Drought Resistance Physiological Responses of Alfalfa to Alternate Partial Root-Zone Drying Irrigation

Abstract

In arid agricultural production, exploring suitable water-saving irrigation strategies and analyzing their water-saving mechanisms are of great significance. Alternating partial root-zone drying irrigation (APRI), a water-saving strategy, enhances the water use efficiency (WUE) of alfalfa (Medicago sativa L.) This paper aims to clarify the physiological mechanisms by which the APRI method enhances the physiological WUE of alfalfa, as well as the differences between this water-saving irrigation strategy, conventional irrigation (CI), and their water deficit adjustments, in order to seek higher water use efficiency for alfalfa production in arid regions. In this experiment, alfalfa was used as the research subject, and three irrigation methods, CI, fixed partial root-zone drying (FPRI), and APRI, were set up, each paired with three decreasing moisture supply gradients of 90% water holding capacity (WHC) (W1), 70% WHC (W2), and 50% WHC (W3). Samples were taken and observed once after every three complete irrigation cycles. Through a comparative analysis of the growth status, leaf water status, antioxidant enzyme activity, and osmotic adjustment capabilities of alfalfa under different water supplies for the three irrigation strategies, the following conclusions were drawn: First, the APRI method, through artificially created periodic wet–dry cycles in the rhizosphere soil, provides pseudo-drought stress that enhances the osmotic adjustment capabilities and antioxidant enzyme activity of alfalfa leaves during the early to middle phases of irrigation treatment compared to CI and FPRI methods, resulting in healthier leaf water conditions. Secondly, the stronger drought tolerance and superior growth conditions of alfalfa under the APRI method due to reduced water availability are key factors in enhancing the water use efficiency of alfalfa under this strategy.

1. Introduction

Water resources are a critical input in global agricultural production, playing an irreplaceable role. The increasing demand for global crops and the expansion of farmland have placed unprecedented pressure on global water and land resources [1,2]. This has raised concerns about whether Earth’s finite water resources can sustain human activities [3]. Alfalfa is currently the most widely planted high-yield, high-quality perennial legume forage in the world, known for its high nutritional value, cutting tolerance, high yield, and strong adaptability [4,5]. With economic development leading to a diversified dietary structure, the increasing demand for quality forage and the escalating conflict between water use for production have continuously heightened the demand for water in artificial grasslands, exacerbating the scarcity of agricultural water resources [6]. Thus, it is imperative to develop water-efficient, water-saving production models in forage production. Among agricultural water-saving measures, enhancing crop water use efficiency through physiological regulation is considered one of the key strategies for achieving water-saving and yield-increasing in crops [7]. APRI is considered one of the most promising water-saving irrigation techniques currently available. It involves irrigating only part of a crop’s root system while leaving the other part in relatively dry soil. To prevent growth inhibition due to prolonged dry conditions in some roots, wet and dry zones are periodically alternated. This technique is derived from deficit irrigation theory and originated with the FPRI developed by Grimes et al. in 1968 in U.S. cotton fields, where irrigation water is applied only to part of the root system while the other part remains dry [8]. FPRI can effectively reduce irrigation quotas and frequency, improving water use efficiency without significantly reducing yield, making it suitable for crops like corn, sorghum, and cotton that are amenable to furrow irrigation. Additionally, the emergence of this technology has fostered the development of root–shoot communication theories, with some scholars revealing that root chemical signals can play a regulatory role when crops are under water stress [9,10]. As the theory of root–shoot communication matured, Davies and Zhang [11] first proposed a complete theory of root–shoot communication, in which a crop experiencing water stress first senses drought stimuli in its roots, producing chemical signals such as abscisic acid (ABA), which are transmitted to the leaves via xylem flow, causing partial stomatal closure and thus reducing stomatal conductance and transpiration to combat drought. In China, Kang Shaozhong et al. [12] first proposed a complete theory of alternate partial root-zone irrigation in 1997 based on the conclusions of Blackman et al., actively creating a dry–wet alternating soil environment in the crop root zone to stimulate the root production of ABA and reduce stomatal conductance and luxurious transpiration, thus enhancing water use efficiency as the main new irrigation method. Subsequently, scholars around the world have conducted extensive research on this strategy at the macro and micro levels, achieving remarkable water-saving effects with the APRI method in perennial woody plants, economically valuable high-water-consuming crops, and perennial herbaceous plants [13,14,15]. Irrigation methods have evolved from alternate furrow irrigation to various forms such as alternate partial root-zone drip irrigation, subsurface alternate partial root-zone drip irrigation, and micro-sprinkler irrigation. Meanwhile, researchers have studied APRI from various perspectives including crop photosynthetic physiology, reactive oxygen species scavenging systems [13], osmotic adjustment capabilities [16], root vitality [17], root hydraulic conductivity [18], and endogenous hormones [19], aiming to reveal the physiological mechanisms by which alternate partial root-zone irrigation improves crop water use efficiency. Alternating partial root-zone drying can reduce ineffective water evaporation by decreasing stomatal aperture in leaves and lowering the transpiration rate, thereby conserving moisture under stress conditions; it enhances drought tolerance in certain crops by increasing the activity of antioxidant enzymes; it also enhances the osmoregulatory substances in some crops, maintaining internal osmotic balance to improve water retention, thereby improving the water use efficiency of crops.

Through a two-year field experiment, we verified that alternate partial root-zone subsurface drip irrigation can enhance water use efficiency during the production of alfalfa by optimizing root morphology and distribution under conditions of full irrigation and mild water deficit, while maintaining stable productivity and nutritional quality of alfalfa over multiple years [20]. However, existing research does not fully explain the physiological responses of alfalfa to APRI and its water deficit strategies. Particularly, it is unclear whether the responses of alfalfa at different growth stages to this irrigation strategy and its water deficits are consistent, and whether these physiological responses have advantages when facing CI, FPRI, and their respective water deficit strategies. This study posits the following hypothesis: Under the APRI method and its associated water deficit compared to traditional irrigation models, the differential water distribution among alfalfa root zones induces pseudo-drought stress, eliciting more active physiological responses to drought. This controlled artificial drought stress interacts with deficit irrigation to achieve enhanced water use efficiency. Therefore, to clarify the physiological mechanisms behind the enhancement of water use efficiency in alfalfa through the APRI method and its water deficit management, as well as to compare its superiority to CI, FPRI, and their respective water deficit strategies, this study investigates the physiological responses of alfalfa at different growth stages to APRI, CI, FPRI, and various water conditions, analyzing the differences between them. This research aims to provide a data foundation for efficient water use under the APRI method, thereby seeking higher water use efficiency for the production of alfalfa in arid regions.

2. Materials and Methods

2.1. Experimental Design

The experiment adopted a two-factor randomized block design. Factor A was the irrigation method, with three types established: CI, FPRI, and APRI. Factor B was irrigation volume, with three levels of water supply, 90% of field capacity (W1), 70% of field capacity (W2), and 50% of field capacity (W3), as the maximum irrigation limits, totaling 9 treatments, each replicated three times. The experiment was conducted in the artificial climate chamber of the College of Grassland Science at Xinjiang Agricultural University from August to October 2023. The conditions were maintained at a constant temperature of 25 °C during the day and 14 °C at night, with a photoperiod of 16 h of light and 8 h of darkness. Humidity was held constant at 70%, and the light source was provided by LED strips with an intensity of 30,000 LUX.

2.2. Test Materials

The test material selected was “Xinmu No. 4” alfalfa (Xinjiang Agricultural University), cultivated in trays using vermiculite as the medium before the experiment. Once the alfalfa seedlings developed a true leaf, seedlings with multiple fibrous roots were transplanted into split-root containers (Figure 1). To ensure uniform growth of the purple alfalfa before irrigation treatment, 60 plants were transplanted. Out of these, 27 plants with uniform growth were selected as the experimental material. The split-root containers were made from PVC pipes and caps, forming the main body of the pot. A PVC divider split the container into two equal volumes, with a V-shaped notch in the center to evenly distribute and secure the alfalfa plant’s root system. Structural adhesive was used at all joints to seal and isolate moisture. The potting medium was a mixture of flower nutrient soil and vermiculite in a 2:1 ratio.

2.3. Irrigation Treatment

Before starting the irrigation treatment, 10 split-root containers were filled with equal volume and weights of dried soil medium and weighed to record the average weight (M₁). All pots were then saturated with water for 24 h and reweighed to record the average weight (M₂). The difference between the two was used to determine the weight of water required for 100% of WHC. When the water absorption of the substrate in the container reaches 100% WHC, the soil moisture content is approximately 40%. After the initiation of the irrigation treatment, all alfalfa potted plants requiring irrigation were weighed before each irrigation session. The average weight (M₃) of the pots treated with equivalent irrigation volumes was recorded. The irrigation quota for each session was calculated using the following formula:

(1)$\mathrm{Single} \mathrm{Irrigation} \mathrm{Quota}={(\mathrm{M}}_2-{\mathrm{M}}_1) \times \mathrm{Irrigation} \mathrm{Gradient} \mathrm{WHC}-({\mathrm{M}}_3-{\mathrm{M}}_1)$

After all alfalfa transplants were completed, from the seedling stage to the branching stage, all treatments were irrigated up to 90% of WHC to meet their water requirements. Once the alfalfa reached the branching stage, irrigation was stopped for all potted alfalfa until the moisture content dropped to 40% WHC, at which point irrigation treatments began. The irrigation cycle was established through a preliminary experiment, which involved recording the time it took for the substrate in pots of ten non-alfalfa plants to decrease from 90% WHC to below 50% WHC. The irrigation cycle was determined to be 6 days, during which the CI-treated alfalfa was uniformly watered on both sides of the pot, with only one irrigation per cycle; FPRI treatment involved watering only one side of the alfalfa’s root system twice per cycle; APRI treatment alternated watering on both sides of the alfalfa’s root system twice per cycle. For both FPRI and APRI treatments, the volume of water applied in each irrigation was half of the calculated irrigation quota prior to irrigation, with two irrigations per cycle to ensure consistency in the total volume of water applied across treatments. The total number of irrigation events and the maximum irrigation limits are shown in Table 1. Samples were taken, and relevant indicators measured every three irrigation cycles, until all treated potted alfalfa grew to the peak flowering stage, at which point the experiment was concluded.

2.4. Measurement Indicators and Methods

2.4.1. Growth Status of Alfalfa

Plant Height (cm) and Stem Thickness (mm): Plant height was measured as the vertical distance from the highest point of the plant to the top of the potting medium using a tape measure. Stem thickness was measured at the base of the stem using a caliper.

Above-ground Biomass (g): After all sampling was completed, the above-ground parts of the plants were cut, leaving a 3 cm stubble. Impurities were removed, and the fresh weight was measured. The samples were then placed in envelopes and dried in an oven at 65 °C until a constant weight was achieved. The dry-to-fresh weight ratio was calculated. The fresh weight of each sample was recorded, and the dry weight is calculated using this ratio. The total above-ground biomass was the sum of the dry weights of sampled and harvested biomass.

Below-ground Biomass (g): After the experiment ended, destructive sampling was conducted. The above-ground parts were cut off at the stem base, and the roots were placed on a mesh screen and washed with running water to remove impurities. The roots were then placed in envelopes and dried in an oven at 65 °C until a constant weight was achieved. The weight of the below-ground biomass for each treatment was recorded.

Water Use Efficiency (%): The WUE is calculated as the ratio of the sum of the dry weights of above-ground and below-ground biomass of alfalfa to the total volume of irrigation water used during the experimental period.

2.4.2. Moisture Status of Alfalfa Leaves

Relative Water Content of Leaves (RWC) (%): After the initiation of the irrigation treatment, every three complete irrigation cycles, on the day following the irrigation treatment, 2–3 functional leaves of similar position and growth from each treatment of alfalfa were selected. Fresh mass (M_f) was measured using an electronic balance. After soaking in distilled water for 8 h, the saturated mass (M_t) was determined. The leaves were then dried in an oven at 65 °C to a constant weight to measure the dry mass (M_d). The calculation formula is as follows [21]:

(2)$\mathrm{RWC}={\displaystyle \frac{({\mathrm{M}}_{\mathrm{f}}-{\mathrm{M}}_{\mathrm{d}})}{{(\mathrm{M}}_{\mathrm{t}}{-\mathrm{M}}_{\mathrm{d}})}}$

Leaf Water Saturation Deficit (WSD) (%): The water saturation deficit of alfalfa leaves was calculated using the relative water content of the leaves. The formula used is

(3)$\mathrm{WSD} (\%)=(1-{\displaystyle \frac{100}{\mathrm{RWC}}}) \times 100$

Relative Electrical Conductivity of Leaves (REC) (%): For each treatment, 0.5 g of healthy alfalfa leaves were taken and washed with deionized water to remove impurities. They were placed in a test tube, and 10 mL deionized water was added and then left at room temperature for 30 min. The electrical conductivity of the solution (C₁) was measured using a conductivity meter (DDS-307, Shanghai Boqu Instrument Co., Ltd., Shanghai, China). The test tube was placed in a boiling water bath for 10 min and cooled to room temperature, any evaporated water was replenished, and the electrical conductivity was measured again (C₂). The relative electrical conductivity of the alfalfa leaves was calculated using the following formula:

(4)$\mathrm{REC}(\%)={\displaystyle \frac{{\mathrm{C}}_1}{{\mathrm{C}}_2}} \times 100$

Photosynthetic Pigments in Leaves (mg·g⁻¹): After every three complete irrigation cycles, alfalfa leaves from similar positions were collected the day after irrigation. Leaves were cut away from the central veins and weighed (0.5 g). The leaves were extracted in 25 mL of 80% acetone solution at 25 °C in the dark for 16 h. The absorbance of the extract was measured at 645 nm and 663 nm using a spectrophotometer (T6, Persee, Beijing, China). The contents of chlorophyll a and chlorophyll b, as well as their ratio, were calculated.

2.4.3. Antioxidant Defense System of Alfalfa Leaves

Antioxidant Enzyme Activities: After every three complete irrigation cycles, healthy alfalfa leaves from similar positions on plants from each treatment were collected the day after irrigation, washed with deionized water, and dried. Leaves were then cut away from the central veins and weighed (0.5 g), placed in a mortar, and ground with 5 mL of phosphate buffer solution in an ice bath. The homogenate was centrifuged at 4 °C and 5000 rpm for 10 min, and the supernatant was collected. The activity of superoxide dismutase (SOD) (U·g⁻¹) in alfalfa leaves was determined using the nitroblue tetrazolium method. Peroxidase (POD) (U·g⁻¹) activity was measured using the guaiacol method, and catalase (CAT) (umol·min⁻¹·g⁻¹)activity was assessed using ultraviolet spectrophotometry [22,23,24].

Malondialdehyde (MDA) (nmol·g⁻¹): A total of 0.1 g of fresh leaves was ground in a mortar with 1.8 mL of 5% trichloroacetic acid in an ice bath to form a slurry and then centrifuged at 12,000 rpm for 20 min. The supernatant was collected, and the MDA content in alfalfa leaves was determined using the thiobarbituric acid reactive substances assay [25].

2.4.4. Regulation of Osmotic Adjustment Capacity in Alfalfa Leaves

After every three complete irrigation cycles, healthy alfalfa leaves from similar positions on plants from each treatment were collected the day after irrigation, washed with deionized water, and dried, and then, 0.5 g was cut, avoiding the central veins of the leaves. A total of 0.1 g of fresh leaves was placed in a test tube with 5 mL of 3% sulfosalicylic acid solution and extracted in a boiling water bath for 10 min, and after cooling to room temperature, the filtrate was analyzed for free proline (Pro) (μg·g⁻¹) content in alfalfa leaves using the acid ninhydrin method. Then, 0.1 g of fresh leaves was placed in a test tube with 12 mL of deionized water and boiled for 20 min, and after cooling and filtering, the supernatant was used to determine the soluble sugar (SS) (mg·g⁻¹) content in alfalfa leaves following the anthrone colorimetric method. Subsequently, 0.1 g of fresh leaves was ground to a powder in a mortar with liquid nitrogen, mixed with 40 mL of deionized water, and centrifuged at 5000 rpm for 10 min, and the supernatant was analyzed for soluble protein (SP) (mg·g⁻¹) content in alfalfa leaves using the Coomassie Brilliant Blue method. Next, 0.2 g of fresh leaves was fully ground in a mortar with 5 mL of deionized water, and the homogenate was transferred to a conical flask, extracted on a shaker for 24 h, and then centrifuged at 20 °C and 10,000 rpm for 20 min; the supernatant was analyzed for betaine (mg·g⁻¹) content in the alfalfa leaves using UV spectrophotometry [26,27,28].

2.5. Data Processing and Statistical Methods

All data were compiled using Microsoft Excel 2021 software. Two-way ANOVA was performed using IBM SPSS 26.0 statistical analysis software, with Duncan’s multiple range test for post hoc analysis. Significant differences were indicated at p < 0.05. Graphs were generated using Origin 2021.

3. Results

3.1. Growth Status of Alfalfa Under Different Irrigation Methods and Irrigation Volume

As shown in Figure 2, the interaction between irrigation methods and volume significantly influenced the initial height of alfalfa plants. Both irrigation method and volume significantly influenced alfalfa height in the early and middle stages of treatment. Early in the treatment (Figure 2a), alfalfa height significantly decreased with reduced irrigation, but mid-treatment (Figure 2b), alfalfa height under the APRI method was significantly higher than under FPRI and CI, while all irrigation methods showed a significant decrease in height with reduced water supply. Late in the treatment (Figure 2c), alfalfa height under the APRI method was significantly higher than under FPRI and CI and significantly decreased with reduced irrigation. Irrigation method and volume significantly affected stem thickness across treatments, though no significant interaction was observed. Early in the treatment (Figure 2d), alfalfa stem thickness under the CI irrigation strategy was significantly lower than other treatments, and in all strategies, stem thickness significantly decreased with reduced water supply. In the middle and late stages of treatment (Figure 2e,f), alfalfa stem thickness under the FPRI method was significantly lower than that for the other treatments, while under the APRI method, stem thickness showed no significant differences across irrigation gradients.

The effects of irrigation methods and volumes on the above-ground and below-ground biomass of alfalfa, as well as WUE, are shown in Figure 3. Alfalfa using the APRI irrigation strategy at the W1 water level had the highest above-ground and below-ground biomass, significantly higher than those under the same irrigation volume using the CI and FPRI methods. Across all irrigation methods, both the above-ground and below-ground biomass of alfalfa significantly decreased as water supply was reduced. The interaction between irrigation methods and irrigation volumes significantly affected the irrigation water use efficiency of alfalfa. Alfalfa irrigated using the APRI method exhibited the highest water use efficiency at the W1 level, significantly outperforming the CI and FPRI methods at the same irrigation volume. At the W2 level, the WUE of the FPRI and APRI methods showed no significant difference from the CI method but was significantly higher than that for the CI method at the W3 level. Under the APRI method, the WUE of alfalfa increased by 14.65% and 14.52% compared to those of the CI and FPRI methods, respectively.

Figure 3

The effects of irrigation method and volume on the above-ground and below-ground biomass, and WUE of alfalfa ((a) Above-ground biomass of alfalfa after irrigation treatment; (b) Below-ground biomass of alfalfa after irrigation treatment; (c) Total water use efficiency).

[Figure omitted. See PDF]

3.2. Water Status of Alfalfa Leaves Under Different Irrigation Methods and Irrigation Volume

The effects of irrigation methods and volume on the water status of alfalfa leaves are shown in Figure 4. The interaction between irrigation methods and volume significantly affected the relative water content of alfalfa leaves at each observation period. At the beginning of the irrigation treatment (Figure 4a), the relative water content of alfalfa leaves at the W1 level under the APRI and CI methods was significantly higher than that under FPRI method, while at the W3 level under the APRI and FPRI methods, it was significantly higher than that under the CI method. During the mid-treatment period (Figure 4b), the relative water content of alfalfa leaves significantly decreased under each irrigation method and at each irrigation level, with the highest relative water content at the W1 level under the APRI treatment, significantly higher than that at the same level under the CI and FPRI treatments; under the APRI treatment, the RWC of the leaves increased by 8.2% to 25.57% compared to that of the CI treatment. In the late phase of irrigation treatment (Figure 4c), the relative water content of alfalfa leaves at the W1 level under the APRI treatment was the highest, significantly higher than that at the same level under the CI and FPRI treatments; under the APRI treatment, the RWC of the leaves increased by 32.29% and 23.72% compared to those of the CI and FPRI treatments, respectively. The interaction between irrigation methods and volume significantly affected the deficit of leaf water saturation in alfalfa during each observation period. The trend of leaf water saturation deficit was consistent across different treatment periods, with a significant increase as water supply decreased under all three irrigation treatments. In the early stage of treatment (Figure 4d), the leaf water saturation deficit at the W1 level under the APRI treatment was the lowest, significantly lower than that at the same level under the FPRI treatment. Under the CI treatment, the leaf water saturation deficit at the W3 level was the highest, significantly higher than that at the same level under the FPRI and APRI treatments. During the mid and late stages of irrigation treatment (Figure 4e,f), the leaf water saturation deficit at the W1 level under the APRI treatment was the lowest, significantly lower than that at the same level under the FPRI and CI treatments.

Figure 4

The effects of irrigation method and volume on the relative water content of alfalfa leaves at different observation stages ((a,d) Irrigation treatment on day 18; (b,e) Irrigation treatment on day 36; (c,f) Irrigation treatment on day 54).

[Figure omitted. See PDF]

The effects of irrigation methods and volume on the relative electrical conductivity of alfalfa leaves are shown in Figure 5. Both irrigation methods and volume significantly affected the relative electrical conductivity of leaves in the initial treatment period, but there was no significant interaction between them. The interaction between the two significantly affected the relative electrical conductivity of leaves during the mid and late treatment periods. In the initial phase of treatment, under the CI and FPRI irrigation methods, the relative electrical conductivity of leaves significantly increased as water supply decreased at each level. In the APRI irrigation method, there was no significant difference in relative electrical conductivity between the W1 and W2 levels, but it significantly increased at the W3 level. Under the APRI treatment, the relative electrical conductivity of leaves was significantly lower than under the CI and FPRI irrigation methods. During the mid and late stages of treatment, the trend in relative electrical conductivity of leaves was consistent, significantly increasing as the water supply decreased under each irrigation method. During these two observation periods, under the ARRI treatment, the relative electrical conductivity of leaves at the W1 and W3 levels was significantly lower than under the CI and FPRI treatments. At the W2 level, there was no significant difference in relative electrical conductivity between FPRI and APRI, but both were significantly lower than under the CI treatment.

Figure 5

The effects of irrigation method and volume on the relative electrical conductivity of alfalfa leaves at different observation stages ((a) Irrigation treatment on day 18; (b) Irrigation treatment on day 36; (c) Irrigation treatment on day 54).

[Figure omitted. See PDF]

The effects of irrigation methods and volume on the MDA content in alfalfa leaves are shown in Figure 6. Irrigation methods significantly affected the MDA content in leaves during the initial phase of treatment. Irrigation volume significantly affected the MDA content in leaves during the initial and late phases of treatment. The interaction between them significantly affected the MDA content in the leaves during the mid-phase of treatment. In the initial phase of irrigation treatment, under the CI and APRI methods, the MDA content in the leaves at the W3 level significantly increased as water supply decreased. Under the CI method, MDA accumulation in the leaves was significantly higher than under the FPRI and APRI methods. Under the FPRI method, there was no significant difference in MDA content between the different irrigation levels. During the mid-phase of irrigation treatment, only under the CI method at the W3 level was MDA accumulation significantly higher than that in other treatments. There were no significant differences in MDA content between the levels under other irrigation methods. In the late phase of irrigation treatment, the MDA accumulation in the leaves at the W3 level was the highest under all irrigation methods, significantly higher than that in other irrigation levels. However, there were no significant differences in MDA content between the different irrigation methods at the same level.

Figure 6

The effects of irrigation method and volume on MDA content in alfalfa leaves at different observation stages ((a) Irrigation treatment on day 18; (b) Irrigation treatment on day 36; (c) Irrigation treatment on day 54).

[Figure omitted. See PDF]

The effect of irrigation methods and volume on the chlorophyll content and the ratio of chlorophyll a to b in alfalfa leaves is shown in Figure 7. The interaction between irrigation methods and volume had a significant effect on the chlorophyll content of alfalfa leaves in the early stage of treatment. Irrigation method and volume significantly affected the chlorophyll content of alfalfa leaves during the mid and late stages of irrigation treatment. In the early stage of irrigation treatment (Figure 7a), there was no significant difference in the chlorophyll content of the alfalfa leaves between the water supply gradients under APRI and CI treatments, but both were significantly higher at the W1 level compared to under the FPRI treatment. During the mid-stage of irrigation treatment (Figure 7b), the chlorophyll content under the APRI and CI treatments was significantly higher than under FPRI. In the late stage of irrigation treatment (Figure 7c), the chlorophyll content under the APRI and FPRI treatments was significantly higher than under the CI treatment. The chlorophyll content of alfalfa leaves at each observation period significantly decreased with the reduction in water supply under each irrigation method. Irrigation method and volume had a significant effect on the ratio of chlorophyll a to b in the early and mid-stages of irrigation treatment. In the early stage of irrigation treatment (Figure 7d), the ratio of chlorophyll a to b under the APRI method was significantly higher than under CI and FPRI; during the mid-stage of treatment (Figure 7e), the chlorophyll a/b ratio under the APRI treatment was significantly lower than under the CI and FPRI treatments.

Figure 7

The effects of irrigation method and volume on the chlorophyll content in alfalfa leaves at different observation stages ((a,d) Irrigation treatment on day 18; (b,e) Irrigation treatment on day 36; (c,f) Irrigation treatment on day 54).

[Figure omitted. See PDF]

3.3. Antioxidant Enzyme Activity of Alfalfa Leaves Under Different Irrigation Methods and Irrigation Volume

The effects of irrigation methods and volume on the antioxidant enzyme activity in alfalfa leaves are shown in Figure 8, Figure 9 and Figure 10. Irrigation methods and volume separately had significant effects on SOD enzyme activity in the leaves during the initial and mid phases of treatment. The interaction between the two significantly affected SOD enzyme activity in the late phase of treatment. In the initial and mid phases of treatment, SOD enzyme activity in the leaves under the APRI irrigation method was significantly higher than under the CI and FPRI methods; under the APRI method, the SOD enzyme activity in leaves increased by 18.88% and 14.67% compared to CI and FPRI, respectively, in the first period, and by 12.19% and 30.51% in the second period, and it significantly increased with reduced water supply under all irrigation methods. In the late phase of treatment, SOD enzyme activity in the leaves under the CI method was significantly higher at all irrigation levels compared to the same levels under the FPRI and APRI methods. Under the FPRI method, there was no significant difference in SOD enzyme activity across different water supply levels, while under the other two methods, it significantly increased as the water supply decreased.

Figure 8

The effects of irrigation method and volume on the SOD activity in alfalfa leaves at different observation stages ((a) Irrigation treatment on day 18; (b) Irrigation treatment on day 36; (c) Irrigation treatment on day 54).

[Figure omitted. See PDF]

Figure 9

The effects of irrigation method and volume on POD activity in alfalfa leaves at different observation stages ((a) Irrigation treatment on day 18; (b) Irrigation treatment on day 36; (c) Irrigation treatment on day 54).

[Figure omitted. See PDF]

Figure 10

The effects of irrigation method and volume on CAT activity in alfalfa leaves at different observation stages ((a) Irrigation treatment on day 18; (b) Irrigation treatment on day 36; (c) Irrigation treatment on day 54).

[Figure omitted. See PDF]

Irrigation volume significantly affected POD enzyme activity in alfalfa leaves at the initial stage of treatment, irrigation strategy significantly affected it during the mid-phase, and their interaction significantly affected it in the late phase of treatment. At the initial stage of treatment, there was no significant difference in POD enzyme activity in the leaves under the three irrigation methods at water levels W1 and W2, but it significantly increased at water level W3. During the mid-phase, POD enzyme activity in the leaves under the APRI treatment was significantly higher than under the CI and FPRI treatments; relative to CI and FPRI, there was an increase of 16.25% and 46.16%, respectively. In the late phase, there was no significant difference in POD enzyme activity in the leaves under the CI and FPRI irrigation methods at water levels W1 and W2, but it was significantly lower than at water level W3. Under the APRI irrigation method, POD enzyme activity significantly increased at all water levels.

Irrigation methods and volume separately had significant effects on CAT enzyme activity in alfalfa leaves at the initial stage of treatment. The interaction between them significantly affected CAT enzyme activity during the mid and late stages of treatment. At the initial stage of treatment, CAT enzyme activity in the leaves under the APRI irrigation method was significantly higher than under the CI and FPRI methods; compared to CI and FPRI, there was an increase of 24.59% and 11.51%, respectively. Under the CI and FPRI methods, there was no significant difference in CAT enzyme activity across the different water supply gradients. During the mid-phase, CAT enzyme activity in the leaves under the CI and FPRI methods significantly increased as the water supply decreased. Under the APRI method, CAT enzyme activity at water level W2 was not significantly different from W1 and was significantly lower than at W3. At water level W1, CAT enzyme activity under APRI was significantly higher than the same level under the CI and FPRI methods.

3.4. The Osmotic Regulation Capacity of Alfalfa Leaves Under Different Irrigation Methods and Irrigation Volume

The effects of irrigation methods and volume on osmoregulatory substances in alfalfa leaves are shown in Figure 11, Figure 12, Figure 13 and Figure 14. These factors significantly affected the Pro content in the leaves during the early and middle phases of treatment, and their interaction significantly affected Pro content in the late phase. At the initial stage of treatment, Pro content in the leaves under APRI and FPRI irrigation strategies was significantly higher than under the CI method; compared to CI, there was an increase of 11.03% and 13.79%, respectively. Under the CI irrigation method, Pro content in the leaves significantly increased as water decreased. There was no significant difference in Pro content among the different irrigation levels under the FPRI and APRI methods. During the mid-phase of irrigation treatment, Pro content in the leaves under the APRI method was significantly higher than under the CI and FPRI methods, increasing by 10.29% and 20.35%, respectively. In the late phase, there was no significant difference in Pro content in the leaves under the APRI and CI methods across different irrigation levels, while Pro content in the leaves under the FPRI method was significantly lower than under CI and APRI at all irrigation levels.

Figure 11

The effects of irrigation method and volume on Pro content in alfalfa leaves at different observation stages ((a) Irrigation treatment on day 18; (b) Irrigation treatment on day 36; (c) Irrigation treatment on day 54).

[Figure omitted. See PDF]

Figure 12

The effects of irrigation method and volume on SS content in alfalfa leaves at different observation stages ((a) Irrigation treatment on day 18; (b) Irrigation treatment on day 36; (c) Irrigation treatment on day 54).

[Figure omitted. See PDF]

Figure 13

The effects of irrigation method and volume on SP content in alfalfa leaves at different observation stages ((a) Irrigation treatment on day 18; (b) Irrigation treatment on day 36; (c) Irrigation treatment on day 54).

[Figure omitted. See PDF]

Figure 14

The effects of irrigation method and volume on betaine content in alfalfa leaves at different observation stages ((a) Irrigation treatment on day 18; (b) Irrigation treatment on day 36; (c) Irrigation treatment on day 54).

[Figure omitted. See PDF]

The interaction between irrigation methods and volume significantly affected SS content in alfalfa leaves during the mid and late phases of treatment. Irrigation volume significantly affected SS content in the leaves during the initial phase of treatment. At the initial stage of treatment, there was no significant difference in SS content in the leaves between different irrigation levels under the CI and APRI methods. Under the FPRI method, SS content significantly increased as water supply decreased. During the mid-phase, SS content in the leaves under the APRI method at the W1 level was significantly higher than under the CI and FPRI methods. At the W2 water level, SS content under FPRI was significantly lower than under CI and APRI. At the W3 level, there was no significant difference in SS content among the irrigation methods. In the late phase, SS content in the leaves under all irrigation methods showed an increasing trend as water supply decreased. SS content under FPRI at all irrigation levels was significantly lower than under CI and APRI at the same levels.

Irrigation method significantly affected SP content in alfalfa leaves during the initial phase. Both irrigation method and volume significantly affected SP content during the mid-phase, and their interaction significantly affected SP content in the late phase. In the initial phase, SP content in the leaves under the FPRI irrigation method was significantly higher than under the CI and APRI methods. During the mid-phase, SP content in the leaves under the APRI method was significantly higher than under the CI and FPRI methods, increasing by 16.46% and 45.88%, respectively. Under CI and FPRI methods, SP content significantly increased across all water levels. Under APRI, there was no significant difference in SP content at the W1 and W2 levels, but it significantly increased at the W3 level. In the late phase, SP content in the leaves under the FPRI method at all irrigation levels was significantly lower than under the CI and APRI methods. There was no significant difference in SP content between the CI and APRI methods across different irrigation levels.

Irrigation method and volume significantly affected the betaine content in alfalfa leaves during the initial phase. The interaction between them significantly affected betaine content during the mid and late phases. In the initial phase, betaine content in the leaves under the APRI method was significantly higher than under the CI and FPRI methods, increasing by 30.02% and 30.46%, respectively. During the mid-phase, betaine content in the leaves under APRI at the W1 level was significantly higher than under CI and FPRI. At the W2 and W3 levels, there was no significant difference in betaine content between CI and APRI, but both were significantly higher than FPRI at the same levels. In the late phase, betaine content in the leaves under APRI at the W1 level was significantly lower than under CI and at W3 level was significantly higher than under CI. Under FPRI, betaine content at all levels was significantly lower than under CI and APRI.

3.5. Correlation Analysis Between Various Indicators, Principal Component Analysis, and Polynomial Regression Fitting

The results of the Pearson correlation analysis, presented in Figure 15, reveal the relationships between various growth and leaf water conditions of alfalfa under different irrigation methods and volume, and the activity of antioxidant enzymes and osmoregulatory substances in alfalfa leaves. During the three different irrigation treatment periods, the activity of antioxidant enzymes and the levels of osmoregulatory substances exhibited a negative correlation with plant height, stem thickness, RWC of leaves, total chlorophyll content, the ratio of chlorophyll a to b, above-ground biomass, and below-ground biomass. Conversely, these parameters showed a positive correlation with leaf water saturation deficit and relative electrical conductivity.

The principal component analysis was conducted on 17 indicators including plant height, stem thickness, above-ground biomass, below-ground biomass, relative water content of leaves, leaf water saturation deficit, relative electrical conductivity, chlorophyll content, chlorophyll a to b ratio, MDA, SOD enzyme activity in leaves, POD enzyme activity in leaves, CAT enzyme activity in leaves, Pro content in leaves, SS content in leaves, SP content in leaves, and Betaine content in leaves under different irrigation methods and volume (Figure 16). Based on eigenvalues greater than 1, two principal components were selected, with variance contributions of 66.06% and 17.93%, respectively, and a cumulative contribution rate of 83.99%, representing the majority of information about alfalfa growth status, leaf water condition, antioxidant enzyme activity, and osmoregulatory substances in leaves. The functional expressions for each principal component were constructed based on their characteristic vectors and used as coefficients for each factor as follows:

F₁ = 0.262X₁ + 0.149X₂ + 0.269X₃ − 0.269X₄ − 0.273X₅ + 0.274X₆ + 0.236X₇ − 0.255X₈ + 0.219X₉(5)

+ 0.229X₁₀ + 0.258X₁₁ + 0.251X₁₂ + 0.236X₁₃ + 0.261X₁₄ + 0.193X₁₅ + 0.236X₁₆ + 0.216X₁₇

F₂ = 0.203X₁ + 0.296X₂ + 0.212X₃ − 0.212X₄ − 0.153X₅ + 0.186X₆ + 0.135X₇−0.162X₈ + 0.304X₉(6)

+ 0.295X₁₀ + 0.242X₁₁−0.271X₁₂ + 0.27X₁₃ + 0.223X₁₄ + 0.411X₁₅ + 0.202X₁₆ + 0.188X₁₇

Using the variance contributions of the two selected principal components as weight coefficients, a comprehensive evaluation model was constructed: (Y = 0.326F₁ + 0.263F₂). The comprehensive scores for each irrigation method were calculated, and MathWorks MATLAB R2023b was used to perform three-dimensional interpolation fitting of the principal component comprehensive scores, irrigation methods, and volume based on cubic interpolation. As shown in Figure 17, within the 70% to 90% field capacity range, the APRI irrigation strategy scored higher overall compared to the CI method. The comprehensive score for the APRI method at 72.8% field capacity was comparable to that of the CI method at 82.8% field capacity. Within the 50% to 70% field capacity range, both APRI and FPRI methods outperformed the CI method, with scores at 50% field capacity comparable to those of the CI method at 62% field capacity. This indicates that in fields aiming for higher water use efficiency with mild water deficits, the APRI method offers a broader range of moisture adjustment compared to traditional full-root-zone irrigation; under extreme drought conditions with limited water availability, alfalfa under APRI and FPRI methods shows stronger adaptability to drought stress.

4. Discussion

4.1. The Effect of APRI and Moisture Adjustment on the Growth Characteristics of Alfalfa

Current reports on the impact of alternate partial root-zone irrigation on crop above-ground and below-ground biomass are inconsistent, with significant variations depending on soil environment, irrigation volume, irrigation cycle, or crop type. However, all findings point towards alternate partial root-zone irrigation significantly enhancing water use efficiency in agricultural production [29,30]. Most studies show that APRI can maintain stable production performance and nutritional quality of alfalfa over multiple years. This stability is attributed not only to APRI’s ability to maintain the photosynthetic rate of alfalfa leaves without decay but also to its reduction in ineffective water dissipation in the leaves. Moreover, APRI enhances alfalfa’s root distribution, promotes the development of lateral roots, and stimulates more rhizome bud production, thereby increasing plant height and facilitating greater biomass allocation to roots and stems [31,32]. The above- and below-ground biomass results align with these patterns. Under the ideal alternating dry and wet conditions provided by the experiment, the APRI irrigation strategy achieved higher plant height and both above-ground and below-ground biomass at each irrigation stage compared to CI and FPRI under the same water supply conditions. However, further water reduction did not yield better results. The FPRI irrigation strategy achieved similar effects to APRI in the early stages of treatment but showed weaker growth in alfalfa during the middle and later stages of irrigation due to drought-induced inhibition of lateral root growth, a phenomenon that has been widely confirmed in previous reports [33].

4.2. The Effect of APRI and Moisture Adjustment on the Water Status of Alfalfa Leaves

Evaluating the suitability of water-saving irrigation techniques for a specific crop involves first determining the extent of damage under various water supply conditions. Drought-induced damage is manifested by the production of MDA from the oxidative decomposition of cell membrane structures and increased cell membrane permeability leading to electrolyte leakage. Therefore, MDA content and relative electrical conductivity can be used to assess the extent of damage to the crop’s leaf cell membranes [34]. Photosynthesis, as the most fundamental and complex physiological process, is crucial for all green plants, and a stable supply of chlorophyll is key to its normal functioning [35]. Water deficiency leads to lipid peroxidation of thylakoid membranes and electrolyte leakage, resulting in pigment degradation and reduced chlorophyll content, thereby weakening photosynthesis [36]. In the early and mid-phases of this experiment, the MDA content in alfalfa leaves under the CI method significantly increased due to reduced water supply, while during the same observation period, the MDA content in alfalfa leaves under the FPRI and APRI methods tended to stabilize within each group. The relative electrical conductivity of alfalfa leaves significantly increased at all observed stages with reduced water supply, but during the mid and late stages of treatment under extreme water scarcity, the relative electrical conductivity of alfalfa leaves under the FPRI and APRI methods remained significantly lower than under CI. The experimental results on the relative water content and saturation water deficit of alfalfa leaves corroborate that the APRI method, with adequate water supply in the mid to late treatment stages, results in superior leaf water health and maintains stable chlorophyll content in the leaves. This is consistent with previous studies on the APRI method, which demonstrated its ability to reduce malondialdehyde content [37], maintain chlorophyll stability [38], and promote healthier leaf water status.

4.3. The Effect of APRI and Moisture Adjustment on the Antioxidant Defense System of Alfalfa Leaves

Maintaining a dynamic balance between the production and scavenging of reactive oxygen species (ROS) is crucial for the survival and growth of crops under water stress conditions. To overcome oxidative stress and protect against the toxic effects of ROS, crops have evolved an effective antioxidant defense system, including key antioxidant enzymes such as SOD, POD, and CAT [39]. Specifically, SOD catalyzes the dismutation of the O²⁻ radical into H₂O₂ and O₂, while CAT and POD can convert H₂O₂ into H₂O [40]. Recent studies have shown that alternate partial root-zone irrigation can enhance the activity of antioxidant enzymes under various abiotic stress conditions [41,42]. The results of this experiment indicate that the APRI method increased the antioxidant enzyme activities in alfalfa leaves during the early to mid-stages of irrigation treatment compared to CI and FPRI methods. However, in the later stages of irrigation, the antioxidant enzyme activities under APRI were comparable to those under CI, and even lower in terms of SOD activity when compared at the same irrigation levels; the FPRI method showed significantly lower antioxidant enzyme activities at all irrigation levels compared to CI and APRI, similar to the findings by Abboud where the APRI method resulted in stronger SOD, POD, and CAT activities for enhanced ROS scavenging capability [43]. The reason for this is that the APRI method maintains healthier leaf water status in the later stages of treatment compared to CI, while FPRI leads to concerning growth conditions due to prolonged drought affecting the root system.

4.4. The Effect of APRI and Moisture Adjustment on the Osmotic Regulation Substances in Alfalfa Leaves

Under adverse conditions, plants increase osmotic regulation to maximize the bound water content in their leaves, thereby preventing severe dehydration. Thus, osmotic adjustment substances are fundamental for maintaining basic physiological functions under stress and are indicators of adaptability [44]. Some researchers have found that alternate partial root-zone irrigation can enhance a crop’s water-holding capacity by accumulating osmotic adjustment substances such as Pro, and crops under the APRI method contain higher levels of these substances compared to those under conventional deficit irrigation [41,45]. The results of this experiment show that APRI irrigation increased the content of proline in alfalfa leaves before and during the mid-period of treatment, the content of SP during the mid-period, and the content of betaine at the beginning of the treatment compared to CI, with no significant increases at other irrigation stages. Pro, SP, and betaine are sensitive to reductions in water supply under all three irrigation strategies, showing a significant increase in response to reduced water availability at all observed stages of irrigation. Thus, in this experiment, the osmoregulatory ability of alfalfa leaves in response to the APRI irrigation strategy was observed in the early to mid-stages of irrigation treatment. In the later stages of irrigation treatment, the water status of alfalfa under the APRI method was healthier compared to the CI irrigation strategy, resulting in no accumulation of osmoregulatory substances. This is similar to findings by Abdulwahab et al. [33] in corn, where osmoregulatory substances were more sensitive to the APRI irrigation strategy during the early to mid-stages of crop growth. Meanwhile, the FPRI irrigation strategy only increased the content of certain osmotic adjustment substances at the beginning of treatment. In the mid and later stages, poor growth due to restricted root development resulted in significantly weaker osmotic adjustment capabilities in alfalfa leaves compared to APRI and CI.

5. Conclusions

Under the APRI irrigation strategy, the phased wet–dry cycling environment of the alfalfa root system enhanced the osmoregulation capacity and antioxidant enzyme activity of alfalfa leaves during the mid-stage of irrigation treatment compared to the CI and FPRI methods, maintaining healthier leaf water status and stable photosynthetic rates, consistent with the hypotheses stated earlier. However, in the later stages of irrigation treatment, the interaction of APRI and its water deficit adjustment did not have a more positive effect on the osmoregulation and antioxidant enzyme activity of alfalfa leaves compared to the CI method, which diverges from the earlier hypotheses. In this experiment, the improvement in water use efficiency of alfalfa depended more on the superior leaf water status and biomass under APRI and its water deficit management. Achieving precise control of large-scale wet–dry cycles in the rhizosphere soil of alfalfa in actual field production is challenging. The water demand intensity, root distribution, and irrigation cycles at different growth stages can affect the practical application results. Therefore, in future field trials, it is necessary to combine the water demand patterns of alfalfa to determine the field water-holding capacity thresholds and corresponding water movement patterns that make the APRI method effective and to establish suitable irrigation cycles to further enhance water use efficiency in alfalfa production.

Author Contributions

Conceptualization, Q.S., Y.W.; Methodology, Q.S., Y.W., X.P., Y.A.; Data analysis and Visualization, Q.S., X.G., B.W., S.Z.; Writing—original draft preparation, Q.S., Y.W.; Experimental Procedure Guidance, G.J., Y.Z., S.Z.; all authors contribute to the Writing—review and Editing. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

Not applicable.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author, S.Z., upon reasonable request.

Acknowledgments

We sincerely thank Zhipeng Jiang, Dongjie Zhang, Qikun Yu, and all the students from the research group for their various assistance during the experiments.

Conflicts of Interest

The authors declare that they have no known competing financial interests or personal relationships that could have influenced the work reported in this paper.

Abbreviations

The following abbreviations are used in this manuscript:

APRI	Alternate Partial Root-zone Drying Irrigation
FPRI	Fixed Partial Root-zone Drying Irrigation
CI	Conventional Irrigation
WHC	Water-Holding Capacity
RWC	Relative Water Content
ABA	Abscisic Acid
ROS	Reactive Oxygen Species
MDA	Malondialdehyde
SOD	Superoxide Dismutase
POD	Peroxidase
CAT	Catalase
Pro	Proline
SS	Soluble Sugars
SP	Soluble Protein

Footnotes

Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Figures and Table

Figure 1 Schematic diagram of the alfalfa root-splitting pot experiment setup.

Figure 2 The effects of irrigation method and volume on alfalfa plant height and stem thickness at different observation stages ((a,d) Irrigation treatment on day 18; (b,e) Irrigation treatment on day 36; (c,f) Irrigation treatment on day 54). Note: All values in Figure 2, Figure 3, Figure 4, Figure 5, Figure 6, Figure 7, Figure 8, Figure 9, Figure 10, Figure 11, Figure 12, Figure 13 and Figure 14 represent the mean of replicated trials with standard errors attached. In the figures, uppercase letters indicate significant differences between irrigation methods (IM < 0.05), while lowercase letters indicate significant differences within groups of irrigation amounts under each irrigation method (IV < 0.05). In figures where the interaction between irrigation method and irrigation volume is significant (IM × IV < 0.05), lowercase letters denote significant differences in the simple effects analysis among variables (p < 0.05).

Figure 15 Pearson correlation analysis between the growth status and leaf water condition of alfalfa and the activity of antioxidant enzymes and osmoregulatory substances ((a) Irrigation treatment on day 18; (b) Irrigation treatment on day 36; (c) Irrigation treatment on day 54).

Figure 16 Principal component analysis of agronomic traits, leaf water status, antioxidant enzyme activity, and osmoregulatory substances in purple alfalfa under various irrigation treatments.

Figure 17 Three-dimensional interpolation fitting results of the principal component composite scores for alfalfa treated under different irrigation methods and volumes.

Table 1

Irrigation treatments during the alfalfa experiment period.

Treatments	Water Supply Area	Irrigation Upper Limit
CI1	Bilateral	90% WHC
CI2	Bilateral	70% WHC
CI3	Bilateral	50% WHC
FPRI1	Unilateral	90% WHC
FPRI2	Unilateral	70% WHC
FPRI3	Unilateral	50% WHC
APRI1	Bilateral alternation	90% WHC
APRI2	Bilateral alternation	70% WHC
APRI3	Bilateral alternation	50% WHC

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