Application of bacillus altitudinis B6 with phosphate-solubilizing, potassium-solubilizing and growth-promoting functions in promoting growth of eucalyptus

Abstract

The invention relates to application of bacillus altitudinis B6 with phosphorus and potassium solubilizing and growth promoting functions in promoting the growth of eucalyptus. The strain has various phosphorus and potassium dissolving capacities, activates insoluble phosphorus and potassium in soil, promotes absorption of inorganic phosphorus by plants, improves nutrients of phosphorus and potassium in the plants, also has IAA production capacity, promotes plant root development and plant growth, and has a remarkable growth promoting effect. In the application process, the strain is pollution-free, residue-free and biologically environment-friendly, is a growth-promoting strain with a good application prospect in the field of plant growth promotion, and has an excellent application prospect.

Description

[0001] The invention belongs to the technical field of plant microorganisms, and in particular relates to the application of a Bacillus subtilis B6 with phosphorus- and potassium-solubilizing and growth-promoting functions in promoting the growth of eucalyptus and the application thereof. Background Art

[0002] Eucalyptus is one of the most important and widely cultivated tree species in the world. It boasts rapid growth, excellent wood quality, and strong resistance to pests and diseases. It matures quickly and has a short felling rotation, typically being harvested within 5-8 to 12 years, providing a rapid payback period. It is an excellent species for establishing timber forests and shelterbelts.

[0003] Phosphorus is an essential mineral element for plant growth and development. It is a component of important substances such as nucleic acids, proteins, and phospholipids, and is a key factor in a range of metabolic processes in plants, including photosynthesis, respiration, energy storage and transfer, and cell division. Phosphorus exists in the soil primarily as insoluble, inorganic, or organic compounds. To meet the needs of forestry and agricultural production, traditional phosphate fertilizer application can address phosphorus deficiency in plant growth and development. However, due to its high affinity for soil, much of the phosphorus in phosphate fertilizer combines with metal ions in the soil to form insoluble phosphates, which accumulate in the soil. This significantly reduces the efficiency of plant utilization of the applied phosphate fertilizer, and environmental pollution such as changes in aquatic ecosystems caused by eutrophication of water bodies can lead to soil acidification and damage to soil structure and function, hindering the sustainable development of forestry and agriculture.

[0004] Bacillus altitudinis is a rhizosphere salt-tolerant growth-promoting bacterium that can be used as a preferred strain of microbial fertilizer and has a growth-promoting resistance effect. Bacillus dissolves phosphate and produces IAA. The phosphate solubilization effect of Bacillus produces phosphorus for plants. IAA increases the elongation and surface area of plant roots, thereby promoting plant growth and alleviating heavy metal stress. For example, CN112795521B discloses a strain ZLB C01 that has the functions of solubilizing phosphorus, potassium, and nitrogen. It can colonize in plants, increase the activity of biological enzymes, and play an important role in regulating plant growth and development.

[0005] In view of this, the present invention is proposed. Summary of the Invention

[0006] The purpose of the present invention is to provide a strain of Bacillus subtilis B6 with the functions of solubilizing phosphate and potassium and promoting growth, and application thereof.

[0007] To achieve the above purpose, the present invention adopts the following technical solutions:

[0008] In the first aspect, the present invention provides a growth-promoting Bacillus altissima strain, which is classified and named Bacillus altissima (Bacillus altissima), and is deposited in the General Microbiology Center of the China Culture Collection Administration, located at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with a deposit number of CGMCC NO.: 32591.

[0009] In a second aspect, the present invention provides a method for preparing the above-mentioned Bacillus subtilis with the functions of solubilizing phosphate and potassium and promoting growth.

[0010] In a third aspect, the present invention provides an application of the above-mentioned Bacillus subtilis with the functions of solubilizing phosphate and potassium and promoting growth. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 This is a positive morphological characteristic diagram of Bacillus subtilis B6 grown in LB medium.

[0012] Figure 2 This is the reverse morphological characteristic diagram of Bacillus subtilis B6 grown on LB medium. Figure 3 This is a positive morphological characteristic diagram of Bacillus subtilis B6 grown on inorganic phosphate medium.

[0013] Figure 4 This is the reverse morphological characteristic diagram of Bacillus subtilis B6 grown on inorganic phosphate medium.

[0014] Figure 5 This is a positive morphological characteristic diagram of Bacillus subtilis B6 grown on silicate medium.

[0015] Figure 6 This is the reverse morphological characteristic diagram of Bacillus subtilis B6 grown on silicate medium.

[0016] Figure 7 Growth of eucalyptus seedlings inoculated with B6 fungicide (4 weeks). DETAILED DESCRIPTION

[0017] In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0018] Unless otherwise specified, the instruments and equipment involved in the following examples are all conventional instruments and equipment; the biochemical reagents and raw materials involved are all commercially available conventional products unless otherwise specified; the detection, testing, experimental methods involved are all conventional methods unless otherwise specified.

[0019] A growth-promoting Bacillus subtilis, classified and named B6, was deposited in the General Microbiology Center of the China Culture Collection Administration in November 2024, with the deposit number: CGMCC NO.: 32591.

[0020] Example 1 Screening of Phosphate-Solubilizing and Potassium-Solubilizing Bacillus

[0021] (1) Screening process

[0022] Several strains were isolated using the gradient dilution method. The isolated endophytic bacteria were purified using the three-zone streak method. Purification was confirmed by microscopic examination. Purified bacteria were numbered, and individual colonies were selected for future use. A single strain with excellent phosphate, potassium, and nitrogen-solubilization properties was identified and designated B6 using plate-based assays for phosphate, potassium, and nitrogen-fixing properties.

[0023] (2) Colony characteristics and morphological identification

[0024] The colonies are milky white, dull, round and non-fluid; the diameter of the colonies is 4-5 mm.

[0025] (3) Molecular biological identification

[0026] Comparison of the measured 16S rRNA with sequences in the NCBI database revealed that B6 is on the same branch as Bacillus saltitudinis, and its 16S rDNA sequence is 99.86% similar to Bacillus saltitudinis (MW474842.1). Combining colony morphology, physiological and biochemical characteristics, and 16S rDNA sequence analysis, B6 was identified as Bacillus saltitudinis.

[0027] Example 2 Plate experiment of phosphate- and potassium-solubilizing Bacillus subtilis

[0028] 1. Determination of phosphate solubilization efficiency

[0029] Use inorganic phosphorus bacterial culture medium to test whether the B6 strain has the ability to solubilize phosphate. Weigh an appropriate amount of culture medium, add distilled water or deionized water to 1L, stir and resuspend, heat to promote dissolution, adjust the pH to 7.0±0.2; sterilize at 121℃ for 15min or 115℃ for 20min, mix well, and pour into a plate; inoculate the B6 strain on the culture medium plate, invert it and culture it in an incubator at 30℃ for 3~7 days. Observe the formation of a transparent circle around the colony, such as Figure 1 、 2 shown.

[0030] 2. Potassium-dissolving efficiency test

[0031] Use silicate bacterial culture medium to test the B6 strain's potassium-dissolving ability. Weigh an appropriate amount of culture medium, add distilled water to 1 L, stir thoroughly, and heat to promote dissolution. Adjust the pH to 7.0-7.2. Sterilize the culture medium at 121°C for 15 minutes or 115°C for 20 minutes. Cool the solid culture medium to approximately 50°C, pour the culture medium into a Petri dish, and store at 4°C in the dark until ready for use. Inoculate the B6 strain onto the culture medium plate, invert, and incubate in an incubator at 30°C for 3-7 days. Observe for the formation of a clear ring around the colonies.

[0032] Example 3 Test of the degradation effect of Bacillus subtilis B6 on soil

[0033] 1. Preparation of bacterial suspension

[0034] The activated strain B6 was inoculated into LB liquid culture medium, and then cultured with shaking at 30°C and 180 r / min for 12 h (logarithmic growth phase), and then centrifuged at 4°C and 6000 r / min for 10 min to collect the bacteria; the bacteria were washed three times with PBS buffer, collected, and diluted with PBS buffer to prepare a bacterial suspension, and the bacterial liquid content was adjusted to about OD600 of 2.0, which is the B6 bacterial agent.

[0035] 2. Soil sampling

[0036] Soil samples around the roots of eucalyptus were taken, freeze-dried, ground, sieved, and stored in a -20°C refrigerator. 5.0 g of soil was weighed, added with 10 ml of acetonitrile and vortexed for 1 minute, 2.0 g of NaCl was added and vortexed for 5 minutes, ultrasonicated for 10 minutes, and centrifuged at 6000 r / min for 5 minutes. Then 2 ml of the supernatant was taken and transferred to another centrifuge tube, and 1000 g of anhydrous magnesium sulfate and 50 mg of PSA were added in sequence, vortexed for 2 minutes, and centrifuged at 6000 r / min for 5 minutes. Finally, 1 ml of the supernatant was taken, dried with nitrogen, made up to volume with 1 ml of n-hexane, and filtered through a 0.22 μm filter membrane for injection.

[0037] 3. Biotransformation

[0038] The B6 bacterial agent cultured in Example 3 was adjusted to a certain concentration and then added to the soil.

[0039] Example 3 Test on the Salt Resistance and Growth-Promoting Effect of Bacillus subtilis B6 on Eucalyptus

[0040] All test strains were cultured overnight in a shaker at 37°C, centrifuged at 4500 rpm for 10 minutes, and the supernatant was decanted. The suspensions were washed and resuspended in sterile deionized water, and then mixed in equal volumes according to the inoculum composition. The resuspended suspensions were evenly applied to the roots of eucalyptus seedlings in the treatment group, while sterile deionized water was used as a control. The inoculum was then applied every two weeks, and the growth of the eucalyptus seedlings was observed.

[0041] The commercial bacterial fertilizer (bacterial fertilizer 3: Zhongnong Lv Kangqiwei No. 1) was diluted according to the instructions and used to treat the eucalyptus seedlings. After 4 weeks of growth, the plant height of the eucalyptus seedlings was measured. Among them, the bacterial agent combinations 1, 2, 6, and 9 had a significant growth-promoting effect. The results are as follows Figure 7 shown.

Claims

1. A strain of Bacillus subtilis B6, deposited with CGMCC No.: 32591.

2. A Bacillus subtilis B6 with the function of solubilizing phosphorus and potassium and promoting growth as claimed in claim 1 and its application.

3. The use of the Bacillus subtilis B6 having the function of solubilizing phosphate and potassium according to claim 1 or 2, characterized in that : The application of the Bacillus subtilis in any of the following aspects: for decomposing insoluble phosphorus, for producing auxin, for salt tolerance, for promoting plant seed germination, for increasing soil available phosphorus, microbial carbon and nitrogen, and for reducing soil available heavy metals.

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