Multifunctional bacillus sp. ASH9-3 and application thereof

Abstract

The application discloses a multifunctional Bacillus mobilis ASH9-3 and application thereof, and belongs to the technical field of microorganisms. The Bacillus mobilis ASH9-3 is preserved in the China General Microbiological Culture Collection Center, and the preservation number is CGMCC No. 32327. The strain has the functions of antagonizing four soil-borne diseases such as tomato early blight, producing protease, producing indole acetic acid (IAA), decomposing inorganic phosphorus and fixing iron ions. The fermentation liquor of the strain is applied to soil, so that the occurrence rate of soil-borne diseases can be significantly reduced, the content of soil organic matter and available nutrients can be increased, and the yield of peppers can be increased by 32.4%. The application fills the blank of multifunctional microbial agents in the prior art, and provides an efficient solution for soil-borne disease prevention and control and sustainable management of soil.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a multifunctional motile Bacillus ASH9-3 and its applications. Background Technology

[0002] Soil-borne diseases (such as early blight of tomatoes and blight of peppers) pose a significant threat to agricultural production. Traditional control methods mainly rely on chemical pesticides, but long-term use can easily lead to increased pesticide resistance in pathogens, soil ecological imbalance, and environmental pollution. In recent years, microbial control has attracted attention due to its environmentally friendly characteristics. For example, patent CN107125267A discloses the application of Trichoderma hookeri in the control of soil-borne diseases, but its function is singular, lacking the ability to promote growth and remediate soil. Existing phosphate-solubilizing strains can release phosphorus, but their colonization ability is weak, making it difficult to maintain soil fertility in the long term. In addition, strains with the functions of producing plant hormones (such as IAA), nitrogen fixation, or iron fixation are rarely reported, which limits the comprehensive application effect of microbial preparations.

[0003] Therefore, developing a multifunctional strain that can antagonize diseases, promote plant growth, repair soil, and has strong colonization ability is an urgent need in the field of agricultural microbiology. Summary of the Invention

[0004] The purpose of this invention is to provide a multifunctional Bacillus motility strain ASH9-3 and its applications to solve the problems existing in the prior art. This strain has multiple functions including disease control, soil remediation, and crop yield enhancement.

[0005] To achieve the above objectives, the present invention provides the following solution:

[0006] This invention provides a strain of Bacillus mobilis ASH9-3, which is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 32327.

[0007] The present invention also provides a microbial agent comprising the aforementioned Bacillus motilityis ASH9-3 or its fermentation broth.

[0008] Furthermore, the microbial agent also includes excipients and / or carriers.

[0009] The present invention also provides a soil improvement method, including the step of applying the microbial agent to the soil.

[0010] The present invention also provides a method for promoting plant growth, comprising the step of treating plant seeds or their roots with the aforementioned microbial agent.

[0011] The present invention also provides a method for increasing plant yield, comprising the step of applying the microbial agent to the soil.

[0012] The present invention also provides a microbial preparation for immobilizing iron ions, comprising the aforementioned bacterial agent.

[0013] The present invention also provides a phosphate-solubilizing microbial preparation, comprising the aforementioned microbial agent.

[0014] The present invention also provides a soil remediation composition comprising the fermentation broth of the Bacillus motilityis ASH9-3 and an organic carrier.

[0015] The present invention also provides the application of the Bacillus motilityis ASH9-3 or the bacterial agent, including any one of the following:

[0016] (1) Application in fixing iron ions in soil;

[0017] (2) Application in the dissolution of inorganic phosphorus;

[0018] (3) Applications in promoting plant growth and increasing plant yield;

[0019] (4) Applications in suppressing soil-borne diseases, repairing soil or increasing soil organic matter content;

[0020] (5) Application in converting slow-release potassium into fast-release potassium or promoting the absorption of potassium nutrients by plants.

[0021] (6) Application in the preparation of bio-organic fertilizer and / or compound microbial fertilizer.

[0022] The present invention discloses the following technical effects:

[0023] This invention discloses a strain of Bacillus mobilis ASH9-3 isolated from the root soil sample of *Galium affine* from Qianshan Mountain, Anshan City, Liaoning Province. This strain has been deposited on October 24, 2024, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC No. 32327. The Bacillus mobilis ASH9-3 disclosed in this invention exhibited inhibition rates exceeding 89% against four soil-borne pathogens in plate confrontation experiments, with the highest reaching 98.1%. Its IAA production capacity promotes plant growth, its phosphorus solubilization function releases soil phosphorus, and its iron fixation function improves iron availability. Pot experiments showed that after applying ASH9-3 fermentation broth, the soil organic matter content increased from 9.12% to 17.69%, and available phosphorus and available potassium increased by 44.8% and 60.7%, respectively. In field trials, chili pepper yield increased by 32.4% per acre, and root development was significantly better than the control group. This strain possesses multiple functions, including disease control, soil remediation, and crop yield enhancement, and has broad prospects for agricultural application. This invention fills the gap in existing technologies for multifunctional microbial agents, providing an efficient solution for soil-borne disease control and sustainable soil management. Attached Figure Description

[0024] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the 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.

[0025] Figure 1 Colony morphology (A) and microscopic morphology (B) of Bacillus motility ASH9-3;

[0026] Figure 2 The phylogenetic tree of Bacillus ASH9-3;

[0027] Figure 3 The results of antagonism between Bacillus ASH9-3 and soil-borne pathogens of tomato early blight (A), pepper blight (B), eggplant root rot (C), and tomato gray mold (D) were obtained.

[0028] Figure 4 Results of protease production by Bacillus ASH9-3;

[0029] Figure 5 Results of IAA production by Bacillus ASH9-3; where 1 and 2 are ASH9-3 treatment groups, CK is the blank control group; CK1 is the positive control group;

[0030] Figure 6 The results of phosphorus solubilization of Bacillus ASH9-3;

[0031] Figure 7 Results of potassium solubilization in Bacillus ASH9-3;

[0032] Figure 8 Results of nitrogen fixation by Bacillus ASH9-3;

[0033] Figure 9 Results of iron ion fixation by Bacillus ASH9-3;

[0034] Figure 10 The effects of the CK control group (A) and the treatment group with Bacillus ASH9-3 on plant roots (B) were investigated. Detailed Implementation

[0035] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0036] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0037] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0038] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0039] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0040] Example 1

[0041] 1. Isolation, screening, and identification of bacterial strains

[0042] 1.1 Isolation and screening of strains

[0043] The soil sample was obtained from Qianshan, Anshan City, Liaoning Province. The soil sample was laid flat and air-dried for one week before use. The soil sample was ground and passed through a 60-mesh sieve. 1g of the soil sample was weighed and added to 99mL of sterile physiological saline containing glass beads. The mixture was shaken in 80℃ water for 20 minutes, then removed and allowed to cool to room temperature. It was then diluted 10-fold serially. -3 10 -4 10 -5 100 μL of the dilution solution was evenly spread onto LB medium culture dishes and incubated at 37°C for 1-2 days. Then, different single colonies were picked and streaked onto LB plates for purification to obtain single colonies.

[0044] 1.2 Strain Identification

[0045] 1.2.1 Observation of bacterial cell morphology

[0046] The selected bacterial strain was streaked onto NA medium and incubated at 37°C for 24 hours. Colony morphology was observed on the plates. The colonies of this strain were large, 4-6 mm, milky white, round, dull, and with irregular edges. Microscopic examination revealed the following characteristics: mesospores, large cell size, and... Figure 1 .

[0047] 1.2.2 Determination of physiological and biochemical characteristics

[0048] The physiological and biochemical characteristics of this strain were tested according to the bacterial identification methods in Bergey's Manual of Bacterial Identification and the Manual of Systematic Identification of Common Bacteria. The results are shown in Table 1.

[0049] Table 1. Results of Physiological and Biochemical Identification

[0050]

[0051] Note: "+" indicates positive, and "-" indicates negative.

[0052] 1.2.3 Molecular biological identification

[0053] DNA was extracted from this bacterial strain according to the instructions on the bacterial DNA extraction kit. Sequencing was performed by Shanghai Sangon Biotech Co., Ltd., and the sequencing results were compared with known sequences in GenBank. Preliminary identification was conducted (see phylogenetic tree). Figure 2The results showed that the strain was *Bacillus mobilis*. This invention names it *Bacillus mobilis* ASH9-3. The original sequence of *Bacillus mobilis* ASH9-3 is shown below:

[0054] GCGTGCCTAATACATGCAAGTCGAGCGAATGGATTGAGAGCTTGCTCTCAAGAAGTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCCATAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATAACATTTTGAACTGCATGGTTCGAAATTGAAAGGCGGCTTCGGCTGTCACTTATGGATGGACCCGCGTCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCAACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGCTTTCGGGTCGTAAAACTCTGTTGTTAGGGAAGAACAAGTGCTAGTTGAATAAGCTGGCACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGAATTATTGGGCGTAAAGCGCGCGCAGGTGGTTTCTTAAGTCTGATGTGAAAGCCCACGGCTCAACCGTGGAGGGTCATTGGAAACTGGGAGACTTGAGTGCAGAAGAGGAAAGTGGAATTCCATGTGTAGCGGTGAAATGCGTAGAGATATGGAGGAACACCAGTGGCGAAGGCGACTTTCTGGTCTGTAACTGACACTGAGGCGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGAGGGTTTCCGCCCTTTAGTGCTGAAGTTAACGCATTAAGCACTCCGCCTGGGGAGTACGGCCGCAAGGCTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGAAAACCCTAGAGATAGGGCTTCTCCTTCGGGAGCAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCATCATTAAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACGGTACAAAGAGCTGCAAGACCGCGAGGTGGAGCTAATCTCATAAAACCGTTCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGGGGTAACCTTTTTGGAGCCAGCCGCCTAAGGTGGGACAGATGATTGGGGTGAAGTCGTACAGGG(SEQ ID NO.1).

[0055] 1.2.4 Strain Preservation

[0056] Bacillus mobilis ASH9-3 was deposited on October 24, 2024, at the China General Microbiological Culture Collection Center (CGMCC), Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 32327.

[0057] 2. Antagonism test

[0058] 2.1 Source of pathogens

[0059] The pathogens of four soil-borne diseases—early blight of tomato, blight of pepper, root rot of eggplant, and gray mold of tomato—were all provided by the Liaoning Provincial Microbial Culture Collection Center.

[0060] 2.2 Antagonism Test

[0061] The plate confrontation method was used. Activated pathogens were abrades (6 mm in diameter) and placed on one side of a PDA plate. The isolated and purified *Bacillus motilityis* ASH9-3 was also abrades (6 mm in diameter) and placed on the other side of the PDA plate, with a distance of 20 mm between the two bacteria. The plates were incubated at 28℃ for 5-7 days, and the antagonistic effect was observed. *Bacillus motilityis* ASH9-3 showed antagonistic effects against all four soil-borne disease pathogens. The results are shown in the table below. Figure 3 .

[0062] 3. Protease production assay

[0063] 3.1 Culture medium

[0064] Protease-selective culture medium: 10g skim milk powder, 20g agar, 1L distilled water, sterilized at 121℃ for 20min.

[0065] 3.2 Methods

[0066] Bacillus motility strain ASH9-3 was inoculated into a protease-selective medium and cultured at 37°C for 36 hours. The presence or absence of a clear zone was then observed. Results showed that Bacillus motility strain ASH9-3 could produce a large amount of protease. (See attached image). Figure 4 .

[0067] 4. Indoleacetic acid (IAA) production test

[0068] IAA is a common plant hormone that stimulates various physiological effects through different signal transduction pathways. It participates in the regulation and control of many physiological and biochemical processes, such as plant cell elongation, cell division, vascular tissue differentiation, apical dominance, tropism, transport of assimilates, and leaf and flower abscission.

[0069] 4.1 Culture medium

[0070] LB liquid medium: 10g tryptone, 10g NaCl, 5g yeast extract, 1000mL distilled water, pH 7.0-7.2, sterilized at 121℃ for 25min.

[0071] 4.2 Methods

[0072] Experimental Groups:

[0073] (1) ASH9-3 processing group Figure 5 (1 and 2 in the text): Inoculate strain ASH9-3 into a solution containing L-tryptophan (100 mg·L⁻¹). －1 LB liquid medium;

[0074] (2) Blank control group (CK): Not vaccinated with ASH9-3, containing L-tryptophan (100 mg·L⁻¹) －1 LB culture medium was mixed with standard colorimetric solution;

[0075] (3) Positive control group (CK1): No ASH9-3 was inoculated. 200 μL of indole-3-acetic acid standard solution (0.9 mg / L) was mixed with the standard colorimetric solution at a ratio of 1:1.

[0076] Each treatment group was replicated twice, at 37°C and 180 rpm. －1 After culturing for 3 days, the fermentation broth was subjected to a 10,000 rpm test. －1 Centrifuge for 10 min, and take 200 μL of the supernatant and mix it with an equal volume of Salkowski colorimetric solution (50 mL H2O + 1 mL 0.5 mol·L⁻¹). －1 Mix FeCl3 + 30 mL 98% H2SO4 in a white cuvette, in the dark, and let stand for 30 minutes. Observe the color change. No change in color indicates a negative result, meaning the strain does not produce IAA; a pink or red color indicates a positive result, meaning the strain produces IAA. The results show that the *Bacillus molybdenum* ASH9-3 treatment group can produce the plant hormone IAA. Figure 5 .

[0077] 5. Phosphorus Solubilization Test

[0078] 5.1 Culture medium

[0079] Phosphorus solubilization test medium: glucose 10g, potassium dihydrogen phosphate 0.2g, sodium chloride 0.3g, magnesium sulfate 0.3g, potassium chloride 0.2g, ammonium sulfate 0.5g, 0.5% ferrous sulfate 6mL, 0.5% manganese sulfate 6mL, tricalcium phosphate 5g, agar 15-20g, distilled water 1000mL, pH 7.2-7.4.

[0080] 5.2 Methods

[0081] Bacillus motilityis ASH9-3 was inoculated onto phosphorus-solubilizing inorganic phosphorus medium and incubated at 37℃ for 48-72 hours. The presence of a clear phosphorus-solubilizing zone was observed. Results showed that Bacillus motilityis ASH9-3 exhibited a clear zone, indicating its phosphorus-solubilizing activity. (See attached results). Figure 6 .

[0082] 6. Potassium solubilization test

[0083] 6.1 Culture medium

[0084] Potassium-solubilizing assay medium: 5g sucrose, 0.2g potassium dihydrogen phosphate, 0.5g magnesium sulfate, 0.1g calcium carbonate, 10 drops ferric chloride, 1g potassium aluminum silicate, 15-20g agar, 1000mL distilled water, pH 7.0-7.2. This medium is used to isolate potassium-solubilizing bacteria in soil.

[0085] 6.2 Methods

[0086] Bacillus motility strain ASH9-3 was inoculated onto potassium-solubilizing medium and incubated at 37°C for 48-72 hours. The presence of a clear potassium-solubilizing zone was observed. Results showed that Bacillus motility strain ASH9-3 lacked potassium-solubilizing function. (See attached results). Figure 7 .

[0087] 7 Nitrogen Fixation Test

[0088] 7.1 Culture medium

[0089] Nitrogen fixation detection medium (Ashbe medium): 10g glucose, 0.2g potassium dihydrogen phosphate, 0.2g sodium chloride, 0.2g magnesium sulfate, 0.2g potassium sulfate, 5g calcium carbonate, 15-20g agar, 1000mL distilled water, pH 7.0-7.2. This medium is used to isolate nitrogen-fixing microorganisms in soil.

[0090] 7.2 Methods

[0091] Bacillus motilityis ASH9-3 was picked and inoculated onto nitrogen fixation assay medium and incubated at 37°C for 48-72 hours. The presence or absence of a clear zone was observed. Results showed that Bacillus motilityis ASH9-3 lacked nitrogen fixation function. (See attached results). Figure 8 .

[0092] 8. Fixed iron ion test

[0093] 8.1 Culture medium

[0094] Iron-fixed detection medium: (1) Preparation of CAS detection solution: Dissolve 0.0605g chromeazurol S (CAS) in 50mL deionized water, and add 10mL FeCl3 solution and stir well, labeling it as "Solution A"; then weigh 0.0729g hexadecyltrimethylammonium bromide and dissolve it in 4mL deionized water, labeling it as "Solution B"; finally, slowly pour Solution A into Solution B and stir well. Iron-fixed detection medium is obtained by slowly pouring Solution A into Solution B and stirring well.

[0095] 8.2 Methods

[0096] The results showed that *Bacillus ASH9-3* could fix iron ions, as detailed below. Figure 9 .

[0097] 9. Applications in suppressing soil-borne diseases and improving soil

[0098] 9.1 Culture medium

[0099] Bacillus ASH9-3 fermentation broth / fermentation medium: Inoculate strain ASH9-3 into a medium containing L-tryptophan (100 mg·L⁻¹). －1 LB liquid medium; LB liquid medium (see 4.1 medium).

[0100] Pathogenic bacteria liquid fermentation medium (PDA liquid medium): Peel potatoes, weigh 200g, then cut them into slices and add 1L of water. Boil for 20 minutes, filter with gauze, collect the filtrate, add to 1L, add 20g of glucose, sterilize at 121℃ for 25 minutes, and set aside.

[0101] 9.2 Methods

[0102] 9.2.1 Bacillus ASH9-3 reduces the incidence of soil-borne diseases

[0103] Using 5-gallon pots, 17L of soil was added, with 25 pots per group. The CK0 group received neither Bacillus aggregatibacterium ASH9-3 fermentation broth nor pathogens, but received 2mL of sterile PDA liquid medium and 10mL of sterile LB liquid medium as a negative control. The CK1 group received neither Bacillus aggregatibacterium ASH9-3 fermentation broth nor pathogens, but received 10mL of sterile LB liquid medium and 2mL of pathogens as a positive control. The experimental groups received 2mL of fermentation broth for tomato early blight, pepper blight, eggplant root rot, and tomato gray mold, and 10mL of ASH9-3 fermentation broth, respectively. Watering was done every two days with 500mL of water each time. Results were collected after 3 months (see Table 2). The results showed that adding Bacillus aggregatibacterium ASH9-3 fermentation broth to the soil significantly reduced the incidence of soil-borne diseases in the pot experiment.

[0104] Table 2. Inhibition rate (%) of Bacillus motility ASH9-3 against four soil-borne disease pathogens.

[0105]

[0106] 9.2.2 Plant growth promotion

[0107] Use 5-gallon pots, add 17L of soil, 6 pots per group. The control group (CK) is given 10mL of sterile LB medium, and the experimental groups are given 10mL of Bacillus ASH9-3 fermentation broth. Add the broth every 15 days, and water with 500mL of water every 2 days. Seedlings are transplanted in March to observe the root system. Results are shown below. Figure 10 The results showed that the plants grown in soil containing Bacillus ASH9-3 fermentation liquid had well-developed root systems and vigorous growth.

[0108] 9.3 Testing soil sample composition changes (9.2.1 Soil samples used to reduce the incidence of soil-borne diseases)

[0109] The results showed that the contents of organic matter, total nitrogen, total phosphorus, total potassium, available nitrogen, available phosphorus, and available potassium in the soil of the experimental group all increased. The application of Bacillus ASH9-3 could increase the available nutrients in the soil, increase the organic carbon content in the soil, and improve the soil quality. The results are shown in Table 3.

[0110] Table 3. Effects of Bacillus ASH9-3 (ASH9-3) on increasing soil available components

[0111]

[0112]

[0113] 9.4 Field Trial of Bacillus ASH9-3 Fermentation Broth

[0114] A chili pepper breeding experiment was conducted in Liuguan Township, Kazuo County. The experimental greenhouse was a cold greenhouse, and the crop was chili pepper. One week after transplanting, the experimental group was drip-irrigated with Bacillus ASH9-3 fermentation broth (3600 mL per row, 50 m long and 1.4 m wide), while the control group was drip-irrigated with LB liquid culture medium (3600 mL per row, 50 m long and 1.4 m wide). Both groups were cared for under the same conditions. The results showed that the experimental group yielded 266.32 kg of chili peppers, while the control group yielded 201.15 kg, representing a 32.4% increase per mu (unit of land area) compared to the control group.

[0115] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A strain of Bacillus motility ( Bacillus mobilis ASH9-3, characterized in that, It is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 32327.

2. A microbial agent, characterized in that, It contains Bacillus ASH9-3 of claim 1 or its fermentation broth.

3. The microbial agent according to claim 2, characterized in that, The microbial agent also includes excipients and / or carriers.

4. A soil improvement method, characterized in that, This includes the step of applying the microbial agent as described in claim 2 or 3 to the soil.

5. A method for promoting plant growth, characterized in that, The step includes treating plant seeds or their roots with the fungal agent described in claim 2 or 3.

6. A method for increasing plant yield, characterized in that, The step includes applying the microbial agent of claim 2 or 3 into the soil.

7. A microbial preparation for immobilizing iron ions, characterized in that, Includes the microbial agent as described in claim 2 or 3.

8. A phosphate-solubilizing microbial preparation, characterized in that, Includes the microbial agent as described in claim 2 or 3.

9. The application of the *Bacillus motilityis* ASH9-3 according to claim 1 or the bacterial agent according to claim 2 or 3, characterized in that, Including any of the following: (1) Application in fixing iron ions in soil; (2) Application in the dissolution of inorganic phosphorus; (3) Applications in promoting plant growth and increasing plant yield; (4) Application in suppressing soil-borne diseases or increasing soil organic matter content; (5) Application in converting slow-release potassium into fast-release potassium or promoting the absorption of potassium nutrients by plants. (6) Application in the preparation of bio-organic fertilizers and / or compound microbial fertilizers; The soil-borne diseases mentioned are early blight of tomatoes, blight of peppers, root rot of eggplants, or gray mold of tomatoes.

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