Bacillus altitudinis ZXY01 and application of bacillus altitudinis ZXY01 in improvement of soil available potassium content and soil fertility

Abstract

The invention discloses bacillus altitudinis ZXY01 and application of the bacillus altitudinis ZXY01 in improving the content of rapidly available potassium in soil and improving the fertility of the soil. The bacillus altitudinis ZXY01 is classified and named as bacillus altitudinis, and the preservation number of the bacillus altitudinis ZXY01 is CGMCC (China General Microbiological Culture Collection Center) No.36684. The bacillus altitudinis ZXY01 provided by the invention has a good potassium-dissolving function, the research of soil culture experiments proves that a bacterial liquid prepared from the bacillus altitudinis ZXY01 can be directly used as an agricultural microbial potassium-dissolving bacterial agent fertilizer, and due to the direct action of the bacillus altitudinis ZXY01, the potassium-dissolving effect of the bacterial liquid is greatly improved. In the plant planting process, the content of rapidly available potassium in the soil can be effectively increased, the soil fertility is enriched, the forms of a non-exchange state and a mineral state of potassium in the soil are further changed, a new way is provided for a novel agricultural microbial potassium-dissolving bacterial agent fertilizer, and the application prospect is wide.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a strain of Bacillus hygroscopicus ZXY01 and its application in improving the available potassium content and soil fertility in soil. Background Technology

[0002] Soil is one of the substrates for plant growth, and various elements in the soil directly affect crop growth, development, and yield. Based on plant requirements, soil elements can be divided into macroelements and microelements. Macroelements, including nitrogen, phosphorus, and potassium, play crucial roles in plant photosynthesis, cell division, and protein synthesis. Potassium, in particular, not only participates in water regulation but also helps plants improve stress resistance, promoting crop yield and quality. Elements in soil can exist in various forms, such as exchangeable, non-exchangeable, and mineral forms. Among these forms, exchangeable elements are usually those that plants can directly absorb. They interact with the negative charge of soil colloids to form exchangeable ions, which are easily absorbed by plant roots. Non-exchangeable and mineral elements are generally difficult for plants to absorb directly. Mineral elements need to be transformed into absorbable forms through soil weathering or microbial metabolism. The availability of potassium in soil is a typical example. Most potassium in soil exists in mineral and non-exchangeable forms, which are not easily absorbed by plants. Only through microbial or physical weathering can these potassium elements be gradually transformed into usable potassium. Under suitable environmental conditions, the metabolic activities of microorganisms can accelerate the weathering process of minerals, thereby releasing potassium ions that plants can utilize.

[0003] Melons are a typical potassium-loving crop, with potassium being essential throughout their entire growth cycle and directly determining fruit quality and yield. For example, insufficient potassium during the fruit expansion stage can lead to decreased sugar content and a dry texture, while potassium deficiency during ripening can easily cause fruit cracking and premature aging. However, in actual cultivation, there is a significant discrepancy between the available potassium in the soil and the demand of melons. Simply applying chemical potassium fertilizers can easily result in waste due to "applying too much and using too little." The application of potassium-solubilizing bacteria is a key means to solve this problem. Moreover, the same potassium-solubilizing bacteria is easily affected by environmental conditions in different regions, making it difficult to guarantee its colony activity and potassium-solubilizing effect. Summary of the Invention

[0004] Based on the above shortcomings, the purpose of this invention is to provide a strain of Bacillus hygroscopicus ZXY01 and its application in improving the available potassium content and soil fertility. The Bacillus hygroscopicus ZXY01 or its prepared inoculum can effectively increase the available potassium content and improve soil fertility. It provides a new type of agricultural microbial potassium-solubilizing inoculum fertilizer in the field of plant cultivation and has broad application prospects.

[0005] To achieve the above objectives, the present invention is implemented through the following solution: This invention provides a strain of Bacillus hygroscopicus ZXY01, which is classified as Bacillus hygroscopicus. Bacillus altitudinis Its accession number is CGMCC No.36684.

[0006] Furthermore, the colony morphology of the Bacillus hygroscopicus ZXY01 is pale yellow, opaque, and round with relatively neat edges. The colony morphology is uniform, and the cells are elliptical rod-shaped and arranged in single rows.

[0007] The present invention also provides a potassium-solubilizing agent prepared from the aforementioned Bacillus cereus ZXY01.

[0008] Furthermore, the potassium-solubilizing bacterial agent includes one or more of bacterial liquid, bacterial powder, and bacterial cake.

[0009] The present invention also provides the application of the aforementioned Bacillus cereus ZXY01 or the aforementioned potassium-solubilizing agent in increasing the available potassium content in soil during plant cultivation.

[0010] Furthermore, the dosage of the *Bacillus hygroscopicus* ZXY01 used in soil is 10... 7 ~10 10 CFU / g.

[0011] Furthermore, the plant includes melon.

[0012] The present invention also provides the application of the aforementioned Bacillus cereus ZXY01 or the aforementioned potassium-solubilizing agent in improving soil fertility during plant cultivation.

[0013] Furthermore, the plant includes melon.

[0014] Compared with existing technologies, the beneficial effects of this invention are as follows: This invention provides a strain of *Bacillus hygroscopicus* ZXY01, whose bacterial solutions and other inoculants can be directly used as fertilizers, effectively increasing the content of available potassium in the soil and improving soil fertility during plant cultivation. *Bacillus hygroscopicus* ZXY01 can reduce planting costs, meet the needs of green planting, and synergistically improve the stress resistance and nutrient absorption efficiency of plants, including melons, promoting the absorption of nitrogen, phosphorus, and other nutrients, forming a synergistic effect of "potassium-driven fertilization". Attached Figure Description

[0015] Figure 1 The images show the colony morphology and Gram staining of strain ZXY01, where (a) is the colony morphology and (b) is the Gram staining.

[0016] Figure 2The results show the 16S rDNA amplification of strain ZXY01.

[0017] Figure 3 Phylogenetic tree of strain ZXY01.

[0018] Figure 4 This is a graph showing the soil pot experiment data of Bacillus hygroscopicus ZXY01. Detailed Implementation

[0019] The technical solution of the present invention is not limited to the specific embodiments listed below, but also includes any combination of the specific embodiments. The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0020] The "water" used in this invention for culture media or culture processes, unless otherwise specified, refers to sterile water that has been sterilized at 121°C.

[0021] The following describes the preparation of the culture medium and solutions involved in the embodiments of the present invention: LB liquid medium: 5 g yeast extract, 10 g tryptone, 10 g sodium chloride, pH adjusted to 7.4±0.1, volume brought to 1 L, autoclaved at 121℃ for 20 min.

[0022] Potassium-solubilizing liquid culture medium: 5 g sodium chloride, 10 g peptone, 3 g beef meal, pH adjusted to 7.3±0.1, volume brought to 1L, autoclaved at 121℃ for 20 min.

[0023] Potassium-solubilizing solid culture medium: 10g glucose, 0.2g NaHPO4, 0.2g MgSO4, 0.2g NaCl, 0.2g CaSO4, 5g CaCO3, 2.5g potassium feldspar, 15g agar powder, pH adjusted to 7, volume brought to 1 L, autoclaved at 115℃ for 20 min.

[0024] LB solid medium: 1 g sodium chloride, 1 g tryptone, 0.5 g yeast extract, 1.8 g agar powder, pH adjusted to 7.4±0.1, volume brought to 100 ml, autoclaved at 121℃ for 20 min.

[0025] Preparation of agarose gel: Mix agarose with 1×TAE and heat. After cooling, add Gold View developer and wait for it to cool and solidify in a container.

[0026] Preparation of PM liquid culture medium: 1.8g disodium hydrogen phosphate, 1.7g potassium dihydrogen phosphate, 1.0g ammonium sulfate, pH adjusted to 7, volume brought to 1 L, autoclave at 121℃ for 20 min.

[0027] 1.0 mol / L neutral NH4 OAc solution: Weigh 77.08 g of NH4 OAc and dissolve it in approximately 1 L of water. Adjust the pH to 7.0 and bring the volume to 1 L with water.

[0028] Example 1: Screening, Identification and Preservation of Strains 1. Strains screening Inside a clean bench, accurately weigh 10 g of fresh soil (soil used for melon cultivation in Laixi City, Qingdao, Shandong Province) and place it in an Erlenmeyer flask containing 15 to 20 glass beads. Add 90 mL of sterile water to the flask and place it in a shaker at 180 rpm for 20 min to obtain 10 g of fresh soil. -1 The soil leachate was diluted. Then, the leachate was incubated in a water bath at 70°C for 20 minutes. Next, from 10... -1 Take the supernatant from the diluent after 10 min and perform serial dilution with 90 mL of pre-prepared sterile water. After mixing well, you will get 10. -2 Diluent. Dilute to 10 using this method sequentially. -4 10 -5 10 -6 .

[0029] Sterilize 1 L of pre-prepared potassium-solidifying solid medium at 115°C for 20 min. After cooling to room temperature, transfer to a sterile workbench, add bromothymol blue indicator, mix well, pour into a transparent petri dish, and turn on the UV sterilization lamp until the plate solidifies (20 min).

[0030] The above 10 -4 Up to 10 -6 Take 100 µL of the diluent and spread it evenly on potassium-solubilizing solid medium using a spreader. Perform three parallel sets for each gradient. After standing for 20 min, seal with plastic film and incubate at 30℃ for 3 days. Observe whether hydrolysis zones are formed.

[0031] Select a single bacterial strain ZXY01 that produced a hydrolysis zone, add it to a 20ml Erlenmeyer flask containing LB liquid medium, and incubate it on a constant temperature shaker at 30℃ and 180 rpm for 12h to obtain bacterial suspension of strain ZXY01.

[0032] 2. Strain identification (1) Morphological identification Prepare a bacterial smear on a clean glass slide and fix it by drying. Add an appropriate amount of bacterial suspension to a clean glass slide and allow it to air dry. Add an appropriate amount of crystal violet stain to the ZXY01 bacterial suspension portion and react for 1 minute. Gently rinse with water to remove excess dye; allow the smear to air dry, or use absorbent paper to remove excess moisture. Then cover the smear with a coverslip and observe the staining results under a microscope.

[0033] Combination Figure 1 The left-middle image shows that the colony morphology of strain ZXY01 on the plate is pale yellow, opaque, and round with relatively neat edges. Microscopic examination reveals... Figure 1 (As shown in the middle right figure) The colonies in the field of view are uniform in morphology, and the cells are elliptical rod-shaped and arranged in single rows.

[0034] (2) Gene identification The strain ZXY01 was sequenced and identified using 16S rDNA detection and phylogenetic tree analysis. Specific procedures included: 1) Using universal bacterial primers 27F and 1492R, the 16S rDNA gene sequence was amplified on an agarose gel, such as... Figure 2 As shown, gene sequencing was then performed.

[0035] 2) Based on the determined gene sequences, homology analysis was performed using the BLAST tool in the NCBI nucleic acid database, and the sequences were compared with those of relevant type strains in the RDP database (http: / / rdp.cme.msu.edu). ZXY01 and Bacillus altitudinis The strain JYY-02 is homologous, and the comparison is 100%. The 16S rDNA sequencing results of the strain ZXY01 are shown in SEQ ID No. 1.

[0036] 3) After downloading the sequence of the type strain with high homology, a phylogenetic tree was constructed using the Mega 11 software in the Maximum Parsimony method.

[0037] (3) Through identification, the screened strain ZXY01 was determined to be Bacillus hygroscopicus, and its classification name was Bacillus hygroscopicus. Bacillus altitudinis .

[0038] 3. Preservation of bacterial strains The selected *Bacillus hygroscopicus* ZXY01 strain was deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, on November 20, 2025. Bacillus altitudinis The accession number is: CGMCC No. 36684.

[0039] Example 2: Investigating the potassium-solubilizing ability of Bacillus hygroscopicus ZXY01 1. Laboratory determination of potassium solubilizing ability of bacterial strains This embodiment determines the strength of potassium-solubilizing ability by measuring the potassium-solubilizing effect of Bacillus hygroscopicus ZXY01.

[0040] This embodiment uses the spot test method, in which the bacterial suspension is added dropwise to potassium-solubilizing solid culture medium, and the diameter of the potassium-solubilizing zone of the strain is recorded. By recording the colony diameter, the diameter of the hydrolysis zone, and calculating the solubility index, the potassium-solubilizing effect of the strain is judged.

[0041] (1) Sample processing The bacterial strain stored at -80℃ was streaked onto LB agar plates. A loopful of the activated bacterial strain was inoculated into a 20ml Erlenmeyer flask containing liquid LB medium and cultured on a shaker at 30℃ and 180 rpm for 12 hours to obtain a growth suspension. 10 µL of the bacterial growth suspension was added to potassium-solubilizing agar and the plates were cultured in a constant temperature incubator at 28±2℃ for 7 days. Each treatment was repeated in triplicate.

[0042] (2) Dot test to determine potassium solubilization effect 1) Measurement method Colony diameter (d) and hydrolysis zone diameter (D) were measured using a ruler, and the solubility index was calculated.

[0043] 2) Experimental Results Table 1. Hydrolysis zone diameter, colony diameter, and solubility index data of Bacillus hygroscopicus ZXY01

[0044] Note: Solubility index = diameter of hydrolysis zone D / colony diameter d.

[0045] 3) Recording of experimental results The average of the three groups was calculated repeatedly, and the results are listed below: Table 2 shows the results of the dot experiment with Bacillus hygroscopicus ZXY01.

[0046] Analysis of experimental results: The dot test showed that potassium feldspar powder in the culture medium containing Bacillus hygroscopicus ZXY01 was continuously decomposed, indicating that Bacillus hygroscopicus ZXY01 has potassium solubilization function.

[0047] Example 3: Soil culture experiment of Bacillus hygroscopicus ZXY01 To address the problems of potassium fixation, microbial flora disorder, and fertility decline caused by the application of chemical fertilizers to the soil in melon cultivation, optimizing soil potassium availability through fertilization methods is of great significance for improving overall soil fertility, promoting the proliferation of beneficial microorganisms in the soil, enhancing the balanced absorption of mineral nutrients by melon plants, and improving the quality of melon fruits and fertilizer utilization efficiency.

[0048] The cultured bacterial solution can be directly used as an agricultural microbial potassium-solubilizing fertilizer. Therefore, this embodiment further expands the application of Bacillus hygroscopicus ZXY01 by observing the results of a soil pot experiment. The Rhodopseudomonas palustris involved in this embodiment was obtained through commercial channels.

[0049] (1) Preparation of microbial agents Bacillus hygroscopicus ZXY01 was cultured in LB medium at 37℃ (200 rpm) for 12 h to obtain bacterial fraction. After centrifugation, the bacterial precipitate was collected, washed twice with sterile water, and resuspended in sterile water to obtain the final Bacillus hygroscopicus inoculum. The dosage of the strain in soil was 1×10⁻⁶. 7 CFU / g.

[0050] Rhodopseudomonas palustris was cultured in PM medium under light for 48 hours to obtain bacterial fractions. The bacterial precipitate was collected by centrifugation, washed twice with sterile water, and resuspended in sterile water. This was then combined with Bacillus hygroscopicus ZXY01 inoculum to obtain a compound inoculum. The dosage of both Bacillus hygroscopicus ZXY01 and Rhodopseudomonas palustris strains in soil was 1×10⁻⁶. 7 CFU / g.

[0051] (2) Experimental methods Weigh 500g of soil into flowerpots and set up 3 treatments: CK (blank control), Bacillus hygroscopicus ZXY01 (treated with Bacillus hygroscopicus ZXY01), and ZR (treated with Bacillus hygroscopicus ZXY01 and Rhodopseudomonas palustris). Each treatment was set up in 6 replicates.

[0052] (3) Determination of available potassium content in soil Take 2 g of air-dried soil sample that has passed through a 1 mm sieve and place it in a 50 mL glass Erlenmeyer flask. Add 20 mL of 1.0 mol / L neutral NH4OAc solution, seal the flask with sealing film, and shake it in a constant temperature shaker at 20 °C for 30 min. Filter the solution with qualitative filter paper. Collect the filtrate and measure it together with a series of potassium standard solutions on a flame spectrophotometer. Record the readings and calculate the concentration using a standard curve. The experimental results are shown in Table 3.

[0053] Preparation of potassium standard curve: Dissolve 0.1907 g of dried KCl in 1.0 mol / L neutral NH4 OAc solution and bring the volume to 1 L to prepare a standard solution. Pipette 0, 2.5, 5.0, 10.0, 15.0, 20.0, and 40.0 mL of the standard solution into 100 mL bottles, and bring the volume to 100 mL with 1.0 mol / L neutral NH4 OAc solution to obtain potassium standard curves for 0, 2.5, 5, 10, 15, 20, and 40 mg / L.

[0054] Available potassium in soil (mg / kg, K) = test solution (mg / L, K) × V / m; V – The number of milliliters of extractant added; M — Mass of the dried soil sample (g).

[0055] Table 3 Experimental data for different treatment groups

[0056] Through Table 3 and Figure 4 It can be seen that Bacillus thuringiensis ZXY01 has a good effect on increasing the content of available potassium in the soil, which is significantly higher than that of the CK group (p<0.05), and is 8.65% higher than that of CK.

[0057] Through Table 3 and Figure 4 It can be seen that after adding Rhodopseudomonas palustris, ZR has a good effect on increasing the content of available potassium in the soil, which is significantly higher than that of the CK group (p<0.05). However, although the content of available potassium in the soil increased compared with Bacillus thuringiensis ZXY01, there was no significant difference (p>0.05).

[0058] Because the Bacillus in the highland Bacillus inoculant comes from the local soil, it has a certain positive effect on soil improvement. Its potassium-releasing effect has a great influence on improving soil quality, increasing soil fertility and promoting the absorption of potassium by crops.

[0059] The Bacillus oryzae inoculant fertilizer used in this study is a functional microbial inoculant product with Bacillus oryzae from the local soil as its core. Through systematic research on its impact on soil potassium levels, the aim is to improve soil quality for melon cultivation, regulate the balance of soil nutrient supply and demand, and, through the direct action of potassium-releasing Bacillus, release fixed potassium and related nutrients in the soil that are difficult for melons to absorb and utilize, thereby enhancing the nutrient absorption efficiency of melon plants and providing support for high-quality and high-yield melons.

[0060] Although the embodiments of the present invention have been described above in conjunction with the examples, it should be noted that the described embodiments are only a part of the specific implementation of the present invention and are not intended to limit the scope of protection of the present invention. Any equivalent substitutions or improvements made within the scope of the technical concept and technical method of the present invention shall fall within the scope of protection and disclosure of the present invention.

Claims

1. A strain of Bacillus hygroscopicus ZXY01, characterized in that, The Bacillus hygroscopicus ZXY01 is classified as Bacillus hygroscopicus. Bacillus altitudinis Its accession number is CGMCC No.36684.

2. The Bacillus cereus ZXY01 according to claim 1, characterized in that, The colony morphology of the Bacillus hygroscopicus ZXY01 is pale yellow, opaque, and round with relatively neat edges. The colony morphology is uniform, and the cells are elliptical rod-shaped and arranged in single rows.

3. The potassium-solubilizing agent prepared from Bacillus cereus ZXY01 as described in claim 1.

4. The potassium-solubilizing bacterial agent according to claim 3, characterized in that, The potassium-solubilizing bacterial agent includes one or more of the following: bacterial liquid, bacterial powder, and bacterial cake.

5. The application of the Bacillus cereus ZXY01 of claim 1 or the potassium-releasing agent of claim 3 in increasing the available potassium content in soil during plant cultivation.

6. The application according to claim 5, characterized in that, The dosage of the Bacillus cereus ZXY01 used in soil was 10. 7 ~10 10 CFU / g.

7. The application according to claim 5, characterized in that, The plant mentioned includes melon.

8. The application of the Bacillus cereus ZXY01 of claim 1 or the potassium-solubilizing agent of claim 3 in improving soil fertility during plant cultivation.

Images