A strain of paraoxidizing microbacterium and its application

Abstract

This invention belongs to the field of microbiology, specifically disclosing a strain of *Mcrobacterium paraoxydans* and its applications. The *Mcrobacterium paraoxydans* strain TYQ7 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 27216. The applicant discovered that the aforementioned *Mcrobacterium paraoxydans* can secrete auxin, promoting the growth of cucumber seedlings. It also has the ability to interfere with the directional migration of nematodes and kill nematodes, significantly alleviating root-knot nematode stress. Furthermore, it was found to inhibit the normal growth of *Fusarium oxysporum*, and further exhibits the ability to degrade vegetable straw and solubilize phosphorus. Therefore, the applicant believes that the aforementioned *Mcrobacterium paraoxydans* has significant application prospects in the fields of agriculture and environmental protection.

Description

Technical Field

[0001] This invention belongs to the field of microbiology, and specifically discloses a strain of paraoxidizing microbacterium and its applications. Background Technology

[0002] Plant rhizosphere growth promoters (PGPRs) play a crucial role in the pursuit of sustainable development and high-efficiency production in modern agriculture. Through various mechanisms, such as nitrogen fixation, phosphorus solubilization, and potassium solubilization, PGPRs increase the content of plant-available nutrients in the soil, thereby significantly promoting plant growth. They also secrete various plant hormones, such as auxins, which regulate plant physiological processes, stimulate root development, enhance the plant's ability to absorb water and nutrients, and improve crop yield and quality. Therefore, exploring and utilizing highly efficient PGPR resources is of great significance for improving agricultural production efficiency, reducing the use of chemical fertilizers, and ensuring the safety of agricultural products and the health of the ecological environment.

[0003] Root-knot nematodes (Meloidogyne) and Fusarium oxysporum are two highly destructive plant pathogens that seriously threaten the safe production of crops worldwide. Root-knot nematodes invade plant roots, forming root knots that hinder the absorption of water and nutrients, leading to weakened and stunted plant growth, and in severe cases, even death of the entire plant. Fusarium oxysporum can cause wilt diseases in various plants, rapidly multiplying under suitable conditions, damaging the plant's vascular system, and causing the plant to wilt due to dehydration. Currently, chemical agents are mainly used for control of these two pathogens, but problems such as residual toxicity, poor environmental compatibility, and the risk of drug resistance are becoming increasingly prominent. Although some research has attempted to use antagonistic microorganisms for biological control, most strains have limitations such as unstable efficacy, single target, or unclear interaction mechanisms with plants, making it difficult to meet the needs of sustainable agricultural development.

[0004] Crop straw is a significant byproduct of agricultural production. Traditional straw disposal methods, such as burning, not only waste resources but also generate large amounts of smoke and dust, polluting the atmosphere. The proper degradation of straw and its conversion into organic fertilizer is a crucial step in achieving agricultural ecological cycles. Microorganisms with highly efficient straw-degrading capabilities can break down complex organic matter in straw, such as cellulose, hemicellulose, and lignin, into smaller molecules, such as humic substances, amino acids, and sugars. These substances can improve soil fertility, enhance soil structure, promote the growth and reproduction of soil microorganisms, and strengthen the soil's water and fertilizer retention capacity, creating a favorable soil environment for crop growth.

[0005] While some microorganisms are already being used in agriculture, strains possessing multiple desirable properties simultaneously, such as promoting plant growth, biological control, straw degradation, soil nutrient release, enhancing plant stress resistance, and exhibiting strong adaptability, remain relatively scarce. Therefore, developing and utilizing multifunctional microbial strains has significant practical importance and broad application prospects. The *Parasitic Microbacterium paraoxidans* strain provided in this invention exhibits excellent performance in multiple aspects, and is expected to bring new breakthroughs to agricultural production. Summary of the Invention

[0006] To address the aforementioned problems, this invention discloses a strain of paraoxidizing microbacterium and its applications.

[0007] To achieve the above-mentioned objectives, the technical solution of the present invention is as follows:

[0008] A strain of Microbacterium paraoxydans, strain TYQ7, is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 27216.

[0009] The present invention also discloses a microbial agent containing the above-mentioned *Microbacterium paraoxidans* cells and / or its metabolites and / or its fermentation broth.

[0010] The present invention also discloses the use of the above-mentioned bacteria or bacterial agents in the preparation of formulations that enhance the growth of cucumber seedlings.

[0011] The present invention also discloses the use of the above-mentioned bacteria or bacterial agents in the preparation of formulations that interfere with the migration of southern root-knot nematodes.

[0012] The present invention also discloses the use of the above-mentioned bacteria or bacterial agents in the preparation of formulations for controlling southern root-knot nematodes.

[0013] The present invention also discloses the use of the above-mentioned bacteria or bacterial agents in the preparation of formulations that inhibit the growth of Fusarium oxysporum.

[0014] The present invention also discloses the use of the above-mentioned bacteria or bacterial agents in the preparation of phosphorus-soluble formulations.

[0015] The present invention also discloses the use of the above-mentioned bacteria or bacterial agents in the preparation of straw degradation formulations.

[0016] The present invention has the following beneficial effects:

[0017] This invention discloses a strain of *Pseudomonas krusei* and its applications. The *Pseudomonas paraoxidans* TYQ7 strain disclosed in this invention exhibits growth-promoting properties, including the ability to dissolve inorganic phosphorus and secrete auxin. TYQ7 can utilize tryptophan to produce IAA, with a measured content of 43.98 μg / mL. Furthermore, it significantly promotes cucumber growth, increasing aboveground dry weight by 32.18%, root volume by 64.10%, and seedling vigor index by 36.29%. In addition, TYQ7 shows significant effects on nematode migration and mortality, especially on the lethality of second-instar larvae of root-knot nematodes, where its effect is significantly superior to the control strain. TYQ7 also significantly alleviates... The effects of TYQ7 on root-knot nematode stress showed that, compared with the same root-knot nematode infection, the aboveground and underground dry weight of cucumber increased by 23.81% and 28.89%, respectively, the number of root knots per unit root weight decreased by 40.57%, and the chlorophyll content increased by 160.54%. It also showed strong antagonistic effects in the Fusarium oxysporum growth inhibition experiment. Within two weeks, TYQ7 achieved degradation rates of 66.14%, 37.72%, 26.23%, and 30.18% on tomato, cucumber, pepper, and eggplant straw, respectively. Biolog analysis showed that it could utilize 71 carbon sources and exhibited resistance to nalidixic acid, lithium chloride, and aztreonam, demonstrating broad-spectrum metabolic capacity and environmental adaptability. In conclusion, the TYQ7 strain shows good application potential in promoting plant growth, enhancing plant stress resistance, soil improvement, and environmental remediation.

[0018] The preservation information for the strain is as follows:

[0019] Name of the depository: China General Microbiological Culture Collection Center, China Committee on the Preservation and Management of Microbial Cultures;

[0020] Address of the depository: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing;

[0021] Deposit date: April 27, 2023

[0022] Accession number CGMCC No. 27216

[0023] Taxonomic name: Microbacterium paraoxydans. Attached Figure Description

[0024] Figure 1 The colony morphology of TYQ7 is as follows;

[0025] Figure 2 Phylogenetic tree of TYQ7:

[0026] Figure 3 Effects of TYQ7 inoculation on cucumber plants and roots (image).

[0027] Figure 4 A diagram illustrating the effect of auxin production in TYQ7;

[0028] Figure 5 A schematic diagram of the tactic migration experiment of southern root-knot nematodes after TYQ7 inoculation;

[0029] Figure 6 Figure showing the effect of TYQ7 inoculation on the directional migration of southern root-knot nematodes;

[0030] Figure 7 The effect of TYQ7 inoculation on the directional migration of southern root-knot nematodes is shown in the figure.

[0031] Figure 8 Figure 1 shows the effect of TYQ7 fermentation supernatant stock solution on the in vitro contact killing effect of Southern root-knot nematode.

[0032] Figure 9 This is a diagram showing the in vitro contact killing effect of TYQ7 fermentation supernatant on Southern root-knot nematodes.

[0033] Figure 10 Effect of TYQ7 inoculation on cucumber plants and roots infected with Southern root-knot nematodes (image).

[0034] Figure 11 A graph showing the antagonistic effect of TYQ7 and Fusarium oxysporum on agar plates;

[0035] Figure 12 The graph shows the degradation effect of TYQ7 on four types of vegetable straw.

[0036] Figure 13 The phosphorus solubility characteristics of TYQ7 are shown in the diagram.

[0037] Figure 14 The graph shows the phosphorus solubility of TYQ7 under gradient salt concentrations and gradient pH levels.

[0038] Figure 15 This is a fingerprint of TYQ7's ability to utilize different carbon sources. Detailed Implementation

[0039] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Unless otherwise specified, the following embodiments and features can be combined with each other.

[0040] The detailed description of the embodiments of the present invention provided below is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments of the invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0041] Example 1

[0042] 1.1 Isolation and purification of Microbacterium paraoxydans TYQ7:

[0043] The *Microbacterium paraoxydans* strain TYQ7 of this invention was isolated from the rhizosphere soil of cucumbers infected with root-knot nematodes in the laboratory of China Agricultural University. The isolation method was the soil dilution method. The specific steps are as follows:

[0044] Four 1g mixed soil samples were collected from the rhizosphere of cucumbers infected with root-knot nematodes in the greenhouse vegetable laboratory of China Agricultural University using the soil dilution method. Each sample was placed in a 50ml centrifuge tube containing 9ml of sterile water and shaken at 180rpm for 20 minutes. After serial dilution, 100μL of each sample was plated onto LB agar plates and incubated at 28℃ under light for 2 days. Single colonies were picked and subjected to three streak purification processes.

[0045] 1.2 Identification of Microbacterium paraoxydans TYQ7 strain

[0046] (1) Microbiological characteristics

[0047] The obtained strain was inoculated onto LB agar plates and incubated at 28°C for 2 days. Colony morphology is as follows: Figure 1 As shown, *Microbacterium paraoxidans* colonies are round, pale yellow, and opaque. The surface is smooth and moist, with irregular edges. The central area is slightly raised, and a biofilm naturally forms around the single colony. Gram staining is positive.

[0048] (2) Molecular biological characteristics

[0049] Single colonies were picked and placed in a 1.5 mL centrifuge tube containing 1 mL of LB medium. The culture was shaken at 28 °C and 180 rpm for 24 h. Using the bacterial culture as a template, the 16S rRNA sequence was amplified using primers 27F and 1492R.

[0050] The PCR amplification reaction system consisted of 50 μL of 2xTaq enzyme, 1 μL of 27F primer, 1 μL of 1492R primer, 1 μL of bacterial culture, and 22 μL of ddH2O. The amplification conditions were: 95°C pre-denaturation for 3 min, 94°C denaturation for 25 s, 55°C annealing for 25 s, 72°C extension for 1 min, 32 cycles, followed by a final extension at 72°C for 5 min. The amplified products were stored at 4°C. The amplified products were separated and identified by 1% agarose gel electrophoresis, and the PCR products were sent to Beijing Qingke Biotechnology Co., Ltd. for bidirectional sequencing. The 16S rDNA of strain TYQ7 is shown in SEQ ID NO: 1 below:

[0051]

[0052]

[0053] BLAST homology comparison of the test results showed that this strain had a similarity of 99.86% to *Microbacterium paraoxydans*. Therefore, the TYQ7 phylogenetic tree... Figure 2 The strain identified is a strain of the genus Microbacterium, named Microbacterium paraoxydans TYQ7, and deposited at the China General Microbiological Culture Collection Center with accession number CGMCC.27216.

[0054] Example 2

[0055] The growth-promoting effect of TYQ7 on cucumbers

[0056] Using cucumber as the test material, a pot experiment was conducted to determine the ability of TYQ7 to promote plant growth. The treatments in this example were: ① Conventional control (CK); ② Inoculation with TYQ7 inoculant (TYQ7). The specific implementation steps are as follows:

[0057] The specific implementation steps are as follows:

[0058] 2.1 Seed disinfection

[0059] Select plump cucumber seeds without mechanical damage, and perform surface disinfection according to the following procedure:

[0060] (1) Soak in 75% ethanol for 30 seconds, then rinse with sterile distilled water 3 times;

[0061] (2) Transfer to 3% NaClO solution and soak for 10 minutes, then rinse with sterile distilled water until no residue remains;

[0062] (3) After soaking the seed in sterile water for 6 hours, place it in a petri dish lined with sterile moist filter paper;

[0063] (4) Germination treatment was carried out in a constant temperature incubator at 28℃.

[0064] 2.2 Inoculation of strains

[0065] Sterilized soil preparation

[0066] After collecting and sieving forest soil, it is sterilized at 120℃ for 1 hour, and then sterilized again after an interval of 24 hours.

[0067] Preparation of bacterial suspension

[0068] The TYQ7 strain was inoculated into LB liquid medium and cultured at 28°C with shaking at 180 rpm until the OD600 reached 0.8. The bacterial cells were collected by centrifugation at 8000 rpm for 5 min and resuspended in an equal volume of 10 mM MgSO4 buffer to prepare the bacterial agent.

[0069] Vaccination procedure

[0070] After the radicle germinates, the seedlings are sown in 50-cell trays filled with sterilized soil. Treatment begins when the first true leaf appears: each seedling is watered with 10 mL of bacterial suspension, once a week for 3 consecutive weeks.

[0071] 2.3 Plant Measurement

[0072] Samples were taken 21 days after treatment (n=20), and the following parameters were measured:

[0073] (1) Morphological indicators: Stem diameter (mm) was measured with vernier calipers, and plant height (cm) was measured with a ruler.

[0074] (2) Biomass: Fresh weight of aboveground / belowground parts was measured using a percentile balance.

[0075] (3) Dry matter: After drying at 65℃ to constant weight, the dry weight of each part was measured.

[0076] (4) Root analysis: After scanning with a root scanner, the WinRHIZO system analyzes the total root length, root surface area, average root diameter, and root volume.

[0077] The seedling quality index (SQI) is calculated using the following formula.

[0078] SQI = TDW / (PH / SD + SDW / RDW)

[0079] TDW, SDW, and RDW represent the total dry weight (g), aboveground dry weight (g), and underground dry weight (g), respectively, while PH (cm) and SD (mm) represent the plant height and stem diameter, respectively.

[0080] 2.4 Results Analysis

[0081] The effects of TYQ7 inoculation on cucumber seedling growth indicators and root system are shown in Tables 1-2. The effects of TYQ7 inoculation on cucumber plants and root system are shown in Tables 1-2. Figure 3 The chart shows:

[0082] (1) Stem diameter development: The TYQ7 treatment group showed a significant increase of 9.47% compared to the control group.

[0083] (2) Dry matter accumulation: The dry weight of the aboveground parts and the dry weight of the whole plant increased by 32.18% and 27.7%, respectively.

[0084] (3) Root development: Total root length, root surface area, average root diameter, and root volume increased by 2.66%, 29.40%, 26.31%, and 64.10%, respectively.

[0085] (4) Seedling vigor index: The TYQ7 treatment group showed a 36.29% increase compared to the control group.

[0086] This indicates that TYQ7 has a significant effect on promoting growth and strengthening seedlings in cucumbers.

[0087] Table 1. Effects of TYQ7 inoculation on cucumber seedling growth indicators.

[0088]

[0089] Table 2 Effects of TYQ7 inoculation on cucumber seedling root system

[0090]

[0091] Example 3

[0092] The Salkowski colorimetric method was used to qualitatively and quantitatively analyze the ability of the strain to produce indoleacetic acid (IAA). The specific procedure is as follows:

[0093] 3.1 Qualitative Detection

[0094] 3.1.1 Preparation of colorimetric reagent

[0095] Prepare the Salkowski colorimetric reagent according to the following steps:

[0096] (1) 0.5M FeCl3 solution: 0.811g FeCl3 dissolved in 10mL sterile distilled water;

[0097] (2) Working solution: Take 1 mL of 0.5 M FeCl3 solution and mix it with 49 mL of 35% HClO4. Mix thoroughly and set aside.

[0098] 3.1.2 Bacterial solution treatment

[0099] (1) Activation of bacterial strain: Pick a single colony and inoculate it into 30 mL of LB medium, and culture at 28℃ and 180 rpm with shaking until OD600 = 0.8;

[0100] (2) Induction culture: The bacterial culture was transferred to LB medium containing 500 μg / mL filtered tryptophan at an inoculation rate of 1%, and the uninoculated medium was used as a blank control. The culture was carried out at 28℃ and 180 rpm for 72 h.

[0101] (3) Centrifuge and collect the supernatant: Centrifuge at 10,000 rpm for 10 min and collect the sterile supernatant.

[0102] 3.1.3 Colorimetric Reaction

[0103] Mix 500 μL of the supernatant with 1 mL of the colorimetric reagent and let stand at room temperature for 30 min. See the image showing the auxin production effect of TYQ7. Figure 4 The solution is pink, indicating the presence of IAA.

[0104] 3.2 Quantitative Analysis

[0105] 3.2.1 Establishment of Standard Curve

[0106] (1) Preparation of stock solution: 100 mg / L IAA standard solution;

[0107] (2) Gradient dilution: 0, 10, 20, 30, 40, 50, 60, 80, 100 mg / L series concentrations;

[0108] (3) Standard determination: OD values ​​of each concentration were measured at a wavelength of 530 nm to establish a concentration-absorbance standard curve.

[0109] 3.2.2 Sample Testing

[0110] (1) Reaction system: 500 μL of bacterial supernatant + 4 mL of colorimetric reagent, react in the dark for 30 min;

[0111] (2) Absorption measurement: The OD value of the sample was measured at 530 nm, and a blank control was used for calibration;

[0112] (3) Concentration calculation: IAA content was converted using the standard curve, and the experiment was repeated 3 times.

[0113] 3.3 Experimental Results

[0114] Quantitative analysis showed that the TYQ7 strain could secrete IAA in tryptophan-containing medium, with a yield of 43.98 μg / mL.

[0115] Example 4

[0116] Effects of fermentation supernatant of *Microbacterium paraoxidans* TYQ7 on the directional migration of root-knot nematodes.

[0117] 4.1 Preparation of bacterial suspension

[0118] Refer to the process for preparing bacterial suspension in Example 2.

[0119] 4.2 Cucumber root treatment

[0120] (1) Seed disinfection: Refer to the disinfection procedure in Example 2;

[0121] (2) Cucumber cultivation: After germination, sow the seeds. During the cotyledon expansion period (5-7 days), irrigate with 10 mL of bacterial suspension. When the cotyledons are two leaves and one heart, take the roots for experiments.

[0122] 4.3 Acquisition of Second Instar Larvae

[0123] (1) Egg mass separation: Egg masses were picked from the roots of infected water spinach and washed with 0.6% NaClO by shaking;

[0124] (2) Aseptic incubation: After filtration through a 600-mesh sieve, the egg mass is placed in sterile water containing 0.004 mg / mL gentamicin and incubated at room temperature for 2-3 days;

[0125] (3) Density adjustment: After counting under a microscope, adjust to a 100 larvae / 100μL larval suspension.

[0126] Preparation of 4.423% Pluronic F-127 gel

[0127] Dissolve 23g of powder in 80mL of sterile water at 4℃, stir magnetically until completely dissolved, and store at 4℃ for later use.

[0128] 4.5 Tactical Migration Experiment

[0129] (1) Experimental grouping: A control group without bacterial inoculation (CK) and a TYQ7 treatment group were set up, with 20 replicates for each group;

[0130] (2) Migration apparatus: The roots of the control group (CK) and the treatment group were placed on both sides of the gel plate, and a 0.5 cm diameter Oxford cup hole was pre-placed in the center. The schematic diagram of the experiment is shown below. Figure 5 As shown;

[0131] (3) Nematode inoculation: Add 100 μL of nematode suspension (about 100 nematodes) to the holes and let stand in the dark for 12 hours;

[0132] (4) Data collection: The number of migrating nematodes on both sides was counted under a microscope, and the migration percentage was calculated.

[0133] 4.6 Results Analysis

[0134] The effects and efficacy of TYQ7 inoculation on the directional migration of southern root-knot nematodes are shown in [the table below]. Figure 6 , Figure 7The directional migration results showed that the application of TYQ7 alone significantly affected the directional migration of nematodes. The percentage of nematodes migrating to the root tips of cucumbers in the control group (CK group) reached 5.359%, significantly higher than that on the cucumber root side where TYQ7 was present. This demonstrated a significant influence on the directional migration of nematodes.

[0135] Example 5

[0136] In vitro contact toxicity of root-knot nematodes by fermentation supernatant of *Microbacterium paraoxidans* TYQ7.

[0137] 5.1 Preparation of fermentation broth supernatant

[0138] The tested strain was *Microbacterium paraoxidans* TYQ7, and the bacterial suspension was the same as in Example 2.

[0139] After the bacterial suspension was completely prepared, it was centrifuged at 10,000 rpm for 5 minutes. The supernatant in the centrifuge tube was drawn with a syringe and filtered through a 0.22 μm filter membrane. The solution obtained after filtration is the supernatant of the fermentation broth of Microbacterium paraoxidans TYQ7.

[0140] 5.2 In vitro contact test

[0141] Add 400 μL of fermentation supernatant of TYQ7 and control strains to each well of a 48-well cell culture plate. Add 100 μL of root-knot nematode suspension containing 100 second-instar larvae to each well. The control group is replaced with 400 μL of sterile water.

[0142] After being protected from light, the cells were placed in a 28°C incubator and incubated statically. Survival was observed using a stereomicroscope after 12 hours and 24 hours. The criteria for survival were: after adding 1 mol / L NaOH solution, cells that remained rigid were considered dead, while those that showed movement were considered alive. The number of dead cells was counted, and the corrected mortality rate was calculated.

[0143]

[0144] 5.3 Results Analysis

[0145] The effects and efficacy of TYQ7 fermentation supernatant on the in vitro contact killing effect of southern root-knot nematodes are described in [the original text]. Figure 8 and Figure 9 The results showed that the supernatant of TYQ7 fermentation broth had a significant lethal effect on second-instar larvae of root-knot nematodes, with a corrected mortality rate significantly higher than that of the control group. Over time, the mortality rate increased from 70.31% at 12 h to 83.67% at 24 h, indicating that its metabolites have a highly efficient and sustained lethal effect on second-instar larvae of southern root-knot nematodes.

[0146] Example 6

[0147] Pot experiment on the control of southern root-knot nematodes by Microbacterium paraoxidans TYQ7.

[0148] Cucumber is the vegetable crop most susceptible to root-knot nematodes in southern China. This study uses cucumber as the model crop.

[0149] The treatment setup in this embodiment is as follows: ① Inoculation with root-knot nematodes (CK+N); ② Inoculation with TYQ7 inoculant (TYQ7+N). The specific implementation steps are as follows:

[0150] 6.1 Seed disinfection

[0151] The specific procedure for seed disinfection is the same as in Example 2.

[0152] 6.2 Inoculation of strains

[0153] The specific procedures for inoculating the strain are the same as in Example 2.

[0154] 6.3 Inoculation with second-instar larvae of the southern root-knot nematode

[0155] When cucumber seedlings are in the one-leaf-one-heart stage, begin inoculation with second-instar larvae of the southern root-knot nematode. Use a syringe to poke two small holes about 1 cm deep around the roots of the cucumber seedling, add a suspension of root-knot nematodes into them, and inoculate each seedling with 250 root-knot nematodes.

[0156] 6.4 Plant Measurement

[0157] On day 21 after nematode inoculation, samples were taken to measure and statistically analyze the growth indicators of cucumber plants (plant height, stem diameter, above-ground fresh weight, underground fresh weight, above-ground dry weight, underground dry weight, and chlorophyll content). The root system of cucumber seedlings was scanned using a root scanner and analyzed using the WinRHIZO analysis system software.

[0158] 6.5 Results Analysis

[0159] Tables 3 and 4 show the effects of TYQ7 inoculation on cucumber growth indicators and root system under southern root-knot nematode infection. Figure 10 The effect of TYQ7 inoculation on cucumber plants and roots infected with southern root-knot nematodes is shown in the figure. Figure 10 The results in Tables 3 and 4 show that inoculation with the TYQ7 strain significantly promoted the growth and development of cucumber plants infected with southern root-knot nematodes. Plant height and stem diameter increased by 30.80% and 0.63% respectively compared to the control; aboveground and underground dry weight increased by 23.81% and 28.89% respectively; and chlorophyll content increased by 160.54%. Root morphology indicators were comprehensively optimized, with total root length, root surface area, average root diameter, and root volume increasing by 50.01%, 76.93%, 17.07%, and 10.645% respectively. Simultaneously, the TYQ7 treatment significantly inhibited root-knot nematode infection, reducing the number of root knots per unit root weight by 40.57% compared to the control, indicating that this strain has a significant dual effect of promoting growth and resisting nematodes.

[0160] Table 3. Effects of TYQ7 inoculation on cucumber growth indicators under Southern root-knot nematode infection.

[0161]

[0162]

[0163] Table 4. Effects of TYQ7 inoculation on cucumber root parameters under Southern root-knot nematode infection.

[0164]

[0165] Example 7

[0166] Plate antagonism of TYQ7 against Fusarium oxysporum

[0167] 7.1 Preparation of bacterial suspension

[0168] The specific procedures for preparing the strain suspension are the same as in Example 3.

[0169] 7.2 Preparation of Fusarium oxysporum mycelium plates

[0170] The Fusarium oxysporum strain used in this invention is a strain stored in our laboratory's bacterial bank. The bacterial colony was picked and inoculated onto a solid PDA medium plate and cultured at 28°C for 3 days.

[0171] 7.3 TYQ7 antagonistic experiment against Fusarium oxysporum

[0172] Activated Fusarium oxysporum inoculum was cut into 3×3mm pieces and inoculated onto a new PDA plate at 1 / 4 of its diameter. Using a sterile toothpick, TYQ7 bacterial suspension adjusted to a specific OD value was streaked perpendicular to the diameter of the pathogenic bacterial piece. To ensure sufficient TYQ7 growth, the streaking was repeated 2-3 times. After incubating the plates at 28℃ for 2-3 days, the major and minor axes (elliptical shape) of the Fusarium oxysporum mycelial zones were measured, and the centrifugation rate was calculated to quantify the inhibitory effect.

[0173]

[0174] 7.4 Results Analysis

[0175] The antagonistic effect of TYQ7 and Fusarium oxysporum on agar plates is shown in the figure. Figure 11 The effects of TYQ7 on the growth of Fusarium oxysporum are shown in Table 5. The results showed that after treatment with TYQ7, the morphology of the Fusarium oxysporum mycelium changed from nearly circular to elliptical with an eccentricity >0.5, indicating that its growth direction was significantly blocked. The area of ​​the mycelium mycelium was reduced by 29.02% compared with the control, which directly reflects the antagonistic effect of TYQ7 on Fusarium oxysporum.

[0176] Table 5. Effects of TYQ7 on the growth of Fusarium oxysporum

[0177]

[0178] Note: The eccentricity in the table represents the growth of Fusarium oxysporum. The higher the eccentricity value, the flatter the ellipse, and the lower the eccentricity value, the more round it is.

[0179] Example 8

[0180] TYQ7's degradation characteristics of vegetable straw

[0181] 8.1 Vegetable straw pretreatment

[0182] In this embodiment, four common fruit and vegetable stalks, including cucumber, tomato, pepper and eggplant stalks, were cut into 3-4cm sections, dried in an oven, and then placed into 250ml Erlenmeyer flasks. After sealing with sealing film, they were placed in an autoclave and sterilized at 121℃ for 15 minutes.

[0183] 8.2 Determination of vegetable straw degradation rate

[0184] Add 5g of straw to each Erlenmeyer flask containing 125ml of carbon-deficient liquid culture medium (the formula of which is the same as in Example 3). Then, inoculate the medium with 5ml of overnight activated TYQ7 bacterial solution. Seal the Erlenmeyer flasks with sealing film and place them on a shaker at 28℃ and 180rpm / min for 15 minutes. Collect the straw, wash, dry, and weigh it. Calculate the degradation rate of the four types of straw using the following formula:

[0185]

[0186] 8.3 Results

[0187] The degradation effect of TYQ7 on four types of vegetable straw is shown in the figure. Figure 12 Table 6 shows the degradation rates of TYQ7 on the straws of four vegetables. Analysis indicates that TYQ7 exhibits degradation ability on all four types of vegetable straws, with tomato straw showing the highest degradation rate (66.14%), while cucumber, pepper, and eggplant straws showed relatively lower degradation rates. It is speculated that the higher degree of lignification and lignin content of the latter three may make them less susceptible to degradation.

[0188] Table 6. Degradation rate of TYQ7 on four types of vegetable straw

[0189]

[0190] Example 9

[0191] Phosphorus solubility characteristics of TYQ7

[0192] 9.1 Evaluation of the solubility of TYQ7 for sparingly soluble phosphorus using inorganic phosphorus solid culture medium

[0193] Take 10 μL of TYQ1 bacterial culture that has been cultured overnight onto the surface of inorganic phosphorus solid medium, seal it with sealing film, and incubate it in a 28℃ incubator for 5 days. Observe whether there is a phosphate-solubilizing zone around the colony.

[0194] 9.2 Results

[0195] See the phosphorus solubility characteristic diagram for TYQ7. Figure 13 After 5 days of cultivation on inorganic phosphorus solid medium, TYQ7 can form a distinct transparent phosphorus-dissolving ring around the colony.

[0196] Example 10

[0197] TYQ7's phosphorus solubility under gradient salt concentrations and pH levels

[0198] 10.1 Preparation of Inorganic Phosphorus Liquid Culture Medium with Gradient Salt Concentration and pH

[0199] Inorganic phosphorus liquid culture medium was prepared, and NaCl was added to set up a gradient salt concentration of 0, 2, 4, 6, and 8 g / L. In addition, NaOH and HCl were precisely added using a pH meter to set up a gradient acidity and alkalinity of pH 5, 6, 7, 8, and 9.

[0200] 10.2 Determination of solubility

[0201] The solubility of TYQ7 for poorly soluble phosphorus was assessed using gradient salt concentrations and inorganic phosphorus liquid culture medium: 50 μL of overnight TYQ7 culture was added to 5 ml of inorganic phosphorus liquid culture medium, sealed with sealing film, and incubated at 28°C for 5 days. The phosphorus-solubility of TYQ7 was then quantitatively analyzed using a molybdenum-antimony colorimetric method. The supernatant was used as the test solution. 5 ml of the supernatant was added to 2.5 ml of molybdenum-antimony colorimetric reagent, incubated at room temperature for 30 minutes, and the absorbance was measured at 700 nm.

[0202] 10.3 Results

[0203] The phosphorus solubility graph of TYQ7 under gradient salt concentration and gradient pH is shown below. Figure 14 The results showed that the phosphorus-solubility of TYQ7 under gradient salinity and gradient pH decreased with increasing salt concentration or as the pH deviated from neutral. The maximum phosphorus-solubility of TYQ7 was 77.33 mg / L at a salt concentration of 0 g / L and 74.69 mg / L at a neutral pH of 7.

[0204] Example 11

[0205] TYQ7's carbon source utilization capacity

[0206] 11.1 Biolog GenIII Microplate

[0207] The utilization or oxidation capacity of strain TYQ7 to various pre-selected carbon sources was assessed using Biolog GenIII microplates. This assay was performed by measuring the reaction solution at OD0.05. 590 The absorbance value at nm was used to generate the metabolic fingerprint of TYQ7 for different carbon sources.

[0208] 11.2 Results

[0209] The fingerprint of TYQ7's ability to utilize different carbon sources is shown in the image. Figure 15 The results showed that TYQ7 exhibited varying degrees of utilization ability for all 71 tested carbon sources, with the OD value directly reflecting its metabolic activity. In the chemical resistance test, this strain showed significant insensitivity to nalidixic acid, lithium chloride, and aztreonam, demonstrating strong environmental adaptability and metabolic diversity.

[0210] In summary, it can be seen from the above examples that:

[0211] (1) This strain was isolated from cucumber rhizosphere soil in the laboratory of China Agricultural University. Its classification name is Microbacterium paraoxydans TYQ7. It was deposited at the China General Microbiological Culture Collection Center on April 27, 2023, with the accession number CGMCC.27216.

[0212] (2) The microbiological characteristics of *Microbacterium paraoxidans* TYQ7 are as follows: Colonies are round, pale yellow, and opaque. The surface is smooth and moist, with irregular edges. The central part is slightly raised, and a biofilm naturally forms around each single spot. It is Gram-positive. The optimal growth temperature is 28–37℃.

[0213] (3) By amplifying the 16S rDNA fragment of the strain and performing BLAST homology comparison, the similarity of the strain to Microbacterium paraoxydans reached 99.86%. Therefore, the strain was identified as a strain of the genus Microbacterium and named Microbacterium paraoxydans TYQ7.

[0214] (4) Qualitative and quantitative analysis revealed that TYQ7 can secrete auxin, and the concentration of IAA secreted by it in the presence of tryptophan was 43.98 μg / mL.

[0215] (5) The TYQ7 inoculation experiment on cucumber cultivation showed that it has a significant growth-promoting effect: the aboveground dry weight and the total plant dry weight increased by 32.18% and 27.7% respectively, the total root surface area and volume increased significantly by 29.40% and 64.10% respectively, and the seedling strength index increased by 36.29%.

[0216] (6) TYQ7 interferes with the directional migration of nematodes in plants: When the cotyledons of seedlings unfold, TYQ7 fermentation liquid is poured into the soil around the roots, 10 ml per seedling. This application caused the migration tendency of southern root-knot nematodes to the control root system to reach 53.59%.

[0217] (7) After treating isolated root-knot nematodes with the supernatant of the fermentation broth of TYQ7 strain for 12 h and 24 h, the mortality rate of southern root-knot nematodes reached 70.31% and 83.67%, respectively.

[0218] (8) Experiments on cucumber cultivation with TYQ7 inoculated with Southern root-knot nematodes showed that it had a significant effect in alleviating root-knot nematode stress: the dry weight of the aboveground and underground parts increased by 23.81% and 28.89% respectively, the number of root knots per unit root weight decreased by 40.57%, and the chlorophyll content increased by 160.54%.

[0219] (9) TYQ7 can effectively inhibit the normal growth of Fusarium spp. during the confrontation with the Fusarium spp. plate, and the area of ​​the fungal circle is reduced by 48.58%, which is beneficial to prevent soil-borne diseases caused by Fusarium spp. when applied.

[0220] (10) Within two weeks, TYQ7 achieved degradation rates of 66.14%, 37.72%, 26.23%, and 30.18% on tomato, cucumber, pepper, and eggplant straw, respectively.

[0221] (11) The phosphorus-solubility of TYQ7 decreases with increasing salt concentration or as the pH deviates from neutral. The maximum phosphorus-solubility of TYQ7 is 77.33 mg / L when the salt concentration is 0 g / L, and the maximum phosphorus-solubility is 74.69 mg / L when the neutral pH is 7.

[0222] (12) Biolog analysis of TYQ7 showed that it can utilize 71 carbon sources, exhibits resistance to naridinic acid, lithium chloride and aztreonam, and has broad-spectrum metabolic capacity and environmental adaptability.

[0223] The few preferred embodiments of the invention are described in detail, but they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the inventive concept, and these all fall within the scope of protection of this invention.

Claims

1. A strain of *Microbacterium paraoxidans*, characterized in that, The paraoxidizing microbacteria ( Microbacterium paraoxydans Strain TYQ7 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 27216.

2. A microbial agent containing *Microbacterium paraoxidans* cells as described in claim 1.

3. Use of the strain described in claim 1 or the microbial agent described in claim 2 in the preparation of a formulation that promotes the growth of cucumber seedlings.

4. Use of the strain described in claim 1 or the microbial agent described in claim 2 in the preparation of a formulation that interferes with the migration of southern root-knot nematodes.

5. Use of the strain described in claim 1 or the microbial agent described in claim 2 in the preparation of formulations for controlling southern root-knot nematodes.

6. Use of the strain as described in claim 1 or the microbial agent as described in claim 2 in the preparation of a formulation for inhibiting the growth of Fusarium oxysporum.

7. Use of the strain described in claim 1 or the microbial agent described in claim 2 in the preparation of straw degradation formulations.

8. Use of the strain as described in claim 1 or the microbial agent as described in claim 2 in the preparation of phosphate-solubilizing formulations.

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