Microvesicle bacteria and application of microbial inoculum thereof in saline-alkali soil

Abstract

The invention relates to the field of agricultural microbial technology and soil remediation, in particular to application of microvesicle bacteria and a microbial agent thereof in saline-alkali soil, the name of the microvesicle bacteria is Microbulbifer sp.PM1, the preservation unit is China Center for Type Culture Collection, the preservation acceptance date is January 4, 2026, and the preservation number is CGMCC NO: 37273. Through tests and determination, the microbial agent prepared from the PM1 has important application value in the field of saline-alkali soil improvement, especially in high-quality and yield-increasing cultivation of corn.

Description

Technical Field

[0001] This invention relates to the fields of agricultural microbial technology and soil remediation, specifically to a strain of Microbulbifer sp. PM1 with highly efficient salt and alkali tolerance and growth-promoting function, and its application in improving saline-alkali land and promoting maize growth. Background Technology

[0002] Saline-alkali land is one of the major obstacles restricting global agricultural production. High salt concentrations lead to increased soil osmotic pressure, making it difficult for plant roots to absorb water, while also causing ion toxicity and nutrient imbalance, severely inhibiting crop growth. As a major food crop in my country, maize yields are significantly affected by saline-alkali land.

[0003] Currently, methods for improving saline-alkali land mainly include physical methods (such as irrigation to leach salt), chemical methods (such as applying gypsum or phosphogypsum), and biological methods. Physical and chemical methods are costly and may cause secondary pollution or have short-lived effects. Biological methods, especially those utilizing salt-tolerant plant growth-promoting bacteria, are an environmentally friendly and sustainable strategy. Plant growth-promoting bacteria promote plant growth and enhance their resistance through multiple mechanisms, including nitrogen fixation, phosphorus solubilization, and the secretion of plant hormones.

[0004] However, plant growth-promoting bacteria reported in existing technologies often exhibit poor colonization ability and unstable effects in complex saline-alkali environments. Therefore, isolating and screening superior strains with high efficiency in adapting to saline-alkali stress and developing their application methods are of great significance for utilizing saline-alkali land and ensuring food security. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide a Burkholderia strain with extremely strong salt and alkali resistance. The strain is named Microbulbifer sp. PM1, deposited at the China Center for Type Culture Collection (CGMCC), date of acceptance of deposit: January 4, 2026, accession number: CGMCC NO: 37273.

[0006] Furthermore, the application of the aforementioned microbubble bacteria in improving saline-alkali land, especially in improving and promoting the growth of maize in saline-alkali land, has significant effects.

[0007] Furthermore, the present invention provides a microbial inoculant, the microbial inoculant being a microbubble bacteria strain PM1, and a nutrient adjuvant; preferably, the nutrient adjuvant includes at least one of humic acid, complex amino acids, and trace elements; preferably, the trace elements include at least one of zinc sulfate, boric acid, and sodium molybdate.

[0008] Further, the microbial agent comprises: microbubble bacteria strain PM1, humic acid, complex amino acids, and trace elements; by weight ratio, the content of humic acid is 10% to 20% of the total mass of the composition, the content of the complex amino acids is 5% to 10%, and the content of trace elements is 0.5% to 1%; more preferably, by weight ratio, the content of humic acid is 15% of the total mass of the composition, the content of the complex amino acids is 7.5%, and the content of trace elements is 0.75%.

[0009] Furthermore, the microbubble bacteria strain PM1 is obtained through fermentation, and the effective viable bacteria count in the fermentation solution is ≥1×10⁻⁶. 8 CFU / mL.

[0010] Furthermore, the preparation process of the microbial agent described in this invention is as follows:

[0011] S1: Preparation of Seed Liquid

[0012] Microbubble bacteria strain PM1 was inoculated into LB liquid medium and cultured with shaking to obtain seed liquid A containing strain PM1;

[0013] S2: Preparation of microbial culture

[0014] Dilute seed culture A and then add it to the fermentation broth at a ratio of 0.5-1.5%. Shake and culture to obtain seed culture B containing strain PM1.

[0015] S3: Preparation of microbial inoculants

[0016] Take 1 L of seed liquid B and place it in a container. Then add humic acid, amino acids and trace elements and mix evenly to make a microbial inoculant.

[0017] Furthermore, the preparation process of the microbial inoculant described in this invention is as follows:

[0018] S1: Preparation of Seed Liquid

[0019] Microbubble bacteria strain PM1 was inoculated into LB liquid medium and cultured at 30°C and 180 rpm for 48 h to obtain an effective viable count ≥1×10⁻⁶. 8 Seed culture A, CFU / mL, should be diluted to 1×10⁻⁶ before use. 8 ~ 1×10 10 CFU / mL;

[0020] S2: Preparation of microbial culture

[0021] Seed culture A was inoculated into the fermentation broth at a ratio of 1%, and cultured at 30°C and 180 rpm for 48 hours with shaking to obtain an effective viable count ≥1×10⁻⁶. 8Bacterial solution B with CFU / mL;

[0022] S3: Preparation of microbial inoculants

[0023] Take 1 L of the bacterial solution from step S2, and then mix it evenly with 50-100g of humic acid, 20-60g of amino acids and 2-6g of trace elements to prepare a microbial inoculant.

[0024] A microbial agent was prepared using the above fermentation broth. The microbial agent, calculated in g / L, comprises: 1 part of the bacterial broth obtained from the above fermentation, 50-100 parts of humic acid, 20-60 parts of complex amino acids, and 2-6 parts of trace elements; wherein the trace elements are composed of ZnSO4, H3BO3, and Na2MoO4 in a weight ratio of 1:1:1; more preferably, the microbial agent comprises: 1 part of the bacterial broth as described in any one of claims 6-7, 75 parts of humic acid, 40 parts of complex amino acids, and 4 parts of trace elements, wherein the effective viable count of the microbubble bacteria strain PM1 is 1×10⁻⁶. 8 ~ 1×10 10 CFU / mL.

[0025] The aforementioned compound amino acids include natural or synthetic amino acids. Amino acid fertilizers can be various protein hydrolysates and / or synthetic and / or natural pure amino acids and / or mixtures and / or mother liquors. Amino acids include natural amino acids such as glycine and glutamic acid, or synthetic amino acids such as iminodiacetic acid, or any combination and proportion thereof. Amino acids are common biological nutrients, obtainable through hydrolysis of various plant and animal proteins, or through microbial fermentation.

[0026] Furthermore, the application of the aforementioned microbial inoculant in improving saline-alkali land and promoting maize growth.

[0027] Furthermore, the microbial agent is used to improve saline-alkali land and promote maize growth, and is applied 2 to 5 times during the maize growth cycle, with each application amount being 1 to 5 L / mu.

[0028] Compared with existing technologies, microbial agents containing the microbubble bacteria strain PM1 have achieved unexpected technical effects in improving crops in saline-alkali land. Specifically:

[0029] The microbubble bacteria strain PM1 exhibits significant effects in improving saline-alkali soil and promoting maize growth, as evidenced by pot and field experiments. In pot experiments, treatment with microbubble bacteria strain PM1 effectively promoted maize growth, specifically increasing plant height, stem diameter, leaf SPAD value, root length, above-ground fresh weight, and below-ground fresh weight. It also improved saline-alkali soil properties, regulating soil pH, electrical conductivity, and salinity, and increasing soil organic matter content, available nitrogen content, and available phosphorus content. Field experiments further verified that microbial agents based on this strain not only significantly increased maize thousand-grain weight but also improved saline-alkali soil. In conclusion, microbubble bacteria strain PM1 has significant application value in saline-alkali soil improvement and high-yield maize cultivation. Attached Figure Description

[0030] Figure 1 This is a morphological diagram of the microbubble bacteria strain PM1.

[0031] Figure 2 This study investigated the growth-promoting effect of Microbubble Bacterium strain PM1 on maize in a pot experiment.

[0032] The effect of Microbubble Bacterium strain PM1 on maize growth promotion in a pot experiment. af represents the plant height, stem diameter, leaf SPAD value, root length, aboveground fresh weight, and underground fresh weight of maize in the control group and the B1 treatment group, respectively.

[0033] Figure 3 This study investigates the effect of microbubble bacteria strain PM1 on the improvement of saline-alkali soil in a pot experiment.

[0034] The effect of Microbubble Bacterium strain PM1 on the improvement of saline-alkali soil in a pot experiment. af represents the soil pH, electrical conductivity, salinity, organic matter content, available nitrogen content, and available phosphorus content in the control group and the B1 treatment group, respectively.

[0035] Figure 4 This study investigated the effect of microbial inoculants on increasing the thousand-kernel weight of corn in a field experiment.

[0036] Figure 5 This study demonstrates the effect of microbial agents on improving saline-alkali soil in field experiments.

[0037] In the figure, CK represents the blank control group, B1 represents the treatment group with microbubble bacteria strain PM1, and BF represents the treatment group with microbial inoculants. The af values ​​represent the soil pH, electrical conductivity, salinity, organic matter content, available nitrogen content, and available phosphorus content in the control group and the B1 treatment group, respectively, in the field experiment. Detailed Implementation

[0038] The technical solution of the present invention will be clearly and completely described below with reference to specific embodiments. The described embodiments are merely some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0039] Example 1: Identification of salt and alkali tolerance of Microbubble Bacterium strain PM1

[0040] To identify the salt and alkali tolerance of the strain, solid LB medium with pH values ​​of 8, 9, 10, 11, and 12, as well as solid LB medium with salt contents of 2%, 5%, 8%, and 10%, were prepared. Single-clonal strains were inoculated onto solid LB medium and cultured at 28°C for 48 hours, with naturally growing strains used as controls. The results showed that this highly salt- and alkali-tolerant strain could grow in an environment with a 10% salt concentration and pH of 10, and was named PM1. Figure 1 As shown.

[0041] The LB plate formulation for the salinity gradient consisted of 10-100 g / L sodium chloride, 5 g / L yeast extract, 10 g / L tryptone, and 1 L distilled water, and was sterilized in an autoclave at 121°C for 20 min.

[0042] The alkalinity gradient LB plate formulation consists of 10 g / L sodium chloride, 5 g / L yeast extract, 10 g / L tryptone, and 1 L distilled water, with the pH adjusted using 1 M to 5 M NaOH solution.

[0043] Example 2: Preparation of microbial inoculants

[0044] Seed culture: Microbubble bacteria strain PM1 was inoculated into LB liquid medium and cultured at 30°C and 180 rpm for 48 h with shaking to obtain an effective viable count ≥1×10⁻⁶. 8 The seed culture is CFU / mL, diluted to 1×10⁻⁶ before use. 8 ~ 1×10 10 CFU / mL.

[0045] Microbial culture: The seed culture was inoculated into the fermentation broth at a ratio of 1%, and cultured at 30°C and 180 rpm for 48 hours with shaking to obtain an effective viable count ≥1×10⁻⁶. 8 CFU / mL bacterial suspension.

[0046] Preparation of microbial inoculant: Take 1 L of the above bacterial solution and mix it evenly with 75 g of humic acid, 40 g of amino acids and 4 g of trace elements (ZnSO4, H3BO3 and Na2MoO4 in a weight ratio of 1:1:1) to prepare microbial inoculant.

[0047] The fermentation liquid is made by adding 5% brown sugar to purified water.

[0048] The humic acid is a mineral- or biochemical-derived humic acid that meets the "Standard for Humic Acid for Agricultural Use"; the amino acid is a compound amino acid, which is commercially available and is dissolved in water to obtain a hydrolysate. The total amount of amino acids after hydrolysis is 50%.

[0049] Example 3: Preparation of microbial inoculants

[0050] Seed culture: Microbubble bacteria strain PM1 was inoculated into LB liquid medium and cultured at 30°C and 180 rpm for 48 h with shaking to obtain an effective viable count ≥1×10⁻⁶. 8 Seed culture at CFU / mL. Dilute to 1×10⁻⁶ before use. 8 ~ 1×10 10 CFU / mL.

[0051] Microbial culture: The seed culture was inoculated into the fermentation broth at a ratio of 1%, and cultured at 30°C and 180 rpm for 48 hours with shaking to obtain an effective viable count ≥1×10⁻⁶. 8 CFU / mL bacterial suspension.

[0052] Microbial inoculant: Take 1 L of the above bacterial solution and mix it evenly with 50g of humic acid, 20g of amino acids and 2g of trace elements (the trace element is ZnSO4) to prepare a microbial inoculant.

[0053] The fermentation liquid is made by adding 5% brown sugar to purified water.

[0054] The humic acid is a mineral- or biochemical-derived humic acid that meets the "Standard for Humic Acid for Agricultural Use"; the amino acid is a compound amino acid, which is commercially available and is dissolved in water to obtain a hydrolysate. The total amount of amino acids after hydrolysis is 40%.

[0055] Example 4: Preparation of microbial inoculants

[0056] Seed culture: Microbubble bacteria strain PM1 was inoculated into LB liquid medium and cultured at 30°C and 180 rpm for 48 h with shaking to obtain an effective viable count ≥1×10⁻⁶. 8 Seed culture at CFU / mL. Dilute to 1×10⁻⁶ before use. 8 ~ 1×10 10 CFU / mL.

[0057] Microbial culture: The seed culture was inoculated into the fermentation broth at a ratio of 1%, and cultured at 30°C and 180 rpm for 48 hours with shaking to obtain an effective viable count ≥1×10⁻⁶. 8 CFU / mL bacterial suspension.

[0058] Microbial inoculant: Take 1 L of the above bacterial solution and mix it evenly with 100g of humic acid, 60g of amino acids and 6g of trace elements (ZnSO4, H3BO3 weight ratio is 1:1) to prepare microbial inoculant.

[0059] The fermentation liquid is made by adding 5% brown sugar to purified water.

[0060] The humic acid is a mineral- or biochemical-derived humic acid that meets the "Standard for Humic Acid for Agricultural Use"; the amino acid is a compound amino acid, which is commercially available and is dissolved in water to obtain a hydrolysate. The total amount of amino acids after hydrolysis is 60%.

[0061] Example 5: Potted plant experiment to verify the growth-promoting effect

[0062] 1. Two-week-old corn seedlings were transplanted into flowerpots (15 cm in diameter × 13 cm in bottom diameter × 11 cm in height) containing 500 g of saline-alkali soil (5.10 g / kg salt content, pH 8.05). The treatment groups were named CK-control group and B1-microbubble bacteria strain PM1 treatment group (microbial inoculant prepared in Example 2), with 6 replicates for each group. The B1 treatment group was treated every 7 days with 10 mL of microbubble bacteria strain PM1 bacterial solution (1 × 10⁻⁶). 8 ~ 1×10 10 (CFU / mL), and the same amount of water was added to the CK control group at the same time.

[0063] 2. The above-mentioned experiments were used to study the growth-promoting effects of strain PM1 on maize. After 30 days, maize plant height, stem diameter, leaf SPAD value, aboveground fresh weight, belowground fresh weight, root length, and root activity were measured in both the control group and the B1 treatment group. In addition, soil physicochemical indicators, including pH, electrical conductivity, organic matter, available phosphorus, available potassium, and salinity, were measured. The results are shown in [Figure number missing]. Figure 2 and Figure 3 .

[0064] The method for preparing the microbubble bacteria strain PM1 bacterial culture is as follows: PM1 strain is inoculated into LB liquid medium and cultured at 30°C and 180 rpm for 48 h with shaking to obtain an effective viable count ≥1×10⁻⁶. 8 The bacterial culture was prepared at CFU / mL and then centrifuged at 8000 rpm, 4°C, for 10 min. The supernatant was removed and diluted with distilled water to a final concentration of 1×10⁻⁶. 8 ~ 1×10 10 CFU / mL yields the microbubble bacteria strain PM1 bacterial solution.

[0065] 3. The method for measuring the aforementioned indicators is as follows:

[0066] Plant height, stem diameter, and root length were measured using a measuring tape, and leaf SPAD values ​​were determined using a SPAD-502Plus chlorophyll meter. The plant was separated into aboveground and root parts from the root collar. The surface of the root system was gently washed with clean water to remove any adhering substances, and the surface moisture was blotted dry with filter paper. Immediately afterward, the mass of the aboveground and root systems was weighed separately using an electronic balance and recorded as the fresh weight of the aboveground and underground parts, in grams (g).

[0067] The triphenyltetrazolium chloride (TTC) method was used for determination. Soil pH and electrical conductivity were measured using a pH meter and a conductivity meter in soil-water ratio suspensions of 1:2.5 and 1:5, respectively. Soil organic matter content was determined using the potassium dichromate oxidation method. Available phosphorus was extracted with sodium bicarbonate and then assessed using the molybdenum blue colorimetric method. Available potassium was extracted with ammonium acetate and then determined using the flame photometry method. Soil salinity was quantified by evaporating the soil extract to dryness and weighing the residual weight.

[0068] 4. Statistical Results:

[0069] Compared with the control group, the microbubble bacteria strain PM1 increased maize plant height, stem diameter, leaf SPAD value, aboveground fresh weight, underground fresh weight, and root length by 20.29%, 18.20%, 24.16%, 1.39%, 53.54%, and 38.83%, respectively. Furthermore, compared with the control group, microbubble bacteria strain PM1 also decreased soil pH, electrical conductivity, and salinity by 8.85%, 16.86%, and 7.90%, respectively, and increased organic matter, available nitrogen, and available phosphorus content by 24.74%, 64.42%, and 15.71%, respectively.

[0070] Example 6: Field Experiment Verification

[0071] 1. The experiment will be conducted in Weifang City, Shandong Province, from April to November 2025.

[0072] The experimental field was moderately saline-alkali land with the following basic physicochemical properties: soil salinity 4.82 g / kg, pH 8.21, and organic matter content 10.5 g / kg. The experiment included a control group (CK) and a microbial inoculant treatment group (BF), with six replicates per treatment, totaling 18 experimental plots arranged in a completely randomized block design. Each plot was 30 m² (6 m × 5 m) in size, with a planting density of 60,000 plants / hectare.

[0073] 2. Experimental Procedure

[0074] After corn seedlings emerge, apply microbial inoculant (the microbial inoculant from Example 2), i.e., the BF group, at a rate of 1-5 L / acre, once every 7-10 days, for a total of 2-5 applications. Apart from the inoculant treatment, irrigation, fertilization, and other agricultural management practices remain consistent across all plots.

[0075] 3. During the corn harvest season, relevant indicators should be measured:

[0076] Ten corn plants were randomly selected from each plot, air-dried, threshed, and weighed using an electronic balance to determine the weight of 1000 kernels. Soil index measurements were performed in the same manner as in Example 5.

[0077] 4. Statistical Results Analysis:

[0078] Compared with the control group, the microbial inoculant increased the thousand-grain weight of corn by 22.92%, decreased soil pH, electrical conductivity and salinity by 1.77%, 11.46% and 15.21% respectively, and increased the contents of organic matter, alkaline nitrogen and available phosphorus by 35.95%, 48.01% and 3.70% respectively.

Claims

1. A microbulbifer that promotes maize growth, strain name Microbulbifer sp. PM1, depositary institution: China Center for Type Culture Collection, deposit acceptance date: January 4, 2026, deposit number: CGMCC NO:37273.

2. The microbubble bacteria according to claim 1, characterized in that, The application of the aforementioned microbubble bacteria in improving saline-alkali land.

3. A microbial inoculant, characterized in that, The microbial agent comprises the microbubble bacteria strain PM1 as described in any one of claims 1-2, and a nutrient adjuvant; preferably, the nutrient adjuvant comprises at least one of humic acid, complex amino acids, and trace elements; preferably, the trace elements comprise at least one of zinc sulfate, boric acid, and sodium molybdate.

4. The microbial inoculant according to claim 3, characterized in that, The microbial agent comprises: microbubble bacteria strain PM1, humic acid, complex amino acids, and trace elements; by weight ratio, the content of humic acid is 10% to 20% of the total mass of the composition, the content of the complex amino acids is 5% to 10%, and the content of trace elements is 0.5% to 1%; more preferably, by weight ratio, the content of humic acid is 15% of the total mass of the composition, the content of the complex amino acids is 7.5%, and the content of trace elements is 0.75%.

5. The microbial inoculant according to any one of claims 3-4, characterized in that, The microbubble bacteria strain PM1 was obtained through fermentation, and the effective viable count in the bacterial solution was ≥1×10⁻⁶. 8 CFU / mL.

6. The microbial agent according to claim 5, characterized in that, The preparation process of the aforementioned microbial inoculant: S1: Preparation of Seed Liquid Microbubble bacteria strain PM1 was inoculated into LB liquid medium and cultured with shaking to obtain seed liquid A containing strain PM1; S2: Preparation of microbial culture Dilute seed culture A and then add it to the fermentation broth at a ratio of 0.5-1.5%. Shake and culture to obtain seed culture B containing strain PM1. S3: Preparation of microbial inoculants Take 1 L of seed liquid B and place it in a container. Then add humic acid, amino acids and trace elements and mix evenly to make a microbial inoculant.

7. The microbial agent according to claim 5, characterized in that, The preparation process of the aforementioned microbial inoculant: S1: Preparation of Seed Liquid Microbubble bacteria strain PM1 was inoculated into LB liquid medium and cultured at 30°C and 180 rpm for 48 h to obtain an effective viable count ≥1×10⁻⁶. 8 Seed culture A, CFU / mL, should be diluted to 1×10⁻⁶ before use. 8 ~ 1×10 10 CFU / mL; S2: Preparation of microbial culture Seed culture A was inoculated into the fermentation broth at a ratio of 1%, and cultured at 30°C and 180 rpm for 48 hours with shaking to obtain an effective viable count ≥1×10⁻⁶. 8 Bacterial solution B with CFU / mL; S3: Preparation of microbial inoculants Take 1 L of the bacterial solution from step S2, and then mix it evenly with 50-100g of humic acid, 20-60g of amino acids and 2-6g of trace elements to prepare a microbial inoculant.

8. A microbial inoculant, characterized in that, Calculated in g / L, the microbial agent composition is as follows: 1 part of the bacterial solution as described in any one of claims 6-7, 50-100 parts of humic acid, 20-60 parts of compound amino acids, and 2-6 parts of trace elements; more preferably, the microbial agent composition is as follows: 1 part of the bacterial solution as described in any one of claims 6-7, 75 parts of humic acid, 40 parts of compound amino acids, and 4 parts of trace elements, wherein the effective viable count of the microbubble bacteria strain PM1 is 1×10⁻⁶. 8 ~1×10 10 CFU / mL.

9. The microbial agent according to claim 8, characterized in that, The application of the aforementioned microbial agents in improving saline-alkali land and promoting maize growth.

10. The microbial agent according to claim 8, characterized in that, The microbial agent described herein is used to improve saline-alkali land and promote maize growth. It is applied 2 to 5 times during the maize growth cycle, with each application being 1 to 5 L / mu.

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