Effects of Bacillus amyloliquefaciens QST713 on Growth and Physiological Metabolism in Cucumber Under Low-Calcium Stress

Abstract

Soil acidification from excessive nitrogen and potassium fertilization in protected cucumber systems impairs calcium uptake, triggering physiological calcium deficiency and reducing yield. We investigated whether the plant growth-promoting rhizobacterium Bacillus amyloliquefaciens QST713 could mitigate low-calcium stress in cucumber (‘Jinyou No. 4’). Under controlled nutrient solution irrigation (4, 0.4, and 0 mmol/L Ca²⁺, with or without B. amyloliquefaciens QST713), low-calcium conditions suppressed growth, reduced ion uptake capacity, photosynthetic pigment content, gas exchange (Pn, Gs, Tr), PSII efficiency (ΦPSII, ETR), and decreased carbohydrate (starch, sucrose) accumulation, while disrupting nitrogen balance (decreases in NO₃⁻-N, soluble protein, and amino acids; increase in NH₄⁺-N) and inhibiting key N-assimilation enzymes (NR, GS, GOGAT, GDH). Inoculation with B. amyloliquefaciens QST713 reversed these effects: it enhanced ion acquisition, chlorophyll content, and photosynthetic performance; restored carbohydrate reserves; promoted NO₃⁻ uptake and NH₄⁺ assimilation; and upregulated N-metabolizing enzyme activities. Principal component analysis confirmed strong coupling among growth, photosynthesis, and C-N metabolism. In summary, low-calcium stress markedly inhibited cucumber growth, suppressed photosynthetic activity, and reduced the levels of carbon and nitrogen metabolism. Application of B. amyloliquefaciens QST713 effectively alleviated the physiological damage caused by low-calcium stress, enhancing photosynthetic performance and thereby accelerating the synthesis and turnover of carbon- and nitrogen-containing metabolites. These effects collectively improved cucumber tolerance to low-calcium conditions and promoted plant growth and development. This study provides a preliminary theoretical basis for further exploration of the stress-resistance capacity of B. amyloliquefaciens.

1. Introduction

Cucumber (Cucumis sativus L.), a fast-growing annual vine in the Cucurbitaceae family, is widely valued for its distinctive flavor and nutritional benefits. In recent years, the cucumber industry has experienced rapid global growth, with both cultivated area and total yield consistently ranking first worldwide [1]. In protected cultivation systems, the excessive application of nitrogen and potassium fertilizers has led to elevated concentrations of NH₄⁺ and K⁺ in the rhizosphere, resulting in severe soil acidification. This change in soil chemistry disrupts calcium uptake by plants and has led to increasingly frequent occurrences of physiological calcium deficiency in cucumber [2]. Calcium-deficiency symptoms are now commonly observed in greenhouse-grown cucumbers. During the seedling stage, affected plants typically exhibit stunted growth, pronounced bulging of young central leaves, basal chlorosis between veins in upper leaves, leaf blade reduction, and downward or inward curling of leaf margins. At later developmental stages, symptoms include shortened internodes and apical leaves, curling and necrosis at leaf tips near the shoot apex, and characteristic “parachute-like” or “spoon-shaped” deformations. In severe cases, leaves display marginal chlorosis with translucent white blotches and a “golden edge” phenotype—complete chlorophyll loss along the margins without clear tissue necrosis. Petioles may become brittle and prone to abscission, while shoot tips can exhibit dieback, mucus secretion, or even rot [3]. In some greenhouses, premature plant senescence has been reported, ultimately compromising cucumber flavor, fruit quality, and yield [3]. As cucumber is a priority crop in China’s modern protected agriculture systems [4], addressing calcium-deficiency-related physiological disorders and developing integrated control strategies have become urgent tasks for ensuring stable production.

Calcium is an essential component required for plant cell division, and its deficiency can significantly suppress normal growth and developmental processes in plants [5]. Previous studies have shown that calcium deficiency reduces the activities of enzymes such as amylase and ATPase in photosynthetically active organs like leaves, thereby lowering the conversion efficiency of photosynthetic products at the “source.” This impairs the translocation of photoassimilates and ultimately disrupts normal plant development [6]. Photosynthetically derived substances and energy provide the foundation for plant growth, yet photosynthesis is highly sensitive to environmental stress. Carbon and nitrogen metabolism are fundamental to plant development, and they are closely intertwined with photosynthetic activity. Carbon metabolism encompasses carbon assimilation via photosynthesis, sucrose and starch metabolism, and the transport and utilization of carbohydrates. These carbohydrates may be translocated as sugars or temporarily stored in the form of sugars, starch, or fructans. Subsequently, sugars are resynthesized in darkness and remobilized from leaf tissues for use elsewhere in the plant [7]. Nitrogen metabolism is strongly influenced by carbon metabolism and, in turn, exerts a significant impact on carbon flux, indicating a reciprocal regulatory relationship. This crosstalk plays a pivotal role in plant development and stress resilience [8]. Nitrogen metabolism supports carbon metabolism by providing key components such as enzymes, proteins, and chlorophyll, all of which are indispensable to photosynthetic and carbon metabolic functions. As enzymes and chloroplasts are protein-based, enhanced nitrogen metabolism can promote photosynthetic efficiency and overall plant productivity [9].

With the continuous expansion of protected cucumber cultivation, constraints imposed by greenhouse microenvironments and limited land availability have contributed to pronounced soil acidification. These conditions reduce calcium availability and uptake by cucumber plants, leading to physiological calcium deficiency—a problem that has been progressively worsening in recent years. In response to the negative impacts of excessive chemical fertilizer use, increasing efforts have been directed toward identifying sustainable alternatives to synthetic fertilizers [10,11]. Among these, microbial fertilizers have emerged as a promising solution and have become a central focus of fertilizer research in organic and sustainable agriculture over the past decade [12,13,14]. B. amyloliquefaciens is an aerobic, motile, Gram-positive bacterium belonging to the family Bacillaceae and the genus Bacillus. It shares a high degree of genetic homology with Bacillus subtilis and is widely recognized as a plant growth-promoting rhizobacterium (PGPR) [15]. This species enhances plant growth through multiple mechanisms, including the secretion of siderophores, indole-3-acetic acid (IAA), and phosphate-solubilizing enzymes, all of which improve nutrient availability and promote root development [16].

In recent years, research on B. amyloliquefaciens has primarily focused on its antimicrobial activity, production of cell wall-degrading enzymes, induction of disease resistance, and plant growth-promoting effects. However, studies exploring its role in enhancing plant tolerance to abiotic stresses remain relatively limited. Regarding stress adaptation, Ryu et al. [17] reported that Bacillus subtilis GB03 and B. amyloliquefaciens IN937a enhanced plant tolerance to both drought and salinity. Chen [18] found that B. amyloliquefaciens SQR9 improved plant salt tolerance, while Wang et al. [19] demonstrated that the YTK1 strain could grow well at temperatures up to 60 °C and exhibited strong amylolytic activity. These findings suggest that B. amyloliquefaciens possesses traits associated with drought, salt, and heat tolerance. However, its growth-promoting effects and underlying mechanisms under calcium-deficient stress remain largely unexplored. Previous research by our group demonstrated that B. amyloliquefaciens QST713 improved the physicochemical properties, enzymatic activities, and microbial diversity of the cucumber rhizosphere under low-calcium conditions, thereby enhancing calcium uptake and utilization and increasing plant tolerance to calcium deficiency. Building upon these findings, the present study employed the cucumber cultivar ‘Jinyou No. 4’ to investigate the effects of B. amyloliquefaciens QST713 on plant growth, photosynthetic performance, and carbon–nitrogen metabolism under low calcium stress. This study aims to preliminarily elucidate the calcium stress-mitigation potential and growth-promoting mechanisms of B. amyloliquefaciens under low calcium conditions, providing valuable insights for the further development and application of this beneficial microorganism.

2. Materials and Methods

2.1. Experimental Materials and Experimental Design

The experiment was conducted in Greenhouse No. 2 at the Horticultural Experiment Station of Shanxi Agricultural University. The cucumber variety employed in the study was ‘JinYou NO. 4’, purchased from Tianjin Kerun Agricultural Science and Technology Co., Ltd., located in Tianjin, China; The B. amyloliquefaciens QST713 isolated from the commercial formulation of Zhuorun^®, originated from North America, purchased from Bayer CropScience (China) Co., Ltd., located in Hangzhou, Zhejiang. Uniformly sized cucumber seeds with a full shape were selected and soaked for 6 h. Sterile water-moistened gauze was used to rinse the soaked cucumber seeds several times. Subsequently, the seeds were carefully placed on the gauze for germination in a constant temperature incubator set at 28 °C. Once the seeds germinated and displayed whitening, they were sown in 50-hole trays filled with sterilized coco-coir. When the seedlings reached the two-leaf, one-heart stage and exhibited similar growth potential, they were transplanted into black cultivation bags measuring 30 cm × 30 cm × 30 cm, filled with an equal amount of sterilized coco-coir. After a resting period of 20 days, the experimental treatments were initiated.

Plants were irrigated with Hoagland nutrient solution, and low-calcium stress was simulated by reducing the concentration of Ca(NO₃)₂ in the solution. To compensate for nitrogen loss due to the reduction in Ca(NO₃)₂, an equivalent molar amount of NaNO₃ was added to maintain consistent nitrogen levels across all treatments. Six treatments were established as follows:

(1)

CK: standard calcium concentration (4 mmol/L Ca(NO₃)₂)

(2)

CK+Q: 4 mmol/L Ca(NO₃)₂ + B. amyloliquefaciens QST713

(3)

LCa: low calcium concentration (0.4 mmol/L Ca(NO₃)₂)

(4)

LCa+Q: 0.4 mmol/L Ca(NO₃)₂ + B. amyloliquefaciens QST713

(5)

0Ca: calcium-free (0 mmol/L Ca(NO₃)₂)

(6)

0Ca+Q: 0 mmol/L Ca(NO₃)₂ + B. amyloliquefaciens QST713

The bacterial suspension was prepared at a concentration of 3.0 × 10⁸ CFU/mL and applied twice: on days 1 and 15 of the treatment period. For each application, 0.5 mL of the suspension was diluted 100-fold with sterile water and applied to the root zone via drenching. In control treatments without bacterial inoculation, an equal volume of sterile water was applied. Each treatment consisted of 20 replicate pots. Beginning on the first day of treatment, 2 L of the respective nutrient solution was applied to each pot every two days. After 30 days of treatment, the fully expanded third to fifth functional leaves from the upper part of cucumber plants were collected, with 10–15 leaves sampled each treatment, rinsed with distilled water, air-dried, and de-veined. The cleaned leaf tissues were immediately flash-frozen in liquid nitrogen and stored at –80 °C for subsequent physiological and biochemical analyses.

2.2. Determination of Experimental Indexes

2.2.1. Measurement of Cucumber Growth Parameters

Prior to treatment, three cucumber plants with uniform growth were randomly selected and labeled in each treatment group. Plant height and stem diameter were measured on days 0 (before treatment), 10, 20, and 30 after the initiation of treatment.

Plant height was measured using a measuring tape from the base of the first true leaf on the main stem to the apical tip of the main vine. Measurements were recorded in centimeters (cm), accurate to 0.1 cm.

Stem diameter was measured at the widest internode in the middle section of the main stem using a digital caliper. Measurements were recorded in millimeters (mm).

Determination of Ca²⁺, Mg²⁺, K⁺, and Na⁺ contents: On day 30 of each treatment, upper functional leaves of cucumber plants were collected. Samples were initially heated at 105 °C for 15 min in an electric blast drying oven to inactivate enzymes and were subsequently dried at 75 °C to a constant weight. The dried samples were ground into a fine powder using a grinder, passed through a 60-mesh sieve, and 0.2 g of the powder was accurately weighed into a 100 mL digestion tube. Five milliliters of concentrated sulfuric acid (H₂SO₄) were added, the mixture was shaken thoroughly and then left to stand overnight. Digestion began at 180 °C for approximately 30 min until white fumes of H₂SO₄ appeared; the temperature was then increased to 380 °C and maintained for approximately 90 min, until the solution became a uniform brown-black color. After slight cooling, 5 drops of hydrogen peroxide (H₂O₂) were added, and the mixture was gently boiled for 7–10 min. Following cooling, the addition of H₂O₂ and subsequent digestion were repeated several times with progressively reduced H₂O₂ volumes, until the digest became colorless or clear. The solution was then heated for an additional 10 min to remove any residual H₂O₂. After cooling, the digest was quantitatively transferred to a 100 mL volumetric flask, diluted to volume with deionized water once cooled to room temperature, and filtered through a 0.22 µm aqueous membrane filter. The concentrations of Ca²⁺, Mg²⁺, K⁺, and Na⁺ in the filtrates were determined using a flame atomic absorption spectrophotometer. Each treatment was performed with three replicates.

2.2.2. Measurement of Photosynthetic and Chlorophyll Fluorescence Characteristics in Cucumber Leaves

On day 30 of treatment, chlorophyll a, chlorophyll b, chlorophyll (a+b), and carotenoid contents in cucumber leaves were determined using the 96% ethanol extraction method. Functional leaves from each treatment group were collected, surface-cleaned, cut into small pieces, and homogenized. Approximately 0.1 g of mixed tissue was transferred into a 10 mL centrifuge tube containing 10 mL of 96% ethanol. Samples were extracted overnight at room temperature in the dark with intermittent shaking until the leaf tissue turned white. Absorbance was measured at 665 nm, 649 nm, and 470 nm using a spectrophotometer. Each treatment was performed with three replicates.

On the same day, between 9:00 and 11:00 AM on a clear day, photosynthetic gas exchange parameters of functional cucumber leaves were measured using a LI-6800 portable photosynthesis system (LI-COR Biosciences, Lincoln, NE, USA). The recorded parameters included net photosynthetic rate (Pn), transpiration rate (Tr), stomatal conductance (Gs), and intercellular CO₂ concentration (Ci). The leaf chamber (6800-01A) was set to a light intensity of 350 μmol·m⁻²·s⁻¹, leaf temperature of 28 °C, and relative humidity of 50–70%. Three leaves were measured per treatment.

After dark adaptation, chlorophyll fluorescence parameters were determined using an FMS-2 portable fluorometer (Hansatech Instruments Ltd., King’s Lynn, Norfolk, UK). The actinic light intensity was set to 800 μmol·m⁻²·s⁻¹. Once readings stabilized, the following parameters were recorded: maximum quantum efficiency of PSII (Fv/Fm), effective quantum yield of PSII (ΦPSII), electron transport rate (ETR), photochemical quenching coefficient (qP), and non-photochemical quenching coefficient (NPQ). Three leaves were measured per treatment.

2.2.3. Measurement of Carbohydrate Content in Cucumber Leaves

On day 30 of treatment, 50 mg of dried leaf tissue from each treatment group was accurately weighed. Each sample was extracted with 4 mL of 80% ethanol in a water bath at 80 °C for 30 min. The mixture was centrifuged at 5000 rpm for 10 min, and the supernatant was collected. The residue was re-extracted with 2 mL of 80% ethanol, and the two supernatants were combined. Activated carbon was added to the pooled extract for decolorization in a water bath for 30 min. The final volume was adjusted to 10 mL with distilled water and filtered. The resulting filtrate was used to determine the contents of soluble sugars, sucrose, fructose, and glucose.

The residue was then oven-dried at 80 °C and gelatinized with 2 mL of distilled water for 15 min. After cooling, 2 mL of pre-chilled 9.2 mol/L perchloric acid was added and extracted for 15 min. Subsequently, 4 mL of distilled water was added, mixed thoroughly, and centrifuged at 3000 rpm for 10 min. A second extraction was performed by adding 2 mL of 4.6 mol/L perchloric acid followed by 6 mL of distilled water. After centrifugation under the same conditions, a third extraction was carried out with 8 mL of distilled water. The supernatants from all three extractions were combined and brought to a final volume of 25 mL. This extract was used for starch determination.

Soluble sugar, sucrose, fructose, glucose, and starch contents were quantified using the anthrone–sulfuric acid colorimetric method. Each treatment was performed with three replicates.

2.2.4. Determination of Nitrogen Metabolism-Related Parameters in Cucumber Leaves

Nitrate nitrogen (NO₃⁻-N) content was determined using the salicylic acid method described by Cataldo et al. [20]. Fresh cucumber leaves (0.5 g) were homogenized in a mortar with 3 mL of distilled water. The homogenate was transferred to a water bath at 100 °C and extracted for 30 min. After filtration, the volume of the extract was adjusted to 5 mL with distilled water. For the assay, 0.1 mL of the extract was mixed with 0.4 mL of salicylic acid–sulfuric acid solution and incubated at room temperature for 20 min. Then, 9.5 mL of 0.08 g/mL NaOH solution was added. Absorbance was measured at 410 nm using a spectrophotometer.

Ammonium nitrogen (NH₄⁺-N) content was determined using the indophenol blue colorimetric method described by Solorzano et al. [21]. Fresh cucumber leaves (0.5 g) were homogenized in 5 mL of 10% acetic acid to form a uniform slurry, which was then diluted with distilled water to a final volume of 100 mL and filtered for further analysis. For the assay, 2 mL of the filtrate was mixed with 3 mL of hydrated indophenol reagent and 0.1 mL of 1% ascorbic acid solution. The mixture was heated at 100 °C for 15 min. After cooling, the volume was brought to 10 mL with anhydrous ethanol. The solution was mixed thoroughly, and the absorbance was measured at 580 nm using a spectrophotometer.

Soluble protein content was determined using the Coomassie Brilliant Blue G-250 staining method. Fresh cucumber leaves (0.3 g) were homogenized with 5 mL of distilled water and centrifuged at 3000 rpm for 10 min. A 0.5 mL aliquot of the supernatant was mixed with an equal volume of distilled water and 5 mL of Coomassie Brilliant Blue reagent. After a 2 min reaction, absorbance was measured at 595 nm using a spectrophotometer.

Free amino acid content was determined using the ninhydrin colorimetric method. Fresh cucumber leaves (0.5 g) were homogenized in 5 mL of 10% acetic acid to form a uniform slurry, then diluted with distilled water to a final volume of 100 mL and filtered. A 1 mL aliquot of the filtrate was mixed with 1 mL of ammonia-free distilled water, followed by 3 mL of hydrated ninhydrin reagent and 0.1 mL of 0.1% ascorbic acid. The mixture was heated at 100 °C for 15 min. After cooling and the development of a blue-purple color, the volume was adjusted to 20 mL with 60% anhydrous ethanol. Absorbance was measured at 570 nm.

Nitrate reductase (NR) activity was measured according to the method of Silveira et al. [22]. Fresh cucumber leaves (0.5 g) were homogenized in 4 mL of extraction buffer and centrifuged at 4000 rpm for 15 min. The resulting supernatant was used as the enzyme extract. For the assay, 0.4 mL of enzyme extract was mixed with 1.2 mL of 0.1 mol/L KNO₃ in phosphate buffer and 0.4 mL of NADH solution, and incubated in a water bath at 25 °C for 30 min. The reaction was terminated by the addition of 1 mL of sulfanilamide solution, followed by 1 mL of N-(1-naphthyl)-ethylenediamine for color development. After 15 min, the mixture was centrifuged, and the absorbance of the supernatant was measured at 540 nm.

Glutamine synthetase (GS) activity was determined following the method of Zhang et al. [23]. Fresh cucumber leaves (0.5 g) were ground in 3 mL of extraction buffer and centrifuged at 15,000 rpm for 20 min. The supernatant was used as the crude enzyme extract. A reaction mixture containing 1.6 mL of reaction buffer, 0.7 mL of enzyme extract, and 0.7 mL of ATP solution was incubated at 37 °C for 30 min. The reaction was stopped by adding 1 mL of chromogenic reagent. After mixing and standing briefly, the sample was centrifuged at 5000 rpm for 15 min, and the absorbance of the supernatant was measured at 540 nm.

Glutamate synthase (GOGAT) activity was determined according to the method of Jiao et al. [24]. The crude enzyme was extracted as described for GS. The reaction system consisted of 1.5 mL of Tris-HCl buffer (pH 7.6), 0.1 mL of 10 mmol/L KCl, 0.5 mL of 20 mmol/L α-ketoglutarate, 0.2 mL of 3 mmol/L NADH, 0.3 mL of enzyme extract, and 0.4 mL of 20 mmol/L L-glutamine. After initiating the reaction, absorbance at 340 nm was recorded every 20 s for 10 cycles. Enzyme activity was calculated based on the linear decline in absorbance.

Glutamate dehydrogenase (GDH) activity was measured following the method of Debouba et al. [25]. The crude enzyme extract was prepared as described for GS. The reaction mixture consisted of 2.6 mL of assay buffer, 0.1 mL of ddH₂O, 0.1 mL of 30 mmol/L CaCl₂, 0.1 mL of 6 mmol/L NADH, and 0.1 mL of enzyme extract. Absorbance at 340 nm was recorded every 20 s for 10 cycles, and enzyme activity was quantified based on the linear decrease in absorbance.

2.3. Data Analysis

All measurements were conducted with three independent replicates, and results are expressed as the mean ± standard error (SE). Data were initially organized in Excel 2010, and statistical analyses were performed using SPSS Statistics 27.0. A two-way analysis of variance (ANOVA) was used to assess the effects of calcium concentration and B. amyloliquefaciens QST713 treatment, and Duncan’s test was applied to assess pairwise differences between means at the p < 0.05 significance level. Graphs were generated using GraphPad Prism 8.0.

The integrated evaluation of cucumber growth, photosynthetic performance, and carbon–nitrogen metabolism was performed as follows. Raw data were first standardized using the Z-score method in SPSS Statistics 27.0 to remove differences in measurement scales among variables. The standardized dataset was then subjected to the Kaiser–Meyer–Olkin (KMO) test and Bartlett’s test of sphericity. The results (KMO > 0.5; Bartlett’s test, p < 0.05) indicated strong inter-variable correlations, confirming the suitability of the dataset for principal component analysis (PCA). In the PCA, key factors were extracted using eigenvalues greater than 1 as the selection criterion, and both eigenvalues and variance contributions were calculated. To improve interpretability, the initial factor loading matrix was subjected to varimax rotation, yielding a rotated loading structure. Factor scores were subsequently estimated using the Thompson regression method. Finally, a weighted composite score was derived by combining factor scores with their respective variance contributions as weights. This composite index provided an integrated assessment of cucumber growth, photosynthetic performance, and carbon–nitrogen metabolism across different treatments. The principal component analysis plot was performed using the Metware Cloud, a free online platform for data analysis (https://cloud.metware.cn) (accessed on 2 September 2025).

3. Results

3.1. Cucumber Growth

The growth responses of cucumber plants to different treatments are presented in Figure 1. Prior to treatment, all plants exhibited uniform development, with no significant differences in plant height among groups. However, from day 10 onward, height disparities gradually emerged. By day 30, the CK+Q treatment showed a significant 5.00% increase in plant height compared to CK. In contrast, low-calcium stress markedly inhibited growth, with plant height reduced by 11.40% under LCa and by 15.68% under 0Ca conditions. The application of B. amyloliquefaciens QST713 effectively mitigated this inhibition. At day 30, plant height under LCa+Q was 12.21% greater than under LCa, while 0Ca+Q exceeded 0Ca by 13.13% (Figure 1a).

Likewise, no significant differences in stem diameter were observed across treatments before initiation. From day 10 onward, low calcium stress significantly suppressed stem thickening, with more pronounced effects over time. By day 30, stem diameter declined by 3.15% in LCa and by 6.74% in 0Ca compared to CK. In contrast, B. amyloliquefaciens QST713 application significantly promoted stem thickening throughout the treatment period. On day 30, CK+Q increased stem diameter by 8.21% relative to CK. Under calcium-deficient conditions, LCa+Q and 0Ca+Q increased stem diameter by 5.30% and 5.03%, respectively, compared to their corresponding controls (Figure 1b).

In summary, prolonged exposure to low-calcium stress significantly inhibits cucumber growth, as indicated by marked reductions in plant height and stem diameter. The severity of this inhibition increases under more extreme calcium deficiency. However, application of B. amyloliquefaciens QST713 effectively alleviates the growth suppression, resulting in increased plant height and stem thickening under low-calcium stress.

Low-calcium stress markedly inhibited cucumber growth. Moreover, under severe calcium deficiency (0Ca), calcium deficiency symptoms progressively intensified as the treatment duration increased (Figure 2). On day 10, the earliest symptoms were observed as mild curling of leaf margins and upward cupping of leaves near the shoot apex. By day 20, these symptoms had become more pronounced, characterized by aggravated leaf deformation, reduced size of new leaves, necrosis along the leaf margins, and chlorosis. By day 30, apical growth was severely inhibited, newly emerged leaves showed extensive necrosis, and the shoot apex had completely died.

We further analyzed the concentrations of macronutrient ions (Ca²⁺, Mg²⁺, K⁺, and Na⁺) in cucumber leaves under different treatments (

Table 1. Effect of B. amyloliquefaciens QST713 on macronutrient contents in cucumber leaves under low-calcium stress.

Treatments	Ca2+ (mg·g−1 DW)	Mg2+ (mg·g−1 DW)	K+ (mg·g−1 DW)	Na+ (mg·g−1 DW)	K+/Na+
CK	14.54 ± 0.49 Aa	9.01 ± 0.06 Aa	50.55 ± 1.00 Aa	0.19 ± 0.01 Aa	263.01 ± 12.85 Aa
CK+Q	15.46 ± 0.44 Aa	9.16 ± 0.10 Aa	51.24 ± 0.28 Aa	0.18 ± 0.01 Aa	280.77 ± 13.63 Aa
LCa	10.95 ± 0.22 Bb	8.58 ± 0.01 Aa	48.19 ± 0.98 Aa	0.21 ± 0.00 Aa	233.15 ± 1.20 Ab
LCa+Q	13.12 ± 0.31 Ba	8.89 ± 0.23 ABa	50.00 ± 1.78 Aa	0.19 ± 0.00 ABb	258.61 ± 8.56 Aa
0Ca	8.76 ± 0.12 Cb	7.23 ± 0.17 Bb	39.00 ± 0.80 Bb	0.17 ± 0.00 Ba	230.23 ± 12.17 Ab
0Ca+Q	10.11 ± 0.13 Ca	8.58 ± 0.15 Ba	45.18 ± 0.93 Ba	0.17 ± 0.01 Ba	271.49 ± 5.70 Aa

Five replicates were used for determination, and each value is presented as “mean ± standard error (SE)” (n = 3). Note: Different uppercase letters indicate significant differences among calcium concentrations within the same bacterial treatment, whereas different lowercase letters indicate significant differences between treatments with or without B. amyloliquefaciens QST713 at the same calcium concentration (Duncan’s test, p < 0.05).

). After 30 days of treatment, low-calcium stress significantly reduced leaf Ca²⁺ concentrations, with decreases of 24.69% under LCa and 39.75% under 0Ca compared with the control. Concentrations of Mg²⁺ and K⁺ also declined under low-calcium conditions; in LCa, the reductions were minor and not statistically significant, whereas in 0Ca, Mg²⁺ and K⁺ concentrations were significantly reduced by 19.76% and 22.85%, respectively. Leaf Na⁺ concentrations remained at low levels (approximately 0.20 mg·g⁻¹ DW) with minimal variation among treatments. The K⁺/Na⁺ ratio was reduced under low-calcium stress, primarily due to declines in K⁺ concentration, although this decrease was not statistically significant.

Application of B. amyloliquefaciens QST713 enhanced, to some extent, the uptake of mineral ions. Compared with the control, CK+Q exhibited slight increases in Ca²⁺, Mg²⁺, and K⁺ concentrations, as well as in the K⁺/Na⁺ ratio. Relative to LCa, LCa+Q significantly increased Ca²⁺ concentration by 19.82% and raised Mg²⁺ and K⁺ concentrations by 3.61% and 3.76%, respectively, along with a 10.92% improvement in the K⁺/Na⁺ ratio. Compared with 0Ca, 0Ca+Q significantly increased Ca²⁺, Mg²⁺, and K⁺ concentrations, and the K⁺/Na⁺ ratio, by 15.41%, 18.67%, 15.85%, and 17.92%, respectively.

Overall, low-calcium stress markedly decreased leaf Ca²⁺ concentrations and negatively affected Mg²⁺ and K⁺ levels, while also reducing the K⁺/Na⁺ ratio. Inoculation with B. amyloliquefaciens QST713 alleviated these negative effects by enhancing the uptake of Ca²⁺, Mg²⁺, and K⁺, thereby improving ionic balance under low-calcium stress.

3.2. Photosynthetic Performance in Cucumber Leaves

3.2.1. Photosynthetic Pigments in Cucumber Leaves

To examine the effects of various treatments on the chlorophyll fluorescence characteristics of cucumber leaves, we first quantified the photosynthetic pigment content after 30 days of treatment (Figure 3). Under low-calcium stress, the concentrations of chlorophyll a, chlorophyll b, chlorophyll (a+b), and carotenoids were significantly lower than those in the control group. Specifically, under LCa stress, these pigments decreased by 23.00%, 16.22%, 21.74%, and 19.23%, respectively; under 0Ca stress, the reductions were 47.00%, 45.95%, 47.83%, and 38.46%, respectively.

Application of B. amyloliquefaciens QST713 promoted the accumulation of photosynthetic pigments under calcium-deficient conditions. Compared with LCa stress alone, the LCa+Q treatment significantly increased chlorophyll a and b levels by 24.68% and 19.35%, respectively. Chlorophyll (a+b) content increased by 24.07%, while carotenoid content rose by 19.05%. Likewise, under 0Ca+Q treatment, chlorophyll a and b levels increased by 56.60% and 45.00%, respectively, with chlorophyll (a+b) rising by 55.56% and carotenoids by 43.75%, compared to the 0Ca stress group.

In summary, low-calcium stress significantly reduces the accumulation of photosynthetic pigments in cucumber leaves, with more severe stress causing greater declines. However, the application of B. amyloliquefaciens QST713 markedly improves pigment content, mitigating the detrimental effects of calcium deficiency.

3.2.2. Photosynthetic Parameters in Cucumber Leaves

Photosynthetic parameters of cucumber leaves under different treatments after 30 days are shown in Figure 4. Low-calcium stress significantly inhibited net photosynthetic rate (Pn), stomatal conductance (Gs), and transpiration rate (Tr), with reductions of 22.30%, 36.54%, and 20.28% under LCa stress, and 30.94%, 46.15%, and 29.18% under severe (0Ca) stress, respectively, compared to the control. Conversely, intercellular CO₂ concentration (Ci) increased significantly by 3.95% and 6.57% under LCa and 0Ca stresses, respectively.

Application of B. amyloliquefaciens QST713 markedly enhanced photosynthetic performance. In all treatments, Pn, Gs, and Tr were significantly elevated. Compared to the control, the CK+Q treatment increased these parameters by 40.69%, 38.46%, and 20.58%, respectively. Relative to LCa stress alone, the LCa+Q treatment elevated Pn, Gs, and Tr by 26.34%, 56.06%, and 24.73%, respectively, while the 0Ca+Q treatment increased these parameters by 26.27%, 25.00%, and 15.45% compared to 0Ca stress. Furthermore, Ci was significantly reduced by 3.37% and 2.28% in the LCa+Q and 0Ca+Q treatments compared to their respective stress controls.

Together, these findings demonstrate that low-calcium stress severely impairs photosynthesis in cucumber, with greater inhibition under severe deficiency. Treatment with B. amyloliquefaciens QST713 effectively mitigates this suppression, significantly improving photosynthetic capacity under calcium-limited conditions.

3.2.3. Chlorophyll Fluorescence Parameters in Cucumber Leaves

Chlorophyll fluorescence parameters of cucumber leaves under different treatments after 30 days are presented in

Table 2. Effects of B. amyloliquefaciens QST713 on chlorophyll fluorescence parameters of cucumber leaves under low-calcium stress.

Treatments	Fv/Fm	ΦPSII	ETR	qP	NPQ
CK	0.82 ± 0.00 Aa	0.67 ± 0.00 Ab	56.98 ± 0.08 Ab	0.88 ± 0.00 Ab	0.14 ± 0.01 Ca
CK+Q	0.82 ± 0.00 Aa	0.70 ± 0.00 Aa	59.14 ± 0.05 Aa	0.92 ± 0.00 Aa	0.14 ± 0.03 Ba
LCa	0.82 ± 0.01 Aa	0.63 ± 0.00 Bb	53.84 ± 0.13 Bb	0.86 ± 0.00 Aa	0.23 ± 0.00 Ba
LCa+Q	0.83 ± 0.00 Aa	0.66 ± 0.00 Ba	56.27 ± 0.33 Ba	0.86 ± 0.01 Ba	0.17 ± 0.01 ABb
0Ca	0.83 ± 0.00 Aa	0.64 ± 0.01 Bb	54.00 ± 1.06 Bb	0.86 ± 0.02 Aa	0.32 ± 0.01 Aa
0Ca+Q	0.82 ± 0.01 Aa	0.66 ± 0.01 Ba	55.95 ± 0.54 Ba	0.87 ± 0.01 Ba	0.20 ± 0.00 Ab

Five replicates were used for determination, and each value is presented as “mean ± standard error (SE)” (n = 3). Note: Different uppercase letters indicate significant differences among calcium concentrations within the same bacterial treatment, whereas different lowercase letters indicate significant differences between treatments with or without B. amyloliquefaciens QST713 at the same calcium concentration (Duncan’s test, p < 0.05).

. Compared to the control (CK), low-calcium stresses (LCa and 0Ca) did not significantly affect the maximum quantum yield of PSII (Fv/Fm) but significantly decreased the effective quantum yield of PSII (ΦPSII) by 5.97% and 4.48%, and the electron transport rate (ETR) by 5.51% and 5.23%, respectively. No significant differences were observed in photochemical quenching (qP), while non-photochemical quenching (NPQ) increased significantly by 64.29% and 128.57% under LCa and 0Ca stresses, respectively.

Compared with LCa stress alone, the LCa+Q treatment significantly increased ΦPSII and ETR by 4.76% and 4.51%, respectively, and decreased NPQ by 26.09%. Similarly, relative to 0Ca stress, the 0Ca+Q treatment significantly elevated ΦPSII and ETR by 3.13% and 3.61%, respectively, and reduced NPQ by 37.50%.

In summary, low calcium stress significantly impairs photosystem II (PSII) photochemical function in cucumber leaves. Treatment with B. amyloliquefaciens QST713 effectively alleviates these effects, reducing the extent of PSII damage under calcium-deficient conditions.

3.3. Carbohydrate Metabolism in Cucumber Leaves

Low-calcium stress significantly inhibited the photosynthetic capacity of cucumber, whereas B. amyloliquefaciens QST713 alleviated this suppression. To further elucidate these effects, we analyzed carbohydrate contents in cucumber leaves under different treatments. As shown in Figure 5, after 30 days of treatment, starch, soluble sugars, and sucrose contents were significantly reduced under low-calcium stress. Compared to the control (CK), starch, soluble sugars, and sucrose decreased by 20.19%, 25.40%, and 18.94% under LCa stress, and by 39.70%, 36.92%, and 32.15% under 0Ca stress, respectively, with 0Ca stress causing a greater decline (Figure 5a–c). Conversely, fructose and glucose contents in cucumber leaves increased significantly under low-calcium stress. Relative to CK, fructose and glucose levels rose by 26.93% and 20.94% under LCa, and by 49.23% and 55.08% under 0Ca, respectively (Figure 5d,e).

Application of B. amyloliquefaciens QST713 significantly increased starch, soluble sugars, and sucrose contents. Compared to CK, the CK+Q treatment raised starch, soluble sugars, and sucrose levels by 9.71%, 19.33%, and 19.36%, respectively. Relative to LCa stress alone, LCa+Q treatment increased these carbohydrates by 20.10%, 33.52%, and 20.26%, while 0Ca+Q treatment increased them by 21.80%, 37.56%, and 24.82%, respectively (Figure 5a–c). Meanwhile, B. amyloliquefaciens QST713 reduced fructose and glucose contents in cucumber leaves. Compared to CK, fructose content in the CK+Q treatment showed no significant change, while glucose decreased by 22.46%. Compared to LCa stress, LCa+Q treatment significantly reduced fructose and glucose by 14.61% and 13.49%, respectively. Similarly, compared to 0Ca stress, 0Ca+Q treatment reduced fructose and glucose levels by 21.01% and 22.17%, respectively (Figure 5d,e).

In summary, low-calcium stress significantly disrupts carbohydrate accumulation and metabolism in cucumber, with more severe stress exerting stronger effects. Treatment with B. amyloliquefaciens QST713 effectively mitigates these impacts, enhancing carbohydrate conversion under calcium deficiency.

3.4. Nitrogen Metabolism in Cucumber Leaves

3.4.1. Nitrogenous Compounds in Cucumber Leaves

After 30 days of treatment, the contents of nitrogenous compounds in cucumber leaves under different treatments are shown in Figure 6. Compared to the control (CK), low-calcium stress significantly decreased nitrate nitrogen (NO₃⁻-N)content. Under LCa stress, NO₃⁻-N decreased by 11.11%, while 0Ca stress caused a larger decline of 29.37% (Figure 6a). Conversely, ammonium nitrogen (NH₄⁺-N) accumulated significantly under low-calcium stress, increasing by 26.85% under LCa and 37.43% under 0Ca (Figure 6b). Protein synthesis was inhibited under low-calcium stress, as soluble protein content decreased significantly by 9.43% and 19.19% under LCa and 0Ca stress, respectively (Figure 6c). Free amino acid content decreased by 3.65% under LCa, though not significantly, whereas under 0Ca stress it declined significantly by 24.05% (Figure 6d).

Application of B. amyloliquefaciens QST713 promoted NO₃⁻-N uptake in cucumber. Compared to CK, the CK+Q treatment increased NO₃⁻-N content by 34.13%. Relative to LCa stress alone, LCa+Q treatment raised NO₃⁻-N by 34.82%, and 0Ca+Q treatment increased it by 22.47% compared to 0Ca stress (Figure 6a). Meanwhile, NH₄⁺-N content was significantly reduced by B. amyloliquefaciens QST713. Compared to CK, CK+Q decreased NH₄⁺-N by 5.13%; compared to LCa stress, LCa+Q reduced NH₄⁺-N by 15.57%; and compared to 0Ca stress, 0Ca+Q treatment lowered NH₄⁺-N by 10.27% (Figure 6b). Soluble protein content increased significantly following B. amyloliquefaciens QST713 application. CK+Q treatment increased soluble protein by 7.74% compared to CK; LCa+Q increased it by 14.50% relative to LCa stress; and 0Ca+Q increased it by 17.50% compared to 0Ca stress (Figure 6c). Free amino acid content was also significantly elevated: CK+Q treatment increased it by 33.27% compared to CK; LCa+Q by 18.96% compared to LCa stress; and 0Ca+Q by 10.91% compared to 0Ca stress (Figure 6d).

In summary, low-calcium stress significantly affects the accumulation of nitrogenous compounds in cucumber leaves, characterized by decreases in NO₃⁻-N, soluble protein, and free amino acids, alongside an increase in NH₄⁺-N. Treatment with B. amyloliquefaciens QST713 effectively alleviates the inhibitory effects of low-calcium stress on nitrogen compound synthesis, enhancing NO₃⁻-N, soluble protein, and free amino acid contents while significantly reducing NH₄⁺-N levels, thereby mitigating ammonium toxicity.

3.4.2. Key Nitrogen Metabolism Enzymes in Cucumber Leaves

After 30 days of treatment, the activities of key nitrogen metabolism enzymes in cucumber leaves under different treatments are presented in Figure 7. Compared to the control (CK), low-calcium stress significantly inhibited the activities of nitrogen metabolism-related enzymes. Under LCa stress, nitrate reductase (NR), glutamine synthetase (GS), glutamate synthase (GOGAT), and glutamate dehydrogenase (GDH) activities decreased by 12.59%, 23.13%, 24.01%, and 10.90%, respectively. 0Ca stress exerted stronger inhibitory effects, with reductions of 27.95%, 32.38%, 52.00%, and 25.12% in NR, GS, GOGAT, and GDH activities, respectively (Figure 7).

Application of B. amyloliquefaciens QST713 significantly enhanced the activities of these enzymes in cucumber leaves. Compared to CK, the CK+Q treatment increased NR, GS, GOGAT, and GDH activities by 28.78%, 58.93%, 42.00%, and 30.57%, respectively. Under low-calcium stress, inoculation with B. amyloliquefaciens markedly alleviated the disruption of nitrogen metabolism by significantly elevating enzyme activities. Relative to LCa stress alone, LCa+Q treatment increased NR, GS, GOGAT, and GDH activities by 13.37%, 61.11%, 34.21%, and 19.15%, respectively. Similarly, compared to 0Ca stress, 0Ca+Q treatment increased these enzyme activities by 29.48%, 50.42%, 45.84%, and 30.38%, respectively (Figure 7).

In summary, low-calcium stress disrupts nitrogen metabolism in cucumber, significantly reducing the activities of key nitrogen metabolism enzymes. Treatment with B. amyloliquefaciens QST713 effectively enhances these enzyme activities, mitigating the metabolic disruption caused by calcium deficiency and improving cucumber tolerance to low-calcium stress.

3.5. Principal Component Analysis of Growth, Photosynthesis, and Carbon–Nitrogen Metabolism-Related Parameters in Cucumber

Principal component analysis (PCA) is a dimensionality reduction technique that transforms multiple correlated variables into a smaller set of uncorrelated new variables called principal components. These components capture the majority of the information contained in the original variables. We performed PCA on 28 parameters measured under different cucumber treatments to reduce dimensionality. Based on the criterion of eigenvalues greater than 1, three principal components were extracted, as shown in

Table 3. Eigenvalues, contribution rates, and cumulative contribution rates of principal components.

Principal Component	Eigenvalues	Contribution Rates/%	Cumulative Contribution Rates/%
1	22.928	81.887	81.887
2	1.792	6.399	88.286
3	1.047	3.738	92.024

. These components correspond to three composite indices, accounting for 81.887%, 6.399%, and 3.738% of the variance, respectively, with a cumulative contribution rate of 92.024%. This indicates that the first three principal components capture the majority of the variation across all growth, photosynthetic fluorescence, and carbon–nitrogen metabolism parameters in cucumber. Therefore, these three principal components can effectively represent the original 28 variables and serve as comprehensive indices for evaluating photosynthetic fluorescence and carbon–nitrogen metabolism in cucumber.

Principal component analysis (PCA) of cucumber growth, photosynthetic, and carbon–nitrogen metabolism parameters revealed clear separation among treatments (Figure 8). The first two principal components distinguished the three calcium levels (CK, LCa, and 0Ca), and within each calcium concentration, treatments with or without B. amyloliquefaciens QST713 were also separated, indicating that PCA effectively discriminated among the treatment groups.

The magnitude of factor loadings reflects the contribution of the original variables to the newly constructed composite variables after dimensionality reduction. Therefore, the factor loading matrix can be used to identify the main original variables closely associated with each principal component. The factor loading matrix of the first three principal components for cucumber photosynthetic fluorescence and carbon–nitrogen metabolism-related parameters is presented in

Table 4. Factor loading matrix of principal components.

Index	Principal Component 1	Principal Component 2	Principal Component 3
Plant height	0.957	−0.122	0.012
Stem diameter	0.960	0.092	0.125
chlorophyll a	0.982	0.108	−0.051
chlorophyll b	0.968	−0.075	−0.030
chlorophyll (a+b)	0.986	0.063	−0.046
Carotenoid	0.975	−0.042	−0.051
Pn	0.928	0.176	−0.050
Tr	0.983	0.036	0.062
Gs	0.973	0.076	0.052
Ci	−0.448	0.824	−0.088
Fv/Fm	−0.058	0.520	0.753
ΦPSⅡ	0.889	0.280	−0.236
ETR	0.891	0.277	−0.234
qP	0.682	0.548	−0.440
NPQ	−0.880	0.320	0.025
Starch	0.933	−0.223	0.096
Soluble sugar	0.977	0.033	0.097
Sucrose	0.950	0.013	0.103
Fructose	−0.818	0.119	0.164
Glucose	−0.956	0.172	0.034
NO3−-N	0.942	−0.012	0.150
NH4+-N	−0.954	0.202	0.045
Soluble protein	0.956	−0.160	0.057
Free amino acid	0.920	0.024	0.112
NR	0.938	0.092	0.080
GS	0.928	0.152	0.136
GOGAT	0.973	−0.022	0.064
GDH	0.952	0.070	0.088

. PC1, accounting for 81.887% of the variance, is predominantly determined by large positive loadings from 25 variables: two growth indices—plant height (0.957) and stem diameter (0.960); four photosynthetic pigment indices—chlorophyll a (0.982), chlorophyll b (0.968), chlorophyll (a+b) (0.986), and carotenoid (0.975); three photosynthetic parameters—Pn (0.928), Tr (0.983), and Gs (0.973); two fluorescence parameters—ΦPSII (0.889) and ETR (0.891); three carbohydrate indices—starch (0.933), soluble sugar (0.977), and sucrose (0.950); and seven nitrogen metabolism indices—NO₃⁻-N (0.942), soluble protein (0.956), free amino acid (0.920), NR activity (0.938), GS activity (0.928), GOGAT activity (0.973), and GDH activity (0.952). Negative loadings with large absolute values were observed for NPQ (−0.880), fructose (−0.818), glucose (−0.956), and NH₄⁺-N (−0.954). All 25 variables had absolute factor loadings greater than 0.8, collectively defining principal component 1. PC2, contributing 6.399% of the variance, is primarily influenced by Ci (0.824) and qP (0.548). PC3 accounts for 3.738% of the variance and is mainly determined by Fv/Fm, with a loading of 0.753.

3.6. Comprehensive Evaluation of Growth, Photosynthesis, and Carbon–Nitrogen Metabolism-Related Parameters in Cucumber Under Different Treatments

Three principal components selected through principal component analysis (PCA) retained the main information regarding variations in cucumber growth, photosynthetic fluorescence, and carbon–nitrogen metabolism-related parameters. These three components effectively replaced the original 28 variables, representing the photosynthetic fluorescence and carbon–nitrogen metabolism status of cucumber. Using the variance contribution rates of the first, second, and third principal components—81.887%, 6.399%, and 3.738%, respectively—as weights, a comprehensive evaluation function for cucumber photosynthetic fluorescence and carbon–nitrogen metabolism-related parameters was constructed as follows: F = F1 × 81.887% + F2 × 6.399% + F3 × 3.738%, where F represents the composite evaluation score for cucumber under different treatments, reflecting the overall strength of growth, photosynthetic fluorescence, and carbon–nitrogen metabolism. A higher score indicates stronger photosynthetic fluorescence, enhanced carbon–nitrogen metabolism, and more vigorous growth. The composite scores (F) for cucumbers under different treatments are shown in

Table 5. Composite scores of growth, photosynthesis, and carbon–nitrogen metabolism-related parameters in cucumber under different treatments.

Treatments	Factor Scores			Comprehensive Score F	Ranking
	F1	F2	F3
CK	0.490	−0.179	−1.299	0.341	3
CK+Q	1.102	1.496	0.581	1.020	1
LCa	−0.473	−0.561	−0.248	−0.432	4
LCa+Q	0.922	−0.914	0.319	0.708	2
0Ca	−1.588	1.523	0.618	−1.180	6
0Ca+Q	−0.652	0.563	0.817	−0.467	5

and Figure 9, ranked from highest to lowest as follows: CK+Q, LCa+Q, CK, LCa, 0Ca+Q, and 0Ca. Treatments with B. amyloliquefaciens QST713 significantly improved cucumber growth, photosynthetic fluorescence, and carbon–nitrogen metabolism, effectively mitigating the damage caused by low-calcium stress.

4. Discussion

4.1. Effects of B. amyloliquefaciens QST713 on Cucumber Growth Under Low-Calcium Stress

Studies have shown that calcium deficiency severely inhibits root growth and development in plants. Symptoms initially appear in young leaf buds, including curling and deformation of leaf tips, chlorosis along the leaf margins, and malformed small leaves exhibiting white, hook-like shapes. Under severe calcium deficiency, the growing point may be damaged or necrotic, with white pectic substances appearing at leaf tips and shoot apices [3]. In the present study, the calcium deficiency symptoms observed in cucumber were consistent with these previously reported descriptions (Figure 2). Our experimental results indicate that cucumber growth is significantly suppressed under low-calcium stress, with notable reductions in plant height and stem diameter. These growth parameters decline further as the duration of stress increases. However, application of B. amyloliquefaciens QST713 alleviates the damage caused by calcium deficiency, resulting in significant increases in both plant height and stem diameter. Chen [18] reported that under salt stress, inoculation with B. amyloliquefaciens enhanced biomass accumulation in rice, maize, and Arabidopsis. The bacterium reduces stress-induced ethylene production by producing ACC deaminase and synthesizes substances such as indole-3-acetic acid (IAA) and cytokinins (CTK), which modulate plant hormone levels to promote growth and enhance stress tolerance. In this study, application of B. amyloliquefaciens QST713 under low-calcium stress enhanced cucumber tolerance to calcium deficiency. This effect is likely mediated through the modulation of plant hormone levels, which promotes plant growth, alleviates tissue damage caused by low-calcium stress, and reduces the energy expenditure required for stress defense, thereby ultimately facilitating cucumber growth and development.

In the design of this experiment, partial substitution of Ca(NO₃)₂ with NaNO₃ in the low-calcium treatments was applied to maintain consistent nitrate levels among treatments and thus avoid nitrogen limitation as an additional variable. However, this approach could, in principle, introduce a potential confounding factor, as excessive Na⁺ accumulation may cause ionic or osmotic stress, thereby affecting plant growth and metabolism. To address this concern, we analyzed the ionic composition of cucumber leaves under different treatments. Low-calcium stress significantly impaired ion uptake, as evidenced by a substantial reduction in leaf Ca²⁺ concentrations. Under severe calcium deficiency (0Ca), Mg²⁺ and K⁺ concentrations were also significantly reduced, likely due to root developmental abnormalities that diminished the capacity for mineral nutrient absorption and thereby indirectly lowered leaf Mg²⁺ and K⁺ levels. Importantly, leaf Na⁺ concentrations remained low (0.17–0.21 mg·g⁻¹ DW) across all treatments, with no significant differences among them. Analysis of the K⁺/Na⁺ ratio revealed a slight decrease under low-calcium stress; however, this decline was primarily attributable to reduced K⁺ concentrations rather than elevated Na⁺ levels. These findings indicate that the amount of Na⁺ introduced via NaNO₃ substitution was insufficient to induce sodium toxicity or osmotic stress, and its impact on plant performance can be considered negligible.

Morphological observations further support this conclusion. Under severe calcium deficiency, plants developed progressively severe and characteristic Ca-deficiency symptoms, including leaf margin curling, deformation, necrosis, and death of the shoot apical meristem—features distinct from the chlorosis and necrotic spotting typically associated with sodium injury. Thus, the growth inhibition observed in this study can be attributed primarily to calcium deficiency. Moreover, inoculation with B. amyloliquefaciens QST713 markedly increased leaf Ca²⁺, Mg²⁺, and K⁺ concentrations, and improved the K⁺/Na⁺ ratio under low-calcium conditions. These effects are likely mediated through multiple mechanisms, including modulation of plant hormone levels, promotion of root growth, enhancement of nutrient acquisition efficiency, and maintenance of ionic homeostasis, thereby enhancing cucumber tolerance to low-calcium stress and mitigating associated damage.

4.2. Effects of B. amyloliquefaciens QST713 on the Photosynthetic Fluorescence Characteristics of Cucumber Under Low-Calcium Stress

Chlorophyll a and b are the principal photosynthetic pigments, playing critical roles in the absorption and conversion of light energy during photosynthesis. Carotenoids, meanwhile, are closely involved in dissipating excess excitation energy, thereby enhancing the resistance of photosynthetic organs to high light intensity. Previous studies have demonstrated a strong correlation between calcium availability and both the content of photosynthetic pigments and the efficiency of photosynthesis in plant leaves [26]. Calcium is essential for maintaining the structural integrity and functional stability of chloroplast and mitochondrial membranes. A deficiency in calcium disrupts the grana lamellae within chloroplasts, ultimately leading to reduced photosynthetic performance [27]. Consistent with these findings, the present study observed a significant reduction in chlorophyll content in cucumber seedlings subjected to low-calcium stress, accompanied by a marked decline in photosynthetic capacity. Parameters associated with gas exchange, such as the net photosynthetic rate (Pn) and stomatal conductance (Gs), decreased significantly, while the intercellular CO₂ concentration (Ci) increased. This pattern suggests that the decline in photosynthesis under calcium deficiency is primarily due to non-stomatal limitations. Similar results were reported in Cucumis melo by Long et al. [28], who also attributed the reduced photosynthetic rate under calcium stress to non-stomatal factors. The application of B. amyloliquefaciens QST713 substantially enhanced photosynthetic performance in cucumber under calcium-deficient conditions. Numerous studies have reported that microbial biofertilizers can increase chlorophyll content in various crops, including naked oats [29] and flue-cured tobacco under continuous cropping systems [30]. Wang et al. [31] demonstrated that Bacillus subtilis promotes chlorophyll biosynthesis in wheat, thereby improving its photosynthetic efficiency. Similarly, root drenching with Bacillus polymyxa S960 increased chlorophyll content and the net photosynthetic rate in tomato leaves [32]. Research by Wang et al. [33] indicated that B. amyloliquefaciens HM618 may serve as a protective agent, mitigating cellular damage and enhancing pigment accumulation. In line with these findings, our study suggests that B. amyloliquefaciens QST713 may function as a protective microbial agent under low-calcium stress by alleviating cellular damage, improving the utilization of light energy, and promoting the development of photosynthetic structures. Consequently, chlorophyll accumulation and photosynthetic capacity were enhanced in cucumber seedlings, contributing to greater resilience under calcium-limiting conditions.

Changes in chlorophyll fluorescence can serve as sensitive indicators of plant responses to abiotic stress. Previous studies have shown that calcium deficiency during plant growth reduces the potential activity of PSII and the efficiency of primary photochemical energy conversion. This impairs the function of PSII reaction centers, thereby limiting the effective conversion of absorbed light energy into chemical energy in the leaves [34]. In the present study, cucumber plants subjected to low-calcium stress exhibited no significant changes in Fv/Fm, whereas ΦPSII and ETR were significantly reduced. NPQ increased markedly, while qP decreased slightly. The significant declines in ΦPSII and ETR suggest that the rate of electron transport across the thylakoid membrane downstream of the PSII reaction center was impaired under calcium-deficient conditions. The observed decrease in qP and increase in NPQ indicate that a smaller proportion of light energy absorbed by antenna pigments was utilized for photochemical electron transport, with a greater portion dissipated as heat. Recent studies have reported that microbial inoculants at appropriate concentrations can effectively enhance the ability of plant leaves to utilize light energy [35]. For instance, the application of microbial fertilizers in Cinnamomum camphora has been shown to improve PSII reaction center activity and enhance its reducing capacity [36]. In agreement with these findings, our study demonstrated that treatment with B. amyloliquefaciens QST713 improved Fv/Fm, ΦPSII, ETR, and qP in cucumber leaves under low-calcium stress, while significantly reducing NPQ. These changes indicate a more efficient allocation of absorbed light energy to photochemical reactions, thereby enhancing both electron transport and PSII photochemical efficiency, and ultimately promoting photosynthesis. As a plant growth-promoting bacterium (PGPB), B. amyloliquefaciens QST713 may partially protect PSII reaction centers, mitigating the decline in photochemical efficiency under calcium-deficient conditions. Consequently, it alleviated the adverse effects of calcium stress on photosynthesis and improved the plant’s tolerance to low-calcium environments.

4.3. Effects of B. amyloliquefaciens QST713 on Carbohydrate Metabolism in Cucumber Under Low-Calcium Stress

Carbohydrates are the primary products of photosynthesis, with starch serving as a major storage form and sucrose functioning as the principal transport form, supplying energy and carbon skeletons to various developing organs of the plant [37]. Under normal growth conditions, carbohydrates synthesized in aerial tissues via photosynthesis are translocated through the phloem to the roots, where water and nutrient uptake occurs. These are then transported via the xylem back to the shoots, supporting overall plant metabolism and development. However, under low-calcium stress, calcium deficiency severely disrupts both the biosynthesis and translocation of carbohydrates, thereby impairing normal plant growth and physiological function. In the present study, cucumber leaves under calcium-deficient conditions exhibited significant reductions in starch, sucrose, and soluble sugar contents. The decline in starch may be attributed to reduced photosynthetic activity and the resulting decrease in photoassimilate production. Alternatively, it may result from impaired downward translocation of assimilates, leading to increased consumption of stored starch to maintain metabolic homeostasis under stress. Calcium ions, recognized as secondary messengers in plant signal transduction, are known to regulate carbohydrate metabolism. Previous studies have shown that increasing calcium concentrations in hydroponically grown rice enhances starch and sucrose accumulation, along with the activities of sucrose synthase and starch biosynthetic enzymes [38]. In contrast, calcium deficiency compromises cell wall integrity and increases membrane permeability, thereby suppressing sucrose synthase activity and causing significant reductions in sucrose and other soluble sugars [39]. Application of B. amyloliquefaciens QST713 significantly increased the concentrations of soluble sugars, starch, and sucrose in cucumber leaves under low-calcium conditions. These results suggest that B. amyloliquefaciens QST713 enhances photosynthetic capacity and promotes the accumulation of photoassimilates, thereby alleviating the adverse effects of calcium deficiency. Furthermore, B. amyloliquefaciens QST713 may modulate carbohydrate metabolism under stress by regulating enzyme activities and facilitating the internal transport and conversion of sugars. Nonetheless, the detailed mechanisms by which B. amyloliquefaciens QST713 influences carbohydrate redistribution and transport under low-calcium stress remain to be elucidated. Interestingly, glucose and fructose levels were significantly elevated in cucumber leaves subjected to calcium deficiency, likely due to increased membrane permeability and heightened invertase activity, resulting in greater monosaccharide leakage [40]. This not only reduces photosynthetic efficiency and impairs the tricarboxylic acid cycle and glycolysis but also increases respiratory consumption and diminishes the plant’s capacity to translocate assimilates. Treatment with B. amyloliquefaciens QST713 reduced glucose and fructose accumulation in cucumber leaves, suggesting a role in stabilizing biomembrane structures, regulating key enzyme activities, and promoting the efficient conversion and utilization of sugars under stress conditions.

4.4. Effects of B. amyloliquefaciens QST713 on Nitrogen Metabolism in Cucumber Under Low-Calcium Stress

Nitrogen metabolism is a vital component of plant nutritional physiology, directly influencing the synthesis of proteins and nitrogen-containing organic compounds, and thereby playing a crucial role in plant growth and development [41]. In plants, nitrogen metabolism begins with the uptake of inorganic nitrogen, primarily in the form of nitrate (NO₃⁻-N), followed by its assimilation into organic nitrogen compounds through the action of key enzymes. Among these, nitrate reductase (NR), glutamine synthetase (GS), glutamate synthase (GOGAT), and glutamate dehydrogenase (GDH) are central to the nitrogen assimilation pathway. NR serves as a major rate-limiting enzyme in nitrogen metabolism, catalyzing the reduction of nitrate to nitrite and thereby establishing the biochemical foundation for nitrogen utilization in plants [42]. The GS/GOGAT cycle constitutes the primary route of nitrogen assimilation in higher plants and plays a pivotal role in converting inorganic nitrogen into organic forms [43]. This pathway is responsible for assimilating more than 95% of NH₄⁺-N within plant tissues [44]. Additionally, GDH offers an alternative route for ammonium assimilation and is primarily localized in the mitochondria of plant cells [45].

In this study, low-calcium stress significantly reduced nitrate (NO₃⁻-N) concentrations in cucumber leaves and impaired the assimilation of ammonium (NH₄⁺-N), resulting in its excessive accumulation. This disruption of nitrogen homeostasis was accompanied by pronounced reductions in free amino acid and soluble protein contents. These effects may be attributed to the inhibition of middle lamella formation and cell wall development under calcium-deficient conditions, which interferes with cell division and alters the structural integrity and composition of the cell wall, ultimately impeding nitrate uptake [46]. Moreover, the impaired conversion of NH₄⁺-N likely contributed to its toxic buildup, disrupting metabolic equilibrium and further reducing nitrogen-containing compound levels, thereby limiting plant growth. The activities of four key enzymes involved in nitrogen metabolism—NR, GS, GOGAT, and GDH—were all significantly reduced under low-calcium conditions. The observed decline in NR activity is likely associated with reduced nitrate uptake. Previous studies have shown that calcium deficiency in wheat seedlings decreases NO₃⁻-N absorption and suppresses NR activity, an effect attributed to calcium-dependent inhibition of protein synthesis [47]. Similar patterns were observed in severely calcium-deficient cucumber seedlings, where diminished nitrate uptake led to lower NO₃⁻-N content in the leaves and a corresponding decrease in NR activity [48], consistent with our findings. Ammonium assimilation depends on carbon skeletons derived from photosynthesis; therefore, NO₃⁻-N assimilation is thought to proceed in coordination with CO₂ fixation. When photosynthesis is inhibited, the transformation of nitrogenous compounds is also constrained. In this study, the activities of GS, GOGAT, and GDH were significantly suppressed under low-calcium stress, likely as a result of inhibited photosynthetic performance. Reduced transpiration under calcium-deficient conditions may further limit the supply of energy and assimilates, hinder the transport of water and ions to aerial parts, and impair nitrogen assimilation efficiency. Collectively, these factors contributed to the observed decline in nitrogen-assimilating enzyme activity.

Application of B. amyloliquefaciens QST713 enhanced nitrate (NO₃⁻-N) uptake and promoted the conversion between nitrate and ammonium forms, thereby facilitating the assimilation of ammonium (NH₄⁺-N) into organic nitrogen compounds. This transformation supported the synthesis of free amino acids and soluble proteins. Simultaneously, the activities of key nitrogen-metabolizing enzymes—NR, GS, GOGAT, and GDH—were significantly upregulated, maintaining nitrogen metabolic function, improving ammonium assimilation efficiency, and effectively preventing ammonium toxicity under stress conditions. These findings suggest that B. amyloliquefaciens QST713 enhances nitrogen mobility within plant tissues, ensuring adequate nutrient availability and mitigating growth inhibition caused by nutritional deficiency. Increased enzyme activity further accelerated nitrogen turnover, alleviating the negative impacts of calcium deficiency on nitrogen metabolism and enhancing cucumber tolerance to low-calcium stress. The beneficial effects of B. amyloliquefaciens QST713 may result from its capacity to improve photosynthetic performance, coordinate carbohydrate accumulation with nitrogen assimilation, and balance carbon skeleton supply with ammonium incorporation [49]. It is also plausible that B. amyloliquefaciens QST713 promotes ammonium assimilation in leaves by enhancing the conversion of NH₄⁺ into amides and amino acids, which are subsequently incorporated into nitrogen metabolic pathways, thereby reducing ammonium accumulation in leaf tissues. Supporting evidence from a study by Wang [50] demonstrated that methylotrophic Bacillus strains significantly increased GS and GOGAT activities in cucumber, enhanced C–N metabolic processes, and promoted the accumulation of downstream metabolites, ultimately accelerating plant growth. The results of the present study are consistent with these observations.

Further principal component analysis revealed a strong interconnection among cucumber growth, photosynthesis, and carbon–nitrogen metabolism, underscoring their mutual dependence. Under low-calcium stress, reduced photosynthetic capacity and disrupted carbon and nitrogen metabolism significantly suppressed plant growth. However, treatment with B. amyloliquefaciens QST713 enhanced photosynthetic performance and stimulated carbon and nitrogen metabolic activity under stress conditions, thereby promoting cucumber growth and mitigating the physiological damage induced by calcium deficiency. These findings suggest that B. amyloliquefaciens QST713 improves plant tolerance to low-calcium stress by maintaining photosynthetic efficiency and metabolic homeostasis. Further studies are needed to elucidate the molecular mechanisms through which B. amyloliquefaciens QST713 alleviates calcium deficiency stress in cucumber.

4.5. Limitations and Future Perspectives

In this study, nutrient solution irrigation and tightly controlled environmental conditions were employed to minimize external variability, thereby enabling a focused assessment of the regulatory effects of B. amyloliquefaciens QST713 on cucumber growth, photosynthesis, and carbon–nitrogen metabolism under low-calcium stress. While such a controlled system is valuable for establishing causal relationships between B. amyloliquefaciens QST713 application and plant responses, it represents a simplified model that may not fully reflect the complexity of actual greenhouse or field production environments. Under practical cultivation conditions, the performance of B. amyloliquefaciens QST713 could be influenced by multiple factors, including the composition and temporal dynamics of indigenous soil microbiota (which may interact synergistically or competitively with the introduced strain), environmental fluctuations (e.g., changes in temperature, humidity, and irrigation regimes), and the genetic background of cucumber cultivars, which may vary in their responsiveness to microbial inoculation. These factors have the potential to affect B. amyloliquefaciens QST713’s rhizosphere colonization efficiency, persistence, and its plant growth-promoting and stress-alleviating effects. Therefore, although our findings under controlled conditions provide foundational evidence for the ability of B. amyloliquefaciens QST713 to mitigate low-calcium stress in cucumber, multi-cultivar, and multi-environment trials—conducted in both greenhouse and open-field settings—are required to validate the consistency and scalability of its efficacy, thereby informing strategies for its optimization in sustainable cucumber production.

Furthermore, the present study primarily relied on physiological and biochemical measurements to infer that B. amyloliquefaciens QST713 may alleviate low-calcium stress by modulating specific growth and metabolic processes in cucumber. While these findings provide important insights into its potential mode of action, the precise molecular mechanisms—such as the expression profiles of stress-responsive genes, alterations in key enzymes at the proteomic level, and the involvement of specific signaling pathways—remain to be clarified. Future research integrating transcriptomic, proteomic, and metabolomic approaches will be essential to systematically elucidate the molecular processes through which B. amyloliquefaciens QST713 mitigates calcium deficiency stress, thereby enabling a more comprehensive understanding of its biological functions and guiding its precise application in agricultural practice.

5. Conclusions

In protected cucumber cultivation, low-calcium stress markedly inhibits growth by compromising photosynthetic performance, depleting carbohydrate reserves, and disrupting nitrogen metabolism, with severity escalating under more extreme calcium deficiency. Inoculation with B. amyloliquefaciens QST713 alleviates these effects by enhancing chlorophyll content, gas exchange, and PSII photochemical efficiency, thereby restoring starch and sucrose accumulation and stabilizing carbon supply. Concurrently, QST713 promotes nitrate uptake and upregulated key nitrogen-assimilating enzymes (NR, GS, GOGAT, GDH), facilitating ammonium assimilation, amino acid synthesis, and protein formation; when the GS/GOGAT cycle is impaired, it activates alternative glutamate synthesis pathways to prevent NH₄⁺ toxicity. Principal component analysis confirms that the recovery of plant height and stem diameter is intrinsically linked to these coordinated enhancements in photosynthesis and carbon nitrogen metabolism. In summary, our findings demonstrate that B. amyloliquefaciens QST713 effectively mitigates multiple physiological impairments in cucumber caused by low-calcium stress by enhancing photosynthesis and carbon–nitrogen metabolism. This not only significantly improves plant stress resistance but also deepens our understanding of the stress-alleviating functions of the B. amyloliquefaciens QST713 strain.

Although this study has elucidated the physiological effects and key metabolic pathways through which B. amyloliquefaciens QST713 alleviates low-calcium stress in cucumber, its underlying molecular mechanisms and broader application potential remain to be further explored. Building on the present work, future studies may investigate how QST713 regulates calcium signal transduction, the expression of photosynthesis-related genes, and the transcriptional and activity networks of key enzymes involved in carbon and nitrogen metabolism. Such efforts will provide a more comprehensive understanding of the mechanisms underlying the beneficial functions of QST713.