Effects of Bacillus amyloliquefaciens QST713 on Mineral Nutrient Utilization of Alfalfa (Medicago sativa L.) under Drought Stress

Abstract

Drought stress is one of the major impediments to plant growth. Plant growth-promoting rhizobacteria (PGPR) can mitigate moisture stress in plants by increasing the ability of plant nutrient uptake and transport. In this study, we investigated the root phenotype, mineral nutrients (in leaves, roots, and soil), soil pH, water saturation deficit (WSD), free water content (FWC), and bound water content (BWC) of leaves of two alfalfa varieties, ‘Galalxie Max’ (drought-tolerant) and ‘Saidi 7’ (drought-sensitive), in the presence or absence of Bacillus amyloliquefaciens QST713 under drought stress conditions. The results showed that water stress negatively affected both cultivar root morphology (total root length, average diameter, total surface area, and volume) and the contents of K and Fe in leaves, roots, and soil. It also reduced the Mn and Zn contents in the soil while increasing the content of Na in the leaves and soil. Additionally, alfalfa plants under drought stress exhibited higher levels of soil pH, WSD, and BWC but lower contents of FWC and ratios of BWC/FWC in the leaves of both cultivars. However, QST713 application significantly enhanced the total root length, average root diameter, and the contents of K and Fe in alfalfa leaves, roots, and soil, as well as the BWC/FWC ratio in leaves under drought stress conditions. A significant reduction in the Na content was detected in QST713-treated alfalfa leaves and soil under drought stress. Furthermore, QST713 application noticeably decreased soil pH and WSD. The current findings showed that QST713 enhanced the water stress tolerance of alfalfa plants by ameliorating root morphology, reducing soil pH, and improving the BWC/FWC ratio, consequently promoting the accumulation of mineral nutrients (mainly K and Fe). Overall, Bacillus amyloliquefaciens QST713 can serve as a potential green fertilizer in sustainable agriculture to improve soil nutrients and enhance plant production under increasing drought conditions.

1. Introduction

The productivity and quality of alfalfa (Medicago sativa L.), a superb perennial forage legume crop, are strongly inhibited by drought stress [1]. In an effort to alleviate these undesirable consequences, researchers have achieved some success via conventional irrigation, various breeding approaches, and genetic engineering [2,3]. Nevertheless, these approaches are commonly time-consuming and labor-intensive. The use of beneficial microorganisms, which can mitigate environmental stresses on plant growth, as potential alternative sustainable agricultural practices has attracted increasing interest in recent years [4,5,6].

The colonization of roots by plant growth-promoting rhizobacteria (PGPR) can improve plant tolerance to water stress. Furthermore, these bacteria play an important role in enhancing soil fertility and promoting plant nutrient uptake [7,8]. Bacillus spp., the main species frequently used to improve plant productivity, have been applied commercially worldwide [9]. These bacteria are known to be major phytohormone producers, synthesizing auxin (IAA), gibberellic acid (GA), abscisic acid (ABA), and cytokinins (CK), which play roles in signal transduction and modification of immune responses for optimal growth [10]. Additionally, they can produce exopolysaccharides (EPSs), enhancing rhizosphere soil aggregation and leading to increased water and nutrient availability for plants [11]. Moreover, they can increase the secretion of siderophores and the solubilization and mineralization of nutrients, assisting in fulfilling the iron, phosphorous, and potassium requirements of plants [12,13,14]. Studies have demonstrated that drought stress adversely affects the uptake and assimilation of mineral nutrients, reducing their contents in plant tissues and eventually impairing plant growth and development [15,16,17]. PGPR can increase root hair, lateral root, and root surface area by generating phytohormones, including ethylene, CK, and IAA, which are conducive to increasing the absorption of minerals and water by plants and enhancing their resistance to abiotic stress [18]. In our study, Bacillus amyloliquefaciens QST713 was capable of secreting IAA and EPS, producing siderophores and enhancing alfalfa drought stress resistance [9]. In addition, it exhibits biocontrol and bio-fertilizer traits, and has been applied in fruit and vegetable production [19,20].

Therefore, the aim of the present study was to evaluate the response of alfalfa (both drought-tolerant and drought-sensitive) to Bacillus amyloliquefaciens QST713 on root morphogenesis and nutrient uptake and translocation under drought stress conditions. This study will not only broaden the comprehension of the mechanisms governing the response of plants associated with QST713 under drought conditions, but also lay the groundwork for QST713 as an environmentally safe biological agent to replace synthetic fertilizers in sustainable agriculture.

2. Materials and Methods

2.1. Experimental Protocol

Two alfalfa varieties (‘Galalxie Max’ and ‘Saidi 7’, referred to as ‘drought-tolerant’ and ‘drought-sensitive’, respectively) were used as plant materials to carry out drought pot experiments in an environment-controlled artificial intelligence climate chamber (300 µmol·m⁻²·s⁻¹ light intensity, 60% relative humidity, 14 h at 28/18 °C (day/night)) at Shanxi Agricultural University, China. These alfalfa cultivars were obtained from the Pratacultural Science Group of Shanxi Agricultural University. When the plants grew to 2 months old, they were divided into four treatments: (a) control (CK): plants not treated with bacteria; (b) CK + QST713: plants inoculated with bacteria; (c) MD: plants treated under drought conditions; (d) MD + QST713: plants inoculated with bacteria under drought conditions. The moderate drought condition was 55–60% of the maximum substrate water holding capacity. The inoculated bacteria (10⁸ CFU/mL, 5 mL) treatments were conducted by the root-irrigation method at 1, 11, and 21 days under moderate drought stress. The control treatment consisted of an equivalent volume of sterile distilled water. A total of 96 alfalfa plants per treatment were randomly arranged. The test was repeated 3 times. The specific protocol was described in our previous study [9].

2.2. Soil Sample Collection

The rhizosphere soil samples were collected from the three biological replicate pots. Each collected sample was sieved (2 mm) and air-dried for evaluation of the soil mineral elements and pH. In brief, we lightly shook the roots to remove loosely attached soil, and the soil still tightly adhering to the roots was harvested as rhizosphere soil.

2.3. Determination of Root Morphology Indicators

After the roots were rinsed with deionized water, a desktop root scanner (V850 pro, EPSON Scan, Tokyo, Japan) was used to scan the roots, and the images were saved to a computer. Then, root scanning analysis system software (Win RHIZO 3.1, Regent Instruments Inc., Québec City, QC, Canada) was used for analysis to obtain parameters such as total root length, total surface area, average diameter, and root volume.

2.4. Determination of Mineral Elements

2.4.1. Determination of Mineral Elements of the Plant

The plant samples were cleaned with deionized water, placed in an oven at approximately 105 °C for 15 min, and then at 70 °C until a constant weight was achieved. After grinding the dried samples (roots and leaves) to powder, the nitric acid–perchloric acid (1:2) digestion method was used to determine the contents of elements such as K, Ca, Na, Mg, Fe, Mn, and Zn by an inductively coupled plasma emission spectrometer (Perkin Elmer Optimal 8000, PerkinElmer, Waltham, MA, USA).

2.4.2. Determination of Mineral Elements in the Soil

Air-dried soil samples were passed through a 2 mm sieve for the analyses of soil mineral elements by an atomic absorption spectrophotometer (55B, Agilent, Santa Clara, CA, USA). K, Ca, Na, and Mg were extracted following the methods of Ochoa-Hueso et al. [21]. The method of Lindsay and Norvell [22] was used to extract Fe, Mn, and Zn.

2.5. Determination of Soil pH

The pH was determined in a 1:2 soil–water mixture by a handheld pH meter (OW-3508, CCOWAY, Shanghai, China).

2.6. Determination of Leaf Moisture Content

2.6.1. Determination of Water Saturation Deficit (WSD)

The detailed measurement of leaf relative water content (RWC) was described in our previous study [9], and the water saturation deficit (WSD) was calculated by the following formula [23]:

WSD (%) = 1 − RWC

(1)

2.6.2. Determination of Free Water Content (FWC) and Bound Water Content (BWC)

The fresh weight (FW) of the leaves was determined with an electronic balance, and the leaves were weighed again to determine the dry weight (DW) after they were air-dried under shaded, dry, and ventilated conditions. The detailed measurements of FW and DW were described in our previous study [9]. Then, the samples were dried in an oven at 80 °C until a constant weight was reached, and their oven-dry weight (ODW) was recorded. The free water content (FWC) and bound water content (BWC) were calculated as:

FWC (%) = (FW − DW)/FW × 100

(2)

BWC (%) = (DW − ODW)/FW × 100

(3)

2.7. Statistical Analysis

All the data were analyzed using the SPSS 20.0 program (SPSS Inc., Chicago, IL, USA) with one-way ANOVA. Significant differences were determined using the least significant difference (LSD) test (p < 0.05). Pearson correlation and principal component analysis (PCA) were used to determine the correlations between the different alfalfa parameters on the 30th day. The graphs were drawn using Origin software version 2024.

3. Results

3.1. Effects of Bacterial Strain QST713 on the Root Morphology of Alfalfa Seedlings under Drought Stress

Root morphology indicators of alfalfa plants under drought conditions with and without bacterial inoculation were investigated (Figure 1). The results showed that drought stress reduced the total length, average diameter, total surface area, and volume of roots compared to those of the control (CK), and QST713 inoculation improved the decrease in both alfalfa cultivars. In particular, relative to those of drought-stressed plants, the total root length and average root diameter of plants with bacterial inoculation increased by 22.67–55.22% and 27.45–27.91%, and by 29.20–69.24% and 26.09–31.11%, respectively, in the ‘Galalxie Max’ and ‘Saidi 7’ plants.

3.2. Effects of Bacterial Strain QST713 on the Leaf Mineral Contents of Alfalfa Seedlings under Drought Stress

Variations in leaf mineral elements, namely, K, Ca, Na, Mg, Fe, Mn, and Zn, were measured in alfalfa plants under different treatments (

Table 1. Effect of drought stress and QST713 application on the leaf mineral nutrient content of alfalfa seedlings in two cultivars.

Treated Days	Varieties	Treatments	K (mg/g)	Ca (mg/g)	Na (mg/g)	Mg (mg/g)	Fe (mg/g)	Mn (mg/g)	Zn (mg/g)
10	Galalxie Max	CK	23.53 ± 0.02 b	0.33 ± 0.00 b	0.63 ± 0.00 c	0.17 ± 0.00 b	114.33 ± 0.78 c	55.44 ± 0.7 b	31.83 ± 0.26 a
		CK + QST713	23.62 ± 0.01 a	0.32 ± 0.00 c	0.58 ± 0.01 d	0.17 ± 0.00 c	132.77 ± 1.93 b	37.78 ± 0.25 d	24.15 ± 0.43 c
		MD	23.32 ± 0.04 d	0.34 ± 0.00 b	0.91 ± 0.01 a	0.18 ± 0.00 a	58.05 ± 2.55 d	50.55 ± 0.26 c	23.77 ± 0.59 c
		MD + QST713	23.43 ± 0.01 c	0.38 ± 0.00 a	0.76 ± 0.00 b	0.18 ± 0.00 a	153.1 ± 0.65 a	64.69 ± 0.58 a	28.62 ± 0.53 b
	Saidi 7	CK	23.39 ± 0.01 b	0.41 ± 0.00 b	0.48 ± 0.00 c	0.17 ± 0.00 b	103.25 ± 0.53 b	46.66 ± 0.46 b	31.68 ± 0.67 b
		CK + QST713	23.44 ± 0.01 a	0.32 ± 0.00 c	0.44 ± 0.01 d	0.16 ± 0.00 c	115.55 ± 3.58 a	35.41 ± 0.31 d	22.78 ± 0.63 c
		MD	23.32 ± 0.01 c	0.33 ± 0.00 c	0.58 ± 0.01 b	0.17 ± 0.00 a	85.68 ± 0.99 c	38.3 ± 0.68 c	20.33 ± 0.16 d
		MD + QST713	23.36 ± 0.02 b	0.43 ± 0.00 a	0.61 ± 0.02 a	0.17 ± 0.00 a	104.17 ± 1.53 b	51.43 ± 0.64 a	36.36 ± 0.68 a
20	Galalxie Max	CK	23.58 ± 0.01 b	0.36 ± 0.00 b	0.26 ± 0.00 c	0.16 ± 0.00 c	116.95 ± 2.3 c	40.57 ± 0.22 b	19.30 ± 0.11 b
		CK + QST713	23.64 ± 0.01 a	0.28 ± 0.00 d	0.22 ± 0.00 d	0.16 ± 0.00 d	134.53 ± 1.89 b	33.09 ± 1.36 c	15.87 ± 0.21 c
		MD	23.42 ± 0.02 d	0.38 ± 0.00 a	0.44 ± 0.00 a	0.17 ± 0.00 a	74.23 ± 1.28 d	49.6 ± 0.45 a	20.52 ± 0.34 a
		MD + QST713	23.48 ± 0.01 c	0.34 ± 0.00 c	0.38 ± 0.00 b	0.17 ± 0.00 b	155.8 ± 0.98 a	47.58 ± 0.61 a	18.85 ± 0.25 b
	Saidi 7	CK	23.51 ± 0.02 ab	0.35 ± 0.00 c	0.14 ± 0.00 c	0.16 ± 0.00 b	123.08 ± 1.64 b	38.73 ± 0.91 c	15.74 ± 0.18 b
		CK + QST713	23.54 ± 0.01 a	0.31 ± 0.00 d	0.14 ± 0.00 c	0.15 ± 0.00 c	130.87 ± 2.42 a	32.37 ± 0.97 d	16.53 ± 0.24 b
		MD	23.39 ± 0.01 d	0.36 ± 0.00 b	0.2 ± 0.00 a	0.17 ± 0.00 a	95.93 ± 1.18 d	53.39 ± 0.51 a	19.65 ± 0.49 a
		MD + QST713	23.47 ± 0.01 c	0.38 ± 0.00 a	0.15 ± 0.00 b	0.16 ± 0.00 b	112.42 ± 1.35 c	42.42 ± 0.30 b	16.19 ± 0.24 b
30	Galalxie Max	CK	23.64 ± 0.02 c	0.29 ± 0.00 b	0.21 ± 0.00 b	0.16 ± 0.00 ab	141.77 ± 0.49 c	34.69 ± 0.30 b	19.36 ± 0.32 a
		CK + QST713	23.71 ± 0.01 b	0.28 ± 0.00 bc	0.2 ± 0.00 c	0.16 ± 0.00 a	147.8 ± 0.75 b	33.71 ± 0.97 b	14.95 ± 0.29 b
		MD	23.48 ± 0.01 d	0.31 ± 0.0 a	0.23 ± 0.00 a	0.16 ± 0.00 b	134.52 ± 1.95 d	36.63 ± 0.35 a	14.28 ± 0.51 b
		MD + QST713	23.78 ± 0.03 a	0.27 ± 0.00 c	0.21 ± 0.00 b	0.16 ± 0.00 b	160.37 ± 1.41 a	38.12 ± 0.31 a	14.91 ± 0.13 b
	Saidi 7	CK	23.54 ± 0.02 b	0.33 ± 0.00 a	0.19 ± 0.00 a	0.16 ± 0.00 b	146.75 ± 0.38 b	38.94 ± 0.14 a	16.46 ± 0.37 ab
		CK + QST713	23.64 ± 0.01 a	0.26 ± 0.00 c	0.18 ± 0.00 b	0.16 ± 0.00 c	156.02 ± 1.61 a	36.26 ± 0.30 b	14.15 ± 0.31 c
		MD	23.43 ± 0.02 c	0.31 ± 0.00 b	0.2 ± 0.00 a	0.16 ± 0.00 b	110.02 ± 1.89 d	36.33 ± 0.40 b	15.81 ± 0.54 b
		MD + QST713	23.52 ± 0.01 b	0.32 ± 0.00 ab	0.17 ± 0.00 b	0.17 ± 0.00 a	139.47 ± 0.44 c	39.68 ± 0.26 a	17.7 ± 0.52 a

Data are the mean ± SE (n = 3). Different letters indicate significant differences (p < 0.05).

). The results showed that, under drought conditions, compared with those in the CK treatment, the contents of K and Fe in the leaves of both alfalfa seedlings were noticeably lower, while the addition of bacterial QST713 significantly alleviated this decrease. The contents of K and Fe in the MD + QST713 treatment group were 0.26–1.28% and 19.22–163.74%, respectively, and 0.17–0.38% and 17.19–26.77% greater than those in the MD treatment group for ‘Galalxie Max’ and ‘Saidi 7’, respectively. The content of Na in all the plants significantly decreased after QST713 addition under drought conditions. On the 30th day, compared with those in the MD + QST713 treatment group, the contents of Mg, Mn, and Zn increased significantly in the ‘Saidi 7’ group, while there was no significant difference in the ‘Galalxie Max’ group.

3.3. Effects of Bacterial Strain QST713 on the Root Mineral Nutrients of Alfalfa Seedlings under Drought Stress

Drought caused a decrease in the content of K and Fe in the roots of both alfalfa cultivars, while QST713 alleviated this decrease (

Table 2. Effect of drought stress and QST713 application on the root mineral nutrients of alfalfa seedlings in two cultivars.

Treated Days	Varieties	Treatments	K (mg/g)	Ca (mg/g)	Na (mg/g)	Mg (mg/g)	Fe (mg/g)	Mn (mg/g)	Zn (mg/g)
10	Galalxie Max	CK	23.23 ± 0.01 b	0.21 ± 0.00 a	0.16 ± 0.00 b	0.19 ± 0.00 b	260.58 ± 2.28 b	63.85 ± 0.90c	67.77 ± 0.21 a
		CK + QST713	23.33 ± 0.02 a	0.17 ± 0.00 d	0.18 ± 0.00 a	0.19 ± 0.00 b	316.65 ± 1.35 a	51.51 ± 0.63 d	62.44 ± 1.00 b
		MD	23.06 ± 0.02 d	0.18 ± 0.00c	0.19 ± 0.00 a	0.19 ± 0.00 a	245.64 ± 1.46c	147.25 ± 3.72 a	42.79 ± 0.17 d
		MD + QST713	23.11 ± 0.01c	0.2 ± 0.00 b	0.19 ± 0.00 a	0.19 ± 0.00 a	256.58 ± 1.35 b	116.46 ± 1.95 b	52.22 ± 0.18c
	Saidi 7	CK	23.37 ± 0.02 a	0.22 ± 0.00 a	0.17 ± 0.00 a	0.19 ± 0.00 a	254.28 ± 2.56 b	59.24 ± 0.11 a	43.32 ± 0.71 b
		CK + QST713	23.43 ± 0.01 a	0.13 ± 0.00c	0.17 ± 0.00 a	0.17 ± 0.00c	274.87 ± 3.23 a	30.54 ± 0.73c	33.2 ± 0.37c
		MD	23.08 ± 0.03c	0.23 ± 0.01 a	0.14 ± 0.00 b	0.19 ± 0.00 a	232.67 ± 1.30c	61.18 ± 1.03 a	55.35 ± 0.42 a
		MD + QST713	23.26 ± 0.03 b	0.19 ± 0.00 b	0.14 ± 0.00 b	0.18 ± 0.00 b	247.86 ± 0.39 b	45.27 ± 0.64 b	27.89 ± 0.63 d
20	Galalxie Max	CK	23.28 ± 0.02 ab	0.26 ± 0.00 b	0.14 ± 0.00 ab	0.18 ± 0.00 b	265.67 ± 4.42c	38.97 ± 0.43 d	50.35 ± 0.02 a
		CK + QST713	23.35 ± 0.01 a	0.21 ± 0.00 d	0.14 ± 0.00 b	0.16 ± 0.00c	416.85 ± 2.89 a	47.60 ± 0.25c	52.48 ± 1.20 a
		MD	23.24 ± 0.01 b	0.25 ± 0.00c	0.15 ± 0.00 a	0.19 ± 0.00 a	256.32 ± 2.78c	65.51 ± 0.18 a	33.62 ± 0.93c
		MD + QST713	23.28 ± 0.04 ab	0.29 ± 0.00 a	0.14 ± 0.00 ab	0.18 ± 0.00 b	309.25 ± 3.87 b	52.3 ± 0.46 b	42.65 ± 0.73 b
	Saidi 7	CK	23.06 ± 0.32 a	0.22 ± 0.00c	0.26 ± 0.00 a	0.18 ± 0.00 a	271.87 ± 5.05 b	31.32 ± 0.32c	38.48 ± 0.33 b
		CK + QST713	23.47 ± 0.01 a	0.19 ± 0.00 d	0.26 ± 0.00 ab	0.17 ± 0.00 b	352.17 ± 3.75 a	29.84 ± 0.62c	44.95 ± 0.61 a
		MD	23.14 ± 0.02 a	0.26 ± 0.00 a	0.11 ± 0.00c	0.18 ± 0.00 a	248.71 ± 4.76c	40.76 ± 0.56 a	35.11 ± 0.32c
		MD + QST713	23.29 ± 0.00 a	0.24 ± 0.00 b	0.25 ± 0.00 b	0.18 ± 0.00 a	262.25 ± 0.38 b	35.51 ± 0.22 b	31.86 ± 1.09 d
30	Galalxie Max	CK	23.47 ± 0.01 ab	0.26 ± 0.00 b	0.18 ± 0.00 b	0.15 ± 0.00c	356.42 ± 1.03 b	34.49 ± 0.65 d	25.06 ± 0.13 b
		CK + QST713	23.50 ± 0.02 a	0.25 ± 0.00c	0.20 ± 0.00 a	0.16 ± 0.01 bc	462.3 ± 2.84 a	37.53 ± 0.12c	21.74 ± 0.04c
		MD	23.41 ± 0.02 b	0.30 ± 0.00 a	0.20 ± 0.00 a	0.17 ± 0.00 a	294.08 ± 2.77c	43.32 ± 0.12 a	24.49 ± 0.35 b
		MD + QST713	23.44 ± 0.02 ab	0.30 ± 0.01 a	0.19 ± 0.00 ab	0.16 ± 0.00 ab	348.07 ± 3.54 b	41.27 ± 0.40 b	30.72 ± 0.35 a
	Saidi 7	CK	23.47 ± 0.00 b	0.24 ± 0.00c	0.20 ± 0.00 a	0.16 ± 0.00 ab	362.60 ± 2.18 b	31.59 ± 0.28c	20.55 ± 0.42 b
		CK + QST713	23.56 ± 0.01 a	0.26 ± 0.01c	0.18 ± 0.00 b	0.16 ± 0.00 ab	448.62 ± 3.68 a	36.38 ± 0.92 b	29.93 ± 0.74 a
		MD	23.17 ± 0.02 d	0.43 ± 0.01 a	0.19 ± 0.00 b	0.16 ± 0.00 a	309.55 ± 4.43c	39.84 ± 0.26 a	18.58 ± 0.29c
		MD + QST713	23.30 ± 0.01c	0.38 ± 0.00 b	0.21 ± 0.00 a	0.15 ± 0.00 b	320.32 ± 4.16c	34.86 ± 0.44 b	21.56 ± 0.25 b

Data are the mean ± SE (n = 3). Different letters indicate significant differences (p < 0.05).

). In addition, the Mn content in both cultivars was notably enhanced by drought stress, while QST713 application obviously decreased the Mn content by 4.73–20.91% and 12.50–26.01% in the ‘Galalxie Max’ and ‘Saidi 7’ cultivars, respectively. On the 30th day, compared with those in the MD + QST713 treatment group, the contents of K, Fe, and Zn in the ‘Galalxie Max’ and ‘Saidi 7’ groups improved by 0.13%, 18.36%, and 25.44% and 0.56%, 3.48%, and 16.04%, respectively.

3.4. Effects of Bacterial Strain QST713 on the Soil Mineral Nutrients of Alfalfa Seedlings under Drought Stress

The effect of QST713 on the soil mineral nutrients of both alfalfa cultivar seedlings was observed (

Table 3. Effect of drought stress and QST713 application on the soil mineral nutrients of alfalfa seedlings in two cultivars.

Treated Days	Varieties	Treatments	K (mg/g)	Ca (mg/g)	Na (mg/g)	Mg (mg/g)	Fe (mg/g)	Mn (mg/g)	Zn (mg/g)
10	Galalxie Max	CK	3.65 ± 0.01 b	0.19 ± 0.00 a	1.17 ± 0.00 c	0.15 ± 0.00 a	418.37 ± 0.64 b	16.63 ± 0.24 a	8.08 ± 0.16 a
		CK + QST713	4.16 ± 0.01 a	0.18 ± 0.00 a	1.20 ± 0.01 b	0.16 ± 0.00 a	383.18 ± 5.78 a	14.49 ± 0.20 b	7.4 ± 0.15 ab
		MD	3.20 ± 0.03 c	0.17 ± 0.01 b	1.28 ± 0.00 a	0.15 ± 0.00 a	250.45 ± 4.55 d	9.86 ± 0.07 d	5.33 ± 0.24 c
		MD + QST713	3.55 ± 0.07 b	0.19 ± 0.00 a	1.14 ± 0.01 d	0.15 ± 0.00 a	365.87 ± 3.13 c	13.85 ± 0.23 c	7.11 ± 0.44 b
	Saidi 7	CK	3.4 ± 0.05 b	0.14 ± 0.00 c	1.00 ± 0.02 b	0.15 ± 0.00 a	495.52 ± 3.44 c	14.46 ± 0.27 c	6.10 ± 0.07 a
		CK + QST713	4.49 ± 0.02 a	0.12 ± 0.00 d	1.03 ± 0.01 b	0.14 ± 0.00 b	574.37 ± 6.98 a	15.34 ± 0.24 b	5.38 ± 0.07 b
		MD	2.80 ± 0.05 c	0.2 ± 0.01 a	1.09 ± 0.02 a	0.15 ± 0.00 ab	247.4 ± 5.47 d	15.21 ± 0.06 b	2.82 ± 0.12 d
		MD + QST713	3.32 ± 0.04 b	0.16 ± 0.00 b	0.98 ± 0.02 b	0.15 ± 0.00 a	545.52 ± 11.06 b	16.58 ± 0.18 a	3.74 ± 0.14 c
20	Galalxie Max	CK	4.03 ± 0.03 b	0.26 ± 0.00 a	1.02 ± 0.02 b	0.17 ± 0.00 a	469.4 ± 10.96 b	17.61 ± 0.47 a	6.46 ± 0.12 a
		CK + QST713	5.15 ± 0.06 a	0.25 ± 0.01 a	1.05 ± 0.01 b	0.17 ± 0.00 a	583.88 ± 20.77 a	16.96 ± 0.38 ab	6.23 ± 0.18 a
		MD	3.79 ± 0.04 c	0.22 ± 0.01 b	1.11 ± 0.01 a	0.16 ± 0.00 b	412.73 ± 3.27 c	14.53 ± 0.12 c	3.66 ± 0.28 c
		MD + QST713	3.86 ± 0.03 b	0.20 ± 0.00 b	1.04 ± 0.01 b	0.16 ± 0.00 b	456.13 ± 9.17 b	16.06 ± 0.45 b	4.88 ± 0.25 b
	Saidi 7	CK	4.35 ± 0.07 b	0.24 ± 0.00 a	0.96 ± 0.00 b	0.16 ± 0.00 ab	546.17 ± 5.47 b	17.55 ± 0.14 a	5.81 ± 0.17 a
		CK + QST713	5.10 ± 0.12 a	0.24 ± 0.01 a	0.98 ± 0.02 b	0.16 ± 0.00 a	682.45 ± 9.60 a	17.27 ± 0.13 a	4.37 ± 0.29 b
		MD	3.56 ± 0.05 c	0.20 ± 0.01 b	1.08 ± 0.04 a	0.15 ± 0.00 b	464.17 ± 3.85 c	15.16 ± 0.17 c	3.30 ± 0.19 c
		MD + QST713	4.31 ± 0.02 b	0.20 ± 0.01 b	0.88 ± 0.01 c	0.16 ± 0.00 b	572.65 ± 13.02 b	16.43 ± 0.28 b	3.93 ± 0.13 b
30	Galalxie Max	CK	4.4 ± 0.03 b	0.36 ± 0.00 a	1.02 ± 0.01 b	0.17 ± 0.00 a	783.27 ± 7.13 b	18.32 ± 0.10 a	6.19 ± 0.38 a
		CK + QST713	5.75 ± 0.06 a	0.34 ± 0.01 a	1.04 ± 0.01 b	0.17 ± 0.00 b	871.85 ± 28.20 a	18.32 ± 0.25 a	5.58 ± 0.26 b
		MD	4.16 ± 0.04 c	0.26 ± 0.01 c	1.08 ± 0.01 a	0.16 ± 0.00 b	607.22 ± 5.83 c	16.15 ± 0.44 c	3.26 ± 0.15 c
		MD + QST713	4.44 ± 0.07 b	0.29 ± 0.01 b	0.97 ± 0.00 a	0.16 ± 0.00 b	793.72 ± 10.74 b	17.19 ± 0.35 b	4.53 ± 0.92 a
	Saidi 7	CK	5.05 ± 0.07 b	0.35 ± 0.00 a	0.90 ± 0.00 c	0.17 ± 0.00 a	557.20 ± 7.65 c	18.13 ± 0.13 b	5.60 ± 0.10 a
		CK + QST713	5.44 ± 0.04 a	0.29 ± 0.00 c	0.95 ± 0.01 ab	0.17 ± 0.00 a	814.45 ± 5.39 a	20.35 ± 0.37 a	4.00 ± 0.08 b
		MD	4.34 ± 0.06 d	0.34 ± 0.00 b	0.98 ± 0.01 a	0.17 ± 0.00 a	324.13 ± 14.51 d	16.65 ± 0.15 c	3.39 ± 0.08 c
		MD + QST713	4.67 ± 0.14 c	0.29 ± 0.01 c	0.92 ± 0.02 bc	0.16 ± 0.00 a	703.42 ± 17.97 b	20.17 ± 0.32 a	3.83 ± 0.13 b

Data are the mean ± SE (n = 3). Different letters indicate significant differences (p < 0.05).

). Compared to those in the CK treatment, the contents of K, Fe, Mn, and Zn in the soil of both cultivars of plants significantly decreased under the MD treatment. However, compared with the MD treatment, the MD + QST713 treatment had a positive effect on these contents, with increases of 1.85–10.94%, 10.52–46.09%, 6.44–40.47%, and 33.33–38.96% and 7.60–21.07%, 23.37–120.50%, 8.38–21.14%, and 12.98–32.62% in the soils of the ‘Galalxie Max’ and ‘Saidi 7’ plants, respectively. In addition, the content of Na in both cultivars was notably enhanced by drought stress, while QST713 inoculation obviously decreased the Na content.

3.5. Effects of Bacterial Strain QST713 on the Soil pH of Alfalfa Seedlings under Drought Stress

The effects of QST713 application on soil pH are shown in Figure 2. Compared with that under CK, drought stress markedly increased the soil pH of alfalfa of both cultivars, while inoculation with QST713 significantly decreased the soil pH of ‘Galalxie Max’ by 8.43–9.79% and that of ‘Saidi 7’ by 5.20–9.14% compared with those in the non-inoculated treatment under stressed conditions.

3.6. Effects of Bacterial Strain QST713 on the Water Saturation Deficit (WSD) of Alfalfa Seedlings under Drought Stress

Drought stress significantly increased the WSD in alfalfa leaves, irrespective of the variety (Figure 3). However, the WSD of the plants inoculated with QST713 was lower than that of the plants not inoculated with QST713 under both normal and stressed conditions in both cultivars. Compared with that in the MD treatment group, the WSD in the ‘Galalxie Max’ and ‘Saidi 7’ cultivars significantly decreased after inoculation with QST713: 24.99–44.25% and 21.75–34.58%, respectively.

3.7. Effects of Bacterial Strain QST713 on the Water Content of Alfalfa Seedlings under Drought Stress

Compared with CK, drought stress markedly reduced the free water content (FWC) while increasing the bound water content (BWC), which resulted in a significant increase in the ratio of bound water to free water (BWC/FWC) in both cultivars (Figure 4). However, compared with non-inoculated plants, QST713-inoculated plants had greater FWC and lower BWC under drought conditions. Compared with those in the MD treatment group, the ratios of BWC/FWC in the ‘Galalxie Max’ and ‘Saidi 7’ cultivars decreased by 12.96–22.92% and 10.89–17.53%, respectively, in the MD + QST713 treatment group.

3.8. Correlation Analysis between the Studied Parameters

3.8.1. Result Evaluation through Principal Component Analysis (PCA)

PCA was performed separately, categorized by mineral nutrient uptake region. In the case of nutrient uptake in leaves, the two first principal components (PCs) explained 72.5% of the observed cumulative variance, with 56.4% and 16.1% in the first (PC1) and second (PC2) principal components, respectively (Figure 5A). PC1 was found to be positively associated with Fe, K, FWC, and root morphology indicators (root length, root average diameter, root total surface area, and root volume). PC2 was found to be positively associated with Ca, Mg, Mn, and Zn. These variables were strongly associated with the treatment of plants inoculated with QST713.

In the case of nutrient uptake under root conditions, the first two components of the PCAs accounted for 78.7% (PC1 = 63.6% and PC2 = 15.1%) (Figure 5B). PC1 was found to be positively associated with Fe, K, Zn, FWC, and root morphology indicators, and it displayed a strong correlation with the QST713 treatment. PC2 was found to be positively associated with Mg, Mn, BWC, and BWC/FWC, and it strongly correlated with the MD + QST713 treatment (Galalxie Max).

In the case of nutrient uptake under soil conditions, PC1 alone explained 63.4% of the total variance, and PC2 explained 14.0% of the total variance (Figure 5C). PC1 was found to be positively associated with Fe, K, Mn, Zn, Ca, Mg, FWC, and root morphology indicators and was strongly related to the QST713 treatment. PC2 was found to be positively associated with Na, BWC, and BWC/FWC, and it was strongly related to the MD + QST713 treatment (Saidi 7).

3.8.2. Result Evaluation through Pearson’s Correlation Analysis

Pearson’s correlation analysis revealed that the parameters of alfalfa treated with QST713 under control and stress conditions were correlated (Figure 6). In the leaves, roots, and soil of alfalfa plants, a positive correlation was observed between the mineral nutrients Fe and K and traits such as root length, root average diameter, root total surface area, root volume, and FWC, whereas a negative correlation was observed with traits such as soil pH, WSD, and BWC/FWC. Except for Na, all the soil mineral nutrients were negatively related to soil pH, WSD, BWC, and BWC/FWC.

4. Discussion

Drought negatively affects root growth and metabolism, which in turn restrains the process of plant nutrient uptake and water absorption [24]. PGPR can alter root system morphology and architecture by modulating phytohormones levels, subsequently increasing plant access to water and nutrients from the soil under stress conditions [16,25,26]. Our results showed that QST713 relieved the negative impact of water stress on root growth, improving the length and diameter of roots in both alfalfa cultivars (‘tolerant’ and ‘sensitive’ to drought). Consistent with these results, Mansour et al. [27] demonstrated that inoculation with PGPR under drought stress improved root length, root diameter, and root dry weight. Our previous study revealed that QST713 increased plant biomass (fresh and dry weight of roots and shoots) under water stress conditions [9]. These results indicate that QST713 can increase plant biomass by promoting root growth and consequently helping plants survive water deficit.

Mineral elements are crucial for plant growth and differentiation at various stages of plant development. Water deficit can reduce a plant’s ability to take up mineral nutrients, even affecting soil nutrient availability and soil nutrient adsorption [28,29,30]. It is evident that K and Na play important roles in maintaining osmotic pressure within plant cells [31]. Drought stress can severely decrease K concentrations and increase Na concentrations. This may be because Na might partially replace K as an osmotically active solute, improving the plant water uptake capacity and preventing water loss [32]. However, excessive absorption of Na⁺ can inhibit various enzyme activities in plants and lead to cell swelling, thereby resulting in toxic effects on plant growth [10,33]. It has been demonstrated that inoculation of plants with PGPR strains helps increase the K⁺ concentration, which in turn leads to a high K⁺/Na⁺ ratio, thereby relieving abiotic stresses [34]. Similarly, QST713 application significantly increased the K concentration in alfalfa leaves, roots, and soils under water deficiency (Table 1, Table 2 and Table 3). These results suggest that QST713 could selectively transport more K⁺ than Na⁺ from roots to leaves under drought stress, consequently ensuring that more K⁺ accumulates in leaves for osmotic adjustment.

Fe is essential for plant growth, due to its participation in several vital cellular processes and metabolic pathways as a redox cofactor [35]. The content and availability of Fe depend on the soil water content [36]. With water scarcity, the greater presence of O₂ in soil favors the formation of insoluble complexes of Fe, such as ferric oxides, making it unavailable for plant absorption [37]. In general, PGPR can produce siderophores with a strong ability to chelate Fe; these siderophores are recognized by specific receptor proteins and transported into the cell by their respective permeases [38,39], consequently enhancing rhizospheric iron concentrations and increasing Fe bioavailability in the soil [24]. Similarly, according to our present investigation, drought stress had an obvious adverse effect on the Fe content in the leaves, roots, and soils of both alfalfa cultivars, while QST713 alleviated this decrease (Table 1, Table 2 and Table 3). This could be related to the production of siderophores by QST713 [40]. In addition, increased contents of Mn and Zn were observed in the soil of both alfalfa cultivar seedlings inoculated with QST713 during drought conditions (Table 3). Several studies have reported that the solubilization of mineral nutrients (such as Mn, Mg, and Zn) by PGPR, which release organic and sugar acids to the rhizosphere and create acidic conditions by CO₂ (respiration), increases the availability of these nutrients for plant uptake. This process is considered a mechanism for enhanced plant growth and stress resistance [7,41]. In our study, a decrease in soil pH was observed with the application of QST713 under drought stress (Figure 2). Increasing evidence has indicated that lowering of soil pH by PGPR production of organic acids, such as acid phosphatases, lactate, citrate, succinate, and gluconic and keto gluconic acids, etc. [42,43,44]. However, alfalfa plants inoculated with QST713 showed lower uptake of Mn and greater uptake of Zn in the roots under water deficit conditions (Table 2). Particularly, its accumulation in the leaves of different drought-tolerant varieties showed obvious differences (Table 1). Various studies have reported that PGPR inoculation generally leads to significant increases in K, Ca, Mg, Fe, Cu, Mn, and Zn contents in plant tissues [45,46,47]. These findings indicate that plant responses to PGPR are highly dependent on plant species, strain characteristics, and mode of inoculation [48].

Water saturation deficit (WSD) refers to the degree of tissue dehydration and is a simple phenotypic trait that indicates plant stress tolerance [49,50]. A higher WSD indicates that the plants are subjected to a greater degree of water deficit [23]. In the present study, WSD was significantly induced under drought stress (Figure 3). However, QST713 suppressed the deleterious effects of water stress and decreased the WSD content in both alfalfa cultivars (‘tolerant’ and ‘sensitive’ to drought). In addition, the water in plant tissues exists in two different states: free water and bound water. The intensity of plant metabolism is restricted by the free water content (FWC), while the bound water content (BWC) is closely linked with plant resistance [51]. In general, when the ratio of free water to bound water is high, the metabolic activity and growth of plant tissues or organs are vigorous, but their stress resistance is weak; On the contrary, growth is slower but resistance is stronger. In our study, with increasing drought duration, FWC decreased in the leaves of alfalfa, while BWC and the BWC/FWC ratio increased (Figure 4). This was consistent with previous studies showing that drought stress increased BWC and reduced FWC in plant leaves [52,53]. However, the opposite results were observed in the treatment with QST713 inoculation under drought stress (Figure 4). When the BWC/FWC ratio was low, the metabolic activity of plant tissue increased [53]. These findings suggest that QST713 may enhance the metabolic intensity of plants by reducing BWC and increasing FWC, thereby alleviating the inhibitory effect of drought stress on plant growth.

PCA biplots are suitable for identifying links between the traits under study. A strong positive correlation between the mineral nutrients Fe and K (in QST713-inoculated alfalfa leaves, roots, and soil), FWC, and root morphology indicators was observed. However, negative correlations between the mineral nutrients Fe and K (in QST713-inoculated alfalfa leaves, roots, and soil), soil pH, WSD, and BWC/FWC were detected (Figure 5). Furthermore, Pearson’s correlation analysis also demonstrated these correlations (Figure 6). Higher K, Fe, and root morphology indicators, and lower pH, WSD, and BWC/FWC, were noted in the MD + QST713 treatment. These results are consistent with the findings of previous studies showing that PGPR plays a role in the enhanced uptake of minerals from the rhizosphere under stress conditions, which are interrelated with plant growth indices [10,54]. Therefore, our results, combined with previous research, suggest that QST713 may enhance plant stress resistance through interactions between root morphology, water content, mineral nutrients, and soil pH.

5. Conclusions

This study was designed to understand the effects of bacterial strain QST713 on the utilization of mineral nutrients in various parts (leaf, root, and soil) of alfalfa plants under drought stress conditions. The findings demonstrated that inoculation of alfalfa plants with QST713 was very effective in conferring tolerance against drought stress, possibly through improving the root phenotype (total root length and average root diameter), maintaining a relatively high free water content in the leaves, reducing soil pH as well as through the improvement of elemental profile. Pearson’s correlation analysis confirmed that mineral nutrients Fe and K were positively correlated with root morphology parameters (root length, root average diameter, root total surface area, root volume) and FWC, whereas they were negatively correlated with soil pH, WSD, and BWC/FWC. Thus, the use of QST713 could be considered a valuable approach for increasing drought tolerance in alfalfa plants and for the remediation of infertile soil in arid areas.