Enterobacter sp. Ct2, microbial agent, microbial fertilizer and application of enterobacter sp. Ct2, microbial agent and microbial fertilizer

Abstract

The invention provides an enterobacter bacterium Ct2, a microbial agent, a microbial fertilizer and application of the enterobacter bacterium Ct2, and belongs to the technical field of agricultural microorganisms. The invention provides an enterobacter bacterium Ct2, and the preservation number of the enterobacter bacterium Ct2 is CCTCC (China Center for Type Culture Collection) NO: M 2025569. The enterobacter sp. Ct2 is separated from rhizosphere soil of safflower and has the effects of secreting IAA, efficiently dissolving phosphorus and reducing the pH value. The enterobacter sp. Ct2 can effectively promote the growth and development of safflower with different flower colors, increase the yield of safflower fruit balls, increase the content accumulation of active components in filaments of safflower with different flower colors in different flowering days, and improve the physicochemical properties of safflower rhizosphere soil at the same time. In conclusion, the enterobacter sp. Ct2 provided by the invention can be applied to preparation of microbial fertilizers and microbial agents, and lays a foundation for research and development of special, efficient and green microbial fertilizers.

Description

Technical Field

[0001] The present invention belongs to the technical field of agricultural microorganisms, and in particular relates to an Enterobacter bacterium Ct2, a microbial agent and a microbial fertilizer and applications thereof. Background Art

[0002] Safflower (Carthamus tinctorius L.) is an annual herbaceous plant in the genus Carthamus in the Asteraceae family. It is resistant to drought, cold, salinity, and infertility. Safflower is a traditional Chinese herbal medicine and a new oil and industrial plant. It can also be used as a dye and feed, and has high economic, medicinal, and ecological value.

[0003] In recent years, microbial agents have been widely reported as agents for promoting plant growth and improving plant quality, and these agents have generally shown good results in crop cultivation. However, as a traditional crop, safflower cultivation research is gradually gaining attention, but there are currently no reports of microbial agents that can be used to promote safflower growth and improve its quality. Summary of the Invention

[0004] In view of the defects in the prior art, the purpose of the present invention is to provide an Enterobacter bacterium Ct2, which can significantly promote the growth of safflower and improve the quality of safflower.

[0005] The purpose of the present invention is achieved through the following technical solutions:

[0006] The invention provides an Enterobacter sp. Ct2. The deposit number of the Enterobacter sp. Ct2 is CCTCC NO: M 2025569.

[0007] The present invention provides a microbial agent, comprising the Enterobacter bacteria Ct2 described in the above technical solution.

[0008] The present invention provides a method for preparing the microbial agent described in the above technical solution, comprising:

[0009] The Enterobacter bacteria Ct2 is cultured to obtain a microbial agent.

[0010] Preferably, the culture temperature is 26-37°C, and the culture process is accompanied by shaking; the shaking speed is 150-200 r·min -1 .

[0011] The present invention provides a microbial fertilizer comprising the Enterobacter bacteria Ct2 described in the above technical solution and a carrier.

[0012] The present invention provides a method for using the microbial fertilizer described in the above technical solution, comprising:

[0013] The microbial fertilizer is mixed with water to prepare a fertilizer solution for application.

[0014] The present invention provides the use of the Enterobacter bacteria Ct2 described in the above technical solution in the degradation of insoluble phosphorus.

[0015] The present invention provides the use of the Enterobacter bacteria Ct2 described in the above technical solution in promoting plant growth and / or improving plant quality.

[0016] The present invention provides the use of the Enterobacter bacteria Ct2 described in the above technical solution in improving the physical and chemical properties of soil.

[0017] The present invention provides a method for promoting safflower growth and / or improving safflower quality, comprising:

[0018] The microbial agent or the microbial fertilizer described in the above technical solution is applied during the growth of safflower.

[0019] Beneficial effects of the present invention:

[0020] The present invention provides an Enterobacter sp. Ct2 bacterium, the deposit number of which is CCTCC NO: M 2025569. The Enterobacter sp. Ct2 bacterium is isolated from the rhizosphere soil of safflower and has the functions of secreting IAA, efficiently solubilizing phosphate, and lowering pH. Studies have shown that the Enterobacter sp. Ct2 bacterium can effectively promote the growth and development of safflowers of different colors, increase the yield of safflower cones, and increase the accumulation of active ingredients in filaments of different colors of safflowers with different flowering days. It can also improve the physical and chemical properties of the rhizosphere soil of safflowers. The present invention shows through the results of the examples that the Enterobacter bacteria Ct2 has a high efficiency in degrading different insoluble phosphorus and organic phosphorus; the Enterobacter bacteria Ct2 is applied as a fertilizer to the planting of safflowers of different colors, which can significantly promote the growth of safflowers of different colors, and significantly improve the plant height, leaf number, stem thickness, root length, root fresh weight, total fresh weight, top bud diameter and number of effective balls per plant of safflowers of different colors; the Enterobacter bacteria Ct2 is applied as a fertilizer to the planting of safflowers of different colors and can also significantly increase the content of hydroxysafflower yellow A and kaempferol active ingredients in safflower filaments, thereby improving the quality of safflower; further, the Enterobacter bacteria Ct2 as a fertilizer can also significantly improve the physical and chemical properties of the soil, reduce soil pH, and increase the content of total nitrogen, organic matter, total phosphorus, available potassium and available phosphorus in the soil. In summary, the Enterobacter bacteria Ct2 provided by the present invention can be applied to the preparation of microbial fertilizers and bacterial agents, laying a foundation for the research and development of high-efficiency and green microbial fertilizers that adapt to special characteristics.

[0021] Biological Deposit Description

[0022] The Enterobacter bacterium Ct2, classified as Enterobacter sp., was deposited in the China Center for Type Culture Collection on March 24, 2025, located in Room 211, China Center for Type Culture Collection, Wuhan University, Wuchang District, Wuhan City, Hubei Province, with the deposit number CCTCCNO: M 2025569. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0024] Figure 1 The morphology, phosphate circle and Gram staining of strain Ct2;

[0025] Figure 2 This is the electrophoresis result of PCR product of 16s rDNA of Ct2 strain;

[0026] Figure 3 This is the phylogenetic analysis result of the Ct2 strain;

[0027] Figure 4 The graph shows the phosphate solubilization ability of the Ct2 strain for different insoluble phosphorus compounds and the pH detection results during the solubilization process;

[0028] Figure 5 This is the result of soluble phosphorus content and pH test during the culture of Ct2 strain;

[0029] Figure 6 This is the result of the assay of auxin (IAA) secretion ability of the Ct2 strain;

[0030] Figure 7 This is the phenotypic diagram of safflower 14 days after the application of microbial fertilizer;

[0031] Figure 8 This is the agronomic characteristics of safflower 14 days after the application of microbial fertilizer;

[0032] Figure 9 This is the phenotypic diagram of safflower on the 80th day after the application of microbial fertilizer;

[0033] Figure 10 This is the agronomic characteristics of safflower 80 days after the application of microbial fertilizer;

[0034] Figure 11This is the agronomic trait result diagram of safflower during flowering period after application of microbial fertilizer;

[0035] Figure 12 This is a graph showing the effect of Ct2 on the active ingredients of white safflower at different flowering days;

[0036] Figure 13 This is a graph showing the effect of Ct2 on the active ingredients of yellow safflower at different flowering days;

[0037] Figure 14 This is a graph showing the effect of Ct2 on the active ingredients of red safflower at different flowering days;

[0038] Figure 15 This is the result diagram of the effect of Ct2 on the physical and chemical properties of soil. DETAILED DESCRIPTION

[0039] The invention provides an Enterobacter sp. Ct2. The deposit number of the Enterobacter sp. Ct2 is CCTCC NO: M 2025569.

[0040] The Enterobacter bacterium Ct2 provided herein was isolated from the rhizosphere soil of safflower. The Enterobacter bacterium Ct2 is a facultative aerobe, positive for catalase reaction and VP test, and capable of producing IAA. The 16s rDNA nucleotide sequence of the Enterobacter bacterium Ct2 is shown in SEQ ID NO. 3.

[0041] The Enterobacter bacteria Ct2 provided by the present invention has the ability to solubilize phosphate and has a high efficiency in degrading various insoluble phosphorus and organic phosphorus; the Enterobacter bacteria Ct2 can also significantly reduce the pH value of the soil during the process of degrading phosphorus.

[0042] The present invention provides a microbial agent, comprising the Enterobacter bacteria Ct2 described in the above technical solution. As an optional embodiment of the present invention, the bacterial activity of the Enterobacter bacteria Ct2 in the microbial agent is ≥10 7 CFU / mL, can be 10 7 ~10 9 CFU / mL, or 10 8 As an optional embodiment of the present invention, the bacterial activity of Enterobacter bacteria Ct2 in the bacterial agent is ≥10 7 CFU / g, can be 10 7 ~10 9 CFU / g, or 10 8 CFU / g.

[0043] The present invention provides a method for preparing the microbial agent described in the above technical solution, comprising: culturing the Enterobacter bacteria Ct2 to obtain the agent. As an optional embodiment of the present invention, the culture medium may be LB medium; the culture temperature may be 26-37°C, or may be 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, or 37°C; the culture process may be accompanied by oscillation; and the oscillation speed may be 150-200 r / min. -1 , can also be 150, 160, 170, 180, 190 or 200 r·min -1 The culture of the present invention is preferably cultured until the bacterial liquid concentration reaches 10 8 ~10 9 CFU / mL, and the Enterobacterium Ct2 bacterial liquid was obtained.

[0044] After obtaining the Enterobacter Ct2 bacterial solution, the bacterial solution can be used directly as a microbial inoculant. The present invention also allows the separation of the Enterobacter Ct2 bacterial solution, collection of bacterial cells, and resuspending the cells in water to obtain a bacterial suspension, which can be used as a microbial inoculant. As an optional embodiment of the present invention, the water can be sterile water.

[0045] The present invention provides a microbial fertilizer, comprising the Enterobacter bacteria Ct2 described in the above technical solution and a carrier. As an optional embodiment of the present invention, the bacterial activity of the Enterobacter bacteria Ct2 in the microbial fertilizer is ≥10 7 CFU / g, can be 10 7 ~10 9 CFU / g, or 10 8 As an optional embodiment of the present invention, the carrier comprises diatomaceous earth and skim milk powder; the mass ratio of diatomaceous earth to skim milk powder in the carrier can be (8-9): (2-1), or can be 8:1, 9:1, 8:1 or 9:2.

[0046] The present invention provides a method for using the microbial fertilizer described in the above technical solution, comprising: mixing the microbial fertilizer with water, preparing a fertilizer solution for application. As an optional embodiment of the present invention, the mass volume ratio of the microbial fertilizer and water can be 12g:250mL. The fertilizer described in the present invention can be applied at any stage of plant growth, preferably 10 days after the plant seeds germinate. When applying the fertilizer solution in the present invention, it is preferred to apply the fertilizer solution to the rhizosphere soil. In the present invention, the application amount of the fertilizer is 250mL of fertilizer solution for 4 plants. As an optional embodiment of the present invention, the plant includes safflower; the safflower includes safflower of different colors; the safflower of different colors includes any one or more of white safflower, yellow safflower and red safflower.

[0047] The present invention provides the use of the Enterobacter bacteria Ct2 described in the above-mentioned technical solution for the degradation of insoluble phosphorus. As an optional embodiment of the present invention, the insoluble phosphorus includes calcium phosphate, zinc phosphate, iron phosphate, and organic phosphorus. The present invention demonstrates, through examples, that the Enterobacter bacteria Ct2 can efficiently degrade calcium phosphate, zinc phosphate, iron phosphate, and organic phosphorus during cultivation, thereby increasing the soluble phosphorus content in the culture system. Furthermore, the Enterobacter bacteria Ct2 can significantly reduce the pH of the culture system while degrading the insoluble phosphorus.

[0048] The present invention provides the use of the Enterobacter bacteria Ct2 described in the above technical solution in promoting plant growth and / or improving plant quality. As an optional embodiment of the present invention, the plant includes safflower; the safflower includes safflower of different colors; the safflower of different colors includes any one or more of white safflower, yellow safflower and red safflower. In the present invention, the Enterobacter bacteria Ct2 can significantly promote the growth of safflower and / or improve the quality of safflower. The results of the examples of the present invention show that the Enterobacter bacteria Ct2 can significantly increase the plant height, number of leaves, stem diameter, root length, root fresh weight, total fresh weight, first branch height, top bud diameter and number of effective bulbs per plant of safflower of different colors, thereby promoting the growth of safflower of different colors. The Enterobacter bacteria Ct2 can significantly increase the content of hydroxysafflower yellow A and kaempferol active ingredients in the filaments of safflower of different colors, thereby improving the quality of safflower.

[0049] The present invention provides the use of the Enterobacter bacteria Ct2 described in the above technical solution for soil improvement. The Enterobacter bacteria Ct2 provided by the present invention can significantly reduce the pH value of the soil and increase the content of any one or more of organic matter, total nitrogen, total phosphorus, total potassium, available phosphorus, fast-acting potassium, inorganic phosphorus, and organic phosphorus in the soil, thereby achieving the effect of improving the soil.

[0050] The present invention provides a method for promoting safflower growth and / or improving safflower quality, comprising:

[0051] The microbial agent described in the above technical solution or the microbial fertilizer described in the above technical solution is applied during the growth of safflower. In the present invention, the microbial agent and the microbial fertilizer can be applied at various stages of the growth of safflower. As an optional embodiment of the present invention, the microbial agent or the microbial fertilizer can be applied when the safflower germinates for 10 days. As an optional embodiment of the present invention, when the microbial fertilizer is applied, the microbial fertilizer is mixed with water to prepare a fertilizer solution for application; the mass volume ratio of the microbial fertilizer to water is 12g:250mL; the application amount can be 250mL of fertilizer solution for 4 safflower plants.

[0052] In order to further illustrate the present invention, the technical solution provided by the present invention is described in detail below with reference to the accompanying drawings and embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0053] Example 1 Isolation and purification of strains

[0054] 1. Collection of rhizosphere soil and soil

[0055] Soil: Shihezi Experimental Station (latitude: 44°19′34.4″; longitude: 85°59′44.1″);

[0056] Safflower rhizosphere soil: Safflower experimental field, Shihezi Experimental Station;

[0057] The five-point sampling method was used to sample safflower in the field, and the shaking soil method was used to collect the rhizosphere soil of safflower.

[0058] 2. Isolation and purification of phosphate-solubilizing strains

[0059] The phosphate-solubilizing bacteria in the safflower rhizosphere were screened by shaking soil. 5 g of safflower rhizosphere soil was added with 45 mL of sterile water and placed in an oscillating box for 30 min before serial gradient dilution. 10 -4 , 10 -5 , 10 -6100 μL of the three soil suspension gradients was applied to an inorganic phosphate solid medium (glucose 10.0 g / L, ammonium sulfate 0.5 g / L, NaCl 0.3 g / L, KCl 0.3 g / L, MgSO 4 0.3 g / L, FeSO 4 0.03 g / L, MnSO 4 0.01 g / L, Ca 3 (PO 4) 25.0 g / L, agar 15.0 g / L, pH 7.0). After incubation for 5 days, colonies with phosphate-solubilizing zones were selected, purified, numbered, and stored at -80°C for future use. The activated phosphate-solubilizing bacteria were then inoculated onto the inorganic phosphate solid medium. The phosphate-solubilizing capacity of the strains was preliminarily determined by measuring their D / d (phosphate-solubilizing zone diameter / colony diameter) values. The strain with the highest D / d value was selected for subsequent experiments.

[0060] Results: Fourteen strains with phosphate-solubilizing ability were isolated from the rhizosphere soil of safflower. The phosphate-solubilizing abilities of the corresponding strains are shown in Table 1.

[0061] Table 1 Solubility index of phosphate-solubilizing bacteria

[0062] strain D(cm) d(cm) D / d Ct1 1.750 1.033 1.705±0.161cde Ct2 2.15 0.783 2.748±0.163b Ct3 1.367 1.133 1.206±0.028h Ct4 1.283 1.117 1.150±0.028h Ct5 1.700 0.950 1.792±0.084c Ct6 1.683 1.000 1.704±0.179cde Ct7 1.817 1.033 1.761±0.068cd Ct8 1.883 1.117 1.691±0.095de Ct10 1.250 0.933 1.341±0.045gh Ct11 1.800 1.167 1.544±0.046ef Ct12 1.717 1.150 1.495±0.069fg Ct13 1.850 1.133 1.634±0.049de Ct15 1.250 0.967 1.293±0.027gh

[0063] As shown in Table 1, according to the solubility index analysis of phosphate-solubilizing bacteria, among the 14 phosphate-solubilizing bacteria, Ct2 has the highest solubility index, reaching 2.748, while the solubility indexes of the remaining phosphate-solubilizing bacteria are all below 2. Therefore, based on its higher solubility index, Ct2 strain was selected as the target strain for subsequent experiments. The phosphate-solubilizing circle diagram of Ct2 strain is shown in Figure 1. Figure 1 As shown in A.

[0064] 3. Physiological and biochemical tests were performed on the Ct2 strain. The results are shown in Table 2.

[0065] Table 2 Physiological and biochemical test results of Ct2 strain

[0066]

[0067]

[0068] As shown in Table 2, strain Ct2 is a facultative aerobe, positive in catalase test and VP test, and can produce IAA.

[0069] 4. Identification of Ct2 strain

[0070] (1) Ct2 strain morphology

[0071] The Ct2 strain was streaked onto LB solid medium plates and cultured at 28°C. The colony morphology of the Ct2 strain was as follows: Figure 1As shown in B. The colony is a light yellow round protrusion with neat edges, shiny, opaque and moist. The Ct2 strain was stained with Gram stain. The results of Gram staining are as follows Figure 1 As shown in C. Gram staining results showed that Ct2 was a Gram-negative bacterium with a short rod shape.

[0072] (2) Molecular biological identification of Ct2 strain

[0073] The strain gene was amplified by PCR using universal primers for bacterial 16s rDNA, and the product was sent to Shanghai Sangon Biotechnology Co., Ltd. for sequencing. The obtained sequence was compared with the sequences of known strains by BLAST homology analysis in the NCBI database, and the strain phylogenetic tree was constructed using MEGA11 software to determine its taxonomic status.

[0074] The specific steps are as follows: fresh bacterial samples were sent to Xinjiang Youkang Biotechnology Co., Ltd., bacterial DNA was extracted using the Ezup column bacterial genomic DNA extraction kit (SK8255), and universal primers 27F, as shown in SEQ ID NO.1, specifically: 5'-AGAGTTTGATCCTGGCTCAG-3', and 1492R, as shown in SEQ ID NO.2, specifically: 5'-CGGTTACCTTGTTACGACTT-3', were used for PCR amplification. The reaction procedure and amplification reaction system are shown in Tables 3 and 4.

[0075] Table 3 PCR reaction program

[0076]

[0077] Table 4 PCR reaction system

[0078]

[0079]

[0080] After the PCR reaction is completed, the obtained PCR products are subjected to electrophoresis detection. Electrophoresis detection uses 1% agarose, 150V, 100mA, 20min, and then observed. The electrophoresis detection results are as follows Figure 2

[0081] The assembled sequences were uploaded to the NCBI (National Center for Biotechnology Information, https: / / www.ncbi.nlm.nih.gov) database for sequence alignment. Sequences with high similarity in the database were selected and a phylogenetic tree was constructed using the neighbor joining method using MEGA11.0 software, as shown in the following example: Figure 3 shown.

[0082] Depend on Figure 3 It can be seen that the Ct2 strain and Enterobacter quasihormaechei are clustered in the same branch.

[0083] Combined with the results of physiological and biochemical testing and molecular biological identification, the Ct2 strain was identified as an Enterobacter sp. bacterium, with the taxonomic name Enterobacter sp., and was deposited with the China Center for Type Culture Collection on March 24, 2025, with the deposit number CCTCC NO: M 2025569. The collection is located at Room 211, China Center for Type Culture Collection, Wuhan University, Wuchang District, Wuhan City, Hubei Province.

[0084] Example 2

[0085] 1. Phosphate solubility and pH determination of Ct2 strain for different insoluble phosphorus compounds

[0086] Calcium phosphate inorganic phosphate medium (g / L): glucose 10.0, ammonium sulfate 0.1, MgCl25, KC10.2, MgSO4·7H2O0.25, Ca3(PO4)25.0, pH 7.0~7.5.

[0087] Zinc phosphate inorganic phosphorus medium (g / L): glucose 10.0, ammonium sulfate 0.1, MgCl25, KC10.2, MgSO4·7H2O0.25, Zn3(PO4)25.0, pH 7.0~7.5.

[0088] Ferric phosphate inorganic phosphorus medium (g / L): glucose 10.0, ammonium sulfate 0.1, MgCl25, KC10.2, MgSO4·7H2O0.25, FePO4 5.0, pH 7.0~7.5.

[0089] Montana organophosphorus medium (g / L): glucose 10.0, ammonium sulfate 0.5, NaCl 0.3, KC10.3, FeSO4 0.03, MnSO4 0.03, egg yolk lecithin 0.2, calcium carbonate 5.0, yeast extract powder 0.4, pH 7.0.

[0090] After the Ct2 strain was activated, it was inoculated into the above-mentioned insoluble inorganic phosphorus medium and Montgina organic phosphorus medium and stirred at 180 r·min. -1 , shake in a shaker at 28℃ for 3 days, and use the molybdenum antimony colorimetric method to determine the phosphate solubilization ability and pH of the Ct2 strain in different culture media.

[0091] The phosphate solubilization ability of Ct2 strain for different insoluble phosphorus compounds and the pH test results during the solubilization process are shown in Tables 5-6 and Figure 4 shown.

[0092] Table 5 Phosphate solubilization ability of Ct2 strain for different insoluble phosphorus compounds

[0093]

[0094]

[0095] Table 6 pH detection results during phosphate solubilization of strain Ct2

[0096]

[0097] From Tables 5 to 6 and Figure 4 It can be found that the Ct2 strain can efficiently degrade calcium phosphate, zinc phosphate, iron phosphate and organic phosphorus, and increase the soluble phosphorus content in the culture system. Furthermore, the Ct2 strain can significantly reduce the pH in the culture system while degrading insoluble phosphorus.

[0098] 2. Soluble phosphorus content and pH detection during Ct2 strain cultivation

[0099] After the Ct2 strain was activated, it was inoculated into NBRIP inorganic phosphorus liquid medium and heated at 180 r·min. -1 The culture was carried out in a shaking incubator at 28°C. The available phosphorus content in the supernatant was determined by the molybdenum antimony colorimetric method and the pH was determined by a pH meter every day during the culture process. The test was continued for 7 days.

[0100] The test results are shown in Table 7 and Figure 5 shown.

[0101] Table 7 Changes in soluble phosphorus content and pH during the culture of Ct2 strain

[0102] Number of days 0 1 2 3 4 5 6 7 Soluble phosphorus content 1.99 25.19 126.12 222.22 224.90 232.95 228.00 258.79 pH 7.13 5.57 4.55 4.11 4.18 4.46 4.50 4.40

[0103] From Table 7 and Figure 5It can be found that during the cultivation of the Ct2 strain, the pH in the culture system dropped sharply within 0 to 3 days of cultivation. After 3 days of cultivation, the pH in the culture system dropped slowly and remained between 4 and 5. As the cultivation time increased, the soluble phosphorus content in the culture system showed a significant increase within 0 to 3 days of cultivation. When the culture time was 3 to 7 days, the soluble phosphorus content in the culture system slowly increased. In summary, the Ct2 strain provided by the present invention has the ability to produce acid and solubilize phosphorus during the cultivation process.

[0104] 4. Determination of the secretion capacity of auxin (IAA) of Ct2 strain

[0105] The IAA secretion capacity of the strains was determined using the Salkoeski method.

[0106] The Ct2 strain was inoculated into 50 mL of LB liquid medium containing 200 mg / L L-tryptophan. Three replicates were made for each strain. The culture medium was rotated at 180 r·min. -1 , shake in a 28℃ shaker for 3 days, take the bacterial solution and put it into a 2mL centrifuge tube at 7000r·min -1 Centrifuge for 10 minutes. Add 0.2 mL of bacterial supernatant and 0.2 mL of Salkoeski colorimetric solution to a white porcelain colorimetric plate and let it stand in the dark for 15 minutes before observation. A red color indicates positive, indicating secretion of IAA. The darker the color, the more IAA the strain secretes. No color change indicates negative, indicating no IAA secretion. Figure 6 shown.

[0107] Depend on Figure 6 It can be seen that the secretion of IAA was determined by the Salkoeski method. Qualitative analysis showed that Ct2 was red compared with CK on the white porcelain plate, indicating that Ct2 could secrete IAA.

[0108] Example 3: Growth-promoting effect of Ct2 strain on safflower

[0109] 1. Production of microbial fertilizer

[0110] (1) Preparation of microbial agents

[0111] Take out the Ct2 strain from the -80℃ freezer, streak the Ct2 strain on the LB solid medium plate to activate it, inoculate the activated single colony strain into the LB liquid medium, and rotate it at 180 r·min. -1 , shake the bacteria at 28℃, and wait until the concentration of the bacterial solution reaches 10 8 ~10 9 CFU / mL (OD 600 =1). Take the bacterial solution at 3500 r·min -1 Centrifuge for 5 minutes, discard the supernatant, and resuspend the cells in sterile water until the cell viability is 10 8 ~109 CFU / mL, and obtain the microbial agent.

[0112] (2) Production of bacterial fertilizer

[0113] 50 mL of the microbial agent obtained in step (1) was added to 500 g of the carrier to obtain a bacterial activity of 10 7 ~10 8 CFU / g of microbial fertilizer. The carriers are diatomaceous earth and skim milk powder, and the mass ratio of diatomaceous earth to skim milk powder is 9:1.

[0114] 2. Potted Plant Experiment

[0115] Prepare a fertilizer solution at a ratio of 12 g microbial fertilizer to 250 mL sterile water. Ten days after safflowers with filament colors (white, yellow, and red) germinate, apply 250 mL of the fertilizer solution to each pot of safflower. Plant four safflower seedlings in each pot, and inoculate the control group with the same amount of sterile carrier liquid.

[0116] 3. Agronomic trait determination

[0117] On the 14th and 80th days after microbial fertilizer application, and during the flowering phase, the biomass of three safflower colors (white, yellow, and red) was measured using a ruler. Partial biomass (plant height, leaf number, and stem diameter) was measured directly in the experimental plot, while the remaining biomass (aboveground fresh weight, root fresh weight, and root length) was measured in the laboratory and photographed. Safflower samples were collected and stored in a -80°C ultra-low temperature freezer. During the flowering phase, plant height, stem diameter, first branch height, top bud diameter, and number of fruiting bulbs per plant were measured and counted.

[0118] The phenotype of safflower on the 14th day after the application of microbial fertilizer is as follows Figure 7 The agronomic traits are shown in Table 8 and Figure 8 As shown in the figure. W represents white; Y represents yellow; R represents red, the same below. The phenotypic diagram of safflower on the 80th day after the application of microbial fertilizer is as follows Figure 9 The agronomic traits are shown in Table 9 and Figure 10 The agronomic characteristics of safflower during flowering after application of microbial fertilizer are shown in Table 10 and Figure 11 shown.

[0119] Table 8 Agronomic characteristics of safflower on the 14th day after application of microbial fertilizer

[0120]

[0121]

[0122] Table 9 Agronomic characteristics of safflower on the 80th day after application of microbial fertilizer

[0123]

[0124] Table 10 Agronomic characteristics of safflower during flowering after application of microbial fertilizer

[0125]

[0126] From Tables 8 to 10 and Figures 7-11 It can be obtained that the plant height, number of leaves, stem thickness, root length, root fresh weight and total fresh weight of the three colors of safflower were significantly improved relative to the control group 14d after the application of microbial fertilizer, and the difference was significant. 80d after the application of microbial fertilizer, the plant height, number of leaves, stem thickness, root length, root fresh weight and total fresh weight of the three colors of safflower were significantly improved relative to the control group, and the difference was significant. After the application of microbial fertilizer, the plant height, stem thickness, top bud diameter and number of effective balls per plant of safflower during the flowering period were significantly improved relative to the control group. At the same time, the first branch height of white safflower and red safflower was significantly improved compared with the control group after the application of microbial fertilizer. In summary, the microbial fertilizer provided by the present invention can significantly promote the growth of safflower of different colors, improve the plant height, number of leaves, stem thickness, root length, root fresh weight, total fresh weight, first branch height, top bud diameter and number of effective balls per plant of safflower.

[0127] 4. Effects of Ct2 on the active ingredients of safflowers with different colors and different flowering days

[0128] (1) Effects of Ct2 on the active ingredients of white safflower at different flowering days

[0129] During the flowering period, the white safflower flowers and filaments of the experimental group and the control group were photographed, and the hydroxysafflower yellow A (HSYA) and kaempferol (KF) in the filaments were detected. The results are shown in Tables 11 and Figure 12 As shown in the figure. -4 indicates 4 days before flowering, -3 indicates 3 days before flowering, -2 indicates 2 days before flowering, -1 indicates 1 day before flowering, 0 indicates the day of flowering (the day of flowering refers to the time of arrival at the field around 9:30 am, when some filaments on the bud are fully unfolded, but not all filaments are fully unfolded, indicating that the flower will fully bloom that day), 1 indicates 1 day of flowering, and 2 indicates 2 days of flowering. HSYA and KF were determined using HPLC. Chromatographic conditions: Chromatographic column: Agilent LC Columus, 5 μm, 4.6 mm (inner diameter) × 250 mm; column temperature: 30°C; mobile phase: 0.4% phosphoric acid (D)-methanol (B), gradient elution (0-60 min, 95%-5% D; 60-65 min, 5%-5% D; 65-70 min, 5%-95% A); flow rate: 1 mL / min; detection wavelength: 403, 367 nm, injection volume 10 μL.

[0130] Table 11 HSYA and KF detection results in the filaments of white safflower

[0131]

[0132] From Table 11 and Figure 12 It was found that compared with the CK control, the application of bacterial fertilizer could significantly increase the content of HSYA and KF active ingredients in the filaments of white safflower at different flowering times, and improve the quality of white safflower.

[0133] (2) Effects of Ct2 on the active ingredients of yellow safflower at different flowering days

[0134] During the flowering period, the yellow safflower flowers and filaments of the experimental group and the control group were photographed, and the hydroxysafflower yellow A (HSYA) and kaempferol (KF) in the filaments were detected. The results are shown in Tables 12 and Figure 13 shown.

[0135] Table 12 HSYA and KF detection results in yellow safflower filaments

[0136]

[0137] Depend on Figure 13 It was found that compared with the control CK, the application of bacterial fertilizer could significantly increase the content of HSYA and KF active ingredients in the filaments of yellow safflower at different flowering days, and improve the quality of yellow safflower.

[0138] (3) Effects of Ct2 on the active ingredients of red safflower at different flowering days

[0139] During the flowering period, the red safflower flowers and filaments of the experimental and control groups were photographed, and the hydroxysafflower yellow A (HSYA) and kaempferol (KF) in the filaments were detected. The results are shown in Tables 13 and Figure 14 shown.

[0140] Table 13 HSYA and KF detection results in red safflower filaments

[0141]

[0142] From Table 13 and Figure 14 It was found that compared with the control CK, the application of bacterial fertilizer could significantly increase the content of HSYA and KF active ingredients in the filaments of red safflower at different flowering days, and improve the quality of red safflower.

[0143] 5. Determination of soil indicators

[0144] Ten grams of safflower rhizosphere soil was diluted 10-fold with deionized water, mixed, and allowed to stand for 30 minutes. The soil pH was then measured using a pH meter. Ten grams of soil was added to 40 mL of a 0.5 mol / L K₂SO₄ solution and extracted at 200 rpm in a 30°C constant-temperature shaking incubator for 30 minutes. After a short stand, the supernatant was filtered through a 0.45 μm filter membrane and then tested for total inorganic carbon, total organic carbon, and total carbon using a total organic carbon analyzer. The soil organic matter content was determined by multiplying the soil total organic carbon content by a coefficient of 1.724.

[0145] Soil total phosphorus was determined using the NaOH fusion-molybdenum antimony colorimetric method; total nitrogen, Kjeldahl nitrogen, and available phosphorus were determined using the molybdenum antimony colorimetric method; organic phosphorus, inorganic phosphorus, total potassium, and available potassium were determined using the method described by Wu Jinshui. Wu Jinshui. Soil Microbial Biomass Determination Methods and Their Applications [M]. Beijing: Meteorological Press, 2006.

[0146] The results of soil index determination are shown in Table 14 and Figure 15 Among them, TN is total nitrogen, OM is organic matter, TK is total potassium, TP is total phosphorus, AP is available phosphorus, and AK is available potassium.

[0147] Table 14 Test results of various physical and chemical properties of soil of various safflowers in the experimental group and the control group

[0148]

[0149] From Table 14 and Figure 15 It was found that after applying Ct2 bacteria, the pH value of the rhizosphere soil of each flower color was significantly reduced compared with CK; total nitrogen, organic matter, total phosphorus, available phosphorus, fast-acting potassium and organic phosphorus all increased significantly. The total phosphorus of white safflower, yellow safflower and red safflower increased by 16.10%, 5.10% and 9.10% respectively; the organic matter increased by 24.27%, 15.63% and 24.82% respectively; the total phosphorus increased by 3.16%, 4.61% and 4.58% respectively; the available phosphorus increased by 4.49%, 5.68% and 7.24% respectively; the fast-acting potassium increased by 10.74%, 11.69% and 17.26% respectively; the organic phosphorus increased by 18.77%, 22.67% and 5.40% respectively; except for the red color, the inorganic phosphorus increased significantly, and there was no significant change in the other two colors; the total potassium did not change significantly in each flower color. This shows that the bacterial fertilizer Ct2 has a significant effect in improving soil acid-base balance and enhancing soil fertility, but there are differences in the responses of plants of different colors to the bacterial fertilizer.

[0150] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. An Enterobacter sp. Ct2, characterized in that The deposit number of the Enterobacter bacteria Ct2 is CCTCC NO: M 2025569.

2. A microbial agent, characterized in that: It includes the Enterobacter bacterium Ct2 according to claim 1.

3. The method for preparing the microbial agent according to claim 2, characterized in that: include: The Enterobacter bacteria Ct2 is cultured to obtain a microbial agent.

4. The preparation method according to claim 3, characterized in that The culture temperature is 26-37°C, and the culture process is accompanied by shaking; the shaking speed is 150-200 r·min -1 .

5. A microbial fertilizer, characterized in that: The method comprises the Enterobacter bacterium Ct2 according to claim 1 and a carrier.

6. A method for using the microbial fertilizer according to claim 5, characterized in that: include: The microbial fertilizer is mixed with water to prepare a fertilizer solution for application.

7. Use of the Enterobacter bacterium Ct2 according to claim 1 in the degradation of insoluble phosphorus.

8. Use of the Enterobacter bacterium Ct2 according to claim 1 in promoting plant growth and / or improving plant quality.

9. Use of the Enterobacter bacterium Ct2 according to claim 1 in improving the physical and chemical properties of soil.

10. A method for promoting safflower growth and / or improving safflower quality, characterized in that: include: The microbial agent according to claim 2 or the microbial fertilizer according to claim 5 is applied during the growth of safflower.

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