A high-yield, stress-resistant growth-promoting bacterium, *Bacillus mirabilis* YPR-35, and its applications.

Abstract

This invention belongs to the field of microbial technology, and more specifically, relates to a high-yielding and stress-resistant growth-promoting bacterium, *Bacillus pyrogenes* YPR-35, and its applications. This strain possesses high production capacity of indoleacetic acid (IAA) and siderophores, as well as potassium-solubilizing and phosphorus-solubilizing abilities. It can be used in the production of IAA and in plant growth processes, significantly promoting crop growth and shortening cultivation time. This invention also provides a bio-agent containing this strain. Pot experiments and field trials have verified that this bio-agent can promote potato plant growth and significantly increase the yield of marketable potatoes, achieving the effect of high yield and increased income. Furthermore, this bacterium exhibits excellent stress resistance, including tolerance to heavy metal stress, acid and alkali stress, and salt stress, demonstrating wide adaptability. Therefore, this invention provides a new strain resource for the microbial fertilizer industry, showing strong application value in rhizosphere growth promotion.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology. More specifically, it relates to a highly IAA-producing Bacillus pyrolyticus strain YPR-35 and its applications. Background Technology

[0002] Crops are often influenced by rhizosphere microorganisms during their growth and development. Some rhizosphere microorganisms can metabolize substances that promote plant growth, such as indoleacetic acid (IAA) and gibberellins. These substances do not directly provide nutrients to plants but act as important chemical signals to regulate plant growth and development. Indoleacetic acid (IAA), as a plant hormone, can stimulate plant cell growth and, at appropriate concentrations, significantly promote the growth of roots, stems, and leaves, shortening cultivation time and increasing yield. Rhizosphere microorganisms can also decompose poorly soluble nutrients in the soil, such as insoluble inorganic phosphorus and potassium, into available phosphorus and potassium that plants can directly absorb and utilize, increasing the absorption of phosphorus and potassium elements by plants, reducing the need for phosphorus and potassium fertilizers, and secreting siderophores (also known as iron carriers), thereby improving iron deficiency in crops.

[0003] Bacillus species possess advantages such as spore production and long shelf life, giving them significant advantages in growth-promoting microbial research. For example, Chinese patent document CN111484946A discloses a heat-resistant, IAA-producing Bacillus belesii. This bacterium can promote seed germination, and it can both solubilize phosphorus and fix nitrogen, improving soil fertility, providing sufficient nutrients for plant growth, enhancing crop stress resistance, and also exhibiting heat resistance, which is beneficial for the composting of agricultural waste. Another example is Chinese patent document CN111057665A, which discloses an IAA-producing cellulose-degrading bacterium, *Azotobacter nigra* n3. This bacterium has a strong ability to produce CMC enzymes and can also produce IAA, with CMC enzyme activity reaching a maximum of 24.96 U / mL and IAA secretion reaching a maximum of 19.07 mg / L. This bacterium can be used to prepare straw decomposition-promoting microbial agents that produce IAA and / or CMC enzymes or have growth-promoting functions, thereby being applied to straw decomposition and crop growth promotion, achieving increased straw return efficiency and crop yield. Current research shows that microorganisms have great potential in promoting plant growth.

[0004] Agricultural microbial germplasm resources are of national strategic significance. Fully exploring microbial fertilizer strain resources is crucial for promoting high-yield, high-quality, green, and efficient modern agriculture. While the strains disclosed in the aforementioned patent literature all possess the ability to synthesize IAA (inorganic acid), this ability remains limited. Furthermore, in complex soil environments such as land with excessively high heavy metal content, saline-alkali land, or acidic or alkaline soils with high or low pH, strains with only IAA synthesis capabilities often experience growth impairments, thus affecting their ability to promote plant growth. Therefore, screening for IAA-producing strains with broad environmental applicability remains an urgent need for advancements in plant growth promotion and yield increase. Summary of the Invention

[0005] This invention aims to develop plant growth-promoting bacteria resources with wide applicability and high IAA production, and provides a high IAA-producing Fictibacillus barbaricus YPR-35 and its applications.

[0006] The first objective of this invention is to provide a strain of Bacillus mirabilis YPR-35.

[0007] A second objective of this invention is to provide a microbial preparation containing the aforementioned *Bacillus mirabilis* YPR-35.

[0008] A third objective of this invention is to provide applications of the aforementioned *Bacillus mirabilis* YPR-35.

[0009] The fourth objective of this invention is to provide a method for preparing the plant growth regulator indoleacetic acid.

[0010] The above-mentioned objective of this invention is achieved through the following technical solution:

[0011] This invention screened and obtained a unique strain of *Fictibacillus barbaricus* YPR-35, which was deposited at the China General Microbiological Culture Collection Center (CGMCC) on December 11, 2023, with accession number CGMCC No. 29286. Such strains are rarely reported, especially regarding their plant growth-promoting effects. This strain produces high levels of IAA and siderophores, and possesses potassium-solubilizing and phosphorus-solubilizing abilities. It also exhibits excellent tolerance to heavy metal stress, acid-alkali stress, and salt stress, making it highly valuable for the development and utilization of microbial fertilizers. Therefore, this invention provides the following application schemes:

[0012] The application of the aforementioned Bacillus mirabilis YPR-35 in promoting plant growth.

[0013] The application of the aforementioned Bacillus mirabilis YPR-35 in the preparation of products that promote plant growth.

[0014] The application of the aforementioned Bacillus mirabilis YPR-35 in promoting plant growth under environmental stress.

[0015] The above-mentioned *Bacillus mirabilis* YPR-35 is used in the preparation of products that promote plant growth under environmental stress.

[0016] The environmental stresses include heavy metal stress, acid-base stress, and / or salt stress.

[0017] More specifically, the heavy metal stress includes Mn(II), Zn(II), Fe(II), Cu(II) and / or Cd(II) heavy metal stress.

[0018] The acid and alkali stresses include acid stress with a pH of 5.5–6.5 and alkali stress with a pH of 7.5–9.5. According to existing classifications of soil acidity and alkalinity, soil acidity and alkalinity are divided into nine levels: <4.5 (extremely acidic), 4.5–5.5 (strongly acidic), 5.5–6.0 (acidic), 6.0–6.5 (weakly acidic), 6.5–7.0 (neutral), 7.0–7.5 (weakly alkaline), 7.5–8.5 (alkaline), 8.5–9.5 (strongly alkaline), and >9.5 (extremely alkaline). Therefore, the *Bacillus oryzae* YPR-35 of this invention exhibits a relatively broad range of acid and alkali stress tolerance.

[0019] The salt stress described refers to salt stress with a salinity of 1%-9%. According to existing technology, soil salinity is classified as follows: ① Slightly saline-alkali land (soil salinity content of 0.2%-0.4%, with low surface soil salinity and minimal impact on crop growth), ② Moderately saline-alkali land (soil salinity content of 0.4%-0.8%, with higher surface soil salinity, potentially affecting crop growth), ③ Severely saline-alkali land (soil salinity content of 0.8%-1.6%, with high surface soil salinity and significant impact on crop growth), ④ Extremely severe saline-alkali land (soil salinity content exceeding 1.6%, with extremely high surface soil salinity and a significant impact on crop growth). Therefore, the *Bacillus oryzae* YPR-35 of this invention exhibits excellent salt stress tolerance and is suitable for almost all saline-alkali lands, including extremely severe saline-alkali lands.

[0020] The application of the aforementioned *Bacillus mirabilis* YPR-35 in the production of indoleacetic acid.

[0021] The application of the aforementioned *Bacillus mirabilis* YPR-35 in the preparation of products for the production of indoleacetic acid.

[0022] The application of the aforementioned *Bacillus mirabilis* YPR-35 in the production of siderophores.

[0023] The application of the aforementioned *Bacillus mirabilis* YPR-35 in the preparation of products that produce siderophores.

[0024] The application of the aforementioned *Bacillus mirabilis* YPR-35 in phosphorus solubilization and / or potassium solubilization.

[0025] The above-mentioned *Bacillus mirabilis* YPR-35 is used in the preparation of products with phosphorus-solubilizing and / or potassium-solubilizing capabilities.

[0026] In addition, microbial preparations containing the aforementioned Bacillus mirabilis strain YPR-35 or its fermentation broth, and their applications, should also be within the scope of protection of this invention.

[0027] Based on the research of this invention, this invention also provides a method for preparing a fermentation broth containing the plant growth regulator indoleacetic acid (IAA). This method involves inoculating *Bacillus mirabilis* YPR-35 into a liquid culture medium and culturing it to obtain a fermentation broth containing IAA. Subsequent purification of the fermentation broth to separate and purify IAA yields the final product.

[0028] As an alternative implementation, the liquid culture medium is LB liquid culture medium.

[0029] The present invention has the following beneficial effects:

[0030] The *Bacillus mirabilis* strain YPR-35 provided by this invention has the characteristics of high production of indoleacetic acid (IAA) and siderophores. This bacterium also has the functions of phosphate solubilization and potassium solubilization. It has significant resistance to heavy metal stress (Mn, Zn, Fe, Cu and Cd), salt stress, and acid and alkali stress, and has good application prospects.

[0031] Experimental tests have shown that this bacterium can significantly promote the growth of corn plants. Using this strain with diatomaceous earth to make a potato seed dressing agent can significantly increase the yield of marketable potatoes, achieving the effect of high yield and increased income. It has great application value in promoting crop production.

[0032] The novel fictitious Bacillus provided by this invention can provide a new strain resource for the microbial fertilizer industry, showing potential application prospects in rhizosphere growth promotion and soil phosphorus activation. Attached Figure Description

[0033] Figure 1 A colorimetric image showing the IAA production capacity of Bacillus mirabilis YPR-35.

[0034] Figure 2 A bar chart showing the IAA production of Bacillus mirabilis YPR-35.

[0035] Figure 3 Images show the spore staining and Gram staining results of Bacillus mirabilis YPR-35 (a is Gram staining image, b is spore staining image).

[0036] Figure 4 Phylogenetic tree of Bacillus mirabilis YPR-35.

[0037] Figure 5 The graphs show the effects of Bacillus mirabilis YPR-35 on heavy metal, acid, and salt stress (a) shows the effect on heavy metal stress, b) shows the effect on acid resistance, and c) shows the effect on salt stress.

[0038] Figure 6 A diagram illustrating the siderophore production capacity of Bacillus mirabilis YPR-35.

[0039] Figure 7 This image shows the growth-promoting effect of Bacillus mirabilis YPR-35 in a pot experiment on maize.

[0040] Figure 8 This image shows the growth-promoting effect of Bacillus mirabilis YPR-35 in a potato pot experiment.

[0041] Figure 9 This is a field layout and distribution map of different treatments during a potato field trial. Detailed Implementation

[0042] The present invention will be further described below with reference to the accompanying drawings and specific examples, but the examples do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0043] Unless otherwise specified, the reagents and materials used in the following examples were commercially available.

[0044] The organic fertilizers used in the following examples were purchased from Huizhou Defeng Biotechnology Co., Ltd.

[0045] The compound fertilizers used in the following examples were purchased from Guangzhou Omino Ecological Agriculture Technology Co., Ltd.

[0046] Example 1: Screening, isolation, and purification of strains

[0047] Using maize rhizosphere soil from the farm of South China Agricultural University in Guangzhou as the screening soil sample, accurately weigh 10.0g of soil sample and place it in a 250mL Erlenmeyer flask containing 90mL of sterile water (with 5-7 glass beads). Shake at 180r / min for 30min to fully disperse the soil sample. Let it stand for 20-30s, take 5mL of the supernatant into a test tube, heat at 90℃ for 10min, and then perform serial dilution to 10. -5Dilute 100 μL of the solution and spread it on an LB agar plate. After incubating at 37°C upside down for 24 h, observe the morphology of the cultured single colonies. Collect single colonies with different morphological characteristics and number them (YPR-1~YPR-80, YPF-1~YPF-50). Purify the selected strains using the streak plate method and store them on LB slant medium (10.0 g peptone, 5.0 g yeast extract, 10.0 g NaCl, 18.0 g agar, 1000 mL distilled water, pH 7.0-7.2) and store them at 4°C for later use.

[0048] Qualitative determination of IAA production by the strain: The isolated and preserved strain was subjected to a qualitative IAA test. A single colony from an LB agar plate was picked and placed in 3 mL of LB medium containing 100 mg / L tryptophan, and incubated at 30 °C and 180 rpm for 24 h. 200 μL of the bacterial suspension was mixed with an equal volume of Salkowski colorimetric solution (50 mL of 35% HClO4 and 1 mL of 0.5 mol / L FeCl3, freshly prepared) in a white porcelain plate. 200 μL of uninoculated LB medium containing 100 mg / L tryptophan and 200 μL of IAA solution containing 25 mg / L were used as blank and positive controls, respectively. The plates were incubated in the dark for 30 min. The colorimetric results of strain YPR-35 are shown below. Figure 1 As shown, the mixture turns red, indicating that the strain has the ability to produce IAA.

[0049] Quantitative determination of IAA production by the strain: IAA solutions of 0, 2.5, 5, 10, 15, 20, and 25 mg / L were prepared. 2 mL of each IAA solution was mixed with an equal volume of Salkowski colorimetric solution. After developing the mixture in the dark for 30 min, the OD value of the mixture at a wavelength of 530 nm was measured using a spectrophotometer. A standard curve was plotted with the OD value on the ordinate and the IAA solution concentration on the abscissa.

[0050] Strains with good colorimetric effects (YPR-35, YPR-32, YPR-31, YPR-41, YPR-56, YPR-77, YPF-33, YPF-37, YPF-61, and YPF-45) were inoculated in LB medium and cultured overnight. Their OD values ​​were then measured. 600 Centrifuge the sample, collect the supernatant and mix it with an equal volume of Salkowski colorimetric solution. After developing the mixture in the dark for 30 minutes, measure the OD value of the mixture at 530 nm using a spectrophotometer. Compare with the standard curve to calculate the amount of IAA produced by different strains. The results are as follows: Figure 2 As shown, strain YPR-35 produced the highest amount of IAA, and this strain had the highest OD value. 600The amount of IAA produced at a concentration of 1.0 was 48.79 mg / L. Strains with strong IAA-producing capacity were inoculated into LB liquid medium and cultured overnight. The freshly cultured bacterial solution was then stored at a 1:1 ratio with 50% sterilized glycerol (final glycerol concentration 25%) and preserved in a -80°C cryogenic freezer for further research. Based on growth viability and IAA production capacity, *Fictibacillus barbaricus* YPR-35 was selected from a large pool of strains as a suitable material for subsequent experiments.

[0051] Example 2 Characterization of YPR-35 strain

[0052] (1) Colony morphology characteristics

[0053] YPR-35 colonies formed after culturing on nutrient agar medium (10.0g peptone, 3.0g beef extract, 5.0g NaCl, 18.0g agar, 1000mL distilled water, pH 7.0-7.2) for 24 hours are round or oval, white, moist and smooth, non-sticky and easy to pick up.

[0054] (2) Growth characteristics

[0055] Single colonies from the agar plate were picked and incubated in LB broth at 180 rpm and 37°C for 12 hours. Then, 1% inoculum was added to fresh LB broth and incubated for another 12 hours. The viable count of the fermentation broth was determined to be 3.3 × 10⁻⁶ cells / mL using the plate count method. 9 cfu / mL.

[0056] (3) Gram staining

[0057] Single colonies of YPR-35 were picked and cultured in liquid LB medium at 37°C and 180 rpm for 12 h. 10 μL of sterile water was placed on a glass slide, and 1 μL of bacterial suspension was added to the prepared sterile water. The mixture was then dried and fixed in a flame. Primary staining with crystal violet for 1 min was performed, followed by rinsing with water and air drying. Mordanting with iodine solution for 1 min was performed, followed by rinsing with water and air drying. Decolorization with 95% ethanol for 30 s was performed, followed by rinsing with water and air drying. Counterstaining with 0.5% safranin solution for 1 min was performed, followed by rinsing with water and air drying. The results were observed under a 100x oil immersion microscope. Figure 3 As shown in Figure a, the bacterial cells are blue-purple, indicating that strain YPR-35 is a Gram-positive bacterium.

[0058] (4) Spore staining

[0059] Colonies of YPR-35 cultured on nutrient agar for 48 hours were picked and transferred to a glass slide. 20 μL of sterile water was added to the slide and mixed thoroughly with the strain. The slide was allowed to air dry and then fixed by flame heating. 5% malachite green staining solution was added, and the slide was heated until steam was emitted 3-4 times within 30 seconds. After cooling, the slide was rinsed with tap water for 30 seconds. 0.5% safranin counterstaining solution was added, and the slide was washed with water after 30 seconds. The slide was then allowed to dry and observed under a microscope. The results are as follows: Figure 3 As shown in Figure b, the spores are green and the bacterial cells are red, indicating that strain YPR-35 has the ability to produce spores.

[0060] (5) Molecular biological characteristics

[0061] Pick a single YPR-35 colony and place it in a centrifuge tube containing 100 μL of sterile water. Mix well and heat in a 95°C water bath for 15 min to rupture the cells and release DNA. Finally, aspirate 1 μL of the DNA and add it to the PCR system (25 μL PCR reaction mixture: 1 μL DNA template, 1 μL primer F1 (1 mM) (5'-AGAGTTTGATCCTGGCTCAG-3'), 1 μL primer R1 (1 mM) (5'-TACGGCTACCTTGTTACGACTT-3'), 9.5 μL ddH2O, 12.5 μL 2×Taq PCR Mix). Amplify and verify using a gel electrophoresis.

[0062] PCR amplification system: pre-denaturation at 95℃ for 3 min, followed by thermal cycling; denaturation at 95℃ for 15 s, annealing at 58℃ for 15 s, extension at 72℃ for 1 min, for a total of 35 cycles; extension at 72℃ for 5 min, and stop at 4℃.

[0063] The length and concentration of PCR products were detected by 1.5% agarose gel electrophoresis. PCR amplification products showing bands were sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing. The obtained 16S rDNA sequences were entered into the NCBI (National Center for Bioinformatics) website for BLAST alignment. All sequences in the database were compared and analyzed using the BLAST program. Mega 7.0 software was used for sequence analysis and phylogenetic tree construction. The results are as follows: Figure 4 As shown.

[0064] In summary, strain YPR-35 was identified as *Bacillus mirabilis*, and was deposited on December 11, 2023, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 29286; the deposit address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0065] Example 3: Determination of potassium solubilization capacity of YPR-35

[0066] YPR-35 cells were streaked onto LB agar plates and incubated at 37°C for 12 hours. Single colonies of YPR-35 were then inoculated into test tubes containing LB liquid medium and incubated at 37°C, 180 rpm for 12 hours. The culture was then transferred to fresh LB liquid medium and shaken at 37°C, 180 rpm until the OD value was reached. 600 =0.4. Take 0.5 mL of bacterial culture (based on 1% culture medium) and inoculate it into 50 mL of liquid potassium-solubilizing medium (5.0 g glucose, 0.5 g MgSO4·7H2O, 0.1 g CaCO3, 0.005 g FeCl3, 2.0 g Ca3(PO4)2, 2.0 g potassium feldspar, 1000 mL distilled water, pH 7.0-7.5). Incubate in a 250 mL Erlenmeyer flask at 30℃, 180 rpm, and shake for 7 days. After 7 days of incubation, centrifuge the culture at 8000 rpm for 5 min, collect the supernatant, and determine the water-soluble potassium content using a flame photometer. Use the supernatant of uninoculated potassium-solubilizing medium as a control. The potassium content of the control treatment is the potassium solubility of the strain. Each treatment is repeated 3 times. The results of the water-soluble potassium content test are shown in Table 1.

[0067] Table 1 Potassium solubilization capacity of strain YPR-35

[0068]

[0069] Note: * This indicates a significant difference, and the same applies below.

[0070] Example 4: Determination of the phosphorus solubility of YPR-35

[0071] YPR-35 cells were streaked onto LB agar plates and incubated at 37°C for 12 hours. Single colonies of YPR-35 from the plates were then inoculated into test tubes containing LB liquid medium and incubated at 37°C, 180 rpm, on a shaker for 12 hours. The culture was then transferred to fresh LB liquid medium and shaken at 37°C, 180 rpm until the OD value was reached. 600=0.4. Take 0.2 mL of bacterial culture (based on 1% culture medium) and inoculate it into 20 mL of phosphate-solubilizing medium (10.0 g glucose, 5.0 g ferric phosphate, 5.0 g MgCl2·6H2O, 0.25 g MgSO4·7H2O, 0.2 g KCl, 0.1 g (NH4)2SO4, 1000 mL distilled water, pH 7.0). Incubate at 30℃, 180 rpm for 7 days on a shaker. After 7 days of incubation, take 2 mL of culture medium and centrifuge at 8000 rpm for 5 min. Collect the supernatant and determine the water-soluble phosphorus content using the molybdenum-antimony spectrophotometric method according to the National Environmental Protection Standard of the People's Republic of China (HJ 632-2011). Use no inoculation as a control; the phosphorus content of the control treatment is the phosphate solubility of the strain. Each treatment is repeated 3 times. The results of the water-soluble phosphorus content test are shown in Table 2. The amount of phosphorus dissolved in YPR-35 after 7 days of culture in a medium with calcium phosphate as the phosphorus source was 227.4 mg / L.

[0072] Table 2 Phosphate solubility of strain YPR-35

[0073]

[0074] Example 5: Effect test of YPR-35 on heavy metal, acid and salt stress

[0075] (I) Determination of YPR-35's resistance to heavy metal stress

[0076] Pick a single colony of YPR-35 from an LB agar plate and incubate it overnight at 37°C and 180 rpm. Then, transfer it to 4 mL of LB liquid medium at a 1% inoculum rate and incubate at 37°C and 180 rpm until OD500. 600 =0.4.

[0077] Take 100 μL OD 600 Add the bacterial suspension at a concentration of 0.4% to 4 mL of LB solid medium (0.75% agar powder by volume) at 50-60℃, mix well, and pour onto a 15 mL LB solid medium plate (1.5% agar powder by volume). Blow dry on a clean bench for 20 min. Place sterilized filter paper discs on the plates and add 10 μL of 1M Fe(II), 500 mM Cu(II), 100 mM Mn(II), 100 mM Zn(II), 1M Ni(II), and 100 mM Cd(II) to the discs, respectively. After drying, incubate at 37℃ for 24 h. Use the Bacillus subtilis model strain NCIB 3610 as a control and record the diameter of the clear zone. The experimental results are as follows: Figure 5 As shown in Figure a.

[0078] The experimental results showed that under Mn(II) and Zn(II) heavy metal stress, the diameter of the transparent zone produced by YPR-35 was smaller than that of the model strain NCIB 3610, indicating that the bacterium exhibits resistance to Mn(II) and Zn(II) heavy metals. Compared with the model strain NCIB 3610, YPR-35 also showed significantly greater resistance to Fe(II), Cu(II), and Cd(II) heavy metal stress. These experimental results provide a foundation for future application of this bacterium in soils with heavy metal residues.

[0079] (II) Determination of the acid and alkali stress resistance of YPR-35

[0080] Prepare LB gradient media with pH values ​​ranging from 4.0 to 12.0 (0.5 increments). Inoculate the test colonies (strain YPR-35) from the plates into test tubes containing LB liquid medium and incubate overnight at 37°C and 180 rpm on a shaker. Transfer the bacterial culture to fresh LB liquid medium and shake at 37°C and 180 rpm until OD reaches zero. 600 =0.4. Dispense culture media of different pH values ​​into 24-well plates, 2 mL LB liquid medium per well. Take 20 μL OD... 600 A bacterial suspension of 0.4 g (based on 1% of the culture medium) was placed in a 24-well plate and incubated at 37°C and 180 rpm for 24 h. The growth status of the strain was then measured, and the experiment was repeated three times.

[0081] The experimental results showed that after 24 hours of cultivation in LB medium at different pH levels, YPR-35 could grow well in media with pH values ​​ranging from 5.5 to 9.5 (growth curves within the acceptable pH range are shown in Figure 1). Figure 5 As shown in Figure b), the preferred pH value is 6–8.5 (most preferred pH value is 7.0–7.5). The results show that YPR-35 has good resistance to acid and alkali stress and can be applied to acidic and alkaline soils.

[0082] (III) Determination of salt stress tolerance of YPR-35

[0083] LB solid medium containing 1%, 3%, 5%, 7%, 9%, 11%, and 13% NaCl was prepared. Strain YPR-35 was inoculated onto LB plates and cultured in an incubator at 37°C for 24 hours. The growth of the strain was observed and photographed.

[0084] The results are as follows Figure 5 As shown in Figure c, strain YPR-35 can grow normally when cultured on plates containing 1%-9% NaCl, demonstrating excellent salt stress tolerance and thus making it suitable for saline-alkali land, including extremely saline-alkali land.

[0085] Example 6: Determination of the siderophore production capacity of strain YPR-35

[0086] Inoculate the bacterial colony (strain YPR-35) from the plate into a test tube containing LB liquid medium and incubate overnight at 37°C and 180 rpm on a shaker. Transfer the bacterial culture to fresh LB liquid medium and shake at 37°C and 180 rpm until OD reaches zero. 600 =0.4. OD absorption 600 Add 5 μL of bacterial suspension with a concentration of 0.4% to a CAS plate and incubate at 28°C for 48 h. Observe whether a clear siderophore halo appears around the colony. The presence of a halo indicates that the strain has the ability to produce siderophores.

[0087] Observe the colonies after 48 hours of incubation on CAS test plates. The results are as follows: Figure 6 As shown, halos were found around strain YPR-35, indicating that the bacteria have a certain ability to produce siderophores.

[0088] Example 7: Experiment on the effect of YPR-35 on the growth of potted maize

[0089] (1) Preparation of bacterial suspension:

[0090] ① After activating the bacterial strain, inoculate it into LB liquid medium, incubate overnight, then transfer it to fresh LB liquid medium and shake until OD is reached. 600 =1.0;

[0091] ② Centrifuge the bacterial culture in a centrifuge at 8000 rpm for 5 minutes;

[0092] ③ Resuspend the bacterial cell mass in an equal volume of sterile water and dilute to 10⁻⁶. 9 CFU / mL available for use.

[0093] (2) Corn seedling raising:

[0094] Wash corn seeds with water to promote germination, then soak gauze to retain moisture and place them in a 25℃ biochemical incubator. When the seed sprouts are about 1cm long, transfer them to 50-cell seedling trays, one seed per cell. Water appropriately every day. When the corn seedlings have grown 3 leaves, they are ready for transplanting.

[0095] (3) Experimental Design:

[0096] A comparative experiment was conducted using YPR-35 and a blank control. Two treatment groups were used: the first group received YPR-35 (T1); the second group received an equal volume of sterile water as a blank control (CK). Each treatment was repeated four times. Round flowerpots with an inner diameter of 12.5 cm and a height of 11.5 cm were selected, and each pot was filled with 0.9 kg of potting soil. Before transplanting, a suitable amount of water was evenly applied to the surface of the seedling soil to ensure that the soil remained moist but not sticky during transplanting. Uniformly grown corn seedlings were selected and transplanted into the pots. After covering with soil, 15 mL of the bacterial suspension was applied to the root zone of the corn plants, ensuring that each gram of potting soil contained 10 colonies. 6 CFU. Water appropriately each day, ensuring the same amount of water per pot, taking care not to let water overflow the bottom of the pot to avoid loss of bacterial solution and fertilizer, which could lead to errors. Take photos on the 10th day after transplanting. Harvest samples on the 30th day and measure the plant height, stem diameter, leaf length, leaf width, above-ground fresh weight, and dry weight of the corn plants. After harvesting, blanch the above-ground parts at 105℃ for 30 minutes, then dry them at 65℃ to constant weight before weighing.

[0097] The results are shown in Table 3. On the 10th day after planting, the plant height, stem diameter, leaf length, and leaf width of maize treated with strain YPR-35 and the control group (CK) were 39.29 and 31.02 cm, 4.16 and 3.51 mm, 31.53 and 23.88 cm, and 2.89 and 2.56 cm, respectively. The plant height, stem diameter, and leaf length of the YPR-35 treatment were significantly higher than those of the control group (CK), increasing by 26.66%, 18.52%, and 31.66%, respectively. The leaf width of the YPR-35 treatment was also significantly higher than that of the control group (CK), increasing by 12.1%. The growth-promoting effect of strain YPR-35 in the maize pot experiment is shown in the figure below. Figure 7 Figure a shows that applying this strain can improve the growth rate of corn.

[0098] On day 30 after planting, the plant height, stem diameter, leaf length, leaf width, and above-ground fresh and dry weight of maize plants were measured. The results are shown in Table 3. The plant height, stem diameter, leaf length, leaf width, and above-ground fresh and dry weight of the treatment with strain YPR-35 were 57.52 cm, 9.45 mm, 39.59 cm, 4.18 cm, 36.44 g, and 5.28 g, respectively; while the plant height, stem diameter, leaf length, leaf width, and above-ground fresh and dry weight of the CK treatment were 48.48 cm, 7.90 mm, 30.56 cm, 3.29 cm, 28.40 g, and 3.54 g, respectively. Compared with the control (CK), the application of strain YPR-35 increased the plant height, stem diameter, and aboveground fresh and dry weight of maize plants by 18.65%, 19.62%, 29.55%, 27.05%, 28.31%, and 49.15%, respectively. The growth-promoting effect of strain YPR-35 in a maize pot experiment is shown in the figure below. Figure 7As shown in Figures b and c, the plants treated with strain YPR-35 showed significantly increased plant height and leaf width ratio (CK). In summary, the experimental results indicate that the addition of *Bacillus mirabilis* YPR-35 isolated in this invention can significantly promote the growth of maize plants.

[0099] Table 3 Comparison of growth indicators of potted maize plants under different treatments

[0100]

[0101] Note: The values ​​in the figure are the mean ± standard error. Data with the same letter in the same column indicate that the variance is not significant (Duncan, p < 0.05), and the same applies below.

[0102] Example 8: Effect of YPR-35 on promoting potato growth

[0103] (1) Preparation of inoculum: Five LB solid medium plates were streaked with YPR-35 strain using a simulated spread method and incubated at 37℃ for 24 hours. The bacterial cells on the surface of the plates were scraped off with a sterile cardboard and mixed with 20g of diatomaceous earth to prepare an inoculum containing 10 colonies. 8 A seed dressing agent with cfu / g is prepared for later use. CK is 20g of diatomaceous earth without added inoculant.

[0104] (2) Seed potato preparation: Select F9 seed potatoes with obvious buds, cut them into uniformly sized pieces, each weighing 30±5g, with only one bud on each piece. Perform surface sterilization on the cut pieces by washing with 75% ethanol for 2 minutes, rinsing twice with water, then treating with 3% sodium hypochlorite for 1 minute, rinsing twice with water, and drying.

[0105] (3) Seed dressing: The prepared seed dressing agent was mixed with the seed potato tubers in an alcohol-sterilized plastic basin to ensure that the cut surface of each tuber was evenly coated with the prepared seed dressing agent. Diatomaceous earth without the agent was used as a blank control. There were 4 tubers in each treatment. The prepared tubers were stored in a 4°C refrigerator.

[0106] (4) Potted plant preparation: Select a round flower pot with an inner diameter of 34cm and a height of 21cm. First, fill each pot with 5kg of test soil (about half of the flower pot). Place a potato tuber in each pot and cover it with soil to a depth of 5cm. After the seedlings have grown long enough, cover them with 3kg of soil.

[0107] After 45 days of growth, the plant height, stem diameter, chlorophyll content, and number of leaves of the two treatments were measured. The results are shown in Table 4. The results showed that the potato plants treated with YPR-35 inoculant had a significant growth-promoting effect compared with the blank control (CK). YPR-35 increased the plant height, stem diameter, chlorophyll content, and number of leaves by 30.56%, 29.35%, 31.65%, and 37.63%, respectively. After 100 days of growth, the yield of the potato plants in the two treatments was measured. The yield of the potato plants treated with YPR-35 inoculant was 2.62 kg, while the yield of the blank control was 2.08 kg, representing a yield increase of 25.96%, indicating a significant yield increase.

[0108] The growth-promoting effect of YPR-35 in potato pot trials is as follows: Figure 8 As shown in the figure, the potato plants treated with YPR-35 inoculant had significantly more leaves than the control (CK), and the potato yield of the YPR-35 inoculant treatment was significantly higher than that of the control (CK).

[0109] Table 4. Potato growth traits on day 45

[0110]

[0111] Example 9: Effect of YPR-35 on Potato Yield Promotion

[0112] (1) Experimental materials and planting conditions

[0113] Cut the seed potatoes into pieces: Cut the seed potatoes into pieces, each weighing 25-30g, and each piece contains 1-2 buds.

[0114] Planting specifications: Double-row ridge planting is adopted, with a ridge width of 1.2 meters (including the furrow), a ridge height of 25-30 cm, a plant spacing of 20 cm, a row spacing of 25-30 cm within the ridge, and a planting depth of 5-6 cm.

[0115] Conventional fertilization: 400 kg / mu of commercial organic fertilizer + 120 kg / mu of high-potassium compound fertilizer (15-5-25). After plowing and preparing the land, spread the commercial organic fertilizer and high-potassium compound fertilizer, then ridge and cover with fertilizer. On the 50th day after planting potatoes, apply 25 kg / mu of high-potassium compound fertilizer (15-5-25).

[0116] (2) Experimental Design

[0117] Community area: The community area is determined based on the actual land plot, such as... Figure 9 As shown, each community is approximately 15.84m. 2 Specifically, there are three rows in total, with a total length of 3 × 1.2m × 4.4m = 15.84m. 2Plant two rows per ridge, with 20 tubers per row. Plant a total of 6 × 20 = 120 tubers per plot. Repeat 4 times. Prepare a total of 4 × 120 = 480 tubers per treatment.

[0118] Seed dressing preparation and treatment: Thoroughly mix YPR-35 microbial agent and 250g diatomaceous earth to obtain a seed dressing agent with a microbial content of 3.3×10⁻⁶. 9 The yield was measured at cfu / g, with a commercially available inoculant (70% methyl thiophanate, wettable powder, dosage 1000g / 100kg potato) as the positive control (CK1) and no inoculant added as the blank control (CK2). The yield was measured at harvest on the 100th day after planting.

[0119] Potatoes were harvested and yield measured 100 days after planting. The results are shown in Table 5. Compared with CK1, the treatment with inoculant YPR-35 increased the yield of large potatoes by 13.16% per mu. Compared with CK1 and CK2, the treatment with inoculant YPR-35 significantly increased the yield of medium potatoes by 33.33% and 35.38% per mu, respectively, and increased the yield of small potatoes by 4.17% and 56.25% per mu, respectively. In addition, the yields of marketable potatoes treated with conventional inoculant methyl thiophanate, no inoculant, and inoculant YPR-35 were also significantly higher. The yields per mu were 2178.31 kg, 2153.82 kg, and 2595.10 kg, respectively. Compared with conventional inoculant treatments such as methyl thiophanate and no inoculant treatment, the yields of marketable potatoes treated with inoculant YPR-35 increased by 19.13% and 20.49% respectively, and by 416.79 kg and 441.28 kg respectively. At a market price of 1.6 yuan / kg, the seed dressing agent made by using inoculant YPR-35 and diatomaceous earth can increase income by 650-750 yuan per mu compared with conventional inoculant methyl thiophanate.

[0120] Table 5 Comparison of field potato yield indicators under different treatments

[0121]

[0122] Note: Extra-large potatoes refer to tubers weighing ≥175g, Grade 1 potatoes refer to tubers weighing 75-175g, marketable potatoes refer to tubers weighing ≥75g, and small potatoes refer to tubers weighing <75g. Potatoes are calculated at a market purchase price of 1.6 yuan / kg.

[0123] In this experiment, under conventional fertilization conditions, a seed dressing agent made from inoculant YPR-35 and diatomaceous earth was used to replace the commercially available inoculant methyl thiophanate for potato seed dressing. This can increase the yield of marketable potatoes and increase farmers' income during the potato harvest period.

[0124] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A strain of *Fictibacillus barbaricus* YPR-35, characterized in that, The bacterium was deposited at the China General Microbiological Culture Collection Center on December 11, 2023, with accession number CGMCC No. 29286.

2. A microbial preparation, characterized in that, Contains the *Bacillus mirabilis* strain YPR-35 or its fermentation broth as described in claim 1.

3. The use of the *Bacillus mirabilis* YPR-35 of claim 1 or the microbial preparation of claim 2 in promoting plant growth or in the preparation of products that promote plant growth.

4. The use of the *Bacillus mirabilis* YPR-35 of claim 1 or the microbial preparation of claim 2 in promoting plant growth under environmental stress or in the preparation of products with the ability to promote plant growth under environmental stress, wherein the environmental stress refers to heavy metal stress, acid-base stress and / or salt stress.

5. The application according to claim 4, characterized in that, The heavy metal stress includes Mn(II), Zn(II), Fe(II), Cu(II) and / or Cd(II) heavy metal stress.

6. The application according to claim 4, characterized in that, The acid-base stress includes acid stress with a pH of 5.5 to 6.5 and alkali stress with a pH of 7.5 to 9.5; the salt stress is salt stress with a salinity of 1% to 9%.

7. The use of the *Bacillus mirabilis* YPR-35 of claim 1 or the microbial preparation of claim 2 in the production of indoleacetic acid or in the preparation of a product for the production of indoleacetic acid.

8. The use of the *Bacillus mirabilis* YPR-35 of claim 1 or the microbial preparation of claim 2 in the production of siderophores or in the preparation of products that produce siderophores.

9. The use of the *Bacillus mirabilis* YPR-35 of claim 1 or the microbial preparation of claim 2 in phosphate solubilization and / or potassium solubilization, or in the preparation of products having phosphate solubilization and / or potassium solubilization capabilities.

10. A method for preparing a fermentation broth containing the plant growth regulator indoleacetic acid, characterized in that, The *Bacillus mirabilis* YPR-35 of claim 1 or the microbial preparation of claim 2 is inoculated into a liquid culture medium for cultivation to obtain a fermentation broth containing indoleacetic acid.

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