Pseudomonas indica and application thereof

Abstract

The present application belongs to the field of microbial technology, and relates to a paddy-dwelling Pseudomonas and application thereof. The present application provides a paddy-dwelling Pseudomonas, which is YC362-2-1 and is preserved in the China General Microbiological Culture Collection Center. The present application has important application value for development of agricultural microbial preparations.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology and relates to a species of Pseudomonas oryzae and its applications. Background Technology

[0002] Wheat stem rot (Fusarium crown rot, FCR) is a soil-borne disease, and its main pathogen is Fusarium pseudogranatum (…). Fusarium pseudograminearum The threat of wheat stem base rot cannot be ignored, as it occurs throughout the entire growth and development cycle of wheat. During the development of wheat stem base rot, a series of toxins and secondary metabolites are produced, including but not limited to deoxynivalenol (DON) and nivalenol (NIV), posing a serious threat to human and livestock health.

[0003] Wheat stem rot occurs throughout the entire growth and development of wheat, exhibiting different symptoms at different stages. Germination stage: After the pathogen invades the wheat seeds, it causes seed rot or hinders the normal development of the radicle, thus threatening normal seedling emergence. During the seedling stage, after infection, brown or black lesions appear on the wheat roots. In severe cases, root rot occurs, even leading to seedling death. Tillering and jointing stage: The infection spreads further from the roots to the stem base. The leaf sheaths at the stem base gradually turn dark brown, and in severe cases, the stem base may rot and break. Mature plant stage: The pathogen may further extend to the wheat stem base, causing brown rot lesions, resembling "soy sauce stalks." In humid environments, these brown stem nodes may also breed a large number of pink mycelia. In severe cases, the lesions may spread further to the wheat ears, causing "withered white ears" during the wheat grain-filling period, and resulting in unfilled or even empty grains, which affects the number of grains per ear, grain weight, etc., and seriously reduces wheat yield.

[0004] Currently, the main methods for disease control include breeding disease-resistant varieties, chemical control, and biological control. However, the breeding of disease-resistant varieties faces many challenges; the research process is complex and time-consuming, and to date, no effective varieties resistant to stem rot have been discovered. Chemical control mainly relies on the spraying of chemical pesticides, but frequent and excessive use of chemical pesticides increases the resistance of pathogens, affecting the later control effects and even causing environmental pollution. Biological control, on the other hand, advocates using beneficial microorganisms to control pathogens, utilizing beneficial microorganisms and their metabolites to inhibit the growth of pathogens. It is effective in controlling wheat stem rot and has less impact on the natural environment, showing high application potential.

[0005] Rhizosphere microorganisms can directly or indirectly affect plant health through various pathways. These include directly inhibiting pathogenicity through resource competition, synthesizing antibiotics, or dissolving pathogen structures; and indirectly promoting plant health by activating host resistance gene expression and enhancing immunity. Isolating microorganisms from rhizosphere soil and developing their biocontrol functions has significant application value for the green control of wheat stem rot. Summary of the Invention

[0006] This invention screened a biocontrol strain—YC362-2-1—from wheat rhizosphere soil suspension. This strain has a significant effect on controlling wheat stem base rot caused by Fusarium graminearum.

[0007] On the one hand, the present invention provides a *Pseudomonas oryzae* strain, the preservation information of which is as follows:

[0008] Strain name: *Pseudomonas oryzae*; Category Naming: Pseudomonas oryzihabitans ; Strain number: YC362-2-1; The collection center received the document on April 13, 2026. Date of issuance of preservation certificate: April 27, 2026; Preservation institution: China General Microbiological Culture Collection Center (CGMCC); Accession number: CGMCC No. 38226; Address of the depository: Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Courtyard, Beichen West Road, Chaoyang District, Beijing.

[0009] Furthermore, the nucleotide sequence of the 16S rDNA of the *Pseudomonas oryzae* is shown in SEQ ID NO:1.

[0010] Furthermore, the single colonies of *Pseudomonas oryzae* are round, yellowish-white, with smooth, even edges, a moist and glossy surface, raised, and a creamy texture, consistent with... Pseudomonas oryzihabitans Typical morphological features.

[0011] On the other hand, a microbial agent is provided, wherein the microbial agent contains *Pseudomonas oryzae* as described in this invention.

[0012] Furthermore, the concentration of *Pseudomonas oryzae* in the microbial inoculant is 1 × 10⁻⁶. 1 cfu / mL -1×10 2 cfu / mL.

[0013] Furthermore, the active ingredient of the microbial agent is the cell body of *Pseudomonas oryzae*.

[0014] The invention also provides the application of *Pseudomonas oryzae* or the microbial agent described in this invention in inhibiting the growth of *Fusarium graminearum*.

[0015] The invention also provides the application of *Pseudomonas oryzae* or the microbial agent described in this invention in the prevention and control of wheat stem rot.

[0016] Furthermore, the wheat stem base rot is caused by *Fusarium graminearum*. Fusarium pseudograminearum cause.

[0017] Furthermore, the application period for the *Pseudomonas oryzae* or microbial inoculant is during the wheat seedling stage.

[0018] The invention also provides the application of the *Pseudomonas oryzae* or the microbial agent described herein in promoting wheat growth.

[0019] Compared with the prior art, the technical solution provided by the present invention has at least the following beneficial effects or advantages: (1) The *Pseudomonas oryzae* strain of the present invention is a biocontrol strain obtained by screening from wheat rhizosphere soil. It has good compatibility with the wheat rhizosphere microenvironment and has a significant inhibitory effect on *Fusarium graminearum*, the pathogen of wheat stem base rot. When it is made into a fungal agent and applied, it shows outstanding control effect on wheat stem base rot, with a relative control efficacy of 56.61%. It can significantly reduce the disease severity index, effectively delay the disease occurrence process, maintain the survival rate of wheat plants, and greatly reduce the damage of the disease to wheat.

[0020] (2) The *Pseudomonas oryzae* strain of the present invention has the ability to solubilize phosphorus, synthesize indole-3-acetic acid, secrete siderophores, and form biofilms. Compared with the normal growth of wheat under sterile conditions, the aboveground fresh weight and plant height of wheat plants are significantly increased under the condition of applying this fungal agent.

[0021] (3) The present invention’s Pseudomonas oryzae and its corresponding agents are environmentally friendly biological control resources, which fundamentally avoid the risks to agricultural product quality and safety and ecological environment caused by chemical pesticides, enrich the biocontrol bacteria resource bank for the control of wheat stem base rot, and make up for the lack of existing biocontrol bacteria resources.

[0022] (4) This invention provides a new and effective technical means for the green control of wheat stem base rot, improves the green control technology system for wheat stem base rot, and provides important microbial resource support for the sustainable development of the wheat industry. It has both theoretical significance and practical application value. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art 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.

[0024] Figure 1 The colony morphology of strain YC362-2-1 of this invention is shown in the image obtained by streaking culture on a TSA plate.

[0025] Figure 2 The phylogenetic tree for strain YC362-2-1 and its closely related species is constructed based on the 16S rDNA sequence.

[0026] Figure 3 This image shows the biocontrol effect of strain YC362-2-1 of this invention against wheat stem rot. CK is the blank control group; FP is the pathogen positive control group; YC362-2-1+FP is the experimental group; and YC362-2-1 is the strain-only treatment group.

[0027] Figure 4 This study evaluates the control efficacy of strain YC362-2-1 against wheat stem rot. A is a disease index statistical chart; B is a disease incidence statistical chart; C is the effect on plant height; D is the effect on aboveground fresh weight; E is the effect on underground fresh weight; CK is the blank control group; FP is the pathogen-positive control group; YC362-2-1+FP is the experimental group; YC362-2-1 is the strain-only treatment group; ns represents no significant (…). P >0.05); For significant ( P ≤0.05); Highly significant ( P ≤0.01); Extremely significant ( P ≤0.001).

[0028] Figure 5 The results of the growth-promoting characteristics determination of strain YC362-2-1 of this invention are shown. A represents the results of siderophore production capacity determination; B represents the results of phosphorus solubility determination; C represents the results of biofilm production determination; and D represents the results of IAA production capacity determination. CK represents the blank control group, and YC362-2-1 represents the strain-only treatment group.

[0029] Figure 6This invention presents the inhibitory phenotype of strain YC362-2-1 against *Fusarium graminearum* under iron-deficient co-culture conditions. A represents the control group, cultured on iron-deficient medium with only *Fusarium graminearum* WZ-8A inoculated; B represents the treatment of co-culturing YC362-2-1 and *Fusarium graminearum* WZ-8A on iron-deficient medium. Detailed Implementation

[0030] The technical solution of the present invention will be described below with reference to embodiments. However, the present invention is not limited to the following embodiments. Unless otherwise specified, the experimental methods and detection methods described in each embodiment are conventional methods; unless otherwise specified, the reagents and materials can be purchased commercially.

[0031] The experimental materials, reagents, and culture media used in the examples are summarized below: I. Experimental Materials *Pseudomonas oryzae*: isolated from wheat rhizosphere soil in October 2025.

[0032] Test pathogen: Fusarium graminearum ( Fusarium pseudograminearum WZ-8A, with accession number CGMCC No.28094, was provided by Professor Li Honglian of the College of Plant Protection, Henan Agricultural University in October 2023.

[0033] Test plant material: wheat variety Bainong 207 (a wheat variety susceptible to stem rot).

[0034] II. Culture media and related reagents (a) Culture medium Tryptone-Soybean Broth Medium (TSB): 15g tryptone, 5g soybean peptone, 5g sodium chloride, diluted with distilled water to 1000mL, pH 7.0-7.4.

[0035] Tryptone-Soybean Agar Medium (TSA medium): 15g tryptone, 5g soybean peptone, 5g sodium chloride, diluted to 1000mL with distilled water, 20g agar, pH 7.0-7.4.

[0036] Potato glucose agar medium (PDA medium): 200g peeled potatoes, 20g glucose, 20g agar powder, 1000mL distilled water, pH at natural value. The peeled potatoes are cut into small pieces, boiled for 20 minutes, and then filtered through 3 layers of gauze to obtain the filtrate as the medium component.

[0037] (ii) Reagents 50 μM PBS buffer (pH 7.0), sterile phosphate buffer, 25% glycerol, universal primers for bacterial 16S rDNA (27F, 1492R), 2× Rapid Taq Master Mix, 200 mg / L L-tryptophan, Salkowski colorimetric solution, 100 mg / L indole-3-acetic acid (IAA) standard.

[0038] Example 1: This example describes the separation and purification of YC362-2-1. 1.1 Culture medium All culture media used in the experiment were conventional culture media, and the specific formulas and preparation methods are as follows: Tryptone-Soybean Broth Medium (TSB): Weigh 15g of tryptone, 5g of soybean peptone, and 5g of sodium chloride, and bring the volume to 1000mL with distilled water. Adjust the pH to 7.0-7.4, sterilize, and set aside. Tryptone soybean agar medium (TSA medium): Based on the TSB medium formula, add 20g of agar, bring the volume to 1000mL with distilled water, adjust the pH to 7.0-7.4, sterilize, and then pour the plates for later use. Potato glucose agar medium (PDA medium): Cut 200g of peeled potatoes into small pieces and boil for 20 minutes. Filter the liquid through three layers of gauze and add 20g of glucose and 20g of agar powder to the liquid. Make up to 1000mL with distilled water, set the pH to the natural value, sterilize, and then pour the medium onto a plate for later use.

[0039] 1.2 Collection and processing of rhizosphere soil samples Rhizosphere soil samples were collected from the crop specimen area of ​​Northwest A&F University. This area is a dedicated nursery for wheat stem rot caused by artificial inoculation with *Fusarium graminearum*, and wheat stem rot has been prevalent for several years. Ten wheat varieties were originally planted at this site. Five wheat plants of each variety were randomly selected for sampling. During sampling, the entire wheat plant was dug up with a shovel, and large clumps of soil attached to the roots were removed by vigorous shaking. The above-ground stems were then cut off with sterilized scissors, and the remaining soil-bearing roots were placed in sealed bags, numbered, and placed in a low-temperature storage box before being quickly transported back to the laboratory. First, the wheat sample roots were rinsed with sterile water to remove loosely attached soil. Then, the wheat roots were cut off with sterilized scissors and transferred to 50 mL centrifuge tubes. 25 mL of sterile phosphate buffer was added to the tubes, and the samples were washed at 180 rpm for 15 minutes at room temperature on a shaker. This washing process was repeated three times. The resulting soil suspension was then centrifuged at 4654×g for 5 minutes, and the precipitate was collected and defined as a rhizosphere soil sample, which was then stored at 4℃ for later use.

[0040] 1.3 Isolation and Purification of Bacteria After mixing the rhizosphere soil samples in equal proportions, microbial isolation was performed using the dilution-spreading method. Ten samples were prepared separately. -1 10 -2 10 -3 10 -4 Four gradients of soil suspensions were evenly spread on TSA plates and incubated in the dark at 26°C for 2-4 days. After colonies grew, they were initially distinguished based on characteristics such as colony size, shape, color, transparency, and surface texture. Single colonies with significant morphological differences were selected and transferred to fresh TSA plates for purification. The purified strains were then stored at -80°C using 25% glycerol. Before subsequent experiments, the stored strains were inoculated onto TSA plates for activation and later use.

[0041] 1.4 Screening of biocontrol bacteria The tested pathogen was Fusarium graminearum WZ-8A.

[0042] Shaking inoculum: Pick a single colony of the purified bacteria obtained above and inoculate it into TSB liquid medium. Incubate with shaking at 28℃ and 180 rpm for 12 h. After the culture becomes turbid, centrifuge the culture at 4℃ and 4654×g for 5 minutes, discard the supernatant, and collect the bacterial precipitate. Resuspend the bacterial cells in 50 μM PBS buffer (pH 7.0) and measure their OD... 600 Adjust the value to 1.0 to obtain the required bacterial suspension for later use.

[0043] Preparation of pathogen inoculum: After cooking millet, it was placed in an Erlenmeyer flask and sterilized at 121℃ for 30 minutes to prepare millet culture medium. The activated WZ-8A pathogen was inoculated into the millet culture medium and cultured at 26℃ for 7 days. After the culture was completed, it was dried and ground in a clean bench to obtain the pathogen powder inoculum for later use.

[0044] Initial screening using pot experiments: After disinfecting the seeds of the wheat variety Bainong 207, which is susceptible to stem base rot, sow them into 120g of sterilized soil (12 seeds per pot), and then cover them with 20g of sterilized soil. Set up a treatment that simultaneously inoculates all strains and pathogens (B+FP) and a treatment that only inoculates the pathogens (FP), without repeating between treatments.

[0045] For the "B+FP" treatment, 25 mL of each treatment's bacterial suspension was inoculated into the soil around the seeds. Then, 0.5 g of pathogenic fungal powder and 0.8 g of wheat bran were evenly inoculated onto the soil surface. Finally, 20 g of sterilized soil was placed on top of the pathogenic fungal powder and wheat bran. Additionally, the treatment inoculated only with the pathogen (FP) served as a control, using an equal volume of sterile PBS buffer instead of the bacterial suspension. Disease severity was assessed on day 21 from sowing, and the plant disease index was calculated. Strains corresponding to treatments with 8 or more seedlings after inoculation and disease indices ranging from 0 to 20.00 were selected as potential biocontrol bacteria.

[0046] The initial screening results of biocontrol bacteria showed that the "B+FP" treatment inoculated with YC362-2-1 had a germination rate of more than 8 plants and a disease index of 11.11. Therefore, YC362-2-1 was selected as the subject of subsequent research.

[0047] Example 2: This example describes the identification and preservation of YC362-2-1. 2.1 Identification of biocontrol bacterial strains Identification of bacterial strain morphology: The bacteria YC362-2-1 obtained in Example 1 were inoculated onto TSA medium and streaked. After culturing in a constant temperature incubator at 26℃ for 2 days, the morphology of the colonies was observed. The shape, size, edge, color, transparency and surface texture of the colony edges were observed and recorded by the naked eye and photographed.

[0048] Colony morphology: Single colonies of YC362-2-1 were picked and streaked onto TSA agar plates and incubated at 28°C for 48 hours. Morphology was then observed. Figure 1 As shown, single colonies are round, yellowish-white, with neat and smooth edges, a moist and glossy surface, raised texture, and a creamy consistency.

[0049] Molecular biological identification of the strain: The species of strain YC362-2-1 was identified by 16S rDNA. YC362-2-1 bacterial culture was inoculated into TSB medium and cultured at 28℃ and 180 rpm for 12 h with shaking. This culture was used as a template. PCR amplification was performed using universal primers for bacterial 16S rDNA (27F 5'-AGAGTTTGATCCTGGCTCAG-3' and 1492R 5'-TACGGCTACCTTGTTACGACTT-3'). The PCR reaction volume (20 μL) is shown in Table 1. The target band was approximately 1500 bp.

[0050] Table 1

[0051] PCR reaction conditions: 95℃ pre-denaturation for 10 min; 95℃ denaturation for 20 s, 1 cycle; 56℃ annealing for 30 s; 72℃ extension for 1 min, 36 cycles; finally, 72℃ extension for 5 min.

[0052] The PCR products were detected by 1% agarose gel electrophoresis, and the fragment was approximately 1500 bp compared with the DL 2000 bp marker. The amplified products were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The obtained gene sequences were BLAST-aligned in the NCBI nucleic acid database to determine the species classification of strain YC362-2-1. A phylogenetic tree was constructed using the Neighbor-Joining method in Mega11.0 software.

[0053] Phylogenetic analysis based on 16S rDNA showed that strain YC362-2-1 exhibited 100% expansion support compared to the reference strain. Pseudomonas oryzihabitans Clustered together, the clustering results are as follows Figure 2 As shown, based on morphological and molecular biological identification, YC362-2-1 was determined to be... Pseudomonas oryzihabitans .

[0054] In this embodiment, the identification criteria for strain YC362-2-1 are as follows: (1) its 16S rRNA gene sequence is similar to... Pseudomonas oryzihabitans The sequence of the model strain was compared with the NCBI nucleic acid database by BLAST and the similarity was as high as 99.86%, far exceeding the species threshold (usually ≥99.5%); (2) Phylogenetic analysis showed that the two formed a monophyletic branch with high support. Therefore, YC362-2-1 was determined to belong to Pseudomonas The genus designation is equivalent to identifying it as the only valid species in that genus at present. Pseudomonas oryzihabitans Its 16S rDNA nucleotide sequence is shown in SEQ ID NO:1, totaling 1435 bp, as follows:

[0055] 2.2 Preservation of microbial strains The isolated and purified YC362-2-1 strain was inoculated onto freshly prepared TSA slant medium. This strain was deposited at the China General Microbiological Culture Collection Center on April 13, 2026, with accession number CGMCC No. 38226.

[0056] Example 3: Evaluation of the biocontrol effect of YC362-2-1 against wheat stem rot 3.1 Evaluation of potted plant control efficacy Preparation of pathogen inoculum: After cooking millet, it was placed in an Erlenmeyer flask and sterilized at 121℃ for 30 minutes to prepare millet culture medium. The activated WZ-8A pathogen was inoculated into the millet culture medium and cultured at 26℃ for 7 days. After the culture was completed, it was dried and ground in a clean bench to obtain the pathogen powder inoculum for later use.

[0057] Preparation of YC362-2-1 bacterial suspension: The method is the same as the shaking method in Example 1, 1.4.

[0058] Pot experiment to verify the control efficacy: After disinfecting wheat seeds of the Bainong 207 variety, sow 12 seeds per pot into 120g of sterilized soil, cover with 20g of sterilized soil, then inoculate with 25mL of YC362-2-1 bacterial suspension around the seeds, and evenly inoculate the soil surface with 0.5g of pathogen powder inoculum and 0.8g of wheat bran, and finally cover with 20g of sterilized soil.

[0059] This embodiment sets up the following four treatment groups: Blank control group (CK): sterile PBS was used instead of YC362-2-1 bacterial suspension, and no pathogen was inoculated; Strain-only treatment group (YC362-2-1): inoculated with YC362-2-1 bacterial suspension, but no pathogen was inoculated; Experimental group (YC362-2-1+FP): inoculated with YC362-2-1 bacterial suspension and pathogen; Pathogen positive control group (FP): sterile PBS was used instead of YC362-2-1 bacterial suspension, and only pathogen was inoculated. Each treatment was replicated in 3 pots.

[0060] For the experimental group (YC362-2-1+FP) and the pathogen-positive control group (FP), the incidence and survival rates of each treatment were recorded every two days starting from the 5th day after sowing. On the 21st day, the disease severity was assessed, and the plant disease index and control effect were calculated. For the blank control group (CK) and the strain-only treatment group (YC362-2-1), intact plants were collected on the 21st day. They were gently rinsed with running water to remove all soil particles, then allowed to air dry in a cool place until there was no obvious surface moisture. The plant height was measured, and the plants were precisely divided into above-ground parts and underground root systems using tools, and their fresh weights were measured separately.

[0061] Disease Index (DI) = 100 × ∑ (value of each disease level × number of plants at each disease level) / (total number of plants × 9) Prevention and control effect (%) = (disease index of positive control group of pathogen - disease index of experimental group) / disease index of positive control group of pathogen × 100.

[0062] Greenhouse pot experiments confirmed that YC362-2-1 has a preventive effect against wheat stem rot. For example... Figure 4 China A and Figure 4 As shown in Figure B, compared with the FP treatment group, the YC362-2-1+FP treatment significantly delayed the onset of disease, and its disease index was significantly lower than that of the FP treatment group. Figure 3 As shown, on day 21, the plant growth status of the YC362-2-1+FP treatment group was significantly better than that of the FP treatment group. According to the data in Table 2, the relative control efficacy of the YC362-2-1 treatment reached 56.61%. The above results indicate that the strain YC362-2-1 of this invention can effectively control the development of wheat stem rot and significantly reduce plant mortality.

[0063] Table 2. Control efficacy of YC362-2-1 against wheat stem rot.

[0064] Note: Different letters (a and b) indicate that there is a significant difference between the two groups.

[0065] Evaluation of plant growth promotion effect: The results of plant height, aboveground fresh weight, and underground fresh weight measurements of the blank control group (CK) and the strain-only treatment group (YC362-2-1) are as follows: Figure 4 Figures C, D, and E are shown in Table 3. Compared with the control group (CK), wheat treated with YC362-2-1 showed significantly higher plant height and aboveground fresh weight. These results indicate that the strain YC362-2-1 of this invention has no adverse effects on wheat growth and can significantly promote plant growth.

[0066] Table 3 Effects of YC362-2-1 on wheat growth indicators

[0067] Note: Different letters (a and b) indicate that there is a significant difference between the two groups; the same letter (a and a) indicates that there is no significant difference between the two groups.

[0068] Example 4: Preliminary study on the antibacterial mechanism of YC362-2-1 against Fusarium oxysporum. 4.1 Determination of IAA Production Capacity of YC362-2-1 Prepare and sterilize LB liquid medium containing 200 mg / L L-tryptophan. Inoculate the purified YC362-2-1 strain into this medium and culture it in a shaker at 28℃ and 180 rpm for 2 days. After the culture is completed, centrifuge the bacterial solution at 4℃ and 4000 r / min for 10 min and collect the supernatant for later use. Add 100 μL of the supernatant to a 96-well cell culture plate and add an equal volume of Salkowski colorimetric solution. Incubate in the dark for 30 min for colorimetric reaction. Use 100 mg / L standard IAA solution as a positive control and uninoculated LB medium supernatant containing L-tryptophan as a blank control. After the colorimetric reaction is completed, use a spectrophotometer to measure the absorbance of the colorimetric solution in each well at a wavelength of 530 nm. Calculate the actual IAA yield of strain YC362-2-1 based on the pre-plotted IAA standard curve.

[0069] The results are as follows Figure 5 As shown in Figure D, this indicates that the strain YC362-2-1 of the present invention has the ability to produce IAA.

[0070] 4.2 Determination of the iron-producing capacity of YC362-2-1 Culture medium: Ferrophilic assay medium (CAS solid medium): Modified CAS liquid medium / ferophilic assay liquid medium (kit) produced by Beijing Coollab Technology Co., Ltd. was used. 121g of basal medium and 15g of agar were added, and the volume was adjusted to 800mL with purified water. The solution was dissolved by stirring with a glass rod. The medium was sterilized at 115℃ for 20min, cooled to 50-60℃, and 100mL of 10× buffer solution (preheated to 60℃) and CAS assay solution were slowly added to the basal medium. Plates were then prepared for use.

[0071] Iron-deficient King's B solid medium: 20g tryptone, 1.5g dipotassium hydrogen phosphate, 1.5g magnesium sulfate heptahydrate, 10mL glycerol, 15g agar, add pure water to a final volume of 1L.

[0072] Iron-deficient King's B liquid medium: 20g tryptone, 1.5g dipotassium hydrogen phosphate, 1.5g magnesium sulfate heptahydrate, 10mL glycerol, add pure water to a final volume of 1L.

[0073] Siderophore production capacity determination: YC362-2-1 strain was inoculated into iron-deficient King's B liquid medium and shaken. 3 μL of bacterial suspension was dropped 2 cm from the center of a CAS solid medium plate. This was repeated three times. An equal volume of iron-deficient King's B liquid medium was used as the control (CK). The culture was incubated at 28℃ for 3 days. An orange-red halo appeared around the YC362-2-1 colonies in the CK, while no halo was observed. The results are as follows: Figure 5 As shown in Figure A, this indicates that the strain YC362-2-1 of the present invention has the ability to secrete siderophores.

[0074] Phosphate-solubilizing capacity determination: YC362-2-1 was inoculated into sterile TSB medium, shaken overnight at 28℃ and 200 r / min, and the OD of the bacterial solution was adjusted. 600 The value was 1.0. 2 μL of bacterial culture was inoculated onto Pikovskaya agar medium. The plates were incubated at 28°C for 7 days. A clear halo was observed around the YC362-2-1 colonies in the medium, while no halo was observed in the CK colonies. The results are as follows: Figure 5 As shown in Figure B, this indicates that the strain YC362-2-1 of the present invention possesses phosphate-solubilizing ability.

[0075] Biofilm formation capacity assay: Strain YC362-2-1 was cultured in TSB liquid medium until OD500. 600 The value was 0.6. The bacterial cells were collected by centrifugation, washed with sterile water, and then suspended in biofilm formation medium (MSgg medium) to allow the final OD value to reach 0.6. 600 The value was 1.0. 10 μL of each bacterial suspension was added to 1 mL of MSG G1 medium, placed in a 48-well cell culture plate, and incubated statically at 28°C for 72 h. A film-like substance was observed on the surface of the medium. The results are as follows: Figure 5 As shown in C, this indicates that the strain YC362-2-1 of the present invention has the ability to produce biofilm.

[0076] Co-culture on iron-deficient medium: pathogen (Fusarium pseudogranatum WZ) 8A) After activation, inoculate the strain YC362-2-1 into the center of an iron-deficient King's B solid medium plate. Inoculate the strain YC362-2-1 into an iron-deficient King's B liquid medium and incubate. Place 3 μL of YC362-2-1 bacterial suspension dropwise on either side, 2 cm away from the pathogen. Use the pathogen-only treatment as a control. Incubate at 28°C for 7 days and test the siderophore production capacity of each treatment. The results are as follows: Figure 6 As shown in Figures A and B, a yellow halo appears in the YC362-2-1 treatment area, and *Fusarium graminearum* WZ... The significantly reduced mycelial growth rate of strain 8A indicates that strain YC362-2-1 of this invention may inhibit the growth of Fusarium graminearum by efficiently chelating trace amounts of iron in the environment through the secretion of siderophores.

[0077] As described above, the basic principles, main features, and advantages of the present invention have been well described. The above embodiments and specifications are merely descriptions of preferred embodiments of the present invention, and the present invention is not limited to the above embodiments. Various changes and improvements made to the technical solutions of the present invention by those skilled in the art without departing from the spirit and scope of the present invention should fall within the protection scope defined by the present invention.

Claims

1. *Pseudomonas oryzae* ( Pseudomonas oryzihabitans Its application in inhibiting the growth of *Fusarium graminearum*, preventing wheat stem rot, and promoting wheat growth is characterized by: The *Pseudomonas oryzae* strain is YC362-2-1, and it is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 38226.

2. The application according to claim 1, characterized in that, The nucleotide sequence of the 16S rDNA of *Pseudomonas oryzae* is shown in SEQ ID NO:

1.

3. The application according to claim 1, characterized in that, The wheat stem base rot is caused by Fusarium graminearum.

4. The application according to claim 1, characterized in that, The application period for the *Pseudomonas oryzae* is during the wheat seedling stage.

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