Pseudomonas aeruginosa with boron dissolving characteristic as well as screening method and application of pseudomonas aeruginosa

Abstract

The invention belongs to the technical field of microorganisms and biological products thereof, and particularly relates to Pseudomonas raphanipes with a boron dissolving characteristic as well as a screening method and application of the Pseudomonas raphanipes. The preservation number of the rape pseudomonas is CCTCC (China Center For Type Culture Collection) NO: M 2026072. The Pseudomonas aeruginosa TB-01 provided by the invention can more effectively solve the problem of growth obstacles of crops in a boron-deficient soil or high-boron ore environment through a dual mechanism of activating soil boron and secreting IAA to promote plant growth; the dual goals of efficient utilization of boron elements in soil with insufficient available boron supply or crops with high boron element demand and healthy growth of plants are achieved, and good application potential and market value are achieved.

Description

Technical Field

[0001] This invention relates to the field of microbial and biological products technology, and in particular to a strain of Pseudomonas brassicae with boron-soluble properties, its screening method and application. Background Technology

[0002] Boron is an essential micronutrient for plants, playing a crucial role in cell wall synthesis, carbohydrate transport, reproductive organ development, and photosynthesis. Globally, boron deficiency in soils is widespread, severely impacting the yield and quality of various crops and fruits, including cotton, rapeseed, sugar beets, and citrus. Agricultural production primarily corrects boron deficiency by applying chemical boron fertilizers such as borax. However, boron's behavior in soil is highly dependent on soil pH. In acidic soils, boron is easily fixed by iron and aluminum oxides, while in alkaline or calcareous soils, its availability drops sharply, resulting in extremely low utilization rates of applied boron fertilizers, typically less than 5%. This traditional method of applying chemical fertilizers not only increases production costs but also poses environmental risks due to leaching or excessive application.

[0003] In the field of plant nutrition, utilizing beneficial microorganisms, such as plant rhizosphere growth-promoting bacteria (PGPR), to activate soil-endowed mineral elements that are difficult for plants to directly utilize has proven to be a green and sustainable agricultural strategy. Among these, phosphorus-solubilizing microorganisms, through their excellent phosphorus-solubilizing abilities, can release phosphorus from insoluble phosphates in the soil, thereby increasing the content of soluble phosphorus in the soil. This helps improve the utilization rate of soil phosphorus, and related microbial agents are widely used in agricultural production. However, there are currently no reports on microorganisms with boron-solubilizing and boron-dissolving properties, and no research has been conducted on using microorganisms to improve the utilization rate of available boron in the soil. Therefore, screening for microorganisms capable of dissolving insoluble boron or developing their bioproducts is an important research direction for reducing the use of boron fertilizers and boron resource consumption in boron-deficient soils, improving soil boron utilization efficiency, and promoting the green and sustainable development of agricultural production. Summary of the Invention

[0004] To address the deficiency in existing technologies regarding the lack of boron-dissolving microorganisms, this invention provides a *Pseudomonas brassicae* strain TB-01 with boron-dissolving capabilities. Based on the excellent characteristic of this *Pseudomonas brassicae* strain in promoting plant growth by activating soil boron and secreting auxin, this invention further provides applications of this *Pseudomonas brassicae* strain or microbial agents containing this strain in dissolving boron-containing minerals, increasing the available boron content in soil, and / or promoting crop growth. This invention is specifically achieved through the following technical solutions:

[0005] The first aspect of this invention provides a *Pseudomonas brassicae* strain, with accession number CCTCC NO: M 2026072.

[0006] A second aspect of the present invention provides a microbial inoculant, including *Pseudomonas brassicae* as described above.

[0007] Furthermore, the microbial agent is a liquid agent, and the concentration of *Pseudomonas brassicae* in the liquid agent is expressed as an OD value. 600 The range is 0.5-1.8.

[0008] Furthermore, the preparation method of the microbial agent includes: inoculating the *Pseudomonas brassicae* in a liquid culture medium and culturing it to the logarithmic growth phase, then centrifuging to collect the bacterial cells, washing the bacterial cells with physiological saline, resuspending them in physiological saline, and adjusting the bacterial concentration (OD) of the culture. 600 The value is 0.5-1.8, and the microbial inoculant is obtained.

[0009] Further, the liquid culture medium is LB medium or inorganic salt medium, wherein the inorganic salt medium comprises: MgSO4·7H2O 0.15 g / L, NaH2PO4 0.08 g / L, Na2HPO4 0.09 g / L, (NH4)2SO4 0.065 g / L, CaCl2 0.02 g / L and glucose 2 g / L, with a pH of 6.8-7.2.

[0010] Furthermore, the culture conditions are 28-30℃ and 150-200rpm.

[0011] The third aspect of the present invention provides the application of the *Pseudomonas rapeus* or microbial inoculant as described above, wherein the application is selected from at least one of (1)-(3) below:

[0012] (1) Application in dissolving boron-containing minerals;

[0013] (2) Application in increasing the available boron content in soil;

[0014] (3) Application in promoting crop growth.

[0015] A fourth aspect of the present invention provides a method for promoting crop growth, comprising the following steps:

[0016] Apply the above-mentioned Pseudomonas rapeseed or microbial inoculants to the soil around the roots of the crop or spray them on the leaves, and then cultivate the crop plants.

[0017] Furthermore, the *Pseudomonas rapeus* or the microbial agent is applied to the soil around the crop roots via root irrigation.

[0018] Furthermore, the crop in question is sugar beet.

[0019] Furthermore, the root irrigation dosage is 60-100 mL per plant, and the bacterial concentration is expressed as OD. 600 The range is 0.5-1.8.

[0020] Furthermore, the root irrigation period is the seedling stage, the rapid growth stage of the leaf cluster, the stage of sugar growth in the tuber, and the stage of sugar accumulation in the sugar.

[0021] The fifth aspect of this invention provides a method for screening boron-soluble bacteria, comprising the following steps:

[0022] S1. Soil samples were cultured in LB medium to isolate and purify soil microorganisms;

[0023] S2. The purified soil microorganisms are cultured in an inorganic salt medium containing boron ore;

[0024] S3. Collect the culture medium, filter it, and detect the soluble boron content in the filtrate. Based on the soluble boron content, screen for microorganisms with boron-soluble properties.

[0025] The advantages and positive effects of this invention are as follows:

[0026] The *Pseudomonas aeruginosa* TB-01 provided by this invention can promote plant growth by activating soil boron and secreting growth-promoting substances (IAA), more effectively solving the problem of crop growth obstacles in boron-deficient soils or high-boron ore environments. It achieves the dual goals of efficient utilization of boron and healthy plant growth in soils with insufficient available boron supply or crops with high boron requirements, and has good application potential and market value. Attached Figure Description

[0027] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0028] Figure 1 This is a phylogenetic tree diagram of Pseudomonas rapeseed TB-01 according to an embodiment of the present invention;

[0029] Figure 2 This is a boron solubility curve of Pseudomonas rapeseed TB-01 in an embodiment of the present invention;

[0030] Figure 3 This is a growth curve of Pseudomonas rapeseed TB-01 in an embodiment of the present invention;

[0031] Figure 4 This is a diagram showing the functional identification results of auxin secreted by Pseudomonas rapeseed TB-01 in an embodiment of the present invention;

[0032] Figure 5 This is a statistical result of the yield of field sugar beets in the group treated with boron fertilizer and Pseudomonas rapeus TB-01 inoculum in an embodiment of the present invention.

[0033] Figure 6 The graph shows the statistical results of sugar content in field sugar beets grown using boron fertilizer and Pseudomonas rape TB-01 inoculum in an embodiment of the present invention.

[0034] Figure 7 The graph shows the detection results of effective boron content in the rhizosphere soil of sugar beets in an embodiment of the present invention, where boron fertilizer and Pseudomonas rape TB-01 inoculant were applied. Detailed Implementation

[0035] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. Unless otherwise specified, the equipment and reagents used in the embodiments and experimental examples are commercially available. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention.

[0036] Based on the information contained in this application, various changes to the precise description of the invention can be readily made by those skilled in the art without departing from the spirit and scope of the appended claims. It should be understood that the scope of the invention is not limited to the defined processes, properties, or components, as these embodiments and other descriptions are merely illustrative of specific aspects of the invention. In fact, various modifications to embodiments of the invention that will be apparent to those skilled in the art or related fields are covered within the scope of the appended claims.

[0037] To better understand the invention and not to limit its scope, all figures indicating amounts, percentages, and other numerical values ​​used in this application should, in all cases, be understood to be modified by the word "approximately." Therefore, unless specifically stated otherwise, the numerical parameters listed in the specification and appended claims are approximate values ​​and may vary depending on the desired properties being sought. Each numerical parameter should at least be considered as obtained based on reported significant figures and through conventional rounding methods. Furthermore, the terms "comprising," "including," "containing," "having," and similar words are non-limiting in meaning, allowing for the addition of other steps and components that do not affect the outcome.

[0038] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings.

[0039] This invention provides a strain of *Pseudomonas brassicae*, with accession number CCTCC NO: M 2026072. This strain was deposited on January 12, 2026, at the China Center for Type Culture Collection, located at No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province (Wuhan University). The culture name is *Pseudomonas brassicae* TB-01, and the taxonomic name is *Pseudomonas brassicae*.

[0040] Addressing the practical problems of nutrient deficiency and restricted crop growth in boron-deficient soils, inspired by phosphate-solubilizing microorganisms, this invention actively explores microorganisms capable of activating available boron in the soil or dissolving insoluble boron in the soil. The screening method for soil microorganisms includes the following steps:

[0041] S1. Soil samples were cultured in LB medium to isolate and purify soil microorganisms;

[0042] S2. Cultivate purified soil microorganisms in an inorganic salt culture medium containing boron ore, wherein the amount of boron ore added is 5 g / L.

[0043] S3. Collect the culture medium, filter it, and detect the soluble boron content in the filtrate. Based on the soluble boron content, screen for microorganisms with boron-soluble properties.

[0044] Through systematic screening and functional studies, a boron-soluble microorganism, *Pseudomonas brassicae* TB-01, was isolated from the rhizosphere soil of sugar beets. The boron-soluble bacterium provided by this invention has the following characteristics:

[0045] (1) Highly efficient boron dissolution: The strain TB-01 of this invention was screened from the rhizosphere soil of a boron-efficient sugar beet variety. Its metabolic characteristics determine that the strain has a highly efficient boron dissolution capacity. Through the boron dissolution capacity test of strain TB-01, the strain was fermented for a period of time after adding the insoluble boron-containing mineral—hard boron calcium stone—to an inorganic salt culture medium. The soluble boron content in the fermentation broth was then detected. It was found that the strain could dissolve the boron ore and release the boron element therein, significantly increasing the boron content in the solution. After irrigating sugar beets with the bacterial solution, the effective boron content in the sugar beet rhizosphere soil was detected after the sugar beet harvest. It was found that strain TB-01 could efficiently activate the insoluble boron in the soil and increase the effective boron content in the soil that can be absorbed and utilized by crops. Compared with the control group without bacterial agent, the effective boron content in the soil increased by 25.31%. This is of great significance for improving soil boron utilization, reducing dependence on exogenous boron fertilizer, and achieving healthy and sustainable agricultural economic development in boron-deficient soils or high boron ore environments.

[0046] (2) Highly efficient growth promotion: The strain TB-01 of this invention can secrete plant growth hormones such as auxin (IAA), which can directly stimulate plant cell division and elongation; and through its boron-dissolving properties, it can improve the efficiency of plant absorption and utilization of boron, thereby indirectly promoting plant growth. This synergistic effect enables the strain to exhibit significant and stable growth promotion effects in different types of boron-deficient soils.

[0047] (3) Rhizosphere colonization ability: The strain TB-01 of the present invention was isolated from the rhizosphere soil of plants. It has good rhizosphere competition and colonization ability, can adapt to the rhizosphere microenvironment, and form a dominant bacterial community, thereby providing plants with continuous and long-lasting boron dissolution and growth promotion effects. Its rhizosphere colonization and growth promotion ability has been verified in the field.

[0048] (4) Easy to cultivate: This strain grows rapidly and has a high biomass in conventional microbial fermentation media. The fermentation process and culture conditions are simple and do not rely on expensive special raw materials or complex fermentation conditions. This enables the strain to effectively control production costs during large-scale industrial production, laying a solid foundation for commercial development and market application.

[0049] Another embodiment of the present invention provides a microbial inoculant, including Pseudomonas brassicae TB-01 as described above.

[0050] Based on the stable biological characteristics of the *Pseudomonas rapeus* TB-01 strain of this invention, it can be prepared into various forms of microbial inoculants, such as liquid inoculants, solid inoculants, or bio-fertilizers compounded with organic and inorganic carriers. These product forms are adaptable to existing agricultural farming systems and can be applied through seed coating, root dipping, irrigation, hole application, etc., which is conducive to the rapid promotion and popularization of this technology in practice.

[0051] Optionally, the microbial agent may further include acceptable excipients. Acceptable excipients refer to components that do not interfere with the efficacy of the strain's biological activity and are not significantly toxic to organisms (including humans or animals) or the environment (including soil and water) at the concentration at which they are applied. These include any one or a combination of at least two of solvents, dispersants, diluents, fillers, wetting agents, binders, disintegrants, lubricants, suspending agents, excipients, flavoring agents, and protectants. The use of the above components in microbial preparations is well known in the art.

[0052] Solvents or dispersants may include sterile water, physiological saline, commonly used culture media such as LB medium, and mixtures thereof. Antioxidants may include one or more of benzoic acid, ascorbic acid, 2,6-di-tert-butyl-4-methylphenol, sodium sulfite, or sodium bisulfite. pH adjusters may include one or more of sodium carbonate, sodium bicarbonate, phosphoric acid, dipotassium hydrogen phosphate, sodium hydroxide, and ammonia. Protectants may include lyophilization or cryopreservation protectants such as glycerol, sodium alginate, and polyvinyl alcohol. The aforementioned acceptable excipients can be used to prepare the microbial strains of the present invention into solid or liquid formulations, or into bio-fertilizers combined with organic-inorganic carriers.

[0053] Optionally, the microbial agent is a liquid agent.

[0054] Optionally, in the liquid inoculant, the concentration of the *Pseudomonas brassicae* bacterial solution is expressed as an OD value. 600 The value is calculated to be 0.5-1.8, and more specifically, it can be 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1 or 1.2, etc.; preferably, it is 0.8.

[0055] Optionally, the method for preparing the microbial agent includes: inoculating *Pseudomonas brassicae* into a liquid culture medium, collecting the bacterial cells by centrifugation, washing the bacterial cells with physiological saline, resuspending the bacterial cells in physiological saline, and adjusting the bacterial concentration (OD) of the culture solution. 600 The value is 0.5-1.8, and the microbial inoculant is obtained.

[0056] The liquid culture medium for culturing *Pseudomonas brassicae* uses conventional fermentation media in this field, such as LB medium or inorganic salt medium. The inorganic salt medium consists of: 0.15 g / L MgSO4·7H2O, 0.08 g / L NaH2PO4, 0.09 g / L Na2HPO4, 0.065 g / L (NH4)2SO4, 0.02 g / L CaCl2, and 2 g / L glucose, with a pH of 7.0 ± 0.2.

[0057] The fermentation culture conditions are standard for microbial culture, such as 28-30℃ and 150-200 rpm. The fermentation time is adjusted adaptively according to cell yield and activity, usually harvesting the cells after reaching the logarithmic growth phase to improve cell viability and concentration.

[0058] Another embodiment of the present invention provides the application of the *Pseudomonas rapeus* or microbial inoculant as described above, wherein the application is selected from at least one of the following (1)-(3):

[0059] (1) Application in dissolving boron-containing minerals;

[0060] (2) Application in increasing the available boron content in soil;

[0061] (3) Application in promoting crop growth.

[0062] When dissolving boron-containing minerals, the present invention's *Pseudomonas rapeus* or microbial agent is added to the fermentation medium and co-cultured with the boron-containing minerals. The fermentation supernatant is then harvested, which increases the boron content in the solution.

[0063] When increasing the available boron content in soil, the present invention’s Pseudomonas rapeus or microbial inoculant can be mixed with the soil to increase the available boron content in the soil.

[0064] When promoting crop growth, the Pseudomonas rapeus or microbial agent of the present invention can be applied to the roots or leaves of the crop for spraying, and the crop can be managed and cultivated in a conventional manner to promote crop growth.

[0065] Another embodiment of the present invention provides a method for promoting crop growth, comprising the following steps:

[0066] Apply the above-mentioned Pseudomonas rapeus or microbial inoculants to the soil around the roots of the crop or spray the leaves, and then cultivate the sugar beet plants.

[0067] In practical applications, *Pseudomonas rapeus* or microbial inoculants are applied to the soil around the crop roots via root drenching. The dosage is 60-100 mL, meaning 60-100 mL of *Pseudomonas rapeus* or microbial inoculant solution is applied to each plant. The bacterial concentration is expressed as an OD value. 600 The dosage is calculated to be 0.5-1.8. Furthermore, there is no limit to the number of applications; it can be added multiple times throughout the plant's growth cycle. For example, it can be applied as a root drench with Pseudomonas rapeus or a microbial inoculant solution during four different growth stages: the seedling stage, the rapid leaf growth stage, the root sugar growth stage, and the sugar accumulation stage.

[0068] The present invention will be further illustrated below with reference to specific embodiments. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions, such as those described in *Molecular Cloning: A Laboratory Manual (Fourth Edition)* published by Cold Spring Harbor Laboratory, or generally under the conditions recommended by the manufacturer.

[0069] The formulation of the main culture medium used in the following examples is as follows:

[0070] (1) LB medium: yeast extract 5 g / L, trypsin 10 g / L and NaCl 10 g / L, pH 7.0;

[0071] (2) Modified Bushnell Haas Medium (BHA): MgSO4·7H2O 0.15 g / L, NaH2PO4 0.08 g / L, Na2HPO4 0.09 g / L, (NH4)2SO4 0.065 g / L, CaCl2 0.02 g / L and glucose 2 g / L, pH 7.0±0.2.

[0072] Add 20 g / L of agar to the solid culture medium.

[0073] Example 1: Isolation, culture and identification of boron-soluble bacteria

[0074] Soil samples used to screen target microorganisms were collected from boron-efficient beet rhizosphere soil in the experimental field of Xinmin Village, Harbin City, Heilongjiang Province. By comparing the amount of boron (B) released by soil microorganisms during fermentation in a basal medium containing hard boron calcium stone (provided by Xinyang Mining Group Co., Ltd.), the highly efficient boron-soluble bacterium, Pseudomonas brassicae TB-01, was isolated.

[0075] (1) Isolation of soil microorganisms: 5 g of collected soil sample was dissolved in 45 mL of sterile physiological saline, an appropriate amount of glass beads was added, and the mixture was shaken at 180 rpm for 30 minutes. After standing for 10 minutes, the supernatant was taken and diluted 10 times. 1 mL of the diluted solution was spread on LB agar plates and incubated at 28℃ for 2 days. Single colonies were picked and inoculated onto fresh LB agar plates. The purified strain was obtained through repeated subculturing. The purified strain was stored in a -80℃ ultra-low temperature freezer with 50% glycerol.

[0076] (2) Test of soil microbial boron solubility: Mineral boron is insoluble boron found in boron-containing minerals and cannot be directly absorbed and utilized by plants. In this example, boron-containing mineral—calcium borate—was used as the research object. After grinding the calcium borate, particles between 100-300 mesh were selected, washed with distilled water to remove impurities, and then dried for later use. 0.5 g of calcium borate powder and 100 mL of modified BHA medium were added to a 250 mL Erlenmeyer flask and sterilized at 121℃ for 30 min. The purified soil microbial strains were inoculated into LB medium and cultured at 28℃ and 180 r / min for 12 h for activation. After centrifugation at 6000 r / min for 10 min, the collected bacterial cells were washed with physiological saline and resuspended, washed three times with sterile water, and then the seed culture concentration was adjusted to OD. 600 The value was 0.8. 1 mL of seed culture was added to the culture medium containing borosilicate powder, and an equal volume of inactivated bacterial solution was added to the control group. Each group was repeated in triplicate, and the average value was taken. The conical flasks were incubated with shaking at 150 r / min and 28℃ for 30 days. Samples were taken on days 15 and 30, and the fermentation supernatant was centrifuged to determine the soluble boron content.

[0077] The soluble boron content was determined using the curcumin absorbance method. The principle is that boron in solution reacts with curcumin in the presence of oxalic acid, undergoing dehydration to form a rose-red complex. After dissolving in ethanol, the absorbance is measured at a wavelength of 550 nm, and the absorbance is directly proportional to the boron concentration.

[0078] Curcumin-oxalic acid solution: Weigh 0.04 g of curcumin and 5.00 g of oxalic acid and dissolve them in 100 mL of 95% ethanol.

[0079] Standard stock solution: Weigh 0.5716g of boric acid (H3BO3, purchased from Shanghai Maclean Biochemical Technology Co., Ltd., product number S736754-100g), dissolve it in deionized water, and make up to 1L in a volumetric flask. Transfer the solution to a dry plastic bottle for storage.

[0080] The fermentation supernatant was filtered through a 15-20 μm filter membrane. 1.00 mL of the filtrate was placed in a 50 mL quartz evaporating dish, and 4.00 mL of curcumin-oxalic acid solution was added. The dish was placed in a constant temperature water bath at 55℃±3℃ and evaporated to dryness. Timing was started from the appearance of a rose-red complex, and the dish was baked for another 15 minutes. The quartz evaporating dish was removed and cooled to room temperature. 20 mL of 95% ethanol was added to dissolve the complex. The zeroing point was adjusted with 95% ethanol solution, and the absorbance was measured at 550 nm using a spectrophotometer. The B content of the test solution was obtained by referring to the calibration curve or calculating the regression equation using the absorbance value after blank subtraction.

[0081] Microbial strains with high B content in the fermentation supernatant were selected as candidate boron-soluble bacteria. Biological identification of the strains revealed *Pseudomonas brassicacearum* TB-01.

[0082] Simultaneously, 16S rDNA gene amplification and sequence analysis were performed. TB-01 cells were cultured in LB medium at 28℃ to the logarithmic growth phase, centrifuged at 12000 r / min for 5 min to collect the cells, and genomic DNA was extracted using the bacterial genomic DNA extraction kit (catalog number DP302-02) from Tiangen Biotech Co., Ltd. Using the extracted DNA product as a template, the 16S rDNA gene fragment was amplified from the genomic DNA using the universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1541R (5'-AAGGAGGTGATCCAGCC-3'). The PCR product was sent to Shanghai Sangon Biotech Co., Ltd. for sequencing. The 16S rDNA sequence determination results are shown in SEQ ID NO.1.

[0083] The 16S rDNA gene of TB-01 is shown below:

[0084]

[0085] Sequence alignment analysis was performed using the BLAST program on EzBioCloud software, and a phylogenetic tree of the strain was constructed using MEGA 4.0 software. The results showed that the 16S rDNA sequence of this strain shared over 99% similarity with the published *Pseudomonas brassicacearum*. Based on the combined characteristics of cell morphology, physiological and biochemical properties, cell components, and 16S rDNA gene sequence, the isolated strain was identified as *Pseudomonas brassicacearum*. The phylogenetic tree is shown below. Figure 1 .

[0086] Figure 2 The boron dissolution curve of *Pseudomonas brassicae* TB-01 is shown, with the horizontal axis representing culture time and the vertical axis representing boron content (mg / L). After 30 days of culture, the boron dissolution level of the control group (CK) remained at a low level, indicating that boron release from the culture medium itself or abiotic factors is very limited under non-inoculated conditions. In contrast, the boron-dissolving bacterium *Pseudomonas brassicae* TB-01 exhibited a strong boron-dissolution capacity, with its soluble boron content more than doubling compared to the control group. This indicates that *Pseudomonas brassicae* TB-01 can dissolve boron-containing ores and increase the available boron content, making it a highly efficient boron-dissoluting strain with good application potential.

[0087] Figure 3 The growth curve of *Pseudomonas brassicae* TB-01 is shown. Under the conditions of LB medium, 30℃, and shaking on a shaker at 150 rpm, the OD of the fermentation broth was measured every 2 hours. 600 As can be seen from the growth curve, when cultured for 20-22 hours, the OD of TB-01 bacterial culture... 600 It reaches its highest value and has the advantages of rapid growth and high biomass.

[0088] The aforementioned Pseudomonas brassicacearum was deposited on January 12, 2026, at the China Center for Type Culture Collection (CCTCC), with accession number CCTCC NO: M 2026072, located at No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province (Wuhan University), and its culture classification name is Pseudomonas brassicacearum.

[0089] Example 2: Functional identification of auxin (IAA) secreted by Pseudomonas brassicae TB-01

[0090] Auxin (IAA) was determined using the Salkowski colorimetric method. Preparation of the Salkowski colorimetric solution: Take 7.5 mL of 0.5 mol / L FeCl3·6H2O, then slowly add 150 mL of concentrated sulfuric acid. After the solution cools, add 250 mL of distilled water.

[0091] TB-01 was cultured overnight in LB medium at 28°C. The activated strain was then transferred at a 1% inoculum to Erlenmeyer flasks containing liquid LB medium (150 mg / L L-tryptophan). The flasks were incubated at 30°C with shaking at 150 rpm for 24 hours. After 24 hours, an equal volume of the bacterial culture was mixed with Salkowski colorimetric solution and reacted in the dark for 30 minutes. A red color indicated that the strain had the ability to secrete IAA. Liquid LB medium without the inoculum served as a control (CK).

[0092] The ability of a bacterial strain to produce IAA was determined by the color reaction between the bacterial culture and Salkowski chromogenic solution; the degree of reddening of the bacterial culture represented the strain's IAA production capacity. Results ( Figure 4 This indicates that Pseudomonas brassicae TB-01 has the ability to produce auxin.

[0093] Example 3: Study on the growth-promoting effect of Pseudomonas brassicae TB-01 on field sugar beets.

[0094] The experiment used a sugar beet variety (KWS1197) and adopted a randomized block design with three treatments: a control group (CK) receiving conventional fertilizers (nitrogen, phosphorus, and potassium fertilizers, but no boron fertilizer or boron-soluble bacterial agent); a boron fertilizer control group (B) receiving 15 kg / ha of borax (purchased from Heilongjiang Xianfeng Agricultural Materials Market); and a boron-soluble bacterial agent group (TB-01) receiving Pseudomonas rapeseed TB-01 bacterial solution (nitrogen, phosphorus, and potassium fertilizer amounts were the same as CK). The bacterial agent was applied via root drenching. 80 mL of solution was applied to the roots, and 20 sugar beet plants of uniform growth were randomly selected as replicates. A total of 320 mL of the bacterial agent solution was added at four different growth stages: seedling stage, rapid leaf growth stage, tuber sugar growth stage, and sugar accumulation stage.

[0095] Preparation method of bacterial inoculum solution: Purify TB-01, and after culturing for 3 days, pick single bacteria with an inoculation loop and inoculate them into a sterilized 150 mL Erlenmeyer flask containing 100 mL of LB liquid medium. Incubate in a shaker at 28℃ and 180 rpm for 12 h. Collect the TB-01 fermentation broth, centrifuge at 6000 r / min for 10 min, collect the bacterial cells, wash with physiological saline and resuspend. Wash the seed culture three times with sterile water, and then adjust the seed culture concentration to OD. 600 The concentration was 0.8, which was used as a fungal solution for root irrigation.

[0096] Sugar beets were sown in May 2025, and conventional water and fertilizer management was adopted. The application rates of nitrogen, phosphorus, and potassium fertilizers were kept consistent across treatments: 160 kg / ha urea (purchased from Heilongjiang Xianfeng Agricultural Materials Market), 90 kg / ha diammonium phosphate (purchased from Heilongjiang Xianfeng Agricultural Materials Market), and 120 kg / ha potassium sulfate (purchased from Heilongjiang Xianfeng Agricultural Materials Market), respectively. For the in-situ field trial, the microbial agent was prepared in advance according to the above-mentioned preparation method. On the day of fertilization, the microbial solution was evenly applied to the field in each experimental plot. An equal volume of sterile physiological saline was used as a control, irrigated at the base of the sugar beets. Information for each treatment group is shown in Table 1.

[0097] Table 1 Different treatment groups and their inoculated strains

[0098]

[0099] At harvest time (September 26, 2025), sugar beet yield and sugar content were measured. Representative plants were selected to determine fresh weight, and the yield per unit area (kg / hectare) was calculated. Yield = Total yield (kg) / Planted area (hectares). Statistical results are shown below. Figure 5 , Figure 6 It is evident that the application of boron fertilizer and TB-01 inoculant significantly increased the yield and sugar content of sugar beets. The best yield was achieved after applying boron fertilizer (86,090 kg / ha), followed by the treatment with boron-soluble strain TB-01 (85,910 kg / ha). Sugar production also increased simultaneously, with sugar contents of 13.79% and 13.06% in the boron-fertilized treatment and the treatment with boron-soluble strain TB-01, respectively.

[0100] In addition, after the sugar beets matured, the available boron content in the soil was determined using the methylimine colorimetric method. Available boron in the soil was extracted with boiling water. After the organic matter color was removed with potassium permanganate, the extract was treated with EDTA to eliminate interference from iron and aluminum ions. In a weakly acidic medium, boron reacted with methylimine to form a yellow complex, and the absorbance was measured at a wavelength of 415 nm.

[0101] Methylimine solution: Weigh 1.35g of methylimine and 3.00g of ascorbic acid into 120mL of water and dilute to 150mL.

[0102] Buffer solution: Weigh 50g of ammonium acetate and 2g of disodium EDTA and dissolve them in 50mL of water. After cooling, dilute to 100mL and add 16mL of 1:4 sulfuric acid solution. Shake well.

[0103] Mix the colorimetric reagent: Measure 3 parts by volume of the above methylimine solution and 2 parts by volume of the above buffer solution and mix them. Prepare fresh on the same day.

[0104] Weigh 0.5719 g of boric acid (analytical grade) into a 500 mL beaker, dissolve it in distilled water, and then transfer it to a 1 L volumetric flask and make up to volume. This is a 100 μg / mL standard stock solution, which should be stored in a plastic bottle.

[0105] Pipette 50.00 mL of the above solution into a 500 mL volumetric flask and dilute to volume to obtain a 10 μg / mL boron standard series solution, which should be stored in a plastic bottle.

[0106] Using a 1mL pipette, pipette 0mL, 0.5mL, 1mL, 2mL, 3mL, 4mL, and 5mL into seven 50mL volumetric flasks, respectively, and dilute to volume to obtain a series of boron standard solutions of 0.0 mg / L, 0.1 mg / L, 0.2 mg / L, 0.4 mg / L, 0.6 mg / L, 0.8 mg / L, and 1.0 mg / L. Store these solutions in plastic bottles.

[0107] After the sugar beets were harvested, 10.00 g of air-dried soil sample from the rhizosphere of the sugar beets in the experimental area was weighed and placed in a 50 mL polyethylene bottle. 20.00 mL of magnesium sulfate solution was added, and the sample was shaken well. The bottle was then capped and placed in a 100°C water bath for 65 minutes. The supernatant was then removed and heated at 3500 r·min. -1 Centrifuge for 10 min and then filter. Pipette 4.00 mL of the filtrate into a 50 mL polyethylene bottle, add 0.5 mL of acidic potassium permanganate, shake well, let stand for 3 min, add 0.5 mL of ascorbic acid, shake well, and wait until the purple-red color fades and the brown manganese dioxide precipitate is completely dissolved. Then add 5.00 mL of mixed colorimetric reagent and shake well. Incubate at 25℃ in the dark for 80 min, then read the absorbance at 415 nm using a 2 cm path length cuvette. Adjust the instrument zero point to the zero concentration of the calibration curve and record the result.

[0108] The content of available boron is expressed in (mg / kg) and is calculated using the following formula:

[0109] ;

[0110] w(B) — Soil available boron content, in milligrams per kilogram (mg / kg);

[0111] m1: The boron content in the colorimetric solution is obtained from the calibration curve, in micrograms (μg).

[0112] m: Mass of soil sample, in grams (g);

[0113] 10 3 And 1000—conversion factor;

[0114] D: Dilution factor.

[0115] Figure 7 The available boron content in the rhizosphere soil of sugar beets was shown in different treatment groups. It can be seen that the application of boron fertilizer and TB-01 inoculant can significantly increase the available boron content in the soil. Compared with the CK treatment, the available boron content in the boron fertilizer and TB-01 inoculant groups increased by 26.54% and 25.31%, respectively. This indicates that the boron-dissolving bacterium TB-01 of the present invention can dissolve insoluble boron in the soil and convert it into an available boron form suitable for plant absorption and utilization.

[0116] 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 and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A type of *Pseudomonas brassicae*, characterized in that, The accession number is CCTCC NO: M 2026072.

2. A microbial inoculant, characterized in that, Including Pseudomonas brassicae as described in claim 1.

3. The microbial agent according to claim 2, characterized in that, The microbial inoculant is a liquid inoculant, and the concentration of *Pseudomonas rapeseed* in the liquid inoculant is expressed as OD0.

05. 600 The range is 0.5-1.

8.

4. The microbial agent according to claim 2, characterized in that, The method for preparing the microbial inoculant includes: The *Pseudomonas brassicae* strain was inoculated into a liquid culture medium and cultured until the logarithmic growth phase. The bacterial cells were then collected by centrifugation, washed with physiological saline, resuspended in physiological saline, and the bacterial concentration (OD) was adjusted. 600 The value is 0.5-1.8, and the microbial inoculant is obtained.

5. The microbial agent according to claim 4, characterized in that, The liquid culture medium is LB medium or inorganic salt medium; the inorganic salt medium comprises: MgSO4·7H2O 0.15 g / L, NaH2PO4 0.08 g / L, Na2HPO4 0.09 g / L, (NH4)2SO4 0.065 g / L, CaCl2 0.02 g / L and glucose 2 g / L, with a pH of 6.8-7.2; The culture conditions are 28-30℃ and 150-200rpm.

6. The application of *Pseudomonas rapeus* as described in claim 1 or the microbial agent as described in any one of claims 2-5, characterized in that, The application is selected from at least one of the following (1)-(3): (1) Application in dissolving boron-containing minerals; (2) Application in increasing the available boron content in soil; (3) Application in promoting crop growth.

7. A method for promoting crop growth, characterized in that, Includes the following steps: The Pseudomonas rapeus as described in claim 1 or the microbial agent as described in any one of claims 2-5 is applied to the soil around the roots of the crop or sprayed on the leaves, and then the crop plants are cultured.

8. The method for promoting crop growth according to claim 7, characterized in that, The *Pseudomonas rapeus* or the microbial agent is applied to the soil around the roots of the crop by root irrigation. The root irrigation dosage is 60-100 mL per crop plant, and the bacterial concentration is expressed as OD. 600 The range is 0.5-1.

8.

9. The method for promoting sugar beet growth according to claim 7, characterized in that, The crop in question is sugar beet, and the root irrigation period includes the seedling stage, the rapid growth stage of the leaf cluster, the stage of sugar growth in the tuber, and the stage of sugar accumulation.

10. A method for screening boron-soluble bacteria, characterized in that, The screening for Pseudomonas brassicae as described in claim 1 includes the following steps: S1. Soil samples were cultured in LB medium to isolate and purify soil microorganisms; S2. The purified soil microorganisms are cultured in an inorganic salt medium containing boron ore; S3. Collect the culture medium, filter it, and detect the soluble boron content in the filtrate. Based on the soluble boron content, screen for microorganisms with boron-soluble properties.

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