Bacillus pumilus with salt-tolerant and growth-promoting functions and application thereof

Abstract

The application relates to the technical field of functional microorganism screening and application, and specifically provides a bacillus pumilus (Bacillus pumilus) with salt-tolerant and growth-promoting functions and an application thereof. Bacillus pumilus The preservation number of the bacillus pumilus is CCTCC NO: M20231590, the bacillus pumilus can effectively relieve salt damage of crops, promote growth of the crops, improve crop yield, improve the harm of increasingly serious land salinization to agricultural planting, and guarantee crop yield and quality.

Description

Technical Field

[0001] This invention relates to the field of functional microbial screening and application technology, specifically to a short Bacillus strain with salt-tolerant growth-promoting function and its application. Background Technology

[0002] Saline soil refers to a general term for various types of soil with high concentrations of soluble salt ions, which adversely affect the soil's physical and chemical properties and plant growth. Soil salinization can lead to undesirable soil characteristics and a decline in soil quality, seriously impacting agricultural production. The formation of soil salinization is influenced by multiple factors. Natural factors are the primary cause, but human activities also contribute. In agricultural production, unreasonable irrigation methods, such as heavy irrigation and light drainage, excessive fertilization, and unscientific crop rotation practices, can all lead to soil salinization in arable land.

[0003] Soil salinization alters soil properties, making it prone to compaction, poorly aerated and permeable, with low nutrient utilization, reduced organic matter content, decreased soil fertility, and decreased soil enzyme activity. This, in turn, affects soil microbial activity and the transformation of soil organic matter. Salt stress is one of the major abiotic stresses affecting plant growth and development, severely impacting crop yield, quality, and economic benefits. Increased soil salinity reduces plants' ability to absorb water, leading to physiological dehydration, resulting in stunted growth, slow growth, yellowing and wilting leaves, and even death.

[0004] Currently, the management and control of saline-alkali land has become a widespread concern. Current measures for saline-alkali land improvement mainly fall into four categories: agricultural, chemical, physical, and biological measures. Among these, agricultural engineering, chemical, and physical measures all have drawbacks to varying degrees, such as large engineering workloads, groundwater rise, salinization, and only short-term improvement of the surface saline soil. Saline soils contain abundant microbial resources, which are highly responsive to soil components, physicochemical properties, and microenvironment. Utilizing microorganisms to remediate saline-alkali land has attracted widespread attention. Beneficial microorganisms, during their life activities, produce a large amount of secondary metabolites, organic acids, which release slow-release nitrogen, phosphorus, and potassium from the soil. Through nitrogen fixation, potassium solubilization, and phosphorus solubilization, they promote plant growth; simultaneously, they produce siderophores, IAA, and ACC deaminases, enhancing plant resistance. Studies have shown that microbial agents can alter soil physicochemical properties and the community structure of soil microorganisms, increase soil nutrients, and promote plant growth. Microbial remediation has advantages such as being pollution-free, requiring low investment, being economical, highly efficient, and having a wide range of applications.

[0005] Screening high-performance salt-tolerant microbial strains from saline-alkali soils is of great significance for the widespread application of microbial agents in soil improvement and remediation, enhancing plant salt tolerance, and promoting plant growth under salt-alkali stress. It is also a research hotspot in this field. Summary of the Invention

[0006] The purpose of this invention is to provide a salt-tolerant and growth-promoting Bacillus pumilus strain and its applications. This Bacillus pumilus can effectively alleviate crop salt damage, promote crop growth, increase crop yield, mitigate the harmful effects of increasingly severe soil salinization on agricultural planting, and ensure crop yield and quality.

[0007] In one aspect, this invention provides a Bacillus pumilus BP67, which was deposited on September 4, 2023, at the China Center for Type Culture Collection, Wuhan University, Wuhan, China, with accession number CCTCC NO:M20231590.

[0008] This invention provides, in one aspect, the application of the aforementioned Bacillus pumilus BP67 strain in bio-fertilizer production.

[0009] This invention provides, in one aspect, the application of the aforementioned Bacillus pumilus BP67 strain in the improvement of saline-alkali land.

[0010] The present invention also provides a bio-fertilizer comprising Bacillus pumilus BP67.

[0011] The bio-fertilizer also contains any one or more combinations of Bacillus, Pseudomonas, Agrobacterium, nitrogen-fixing bacteria, Rhizobium, Penicillium, Aspergillus, Rhizopus, and Streptomyces.

[0012] The viable count of Bacillus brevis BP67 in the bio-fertilizer shall not be less than 10. 9 CFU / g.

[0013] The present invention also provides the application of the bio-fertilizer in agricultural planting.

[0014] The application rate of the bio-fertilizer is 1-5 kg / mu.

[0015] The *Bacillus pumilus* BP67 strain screened in this invention exhibits strong tolerance to high-salt environments and can grow on a medium with an 18% NaCl concentration. It produces high levels of indoleacetic acid (IAA) and extracellular polysaccharides (EPS). After 48 hours of fermentation, the IAA content in the fermentation supernatant reached 14.8 mg / L, and after 72 hours of fermentation, the EPS yield was 0.48 g / g. *Bacillus pumilus* BP67 also possesses the ability to produce ACC deaminase, which is beneficial for inhibiting ethylene synthesis in plants, reducing plant sensitivity to stress, and improving plant resistance.

[0016] Bacillus pumilus BP67 has no adverse effects on wheat seed germination and can effectively promote seedling growth, increase enzyme activity in the rhizosphere soil, and promote the absorption and utilization of nutrients such as carbon, nitrogen, and phosphorus by crops. Compared with the control group, the wheat seed germination rate in treatment group 1, which added Bacillus pumilus BP67 bacterial solution, was 100%. The plant height, root length, aboveground fresh weight, and underground fresh weight of wheat seedlings increased by 25.0%, 14.6%, 10.5%, and 14.3%, respectively, indicating better growth. The activities of sucrase, urease, and alkaline phosphatase in the wheat rhizosphere soil increased by 92.9%, 3.5%, and 7.1%, respectively, achieving unexpected technical results.

[0017] Bacillus pumilus BP67 can effectively reduce the impact of salt stress on seed germination and emergence, promoting normal crop growth under salt stress conditions. Compared with salt stress treatment group 2, treatment group 3, which also added Bacillus pumilus BP67 bacterial solution, showed a 26.7% increase in wheat seed emergence rate, and increases in wheat seedling height, root length, aboveground fresh weight, and underground fresh weight by 30.9%, 90.9%, 47.4%, and 77.8%, respectively. The activities of sucrase, urease, and alkaline phosphatase in the wheat rhizosphere soil increased by 14.2%, 37.7%, and 7.7%, respectively, demonstrating a very significant effect.

[0018] Bacillus pumilus BP67 can be widely used in the cultivation of crops in saline-alkali land, significantly improving seedling emergence rate, promoting crop growth, and enhancing quality and yield. Compared with the control group, the treatment group treated with Bacillus pumilus BP67 powder showed a 5.1% increase in corn seedling emergence rate and a 12.2% increase in yield; tomato plant height, plant weight, and yield increased by 15.6%, 23.7%, and 12.57%, respectively, achieving unexpected technical results.

[0019] The *Bacillus pumilus* BP67 provided by this invention can alleviate crop salt damage, promote crop growth, and increase crop yield. It can effectively mitigate the increasingly serious harm of soil salinization to agricultural planting, ensuring crop yield and quality. *Bacillus pumilus* BP67 can also be added to organic fertilizers, chemical fertilizers, and other fertilizers. It can be combined with any one or more of other *Bacillus*, *Pseudomonas*, *Agrobacterium*, nitrogen-fixing bacteria, rhizobia, *Penicillium*, *Aspergillus*, *Rhizopus*, and *Streptomyces*, showing broad application prospects. Attached Figure Description

[0020] Figure 1 This is a colony morphology diagram of Bacillus pumilus BP67.

[0021] Figure 2 The protein spectrum of Bacillus pumilus BP67;

[0022] Figure 3 A comparative diagram showing the effects of Bacillus pumilus BP67 on wheat emergence.

[0023] Figure 4 A comparative graph showing the growth-promoting effects of Bacillus pumilus BP67 on wheat;

[0024] Figure 5 This is a comparison chart showing the effects of Bacillus pumilus BP67 on corn cultivation. Detailed Implementation

[0025] For the specific methods or materials used in the embodiments, those skilled in the art can make conventional substitutions based on existing technologies, and not be limited to the specific descriptions in the embodiments of the present invention.

[0026] The present invention will be further described below with reference to specific embodiments.

[0027] Example 1: Isolation and Screening of Strains

[0028] 1.1 Sample source: Suaeda salsa and rhizosphere soil from the Qingdao tidal flats.

[0029] 1.2 Isolation of salt-tolerant strains

[0030] Take 10g of soil sample and place it in 90mL of sterile water. Shake at 180rpm for 30min to obtain a sample mixture. After serial dilution, spread the sample mixture onto 10% NaCl LB solid medium to isolate salt-tolerant bacterial strains from the sample mixture. Incubate at 37℃ for 48-96h. Select colonies of different morphology, color, and size for multiple purifications to obtain 6 strains, which are numbered NY1-NY6 and preserved in liquid glycerol.

[0031] 1.3 Determination of salt tolerance of strains

[0032] Salt-tolerant bacterial strains were screened by continuously increasing the salinity of the culture medium. LB medium with NaCl concentrations of 10%, 12%, 14%, 16%, and 18% was prepared. The six strains isolated and purified above were inoculated into plates and incubated at 37°C. The presence or absence of bacterial growth was used as the criterion for judging the salt and alkali tolerance of the strains, and the strain with the best salt and alkali tolerance was screened. The results are shown in Table 1.

[0033] Table 1 Salt tolerance of each strain

[0034]

[0035] Note: "+" indicates that the strain has grown, and "-" indicates that the strain has not grown.

[0036] As shown in Table 1, different strains showed different growth characteristics in culture media with different salinities. Among them, strain NY6 had the strongest salt tolerance and could grow in culture media with a salinity of 18% NaCl.

[0037] The applicant named strain NY6 BP67 and conducted further evaluation on it.

[0038] Example 2: Identification of BP67 strain

[0039] 2.1 Colony morphology identification

[0040] Colonies of BP67 strain on LB medium as shown Figure 1 As shown, the color is white, round, with a smooth surface, neat edges, raised and viscous; the Bacillus cells are straight rod-shaped and can produce spores, which are round and Gram stain positive under microscopic examination.

[0041] 2.2 Molecular identification of 16S rRNA

[0042] The genome of strain BP67 was extracted using a kit. Then, using this genome as a template, its 16S rRNA was amplified using specific primers 27F and 1492R.

[0043] 27F: 5'-AGAGTTTGATCATGGCTCAG-3';

[0044] 1492R: 5'-TAGGGTTACCTTACGACTT-3'.

[0045] The PCR system consisted of: 0.7 μl 27F, 0.7 μl 1492R, 4 μl template DNA, 17.5 μl SuperMiX, and 12.1 μl water. The PCR reaction conditions were set as follows: (1) 94℃ for 5 min; (2) 94℃ pre-denaturation for 30 s; (3) 55℃ for 30 s; (4) 72℃ for 1 min; and steps (2) to (4) were repeated for 35 cycles; (5) 72℃ for 10 min. The amplified PCR products were detected by 1% agarose gel electrophoresis. The results showed that the PCR product size was approximately 1500 bp, which met the requirements.

[0046] The PCR amplification product was sent to a sequencing company for sequencing. The obtained sequence SEQ ID NO:1 was compared with the NCBI database using BLAST, and it showed the highest similarity to Bacillus pumilus. Therefore, BP67 strain was preliminarily identified as Bacillus pumilus.

[0047] 2.3 MALDI-TOF-MS protein spectroscopy identification

[0048] A small amount of BP67 single colony was coated onto a target plate in the form of a thin film; 1 μL of lysis buffer from the mass spectrometry sample pretreatment kit was added, and the sample was allowed to air dry at room temperature; 1 μL of matrix solution from the mass spectrometry sample pretreatment kit was added to cover the sample, and the sample was allowed to air dry at room temperature; the sample target was then placed in a mass spectrometer for identification. The identification results showed that the BP67 strain was *Bacillus pumilus*, and its protein spectrum peaks are shown below. Figure 2 As shown.

[0049] In summary, the applicant used two molecular biology methods—16S rRNA identification and MALDI-TOF-MS protein spectrometry—to identify strain BP67, and the results were consistent. Combined with the colony morphology characteristics of strain BP67, the applicant identified the strain as *Bacillus pumilus* and named it *Bacillus pumilusBP67*.

[0050] The applicant deposited the above-mentioned Bacillus pumilus BP67 at the China Center for Type Culture Collection, Wuhan University, Wuhan, China on September 4, 2023, with accession number CCTCC NO:M20231590.

[0051] Example 3 Evaluation of the salt tolerance and growth-promoting characteristics of Bacillus pumilus BP67

[0052] Salt-tolerant microorganisms can adapt to high-salt environments by forming extracellular polysaccharides (EPS) and biofilms. They can also directly regulate and alleviate plant salt stress by producing plant hormone signals IAA and ACC deaminase, thereby improving the acquisition of nutrients and other resources and promoting healthy crop growth.

[0053] 3.1 Ability to produce indoleacetic acid (IAA)

[0054] Bacillus pumilus BP67 was inoculated at a 5% inoculum into LB liquid medium containing 5% NaCl and cultured at 30°C and 180 rpm for 48 h. The fermentation broth was centrifuged at 10,000 rpm for 10 min to remove the bacterial cells. 2 mL of the supernatant was mixed with an equal volume of Salkowski reagent, and the mixture was placed in the dark. After 30 min, the absorbance of the solution was measured at a wavelength of 530 nm. The IAA content in the fermentation broth was calculated based on the IAA standard curve.

[0055] The results showed that after 48 hours of fermentation culture with Bacillus pumilus BP67, the IAA content in the fermentation supernatant reached as high as 14.8 mg / L. The IAA secreted by Bacillus pumilus BP67 can promote the synthesis of IAA in plants, thereby promoting crop growth.

[0056] 3.2 Capacity to produce extracellular polysaccharides (EPS)

[0057] Bacillus pumilus BP67 was inoculated into EPS-producing fermentation medium at a 5% inoculum and cultured at 30°C for 72 h. 10 mL of the fermentation broth was centrifuged at 4000 r / min for 15 min to collect the bacterial cells. The bacterial cells were dried at 105°C to constant weight. The fermentation supernatant was mixed with 30 mL of 95% ethanol and incubated overnight at 4°C for alcohol precipitation. The mixture was centrifuged at 4000 r / min for 30 min to collect the precipitate. The precipitate was dried at 105°C to constant weight to obtain EPS.

[0058] The formula for calculating EPS yield of the strain is as follows:

[0059] EPS yield (g / g) = (dry weight of EPS in fermentation broth - dry weight of EPS in culture medium) / dry weight of strain.

[0060] The results showed that after 72 hours of fermentation culture with Bacillus pumilus BP67, the yield of EPS was 0.48 g / g.

[0061] 3.3 Ability to produce 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase

[0062] Prepare 5 g / L ACC and ammonium sulfate solutions separately, and filter sterilize. Add 5 g / L ACC solution to sterilized SM liquid medium to achieve a final ACC concentration of 0.5 g / L, which is the SMA medium. Similarly, add 5 g / L ammonium sulfate solution to SM medium to achieve a final ammonium sulfate concentration of 0.5 g / L, which is the SMN medium. Inoculate the strain into liquid LB medium and culture for 12-24 h. Centrifuge at 5000 rpm and 4℃ for 10 min, discard the supernatant, collect the bacterial cells, and resuspend them in sterile water. Add 0.5 ml of the bacterial suspension to each of the three liquid media (SM, SMA, and SMN). Incubate at 28℃ and 180 rpm for 12 h on a shaker. Determine the ability to produce ACC deaminase by measuring the absorbance at 535 nm in the three different media. The strain producing ACC deaminase cannot grow in SM medium, but can grow stably in SMA and SMN media.

[0063] The results showed that *Bacillus pumilus* BP67 grew only in small amounts in nitrogen-free SM medium, but could grow in SMA medium with ACC as the sole nitrogen source, exhibiting stronger growth than in SM medium. This ruled out the influence of nitrogen fixation factors, indicating that *Bacillus pumilus* BP67 has the ability to produce ACC deaminase. ACC deaminase can decompose ACC into ammonia and α-butanol, thereby inhibiting ethylene synthesis in plants, reducing plant sensitivity to stress, and improving plant resistance.

[0064] Example 4: Effects of Bacillus pumilus BP67 on wheat seed emergence under salt stress.

[0065] 4.1 Preparation of bacterial culture

[0066] Bacillus pumilus BP67 was activated, and the activated Bacillus pumilus BP67 was inoculated into nutrient broth medium and cultured at 37℃ and 220 r / min for 18 h to obtain a viable count of 10. 8 -10 9 Prepare a bacterial culture at CFU / ml for later use.

[0067] 4.2 Hydroponic Experiment

[0068] Disinfect wheat seeds with 10% sodium hypochlorite solution for 30 minutes, rinse three times with clean water, and then sow 15 seeds per hydroponic bottle. The hydroponic system is 700ml, and the specific groups are as follows:

[0069] Control group (CK): 700ml of water;

[0070] Treatment group 1: 100ml BP67 bacterial culture + 600ml water

[0071] Treatment group 2: 100ml water + 600ml 100mmol / L NaCl aqueous solution;

[0072] Treatment group 3: 100 ml BP67 bacterial culture + 600 ml 100 mmol / L NaCl aqueous solution

[0073] Cultivate in an incubator at 25℃, observe and record regularly, and count seedling emergence and measure plant height after 7 days.

[0074] Table 2. Effects of Bacillus pumilus BP67 on wheat emergence.

[0075] Grouping Handling method Number of seedlings (plants) Emergence rate Plant height CK Clear water 15 100％ 8.8cm Processing Group 1 BP67 bacterial solution 15 100％ 10.2cm Processing Group 2 NaCl aqueous solution 11 73.3％ 5.6cm Processing Group 3 NaCl aqueous solution + BP67 bacterial solution 15 100％ 7.5cm

[0076] From Table 2 and Figure 3 The results showed that, compared with the control group, the wheat seed germination rate in treatment group 1, which included Bacillus pumilus BP67 bacterial solution, was 100%, and the wheat seedlings grew better, with a plant height increase of 15.9%. This indicates that Bacillus pumilus BP67 has no side effects on seed germination and can effectively promote seedling growth.

[0077] Salt stress can severely affect seed germination. In treatment group 2, where NaCl aqueous solution was added, the emergence rate of wheat seeds was only 73.3%, and the plant height was also 36.4% lower than the control group. Compared to treatment group 2, treatment group 3, which also added Bacillus pumilus BP67 bacterial solution, showed a 26.7% increase in emergence rate and a 33.9% increase in plant height, demonstrating a very significant effect. This indicates that Bacillus pumilus BP67 can effectively reduce the impact of salt stress on seed germination and emergence, promote healthy wheat growth, and achieve unexpected technical results.

[0078] Example 5: Evaluation of the growth-promoting effect of Bacillus pumilus BP67 on wheat seedlings under salt stress.

[0079] 5.1 Preparation of bacterial culture

[0080] Bacillus pumilus BP67 was activated, and the activated Bacillus pumilus BP67 was inoculated into nutrient broth medium and cultured at 37℃ and 220 r / min for 18 h to obtain a viable count of 10. 8 -10 9 Prepare a bacterial solution of CFU / ml for later use.

[0081] 5.2 Pot Experiment

[0082] Wheat seeds were surface-sterilized with 10% sodium hypochlorite for 30 minutes, rinsed three times with clean water, and then sown in seedling trays for germination and cultivation. After germination, seedlings with uniform growth and health were selected and transplanted into pots. The soil in the pots was treated as follows:

[0083] Control group (CK): Add 150ml of water;

[0084] Treatment group 1: Add 100ml BP67 bacterial solution + 50ml water;

[0085] Treatment group 2: Add 50 ml of 200 mmol / L NaCl aqueous solution + 100 ml of water;

[0086] Treatment group 3: Add 50 ml of 200 mmol / L NaCl aqueous solution and 100 ml of BP67 bacterial culture;

[0087] Each group was set up in triplicate and cultured in an incubator at 25℃. Samples were taken after 10 days for testing, and plant growth indicators and wheat rhizosphere soil enzyme activities were measured. The activities of alkaline phosphatase, urease, and sucrase in wheat rhizosphere soil were measured using the indophenol colorimetric method, the ninhydrin colorimetric method, and the 3,5-dinitrosalicylic acid colorimetric method, respectively.

[0088] 5.3 Experimental Results

[0089] Table 3. Effects of different treatment groups on wheat seedling growth.

[0090] category Handling method Plant height Root length Fresh weight of aboveground parts underground fresh weight CK Clear water 12cm 4.1cm 3.8g 2.1g Processing Group 1 BP67 bacterial solution 15 cm 4.8cm 4.2g 2.4g Processing Group 2 NaCl aqueous solution 6.8 cm 2.2cm 1.9g 0.9g Processing Group 3 NaCl aqueous solution + BP67 bacterial solution 8.9 cm 4.2cm 2.8g 1.6g

[0091] From Table 3 and Figure 4 The results showed that, compared with the control group, the wheat seedlings in the treatment group 1, which was treated with Bacillus pumilus BP67 bacterial solution, exhibited significantly increased plant height, root length, aboveground fresh weight, and underground fresh weight by 25.0%, 14.6%, 10.5%, and 14.3%, respectively, indicating better seedling growth. This demonstrates that Bacillus pumilus BP67 effectively promotes wheat seedling growth with remarkable results.

[0092] Salt stress inhibits wheat growth. In treatment group 2, with the addition of NaCl aqueous solution, the plant height, root length, aboveground fresh weight, and underground fresh weight of wheat seedlings were significantly lower than those in the control group. Compared with treatment group 2, treatment group 3, with the addition of Bacillus pumilus BP67 bacterial solution, showed increases in plant height, root length, aboveground fresh weight, and underground fresh weight of wheat seedlings by 30.9%, 90.9%, 47.4%, and 77.8%, respectively. This indicates that Bacillus pumilus BP67 can effectively reduce the inhibitory effect of salt stress and promote the recovery of normal crop growth.

[0093] Table 4 Effects of different treatments on soil enzyme activity

[0094]

[0095] As shown in Table 4, compared with the control group, the activities of sucrase, urease, and alkaline phosphatase in the rhizosphere soil of wheat in treatment group 1 (containing Bacillus pumilus BP67 bacterial solution) increased by 92.9%, 3.5%, and 7.1%, respectively. Salt stress significantly reduced the activities of these three enzymes, and the activities of all three enzymes in the rhizosphere soil of wheat in treatment group 2 also decreased significantly. Compared with treatment group 2, the activities of sucrase, urease, and alkaline phosphatase in the rhizosphere soil of wheat in treatment group 3 (containing Bacillus pumilus BP67 bacterial solution) increased by 14.2%, 37.7%, and 7.7%, respectively, showing a very significant effect.

[0096] Sucrase participates in the soil organic carbon cycle, effectively improving soil carbon utilization; urease is a key enzyme in soil nitrogen transformation, and its increased activity can promote the conversion of available nitrogen; soil phosphatase plays an important role in plant phosphorus nutrition by mediating the release of inorganic phosphorus from organically bound phosphorus. Therefore, Bacillus pumilus BP67 can effectively improve soil enzyme activity, promote crop absorption and utilization of nutrients such as carbon, nitrogen, and phosphorus, and promote normal crop growth under salt stress conditions.

[0097] Example 6: Application of Bacillus pumilus BP67 in maize cultivation in saline-alkali land

[0098] 6.1 Experimental site and crop varieties:

[0099] The corn variety used in the saline-alkali land planting base in the Agricultural High-tech Zone of Dongying City, Shandong Province is Meikang 68.

[0100] 6.2 Field Experiment:

[0101] The experimental area was 4 mu (approximately 0.67 hectares), divided into two groups, with each group having a treatment area of ​​2 mu (approximately 0.33 hectares). The soil salinity of the experimental plots was 3‰. The treatment groups received Bacillus pumilus BP67 inoculum powder (10 billion CFU / g), applied at 1 kg / mu (approximately 0.67 hectares) as basal fertilizer during tilling and again after 20 days via irrigation. The control group (CK) received conventional fertilization without inoculum. Field management practices were kept consistent. Emergence rate was assessed at the seedling stage, and growth indicators and yield were assessed at harvest.

[0102] 6.3 Results

[0103] Table 5. Effects of Bacillus pumilus BP67 on maize.

[0104]

[0105] From Table 5 and Figure 5 The experimental results showed that, compared with the control group, the corn emergence rate increased by 5.1% and the yield increased by 12.2% in the treatment group treated with Bacillus pumilus BP67 powder. This demonstrates that Bacillus pumilus BP67 can effectively promote the emergence and growth of corn in saline-alkali soil, achieving unexpected technical results.

[0106] Example 7: Application of Bacillus pumilus BP67 in tomato cultivation in saline-alkali soil

[0107] 7.1 Experimental site and crop varieties:

[0108] The tomato greenhouses in Liuhe Street, Dongying City, Shandong Province, are planted with the Provence variety.

[0109] 7.2 Field Experiment:

[0110] The soil salinity of the experimental plot was 2‰. Each experimental plot measured 10m × 8m, containing 10 rows of tomato plants, approximately 400±20 plants per plot. Treatment groups were treated with 10 billion CFU / g of BP67 inoculum powder, while other fertilizer, water, and pesticide management remained consistent. Each treatment group was replicated three times, for a total of nine experimental plots, as follows:

[0111] Control group (CK): No bacterial agent was used; the fungicide was applied by watering.

[0112] Treatment Group 1: Apply 500g / mu each time by watering after transplanting and by fertigation 10 days after transplanting.

[0113] Treatment Group 2: Apply 1 kg / mu each time by watering after transplanting and by irrigation 10 days after transplanting.

[0114] During the tomato growing season, plant height, plant weight, and yield were investigated and statistically analyzed to calculate the yield increase effect. Samples were taken to test the soluble sugar and vitamin C content of tomatoes and to evaluate the impact on quality.

[0115] 7.3 Experimental Results

[0116] Table 6. Application effects of Bacillus pumilus BP67 on tomatoes

[0117]

[0118] As shown in Table 6, the application of Bacillus pumilus BP67 powder can significantly promote tomato growth in saline-alkali soil. The plant height, plant weight, and yield of the treated group were increased by 15.6%, 23.7%, and 12.57% respectively compared with the control group. At the same time, the quality of the treated group tomatoes was significantly improved, with soluble sugar and vitamin C content increasing by 13.3% and 15.8% respectively, achieving unexpected technical results.

[0119] In summary, the Bacillus pumilus BP67 provided by this invention can be widely used in the cultivation of crops in saline-alkali land. It can alleviate crop salt damage, promote crop growth, and increase crop yield, effectively mitigating the increasingly serious harm of soil salinization to agricultural planting and ensuring crop yield and quality. Bacillus pumilus BP67 can also be added to organic fertilizers, chemical fertilizers, and other fertilizers, and can be combined with any one or more of other Bacillus species, Pseudomonas, Agrobacterium, nitrogen-fixing bacteria, Rhizobium, Penicillium, Aspergillus, Rhizopus, and Streptomyces, showing broad application prospects.

Claims

1. A short-lived Bacillus species, characterized in that, The preservation number of the Bacillus pumilus is CCTCC NO: M20231590.

2. The application of Bacillus pumilus as described in claim 1 in the production of bio-fertilizer.

3. The application of the Bacillus pumilus according to claim 1 in the improvement of saline-alkali land.

4. A bio-fertilizer, characterized in that, The bio-fertilizer comprises the Bacillus simulans described in claim 1.

5. The bio-fertilizer as described in claim 4, characterized in that, The bio-fertilizer also contains any one or more combinations of Bacillus, Pseudomonas, Agrobacterium, nitrogen-fixing bacteria, Rhizobium, Aspergillus, Rhizopus, and Streptomyces.

6. The bio-fertilizer as described in claim 4 or 5, characterized in that, The viable count of Bacillus pumilus in the bio-fertilizer is not less than 10. 9 CFU / g.

7. The application of the bio-fertilizer according to any one of claims 4-6 in agricultural planting.

8. The application as described in claim 7, characterized in that, The application rate of the bio-fertilizer is 1-5 kg / mu.

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