The experiment period runs from October 2022 to June 2023. The field experiment was conducted in Huanggang village, Maji town, Luhe District, Nanjing city, Jiangsu Province, China (N 32° 02’ 00”, E 118° 49’ 12”). This area is characterized by a subtropical monsoon climate, featuring high temperatures and heavy rainfall in summer and low temperatures with minimal rainfall in winter. The average annual temperature is 15.1°C, with a total precipitation of 979.5 mm and a frost-free period of 226 days. The average monthly temperature and precipitation during the experiment are shown in Figure 1. The soil type at the experimental site is yellow–brown soil. The basic physical and chemical properties of the 0–20 cm soil layer are as follows: pH, 5.76 ± 0.02; soil organic matter (SOM), 10.78 ± 0.10 g kg⁻¹; total nitrogen, 0.59 ± 0.01 g kg⁻¹; total phosphorus, 0.37 ± 0.02 g kg⁻¹; total potassium, 8.36 ± 0.12 g kg⁻¹; alkali-hydrolyzed nitrogen (AN), 105.42 ± 0.78 mg kg⁻¹; and rapidly AP, 10.39 ± 0.41 mg kg⁻¹.

The community experiment consisted of five treatments: conventional monoculture of winter wheat (Triticum aestivum L., CK), intercropping of winter wheat with Chinese milk vetch (Astragalus sinicus L., IM), intercropping with crimson clover (Trifolium incarnatum L., IC), intercropping with smooth vetch (Vicia villosa Roth., IS), and intercropping with common vetch (Vicia sativa L., IV). Each treatment was replicated three times, resulting in a total of 15 communities. Each community covered an area of 6 m² (2 m × 3 m) and was separated by 50 cm wide and 30 cm high drainage ditches. The wheat was sown on October 20, 2022. In the monoculture wheat treatment, the sowing density was 2.7 × 10⁶ plants per hectare with a row spacing of 25 cm. The intercropping of leguminous green manure was implemented by alternating one row of wheat with one row of green manure, with a row spacing of 25 cm. The sowing density for each wheat row was twice that of the monoculture wheat plot. The sowing densities for Chinese milk vetch, crimson clover, smooth vetch, and common vetch were each 1.005 × 10⁶ plants/hm². The sowing densities for both monoculture wheat and intercropped leguminous green manure were standardized. The wheat was planted in an east-west direction. In the monoculture wheat plot, 12 rows of wheat were planted. In the intercropping green manure plot, wheat and green manure were planted in alternating rows, resulting in a total of 6 rows of wheat and 6 rows of green manure. The distribution and planting methods for each plot are illustrated in Figure 2.

During the growth period of monoculture wheat, 270 kg of pure nitrogen (N), 105 kg of phosphorus pentoxide (P₂O₅), and 150 kg of potassium oxide (K₂O) were applied per hectare. In the wheat intercropping system, the application of leguminous green manure was reduced by 30% compared with that of monoculture wheat, resulting in the application of 189 kg of pure N, 73.5 kg of P₂O₅, and 105 kg of K₂O per hectare. The fertilizers used included a compound fertilizer (N₂:P₂O₅:K₂O = 18:18:8), urea (containing 46.4% N), and potassium sulfate (containing 52% K₂O). All the compound and potassium fertilizers were applied as base fertilizers, with 60% of the nitrogen fertilizer applied at planting and 40% applied as topdressing after the wheat booting stage. Irrigation was conducted as necessary on the basis of soil moisture to manage diseases, pests, and weeds.

Soil samples were collected randomly from the topsoil (0–20 cm) using the S-shaped sampling method during the overwintering (S1), jointing (S2), heading (S3), and ripening (S4) stages of wheat, with each stage sampled three times. A portion of each sample was sieved through a 2 mm sieve to evaluate the soil physicochemical properties, whereas another portion was stored in a -80°C freezer for microbial community analysis. One meter of uniformly growing wheat plants was randomly selected from each community for yield testing. Additionally, ten uniformly growing wheat plants were randomly chosen to assess agronomic traits and nutrient absorption and utilization indicators.

The data in this study were organized via Microsoft Excel and analyzed for normality and variance via SPSS version 25.0 (IBM, USA). One-way analysis of variance (ANOVA) and Duncan’s honestly significant difference (HSD) test (P < 0.05) were employed to assess differences in soil physicochemical properties, enzyme activity, wheat yield, and constituent factors across various intercropping treatments.

The original microbiome sequences were processed via QIIME 2 (2019) software. This included quality filtering, merging, and optimizing the raw sequencing data. For the 16S rRNA gene analysis, the default reference was the Silva 138 rRNA database (Edgar, 2013). Species classification information corresponding to each amplicon sequence variant (ASV) was obtained by aligning ASV sequences with the Silva bacterial database.

QIIME 2 software was used to generate rarefaction curves, which helped evaluate whether the current sample size accurately reflected changes in the bacterial community structure. The diversity indices of the soil bacterial communities were calculated, with a focus on alpha diversity through the Chao1 and Shannon indices. These indices indicate the richness and diversity of bacterial communities, respectively. Nonmetric multidimensional scaling (NMDS) analysis was performed via the vegan package in R, whereas statistical tests were conducted via permutation multivariate analysis of variance (PERMANOVA). Additionally, redundancy analysis (RDA) was used to demonstrate the associations between the soil bacterial community composition and physicochemical factors.

In the present study, intercropping of different legume green manure treatments significantly increased the number of grains and thousand kernel weight of wheat spikes while decreasing the number of spikes and tillers compared to monoculture wheat. This may be due to the good long-term synergistic effect between ecological factors such as water, fertilizer, gas, light, temperature, etc. in the near-surface layer under intercropping cultivation, where crops are able to utilize natural conditions such as land resources, light, water and nutrients more effectively. It was also found that the intercropping legume green manure kept the sabre leaves of the wheat bright green during the later stages of growth, thus providing sustained productivity for continued photosynthesis and facilitating the transfer of more nutrients to the wheat grain, which in turn increased the number of spikes and thousand kernel weight of the wheat (Nasar et al., 2022). We increased the seeding density of wheat per row in the intercropping pattern, and too high seeding density leads to ineffective tillering, more pollen abortion, wasted nutrient and water use, and shading that leads to premature plant senescence (Jiang et al., 2023), which affects tillering and spike formation in wheat. In addition, wheat yields of both the intercropped smooth vetch and intercropped common vetch treatments were reduced compared to the wheat monoculture, probably because both legume green manures are climbers (Li et al., 2021), which would entangle on the wheat stalks during the flowering period, interfering with wheat irrigation and ultimately leading to reduced yields. Wheat intercropped with Chinese milk vetch and wheat intercropped with crimson clover treatments were essentially the same in terms of yield compared to wheat monoculture, suggesting that wheat is not affected by stem entanglement of these two legume green manures in the later stages of growth (Bista and Dahal, 2018). Competition between plants became more intense compared to the control, which weakened their ability to regenerate tillers and led to fragmentation of nutrient distribution. At the same time, pollen abortion increased and grain yield and thousand kernel weight decreased.

In intercropping systems, different crop combinations have complementary effects on nutrient absorption and utilization in terms of time and space (Zhang and Li, 2003). Compared with monocultures, intercropping enhances nutrient absorption capacity, thereby increasing yield (Zhang et al., 2015). In our study, the IM, IC, IS, and IV treatments significantly increased aboveground nitrogen accumulation (P < 0.05) relative to that of monoculture wheat. This increase may result from the strong nitrogen fixation ability of leguminous green manure, which increases the nutrient pool in the soil and increases nitrogen availability. This, in turn, increases the nitrogen content and uptake in intercropped grass crops (Dos Santos Sousa et al., 2022). Research has indicated that intercropping corn with alfalfa can increase nitrogen accumulation in the aboveground parts of corn and improve its nitrogen absorption and utilization (Shao et al., 2020). Additionally, Van Deynze et al. (2018) reported that, in intercropping systems, leguminous crops transfer nitrogen to gramineous crops through various mechanisms, accounting for approximately 20% to 82% of total nitrogen uptake by those crops. This finding aligns with our results.

Leguminous green manure provides a direct nitrogen source for crops and significantly enhances soil nitrogen utilization efficiency and retention (Ashraf et al., 2004). This occurs through two key mechanisms: it increases nitrogen absorption directly and increases both AN and AP levels in the soil (Gao et al., 2024). This study revealed that, compared with monoculture wheat, intercropping with leguminous green manure reduced the soil AN content. As a result, it led to decreased nitrogen and phosphorus utilization efficiency in wheat. Research on maize–soybean intercropping has shown that such practices increase nitrogen content, uptake, and use efficiency in maize, especially with optimized nitrogen fertilizer applications. This improvement is largely due to better rhizosphere conditions and nutrient contributions from soybean (Yong et al., 2018). Additionally, other studies have indicated that planting leguminous green manure can increase the soil AP content and increase phosphorus uptake in crops, leading to improved phosphorus absorption efficiency (Pavinato et al., 2017). Our experiment supports this view, showing that, compared with monoculture, all intercropping treatments with different leguminous green manures enhanced phosphorus absorption efficiency.

Previous studies have shown that intercropping systems can increase nutrient contents (Wang et al., 2022). Our results confirmed that the contents of AP and SOM were higher in wheat intercropped with a leguminous green manure system than in monocropped wheat during S1, S2, S3, and S4. These findings indicate that intercropping influences nutrient dynamics by increasing soil organic carbon in the rhizosphere and changing the soil phosphorus composition. This process converts more stable phosphorus into its active form (Yang et al., 2022), thus increasing both the SOM and AP contents (Inal et al., 2007; Singh et al., 2021). Moreover, intercropping with leguminous green manure can increase soil phosphatase activity (Chamkhi et al., 2022) or activate phosphorus through the root exudates of green manure. This leads to increased phosphorus availability in the soil. In this research, we found that the soil’s AN content in the IM, IS, and IV treatments exhibited a decreasing trend at S4 compared to monocropped wheat. This decline is primarily attributed to the fact that, at this stage—when both green manure and wheat crops are at their peak growth—wheat plants have a significant demand for nitrogen, which depletes the soil’s alkaline dissolved nitrogen content (Du and Xue, 2017). Similar conclusions have been reached in other studies. Ding et al. (2021) reported that green manure application significantly increased the soil pH and effectively reduced soil acidification. In this experimental study, it was found that intercropping with leguminous green manure resulted in an increase in soil pH for wheat during the S1, S2, S3, and S4 periods compared to monocropping wheat. Among the treatments, the IS treatment exhibited the most significant increase in pH. This effect may be attributed to the greater biomass of sweet potato than of other leguminous green manures, which enhances the field water-holding capacity and improves the buffering ability of the soil against carbonates (Odlare et al., 2008), promoting a shift in soil pH toward neutrality.

Enzymes, as key factors influencing soil nutrient cycling, have activities that are closely related to plant growth (Ren et al., 2021). The experimental findings revealed that, compared to monoculture wheat, intercropping with leguminous green manure during the S1, S2, S3, and S4 periods increased the activities of URE, SUC, and ACP. This improvement may result from alterations in the physical and chemical properties of the soil due to the use of green manure. These changes facilitate the acquisition of carbon and nitrogen sources, stimulating an increase in soil microbial biomass and enzyme activity (Piotrowska and Wilczewski, 2012). A positive correlation between soil enzyme activity and SOM content has been established (Wei et al., 2015), an association confirmed in this study. Additional research indicates that intercropping leguminous green manure in rubber plantations enhances urease, catalase, polyphenol oxidase, and sucrase activities. This significantly improves soil quality and promotes rubber tree growth (Shamshuddin et al., 2004). Collectively, these results suggest that, compared with monoculture practices, intercropping systems increase soil enzyme activity and nutrient contents (Wang et al., 2014).

Planting and utilizing green manure can increase the soil nutrient content and alter microbial diversity and stability, making it a valuable agricultural strategy (Özbolat et al., 2023). This study revealed that, compared with wheat monoculture, intercropping with smooth vetch or common vetch increased both the Chao 1 index and the Shannon index. Among them, intercropping with smooth vetch significantly increased the Chao 1 and Shannon indices the most. This might be related to the fact that it had the highest soil organic matter content in each period, provided a rich source of carbon to the soil. thereby indirectly promoting the improvement of soil bacterial community diversity (Xu et al., 2023). These findings indicate that intercropping with leguminous green manure enhances soil bacterial species richness and diversity. During the maturity stage of wheat, leguminous green manure approaches the end of its life cycle, initiating decomposition. The breakdown of green manure residue improves the soil aggregate structure, loosens the soil, and creates space for microbial activity (Zhang et al., 2023c). The primary components of decomposed green manure include cellulose, lignin, and hemicellulose. Beneficial microorganisms, such as Pseudomonas and Arthrobacter, can hydrolyze these components into smaller molecular compounds or monomers with enzymes such as cellulase. This process further promotes nutrient release from straw (Guo et al., 2020; Kielak et al., 2016). Consequently, the relative abundance of these beneficial bacteria and the soil nutrient content increase during decomposition (He et al., 2020). However, a sufficient nutrient supply during this process is vital for the growth of less common bacteria, which may increase bacterial community diversity.

This study revealed that Proteobacteria, Acidobacteria, Chloroflexia, and Bacteroidetes were the most abundant bacterial phyla across various intercropping systems, which is consistent with previous findings (Bai et al., 2022; Huang et al., 2022). Notably, in intercropping systems—particularly in the IC treatment—Proteobacteria levels peaked (Figure 5A). Many nitrogen-fixing bacteria belong to the Proteobacteria phylum (Rahav et al., 2016). Therefore, we speculate that leguminous green manure may utilize these specific bacterial communities to increase nitrogen acquisition. Acidobacteria are primarily acidophilic, thrive in acidic environments, and constitute approximately 20% of the total bacterial population (Jones et al., 2009). Research has shown an inverse correlation between Acidobacteria abundance and soil pH within a certain range (Lauber et al., 2008), with greater relative abundance in acidic soils. Our experiment confirmed this trend, as the experimental soil remained acidic throughout the study period, resulting in a relatively high relative abundance of Acidobacteria. Moreover, the dominant bacterial genera across all the treatments included Subgroup_7, Sphingomonas, Vicinamibacteraceae, and KD4-96. Sphingomonas is crucial for phosphate solubilization (Daniel et al., 2009). Its enrichment can improve soil nutrients and stimulate plant growth. Therefore, intercropping with various leguminous green manures can increase the abundance of beneficial bacterial phyla and genera, creating a more favorable micro-ecological environment for crops.