A Bacillus zhangzhouensis and its application

Abstract

The present invention discloses Bacillus zhangzhouensis BG30 isolated from the roots of Robinia pseudoacacia cv. Hongsenhuai, which was deposited at the Guangdong Microbial Culture Collection Center (GDMCC) on December 30, 2022, and the strain preservation number is GDMCC NO. 63104. Bacillus zhangzhouensis BG30 of the present application can effectively dissolve insoluble phosphorus and potassium in the rhizosphere soil of plants, can greatly increase the content of available phosphorus and potassium in the soil, improve the utilization rate of phosphorus and potassium in the soil by plants, thereby promoting plant growth, reducing the usage amount of chemical fertilizers, and increasing crop yields.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a Bacillus zhangzhouensis and its application. Background Art

[0002] The forms of phosphorus in soil are two types: organic phosphorus such as inositol phosphate, phosphoprotein, nucleotide, etc. and inorganic phosphorus such as phosphatic minerals limestone, secondary phosphate, etc. When the environment is acidic, phosphorus in the soil combines with Fe 3+ and Al 3+ to form insoluble phosphates; when the environment is alkaline, phosphorus in the soil combines with Ca 2+ to form insoluble phosphates. Most of the phosphate fertilizers applied to the soil are converted into insoluble phosphates, and the utilization rate by plants is very low. Through biological transformation, the utilization rate of phosphate fertilizers can be partially increased. In addition to the production of acidic substances by plant roots under phosphorus stress, microorganisms can also produce acidic substances through their own metabolic activities to dissolve insoluble phosphates. Non-water-soluble potassium ore resources represented by potassium feldspar are rich. Because of their easy mining and excellent quality, they are the main way of potassium fertilizer supply. Traditional potassium release methods include melting leaching method, sintering method, high-temperature melting method, hydrothermal method, low-temperature decomposition method, ion exchange method, etc. These physical and chemical methods have high energy consumption, high pollution, low conversion rate and are difficult to industrialize. Using microorganisms to release potassium effectively solves the disadvantages of traditional methods and conforms to the principle of sustainable development of agriculture. Potassium-releasing bacteria can have a biochemical reaction with potassium feldspar powder, and the combined action of its low-molecular-weight acidic metabolites and the released extracellular polysaccharides or the organic acids and enzymes produced can destroy the crystal structure of potassium feldspar and decompose it into soluble potassium that can be directly absorbed and utilized in the processes of plant growth and metabolism.

[0003] Therefore, isolating and screening phosphorus-dissolving and potassium-releasing functional strains from the rhizosphere of plants and studying their growth-promoting effects on plants are of great significance for reducing the use of potassium fertilizers and increasing crop yields. Summary of the Invention

[0004] The present invention aims to solve at least one of the problems in the related technologies to some extent. For this reason, the object of the present invention is to provide a Bacillus zhangzhouensis BG30, which can promote plant growth, reduce the usage amount of chemical fertilizers, increase crop yields, and has important significance in agricultural production.

[0005] In order to achieve the above-mentioned purpose, the present application adopts the following technical scheme: The Bacillus zhangzhouensis BG30 described in the present invention has been deposited in the Guangdong Provincial Microbiological Culture Collection Center (GDMCC) on December 30, 2022, with the culture collection number GDMCCNO.63104, and the classification name is Bacillus szhangzhouensis. The storage address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0006] The present invention isolates Bacillus zhangzhouensis BG30 from the root of Sophora japonica, and conducts morphological, physiological and biochemical characteristics and genetic identification on it. The 16S rDNA identification result verifies that it is Bacillus zhangzhouensis. By measuring the phosphorus and potassium contents, the results show that the strain can effectively dissolve the insoluble phosphorus and potassium in the rhizosphere soil of plants, can greatly increase the content of available phosphorus and potassium in the soil, improve the utilization rate of phosphorus and potassium in the soil by plants, and then promote plant growth, reduce the use of chemical fertilizers, and increase crop yields.

[0007] The Bacillus zhangzhouensis BG30 strain of the present application has the following morphological and physiological characteristics:

[0008] a. Bacterial morphological characteristics: Bacillus zhangzhouensis BG30 strain is rod-shaped.

[0009] b. Colony morphology: It grows fast on LB agar plates. The colonies are round, light yellow, convex in the middle, and moist and neat at the edges (30°C, 48 hours of growth). The colony diameter is 1.3-2.4 cm. The optimal growth temperature is 30°C and the optimal growth pH is 7.

[0010] The Bacillus zhangzhouensis BG30 strain has the following morphological and physiological characteristics:

[0011] a. Bacterial morphological characteristics: Bacillus zhangzhouensis BG30 strain is rod-shaped.

[0012] b. Colony morphology: It grows fast on LB agar plates. The colonies are round, light yellow, convex in the middle, and moist and neat at the edges (30°C, 48 hours of growth). The colony diameter is 1.3-2.4 cm. The optimal growth temperature is 30°C and the optimal growth pH is 7.

[0013] The present application also provides an application of Bacillus zhangzhouensis BG30 in increasing the available potassium content in the rhizosphere soil of plants.

[0014] The present application also provides an application of Bacillus zhangzhouensis BG30 in increasing the available phosphorus content in the rhizosphere soil of plants.

[0015] The present application also provides an application of Bacillus zhangzhouensis BG30 in dissolving the insoluble potassium in the rhizosphere soil of plants.

[0016] The present application also provides an application of Bacillus zhangzhouensis BG30 in dissolving the insoluble phosphorus in the rhizosphere soil of plants.

[0017] The present application also provides an application of Bacillus zhangzhouensis BG30 in the preparation of a preparation for promoting plant growth.

[0018] The present application also provides an application of Bacillus zhangzhouensis BG30 in the preparation of a preparation for increasing the plant height.

[0019] The present application also provides an application of Bacillus zhangzhouensis BG30 in the preparation of a preparation for increasing the potassium content in plants.

[0020] The present application also provides an application of Bacillus zhangzhouensis BG30 in the preparation of a preparation for increasing the phosphorus content in plants.

[0021] The above technical solutions provided in the embodiments of the present application have the following advantages compared with the prior art: Bacillus zhangzhouensis BG30 provided in the present application can effectively dissolve the insoluble phosphorus and potassium in the rhizosphere soil of plants, can greatly increase the content of available phosphorus and potassium in the soil, improve the utilization rate of phosphorus and potassium in the soil by plants, thereby promoting plant growth, reducing the usage amount of chemical fertilizers, and increasing the crop yield. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 It is a colony map after the strain BG30 is diluted and spread on an LB solid medium and cultured for 48 hours;

[0023] Figure 2 It is a phosphorus-dissolving circle formed by the strain BG30;

[0024] Figure 3 It is the potassium-dissolving circle formed by strain BG30. Specific implementation manners

[0025] The following examples are used to illustrate the present invention, but are not used to limit the scope of the present invention. Without departing from the spirit and essence of the present invention, any modification or replacement made to the methods, steps or conditions of the present invention shall fall within the protection scope of the present invention. If not specifically specified, the experimental materials, reagents, instruments, etc. used in the examples of the present invention can be obtained commercially; if not specifically specified, all technical means in the examples of the present invention are conventional means well known to those skilled in the art.

[0026] Example 1 Isolation and purification of strain

[0027] The nitrogen-fixing microorganisms were isolated and purified from Robinia pseudoacacia var. deeringiana. The specific isolation and purification steps are as follows: The medium BPA medium (1.0 L) used for isolation: beef extract 5.0 g, peptone 5.0 g, glucose 10.0 g, yeast extract 1.0 g, agar 20.0 g (solid), pH 7.0, autoclaved at 121 °C for 15 min.

[0028] The roots of Robinia pseudoacacia var. deeringiana were respectively cut into 3-cm segments with sterile scissors, the surface dust was washed with sterile water, soaked in 70% alcohol for 5 min, washed once with sterile water, soaked in 0.1% mercuric chloride for 5 min. After surface disinfection, it was washed 10 times with sterile water, 10 min each time. The sterile water of the last wash was spread on the LB plate as a control to determine whether the disinfection was thorough. Cut off 1 cm from both ends of the disinfected roots, and inoculate them into the solid BPA medium respectively. Repeated subculture was carried out on the BPA medium by the streak plate method and the dilution plate method until the color, shape, size, texture and transparency of the colonies were the same. Finally, through simple staining (carbol fuchsin staining) and microscopic examination (oil immersion lens), its morphology was further observed, and the purification standard of the strain was that the length was uniform, the width was consistent, and the staining condition was unified. The purified strain BG30 was stored at -20 °C and -80 °C in 15% glycerol.

[0029] Example 2 Physiological and biochemical characteristics and classification of the strain

[0030] 1. Morphological characteristics

[0031] The strain BG30 was prepared into a bacterial suspension, diluted and spread on the LB medium, and the colony and cell morphology were observed after culturing at 30 °C for 24 h.

[0032] The results showed that the bacteria were Gram-negative. After culturing on the LB medium for 24 h, the colonies were round, with serrated edges, milky white, opaque and without luster.

[0033] 2. Physiological and biochemical characteristics

[0034] The physiological and biochemical characteristics of strain BG30, such as VP test, MR test, indole test, etc., were determined with reference to the bacterial identification manual. The results showed that the urease test and nitrate reduction test of this strain were negative, while the ammonia production test, gelatin liquefaction test, catalase test, VP test and MR test were positive.

[0035] 3. 16S rDNA sequencing

[0036] The genomic DNA of strain BG30 was extracted using a bacterial genomic DNA extraction kit. The 16S rDNA was amplified with the bacterial universal primers 27F (5’-AGAGTTTGATCCTGGCTCAG) and 1492R (5’-TACCTTGTTACGACTT). After electrophoresis detection, it was sent to the company for sequencing, and the sequence was aligned in the NCBI database. The results showed that the similarity between strain BG30 of the present invention and the type strain DW5-4 of Bacillus zhangzhouensis was 99.5%. Therefore, strain BG30 of the present invention should be classified into Bacillus zhangzhouensis.

[0037] Example 3: Determination of the phosphate-solubilizing ability of the strain

[0038] After purifying strain BG30, it was inoculated on the PKO medium to observe the phosphate-solubilizing transparent circle. Colonies were picked and cultured in LB liquid medium at 28 °C for 24 h. 1 mL of the above-mentioned bacterial suspension was pipetted into 50 mL of potassium feldspar liquid medium, and 1 mL of LB medium was used instead of the bacterial suspension as a control. Each treatment was set with 3 replicates. After culturing for 3 days at 28 °C and 200 rpm, the content of available phosphorus in the supernatant of the phosphate-solubilizing bacteria culture solution was determined by the molybdenum antimony anti-colorimetric method.

[0039] The test results showed that strain BG30 had a phosphate-solubilizing circle, and the content of soluble available phosphorus in the culture solution was 83.68 mg / L. The phosphate-solubilizing circle formed by strain BG30 is as Figure 2 shown.

[0040] Example 4: Determination of the potassium-solubilizing ability of the strain

[0041] After purifying the potassium-solubilizing strains with potassium-solubilizing halos, they were re-inoculated onto potassium-solubilizing medium to observe the potassium-solubilizing transparent halos. Colonies with D / d greater than 2 were picked and cultured in LB liquid medium at 28 °C for 24 h. 1 mL of the above bacterial suspension was pipetted into 50 mL of potassium feldspar liquid medium, and 1 mL of LB medium was used instead of the bacterial suspension as a control. Each treatment was set with 3 replicates. After culturing for 3 days at 28 °C and 200 rpm, the content of available potassium in the supernatant of the potassium-solubilizing bacteria culture solution was determined by the sodium tetraphenylborate method. The test results showed that the strain numbered J16 (i.e., CCNWSX1901) had a potassium-solubilizing halo, and the content of soluble available potassium in the culture solution was 33.45 mg / L. The potassium-solubilizing halo formed by strain BG30 was as Figure 3 shown.

[0042] Example 5 Rice growth promotion test

[0043] Rice seed germination test: Strain BG30 was activated in LB liquid medium and cultured with shaking at 150 rpm and 30 °C for 48 h. After culturing, a bacterial suspension with an OD 600 of 1 was prepared. Disinfection of rice seeds: Rice seeds were soaked in 0.1% HgCl2 solution for 3 min and rinsed 7 times with sterile water. The disinfected rice seeds were immersed in 20 mL of the bacterial suspension for 24 h, with 20 seeds in each group. Using sterile water as a control, after soaking, they were placed in a petri dish lined with filter paper and germinated in a dark incubator at 28 °C for 7 d. 10 mL of distilled water was added every 24 h to keep the filter paper moist. After germination, 3 plants were randomly selected to measure data.

[0044] Rice growth promotion test: Uniform-sized rice seedlings germinated by soaking in sterile water for 10 d were transplanted into plastic seedling pots containing sterile soil. 200 mL of distilled water was poured in to moisten the soil. 100 mL of the bacterial suspension was poured in on the 1st / 6th / 11th day respectively. The control group was poured with 100 mL of LB liquid medium without inoculated strains. 100 mL of sterile water was poured every 3 days during the test. Each treatment had 3 replicates. Data were measured after the rice grew for 30 d.

[0045] The growth promotion effect of strain BG30 on seedlings was as follows: the root length of the longest root increased by about 36.67%, and the stem and leaf length increased by 32.71%; the growth promotion effect of strain BG30 on seedlings was as follows: the root length of the longest root increased by about 55.86%, and the stem and leaf length increased by 103.14%.

[0046] The Bacillus zhangzhouensis BG30 was isolated from the roots of Robinia pseudoacacia cv. Hongsen, and its morphological, physiological and biochemical characteristics and genetics were identified. The 16S rDNA identification results verified that it was Bacillus zhangzhouensis. By measuring the phosphorus and potassium contents, the results showed that this strain could effectively dissolve the insoluble phosphorus and potassium in the rhizosphere soil of plants, greatly increase the contents of available phosphorus and potassium in the soil, improve the utilization rate of phosphorus and potassium in the soil by plants, thereby promoting plant growth, reducing the usage amount of chemical fertilizers and increasing crop yields.

[0047] It can be understood that the above embodiments only express the preferred embodiments of the present invention, and the description thereof is relatively specific and detailed, but it should not be construed as a limitation to the scope of the patent of the present invention; it should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, the above technical features can be freely combined, and several deformations and improvements can also be made, which all belong to the protection scope of the present invention; therefore, all equivalent transformations and modifications made to the scope of the claims of the present invention should fall within the scope covered by the claims of the present invention.

Claims

1. A Bacillus zhangzhouensis BG30, characterized in that, it was deposited at the Guangdong Microbial Culture Collection Center on December 30, 2022, with the deposit number GDMCC NO.63104.

2. Use of the Bacillus zhangzhouensis BG30 according to claim 1 in increasing the available potassium content in the rhizosphere soil of plants.

3. Use of the Bacillus zhangzhouensis BG30 according to claim 1 in increasing the available phosphorus content in the rhizosphere soil of plants.

4. Use of the Bacillus zhangzhouensis BG30 according to claim 1 in dissolving the insoluble potassium in the rhizosphere soil of plants.

5. Use of the Bacillus zhangzhouensis BG30 according to claim 1 in dissolving the insoluble phosphorus in the rhizosphere soil of plants.

6. Use of the Bacillus zhangzhouensis BG30 according to claim 1 in the preparation of a preparation for promoting plant growth.

7. Use of the Bacillus zhangzhouensis BG30 according to claim 1 in the preparation of a preparation for increasing the plant height.

8. Use of the Bacillus zhangzhouensis BG30 according to claim 1 in the preparation of a preparation for increasing the potassium content of plants.

9. Use of the Bacillus zhangzhouensis BG30 according to claim 1 in the preparation of a preparation for increasing the phosphorus content of plants.

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