Compound Microbial Strains and Humic Acid Improve Physicochemical Properties of Salinized Soil and Physiological Characteristics of Oil Sunflower: An Experimental Investigation

Abstract

Soil salinization commonly prevails in global arid and semi-arid areas, shrinking farmland and endangering ecological, agricultural and social sustainability. Therefore, it is essential to develop effective strategies for salinized soil remediation. In this study, soil samples were collected from Nanliang Farm in Yinchuan, China. Compound microbial strains (CMS) and humic acid (HA) were selected as soil amendments. A total of eight treatments with different application rates of CMS and HA were set up in pot cultivation experiments, where oil sunflower was planted. The results showed that both amendments effectively elevated soil water content and chlorophyll content, as well as multiple physiological indices of sunflower. Meanwhile, they decreased soil total salinity, proline content and malondialdehyde (MDA) content. For single humic acid treatments, Treatment F1 achieved the optimal amelioration effect: it reduced soil total salinity by an average of 24.34%, and increased sunflower plant height, leaf area and aboveground fresh weight by 5.84%, 95.01% and 77.40%, respectively. Among the single CMS treatments, Treatment S3 performed best, with an average reduction of 31.04% in soil total salinity, and increases of 5.66%, 2.85% and 8.16% in plant height, leaf area and aboveground fresh weight correspondingly. Notably, among all eight groups, the control group CK1 exhibited the most prominent improvement effect, which was significantly superior to F1 and S3. This finding suggests that long-term application (one year or more) of CMS can produce an especially strong ameliorative effect on salinized soil.

1. Introduction

Salinized soil refers collectively to saline soils, alkaline soils, and soils affected by varying degrees of salinization and alkalization. Soil salinization has become one of the most severe environmental challenges globally and a major research hotspot in related fields [1]. According to incomplete statistics, more than 9.5 × 10⁸ hm² of land in the world is affected by soil salinization, which keeps deteriorating with climate change and irrational irrigation [2,3]. In China, the saline–alkaline land with agricultural utilization prospects totals about 6.67 × 10⁶ hm², and is predominantly distributed in North China [4]. Ningxia Hui Autonomous Region, as an inland region in the northwest of China, has a very dry climate and low precipitation, with an average annual rainfall of only about 200 mm. However, due to high evaporation rates and a shallow water table, the water carries a lot of salts to the soil surface through capillary action, which leads to soil secondary salinization. Consequently, saline and alkaline soils are widely distributed across the Ningxia Yellow River Irrigation Area.

Saline and alkaline conditions not only degrade soil quality and decrease soil permeability, but also lower agricultural productivity [5]. Therefore, it is of great practical significance to study how to improve and utilize saline–alkaline soils for the sustainable development of agriculture, economy and society.

To mitigate ongoing soil salinization, numerous management and remediation strategies involving different soil amendments have been developed by researchers [6,7,8]. Guo et al. used three soil amendments in field experiments and found that the addition of bio-amendments increased the soil organic matter and nutrient content, increased crop yields and significantly affected the soil [9]. Saidimoradi et al. studied the effect of HA on two strawberry varieties at different salinity levels, and found that after adding HA, the sodium content of the plant decreased, but a lot of indices, including the potassium content, the salinity tolerance, the relative water content of the leaves, and the membrane stability, etc., increased [10]. Sánchez et al. applied almond shells (AS) and almond shell biochar (ASB) to saline soils planted with arugula and found that the addition of a low percentage of AS and ASB can decrease soil pH and conductivity values, and can increase the soil moisture, organic matter and organic carbon [11].

In addition to the use of the above physical and chemical improvers, numerous bio-organic soil amendments have been found. They can improve soil quality, health, growth, yield and crop quality, and exert no adverse effects on the environment [12]. Lastochkina et al. reported that when Bacillus subtilis was applied to saline soils, the content of proline and MDA in wheat leaves decreased significantly compared with the control group and wheat growth was enhanced [13]. Jiang et al. investigated the effects of four fertilization methods on bacterial communities in saline–alkali soils along the coastal region of Jiangsu, China. The bio-organic fertilizer used in their study was a composite microbial inoculant containing 10⁷ beneficial microorganisms. Their results indicated that a combination of 70% bio-organic fertilizer and 30% chemical fertilizer was the optimal formulation for tomato cultivation in coastal saline–alkali areas [14].

Numerous studies have investigated the improvement effects of humic acid (HA) and microbial consortia on saline–alkali soils, with their efficacy commonly evaluated via crop growth indicators. Nevertheless, most of these investigations were conducted in specific regions, and the practical performance of the above amendments in the Yellow River Irrigation Area of Ningxia has not been fully validated.

Compound microbial strains (CMS) were adopted in this study to continue the research achievements of our senior team members and further explore their capacity to improve saline–alkali soils. Additionally, a literature review showed that relevant research on CMS application in Ningxia is insufficient: most studies concentrated on plant growth promotion by microbial inoculants, while few examined their remediation effects on saline–alkali soils.

Herein, HA and CMS were selected as soil amendments. A pot experiment was carried out to compare their ameliorative effects on saline–alkali soil sampled from Team 1 of Nanliang Farm (Xixia District, Yinchuan, Ningxia) and their influences on the growth of sunflower seedlings.

This study delivers multiple tangible contributions to agricultural, ecological and regional sustainability against the backdrop of widespread soil salinization in arid and semi-arid regions worldwide.

Soil salinization continuously degrades arable land, reduces crop productivity and undermines the stability of terrestrial ecosystems, posing long-term threats to food security and ecological sustainability. In this study, the remediation effects of CMS and HA on salinized soil are investigated. The two eco-friendly amendments can effectively reduce soil total salinity, improve soil water retention and optimize soil physicochemical properties, which helps rehabilitate degraded saline land, reuse problematic farmland, and control the trend of soil degradation, thereby safeguarding the sustainable utilization of soil resources.

From the perspective of crop production sustainability, salinization triggers salt stress in crops, inhibiting growth and disrupting physiological metabolism. Our research results demonstrate that CMS and HA can significantly increase chlorophyll content, plant height, leaf area and aboveground biomass of oil sunflower, while lowering the accumulation of proline and MDA induced by salt stress. Therefore, CMS and HA can effectively alleviate salt damage to crops, and improve crop stress tolerance and yield performance. Promoting this green regulation technology helps maintain stable crop production in saline areas, guarantee regional food supply and support the sustainable development of local agriculture.

In addition, in terms of environmental sustainability, CMS and HA are natural, low-cost and pollution-free soil amendments, differing from chemical improvers that may cause secondary soil pollution. The combined application of these biological and organic materials follows the concept of green agriculture and circular ecology. In particular, our research can further confirm that long-term application of CMS presents superior amelioration performance. It provides a novel, durable and environmentally friendly technical option for the long-term management of salinized farmland. The popularization of this technology can reduce the reliance on chemical inputs in saline land management, mitigate agricultural non-point source pollution, and maintain the health and stability of regional agro-ecosystems.

In summary, the research results can not only enrich the theoretical research on saline soil remediation and crop salt resistance regulation, but also offer a practical, sustainable technical solution for the improvement and sustainable utilization of salinized soil in arid and semi-arid zones. It integrates soil protection, crop production and ecological conservation, and complies with the core goals of environmental and agricultural sustainability.

2. Materials and Methods

2.1. Overview of the Test Area

Experiments were carried out from May to September 2023 in the outdoor experimental shed of the Fluid Mechanics Laboratory at North Minzu University. By analyzing the meteorological data from the laboratory weather station during the seedling period of oil sunflower (from May to July), the average air temperature at the experimental site was found to be 24.83 °C, and the minimum and maximum air temperatures were 8.31 °C and 38.93 °C, respectively, with a large temperature difference between day and night. The average air humidity, sunshine hours and evapotranspiration rate were 35.16%, 251.26 min, and 52.14 mm, respectively.

2.2. Test Materials

The saline–alkali soil used in this experiment was collected from the 0–20 cm surface layer at the Experimental Station of Team 1, Nanliang Farm, Yinchuan City, Ningxia Hui Autonomous Region. The sampling coordinates were 38°1′10″ N, 106°13′35″ E, and the sample was collected in September 2022. The soil is a sandy loam and is classified as moderately saline–alkali, with a dry bulk density of 1.51 g/cm³. The following two soil amendments were used in this study.

• CMS is a yellowish-brown liquid, and a bacterial preparation independently developed by the research group led by Professor Xinhui Zhang at Ningxia Medical University, China. It is formulated from Bacillus subtilis and Bacillus cereus in a 1:1 ratio (i.e., a preparation composed of these two bacterial species). Bacillus subtilis strain CGMCC No. 16879 was previously isolated, purified, and identified from licorice root by the research group; Bacillus cereus was prepared in the laboratory by the same group. Bacteria were cultured on Nutrient Agar at 30 °C. The total viable count of fermentation broth was 10⁸–10⁹ CFU/mL. The inoculant was produced via strain activation and submerged liquid fermentation followed by sterile mixing. Liquid inoculants were stored at 4 °C, and solid preparations were preserved at room temperature in cool and dry conditions with a shelf life over 6 months. The survival rate in soil reached 60–85% within 7 days, and the strains maintained long-term colonization relying on endospores. Species identification was verified by 16S rRNA gene sequencing. Both are Gram-positive spore-forming bacilli with stable phenotypic traits. The strains exhibit prominent capacities of phosphate solubilization, ACC deaminase activity and EPS synthesis, acting as typical plant-growth-promoting rhizobacteria (PGPR). The used Bacillus cereus is a non-pathogenic rhizosphere strain without hemolytic activity or pathogenicity, which is safe for field application.
• HA is a black-brown granular product manufactured by Shenzhen Dugao Bio-New Technology Co., Ltd., Shenzhen, China. As a soil amendment, it contains humic acid (HA) ≥ 65%, fulvic acid (FA) ≥ 30% and potassium oxide (K₂O) ≥ 12%, with a pH value ranging from 9 to 11.3.

The compound fertilizer used in this study was mainly composed of nitrogen (N), phosphorus pentoxide (P₂O₅) and potassium oxide (K₂O), with respective contents of 15%, 15% and 15%.

The test plant is an oil sunflower, whose variety number is LD1003, and seeds were provided by Beijing Kafry Technology Co., Ltd., Beijing, China.

2.3. Experimental Design

In the pot experiments, the oil sunflower was chosen as the planting crop, and seven treatments and a control group (Case CK) were designed, as shown in

Table 1. Application rates of CMS and HA in soil.

Cases	Soil Amendments	Application Rate of the Amendments
CK	the control group without using any soil amendments	0
CK1	using the soil sample that has been improved by CMS for one year	2.0 g/kg
F1	the group using the soil amendment of HA	1.5 g/kg
F2	the group using the soil amendment of HA	3.0 g/kg
F3	the group using the soil amendment of HA	4.5 g/kg
S1	the group using the soil amendment of CMS	1.5 g/kg
S2	the group using the soil amendment of CMS	3.0 g/kg
S3	the group using the soil amendment of CMS	4.5 g/kg

Note: “CK” stands for “control group without using any soil amendments”; “CK1” stands for “control group using the soil sample that has been improved by compound microbial strain for one year”, “F” stands for “the group using the soil amendment of HA”, and “S” stands for “the group using the soil amendment of CMS”.

. Each case was repeated three times, so there were 24 pots used for the test, and all the pots were randomly placed in the outer experimental shed. The pots were 19 cm in diameter and 20 cm in depth, and the bottoms of the pots were fitted with pot holders. Each pot was filled with 7.5 kg of air-dried soil passed through a 2.5 mm sieve, packed to a dry bulk density of 1.41 g/kg. The basal fertilizer used was farmyard manure (cow manure) at a rate of 10 g/kg; compound fertilizer was selected as the seed fertilizer at a rate of 0.1 g/kg. The farmyard manure was applied as a basal fertilizer one week prior to the experiment, while the seed fertilizer was applied at the time of sowing; no additional fertilizers were applied thereafter. After sowing, watering was performed every 2–5 days depending on daily temperatures, using fresh water at a volume of 400–550 mL per application. Thinning and final selection were conducted when the second pair of leaves on the seedlings had fully unfolded (27 May 2023), retaining three sunflower plants of consistent growth per pot. Physiological measurements of the sunflowers were taken on 20 June 2023, including total soil salinity, nutrient content, stem diameter, fresh aboveground weight, leaf area, number of leaves, chlorophyll content, and MDA and proline content.

2.4. Measurements and Methods

• Soil physical and chemical properties

In 2023, about 30 d before plowing, the soil was sampled by the five-point sampling method, and the average value was taken as the index of soil physical and chemical properties of the experimental field in the current year, as shown in

Table 2. Physical and chemical properties of the soil samples.

P (mg/kg)	K (mg/kg)	TN (%)	pH	OM (g/kg)	Total Salt (g/kg)	Salt Ion Content in Soil (g/kg)
						Na+	Mg2+	K+	Ca2+	Cl−	SO42−	CO32−	HCO3−
38.3	196	0.028	8.1	3.76	5.6	1.2	0.09	0.06	0.19	0.8	2.0	0.034	0.14

Note: 1. TN: total nitrogen, OM: organic matter. 2. Phosphorus in the table was measured using the Kjeldahl method.

.

2.

Measurement of the dry bulk density and water content in the soil

Cutting rings were employed to take soil samples, and the dry bulk of the soil density was calculated as follows:

$${\rho }_b={\displaystyle \frac{M-M_0}{V}},$$

(1)

where ${\rho }_b$ is the dry bulk weight of the soil (g/cm³); $M$ is the total mass of the cutting ring and the dry soil (g); $M_0$ is the mass of the cutting ring (g); and V is the volume of the cutting ring (cm³).

Soil water content was measured by the drying method. The soil sample was weighed and then weighed again after being placed in an oven at 105–110 °C to dry for more than 8 h. The soil water content can be calculated using the following formula:

$${\theta }_m={\displaystyle \frac{g_w-g_s}{g_s}},$$

(2)

where $g_w$ is the wet weight of the soil sample and $g_s$ is the dry weight of the soil sample.

3.

Soil moisture characteristic curves

Soil moisture characteristic curves were measured at the Fluid Flow Laboratory of the University of Northern Nationalities (Figure 1) using a pressure film instrument consisting of three parts, pressure chamber, drainage and measurement system, and pressure regulating system, with a maximum pressure of 1500 kPa. The soil samples taken were passed through a sieve and the appropriate improvers were added, and then they were loaded into a ring cutter and placed on a terracotta plate at 1.41 g/cm³ to begin the measurements after they had been fully saturated.

4.

Salinity and nutrient measurement

The soil salinity and nutrient levels mentioned in this paper were measured using soil sensors from a soil moisture monitoring instrument. To take a reading, simply insert the soil sensor into the soil; various soil data, including soil salinity and nutrient levels, can then be viewed in real time on a mobile device or computer. This soil monitoring instrument was purchased from Beijing Mengchuang Weiye Technology Co., Ltd., Beijing, China.

5.

Chlorophyll, proline, and MDA measurement

(a)

Chlorophyll: Fresh leaf samples were thoroughly ground in the dark using 80% acetone and centrifuged at 9000× g for 10 min at 4 °C. Absorbance readings at 645 nm and 663 nm in the collected supernatant were used to estimate total chlorophyll content.

(b)

MDA: Measured spectrophotometrically using the thiobarbituric acid (TBA) assay, with absorbance readings at 450 nm, 532 nm, and 600 nm using a UV spectrophotometer, which was manufactured by Shanghai Mapada Instruments Co., Ltd., Shanghai, China.

(c)

Proline: Determined using the acidic ninhydrin colorimetric method. Absorbance was measured at wavelengths of 520 nm and 625 nm using the UV spectrophotometer with pure proline as the standard.

6.

Indicators to evaluate oil sunflower

(a)

Overground fresh weight

The overground fresh weight of the sunflowers was measured by the following steps: firstly, cut the overground parts of the sunflowers with scissors; secondly, wash them with distilled water; thirdly, dry them in an oven at 105 °C for 15 min; fourthly, dry them in the oven at 75 °C for 10 h; finally, weigh them with an electronic balance.

(b)

Plant height

The plant height was measured with a band tape, from the bottom to the top of the plant.

(c)

Stem thickness

Stem thickness of each treatment was measured with a vernier caliper.

(d)

Leaf parameters

Leaf parameters include number of leaves, leaf length, leaf width and leaf area. The number of leaves of the sunflower can be directly counted; the leaf length is the length from the base to the tip of the leaf; the leaf width is the length at the widest part of the leaf; and the leaf area is calculated by following formula:

$$A=L\times W\times 0.65,$$

(3)

where $A$ is the area of a single leaf in cm², $L$ is the leaf length in cm, and $W$ is the leaf width in cm.

2.5. Data Processing

IBM SPSS Statistics 27.0.1 was used to perform a one-way analysis of variance (ANOVA) on the data and to conduct a significance test, with a sample size of n = flower pots; Origin 2022 was used for graphing. In Section 3.4, nutrient content was measured using a soil moisture monitoring system.

3. Experimental Results

3.1. Soil Moisture Characteristic Curves

Soil moisture characteristic curves reflect the relationship between soil water potential and soil volumetric water content, and can indirectly reflect the water retentiveness and the porosity of the soil. This relationship is one of the most important soil hydraulic properties, and the key to studying the improvement of soil salinization [15]. In this study, the soil moisture characteristic curves were measured by a pressure film instrument at the Fluid Mechanics Laboratory in North Minzu University (Figure 1).

Under the condition of the same water content, the further right the curve is located, the higher the soil water content, which indicates that the soil water-holding capacity is better, and vice versa. Figure 2 shows the soil moisture characteristic curves measured by the pressure film instrument for different treatments. It can be seen from Figure 2 that the soil moisture characteristic curves with different treatments have a similar change trend, and the soil water content decreases with increasing water suction. After using CMS (Cases S1–S3), the water-holding capacity of the soil was significantly improved, and was significantly greater than that of control group. The water-holding capacity when using HA (Cases F1–F3) was better than that of Case CK, when the water suction was below 600 kPa. The water-holding capacity of the soil also decreased with the increased use of HA. It can also be found from Figure 2 that the enhancement effect of using CMS on the water-holding capacity of the salinized soil used in this experiment is significantly better than that using HA.

Soil moisture characteristic curves can reflect the soil structure, such as soil porosity. It can be concluded that the content of clay particles in the soil increased significantly after using CMS and HA, and the small pores in the soil developed continuously and were distributed more uniformly, which led to the continuous enhancement of soil water-holding capacity. HA contains hydrophilic functional groups, which can effectively adsorb water molecules in the soil, while the acid produced by CMS in the soil can dissolve the metal ions on the surface of the minerals; as a result, the soil micropores decrease, and the soil structure improves, which strengthens the soil water-holding properties. However, the water-holding performance decreases with increased use of the above two soil improvements. The excessive use of HA can make the soil over-dispersed and reduce the soil cohesion, so that water cannot be effectively retained. Similarly, excessive use of CMS can increase the negative charge on the surface of soil particles, which can increase the pore diameter, and can lead to soil water being easily lost.

3.2. Soil Water Content and Salinity

Water content and total salinity of the soil were measured at different depths of the pots the day after each irrigation for three measurements. The water content of the soil at the surface layer (0–10 cm) and the bottom layer (10–20 cm) increased with increasing use of the improver HA, and it was significantly greater than Case CK (Figure 3a). The soil water content after use of CMS had a similar pattern to the above group. In the surface layer, the soil water content difference among various soil treatments was small, but at the bottom layer (10–20 cm) of the pots, the difference was large. At the bottom layer, compared to Case CK, the water content increased by 40.1%, 57.95%, and 75.21% for the group using HA, i.e., Cases F1, F2, and F3, respectively, but it increased by 19.35%, 23.63%, and 35.53% for the group using CMS, i.e., Cases S1, S2, and S3, respectively.

At the surface layer, the total salt of the two treatments (CMS and HA) was significantly reduced (Figure 3b), in which Case CK1 was the most significant, and the total salt was reduced by 82.98%. As shown by Figure 3b, for the treatment group using HA, Case F1 had the best desalinization effect and the total salt decreased by 29.28% compared with Case CK. For the group using CMS, Case S3 had the best desalinization effect, which reduced the total salt by 37.24% compared with Case CK. With the increasing use of the two improvers, the total soil salt of the group using HA also increased, while that of the group using CMS (Cases S1–S3) decreased.

At the bottom soil layer, the total soil salt of all treatments was lower than that of Case CK except for Case F3 (Figure 3b). Among them, Case CK1 had the best effect of desalinization, reducing the total salt by 62.22% compared with Case CK, and the effect was very significant. Except for Case CK1, the best treatment that can decrease the total soil salt is Case S3 (application rate of CMS is 4.5 g/kg).

3.3. The Soil Nutrients

From Figure 4, it can be found that the contents of nitrogen (N), phosphorus (P), potassium (K), ammoniacal nitrogen (AN) and nitrate nitrogen (NN) at both 0–10 cm and 10–20 cm soil layers of the pots increased significantly after using CMS and HA, and the soil nutrients at the bottom layer were significantly higher than in the surface layer. The reason is that the bottom layer of the soil has more organic matter than that the surface layer after planting the sunflowers, and the organic matter can easily develop into soil nutrients.

In order to facilitate the analysis, the growth rates of soil nutrient contents at different depths of each treatment group were calculated and listed in

Table 3. Nutrient growth rate at different depths for each treatment group.

Treatments	Depths/cm	N/%	P/%	K/%	AN/%	NN/%
CK1	0–10	27.82 ± 1.24	22.41 ± 1.89	28.29 ± 2.35	27.97 ± 1.21	28.14 ± 2.41
	10–20	35.64 ± 5.51	34.74 ± 2.37	34.43 ± 3.81	36.60 ± 4.56	35.88 ± 6.01
F1	0–10	8.56 ± 2.01	8.57 ± 3.50	8.56 ± 4.28	8.54 ± 7.20	8.56 ± 5.27
	10–20	8.92 ± 3.30	8.89 ± 5.21	8.91 ± 5.44	8.91 ± 6.57	8.92 ± 8.32
F2	0–10	17.65 ± 1.77	17.65 ± 2.64	17.65 ± 7.45	17.67 ± 5.52	17.64 ± 4.22
	10–20	21.88 ± 3.36	21.87 ± 3.69	21.88 ± 7.52	21.90 ± 4.32	21.88 ± 4.47
F3	0–10	23.72 ± 4.28	23.74 ± 3.91	23.72 ± 4.58	23.70 ± 6.54	23.71 ± 4.35
	10–20	50.66 ± 2.54	50.64 ± 2.58	50.65 ± 1.26	50.68 ± 7.56	50.66 ± 4.54
S1	0–10	21.95 ± 4.21	21.96 ± 2.24	21.95 ± 2.68	21.94 ± 4.52	21.95 ± 1.89
	10–20	19.28 ± 1.25	19.26 ± 4.20	19.28 ± 3.22	19.29 ± 3.57	19.28 ± 5.68
S2	0–10	34.13 ± 2.28	34.13 ± 5.30	34.13 ± 2.55	34.17 ± 2.69	34.12 ± 4.67
	10–20	26.19 ± 2.87	31.18 ± 2.36	24.76 ± 3.64	38.67 ± 2.51	26.46 ± 2.98
S3	0–10	39.94 ± 2.14	38.94 ± 2.18	44.44 ± 3.66	40.69 ± 2.85	39.61 ± 3.65
	10–20	58.44 ± 1.11	48.51 ± 1.84	35.61 ± 5.46	61.60 ± 7.55	51.66 ± 6.54

. Tt can be seen that in the surface layer, the highest increase of nitrogen, phosphorus and potassium content was in Case S3, while the lowest increase was in Case F1; in bottom layer, the highest increase in nitrogen content was also in Case S3, while the lowest increase was in Case F1. For the content of phosphorus and potassium, the highest increase was in Case F3, and the lowest increase was in Case F1. For both improvers at the same application rate, the N, P and K contents were higher after using CMS in all treatments, except for Case S3 which had slightly lower phosphorus and potassium contents at 10–20 cm than HA treatment. It can be seen that CMS can provide more N, P and K than HA at the same application rate.

AN and NN are two common nitrogen fertilizers that can be directly absorbed and utilized by plants, and they are both essential nutrients for plant growth. At the surface layer, the contents of ammoniacal nitrogen and nitrate nitrogen increased under each treatment compared to Case CK (Figure 4), where the contents of ammoniacal nitrogen increased by 27.97%, 8.54%, 17.67%, 23.70%, 21.94%, 34.17%, and 40.69%, respectively, while the content of nitrate nitrogen increased by 28.14%, 8.56%, 17.64%, 23.71%, 21.95%, 34.12%, and 39.61%, for CK1, F1, F2, F3, S1, S2, and S3, respectively. At the bottom layer, the content of soil ammoniacal nitrogen for Cases CK1, F1, F2, F3, S1, S2, and S3 increased by 36.60%, 8.91%, 21.90%, 50.68%, 19.29%, 38.67%, 61.60%, and nitrate nitrogen increased by 35.88%, 8.92%, 21.88%, 50.66%, 19.28%, 26.46%, 51.66%, respectively. Therefore, it can be concluded that HA and CMS can promote ammoniacal nitrogen and nitrate nitrogen in the salinized soil, and CMS is superior to HA in terms of the nutrients.

3.4. The Growth Indicators of Oil Sunflower

Leaf length, width, and area are three important indicators of oil sunflower growth, and leaf area is more representative than leaf length or leaf width. Therefore, leaf area was chosen, along with plant height, stem width, number of leaves, and aboveground fresh weight, as a key indicator to discuss oil sunflower’s growth features.

During the seedling stage of the oil sunflower growth, with the increasing use of HA, the height of the oil sunflower plant decreased continuously (

Table 4. Seedling physiological indicators of sunflower under different treatments.

Treatments	Plant Height (cm)	Stem Thickness (mm)	Number of Leaves (Piece)	Leaf Length (cm)	Leaf Width (cm)	Leaf Area (cm2)
CK	16.09 ± 1.16 bc	3.12 ± 0.07 c	6.33 ± 1.25 b	4.93 ± 0.36 bc	3.08 ± 0.18 c	9.83 ± 1.3 c
CK1	45.47 ± 3.74 a	6.65 ± 0.12 a	8.86 ± 0.15 a	9.16 ± 0.15 a	5.98 ± 0.18 a	35.72 ± 1.05 a
F1	17.03 ± 1.58 b	4.00 ± 0.7 b	7.67 ± 1.25 ab	5.90 ± 0.99 bc	4.13 ± 0.82 b	19.17 ± 2.89 b
F2	11.63 ± 3.23 c	2.72 ± 0.5 c	7.17 ± 0.94 ab	4.54 ± 0.65 c	2.88 ± 0.61 c	8.78 ± 3.21 c
F3	11.27 ± 2.66 c	2.81 ± 0.36 c	5.83 ± 1.12 b	4.27 ± 0.86 c	2.61 ± 0.41 c	7.44 ± 2.52 c
S1	13.93 ± 1.72 bc	2.83 ± 0.28 c	6.13 ± 0.12 b	4.47 ± 0.27 c	2.74 ± 0.13 c	7.98 ± 0.79 c
S2	16.20 ± 1.02 bc	3.18 ± 0.23 bc	6.33 ± 0.24 b	4.78 ± 0.12 bc	3.08 ± 0.15 c	9.59 ± 0.74 c
S3	17 ± 1.27 b	3.29 ± 0.3 bc	7.17 ± 0.62 ab	4.97 ± 0.39 bc	3.13 ± 0.25 c	10.11 ± 1.54 c

Note: Different alphabet notations indicate significance at p < 0.05 by default. Letter a, b, c on the bars indicate significant differences (p < 0.05). The letter a is assigned to the group with the maximum mean value (the optimal group), followed by b, c. A group marked with “ab” means it shows no significant difference from all groups labeled “a, b”.

), from 17.03 cm for Case F1 to 11.27 cm for Case F3, with only Case F1 increasing 5.84% in comparison with Case CK. In the CMS treatment group, the plant height of Case S2 and Case S3 increased by 5.66% and 0.68%, respectively, but Case S1 decreased by 13.42% compared to Case CK. From the table, it can also be seen that the plant height of Case CK1 increased by 182.6% compared to Case CK, which was very significant (p < 0.05).

Cases CK1, F1, S2 and S3 can promote the stem width of the sunflower, where the stem thickness increased by 113.14% for Case CK1, and it increased by 28.21%, 5.45% and 1.92% for Cases F1, S3 and Case S2, respectively. However, under other treatments (Cases F2, F3 and S1), the stem width of the sunflower decreased compared to Case CK, where Case F2 decreased by 12.82%, Case F3 decreased by 9.94% and Case S1 decreased by 9.29%. It can be concluded that except for Case CK1, the treatments can mildly promote or inhibit the growth of the sunflower based on the stem width.

Leaf area is very important for the growth of plants, and a large leaf area indicates that the chlorophyll content in the leaves is relatively high, so the plant can photosynthesize better and accumulate more organic matter. From Table 4, it can be found that the number of leaves and leaf area in Case CK1 increased significantly (p < 0.05). In Case F1, the number of leaves increased by 21.17% and leaf area increased by 95.01%, and the number of leaves and leaf area in Case S3 also increased a little. The remaining treatments showed a decrease in number of leaves and leaf area except for Case F2 which showed an increase in number of leaves; the least number of leaves and lowest leaf area were in Case F3 which showed a decrease of 7.9% and 24.31%, respectively, compared to the control group. With the increase in the two soil amendments, the changes in leaf number and leaf area had the same pattern as plant height and stem thickness; i.e., HA was increasing and leaf number and leaf area were decreasing, whereas it was the opposite in the CMS treatment group.

Aboveground fresh weight is one of the most important physiological indicators of plants. In this study, a histogram was created by measuring the aboveground fresh weight of oil sunflower at seedling stage, as shown by Figure 5. As can be seen from the figure, compared to Case CK, only Cases CK1 and F1 showed a significant increase in aboveground fresh weight (p < 0.05). Case CK1 was about 4.3 times the aboveground fresh weight of the control group, with an increase of 324.8%; after one year of the saline soil improved by CMS, there was an exceptionally significant effect on the aboveground fresh weight of the oil sunflower. And in an experimental period, CMS had little effect on the aboveground fresh weight of oil sunflower, and the difference was not significant (p < 0.05). However, HA can significantly increase aboveground fresh weight of oil sunflower. Compared to Case CK, Case F1 increased the aboveground fresh weight by 77.4%, while Case F2 and Case F3 decreased the weight by 42.22% and 45.45%, respectively.

It is concluded that FA applied at 1.5 g/kg (Case F1) exerts a better promoting effect on sunflower growth than CMS. With the increase in FA application rate (Cases F2 and F3), sunflower physiological indicators such as plant height, stem thickness, leaf number, leaf length, leaf width and leaf area all decline. On the contrary, these indicators increase as CMS application rate rises.

3.5. The Chlorophyll, MDA, and Proline of the Oil Sunflower

Chlorophyll is a key molecule for plants to carry out photosynthesis, and it can convert light energy into chemical energy and promote plants to carry out photosynthesis to produce oxygen and organic matter, while proline and MDA can reflect the physiological state and emergency response of plants to a certain extent, so they can reflect the improvement effect of the soil amendment. With increasing HA, the chlorophyll decreased continuously (Figure 6a). In Case F1, the chlorophyll was slightly higher than that of Case CK, while in Cases F2 and F3, it was lower than that of Case CK. In CMS treatments, the chlorophyll of Case S2 was less than that of Case CK, but in Cases S1 and S3, it was higher than that of Case CK. Therefore, CMS can improve the content of chlorophyll, and its effect is better than that of HA treatments. Figure 6b,c show that the change trend of proline and MDA content is the same, and their order is CK1 < S3 < S2 < F1 < S1 < F2 < CK < F3. Furthermore, the content of proline and MDA in the CMS treatment group was lower than that of Case CK, whereas in the HA treatment group, except for in Case F3, where it was slightly higher than that of Case CK, it was lower than that of Case CK.

The above phenomenon indicates that both soil amendments can basically reduce the content of proline and MDA in plants and effectively alleviate the adverse effects of salt stress on plants, and the effect of CMS treatment was significantly better than that of HA treatment.

4. Discussion

4.1. Soil Structure and Water-Holding Capacity

Good soil structure is the key to improving crop yields in degraded soil [16]. The soil used in this study was a mildly salinized sandy loam, which is highly susceptible to scouring and has poor water-holding capacity. After using the soil amendments in suitable amounts, the water-holding capacity of the soil was greatly improved due to the improvement of the soil structure. HA is an organic substance that can adhere to soil particles and increase the stability of aggregates, which makes the soil porosity increase and the weight capacity decrease, allowing more water to be stored in the small pores and slowly released for plant use [17]. Furthermore, HA possesses numerous hydrophilic functional groups, enabling it to absorb water and greatly improve the water retention capacity of soil [18]. In this study, HA application markedly raised soil water content, particularly in the 10–20 cm layer, with increases of 40.1%, 57.95% and 75.21% for the three treatments, respectively (Figure 3a). Plant root systems play an important role in promoting soil aggregate formation. HA can effectively improve root development. In addition, HA benefits soil fungal mycelium, which is another driver of aggregate formation [19]. It has been proved that soil fungal mycelium can combine tiny agglomerates to form large agglomerates, and the vertical migration of HA can promote the plant root system and the fungal mycelium in the soil profile, leading to increased agglomerates [20].

CMS markedly enhances soil water-holding capacity and optimizes soil pore structure. Research indicates that Bacillus subtilis improves soil porosity: the polysaccharides and glycoproteins secreted by CMS coat soil particles, bind them together, and form stable soil aggregates [21]. In this study, soil water content generally increased after using CMS, which can decompose organic matter in the soil, improve the soil structure and increase soil water retention (Figure 3a). In arid conditions, Bacillus strains within CMS conserve soil water through metabolism and sustain higher water use efficiency [22].

4.2. Soil Salinity and Nutrients

Excessive soil salinity accumulates harmful ions, leading to soil crusting and nutrient leaching. As eco-friendly organic amendments, HA and CMS have been widely used for the remediation of saline soils in recent years, owing to their excellent amelioration performance. Rich in organic acids, HA neutralizes soil alkalinity and reduces soil pH. Furthermore, HA contains diverse organic acids with abundant active functional groups (carboxyl, hydroxyl and amide groups), which can boost soil cation exchange capacity, adjust acid–base equilibrium and decrease soil salinity [23]. HA is also rich in organic matter, which increases soil aggregation and water-holding capacity, increases soil water content, and reduces evaporation and drainage losses, and thus is able to alleviate salt stress. However, overuse of HA is detrimental. It fails to alleviate soil salinity and may lead to adverse impacts as well as unnecessary waste. The results in this study show that using HA in the soil can lead to a significant increase in soil nutrient content (Figure 4), which is attributed to the higher organic matter content of HA. In addition, HA can promote the formation of large aggregates, which segregates soil nutrients, prevents microbial decomposition and leaching, and improves the overall efficiency of nutrient sources, which is one of the main reasons for the increase in soil nutrients [24]. Moreover, HA features a higher cation exchange capacity (CEC) and a wider variety of functional groups. These favorable properties enable soil to retain essential nutrients including potassium, calcium and magnesium, making them more readily available for plant uptake [25].

In this study, the contents of N and P increase significantly after using CMS (Figure 4). This is because it releases nitrogen to optimize the nitrogen conditions of saline soil, and indirectly modulates bacterial activity, thereby boosting nitrogen and phosphorus transformation [26]. In addition, CMS participates in the decomposition of organic matter. This process releases nutrients such as N, P and K for plants to absorb and utilize. CMS is widely used as a biofertilizer thanks to its strong nitrogen-fixing capacity. It reduces nutrient loss and replenishes the pool of organic nitrogen in soil [27]. With the increase in CMS, the salinity of the soil at different depths kept decreasing (Figure 3), which indicates that CMS can effectively reduce the soil salinity. Studies have demonstrated that inoculation with rhizosphere bacteria can promote plant growth and yield under normal soil conditions. Meanwhile, these bacteria mitigate salt-induced damage by lowering ethylene production under salt stress, which is likely a key factor behind the marked reduction in salt accumulation in plants [28]. Xiao et al. found that strain G2 can effectively promote carbohydrate accumulation and enhance salt tolerance, thereby facilitating the growth of Citrus aurantium seedlings under salt stress. CMS is rich in Bacillus sp. G2, a highly salt-tolerant strain. It can survive and proliferate in high-salinity environments and exhibits strong salt stress alleviation capacity [29].

4.3. Physiological Indicators of Oil Sunflower

High salt levels disrupt cell wall metabolism in plants, lowering cell wall elasticity, stiffening cell walls and gradually stunting plant growth [30]. Plant height, leaf area and aboveground fresh weight in Case F1 (HA 1.5 g/kg) increased significantly by 5.84%, 95.01% and 77.4%, respectively, relative to Case CK (Table 4). Similar results were reported by Wu Yun et al., who observed that HA effectively improved lily plant height, leaf area and stem thickness [31]. The effect may be attributed to HA addition: it lowers soil bulk density and enlarges soil pores, thereby promoting water and nutrient uptake in oil sunflower. In addition, HA increases the permeability of cell membranes, which increases water and nutrient uptake and increases the ability of roots to absorb water and nutrients, thus enhancing plant growth and fresh weight. In the CMS treatment group, aboveground fresh weight increased by 324.8% (Case CK1) and 8.16% (Case S3) (Figure 5). This indicated that CMS could promote the growth of oil sunflower, and the effect was more significant after one year of application (Case CK1). This is because Bacillus requires a period of time to grow and proliferate in soil. After one year of CMS application, the salinized soil was completely ameliorated, creating a favorable environment for sunflower cultivation. By producing plant-growth-stimulating compounds and activating soil nutrients, Bacillus enhances nutrient uptake in oil sunflower and accelerates its growth [32].

4.4. Chlorophyll, Proline and MDA

Chlorophyll is the key for photosynthesis in plants, and its amount is related to the efficiency of the plant in producing organic matter, while proline and MDA can reflect the physiological status and emergency response of the plant to some extent. Soil amendment with HA elevated chlorophyll levels in sunflower leaves (Figure 6a), possibly by stimulating chlorophyll synthesis and retarding its degradation [33]. HA is rich in amino acids, and the increased chlorophyll level may also be due to the stimulatory effect of amino acids on chlorophyll biosynthesis and the decrease in chlorophyll degradation, and amino acids may inhibit the peroxidation and degradation of cellular constituents (especially chlorophyll), which prolongs the cellular lifespan [34]. As shown in Figure 6a, CMS application increased the chlorophyll content of oil sunflower by 16.06% in CK1, 15.69% in S1 and 7.22% in S3. This phenomenon is probably attributed to the fact that inoculation with Bacillus subtilis inhibited chlorophyll degradation induced by water stress [35]. Furthermore, CMS can increase plant stomatal conductance, which is one of the major factors influencing photosynthetic efficiency under drought stress [36].

When plants grow in a high-salinity environment, the levels of proline and MDA rise. The reason is that a high-salt environment reduces the water potential of the plant roots, which cannot absorb enough water. In order to overcome the above difficulty, the plant accumulates proline to maintain the osmotic pressure of the cells. At the same time, a high-salt environment also leads to oxidative stress, and the plant increases the production of MDA to solve the problem. In this study, the application of HA and CMS resulted in a significant decrease in proline and MDA for all treatments except for Case F3 (application rate of HA was 4.5 g/kg). High accumulation of HA plays a major role in osmoregulation. Under salt stress, exogenous HA application increases stomatal conductance and transpiration rate in plants, which optimizes plant water status and alleviates cellular osmotic stress. Consequently, the accumulation of proline is reduced. The decreased contents of proline and MDA in CMS can be explained as follows. First, Bacillus sp. G2 upregulates the activities of superoxide dismutase (SOD) and peroxidase (POD), which in turn lowers MDA levels. This bacterium effectively mitigates salt stress and reduces leaf osmotic potential [37]. Second, Bacillus regulates tissue water homeostasis and promotes protein turnover, thereby effectively reducing proline accumulation [38]. In addition, Bacillus subtilis in CMS can help plants initially adapt to subsequent stress [13].

5. Conclusions

In order to study the effect of two soil amendments, i.e., CMS (CMS) and humic acid (HA), on salinized soil in Ningxia Province, China, eight treatments were conducted in a series of pot experiments with sunflowers planted in the pots. It can be concluded that the two soil amendments can not only effectively increase soil water content, chlorophyll concentration, and various physiological indicators of sunflower plants, but also reduce total soil salinity, proline, and malondialdehyde (MDA). The following conclusions can be obtained.

(1)

Appropriate amounts of HA and CMS can improve saline soil structure and porosity, which effectively enhances the water-retaining capacity of the salinized soil, and the enhancement effect of using CMS on the water-retaining capacity is significantly better than that using HA. However, the excessive use of HA can make the soil over-dispersed and reduce the soil cohesion, so that the water cannot be effectively retained. Similarly, excessive use of CMS can increase the negative charge on the surface of soil particles, which can increase the pore diameter, and can lead to easily losing soil water.

(2)

Moderate amounts of HA and CMS can effectively reduce the contents of salt, proline and MDA of the soil. Among all cases for soil treatment using HA and CMS, the effect of reducing soil salinity with CMS is better than that of HA, and Case CK1 has the best effect of desalinization. However, this treatment used soil that had been improved by CMS (2 g/kg) for one year, so it cannot be compared with other treatments. Among other treatments, Case S3 (application rate of CMS is 4.5 g/kg) has the best effect of desalinization, being able to reduce the total salt by 37.24% compared with the control group (Case CK).

(3)

The application of HA and CMS can effectively improve the content of nutrients including N, P, K, AN, and NN in the salinized soil. Generally speaking, CMS can increase nutrient content more effectively than HA. Among these treatments, it can be found that Case S3 (application rate of CMS is 4.5 g/kg) is the best treatment, being able to increase the nutrient content of N, P, K, AN and NN in the soil by about 40–50%.

(4)

Moderate application rate of HA and CMS can increase growth indices such as plant height, stem thickness, number of leaves, leaf length, leaf width, leaf area, aboveground fresh weight, and chlorophyll content of the oil sunflower. Except for Case CK1, Case F1 (application rate of FA is 1.5 g/kg) can greatly increase the oil sunflower’s growth indices. However, it can be found that excessive use of HA (application rate of FA is more than 3.0 g/kg, Cases F2 and F3) is harmful to the soil physicochemical properties, specifically increasing soil salt content and inhibiting the growth of oil sunflower.