Bacillus tokyoensis, microbial inoculum, preparation method and application

Abstract

The present application belongs to the technical field of microbial seed coating agent, and particularly relates to a Bacillus toyonensis, a microbial agent, a preparation method and application. The present application provides a Bacillus toyonensis PSLXL1 with a preservation number of CCTCC NO.M20221919 。 The microbial seed coating agent prepared by the Bacillus toyonensis PSLXL1 can effectively improve the germination rate, emergence rate and germination potential of the rhizoma polygonati seeds. It can be seen that the Bacillus toyonensis has a good effect of seedling protection and growth promotion on the seedlings of rhizoma polygonati in the rhizoma polygonati planting process.

Description

Technical Field

[0001] This invention belongs to the field of microbial seed coating technology, specifically relating to a strain of Bacillus tonkinensis, a bacterial agent, a preparation method, and its application. Background Technology

[0002] Seed coating agents are thin films that coat the surface of plant seeds. They are made using plant growth promoters or microorganisms as active ingredients, and film-forming agents, suspending agents, and stabilizers as auxiliary ingredients, through a special processing technique. They can be directly coated onto the seed surface to form a protective film with a certain degree of air permeability and strength. Seed coating can promote plant emergence, root development, seedling growth, and control soil-borne diseases.

[0003] In agriculture, the long-term and excessive application of chemical pesticides and seed coating agents, whose main functional components are chemicals, will lead to soil compaction, reduced soil fertility, and increasingly serious soil pesticide damage, significantly harming soil quality and the quality of medicinal materials. While a large number of chemical seed coating agents are available on the market, bringing convenience to agricultural production, they also have limitations, such as limited effectiveness in improving plant disease resistance and immunity. Improper use can also easily cause seed and seedling toxicity.

[0004] Polygonatum is a valuable traditional Chinese medicine that can be used both as food and medicine. During artificial cultivation, the low germination rate, poor seedling emergence rate, and weak germination vigor of Polygonatum seeds severely restrict the large-scale development of the Polygonatum industry, keeping its price high and limiting its consumption by ordinary consumers.

[0005] Existing technologies for breaking the dormancy of Polygonatum seeds mainly utilize stratification and factors such as hormones, growth regulators, and magnetic fields. For example, Chinese patent CN103858769A describes a rapid propagation technique for Polygonatum, using gibberellin and kinetin to break seed dormancy and promote germination. Another example is Chinese patent CN109566371A, a method for cultivating Polygonatum seeds, which promotes germination by damaging the seed surface, low-temperature refrigeration, and layering with a substrate. Yet another example is Chinese patent CN111557219A, a method for cultivating multiflora Polygonatum seedlings, which uses plant growth regulators (gibberellin and naphthaleneacetic acid) to promote seed germination. A method for sexual reproduction of Hubei Polygonatum (CN104396493A) uses the broad-spectrum fungicide carbendazim to disinfect the seeds, followed by stratification to promote germination. Another example is a seedling propagation method for Polygonatum multiflorum seeds (CN110115131A) in Chinese patent. This method primarily targets Polygonatum multiflorum, treating the fruits with ethephon followed by alternating temperature treatment to remove the pericarp. The seeds are then treated with gibberellin and sown in a self-made substrate to promote seed germination and seedling cultivation. Currently, there are no reports on seed coating agents prepared from microbial strains that improve the germination rate of Polygonatum seeds. Summary of the Invention

[0006] The purpose of this invention is to provide a strain of Bacillus toyonensis PSLXL1, which can improve the germination rate, emergence rate and germination potential of Polygonatum seeds by preparing a microbial seed coating agent using this strain.

[0007] This invention provides a strain of Bacillus toyonensis PSLXL1, with accession number CCTCCNO.M20221919.

[0008] The present invention provides a bacterial agent containing Bacillus toyonensis PSLXL1 as described in the above technical solution, characterized in that the Bacillus toyonensis exists in the form of bacterial liquid, spores, mutants or metabolites.

[0009] Preferably, the microbial agent includes a seed coating agent.

[0010] Preferably, the seed coating agent includes a rhizosphere growth-promoting seed coating agent or a rhizosphere growth-promoting and insecticidal seed coating agent.

[0011] Preferably, by weight, the rhizosphere seed coating agent comprises 1-35 parts of Bacillus tonkinensis, 10-20 parts of adsorbent, 1-8 parts of binder, and 3-12 parts of sodium polyacrylate; or 1-35 parts of Bacillus tonkinensis, 1-8 parts of binder, and 1-15 parts of polyethylene glycol;

[0012] The adsorbent includes maifanite and / or biochar;

[0013] The adhesive comprises one or more of polyvinylpyrrolidone, dextrin, and chitosan.

[0014] Preferably, the rhizosphere growth-promoting and insecticidal seed coating agent, by weight, comprises 1-35 parts of Bacillus thomsonii, 10-20 parts of adsorbent, 1-8 parts of binder, 3-12 parts of sodium polyacrylate, and 3-18 parts of insecticidal component; or 1-35 parts of Bacillus thomsonii, 1-8 parts of binder, 1-15 parts of polyethylene glycol, and 3-18 parts of insecticidal component.

[0015] Preferably, the insecticidal ingredient comprises imidacloprid and / or thiamethoxam in parts by weight.

[0016] This invention provides a method for preparing the microbial agent described in the above technical solution, comprising the following steps:

[0017] The seed coating agent is obtained by mixing the raw material components.

[0018] The application of Bacillus tonkinensis described in the above technical solution or the bacterial agent described in the above technical solution or the bacterial agent prepared by the preparation method described in the above technical solution in one or more of the following (1) to (3);

[0019] (1) Improve the germination rate of Polygonatum seeds;

[0020] (2) Improve the germination potential of Polygonatum seeds;

[0021] (3) Improve the germination rate of Polygonatum seeds.

[0022] Preferably, the seeds of Polygonatum are coated with a seed coating agent to obtain coated Polygonatum seeds, wherein the mass ratio of the seed coating agent to the Polygonatum seeds is 1:30 to 1:65.

[0023] The beneficial effects of this invention: This invention provides a strain of Bacillus toyonensis (B. toyonensis) PSLXL1, with the preservation number CCTCC NO. M20221919. The microbial seed coating agent prepared using Bacillus toyonensis in this invention can effectively improve the germination rate, emergence rate, and germination potential of Polygonatum sibiricum seeds. Therefore, the Bacillus toyonensis and its preparation of this invention have a significant effect on improving the germination rate, emergence rate, and germination potential of Polygonatum sibiricum seeds during the cultivation process.

[0024] Biological Preservation Instructions

[0025] Bacillus toyonensis PSLXL1 was deposited at the China Center for Type Culture Collection (CCTCC) on December 9, 2022, with accession number CCTCC NO.M20221919. The address of the depository is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province. Attached Figure Description

[0026] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0027] Figure 1 The endophytic bacteria PSLXL1 of Polygonatum rhizome was isolated by the Polygonatum rhizome tissue block culture method in Example 1;

[0028] Figure 2 The 16S rDNA sequence of the endophytic bacteria PSLXL1 isolated in Example 1 was compared with the EZbiocloud database using BLAST.

[0029] Figure 3 The 16S rDNA gene sequence of the endophytic bacterium PSLXL1 in Polygonatum sibiricum from Example 1 was compared with the NCBI database.

[0030] Figure 4 Gram staining and microscopic examination results (1000×) of the endophytic bacteria PSLXL1 in Polygonatum sibiricum in Example 1;

[0031] Figure 5 The diameter of the bacterial plaques of strain Bacillus toyonensis PSLXL1 under different acid and alkaline conditions in Example 1;

[0032] Figure 6 The diameter of the bacterial plaques of strain Bacillus toyonensis PSLXL1 under different salinity conditions in Example 1;

[0033] Figure 7 The changes in viable bacterial counts of the coated Polygonatum seeds prepared by the seed-coating agents A to C in Example 5 over 4 weeks are shown. Detailed Implementation

[0034] This invention provides a strain of Bacillus toyonensis PSLXL1, with accession number CCTCC NO. M20221919. The preferred 16S rDNA gene sequence of the Bacillus toyonensis described in this invention is shown in SEQ ID NO. 1.

[0035]

[0036] The *Bacillus thomsonii* strain described in this invention is preferably isolated from wild *Polygonatum sibiricum* samples. It exhibits excellent germination-promoting effects on *Polygonatum sibiricum* seeds. This *Bacillus thomsonii* is a growth-promoting bacterium, effectively improving the germination rate, germination potential, and seedling emergence rate of *Polygonatum sibiricum* seeds. The colony morphology of the *Bacillus thomsonii* strain described in this invention is characterized by large, round, yellow, opaque colonies with a moist surface. Under a microscope, the bacteria appear rod-shaped, and preliminary Gram staining analysis confirms a positive result.

[0037] The Tokyo Bacillus of the present invention has the following advantages and benefits compared with the Tokyo Bacillus already existing in the art: (1) Tokyo Bacillus has a strong ability to resist acid, alkali and salt stress and can be used in soils with different pH levels.

[0038] (2) Bacillus tonkinensis has strong compatibility with chemical drugs. When used in combination with other chemical seed coating agents, the strain has good compatibility with chemical drugs.

[0039] (3) Bacillus tonkinensis can effectively break the dormancy of Polygonatum seeds, promote the differentiation and development of Polygonatum seed embryo structure, and effectively improve the germination rate, germination potential and emergence rate of Polygonatum.

[0040] The present invention provides a bacterial agent containing Bacillus toyonensis PSLXL1 as described in the above technical solution, wherein the Bacillus toyonensis exists in the form of bacterial liquid, spores, mutants or metabolites.

[0041] In this invention, the microbial agent preferably includes a seed coating agent, and the seed coating agent preferably includes a rhizosphere growth-promoting seed coating agent or a rhizosphere growth-promoting and insecticidal seed coating agent.

[0042] In this invention, the rhizosphere growth-promoting seed coating agent provided by this invention preferably comprises 1-35 parts of Bacillus tonkinensis, 10-20 parts of adsorbent, 1-8 parts of binder, and 3-12 parts of sodium polyacrylate, by weight; or 1-35 parts of Bacillus tonkinensis, 1-8 parts of binder, and 1-15 parts of polyethylene glycol.

[0043] The adsorbent described in this invention preferably includes maifanite and / or biochar;

[0044] The adhesive of the present invention preferably includes one or more of polyvinylpyrrolidone, dextrin and chitosan.

[0045] In one seed coating agent product of the present invention, the rhizosphere growth-promoting seed coating agent provided by the present invention preferably comprises 1 to 35 parts of Bacillus thomsonii, more preferably 10 to 33 parts, and even more preferably 30 parts. In the present invention, the Bacillus thomsonii is preferably added in the form of Bacillus thomsonii bacterial suspension. The preparation method of the Bacillus thomsonii bacterial suspension of the present invention is not particularly limited, and conventional methods can be used.

[0046] The preferred method for preparing the *Bacillus toyonensis* bacterial culture of this invention includes the following steps: *Bacillus toyonensis* PSLXL1 original strain is streaked in three zones on LB solid medium (10 g / L trypsin, 5 g / L yeast extract, 10 g / L sodium chloride, 15 g / L agar, pH 7.0) at 37°C for 10–14 h. Single colonies are then picked from the medium and incubated in 50 mL of LB liquid medium at 37°C for 12 h to obtain the *Bacillus toyonensis* bacterial culture. In this invention, the *Bacillus toyonensis* bacterial culture is added to LB liquid medium for fermentation. Preferably, the amount of *Bacillus toyonensis* bacterial culture added is 4%–6% of the volume of LB liquid medium, more preferably 5%. The fermentation temperature of the present invention is preferably 30℃~40℃, more preferably 36℃; the fermentation speed is preferably 110~130 r / min, more preferably 120 r / min; the aeration rate is preferably 1.5~1.9 VVM, more preferably 1.7 VVM; and the fermentation time is preferably 2~4 days, more preferably 3 days. The fermentation of the present invention is preferably carried out in a fermenter. The effective viable count of *Bacillus tonkinensis* bacterial suspension prepared by the present invention is preferably ≥1×10⁻⁶. 9 cfu / mL, more preferably 1×10⁻⁶ 9 cfu / mL.

[0047] Based on the mass fraction of Bacillus Tokyois, the rhizosphere seed coating agent provided by this invention preferably includes 10-20 parts of adsorbent, more preferably 15-20 parts, and even more preferably 20 parts. In this invention, the adsorbent preferably includes maifanite and / or biochar, more preferably maifanite or biochar, and even more preferably biochar. The adsorbent of this invention serves to prolong the survival period of the bacterial strain under immobilized conditions, and also improves the rhizosphere soil structure and provides physical protection for the bacterial strain.

[0048] Based on the mass fraction of Bacillus tonkinensis, the rhizosphere seed coating agent provided by this invention preferably comprises 1 to 8 parts of an adhesive, more preferably 2 to 6 parts, and even more preferably 4 parts. In this invention, the adhesive preferably comprises one or more of polyvinylpyrrolidone, dextrin, and chitosan, more preferably dextrin and chitosan. The adhesive of this invention functions to ensure that the bacterial strain adheres evenly to the outer surface of the seed, while effectively guaranteeing normal seed respiration and improving the soil microenvironment surrounding the seed.

[0049] Based on the mass fraction of Bacillus tonkinensis, the rhizosphere seed coating agent provided by this invention preferably comprises 3-12 parts of sodium polyacrylate, more preferably 5-8 parts, and even more preferably 6 parts. The sodium polyacrylate described in this invention is a water-retaining agent. The function of this water-retaining agent is to regulate the moisture content of the soil surrounding the seed, thereby improving the effective colonization of the fungus and the germination rate of Polygonatum sibiricum seeds under drought conditions.

[0050] In another seed coating agent product of the present invention, the rhizosphere growth-promoting seed coating agent provided by the present invention preferably comprises 1-35 parts of Bacillus tonkinensis, 1-8 parts of binder, and 1-15 parts of polyethylene glycol. The rhizosphere growth-promoting seed coating agent provided by the present invention preferably comprises 1-35 parts of Bacillus tonkinensis, more preferably 20-33 parts, and even more preferably 30 parts. In the present invention, the Bacillus tonkinensis is preferably added in the form of a Bacillus tonkinensis bacterial suspension. The preparation method and viable cell count of the Bacillus tonkinensis bacterial suspension are the same as described above and will not be repeated here.

[0051] Based on the mass fraction of Bacillus tonkinensis, the rhizosphere growth-promoting seed coating agent provided by the present invention preferably comprises 1 to 8 parts of adhesive, more preferably 3 to 7 parts, and even more preferably 4 parts. The adhesive described in the present invention has been described above and will not be repeated here.

[0052] Based on the mass fraction of Bacillus tonkinensis, the rhizosphere growth-promoting seed coating agent provided by the present invention preferably comprises 1 to 15 parts of polyethylene glycol, more preferably 10 to 14 parts, and even more preferably 12 parts. The polyethylene glycol described in the present invention is a dispersant, and its function is to ensure good compatibility of the film-forming substance, while its moisturizing effect also maintains the activity of the bacterial strain.

[0053] After obtaining the rhizosphere growth-promoting seed coating agent, the present invention preferably adds an insecticidal component to the rhizosphere growth-promoting seed coating agent to obtain a rhizosphere growth-promoting and insecticidal seed coating agent. By weight, the rhizosphere growth-promoting and insecticidal seed coating agent provided by the present invention comprises 1-35 parts of Bacillus tonkinensis, 20-40 parts of adsorbent, 1-8 parts of binder, 3-12 parts of sodium polyacrylate, and 3-18 parts of insecticidal component; or 1-35 parts of Bacillus tonkinensis, 1-8 parts of binder, 1-15 parts of polyethylene glycol, and 3-18 parts of insecticidal component.

[0054] Bacillus tonkinensis, adsorbents, adhesives, sodium polyacrylate, and polyethylene glycol have been discussed above and will not be repeated here.

[0055] Based on the mass fraction of Bacillus tonkinensis, the rhizosphere growth-promoting seed coating agent provided by this invention preferably also includes 3 to 18 parts of an insecticidal ingredient, more preferably 3 to 8 parts, and even more preferably 4 parts. This invention does not specifically limit the insecticidal ingredient; conventional insecticidal ingredients can be used. The insecticidal ingredient of this invention preferably includes imidacloprid and / or thiamethoxam.

[0056] The mechanisms of dormancy and germination in Polygonatum seeds are not well understood in existing technologies. This invention utilizes the colonization of Bacillus toyonensis PSLXL1 on the seed surface to effectively construct a microbial community on the seed surface. Simultaneously, this bacterium may secrete substances that promote seed germination, effectively resolving dormancy in Polygonatum seeds and promoting the differentiation and development of the seed embryo structure, thereby increasing the seedling emergence rate.

[0057] This invention provides a method for preparing the microbial agent described in the above technical solution, comprising the following steps:

[0058] The seed coating agent is obtained by mixing the above raw material components. This invention does not have any special limitations on the mixing process; conventional methods can be used.

[0059] The present invention provides the application of Bacillus tonkinensis or the bacterial agent or the bacterial agent prepared by the above-described technical solution in one or more of the following (1) to (3);

[0060] (1) Improve the germination rate of Polygonatum seeds;

[0061] (2) Improve the germination potential of Polygonatum seeds;

[0062] (3) Improve the germination rate of Polygonatum seeds.

[0063] The seeds of Polygonatum sibiricum described in this invention are preferably those of Polygonatum sibiricum.

[0064] The present invention preferably uses a seed coating agent to coat the seeds of Polygonatum sibiricum to obtain coated Polygonatum sibiricum seeds. The mass ratio of the seed coating agent to the Polygonatum sibiricum seeds is preferably 1:30 to 1:65, more preferably 1:35 to 1:55, and even more preferably 1:40.

[0065] When the seed coating agent of the present invention preferably includes Bacillus tonkinensis, an adsorbent, a binder, and a water-retaining agent, the process of coating Polygonatum sibiricum seeds with the seed coating agent of the present invention preferably includes: mixing Bacillus tonkinensis, the binder, the water-retaining agent, and the adsorbent to obtain the seed coating agent, and mixing the seed coating agent with Polygonatum sibiricum seeds to obtain coated Polygonatum sibiricum seeds. When the seed coating agent of the present invention preferably includes Bacillus tonkinensis, an adsorbent, a binder, and a water-retaining agent, the coating of Polygonatum sibiricum seeds is preferably carried out under rotational conditions to ensure thorough mixing of the seed coating agent and the Polygonatum sibiricum seeds. The present invention does not have special limitations on the mixing parameters and conventional parameters can be used. The rotational speed of the rotational conditions described in the present invention is preferably 600-1150 r / min, more preferably 1100 r / min.

[0066] In a seed coating agent product provided by the present invention, it preferably includes 30 parts of Bacillus tonkinensis bacterial solution, 1.5 parts of chitosan binder, 3 parts of sodium polyacrylate water-retaining agent, and 20 parts of maifanite adsorbent.

[0067] In another seed coating agent product provided by the present invention, it preferably includes 30 parts of Bacillus tonkinensis bacterial solution, 1.5 parts of binder dextrin, 3 parts of sodium polyacrylate and 20 parts of biochar.

[0068] In another seed coating agent product provided by the present invention, it preferably includes 30 parts of Bacillus tonkinensis bacterial solution, followed by the addition of 2 parts of polyvinylpyrrolidone and 1 part of polyethylene glycol.

[0069] The insecticide seed coating agent provided by the present invention preferably includes 30 parts of Bacillus tonkinensis bacterial solution, 2 parts of polyvinylpyrrolidone, 1 part of polyethylene glycol and 4 parts of imidacloprid.

[0070] Another insecticide seed dressing agent provided by the present invention preferably includes 30 parts of Bacillus tonkinensis bacterial solution, 2 parts of polyvinylpyrrolidone, 1 part of polyethylene glycol, and 4 parts of thiamethoxam.

[0071] When the seed coating agent of the present invention preferably includes Bacillus tonkinensis, an adhesive and polyethylene glycol; the process of coating Polygonatum seeds with the seed coating agent of the present invention preferably includes: obtaining a mixture of Bacillus tonkinensis, an adhesive and polyethylene glycol, and then adding Polygonatum seeds for coating to obtain coated Polygonatum seeds.

[0072] The diameter of the coated Polygonatum seeds of this invention is preferably 7-9 mm, more preferably 7.8-8.2 mm, and even more preferably 8 mm. The preferred effective viable bacterial count of the coated Polygonatum seeds of this invention is 1 × 10⁻⁶. 4 CFU / seed ~ 2.5 × 10 8 cfu / seed.

[0073] This invention isolates and identifies root microorganisms from *Polygonatum odoratum*, a species of *Polygonatum*, and screens a strain of *Bacillus tonkinensis* that helps break dormancy in *Polygonatum* seeds, increasing germination rate. This strain is then used to form a seed coating agent. During *Polygonatum* cultivation, applying this seed coating agent effectively breaks dormancy, promotes embryo differentiation and development in *Polygonatum* seeds, and improves germination rate. The application of this seed coating agent significantly improves the germination rate, germination potential, and germination rate of *Polygonatum* seeds.

[0074] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0075] The Polygonatum seeds used in the embodiments of this invention are all Polygonatum sibiricum seeds.

[0076] Example 1

[0077] 1. Isolation and purification of endophytic bacteria

[0078] The experimental method used in this invention to isolate endophytic bacteria is the tissue block culture method (see...). Figure 1 The process involves thoroughly cleaning the Solomon's Seal rhizome with detergent water, then rinsing it under running water for 1 hour. The rinsed rhizome is then placed in a sterilized 100mL Erlenmeyer flask, and a 75% ethanol solution is added for 2 minutes. After removing the ethanol solution, it is rinsed four times with sterile water. Next, it is treated with a 0.1% HgCl2 solution for 8 minutes. After removing the HgCl2 solution, it is rinsed six times with sterile water, and any residual sterile water is absorbed. Finally, the rhizome is cut into 1cm pieces using a sterile blade. 3 Small pieces of the tubers were placed in LB agar to isolate endophytic bacteria. Three tubers were inoculated onto each plate as the experimental group. The sterile water from the final rinse was inoculated onto LB agar as the control group. Both the experimental and control agar were cultured at a constant temperature of 30°C for 2–3 days. After bacteria grew from the cut surfaces of the tubers in the experimental group, colonies were picked up with an inoculation loop and purified onto LB agar. Colonies purified three times were considered pure cultures. One strain was labeled as PSLXL1 and the pure endophytic bacteria PSLXL1 was stored on LB slant agar at 4°C for later use.

[0079] 2. Molecular identification of endophytes

[0080] (1) Extraction of PSLXL1 genome from Polygonatum sibiricum endophytes

[0081] Genomic DNA was extracted from the bacterial strain using the SDS method, following these steps: ① The strain, stored at 4℃, was streaked onto LB agar plates and incubated upside down at 37℃ for 12–16 h until the colony diameter reached 1–2 mm; ② A single, normally growing colony was picked and inoculated into 35 mL of liquid LB medium and incubated at 37℃ with shaking (≥120 rpm) for 14 h; ③ 2.0 mL of the bacterial culture was transferred to a 2.0 mL centrifuge tube and centrifuged at 12000 rpm for 1 min. Collect bacterial cells; ④ Add 600 μL of 1% SDS bacterial lysis buffer, incubate at 37℃ for 30 min, and mix by inversion every 5 min; then add 600 μL of 3 mol / L NaCl solution, mix by inversion; then add 300 μL of LCTAB (10% CTAB), mix by inversion, incubate at 65℃ for 20 min, and centrifuge at 12000 r / min for 10 min. Obtain the supernatant after centrifugation. ⑤ Transfer the supernatant to a 2 mL centrifuge tube, add an equal volume of equilibrated phenol-chloroform-isoamyl alcohol mixture (the volume ratio of phenol, chloroform, and isoamyl alcohol in the phenol-chloroform-isoamyl alcohol mixture is 25:24:1), invert several times, let stand for a moment, and centrifuge at 12000 r / min for 10 min; ⑥ Transfer the supernatant to a 1.5 mL centrifuge tube, add 1.5 mL of chloroform and isoamyl alcohol mixture (the volume ratio of chloroform and isoamyl alcohol is 24:1), invert several times, let stand for a moment, and centrifuge at 12000 r / min for 10 min. Centrifuge for 1 minute to obtain the supernatant; ⑦ Transfer the supernatant to a 1.5 mL centrifuge tube, add 1 / 9 of the volume of the supernatant with 3 mol / L NaAc solution (pH = 8.0) to make the final concentration of NaAc solution 0.3 mol / L, mix well, add 2 volumes of pre-cooled anhydrous ethanol, mix well, and place at -20℃ for 30 min to 1 h; ⑧ Centrifuge at 4℃, 12000 r / min for 15 min, discard the supernatant, drain, wash 2 to 3 times with 75% ethanol, invert the centrifuge tube, blow dry, dissolve in 50 μL ddH2O, and store at -20℃ for later use.

[0082] (2) PCR amplification of 16S rDNA

[0083] Using the genomic DNA extracted from the strain as a template, 16S rDNA clones were amplified by PCR using universal primers 27F (5′-AGAGTTTGATCCTGGCTCAG-3′, SEQ ID NO.2) and 1492R (5′-GGTTACCTTGTTACGACTT-3′, SEQ ID NO.3). The PCR reaction mixture consisted of: primer 27F: 1 μL, primer 1492R: 1 μL, 2×TaqMix: 25 μL, template: 0.8 μL, and ddH2O added to a final volume of 50 μL. The PCR cycling system consisted of: pre-denaturation at 94℃ for 5 min; denaturation at 94℃ for 40 s; annealing at 59℃ for 30 s; extension at 72℃ for 100 s; 32 cycles; and final extension at 72℃ for 10 min.

[0084] (3) Recovery of 16S rDNA

[0085] 16S rDNA was recovered using a SanPrep column DNA gel recovery kit purchased from Sangon Biotech (Shanghai) Co., Ltd.

[0086] (4) Preparation of highly efficient competent cells

[0087] Competent cells were prepared using a high-efficiency competent cell preparation kit purchased from Sangon Biotech (Shanghai) Co., Ltd.

[0088] (5) Ligation of 16S rDNA with pUCm-T vector and transformation

[0089] The following ligation system was prepared in a 0.2 mL PCR tube to ligate 16S rDNA with the pUCm-T vector: purified 16S rDNA: 1 μL, pUCm-T Vector (50 ng / μL): 1 μL, 10× Ligation Buffer: 1 μL, 50% PEG4000: 1 μL, T4 DNA Ligase: 1 μL, and sterilized ddH2O (sterile double-distilled water) to a final volume of 10 μL. The ligation was carried out overnight at 16°C.

[0090] (6) Transformation

[0091] ① After thawing 100 μL of competent cells on ice, add 10 μL of the above ligation solution, mix gently, place on ice for 30 min, heat shock in a 42℃ water bath for 90 s, and place on ice for 5 min.

[0092] ② Add 900 μL LB liquid culture medium and incubate at 37°C with shaking for 1 hour;

[0093] ③ Centrifuge at 4000 rpm for 3 min, remove 750 μL of supernatant with a pipette tip, and suspend the cells in the remaining culture medium;

[0094] ④ Spread the bacterial suspension evenly on an LB agar plate containing 20 μL IPTG (100 mM) and 100 μL X-gal (20 mg / mL) resistant to ampicillin;

[0095] ⑤ Incubate the plates at 37°C, placing the plates upright for 1 hour, then inverting them and incubating overnight;

[0096] ⑥ Pick white colonies with a toothpick and incubate them overnight at 37°C in LB liquid medium containing ampicillin. Extract plasmids and perform enzyme digestion for identification. Plasmid extraction and identification of positive clones: Plasmid extraction: Extract plasmids using the SanPrep column-based plasmid DNA mini-extraction kit purchased from Sangon Biotech (Shanghai) Co., Ltd.; Enzyme digestion identification of positive clones used a 20 μL digestion system: 5 μL recombinant plasmid, EcoR-Ⅰ: 0.9 μL, Pst-Ⅰ: 0.9 μL, ddH2O: 13.2 μL, to identify positive clones.

[0097] (7) 16S rDNA sequencing and phylogenetic analysis

[0098] The PSLXL1 strain, identified as a positive clone by enzyme digestion, was sent to Sangon Biotech for sequencing. The obtained 16S rDNA sequence was then BLAST-aligned using the EZbiocloud database (https: / / www.ezbiocloud.net / ) to retrieve its homologous sequences. Figure 2 The 16S rDNA gene sequence of the endophytic bacterium PSLXL1 in Example 1 was compared with the NCBI database. Figure 3 .

[0099] (8) Gram staining identification and analysis

[0100] Gram staining was performed according to the method in the *Handbook of Systematic Identification of Common Bacteria*. A small amount of bacterial growth was picked up with an inoculation needle and spread onto a clean glass slide in a drop of sterile water or distilled water. The slide was air-dried, then stained with a mixture of crystal violet for 1 minute, washed with water, reacted with iodine solution for 1 minute, washed with water, blotted dry, and decolorized with 95% ethanol solution. The staining continued until the eluent was colorless for approximately 30 seconds. Finally, it was stained with safranin solution for 2–3 minutes, washed with water, and air-dried. The staining was observed under a microscope and photographed. Figure 4 .according to Figure 4 It can be seen that the Gram staining of the endophytic bacterium PSLXL1 of Polygonatum sibiricum is purple, indicating that PSLXL1 is a Gram-positive bacterium.

[0101] (9) Physiological and biochemical analysis of endophytic bacteria PSLXL1

[0102] Physiological and biochemical experiments were conducted according to the bacterial physiological and biochemical identification methods in Bergey's Manual of Bacterial Identification and Experimental Microbiology. The isolated PSLXL1 strain was inoculated into the bacterial biochemical micro-identification tubes using an inoculation needle and labeled accordingly.

[0103] The physiological and biochemical experiments included: ① Carbon source utilization capacity experiment: using biochemical tube methods to identify the strain's ability to utilize glucose, lactose, glucosamine, sucrose, arabinose, muscarine, mannose, inositol, fructose, cellobiose, maltose, and mannitol. ② O-F experiment. ③ Peptone hydrolysis (indole) experiment. ④ Nitrate reduction. ⑤ Arginine double hydrolysis. ⑥ Gelatin liquefaction test. ⑦ Malonate utilization. ⑧ Citrate utilization. ⑨ Methyl red (MR) test. ⑩ V-P experiment. Hydrolyzed aescin Oxidase test. The results of the physiological and biochemical experiments are shown in Table 1 below.

[0104] Table 1. Results of physiological and biochemical experiments on endophytic bacteria PSLXL1

[0105]

[0106]

[0107] According to Table 1, Figure 2 and Figure 3 It can be seen that this endophytic strain PSLXL1 was identified through the EZBioCloud database (https: / / www.ezbiocloud.net / ) ( Figure 2 ) and the NCBI database (https: / / www.ncbi.nlm.nih.gov / ) Figure 3 The sequence similarity between PSLXL1 strain and Bacillus. Tokyoensis was 98.54% and 98.41%, respectively, according to the search and comparison. Combined with physiological and biochemical analysis (Table 1), it was identified as Bacillus. Tokyoensis, and the characteristic sequence used for identification is shown in SEQ ID NO: 1. This strain was deposited on December 9, 2022, at the China Center for Type Culture Collection (CCTCC), Wuhan University, with accession number CCTCC NO. M20221919.

[0108] 3. Determination of the acid and alkali resistance of Bacillus toyonensis PSLXL1

[0109] The acid and alkali resistance of Bacillus toyonensis PSLXL1 was determined: Preparation of culture media with different pH values: LB medium was used as the base medium, and sodium hydroxide or hydrochloric acid solution was added to adjust its pH value to prepare a series of culture media with pH values ​​of 3.0, 5.0, 7.0, 9.0 and 11.0. After sterilization at 121℃ for 30 min, the media were plated and ready for use.

[0110] Preparation of salinity gradient culture media: Preparation of culture media with different salinity: Using LB medium as the base medium, sodium chloride was added to prepare a series of culture media with sodium chloride mass concentrations of 1.0%, 5.0%, 10.0%, 15.0% and 20%, respectively. After sterilization at 121℃ for 30 min, the media were plated and ready for use.

[0111] Bacillus Tokyo was spread on LB agar plates with different pH values ​​and salinities, with three replicates for each gradient. After inoculation, the plates were incubated at 28°C for 4 days, and the plaque size was recorded. Figure 5 , Figure 6 Tables 2 and 3 show that *Bacillus tonkinensis* can grow well under conditions of pH 3–11 and sodium chloride concentration of 1%–15%, without significant inhibition. *Bacillus tonkinensis* exhibits strong salt and acid / alkali tolerance.

[0112] Table 2. Plaque diameter of Bacillus toyonensis PSLXL1 under different pH conditions.

[0113]

[0114]

[0115] Table 3. Plaque diameter of Bacillus toyonensis PSLXL1 under different salinity conditions.

[0116]

[0117] Example 2

[0118] The manufacturer of maifanite powder is Mengyin Maifanite Factory; the manufacturer of dextrin is Shenzhen Honghai Food Technology Co., Ltd.; the manufacturer of sodium polyacrylate is Hangzhou Juhe Biotechnology Co., Ltd.; and the manufacturer of chitosan is Shenzhen Honghai Food Technology Co., Ltd.

[0119] The preparation method of this Bacillus tonkinensis bacterial culture is as follows:

[0120] The original strain of Bacillus toyonensis (PSLXL1) was streaked in three zones on LB solid medium and cultured at 37°C for 14 h. Then, single clones were picked from the medium and cultured in 50 mL LB liquid medium at 37°C for 12 h to obtain the bacterial culture of Bacillus toyonensis.

[0121] Bacillus tonkinensis culture was added to LB liquid medium at a volume ratio of 5%. Fermentation conditions were: stirring speed 120 r / min, aeration rate maintained at 1.6 VVM, and fermentation temperature 30℃ for 3 days in a fermenter. The effective viable count of the fermentation broth was 1 × 10⁻⁶. 9 cfu / mL.

[0122] Preparation method and application of seed coating agent: Weigh 30g of Bacillus thomsonii bacterial solution. The effective viable count of Bacillus thomsonii bacterial solution is 1×10⁻⁶. 9 Add 1.5g of chitosan (cfu / mL) as a binder, 3g of sodium polyacrylate (water-retaining agent), and 20g of maifanite (adsorbent) as an adsorbent. Mix thoroughly and dissolve to obtain a solution. Rotate the *Polygonatum sibiricum* seeds at 1100 rpm at a mass ratio of 1:30 (coating agent: naked seed). While rotating the seeds, slowly add the solution. Repeat this process every 8 seconds until the naked *Polygonatum sibiricum* seeds are coated with a diameter of 8.0–8.2 mm.

[0123] Example 3

[0124] The biochar powder was manufactured by Jiangsu Chengsen Charcoal Industry Technology Co., Ltd.; the manufacturers of dextrin and sodium polyacrylate were the same as in Example 2.

[0125] The preparation method of Bacillus tonkinensis bacterial suspension is the same as in Example 2.

[0126] Preparation and application of seed coating agent: Weigh 30g of Bacillus tonkinensis bacterial solution, then add 1.5g of binder dextrin, 3g of sodium polyacrylate, and 20g of biochar. Mix thoroughly and dissolve to obtain a mixture. Rotate naked Polygonatum seeds at 1100r / min at a mass ratio of 1:40 (coating agent: naked seed). During the rotation, slowly add the mixture. Repeat the above steps every 8 seconds until the naked Polygonatum seeds are coated to a size of 8.0–8.2mm.

[0127] Example 4

[0128] The manufacturer of polyvinylpyrrolidone is Runyou Chemical; the manufacturer of polyethylene glycol is Sinopharm Group.

[0129] The preparation method of Bacillus tonkinensis bacterial suspension is the same as in Example 2.

[0130] Preparation and application of Polygonatum sibiricum seed coating agent: Weigh 30g of Bacillus tonkinensis bacterial solution, then add 2g of polyvinylpyrrolidone and 1g of polyethylene glycol, mix thoroughly to obtain 33g of seed coating agent. Mix 1g of this seed coating agent with 30g of naked Polygonatum sibiricum seeds at a mass ratio of 1:30 for coating, so that Bacillus tonkinensis can be uniformly adsorbed on the Polygonatum sibiricum seeds. The effective viable bacteria count of each coated Polygonatum sibiricum seed is 1×10⁻⁶. 4 CFU / seed ~ 2.5 × 10 8 cfu / seed.

[0131] Example 5

[0132] The method for preparing the bacterial culture of Bacillus Tokyo is the same as in Example 2.

[0133] Two insecticides, imidacloprid and thiamethoxam, were selected and their compatibility with a biological seed coating agent made from Bacillus toyonensis PSLXL1 was tested on Polygonatum seeds.

[0134] The control group consisted of a seed coating agent A for promoting the growth of Polygonatum sibiricum seeds. 30g of Bacillus tonkinensis bacterial suspension was weighed, then 2g of polyvinylpyrrolidone and 1g of polyethylene glycol were added and mixed thoroughly to obtain 33g of seed coating agent. 1g of this seed coating agent was then mixed with 30g of naked Polygonatum sibiricum seeds at a mass ratio of 1:30 for coating, allowing Bacillus tonkinensis to be evenly adsorbed onto the seeds, resulting in coated Polygonatum sibiricum seeds. After coating, the effective viable bacteria count on each seed was 1×10⁻⁶. 4 CFU / seed ~ 2.5 × 10 8 cfu / seed.

[0135] Preparation of Seed Coating Agent B for Promoting the Growth of Polygonatum sibiricum Containing the Insecticide Imidacloprid: 30g of Bacillus tonkinensis bacterial solution was weighed, then 2g of polyvinylpyrrolidone, 1g of polyethylene glycol, and 4g of imidacloprid were added and mixed thoroughly to obtain 37g of seed coating agent. 1g of this seed coating agent was mixed with 30g of naked Polygonatum sibiricum seeds at a mass ratio of 1:30 for coating, allowing imidacloprid and Bacillus tonkinensis to be evenly adsorbed onto the Polygonatum sibiricum seeds, resulting in coated Polygonatum sibiricum seeds. The effective viable bacteria count on each Polygonatum sibiricum seed was 1×10⁻⁶. 4 CFU / seed ~ 2.5 × 10 8 cfu / seed.

[0136] Preparation of Seed Coating Agent C for Promoting the Growth of Polygonatum sibiricum Containing the Insecticide Thiamethoxam: 30g of Bacillus tonkinensis bacterial solution was weighed, then 2g of polyvinylpyrrolidone, 1g of polyethylene glycol, and 4g of thiamethoxam were added and mixed thoroughly to obtain 37g of seed coating agent. 1g of this seed coating agent was mixed with 30g of naked Polygonatum sibiricum seeds at a mass ratio of 1:30 for coating, allowing thiamethoxam and Bacillus tonkinensis to be uniformly adsorbed onto the Polygonatum sibiricum seeds, resulting in coated Polygonatum sibiricum seeds. The effective viable bacteria count on each Polygonatum sibiricum seed was 1×10⁻⁶. 4 CFU / seed ~ 2.5 × 10 8 CFU / seed. Weekly sampling of coated seeds was conducted to measure changes in bacterial count.

[0137] In the compatibility test between imidacloprid and thiamethoxam, the changes in viable bacterial counts in coated Polygonatum seeds were tested over a total of 4 weeks. Results are shown below. Figure 7 As shown in Table 4, when *Bacillus thomsonii* was mixed with imidacloprid and thiamethoxam for seed coating, the bacterial count on the seeds remained stable at ≥2.00E+06 cfu / seed for 4 weeks. Compared with control A, the bacterial count of *Bacillus thomsonii* decreased slightly in both treatments, and the count remained at ≥2.0E+06 cfu / seed for 4 weeks. Therefore, *Bacillus thomsonii* exhibits good compatibility with imidacloprid and thiamethoxam.

[0138] Table 4. Changes in viable bacterial counts of Polygonatum sibiricum seeds prepared with seed coating agents A-C over 4 weeks.

[0139]

[0140] Example 6: Effect of Seed Coating Agent A on Germination of Polygonatum Seeds

[0141] Fine river sand was sterilized in an autoclave at 121°C for 20 minutes and set aside. Coated Polygonatum seeds prepared using coating agent A (prepared in Example 5) were stratified in a germination box at a volume ratio of 1:3 to 3. Uncoated Polygonatum seeds were also stratified in a 1:3 volume ratio with sand. The germination box was placed in a constant temperature incubator at 20°C for 20 days. Germinated or emerging Polygonatum seeds were picked out every 3 days, and the number was recorded, repeated 3 times. Germination was defined as the radicle breaking through the seed coat and exceeding the length of the seed itself, and emergence was defined as the emergence of the first true leaf. The germination rate and germination potential were calculated after 40 days, and the emergence rate was calculated after 80 days using the following formula.

[0142] Germination rate = (Total number of germinated Polygonatum seeds / Total number of Polygonatum seeds tested) × 100%;

[0143] Germination potential = (Number of normally germinated Polygonatum seeds on the peak day / Total number of Polygonatum seeds tested) × 100%;

[0144] Germination rate = (Total number of germinated Polygonatum seeds / Total number of Polygonatum seeds tested) × 100%.

[0145] The results are shown in Table 4. According to Table 4, coating the seeds with seed coating agent can significantly improve the germination rate, emergence rate and germination potential of Polygonatum seeds. The germination rate, emergence rate and germination potential in Table 1 are average values.

[0146] Table 4. Effects of coating agent A on germination and emergence of Polygonatum seeds.

[0147]

[0148] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A strain of Bacillus tonkinensis Bacillus toyonensis PSLXL1, characterized in that, Its accession number is CCTCCNO.M20221919.

2. A type of organism containing the Bacillus tonkinensis of claim 1 Bacillus toyonensis The bacterial agent of PSLXL1 is characterized by, The inoculant is a seed coating agent.

3. The microbial agent according to claim 2, characterized in that, The seed coating agent includes a rhizosphere growth-promoting seed coating agent or a rhizosphere growth-promoting and insecticidal seed coating agent.

4. The microbial agent according to claim 3, characterized in that, By weight, the rhizosphere seed coating agent comprises 1-35 parts of Bacillus tonkinensis, 10-20 parts of adsorbent, 1-8 parts of binder, and 3-12 parts of sodium polyacrylate; or 1-35 parts of Bacillus tonkinensis, 1-8 parts of binder, and 1-15 parts of polyethylene glycol. The adsorbent includes maifanite and / or biochar; The adhesive comprises one or more of polyvinylpyrrolidone, dextrin, and chitosan.

5. The microbial agent according to claim 3, characterized in that, By weight, the rhizosphere growth-promoting and insecticidal seed coating agent includes 1-35 parts of Bacillus thomsonii, 10-20 parts of adsorbent, 1-8 parts of binder, 3-12 parts of sodium polyacrylate, and 3-18 parts of insecticidal ingredient; or 1-35 parts of Bacillus thomsonii, 1-8 parts of binder, 1-15 parts of polyethylene glycol, and 3-18 parts of insecticidal ingredient.

6. The microbial agent according to claim 5, characterized in that, The insecticidal ingredients, by weight, include imidacloprid and / or thiamethoxam.

7. The method for preparing the microbial agent according to any one of claims 2 to 6, characterized in that, The process includes the following steps: mixing the raw material components of the fungal agent according to any one of claims 2 to 6 to obtain a seed coating agent.

8. The use of Bacillus tonkinensis according to claim 1, or the bacterial agent according to any one of claims 2 to 6, or the bacterial agent prepared by the preparation method according to claim 7, in one or more of the following (1) to (3); (1) Improve the germination rate of Polygonatum seeds; (2) Improve the germination potential of Polygonatum seeds; (3) Improve the germination rate of Polygonatum seeds.

9. The application according to claim 8, characterized in that, The seeds of Polygonatum were coated with a seed coating agent to obtain coated Polygonatum seeds. The mass ratio of the seed coating agent to the Polygonatum seeds was 1:30 to 1:65.

Images