A rhizosphere growth-promoting bacterium for promoting crop growth and its application

Abstract

The present invention discloses a rhizosphere growth-promoting bacterium for promoting crop growth. The rhizosphere growth-promoting bacterium (Streptomyces p.) is Streptomyces SwB1, which was deposited in the China Center for Type Culture Collection on March 14, 2023, with a deposit number of CCTCCNO: M2023321, a deposit address of Wuhan University, Wuhan, China, and a classification name of Streptomyces p. SwB1. The rhizosphere growth-promoting bacterium can be used to prepare a biological preparation for promoting crop growth, and has the functions of nitrogen fixation, phosphorus solubilization, potassium solubilization, IAA secretion, and iron carrier production.

Description

Technical Field

[0001] The present invention relates to the technical field of screening rhizosphere growth-promoting bacteria, and more specifically to rhizosphere growth-promoting bacteria for promoting crop growth and applications thereof. Background Art

[0002] Swida wilsoniana is a deciduous shrub or small tree belonging to the genus Swida of the family Cornaceae of the dicotyledonous plant class. It has strong adaptability and can grow in all kinds of soils, including neutral, slightly acidic and slightly saline-alkali soils, becoming a pioneer tree species for rocky desertification afforestation. Due to the high oil content of the whole fruit, Swida wilsoniana can be used as an important ecological and economic oil tree species, and has significant development potential in many fields such as plant oils and fats.

[0003] Bacteria that grow in the rhizosphere of plants and are beneficial to plant growth are called plant rhizosphere growth-promoting bacteria. Plant rhizosphere growth-promoting bacteria can prevent and control pathogens in the soil, reduce the harm of pathogens to plants, improve the efficiency of plants in absorbing and utilizing mineral nutrients, produce metabolites that are beneficial to plant growth, thereby promoting plant growth, increasing yields, and improving the ability of plants to resist biotic and abiotic stresses. The main growth-promoting effects of plant rhizosphere growth-promoting bacteria are that they can fix nitrogen in the soil, metabolize to produce organic acids, promote the absorption of elements such as phosphorus and potassium by plants, and promote plant growth and development by secreting auxins.

[0004] For a long time, chemical fertilizers have met the necessary nutrient needs of agricultural production and played an important role in increasing agricultural production. However, the application efficiency of chemical fertilizers is very low, and excessive use of chemical fertilizers can lead to soil degradation and soil salinization. In addition, when too much chemical fertilizer is used, the abundance and diversity of soil microorganisms will decrease. Using plant rhizosphere growth-promoting bacteria and organic fertilizers instead of chemical fertilizers, protecting the ecology of farmland soil, and reducing soil degradation and salinization are important directions for the development of modern agriculture.

[0005] Therefore, the development and application of rhizosphere growth-promoting bacteria isolated from the pioneer tree species Lonicera japonica in promoting crop growth and development has great practical value and economic potential. Summary of the invention

[0006] In view of this, the present invention provides a rhizosphere growth-promoting bacterium for promoting crop growth, which has the functions of fixing nitrogen, solubilizing phosphorus and potassium, secreting IAA, and producing iron carriers, and can provide the nutrients and growth-promoting substances needed by plants in a long-term manner, thereby reducing the harm of chemical fertilizers to the soil.

[0007] In order to achieve the above object, the present invention adopts the following technical solution:

[0008] A rhizosphere growth-promoting bacterium that promotes crop growth. The rhizosphere growth-promoting bacterium (Streptomyces sp.) is Streptomyces SwB1, which was deposited in the China Center for Type Culture Collection on March 14, 2023, with a collection number of CCTCC NO: M2023321, and a collection address of Wuhan University, Wuhan, China. It is classified and named Streptomyces sp. SwB1.

[0009] As an inventive concept identical to the above technical solution, the present invention also seeks to protect the use of the Streptomyces SwB1 in the preparation of biological agents for promoting crop growth.

[0010] Preferably, the Streptomyces SwB1 has the functions of solubilizing phosphate, potassium, fixing nitrogen, producing IAA and producing siderophores.

[0011] As an inventive concept identical to the above technical solution, the present invention also requests protection for a biological agent for promoting crop growth, including a Streptomyces SwB1 agent.

[0012] Through the above technical scheme, it can be known that the present invention provides a multi-functional rhizosphere growth-promoting bacterium of the strychnos nux vomica, which has the functions of nitrogen fixation, phosphorus solubilization, potassium solubilization, IAA secretion, and iron carrier production, and can provide the nutrients and growth-promoting substances needed by plants for a long time, thereby reducing the harm of chemical fertilizers to the soil. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0014] Figure 1 The accompanying drawing is a colony diagram of Streptomyces SwB1 of the present invention;

[0015] Figure 2 The accompanying drawing is a phylogenetic tree of Streptomyces SwB1 of the present invention;

[0016] Figure 3 The accompanying drawing is a comparison diagram of the phosphate solubilization ability of Streptomyces SwB1 of the present invention;

[0017] Figure 4 The accompanying drawing is a growth curve diagram of Streptomyces SwB1 of the present invention;

[0018] Figure 5 The accompanying drawing is a diagram for identifying the nitrogen fixation ability of Streptomyces SwB1 of the present invention;

[0019] Figure 6The accompanying drawing is a diagram for identifying the potassium-solubilizing ability of Streptomyces SwB1 of the present invention;

[0020] Figure 7 The attached figure is a diagram for identifying the ability of Streptomyces SwB1 to produce siderophores of the present invention. DETAILED DESCRIPTION

[0021] The following will be combined with the drawings in 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.

[0022] Example 1 Screening of phosphate-solubilizing bacteria

[0023] The rhizosphere soil samples were taken from the rhizosphere soil of the safflower tree planting base at the Hunan Academy of Forestry Experimental Forest Farm. The rhizosphere soil was obtained by the shaking soil method. Weigh 1g of rhizosphere soil and put it into a triangular flask containing 99mL of sterile water. Shake it for 5 minutes to disperse the bacteria. In this way, the sample was diluted to 10 -2 Then dilute to 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 , 10 -8 . The bacterial suspension was evenly spread on the inorganic phosphorus selective medium and inverted for 7 days at 28°C. The isolated phosphate-solubilizing bacteria were purified by streaking on the plate to obtain pure colonies of the bacteria. The bacteria with the best phosphate-solubilizing effect were selected and named SwB1. Figure 1 As shown. The colony is round, dry, not easy to pick up, opaque, 2-3mm in diameter; the hyphae are white and radial. Microscopic examination results show that the hyphae are whorled and the spores are spiral-shaped. It is preliminarily identified as Streptomyces.

[0024] The inorganic phosphorus selective medium formula is: glucose 10g, ammonium sulfate 0.1g, magnesium sulfate heptahydrate 0.25g, potassium chloride 0.2g, magnesium chloride hexahydrate 5g, calcium phosphate 5g, agar 15g, pH 7.0, deionized water 1000mL, 1×10 5 Pa sterilization for 25 minutes.

[0025] Example 2 Identification of phosphate-solubilizing bacteria

[0026] Pick the spores of SwB1 in NB liquid medium and activate them for 24 hours. Take 50μL of bacterial solution in a centrifuge tube and lyse it at 100℃ for 15 minutes using a PCR instrument. Use bacterial 16S rDNA universal primers for PCR amplification. The universal primers are 27F (5'-AGAGTTTGATCCT-GGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3'), which are shown in SEQ ID NO.2 and SEQ ID NO.3 respectively; the amplification system uses a 40μL system: 4μL DNA template, 20μL 2×T5 SuperPCR Mix, 1.6μL primers each, and 12.8μL ddH2O.

[0027] The NB liquid culture medium formula is: peptone 10g, sodium chloride 5g, beef powder 3g, pH 7.0, deionized water 1000mL, 1×10 5 Pa sterilization for 25 minutes.

[0028] PCR reaction conditions: 98℃ pre-denaturation for 2min, 98℃ denaturation for 10s, 55℃ annealing for 10s, 72℃ extension for 25s, 34 cycles, then 72℃ extension for 2min, and storage at 12℃. After PCR was completed, 2μL of PCR stock solution was taken for electrophoresis detection and 16SrDNA sequence determination. The sequencing sequence of the SwB16 strain is as follows:

[0029] >Streptomyces sp.SwB1

[0030]

[0031] The 16S rDNA sequence of the strain was blasted against the model strain nucleic acid database on the NCBI website (www.ncbi.nLm.nih.gov / blast), and the phylogenetic tree was constructed using the neighbor-joining method using the MEGA11 software. The results are as follows: Figure 2 As shown. Figure 2 It can be seen that the strain belongs to the same branch as OP630764.1. The results are consistent with the morphological identification and belong to the genus Streptomyces. The screened strain was named Streptomyces SwB1, and was deposited in the China Center for Type Culture Collection on March 14, 2023, with the deposit number CCTCC NO: M 2023321, the deposit address is Wuhan University, Wuhan, China, and the classification name is Streptomyces cessp. SwB1.

[0032] Example 3 Phosphate-solubilizing ability of phosphate-solubilizing bacteria

[0033] Pick a pure colony of SwB1, and after culturing in liquid culture medium containing calcium phosphate, iron phosphate, and aluminum phosphate for 7 days, take 2 mL of the culture medium into a 50 mL volumetric flask and dilute it 15 times with water. Add 2-3 drops of dinitrophenol indicator, and adjust the solution to just slightly yellow with 10% sodium carbonate solution or 5% sulfuric acid solution. Accurately add 5 mL of molybdenum antimony anti-color developer, shake well, and add water to make up to volume. Leave it at room temperature above 15°C for 30 minutes.

[0034] The colorimetric sample solution is placed on a spectrophotometer, and the instrument zero point is adjusted using a liquid culture medium without bacteria as the reference solution. The wavelength is 700 nm for colorimetric determination, and the absorbance is read. The corresponding phosphorus content is obtained from the standard curve, such as Figure 3 As shown, the calcium phosphate solubilizing ability of Streptomyces SwB1 is 244.59 mg / L, the aluminum phosphate solubilizing ability is 121.62 mg / L, and the iron phosphate solubilizing ability is 53.21 mg / L.

[0035] Example 4 Growth curve of SwB1

[0036] Spores of SwB1 were picked from the plate and prepared into spore suspension, which was activated for 24 h. The activated SwB1 suspension was gradiently diluted to 1×10 6 cfu / mL, take 5 μL of the bacterial solution into a conical flask containing 50 mL of NB medium, shake and mix appropriately. Incubate in a shaker at 28°C and 150 rpm for 24 hours, measure the absorbance every 2 hours, the wavelength is 600 nm, and the growth curve is shown in Figure 4 .

[0037] Example 5 Growth-promoting effect of SwB1

[0038] (1) Phosphate solubilization

[0039] As shown in Example 3.

[0040] (2) Nitrogen fixation

[0041] SwB1 was inoculated on nitrogen-free Ashby medium by streaking and incubated at 28°C for 48 h. Figure 5 It can be seen that the bacteria grow well and have the ability to fix nitrogen.

[0042] The nitrogen-free Ashby medium formula is as follows: nitrogen-free Ashby medium: weigh 10 g mannitol, 0.2 g KH2PO4, 0.2 g NaCl, 0.1 g CaSO4, 5 g CaCO3, 20 g agar, 1000 mL deionized water, pH 7.0±0.2, 1×10 5 Pa, moist heat sterilization for 25 minutes.

[0043] (3) Potassium-dissolving effect

[0044] SwB1 was inoculated on the potassium-solubilizing capacity test medium by streaking the plate and incubated at 28°C for 48 hours. Figure 6 It can be seen that a transparent zone is produced around the colony of SwB1, which can dissolve insoluble potassium and has the ability to dissolve potassium.

[0045] The potassium-solubilizing ability test medium formula is: 2.5 mg ferric chloride, 250 mg magnesium sulfate heptahydrate, 2.5 g sucrose, 0.1 g CaCO3, 1 g disodium hydrogen phosphate, 7.5 g agar powder, 500 mg potassium feldspar powder, 500 mL deionized water, pH 7.0±0.2, 121°C, 1×10 5 Pa sterilization for 20 minutes.

[0046] (4) IAA production detection

[0047] SwB1 was inoculated into the IAA detection medium and cultured at 28°C and 180 rpm for 48 hours. The colorless medium turned orange-yellow, indicating that SwB1 can produce IAA. The IAA production capacity calculated by the standard curve was 16.82 mg / L.

[0048] The IAA detection medium formula is: yeast extract powder 1g, mannitol 10g, potassium dihydrogen phosphate 0.5g, magnesium sulfate 0.2g, sodium chloride 0.1g, calcium carbonate 1g, L-tryptophan 2g, deionized water 1000mL, pH 6.8±0.2, 1×10 5 Pa sterilization for 25 minutes.

[0049] (5) Siderophore production detection

[0050] SwB1 was inoculated on the iron-free NB medium by streaking the plate and incubated at 28°C for 48 hours. Then the iron carrier detection solution was covered in the NB medium and the color was developed for 30 minutes. Figure 7 It can be seen that a red secretion circle is produced around the SwB1 colony, indicating that iron carriers are produced.

[0051] The iron carrier detection solution formula is: Solution A: weigh 0.079g CAS and dissolve it in 50mL deionized water, then add 10mL 1mmol / L FeCl3 solution (prepared with HCl); Solution B: dissolve 0.069g hexadecyltrimethylammonium bromide (HDTMA) in 40mL deionized water; slowly add solution A to solution B along the wall of the beaker, stir and mix to obtain 100mL CAS blue detection solution.

[0052] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0053] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A rhizosphere growth-promoting bacterium for promoting crop growth, characterized in that: The rhizosphere growth-promoting bacteria is Streptomyces sp. SwB1, which was deposited in the China Center for Type Culture Collection on March 14, 2023, with the collection number CCTCC NO: M 2023321, the collection address is Wuhan University, Wuhan, China, and the classification name is Streptomyces sp. SwB1.

2. Use of the Streptomyces SwB1 according to claim 1 in the preparation of a biological agent for promoting crop growth.

3. A biological agent for promoting crop growth, characterized in that: A bacterial agent comprising the Streptomyces SwB1 according to claim 1.

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