric Ratios and Homeostasis in Northwestern China’s Desert Regions

TL;DR

This study explores how the growth of desert plants called Pugionium Gaertn. affects soil microbes' nutrient balance, revealing how these plants influence soil health in arid regions.

Contribution

The study reveals stage-dependent regulation of microbial C:N:P stoichiometry by Pugionium Gaertn. and species-specific responses to phosphorus limitation.

Findings

Pugionium Gaertn. growth increases microbial biomass C, N, and P during vigorous growth stages.

Phosphorus limitation intensifies during the plant's reproductive stage, affecting microbial stoichiometric ratios.

Soil nitrogen, phosphorus, and extracellular enzymes regulate microbial stoichiometry through bacterial diversity.

Abstract

Understanding how desert plants interact with their soil environment is crucial for ecosystem restoration. This study investigated how the growth of Pugionium Gaertn. plants, a genus native to the deserts of northwestern China, affects the balance of carbon (C), nitrogen (N), and phosphorus (P) in surrounding soil microbes. We aimed to identify the key factors driving these nutrient dynamics. Our results show that plant growth significantly increased microbial nutrient levels. However, the ratios of C:N:P in microbes shifted periodically, revealing a strong limitation of phosphorus, especially during the plant’s reproductive stage. We found that soil nitrogen, phosphorus, and the activity of key enzymes control these microbial nutrient ratios by influencing bacterial diversity. Furthermore, different Pugionium Gaertn. triggered distinct responses in fungi and bacteria to phosphorus shortage. These findings provide new insights into plant–microbe interactions in deserts and offer a scientific basis for improving vegetation restoration and soil health in these fragile ecosystems.

Microbial stoichiometry serves as a fundamental indicator of nutrient limitations in microbial communities. However, the dynamic effects of Pugionium Gaertn. growth on soil microbial C:N:P stoichiometric ratios and their primary driving factors in native desert ecosystems remain poorly understood. This study aimed to clarify the stage-dependent regulation of microbial C:N:P stoichiometry by Pugionium Gaertn. in native desert ecosystems. This study examined representative Pugionium Gaertn. (P. cornutum and P. dolabratum) in northwestern China’s desert regions, based on investigations conducted during 2022–2023, conducting systematic analysis of variations in rhizosphere soil microbial biomass C, N, and P levels, C:N:P stoichiometric ratios, fungal and bacterial diversity, soil physicochemical properties, and extracellular enzyme activities (EEAs) across different phenological stages. Results demonstrated that Pugionium Gaertn. growth significantly enhanced microbial biomass C, N, and P accumulation during vigorous growth stages. Simultaneously, stoichiometric ratios (C:N, C:P, N:P) exhibited periodic fluctuations, with P limitation characteristics becoming substantially intensified during the reproductive stage. Total soil nitrogen, total phosphorus, and EEAs significantly regulated microbial C:N:P stoichiometric ratios through their effects on bacterial diversity. In P. dolabratum, distinct response pathways were observed between fungi and bacteria to P limitation, indicating species-specific regulatory mechanisms. These findings provide novel insights into the relationship between Pugionium Gaertn. and soil elemental stoichiometry, as well as its influence on elemental dynamic balance at microbial and community levels. Furthermore, the results support ecological adaptation strategies of Pugionium Gaertn. communities in native habitats, offering scientific evidence for vegetation restoration and soil improvement in desert regions.

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1. Introduction

Carbon (C), nitrogen (N), and phosphorus (P) are universally acknowledged as fundamental elements essential for sustaining terrestrial life. The stoichiometric ratios of C:N:P within vegetation, soils, and microbial communities exert significant influence over primary productivity, nutrient turnover, and trophic interactions in terrestrial systems [1]. Moreover, ratios of soil extracellular enzyme activities (EEAs) have been regarded as functional markers reflecting microbial strategies for nutrient acquisition [2]. Resource assimilation by soil microorganisms is primarily mediated through the excretion of specific enzymes for biomass synthesis, simultaneously modulating C, N, and P fluxes, thereby exerting profound effects on ecosystem-level functions and productivity [3]. The stoichiometric ratios of microbial biomass C:N:P have been widely utilized to infer microbial strategies in resource assimilation as well as to elucidate the extent and nature of nutrient constraints [2,4]. Arid and hyper-arid desert zones collectively constitute one-fifteenth (33.7 × 10^6^ km^2^) of the Earth’s terrestrial area [5]. China is among the nations most profoundly impacted by desertification on a global scale. Nevertheless, anthropogenic disturbances, including overreclamation, overgrazing, and the progression of climate warming, have intensified ecosystem degradation, resulting in accelerated desertification, reduced biodiversity heterogeneity, and substantial socio-environmental repercussions. Thus, it is essential to pursue a nuanced and systematic understanding of desert ecosystem dynamics and to enhance the protection of their ecological integrity and biodiversity.

As microbial C:N:P stoichiometric ratios function as a reflection of nutrient constraints and fundamental ecological processes within ecosystems, assessing its temporal variation and the mechanisms influencing it throughout different stages of plant development is considered essential [6]. According to stoichiometric homeostasis theory, soil microorganisms modulate the elemental makeup of their biomass as an adaptive response to environmental fluctuations, which constitutes a principal mechanism regulating soil microbial C:N:P patterns [1,7,8]. Despite the fact that ambient conditions frequently fail to satisfy microbial nutritional requirements, substantial homeostasis can still be sustained through adjustments in metabolic regulation [9]. More precisely, microbial communities may buffer environmental variability by storing surplus quantities of C, N, and P, thereby achieving regulatory balance [10]. Evidence from other investigations indicates that microbial biomass C, N, and P levels function as effective indicators for evaluating plant C, N, and P use efficiency across distinct phenological stages [11,12]. The progression of plant growth stages also directly affects the uptake and allocation of C, N, and P [13]. Due to minimal soil organic matter content, elevated pH levels, and restricted water and nutrient availability, desert ecosystems remain particularly fragile. These environmental features offer a valuable framework for investigating C:N:P stoichiometric links between desert plants and soil microbial biomass. Nevertheless, a comprehensive understanding of the dynamic mechanisms by which indigenous desert vegetation modulates microbial C:N:P stoichiometric ratios throughout various growth stages is still insufficient. Thus, integrating regional habitat conditions in analyses of microbial C:N:P transformations during plant development and their associations with edaphic factors will contribute to a deeper understanding of nutrient cycling dynamics and adaptive plant strategies, thereby offering theoretical guidance for the maintenance of desert ecosystem stability.

Throughout plant growth and developmental stages, modifications in soil physicochemical conditions, encompassing moisture levels, pH, nutrient status, and organic matter inputs, are frequently induced, thereby influencing both microbial biomass and soil microorganism community structure [14]. Such alterations in edaphic properties are known to substantially impact the functional dynamics within the plant-soil-microbe interface, including plant productivity, biodiversity, soil acidification, and eutrophication processes, as well as microbial assemblage structures [15]. In such contexts, microbial biomass often sustains stoichiometric homeostasis as a strategy to accommodate variability in nutrient availability. In environments characterized by resource scarcity, microorganisms often employ the secretion of specific EEAs to obtain limiting nutrients, thereby maintaining the stoichiometric equilibrium between metabolic demands and accessible nutrients. For example, enzymes such as sucrase (Suc), urease (Ure), and acid phosphatase (Aph) are functionally associated with C, N, and P cycling, respectively, within the soil matrix [16,17]. The stoichiometric ratios of EEAs have been recognized as a reflection of microbial responsiveness to complex resource constraints and overall soil nutrient status [18]. Thus, the equilibrium of elemental stoichiometry in soil microbial biomass is shaped not only by internal metabolic mechanisms but also by its interactive dynamics with EEAs. However, the integrated responses of native desert vegetation under natural conditions, considering soil attributes, enzymatic activity, and microbial C:N:P stoichiometry across developmental stages, remain insufficiently understood.

The genus Pugionium Gaertn., classified within the Brassicaceae family, comprises biennial herbaceous plants geographically restricted to the sandy ecosystems of the Mongolian Plateau in Central Asia [19]. This genus includes two species: P. cornutum, endemic to China, and the endangered P. dolabratum, distributed in sandy areas surrounding major lake basins in China and Mongolia [19]. These species possess well-developed root systems and strong resistance to sand burial, enabling effective dune stabilization. In addition to their sand-fixing and soil-stabilizing functions, Pugionium Gaertn. species are also valued for their food and medicinal uses [20,21,22]. These plants are widely recognized as essential ecological and resource components in local arid environments [23]. However, in recent decades, their population abundance has declined markedly and their distribution range has contracted due to climate change, desertification, overexploitation, and human disturbances, raising concerns over genetic resource loss. Hence, elucidating temporal shifts in soil microbial biomass C, N, and P (C:N:P) stoichiometric ratios across the growth stages of Pugionium Gaertn. in situ is essential for interpreting plant–soil interactions and supporting ecological restoration and conservation in the arid regions of northwestern China. In pursuit of this objective, representative desert areas in Inner Mongolia were selected for a systematic investigation of the distinct developmental stages of Pugionium Gaertn. influence key ecological indicators, including rhizosphere microbial biomass C, N, and P content, C:N:P stoichiometric ratios, soil physicochemical attributes, EEAs, and microbial community diversity. Three hypotheses were formulated: (1) That different growth stages would markedly alter microbial biomass C, N, and P levels in soil, with pivotal transitions occurring across stages; (2) That microbial C:N:P stoichiometric ratios would be regulated across growth stages, with microbial community composition contributing to stoichiometric homeostasis under nutrient constraints; (3) That reciprocal interactions between soil properties and microbial communities would collaboratively mediate fluctuations in microbial C:N:P ratios. This study aims to elucidate the stage-dependent dynamics of soil microbial biomass C, N, and P stoichiometry in Pugionium Gaertn. and provides a novel integrative perspective by linking plant developmental progression with microbial stoichiometric regulation and soil microecosystem functioning. The outcomes of this research not only advance the current understanding of the environmental adaptability of Pugionium Gaertn. but also offer a theoretical foundation and practical guidance for their conservation and for implementing vegetation restoration in northwestern China’s desert regions.

2. Materials and Methods

2.1. Study Site

The study site is positioned within the Dalad Banner of the Inner Mongolia Autonomous Region in northwestern China, specifically located along the eastern periphery of the Kubuqi Desert (40.24° N, 109.87° E) (Figure 1). Geographically, the area lies on the northern edge of the Ordos Plateau, adjacent to the southern bank of the Yellow River’s great bend. The topography is primarily composed of shifting and semi-stabilized sand dunes exhibiting a variety of morphological forms. The local climate conforms to a temperate continental classification, receiving yearly precipitation between 250 and 350 mm, concentrated during July and August. Average annual temperatures fluctuate between 6.0–7.5 °C, while the frost-free periods extend from 130 to 150 days per year. Northwest winds prevail throughout the year, with yearly wind velocities averaging 3–4 m/s. The predominant soil type is aeolian sandy soil, distinguished by its loose structure, minimal organic matter content, and limited nutrient levels.

2.2. Experimental Design and Sample Collection

Sampling procedures were categorized into four developmental stages: A (vigorous growth; July 2022), B (senescence; September 2022), C (budding; July 2023), and D (fruiting; September 2023). These stages were defined according to the local phenological cycle of Pugionium species as a biennial plant across the 2022–2023 growth period in the study area. Rhizosphere soil associated with Pugionium Gaertn. (including P. cornutum and P. dolabratum) was collected at these stages during 2022–2023. During each sampling interval, the root-shaking technique was applied to acquire rhizosphere soil, after which soils from identical plots were thoroughly homogenized into a composite sample. Five biological replicates were established for each developmental stage. All collected soil samples were evenly partitioned into three subsamples and stored in sterile, enzyme-free centrifuge tubes. One subsample was allocated for microbial DNA extraction; another was designated for quantifying soil microbial biomass C, N, and P levels along with enzyme activity; the third was left to air-dry under natural conditions, subsequently filtered through a 2 mm mesh to eliminate visible plant materials, stones, and other particulate matter, and ultimately employed for analyzing soil physicochemical properties. The soil present at the study location was identified as sandy soil, with its particle size composition determined in accordance with the United States Department of Agriculture’s classification system [24] (Table 1).

2.3. Soil Physicochemical Properties and Enzyme Activity

Soil pH was assessed utilizing a glass electrode meter (PHS-3C, Lexi, Shanghai, China) within a soil-to-water mixture at a standardized ratio of 1:25. The concentration of soil organic carbon (SOC; g·kg^−1^) was quantified through the potassium dichromate oxidation procedure [25]. Total nitrogen (TN; g·kg^−1^) and total phosphorus (TP; g·kg^−1^) levels were measured via the Kjeldahl technique [26] and the H_2_SO_4_-HClO_4_ digestion approach [27], respectively. Details regarding soil physicochemical attributes are depicted in Table S1. In addition, three representative soil enzyme activities were selected for determination, including sucrase (Suc), urease (Ure), and acid phosphatase (Aph). Sucrase activity (Suc; U·g^−1^) was determined using the 3,5-dinitrosalicylic acid colorimetric method, urease activity (Ure; U·g^−1^) was measured using a colorimetric assay based on ammonium nitrogen production, and acid phosphatase activity (Aph; U·g^−1^) was assessed by the disodium phenyl phosphate colorimetric method.

2.4. Soil Microbial Biomass and Microbial Diversity

The determination of soil microbial biomass carbon (MBC; mg·kg^−1^) and microbial biomass nitrogen (MBN; mg·kg^−1^) employed the chloroform fumigation extraction technique with fresh soil specimens [28,29]. The carbon concentrations in K_2_SO_4_ extracts procured from fumigated and non-fumigated soil specimens were quantified utilizing a Phoenix 8000 analyzer (Tekmar Dohrmann, Mason, OH, USA). The nitrogen level in these extracts was assessed through a flow injection nitrogen analyzer (FIAstar 5000 Foss, Hillerød, Denmark). The measurement of microbial biomass phosphorus (MBP; mg·kg^−1^) incorporated 0.5 M NaHCO_3_ extraction coupled with molybdenum-antimony colorimetric analysis [30]. The calculation coefficients utilized for MBC, MBN, and MBP determinations corresponded to 0.45, 0.45, and 0.40, respectively [31].

Soil DNA isolation was executed utilizing the Mag-Bind^®^ Soil DNA Kit (Omega Bio-tek, Norcross, GA, USA). Following extraction, DNA level and purity measurements were completed, while DNA integrity underwent verification through 1% agarose gel electrophoresis. DNA fragmentation occurred via the Covaris M220 system (Gene Company, Hong Kong, China), with fragments of roughly 400 bp selected for paired-end library construction. The library preparation process utilized the NEXTFLEX Rapid DNA-Seq protocol (Bioo Scientific, Austin, TX, USA), succeeded by sequencing on the Illumina NovaSeq platform. Gene sequences procured from all specimens underwent clustering analysis through CD-HIT [32] (http://www.bioinformatics.org/cd-hit/ (accessed on 4 February 2026), version 4.6.1) with parameters set at 90% sequence identity and 90% coverage. Within each resulting cluster, the longest sequence was designated as the representative to generate a non-redundant gene catalog. Microbial community diversity was characterized through the calculation of the Shannon diversity index, which was computed using the corresponding algorithm embedded in Mothur software (version 1.30.2, https://mothur.org/wiki/calculators/ (accessed on 4 February 2026)).

2.5. Homeostatic Regulation Coefficient

In accordance with the commonly referenced approach proposed by Sterner and Elser [1], the regulation coefficient for microbial biomass stoichiometric homeostasis is computed as follows:

$$[eqn]$$

where y denotes the microbial biomass C:N:P stoichiometric ratios, x signifies the stoichiometric ratios of available nutrients (C:N, C:P, N:P), and c represents a constant term. Therefore, 1/H corresponds to the slope derived from the regression of log(y) against log(x). When the regression slope lacks statistical significance (p > 0.05), the system is interpreted as exhibiting strong stoichiometric homeostasis. In contrast, when the slope is significant (p < 0.05), and 0 < 1/H < 1, the response can be split into four categories per the value of 1/H: homeostasis (0 < 1/H < 0.25), weak homeostasis (0.25 < 1/H < 0.5), weak plasticity (0.5 < 1/H < 0.75), and plasticity (1/H > 0.75).

2.6. Statistical Analyses

All statistical procedures were conducted utilizing GraphPad Prism (Version 10.4.1). To assess the influence of varying growth stages of Pugionium Gaertn. on microbial biomass, stoichiometric ratios, enzyme activity, microbial diversity, and physicochemical characteristics of desert soils, one-way ANOVA was applied. Significant variations among groups were assessed utilizing Duncan’s multiple range test, applying a significance level of p < 0.05. The association between microbial biomass C:N:P stoichiometric ratios and soil enzyme activities was examined through linear regression analysis. Redundancy analysis (RDA) was carried out via R Studio 4.5.0 (2025-04-11) to assess the extent to which soil attributes and microenvironmental variables affect microbial biomass C:N:P ratios. During RDA modeling, backward stepwise regression was implemented to remove non-significant explanatory factors. Structural equation modeling (SEM) was also executed in R Studio to further delineate both direct and indirect pathways by which environmental parameters impact microbial biomass C:N:P stoichiometric ratios.

3. Results

3.1. Changes in Soil Microbial Biomass, Stoichiometric Ratios, and Community Diversity

The concentrations of MBC, MBN, and MBP within the rhizosphere soil of P. cornutum was observed to peak during stage A, corresponding to the vigorous growth stage (Figure 2a–c). At stage B (senescence stage) and stage C (budding stage), MBC showed a significant decline compared with the preceding stage (p < 0.05). In comparison to stage A, stage D (fruiting stage) exhibited reductions of 47.3%, 35.2%, and 59.3%, respectively (p < 0.05). Statistically significant variations were identified in the stoichiometric ratios of MBC:MBN, MBC:MBP, and MBN:MBP across growth stages (p < 0.05) (Figure 2d–f). A decreasing trend in the MBC:MBN ratio was detected over time, with notably lower values recorded in stages C (budding stage) and D, showing declines of 22.6% and 23.3%, respectively (p < 0.05). The MBC:MBP ratio initially decreased and subsequently increased; relative to stage C, which had the lowest value, stage D showed a 27.7% increase (p < 0.05), yet there was no notable variation detected between stages A and D (p > 0.05). In contrast, the MBN:MBP ratio displayed a reverse trend, increasing by 42.5% in stage D (p < 0.05). The arrangement characteristics of bacterial and fungal assemblages exhibited distinct developmental paths (Figure 2g,h). Bacterial diversity levels were markedly elevated in stages B (senescence stage) and D compared to other stages (p < 0.05), whereas fungal diversity increased initially and declined thereafter throughout plant development. Notably, both bacterial and fungal communities attained their peak Shannon diversity indices during stage B.

In the rhizosphere soil of P. dolabratum, the concentrations of MBC, MBN, and MBP exhibited trends generally comparable to those observed in P. cornutum, with peak values detected at stage A and subsequent reductions of 37.9%, 22.3%, and 32.3%, respectively, during stage D (p < 0.05). At stage B, MBC and MBP decreased significantly compared with stage A (p < 0.05), whereas no significant differences in microbial biomass were detected between stages B and C (p > 0.05). Among these variables, no substantial distinction in MBN emerged between stages A and B (p > 0.05). The MBC:MBP ratio reached its maximum during stage D, exceeding those of stages A and C by 36.2% and 39.1%, respectively. A comparable trend was noted in the MBN:MBP ratio, which was markedly greater in stage D than in stages A and C by 49.4% and 42.3%, respectively. Relative to P. cornutum, no significant changes were recorded in the MBC:MBN ratio (p > 0.05). The patterns of bacterial and fungal community diversity fluctuations appeared similar (Figure 2g,h), with the highest Shannon index observed in stage D (p < 0.05), while differences between stages B and D were statistically insignificant (p > 0.05). Notably, when growth-stage-specific data for P. cornutum and P. dolabratum were analyzed separately, significant interspecies differences were identified in MBC, MBN, and the MBC:MBN ratio (p < 0.05), potentially attributable to species-specific physiological traits.

3.2. Changes in Soil Enzyme Activity

The soil Aph activity of P. cornutum and P. dolabratum showed differentiated fluctuations across different growth stages (Figure 3a). Among them, the Aph of P. cornutum was higher than that of P. dolabratum in all four stages. The Aph of both P. cornutum and P. dolabratum reached the highest levels in stage A, then markedly decreased in stage B by 31.3% and 19.38%, respectively (p < 0.05). In stage C, the Aph of P. cornutum markedly increased, with a 34.4% increase compared to stage B (p < 0.05), and stage D was notably elevated versus stage C (p < 0.05). In contrast, although the Aph of P. dolabratum increased in stages C and D, the increases were not significant (p > 0.05).

The activity of Ure in the soil associated with both P. cornutum and P. dolabratum was found to be at its minimum during stage B (Figure 3b). In the case of P. cornutum, Ure activity decreased markedly by 55.96% in stage B relative to stage A (p < 0.05), followed by a marked increase during stage C. However, a subsequent reduction of 16.16% was recorded in stage D compared with stage C (p < 0.05). For P. dolabratum, Ure activity also exhibited a notable reduction of 51.18% in stage B versus stage A (p < 0.05), after which a continuous upward trend was observed during stages C and D.

The soil Suc activity in both P. cornutum and P. dolabratum exhibited a pattern of initial reduction followed by a gradual rise (Figure 3c). For P. cornutum, the lowest Suc activity was recorded at stage C, with reductions of 41.74% and 35.83% observed in comparison to stages A and B, respectively (p < 0.05). Although an increase in Suc was noted during stage D relative to stage C, the change was not statistically significant. In P. dolabratum, a slight, non-significant decrease of 1.38% was measured in stage B (p > 0.05). A significant decline occurred in stage C, where Suc dropped by 33.22% relative to stage B (p < 0.05). By stage D, a statistically significant recovery was evident, with Suc increasing by 24.24% compared to stage C (p < 0.05).

3.3. Stoichiometric Homeostasis

To assess stoichiometric homeostasis, linear regression was performed in log–log space between microbial biomass stoichiometry and resource stoichiometry, following the stoichiometric homeostasis framework proposed by Sterner and Elser [1], as shown in Figure 3d–f, the slopes of the regression relationships between log(Cm:Nm, Cm:Pm, Nm:Pm) and log(Cs:Ns, Cs:Ps, Ns:Ps) did not differ significantly from zero (all p > 0.05), indicating that microbial biomass stoichiometry in P. cornutum and P. dolabratum maintained strong homeostasis throughout the growth and developmental stages. These findings suggest that nutrient stoichiometric homeostasis at the community level was maintained throughout the developmental stages of Pugionium Gaertn. Moreover, when data from both P. cornutum and P. dolabratum were collectively analyzed, stoichiometric homeostasis was likewise observed (upper right corner of Figure 3).

3.4. Relationships Between Soil Microbial Biomass Stoichiometry, Soil Enzyme Activities, Soil Physicochemical Properties, and Microbial Community Diversity

RDA indicated that variations in soil microbial biomass were primarily governed by soil enzyme activities and physicochemical attributes (RDA Figure 4a,b). In the case of P. cornutum, the RDA1 and RDA2 axes explained 29.19% and 19.7% of the environmental variance, respectively. For P. dolabratum, the corresponding explanatory power of RDA1 and RDA2 was 36.94% and 26.47%, respectively.

In the rhizosphere soil of P. cornutum, TN and TP demonstrated strong positive correlations with the MBC:MBN ratio. In contrast, both TN and TP were strongly negatively linked to the MBC:MBP and MBN:MBP ratios. Among the influencing factors, fungal communities displayed a clear positive association with the MBC:MBN ratio.

Within the rhizosphere soil of P. dolabratum, TN and SOC were found to be strongly and inversely associated with the MBC:MBN ratio. In contrast, both TN and SOC exhibited strong positive relationships with MBC:MBP and MBN:MBP. Additionally, fungal and bacterial community diversity exhibited positive connections to MBC:MBP and MBN:MBP, but displayed negative associations with MBC:MBN.

3.5. Relative Contribution of Environmental Factors to Soil Microbial Biomass Stoichiometry

As illustrated in Figure 5a, the SEM analysis revealed that bacterial diversity (B-d) exerted a strong positive direct influence (2.55) on the MBC:MBN ratio in P. cornutum, whereas Ure activity displayed a direct positive effect (0.70) on the MBN:MBP ratio. Other factors were found to impact microbial C:N:P stoichiometric ratios through indirect pathways. Both direct and indirect influences encompassed positive and negative contributions. The total absolute effect was computed as the aggregate of these two components. Upon integration of direct and indirect contributions, it was evident that SOC, Ure, Suc, and fungal diversity (F-d) contributed positively to the MBC:MBN ratio, while Aph exhibited an overall suppressive effect. Regarding MBC:MBP, SOC, Ure, and Aph collectively contributed positively, whereas TN, Suc, B-d, and F-d were associated with net negative impacts. For MBN:MBP, positive total effects were observed for SOC and Aph, while negative total effects were linked to TN, Ure, Suc, and F-d. Collectively, SOC demonstrated a positive cumulative influence on microbial biomass C:N:P stoichiometric ratios in P. cornutum, whereas TN exhibited a negative cumulative effect.

According to the SEM (Figure 5b), SOC demonstrated a direct negative influence (−1.08) on the MBC:MBN ratio in P. dolabratum, whereas Aph exerted a positive direct effect (0.97). Additionally, both B-d and Suc were found to positively affect the MBC:MBP ratio, with corresponding values of 0.97 and 0.92. Regarding the MBN:MBP ratio, Aph, F-d, and TN demonstrated positive direct contributions (1.08, 2.08, and 0.45, respectively), while negative direct effects were associated with Suc (−1.23), Ure (−2.33), SOC (−0.95), and B-d (−0.71). When total effects, comprising both direct and indirect pathways, were considered, TN, Aph, and F-d exhibited net positive influences on MBC:MBN, whereas overall negative effects were attributable to SOC and Ure. In the case of MBC:MBP, SOC, TN, Suc, B-d, and F-d acted as positive contributors, while Ure and Aph were associated with cumulative negative effects. For MBN:MBP, net positive outcomes were attributed to SOC, TN, Ure, and Suc, whereas Aph and B-d exerted net negative effects. Collectively, TN and Suc demonstrated positive total absolute effects on the C:N:P stoichiometric ratios of microbial biomass in P. dolabratum.

4. Discussion

4.1. Effects of Pugionium Gaertn. at Different Growth Stages on Soil Microbial C:N:P Stoichiometric Ratios and Community Diversity Under Natural Habitat Conditions

The dynamic changes in soil microbial biomass were quantitatively assessed, confirming that the growth and developmental stages of Pugionium Gaertn. exert significant regulatory influences on the microbial biomass and C:N:P stoichiometric ratios of rhizosphere soils (Figure 2). The findings demonstrated that MBC, MBN, and MBP levels in the rhizosphere of P. cornutum and P. dolabratum exhibited stage-dependent fluctuations throughout the plant lifecycle, with maximum concentrations observed during the vigorous growth stage. This outcome aligns with prior research indicating that plant growth stages are pivotal ecological drivers modulating the structure and operational patterns of soil microbial communities [33]. Subsequent analysis revealed that during the senescence stage (B), an increase in soil organic matter was facilitated by root exudation and litter deposition from Pugionium Gaertn., thereby supplying abundant carbon sources to microbial populations. As microbial metabolic activity escalated, mineralization processes released C, N, P, and additional nutrients from microbial residues, ultimately augmenting microbial biomass potential [34]. In contrast, during the fruiting stage, significant reductions in MBC, MBN, and MBP were detected, implying that Pugionium Gaertn. limited nutrient availability necessary for sustaining microbial proliferation at this stage. These results suggest that Pugionium Gaertn. imposes distinct stage-specific regulatory effects on rhizosphere microbial communities across developmental transitions [1,35].

This study revealed a substantial disruption in the microbial biomass C:N:P stoichiometric ratios during distinct developmental stages of Pugionium Gaertn., aligning with earlier shrubland ecosystem findings where plant growth stages markedly influenced stoichiometric ratios [13]. The MBC:MBN ratios in P. cornutum and P. dolabratum exhibited a consistent decline throughout growth progression. This phenomenon could be linked to the slow decomposition rate of soil organic matter during the period of vigorous vegetative development, which facilitated increased microbial nitrogen input to sustain nitrogen nutrient levels [36]. A reduced carbon-phosphorus ratio suggested enhanced mineralization potential and greater phosphorus availability in soil [37]. In the present investigation, relatively low MBC:MBP ratios were detected in rhizosphere soils during the plants’ vigorous growth stage, indicating heightened P mineralization efficiency and availability at this stage, and a reduced probability of microbial phosphorus limitation during organic matter degradation. Conversely, both MBC:MBP and MBN:MBP ratios rose markedly during the fruiting stage, signifying an emergent phosphorus constraint for soil microbes. It has been demonstrated that microbial communities can sustain basic functionality under P-deficient conditions by altering their internal community composition [38,39]. While certain variations in MBC:MBN, MBC:MBP, and MBN:MBP were detected between P. cornutum and P. dolabratum at different developmental stages, the fitted distinctions in MBC:MBP and MBN:MBP across their growth cycles remained marginal. This outcome diverged from prior studies, which reported considerable discrepancies across vegetation types [40]. Such incongruity may stem from the two species’ shared genus, endowing them with comparable capabilities to promote invertebrate activity and microbial stimulation during growth [41], ultimately resulting in parallel temporal dynamics in rhizosphere MBC:MBP and MBN:MBP. In this context, a notable increase in bacterial community diversity was observed during the fruiting stage for both P. cornutum and P. dolabratum, implying that microbial adaptation to phosphorus scarcity might predominantly occur via restructuring of the bacterial community. In contrast, both fungal and bacterial diversities exhibited significant enhancement during the fruiting period in P. dolabratum, indicating a synergistic response involving both microbial domains to cope with phosphorus limitation. Given the elevated phosphorus requirement during rapid bacterial proliferation, inadequate phosphorus supply could suppress bacterial productivity, consequently restrain diversity expansion [42]. Furthermore, a significant elevation in soil Aph activity during the fruiting stage was identified, underscoring the central regulatory function of phosphatases in organic P mineralization at this stage, closely coupled with bacterial community shifts [43].

4.2. Stoichiometric Dynamic Equilibrium of Microbial Communities During Different Growth and Development Processes of Pugionium Gaertn.

In line with the second hypothesis, the findings demonstrated that microbial communities sustained a stable dynamic equilibrium throughout the developmental stages. This was particularly reflected in the observation that the regression slopes relating microbial C:N:P stoichiometric ratios (log (C:Nm, C:Pm, and N:Pm)) to soil resource variables (log(Cs:Ns, Cs:Ps, Ns:Ps)) did not markedly deviate from zero (p > 0.05) (Figure 3d–f). These results reinforced the prevailing perspective that microbial stoichiometry tends to be homeostatic [44], suggesting that microorganisms in soil systems are capable of preserving proportional element balances, namely carbon, nitrogen, and phosphorus, within their biomass by modulating nutrient acquisition rates. This outcome implied that, even under the constraints of arid desert conditions, the maintenance of microbial biomass was not exclusively dependent on the availability of soil nutrients. Rather, it partially reflected the intrinsic homeostatic and ecological adaptation mechanisms of Pugionium Gaertn. within its natural ecological niche. It is conceivable that microorganisms modulate both the allocation patterns and turnover dynamics of elemental resources within their biomass in response to fluctuating nutrient conditions, thereby attaining stoichiometric balance [45].

4.3. Driving Factors of Soil Microbial C:N:P Stoichiometric Ratios During Different Growth and Development Stages of Pugionium Gaertn.

Consistent with the third hypothesis, the outcomes revealed the modulation of soil microbial C:N:P stoichiometric ratios by Pugionium Gaertn. at various developmental stages were intimately linked with soil enzymatic functions, physicochemical conditions, as well as fungal and bacterial diversity. This study provides new evidence for the stage-dependent regulation of microbial C:N:P stoichiometry by Pugionium Gaertn. under natural desert conditions. The RDA analysis further verified that TN and TP within the rhizosphere of P. cornutum served as a pivotal factors markedly influencing alterations in microbial biomass stoichiometry (Figure 4). Soil nutrient availability has been identified as a determinant directly impacting plant performance, agricultural yield, and ecosystem stability [46], as it stimulates microbial proliferation and thereby governs stoichiometric balance. This observation diverges from earlier assertions that MBC:MBN ratios remain uninfluenced by soil EEAs [47]. Given the overall homeostatic behavior of soil microbial C:N:P stoichiometric ratios detected in this investigation, it is plausible that microorganisms are capable of adjusting to environmental variations in nutrient supply and demand through finely tuned regulation of EEAs synthesis and excretion, thereby preserving elemental proportionality [48].

Compared to other developmental stages, significant reductions in Aph and Ure enzymatic activities were observed in both P. cornutum and P. dolabratum during the senescence stage. Additionally, Suc activity was markedly lower during both the budding and fruiting stages relative to other growth periods. Among these enzymes, Aph serves a pivotal function in microbial phosphorus turnover and serves as a mediator of soil organic phosphorus mineralization processes [49]. A pronounced decline in Aph activity substantially impaired phosphorus transformation efficiency, thereby placing microorganisms under phosphorus-restricted conditions. In contrast, the decreased Suc and Ure activities might be associated with a reduction in fungal community diversity, which in turn may have contributed to a simplified microbial structure and the stabilization of nutrient release patterns [50]. As Pugionium Gaertn. progressed through their growth cycle, disruptions in soil microbial community composition were induced, which subsequently led to modifications in soil properties. Meanwhile, the evolving microbial assemblages interacted with these altered soil properties, eventually affecting the activities of soil enzymes. In addition, soil properties may also be influenced by mechanical treatment and variations in soil moisture, and these factors warrant further distinction and investigation in future studies. Notably, Suc, Ure, and Aph activities in soils associated with P. dolabratum exhibited signs of functional recovery during the fruiting stage, which might be attributable to heightened microbial demands for metabolic energy and structural components, consequently stimulating the production of catalytic and transformation-related enzymes. As metabolically active microorganisms and their residual biomass accumulated within the soil matrix, elemental concentrations increased accordingly, leading to elevated C:P and N:P ratios in the soil microbial biomass (Figure 2d–f). Of particular importance, the considerable enhancement in Aph activity in the rhizosphere of both P. cornutum and P. dolabratum during the fruiting stage was instrumental in improving phosphorus turnover and mitigating microbial phosphorus deficiency.

SEM analysis indicated that microbial C:N:P stoichiometric ratios were influenced by soil physicochemical attributes and enzymatic activities, primarily through their regulation of microbial diversity. In this investigation, bacterial community variation exerted a more substantial direct effect on microbial biomass stoichiometry than did fungal communities, particularly within the rhizosphere of P. dolabratum. For P. cornutum, bacterial influence on MBC:MBN ratios appeared especially prominent (Figure 5). This observation diverged from prior findings, which suggested that bacterial impact on soil microbial C:N:P stoichiometric ratios was relatively minor [47]. Pronounced impacts of microbial activity on enzyme functions were strongly linked to shifts in C:N and C:P ratios. Earlier studies have proposed that soil EEAs may serve as reliable proxies for evaluating microbial metabolic functionality, soil health conditions, and nutrient turnover in the context of land use transitions [45]. It has been shown that bacteria are capable of modulating enzymatic stoichiometry. Under nitrogen-limited conditions, a preferential synthesis of nitrogen-acquiring enzymes over phosphorus-acquiring counterparts occurs, reflecting adaptive responses to variable nutrient regimes shaped by fertilization inputs and plant-derived exudates [51]. Specifically, microbial biomass C:N:P ratios provide valuable insights into the impacts of N and P availability on bacterial community configuration [52], and offer novel perspectives for elucidating the interactions between nutrient cycling processes and microbial dynamics. It should be emphasized that the stoichiometric ratios of microbial biomass are intricately linked to the plant species present within a given ecosystem [53]. This assertion aligns with the findings of the current study, in which distinct differences in microbial stoichiometric ratios were observed between P. cornutum and P. dolabratum Collectively, the findings indicated that bacterial communities and EEAs played essential roles in maintaining stoichiometric balance in microbial biomass C:N:P across the developmental stages of Pugionium Gaertn.

5. Conclusions

This study systematically elucidated the dynamic changes in rhizosphere soil microbial biomass and C:N:P stoichiometric ratios associated with P. cornutum and P. dolabratum. The findings revealed that MBC, MBN, and MBP contents displayed distinct stage-specific fluctuations throughout plant ontogeny, reaching maximal levels during the vigorous growth stage and subsequently declining during the fruiting period. These patterns reflect the differential modulation of rhizosphere microbial communities by host plants across developmental stages. Additional analyses indicated that the progression of Pugionium Gaertn. growth markedly impacted microbial biomass stoichiometric ratios. Specifically, a gradual reduction in the MBC:MBN ratio was observed with advancing growth stages, primarily attributable to the sluggish decomposition of soil organic matter during vigorous growth, which stimulated nitrogen assimilation by microbes to maintain internal nutrient equilibrium. Concurrently, soil microbial biomass C:N:P stoichiometric ratios were found to be tightly associated with enzymatic activity levels, physicochemical parameters, and both fungal and bacterial diversity, suggesting coordinated response mechanisms among diverse rhizosphere ecosystem components (Figure 6). Notably, species-specific differences in microbial biomass content and stoichiometry were detected between P. cornutum and P. dolabratum. In conclusion, bacterial consortia and EEAs were identified as key regulatory agents in sustaining the stoichiometric homeostasis of rhizosphere microbial biomass during the developmental trajectory of Pugionium Gaertn. These findings provide a theoretical framework for advancing the understanding of functional evolution in desert plant rhizosphere micro-ecosystems and offer valuable perspectives for investigating adaptive strategies underpinning plant–microbe–soil interactions in arid environments. Future studies involving multi-environmental comparisons are warranted to evaluate the generalizability of these outcomes.