Bacillus paralicheniformis and application thereof

Abstract

The present application discloses a strain of Bacillus paralicheniformis and its application, and belongs to the technical field of microorganisms. The strain has been preserved in the China General Microbiological Culture Collection Center on December 25, 2022, and its biological preservation number is CGMCC No. 26241. The Bacillus paralicheniformis SYN-191 has the functions of antagonizing ginger root rot pathogen, decomposing cellulose, decomposing potassium, decomposing protein and significantly promoting growth on ginger. The Bacillus paralicheniformis screened in the present application can promote the growth of ginger in healthy soil and continuous cropping obstacle soil, improve the rhizosphere microbial community of ginger, and help crops to better utilize the nutrients in the soil. The ginger is treated by drenching roots with the Bacillus paralicheniformis, so that the plant height of the ginger is increased, the growth of the ginger is promoted, the dry and fresh weight of the stems and leaves of the ginger is increased, and thus the plant biomass is improved. Meanwhile, the strain resource of plant growth promoting bacteria is enriched, and therefore the present application has good practical application value.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a strain of Bacillus paralicheniformis and its applications. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Ginger, as one of my country's major economic fruits and vegetables, is widely cultivated in northern my country, primarily in Shandong province. my country has become the world's largest producer and cultivator of ginger. However, with the continuous expansion of cultivation, many problems have gradually emerged. Various soil-borne diseases affecting ginger are becoming increasingly serious. Long-term use of chemical pesticides has exacerbated soil compaction, severely damaging the soil microbial community and making restoration difficult. Decreased soil fertility further hinders the supply of sufficient nutrients to plants and contributes to soil and water pollution. Therefore, there is an urgent need to find a strain of bacteria to improve soil nutrients and promote ginger growth.

[0004] Microbial inoculants are an environmentally friendly fertilizer. The microbial strains used are all beneficial microorganisms derived from plant tissues and rhizosphere soil. They can promote plant growth, improve crop quality and yield, effectively control plant diseases and pests, and thus improve soil nutrient structure and physicochemical properties. However, there are still relatively few existing microbial inoculants specifically designed to promote ginger growth, and the technology is not yet mature. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the purpose of this invention is to provide a strain of Bacillus paralichrysum and its applications. Experiments have shown that the Bacillus paralichrysum provided by this invention can effectively improve soil structure, thereby solving problems such as difficulties in ginger cultivation, low yield, and soil-borne diseases.

[0006] To achieve the above objectives, the technical solution of the present invention is as follows:

[0007] In a first aspect, the present invention provides a strain of Bacillus paralicheniformis (B. paralicheniformis) Bacillus paralleheniformis SYN-191, this strain was deposited on December 25, 2022 at the China General Microbiological Culture Collection Center (address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing), with the biological accession number CGMCC No. 26241.

[0008] A second aspect of the present invention provides a microbial inoculant, characterized in that it contains the aforementioned *Bacillus paralicheniformis* (…). Bacillus paralleheniformis SYN-191 and its fermentation products.

[0009] Specifically, the microbial agent is a microbial inoculant. More specifically, the number of effective viable bacteria in the microbial agent is ≥10. 8 cfu / mL. The microbial inoculant of this invention, when applied as a root drenching treatment after ginger germination and seedling establishment, can increase the plant height of ginger, significantly promote ginger growth, increase the dry and fresh weight of ginger stems and leaves, and thus improve plant biomass.

[0010] Specifically, the microbial agent is obtained by inoculating Bacillus paralichrysiformis SYN-191 into a culture medium and then culturing it.

[0011] A third aspect of the present invention provides the application of the above-mentioned Bacillus paralichrysiformis SYN-191 and / or microbial agents in promoting crop growth.

[0012] Specifically, the crop is ginger, and promoting ginger growth includes:

[0013] One or more of the following can enhance the root vitality of ginger, promote the growth of ginger plant height, increase the dry and fresh weight of ginger stems and leaves, and increase ginger biomass.

[0014] In a fourth aspect, the present invention provides a method for promoting ginger growth, the method comprising soaking ginger seeds in the above-mentioned Bacillus paralichrysiformis SYN-191 and / or the above-mentioned microbial inoculant;

[0015] Alternatively, after the ginger sprouts and seedlings are established, the above-mentioned Bacillus paralichrysum SYN-191 and / or microbial inoculant can be applied to the rhizosphere soil of the ginger plants; more specifically, the application method is to perform root irrigation treatment with Bacillus paralichrysum SYN-191 and / or microbial inoculant.

[0016] Compared with the prior art, the present invention has achieved the following beneficial effects:

[0017] This invention reports for the first time a *Bacillus paralichrysum* species that promotes ginger growth. It simultaneously exhibits antagonistic effects against ginger root rot pathogens, decomposes cellulose, solubilizes potassium, degrades proteins, and significantly promotes ginger growth. Pot experiments verified that the *Bacillus paralichrysum* species screened in this invention can promote ginger growth, improve the rhizosphere microbial community, and help the crop better utilize soil nutrients. Root irrigation with this species increases ginger plant height, promotes growth, and increases the dry and fresh weight of ginger stems and leaves, thereby improving plant biomass. It also enriches the strain resources of plant growth-promoting bacteria, thus possessing significant practical application value.

[0018] The microbial agent containing Bacillus paralichrysogenum SYN-191 of this invention is pollution-free and has no toxic side effects. Repeated verification has shown that the strain colonizes rapidly on ginger, grows quickly, and is easy to preserve; it can also effectively improve soil structure, thereby effectively meeting the nutritional needs of crop growth, and has good practical application value. Attached Figure Description

[0019] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0020] Figure 1 The images show colony diagram (A) and microscopic examination of the bacterial cells of Bacillus paralichrysogenum SYN-191, which are part of this invention.

[0021] Figure 2 This is a plate antagonistic effect diagram of the *Bacillus paralichrysiformis* SYN-191 strain of the present invention against the pathogen causing ginger root rot;

[0022] Figure 3 This is a diagram of the cellulose decomposition process by the *Bacillus paralichrysiformis* SYN-191 strain of the present invention.

[0023] Figure 4 This is a diagram showing the potassium solubility of the *Bacillus paralichrysiformis* SYN-191 strain of the present invention.

[0024] Figure 5 This is a diagram of the degradation proteins of the *Bacillus paralichrysiformis* SYN-191 strain of the present invention;

[0025] Figure 6 This is a diagram showing the growth-promoting effect of microbial inoculants in a pot experiment (healthy soil) in an embodiment of the present invention.

[0026] Figure 7 This is a diagram showing the growth-promoting effect of microbial inoculants in a pot experiment (continuous cropping obstacle soil) in an embodiment of the present invention.

[0027] Figure 8 The growth curve of the Bacillus paralichrysiformis strain SYN-191 is shown in this invention. Detailed Implementation

[0028] In one specific embodiment of the present invention, a strain of Bacillus paralicheniformis (B. paralicheniformis) is provided. Bacillus paralleheniformis SYN-191, this strain was deposited at the China General Microbiological Culture Collection Center on December 25, 2022, with the biological accession number CGMCC No. 26241.

[0029] In this invention, by determining the 16S rDNA gene sequence and whole genome sequence of the strain, and through morphological characteristics and physiological and biochemical indicators, it was finally determined that the strain belongs to *Bacillus paralichrysogenum* (B. paralichrysogenum). Bacillus paralleheniformis ).

[0030] Specifically, the aforementioned Bacillus paralicheniformis ( Bacillus paralleheniformis SYN-191 was cultured on LB medium (5 g yeast extract, 10 g peptone, 10 g NaCl, brought to 1000 mL, sterilized at 118 °C for 20 min, pH 7.0) (culture conditions: 37 °C, 24 h, rotation speed: 180 rpm).

[0031] In another specific embodiment of the present invention, a microbial inoculant is provided, which contains the above-mentioned Bacillus paralicheniformis (… Bacillus paralleheniformis SYN-191 and its fermentation products.

[0032] Specifically, the microbial agent is a microbial inoculant. More specifically, the number of effective viable bacteria in the microbial agent is ≥10. 8 cfu / mL.

[0033] In another specific embodiment of the present invention, the microbial agent further includes a carrier. The carrier is a solid carrier or a liquid carrier.

[0034] In another specific embodiment of the present invention, the solid carrier is a mineral material, a biological material, or a polymer compound; the mineral material is at least one selected from clay, talc, kaolin, montmorillonite, white carbon, zeolite, silica, peat moss, and diatomaceous earth; the biological material is at least one selected from various crop straws, pine shells, rice straw, peanut shells, corn flour, soybean flour, starch, peat moss, and animal excrement; the polymer compound is polyvinyl alcohol and / or polyethylene glycol.

[0035] In another specific embodiment of the present invention, the liquid carrier may be an organic solvent, vegetable oil, mineral oil or water; the organic solvent may be decane and / or dodecane.

[0036] In another specific embodiment of the present invention, the dosage form of the microbial agent can be a variety of dosage forms, such as liquid, emulsion, suspension, powder, granules, wettable powder or water-dispersible granules.

[0037] In another specific embodiment of the present invention, the application of the above-mentioned Bacillus paralichrysiformis SYN-191 and / or microbial inoculants in promoting crop growth is provided.

[0038] In another specific embodiment of the present invention, the crop is ginger, and promoting ginger growth includes:

[0039] One or more of the following can enhance the root vitality of ginger, promote the growth of ginger plant height, increase the dry and fresh weight of ginger stems and leaves, and increase ginger biomass.

[0040] In another specific embodiment of the present invention, a method for promoting ginger growth is provided, the method comprising soaking ginger seeds in the above-mentioned Bacillus paralichrysiformis SYN-191 and / or microbial inoculant;

[0041] Alternatively, after ginger sprouts and seedlings are established, apply the aforementioned Bacillus paralichrysum SYN-191 and / or microbial inoculants to the rhizosphere soil of the ginger plants. Application methods include root drenching. More specifically, the application method involves drenching the roots with Bacillus paralichrysum SYN-191 and / or the microbial inoculant.

[0042] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0043] Example 1: Strain Identification

[0044] This invention identifies a bacterial strain that promotes ginger growth, obtained through screening, as *Bacillus paralicheniformis*, through 16S rDNA analysis, whole-genome sequencing, biocontrol function testing, and physiological and biochemical index identification. Bacillus paralleheniformis ).like Figure 1 , 2 3, 4, 5 and Table 1 are shown.

[0045] Table 1 Results of physiological and biochemical index determination of strains

[0046] feature SYN-191 gelatin √ Rhamnose √ sucrose √ glucose √ Tryptophan Broth × Sorbitol √ Raffinose × ONPG √ D-cellulose disaccharide √ 3% catalase √ Mannitol √ Trehalose √ fructose √ maltose √ lactose × Methyl red (MR) √ VP Measurement × Nitrate reduction √ Xylose × Arabic sugar √

[0047] Example 2: Growth-promoting effect of microbial inoculants on ginger (healthy soil)

[0048] Preparation of a single microbial inoculum: The *Bacillus paralichrysogenus* SYN-191 strain preserved on an agar slant was transferred to a test tube containing 5 mL of LB liquid medium and incubated in a constant-temperature shaker for 12 h at 37 ℃ and 180 rpm. The activated bacterial solution was then inoculated into 50 mL of LB liquid medium at a rate of 1 mL and incubated in a shaker for 24 h at 37 ℃ and 180 rpm until the late logarithmic growth phase, maintaining a viable count ≥10⁻⁶. 8 cfu / mL.

[0049] Uniformly growing ginger seedlings were transplanted into healthy soil. After the seedlings were established, 2 mL of microbial inoculant was diluted 100 times, and then 200 mL of the prepared inoculant was applied to each pot of ginger for root drenching (for the control group, 2 mL of sterile LB liquid medium was diluted to 200 mL and then applied to the roots). The growth-promoting effect on ginger was assessed after 40 days of cultivation, and the results are as follows: Figure 6 As shown in Table 2, it can be seen that, after pot experimentation, the single microbial agent of this invention, when applied to the roots of ginger during the early growth stage under outdoor soil conditions, can produce good growth-promoting effects on various indicators of ginger. Compared with the control group, ginger plant height, stem diameter, maximum root length, above-ground fresh weight, and underground fresh weight all showed growth-promoting effects.

[0050] Table 2. Growth-promoting effect on potted plants

[0051] Physiological plant height Stem thick Root length Fresh weight on the ground underground fresh weight CK 14.3±6.25 5.2±0.62 1.93±0.6 1.3±0.4 1.2±0.08 SYN-191 25.3±4.01 7.1±0.71 2.52±1.26 1.9±0.38 2.7±1.06

[0052] Example 3: Growth-promoting experiment of microbial inoculants on ginger (continuous cropping obstacle soil, taken from a field with continuous cropping obstacles for ginger cultivation)

[0053] Preparation of a single microbial inoculum: The *Bacillus paralichrysogenus* SYN-191 strain preserved on an agar slant was transferred to a test tube containing 5 mL of LB liquid medium and incubated in a constant-temperature shaker for 12 h at 37 ℃ and 180 rpm. The activated bacterial solution was then inoculated into 50 mL of LB liquid medium at a rate of 1 mL and incubated in a shaker for 24 h at 37 ℃ and 180 rpm until the late logarithmic growth phase, maintaining a viable count ≥10⁻⁶. 8 cfu / mL.

[0054] Uniformly growing ginger seedlings (to ensure survival rate, the seedling cultivation time in pots was longer than that in healthy soil) were transplanted into continuous cropping obstacle soil (infected by Fusarium graminearum, the pathogen causing ginger root rot). After the ginger seedlings were established, 2 mL of microbial inoculant was diluted 100 times, and then 200 mL of the prepared inoculant was applied to each pot of ginger for root drenching (for the control group, 2 mL of sterile LB liquid medium was diluted to 200 mL before root drenching). The growth-promoting effect on ginger was tested after 40 days of cultivation, and the results are as follows: Figure 7 As shown in Table 3, it can be seen that, after pot experimentation, the single microbial agent of this invention, when applied to the roots of ginger during the early growth stage under outdoor soil conditions, can produce good growth-promoting effects on various indicators of ginger. Compared with the control group, ginger plant height, stem diameter, above-ground fresh weight, and underground fresh weight all showed growth-promoting effects.

[0055] Table 3. Growth-promoting effect on potted plants

[0056] Physiological plant height Stem thick Fresh weight on the ground underground fresh weight CK 19.83±1.87 7.22±1.37 3.29±0.14 1.72±0.61 SYN-191 28.04±5.5 8.04±1.8 6.39±0.96 3.33±0.7

[0057] This invention studies the growth-promoting effect of this microbial agent on ginger under outdoor potted conditions. The experimental method used closely resembles actual application conditions. By directly planting ginger in soil as an experiment, agricultural ginger cultivation conditions are simulated, demonstrating the good effect of adding this microbial agent. Simultaneously, this experiment measured the plant height, maximum root length, above-ground fresh weight, and underground fresh weight of potted ginger, strongly proving the growth-promoting effect of this strain.

[0058] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. Bacillus paralicheniformis ( Bacillus paralleheniformis The application of SYN-191 and / or microbial agents in promoting ginger growth and antagonizing one or more of the root rot pathogens of ginger, Fusarium oxysporum; The *Bacillus paralichrysogenus* SYN-191 strain was deposited at the China General Microbiological Culture Collection Center (CGMCC) on December 25, 2022, with accession number CGMCC No. 26241. The microbial agent contains Bacillus paralicheniformis SYN-191 and its fermentation products.

2. The application as described in claim 1, characterized in that, The microbial agent is a microbial inoculant.

3. The application as described in claim 1, characterized in that, The number of effective viable bacteria in the microbial agent is ≥10. 8 cfu / mL.

4. The application as described in claim 1, characterized in that, The microbial agent was obtained by inoculating Bacillus paralichrysum SYN-191 into a culture medium and then culturing it.

5. The application as described in claim 4, characterized in that, The culture media include LB medium, potato dextrose agar (PDA) medium, bean sprout juice medium, casein medium, silicate bacteria medium, and cellulose medium.

6. The application as described in claim 4, characterized in that, The culture medium is LB medium.

7. The application as described in claim 4, characterized in that, The specific fermentation conditions are: 30-40℃, 20-30h, rotation speed: 160-200rpm.

8. The application as described in claim 4, characterized in that, The fermentation conditions were: 37℃ for 24 hours and 180 rpm.

9. The application as described in claim 1, characterized in that, The microbial agent also includes a carrier.

10. The application as described in claim 9, characterized in that, The carrier can be a solid carrier or a liquid carrier.

11. The application as described in claim 1, characterized in that, The microbial inoculants are available in liquid, powder, or granule forms.

12. The application as described in claim 1, characterized in that, Promoting ginger growth includes: It can enhance the root vitality of ginger, promote the growth of ginger plants, and increase the dry and fresh weight of ginger stems and leaves, among other things.

13. A method for promoting ginger growth, characterized in that, The method includes soaking ginger seeds in Bacillus paralicheniformis SYN-191 as described in claim 1 and / or the microbial agent as described in any one of claims 2-11; Alternatively, after the ginger has sprouted and been established, apply the Bacillus paralicheniformis SYN-191 as described in claim 1 and / or the microbial agent as described in any one of claims 2-11 to the rhizosphere soil of the ginger plants.

14. The method as described in claim 13, characterized in that, The application method includes root irrigation.

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