Bacterium capable of degrading pesticide tebuconazole and having growth promoting ability and use thereof

Abstract

The application discloses a bacterium capable of degrading pesticide tebuconazole and having growth promotion ability and application thereof. The application provides Pseudomonas hunanensis, a strain of which is HCL-7, and the strain is registered in the China General Microbiological Culture Collection Center with a registration number of CGMCC No.31920. The strain HCL-7 provided by the application has potential application prospects in relieving the growth stress of tebuconazole on Chinese cabbage, promoting the growth of Chinese cabbage and improving the yield, and has good application prospects in developing a microbial agent for relieving the growth stress of chemical pesticides on crops and a special microbial fertilizer for relieving the stress of tebuconazole on Chinese cabbage.

Description

Technical Field

[0001] This invention belongs to the fields of environmental science and agricultural production technology, specifically relating to a strain of pesticide tebuconazole and a bacterium with growth-promoting ability, and their uses. Background Technology

[0002] Tebuconazole is a highly effective, broad-spectrum, systemic triazole fungicide, mainly used to control various fungal diseases on crops such as wheat, rice, peanuts, vegetables, bananas, apples, pears, corn, and sorghum. Its half-life in soil is approximately 20-90 days. However, long-term and excessive use of pesticides inevitably increases their residue levels in the soil, causing phytotoxicity to crops. Therefore, reducing pesticide residue levels in the soil and alleviating the stress of pesticide residues on crop growth is of great significance for ensuring healthy crop growth, improving the quality and safety of agricultural products, protecting the ecological environment, and promoting sustainable agricultural development.

[0003] Plant growth-promoting rhizobacteria (PGPR) are a class of bacteria that promote plant growth within the rhizosphere. The rhizosphere microbiome, as the second genome of crops, is the engine for the migration and transformation of biogenic elements and micronutrients in the soil-plant system. The decomposition and accumulation of organic matter in the soil, nitrogen transformation, the release of nutrients such as phosphorus, potassium, iron, and zinc, the supply of nutrients to plants to stimulate growth, and the processes of inhibiting pathogens and improving soil are all closely related to microbial activity.

[0004] With the continuous development of agricultural biotechnology and the increasing emphasis on environmental protection, strains capable of degrading various pesticides and promoting growth will have broader application prospects in the agricultural field. These multifunctional strains not only play a vital role in degrading pesticides, reducing pesticide pollution, protecting the ecological environment, and improving crop yield and quality, but also contribute to promoting green agricultural development and improving agricultural production efficiency. Summary of the Invention

[0005] The purpose of this invention is to provide a strain of bacteria that degrades the pesticide tebuconazole and has growth-promoting capabilities, as well as their uses.

[0006] Firstly, this invention claims protection for a strain of *Pseudomonas hunanense*.

[0007] The *Pseudomonas hunanensis* strain claimed in this invention is strain number HCL-7, and its registration number at the China General Microbiological Culture Collection Center (CGMCC, address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing) is CGMCC No. 31920. This strain is capable of phosphate solubilization, producing hematophilic acid, producing IAA, and degrading the pesticide tebuconazole. It has growth-promoting functions and alleviates the stress of tebuconazole on plant growth.

[0008] Secondly, this invention claims protection for cultures of *Pseudomonas hunanensis* as described in the first aspect above. The culture is the substance obtained by culturing *Pseudomonas hunanensis* as described in the first aspect above in a bacterial culture medium (all substances within the culture container).

[0009] The substances in the above-mentioned cultures include *Pseudomonas hunanensis* (the bacterial cell itself) and its metabolites as described in the first aspect above.

[0010] The term "metabolite" refers to the primary and / or secondary metabolites produced during microbial metabolism. Primary metabolism refers to the process by which microorganisms absorb various nutrients from the external environment and generate substances and energy to sustain life activities through catabolism and anabolism. The products of primary metabolism are called primary metabolites, such as monosaccharides or monosaccharide derivatives, nucleotides, vitamins, amino acids, fatty acids, and various macromolecular polymers composed of them, such as proteins, nucleic acids, polysaccharides, and lipids. Secondary metabolism refers to the synthesis of secondary metabolites by microorganisms using primary metabolites as precursors during a certain growth stage. These are mostly compounds with relatively complex molecular structures. Based on their functions, they can be classified into types such as antibiotics, hormones, alkaloids, and toxins.

[0011] In the above-mentioned cultures, the bacterial culture medium can be a solid culture medium or a liquid culture medium.

[0012] The term "culture" refers to any liquid or solid culture medium that has grown a microbial community after artificial inoculation and cultivation. It is the product obtained by growing and / or amplifying microorganisms; it can be a biologically pure culture of microorganisms, or it can contain a certain amount of culture medium, metabolites, or other components produced during the cultivation process. The term "culture" also includes passaged cultures obtained by subculturing microorganisms; these can be cultures of a single generation or mixtures of several generations.

[0013] Thirdly, this invention claims protection for a microbial agent.

[0014] The microbial agent claimed in this invention contains *Pseudomonas hunanensis* as described in the first aspect above, metabolites of *Pseudomonas hunanensis*, and / or the culture described in the second aspect above.

[0015] The microbial agent is a biodegradable pesticide tebuconazole and / or a microbial agent that promotes plant growth.

[0016] In the aforementioned microbial agent, the active ingredient may be *Pseudomonas aeruginosa*, its metabolites, and / or its culture as described in the first aspect above. The active ingredient may also contain other biological and / or non-biological components. Other active ingredients of the microbial agent can be determined by those skilled in the art based on their ability to degrade the pesticide tebuconazole and / or promote plant growth.

[0017] In the aforementioned microbial agent, in addition to the active ingredient, a carrier is also included. The carrier can be a commonly used and biologically inert carrier in the pesticide field. The carrier can be a solid or liquid carrier; the solid carrier can be a mineral material, plant material, or polymer compound; the mineral material can be at least one of clay, talc, kaolin, montmorillonite, white carbon, zeolite, silica, and diatomaceous earth; the plant material can be at least one of corn flour, soybean flour, and starch; the polymer compound can be polyvinyl alcohol and / or polyethylene glycol; the liquid carrier can be an organic solvent, vegetable oil, mineral oil, or water; the organic solvent can be decane and / or dodecane.

[0018] The above-mentioned microbial agents can be in various formulations, such as liquid, emulsion, suspension, powder, granules, wettable powder or water-dispersible granules.

[0019] Depending on the requirements, surfactants (such as Tween 20, Tween 80, etc.), binders, stabilizers (such as antioxidants), pH adjusters, etc. may also be added to the bacterial agent.

[0020] Fourthly, the present invention claims protection for any of the following applications of *Pseudomonas hunanensis* or its metabolites or cultures described in the first aspect above, or the bacterial agent described in the second aspect above:

[0021] (A1) Phosphorus dissolution (including dissolution of organic phosphorus and dissolution of inorganic phosphorus);

[0022] (A2) Preparation of phosphorus-soluble products;

[0023] (A3) produces heptaphilin;

[0024] (A4) Prepare products that produce ferrophiles;

[0025] (A5) Produces IAA;

[0026] (A6) Prepare products that produce IAA;

[0027] (A7) Promotes plant growth;

[0028] (A8) Prepare products that promote plant growth.

[0029] Fifthly, the present invention claims protection for any of the following applications of *Pseudomonas hunanensis* or its metabolites or cultures described in the first aspect above, or the bacterial agents described in the second aspect above:

[0030] (A9) Degrades tebuconazole;

[0031] (A10) Prepare a product for the degradation of tebuconazole.

[0032] Sixthly, the present invention claims protection for any of the following applications of *Pseudomonas hunanensis* or its metabolites or cultures described in the first aspect above, or the bacterial agents described in the second aspect above:

[0033] (B1) Promotes plant growth under tebuconazole stress;

[0034] (B2) Preparation of products that promote plant growth under tebuconazole stress;

[0035] (B3) Relieves the stress of tebuconazole on plant growth;

[0036] (B4) Prepare products to alleviate the stress of plant growth caused by tebuconazole.

[0037] Seventhly, the present invention claims a method for degrading tebuconazole.

[0038] The method for degrading tebuconazole claimed in this invention may include the following steps: treating the substance to be degraded with *Pseudomonas aeruginosa* as described in the first aspect above, or its metabolites, or the culture as described in the second aspect above, or the bacterial agent as described in the third aspect above; thereby achieving the degradation of tebuconazole.

[0039] Eighthly, the present invention claims a method for promoting plant growth and / or alleviating the stress of tebuconazole on plant growth under tebuconazole stress.

[0040] The method for promoting plant growth and / or alleviating the stress of tebuconazole on plant growth under tebuconazole stress claimed in this invention may include the following steps: applying *Pseudomonas aeruginosa* or its metabolites, or the culture, or the inoculant, as described in the first aspect above, to a plant growth substrate contaminated with tebuconazole, thereby promoting plant growth and / or alleviating the stress of tebuconazole on plant growth under tebuconazole stress.

[0041] In the aforementioned aspects, promoting plant growth is manifested in increasing plant height and / or the fresh weight of the above-ground parts.

[0042] In one embodiment of the present invention, the tebuconazole stress is 50 mg of tebuconazole per 1 kg of growth substrate (such as soil).

[0043] In the aforementioned related aspects, the product may specifically be a microbial inoculant or a microbial fertilizer.

[0044] In the aforementioned relevant aspects, the plant may be any of the following:

[0045] (C1) Crops;

[0046] (C2) Vegetables;

[0047] (C3) Leafy vegetables;

[0048] (C4) Cabbage.

[0049] Experiments have demonstrated that the *Pseudomonas hunanensis* HCl-7 (CGMCC No. 31920) provided in this invention can solubilize phosphorus, produce hematophilic acid and IAA, promote growth, and effectively degrade the pesticide tebuconazole. Soil greenhouse cultivation experiments have shown that under 50 mg / kg tebuconazole pollution, the application of strain HCL-7 can increase the plant height and above-ground fresh weight of Chinese cabbage. Therefore, strain HCL-7 provided in this invention has potential application prospects in alleviating the growth stress of tebuconazole on Chinese cabbage, promoting Chinese cabbage growth, and increasing yield. It also shows good application prospects in developing microbial agents for crop growth under chemical pesticide stress and special microbial fertilizers for alleviating tebuconazole stress on Chinese cabbage.

[0050] Preservation Instructions

[0051] Classification and naming: Pseudomonas hunanensis;

[0052] Biological material from ginseng: HCl-7;

[0053] Preservation institution: China General Microbiological Culture Collection Center, China Committee on the Preservation and Management of Microbial Cultures;

[0054] The abbreviation for the depository institution is CGMCC.

[0055] Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing;

[0056] Deposit date: September 10, 2024;

[0057] Registered with the China National Collection Center (CGMCC) No. 31920. Attached Figure Description

[0058] Figure 1This is a diagram showing the colony morphology of strain HCL-7 on a plate.

[0059] Figure 2 Gram-stained microscopic image of strain HCL-7.

[0060] Figure 3 The graph shows the organophosphate solubility of strain HCl-7.

[0061] Figure 4 The graph shows the inorganic phosphorus solubility of strain HCl-7.

[0062] Figure 5 The graph shows the ability of strain HCL-7 to produce heptaphiles.

[0063] Figure 6 The graph shows the ability of strain HCl-7 to degrade tebuconazole.

[0064] Figure 7 Figure showing how strain HCl-7 alleviates the growth stress of tebuconazole on Chinese cabbage. Detailed Implementation

[0065] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0066] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0067] The culture media used in the following examples:

[0068] Inorganic salt culture medium: 2.8g disodium hydrogen phosphate, 1g potassium dihydrogen phosphate, 0.5g ammonium sulfate, 53mg magnesium chloride, 50mg calcium chloride tetrahydrate, 0.5mg disodium EDTA, 0.2mg ferrous sulfate heptahydrate, 0.01mg zinc sulfate heptahydrate, 0.003mg manganese chloride tetrahydrate, 0.03mg boric acid, 0.02mg cobalt chloride hexahydrate, 0.001mg copper chloride dihydrate, 0.002mg nickel chloride hexahydrate, 0.003mg sodium molybdate dihydrate, 1000mL distilled water, adjusted to pH 7.0.

[0069] Beef extract peptone medium: 3g beef extract, 10g peptone, 5g sodium chloride, 15-20g agar, 1000mL distilled water, adjust pH to 7.0.

[0070] Example 1: Enrichment, domestication, isolation, and identification of strain HCL-7

[0071] I. Enrichment and domestication of strains

[0072] A 10g soil sample (from Zhijiang, Hubei Province, where chemical pesticides tebuconazole, butachlor, and chlorantraniliprole have been used for many years) was added to a 250mL Erlenmeyer flask containing 100mL of inorganic salt medium and cultured on a shaker at 30℃ and 150rpm for one week. The initial concentration of each pesticide in the liquid medium was 20mg / L (tebuconazole, butachlor, and chlorantraniliprole were mixed, each at a concentration of 20mg / L). Every week, 10% of the liquid was transferred to the next culture flask, for a total of 4 transfers. Each time the concentration of each pesticide was increased, the concentrations were 40, 60, 80, and 100mg / L, respectively. The acclimatization process was completed after 35 days.

[0073] II. Isolation of bacterial strains

[0074] Under aseptic conditions, 10 mL of liquid was drawn from a 100 mg / L enrichment and acclimatization culture medium and added to 90 mL of sterile water containing glass beads. The mixture was shaken thoroughly at 150 rpm for 30 min to obtain the mother culture suspension. The above bacterial suspension was then diluted with sterile water to a concentration of 10 mL / L. -1 10 -2 10 -3 10 -4 and 10 -5 Gradient, set to 10 -3 10 -4 10 -5 Three consecutive 100 μL dilutions were plated on prepared beef extract peptone agar plates, with three replicates for each dilution. Sterile distilled water was used as a blank control. All plates were incubated at 30°C. After 3 days, single colonies of bacteria with different colors and morphologies were streaked onto plates, purified, and transferred to glycerol tubes for storage. One of the bacterial strains was identified as HCl-7.

[0075] III. Identification of strain HCl-7

[0076] 1. Bacterial genetic identification and phylogeny

[0077] DNA was extracted from the isolated strains using a bacterial genomic DNA extraction kit (TIANGEN). PCR primers were the universal 16S rDNA primers 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-TACGGTTACCTTGTTACGACTT-3′), with a DNA fragment size of approximately 1500 bp (SEQ ID No. 1), synthesized by Shanghai Sangon Biotech Co., Ltd. The PCR amplification reaction system consisted of 3 μL of DNA template, 1 μL each of 27F and 1492R primers (10 μmol / L), 12.5 μL of 2×mixTaq enzyme (GenStar), and 7.5 μL of ddH2O, diluted to a final volume of 25 μL. The PCR amplification program was as follows: 94℃ pre-denaturation for 10 min; 94℃ denaturation for 30 s, 55℃ annealing for 1 min, and 72℃ extension for 1.5 min, for 30 cycles; a final extension at 72℃ for 10 min, followed by storage at 4℃. The PCR products were sent to Sangon Biotech for sequencing.

[0078] The sequencing results of the bacteria were uploaded to the database https: / / www.ezbiocloud.net / for online comparison. It was found that strain HCL-7 had the highest similarity with Pseudomonas hunanensis, at 99.65%.

[0079] 2. Morphological observation and Gram staining of strain HCl-7

[0080] The obtained strain HCL-7 was streaked onto beef extract peptone medium using an inoculation loop and incubated upside down at 30°C for 24 hours. The basic morphology of the colonies was observed. Sterilized slides were prepared in a clean bench. A drop of sterile water was placed on the slide, and a small amount of bacterial cells was dipped in and spread into a uniform thin layer using an inoculation loop. With the specimen side up, the slide was held by one end and carefully heated slightly over an alcohol lamp to evaporate the water. After cooling, staining began. Add 1-2 drops of ammonium oxalate crystal violet to the smear film to cover the smear with the staining solution. Stain for about 1 minute. Tilt the slide and rinse under a small stream of tap water until the rinsing water is colorless. Take 300 μL of iodine solution and apply it to the smear film to cover the smear with the staining solution. Stain for about 1 minute. Tilt the slide and rinse under a small stream of tap water until the rinsing water is colorless. Tilt the slide and add 95% ethanol to decolorize until the ethanol no longer appears purple. Rinse with water after about 20-30 seconds. Add 1-2 drops of safranin stain to the smear film to cover the smear with the staining solution. Stain for about 1 minute. Tilt the slide and rinse under a small stream of tap water until the rinsing water is colorless. Absorb the water droplets with absorbent paper and observe the specimen under a microscope after it dries.

[0081] The results showed that strain HCL-7 formed round, convex, and smooth, opaque white colonies on beef extract peptone medium, with regular edges. Figure 1 Under a microscope, the colonies appear red, indicating they are Gram-negative bacteria. Figure 2 ).

[0082] 3. Determination of physiological and biochemical characteristics of strain HCL-7

[0083] (1) Inoculate strain HCL-7 into tryptone soybean agar medium (TSA, 15g tryptone, 5g soybean peptone, 5g sodium chloride, 15g agar powder, 1000mL distilled water, pH=7.3) and culture for no more than 16h to avoid the formation of spores.

[0084] (2) Place the turbidimeter on a stable surface, calibrate and adjust the turbidimeter reading to 100, and use an Inoculatorz swab to collect a colony with a diameter of approximately 3 mm from the prepared plate and inoculate it into the IF-A inoculation solution. During the inoculation process, gently smear the swab on the wall of the inoculation bottle, shake the inoculation tube several times to ensure even inoculation of the strain, repeat several times, and adjust the turbidity of the inoculation solution to 90-98%.

[0085] (3) Pour the bacterial suspension containing the colonies into a sterile V-shaped sample loading trough. Using an 8-channel pipette, carefully add 100 μL of the bacterial suspension to each well in sequence. Cover the microplate with the cap, being careful not to touch the top and bottom surfaces to avoid inaccurate measurements.

[0086] (4) Place the Biolog GENEIII 96-well plate with the added samples directly into the OmniLog incubator and incubate at 33℃ for 24-48 hours. Then, read the data and perform identification. The results are shown in Table 1.

[0087] Table 1. Determination of the unique carbon source utilization of strain HCl-7 using BIOLOG GENIII reagent strips

[0088]

[0089]

[0090]

[0091] Note: + indicates positive; - indicates negative; w indicates weak positive.

[0092] Based on the above identification, strain HCL-7 was identified as *Pseudomonas hunanensis*, which was deposited on September 10, 2024, at the China General Microbiological Culture Collection Center (CGMCC, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing), with accession number CGMCC No. 31920. Hereinafter referred to as strain HCL-7.

[0093] Example 2: Determination of the growth-promoting ability of strain HCL-7

[0094] I. Determination of Organophosphorus Solubility

[0095] The culture medium for determination consisted of: 0.03 g manganese sulfate tetrahydrate, 10.0 g glucose, 5.0 g calcium carbonate, 0.3 g sodium chloride, 0.5 g ammonium sulfate, 0.3 g magnesium sulfate heptahydrate, 0.03 g ferrous sulfate heptahydrate, 0.3 g potassium chloride, 0.2 g lecithin, and 16.0 g agar, dissolved in 1000 mL distilled water, with a pH of 7.0-7.2.

[0096] Strain HCL-7 was inoculated onto the above-mentioned test medium and incubated at 28°C for 3 days. The appearance of a clear zone around the colony was observed and photographed. The ability of the strain to dissolve organophosphates was determined based on the size of the clear zone's Dc value. Dc = diameter of the clear zone (D, cm) / colony diameter (d, cm).

[0097] The results are as follows Figure 3 As shown in Table 2, the results indicate that a clear zone can form around strain HCL-7 on plates containing organophosphates, suggesting that the strain has the ability to dissolve organophosphates.

[0098] II. Determination of Inorganic Phosphorus Solubility

[0099] The culture medium for determination was prepared as follows: 10.0 g calcium phosphate, 0.5 g ammonium sulfate, 10.0 g glucose, 0.3 g magnesium sulfate heptahydrate, 0.3 g potassium chloride, 0.3 g sodium chloride, 0.03 g manganese sulfate tetrahydrate, and 16.0 g agar, dissolved in 1000 mL distilled water, with a pH of 7.0-7.2.

[0100] Strain HCl-7 was inoculated onto the above-mentioned test medium and incubated at 28°C for 3 days. The appearance of a clear zone around the colony was observed and photographed. The ability of the strain to dissolve inorganic phosphorus was determined based on the size of the clear zone's Dc value. Dc = diameter of the clear zone (D, cm) / colony diameter (d, cm).

[0101] The results are as follows Figure 4As shown in Table 2, the results indicate that a clear zone can form around strain HCL-7 on plates containing inorganic phosphorus, suggesting that the strain has the ability to dissolve inorganic phosphorus.

[0102] III. Determination of Ferrophilic Production Capacity

[0103] CAS assay medium: Chromium azurite S (CAS) 60.5 mg; hexadecyltrimethylammonium bromide (HDTMA) 72.9 mg; ferrous chloride hexahydrate 2.645 mg; agar 9.0 g; 0.1 mol / L phosphate buffer 50 mL; The following is the formula for 0.1 mol / L phosphate buffer: sodium dihydrogen phosphate dihydrate 295.25 mg; disodium hydrogen phosphate dodecahydrate 1213.5 mg; ammonium chloride 125 mg; potassium dihydrogen phosphate 37.5 mg; sodium chloride 62.5 mg; dissolved in 1000 mL distilled water; pH 6.6-7.0.

[0104] Strain HCL-7 was inoculated onto the CAS detection medium described above and incubated at 28°C for 3 days. The appearance of a clear zone around the colony was observed and photographed. The ability of the strain to produce siderophiles was determined based on the size of the clear zone's Dc value. Dc = diameter of the clear zone (D, cm) / colony diameter (d, cm).

[0105] The results are as follows Figure 5 As shown in Table 2, the results indicate that a clear zone forms around strain HCL-7 on the CAS test plate, suggesting that the strain has the ability to produce ferropterins.

[0106] IV. IAA Production Capacity Measurement

[0107] Standard curve construction: Prepare standard IAA solutions with concentrations of 10, 20, 30, 40, and 50 μg / mL in volumetric flasks. Add 100 μL of each concentration of standard IAA solution to a 96-well plate (with an equal volume of Salkowski colorimetric solution added beforehand), in triplicate. Quickly place the plates in the dark for 30 min. Add 100 μL of NA liquid culture medium to 100 μL of Salkowski colorimetric solution as a negative control, and add 100 μL of 50 μg / mL standard IAA solution to 100 μL of Salkowski colorimetric solution as a positive control. Measure the OD530 absorbance, and take the average of the three measurements. Plot the standard curve with IAA concentration on the x-axis and OD530 nm value on the y-axis.

[0108] Strain HCL-7 was inoculated in NA liquid medium (containing 500 mg / L L-tryptophan) and cultured at 180 r / min at 30 ℃ for 48 h. 1 mL of bacterial culture was transferred to a centrifuge tube, centrifuged at 8000 r / min for 5 min, and 100 μL of the supernatant was added to a 96-well plate. This process was repeated three times. 100 μL of Salkowski colorimetric solution was added, and the plate was immediately in the dark for 30 min. The OD530 absorbance was measured, and the average of the three measurements was substituted into the standard curve to obtain the IAA content.

[0109] The results are shown in Table 2. It can be seen that the IAA content of strain HCL-7 is 8.62±1.97μg / mL.

[0110] Table 2. Growth-promoting ability of strain HCl-7

[0111]

[0112] Example 3: Determination of pesticide degradation ability of strain HCL-7

[0113] Strawberry strain HCL-7 was aseptically streaked onto beef extract peptone medium and incubated at 30°C for 2 days. Then, a small amount of bacterial cells was inoculated into beef extract peptone liquid medium using a sterile inoculation loop and cultured at 30°C and 180 rpm on a shaker until the logarithmic growth phase. The culture was centrifuged at 8000 rpm for 5 min, the supernatant was removed, and the culture was washed twice with sterile water and resuspended to prepare a seed culture. 10 mL of inorganic salt culture base was added to a 50 mL centrifuge tube, and tebuconazole was added to achieve a concentration of 30 mg / L. The seed culture was inoculated at a rate of 10% (v / v), with a blank control (CK) also included. The culture was incubated at 37°C and 180 rpm for one week, and the degradation rate was measured. 1 mL of the degraded bacterial culture, filtered through a 0.22 μm filter, was placed in a 2 mL amber chromatographic vial. The content of tebuconazole was determined using an Athena C18 (4.6×250mm, 5μm) column and a 1290 high-performance liquid chromatography instrument. The mobile phase was methanol:water = 80:20 (V / V), the flow rate was 1mL / min, the detection wavelength was 223nm, the column temperature was 30℃, and the injection volume was 10μL.

[0114] The results are as follows Figure 6 As shown in Table 3, the addition of strain HCl-7 significantly promoted the degradation of tebuconazole compared with the CK group (P<0.05).

[0115] Table 3. Degradation capacity of pesticide tebuconazole by strain HCl-7 (three replicates)

[0116]

[0117] Example 4: Effects of strain HCl-7 on the growth of Chinese cabbage under tebuconazole stress

[0118] Unless otherwise specified, all materials and reagents used in the following examples are commercially available. The required materials are as follows:

[0119] (1) Cabbage variety: Four Seasons Little Concubine Fast Cabbage (a new variety of very early maturing cabbage), provided by Henan Zhuyou Seed Industry Co., Ltd.

[0120] (2) Potting materials: disposable aviation cup (7cm high, 7cm mouth diameter, 5cm bottom diameter).

[0121] (3) Tebuconazole pesticide: Tebuconazole, a fungicide with an active ingredient of 43% (% means g / 100mL), was provided by Nanjing Nanong Pesticide Technology Development Co., Ltd.

[0122] (4) Test strain: strain HCL-7.

[0123] (5) Beef extract peptone liquid culture medium: 3g beef extract, 10g peptone, 5g sodium chloride, 1000mL distilled water, adjust pH=7.0.

[0124] (6) Experimental soil: nutrient soil.

[0125] Experimental treatment:

[0126] Treatment 1 was clean soil (control);

[0127] Treatment 2 involved soil contaminated with 50 mg / kg tebuconazole (50 mg / kg tebuconazole);

[0128] Treatment 3 consisted of soil contaminated with 50 mg / kg tebuconazole and bacterial strain HCl-7 (50 mg / kg tebuconazole + HCl-7).

[0129] Each process is set to 3 repetitions.

[0130] Soil preparation: Soil contaminated with the pesticide tebuconazole at a concentration of 50 mg / kg was prepared. The pesticide dosage was calculated, and the measured amount of pesticide was diluted with 250 ml of water and stirred thoroughly. This solution was then added to 500 g of soil to ensure even wetting. The soil was left to stand for one day, and then dispensed into individual aviation containers the following day. Simultaneously, clean soil without pesticide was prepared.

[0131] Cabbage seed germination and sowing: Place two layers of filter paper in a petri dish, then add 10-15 cabbage seeds that have been soaked in water for 2 hours. Place the dishes in a 25℃ constant temperature incubator to germinate for 2 days. When sowing, use tweezers to sow the large and plump germinated cabbage seeds into the aviation cup.

[0132] Preparation and inoculation of bacterial culture: Using a sterile inoculation loop, take a small amount of HCl-7 cells and inoculate them into beef extract peptone liquid medium. Incubate at 30°C and 180 rpm on a shaker until the logarithmic growth phase. Inoculate 10 mL of bacterial culture into each aero-cup.

[0133] The results are as follows Figure 7 As shown in Table 4, compared with clean soil (control), the 50 mg / kg tebuconazole-contaminated soil treatment significantly inhibited the plant height of Chinese cabbage, resulting in a significant reduction of 40.21% in plant height. Compared with clean soil (control), the 50 mg / kg tebuconazole-contaminated soil treatment also significantly reduced the above-ground fresh weight of Chinese cabbage, resulting in a reduction of 34.83% in above-ground fresh weight. Compared with the 50 mg / kg tebuconazole-contaminated soil treatment, the addition of HCl-7 strain increased the taproot diameter, plant height, and above-ground fresh weight of Chinese cabbage, by 7.8%, 20.08%, and 50.57%, respectively.

[0134] Table 4. Biomass of Chinese cabbage under different treatments

[0135]

[0136]

[0137] Note: Different lowercase letters in the same column indicate significant differences (P<0.05).

[0138] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. *Pseudomonas hunanense*, characterized by: The *Pseudomonas hunanensis* strain mentioned is strain HCL-7, and its registration number at the China General Microbiological Culture Collection Center is CGMCC No. 31920.

2. The culture of *Pseudomonas hunanensis* according to claim 1 is a substance obtained by culturing *Pseudomonas hunanensis* according to claim 1 in a bacterial culture medium.

3. A microbial agent, characterized in that: The bacterial agent contains the Hunan Pseudomonas as described in claim 1 or the culture as described in claim 2.

4. The microbial agent according to claim 3, characterized in that: The microbial agent is a microbial agent that degrades the pesticide tebuconazole and / or promotes plant growth.

5. Any of the following applications of the *Pseudomonas hunanensis* according to claim 1, the culture according to claim 2, or the bacterial agent according to claim 3 or 4: (A1) Soluble phosphorus; (A2) Preparation of phosphorus-soluble products; (A3) produces heptaphilin; (A4) Prepare products that produce ferrophiles; (A5) Produces IAA; (A6) Prepare products that produce IAA.

6. Any of the following applications of the *Pseudomonas hunanensis* according to claim 1, the culture according to claim 2, or the bacterial agent according to claim 3 or 4: (A9) Degrades tebuconazole; (A10) Prepare a product for the degradation of tebuconazole.

7. Any of the following applications of the *Pseudomonas hunanensis* according to claim 1, the culture according to claim 2, or the bacterial agent according to claim 3 or 4: (B1) Promotes the growth of Chinese cabbage under tebuconazole stress; (B2) Preparation of a product that promotes the growth of Chinese cabbage under tebuconazole stress; (B3) Relieves the stress of tebuconazole on the growth of Chinese cabbage; (B4) Prepare a product to alleviate the growth stress of tebuconazole on Chinese cabbage.

8. A method for degrading tebuconazole, comprising the following steps: treating the substance to be degraded with the *Pseudomonas aeruginosa* of claim 1, the culture of claim 2, or the bacterial agent of claim 3 or 4; thereby achieving the degradation of tebuconazole.

9. A method for promoting plant growth and / or alleviating the stress of tebuconazole on plant growth under tebuconazole stress, comprising the following steps: applying the *Pseudomonas aeruginosa* of claim 1, the culture of claim 2, or the inoculant of claim 3 or 4 to a plant growth substrate contaminated with tebuconazole, thereby promoting plant growth and / or alleviating the stress of tebuconazole on plant growth under tebuconazole stress; wherein the plant is Chinese cabbage.

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