Enterobacter cloacae and application thereof in tobacco growth promotion

Abstract

The application discloses an Enterobacter cloacae and application thereof in tobacco growth promotion, and belongs to the technical field of microorganisms. The Enterobacter cloacae FBH1 is preserved in the China Center for Type Culture Collection (CCTCC), the preservation address is Wuhan University, Wuhan, China, the preservation number is CCTCC NO: M 20241838, and the preservation time is August 23, 2024. The Enterobacter cloacae (FBH1) is isolated for the first time. Enterobacter cloacae The strain FBH1 has good abilities of phosphorus solubilization, potassium solubilization, IAA secretion, iron carrier production and ACC deaminase production, can significantly improve the seed germination rate, germination potential, seedling length and root length, thereby promoting seed germination, can significantly promote plant growth, can reduce the enrichment of phosphorus and potassium elements in tobacco planting layer soil, and can improve the fertilizer utilization efficiency, and therefore has good application prospect.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a strain of Enterobacter cloacae and its application in tobacco growth promotion. Background Technology

[0002] Flue-cured tobacco is an important economic crop in my country, ranking among the world's top in terms of total planting area, total output, and total sales. Hubei Province has a long history of tobacco cultivation and is one of the main tobacco-producing areas in the country. In recent years, the long-term use of chemical fertilizers in Hubei's tobacco-growing areas has led to an imbalance in the soil microbial community structure, resulting in the enrichment of nutrients such as nitrogen, phosphorus, and potassium in the soil, leading to low fertilizer utilization and a decline in tobacco yield and quality.

[0003] Plant growth-promoting bacteria can decompose insoluble elements such as phosphorus and potassium in the soil, secrete plant hormones, and produce siderophores and ACC deaminase, thus playing an important role in promoting plant growth. Specifically, phosphorus-solubilizing microorganisms in plant growth-promoting bacteria can convert insoluble phosphorus into soluble phosphorus through acidification, chelation, and exchange reactions, releasing it into the soil for plant absorption and utilization. Potassium-solubilizing bacteria in plant growth-promoting bacteria produce organic and inorganic acids that can directly promote the weathering of potassium-containing minerals by lowering the pH of their surrounding environment, leading to the slow release of exchangeable potassium, increasing the content of available potassium in the environment, and thus promoting plant growth. Microorganisms in plant growth-promoting bacteria can directly secrete plant hormones (IAA), thereby promoting plant growth. Microorganisms in plant growth-promoting bacteria can produce siderophores to form iron chelates, effectively absorbing iron with very low solubility in the environment to promote plant growth. Microorganisms in plant growth-promoting bacteria produce ACC deaminase, which decomposes ACC in ethylene synthesis into α-butanone and NH3, reducing the plant's ethylene level, alleviating plant stress, and thus promoting plant growth.

[0004] Currently, there are many types of growth-promoting microbial agents on the market, with various strains. However, since the activity of microorganisms is affected by factors such as temperature, humidity, and soil conditions, and given the wide distribution of tobacco-growing areas in China and the significant differences in natural conditions, screening suitable growth-promoting bacteria for tobacco-growing areas in Hubei and reconstructing beneficial rhizosphere microbial communities are key to their large-scale promotion and application.

[0005] However, there are few reports of Enterobacter cloacae simultaneously possessing functions such as phosphorus solubilization, potassium solubilization, IAA secretion, siderophore production, ACC deaminase production, seed germination promotion, and plant growth promotion. Summary of the Invention

[0006] The purpose of this invention is to provide a strain of *Enterobacter cloacae* and its application in promoting tobacco growth. The *Enterobacter cloacae* FBH1 provided by this invention exhibits good abilities in phosphorus and potassium solubilization, IAA secretion, siderophore production, ACC deaminase production, and promotion of seed germination and plant growth. Therefore, it has promising applications in improving fertilizer utilization and / or promoting plant growth.

[0007] In a first aspect, the present invention provides a strain of Enterobacter cloacae (… Enterobacter cloacae FBH1, Enterobacter cloacae ( Enterobacter cloacae The accession number of FBH1 is CCTCC NO:M 20241838.

[0008] This strain FBH1 was isolated from the rhizosphere soil of flue-cured tobacco in a typical tobacco-growing area of ​​Dongping Village, Yeren Valley Town, Fang County, Hubei Province. When cultured on LB agar plates at 28°C for 1-2 days, strain FBH1 produced round, white colonies with a diameter of 2-3 mm, smooth surface, non-sticky texture, easy to pick up, opaque colonies with clear edges, and a fermented odor.

[0009] Furthermore, the 16S rDNA of strain FBH1 was sequenced, and its 16S rDNA sequence was BLASTed and aligned using NCBI (https: / / www.ncbi.nlm.nih.gov / ). A phylogenetic tree was constructed, and the results showed that strain FBH1 had high homology with Enterobacteriaceae and MK188868.1. Enterobacter cloacae strain EC-3 belongs to the same branch; based on morphological characteristics and molecular biological identification results, strain FBH1 was named Enterobacter cloacae (Enterobacter cloacae). Enterobacter cloacae FBH1.

[0010] In a second aspect, the present invention provides a biological agent comprising the aforementioned Enterobacter cloacae (… Enterobacter cloacae FBH1.

[0011] In some implementations, the biological agent is a liquid agent.

[0012] In some implementations, the liquid bacterial agent contains Enterobacter cloacae (… Enterobacter cloacae The content of FBH1 is ≥1×10 7 cfu / mL.

[0013] Understandably, biological agents can be selected from conventional formulations in the prior art according to actual usage needs. For example, they can also be solid agents, and solid agents can be prepared using conventional methods in the art.

[0014] In a third aspect, the present invention provides a method for preparing the above-described biological agent, comprising the following steps: taking the above-described Enterobacter cloacae (… Enterobacter cloacae FBH1 was inoculated into liquid culture medium and cultured. The resulting culture was separated and resuspended in a solvent to obtain a biological agent.

[0015] The preparation method of the biological agent provided by this invention is simple, environmentally friendly, and has good biosafety.

[0016] Understandably, the liquid culture medium can be any common culture medium in the art, as long as it meets the requirements of this Enterobacter cloacae (…). Enterobacter cloacae Normal growth of FBH1 is sufficient. Conventional culture conditions can be selected. In this invention, the liquid culture medium is preferably LB medium.

[0017] In some implementations, separation includes centrifugation for 3-10 minutes at 5000-7000 r / min.

[0018] In some implementations, the solvent includes at least one of PBS buffer and sterile water.

[0019] In a fourth aspect, the present invention provides Enterobacter cloacae as described above (… Enterobacter cloacae FBH1, any of the above-mentioned biological agents or the biological agents obtained by the above preparation methods, are used in at least one of the following: A1) phosphorus solubilization; A2) potassium solubilization; A3) secretion of IAA; A4) production of siderophores; A5) production of ACC deaminase.

[0020] In a fifth aspect, the present invention provides Enterobacter cloacae as described above (… Enterobacter cloacae FBH1, the application of any of the above-mentioned biological agents or biological agents prepared by the above-mentioned methods in promoting seed germination.

[0021] In some implementation schemes, the seeds include at least one of rapeseed seeds, bok choy seeds, rice seeds, and flue-cured tobacco seeds.

[0022] It is understood that the seeds can be conventional plant seeds in the prior art. In this invention, the seeds preferably include at least one of rapeseed seeds, Chinese cabbage seeds, rice seeds, and flue-cured tobacco seeds.

[0023] In a sixth aspect, the present invention provides Enterobacter cloacae as described above (… Enterobacter cloacae FBH1, the application of any of the above-mentioned biological agents or biological agents prepared by the above-mentioned methods in improving fertilizer utilization and / or promoting plant growth.

[0024] In some implementations, the plant includes tobacco.

[0025] Understandably, the plant can be any plant conventional in the prior art, and in this invention, the plant preferably includes tobacco.

[0026] The beneficial effects of this invention are: unlike the prior art, this invention is the first to isolate a strain of Enterobacter cloacae (…). Enterobacter cloacaeFBH1 strain exhibits good abilities in phosphorus and potassium solubilization, IAA secretion, siderophore production, and ACC deaminase production. It promotes seed germination by significantly increasing seed germination rate, germination potential, seedling length, and root length, and can also significantly promote plant growth. At the same time, it can reduce the accumulation of phosphorus, potassium, and other elements in the topsoil of tobacco-growing areas, thereby improving fertilizer utilization efficiency. Therefore, it has good application prospects. Attached Figure Description

[0027] Figure 1 This is a colony morphology diagram of strain FBH1 in Example 1 of the present invention on an LB plate;

[0028] Figure 2 This is a diagram of the phosphorus-releasing rings formed by strain FBH1 in Example 1 of the present invention after being cultured on an inorganic phosphorus solid medium;

[0029] Figure 3 This is a diagram of the potassium-solubilizing circles formed by strain FBH1 in Example 1 of the present invention after being cultured on M1997 potassium-solubilizing bacteria solid medium;

[0030] Figure 4 This is an image of the orange halo formed after strain FBH1 in Example 1 of the present invention was cultured on CAS detection solid medium;

[0031] Figure 5 This is the phylogenetic tree diagram constructed in Embodiment 1 of the present invention;

[0032] Figure 6 This is a color reaction diagram of the supernatant of strain FBH1 in Example 5 of the present invention after adding Salkowski reagent colorimetric solution, wrapping it with tin foil, and reacting at room temperature in the dark for 30 min.

[0033] Figure 7 This is a graph showing the ACC deaminase activity results of strain FBH1 in Example 6 of the present invention;

[0034] Figure 8 This is a graph showing the seed germination trend of strain FBH1 in Example 7 of the present invention;

[0035] Figure 9 This is a graph showing the fresh and dry weight results of the aboveground and underground parts of flue-cured tobacco after growth promotion by strain FBH1 in Example 8 of the present invention.

[0036] Biological Preservation

[0037] The strain provided by this invention is deposited at the China Center for Type Culture Collection (CCTCC), located at Wuhan University, Wuhan, China, with accession number CCTCC NO: M 20241838, deposited on August 23, 2024, and classified as *Enterobacter cloacae*. Enterobacter cloacaeFBH1. Detailed Implementation

[0038] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0039] Experimental methods not specifically described in the examples are generally performed according to conventional experimental methods in the field of molecular biology, including but not limited to those described in *Molecular Cloning: A Laboratory Manual* by M.R. Green and *Molecular Biology* by Robert F. Weaver, or according to the experimental methods recommended by the reagent kit and instrument manufacturers. Unless otherwise specified, all reagents and biological materials used in the examples are commercially available.

[0040] The culture medium used in this invention is as follows:

[0041] The LB liquid culture medium formula is: 10.0g tryptone, 5.0g yeast extract, 10.0g sodium chloride, and 1000mL distilled water.

[0042] The formula for inorganic phosphorus solid culture medium is as follows: 10.0g glucose, 0.5g ammonium sulfate, 0.5g yeast extract, 0.3g sodium chloride, 0.3g potassium chloride, 0.3g magnesium sulfate, 0.03g ferrous sulfate, 0.03g manganese sulfate, 5.0g tricalcium phosphate, 15g agar, and 1000mL distilled water.

[0043] The formula for M1997 potassium-solubilizing bacteria solid culture medium is as follows: 0.5g magnesium sulfate, 0.1g calcium carbonate, 2.0g potassium aluminum silicate, 5.0g glucose, 0.005g ferric chloride, 2.0g calcium carbonate, 15g agar, and 1000mL distilled water.

[0044] The formula for silicate solid culture medium is as follows: 5.0g glucose, 0.5g magnesium sulfate, 0.1g calcium carbonate, 2.0g disodium bicarbonate, 0.005g ferric chloride, 1.0g glass powder, 15g agar, and 1000mL distilled water.

[0045] The LB liquid medium formula containing 0.5 g / L L-tryptophan is as follows: 0.5 g L-tryptophan, 10.0 g tryptone, 5.0 g yeast extract, 10.0 g sodium chloride, and 1000 mL distilled water.

[0046] The CAS detection medium formula is as follows: Chromium Azurite S (CAS) 60.5 mg, hexadecyltrimethylammonium bromide (HDTMA) 72.9 mg, ferric chloride hexahydrate 2.645 mg, 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, agar 9000 mg, pH adjusted to 6.8±0.1, and distilled water 1000 mL.

[0047] The MKB iron-limiting culture medium formula is as follows: ① 50g casein amino acids, 15mL glycerol (glycerol) and 785mL deionized water; ② 2.5g dipotassium hydrogen phosphate dissolved in 100mL deionized water; ③ 2.5g magnesium sulfate heptahydrate dissolved in 100mL deionized water. After preparation, sterilize each separately and adjust the pH to 7.2. When using, mix ①②③ together.

[0048] The nitrogen-free DF medium formula is as follows: 4g potassium dihydrogen phosphate, 6g disodium hydrogen phosphate, 0.2g magnesium sulfate heptahydrate, 2g glucose, 2g gluconic acid, 2g citric acid, 0.1mL trace element stock solution ①, 0.1mL trace element stock solution ②, and 1000mL distilled water. The components of trace element stock solution ① are as follows: per 100mL, it contains 10mg boric acid, 11.2mg magnesium sulfate, 124.6mg zinc sulfate, 78.2mg copper sulfate, and 10mg molybdenum trioxide, and is stored at 4℃. The components of trace element stock solution ② are as follows: per 10mL, it contains 100mg ferrous sulfate heptahydrate, and is stored at 4℃.

[0049] In this invention, the preparation method of the CAS detection solution is as follows:

[0050] First, prepare the following solution:

[0051] 1mM ferric chloride stock solution: Weigh 0.2703g of ferric chloride hexahydrate and dissolve it in 1L of 10mM hydrochloric acid;

[0052] CAS stock solution: Weigh 0.2421g of CAS (chromium azurite) and dissolve it in 200mL of deionized water;

[0053] HTDMA (hexadecyltrimethylammonium bromide) solution: Weigh 0.0219 g of HTDMA and dissolve it in 50 mL of water;

[0054] Piperazine buffer: Weigh 4.3079 g of anhydrous piperazine and dissolve it in 30 mL of water, then adjust the pH to 5.6 with hydrochloric acid;

[0055] Then, take 1.5 mL of 1 mM ferric chloride stock solution and add 7.5 mL of CAS stock solution to it and mix well. While stirring, continue to add 50 mL of HTDMA solution, then add 30 mL of piperazine buffer, and finally add deionized water to prepare 100 mL of CAS detection solution.

[0056] In this invention, the Salkowski reagent colorimetric solution consists of 1 mL of solution A and 50 mL of solution B, mixed thoroughly and used immediately; wherein, the preparation of solution A and solution B is as follows:

[0057] Solution A (0.5M FeCl3): Weigh 4.055g of FeCl3 and add water to make up to 50mL;

[0058] Solution B (35% HClO4): Measure 50 mL of HClO4 and add water to make up to 100 mL.

[0059] 100 mg / L IAA Standard Stock Solution: Weigh 10 mg of IAA, dissolve it in a small amount of ethanol, and then dilute to 100 mL with deionized water to obtain an IAA standard stock solution with a concentration of 100 mg / L.

[0060] Example 1: Screening and Identification of Strains FBH1

[0061] 1.1 Sample Source

[0062] Rhizosphere soil from flue-cured tobacco in a typical tobacco-growing area of ​​Dongping Village, Yeren Valley Town, Fang County, Hubei Province was selected. A five-point random sampling method was used. The entire tobacco plant was pulled out, and the loose soil at the roots was shaken off. The remaining soil was scraped into a numbered self-sealing bag with a brush. The soil samples were mixed and transported to the laboratory with dry ice and stored at -80℃.

[0063] 1.2 Screening of strain FBH1

[0064] Prepare a sterilized 150mL Erlenmeyer flask, weigh 1.00g of tobacco rhizosphere soil into it, add 99mL of sterile water, and shake in a constant temperature shaker at 28℃ and 180r / min for 30min to obtain 10 -2 Dilute the soil bacterial suspension using a dilution gradient. Using a pipette, extract the supernatant from the settled soil bacterial suspension and dilute it according to a 10:10 dilution gradient. -3 10 -4 10 -5Diluents were prepared. 100 μL of each serial dilution was applied evenly to inorganic phosphate solid medium using a glass spreader sterilized by flame burning with an alcohol lamp. Three replicates were performed for each dilution. The culture was incubated upside down at 28°C for 5 days. After 5 days, colonies with a clear phosphate-solubilizing zone were picked and streaked onto inorganic phosphate solid medium for purification. The culture was then incubated upside down at 28°C for 3 days. The streak purification process was repeated until single colonies were obtained. The purified single colonies were inoculated into a mixture of 1 mL glycerol and 1 mL LB liquid medium and stored at -80°C for later use.

[0065] The obtained strains were inoculated into M1997 potassium-solubilizing bacteria solid medium and silicate solid medium, and cultured upside down in a 28℃ incubator for 3 days. The growth of the strains and the formation of clear zones were observed to preliminarily determine their potassium-solubilizing ability. Twelve strains with both phosphorus and potassium solubilizing properties were obtained. These 12 strains were inoculated into CAS detection medium and cultured upside down at 28℃ for 3 days. A strain with an orange-yellow halo was obtained and named FBH1.

[0066] The colony morphology of strain FBH1 after incubation on LB solid medium at 28°C for 1-2 days is shown in the figure below. Figure 1 As shown.

[0067] from Figure 1 As can be seen, strain FBH1 is round, white, 2-3 mm in diameter, with a smooth surface, non-sticky texture, easy to pick up, with opaque colonies, clear edges, and a fermented smell.

[0068] The phosphorus solubilization effect of strain FBH1 after culturing at 28℃ for 5 days on inorganic phosphorus solid medium is as follows: Figure 2 As shown.

[0069] from Figure 2 As can be seen, strain FBH1 formed a phosphorus-solubilizing zone after being cultured on inorganic phosphorus solid medium, indicating that strain FBH1 has good phosphorus-solubilizing ability.

[0070] The potassium-solubilizing effect of strain FBH1 after culturing at 28°C for 3 days on M1997 potassium-solubilizing bacteria solid medium is as follows: Figure 3 As shown.

[0071] from Figure 3 As can be seen, strain FBH1 formed potassium-solubilizing zones after being cultured on M1997 potassium-solubilizing bacteria solid medium, indicating that strain FBH1 has good potassium-solubilizing ability.

[0072] The siderogenic effect of strain FBH1 after culturing at 28℃ for 3 days on CAS detection medium is as follows: Figure 4 As shown.

[0073] from Figure 4 As can be seen, strain FBH1 formed an orange-yellow halo after being cultured on CAS detection medium, indicating that strain FBH1 has a good ability to produce siderophores.

[0074] 1.3 Biological identification of strain FBH1

[0075] The selected strain FBH1 was sent to Guangdong Megagene Technology Co., Ltd. for sequencing. After obtaining the genomic DNA of the target strain, it was amplified using appropriate primers (e.g., 27F / 1492R primers for 16S rDNA fragments). The amplified products were sequenced using an ABI 3730 sequencing platform.

[0076] The sequencing sequence (SEQ ID NO: 1) was BLASTed in NCBI (https: / / www.ncbi.nlm.nih.gov / ) and a phylogenetic tree was constructed. The results are as follows: Figure 5 As shown.

[0077] from Figure 5 As can be seen, strain FBH1 has a high degree of homology with Enterobacteriaceae and MK188868.1. Enterobacter cloacae strain EC-3 belongs to the same branch; based on morphological characteristics and molecular biological identification results, strain FBH1 was named Enterobacter cloacae (Enterobacter cloacae). Enterobacter cloacae FBH1.

[0078] Example 2: Quantitative determination of phosphorus solubilization ability of strain FBH1

[0079] Strain strain FBH1 was inoculated into LB liquid medium and cultured on a shaker at 28℃ and 180 rpm for 24 h. Then, 1 mL of the bacterial culture was added to 100 mL of inorganic phosphorus liquid medium (no agar was added to the inorganic phosphorus solid medium). Each treatment was performed in triplicate, and a control group was set up without inoculation with strain FBH1. The culture was carried out on a shaker at 28℃ and 180 rpm for 5 days. On the 5th day, samples were taken, centrifuged at 10000 rpm for 5 min, and the amount of phosphorus solubilized was determined by the molybdenum antimony colorimetric method.

[0080] Using phosphorus standard solution as the standard sample, a standard curve was obtained, with the equation: y = 0.3962x + 0.3669 (R0). 2 =0.9968).

[0081] The phosphorus solubility of strain FBH1 was calculated to be 93.02 mg / L using the standard curve described above.

[0082] Example 3: Quantitative determination of potassium solubilization ability of strain FBH1

[0083] The FBH1 strain was inoculated into LB liquid medium and cultured at 28℃ and 180 r / min for 24 h. Then, 1 mL of the bacterial culture was added to 100 mL of M1997 potassium-solubilizing bacteria liquid medium (M1997 potassium-solubilizing bacteria solid medium can be prepared without agar). Each treatment was repeated in triplicate, and a control group was set up without inoculation with the FBH1 strain. After culturing at 28℃ and 180 r / min for 5 days, samples were taken, centrifuged at 10000 r / min for 5 min, and the potassium solubility was determined by flame photometry.

[0084] Using potassium standard solution as the standard sample, a standard curve was obtained, with the equation: y = 0.0342x - 2.8619 (R² - 2.8619)². 2 =1.000).

[0085] The potassium solubilizing capacity of strain FBH1 was calculated to be 6.30 mg / L using the standard curve described above.

[0086] Example 4: Quantitative determination of siderophore production capacity of strain FBH1

[0087] The FBH1 strain was activated and inoculated at 1% in MKB iron-limiting medium, with three replicates for each strain. The cultures were incubated at 28°C and 180 rpm for 48 h in a shaker. After 48 h, the culture was centrifuged for 15 min at 3500 rpm, and 3 mL of the supernatant was added to the CAS detection solution and mixed thoroughly. One h later, the absorbance (As) at 630 nm was measured using a spectrophotometer, with double-distilled water used as a control for zeroing. Separately, 3 mL of the CAS detection solution was mixed thoroughly with 3 mL of uninoculated MKB liquid medium supernatant, and the absorbance was measured using the same method. This absorbance was used as the reference value (Ar), and the As / Ar ratio was determined.

[0088] The bacteria producing siderophores react with the reaction solution to turn orange-yellow or purplish-red; if no siderophores are produced, the color remains unchanged, remaining blue. Sterile water was used as a control in the experiment. Because the absorbance (Ar) value of the test solution is relatively high, the lower the As / Ar value, the stronger the siderophore-producing ability of the strain. General reference standards are as follows: As / Ar values ​​are in the following ranges: 0-0.25, 0.25-0.5, 0.5-0.75, 0.75-1.0, >1.0, corresponding to ++++, +++, ++, +, and - respectively.

[0089] The results of the siderophore production capacity of strain FBH1 are shown in Table 1 below.

[0090] Table 1 Results of siderophore production capacity of strain FBH1

[0091]

[0092] As can be seen from Table 1, strain FBH1 has a good ability to produce siderophores.

[0093] Example 5: Quantitative determination of IAA production capacity of strain FBH1

[0094] Accurately pipette a measured amount of IAA standard stock solution into a 100 mL volumetric flask, dilute to the mark with deionized water to obtain a series of IAA standard solutions with standard concentrations of 0, 20, 40, 60, 80, 100, and 200 mg / L. Store the prepared solutions in the dark. Take 3 mL of each concentration standard solution and add an equal volume of Salkowski reagent. Incubate in the dark for 30 min, and measure the absorbance at 530 nm using a spectrophotometer. Plot an IAA standard curve with the IAA standard concentration on the x-axis and the absorbance on the y-axis. The equation of the standard curve is: y = 0.0087x + 0.0052(R² / L). 2 =0.997).

[0095] The FBH1 strain was activated and inoculated at 1% in LB medium supplemented with 0.5 g / L tryptophan. After culturing at 28°C and 180 rpm for 3 days, the culture was ultracentrifuged at 10,000 rpm for 10 min. 1 mL of the supernatant was pipetted into a 24-well plate, and an equal volume of Salkowski reagent was added. The plate was then wrapped in aluminum foil and reacted at room temperature in the dark for 30 min, after which the color reaction was observed. Simultaneously, 3 mL of the supernatant was added to an equal volume of Salkowski reagent in a 10 mL centrifuge tube and reacted at room temperature in the dark for 30 min. Figure 6 After the reaction, the absorbance at 530 nm was measured using a spectrophotometer.

[0096] The IAA production capacity of strain FBH1 was calculated to be 206.37 μg / mL based on the IAA standard curve described above.

[0097] Example 6 Quantitative determination of the ACC deaminase production capacity of strain FBH1

[0098] Strain strain FBH1 was inoculated into an Erlenmeyer flask containing 20 mL of LB liquid medium and cultured with shaking at 28 °C and 200 rpm for 12 h. The culture was then centrifuged at 4 °C and 8000 rpm for 10 min. The cells were resuspended in 7.5 mL of nitrogen-free DF medium, and 45 μL of filtered sterile 0.5 mol / L ACC solution was added. The culture was incubated with shaking at 28 °C and 200 rpm for 24 h to induce ACC deaminase activity. The cells were centrifuged at 4 °C and 8000 rpm for 10 min, the supernatant was removed, and the bacterial pellet was collected. The pellet was resuspended in 5 mL of 0.1 mol / L Tris-HCl buffer (pH=7.6) and centrifuged at 4 °C and 8000 rpm for 10 min. This step was repeated three times to completely remove the DF medium. 1 mL of 0.1 mol / L Tris-HCl buffer (pH=7.6) was added to the cells, and the culture was centrifuged at 16000 rpm for 5 min. The cells were then resuspended in 600 μL of DF medium. Dissolve the bacteria in 0.1 mol / L Tris-HCl buffer (pH=8.5) with 30 μL of toluene, and vortex for 30 s to lyse the cells. After incubating at 4°C for 1 h, centrifuge at 12000 rpm for 10 min. Divide the supernatant into two 2 mL centrifuge tubes, 200 μL in each. Store one tube at 4°C for protein assays, and immediately use the other tube for ACC deaminase activity assays.

[0099] ACC deaminase activity assay:

[0100] Construction of the standard curve: α-Butyric acid was prepared into gradient solutions of 0.02, 0.04, 0.06, 0.08, and 0.1 μmol / L using 0.1 mol / L Tris-HCl buffer (pH=8.5). 300 μL of 2,4-dinitrophenylhydrazine was added to each solution, and the mixture was thoroughly mixed and incubated at 30°C for 30 min. Finally, 2 mL of 2 mol / L NaOH was added for color development. After stabilization, the absorbance was measured at 540 nm. A Tris-HCl solution (pH=8.5) was used as a blank control. A standard curve was constructed based on the concentrations of α-butyric acid and their corresponding absorbance values. The equation is: y = 0.1739x - 0.0057 (R² + π / 4)² ... 2 =0.9905).

[0101] Take 200 μL of the lysed bacterial culture, add 20 μL of 0.5 mol / L ACC solution, mix well, and incubate in a water bath at 37 °C for 15 min. Then add 1 mL of 0.56 mol / L HCl, centrifuge at 25 °C and 16000 rpm for 5 min, take 1 mL of the supernatant, add 800 μL of 0.56 mol / L HCl, mix well, add 300 μL of 2,4-dinitrophenylhydrazine, and incubate in a water bath at 30 °C for 30 min. Finally, add 2 mL of 2 mol / L NaOH for color development, and measure the absorbance at 540 nm. Use distilled water as a blank control.

[0102] OD of the sample 540 Substitute these values ​​into the above equation to calculate the content of α-butanone, and then calculate the amount (μmol) of α-butanone produced by ACC deaminase per unit time, which is the enzyme activity per unit U.

[0103] Protein detection:

[0104] Preparation of Standard Curve: For the 0~100μg / mL standard curve: Take six clean, stoppered test tubes (10mL each) and sample according to Table 2. After stoppering, invert each tube to mix the solutions vertically. After standing for 2 minutes, measure the optical density (OD) at 595nm using a 1cm cuvette. Record the measured OD of each tube. 595nm A standard curve was constructed, with the equation: y = 0.188x - 0.1241 (R²). 2 =0.9905).

[0105] Table 2 Sampling of Standard Curve

[0106]

[0107] Take two more 10mL stoppered test tubes, and pipette 1mL of the extract (make one replicate) into each stoppered graduated test tube. Add 5mL of Coomassie Brilliant Blue G-250 protein reagent, mix thoroughly, and let stand for 2 minutes. Then, measure the optical density (OD) at 595nm using a 1cm path length cuvette. 595nm The protein content in the extract of the sample to be tested was calculated using a standard curve, with test tube 1 of the standard curve serving as a blank.

[0108] The specific activity (U / mg) is obtained by dividing the unit enzyme activity U by the total protein mass, which represents the ACC deaminase activity of strain FBH1.

[0109] The calculated ACC deaminase activity of strain FBH1 was 11.60 U / mg. Figure 7 ).

[0110] In summary, strain FBH1, after culturing at 28°C for 5 days in inorganic phosphorus liquid medium, produced 93.02 mg / L of phosphorus; after culturing at 28°C for 5 days in M1997 potassium-solubilizing bacteria liquid medium, produced 6.30 mg / L of potassium; after culturing at 28°C for 2 days in MKB iron-limited medium, its As / Ar ratio was 0.914, indicating the production of siderophores; after culturing at 28°C for 3 days in LB medium containing 0.5 g / L tryptophan, it produced as much as 206.37 μg / mL of IAA; and after culturing at 28°C for 2 days in nitrogen-free DF medium, it produced 11.60 U / mg of ACC deaminase.

[0111] Example 7: Seed germination promotion test of strain FBH1

[0112] Strain preparation: Activate the FBH1 strain and wait for colonies to grow. Inoculate selected colonies into 10 mL of LB liquid medium and incubate at 28℃ and 180 rpm for 16-24 h with shaking. Count the bacterial count using the hemocytometer method to determine if the bacterial count is ≥1×10⁻⁶. 7 cfu / mL. Centrifuge the FBH1 bacterial culture at 6000 r / min for 5 min, remove the supernatant, and resuspend in 100 mL of sterile water to obtain the FBH1 bacterial agent.

[0113] The experimental seeds used were Shanghai bok choy (original variety), Zhongshuang 9 rapeseed, Hanyou 73 rice, and Yunyan 87 tobacco.

[0114] After rapid disinfection with 75% ethanol, the seeds were soaked in 1% sodium hypochlorite for 15 minutes and then rinsed 3-5 times with sterile water. The disinfected seeds were then soaked in FBH1 inoculant for 30 minutes. The control (CK) consisted of untreated seeds soaked in sterile water. Filter paper was placed in sterile petri dishes, moistened with a suitable amount of sterile water, and the treated seeds were evenly spread on the filter paper, 100 seeds per dish (50 rice seeds per dish). Each treatment was repeated 3 times. The seeds were cultured at room temperature for 144 hours (240 hours for tobacco). Seed germination was observed every 12 hours. On day 7 (day 10 for tobacco), seedling length, root length, germination rate, and germination potential were measured. The results are shown in Table 3 below. Figure 8 As shown.

[0115] Table 3 Germination Index

[0116]

[0117] From Table 3 and Figure 8 As can be seen, FBH1 inoculant treatment significantly promoted the growth of all four crops.

[0118] Specifically, regarding germination potential, the FBH1 inoculant treatment group showed increases of 29.34%, 22.00%, 36.67%, and 17.67% in germination potential compared to the CK control group, respectively. This indicates that the FBH1 inoculant treatment has a significant promoting effect on crop germination in the early stages, especially on budding cabbage and rice.

[0119] In terms of germination rate, the FBH1 inoculant treatment also showed advantages. Compared with the CK control group, the germination rates of Chinese cabbage, rapeseed, rice, and flue-cured tobacco increased by 8.67%, 2.33%, 9.33%, and 23.50%, respectively, under the FBH1 inoculant treatment. This result further confirms the positive effect of FBH1 inoculant treatment on improving crop germination rate.

[0120] FBH1 inoculant treatment also significantly promoted crop growth. The seedling length of Chinese cabbage, rapeseed, and rice under FBH1 treatment increased by 9.82 mm, 8.15 mm, and 17.21 mm, respectively, compared with the CK control group. The increase in seedling length of flue-cured tobacco was smaller (only 1.10 mm), but it still reached a statistically significant level.

[0121] Regarding root length, FBH1 inoculant treatment also had a positive impact on root growth in the four crops. Compared with the control group (CK), the root lengths of Chinese cabbage, rapeseed, rice, and flue-cured tobacco increased by 8.26 mm, 25.14 mm, 11.90 mm, and 3.34 mm, respectively, under FBH1 treatment. This result indicates that FBH1 treatment not only promoted the aboveground growth of crops but also significantly enhanced root development.

[0122] In summary, FBH1 inoculant treatment significantly promoted the germination potential, germination rate, seedling length, and root length of four crops: Chinese cabbage, rapeseed, rice, and flue-cured tobacco.

[0123] Example 8: Experiment on the growth-promoting effect of strain FBH1 on flue-cured tobacco

[0124] The experiment used polyethylene plastic flowerpots, each containing 1.5 kg of soil. Yunyan No. 87 tobacco seedlings were used as the test subject. The inoculant prepared in Example 7 was used to drench the roots of tobacco seedlings, with an equal volume of sterile water used as a control. Each seedling was treated with 50 mL of sterile water at 7-day intervals. Deionized water was used for irrigation throughout the experiment.

[0125] Group CK: Yellow-brown soil from Huazhong Agricultural University;

[0126] FBH1 group: Yellow-brown soil from Huazhong Agricultural University + FBH1 inoculant;

[0127] For each treatment group, plants were harvested on the 30th day of growth. On the day of sampling, plant height, maximum leaf length, maximum leaf width, aboveground fresh weight, aboveground dry weight, underground fresh weight, and underground dry weight were measured. The results are shown in Table 4 below. Figure 9 As shown.

[0128] Table 4. Results of the effect of FBH1 inoculant on the growth of flue-cured tobacco.

[0129]

[0130] From Table 4 and Figure 9 The results show that the FBH1 inoculant has a significant effect on the growth of flue-cured tobacco (P < 0.05). The drenching treatment strain first attaches to the root system of the tobacco plant, thereby promoting plant growth and increasing plant biomass. Plant height increased by 72.85% compared to the control, maximum leaf length increased by 8.71%, and maximum leaf width increased by 11.31%. Aboveground fresh weight increased by 44.40%, underground fresh weight increased by 44.81%, aboveground dry weight increased by 42.38%, and underground dry weight increased by 50.00%. These results indicate that the FBH1 inoculant has a significant growth-promoting effect on tobacco.

[0131] In summary, the Enterobacter cloacae provided by this invention ( Enterobacter cloacae FBH1 has good abilities to solubilize phosphorus and potassium, secrete IAA, produce siderophores, and produce ACC deaminase. It promotes seed germination by significantly improving seed germination rate, germination potential, seedling length and root length, and can also significantly promote plant growth.

[0132] It should be noted that all the above embodiments belong to the same inventive concept, and the descriptions of each embodiment have different focuses. Where the description in a particular embodiment is not detailed, please refer to the description in other embodiments.

[0133] The embodiments described above are merely illustrative of implementation methods of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A strain of Enterobacter cloacae ( Enterobacter cloacae FBH1, characterized in that, The Enterobacter cloacae FBH1 strain is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO:M 20241838.

2. A biological agent, characterized in that, Includes Enterobacter cloacae FBH1 as described in claim 1.

3. The biological agent according to claim 2, characterized in that, The biological agent is a liquid agent.

4. The biological agent according to claim 3, characterized in that, The liquid bacterial agent contains ≥1×10⁻⁶ Enterobacter cloacae FBH1. 7 cfu / mL.

5. A method for preparing a biological agent as described in any one of claims 2-4, characterized in that, The process includes the following steps: inoculating the Enterobacter cloacae FBH1 of claim 1 into a liquid culture medium for cultivation, and then separating and resuspending the resulting culture in a solvent to obtain a biological agent.

6. The use of *Enterobacter cloacae* FBH1 as described in claim 1, the biological agent as described in any one of claims 2-4, or the biological agent prepared by the method of claim 5 in at least one of the following: A1) Phosphorus solubilization; A2) Potassium solubilization; A3) IAA secretion; A4) Siderophore production; A5) ACC deaminase production.

7. The application of Enterobacter cloacae FBH1 as described in claim 1, the biological agent as described in any one of claims 2-4, or the biological agent prepared by the method in claim 5 in promoting seed germination.

8. The application according to claim 7, characterized in that, The seeds are at least one of rapeseed seeds, bok choy seeds, rice seeds, and flue-cured tobacco seeds.

9. The application of Enterobacter cloacae FBH1 as described in claim 1, the biological agent as described in any one of claims 2-4, or the biological agent prepared by the method in claim 5 in improving fertilizer utilization and / or promoting plant growth.

10. The application according to claim 9, characterized in that, The plant in question is tobacco.

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