Bacterium capable of degrading pesticide tebuconazole and promoting growth and application thereof

Abstract

The invention discloses a bacterium capable of degrading pesticide tebuconazole and promoting growth and application of the bacterium. The invention provides Pseudomonas Hunanensis, which is Pseudomonas Hunanensis, the strain number of the Pseudomonas Hunanensis is HCL-7, and the registration number of the Pseudomonas Hunanensis in the China General Microbiological Culture Collection Center (CGMCC) is CGMCC No.31920. The Pseudomonas Hunanensis provided by the invention has the advantages that the Pseudomonas Hunanensis can be used for preparing the Pseudomonas Hunanensis; the strain HCL-7 provided by the invention has potential application prospects in the aspects of relieving the growth stress of tebuconazole on Chinese cabbages, promoting the growth of the Chinese cabbages and improving the yield, and has good application prospects in the aspects of developing a microbial agent for developing the growth of crops stressed by chemical pesticides and a special microbial fertilizer for relieving the stress of tebuconazole on the Chinese cabbages.

Description

Technical Field

[0001] The invention belongs to the field of environmental science and agricultural production technology, and specifically relates to a bacterium capable of degrading the pesticide tebuconazole and promoting growth, and uses thereof. Background Art

[0002] Tebuconazole is a highly effective, broad-spectrum, systemic triazole fungicide pesticide, mainly used to control a variety of fungal diseases on crops such as wheat, rice, peanuts, vegetables, bananas, apples, pears, corn and sorghum, with a half-life in the soil of about 20-90 days. However, the long-term and large-scale use of pesticides will inevitably increase their residual levels in the soil, causing crop damage. Therefore, reducing the level of pesticide residues in the soil and alleviating the stress of pesticide residues on crop growth are of great significance to ensuring the healthy growth of crops, improving the quality and safety of agricultural products, protecting the ecological environment, and promoting the sustainable development of agriculture.

[0003] Plant growth promoting rhizobacteria (PGPR) refers to a type of bacteria that can promote plant growth within the rhizosphere of plants. As the second genome of crops, the rhizosphere microbiome 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 plant nutrition to stimulate plant growth, and the inhibition of pathogens and improvement of soil are all closely related to the activities of microorganisms.

[0004] With the continuous development of agricultural biotechnology and people's increasing attention to environmental protection, the application prospects of strains that can degrade multiple pesticides and promote growth in the agricultural field will be broader. This multifunctional strain not only plays an important role in degrading pesticides, reducing pesticide pollution, protecting the ecological environment, and improving crop yields and quality, but also helps promote green agricultural development and improve agricultural production efficiency. Summary of the invention

[0005] The purpose of the present invention is to provide a bacterium capable of degrading the pesticide tebuconazole and promoting growth and use thereof.

[0006] In a first aspect, the present invention claims protection for a strain of Hunan Pseudomonas.

[0007] The Hunan Pseudomonas protected by the present invention is Pseudomonas hunanensis, whose strain number is HCL-7, and whose registration number in the General Microbiological Center of China Microorganism Culture Collection Committee (referred to as CGMCC, address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing) is CGMCC No. 31920. The strain can dissolve phosphate, produce siderophore, produce IAA, and degrade the pesticide tebuconazole, has a growth-promoting function and alleviates the stress of tebuconazole on plant growth.

[0008] In the second aspect, the present invention claims protection for the culture of the Hunan Pseudomonas described in the first aspect above. The culture is a substance (all substances in the culture container) obtained by culturing the Hunan Pseudomonas described in the first aspect above in a bacterial culture medium.

[0009] In the above culture, the substances include the Hunan Pseudomonas (the bacteria itself) and its metabolites as described in the first aspect above.

[0010] The term "metabolite" refers to the primary metabolites and / or secondary metabolites produced during the metabolism of microorganisms. Primary metabolism refers to the process by which microorganisms absorb various nutrients from the outside world and generate substances and energy to maintain life activities through catabolism and anabolism. The products of primary metabolism are primary metabolites, such as monosaccharides or monosaccharide derivatives, nucleotides, vitamins, amino acids, fatty acids and other monomers, as well as various macromolecular polymers composed of them, such as proteins, nucleic acids, polysaccharides, lipids, etc. Secondary metabolism refers to the synthesis of secondary metabolites by microorganisms in a certain growth period, using primary metabolites as precursors. Most of them are compounds with relatively complex molecular structures. According to their effects, they can be divided into types such as antibiotics, hormones, alkaloids, toxins, etc.

[0011] In the above culture, the bacterial culture medium may be a solid culture medium or a liquid culture medium.

[0012] The term "culture" refers to a general term for liquid or solid culture media that have a microbial population after artificial inoculation and culture. That is, the product obtained by growing and / or amplifying microorganisms, which 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 culture process. The term "culture" also includes a subculture obtained by subculturing microorganisms, which can be a culture of a certain generation or a mixture of several generations.

[0013] In a third aspect, the present invention claims protection for a bacterial agent.

[0014] The bacterial agent claimed in the present invention contains the Hunan Pseudomonas described in the first aspect above, metabolites of the Hunan Pseudomonas and / or the culture described in the second aspect above.

[0015] Wherein, the bacterial agent is a bacterial agent that can degrade the pesticide tebuconazole and / or promote plant growth.

[0016] In the above-mentioned microbial agent, the active ingredient of the microbial agent may be the Hunan Pseudomonas, its metabolites and / or its culture as described in the first aspect above, and the active ingredient of the microbial agent may also contain other biological components and / or non-biological components. Those skilled in the art may determine the other active ingredients of the microbial agent based on the ability to degrade the pesticide tebuconazole and / or promote plant growth.

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

[0018] The above-mentioned bacterial agents may be in various dosage forms, such as liquid, emulsion, suspension, powder, granule, wettable powder or water dispersible granule.

[0019] As required, a surfactant (such as Tween 20, Tween 80, etc.), a binder, a stabilizer (such as an antioxidant), a pH adjuster, etc. may also be added to the bacterial agent.

[0020] In a fourth aspect, the present invention claims protection for any of the following uses of the Hunan Pseudomonas or its metabolites or its culture described in the first aspect above or the bacterial agent described in the second aspect above:

[0021] (A1) dissolved phosphorus (including dissolved organic phosphorus and dissolved inorganic phosphorus);

[0022] (A2) preparing a phosphate-dissolving product;

[0023] (A3) siderophore production;

[0024] (A4) preparing a siderophore-producing product;

[0025] (A5) produces IAA;

[0026] (A6) preparing an IAA-producing product;

[0027] (A7) promoting plant growth;

[0028] (A8) Preparation of products for promoting plant growth.

[0029] In a fifth aspect, the present invention claims protection for any of the following uses of the Hunan Pseudomonas or its metabolites or its culture described in the first aspect above or the bacterial agent described in the second aspect above:

[0030] (A9) degradation of tebuconazole;

[0031] (A10) preparing a product for degrading tebuconazole.

[0032] In a sixth aspect, the present invention claims protection for any of the following uses of the Hunan Pseudomonas or its metabolites or its culture described in the first aspect above or the bacterial agent described in the second aspect above:

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

[0034] (B2) preparing a product for promoting plant growth under tebuconazole stress;

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

[0036] (B4) preparing a product that alleviates the stress induced by tebuconazole on plant growth.

[0037] In a seventh aspect, the present invention claims a method for degrading tebuconazole.

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

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

[0040] The method for promoting plant growth under tebuconazole stress and / or alleviating the stress of tebuconazole on plant growth claimed in the present invention may include the following steps: applying the Hunan Pseudomonas described in the first aspect above or the metabolites of the Hunan Pseudomonas or the culture described in the second aspect above or the bacterial agent described in the third aspect above to the plant growth matrix contaminated with tebuconazole, thereby promoting plant growth under tebuconazole stress and / or alleviating the stress of tebuconazole on plant growth.

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

[0042] In the above-mentioned related aspects, in one embodiment of the present invention, the tebuconazole stress contains 50 mg of tebuconazole per 1 kg of growth medium (such as soil).

[0043] In the above-mentioned related aspects, the product may specifically be a microbial agent or a microbial fertilizer.

[0044] In the above related 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 shown that the Hunan Pseudomonas (Pseudomonas hunanensis) HCL-7 (CGMCC No. 31920) provided by the present invention can dissolve phosphorus, produce siderophore and IAA, has a growth-promoting effect, and can effectively degrade the pesticide tebuconazole. Soil greenhouse cultivation test studies have shown that under 50 mg / kg tebuconazole contamination, the use of strain HCL-7 can increase the plant height and fresh weight of the aboveground part of cabbage. Therefore, the strain HCL-7 provided by the present invention has potential application prospects in alleviating the growth stress of tebuconazole on cabbage, promoting cabbage growth and increasing yield, and has good application prospects in developing microbial agents for the growth of crops under chemical pesticide stress and special microbial fertilizers for alleviating the stress of tebuconazole on cabbage.

[0050] Collection Instructions

[0051] Taxonomic nomenclature: Pseudomonas hunanensis;

[0052] Reference biological materials: HCL-7;

[0053] Depository: General Microbiology Center, China Microbiological Culture Collection Administration;

[0054] Abbreviation of depository institution: CGMCC;

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

[0056] Deposit date: September 10, 2024;

[0057] The registration number of the Collection Center is: CGMCC No.31920. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1The colony morphology of strain HCL-7 on the plate.

[0059] Figure 2 This is the Gram staining microscopic examination of strain HCL-7.

[0060] Figure 3 This is the graph of the ability of strain HCL-7 to dissolve organic phosphorus.

[0061] Figure 4 This is the graph of the ability of strain HCL-7 to dissolve inorganic phosphorus.

[0062] Figure 5 This is a graph showing the siderophore-producing ability of strain HCL-7.

[0063] Figure 6 This is the graph showing the ability of strain HCL-7 to degrade tebuconazole.

[0064] Figure 7 This is a diagram showing that strain HCL-7 alleviates the growth stress of cabbage induced by tebuconazole. DETAILED DESCRIPTION

[0065] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0066] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0067] The culture medium used in the following examples is:

[0068] Inorganic salt culture medium: 2.8g disodium hydrogen phosphate, 1g potassium dihydrogen phosphate, 0.5g ammonium sulfate, 53mg magnesium chloride, 50mg calcium salt 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 cupric chloride dihydrate, 0.002mg nickel chloride hexahydrate, 0.003mg sodium molybdate dihydrate, 1000mL distilled water, adjust pH = 7.0.

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

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

[0071] 1. Enrichment and domestication of strains

[0072] 10g of soil sample (from Zhijiang, Hubei, where chemical pesticides such as tebuconazole, butachlor, and chlorantraniliprole were used for many years) was added to a 250mL triangular flask containing 100mL of inorganic salt culture medium and cultured on a shaker at 30°C and 150rpm for one week. The initial concentration of each pesticide in the liquid culture medium was 20mg / L (tebuconazole, butachlor, and chlorantraniliprole were mixed, and each concentration was 20mg / L). 10% of the liquid was transferred to the next culture bottle every other week, and the transfer was 4 times. The concentration of each pesticide increased with each transfer, which was 40, 60, 80, and 100mg / L, respectively. The acclimation was completed after 35 days.

[0073] 2. Isolation of strains

[0074] Under aseptic operation, 10 mL of liquid was drawn from the enriched acclimation culture solution with a concentration of 100 mg / L, added to 90 ml of sterile water containing glass beads, and fully shaken at 150 r / min for 30 min to obtain the mother liquid bacterial suspension; the above bacterial suspension was diluted with sterile water to 10 -1 , 10 -2 , 10 -3 , 10 -4 and 10 -5 Gradient, take 10 -3 , 10 -4 , 10 -5 Three consecutive dilutions of 100 μL were spread on the prepared beef extract peptone medium plates, with three replicates for each gradient. Sterile distilled water was used as a blank control, and all plates were placed in a 30°C constant temperature incubator for culture. After 3 days, bacterial colonies with different colors and shapes were selected for plate streaking, and after purification, they were transferred to glycerol tubes for storage. One of the strains was HCL-7.

[0075] 3. Identification of strain HCL-7

[0076] 1. Bacterial genetic identification and phylogeny

[0077] The bacterial genomic DNA extraction kit (TIANGEN) was used to extract the DNA of the isolated strains. The PCR amplification primers were 16S rDNA universal primers 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-TACGGTTACCTTGTTACGACTT-3′), with a DNA fragment size of about 1500 bp (SEQ ID No. 1), synthesized by Shanghai Shenggong Biological Co., Ltd. The PCR amplification reaction system included 3 μL of DNA template, 1 μL of 27F and 1492R primers (10 μmoL / L), 12.5 μL of 2×mixTaq enzyme (GenStar product), and 7.5 μL of ddH2O, making a 25 μL system. The PCR amplification reaction program was as follows: pre-denaturation at 94°C for 10 min; denaturation at 94°C for 30 s, annealing at 55°C for 1 min, and extension at 72°C for 1.5 min, for 30 cycles; finally, extension at 72°C for another 10 min, and storage at 4°C. The PCR products were sent to Sangon Biotechnology for sequencing.

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

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

[0080] The obtained strain HCL-7 was streaked on beef extract peptone medium with an inoculation loop, incubated upside down in a 30℃ constant temperature incubator for 24 hours, and the basic morphology of the colony was observed. Take a sterilized slide and operate in a clean bench. Pipette a drop of sterile water on the slide, dip a trace of bacteria, and spread it into a uniform thin layer with an inoculation loop. With the specimen facing up, hold one end of the slide and carefully heat it slightly above an alcohol lamp to evaporate the water, and start staining after cooling. Add 1-2 drops of ammonium oxalate crystal violet to the smear film, so that the staining solution covers the smear, stain for about 1 minute, tilt the slide, and rinse with a small stream of water under the tap until the washed water is colorless; draw 300μL of iodine droplets and apply them to the smear film, so that the staining solution covers the smear, stain for about 1 minute, tilt the slide, and rinse with a small stream of water under the tap until the washed water is colorless; tilt the slide, add 95% ethanol to decolorize until the outflowing ethanol is no longer purple, and rinse with water after about 20-30 seconds; add 1-2 drops of safranin dye solution to the smear film, so that the staining solution covers the smear, stain for about 1 minute, tilt the slide, and rinse with a small stream of water under the tap until the washed water is colorless; absorb the water droplets with absorbent paper, and observe under a microscope after the specimen is dry.

[0081] The results showed that the single colony of strain HCL-7 on beef extract peptone medium was round, with a raised center, neat edges, white, smooth and opaque ( Figure 1 ). The colonies appear red under the microscope and are Gram-negative bacteria ( Figure 2 ).

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

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

[0084] (2) Place the turbidity meter steady, calibrate and adjust the turbidity meter reading to 100, use an Inoculatorz cotton swab to pick up a colony with a diameter of about 3 mm from the streaked plate and inoculate it into the IF-A inoculum. During the inoculation process, gently smear the cotton swab on the wall of the inoculation bottle, shake the inoculation tube several times to ensure uniform inoculation of the strain, repeat several times, and adjust the turbidity of the inoculation solution to 90-98%.

[0085] (3) Pour the inoculated bacterial suspension into a sterile V-shaped sample trough. Use an 8-channel pipette to carefully add 100 μL of the bacterial suspension to all wells in sequence. Cover the microplate lid, taking care not to touch the upper and lower surfaces to avoid inaccurate measurements.

[0086] (4) The Biolog GENEIII 96-well plate with the sample was directly placed in the OmniLog incubation reader. After constant temperature incubation at 33°C for 24-48 hours, the data results were read and identified. The results are shown in Table 1.

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

[0088]

[0089]

[0090]

[0091] Note: +, positive; -, negative; w, weakly positive.

[0092] Through the above identification, strain HCL-7 was identified as Pseudomonas hunanensis (Chinese name: Hunan Pseudomonas), which was deposited in China General Microbiological Culture Collection Center (CGMCC, address: No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing) on ​​September 10, 2024, with a deposit number of CGMCC No. 31920. Hereinafter referred to as strain HCL-7.

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

[0094] 1. Determination of the ability to dissolve organic phosphorus

[0095] Assay medium: 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, 16.0 g agar, dissolved in 1000 mL distilled water, pH 7.0-7.2.

[0096] Inoculate strain HCL-7 onto the above assay medium and culture at 28°C for 3 days. Observe the appearance of transparent circles around the colonies and take photos. The ability of the strain to dissolve organic phosphorus is determined by the value of the transparent circle Dc. Dc = transparent circle diameter (D, cm) / colony diameter (d, cm).

[0097] The results are as follows Figure 3 As shown in Table 2, the results showed that on the plate containing organophosphate, an obvious transparent zone could be formed around the strain HCL-7, indicating that the strain had the ability to dissolve organophosphate.

[0098] 2. Determination of the ability to dissolve inorganic phosphorus

[0099] Assay medium: calcium phosphate 10.0 g, ammonium sulfate 0.5 g, glucose 10.0 g, magnesium sulfate heptahydrate 0.3 g, potassium chloride 0.3 g, sodium chloride 0.3 g, manganese sulfate tetrahydrate 0.03 g, agar 16.0 g, dissolved in distilled water 1000 mL, pH 7.0-7.2.

[0100] Inoculate strain HCL-7 onto the above assay medium and culture at 28°C for 3 days. Observe the appearance of a transparent zone around the colony and take photos. The ability of the strain to dissolve inorganic phosphorus is determined by the value of the transparent zone Dc. Dc = transparent zone diameter (D, cm) / colony diameter (d, cm).

[0101] The results are as follows Figure 4As shown in Table 2, the results showed that on the plate containing inorganic phosphorus, an obvious transparent circle could be formed around the strain HCL-7, indicating that the strain had the ability to dissolve inorganic phosphorus.

[0102] 3. Determination of siderophore production ability

[0103] CAS assay medium: chrome azurol 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 of 0.1 mol / L phosphate buffer: sodium dihydrogen phosphate dihydrate 295.25 mg; sodium dihydrogen phosphate dodecahydrate 1213.5 mg; ammonium chloride 125 mg; potassium dihydrogen phosphate 37.5 mg; sodium chloride 62.5 mg; dissolved in distilled water 1000 mL; pH 6.6-7.0.

[0104] Inoculate strain HCL-7 onto the above CAS test medium, culture at 28°C for 3 days, observe the appearance of transparent circles around the colonies, and take photos. The ability of the strain to produce siderophores is determined based on the value of the transparent circle Dc. Dc = transparent circle diameter (D, cm) / colony diameter (d, cm).

[0105] The results are as follows Figure 5 As shown in Table 2, the results showed that on the CAS test plate, an obvious transparent circle could be formed around the strain HCL-7, indicating that the strain had the ability to produce siderophore.

[0106] 4. Determination of IAA production capacity

[0107] Drawing of the calibration curve: prepare standard IAA solutions with concentrations of 10, 20, 30, 40, and 50 μg / mL in volumetric flasks, and add 100 μL of standard IAA solutions of different concentrations into a 96-well plate (with an equal volume of Salkowski colorimetric solution added in advance) in triplicate, quickly place in a dark environment for 30 min, and add 100 μL of NA liquid culture medium to 100 μL of Salkowski colorimetric solution as a negative control, and add 100 μL of standard IAA solution with a concentration of 50 μg / mL to 100 μL of Salkowski colorimetric solution as a positive control, measure the OD530 absorbance, take the average of the three times, and draw the calibration curve with IAA concentration as the horizontal axis and OD530nm value as the vertical axis.

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

[0109] The results are shown in Table 2, which shows that the IAA content of strain HCL-7 was 8.62±1.97 μg / mL.

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

[0111]

[0112] Example 3: Determination of the ability of strain HCL-7 to degrade pesticides

[0113] Take strain HCL-7, and inoculate it on beef extract peptone medium by streaking on a plate under aseptic operation, incubate it upside down at 30℃ in a constant temperature box for 2 days, then use a sterile inoculation loop to pick up a small amount of bacteria and inoculate it in beef extract peptone liquid medium, and shake and culture it on a shaker at 30℃ and 180rpm until the logarithmic phase. Centrifuge at 8000rpm for 5min, remove the supernatant, wash it twice with sterile water and resuspend it to make a seed liquid. Add 10mL of inorganic salt culture medium to a 50mL centrifuge tube, add tebuconazole to make its concentration reach 30mg / L; inoculate the seed liquid at an inoculum of 10% (volume percentage), and set up a blank control (CK) without inoculation. After shaking and culturing on a shaker at 37℃ and 180rpm for one week, determine the degradation rate. Take 1mL of the degraded bacterial solution filtered by a 0.22μm filter membrane and put it in a 2mL brown chromatographic vial. Athena C18 (4.6×250mm, 5μm) chromatographic column and 1290 high performance liquid chromatography instrument were used to detect the content of tebuconazole. The mobile phase was methanol: water = 80:20 (V / V), the flow rate was 1 mL / min, the detection wavelength was 223 nm, the column temperature was 30°C, and the injection volume was 10 μL.

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

[0115] Table 3. Degradation ability of the pesticide tebuconazole by strain HCL-7 (three replicates)

[0116]

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

[0118] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources. The required materials are as follows:

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

[0120] (2) Potting material: disposable airline cup (7 cm high, 7 cm in diameter at the mouth, 5 cm in diameter at the bottom).

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

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

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

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

[0125] Experimental treatment:

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

[0127] Treatment 2 was 50 mg / kg tebuconazole-contaminated soil (50 mg / kg tebuconazole);

[0128] Treatment 3 was 50 mg / kg tebuconazole-contaminated soil + strain HCL-7 (50 mg / kg tebuconazole + HCL-7).

[0129] Three replicates were set for each treatment.

[0130] Experimental soil preparation: Prepare soil contaminated with 50mg / kg of the pesticide tebuconazole, calculate the amount of pesticide, add the measured pesticide to water (250ml) and stir evenly, then add it to the soil (500g) to evenly moisten the soil. Let it stand for one day, and dispense it into each airline cup the next day. At the same time, prepare clean soil without pesticide.

[0131] Cabbage seed germination and sowing: Put two layers of filter paper in the culture dish, then put the cabbage seeds soaked in water for 2 hours, put 10-15 seeds in each dish, and germinate in a 25℃ constant temperature incubator for 2 days. When sowing, use tweezers to sow large and plump sprouted cabbage seeds into the aviation cup.

[0132] Preparation and inoculation of bacterial solution: Use a sterile inoculation loop to pick up a small amount of HCL-7 bacteria and inoculate it into beef extract peptone liquid medium. Shake and culture on a shaker at 30°C and 180 rpm until the logarithmic phase. When inoculating, inoculate 10 mL of bacterial solution into each aviation cup.

[0133] The results are as follows Figure 7 From Table 4, it can be seen that: compared with clean soil (control), 50 mg / kg tebuconazole contaminated soil treatment can significantly inhibit the plant height of cabbage, which significantly reduced the plant height of cabbage by 40.21%; compared with clean soil (control), 50 mg / kg tebuconazole contaminated soil treatment can significantly reduce the aboveground fresh weight of cabbage, which reduced the aboveground fresh weight of cabbage by 34.83%. Compared with 50 mg / kg tebuconazole contaminated soil, under the treatment of adding HCL-7 strain, the taproot thickness, plant height and aboveground fresh weight of cabbage were all increased, which increased 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. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims

1. Hunan Pseudomonas, characterized in that: The Hunan Pseudomonas is Pseudomonas hunanensis, its strain number is HCL-7, and its registration number in the General Microbiological Center of China National Microbiological Culture Collection is CGMCC No.31920.

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

3. A microbial agent, characterized in that: The bacterial agent contains the Hunan Pseudomonas according to claim 1, the metabolite of the Hunan Pseudomonas according to claim 1 and / or the culture according to claim 2.

4. The bacterial 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 uses of the Hunan Pseudomonas according to claim 1 or the metabolites of the Hunan Pseudomonas, the culture according to claim 2, or the bacterial agent according to claim 3 or 4: (A1) Phosphate dissolution; (A2) preparing a phosphate-dissolving product; (A3) siderophore production; (A4) preparing a siderophore-producing product; (A5) produces IAA; (A6) preparing an IAA-producing product; (A7) promoting plant growth; (A8) Preparation of products for promoting plant growth.

6. Any of the following uses of the Hunan Pseudomonas according to claim 1 or the metabolites of the Hunan Pseudomonas, the culture according to claim 2, or the bacterial agent according to claim 3 or 4: (A9) degradation of tebuconazole; (A10) preparing a product for degrading tebuconazole.

7. Any of the following uses of the Hunan Pseudomonas according to claim 1 or the metabolites of the Hunan Pseudomonas, the culture according to claim 2, or the bacterial agent according to claim 3 or 4: (B1) Promote plant growth under tebuconazole stress; (B2) preparing a product for promoting plant growth under tebuconazole stress; (B3) Alleviate the stress of tebuconazole on plant growth; (B4) preparing a product that alleviates the stress induced by tebuconazole on plant growth.

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

9. A method for promoting plant growth under tebuconazole stress and / or alleviating the stress of tebuconazole on plant growth, comprising the following steps: applying the Hunan Pseudomonas according to claim 1 or the metabolite of the Hunan Pseudomonas or the culture according to claim 2 or the bacterial agent according to claim 3 or 4 to a plant growth matrix contaminated with tebuconazole, thereby promoting plant growth under tebuconazole stress and / or alleviating the stress of tebuconazole on plant growth.

10. The bacterial agent, use or method according to any one of claims 4 to 9, characterized in that: The plant is any of the following: (C1) Crops; (C2) Vegetables; (C3) Leafy vegetables; (C4) Cabbage.

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