Saline-alkali-tolerant bacillus deserticola BW-13 with biocontrol and growth promoting functions and application thereof

Abstract

The invention relates to the technical field of microorganisms, and particularly discloses a saline-alkaline tolerant bacillus deserticola BW-13 with biocontrol and growth promoting functions and application thereof. The bacillus safensis BW-13 is preserved in the China Center for Type Culture Collection on January 13, 2025, the classification name of the bacillus safensis BW-13 is bacillus safeensis, and the preservation number of the bacillus safensis BW-13 is CCTCC NO: M 2025108. The strain BW-13 has relatively high salt tolerance, and still keeps good resistance under various stress conditions; meanwhile, the plant growth promoting agent has various plant growth promoting characteristics including nitrogen fixation, phosphorus solubilization and cellulase and protease secretion capacity, and pot experiment verifies that the plant growth promoting agent can improve the salt resistance of cucumber seedlings and promote plant growth; the strain has an antagonistic effect on a plurality of phytopathogens, and especially has a remarkable prevention and treatment effect on plant diseases such as apple ring spot, polygonatum kingianum anthracnose, pear tree rot and tomato gray mold caused by ascomycophyta pathogenic fungi.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to the salt-tolerant Bacillus salsa-13, which has both biocontrol and growth-promoting functions, and its uses. Background Technology

[0002] Saline-alkali land is a general term for soil salinization and alkalization, encompassing saline soil, alkaline soil, and various types of salinized and alkalized soils. This type of soil is characterized by excessive accumulation of soluble salts, and its high salinity and alkalinity significantly inhibits crop growth and development. Simultaneously, high salinity and alkalinity stress severely suppresses the activity and abundance of beneficial soil microorganisms, leading to microecological imbalance and increasing the frequency and severity of soil-borne diseases. Furthermore, salinized soil environments significantly reduce the availability of fertilizer nutrients and crop utilization rates.

[0003] Saline-alkali environments contain abundant salt-tolerant / halophilic microbial resources. Plant growth-promoting rhizobacteria (PGPRs) are microorganisms that colonize the rhizosphere of plants and can directly or indirectly promote plant growth, increase crop yield, and control pests and diseases. Compared to other soil microorganisms, PGPRs have a stronger colonization ability in the plant rhizosphere and exhibit antagonistic effects against a variety of harmful microorganisms. Therefore, compared to methods of saline-alkali land improvement such as physical extraction, chemical neutralization, and breeding of resistant varieties, utilizing salt-tolerant PGPRs is considered an economical and effective biological strategy for improving the fertility of saline soils, assisting plants in resisting multiple stresses (such as diseases and salt damage), and promoting plant growth. Currently reported PGPRs include those from the genus *Pseudomonas* (…). Pseudomonas ), Bacillus spp. ( Bacillus Agrobacterium ( ) Agrobacterium More than 20 species, including *Bacillus safortifolius*. Bacillus safensis As a type of Gram-positive bacterium widely distributed in the natural environment, *Bacillus sabina* is known for its ability to produce highly resistant endophytic spores, resulting in live bacterial biocontrol agents that typically exhibit good storage stability and a long shelf life. This strain effectively inhibits plant pathogenic fungi through multiple mechanisms, including competition for nutrients and ecological niches, secretion of antimicrobial metabolites, and induction of systemic resistance in plants. Based on these characteristics, *Bacillus sabina* is considered a promising microbial resource for biocontrol.

[0004] In recent years, research on microorganisms in extreme environments has become increasingly extensive; however, research on screening microbial resources with both disease resistance and life-promoting properties from saline-alkali environments remains relatively limited. Therefore, discovering viable microorganisms with application potential from saline-alkali environments is a crucial step in constructing an integrated management system for soil salinization. Clarifying the characteristics and scope of action of such strains will provide important basis for their subsequent development and utilization. Summary of the Invention

[0005] The purpose of this invention is to address the shortcomings of the prior art by providing a salt-tolerant Bacillus salsa BW-13 strain with both biocontrol and growth-promoting functions, and its applications. The strain BW-13 possesses characteristics of biological control of plant diseases, plant growth promotion, and tolerance to multiple environmental stresses, and shows promise in the application of microbial fungicides and agricultural soil inoculants.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: The first aspect of the present invention is to provide *Bacillus salsa* BW-13, which was deposited at the China Center for Type Culture Collection on January 13, 2025, and is classified and named as follows: Bacillus safensis The accession number is CCTCC NO: M 2025108.

[0007] A second aspect of the present invention is to provide a microbial agent comprising the fermentation broth of Bacillus salsa BW-13 as described in claim 1 and / or Bacillus salsa BW-13.

[0008] A third aspect of the present invention is to provide the application of the above-mentioned Bacillus salsa BW-13 or its inoculum, including any of the following applications: A1) Application in improving saline-alkali soil; A2) Applications in promoting plant growth; A3) Application in suppressing plant pathogens; A4) Application in the preparation of products that inhibit plant pathogens.

[0009] Furthermore, the mass concentration of inorganic salts in the saline-alkali soil ranges from 0.5% to 12%, and the inorganic salts include one or more of NaCl, KCl, MgCl2, and CaCl2.

[0010] Furthermore, the pH range of the saline-alkali soil is 5 to 10.

[0011] Furthermore, the growth temperature range of the *Bacillus salsa* BW-13 is 15~45℃.

[0012] Furthermore, the promotion of plant growth is manifested in all or part of the following: nitrogen fixation capacity, inorganic phosphorus dissolution capacity, cellulase secretion capacity, protease secretion capacity, and / or increased plant height growth rate.

[0013] Furthermore, the plant pathogens include at least one of Botryosphaeria dothidea, Botrytis cinerea, Valsa ambiens, Phytophthorainfestans, Fusarium graminearum, or Colletotrichum paethianum.

[0014] A fourth aspect of the present invention is to provide a method for promoting plant growth, comprising the steps of applying the above-mentioned Bacillus sabriophyte BW-13 or its agent to plant plants, plant seeds, or plant rhizosphere soil.

[0015] A fifth aspect of the present invention is to provide a method for improving soil, comprising the steps of: applying the above-mentioned Bacillus salsa BW-13 or the above-mentioned bacterial agent to the soil to be improved; wherein the improved soil exhibits salt tolerance and promotes growth.

[0016] Compared with the prior art, the beneficial effects of the technical solution provided by the present invention are as follows: (1) The Bacillus salsa strain BW-13 obtained by screening in this invention has high salt tolerance and maintains good resistance under various stress conditions. This strain also has a variety of plant growth-promoting characteristics, including nitrogen fixation, phosphorus solubilization and secretion of cellulase and protease. Pot experiments have verified that it can improve the salt tolerance of cucumber seedlings and promote plant growth. At the same time, strain BW-13 has antagonistic effects on a variety of plant pathogens, especially on plant diseases such as apple ring rot, Polygonatum anthracnose, pear rot and tomato gray mold caused by Ascomycete pathogens.

[0017] (2) The Bacillus salsa BW-13 of the present invention has the characteristics of biological control of plant diseases, plant growth promotion and tolerance to multiple environmental stresses. The fermentation agent is simple to make and low in cost. It can be used to develop a broad-spectrum, safe, efficient microbial bactericide or soil conditioner that is adapted to complex soil conditions, especially in the field of ecological restoration and disease control of saline soil. Attached Figure Description

[0018] Figure 1 The *Bacillus sarfusca* isolated in this invention Bacillus safensis Morphological characteristics of BW-13; A: Colony morphology on LB solid medium; B: Gram staining results; C: 1μm scanning electron microscope image; Figure 2 For Bacillus salsa BW-13 16S rDNA Genetic phylogenetic tree diagram; Figure 3 For Bacillus salsa BW-13 gyrB Genetic phylogenetic tree diagram; Figure 4 Figure 1 shows the growth of Bacillus salsa BW-13 under different concentrations of sodium chloride stress. Figure 5 Figure showing the growth of Bacillus sabovellae BW-13 under different pH stress conditions; Figure 6 Figure showing the growth of Bacillus sabovellae BW-13 under different temperature stress conditions; Figure 7 Figure showing the growth of Bacillus sabinatus BW-13 under different inorganic salt stress conditions; Figure 8 Plate diagrams for identifying the growth-promoting characteristics of Bacillus sabovellae BW-13; A represents nitrogen fixation; B represents phosphate solubilization; C represents secreted protease; D represents secreted cellulase. Figure 9 Comparison of the salt tolerance and growth-promoting effects of Bacillus salsa BW-13 bacterial suspension on potted plants; Figure 10 A plate confrontation antagonism diagram of Bacillus sabovellae BW-13 against six plant pathogenic fungi; Figure 11 The control effect of Bacillus sabensis BW-13 treatment on Polygonatum leaves infected with anthracnose. Detailed Implementation

[0019] To make the objectives, technical solutions, and advantages of this invention clearer, the specific embodiments and accompanying drawings are described in further detail below. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0020] The culture media and reagents involved in the embodiments of this invention are as follows: LB medium: 10 g peptone, 5 g yeast extract, 2 g sodium chloride, 18 g agar, pH natural.

[0021] PDA medium: 200 g potato, 20 g glucose, 18 g agar, pH natural.

[0022] Ashbee medium: potassium dihydrogen phosphate 0.2 g, magnesium sulfate 0.2 g, sodium chloride 0.2 g, calcium carbonate 5 g, mannitol 10 g, calcium sulfate 0.1 g, agar 18 g, pH 7.

[0023] Inorganic phosphorus bacteria culture medium: sodium chloride 0.3 g, magnesium sulfate heptahydrate 0.3 g, potassium chloride 0.3 g, ammonium sulfate 0.5 g, ferric sulfate 0.003 g, manganese sulfate 0.003 g, tricalcium phosphate 10 g, glucose 10 g, agar 18 g, pH 7.

[0024] Protease assay medium: 15 g skim milk, 18 g agar, natural pH.

[0025] Carboxymethyl cellulose medium: 1 g potassium dihydrogen phosphate, 2 g ammonium sulfate, 0.5 g magnesium sulfate heptahydrate, 2 g sodium carboxymethyl cellulose, 0.5 g sodium chloride, 0.4 g Congo red, 18 g agar, natural pH.

[0026] The Staphylococcus aureus used in the embodiments of this invention ( Botryosphaeria dothidea ), Botrytis cinerea ( Botrytis cinerea ), pear black rot skin fungus ( Valsa ambiens ), pathogenic fungus ( Phytophthora infestans Fusarium graminearum ( ), Fusarium graminearum ) or Polygonatum anthrax bacteria ( Colletotrichum spaethianum (From Hubei Provincial Engineering Research Center for Biological Pesticides)

[0027] Example 1 Isolation, identification and preservation of Bacillus salsa BW-13.

[0028] 1. Isolation, purification and screening of Bacillus salsa BW-13 Fresh soil was obtained in Zhangye, Gansu (38.97°N, 100.07°E) using a random sampling method.

[0029] Weigh 10 g of fresh soil sample and place it in a sterile Erlenmeyer flask containing 90 mL of sterile physiological saline. Place the flask in a constant temperature shaking incubator and shake at 30℃ and 180 rpm for 20 min. After standing for 30 min, collect the supernatant to obtain 10 g of fresh soil sample. - 1 A soil suspension of g / mL was prepared. A 10-fold serial dilution method was used to dilute the above 10... -1 The g / mL soil suspension was successively diluted to 10 g / mL -6 A concentration gradient of g / mL was established. Each dilution gradient (10 g / mL) was then sampled separately. -4 10 -5 10 -60.1 mL of a suspension (g / mL) was evenly spread onto the surface of LB agar plates containing 2% (w / v) NaCl at pH 8. Three replicates were performed for each dilution. The plates were incubated upside down at 28°C for 5 days. Single colonies of varying morphology and size were picked using a sterile inoculation loop and streaked onto fresh LB agar plates containing 2% (w / v) NaCl for purification until uniformly morphologically consistent and characteristically stable single colonies were obtained on the plates.

[0030] Salt tolerance re-screening: The purified single colonies were sequentially transferred to LB agar plates with increasing NaCl concentration gradients (4%, 6%, 8%, 10%, 12%, w / v). Growth was observed at 28°C. A salt-tolerant strain, designated BW-13, was obtained that could still grow well at 12% NaCl. This strain underwent at least five consecutive subcultures to ensure its genetic stability. Finally, the purified and stable strain BW-13 was transferred to LB agar slant and stored at 4°C.

[0031] Strain activation: A slant culture of Bacillus sabolicus BW-13, preserved at 4℃, was streaked onto LB agar plates. The plates were then incubated at 28℃ for 24 h.

[0032] Seed culture preparation: Pick a single colony and inoculate it into an Erlenmeyer flask containing 50 mL of LB liquid medium. Incubate at 28℃ and 200 r / min for 16 h with shaking to obtain the seed culture.

[0033] Fermentation broth preparation: The obtained seed culture was transferred to an Erlenmeyer flask containing 100 mL of fresh LB liquid medium at an inoculation rate of 2% (v / v). The flask was then incubated with shaking at 28℃ and 200 r / min for 24 h to obtain the fermentation broth.

[0034] Preparation of bacterial suspension: Centrifuge the fermentation broth, discard the supernatant, collect the bacterial cells, wash them, and resuspend the bacterial cells in sterile water to a concentration of 1×10⁻⁶. 8 CFU / mL was used to obtain a bacterial suspension.

[0035] 2. Strain identification 2.1 Identification of the morphological and physiological-biochemical characteristics of strain BW-13 Referencing the "Manual of Systematic Identification of Common Bacteria" and the "Bergey's Manual of Bacterial Identification," observe and describe the colony morphology, color, and physiological and biochemical assays of strain BW-13.

[0036] Colony morphology: After 48 hours of cultivation on solid LB medium, strain BW-13 turned pale yellow, with nearly circular colonies featuring intact edges and a flattened appearance when viewed from the side. The surface was smooth, glossy, moist, easily picked up, opaque, and had protrusions. Wrinkles appeared on the colonies after approximately 72 hours, with a diameter of approximately 0.5–2 mm. Figure 1 As shown in Figure A. Scanning electron microscopy revealed that the bacterial cells were short, rod-shaped, and approximately 0.5 μm × (1~2) μm in size. Figure 1 As shown in C.

[0037] Physiological and biochemical characteristics: Gram staining results of strain BW-13 indicate that this strain is Gram-positive. Figure 1 As shown in Figure B. The physiological and biochemical identification results are shown in Table 1.

[0038] Table 1. Physiological and biochemical identification results

[0039] 2.2 Molecular identification of strain BW-13 A single colony of strain BW-13 was picked and thoroughly rinsed and mixed in a 0.2 mL centrifuge tube containing 10 μL of sterile deionized water. The resulting bacterial suspension was used as a PCR template. 16S rDNA and gyrB The target gene was identified. PCR amplification was performed using primers listed in Table 2. Electrophoresis was performed on a 1% agarose gel at 150 V and 100 mA for 20 min, followed by EB staining for observation. Samples were sequenced by Shanghai Sangon Biotech Co., Ltd. Nucleotide homology was compared with sequences in GenBank using the BLAST program, and a phylogenetic tree was constructed using MEGA 11.0. The 16S rDNA sequence of strain BW-13 was compared with... Bacillus safensis subsp. osmophilus The sequence homology was highest, reaching 99.93%. Based on its morphological characteristics and physiological and biochemical properties, strain BW-13 was preliminarily identified as *Bacillus safranin*. Further differentiation of closely related species was conducted... gyrB Gene sequence analysis was performed. The comparison results showed that strain BW-13 is similar to known Bacillus safranin. gyrB The gene sequence homology is greater than 99%. Based on comprehensive analysis, strain BW-13 was confirmed to belong to the genus *Bacillus safranin*. Bacillus safensis Its 16S rDNA gene sequence is shown in SEQ ID NO: 1. gyrB The sequence is shown in SEQ ID NO: 2.

[0040] Table 2. Primer sequences for gene amplification

[0041] 3. Preservation of bacterial strains Bacillus salsa BW-13, its classification name is Bacillus safensis It was deposited on January 13, 2025 at the China Center for Type Culture Collection, located at Wuhan University, Luojia Mountain, Bayi Road, Wuchang District, Wuhan, Hubei Province, China, with accession number CCTCC NO: M 2025108.

[0042] Example 2 The salt and acid / alkali tolerance of strain BW-13 was investigated.

[0043] Seed culture of *Bacillus sabolicii* BW-13 was inoculated at a rate of 2% (v / v) into 100 mL Erlenmeyer flasks containing LB liquid medium. The mediums were prepared with the following NaCl concentration gradients: 0.5%, 2%, 4%, 6%, 8%, 10%, 12% (w / v); and pH gradients: 4, 5, 6, 7, 8, 9, 10. Uninoculated medium was provided as a blank control. Each treatment was repeated three times, and the medium was incubated at 28°C with shaking at 200 rpm. Samples were taken periodically to measure the OD (oxidative stress). 600 value.

[0044] The results are as follows Figure 4 and Figure 5 As shown, strain BW-13 can grow in LB medium with NaCl concentrations ranging from 0.5% to 12% (w / v) and pH values ​​ranging from 5 to 10.

[0045] Example 3 The ability of strain BW-13 to withstand different temperatures was investigated.

[0046] The seed culture of BW-13 was inoculated at a rate of 2% (v / v) into Erlenmeyer flasks containing 100 mL of LB liquid medium. The flasks were then placed in constant-temperature shakers at 4℃, 15℃, 28℃, 37℃, and 45℃, and cultured at 200 rpm. Uninoculated LB medium was used as a blank control at the corresponding temperatures. Each treatment was repeated three times. After 24 h of culture, the OD of the culture medium was measured. 600 value.

[0047] The results are as follows Figure 6 As shown, strain BW-13 can grow in a temperature range of 15℃ to 45℃.

[0048] Example 4 The ability of strain BW-13 to withstand different types of inorganic salt stress was investigated.

[0049] Add the following seven inorganic salts to LB liquid basal medium (containing no inorganic salts): NaNO₂, Na₂CO₃, NaHCO₃, NaCl, KCl, MgCl₂, and CaCl₂. Adjust the amount of each salt added to achieve the desired concentration of the target ion (Na₂NO₂, Na₂CO₃, NaHCO₃, NaCl, KCl, MgCl₂, and CaCl₂) in the medium. + or Cl - The final concentration of all samples was 0.34 mol / L. The seed culture of strain BW-13 was inoculated at a rate of 2% (v / v) into Erlenmeyer flasks containing 100 mL of the above-mentioned medium containing specific inorganic salts. Medium with the corresponding inorganic salts added but not inoculated served as a blank control. Each group was treated three times. After incubation at 28°C and 200 rpm with shaking for 24 hours, the OD of the culture medium was measured. 600 value.

[0050] The results are as follows Figure 7 As shown, strain BW-13 can grow in media containing CaCl2, MgCl2, and KCl, indicating its tolerance to ion stress caused by these salts, while its growth is significantly inhibited in media containing high concentrations of Na2CO3, NaHCO3, and NaNO2.

[0051] Example 5 The growth-promoting ability of strain BW-13 was investigated.

[0052] Identification of growth-promoting culture medium plates: The bacterial suspension of strain BW-13 was inoculated onto each identification plate, such as... Figure 8 As shown, the results indicate that strain BW-13 can stably grow distinct colonies on Assumption medium, demonstrating its nitrogen-fixing ability. Strain BW-13 also produces distinct clear zones after being cultured on inorganic phosphorus bacteria medium, skim milk medium, and carboxymethyl cellulose medium, indicating its ability to solubilize phosphorus and produce cellulase and protease.

[0053] Example 6 The salt tolerance and growth-promoting ability of strain BW-13 on cucumber was investigated.

[0054] Seed pretreatment: Select plump cucumber seeds with no surface damage, soak them in a 5% sodium hypochlorite solution for 3 minutes for disinfection, and rinse them three times with sterile distilled water. Bury them in a sterilized nutrient soil culture box until they germinate and grow two cotyledons.

[0055] Seedling treatment and grouping: Healthy cucumber seedlings with uniform growth were selected for treatment, with 30 seedlings per group. 10 mL of bacterial suspension or 10 mL of sterile water was added to each culture box. After 2 days of colonization, the seedlings were watered with sterile water and 2.9 g / L NaCl solution, respectively. The resulting groups were: unsalted-infected group, unsalted-sterile group, salinized-infected group, and salinized-sterile group. The seedlings were cultured at room temperature, with each culture pot sprayed with an appropriate amount of sterile water daily, and 10 mL of NaCl solution sprayed onto each culture box every 4 days.

[0056] Growth index determination: After 14 days of cultivation, the height of cucumber seedlings was measured; the stem diameter and root length of the seedlings were measured using vernier calipers; the dry weight and fresh weight were weighed using an electronic balance; and the maximum leaf area was measured. The maximum leaf area was calculated using the leaf area coefficient method, with the formula s=klw (s is the area, l is the length of the leaf, w is the width of the leaf, and k is the coefficient). In this invention, k is taken as 1 / 3.

[0057] The results are as follows Figure 9 As shown, salinization inhibits cucumber growth. Compared with the control group, the addition of bacterial suspension of strain BW-13 promoted seedling growth and improved its salt tolerance. Table 3 shows the measurement results of growth indicators of cucumber seedlings in the control and experimental groups in the pot experiment. According to the data in the table, compared with the control group, the plant height increased by up to 11.85%, the root length increased by up to 9.72%, the maximum leaf area increased by up to 6.77%, the dry weight increased by up to 25.53%, and the fresh weight increased by up to 15.72%; there was no significant effect on stem diameter.

[0058] Table 3. Effects of strain BW-13 on morphological parameters of cucumber seedlings

[0059] Note: Different letters in the table indicate significant differences between data in the same row (p<0.05).

[0060] Example 7 The anti-pathogenic activity of strain BW-13 was investigated.

[0061] The antibacterial spectrum of *Bacillus salsa* BW-13 was determined using the streak plate confrontation method. Six plant pathogens were cultured for 5-7 days, and holes were punched using a 5 mm diameter sterile punch. Mycelial cakes were inoculated onto the center of PDA plates, and two points were marked 2 cm away from the cakes to form a straight line for inoculation with *Bacillus salsa* BW-13. A control group inoculated only with plant pathogens was used. Each treatment was repeated three times. Figure 10As shown, the results indicate that it has a significant direct antagonistic effect on the growth of six plant pathogenic fungi, with mycelial growth inhibition rates ranging from 54.81% to 91.57%. The inhibition rates are shown in Table 4 below. The inhibition rate was calculated as follows: Inhibition rate = (Coronation diameter of control group - Coronation diameter of experimental group) / (Coronation diameter of control group - 0.5).

[0062] Table 4. Antagonistic and inhibitory effects of strain BW-13 against various plant pathogens

[0063] Note: Different letters in the table indicate significant differences (p<0.05) between data in the same column.

[0064] Example 8 The effect of strain BW-13 on the control of anthracnose in Polygonatum leaves inoculated with Polygonatum anthracnose was investigated.

[0065] 1. Preparation of experimental materials Select healthy, uniformly growing leaves of Polygonatum sibiricum, wash them with sterile water, disinfect the surface with 75% alcohol, rinse them 2-3 times with sterile water, and place them in a laminar flow hood to air dry. Use a disposable sterile syringe to puncture the surface of the leaf in the center to create a wound, and wrap the petiole with a cotton ball soaked in sterile water to keep it moist.

[0066] In the following procedures, "inoculation with fungal cake" refers to inoculating the leaf wound with a 5 mm diameter *Polygonatum sibiricum* anthracnose fungal cake. "Spraying" involves using a sterile, defatted cotton ball to apply BW-13 bacterial solution evenly to both sides of the leaf, then allowing it to air dry. "Volatile organic compounds (VOCs)" refers to placing the inoculated fungal cake in a sealed polyethylene film bag, along with three bags coated with 10 mm of solution. 8 Plates of CFU / mL BW-13 bacterial suspension.

[0067] 2. Processing group design Four experimental groups were set up, with 20 leaves treated in each group. The groups were: blank control (inoculated with mycelium 24 h after spraying with water), VOCs treatment, and mycelium inoculation 24 h after spraying with bacterial solution. All were stored at 28℃.

[0068] 3. Disease assessment methods Disease incidence was recorded regularly, and the disease index and relative control efficacy were calculated after 5 days. The leaf quality grading standards are shown in Table 5, and the calculation formulas for the disease index and relative control efficacy are as follows: G =[Σ( a × t ) / ( A × T )]×100. Where: G Indicates the disease index, a This indicates the number of blades of each quality grade in each repetition. tA representative numerical value indicating the quality grade of the blade. A This indicates the total number of leaves in each repetition; T The representative value representing the highest grade of leaf quality is: relative efficacy (%) = (control disease index - treatment disease index) / control disease index × 100.

[0069] Table 5. Quality Grading Standards for Polygonatum Leaf

[0070] The results show that, according to Table 6 and Figure 11 It was observed that on the 5th day after treatment, the blank control group showed no lesions except for natural aging, while the water-treated group developed large, dark brown lesions, and the leaves became soft and rotten. The lesion area treated with BW-13 bacterial solution and VOCs was significantly smaller than the control, and the disease index was reduced. The relative control efficacy reached 54.87% and 63.40%, respectively. Therefore, strain BW-13 has a good control effect on anthracnose infection in Polygonatum sibiricum, and its volatile gaseous products can effectively inhibit the infection of Polygonatum sibiricum leaves by the anthracnose pathogen.

[0071] Table 6. Disease control effect of strain BW-13 on Polygonatum leaves

[0072] Note: Different letters in the table indicate significant differences between data in the same column (p<0.05).

[0073] In summary, the *Bacillus sabinatus* BW-13 provided by this invention has the following outstanding advantages: broad-spectrum antibacterial activity: it exhibits significant antagonistic effects against a variety of plant pathogenic fungi, especially ascomycetes; environmental adaptability: it tolerates 0-12% NaCl, pH 5-10, 15-45℃, and various inorganic salt stresses; multifunctional growth-promoting properties: plate identification has confirmed that it possesses nitrogen-fixing, phosphorus-solubilizing, cellulase-producing, and protease-producing activities; diverse applications: direct application of the bacterial suspension can alleviate salt stress and promote crop growth; industrialization potential: simple cultivation conditions, suitable for developing biocontrol agents, soil conditioners, and stress-resistant growth-promoting agents. This strain has application value in the fields of saline-alkali soil bioremediation, plant disease control, and crop yield enhancement.

[0074] Where there is no conflict, the above embodiments and features described herein can be combined with each other.

[0075] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A salt-tolerant Bacillus sacchariformis strain with both biocontrol and growth-promoting functions, characterized in that, The *Bacillus sabolicii* strain mentioned is *Bacillus sabolicii* BW-13, which was deposited at the China Center for Type Culture Collection on January 13, 2025, and is classified and named as follows: Bacillus safensis The accession number is CCTCC NO: M 2025108.

2. A microbial agent, characterized in that, The inoculum comprises the fermentation broth of Bacillus salsa BW-13 as described in claim 1 and / or Bacillus salsa BW-13.

3. The application of Bacillus salsa BW-13 according to claim 1 or the bacterial agent according to claim 2, characterized in that, Includes any of the following applications: A1) Application in improving saline-alkali soil; A2) Applications in promoting plant growth; A3) Application in suppressing plant pathogens; A4) Application in the preparation of products that inhibit plant pathogens.

4. The application according to claim 3, characterized in that, The mass concentration of inorganic salts in the saline-alkali soil ranges from 0.5% to 12%, and the inorganic salts include one or more of NaCl, KCl, MgCl2 and CaCl2.

5. The application according to claim 4, characterized in that, The pH range of the saline-alkali soil is 5 to 10.

6. The application according to claim 3, characterized in that, The growth temperature range of the *Bacillus salsa* BW-13 is 15~45℃.

7. The application according to claim 3, characterized in that, The promotion of plant growth is manifested in all or part of the following: nitrogen fixation capacity, inorganic phosphorus dissolution capacity, cellulase secretion capacity, protease secretion capacity, and / or increased plant height growth rate.

8. The application according to claim 3, characterized in that, The plant pathogens include Staphylococcus aureus (… Botryosphaeria dothidea ), Botrytis cinerea ( Botrytis cinerea ), pear black rot skin fungus ( Valsa ambiens ), pathogenic fungus ( Phytophthora infestans Fusarium graminearum ( ), Fusarium graminearum ) or Polygonatum anthrax bacteria ( Colletotrichum spaethianum At least one of the following.

9. A method for promoting plant growth, characterized in that, The procedure includes the following steps: applying the Bacillus sabensis BW-13 of claim 1 or the inoculum of claim 2 to the plant plant, plant seed or plant rhizosphere soil.

10. A method for improving saline-alkali soil, characterized in that, The process includes the following steps: applying the Bacillus salsa BW-13 of claim 1 or the bacterial agent of claim 2 to the saline-alkali soil to be improved; wherein the improved saline-alkali soil exhibits salt tolerance and promotes growth.

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