Enterobacter cloacae and application thereof in tobacco growth promotion

Abstract

The invention discloses 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 obtained through separation for the first time, the strain FBH1 has the good capacity of dissolving phosphorus and potassium, secreting IAA, producing siderophores and producing ACC deaminase, seed germination is promoted by remarkably improving the germination rate, germination potential, seedling length and root length of seeds, plant growth can be remarkably promoted, and the yield of plants is increased. Meanwhile, enrichment of phosphorus, potassium and other elements in tobacco planting plough layer soil can be reduced, and the utilization efficiency of the fertilizer is improved, so that the application prospect is good.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and particularly relates to a strain of Enterobacter cloacae and application thereof in tobacco growth promotion. Background Art

[0002] Flue-cured tobacco is an important cash crop in my country, with its total planting area, total output and total sales ranking among the highest in the world. Hubei Province has a long history of tobacco cultivation and is one of the main tobacco producing areas in China. In recent years, the long-term application of chemical fertilizers in Hubei tobacco-growing areas has led to an imbalance in the structure of soil microbial communities, which in turn has caused the enrichment of nutrients such as nitrogen, phosphorus and potassium in the soil of tobacco-growing areas, resulting in low fertilizer utilization and reduced tobacco yield and quality.

[0003] Growth-promoting bacteria can decompose insoluble elements such as phosphorus and potassium in the soil, secrete plant hormones, produce siderophores and ACC deaminase, and therefore play an important role in promoting plant growth. Specifically, the phosphate-dissolving microorganisms in the growth-promoting bacteria can convert insoluble phosphorus into soluble phosphorus through acidification, chelation and exchange reactions and release it into the soil for plant absorption and utilization. The organic and inorganic acids produced by the potassium-dissolving bacteria in the growth-promoting bacteria can directly promote the weathering of potassium-containing minerals by lowering the pH of the surrounding environment, resulting in the slow release of exchangeable potassium and increasing the environmental available potassium content, thereby promoting plant growth. The microorganisms in the growth-promoting bacteria can promote plant growth by directly secreting plant hormones (IAA). The microorganisms in the growth-promoting bacteria can produce siderophores to form iron chelates, so as to effectively absorb the low-solubility iron in the environment to promote plant growth. The microorganisms in the growth-promoting bacteria produce ACC deaminase, which decomposes ACC in the synthetic ethylene into α-ketobutyric acid and NH3, reducing the ethylene level of the plant, alleviating the stress on the plant, and promoting plant growth.

[0004] At present, there are many types of growth-promoting microbial agents on the market, with different strains. However, since the activity of microorganisms is affected by factors such as temperature, humidity, and soil conditions, and China's tobacco-growing areas are widely distributed and the natural conditions vary greatly, therefore, screening growth-promoting bacteria suitable for Hubei tobacco-growing areas and reconstructing the beneficial rhizosphere microbial flora are the keys to their large-scale promotion and application.

[0005] However, there are few reports that Enterobacter cloacae has the functions of solubilizing phosphate and potassium, secreting IAA, producing siderophores, producing ACC deaminase, promoting seed germination, and promoting plant growth. Summary of the invention

[0006] The purpose of the present invention is to provide a strain of Enterobacter cloacae and its application in tobacco growth promotion. The Enterobacter cloacae FBH1 provided by the present invention has good ability to solubilize phosphorus and potassium, secrete IAA, produce siderophores, produce ACC deaminase, promote seed germination and promote plant growth, and therefore has good application prospects 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 deposit number of FBH1 is CCTCC NO:M 20241838.

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

[0009] Furthermore, the 16S rDNA of the strain FBH1 was sequenced, and its 16S rDNA sequence was subjected to BLAST multiple sequence alignment in NCBI (https: / / www.ncbi.nlm.nih.gov / ), and a phylogenetic tree was constructed. The results showed that the strain FBH1 had a high homology with Enterobacter and MK188868.1. Enterobacter cloacae strain EC-3 belonged to the same branch; combined with the morphological characteristics and molecular biological identification results, strain FBH1 was named Enterobacter cloacae ( Enterobacter cloacae )FBH1.

[0010] In a second aspect, the present invention provides a biological bacterial agent, including the above-mentioned Enterobacter cloacae ( Enterobacter cloacae )FBH1.

[0011] In some embodiments, the bioagent is a liquid agent.

[0012] In some embodiments, the liquid bacterial agent contains Enterobacter cloacae ( Enterobacter cloacae ) FBH1 content ≥1×10 7 cfu / mL.

[0013] It is understandable that the biological bacterial agent can be selected according to the actual use needs to select the conventional dosage form in the prior art, for example, it can also be a solid bacterial agent, and the solid bacterial agent can be prepared by conventional methods in the art.

[0014] In a third aspect, the present invention provides a method for preparing the above-mentioned biological agent, comprising the following steps: Enterobacter cloacae )FBH1 was inoculated into a liquid culture medium for cultivation, and the obtained culture was separated and resuspended in a solvent to obtain a biological agent.

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

[0016] It is understood that the liquid culture medium can be a common culture medium in the art, as long as it can meet the requirements of the Enterobacter cloacae ( Enterobacter cloacae ) The normal growth of FBH1 is sufficient. The culture conditions can be selected from conventional conditions. In the present invention, the liquid culture medium is preferably LB culture medium.

[0017] In some embodiments, the separation comprises: centrifugation at 5000-7000 r / min for 3-10 min.

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

[0019] In a fourth aspect, the present invention provides the above-mentioned Enterobacter cloacae ( Enterobacter cloacae ) Use of FBH1, any of the above biological agents or the biological agent obtained by the above preparation method in at least one of the following: A1) solubilizing phosphate; A2) solubilizing potassium; A3) secreting IAA; A4) producing iron carriers; A5) producing ACC deaminase.

[0020] In a fifth aspect, the present invention provides the above-mentioned Enterobacter cloacae ( Enterobacter cloacae ) Application of FBH1, any of the above-mentioned biological agents or the biological agents obtained by the above-mentioned preparation methods in promoting seed germination.

[0021] In some embodiments, the seeds include at least one of rapeseed seeds, Chinese cabbage seeds, rice seeds, and flue-cured tobacco seeds.

[0022] It is understandable that the seeds may be conventional plant seeds in the prior art. In the present 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 the above-mentioned Enterobacter cloacae ( Enterobacter cloacae ) Application of FBH1, any of the above biological agents or the biological agents obtained by the above preparation methods in improving fertilizer utilization and / or promoting plant growth.

[0024] In some embodiments, the plant comprises tobacco.

[0025] It can be understood that the plant can be any conventional plant in the prior art. In the present invention, the plant preferably includes tobacco.

[0026] The beneficial effects of the present invention are as follows: Different from the prior art, the present invention is the first to isolate a strain of Enterobacter cloacae ( Enterobacter cloacae)FBH1, the strain FBH1 has good ability to solubilize phosphorus and potassium, secrete IAA, produce siderophore, and produce ACC deaminase. It promotes seed germination by significantly improving the germination rate, germination potential, seedling length and root length of seeds, and can significantly promote plant growth. At the same time, it can reduce the enrichment of elements such as phosphorus and potassium in the tobacco planting topsoil and improve the utilization efficiency of fertilizers. Therefore, it has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is a colony morphology diagram of the strain FBH1 on the LB plate in Example 1 of the present invention; Figure 2 This is a diagram of the phosphate solubilization circle formed after the strain FBH1 in Example 1 of the present invention is cultured on an inorganic phosphorus solid medium; Figure 3 This is a potassium-dissolving circle diagram formed after the strain FBH1 in Example 1 of the present invention is cultured on the M1997 potassium-dissolving bacteria solid culture medium; Figure 4 This is an orange halo image formed after the strain FBH1 in Example 1 of the present invention is cultured on a CAS detection solid medium; Figure 5 The phylogenetic tree diagram constructed in Example 1 of the present invention; Figure 6 This is a color reaction diagram after adding Salkowski reagent colorimetric solution to the supernatant of strain FBH1 in Example 5 of the present invention, wrapping it with tin foil and reacting it at room temperature in the dark for 30 minutes; Figure 7 This is a graph showing the results of ACC deaminase activity produced by strain FBH1 in Example 6 of the present invention; Figure 8 This is a trend diagram of the strain FBH1 promoting seed germination in Example 7 of the present invention; Fig. 9 This is a graph showing the fresh weight and dry weight of the aboveground and underground parts of flue-cured tobacco after growth promotion by strain FBH1 in Example 8 of the present invention.

[0028] Biological Deposit The strain provided by the present invention is deposited in China Center for Type Culture Collection (CCTCC), deposit address: Wuhan University, Wuhan, China, deposit number: CCTCC NO:M 20241838, deposit time: August 23, 2024, classification name: Enterobacter cloacae ( Enterobacter cloacae )FBH1. DETAILED DESCRIPTION

[0029] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0030] The experimental methods without specific conditions in the examples are usually carried out according to conventional experimental methods in the field of molecular biology, including but not limited to the experimental methods described in Molecular Cloning: A Laboratory Manual by MR Green, Molecular Biology by Robert F. Weaver, etc., or according to the experimental methods recommended by the kit and instrument manufacturers. The reagents and biological materials used in the examples can be obtained from commercial channels unless otherwise specified.

[0031] In the present invention, the culture medium used is as follows: The formula of LB liquid culture medium is: 10.0 g tryptone, 5.0 g yeast extract powder, 10.0 g sodium chloride, and 1000 mL distilled water.

[0032] The formula of inorganic phosphorus solid culture medium is: 10.0g glucose, 0.5g ammonium sulfate, 0.5g yeast extract powder, 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.

[0033] The formula of M1997 potassium-soluble bacterial solid culture medium is: 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.

[0034] The formula of silicate solid culture medium is: 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.

[0035] The formula of LB liquid culture medium containing 0.5 g / L L-tryptophan is: 0.5 g L-tryptophan, 10.0 g tryptone, 5.0 g yeast extract powder, 10.0 g sodium chloride, and 1000 mL distilled water.

[0036] The formula of CAS detection medium is: chrome azuro blue 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, adjust pH to 6.8±0.1, and distilled water 1000 mL.

[0037] The formula of MKB iron-limited culture medium is: ① 50g of casein amino acids, 15mL of glycerol (propylene glycol) plus 785mL of deionized water; ② 2.5g of potassium dihydrogen phosphate dissolved in 100mL of deionized water; ③ 2.5g of magnesium sulfate heptahydrate dissolved in 100mL of deionized water; after separate preparation, sterilize them separately, adjust the pH to 7.2, and mix ①, ②, and ③ together before use.

[0038] The formula of DF culture medium without nitrogen source is: 4 g potassium dihydrogen phosphate, 6 g disodium hydrogen phosphate, 0.2 g magnesium sulfate heptahydrate, 2 g glucose, 2 g gluconic acid, 2 g citric acid, 0.1 mL of trace element mother solution ①, 0.1 mL of trace element mother solution ②, and 1000 mL of distilled water; among them, the components of trace element mother solution ① are as follows: 10 mg boric acid, 11.2 mg magnesium sulfate, 124.6 mg zinc sulfate, 78.2 mg copper sulfate and 10 mg molybdenum trioxide per 100 mL, stored at 4°C; the components of trace element mother solution ② are as follows: 100 mg ferrous sulfate heptahydrate per 10 mL, stored at 4°C.

[0039] In the present invention, the preparation method of CAS detection solution is as follows: First, prepare the following solution: 1mM ferric chloride stock solution: weigh 0.2703g ferric chloride hexahydrate and dissolve in 1L 10mM hydrochloric acid; CAS stock solution: weigh 0.2421 g CAS (chrome azurol) and dissolve it in 200 mL deionized water; HTDMA (hexadecyltrimethylammonium bromide) solution: weigh 0.0219 g HTDMA and dissolve it in 50 mL water; Piperazine buffer: Weigh 4.3079 g of anhydrous piperazine and dissolve it in 30 mL of water. Adjust the pH to 5.6 with hydrochloric acid. Then, take 1.5 mL of 1 mM ferric chloride stock solution, add 7.5 mL of CAS stock solution to it and mix well, continue to add 50 mL of HTDMA solution while stirring, then continue to add 30 mL of piperazine buffer, and finally add deionized water to finally prepare 100 mL of CAS detection solution.

[0040] In the present invention, the components of the Salkowski reagent colorimetric solution are: 1 mL of solution A and 50 mL of solution B are mixed and used immediately after preparation; wherein, the preparation of solution A and solution B is as follows: Solution A (0.5M FeCl3): weigh 4.055g FeCl3 and add water to make up to 50mL; Solution B (35% HClO4): Measure 50 mL of HClO4 and add water to make up to 100 mL.

[0041] 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.

[0042] Example 1 Screening and identification of strain FBH1 1.1 Sample source The rhizosphere soil of flue-cured tobacco in a typical tobacco-growing area of ​​Dongping Village, Yerengu Town, Fang County, Hubei Province was selected, and random sampling was performed using the five-point method. The tobacco plants were pulled out whole, and the loose soil at the roots was shaken off. The remaining part was scraped into numbered ziplock bags with a brush. The soil samples were mixed and transported to the laboratory with dry ice and stored at -80℃.

[0043] 1.2 Screening of strain FBH1 Prepare a sterilized 150 mL Erlenmeyer flask, weigh 1.00 g of tobacco rhizosphere soil, add 99 mL of sterile water, and shake at 28 °C, 180 r / min for 30 min to obtain 10 -2 Dilution gradient soil suspension. Use a pipette to absorb the supernatant of the soil bacterial suspension after standing, and dilute it according to the gradient to make a dilution gradient of 10 -3 , 10 -4 , 10 -5 Dilution. Take 100 μL of gradient dilution and use the alcohol burner flame to burn the sterilized glass coating rod, evenly spread it on the inorganic phosphorus solid culture medium, do three replicates for each gradient, and invert and culture in a 28℃ constant temperature incubator for 5 days. After 5 days, pick the colonies with obvious phosphorus decomposition rings, streak and purify them on the inorganic phosphorus solid culture medium, and invert and culture in a 28℃ constant temperature incubator for 3 days. Repeat the streak purification steps until a single colony is obtained. Inoculate the purified single colony into a mixture of 1mL glycerol + 1mL LB liquid culture medium and store it in a -80℃ refrigerator for later use.

[0044] The strains obtained above were inoculated in M1997 potassium-dissolving bacteria solid culture medium and silicate solid culture medium, and inverted in a constant temperature incubator at 28°C for 3 days. The growth of the strains and the production of transparent circles were observed to preliminarily determine the potassium-dissolving ability of the strains, and 12 strains with both phosphorus and potassium dissolving abilities were obtained. The 12 strains were inoculated in CAS detection medium and inverted at 28°C for 3 days to obtain a strain with an orange halo, which was named FBH1.

[0045] The colony morphology of the strain FBH1 after being cultured on LB solid medium at 28°C for 1-2 days is shown in the figure below. Figure 1 shown.

[0046] from Figure 1 It can be seen that strain FBH1 is round, white, 2-3 mm in diameter, smooth in surface, non-sticky, easy to pick up, opaque in colony, with clear edges and a fermented smell.

[0047] The phosphate solubilization effect of the strain FBH1 after being cultured on inorganic phosphorus solid medium at 28°C for 5 days is as follows Figure 2 shown.

[0048] from Figure 2 It can be seen that strain FBH1 formed a phosphate-solubilizing circle after being cultured on inorganic phosphorus solid culture medium. The results showed that strain FBH1 had good phosphate-solubilizing ability.

[0049] The potassium-dissolving effect of the strain FBH1 after being cultured on M1997 potassium-dissolving bacteria solid medium at 28°C for 3 days is as follows Figure 3 shown.

[0050] from Figure 3 It can be seen that the strain FBH1 formed a potassium-solubilizing circle after being cultured on the M1997 potassium-solubilizing bacteria solid culture medium. The results showed that the strain FBH1 has a good potassium-solubilizing ability.

[0051] The iron carrier production effect of the strain FBH1 after culturing at 28°C for 3 days on CAS test medium is as follows Figure 4 shown.

[0052] from Figure 4 It can be seen that strain FBH1 formed an orange halo after being cultured on the CAS detection medium. The results showed that strain FBH1 had a good ability to produce siderophores.

[0053] 1.3 Biological identification of strain FBH1 The strain FBH1 obtained by screening was sent to Guangdong Meige Gene Technology Co., Ltd. for sequencing. After obtaining the genomic DNA of the target strain, the corresponding primers (such as 27F / 1492R primers for 16S rDNA fragments) were used for amplification. The amplified product was sequenced using the ABI3730 sequencing platform.

[0054] The sequence (SEQ ID NO: 1) was aligned by BLAST in NCBI (https: / / www.ncbi.nlm.nih.gov / ), and a phylogenetic tree was constructed. The results are shown in Figure 5 shown.

[0055] from Figure 5 It can be seen that the strain FBH1 has a high homology with Enterobacter and MK188868.1 Enterobacter cloacae strain EC-3 belonged to the same branch; combined with the morphological characteristics and molecular biological identification results, strain FBH1 was named Enterobacter cloacae ( Enterobacter cloacae )FBH1.

[0056] Example 2 Quantitative determination of phosphate solubilization ability of strain FBH1 The strain FBH1 was inoculated into LB liquid culture medium, and cultured in a shaker at 28°C and 180 r / min for 24 hours. Then, 1 mL of the bacterial solution was added to 100 mL of inorganic phosphorus liquid culture medium (agar is not required in the inorganic phosphorus solid culture medium). Each treatment was repeated 3 times, and a control group was set up without inoculating the strain FBH1. The culture was shaken in a shaker at 28°C and 180 r / min for 5 days. Samples were taken on the 5th day, and the amount of solubilized phosphorus was determined by the molybdenum antimony colorimetric method after centrifugation at 10,000 r / min for 5 minutes.

[0057] Among them, the phosphorus standard solution is used as the standard sample to obtain the standard curve, and its equation is: y=0.3962x+0.3669(R 2 =0.9968).

[0058] The phosphate solubilizing capacity of strain FBH1 was calculated to be 93.02 mg / L using the standard curve above.

[0059] Example 3 Quantitative determination of potassium solubilization ability of strain FBH1 The FBH1 strain was inoculated into LB liquid culture medium, and after shaking and culturing at 28°C and 180 r / min for 24 h, 1 mL of the bacterial solution was added into 100 mL of M1997 potassium-soluble bacterial liquid culture medium (agar is not required in the M1997 potassium-soluble bacterial solid culture medium), with 3 replicates for each treatment. At the same time, a treatment without inoculation of strain FBH1 was set as a control group. After shaking and culturing at 28°C and 180 r / min for 5 days, samples were taken, and after centrifugation at 10,000 r / min for 5 min, the amount of potassium dissolved was determined by flame photometry.

[0060] Among them, potassium standard solution is used as the standard sample to obtain the standard curve, and its equation is: y=0.0342x-2.8619 (R 2 =1.000).

[0061] The potassium-solubilizing capacity of strain FBH1 was calculated to be 6.30 mg / L using the standard curve above.

[0062] Example 4 Quantitative determination of the ability of strain FBH1 to produce siderophore The strain FBH1 was activated and inoculated into the MKB iron-limited medium at 1% of the amount. Each strain was replicated 3 times and cultured in a shaker at 28°C and 180rpm for 48h. After 48h, centrifuge for 15min at a speed of 3500r / min, take 3mL of the supernatant and add it to the CAS detection solution and mix thoroughly. After 1h, the absorbance value (As) at a wavelength of 630nm was measured by a spectrophotometer, and double distilled water was used as a control to adjust the zero. Take another 3mL of CAS detection solution and mix it thoroughly with 3mL of uninoculated MKB liquid culture medium supernatant, and measure it in the same way. The absorbance value is the reference value (Ar), and the As / Ar value is measured.

[0063] Among them, bacteria producing iron carriers can react with the reaction solution to show orange or purple red. If they do not produce, the color will not change and they will remain blue. Sterile water was used as a control in the experiment. Since the absorbance value (Ar) of the test solution is large, the smaller the As / Ar value, the stronger the ability of the strain to produce iron carriers. The general reference standard is: As / Ar values ​​are as follows: 0-0.25, 0.25-0.5, 0.5-0.75, 0.75-1.0, >1.0, which correspond to ++++, +++, ++, +, - respectively.

[0064] The results of the siderophore production ability of strain FBH1 are shown in Table 1 below.

[0065] Table 1 Results of the ability of strain FBH1 to produce siderophores

[0066] As can be seen from Table 1, strain FBH1 has a good ability to produce siderophores.

[0067] Example 5 Quantitative determination of IAA production capacity of strain FBH1 Accurately pipette a quantitative amount of IAA standard stock solution into a 100mL volumetric flask, dilute to volume with deionized water, and obtain a series of IAA standard solutions with standard concentrations of 0, 20, 40, 60, 80, 100, and 200 mg / L, respectively. Store in a dark place after preparation. Take 3mL of each concentration standard solution, add an equal volume of Salkowski reagent colorimetric solution, and treat in a dark place for 30 minutes. Use a spectrophotometer to detect the absorbance at 530nm. Use the IAA standard concentration as the horizontal coordinate and the absorbance as the vertical coordinate to draw the IAA standard curve. The standard curve equation is: y=0.0087x+0.0052(R 2 =0.997).

[0068] The strain FBH1 was activated and inoculated into LB medium supplemented with 0.5g / L tryptophan at 1% concentration. After culturing at 28°C and 180rpm for 3 days, ultracentrifuged at 10000rpm for 10min. After centrifugation, 1mL of supernatant was taken out with a pipette and placed in a 24-well plate. An equal volume of Salkowski reagent colorimetric solution was added and wrapped with tin foil. The plate was reacted at room temperature in the dark for 30min and the color reaction was observed. At the same time, 3mL of supernatant was added to an equal volume of Salkowski reagent colorimetric solution in a 10mL centrifuge tube. The plate was also reacted at room temperature in the dark for 30min ( Figure 6 After the reaction, the absorbance at 530 nm was measured using a spectrophotometer.

[0069] The IAA production capacity of strain FBH1 was calculated to be 206.37 μg / mL using the above IAA standard curve.

[0070] Example 6 Quantitative determination of the ability of strain FBH1 to produce ACC deaminase The strain FBH1 was inoculated into a flask containing 20 mL of LB liquid medium, and cultured at 28°C and 200 rpm for 12 h. The culture was centrifuged at 4°C and 8000 rpm for 10 min. The cells were resuspended in 7.5 mL of DF medium without nitrogen source, and 45 μL of filtered sterilized 0.5 mol / L ACC solution was added. The cells were cultured at 28°C and 200 rpm for 24 h to induce the deaminase activity of ACC. The cells were centrifuged at 4°C and 8000 rpm for 10 min, the supernatant was removed, and the cell pellet was collected. The cells were resuspended in 5 mL of 0.1 mol / L Tris-HCl buffer (pH = 7.6), and the pellet was collected after centrifugation at 4°C and 8000 rpm for 10 min. This step was repeated 3 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 cells were centrifuged at 16000 rpm for 5 min. The cells were then resuspended in 600 μL 0.1mol / L Tris-HCl buffer (pH=8.5), add 30μL toluene, and oscillate on a vortex shaker for 30s to break the bacteria; after standing at 4℃ for 1h, centrifuge at 12000rpm for 10min. Transfer the supernatant into two 2mL centrifuge tubes, taking 200μL of each. One portion is stored in a 4℃ refrigerator for protein detection, and the other portion is immediately used for ACC deaminase activity detection.

[0071] ACC deaminase activity assay: Drawing of the standard curve: Use 0.1mol / L Tris-HCl buffer (pH=8.5) to prepare α-ketobutyric acid into gradient solutions of 0.02, 0.04, 0.06, 0.08, and 0.1μmol / L, and add 300μL of 2,4-dinitrophenylhydrazine respectively. After mixing evenly, water bath at 30℃ for 30min. Finally, add 2mL 2mol / L NaOH for color development. After stabilization, measure its absorbance at 540nm, and use Tris-HCl (pH=8.5) solution as blank control. The standard curve was prepared according to the concentration of α-ketobutyric acid solution and its corresponding absorbance value. The equation is: y=0.1739x-0.0057(R 2 =0.9905).

[0072] Pipette 200 μL of broken bacterial liquid, add 20 μL of 0.5mol / L ACC solution, mix well and incubate in a water bath at 37°C for 15 min, then add 1 mL of 0.56mol / L HCl, centrifuge at 25°C and 16000 rpm for 5 min, take 1 mL of the supernatant, add 800 μL of 0.56mol / L HCl, mix well and add 300 μL of 2,4-dinitrophenylhydrazine, incubate in a water bath at 30°C for 30 min, finally add 2 mL of 2mol / L NaOH for color development, measure the absorbance at 540 nm, and use distilled water as the blank control.

[0073] The OD of the sample 540 Substitute the above equation to calculate the content of α-ketobutyric acid, and then calculate the amount (μmol) of ACC deaminase-catalyzed ACC to produce α-ketobutyric acid per unit time, that is, the unit enzyme activity U.

[0074] Protein detection: Preparation of standard curve: Preparation of 0~100μg / mL standard curve: Take 6 clean 10mL stoppered test tubes and take samples according to Table 2. After plugging, invert the solution in each test tube vertically to mix, let it stand for 2 minutes, and then use a 1cm optical diameter colorimetric cup at a wavelength of 595nm to compare the color, and record the optical density OD of each tube. 595nm , and make a standard curve, the equation of which is: y=0.188x-0.1241 (R 2 =0.9905).

[0075] Table 2 Standard curve sampling

[0076] Take another 2 10mL stoppered test tubes, draw 1mL of the extract (do one repeat), put it into the stoppered graduated test tube, add 5mL of Coomassie Brilliant Blue G-250 protein reagent, mix thoroughly, let it stand for 2 minutes, and then use a 1cm optical path colorimetric cup to compare the color at 595nm, and record the optical density OD 595nm The protein content in the sample extract to be tested is calculated through the standard curve, and test tube No. 1 of the standard curve is used as the blank.

[0077] The specific activity (U / mg) was obtained by dividing the unit enzyme activity U by the total protein mass, which represents the ACC deaminase activity of strain FBH1.

[0078] The ACC deaminase activity of strain FBH1 was calculated to be 11.60 U / mg ( Figure 7 ).

[0079] In summary, the amount of phosphorus solubilized by strain FBH1 after being cultured at 28℃ for 5 days in inorganic phosphorus liquid medium was 93.02 mg / L; the amount of potassium solubilized after being cultured at 28℃ for 5 days in M1997 potassium-soluble bacteria liquid medium was 6.30 mg / L; after being cultured at 28℃ for 2 days in MKB iron-limited medium, its As / Ar=0.914, and iron carriers could be produced; the amount of IAA produced after being cultured at 28℃ for 3 days in LB medium containing 0.5 g / L tryptophan was as high as 206.37 μg / mL; the activity of ACC deaminase produced after being cultured at 28℃ for 2 days in DF medium without nitrogen source was 11.60 U / mg.

[0080] Example 7 Experiment on promoting seed germination by strain FBH1 Preparation of strains: Activate the FBH1 strain, wait for colonies to grow, inoculate the selected colonies into 10 mL LB liquid medium, shake at 28°C, 180 r / min, for 16-24 h, and count the number of bacteria by hemocytometer to ≥ 1 × 10 7 The FBH1 bacterial solution was centrifuged at 6000r / min for 5min, the supernatant was removed and resuspended in 100mL sterile water to prepare the FBH1 bacterial agent.

[0081] The experimental Chinese cabbage seeds used were the original Shanghai Qing, the rapeseed seeds used were Zhongshuang No. 9, the rice seeds used were Hanyou No. 73, and the tobacco seeds used were Yunyan No. 87.

[0082] After the above seeds were quickly disinfected with 75% ethanol, they were soaked in 1% sodium hypochlorite for 15 minutes and then rinsed with sterile water for 3-5 times for later use. The disinfected seeds were soaked in FBH1 bacterial agent for 30 minutes. The control (CK) was uninoculated seeds soaked in sterile water. Filter paper was laid in a sterile culture dish, moistened with an appropriate amount of sterile water, and the treated seeds were evenly spread on the filter paper, with 100 seeds per dish (50 rice seeds per dish). Each treatment was repeated 3 times, and cultured at room temperature for 144 hours (tobacco cultured for 240 hours). The seed germination was observed every 12 hours, and the seedling length, root length, germination rate, and germination potential were measured on the 7th day (tobacco on the 10th day). The results are shown in Tables 3 and 4. Figure 8 shown.

[0083] Table 3 Germination index

[0084] From Table 3 and Figure 8 It can be seen that the FBH1 bacterial agent treatment had a significant promoting effect on the growth of the four crops.

[0085] Specifically, the germination potential of Chinese cabbage, rapeseed, rice and flue-cured tobacco in the FBH1 treatment group was 29.34%, 22.00%, 36.67% and 17.67% higher than that of the CK control group, respectively. This shows that the FBH1 treatment has a significant promoting effect in the early stage of crop germination, especially for Chinese cabbage and rice.

[0086] In terms of germination rate, the FBH1 treatment also showed advantages. Compared with the CK control group, the germination rates of Chinese cabbage, rapeseed, rice and flue-cured tobacco under the FBH1 treatment increased by 8.67%, 2.33%, 9.33% and 23.50%, respectively. This result further confirmed the positive effect of FBH1 treatment on improving the germination rate of crops.

[0087] The FBH1 treatment also significantly promoted the growth of crops. The seedling length of Chinese cabbage, rapeseed and rice increased by 9.82mm, 8.15mm and 17.21mm respectively under the FBH1 treatment compared with the CK control group. The growth of flue-cured tobacco seedling length was smaller (only 1.10mm), but still reached a statistically significant level.

[0088] In terms of root length, the FBH1 inoculant treatment also had a positive effect on the root growth of the four crops. Compared with the CK control group, the root length of Chinese cabbage, rapeseed, rice and flue-cured tobacco increased by 8.26mm, 25.14mm, 11.90mm and 3.34mm respectively under the FBH1 treatment. This result shows that the FBH1 treatment not only promotes the growth of the aboveground part of the crops, but also significantly enhances the root development of the crops.

[0089] In summary, the FBH1 inoculant treatment had a significant promoting effect on the growth indicators of four crops, namely, pakchoi, rapeseed, rice and flue-cured tobacco, including germination potential, germination rate, seedling length and root length.

[0090] Example 8 Experiment on the Growth-Promoting Effect of Strain FBH1 on Flue-cured Tobacco The test used polyethylene plastic flower pots, each containing 1.5 kg of soil. Yunyan 87 was used as the test object. The tobacco seedlings were treated with the bacterial agent prepared in Example 7, and the same amount of sterile water was used as the control. The amount per pot was 50 mL, and the treatment was performed once every 7 days. Deionized water was used for irrigation during the test, wherein: CK group: yellow-brown soil from Huazhong Agricultural University; FBH1 group: yellow-brown soil from Huazhong Agricultural University + FBH1 bacterial agent; Each treatment group was harvested on the 30th day of plant growth. The plant height, maximum leaf length, maximum leaf width, aboveground fresh weight, aboveground dry weight, underground fresh weight, and underground dry weight were measured on the harvest day. The results are shown in Tables 4 and Fig. 9 shown.

[0091] Table 4 Effects of FBH1 bacterial agent on flue-cured tobacco growth

[0092] From Table 4 and Fig. 9 It can be seen that FBH1 has a significant effect on the growth of flue-cured tobacco (P < 0.05). The root irrigation treatment strain first attaches to the root system of the tobacco plant, thereby promoting plant growth, thereby increasing the biomass of the plant. The plant height increased by 72.85% compared with the control, the maximum leaf length increased by 8.71% compared with the control, and the maximum leaf width increased by 11.31% compared with the control. The aboveground fresh weight increased by 44.40% compared with the control, the underground fresh weight increased by 44.81% compared with the control, the aboveground dry weight increased by 42.38% compared with the control, and the underground dry weight increased by 50.00% compared with the control. The results show that FBH1 has a significant growth-promoting effect on tobacco.

[0093] In summary, the Enterobacter cloacae provided by the present invention ( Enterobacter cloacae ) FBH1 has good ability to solubilize phosphate and potassium, secrete IAA, produce siderophore, and produce ACC deaminase. It promotes seed germination by significantly improving seed germination rate, germination potential, seedling length and root length, and can significantly promote plant growth.

[0094] It should be noted that the above embodiments all belong to the same inventive concept, and the description of each embodiment has its own focus. For matters that are not described in detail in some embodiments, reference may be made to the description in other embodiments.

[0095] The above-mentioned embodiments only express the implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A strain of Enterobacter cloacae FBH1, characterized in that: The deposit number of the Enterobacter cloacae FBH1 is CCTCC NO: M20241838.

2. A biological bacterial agent, characterized in that: It includes the Enterobacter cloacae FBH1 described in claim 1.

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

4. The biological agent according to claim 3, characterized in that: The content of Enterobacter cloacae FBH1 in the liquid bacterial agent is ≥1×10 7 cfu / mL.

5. A method for preparing a biological agent as claimed in any one of claims 2 to 4, characterized in that: The method comprises the following steps: inoculating the Enterobacter cloacae FBH1 described in claim 1 into a liquid culture medium for cultivation, separating the obtained culture and resuspending it with a solvent to obtain a biological bacterial agent.

6. Use of the Enterobacter cloacae FBH1 according to claim 1, the biological agent according to any one of claims 2 to 4, or the biological agent obtained by the preparation method according to claim 5 in at least one of the following: A1) Solubilizes phosphate; A2) Solubilizes potassium; A3) Secretes IAA; A4) Produces siderophore; A5) Produces ACC deaminase.

7. Use of the Enterobacter cloacae FBH1 according to claim 1, the biological agent according to any one of claims 2 to 4, or the biological agent obtained by the preparation method according to claim 5 in promoting seed germination.

8. The use according to claim 7, characterized in that: The seeds include at least one of rapeseed seeds, Chinese cabbage seeds, rice seeds, and flue-cured tobacco seeds.

9. Use of the Enterobacter cloacae FBH1 according to claim 1, the biological agent according to any one of claims 2 to 4, or the biological agent obtained by the preparation method according to claim 5 in improving fertilizer utilization and / or promoting plant growth.

10. The use according to claim 9, characterized in that: The plants include tobacco.

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