Effect of Winter Cropping Forage on Soil Aggregate Distribution and Stability

Abstract

Soil structure is crucial for maintaining soil health and can be improved through winter cropping. This study evaluated the effects of winter cropping Italian ryegrass (WI), rye (WR), oat (WO), and winter fallow (CK) on soil aggregate structure and explored the role of soil-cementing materials and arbuscular mycorrhizal fungi (AMF) communities in regulating soil aggregate distribution and stability. Compared to CK, the WI and WR treatments increased the proportion of water-stable large macroaggregates (>2 mm diameter) by 45.7% and 41.5%, respectively. Both WI and WR treatments enhanced the mean weight diameter and geometric mean diameter of soil aggregates, while soil porosity increased by 15.7% and 21.7%, respectively. The contents of amorphous iron oxide, humic acid, and fulvic acid were significantly higher in the WI and WR treatments. The WR treatment improved the Shannon index of AMF communities by 14.6%, and the relative abundances of Claroideoglomus increased by 55.3%, 51.3%, and 43.5% in the WI, WR, and WO treatments, compared to CK, respectively. Dominant AMF genera had a substantial impact on soil aggregate distribution. The partial least squares path model indicated that distinct AMF communities contributed to variations in soil aggregate distribution following winter cropping forages. Both Italian ryegrass and rye showed the greatest potential for enhancing soil structure and are recommended for winter cropping in Southern China. These findings suggest that winter cropping forages can improve soil aggregate structure primarily by enhancing AMF communities, providing a promising strategy for improving soil health.

1. Introduction

Soil aggregates form the foundation of soil structure and play a crucial role in regulating soil quality and ecological functions [1]. The uptake and utilization of nutrients by plant roots, as well as key soil properties such as pore size, enzyme activity, and microbial composition, are closely linked to soil aggregate structure [2,3]. Aggregates of different particle sizes impact the dynamics of soil nutrients through their specific particle characteristics [4,5]. For example, macroaggregates (>2 mm), which typically contain a higher proportion of labile substrates derived from plant residues, facilitate gas exchange and contribute significantly to the accumulation of soil organic carbon [6,7]. The composition and stability of soil aggregates are typically affected by environmental factors (e.g., parent material, topography, climate), biological factors (e.g., soil fauna, plant roots, microbial activity), and human activities (e.g., tillage, mechanical compaction) [1,8,9].

Cementing materials serve as binding agents in aggregate formation [10,11]. Inorganic cementing materials primarily comprise silicic acid, clay, and metal oxides (e.g., alumina and iron oxides) [12,13]. Metal oxides such as calcium and magnesium enhance soil aggregate adhesion through ion bridging and cation polarization effects [14]. Organic cementing materials mainly include plant roots, proteins, polysaccharides, and other organic compounds [15,16]. Plant roots regulate various physical and chemical processes during soil aggregate agglomeration. Specifically, roots alter soil density through root infiltration and influence hydrological processes via evapotranspiration, thereby affecting soil aggregate formation [17,18]. Root-derived chemical exudates also facilitate soil aggregation [8,11]. Furthermore, complex root systems and symbiotic fungal mycelium exert a binding effect on soil aggregates [15]. Arbuscular mycorrhizal fungi (AMF) secrete highly cohesive substances that contribute to soil aggregate formation [19,20].

Numerous factors, including soil type, nutrient levels, and fertilizer application, exert a significant influence on soil aggregate structure [9,21,22]. Conventional tillage practices can disrupt the formation and stability of soil aggregates and alter the content of soil-cementing materials, such as organic matter [23,24]. In contrast, appropriate tillage practices can enhance the physical structure of soil aggregates. Crop rotation systems are effective in addressing issues related to soil degradation and continuous cropping, as well as enhancing soil fertility and increasing crop yields [25,26]. Crop rotation influences pore space distribution within soil aggregates and drives organic carbon input, serving as an effective soil management strategy [27]. For example, the inclusion of legumes in rotation significantly enhances soil aggregation through differential organic carbon accumulation, which promotes soil aggradation and improves aggregate stability [28]. Additionally, plant rhizosphere effects play a key role in improving aggregate stability, where the aggregate stability is stronger in fibrous roots than in tap roots [29]. Italian ryegrass-rice rotation has been demonstrated to improve the proportion of soil aggregates, significantly increasing total soil porosity and non-capillary porosity [30]. However, the key mechanisms underlying improvements in soil structure under winter cropping systems remain poorly understood.

In this study, four treatments of winter cropping Italian ryegrass (WI), ryegrass (WR), oat (WO), and winter fallow (CK) were established. We compared the differences in aggregate distribution and stability, cementing material contents, and AMF communities in the soil to explore: (1) the effects of different winter forages on the composition and stability of soil aggregates; (2) the effects of different winter forages on soil-cementing materials and AMF communities; and (3) the key factors influencing soil aggregate formation. These findings would provide a basis for selecting suitable winter forages to improve soil structure in Southern China, offering a scientific foundation for enhancing soil quality.

2. Materials and Methods

2.1. Field Site Description

The experimental site is located in Conghua, Guangzhou, Guangdong Province, China (23.55° N, 113.56° E). This region experiences a subtropical monsoon climate, with an average annual temperature of 21.7–23.1 °C and average annual precipitation of 1923 mm. The soil type is acidic red soil (Ferralic Cambisol). Prior to the experiment, the field had been under long-term double-cropping rice cultivation. The initial soil physicochemical properties were as follows: pH 6.19, soil organic matter (SOM) 18.97 g kg⁻¹, total phosphorus (TP) 0.67 g kg⁻¹, available phosphorus (AP) 39.20 mg kg⁻¹, total nitrogen (TN) 1.28 g kg⁻¹, available nitrogen (AN) 127.82 mg kg⁻¹, total potassium (TK) 16.19 g kg⁻¹, and available potassium (AK) 86.54 mg kg⁻¹.

2.2. Experimental Material and Design

Italian ryegrass (Lolium multiflorum Lam., cv. Tetila; Beijing Best Grass Industry Co., Ltd., Beijing, China), rye (Secale cereale L., cv. Wintergrazer 70; Huifa Animal Husbandry online store, Tianjing, China), and oat (Avena sativa L., cv. Monico; Beijing Best Grassland Co., Ltd., Beijing, China) were selected for the experiment, with sowing rates of 22.5 kg hm⁻², 112.5 kg hm⁻², and 180 kg hm⁻², respectively. These sowing rates align with optimal reference seeding rates known to yield the best comprehensive effects in actual field production.

The experimental area comprised 20 plots (12 m × 1.5 m), arranged in a completely randomized block design. Four treatments were applied: winter cropping Italian ryegrass (WI), winter cropping rye (WR), winter cropping oat (WO), and winter fallow treatment (CK), each with 3 replicates. All forages were sown in December 2021, following rotary tilling of each plot after the late rice harvest. No fertilizer was applied during the winter cropping phase. Forage was harvested twice, on 27 February and 30 March 2022, respectively. After the second harvest, roots were collected to determine root morphological traits. During sampling, sufficient, soil samples (0–20 cm soil layer) were collected using a five-point sampling method. These samples were thoroughly homogenized and then subdivided into equal portions to serve as individual biological replicate. A portion of the soil samples was air-dried to determine the proportion and stability of soil aggregates. Another portion was used to quantify soil physicochemical properties, cementing material contents, and pore characteristics. The remaining samples were immediately frozen at −80 °C for DNA extraction and AMF sequencing.

2.3. Determination of Root Morphological Traits

Fresh forage roots were scanned using an EPSON L4160 scanner (Seiko Epson Corporation, Shenzhen, China). The scanning images were imported into the Wanshen-LA-S root analysis software (version 2.6.5.1) for analysis. The following root traits were obtained: root length, root surface area, root fractal dimension, root mean diameter, total length and proportion of root (R1) with a diameter of <0.5 mm, and total length and proportion of root (R2) with a diameter of 0.5–2 mm.

2.4. Aggregate Fractionation Scheme

The fractionation of mechanically stability aggregates was determined using the dry sieving method, whereas water-stable aggregates was measured using the wet sieving method. The dry sieving method is primarily applied to analyze aggregate distribution in the natural state, while the wet sieving method simulates aggregate distribution under field conditions [31]. For dry sieving analysis, soil samples were gently broken along natural cracks and air-dried in a cool environment prior to separation. The samples were then passed through screening sieves of sizes 2 mm, 0.25 mm, and 0.053 mm to obtain four size classes of mechanically stable aggregates: large macroaggregates (>2 mm diameter), small macroaggregates (0.25–2 mm diameter), microaggregates (0.053–0.25 mm diameter), and mineral fractions (<0.053 mm diameter). Then the weight of each fraction was weighed, and its proportion relative to the total weight was calculated. For wet sieving analysis, a 50 g soil sample was prepared according to the proportions of each particle size obtained from dry sieving. The samples were subjected to wet sieving for 10 min at 30 cycles per minute with a 5 cm amplitude using a soil aggregate structure analyzer (LvBo LBF-100D, Guangzhou Taoyi Technology Co., Ltd., Hangzhou, China) equipped with a nested sieve assembly of 2 mm, 0.25 mm and 0.053 mm sieve assembly. The top sieve was kept submerged in the water throughout the process. All fractions were collected into beakers, dried in an oven at 70 °C, and weighed to calculate the percentage. Additionally, to measure physicochemical properties of aggregates, another portion of samples was sieved and subsequently air-dried under natural conditions to minimize thermal alteration of their chemical composition. All aggregate fractions provided sufficient soil material for subsequent analyses. For size classes with relatively low recovery, replicate sub-samples from the same homogenized soil plots were pooled to ensure that the minimum requirements for physicochemical analyses were met.

2.5. Determination of Soil Aggregate Stability Indices and Pore Characteristics

The stability indicators of soil aggregates were calculated using size-classes data obtained from the wet sieving method. The calculation formulas were as follows [32]:

(1)

Mean weight diameter (MWD, mm) and geometric mean diameter (GMD, mm):

$$\mathrm{MWD}=\sum _{\mathrm{i}=1}^{\mathrm{n}}{\overline{\mathrm{d}}}_{\mathrm{i}}{\mathrm{w}}_{\mathrm{i}}$$

(1)

$$\mathrm{GMD}=\exp \left[{\displaystyle \frac{\sum _{\mathrm{i}=1}^{\mathrm{n}}\left({\mathrm{w}}_{\mathrm{i}}\ln {\overline{\mathrm{d}}}_{\mathrm{i}}\right)}{\sum _{\mathrm{i}=1}^{\mathrm{n}}{\mathrm{w}}_{\mathrm{i}}}}\right]$$

(2)

where ${\overline{\mathrm{d}}}_{\mathrm{i}}$ indicates the mean diameter (mm) of each size fraction, ${\mathrm{w}}_{\mathrm{i}}$ indicates the weight percentage of the aggregates in each size fraction, and n denotes the number of sieves.

(2)

Water-stable aggregates > 0.25 mm (R_0.25) content:

$${\mathrm{R}}_{0.25}={\displaystyle \frac{{\mathrm{M}}_{\mathrm{d}0.25}}{{\mathrm{M}}_{\mathrm{T}}}}\times 100\%$$

(3)

where ${\mathrm{M}}_{\mathrm{d}0.25}$ indicates the weight (g) of water-stable aggregates with >0.25 mm size, ${\mathrm{M}}_{\mathrm{T}}$ indicates the total weight (g) of water-stable aggregates.

(3)

Percentage of aggregate destruction (PAD):

$$\mathrm{PAD}={\displaystyle \frac{{\mathrm{w}}_{\mathrm{x}0.25}-{\mathrm{w}}_{\mathrm{d}0.25}}{{\mathrm{w}}_{\mathrm{x}0.25}}}\times 100\%$$

(4)

where ${\mathrm{w}}_{\mathrm{x}0.25}$ indicates the weight (g) of mechanically stable aggregates with >0.25 mm size, ${\mathrm{M}}_{\mathrm{d}0.25}$ indicates the weight (g) of water-stable aggregates with >0.25 mm size.

(4)

Fractal dimension (Dm) was calculated using the formula proposed by Tyler and Wheatcraft [33]:

$$\left(3\text{-}\mathrm{Dm}\right)\lg \left[{\displaystyle \frac{{\mathrm{d}}_{\mathrm{i}}}{{\mathrm{d}}_{\max }}}\right]=\lg \left[{\displaystyle \frac{{\mathrm{M}}_{\mathrm{d} < {\mathrm{x}}_{\mathrm{i}}}}{{\mathrm{M}}_{\mathrm{T}}}}\right]$$

(5)

where ${\mathrm{d}}_{\mathrm{i}}$ indicates the mean diameter (mm) of aggregates within each size, d_max indicates the average diameter (mm) of the maximum particle size water-stable aggregates, M_{d < xi} is the total weight (g) of aggregates with particle size below classes i, M_T is the total weight (g) of all water-stable aggregates. A linear regression was performed Using $\lg \left[{\displaystyle \frac{{\mathrm{M}}_{\mathrm{d} < {\mathrm{x}}_{\mathrm{i}}}}{{\mathrm{M}}_{\mathrm{T}}}}\right]$ as the ordinate against $\lg \left[{\displaystyle \frac{{\mathrm{d}}_{\mathrm{i}}}{{\mathrm{d}}_{\max }}}\right]$ as the abscissa, then the slope of the fitted line corresponds to 3-Dm.

Soil pore characteristics were determined at the bulk soil level using the mercury intrusion method [34]. Liquid mercury at room temperature and pressure an external intrusion pressure to overcome surface tension and enter soil pores. This pressure directly determines the smallest pore diameter accessible to mercury: higher pressures enable entry into smaller pores, thereby controlling the volume of mercury intruded. Mercury injection curves and related data were obtained using a mercury injection instrument (AutoPore V 9600, Micromeritics Instrument Corporation, Norcross, GA, USA), at a pressure range of 0.5–33,000 psi, corresponding to a measurable pore diameter range of 350 μm to 5 nm.

Soil pores were classified as follow: large pores (>1000 nm), medium pores (100–1000 nm), and small pores (10–100 nm). The following parameters were calculated: soil porosity, bulk density, apparent density, average pore diameter, total pore area, mercury removal efficiency, total pore volume of each pore grade, and the proportion of pore volume.

$$\mathrm{d} =-{\displaystyle \frac{4\mathsf{\gamma }\cos \mathsf{\theta }}{\mathrm{p}}}$$

(6)

where d represents the pore diameter (μm), γ denotes the surface tension coefficient of liquid mercury (0.485 N m⁻¹), θ (the contact angle between liquid mercury entering and the sample) is 130°, and p indicates the mercury intrusion pressure (MPa).

2.6. Determination of Soil Properties

Soil pH was determined by a PHS-3C pH meter (INESA Scientific Instrument Co., Ltd., Shanghai, China) with fresh soil suspensions prepared at a 1: 2.5 (w/v) soil-to-water ratio. SOM was determined by combustion using a CNS elemental analyzer (CNS-2000, LECO Corporation, St. Joseph, MI, USA) [34]. TN was measured using the Kjeldahl digestion method [35]. AN was measured by the alkaline hydrolysis diffusion method with 1 M NaOH [35]. TK and AK were determined using a flame spectrophotometer method [36]. TP was measured after digesting 0.5 g soil with concentrated H₂SO₄-HClO₄, and AP was extracted from soil with 0.5 M NaHCO₃ solution (pH 8.5) at a 1: 20 (w/v) ratio for 30 min, respectively. Both TP and AP contents were measured by molybdenum-blue colorimetry [36].

2.7. Determination of Soil-Cementing Materials’ Contents

The contents of soil-cementing materials were measured in different winter forage treatment groups at the bulk soil level. Soil polysaccharide (Pol) content was determined according to the method described by Rimada and Abraham [37]. Briefly, 0.05 g soil was added to 1 mL water and heated in a water bath at 100 °C for 2 h. After cooling, the mixture was centrifuged with a centrifuge at 10,000× g for 10 min. Subsequently, 0.2 mL supernatant was collected and slowly mixed with 0.8 mL anhydrous ethanol. The mixture was vortex-mixed thoroughly using a vortex mixer (Vortex-Genie 2, Scientific Industries, Inc., Bohemia, NY, USA) and incubated at 4 °C overnight. Following incubation, 1 mL water was added to thoroughly dissolve the precipitate. Next, 200 µL supernatant was transferred and mixed with 100 µL phenol solution and 0.5 mL concentrated sulfuric acid. The mixture was vortex-mixed and heated in a water bath at 90 °C for 20 min. After heating, 200 µL of the solution was cooled under running water and transferred to an enzyme-labeled plate to measure absorbance at 490 nm. Finally, the Pol content was calculated based on the absorbance values using an appropriate calibration curve.

Soil humic acid (HA) and fulvic acid (FA) were isolated and measured according to the method described by Zhang et al. [38]. Briefly, soil samples were treated with 0.1 M Na₄P₂O₇ and 0.1 M NaOH to obtain the alkaline extract and alkali-insoluble solid residue. Alkaline extracts were then separated into acid-insoluble HA and acid-soluble FA using 0.5 M H₂SO₄. The alkali-insoluble solid residue (soil humus) was determined by the K₂CrO₇-H₂SO₄ digestion method. HA and FA contents were measured with a TOC-VCSH analyzer (Shimadzu Corporation, Kyoto, Japan).

Free iron oxide (FIO) and free aluminum (FAO) oxide were extracted according to the method reported by Lu [35]. Briefly, 0.3 g air-dried soil was placed in a 50 mL centrifuge tube with 20 mL 0.3 M sodium citrate and 2.5 mL 1 M sodium bicarbonate were added. When the water bath reached 80 °C, 0.5 g sodium dithionite was added with constant stirring for 15 min. After cooling and centrifugation, the supernatant was transferred into a 250 mL volumetric flask. Residual material was rinsed into the flask with 1 M NaCl. Amorphous iron oxide (AIO) and amorphous aluminum oxide (AAO) were extracted using the ammonium oxalate method [35]. Here, 0.5 g air-dried soil was placed into a 50 mL centrifuge tube with 25 mL 0.2 M ammonium oxalate. After 2 h of shaking with a orbital shaker (Neo 230, Labwit Scientific, Shenzhen, China) in the dark, samples were centrifuged at 4000 rpm, and the supernatant was collected and diluted 5 times. Complexed iron oxide (CIO) and complexed aluminum oxide (CAO) were extracted by adding 3.0 g air-dried soil with 60 mL 0.1 M sodium pyrophosphate solution for 2 h, followed by centrifugation. Iron and aluminum contents in all extracts were determined using an Inductively Coupled Plasma Optical Emission Spectrometer (Agilent 5110, Agilent Technologies, Santa Clara, CA, USA).

2.8. DNA Extraction and Soil AMF Sequencing

Soil DNA was extracted from bulk soil samples stored at −80 °C using the E.Z.N.A.^® Soil Kit (Omega Bio-tek, Norcross, GA, USA). DNA concentration and purity were assessed using a NanoDrop2000 UV-vis spectrophotometer (Thermo Scientific, Wilmington, NC, USA), and the quality of the DNA extraction was evaluated by 1% agarose gel electrophoresis. The primer pairs AML1 (5′-ATCAACTTTCGATGGTAGGATAGA-3′)/AML2 (5′-GAACCCAAACACTTTGGTTTCC-3′) and AMV4-5NF (5′-AAGCTCGTAGTTGAATTTCG-3′)/AMDGR (5′-CCCAACTATCCCTATTAATCAT-3′) were used to amplify the fungal 18S rDNA gene via nested PCR. The amplified fragment length was about 800 bp and 300 bp, respectively. The PCR products were excised from a 2% agarose gel, purified using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union, CA, USA), and quantified using QuantiFluor™-ST (Promega Corporation, Madison, WI, USA). Purified amplicons were sequenced on the Illumina MiSeq platform [39].

2.9. Statistical Analysis

All data were organized in Excel 2019. One-way analysis of variance (ANOVA) was performed in SPSS 26.0 (IBM, Armonk, NY, USA), followed by post hoc tests using LSD’s test at the 5% confidence level. Graphs were generated using Origin version 2021 (OriginLab Inc., Northampton, MA, USA). Principal component analysis (PCA) was conducted to differentiate the composition of the AMF community using the “vegan” packages in R version 4.3.3. Redundancy analysis (RDA) was performed using the “vegan” packages in R version 4.3.3. Spearman correlation analysis was conducted using R version 4.3.3. Partial mantel test was executed using the “linkET” package in R version 4.3.3. Partial least squares path model (PLS-PM) was constructed using the “plspm” package in R version 4.3.3 to explore the effects of both biotic and abiotic factors on soil aggregate distribution and stability.

3. Results

3.1. Distribution and Stability of Soil Aggregates

The proportion of mechanically stable aggregates in the four treatments followed a consistent trend: large macroaggregates > small macroaggregates > microaggregates > mineral fractions (Figure 1). The proportion of large macroaggregates ranged from 61.1% to 70.6%, significantly higher than that of other size classes in all treatments (p < 0.05). In contrast, mineral fractions accounted for the smallest proportion (0.6–1.4%). Compared to the CK, the proportions of microaggregates in the WI treatment were significantly increased by 30.6%, while the proportions of microaggregates and mineral fractions in the WO treatment were significantly increased by 60.7% and 119.0%, respectively (p < 0.05).

For water-stable aggregates, the CK showed a proportion trend of small macroaggregates > large macroaggregates > microaggregates > mineral fractions, whereas the WI, WR, and WO treatments followed the order: large macroaggregates > small macroaggregates > microaggregates > mineral fractions (Figure 1). Compared to the CK, WI and WR treatments significantly increased the proportions of water-stable large macroaggregates by 45.7% and 41.5%, respectively, while significantly decreased the proportions of water-stable small macroaggregates (p < 0.05). Additionally, the proportions of water-stable microaggregates and mineral fractions in the WO treatment were significantly increased by 39.9% and 82.7%, respectively, compared to the CK (p < 0.05).

Winter cropping forage significantly affected soil aggregate stability (

Table 1. Effect of winter cropping forage on soil aggregate stability.

Treatment	Mean Weight Diameter (mm)	Geometric Mean Diameter (mm)	Percentage of Aggregates Destruction (%)	Content of Water-Stable Aggregates > 0.25 mm (%)	Fractal Dimension
CK	2.41 ± 0.12 b	1.07 ± 0.05 b	12.24 ± 1.71 b	77.23 ± 1.12 a	2.36 ± 0.03 b
WI	3.14 ± 0.06 a	1.48 ± 0.06 a	6.31 ± 1.44 b	80.21 ± 1.51 a	2.34 ± 0.05 b
WR	3.06 ± 0.09 a	1.42 ± 0.11 a	7.06 ± 1.22 b	79.37 ± 2.74 a	2.37 ± 0.04 b
WO	2.39 ± 0.05 b	0.81 ± 0.07 c	19.48 ± 3.87 a	65.36 ± 2.08 b	2.51 ± 0.05 a

Note: CK: winter fallow; WI: winter cropping Italian ryegrass; WR: winter cropping rye; WO: winter cropping oat. Error bars show standard errors (n = 3). Different lowercase letters indicate significant difference (p < 0.05) among treatments.

). The MWD in the WI and WR treatments was 30.0% and 27.0% greater than that in the CK, respectively (p < 0.05). The GMD was significantly higher in the WI (1.5 mm) and WR (1.4 mm) treatments compared to the CK, but significantly lower in the WO treatment (p < 0.05). The WO treatment exhibited significantly higher PAD and Dm values, but a lower R_0.25 compared to the CK (p < 0.05).

3.2. Soil Chemical Properties

The chemical properties of soil aggregates in different treatments are showed in Table S1. Within each treatment, the contents of SOM, TN, TP, AN, AK, and AP were lowest in microaggregates and highest in mineral fractions. In the WR treatment, the SOM and TN contents in mineral fractions were 28.5 g kg⁻¹ and 1.6 g kg⁻¹, respectively, significantly higher than those in the CK (p < 0.05). The AP content in small macroaggregates in the WR treatment and in mineral fractions in both the WI and WR treatments was significantly higher than that in the WO treatment (p < 0.05). Compared to the CK, the WI, WR, and WO treatments exhibited a slight reduction in TK content in soil aggregates. Soil pH remained stable across different sizes and treatments.

3.3. Root Traits of Forage Grasses

The three forage species studied were grasses, sharing generally similar fibrous root system morphologies (Figure S1). The root traits of different forage grasses are shown in

Table 2. Root trait characteristics of different forage.

Treatment	Root Length (cm)	Root Surface Area (cm2)	Fractal Dimension of Root System	Average Root Diameter (mm)	Total Length of R1 (cm)	Proportion of R1 (%)	Total Length of R2 (cm)	Proportion of R2 (%)
I	539.51 ± 18.6 b	130.12 ± 12.46 b	1.61 ± 0.01 a	0.83 ± 0.08 a	187.67 ± 7.88 b	34.96 ± 2.61 b	335.17 ± 20.77 a	62.01 ± 1.73 a
R	1021.51 ± 114.82 a	184.7 ± 18.79 a	1.6 ± 0.03 a	0.6 ± 0.01 b	586.23 ± 68.97 a	57.31 ± 0.34 a	415.07 ± 44.35 a	40.69 ± 0.3 b
O	341.38 ± 9.59 b	60.53 ± 4.87 c	1.48 ± 0.02 b	0.63 ± 0.06 ab	213.73 ± 25.27 b	62.3 ± 5.63 a	117.4 ± 13.85 b	34.66 ± 4.96 b

Note: I: Italian ryegrass; R: rye; O: oat. Error bars show standard errors (n = 3). Different lowercase letters indicate significant difference (p < 0.05) among treatments.

. Rye exhibited the longest total root length (1021.5 cm), which was significantly greater than that of Italian ryegrass and oat (p < 0.05). Rye had a 41.9% larger root surface area than Italian ryegrass, while Italian ryegrass had a 115.0% larger root surface area than oat (p < 0.05). The fractal dimension of the forage root system was 8.7% higher in Italian ryegrass and 8.2% higher in rye compared with oat. The average root diameter of Italian ryegrass (0.8 mm) was significantly greater than that of rye (0.6 mm). Rye had a significantly higher total length of R1 roots than both Italian ryegrass and oat. Similarly, the total length of R2 roots was significantly greater in Italian ryegrass and rye than in oat (p < 0.05). The proportion of R1 roots was higher in rye and oat than in Italian ryegrass, whereas the proportion of R2 roots was lower (p < 0.05).

3.4. Soil Pore Characteristics

Higher increments in mercury intrusion were observed within the pore size diameter range of 77–151 nm, 226–283 nm, and 2052 nm across all treatments, suggesting a larger pore volume in these ranges (Figure S2). Total pore volumes for macropore, mesopore and micropore were 0.074–0.104 mL g⁻¹, 0.080–0.097 mL g⁻¹, and 0.054–0.061 mL g⁻¹, respectively (Figure S2). Compared with the other treatments, the WR treatment had the highest pore volumes across all pore sizes. Except in the CK, total macropore and mesopore volumes in the WI, WR, and WO treatments were significantly higher than those of micropore (p < 0.05) (Figure S2). The WO treatment exhibited the highest macropore proportion (40.89%). However, in all treatments, the proportions of macropore and mesopore were significantly higher than that of micropore (p < 0.05) (Figure S2).

The effects of winter cropping forages on soil pore characteristics are shown in Table S2. Compared to the CK, the WI and WR treatments significantly increased soil porosity (Po) by 15.7% and 21.7%, respectively (p < 0.05). Similarly, the WR treatment significantly decreased soil bulk density (BD) while increased total pore area (p < 0.05).

3.5. Soil-Cementing Material Contents

Winter cropping forage influenced the contents of soil-cementing materials (Figure 2). Soil HA contents were 2.1–2.5 g kg⁻¹, while FA contents were 0.9–1.5 g kg⁻¹. Compared to the CK, the WR treatment had a higher HA content (2.5 g kg⁻¹), and the WI treatment exhibited a significantly higher FA content (2.5 g kg⁻¹) (p < 0.05). The WI and WR treatments significantly increased total contents of FA and HA by 16.7% and 8.9%, respectively (p < 0.05).

Compared to the CK, the content of CIO in the WI treatment was significantly decreased, while it was significantly increased in the WR and WO treatments (p < 0.05). Compared to the other treatments, the WO treatment showed a significantly higher FIO content of 15.8 g kg⁻¹ (p < 0.05). The total contents of iron oxide in the WI and WO treatments were 6.4% and 11.2% higher than those in the CK, respectively (p < 0.05). In comparison with the CK, the WI and WR treatment significantly increased AIO contents by 24.8% and 14.3%, but decreased AAO contents by 46.1% and 34.4%, respectively (p < 0.05). Moreover, the WI treatment had the lowest total aluminum oxide content of 3.1 g kg⁻¹ (p < 0.05).

3.6. AMF Community Diversity and Composition

The diversity of AMF community in different treatments is shown in Figure 3. The α diversity indices (measured by the Shannon index) were similar in the WI and WO treatments, whereas the WR treatment exhibited a significant 14.6% increase compared with the CK (p < 0.05) (Figure 3a). Principal component analysis (PCA), based on Bray–Curtis distances, revealed significant differences in AMF community structure among the treatments. Winter cropping forage treatments shared similar AMF community structures, which distinctly differed from the CK (Figure 3b).

At the genus level, AMF community composition significantly differed among the CK, WI, WR, and WO treatments (Figure 3c). The dominant genus in the CK was Glomus, while Claroideoglomus was the dominant genus in the other treatments. Winter cropping forage treatments significantly altered soil AMF community composition. The relative abundances of Claroideoglomus, Paraglomus, and Acaulospora were significantly increased. The WI, WR, and WO treatments exhibited higher relative abundances of Claroideoglomus (55.3%, 51.3%, and 43.5%, respectively), compared to the CK (9.2%). In contrast, Glomus had the highest relative abundance (53.9%) in the CK, compared with the other treatments.

3.7. Correlations Between Soil-Cementing Material and Soil Aggregate Characteristic

RDA was conducted to investigate the correlations between soil-cementing materials, AMF communities, and soil aggregate characteristics (Figure 4a). The first and second axes explained 34.57% and 33.32% of the total variance in aggregate proportions of different particle sizes, respectively. The contents of HA+FA, CAO, Acaulospora, and Glomus and the Shannon index had the greatest impacts on soil aggregate proportions (p < 0.05). For soil aggregate stability, the first and second axes together explained 93.27% of the total variance. AAO content exerted the greatest impact on soil aggregate stability, followed by AIO content, total aluminum oxide content, and Claroideoglomus (p < 0.05).

A Mantel test was used to reveal the correlations among pairwise distance matrices: soil aggregate distribution and stability distances based on Bray–Curtis distance, and soil-cementing material and AMF community distances based on Euclidean distance (Figure 4b). The contents of Pol, HA + FA, and CAO were significantly positively correlated with soil aggregate distribution, while FIO and total iron oxide contents were significantly positively correlated with aggregate stability (p < 0.05). The relative abundances of Acaulospora and Glomus, as well as Shannon index, were significantly positively correlated with soil aggregate distribution (p < 0.05), while there was no significant correlation between the AMF community and soil aggregate stability.

PLS-PM was conducted to elucidate causal relationships among winter cropping forage, soil physicochemical properties, soil-cementing materials, AMF communities, soil aggregate distribution, and soil aggregate stability (Figure S3). The PLS-PM explained 81% and 63% of the total variance in soil aggregate distribution and stability during winter cropping forage, respectively. Winter cropping forage exhibited a direct negative effect (p < 0.05) on soil physicochemical properties, but a direct positive effect on the AMF community (p < 0.05). The AMF community exhibited a strong positive effect on soil aggregate distribution (p < 0.01). These results suggest that winter cropping forage can improve soil aggregate distribution primarily by regulating the soil AMF community.

4. Discussion

4.1. Winter Cropping Forage Improves Soil Physicochemical Properties

Soil aggregates are considered the basic unit of soil structure, reflecting soil fertility and structural stability [1,40]. The physicochemical properties of aggregate with different particle sizes vary considerably due to the differences in cementing materials, microbial communities, and aggregate biomass [5,41]. Nutrient contents peaked in mineral fractions, consistent with the findings of Han et al. [4], where soil total carbon, TN, and SOM contents also peaked alongside microbial diversity, functional gene abundance, and enzyme activity in aggregates of the same particle size. Winter cropping forage significantly increased SOM and TN content, with the WR treatment having a positive effect on soil AP. Compared to monoculture, crop rotation improved SOM and TN content across aggregate sizes, likely due to increased soil microbial activity stimulated by crop diversity [42]. Additionally, organic acids from root exudates stimulate the activation and release of soil nutrients through a series of biochemical reactions [43].

The pores within or between soil aggregates form the pore system, a critical indicator of soil physical structure. Pores within aggregates are generally thinner and more tortuous, which retars the flow of water and nutrients [44]. In addition to the structural composition of aggregates, their packing arrangement also influences soil pores. This can reduce inter-aggregate spaces, slowing gas flow, and impeding root penetration [45]. Higher soil porosity and total pore area observed after winter cropping forage suggest an enhancement in soil physical properties. The observed increases in soil porosity and total pore area may be driven by an increased proportion of water-stable macroaggregates, which could enhance the proportion of pores. The total pore volumes of fractions with diameters <9 μm and 9–1000 μm increased with the increasing proportion of water-stable macroaggregates, whereas mechanical stability aggregates with diameter >1 mm primarily influenced pore size distributions, rather than overall soil porosity [10]. Macroaggregate fragmentation may also reduce soil porosity [45,46]. Therefore, soil aggregate distributions resulting from different winter cropping forage systems are closely related to soil pore characteristics, which also alter porosity and connectivity in complex ways.

4.2. Winter Cropping Forage Promotes Soil Aggregate Structure

The stability and quantity of soil aggregates significantly impact soil properties and nutrient storage. A higher stability and macroaggregate content are key indicators of high-quality soil structure, which helps preserves SOM and enhances soil fertility [9,47]. Compared to winter fallow fields, winter cropping forages increased the proportion of water-stable large macroaggregates and reduced small macroaggregates in this study. This confirms that winter cropping forage alters the composition of water-stable aggregates. Specifically, winter cropping forage promoted the proportion of water-stable large macroaggregates in farmland soils. An increased proportion of macroaggregates helps reduce nutrient loss associated with microaggregate retention [48].

Legumes–grass rotations tend to improve soil structure and increase the proportion of water-stable aggregates [25,28]. Significantly higher MWD and GMD in the WI and WR treatments indicate that winter cropping with Italian ryegrass and rye enhances soil stability against erosion and tillage. Similar enhancements in aggregate stability have been observed after planting alfalfa forage [49,50]. The enhanced resistance to tillage likely results from increased organic matter, which buffers aggregate disruption during tillage via forage root systems. Additionally, the complex root networks of green forages strengthen aggregate formation and stability through binding mechanisms [51]. Plant root exudates, including organic compounds (e.g., polysaccharides and humic acids) that easily bind with soil particles, further enhance aggregate stability by facilitating water-stable aggregate formation [43,52]. Notably, the WO treatment showed a negative effect on soil aggregate stability, indicating that crop type also affects soil aggregates stability [52].

4.3. Soil-Cementing Materials Regulate Aggregate Distribution and Stability

As a critical component of plants, root system strongly influences the composition and structure of soil aggregates through network connections, root-soil adhesion, and root biochemical processes [53]. It is worth noting that the extent of this influence largely depends on root morphology [29,54]. Well-developed root systems play a central role in physically interweaving and binding soil aggregates, thereby exerting a stronger rhizosphere effect and significantly enhancing aggregate stability [29]. Our results revealed that Italian ryegrass and rye possess superior root systems, characterized by greater root length, surface area, and fractal dimension, which strengthen their rhizosphere effects and thereby significantly enhance soil aggregate stability. On the one hand, their fine fibrous roots substantially increase the root–soil contact interface, promoting both the physical entanglement of aggregates and the localized secretion of organic cementing substances [43,52]. Furthermore, their thicker and longer roots provide additional symbiotic sites for AMF hyphae, which unite soil particles via physical entanglement [3,55].

Polysaccharides, originating from plant roots and microbial secretions, typically constitute 50–70% of plant root exudates [16]. Our findings indicate a modest effect of different winter cropping forages on soil polysaccharide content, which may be attributed to variations in root morphology and secretion characteristics among forage species. In contrast, elevated levels of FA, HA, and certain iron oxides partially explain the enhanced soil aggregate stability observed in winter cropping forage systems. Humic substances (including FA and HA) are primarily supplemented through the decomposition of plant residues [56,57]. As primary cementing materials in red soil, iron and aluminum oxides play vital roles in soil aggregate formation and are closely associated with plant root exudates [58,59]. For example, citric acid in exudates facilitates iron/aluminum oxide formation, while oxalic acid dissolves these oxides and thereby leading to soil aggregate fragmentation [60]. Variations in the specific surface areas and functional group structures of these oxides result in differing cementation abilities [61]. The significant positive correlation between aggregate distribution and CAO content supports the idea that aluminum oxide contributes more substantially to aggregate formation than iron oxide. Notably, AIO exhibit stronger cementation than FIO, due to the abundant surface hydroxyl groups that facilitate binding with clay minerals [62]. This partly explains the positive correlations between AIO content and water-stable large macroaggregates in winter cropping forages. Furthermore, aggregate stability likely relies on iron oxides, as indicated by positive correlations with FIO and TIO contents. Iron oxides with large specific surface areas significantly contribute to the formation and stabilization of small-size aggregates [10], by firmly binding soil particles and providing adsorption sites for organic matter, thereby enhancing aggregate stability [63].

4.4. Winter Cropping Forage Impacts Soil Aggregates by Improving the AMF Community

In this study, the AMF community was a stronger determinant of soil particle aggregation than soil-cementing materials and forage physiology. The principal role of AMF in soil structure is well-established, with AMF colonization significantly increasing macroaggregate content [64]. Fungal hyphae (especially AMF-derived hypha) act as a “sticky string bag”, which is essential for macroaggregate formation [65]. Hyphal entanglement and adhesive secretions (e.g., polysaccharides, glomalin) physically bind soil particles and microaggregates [65]. Additionally, AMF promote root exudate production, which further binds soil particles, stabilizing macroaggregates [15]. Saprophytic fungi contribute aggregate-forming substrates through degradation of plant-derived organic matter [66]. AMF also indirectly stimulates organic matter decomposition and extracellular enzyme abundance by promoting saprophytic fungi growth, providing a potential mechanism for AMF-mediated soil aggregation [67]. Furthermore, the effects of AMF hyphae on soil organic carbon distribution and aggregate stability are moderated by soil fauna, such as nematodes [68].

The influence of AMF community on soil aggregates follows a species-dependent manner. The diversity and composition of the AMF community vary with host plant species [69,70]. In this study, winter cropping forage increased the Shannon index of AMF community, potentially due to sufficient colonization sites on well-developed forage roots [71]. Different AMF species differentially affect soil aggregation, mediated by variations in extraradical hyphae, organic aggregating materials, hyphal length, and hyphal spread rate [72,73]. A higher relative abundance of Claroideoglomus in winter cropping forage treatments improved soil aggregate stability. The promotion of soil structure and aggregate stability likely results from Claroideoglomus’ faster hyphal spread rate [74]. Hyphal spread rate determines AMF-mediated soil aggregate formation, enabling hyphae to extend farther from the roots to form more water-stable aggregates and bind more soil particles [75]. Glomus also promotes soil aggregate distribution, as its longer hyphae more effectively reorganize soil particles into stable aggregates [73,76]. The dominant relative abundances of Glomus and Claroideoglomus also drive their aggregate-forming influence, potentially reflecting high environmental adaptability [77].

However, host plant species decisively influence the impact of AMF on soil aggregation, given the variation in AMF communities across different host plants [73,78]. Plant root morphology and exudate diversity affect AMF recognition and infection, thereby shaping AMF community composition and diversity [79]. Additionally, plant roots alter AMF community structure and diversity by modifying soil physical properties [80]. The varying nutrient and resource requirements among plant species during development further influence symbiotic AMF domestication [81,82]. For example, polypeptide small molecules may facilitate Acaulospora mycelium growth during legume–rhizobia symbiosis [83], while flavonoids in ryegrass root exudates potentially drive Glomus prevalence after winter planting [84]. AMF-plant interactions modulate AMF functionality, with AMF effects on aggregate formation and stability varying according to hyphal length and morphology [69,70]. Some studies confirm that AMF-host species combinations differentially regulate water-stable soil aggregate distribution [72,73,85]. In this study, the enhancement of the AMF community associated with various forage grass demonstrates the potential for winter cropping to improve soil aggregates. Therefore, further investigations into precisely establishing distinct AMF communities through winter cropping forage are promising for improving soil structure and stability.

5. Conclusions

In summary, winter cropping forage significantly improved soil structure and stability by increasing the proportion of water-stable large macroaggregates, soil porosity, total pore area, MWD, and GMD of aggregates. These improvements were closely correlated with enhanced formation of soil-cementing materials. Distinct AMF communities established by different winter cropping forages were positively associated with soil aggregate proportion and stability, with dominant AMF genera showing significant positive correlations with aggregate formation. Winter cropping forage stimulates improvements in soil aggregate distribution and stability through modifying AMF communities in a forage-species dependent manner, with the highest proportion of water-stable large macroaggregates and MWD and GMD values achieved by Italian ryegrass and rye. Therefore, integrating Italian ryegrass and rye as winter forage crops is recommended not only in Southern China but also in other regions with comparable humid subtropical and mild temperate climates, such as Southeast Asia, southern Japan, the southeastern United States, and parts of southern Europe and eastern Australia. Incorporating these forages into annual crop rotations offers a promising sustainable strategy for enhancing soil quality, reducing erosion, and strengthening ecosystem resilience in cropping systems globally.