Drought-resistant salt-tolerant growth-promoting strain and application thereof

Abstract

The invention discloses a drought-resistant salt-tolerant growth-promoting strain and application thereof. The bacterial strain is bacillus parlicheniformis XT33, and the preservation number of the bacterial strain is CCTCC (China Center For Type Culture Collection) NO: M 20252789. The strain has remarkable drought resistance, salt resistance and multiple growth promoting functions, and can still grow well under the stress of 20% PEG and 5% NaCl; the strain can secrete heteroauxin, produce exopolysaccharide and form a biological membrane, and has the capabilities of dissolving phosphorus, producing siderophores and the like. The invention also provides a microbial agent containing the strain, a preparation method of the microbial agent, and a method for promoting seed germination and seedling growth of plants under drought and / or salt stress by soaking seeds with the microbial agent. The strain has composite functions and strong stress resistance, and provides an excellent strain resource for developing a green microbial inoculant for agricultural production in arid and saline-alkali areas.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a drought-resistant and salt-tolerant growth-promoting strain and its application. Background Technology

[0002] Drought and soil salinization are major abiotic stressors constraining global agricultural production and ecological restoration. Against the backdrop of climate change, their frequency and intensity continue to increase, severely inhibiting crop seed germination, growth, development, yield, and quality, threatening food security and ecological sustainability. Current countermeasures mainly include engineering improvements, large-scale irrigation for salt leaching, and the application of chemically synthesized growth regulators. However, these methods generally suffer from high costs, large water consumption, water waste, secondary salinization, or environmental pollution, making them insufficient to meet the demands of green and sustainable agriculture.

[0003] In recent years, enhancing plant stress resistance using plant rhizosphere growth-promoting bacteria (PGPR) has become a research hotspot. Bacillus microorganisms are considered ideal resources for developing microbial inoculants due to their ability to form stress-resistant spores and their ease of cultivation and storage. Some studies have reported Bacillus strains with single or partial growth-promoting functions, such as nitrogen fixation, phosphorus solubilization, secretion of plant hormones, or production of siderophores. However, in practical applications, especially in complex field stress environments where drought and salinity coexist and fluctuate drastically, existing strains and preparations still have significant limitations: First, most strains have relatively singular functions, either focusing on growth promotion while lacking sufficient stress resistance, or possessing some stress resistance but weak growth promotion ability, making it difficult to exert stable and synergistic effects under combined stress; Second, the colonization ability and survival rate of strains in severely stressed soils are often low, resulting in their growth-promoting and stress-resistance effects not being sustainable; Third, multifunctional compound strains that combine high drought and salt tolerance and can comprehensively promote seed germination, seedling establishment, and plant growth through multiple mechanisms (such as secreting extracellular polysaccharides, forming biofilms, and systematically inducing the plant's own stress resistance physiological response) are still relatively scarce.

[0004] Therefore, there is an urgent need to isolate and screen novel Bacillus strains that can simultaneously tolerate high-intensity drought and salinity stress, effectively colonize in adverse soils, and possess multiple significant growth-promoting and stress-resistance-inducing functions. Developing highly efficient microbial inoculants based on such strains has significant practical implications and application value for promoting green, low-cost agricultural production and ecological restoration in arid and saline-alkali regions. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a drought- and salt-tolerant growth-promoting strain and its application. This strain and its microbial agents can systematically enhance the physiological resistance of plants under drought and salt stress, promote growth and seed germination through multiple synergistic mechanisms, providing an efficient and green microbial solution for agricultural production in arid and saline-alkali areas.

[0006] This invention is achieved through the following technical solution:

[0007] A drought-resistant and salt-tolerant growth-promoting bacterial strain, namely Bacillus paralicheniformis XT33, has been deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M20252789.

[0008] Preferably, the nucleotide sequence of the 16S rDNA of the strain is shown in SEQ ID NO.1.

[0009] The above-mentioned drought-resistant and salt-tolerant growth-promoting strains are used in the preparation of microbial inoculants.

[0010] A microbial inoculant comprising the aforementioned drought-resistant, salt-tolerant, and growth-promoting bacterial strains, wherein the viable count of the inoculant is 1 × 10⁻⁶. 8 ~1×10 11 CFU / mL, pH value 6~8.

[0011] The above-mentioned method for preparing microbial inoculants includes the following steps:

[0012] Step 1) Seed culture: The above-mentioned drought-resistant and salt-tolerant growth-promoting strains were inoculated into LB medium and cultured with shaking at 37℃ and 180 rpm for 18-24 h to obtain OD. 600 Seed fermentation broth >1.0;

[0013] Step 2) Fermentation culture: The seed fermentation broth obtained in step 1) was inoculated into M9 medium and cultured with shaking at 37℃ and 180 rpm for 12-18 h to obtain OD. 600 The fermentation broth has a concentration of 0.8 to 1.2, which is the final product.

[0014] The application of the above-mentioned drought-resistant and salt-tolerant growth-promoting strains, or the above-mentioned microbial agents, in promoting plant growth.

[0015] The application of the above-mentioned drought-resistant and salt-tolerant growth-promoting strains, or the above-mentioned microbial agents, in improving the drought resistance and / or salt tolerance of plants.

[0016] A method for promoting plant seed germination and seedling growth includes the following steps:

[0017] Under drought and / or salt stress conditions, the above-mentioned microbial inoculants are mixed with plant seeds at a weight ratio of 1:(1~3) and soaked for 4~6 hours. After the microbial soaking is completed, the seeds are sown in the soil.

[0018] Preferably, the method is carried out under non-biological stress conditions.

[0019] Preferably, the plant is cucumber or jute.

[0020] The beneficial effects of this invention are as follows:

[0021] (1) The *Bacillus paralichrysogenus* XT33 isolated and screened from extremely arid and saline habitats in this invention has a strong tolerance to drought and salt stress and can grow normally under severe drought simulated by 30% PEG and high salt concentration of 10% NaCl. This characteristic derived from harsh environment gives it a natural advantage in surviving, colonizing and functioning in complex adverse soils with both drought and salt stress in the field, overcoming the limitations of many existing growth-promoting strains with low survival rate and unstable effect under single or combined stress.

[0022] (2) The strain of this invention not only possesses traditional growth-promoting functions such as secreting auxin (IAA), producing siderophores, solubilizing phosphorus and potassium, and fixing nitrogen, but also synthesizes large amounts of extracellular polysaccharides (EPS), forms biofilms, and exhibits significant ACC deaminase activity and proline production capacity. This synergistic effect of multiple mechanisms—"growth promotion (nutrient activation and hormone regulation) + stress resistance (osmotic protection, ethylene regulation and physical barrier)"—enables the strain to not only directly promote plant growth but also systematically enhance the plant's adaptability and resistance to adverse conditions, making its functions more comprehensive and efficient.

[0023] (3) By inoculating the microbial agent of the present invention, the activity of antioxidant enzymes (SOD, POD, CAT) in plants can be significantly increased, accelerating the removal of reactive oxygen species, reducing the accumulation of malondialdehyde (MDA), a product of membrane lipid peroxidation, and protecting the integrity of cell membrane structure; at the same time, it increases the content of osmotic regulators such as proline and chlorophyll in plants. This multi-level physiological regulation from "antioxidant damage" to "maintaining osmotic and photosynthetic balance" fundamentally enhances the drought and salt tolerance of plants and promotes normal growth and development under stress conditions.

[0024] (4) The microbial agent preparation process of this invention is mature and stable, with a high viable count. Its core application method—seed soaking treatment—is simple to operate, requires a small amount of agent, is low in cost, and is easy to promote on a large scale in agricultural production. This method helps the strains preferentially colonize the root system in the early stage of seed germination, establish a dominant microbial community, and thus provide continuous protection and growth promotion during the most vulnerable seedling stage of the plant. Practice has proven that this treatment can significantly improve the germination rate, vigor index, and seedling biomass of cucumber, alfalfa, and other seeds under drought and salt stress, with a high input-output ratio, providing an environmentally friendly and sustainable microbial solution for stable and increased crop yields and green production in arid and saline-alkali areas. Attached Figure Description

[0025] Figure 1The colony morphology of Bacillus paralichrysiformis XT33 on LB plates in Example 1;

[0026] Figure 2 Gram staining of Bacillus paralichrysiformis XT33 in Example 1;

[0027] Figure 3 This is the phylogenetic tree of Bacillus paralicheniformis XT33 in Example 1. Detailed Implementation

[0028] The present invention will now be described in further detail with reference to the accompanying drawings and specific embodiments.

[0029] Unless otherwise specified, the technical means used in the following embodiments are all conventional means well known to those skilled in the art, and the experimental methods without specific conditions are all conventional methods in the art.

[0030] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0031] Example 1: Screening of Bacillus paralichrysiformis XT33

[0032] 1. Sample Source

[0033] Soil samples were taken from the rhizosphere of Haloxylon ammodendron in Tarim No. 2 Ranch, Shaya County, Aksu Prefecture, Xinjiang Uygur Autonomous Region (E: 82°46′21″, N: 41°12′47″), placed in sterile bags, and stored at 4°C.

[0034] 2. Strains screening

[0035] Weigh 1 g of soil sample into a 50 mL centrifuge tube, add 9 mL of sterile water, and shake at 180 rpm for 30 min. Transfer 100 μL of the shaken solution to a centrifuge tube containing 10 mL of LB liquid medium and incubate at 37℃ and 180 rpm for 24 h. Then, perform 10... -2 ~10 -7 Serial dilutions were performed, with 50 μL pipetteed onto LB agar plates, evenly spread and labeled accordingly. Incubation was carried out at 37°C for 24–48 h. Colonies on the plates were observed, and single colonies with different appearances and morphologies were picked and streaked for purification until pure single colonies were obtained. The selected single colonies were then inoculated into LB liquid medium, and the OD values ​​were allowed to develop. 600 When the pH value is 0.8~1.2, glycerol is used for preservation, and the strain is temporarily stored at -20℃. Through comprehensive evaluation of the strain's effects on drought resistance, salt tolerance, and growth promotion, strain XT33 was finally obtained. Figure 1 ).

[0036] LB medium (pH=7.0): 10 g peptone, 5 g yeast extract, 10 g NaCl, 20 g agar powder, 1000 mL water.

[0037] 3. Observation of colony morphology of strain XT33

[0038] The selected XT33 strain was cultured on LB solid medium at 37°C for 3 days, followed by Gram staining. Microscopic observation revealed that the vegetative cells were single-celled, rod-shaped. After culturing at 37°C for 12 hours in the same medium, the bacterial cells grew in large quantities. Figure 2 As shown, the colonies are irregular in shape, dirty white or slightly yellow, with a certain luster, and the surface is rough, opaque, and wrinkled.

[0039] 4. Physiological and biochemical characteristics (Table 1)

[0040] Table 1 Physiological and Biochemical Characteristics

[0041]

[0042] 5. Strain identification (16S rDNA sequence analysis)

[0043] (1) The universal primers for bacterial 16S rDNA amplification were as follows:

[0044] 27F (SEQ ID NO.2): 5'-AGAGTTTGATCMTGGCTCAG-3';

[0045] 1492R (SEQ ID NO. 3): 5'-GGTTACCTTGTTACGACTT-3'.

[0046] (2) PCR reaction system (2.5 μL): 5× amplification buffer (5 μL), genomic DNA (0.1 μL), dNTP (2 μL), primer 27F (0.5 μL), primer 1492R (0.5 μL), Taq DNA polymerase (0.25 μL), deionized water (16.65 μL).

[0047] Reaction conditions: 98℃ pre-denaturation for 10 min, 98℃ denaturation for 10 s, 58℃ annealing for 10 s, 72℃ extension for 90 s, for a total of 30 cycles, followed by 72℃ extension for 10 min.

[0048] (3) Result of the appraisal

[0049] The majority of the 16S rDNA sequence was determined, as shown in SEQ ID NO.1. The determined sequence was aligned using BLAST from the NCBI website, and a phylogenetic tree based on the full 16S rDNA sequence was constructed. Figure 3 As shown, strain XT33 was identified as Bacillus paralicheniformis.

[0050] This strain has been deposited at the China Center for Type Culture Collection (CCTCC), located at No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province. Its classification name is Bacillus paralicheniformis XT33, its accession number is CCTCC NO: M20252789, and its deposit date is December 5, 2025.

[0051] Example 2: Drought resistance test of Bacillus paralichrysiformis XT33

[0052] 1. Experimental Procedure

[0053] Strain XT33 was inoculated into LB medium and fermented at 37°C and 180 rpm for 24 h to obtain OD. 600 The fermentation broth was prepared with a concentration of 1.2. Then, it was inoculated into LB medium containing different concentrations (10%, 20%, 30%) of PEG (polyethylene glycol) to simulate drought conditions. Fermentation was carried out at 37°C and 180 rpm for 24 h with shaking. The OD value was then measured by turbidity method. 600 The value was used to observe the survival ability of strain XT33 in arid habitats.

[0054] 2. Experimental Results

[0055] Table 2 Drought Resistance Test

[0056]

[0057] Table 2 shows that *Bacillus paralichrysum* XT33 maintained good growth under PEG-simulated drought stress. Its OD values ​​were [not specified] under 20% and 30% PEG conditions. 600 The values ​​remained at approximately 0.93 and 0.58, indicating that strain XT33 possesses good drought resistance under moderate to high drought stress.

[0058] Example 3 Salt tolerance test of Bacillus paralichrysiformis XT33

[0059] 1. Experimental Procedure

[0060] Strain XT33 was inoculated into LB medium and fermented at 37°C and 180 rpm for 24 h to obtain OD. 600 The fermentation broth was diluted to 1.2. Then, it was inoculated into LB liquid medium containing different concentrations (3%, 5%, 10%) of NaCl and cultured at 37°C and 180 rpm for 24 h in a shaker. The OD was then measured by turbidity method. 600The value was used to observe the survival ability of strain XT33 in saline-alkali habitats.

[0061] 2. Experimental Results

[0062] Table 3 Salt Tolerance Test

[0063]

[0064] Table 3 shows that *Bacillus paralichrysum* XT33 maintained good growth under NaCl salt stress. Its OD value under 5% NaCl salt stress was... 600 The OD value of 10% NaCl reached 1.76. 600 The value remained at 0.61, indicating that strain XT33 possesses good salt tolerance in high-salt-alkali environments.

[0065] Example 4: Growth-promoting properties test of Bacillus paralichrysiformis XT33

[0066] 1. Experimental Procedure

[0067] (1) Test of the strain's ability to produce indoleacetic acid (IAA)

[0068] After activating strain XT33, it was inoculated at 1% onto LB liquid medium containing L-tryptophan (100 mg / L) and cultured at 37°C and 180 rpm for 3 days. The bacterial culture was centrifuged at 8000 rpm for 10 min, and 2 mL of the supernatant was collected. An equal volume of this supernatant was mixed with Salkowski chromogenic reagent, and the mixture was reacted in the dark for 30 min. The OD value was then measured. 530 Value. Simultaneously, an IAA standard solution was prepared, and an IAA standard curve was obtained. The concentration of IAA produced by the strain was then determined based on the standard curve.

[0069] Salkowski reagent: Mix 1 mL of 0.5 M FeCl3 with 49 mL of 35% HClO4.

[0070] (2) Test of the strain's ability to produce extracellular polysaccharides (EPS)

[0071] Strain XT33 was inoculated into LB medium and fermented at 37°C and 180 rpm for 24 h to obtain OD. 600 The fermentation broth was diluted to 1.2 mg / L, then centrifuged at 8000 rpm for 10 min, and the supernatant was collected. 5 mL of the supernatant was added to 3 times its volume of 95% ethanol, and the mixture was allowed to stand overnight at 4°C. The mixture was then centrifuged at 8000 rpm for 10 min, and the precipitate was collected. The precipitate was dissolved in 10 mL of distilled water, and 3 times its volume of 95% ethanol was added again. The mixture was allowed to stand overnight at 4°C, and the supernatant was discarded. The precipitate was dissolved and the volume was adjusted to 100 mL. The OD of the EPS was determined using the sulfuric acid-phenol method.490 The value was then used to calculate the polysaccharide content based on the standard curve of glucose.

[0072] (3) Test of phosphate solubilization ability of strains

[0073] Strain strain XT33 was inoculated into 100 mL of NBRIP inorganic phosphorus medium (2% inoculation amount), with no inoculation as a control. Three replicates were set for each group. The culture was incubated at 37℃ and 180 rpm for 5 days. The culture was then centrifuged at 8000 rpm for 20 min. The supernatant was used to determine the OD using the molybdenum blue colorimetric method. 700 The value is used to calculate the soluble phosphorus content based on the phosphorus standard curve.

[0074] NBRIP Inorganic Phosphorus Medium (pH=7.0): 10 g glucose, 5 g calcium phosphate, 5 g magnesium chloride, 0.25 g magnesium sulfate, 0.2 g potassium chloride, 0.1 g ammonium sulfate, 18 g agar powder, 1000 mL distilled water.

[0075] (4) Test of potassium solubilization ability of strains

[0076] Strain XT33 was inoculated into potassium-solubilizing medium and cultured at 37℃ and 180 rpm for 7 days. Uninoculated medium served as a control. Each group was divided into three replicates. After centrifugation at 8000 rpm for 20 min, the supernatant was appropriately diluted, and the potassium content of the supernatant was determined by atomic absorption spectrometry. + concentration.

[0077] Potassium-solubilizing medium (pH=7.0): 5 g sucrose, 2 g disodium hydrogen phosphate, 0.005 g ferric chloride, 0.1 g calcium carbonate, 0.5 g magnesium sulfate heptahydrate, 1 g potassium feldspar powder, 0.1 g bromothymol blue, 18 g agar powder, 1000 mL distilled water.

[0078] (5) Test of nitrogen fixation capacity of strains

[0079] Strain strain XT33 was inoculated into Ashby medium and cultured at 37°C and 180 rpm for 7 days. Uninoculated medium served as a control. Each group was divided into three replicates. The culture was centrifuged at 8000 rpm for 20 min, and the supernatant was appropriately diluted. The total nitrogen content of the supernatant was determined using the acetylene reduction method.

[0080] Ashby medium (pH=7.0): mannitol 10 g, dipotassium hydrogen phosphate 0.2 g, magnesium sulfate heptahydrate 0.2 g, sodium chloride 0.2 g, calcium sulfate heptahydrate 0.1 g, calcium carbonate 5.0 g, 0.5% Congo red solution 10 mL, agar powder 18 g, distilled water 1000 mL.

[0081] (6) ACC deaminase activity test of the strain

[0082] Strain XT33 was inoculated at 1% in SMN liquid medium and cultured with shaking for 20 h. After centrifugation at 4°C, the supernatant was removed and the bacterial cells were collected. The cells were washed twice with SM medium and centrifuged again, resuspending the cells in SM medium. This was then inoculated at 5% in SMA liquid medium and cultured at 28°C for 2 days. Using uninoculated SM medium as a zero-control, the OD was determined by the α-ketobutyric acid colorimetric method. 540 The content of α-ketobutyric acid was calculated based on the standard curve, and the protein content in the remaining strain suspension was determined. Finally, the ACC deaminase activity was calculated.

[0083] SM medium (pH=7.2): 5 g glucose, 1 g ammonium nitrate, 0.5 g potassium dihydrogen phosphate, 1.5 g disodium hydrogen phosphate, 1 g sodium chloride, 0.2 g magnesium sulfate heptahydrate, 1000 mL distilled water.

[0084] Add 0.5 g / L of (NH4)2SO4 to SM medium to obtain SMN liquid medium.

[0085] Add 0.5 g / L of 1-aminocyclopropane-1-carboxylic acid (ACC) to SM liquid medium to obtain SMA liquid medium.

[0086] (7) Test of the activity of the strain in producing ferophiles

[0087] Strain strain XT33 was inoculated into MKB medium at a 2% inoculum and cultured at 37℃ and 180 rpm for 48 h. After centrifugation at 8000 rpm for 20 min, the supernatant was mixed with CAS detection solution at a 1:1 volume ratio. After standing for 1 h, the OD was measured using a UV spectrophotometer. 630 The A / Ar ratio was used to determine the siderophore production capacity of the strain, with distilled water as a control for zeroing and uninoculated sterile MKB medium as a reference.

[0088] MKB medium (pH=7.2): 5 g acid-hydrolyzed casein, 2.5 g magnesium sulfate heptahydrate, 2.5 g dipotassium hydrogen phosphate, 15 mL glycerol, 1000 mL distilled water.

[0089] (8) Biofilm content test of strain

[0090] Strain strain XT33 was inoculated into LB medium containing crystal violet, and the OD was measured using a spectrophotometer. 570 The absorbance values ​​were used to verify the biofilm production capacity and to calculate the biofilm content.

[0091] (9) Proline content test of strains

[0092] Strain strain XT33 was inoculated into LB broth at a 1% inoculum and cultured at 28°C and 180 rpm for 72 h. The supernatant was collected by centrifugation at 7000 rpm for 20 min. 1 mL of the supernatant was added to 2 mL of 3% sulfosalicylic acid and incubated for 30 min. The supernatant was collected by centrifugation at 7000 rpm for 20 min. 1 mL of the supernatant was then added to 2 mL each of glacial acetic acid and acidic ninhydrin. The reaction was carried out in a boiling water bath for 1 h, and the reaction was terminated by placing the test tube on ice. 4 mL of toluene was added to extract the pigment generated in the reaction, and the OD value of toluene containing the pigment was measured at 520 nm, using toluene as a control. The proline content (mg / L) was calculated based on the standard curve.

[0093] 2. Experimental Results

[0094] Table 4. Growth-promoting characteristic test

[0095]

[0096] Table 4 shows that the phosphorus-solubilizing, potassium-solubilizing, and nitrogen-fixing capacities of *Bacillus paralichrysum* XT33 were 5.46 mg / L, 10.22 mg / L, and 8.17 mg / L, respectively, indicating that its presence in stress-resistant environments can enhance plant nutrient uptake from the soil. The extracellular polysaccharide yield and biofilm mass of *Bacillus paralichrysum* XT33 were 40 mg / L and 0.94 mg / cm³, respectively. 2 This indicates that it easily forms biofilms, allowing for better attachment and colonization on plants. The IAA yield, siderophore content, deaminase activity, and proline content in *Bacillus paralichrysum* XT33 were 31 mg / L, 81%, 0.91 mg / L, and 174.58 mg / L, respectively, suggesting that during its metabolism, it readily secretes more growth-promoting substances, thereby directly or indirectly promoting plant growth and enhancing plant stress resistance.

[0097] Example 5: Verification of the seed germination-promoting effect of Bacillus paralichrysiforme XT33

[0098] 1. Experimental Procedure

[0099] Bacillus paralichrysogenum XT33 was inoculated into LB medium for activation, and fermented at 37°C with shaking at 180 rpm for 24 h to obtain OD. 600 =1.3% seed fermentation broth. The fermentation broth was then inoculated into M9 medium and cultured at 37℃ and 180 rpm for 18 h to obtain OD. 600 The fermentation broth was prepared with a ratio of 1.2, and then the fermentation broth was mixed with cucumber seeds at a weight ratio of 1:1.5. The cucumber seeds were then soaked in the bacterial solution for 6 hours.

[0100] LB medium (pH=7.0): 10 g peptone, 5 g yeast extract, 10 g NaCl, 20 g agar powder, 1000 mL water.

[0101] M9 medium (pH=7.0): 6.78 g disodium hydrogen phosphate, 3 g potassium dihydrogen phosphate, 0.5 g sodium chloride, 1 g ammonium chloride, 4 g glucose, 0.01 g calcium ions, 1000 mL distilled water.

[0102] Seed germination was performed using the petri dish germination method. The inoculated cucumber seeds were placed in petri dishes, and 15 mL of water, 30% PEG, and 1% NaCl solution were added. Each group had three replicates, with 30 seeds placed in each petri dish. Germination rate was recorded, with the embryo breaking through the seed coat as the criterion. Germination was completed after 7 days. Germinated seeds were collected, and five plants from each treatment were randomly selected. Root length, shoot length, germination index, and vigor index were measured using calipers. The following experimental groups were set up:

[0103] Group CK: Cucumber seeds + water;

[0104] CK0 group: cucumber seeds + XT33 + water;

[0105] A0 (drought stress) group: cucumber seeds + 30% PEG;

[0106] Group A1 (drought stress): cucumber seeds + XT33 + 30% PEG;

[0107] Group B0 (salt stress): cucumber seeds + 1% NaCl;

[0108] Group B1 (salt stress): cucumber seeds + XT33 + 1% NaCl.

[0109] 2. Experimental Results

[0110] Table 5. Effects of Bacillus paralichrysiformis XT33 on cucumber seed germination under abiotic stress.

[0111]

[0112] Table 5 shows that under normal conditions, Bacillus paralichrysiformis XT33 treatment (CK0) had no negative impact on cucumber seed germination, and all indicators were comparable to the water control (CK). However, under drought (30% PEG) and salt (1% NaCl) stress, the germination of untreated seeds was significantly inhibited (A0 and B0 groups), with a significant decrease in root length, shoot length, germination rate, germination potential, and vigor index. In particular, under drought stress, the germination potential and vigor index plummeted to 3.33% and 3.21, respectively. After treatment with strain XT33, significant alleviating effects were observed under both stresses: under drought stress (A1 group), the germination rate, germination potential, and vigor index increased by 21.7%, 400%, and 86.0% compared to the stress control (A0), respectively; under salt stress (B1 group), the germination rate increased from 73.33% to 100% (an increase of 36.4%), and the vigor index was also improved.

[0113] The experimental results of this embodiment show that Bacillus paralicheniformis XT33 can effectively enhance the germination ability and early seedling vigor of cucumber seeds under drought and salt stress, which is a key initial step in its systematic improvement of plant stress resistance.

[0114] Example 6: Verification of the effect of Bacillus paralichrysiforme XT33 on promoting cucumber growth under drought stress

[0115] 1. Experimental Procedure

[0116] Bacillus paralichrysogenum XT33 was inoculated into LB medium for activation, and fermented at 37°C and 180 rpm for 20 h to obtain OD. 600 =1.0 of seed fermentation broth. The fermentation broth was then inoculated into M9 medium and cultured at 37℃ and 180 rpm for 18 h to obtain OD. 600 The fermentation broth was prepared with a ratio of 1.2, and then the fermentation broth was mixed with cucumber seeds at a weight ratio of 1:1.5. The cucumber seeds were then soaked in the bacterial solution for 6 hours.

[0117] The inoculated cucumber seeds were then used in pot experiments for verification. Pots with a diameter of 140 mm and a height of 115 mm were used, each containing 400 g of soil under moderate drought stress (soil moisture content 40%). The plants were weighed daily, and water was added according to the weight of the lost water. Three cucumber seeds were sown in each pot, and each treatment was replicated three times. After 20 days of growth, morphological growth indicators such as root length, plant height, fresh weight, dry weight, and root activity were measured, and the plant's stress resistance was assessed. The control group (CK) received no inoculant treatment; group A was the inoculant-treated group. The stress resistance was measured as follows:

[0118] (1) Determination of malondialdehyde (MDA) content

[0119] Prepare 250 mL of 5% trichloroacetic acid (TCA) and 100 mL of 0.67% thiobarbituric acid (TBA) (dissolved in a small amount of 1 mol / L sodium hydroxide and then diluted to 100 mL with 10% TCA). Take about 0.5 g of plant sample (leaves), add 5 mL of 5% TCA, grind and centrifuge the resulting homogenate at 3000 rpm for 10 min. Slowly pipette 2 mL of the supernatant (do not pipette too quickly to avoid aspirating plant material), add 2 mL of 0.67% TBA, mix, boil in a 100℃ water bath for 15 min, cool and centrifuge again (10 min). Measure the absorbance of the supernatant at 450 nm, 532 nm and 600 nm, using 2 mL of distilled water with 2 mL of 0.67% TBA as a control.

[0120]

[0121] In the formula: A 532 A 600 A 450 This indicates the absorbance value for the corresponding wavelength.

[0122] (2) Assay of antioxidant enzyme activity

[0123] The activity of catalase (CAT) was determined by ultraviolet spectrophotometry using an activity assay kit, and the activities of peroxidase (POD) and superoxide dismutase (SOD) were determined by visible spectrophotometry.

[0124] (3) Determination of proline content

[0125] Accurately weigh 0.5 g of each crop leaf and place them in separate stoppered colorimetric tubes. Add 5 mL of 3% sulfosalicylic acid solution to each tube and incubate in a boiling water bath for 10 min (shaking frequently during the process). After cooling, filter the solution into a clean test tube; the filtrate is the proline extract. Pipette 2 mL of the filtrate into a clean stoppered colorimetric tube, add 2 mL of glacial acetic acid and 2 mL of acidic ninhydrin reagent, and heat in a boiling water bath for 30 min until the solution turns red. After cooling, add 4 mL of toluene, shake for 30 s, let stand for a moment, and transfer the supernatant to a 10 mL centrifuge tube. Centrifuge at 3000 rpm for 5 min. Pipette the red proline toluene solution into a cuvette, using toluene as a blank control, and measure the color at 520 nm using a UV spectrophotometer.

[0126]

[0127] In the formula: C is the mass concentration of proline in the test solution (μg / mL) calculated based on the proline standard curve; V is the volume of the extract (mL); and W is the fresh weight of the plant (g).

[0128] (4) Determination of relative chlorophyll content

[0129] Weigh 0.1 g of fresh leaves using an analytical balance, cut them into small pieces, place them in a 15 mL capped centrifuge tube, add 5 mL of 95% ethanol, and store in the dark until the leaves turn white. Take the supernatant, dilute it 10 times, and measure the absorbance at 649 nm and 665 nm. Repeat three times. Calculate the chlorophyll content using the following formula:

[0130]

[0131]

[0132]

[0133] In the formula: A 665 A 649 This indicates the absorbance value for the corresponding wavelength.

[0134] 2. Experimental Results

[0135] Table 6. Effects of Bacillus paralichrysiforme XT33 on cucumber phenotypic traits under drought stress.

[0136]

[0137] Table 6 shows that under drought stress, cucumber plants treated with Bacillus paralichrysum XT33 (Group A) showed significantly higher root length, plant height, leaf area, fresh weight, dry weight, and root activity compared to the untreated control group (CK group) (increased by 101.3%, 69.6%, 31.3%, 29.7%, 115.7%, and 36.9%, respectively). All differences between the groups were statistically significant (marked with different letters). This indicates that Bacillus paralichrysum XT33 can effectively alleviate the inhibitory effect of drought stress on cucumber growth by significantly promoting root development, enhancing plant growth potential and nutrient accumulation, and increasing root metabolic activity, thereby systematically improving the drought adaptability of cucumber.

[0138] Table 7. Effects of Bacillus paralichrysiformis XT33 on physiological parameters of cucumber under drought stress.

[0139]

[0140] Table 7 shows that under drought stress, the key physiological indicators of cucumber plants treated with Bacillus paralichrysum XT33 (Group A) underwent systematic optimization: Malondialdehyde (MDA) is one of the products of plant cell membrane peroxidation, and its increased content can damage the cell membrane system. Under the action of strain XT33, the content of MDA, an indicator of membrane lipid peroxidation damage, decreased significantly by 60.6%. Proline is an osmotic regulator in plants, and catalase (CAT), peroxidase (POD), and superoxide dismutase (SOD) are all antioxidant enzymes. Under the action of strain XT33, the content of proline, as an osmotic regulator, increased significantly by 63.8%; the activities of the three protective enzymes responsible for scavenging reactive oxygen species—CAT, POD, and SOD—were significantly increased by 132.3%, 389.8%, and 29.2%, respectively. Simultaneously, the total chlorophyll content in cucumbers in Group A treated with strain XT33 also increased by 13.9% compared to the CK group. The above data indicate that Bacillus paralichrysum XT33 can systematically enhance the physiological adaptability and resistance of cucumber plants to drought stress by effectively inducing the plant's own antioxidant defense system, enhancing osmotic regulation capacity, and reducing membrane system damage.

[0141] Example 7: Verification of the effect of Bacillus paralichrysum XT33 on promoting the growth of velvetleaf under drought and salt stress.

[0142] 1. Experimental Procedure

[0143] Bacillus paralichrysogenum XT33 was inoculated into LB medium for activation, and fermented at 37°C with shaking at 180 rpm for 24 h to obtain OD. 600 =1.0 of seed fermentation broth. The fermentation broth was then inoculated into M9 medium and cultured at 37℃ and 180 rpm for 18 h to obtain OD. 600 The fermentation broth was prepared with a ratio of 1.2, and then mixed with velvet seeds at a weight ratio of 1:1.5. The velvet seeds were then soaked in the bacterial solution for 6 hours.

[0144] The inoculated jute seeds were then used in pot experiments for verification. The pots used were 140 mm in diameter and 115 mm high, each containing 400 g of soil. The plants were subjected to moderate drought stress (soil moisture content 40%) and a salinity of 3‰. The plants were weighed daily, and water was added according to the weight of the lost water. Three jute seeds were sown in each pot, and each treatment was repeated three times. After 20 days of growth, morphological growth indicators such as root length, plant height, fresh weight, dry weight, and root activity were measured, and the plant's stress resistance was assessed. The control group (CK) received no inoculant treatment; group A was the inoculant-treated group. The stress resistance assessment was conducted in the same manner as in Example 6.

[0145] 2. Experimental Results

[0146] Table 8 Effects of Bacillus paralichrysum XT33 on phenotypic traits of Abutilon theophrasti under drought and salt stress

[0147]

[0148] Table 8 shows that under drought and salt stress, the phenotypic traits of *Abutilon theophrasti* plants treated with *Bacillus paralichrysum* XT33 (Group A) were comprehensively and significantly improved: root length, plant height, number of leaves per plant, leaf area, fresh weight, dry weight, and root activity increased by 39.7%, 15.7%, 59.3%, 66.1%, 110.9%, 97.6%, and 27.5% respectively compared to the control group, and the differences were statistically significant (marked with different letters). The results indicate that strain XT33 can effectively alleviate the inhibition of *Abutilon theophrasti* growth by drought and salt stress, especially in promoting biomass accumulation (fresh weight and dry weight nearly doubling or more) and leaf area expansion, systematically enhancing the growth adaptability and productivity of *Abutilon theophrasti* under combined adversity.

[0149] Table 9. Effects of Bacillus paralichrysogenum XT33 on physiological parameters of Abutilon theophrasti under drought and salt stress.

[0150]

[0151] Table 9 shows that under drought and salt stress, the physiological indicators of *Abutilon theophrasti* plants treated with *Bacillus paralichrysum* XT33 underwent profound and systematic changes: the content of malondialdehyde (MDA), a membrane lipid peroxidation product, decreased significantly by 44.7%; the content of proline, an osmotic regulator, increased dramatically by 148.1%; the activities of core antioxidant enzymes were significantly enhanced, with catalase, peroxidase, and superoxide dismutase activities increasing by 2124.2%, 69.2%, and 129.2%, respectively; and the total chlorophyll content also increased by 10.3%. This indicates that strain XT33 can strongly activate the multiple stress-resistance physiological mechanisms of *Abutilon theophrasti*, significantly enhancing its reactive oxygen species scavenging ability, accumulating osmotic protective substances, and effectively maintaining photosynthetic pigment levels, thereby synergistically mitigating oxidative damage and osmotic stress, and systematically endowing *Abutilon theophrasti* with strong physiological adaptability and tolerance to drought and salt stress.

[0152] The embodiments described above are only some, not all, of the embodiments of the present invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments. The scope of protection of the present invention is determined by the scope claimed in the claims. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

Claims

1. A drought-resistant, salt-tolerant, and growth-promoting bacterial strain, characterized in that, The strain is Bacillus paralicheniformis XT33, which has been deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M20252789.

2. The drought-resistant and salt-tolerant growth-promoting bacterial strain according to claim 1, characterized in that, The nucleotide sequence of the 16S rDNA of the strain is shown in SEQ ID NO.

1.

3. The application of the drought-resistant and salt-tolerant growth-promoting strains as described in claim 1 or 2 in the preparation of microbial inoculants.

4. A microbial inoculant, characterized in that, Containing the drought-resistant and salt-tolerant growth-promoting bacterial strain as described in claim 1 or 2, wherein the viable count of the bacterial agent is 1 × 10⁻⁶. 8 ~1×10 11 CFU / mL, pH value 6~8.

5. A method for preparing a microbial inoculant as described in claim 4, characterized in that, Includes the following steps: Step 1) Seed culture: The drought-resistant and salt-tolerant growth-promoting strain as described in claim 1 or 2 is inoculated into LB medium and cultured with shaking at 37°C and 180 rpm for 18-24 h to obtain OD. 600 Seed fermentation broth >1.0; Step 2) Fermentation culture: The seed fermentation broth obtained in Step 1) was inoculated into M9 medium and cultured with shaking at 37℃ and 180 rpm for 12-18 h to obtain OD. 600 The fermentation broth has a concentration of 0.8 to 1.2, which is the final product.

6. The application of the drought-resistant and salt-tolerant growth-promoting strain as described in claim 1 or 2, or the microbial agent as described in claim 4, in promoting plant growth.

7. The application of the drought-resistant and salt-tolerant growth-promoting strains as described in claim 1 or 2, or the microbial agents as described in claim 4, in improving the drought resistance and / or salt tolerance of plants.

8. A method for promoting plant seed germination and seedling growth, characterized in that, Includes the following steps: Under drought and / or salt stress conditions, the microbial agent as described in claim 4 is mixed with plant seeds at a weight ratio of 1:(1~3) and soaked for 4~6 h. After the microbial soaking is completed, the seeds are sown in the soil.

9. The method according to claim 8, characterized in that, The method is implemented under abiotic stress conditions.

10. The method according to claim 8, characterized in that, The plant in question is either cucumber or velvetleaf.

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