A soybean endophytic bacterium hml5368, use thereof, and plant leaf spraying agent

Abstract

The invention discloses soybean endophyte HML5368, application of the soybean endophyte HML5368 and a plant leaf spraying agent, the soybean endophyte HML5368 is named as Bacillus cereus according to taxonomy and is preserved in China General Microbiological Culture Collection Center on September 23, 2024, and the preservation number of the soybean endophyte HML5368 is CGMCC NO: 32043. In the invention, the soybean endophyte HML5368 is found and separated by the inventor for the first time, the secreta of the soybean endophyte HML5368 serves as an effective component of the plant leaf spraying agent, the benefit of the soybean endophyte HML5368 on plant growth is reserved, the soybean endophyte HML5368 can be used for other plants except soybeans, the requirement of the endophyte on specificity is avoided, and the application prospect is wide. The endophyte has important significance in popularization and application of the endophyte in crops.

Description

(19) State Intellectual Property Office (12) Invention Patent Application (10) Application Publication Number (43) Application Publication Date (21) Application Number 202411461785.2 (22) Application Date 2024.10.18 (83) Biological Preservation Information CGMCC NO: 32043 2024.09.23 (71) Applicant The Chinese University of Hong Kong Address Sha Tin, New Territories, Hong Kong, China (72) Inventors Lin Hanming Gu Qishan (74) Patent Agency Shenzhen Runlin Chuangyou Patent Agency Co., Ltd. 44223 Patent Attorney Liu Li (51) Int.Cl. C12N 1 / 205 (2026.01) C12N 1 / 20 (2026.01) C12N 1 / 02 (2006.01) A01N 63 / 22 (2020.01) A01P 21 / 00(2006.01) A01P 1 / 00(2006.01) C12R 1 / 07(2006.01) (54) Invention Title: A Soybean Endophytic Bacterium HML5368 and Its Application, Plant Leaf Sprayer (57) Abstract: This invention discloses a soybean endophytic bacterium HML5368 and its application, as well as a plant leaf sprayer. The taxonomic name of the soybean endophytic bacterium HML5368 is Bacillus cereus. It was deposited at the China General Microbiological Culture Collection Center on September 23, 2024, with the accession number CGMCC NO:32043. In this invention, the inventors discovered and isolated soybean endophytic fungus HML5368 for the first time. The secretions of this soybean endophytic fungus HML5368, as an effective component of a plant foliar spray, retain the benefits of soybean endophytic fungus HML5368 for plant growth and can be used on other plants besides soybeans, eliminating the need for specificity in endophytic fungi. This is of great significance for the promotion and application of endophytic fungi in crops. Claims: 1 page; Description: 4 pages; Drawings: 2 pages; CN 121914906 A 2026.04.24 CN 1 21 91 49 06 A 1. A soybean endophytic fungus HML5368, characterized in that the soybean endophytic fungus HML5368 is a new species of Bacillus sp., deposited on September 23, 2024 at the China General Microbiological Culture Collection Center, with accession number CGMCC NO:32043. 2. A plant foliar spray, characterized in that the plant foliar spray contains secretions from the soybean endophytic bacterium HML5368 as described in claim 1. 3. An application of the soybean endophytic bacterium HML5368 as described in claim 1 in the preparation of a plant foliar spray, characterized in that the secretions from the soybean endophytic bacterium HML5368 are extracted and used as the active ingredient in the plant foliar spray. 4.The application as described in claim 3, characterized in that the application includes the following steps: S1, isolation of soybean endophytic bacteria HML5368 from leaves; including the following steps: S11, surface disinfection of wild soybean leaves; S12, under aseptic conditions, grinding the leaves treated in step S11 into juice, and coating the juice onto an agar plate for culture; S13, isolating single colonies of bacteria growing on the agar plate; S2, extracting secretions of soybean endophytic bacteria HML5368: inoculating the single colonies isolated in step S13 into a culture medium for culture; S3, centrifuging the bacterial solution after culture in step S2, taking the supernatant, filtering the supernatant, and obtaining the filtrate as a plant leaf spray agent. 5. The application as described in claim 4, characterized in that the components of the agar plate in step S12 include: 5 g / L tryptone, 3 g / L yeast extract, 0.75 g / L CaCl2 and 1.5% (w / v) agar. 6. The application as described in claim 4, characterized in that the culture in step S12 involves incubating the agar plate at 28-30°C for 20-24 hours. 7. The application as described in claim 4, characterized in that the culture medium in step S2 comprises: 15 g / L glycerol, 1 g / L (NH4)H2PO4, 0.1 g / L MgSO4·7H2O, 0.005 g / L FeSO4·7H2O, and 0.0035 g / L MnSO4·7H2O, with a pH of 7. 8. The application as described in claim 4, characterized in that the culture in step S2 refers to a shaking culture at 28-30°C for 44-48 hours. 9. The application as described in claim 4, characterized in that the centrifugation in step S3 refers to centrifuging the bacterial solution at 1000 (× g) at 4°C for 5 minutes. 10. The application as described in claim 4, characterized in that the filtration in step S3 refers to using 0.Filtration is performed using a 22μm filter membrane. Claims 1 / 1 page 2 CN 121914906 A Soybean Endophytic Bacterium HML5368 and its Application, Plant Leaf Spraying Agent Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a soybean endophytic bacterium HML5368 and its application, and a plant leaf spraying agent. Background Art

[0002] Green farming is a trend in agricultural development. Microbial-based fertilizers and pesticides can increase crop yields while reducing environmental pollution. Bacteria that cooperate with plants are broadly classified into two categories: ectophytes and endophytes. The former interacts with plants at the edge of the plant, while the latter lives symbiotically within the plant. At present, ectophytes are more commonly used, while the cooperation between endophytes and host plants requires the specificity of endophytes and host plants, making it difficult to popularize their use and limiting the application of endophytes in crops. Summary of the Invention

[0003] In order to overcome the shortcomings of the prior art, this invention proposes a soybean endophytic bacterium HML5368 and its application, and a plant leaf spraying agent.

[0004] In a first aspect, the present invention provides a soybean endophyte HML5368, wherein the soybean endophyte HML5368 is Bacillus sp.A new species of this species was deposited on September 23, 2024, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC NO:32043.

[0005] In a second aspect, the present invention provides a plant foliar spray containing the secretions of the soybean endophytic fungus HML5368 described in the first aspect.

[0006] In a third aspect, the present invention provides the application of the soybean endophytic fungus HML5368 in the preparation of a plant foliar spray, wherein the secretions of the soybean endophytic fungus HML5368 are extracted and used as the effective ingredient of the plant foliar spray.

[0007] The beneficial effects of the present invention include:

[0008] 1. In the present invention, the inventors have for the first time discovered and isolated the soybean endophytic fungus HML5368.

[0009] 2. The secretions of the soybean endophytic fungus HML5368 in the present invention, as the effective ingredient in the plant foliar spray, can significantly reduce the degree of yellowing of the leaves after being sprayed on the plant leaves.

[0010] 3. This invention uses the secretion of soybean endophytic bacteria HML5368 as the effective component of plant leaf spray, retaining the benefits of soybean endophytic bacteria HML5368 to plant growth, and can be used on other plants besides soybeans, eliminating the need for specificity of endophytic bacteria, which is of great significance for the promotion and application of endophytic bacteria in crops. Brief Description of the Drawings

[0011] Figure 1 is a comparison diagram of Arabidopsis thaliana leaves after being infected with tomato bacterial spot disease, after being sprayed with culture medium and the secretion of soybean endophytic bacteria HML5368 respectively.

[0012] Figure 2 is a morphological diagram of soybean endophytic bacteria HML5368 growing on an agar plate in Example 1 of this invention. Specification 1 / 4 pages 3 CN 121914906 A Detailed Description of the Embodiments

[0013] In order to make the inventive purpose, technical solution and technical effects of this invention clearer, the invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the specific embodiments described in this specification are only for explaining the invention and are not intended to limit the invention.

[0014] Unless otherwise specified, all experimental materials and reagents used are commercially available consumables and reagents.

[0015] A specific embodiment of the present invention provides a soybean endophyte HML5368, wherein the soybean endophyte HML5368 is Bacillus sp.A new species of Bacillus cereus, whose Chinese name is Bacillus cereus and genus name is Bacillus, was deposited on September 23, 2024, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC NO:32043, and deposit address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0016] A specific embodiment of the present invention also provides a plant leaf spray containing the secretions of the soybean endophytic bacterium HML5368.

[0017] A specific embodiment of the present invention also provides the application of the soybean endophytic bacterium HML5368 in the preparation of a plant leaf spray, wherein the secretions of the soybean endophytic bacterium HML5368 are extracted and used as the effective component of the plant leaf spray.

[0018] In some embodiments, the application includes the following steps:

[0019] S1, Isolation of soybean endophytic bacteria HML5368 from leaves; including the following steps:

[0020] S11, Surface disinfection of wild soybean leaves;

[0021] S12, Under aseptic conditions, grinding the leaves treated in step S11 into juice, and coating the juice onto an agar plate for culture;

[0022] S13, Isolating single colonies of bacteria growing on the agar plate;

[0023] S2, Extracting secretions of soybean endophytic bacteria HML5368: Inoculating the single colonies isolated in step S13 into a culture medium for culture;

[0024] S3, Centrifuging the bacterial solution after culture in step S2, taking the supernatant, filtering the supernatant, and obtaining the filtrate as a plant leaf spray.

[0025] In some embodiments, the components of the agar plate in step S12 include: 5 g / L tryptone, 3 g / L yeast extract, 0.75 g / L CaCl2, and 1.5% (w / v) agar.

[0026] In some embodiments, the culture in step S12 involves placing the agar plate at 28-30°C for 20-24 hours.

[0027] In some embodiments, in step S2, the culture medium includes: 15 g / L glycerol, 1 g / L (NH4)H2PO4, 0.1 g / L MgSO4·7H2O, 0.005 g / L FeSO4·7H2O, and 0.0035 g / L MnSO4·7H2O, with a pH of 7.

[0028] In some embodiments, the culture in step S2 refers to shaking culture at 28-30°C for 44-48 hours.

[0029] In some embodiments, the centrifugation in step S3 refers to centrifuging the bacterial solution at 1000 (×g) at 4°C for 5 minutes.

[0030] In some embodiments, the filtration in step S3 refers to using 0.Filtration is performed using a 22μm filter membrane.

[0031] Endophytic bacteria can promote plant growth, but they require specificity with the host plant, so their use is difficult to popularize. Moreover, bacteria produce different metabolites at different growth stages, and these metabolites are not necessarily beneficial to plant growth, and may even be harmful. In a specific embodiment of the present invention, the secretion of soybean endophytic bacteria HML5368 is used as the effective ingredient of plant leaf spray, which retains the benefits of soybean endophytic bacteria HML5368 to plant growth and can be used on other plants besides soybeans, eliminating the need for specificity of endophytic bacteria. In addition, the present invention extracts the secretion of soybean endophytic bacteria HML5368 at a specific growth stage (i.e., step S2 in the above embodiment of the present invention), which can control the composition of the secretion, thereby ensuring the benefits of soybean endophytic bacteria HML5368 to plant growth.

[0032] The present invention will be described in detail below through a specific example.

[0033] Example 1

[0034] Application of soybean endophytic bacteria HML5368 in the preparation of plant leaf spray, including the following steps:

[0035] Step 1: Isolation of soybean endophytic bacteria HML5368 from leaves, specifically including:

[0036] Step 11: Harvesting wild soybean leaves from soybean fields and performing surface disinfection. Surface disinfection process: First, rinse the leaves with 50% (v / v) household bleach containing 0.01% (v / v) TWEEN-20 for one minute; then rinse with 70% (v / v) ethanol; finally rinse with sterile pure water.

[0037] Step 12: Under sterile conditions, grind the leaves with a pestle, and then apply the juice to an agar plate. The agar plate consisted of 5 g / L tryptone, 3 g / L yeast extract, 0.75 g / L CaCl2, and 1.5% (w / v) agar. After coating with the liquid, the agar plate was incubated at 28°C for one day. Figure 2 shows the morphology of soybean endophytic bacteria HML5368 growing on the agar plate.

[0038] Step 13: The grown bacteria were isolated as single colonies by streaking.

[0039] Step 2: Extract the secretions of soybean endophytic bacteria HML5368

[0040] The single colonies isolated in Step 1 were inoculated into a culture medium: 15 g / L glycerol, 1 g / L (NH4)H2PO4, 0.1 g / L MgSO4·7H2O, 0.005 g / L FeSO4·7H2O and 0.0035 g / L MnSO4·7H2O, pH 7, and cultured at 28°C with shaking for two days.

[0041] Step 3: The bacterial solution was centrifuged at 4°C for 5 minutes, 1000 (×g), and the supernatant was collected, then treated with 0.The supernatant was filtered through a 22μm filter membrane to obtain the plant leaf spray.

[0042] BIOLOG Microbial Identification Test

[0043] Using 94 carbon sources on a 96-well microplate, the Biolog database was used for comparison: GENIII AEROBIC BACTERIA (1568 taxa). Specifically, HML 5368 cells were cultured on agar plates (5 g / L tryptone, 3 g / L yeast extract, 0.75 g / L CaCl2, 1.5% (w / v) agar) at 28°C for 16 hours. Then, the cells were seeded into inoculum IF-B (Biolog) according to the manufacturer's (Biolog) instructions. After mixing, 100 μl of the liquid was added to each well of the 96-well plate (GEN III microplate, Biolog). According to the manufacturer's instructions, each well was coated with a different carbon metabolite. The plate containing the liquid was incubated at 28°C for 22 hours. The color signal generated in each well was detected by the PM System (Biolog) system. The similarity between HML 5368 and Bacillus cereus was >60%.

[0044] The secretion of soybean endophytic fungus HML5368 was used as an effective ingredient in plant leaf spray to reduce leaf yellowing in an experiment.

[0045] Using the simulated plant Arabidopsis thaliana as a model, as shown in Figure 1, after the leaves of Arabidopsis thaliana were infected with tomato bacterial spot fungus (Pseudomonas syringae pv. tomato DC3000), they were sprayed with culture media (15 g / L glycerol, 1 g / L (NH4)H2PO4, 0.1 g / L MgSO4·7H2O, 0.005 g / L FeSO4·7H2O and 0.0035 g / L MgSO4·7H2O). MnSO4·7H2O, pH 7 (as a control group) or the secretion of soybean endophytic bacteria HML5368 (i.e., the plant leaf spray obtained above) were used in the experiment. The experiment was conducted by saturating the dosage, fully wetting the leaves, and spraying once. The results shown in Figure 1 were obtained 3 days after spraying. The experimental data showed that Arabidopsis leaves were infected with tomato bacterial spot fungus (Pseudomonas syringae pv.).After processing with tomato DC3000, the secretions of soybean endophytic bacteria HML5368 effectively reduced the damage of this bacteria to the leaves. Compared with the control group (culture medium), the secretions of soybean endophytic bacteria HML5368 reduced the degree of yellowing of the leaves.

[0046] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention should be considered equivalent substitutions and are included within the protection scope of the present invention. Instruction manual 4 / 4 pages 6 CN 121914906 A Figure 1 Instruction manual figure 1 / 2 pages 7 CN 121914906 A Figure 2 Instruction manual figure 2 / 2 pages 8 CN 121914906 A Abstract Abdominal ultrasound examination method, system and device A soybean endophytic bacterium HML5368 and its application, plant foliar spray This invention discloses a soybean endophytic bacterium HML5368 and its application, as well as a plant foliar spray. The taxonomic name of the soybean endophytic bacterium HML5368 is Bacillus cereus, which was deposited at the China General Microbiological Culture Collection Center on September 23, 2024, with the accession number CGMCC NO:32043. In this invention, the inventors have for the first time discovered and isolated soybean endophytic bacterium HML5368. The secretions of this soybean endophytic bacterium HML5368, as an effective component of a plant leaf spraying agent, retain the benefits of soybean endophytic bacterium HML5368 for plant growth and can be used on other plants besides soybeans. This eliminates the need for specificity in endophytic bacteria and is of great significance for the promotion and application of endophytic bacteria in crops.The soybean endophytic bacterium HML5368 is taxonomically designated as Bacillus cereus, which was deposited at the China General Microbiological Culture Collection Center on September 23, 2024, with the deposit number CGMCC NO: 32043. In the present invention, the inventors have, for the first time, discovered and isolated the soybean endophytic bacterium HML5368. A secretion of the soybean endophytic bacterium HML5368 is used as an active ingredient of the plant leaf spraying agent, thereby retaining the beneficial effects of the soybean endophytic bacterium HML5368 on plant growth. The agent can be applied to plants other than soybean, eliminating the requirement for specificity of endophytic bacteria. The present invention is of great significance for the promotion and application of endophytic bacteria in agricultural crops.

Claims

1. A soybean endophytic bacterium HML5368, characterized in that, The soybean endophytic fungus HML5368 is a new species of Bacillus sp., which was deposited on September 23, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO:32043.

2. A plant foliar spray, characterized in that, The plant leaf spray contains secretions from the soybean endophytic bacteria HML5368 as described in claim 1.

3. The application of the soybean endophytic bacteria HML5368 as described in claim 1 in the preparation of plant foliar sprays, characterized in that, The secretions of the soybean endophytic bacteria HML5368 were extracted and used as an effective ingredient in a plant foliar spray.

4. The application as described in claim 3, characterized in that, The application includes the following steps: S1. Isolation of soybean endophytic bacteria HML5368 from leaves; including the following steps: S11. Disinfect the surface of wild soybean leaves; S12. Under sterile conditions, the leaves treated in step S11 are ground into juice, and the juice is spread on an agar plate for culture. S13. Isolate single colonies of bacteria grown on the agar plate; S2. Extract the secretions of soybean endophytic bacteria HML5368: Inoculate the single colony isolated in step S13 into the culture medium for culture; S3. Centrifuge the bacterial culture after step S2, take the supernatant, filter the supernatant, and the resulting filtrate is a plant leaf spray.

5. The application as described in claim 4, characterized in that, The components of the agar plate described in step S12 include: 5 g / L tryptone, 3 g / L yeast extract, 0.75 g / L CaCl2 and 1.5% (w / v) agar.

6. The application as described in claim 4, characterized in that, The culture described in step S12 involves placing the agar plate at 28-30°C and culturing it for 20-24 hours.

7. The application as described in claim 4, characterized in that, In step S2, the culture medium comprises: 15 g / L glycerol, 1 g / L (NH4)H2PO4, 0.1 g / L MgSO4·7H2O, 0.005 g / L FeSO4·7H2O and 0.0035 g / L MnSO4·7H2O, with a pH of 7.

8. The application as described in claim 4, characterized in that, The cultivation mentioned in step S2 refers to cultivation at 28-30℃ with shaking for 44-48 hours.

9. The application as described in claim 4, characterized in that, The centrifugation mentioned in step S3 refers to centrifuging the bacterial solution at 1000 (×g) at 4°C for 5 minutes.

10. The application as described in claim 4, characterized in that, The filtration mentioned in step S3 refers to filtration using a 0.22μm filter membrane.

Images