Pseudomonas fluorescens Ct9 and application thereof

Abstract

The invention provides pseudomonas Ct9 and application thereof, and belongs to the technical field of agricultural microorganisms. The invention provides a pseudomonas Ct9, and the preservation number of the pseudomonas Ct9 is CCTCC (China Center For Type Culture Collection): M 2025570. The pseudomonas Ct9 can secrete IAA (Indoleacetic Acid), has efficient phosphate solubilizing capacity and can also remarkably reduce the pH value of soil. The pseudomonas Ct9 has efficient degradation capacity on different insoluble phosphorus and organophosphorus, and can obviously promote the growth of the safflower, improve the quality of the safflower and improve the physicochemical properties of soil. The pseudomonas Ct9 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 particularly relates to Pseudomonas fluorescens Ct9 and applications thereof. Background Art

[0002] Phosphate-solubilizing microorganisms are microorganisms that can convert insoluble phosphorus into soluble phosphorus, promote the dissolution and conversion of inorganic and organic phosphorus in the soil, provide phosphorus that can be directly absorbed and utilized for plant growth and development, and thus increase crop yields. This type of rhizosphere microorganism that is beneficial to plant growth is called plant rhizosphere growth-promoting bacteria, which is of great significance to promoting plant growth and maintaining soil ecological health.

[0003] Safflower (Carthamus tinctorius L.) is an annual herbaceous plant of the genus Carthamus in the Asteraceae family. It is drought-resistant, cold-resistant, salt-alkali-tolerant, and barren-resistant. Safflower is a traditional Chinese herbal medicine in my country, as well as a new oil-producing and industrial plant. It can also be used as a dye, feed, etc., and has high economic, medicinal, and ecological value in production. Safflower filaments are the main medicinal part, and more than 200 compounds including flavonoids, alkaloids, fatty acids, and polyacetylenes have been isolated and reported. Flavonoids are the main medicinal ingredients, with pharmacological effects such as vasodilation, blood pressure lowering, anti-oxidation, anti-cerebral ischemia and neuroprotection, and anti-tumor effects.

[0004] Currently, there are relatively many reports on phosphate-solubilizing microorganisms, but there are no reports on phosphate-solubilizing microorganisms that can significantly promote safflower growth and improve safflower quality. Summary of the Invention

[0005] In view of the defects in the prior art, the purpose of the present invention is to provide a Pseudomonas Ct9 that can significantly promote the growth of safflower and improve the quality of safflower and increase the active ingredients in safflower.

[0006] The purpose of the present invention is achieved through the following technical solutions:

[0007] The invention provides a Pseudomonas sp. Ct9. The preservation number of the Pseudomonas sp. Ct9 is CCTCC NO: M 2025570.

[0008] The present invention provides a bacterial agent, comprising the Pseudomonas Ct9 described in the above technical solution.

[0009] The present invention provides a method for preparing the bacterial agent described in the above technical solution, comprising:

[0010] The Pseudomonas sp. Ct9 is cultured to obtain a bacterial agent.

[0011] 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 .

[0012] The present invention provides a bacterial fertilizer comprising the Pseudomonas Ct9 described in the above technical solution and a carrier.

[0013] The present invention provides a method for using the bacterial fertilizer described in the above technical solution, comprising:

[0014] The bacterial fertilizer is mixed with water to prepare a bacterial fertilizer solution for application.

[0015] The present invention provides the use of Pseudomonas Ct9 described in the above technical solution in the degradation of insoluble phosphorus.

[0016] The present invention provides the application of Pseudomonas Ct9 described in the above technical solution in safflower cultivation.

[0017] The present invention provides the use of Pseudomonas Ct9 described in the above technical solution in improving the physical and chemical properties of soil.

[0018] The present invention provides a method for promoting safflower growth and / or improving safflower quality, comprising:

[0019] The bacterial agent or the bacterial fertilizer described in the above technical solution is applied during the growth of safflower.

[0020] Beneficial effects of the present invention:

[0021] The present invention provides Pseudomonas sp. Ct9, which has a deposit number of CCTCC NO: M 2025570. Pseudomonas sp. Ct9 has a highly efficient phosphate solubilization capability, significantly reducing soil pH while simultaneously solubilizing phosphate. Pseudomonas sp. Ct9 can also secrete IAA. Studies have shown that Pseudomonas sp. Ct9 can effectively promote the growth and development of safflowers of different colors, increase safflower cone yield, and enhance the accumulation of active ingredients in filaments of safflowers of different colors and flowering days. The present invention shows through the results of the examples that the Pseudomonas Ct9 has an efficient degradation ability for different insoluble phosphorus and organic phosphorus; the Pseudomonas Ct9 is applied as a bacterial fertilizer in safflower planting, which can significantly promote the growth of safflower 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 safflower; the Pseudomonas Ct9 is applied as a bacterial fertilizer in safflower planting 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 Pseudomonas Ct9 as a bacterial fertilizer can also significantly improve the physical and chemical properties of the soil, and increase the content of total nitrogen, organic matter, total phosphorus and available phosphorus in the soil. In summary, the Pseudomonas Ct9 provided by the present invention can be applied to the preparation of microbial fertilizers and bacterial agents, laying the foundation for the research and development of high-efficiency and green microbial fertilizers that are adaptable to special features.

[0022] Biological Deposit Description

[0023] Pseudomonas Ct9, classified as Pseudomonas 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 being CCTCC NO: M 2025570. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] 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.

[0025] Figure 1 The colony morphology, phosphate circle and Gram staining of the Ct9 strain;

[0026] Figure 2 This is the electrophoresis result of PCR product of 16s rDNA of Ct9 strain;

[0027] Figure 3 This is the phylogenetic analysis result of Ct9 strain;

[0028] Figure 4 The graph shows the phosphate solubilization ability of the Ct9 strain for different insoluble phosphorus compounds and the pH detection results during the solubilization process;

[0029] Figure 5 This is a graph showing the soluble phosphorus content and pH test results during the culture of the Ct9 strain;

[0030] Figure 6 This is the result of the assay of auxin (IAA) secretion ability of the Ct9 strain;

[0031] Figure 7 This is the phenotypic diagram of safflower on the 14th day after the application of bacterial fertilizer;

[0032] Figure 8 This is the agronomic characteristics of safflower 14 days after the application of bacterial fertilizer;

[0033] Figure 9 This is the phenotypic diagram of safflower on the 80th day after the application of bacterial fertilizer;

[0034] Figure 10 This is the agronomic characteristics of safflower on the 80th day after the application of bacterial fertilizer;

[0035] Figure 11 This is the agronomic trait result diagram of safflower flowering period;

[0036] Figure 12 This is a graph showing the effect of Ct9 on the active ingredients of white safflower at different flowering days;

[0037] Figure 13 This is a graph showing the effect of Ct9 on the active ingredients of yellow safflower at different flowering days;

[0038] Figure 14 This is a graph showing the effect of Ct9 on the active ingredients of red safflower at different flowering days;

[0039] Figure 15 This is the result of the effect of Ct9 on the physical and chemical properties of soil. DETAILED DESCRIPTION

[0040] The invention provides a Pseudomonas sp. Ct9. The preservation number of the Pseudomonas sp. Ct9 is CCTCC NO: M 2025570.

[0041] The Pseudomonas sp. Ct9 provided herein was isolated from the rhizosphere soil of safflower. Pseudomonas sp. Ct9 is a facultative aerobic, Gram-negative bacterium that is catalase-positive and capable of producing IAA. The 16s rDNA nucleotide sequence of Pseudomonas sp. Ct9 is shown in SEQ ID NO. 3.

[0042] The Pseudomonas Ct9 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 Pseudomonas Ct9 can also significantly reduce the pH value of the soil during the process of degrading phosphorus.

[0043] The present invention provides a bacterial agent, comprising the Pseudomonas Ct9 described in the above technical solution. As an optional embodiment of the present invention, the bacterial activity of Pseudomonas Ct9 in the bacterial 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 Pseudomonas Ct9 in the bacterial agent is ≥10 7 CFU / g, can be 10 7 ~10 9 CFU / g, or 10 8 CFU / g.

[0044] The present invention provides a method for preparing the bacterial agent described in the above technical solution, comprising: culturing the Pseudomonas sp. Ct9 to obtain a bacterial agent. As an optional embodiment of the present invention, the culture medium includes LB medium; the culture temperature can be 26-37°C, or can 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 is accompanied by oscillation; the oscillation speed can 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 obtain Pseudomonas Ct9 bacterial liquid.

[0045] After obtaining the Pseudomonas Ct9 bacterial solution, the bacterial solution can be directly used as a bacterial agent. The present invention can also separate the Pseudomonas Ct9 bacterial solution, collect the bacterial bodies, resuspend the bacterial bodies in water to obtain a bacterial suspension, and use the obtained bacterial suspension as a bacterial agent.

[0046] The present invention provides a bacterial fertilizer, comprising the Pseudomonas Ct9 described in the above technical solution and a carrier. As an optional embodiment of the present invention, the bacterial activity of Pseudomonas Ct9 in the bacterial fertilizer is ≥10 7 CFU / g, can be 10 7 ~10 9 CFU / g, or 10 8As 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 8:2, 9:1, 8:1 or 9:2.

[0047] The present invention provides a method for using the bacterial fertilizer described in the above technical solution, comprising: mixing the bacterial fertilizer with water, preparing a bacterial fertilizer solution and applying it. As an optional embodiment of the present invention, the mass volume ratio of the bacterial fertilizer and water can be 12g:250mL. The bacterial fertilizer of the present invention can be applied at any stage of plant growth, preferably 10 days after the plant seeds germinate. When applying, the present invention is preferably applied to the rhizosphere soil. In the present invention, the application amount of the bacterial fertilizer is 250mL of bacterial fertilizer solution for 4 plants. As an optional embodiment of the present invention, the plants include safflowers of different colors.

[0048] The present invention provides the use of Pseudomonas sp. Ct9 described in the above-mentioned technical solution for the degradation of insoluble phosphate. As an optional embodiment of the present invention, the insoluble phosphate includes calcium phosphate, zinc phosphate, iron phosphate, and organic phosphorus. The present invention demonstrates, through examples, that the Pseudomonas sp. Ct9 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 Ct9 strain can significantly reduce the pH of the culture system while degrading the insoluble phosphate.

[0049] The present invention provides the application of Pseudomonas Ct9 described in the above technical solution in safflower cultivation. As an optional embodiment of the present invention, the safflower includes safflowers of different colors; the safflowers of different colors include any one or more of white safflowers, yellow safflowers and red safflowers. In the present invention, the Pseudomonas Ct9 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 Pseudomonas Ct9 can significantly increase the plant height, number of leaves, stem thickness, root length, root fresh weight, total fresh weight, top bud diameter and number of effective bulbs per plant of safflowers of different colors, thereby promoting the growth of safflowers of different colors. The Pseudomonas Ct9 can significantly increase the content of hydroxysafflor yellow A and kaempferol active ingredients in the filaments of safflowers of different colors, thereby improving the quality of safflowers of different colors.

[0050] The present invention provides the use of the Pseudomonas Ct9 described in the above technical solution for improving soil physical and chemical properties. The Pseudomonas Ct9 provided by the present invention can significantly reduce soil pH 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 improving the soil.

[0051] The present invention provides a method for promoting safflower growth and / or improving safflower quality, comprising:

[0052] During the growth of safflower, the bacterial agent described in the above technical solution or the bacterial fertilizer described in the above technical solution is applied. In the present invention, the bacterial agent and bacterial fertilizer can be applied at various stages of the growth of safflower. As an optional embodiment of the present invention, the bacterial agent and bacterial fertilizer can be applied when the safflower germinates for 10 days. As an optional embodiment of the present invention, when the bacterial fertilizer is applied, the bacterial fertilizer is mixed with water to prepare a bacterial fertilizer solution for application; the mass volume ratio of the bacterial fertilizer to water is 12g:250mL; the application amount can be 250mL of bacterial fertilizer solution for 4 safflower plants. As an optional embodiment of the present invention, the safflower includes safflowers of different colors; the safflowers of different colors include any one or more of white safflower, yellow safflower and red safflower.

[0053] 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.

[0054] Example 1 Isolation and purification of strains

[0055] 1. Collection of rhizosphere soil and soil

[0056] Soil: Shihezi Experimental Station (latitude: 44°19′34.4″; longitude: 85°59′44.1″);

[0057] Safflower rhizosphere soil: Safflower experimental field, Shihezi Experimental Station;

[0058] 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.

[0059] 2. Isolation and purification of phosphate-solubilizing strains

[0060] 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 each soil suspension from each of the three gradients was applied to an inorganic phosphate solid medium. After incubation for 5 days, colonies exhibiting a phosphate-solubilizing zone were selected, purified, numbered, and stored at -80°C for future use. The activated phosphate-solubilizing bacteria were then inoculated onto 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). The phosphate-solubilizing ability 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.

[0061] 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.

[0062] Table 1 Solubility index of phosphate-solubilizing bacteria

[0063] strains 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 Ct9 2.183 0.683 3.198±0.095a 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

[0064] As shown in Table 1, according to the solubility index analysis of phosphate-solubilizing bacteria, among the 14 phosphate-solubilizing bacteria, Ct9 has the highest solubility index, reaching 3.198, while the solubility indexes of the remaining phosphate-solubilizing bacteria are all lower than 3. Therefore, based on its higher solubility index, Ct9 strain was selected as the target strain for subsequent experiments. The phosphate-solubilizing circle diagram of Ct9 strain is shown in Figure 1. Figure 1 As shown in A.

[0065] 3. Physiological and biochemical tests were performed on the Ct9 strain. The results are shown in Table 2.

[0066] Table 2 Physiological and biochemical test results of Ct9 strain

[0067] project Ct9 Facultativeaerobic + Gram staining - Contactenzymetest + Methyl red test - VP test V-Ptest - Urease test - IAA production test + Indole production test - Tryptophan deaminase - Hydrolysis of starch - Gelatin liquefaction - <![CDATA[H2S production test]]> -

[0068] As shown in Table 2, strain Ct9 is a facultative aerobic Gram-negative bacterium, which is positive in the catalase test and can produce IAA.

[0069] 4. Ct9 strain identification

[0070] (1) Ct9 strain morphology

[0071] The Ct9 strain was streaked onto LB solid medium plates and cultured at 28°C. The colony morphology of the Ct9 strain was as follows: Figure 1 As shown in B. The colonies are round, convex, shiny and opaque. Gram staining of Ct9 strain was performed, and the results of Gram staining are shown in Figure 1As shown in C. Gram staining results showed that Ct9 was a Gram-negative bacterium with a short rod shape.

[0072] (2) Molecular biological identification of Ct9 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] 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 Then, the DNA fragments were recovered from the agarose gel using a SanPrep column DNA gel recovery kit and sequenced to obtain the 16s rDNA sequence of the Ct9 strain, as shown in SEQ ID NO.3, specifically:

[0080]

[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 As shown. Figure 3 It was found that strain Ct9 and Pseudomonas fluorescens were clustered in the same branch.

[0082] Combined with the results of physiological and biochemical tests and molecular biological identification, the Ct9 strain was identified as Pseudomonas sp., with the taxonomic name Pseudomonas sp., and was deposited with the China Center for Type Culture Collection on March 24, 2025, with the deposit number CCTCC NO: M 2025570. The collection center is located at Room 211, China Center for Type Culture Collection, Wuhan University, Wuchang District, Wuhan City, Hubei Province.

[0083] Example 2

[0084] 1. Phosphate solubility and pH determination of Ct9 strain for different insoluble phosphorus compounds

[0085] 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.

[0086] 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.

[0087] 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.

[0088] 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.

[0089] After the Ct9 strain was activated, it was inoculated into the above-mentioned insoluble inorganic phosphorus medium and Montgina organic phosphorus medium and cultured at 180 r·min. -1 , shake in a shaker at 28°C for 3 days, and use the molybdenum antimony colorimetric method to determine the phosphate solubilization ability and pH of the Ct9 strain in different culture media.

[0090] The phosphate solubilization ability of Ct9 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.

[0091] Table 5 Phosphate solubility test results of Ct9 strain for different insoluble phosphorus compounds

[0092]

[0093] Table 6 pH detection results during phosphate solubilization of strain Ct9

[0094]

[0095]

[0096] From Tables 5 to 6 and Figure 4 It can be found that the Ct9 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 Ct9 strain can significantly reduce the pH in the culture system while degrading insoluble phosphorus.

[0097] 2. Soluble phosphorus content and pH detection during Ct9 strain cultivation

[0098] After the Ct9 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.

[0099] The test results are shown in Table 7 and Figure 5 shown.

[0100] Table 7 Results of soluble phosphorus content and pH test during the culture of Ct9 strain

[0101] Number of days 0 1 2 3 4 5 6 7 Soluble phosphorus content 1.99 24.43 128.90 206.21 232.81 251.51 276.94 258.11 pH 7.13 5.65 4.55 4.30 4.19 4.14 4.10 4.22

[0102] From Table 7 and Figure 5It can be found that during the cultivation of the Ct9 strain, the pH in the culture solution dropped sharply within 0 to 2 days of cultivation. After 2 days of cultivation, the pH of the culture solution began to slowly decrease and remained between 4 and 5. As the cultivation time increased, the soluble phosphorus content in the culture system gradually increased within 0 to 6 days of cultivation. After 6 days of cultivation, the soluble phosphorus content in the culture system reached the highest level. In summary, the Ct9 strain provided by the present invention has the ability to produce acid and solubilize phosphorus during the cultivation process.

[0103] 4. Determination of the secretion capacity of auxin (IAA) of Ct9 strain

[0104] The IAA secretion capacity of the strains was determined using the Salkoeski method.

[0105] The Ct9 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 at 28℃ for 3 days, take the bacterial solution and put it into 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 As shown. Figure 6 It can be seen that the IAA secretion was determined by the Salkoeski method. Qualitative analysis showed that Ct9 was pink compared with CK on the white porcelain plate, indicating that Ct9 can secrete IAA.

[0106] Example 3 Growth-promoting effect of Ct9 strain on safflower

[0107] 1. Production of bacterial fertilizer

[0108] (1) Preparation of microbial agents

[0109] Take out the Ct9 strain from the -80℃ freezer, streak the Ct9 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 (OD600=1). Take 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 ~10 9 CFU / mL, and the bacterial agent was obtained.

[0110] (2) Production of bacterial fertilizer

[0111] 50 mL of the bacterial 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 bacterial fertilizer. The carriers are diatomaceous earth and skim milk powder, and the mass ratio of diatomaceous earth to skim milk powder is 9:1.

[0112] 2. Potted Plant Experiment

[0113] Prepare a bacterial fertilizer solution at a ratio of 12 g of bacterial fertilizer to 250 mL of sterile water. 10 days after safflowers with filament colors (white, yellow, and red) germinate, apply 250 mL of bacterial fertilizer solution to each pot of safflower. Plant 4 safflower seedlings in each pot, and inoculate the control group with an equal amount of sterile carrier liquid.

[0114] 3. Agronomic trait determination

[0115] On days 14 and 80 after the application of the microbial fertilizer, as well as during the flowering phase, the biomass of the 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 plots, 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.

[0116] The phenotype of safflower on the 14th day after the application of bacterial 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 bacterial fertilizer is as follows Figure 9 The agronomic traits are shown in Table 9 and Figure 10 The agronomic characteristics of safflower during flowering are shown in Table 10 and Figure 11 shown.

[0117] Table 8 Agronomic characteristics of safflower on the 14th day after application of microbial fertilizer

[0118]

[0119] Table 9 Agronomic characteristics of safflower on the 80th day after application of microbial fertilizer

[0120]

[0121] Table 10 Agronomic characteristics of safflower during flowering after application of microbial fertilizer

[0122]

[0123] From Tables 8 to 10 and Figures 7-11It can be obtained that the plant height, leaf number, root length, root fresh weight and total fresh weight of the three colors of safflower were significantly increased relative to the control group 14 days after the application of the bacterial manure, and the difference was significant. 80 days after the application of the bacterial manure, the plant height, leaf number, stem thickness, root length, root fresh weight and total fresh weight of the three colors of safflower were significantly increased relative to the control group, and the difference was significant. After the application of the bacterial manure, the plant height, stem thickness, top bud diameter and number of effective balls per plant of safflower during the flowering period were significantly increased relative to the control group. In summary, the bacterial manure provided by the present invention can significantly promote the growth of safflower of different colors, and 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 safflower.

[0124] 4. Effects of Ct9 on the active ingredients of safflowers with different colors and different flowering days

[0125] (1) Effects of Ct9 on the active ingredients of white safflower at different flowering days

[0126] 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.

[0127] Table 11 HSYA and KF detection results in the filaments of white safflower

[0128]

[0129] From Table 11 and Figure 12 The results showed that the application of bacterial fertilizer could significantly increase the content of HSYA and KF in the silk of white safflower at different flowering days compared with no application of bacterial fertilizer, and improve the quality of white safflower.

[0130] (2) Effects of Ct9 on the active ingredients of yellow safflower at different flowering days

[0131] 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.

[0132] Table 12 HSYA and KF detection results in yellow safflower filaments

[0133]

[0134]

[0135] Depend on Figure 13 It can be seen that the application of bacterial fertilizer can significantly increase the content of HSYA and KF active ingredients in the filaments of yellow safflower at different flowering days compared with no application of bacterial fertilizer, and improve the quality of yellow safflower.

[0136] (3) Effects of Ct9 on the active ingredients of red safflower at different flowering days

[0137] 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.

[0138] Table 13 HSYA and KF detection results in red safflower filaments

[0139]

[0140] From Table 13 and Figure 14 The results showed that the application of bacterial fertilizer could significantly increase the content of HSYA and KF in the filaments of red safflower at different flowering days compared with no application of bacterial fertilizer, and improve the quality of red safflower.

[0141] 5. Determination of various soil indicators

[0142] Ten grams of safflower rhizosphere soil were 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 were 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 period of simmering, the supernatant was filtered through a 0.45 μm filter membrane and then analyzed using a total organic carbon analyzer for total inorganic carbon, total organic carbon, and total carbon. The soil organic matter content was determined by multiplying the total organic carbon content by a factor of 1.724. Soil total phosphorus was determined using the NaOH molten-molybdenum antimony colorimetric method; total nitrogen was determined using Kjeldahl determination; available phosphorus was determined using the molybdenum antimony colorimetric method; and 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.)

[0143] 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.

[0144] Table 14 Test results of various physical and chemical properties of soil of various safflowers in the experimental group and the control group

[0145]

[0146]

[0147] From Table 14 and Figure 15 After applying the microbial fertilizer, the pH value of the rhizosphere soil of all flower colors decreased compared to the control. Total nitrogen, organic matter, total phosphorus, and available phosphorus all increased significantly. Total phosphorus increased by 18.56%, 7.64%, and 11.85% in white safflower, yellow safflower, and red safflower, respectively; organic matter increased by 27.05%, 15.74%, and 29.94%, respectively; total phosphorus increased by 2.84%, 5.25%, and 3.90%, respectively; and available phosphorus increased by 5.48%, 6.14%, and 8.32%, respectively. Furthermore, organic phosphorus in the soil of white safflower and yellow safflower increased by 19.06% and 18.06%, respectively. Available potassium increased significantly in the soil of safflower and white safflower; organic phosphorus increased significantly in the soil of both yellow and white safflower; inorganic phosphorus increased significantly in the soil of red safflower, while there was no significant change in the soil of yellow and white safflower. Total potassium did not change significantly in the soil of any flower color. This shows that the bacterial fertilizer Ct9 has a significant effect in improving soil acid-base balance and enhancing soil fertility, but there are differences in the responses of plants with different flower colors to the bacterial fertilizer.

[0148] 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. A Pseudomonas sp. Ct9, characterized in that The deposit number of the Pseudomonas sp. Ct9 is CCTCC NO: M 2025570.

2. A bacterial agent, characterized in that The invention comprises the Pseudomonas Ct9 according to claim 1.

3. The method for preparing the microbial agent according to claim 2, characterized in that: include: The Pseudomonas sp. Ct9 is cultured to obtain a bacterial 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 bacterial fertilizer, characterized in that: The invention comprises the Pseudomonas Ct9 according to claim 1 and a vector.

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

7. Use of the Pseudomonas sp. Ct9 according to claim 1 in the degradation of insoluble phosphorus.

8. Use of the Pseudomonas Ct9 according to claim 1 in safflower cultivation.

9. Use of the Pseudomonas sp. Ct9 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 bacterial agent according to claim 2 or the bacterial fertilizer according to claim 5 is applied during the growth of safflower.

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