Phosphorus Fertilization Overrides Intercropping-Induced Shifts in Microbial Stoichiometry to Increase Forage Yield

Abstract

Legume–grass intercropping and phosphorus (P) fertilization are recognized strategies for enhancing forage productivity, but their interactive effects on soil microbial processes and plant phosphorus nutrition in a semi-arid climate remain poorly understood. We conducted a field experiment with common vetch (Vicia sativa) and oat (Avena sativa) under two monocultures and three intercropping treatments (legume–grass ratios of 1:3, 2:3, and 1:1), combined with three P fertilization rates (0, 60, and 120 kg P ha⁻¹). The results showed that common vetch/oat intercropping with moderate legume–grass proportions (1:3 and 2:3) significantly outyielded monocultures across all P levels and exhibited a stronger net biodiversity effect than the 1:1 intercropping at P fertilization. Plant P concentration was primarily increased by P fertilization. Crucially, all intercropping treatments showed a significantly lower microbial biomass carbon/phosphorous ratio than the monoculture in the absence of P fertilization. However, this difference disappeared when P was applied, indicating P fertilization overrode the intercropping-induced stoichiometric shift. Correlation analyses further showed that forage yield and plant P uptake were positively linked to microbial biomass P and negatively to the microbial biomass carbon/phosphorous ratio. Together, our findings reveal that a common vetch/oat intercropping system combined with P fertilization may improve the nutrient use efficiency through microbial pathway. This improvement in nutrient use efficiency leads to higher nutrient uptake by plants, thereby causing more rapid soil reserve depletion.

1. Introduction

Grassland ecosystems are under mounting pressure from land degradation, soil nutrient depletion, and climate variability, posing significant challenges to sustainable forage production and plant nutrient uptake [1,2]. In this context, developing management strategies that enhance forage yield while maintaining or improving soil fertility is a critical global priority. Among various approaches, the intercropping of legumes and grasses has received growing attention for its potential to increase biodiversity, improve resource use efficiency, and stabilize productivity in forage systems [3,4,5].

Legume–grass intercropping can substantially increase the pool of plant-available soil nitrogen, leading to more efficient resource allocation, greater nutrient–use efficiency, and higher forage yield for sustainable livestock production [6,7]. These systems also promote soil biological activity. For example, co-cultivation of Leymus chinensis and alfalfa has been shown to elevate levels of extracellular enzymes (e.g., alkaline phosphatase, catalase), microbial biomass carbon, nitrogen, and phosphorus, soil organic matter, and available phosphorus and potassium compared with monocultures [8]. Legume–grass intercropping can further enrich beneficial microbial communities, including plant growth promoting rhizobacteria, thereby enhancing nitrogen nutrition and forage yield [9]. In arid regions, intercropping at a legume/grass ratio of 1:1 was reported to significantly increase mineralizable carbon and nitrogen and total microbial biomass, thereby improving both soil health and plant nitrogen uptake [10]. Beyond these benefits, when compared with monocultures, increased plant diversity in intercropping can also mitigate drought stress in water-limited environments [11].

The productivity of legume–grass intercropping is sensitive to the legume proportion. Forage yield typically peaks at a moderate legume proportion (e.g., 39%), beyond which further increases can lead to a linear decline in forage yield [12]. At an optimal legume proportion of between 33% and 41%, intercropping can dramatically increase nitrogen acquisition from both atmospheric and soil sources, with soil-derived nitrogen yield substantially exceeding that of pure grass or legume stands [5]. Intercropping oats with legumes can also improve water use efficiency and light interception [13,14]. Although optimal legume proportions have been reported in some regions, the interactive effects of legume proportion and fertilization regimes, particularly in temperate arid and semi-arid climates, remain poorly understood.

Phosphorus (P) availability frequently limits plant growth in grasslands. Although total soil P is often abundant, only a small fraction is readily available for plant uptake. P fertilization is commonly used to overcome this limitation, but a large proportion of applied P can become fixed to soil particles, reducing its bioavailability [15,16]. Phosphorus addition enhances plant P acquisition and promotes biomass accumulation, as plant P concentrations increase with greater soil P availability [17,18,19,20]. Soil microorganisms play a key role in P cycling. Through the release of organic acids and enzymes such as phosphatases and phytases, microbes solubilize insoluble inorganic P and mineralize organic P, thereby increasing plant-available P [21]. Moreover, practices that increase soil organic matter (e.g., intercropping) can enhance P availability and uptake efficiency by supporting P-solubilizing microbial communities [22]. In acidic soils, the combination of microbial consortia and P fertilization has been shown to act synergistically, improving both crop yield and P uptake [23]. Compared with monocultures, diverse plant cultivation can stimulate soil phosphatase activity and increase available P, thereby improving plant P nutrition [24]. These observations suggest that intercropping combined with P fertilization could support favorable conditions for microbially mediated P mobilization, offering a pathway to forage yield and plant P uptake.

Despite growing evidence for the individual benefits of intercropping and P fertilization, few studies have systematically examined their combined effects on forage productivity, plant P concentration, soil physicochemical properties, and microbial biomass stoichiometry. In particular, the ecological mechanisms that underline the interaction between legume proportion, P input, and soil microbial processes remain poorly understood in temperate semi-arid intercropping systems. To address these knowledge gaps, we conducted a field experiment with common vetch and oat under two monocultures and three intercropping treatments (legume–grass ratios of 1:3, 2:3, and 1:1), combined with three P fertilization rates (0, 60, and 120 kg P ha⁻¹) in the arid agro–pasture ecotone of China. Our objectives were to (1) assess the effects of legume/grass ratio and P input on forage yield and plant P concentration, (2) identify the key soil and microbial indicators driving these responses, and (3) elucidate the relationship between forage yield, plant P concentration and microbial biomass stoichiometry. This study will provide mechanistic insights into how legume/grass intercropping and P fertilization can be co-optimized to enhance sustainable forage production and improve P management in arid intercropping systems.

2. Materials and Methods

2.1. Study Site

The study was conducted in the Zhengxiangbai Banner of Inner Mongolia in northern China (115°24′ E, 42°9′ N). This area has a temperate arid continental climate, with an annual mean temperature of 1.9 °C and annual mean precipitation of 351 mm, most of which falls during the summer. The soil is classified as Katsanoses soil according to the world reference base [25], with an initial soil pH of 7.72, total nitrogen content of 3.1 g kg⁻¹, and available phosphorus content of 10.1 mg kg⁻¹. The soil texture consists of sand (28.2%), silt (63.7%), and clay (8.1%) [25].

2.2. Experimental Design and Field Management

A randomized complete-block design with four replicates was established in June 2023. The experiment included two monocultures (common vetch, Vicia sativa L. and oat, Avena sativa L.) and three legume–grass intercropping treatments with seeding ratios (common vetch: oat) of 1:3, 2:3, and 1:1. Each intercropping and monoculture treatment was combined with three phosphorus (P) fertilization rates: 0, 60, and 120 kg P ha⁻¹ (supplied as single superphosphate). This experiment consisted of 15 treatments, each replicated 4 times, resulting in a total of 60 plots, which were randomized within blocks.

Individual plots (4 m × 4 m) were bordered by 25 cm high ridges, with 0.8 m spacing between plots along north–south rows and 0.45 m along east–west rows. All plots were plowed and leveled before sowing. Seeds were sown on the same day using a drill seeder (2BF-6, Shangjie Co., Ltd. Shijiazhuang, China) at a depth of 4–5 cm with 25 cm row spacing. In intercropping plots, the target legume proportions (25%, 40%, and 50% for the 1:3, 2:3, and 1:1 ratios, respectively) were achieved by adjusting the seed weight of each species while maintaining a total stand density equivalent to local practice. Monoculture seeding rates were 90 kg ha⁻¹ for common vetch and 150 kg ha⁻¹ for oat. Farmyard manure was used as a basal fertilizer and applied according to local recommendations prior to sowing, at an application rate of 15,000 kg ha⁻¹ [3]. Plots were irrigated when needed during the growing season, and manual weeding was performed twice.

2.3. Plant and Soil Sampling

Forage yield was determined in late August 2023 by harvesting all aboveground biomass within a randomly placed 1 m × 1 m quadrat per plot. Plants were cut 3 cm above the soil surface, dried at 65 °C to constant weight, and weighed [3]. Dried plant samples were ground to pass a 1 mm sieve, and plant phosphorus concentration was determined using the molybdenum blue colorimetric method after acid digestion. After plant sampling in each plot, five soil cores were randomly collected from the 0–20 cm layer using a 3 cm diameter auger and mixed as a composite sample. Each soil sample was sieved to <2 mm to remove any coarse material. After homogenizing, one part was stored at 4 °C for microbial biomass, enzyme activities and soil chemical analysis, and the other was air–dried for soil properties analyses.

2.4. Soil and Microbial Analyses

Soil pH was measured in water at the ratio of 1:5 (5 g of dry soil and 25 mL of deionized water) [7]. Soil organic matter (SOM) was measured by the potassium dichromate external heating method [7]. Total C and N were measured using 150 mg of ground soil sample with an elemental analyzer (Vario MACRO cube, organic elemental analyzer (Elementar, Langenselbold, Germany) [7]. Soil-available phosphorus (AP) was extracted using 0.5 M NaHCO₃ solution and measured with an atomic absorption spectrometer [7]. Ammonium–N (NH₄⁺–N) and nitrate–N (NO₃⁻–N) concentrations were determined by using a continuous flow analyzer (Auto Analyzer 3, SEAL Analytical, Norderstedt, Germany) [7]. Urease activity was measured using the indophenol blue method. Briefly, fresh soil (5 g) was incubated with 0.1 M urea in borate buffer at 37 °C for 2 h. The ammonium released was extracted with 2 M KCl and quantified colorimetrically at 690 nm. Nitrate reductase activity (NR), acid phosphatase activity (ACP) and alkaline phosphatase activity (ALP) were measured using a commercial kit (Solarbio, Beijing, China). Microbial biomass carbon (MBC), nitrogen (MBN), and phosphorus (MBP) were determined by the chloroform fumigation extraction method [9]. Briefly, fresh soil was fumigated with ethanol-free chloroform for 24 h at 25 °C. After fumigation, MBC and MBN were extracted with 0.5 M K₂SO₄ and analyzed using a TOC analyzer. MBP was extracted with 0.5 M NaHCO₃ and measured colorimetrically. MBC, MBN, and MBP were calculated as the difference between fumigated and non-fumigated samples using conversion factors of 0.45, 0.54, and 0.40, respectively. The microbial biomass stoichiometry was calculated as MBC/MBN, MBC/MBP, and MBN/MBP. The soil C/N ratio was the ratio of soil organic carbon to total nitrogen.

2.5. Calculation of Net Biodiversity Effect (NE)

We calculated NE based on the total yield of each mixture to quantify biodiversity effects as follows:

$$\mathrm{N}\mathrm{E}=Y_{obs}-Y_{exp}$$

The NE quantifies the overyielding of a mixture relative to the expected yield, where $Y_{obs}$ and $Y_{exp}$ represent the observed and expected hay yield of the mixture, respectively.

2.6. Statistical Analysis

First, the effects of fertilization and mixed sowing on soil and microbial variables were evaluated using a two-way analysis of variance (ANOVA). Before performing the ANOVA, homogeneity of variances was tested using Bartlett’s method, and normality was assessed using the Shapiro–Wilk test. Data transformations were applied to meet normality assumptions. Specifically, logarithmic transformation was used for SOM, AP, the C/N ratio, and alkaline phosphatase activity; reciprocal transformation for NO₃⁻–N, NH₄⁺–N, and the MBC/MBN ratio; square root of reciprocal transformation for pH; and power transformation for MBP, MBC, nitrate reductase activity, and urease activity using the powerTransform function. After transformation, normality was rechecked, and all transformed data conformed to a normal or approximately normal distribution. Second, we tested for the impact of the ratio of legumes to grass, phosphorus fertilizer, and their interactions on herbage yield, NE, soil properties, and microbial variables. This was undertaken using a linear mixed-effects model, where “ratio of legumes to grass” and “phosphorus fertilizer” were treated as fixed factors, and block was treated as a random factor (“lme” function, NLME package). Post–hoc comparisons were performed using the emmeans test, and differences were considered significant at p < 0.05. All statistical analyses were conducted in R version 4.4.3 [26].

3. Results

3.1. Forage Yield, Net Plant Diversity Effect and Plant Phosphorus Concentration

Common vetch/oat intercropping at legume–grass ratios of 1:3 and 2:3 significantly outyielded monocultures across all P fertilization levels (p < 0.05; Figure 1a). This yield advantage was supported by a correspondingly greater NE in the 1:3 and 2:3 mixtures compared with the 1:1 mixture at P rates of 60 and 120 kg ha⁻¹ (Figure 1b). While the intercropping ratio governed yield and NE responses, plant P concentration was primarily increased by P fertilization, with both application rates resulting in significantly higher concentrations than the unfertilized control (Figure 1c). However, intercropping and phosphorus addition have no interactive effect on the above three variables (Table 1).

Figure 1. Forage yield (a), net biodiversity effect (b), and plant P concentration (c) under different intercropping treatments and P fertilization levels. Monoculture represents the average of common vetch and oat single cultivation. Intercropping treatments represent the common vetch/oat at sowing ratios of 1:3, 2:3, and 1:1. P0, 0 kg ha⁻¹; P1, 60 kg ha⁻¹; P2, 120 kg ha⁻¹. Different lowercase letters above boxes indicate significant differences among intercropping treatments (p < 0.05) and different uppercase letters indicate significant differences among P fertilization levels (p < 0.05).

Table 1. Effects of mixed sowing and fertilization and their interaction on yield, plant phosphorus content, soil physicochemical properties, microorganisms and enzyme activities. *, p < 0.05; **, p < 0.01; ***, p < 0.0001.

	Fertilization		Variety		Fertilization × Variety
	F Value	p Value	F Value	p Value	F Value	p Value
Forage yield	2.080	0.14	38.469	2.54 × 10−11 ***	1.943	0.10
Net effect of plant diversity	4.018	0.0297 *	15.402	3.44 × 10−5 ***	0.883	0.4872
Plant phosphorus concentration	9.355	0.000535 ***	1.088	0.366720	0.779	0.592029
Soil total nitrogen	6.791	0.00315 **	2.129	0.11362	0.257	0.95304
Soil organic matter	0.332	0.71939	4.939	0.00565 **	0.202	0.97404
Available phosphorus	0.242	0.786	0.304	0.822	1.152	0.353
Nitrate nitrogen	8.742	0.000804 ***	4.425	0.009524 **	3.24	0.011945 *
Ammonium nitrogen	0.763	0.4738	3.535	0.0242 *	1.16	0.3491
C/N ratio	0.505	0.608	3.36	0.0292 *	0.45	0.840
pH	7.386	0.00205 **	20.385	6.82 × 10−8 ***	1.13	0.36472
Microbial biomass carbon	3.224	0.0515	10.998	2.86 × 10−5 ***	1.155	0.3515
Microbial biomass nitrogen	3.025	0.061	10.128	0.000056 ***	0.417	0.863
Microbial biomass phosphorus	11.005	0.000186 ***	7.441	0.000532 ***	1.406	0.239333
MBC/MBN ratio	2.176	0.128	11.543	1.89 × 10−5 ***	0.181	0.98
MBN/MBP ratio	4.422	0.0192 *	2	0.1313	2.002	0.091
MBC/MBP ratio	14.75	2.09 × 10−5 ***	10.218	5.22 × 10−5 ***	5.492	0.000404 ***
Urease	0.702	0.5024	3.596	0.0227 *	0.662	0.6802
Nitrate reductase	11.148	0.000171 ***	1.613	0.203453	0.391	0.879906
Acid Phosphatase	1.677	0.201	1.069	0.374	0.551	0.766
Alkaline phosphatase	4.294	0.0213 *	1.487	0.2345	0.804	0.5732

3.2. Soil Physicochemical Properties

Phosphorus fertilization significantly increased total soil nitrogen but decreased soil pH (p < 0.05; Figure 2a,g; Table 1). In parallel, intercropping significantly enhanced SOM, NH₄⁺–N, and the soil C/N ratio when compared with monoculture (Figure 2b,e,f). Notably, a significant interaction between intercropping and P fertilization was observed for soil NO₃⁻–N, with the common vetch/oat 1:3 treatment under high P (120 kg ha⁻¹) showing the highest NO₃⁻–N content (Figure 2d; Table 1). In contrast, soil AP content was not significantly affected by either intercropping or P fertilization (Figure 2c).

Figure 2. Soil physicochemical properties under different intercropping treatments and P fertilization levels. Panels (a)–(g) present soil properties for soil total nitrogen content (STN), soil organic matter content (SOM), soil-available phosphorus content (AP), soil nitrate nitrogen content (NO₃⁻–N), soil ammonium nitrogen content (NH₄⁺–N), the ratio of soil organic carbon to total nitrogen (C/N ratio), and soil pH. Monoculture represents the average of common vetch and oat single cultivation. Intercropping treatments represent the common vetch/oat at sowing ratios of 1:3, 2:3, and 1:1. P0, 0 kg ha⁻¹; P1, 60 kg ha⁻¹; P2, 120 kg ha⁻¹. Different lowercase letters above boxes indicate significant differences among intercropping treatments (p < 0.05) and different uppercase letters indicate significant differences among P fertilization levels (p < 0.05).

3.3. Microbial Biomass and Enzyme Activity

Intercropping significantly increased MBC and MBN relative to monoculture (p < 0.05; Figure 3a,b). In contrast, P fertilization significantly increased MBP (p < 0.05; Figure 3c). These shifts in microbial biomass were accompanied by distinct changes in microbial stoichiometry. Intercropping significantly lowered the MBC/MBN ratio, while P fertilization significantly reduced both the MBN/MBP and MBC/MBP ratios (p < 0.05; Figure 3d–f). A particularly notable interaction was observed for the MBC/MBP ratio, with all intercropping treatments showing a significantly lower ratio than the monoculture in the absence of P fertilization. However, this difference disappeared when P was applied at either 60 or 120 kg ha⁻¹, indicating that P fertilization overrode the intercropping-induced stoichiometric shift (Table 1).

Figure 3. Microbial biomass carbon (MBC), microbial biomass nitrogen (MBN), microbial biomass phosphorus (MBP), and microbial enzyme activities under different intercropping treatments and P fertilization levels. Panels (a)–(j) present MBC, MBN, MBP, MBC/MBN, MBN/MNP, MBC/MBP, acid phosphatase activity (ACP), alkaline phosphatase activity (ALP), nitrate reductase activity (NR), and Urease. Monoculture represents the average of common vetch and oat single cultivation. Intercropping treatments represent the common vetch/oat at sowing ratios of 1:3, 2:3, and 1:1. P0, 0 kg ha⁻¹; P1, 60 kg ha⁻¹; P2, 120 kg ha⁻¹. Different lowercase letters above boxes indicate significant differences among intercropping treatments (p < 0.05) and different uppercase letters indicate significant differences among P fertilization levels (p < 0.05).

Enzyme activities responded differently to the two management factors. Intercropping significantly enhanced urease activity (p < 0.05; Figure 3j); P fertilization significantly suppressed ALP activity but stimulated NR activity (Figure 3h,i); and ACP activity, in contrast, remained unaffected by either intercropping or P fertilization (Figure 3g).

3.4. Key Drivers of Forage Yield and Plant P Concentration

Forage yield was positively associated with the NE, SOM, MBN, MBP, and urease activity (p < 0.05), whereas it showed negative correlations with soil pH, the MBC/MBN ratio, and the MBC/MBP ratio (p < 0.05; Figure 4). Notably, the NE declined as the legume proportion increased in the intercropping system (p < 0.05; Figure 4).

Figure 4. Contribution of the net biodiversity effect (a,b), soil physicochemical properties (c,d), and microbial indicators (e–i) to forage yield. NE, net biodiversity effect; SOM, soil organic matter; MBC, microbial biomass carbon; MBN, microbial biomass nitrogen; MBP, microbial biomass phosphorus. The solid lines indicate a significant linear relationship (p < 0.05) the shaded area represents the 95% confidence interval.

Plant phosphorus concentration exhibited a distinct set of linkages. It was positively related to soil total nitrogen, soil C/N ratio, NO₃⁻–N, AP, MBP, and NR activity, but negatively related to the MBC/MBP ratio (p < 0.05; Figure 5). Together, these correlation patterns underscore the central role of microbially mediated phosphorus cycling in coupling the effects of intercropping and phosphorus fertilization to forage productivity and plant phosphorus uptake.

Figure 5. Contribution of soil physicochemical properties (a–d) and microbial indicators (e–g) to plant P concentration. STN, soil total nitrogen content; C/N ratio, the ratio of soil organic carbon and total nitrogen; NO₃⁻–N, nitrate nitrogen content; AP, soil-available phosphorus content; MBP, microbial biomass phosphorus; NR, nitrate reductase activity. The solid lines indicate a significant linear relationship (p < 0.05) the shaded area represents the 95% confidence interval.

4. Discussion

4.1. Effects on Forage Yield and Net Diversity Effects

Higher plant diversity in legume–grass mixtures generally enhances forage yield, with the NE serving as a key indicator of diversity–ecosystem functioning [27,28]. Previous studies have reported optimal legume proportions for maximizing productivity in the range of 40–50% [29], and even a 30% legume seeding ratio combined with phosphorus fertilization has been shown to improve forage production [3]. In contrast, our results revealed that a lower legume proportion (25%) produced the highest hay yield and NE across most P fertilization levels, suggesting a shifted optimum in this semi-arid alkaline grassland. This divergence is likely context-dependent. Under water-limited and alkaline conditions, a moderate legume proportion may provide sufficient nitrogen facilitation while minimizing competition for soil moisture and phosphorus, thereby maximizing complementarity and NE. Consistent with this interpretation, the common vetch/oat 1:3 treatment maintained higher NE and yield than the 1:1 treatment, supporting the view that excessive legume dominance can weaken interspecific complementarity. Additionally, phosphorus fertilization contributed to a slight decline in soil pH, a shift that is favorable for soil organic matter accumulation [30,31]. In this study, this concurrent increase in soil organic matter and urease activity, particularly in the common vetch/oat 1:3 treatment, suggests a more active nitrogen cycle driven by biological nitrogen fixation of common vetch, which may partly explain the overyielding observed at a legume proportion of 25% [32,33].

4.2. Effects on Plant Phosphorus Concentration

Phosphorus fertilization significantly increased plant P concentration, an effect closely associated with elevated microbial biomass phosphorus and reduced soil pH. The decline in soil pH induced by P addition likely enhanced P solubility and stimulated phosphate-solubilizing microorganisms, thereby expanding the microbial P pool [34,35]. The combined application of P fertilizer with organic acids has also been reported to increase plant P concentration [16]. Meanwhile, greater plant diversity, reflected by a higher net biodiversity effect, may have increased the diversity of root exudates, thereby promoting microbial functional differentiation and enhancing P mobilization [36]. Because available P is the direct source for plant P uptake, an increase in available P directly contributes to improved plant P concentration [37]. In addition, improved soil nitrogen status, evident from the positive correlations among total soil nitrogen, nitrate–N, and plant P, may further facilitate P acquisition, consistent with the coupling of nitrogen supply and P demand during rapid biomass accumulation.

4.3. Soil and Microbial Responses to Intercropping and Fertilization

Phosphorus application indirectly influenced forage yield by increasing total soil nitrogen and enhanced plant phosphorus concentration through a significant increase in nitrate reductase activity and soil nitrate levels. These effects likely reflect the way that P input promoted root growth and nitrogen fixation in common vetch, thereby improving soil nitrogen status [38,39]. Notably, the NE responded non-linearly to P addition, suggesting threshold effects and the involvement of multiple concurrent processes. Intermediate P supply may have coincided with transient P immobilization or adsorption, alongside a rebalancing of legume–grass competition that reduced complementarity, whereas higher P more fully alleviated P limitation and reactivated facilitative interactions and microbial turnover pathways [3]. The reduction in yield and NE at 60 kg P ha⁻¹ compared with both the unfertilized control and the 120 kg P ha⁻¹ treatment may therefore reflect a threshold response, short-term P immobilization or shifts in competitive dynamics under intermediate P availability.

The contrasting responses of soil organic matter and ammonium–N between the common vetch/oat 1:3 and 2:3 treatments at 60 kg P ha⁻¹ suggest that the 1:3 mixture may require higher P input to fully realize its facilitative effects, whereas the 2:3 mixture can already benefit from moderate P supply. Intercropping of common vetch and oat significantly reshaped microbial nutrient pools and stoichiometry. MBN and MBP were positively correlated with hay yield, while the MBC/MBN, and MBC/MBP ratios were negatively correlated, indicating that mixtures promoted more efficient microbial turnover and improved N and P supply to plants [40,41]. Phosphorus fertilization further increased MBC and, more notably, MBP, suggesting enhanced microbial P cycling and soil fertility, likely driven by legume root growth and N fixation that also supported plant P acquisition [42,43,44]. Enzymatic responses were also dependent on P status. Alkaline phosphatase activity declined under P addition, whereas acid phosphatase activity remained unchanged. This pattern is consistent with ecosystem-specific regulation influenced by background climate and C–N availability [45,46]. Collectively, these results support a coupled pathway whereby intercropping and P supply expand microbial N and P pools, optimize microbial stoichiometry, and regulate phosphatases, thereby sustaining forage productivity [47,48,49,50].

5. Conclusions

We found that common vetch/oat intercropping with a 25–40% legume proportion outyielded monocultures across all P levels and exhibited stronger net biodiversity effects than the 1:1 intercropping. Forage yield and plant P concentration were positively correlated with MBP and negatively with the MBC/MBP ratio. Intercropping increased microbial biomass C and N, whereas P fertilization specifically enhanced MBP, followed by reductions in the MBN/MBP and MBC/MBP ratios. Notably, a significant interaction between intercropping and P addition was observed for the MBC/MBP ratio. These findings suggest that P supply can buffer intercropping-induced stoichiometric shifts. Overall, optimizing legume–grass intercropping ratios with moderate P fertilization synergistically enhances forage yield and plant P uptake in semi-arid grasslands. Collectively, this study provides a mechanistic framework for optimizing nutrient management in diversified forage systems and underscores the critical role of microbial phosphorus cycling in mediating plant–soil interactions under integrated management practices.