Alfalfa Plant Age (3 to 8 Years) Affects Soil Physicochemical Properties and Rhizosphere Microbial Communities in Saline–Alkaline Soil

Abstract

Increasing soil salinization can severely restrict local agricultural production. Planting alfalfa is considered an effective measure to ameliorate saline–alkali soil. However, it remains unclear how alfalfa planting years affect the sustained impact on soil and rhizosphere microecology. This study analyzed the effects of alfalfa planted 3, 6, and 8 years ago on soil physicochemical properties and key soil enzyme activities and investigated the rhizosphere microbial community structure and diversity. The results indicate that cultivating alfalfa plants for six years can improve soil physicochemical properties and enhance soil fertility to a certain extent. This is attributed to a higher abundance of plant growth-promoting bacteria, such as Bradyrhizobium and Allorhizobium, as well as degradation bacteria, such as Flavobacterium, Stenotrophomonas, Brevundimonas, and Massilia, in the rhizosphere of alfalfa plants. These microorganisms promote alfalfa growth, improve soil quality, and inhibit the accumulation of autotoxins. This not only maintains high alfalfa yields but also optimizes soil physicochemical properties and enzyme activity, facilitating more effective nutrient cycling and metabolic processes in the soil. However, extending plant growth to 8 years is not beneficial.

1. Introduction

Soil salinization is an increasingly severe global environmental issue and a primary factor contributing to land degradation and constraints on agricultural production [1,2]. Over one billion hectares of land worldwide are affected by primary salinization, with approximately 94 million hectares impacted by secondary salinization [3,4]. The accumulation of soluble salts on the soil surface leads to poor physical and structural characteristics, resulting in reduced soil aeration and permeability. Consequently, this can lead to decreased soil enzyme activity and a reduction in soil fertility. Currently, the total area of saline–alkaline including secondary affected soils in China has accumulated to 350 million hectares [5], with approximately 13 million hectares of saline–alkaline soils holding the potential for agricultural production [6]. Further improvement, development, and utilization of these saline–alkaline land resources could significantly contribute to the sustainable development of agriculture and animal husbandry in China. In recent years, numerous studies have revealed that halophytic and salt-tolerant plants, due to their ability to overcome the adverse effects of saline–alkaline stress through specific adaptive mechanisms, exhibit superior performance in soil reclamation and amelioration of saline–alkaline soils [7,8]. Among these, various types of forage grasses with differing salt tolerance have garnered significant attention. They can improve the physicochemical properties and structure of the soil, reduce surface moisture evaporation, enhance aeration and water retention; and by direct absorption of salts, the plants maintain soil desalination and improve the salt balance, thereby contributing to the amelioration and reclamation of salt-affected grasslands [9,10].

Alfalfa is a perennial leguminous forage and is one of the most important and widely cultivated forages worldwide [11]. In terms of planting area, China is the second largest producer of alfalfa in the world, with production mainly concentrated in arid and saline–alkaline inland areas in northern China, such as Gansu and Inner Mongolia [12]. Alfalfa is known for its high yield and high nutritional quality, and its nutritional value makes it a high-quality choice for animal feed, ensuring high economic benefits [13]. Additionally, alfalfa demonstrates moderate salt and alkali tolerance, enabling growth in saline–alkaline soils. Importantly, it contributes to local ecological enhancement, soil structure improvement, and increased soil fertility [14]. However, it is worth noting that the age of alfalfa plants has an impact on their productivity. It has been described that prolonged continuous cultivation (beyond 4 years) results in a gradual decline in productivity [15]. This decline is characterized by a decrease in root vitality, a year-on-year reduction in forage yield, and an increasing incidence of root rot [16]. Furthermore, the years of cultivation also influence soil moisture, other physicochemical properties, nutrient content, and soil enzyme activity [17]. The limitations related to plant age and culture duration conflict with its sustainable high yield and soil improvement capacity.

Soil microorganisms serve as vital biological indicators of soil quality and health, with the diversity and functionality of their community structure reflecting the ecological environment’s functional changes and its impact on the environment [18]. Studies have shown that continuous cultivation of alfalfa on the Loess Plateau for over 10 years resulted in a significant decline in alfalfa yield and led to severe land degradation [19]. This phenomenon is attributed to the accumulation of allelopathic substances in the rhizosphere soil, leading to an increase in harmful microorganisms and a reduction in beneficial members. Consequently, this imbalance disrupts the microbial community structure and causes ecological degradation of the soil [20]. In general, the root exudates of perennial plants affect the quantity, structure, and function of soil microorganisms [21]. With increasing years of cultivation, both plant yield and rhizosphere microbial diversity significantly decrease [22]. For instance, as highlighted by Zhao et al. (2018), continuous coffee cultivation reduces the potential beneficial microorganisms in the soil while increasing the prevalence of pathogens, resulting in reduced yields and soil degradation [23]. Deep-rooting alfalfa plants also influence the population structure of soil microorganisms in the years following planting. It has been shown that, with an increase in alfalfa plant age, autotoxins gradually accumulate in the rhizosphere soil, leading to a gradual shift with more harmful and fewer beneficial microorganisms [24]. However, there is currently limited in-depth research on this effect, and there is a lack of data on the response of soil indicators to these microbial changes.

Maintaining sustainable yields and soil improvement is key to planting perennial alfalfa in saline–alkali soils. However, little is known about the impact of rhizosphere microorganisms on soil fertility related to different planting years. Given the important role of alfalfa cultivation in the utilization and improvement of saline–alkali land, understanding the changes in rhizosphere microorganisms, soil physicochemical properties, and soil enzyme activity of alfalfa planted in saline–alkali land with increasing plant age is of great significance because it can clarify the dynamic response of alfalfa rhizosphere microorganisms and soil quality in response to planting practices. A comparative analysis of alfalfa planting strategies, including soil analysis and the isolation of beneficial microorganisms, is needed. Therefore, this study sequenced 16S rRNA amplicons of rhizosphere bacteria from alfalfa planted in saline–alkali soil 3, 6, and 8 years earlier and compared the soil physicochemical properties and soil enzyme activity of the plots. We focused on exploring (1) changes in the physicochemical properties and enzyme activity of the rhizosphere soil of alfalfa with increasing planting years; (2) changes in the diversity and species composition abundance of alfalfa rhizosphere soil microbial community, thereby identifying key differential bacteria and their functions in the alfalfa rhizosphere at different planting years; (3) the correlation between alfalfa rhizosphere microorganisms and soil physicochemical properties and soil enzyme activity. Our aim was to understand the evolution of soil quality over time in alfalfa planting areas and to study the effects of rhizosphere microorganisms of alfalfa at different planting ages on these parameters. Our research provides a theoretical reference for scientifically evaluating the production capacity of alfalfa in the saline–alkali areas of northern China, understanding its rhizosphere microecology, soil improvement effects, and reasonable rotation cycles.

2. Materials and Methods

2.1. Description of the Study Area

The experimental site was located in Tumet Left Banner (40°67′95.58″ N, 111°35′64.83″ E), Hohhot City, in the Inner Mongolia Autonomous Region of China (Figure 1). This location has an altitude of 1011 m, an annual average temperature of 8 °C with an average maximum temperature of 23 °C from July to August, a frost-free period of about 130 days, and an annual average rainfall of 346 mm. The moderate saline–alkaline soil had a pH of 8.6, and prior to the experiment, it contained 18.42 mg/kg available nitrogen (AN), 12.84 mg/kg available phosphorus (AP), 110.44 mg/kg available potassium (AK), and 9.22 mg/kg organic matter (OM). The salt content was 4.65 g/kg, and the electrical conductivity (EC) was 4.29 ds/m.

2.2. Experimental Design

The experiments were conducted with the locally commonly used alfalfa variety Medicago varia Martin ‘Caoyuan No.3′ (CY3) that was provided by Inner Mongolia Agricultural University. This variety was planted in the experimental area in 2015, 2017, and 2020, with a plot area of 18 m² and triplicate plots per planting year. During the growth period of alfalfa, field management, such as irrigation, fertilization, and harvesting, was carried out in accordance with common agricultural production practices.

In June 2023, when all plants were in the budding stage, we collected soil samples from rhizosphere soil that was closely attached to the surface of alfalfa roots using the method described by Edwards et al. [25]. For this, we randomly selected 9 evenly growing and healthy alfalfa plants from each plot. Their roots were laid bare with sterile shovels, and loose soil was manually removed while wearing sterile gloves, after which the rhizosphere soil attached to the root surface was carefully collected using sterile brushes. The rhizosphere soil of the 9 plants per plot was mixed to give a rhizosphere soil sample of the alfalfa in this plot. The treatment was the same for each experimental plot, giving three replicates per plant age. Bulk soil (BS) was taken as a control at a distance of 20 cm from the roots. Thus, a total of 12 soil samples were obtained. Subsequently, each soil sample was divided into two sub-samples. One of these was placed in a 5 mL sterile tube, rapidly frozen in liquid nitrogen, and immediately transported to the laboratory on dry ice. It was stored at −80 °C, and rhizosphere microbial DNA extraction was completed within 24 h [26]. The other part was placed in a plastic bag, transported to the laboratory at 4 °C, and stored at −20 °C for subsequent determination of soil physicochemical properties and enzyme activity.

2.3. Analytical Methods

2.3.1. Determination of Soil Physicochemical Properties and Soil Enzyme Activity

The soil physicochemical properties were determined with reference to the literature [27]. The pH was determined using an emulsion in water (water-to-soil ratio 2.5:1, g:g), and the EC with a water-to-soil ratio of 5:1. The heating potassium dichromate volumetric method was used for OM content, the Kjeldahl distillation method was applied for total nitrogen (TN), hydrofluoric acid-perchloric acid digestion and molybdenum antimony anticolorimetry was used for total phosphorus (TP), the flame photometric method produced total potassium (TK) levels, and the alkaline diffusion method was employed for available nitrogen (AN). The available phosphorus (AP) was extracted with 0.5 mol/L NaHCO₃ (water-to-soil ratio 20:1) and then determined using Mo-Sb anti-colorimetry, and available potassium (AK) was extracted with ammonium acetate and then determined using flame photometry.

The enzyme activities of α-glucosidase, β-glucosidase, β-xylosidase, cellobiosidase, urease, alkaline protease, dehydrogenase, alkaline phosphatase, and N-acetyl-β-D-glucosidase were determined with enzyme activity assays (Solarbio, Beijing, China, catalog numbers BC3085, BC0165, BC0165, BC4035, BC0125, BC0395, BC0280, BC4000, and BC4120, respectively) according to the instructions provided in the assay kit manuals.

2.3.2. DNA Extraction and Amplification

The collected soil was passed through a 2 mm sieve and total microbial genomic DNA was extracted from 0.5 g soil using an E.Z.N.A.^® soil DNA Kit (Omega Bio-tek, Norcross, GA, USA). After checking the concentration using NanoDrop2000 ultra-micro ultraviolet spectrophotometry and assessing the quality using visual inspection after 1% agarose gel electrophoresis, the DNA was used as a template to amplify the V3–V4 region of bacterial the 16S rRNA gene with universal primers (338F, 5′-ACTCCTACGGGAGGCAGCAG and 806R, 5′GGACTACHVGGGTWTCTAAT) using a published PCR protocol [28]. The amplicon of 468 bp was checked on a 2% agarose gel, excised, and purified using Axy Prep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, USA) and QuantiFluor TM-ST (Promega, Madison, WI, USA).

2.3.3. High-Throughput Sequencing and Raw Data Analysis

Equimolar and double-ended sequence reads were obtained on an Illumina MiSeq platform according to standard procedures at Biomarker Technologies Co., Ltd., Beijing, China. Raw sequence data were quality-controlled and spliced, low-quality reads were filtered out, and the obtained double-ended sequences were separated into tags. Low-quality tags and chimeras were removed to obtain high-quality data using standard procedures. The high-quality sequences were clustered using UPARSE (v7.1), and operational taxonomic units (OTUs) were called at a cutoff of 97% sequence similarity and taxonomically annotated based on the Silva16S rRNA database (v138) with a confidence threshold of 70%.

2.3.4. Statistical and Bioinformatics Analyses

Statistical analyses performed with SPSS software (v.26.0.0.2), included ANOVA analyses between different groups. Bar plots were produced with Prism 9.5 (GraphPad Software, LLC). Alpha diversity indexes were calculated using QIIME (v.1.9.1), and their differences were evaluated by t-tests. The Bray–Curtis distance was adopted to calculate the distance between samples, and ANOSIM was applied for principle coordinate analysis (PCoA). Linear discriminant analysis effect size (LefSe) was applied with a logarithmic LDA score of 3.0 to reveal statistical significance (p < 0.05), and the functional potential of the identified bacterial communities was predicted by PICRUSt2; these analyses were performed by use of the BMK Cloud platform (http://www.biocloud.net/, accessed on 10 September 2023).

3. Results

3.1. Effects of the Plant’s Age on Physical and Chemical Properties of Alfalfa Rhizosphere Soil

The rhizosphere soil collected from alfalfa plants of different ages (3, 6, and 8 years of cultivation) was analyzed for physical and chemical properties, and this was compared with control soil (BS) collected from a vegetation-free area of the same plots. The results are summarized in Figure 2. The pH of the soil was alkaline, ranging from 8.13 to 9.11. A significant difference was noted between the soils of the differently aged plants (p < 0.05), with the lowest pH determined in the rhizosphere soil of 6-year-old plants and the highest in plants planted 8 years earlier. The EC was the lowest (1.81 ds/m) in soil collected from plants that had grown for 3 years. The contents of TN and TP in the soil decreased with an increase in plant age and reached the lowest levels at 1.9 g/kg and 1.24 g/kg, respectively, in 8-year-old plants. There was no significant difference in TK content (p > 0.05). The contents of AP, AK, and OM were the highest at a plant age of 3 years (24.84 mg/kg, 112.56 mg/kg, and 43.27 mg/kg, respectively), and a decreasing trend was observed with plant age, with a significant difference between 3 and 8 years of age.

3.2. Effects of the Plant’s Age on Enzyme Activity in the Rhizosphere Soil

The age of the plants also resulted in differences in the enzyme activity of their rhizosphere soil (Figure 3). By far, the strongest effect was seen for α-glucosidase: its activity was highest in the soil of 3-year-old plants and strongly decreased with plant age. In contrast, β-glucosidase was slightly higher in soil associated with 6-year-old plants compared to 3-year-old and 8-year-old plants. Similarly, β-xylosidase was most active in the soil of 6-year-old plants, and here the difference was larger. Cellobiosidase activity was strikingly higher in the soil collected from the oldest plants than in the other two age groups. All these enzymes are involved in carbon cycling.

Of the enzymes involved in nitrogen cycling, the activity of N-acetyl-β-D-glucosidase, alkaline protease, and dehydrogenase was the highest in 6-year-old plants. Alkaline phosphatase, on the other hand, was least active in the soil of these plants. Last, urease only slightly varied and was least active in the soil collected from the oldest plants (Figure 2).

3.3. Effects of Plant Age on Microbial Community Characteristics in the Rhizosphere

3.3.1. Sequencing Reads Information, Data Statistics, and OTU Analysis

Amplicon sequencing of the bacterial 16S rRNA V3–V4 region resulted in a total of 958,186 high-quality sequences from the 12 soil samples. These sequences were clustered into 2608 bacterial OTUs at 97% sequence similarity. The number of obtained sequences reached saturation for all four sample types (Figure 4A). The total number of OTUs was considerably lower in the rhizosphere soil of the youngest plants (1119), while the other two rhizospheres produced more OTUs than the control. Thus, the number of bacterial OTUs in the alfalfa rhizosphere increased with the age of the plants, and the strongest increase was seen between the ages of 3 and 6 years. A Venn diagram (Figure 4B) illustrates that 274 OTUs (10.5% of the total) were shared by all samples. Each sample type contained unique OTUs, which accounted for 25.7% of the OTUs detected in the soil of 3-year-old plants (288 of 1119), 18.1% for the 6-year-old plants (254/1402), and 20.4% (288/1409) for the 8-year-old plants.

3.3.2. Alpha Diversity and PCoA Analysis

To ensure the effectiveness and accuracy of data analysis, all samples were rarefied to the minimum sequence count, which retained 37,324 high-quality sequences for subsequent analysis. The Shannon and Chao1 indices were used as metrics to compare microbial diversity and richness, respectively. No difference was found for these indices between the control and the soil associated with the roots of 3-year-old plants, but both the richness and diversity of the detected bacterial communities were higher in the soil associated with the roots of plants aged 6 years and 8 years (Figure 5A).

Based on Bray–Curtis distances in combination with ANOSIM tests, the community structure differences were visualized in a PCoA plot (Figure 5B). This revealed that, despite their similarity in richness and diversity index, the soil of BS and CY3T was quite different for the first principle component, and both were distant from CY3S and CY3E. This indicates a low similarity in species classification and richness among the samples of the different plots. In particular, CY3T was completely separated from the other two alfalfa soil types, and its three replicates were much closer together than the replicates of the other two soil types. The largest distance was observed between the bacterial community of plants planted 3 and 8 years ago, indicating that the bacterial community composition changed significantly during this time.

3.3.3. Bacterial Community Composition and Structure

Based on 97% similarity, the sequences could be attributed to 22 phyla, 47 classes, 115 orders, 204 families, and 368 genera. There were differences in the relative abundance of bacterial compositions among the analyzed rhizosphere soils of the different plant age treatments. The top three phyla with the highest relative abundance (r.a.) were Proteobacteria, Acidobacteriota, and Bacteroidota, but their fractions varied considerably between the samples (Figure 6A). We observed that in comparison with BS, the rhizosphere of alfalfa was enriched for Proteobacteria and Bacteroidota. However, with increasing plant age, the r.a. of these two phyla gradually decreased. In contrast, the alfalfa rhizosphere contained lower fractions of Acidobacteriota, Actinobacteriota, Gemmatimonadota, and Chloroflexi compared to the BS, especially in the case of 3-year-old plants, while the r.a. of these phyla increased with the plant’s age (Figure 5A).

The three most relative abundance genera were Sphingomonas, unclassified Vicinamibacterales, and unclassified_Gemmatimonadaceae and these three genera together accounted for 25% of the reads in the control but less than 20% in the alfalfa soils (Figure 6B). In particular, the unclassified Vicinamibacterales and unclassified Gemmatimonadaceae were relatively low in CY3T. The relative abundances of Sphingomonas, Flavobacterium, Luteimonas, Brevundimonas, Stenotrophomonas, and Massili were all higher in CY3T than in the other soil types. The r.a. of the top 30 genera were compared in a heatmap (Figure 6C), which further demonstrated the difference in the soil of BS and the alfalfa plots. In particular, the strongest differences (represented by the darkest colors) were noted between BS and CY3T, while over time, these differences became less striking (weaker colors indicative of smaller differences). Nevertheless, the microbial community of the soil of 8-year-old alfalfa plants was still quite distinct from that of vegetation-free soil.

ANOVA analysis was used to identify key genera that were significantly different in the rhizosphere of alfalfa of different ages. This identified an Acidobacteria genus (Acidobacteria_bacterium_SCN_69-37), Bradyrhizobium, Gaiella, and an unclassified_Blastocatellaceae, which were all most abundant in the soil of 6-year-old plants and very low or even undetectable in the soil of 8-year-old plants (Figure 7A).

Further, LEfSe analysis was performed to search for dominant bacterial genera in the rhizosphere of alfalfa at a particular age. This identified seven dominant bacterial genera that were overrepresented only in CY3T, including Chryseobacterium, Pantoea, and Acidovorax; six that were exclusively overrepresented in CY3S, including unclassified Blastocatellaceae, Bradyrhizobium, and Nordella, and six that were exclusively overrepresented in CY3E, including Nitrospira, unclassified_Subgroup-7 and Mesorhizobium (Figure 7B). Additionally, Picrust2 was adopted to predict possible functional differences of these alfalfa rhizosphere bacteria. A significant difference was found in carbon metabolism functions, which were more abundant in the CY3T rhizosphere bacteria compared with that of CY3E, whereas two-component systems were less abundant (Figure 7C).

3.4. Correlation Analysis of Soil Physical and Chemical Properties with Enzyme Activity and Microbial Community Composition

A correlation analysis was first performed to assess correlations between the determined enzyme activity in the soils and their physical and chemical properties (

Table 1. Spearman correlation analysis of soil physical and chemical properties with determined enzyme activity.

	pH	EC	TN	TP	TK	AN	AP	AK	OM
α-GC	−0.993 **	−0.913 **	0.992 **	0.978 **	0.837	−0.579	−0.802	0.078	0.864 *
β-GC	−0.993 **	−0.912 **	0.992 **	0.978 **	0.837	−0.579	−0.803	0.078	0.865 *
β-XYS	−0.968 *	−0.932 **	0.952 *	0.949 *	0.819	−0.512	−0.729	−0.006	0.776
CB	−0.975 *	−0.941 **	0.976 *	0.960 **	0.871	−0.537	−0.741	0.009	0.791
ALPT	−0.845	−0.839	0.849	0.726	0.654	−0.364	−0.635	−0.107	0.4
NAG	−0.944 *	−0.939 **	0.954 *	0.915 *	0.777	−0.482	−0.722	−0.018	0.649
ALP	−0.972 **	−0.938 **	0.973 **	0.940 **	0.797	−0.53	−0.733	0.015	0.826
UE	−0.997 **	−0.934 **	0.996 **	0.991 **	0.866	−0.609	−0.822	0.085	0.873 **
DHA	−0.986 **	−0.919 **	0.983 **	0.965 **	0.825	−0.555	−0.774	0.037	0.821

Abbreviations: α-GC: α-glucosidase, β-GC: β-glucosidase, β-XYS: β-xylosidase, CB: cellobiosidase, ALPT: alkaline protease, NAG: N-acetyl-β-D-glucosidase, ALP: alkaline phosphatase, UE: urease, DHA: dehydrogenase, EC: electric conductivity TN: total N, TP: total P, TK: total K, AN: available N, AP: available P, AK, available K, OM: organic matter. Note: Different significance levels of correlation analyses are marked with asterisks (*, p < 0.05 and **, p < 0.01).

). Strong negative correlations (p < 0.01) were identified between pH and activity of glucosidases, alkaline phosphatase, urease, and dehydrogenase. The other enzymes, except for protease, correlated negatively with pH at a lower significance (p < 0.05). EC strongly (p < 0.01) negatively correlated with all enzymes except for protease. TN strongly (p < 0.01) negatively correlated with both glucosidases, alkaline phosphatase, urease, and dehydrogenase, and at a lower significance (p < 0.05) with N-acetyl-β-D-glucosidase, β-xylosidase, and β-cellobiase. TP followed the same trend, although its correlation with β-cellobiase was stronger (p < 0.01). There was no significant correlation between TK, AN, AP, and AK with enzyme activity. The level of OM strongly (p < 0.01) positively correlated with urease activity and weaker (p < 0.05) with the two glucosidases (Table 1).

Correlations between these soil characteristics and the abundance of the top 20 genera identified in the microbial communities of the soil types were graphically captured in a heatmap (Figure 8A). No significant correlations were identified for soil pH or EC in alfalfa. However, rhizosphere bacteria significantly correlated with soil nutrients, and in particular, the abundance of Brevundimonas and Devosia positively correlated with all nutrient indicators. The abundance of Massillia and unclassified Comamonadaceae correlated with all nutrient indicators except for TN, while Dyadobacter and Stenotrophomonas correlated with all except for TK. The presence of Allorhizobium and Sphingobacterium correlated with TN and AN, contrasting with Sphingomonas, which correlated with TN, TK, and AK. Last, Flavobacterium correlated with TP.

A similar heatmap was constructed for the determined enzyme activities (Figure 8B). This identified multiple correlations for urease, which positively correlated with Massilia, unclassified Comamonadaceae, Brevundimonas, Devosia, Dyadobacter, and Stenotrophomonas, and negatively with unclassified Vicinamibacteralis. Dehydrogenase activity positively correlated with Pseudoxanthomonas and unclassified_Micrococcaceae and negatively with Sphingomonas. Last, β-glucosidase activity strongly positively correlated with unclassified_Micrococcaceae.

4. Discussion

4.1. Effects of Alfalfa Plant’s Age on Physical and Chemical Properties of Alkaline Soil

Planting alfalfa is beneficial for soil improvement, as it can enhance soil structure, its physicochemical properties, nutrient content, and enzyme activity [9,29,30,31]. However, this effect is not sustained over time. With the increasing age of alfalfa plants, yields decrease, and negative impacts on soil quality may also become apparent [32]. As previously reported, alfalfa reaches its peak yield in the third and fourth year of growth, after which it gradually declines [24]. Those authors concluded that the optimal utilization age of alfalfa should not exceed six years, as prolonged cultivation would reduce overall soil nutrient content [24]. Our research results confirm this. We observed that pH and EC values decreased after three and six years of cultivation, but they increased after eight years, especially in the case of pH, which even surpassed the levels in unplanted soil. This exacerbates the salinization and alkalization of the soil. It is noteworthy that alfalfa plants can, to some extent, reduce soil pH and EC, although this effect disappears after six years of growth. This is attributed to the nitrogen-fixing symbiosis between alfalfa and its rhizobia. Alfalfa can enhance the absorption and utilization of soil nutrients and effective nutrients by acidifying the soil, thereby reducing soil pH and salt content [33]. However, with an increase in the cultivation period, the interactive effect weakens, leading to a bouncing rise in soil pH and EC again. The variations in soil nutrient content determine the quality of the plant growth environment. The levels of soil N, P, K, and OM content are pivotal factors representing the soil nutrient status [34]. Research indicates that planting alfalfa in low-productivity saline–alkali soil can gradually increase those nutrient levels, particularly N, through symbiotic nitrogen fixation, thereby restoring soil health [35]. However, due to variations in nutrient consumption by alfalfa at different ages, the nutrient content, including N, in the soil undergoes significant changes over time following alfalfa planting [36]. In this study, the content of TN and AN exhibited significant fluctuation with increasing planting year, peaking in the 3rd and 6th year and significantly decreasing in the 8th year (Figure 2). This pattern can be attributed to the vigorous growth of alfalfa, leading to enhanced nitrogen-fixing ability and an increase in soil nitrogen content. However, with prolonged plant cultivation, the diminished nitrogen-fixing capacity of its rhizobia results in a reduction in soil nitrogen content [37]. Similar trends were observed for AP and AK. Furthermore, research indicated that the plant’s age can impact the content of surface litter in the soil. When alfalfa grows for 3–6 years, a substantial accumulation of surface litter occurs, leading to an increase in soil OM. This not only provides a favorable environment for microbial activity but also adds more C and N into the soil [38]. However, when the plants exceed an age of 6 years, the surface litter in the soil significantly decreases, resulting in a reduction in soil OM content and affecting the activity of rhizosphere soil microbes. In this experiment, the influence of surface litter on soil nutrient indicators was not investigated since the experimental site is located in a saline–alkali area with poor ecological conditions and significant wind and sand movements, making it challenging to collect surface litter in a timely manner and thus precluding that type of research in this area.

Soil enzyme activity reflects the biological activity and biochemical reaction intensity of the soil and serves as a crucial indicator to evaluate soil fertility, quality, and health [39]. For instance, soil urease reflects the ability of the soil to convert organic nitrogen into available nitrogen and provides a measure for the supply capacity of inorganic nitrogen [40]. Phosphatase, on the other hand, catalyzes the hydrolysis of soil phosphates to form inorganic phosphorus, and its activity is used to evaluate the soil phosphate status [41]. Our study reveals that younger alfalfa exhibits relatively higher rhizosphere soil enzyme activity, particularly urease and alkaline phosphatase associated with nitrogen cycling (Figure 3). The maximum root mass of this perennial forage grass is reached after 6 years of growth. The high vitality and root mass of 6-year-old alfalfa roots are correlated with soil enzyme activity, as root exudates are significantly correlated with soil enzyme activity [42,43]. An increase in root exudates enhances soil enzyme activity, leading to improved soil quality and nutrient availability. However, when the plants are older than 6 years, clear signs of land degradation appear, partly due to the weakened nitrogen-fixing ability of aging alfalfa. In addition, plants with extensive and deep root systems, such as alfalfa, can absorb nutrients from deeper soil profiles [44], depleting soil nutrient content over time. Additionally, the composition and quantity of rhizosphere exudate from older alfalfa undergo changes, leading to the secretion and accumulation of autotoxins, such as phenolic acids, into the rhizosphere [24]. This imbalance in soil nutrient proportions, combined with other factors, contributes to the observed reduction in soil enzyme activity and nutrient content in the 8-year-old alfalfa field.

4.2. Effects of Alfalfa Plant’s Age on Rhizosphere Microbial Community

The number of years of alfalfa plant cultivation can influence the diversity and community composition of their rhizosphere soil microbiota, a phenomenon corroborated in numerous studies where significant differences in the rhizosphere microbial communities of older plants compared to younger ones have been established [45,46]. While many studies suggest a continuous decline in plant rhizosphere microbial diversity with increasing plant age [24,47], our results produced a different outcome: we observed differences but not necessarily decreases in diversity in the rhizosphere microbial structure and composition over time. Interestingly, as the age of alfalfa increased, the richness and diversity of their rhizosphere bacteria also increased. This phenomenon has also been observed in long-term blueberry cultivation [48]. It may be attributed to the fact that with increasing age, plant roots not only need to resist the effects of salinity and alkalinity but also need to suppress the harm caused by pathogenic bacteria and fungi [49]. Therefore, more microorganisms are required to exert beneficial effects. Additionally, the influence of plants on their rhizosphere microorganisms depends on the types of root exudates produced by the plants, including sugars, amino acids, and organic acids. These substances vary with plant species or growth stages, thereby influencing the types and quantities of recruited microorganisms [24]. Previous studies have indicated that variations in the root exudates of plants at different ages can lead to changes in the composition of rhizosphere microbial communities [50]. With increasing plant age, differences in the composition, quantity, and quality of root exudates can alter the soil microenvironment, directly or indirectly influencing the composition of microbial communities [51]. In our study, we take into account that the long-term cultivation of the plants results in a greater variety and complexity of root exudates, expanding the enrichment range and intensity of the rhizosphere microbial community, thereby further enhancing microbial diversity.

The microbial community structure and relative species abundance differed significantly among the plants of different ages (Figure 6). With the increasing age of alfalfa, the relative fractions of Acidobacteriota, Actinobacteriota, and Chloroflexi were increasing at the expense of Proteobacteria and Bacteroidota (Figure 6A). Previous studies have suggested that Acidobacteriota and Actinobacteriota play a strong inhibitory role against abiotic stress and plant pathogens [52], while Chloroflexi participates in nitrogen and carbon cycling through nitrite oxidation, carbon fixation, fermentation, and sugar respiration [53]. This implies that older alfalfa is subjected to a greater extent of various stresses compared with younger alfalfa. Among the more common rhizosphere microorganisms Bacillus and Pseudomonas have been extensively studied because of their beneficial effects on plant growth [54]. Some members of these genera have been employed for biological control of crop diseases or regulation of soil microecological environments [55,56]. Our study did not observe a significant enrichment of these bacterial genera, but the observations may be influenced by the soil type [57]. During the 8 years of alfalfa cultivation, the relative abundance of bacteria involved in autotoxin degradation, such as Flavobacterium, Stenotrophomonas, Brevundimonas, and Massilia [58,59,60], notably decreased in the rhizosphere soil (Figure 6B). Degrading bacteria are known for their crucial ecological functions, breaking down and decomposing organic matter, promoting soil nutrient cycling, and maintaining soil ecological balance [61]. The reduction in the relative abundance of these bacteria may be a key factor contributing to the weakened ability of disease suppression in the rhizosphere after prolonged alfalfa cultivation and the development of obstacles associated with continuous cropping. Intensive saline–alkali stress significantly reduces the relative abundance of key bacteria, such as Gemmatimonas, Sphingomonas, and Bradyrhizobium, leading to a decreased utilization of carbohydrates and amino acid carbon sources [62], and we observed similar changes: in the rhizosphere of alfalfa cultivated for 3 and 6 years, the relative abundance of Bradyrhizobium and Allorhizobium was significantly higher than in that of 8-year-old plants. Especially in the rhizosphere of 6-year-old alfalfa, Gaiella and Acidobacterium were enriched (Figure 6C and Figure 7A), which is consistent with previously described results [24], and these genera have multiple beneficial ecological functions [63]. Bradyrhizobium, a diazotrophic soil bacterium, plays a crucial role in the formation of root nodules, ammonia production, and nitrogen-fixing symbiosis in leguminous plants [64]. The relative abundance of Bradyrhizobium varied significantly among the three different plant ages. In comparison, its relative abundance was highest in the rhizosphere of 6-year-old alfalfa plants, while it was absent after 8 years of cultivation (Figure 6B and Figure 7A). This may be a key factor contributing to the decline in yield and soil quality after 8 years of alfalfa cultivation. Additionally, the relative abundance of the bacterium RB41 gradually increased with plant age. Under long-term low-nutrient stress, acidophilic bacteria may play a vital role in maintaining soil metabolism and possibly degrade polymers of plant residues [65]. This could be another factor involved in sustaining the continuous growth of mature alfalfa. Therefore, with the increasing age of alfalfa plants, functionally relevant rhizosphere microorganisms may be assembled specifically to meet the functional requirements of mature plants, resulting in changes in community structure composition.

4.3. Relationship between Soil Microbial Community Diversity and Physicochemical Properties

Plant growth, soil physicochemical properties, enzyme activity, nutrient levels, and rhizosphere microorganisms are all closely interconnected, exerting significant mutual influences [66]. The composition of the microbial community and enzyme activity are regulated by the physicochemical properties of the soil (pH, EC) and by soil nutrients (the content of C, N, and P) and are associated with soil health, further impacting plant growth. Conversely, soil microorganisms can influence soil physicochemical properties and plant growth [24,48]. Our results similarly indicate a strong correlation between soil physicochemical properties and bacterial community structure. pH and EC exhibited a significant negative correlation with soil enzyme activity and the rhizosphere microorganisms of alfalfa, while N and P levels produced a significant positive correlation with soil enzyme activity and most rhizosphere bacteria (Figure 8). We have confirmed that cultivating alfalfa can effectively improve soil quality to a certain extent. Over a 6-year period of alfalfa cultivation, soil pH and EC decreased significantly, while AN and AP showed a noticeable increase (Figure 2). This improvement is attributed to the vigorous microbial activity in the alfalfa rhizosphere. During this period, OM content was higher (Figure 2), and OM is crucial for soil ecology as it provides nutrients for plants and microorganisms [67]. Root nodule bacteria such as Bradyrhizobium and Allorhizobium (Figure 6C) can form a symbiotic relationship with alfalfa roots, and by nitrogen fixation they increase the soil nitrogen content (Figure 2). Simultaneously, they enhance soil pH through acidification and influence the availability of other nutrients, thereby altering the effectiveness of soil nutrients [68]. However, beyond 6 years of cultivation, the soil tends to deteriorate, particularly evident in the observed increase in pH. Soil pH has been identified as a crucial factor influencing the bacterial community in agricultural soils [69]. Due to differences in microbial growth tolerance, the pH can directly impact the dominance of certain bacteria. For instance, members of Proteobacteria that are primarily involved in the decomposition of OM and the promotion of plant growth [70] generally exhibit a negative correlation with pH [71]. On the other hand, Actinobacteria and Chloroflexi tend to accumulate in large quantities when plants face intense stress, often serving as beneficial biocontrol agents with functions such as phosphate solubilization and the secretion of secondary metabolites for organic matter degradation [72]. Typically, these groups show a positive correlation with pH [73], aligning with our research findings. Moreover, the EC values of the soil also follow a similar trend, as salt and alkalinity often co-occur, exerting significant influences on soil microbial communities [74]. In addition, we observed that bacteria that are abundant during the early growth stages of the plants, such as growth-promoting Bradyrhizobium and Massilia, decline after 6 years of cultivation, disappearing by the 8th year (Figure 6C and Figure 7A). As a consequence, the suppletion of nutrients in the alfalfa rhizosphere, such as AN, AP, and AK, is lower than their utilization, leading to continuous depletion of soil nutrients (Figure 2). Simultaneously, this has a profound impact on the composition of the rhizosphere microbial community. More critically, the rhizosphere continuously accumulates complex autotoxins that cannot be decomposed, further contributing to the occurrence of continuous cropping obstacles and alfalfa diseases, such as root rot [24]. This has significant adverse effects on forage production and soil ecology.

4.4. Utilizing Alfalfa to Improve Saline Alkali Soil Requires Reasonable Planting and Management Measures

In northern China, extensive areas are characterized by saline–alkali soils with a pH generally exceeding 8. Moreover, these soils exhibit low levels of organic matter, total nitrogen, total phosphorus, and available nutrients, making them less suitable for agricultural development. Currently, China encourages the cultivation of forage crops rather than cereal crops in saline–alkali soils. Alfalfa, as a leguminous plant, possesses good salt and alkali tolerance and plays a crucial role in biological nitrogen fixation. It effectively increases soil nitrogen content, enhances soil fertility, and serves the dual purpose of soil improvement and providing high-quality forage, thus holding significant ecological and agricultural value. However, as demonstrated by our study, although alfalfa can improve soil quality to a certain extent, this positive effect diminishes with increasing plant age. After six years of growth, this beneficial impact weakens, and extended cultivation may even contribute to soil degradation. This limitation poses a challenge to utilizing alfalfa for soil reclamation. Therefore, a scientifically sound alfalfa cultivation management system is essential for balancing forage production and soil improvement, with due consideration to the optimal alfalfa planting duration being a key factor in this equation.

Crop rotation is a viable measure, as research indicates a significant decline in alfalfa yield when cultivated as a sole crop, starting from the sixth year. In contrast, the soil fertility index and alfalfa yield in a wheat–corn–alfalfa rotation regime are higher than those in alfalfa cultivation alone [75]. Furthermore, with the extension of the cultivation period, the levels of soil organic carbon and TN, as well as nutrients such as ammonium nitrogen, available phosphorus, and available potassium, show varying degrees of increase. This can markedly reduce nutrient loss in semi-arid soil [76]. Additionally, intercropping is another favorable measure. Intercropping with alfalfa can reduce soil pH and improve the functionality and diversity of its rhizosphere microbial community, ensuring growth while providing resistance to adversity and pathogens [77]. Moreover, in recent years, microbial fertilizers have emerged as a green approach with immense potential in promoting plant growth, soil improvement, and disease suppression [78,79]. Our study provides valuable targets for the isolation and cultivation of these functional microbial groups. In summary, based on our experimental results, continuous cultivation of alfalfa for six years in saline–alkali soil effectively improves soil physicochemical properties and enhances soil nutrient levels. Additionally, it is advisable to consider crop rotation or intercrop to mitigate the negative impacts associated with a monoculture of alfalfa beyond six years. Future efforts can focus on isolating and cultivating salt-tolerant rhizospheric plant growth-promoting bacteria, developing microbial fertilizers to enhance alfalfa productivity, and thereby achieving effective reclamation of saline–alkali soil.

5. Conclusions

This study revealed how alfalfa plants cultivated in saline–alkaline soil for a variable number of years since planting affected soil physicochemical properties and rhizosphere microbial communities. The soil pH value and electrical conductivity were higher for fields occupied by the oldest plants. The results indicated that culturing alfalfa plants for a period of 6 years was optimal for soil improvement. This leads to a higher abundance of beneficial bacteria in the rhizosphere of alfalfa cultivated for 6 years, such as Bradyrhizobium, Acidobacterium, and others. This can optimally improve soil physicochemical properties and enzyme activity and maintain higher metabolic capacity. Growing alfalfa for more than 6 years can alter the diversity and composition of soil microbial communities in an unfavorable way. Soil pH and EC values negatively correlated with most soil enzyme activities, and OM positively correlated with urease and glucosidase activity, indicating that the longer the plants grow, the higher the pH and EC values in the soil become, with a lower OM content and more serious levels of soil salinization. The results of this study provide a foundation for more optimal alfalfa cultivation in the saline–alkaline lands of Northern China, as it explored the relationship between alfalfa growth duration over the years and the presence of microorganisms in the soil. The results provide a scientific basis for further research on the relationship between the accumulation of effective components of alfalfa and its rhizosphere microbial environment and hints at control measures to avoid plant diseases and pests. In all, the study is conducive to promoting sustainable and economical cultivation of alfalfa in saline–alkaline soils.