Soybean endophyte HML5368, application 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

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a soybean endophytic bacterium HML5368 and its application, as well as a plant leaf spray. Background Technology

[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 interact with plants at their edges, while the latter live symbiotically within the plant. Currently, ectophytes are more widely used, while the cooperation between endophytes and their host plants requires specificity between the endophytes and the host plant, making their application more difficult and limiting their use in crops. Summary of the Invention

[0003] To overcome the shortcomings of existing technologies, this invention proposes a soybean endophytic bacterium HML5368 and its application, as well as a plant leaf spray.

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

[0005] In a second aspect, the present invention provides a plant leaf spray containing secretions from the soybean endophytic bacteria HML5368 described in the first aspect.

[0006] A third aspect of the present invention provides an 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 an effective component of the plant leaf spray.

[0007] The beneficial effects of this invention include:

[0008] 1. In this invention, the inventors discovered and isolated soybean endophytic bacteria HML5368 for the first time.

[0009] 2. The secretions of soybean endophytic bacteria HML5368 in this invention, as an effective component in plant leaf spray, can significantly reduce the degree of yellowing of leaves after being sprayed on plant leaves.

[0010] 3. This invention uses the secretions of soybean endophytic bacteria HML5368 as the effective ingredient of plant foliar spray, which retains the benefits of soybean endophytic bacteria HML5368 to plant growth and can be used on other plants besides soybeans. It eliminates the need for specificity of endophytic bacteria and is of great significance for the promotion and application of endophytic bacteria in crops. Attached Figure Description

[0011] Figure 1 The image shows a comparison of Arabidopsis thaliana leaves from Example 1 of this invention after they were infected with tomato bacterial spot disease and sprayed with culture medium and secretions from soybean endophyte HML5368, respectively.

[0012] Figure 2 This is a morphological diagram of the soybean endophytic bacteria HML5368 grown on an agar plate in Example 1 of this invention. Detailed Implementation

[0013] To make the objectives, technical solutions, and effects of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described in this specification are merely 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] This invention provides a soybean endophytic bacterium, HML5368, which is a new species of Bacillus sp., with the Chinese name Bacillus cereus and the genus name Bacillus. It was deposited on September 23, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCCNO:32043. The deposit address is: 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, wherein the plant leaf spray contains the secretions of the soybean endophytic bacteria HML5368.

[0017] The present invention also provides an application of the soybean endophytic bacterium HML5368 in the preparation of plant leaf sprays, 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 implementations, the application includes the following steps:

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

[0020] S11. Disinfect the surface of wild soybean leaves;

[0021] 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.

[0022] S13. Isolate single colonies of bacteria grown on the agar plate;

[0023] S2. Extract the secretions of soybean endophytic bacteria HML5368: Inoculate the single colony isolated in step S13 into the culture medium for culture;

[0024] S3. Centrifuge the bacterial culture after step S2, take the supernatant, filter the supernatant, and the resulting filtrate is 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 and culturing it for 20-24 hours.

[0027] In some embodiments, 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.

[0028] In some embodiments, the culture in step S2 refers to a 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 filtration using a 0.22 μm filter membrane.

[0031] Endophytic bacteria can promote plant growth, but they require specificity with their host plant, making their widespread use difficult. Furthermore, bacteria produce different metabolites at different growth stages, which may not necessarily be beneficial to plant growth and could even be harmful. This invention specifically uses the secretions of soybean endophytic bacteria HML5368 as the effective ingredient in a foliar spray, preserving the benefits of HML5368 for plant growth and allowing its application to other plants besides soybeans, thus eliminating the requirement for endophytic bacteria specificity. In addition, this invention extracts the secretions at a specific growth stage of soybean endophytic bacteria HML5368 (i.e., step S2 in the above embodiment), allowing control over the composition of the secretions and ensuring the benefits of soybean endophytic bacteria HML5368 for plant growth.

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

[0033] Example 1

[0034] The application of soybean endophytic bacteria HML5368 in the preparation of plant foliar sprays includes the following steps:

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

[0036] Step 11: Harvest the leaves of wild soybeans from the soybean field and perform 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, then spread the juice onto an agar plate. The agar plate consists of 5 g / L tryptone, 3 g / L yeast extract, 0.75 g / L CaCl2, and 1.5% (w / v) agar. After spreading the juice, incubate the agar plate at 28°C for one day. Figure 2 This is the morphology of soybean endophytic bacteria HML5368 growing on an agar plate.

[0038] Step 13: Isolate single colonies of the grown bacteria by drawing lines.

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

[0040] The single colonies isolated in step 1 were inoculated into a culture medium consisting of 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 incubated at 28°C with shaking for two days.

[0041] Step 3: Centrifuge the bacterial solution at 4℃ for 5 minutes, 1000 (×g), take the supernatant, and then filter the supernatant through a 0.22μm filter membrane to obtain the plant leaf spray.

[0042] BIOLOG Microbial Identification Test

[0043] Ninety-four carbon sources were used in 96-well microplates, compared using the Biolog database: 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. Cells were then 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 a 96-well plate (GEN III microplate, Biolog). Each well was coated with a different carbon metabolite according to the manufacturer's instructions. The plates containing the liquid were incubated at 28°C for 22 hours. The color signal generated in each well was recorded by... According to PM System (Biolog) analysis, HML 5368 has a similarity of >60% to Bacillus cereus.

[0044] Experiments were conducted on the use of secretions from soybean endophytic bacteria HML5368 as an active ingredient in plant foliar sprays to reduce leaf yellowing.

[0045] Using the mimicry plant Arabidopsis thaliana as a model, such as Figure 1 As shown, Arabidopsis thaliana plants, after their leaves were infected with Pseudomonas syringae pv. tomato DC3000, were sprayed with either 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) (as a control group) or secretions from soybean endophyte HML5368 (i.e., the plant foliar spray obtained above). The experiment was conducted using a saturated dosage, fully wetted leaves, and a single spraying method. Results were obtained 3 days after spraying. Figure 1 The results show that, after Arabidopsis leaves were infected with Pseudomonas syringae pv. tomato DC3000, the secretions of soybean endophytic bacteria HML5368 could effectively reduce the damage of the bacteria to the leaves. Compared with the control group (culture medium), the secretions of soybean endophytic bacteria HML5368 could reduce 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 shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

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.

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