Bacillus marinus DXGZ2-6, a bacterial agent and application thereof

Abstract

The application belongs to the technical field of microorganisms, and specifically discloses a Bacillus haynesii DXGZ2-6, a bacterial agent thereof and application thereof. The Bacillus haynesii DXGZ2-6 has a preservation number of CGMCC No.33710. The Bacillus haynesii can produce various enzymes, effectively promote plant growth, slow down the decomposition or transfer of chlorophyll in plant leaves, and effectively inhibit various plant pathogenic bacteria.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a Bacillus hainanensis DXGZ2-6, its inoculum and applications. Background Technology

[0002] Rhizosphere bacteria live in the soil or are epiphytic on plant roots, promoting the absorption of nutrients and thus plant growth. Under their influence, nutrients in the soil can be transformed from a state where they cannot be directly utilized by plants into a state that can be absorbed and utilized. Some rhizosphere bacteria can also synthesize plant growth regulators.

[0003] Applying plant rhizosphere growth-promoting bacteria to the plant growth process can improve the efficiency of crops in utilizing soil nutrients, help reduce the use of chemical fertilizers, and play an important role in developing ecological and healthy agriculture and increasing crop yields. Therefore, it is urgent to explore and utilize more plant rhizosphere growth-promoting bacteria that can effectively promote crop growth in order to promote high-yield and healthy crop cultivation. Summary of the Invention

[0004] In view of the above, it is necessary to provide a strain that can produce a variety of enzymes, effectively promote plant growth, slow down the decomposition or transfer of chlorophyll in plant leaves, and effectively inhibit a variety of plant pathogens, so as to promote high-yield and healthy crop cultivation.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0006] This invention provides a Bacillus haynesii DXGZ2-6, which is classified as Bacillus haynesii. It is deposited at the China General Microbiological Culture Collection Center (CGMCC) at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, on March 3, 2025, with accession number CGMCC No. 33710.

[0007] Furthermore, the Bacillus hainanensis DXGZ2-6 has the function of producing amylase, cellulase, ligninase and protease.

[0008] The present invention also provides a bacterial agent containing the above-mentioned Bacillus hainanensis DXGZ2-6.

[0009] The present invention also provides the application of the above-mentioned Bacillus hainanensis DXGZ2-6 or the above-mentioned bacterial agent in promoting plant growth.

[0010] Furthermore, the promotion of plant growth includes increasing plant height, stem diameter, dry weight, and / or fresh weight.

[0011] The present invention also provides the application of the above-mentioned Bacillus hainanensis DXGZ2-6 or the above-mentioned bacterial agent in slowing down the decomposition or transfer of chlorophyll in plant leaves.

[0012] Furthermore, the plants include chili peppers and lettuce.

[0013] The present invention also provides the application of the above-mentioned Bacillus hainanensis DXGZ2-6 or the above-mentioned inoculum in inhibiting plant pathogens.

[0014] Furthermore, the plant pathogen is *Smutella smutella*, which causes corn smut.

[0015] The present invention has the following beneficial effects:

[0016] 1. The Bacillus haynesii DXGZ2-6 of the present invention can produce a variety of enzymes, including amylase, cellulase, ligninase and protease, which can decompose organic matter in the soil, promote the transformation of soil organic matter, decompose organic matter into mineral elements that can be used by plants, participate in N2 fixation, and participate in nitrification and denitrification processes. Therefore, its application to the soil helps to activate soil nutrients and promotes crop growth by promoting the absorption and utilization of nutrients by crops.

[0017] 2. The Bacillus haynesii DXGZ2-6 of the present invention and the inoculant prepared using Bacillus haynesii can be applied to plant cultivation, effectively promoting plant growth, while slowing down the decomposition or transfer of chlorophyll in old leaves, thereby improving photosynthesis in plant leaves and delaying plant senescence. In addition, it can also inhibit a variety of plant pathogenic fungi, especially Smut fungus that causes corn smut. Attached Figure Description

[0018] Figure 1 This is a colony diagram of the Bacillus haynesii DXGZ2-6 strain on a plate.

[0019] Figure 2 This is a microscopic image of the Bacillus haynesii DXGZ2-6 strain of the present invention.

[0020] Information on the preservation of biological materials

[0021] The strain information deposited in this application is: Bacillus haynesii DXGZ2-6, classified as Bacillus haynesii, deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, on March 3, 2025, with accession number CGMCC No. 33710. Detailed Implementation

[0022] To make the technical problems, technical solutions and advantages of the present invention clearer, a detailed description will be given below in conjunction with specific embodiments.

[0023] All features disclosed in this specification, or all steps in all disclosed methods or processes, may be combined in any way, except for mutually exclusive features and / or steps.

[0024] Unless otherwise stated, each feature disclosed in this specification (including any appended claims and abstract) is merely one example of a series of equivalent or similar features.

[0025] Example 1

[0026] This embodiment provides a Bacillus haynesii DXGZ2-6 strain, which was isolated from unfertilized sugarcane rhizosphere soil samples taken from G363 National Highway in Changming Township, Daxin County, Chongzuo City. The specific isolation and screening method is as follows:

[0027] (1) Strain isolation and screening

[0028] Soil samples were collected from the rhizosphere of unfertilized sugarcane along the G363 National Highway in Changming Township, Daxin County, Chongzuo City. The samples were placed in sterile bags and brought back to the laboratory, where they were stored at 4℃. 10g of fresh soil sample was weighed and added to 90mL of sterile physiological saline. The mixture was shaken at 180 rpm for 30 min and then incubated at 28℃ for 24 h. After enrichment, the supernatant was diluted to 10⁻⁶. -3 10 -4 10 -5 Three gradients were used, with 100 μL of liquid from each dilution spread onto LB solid medium. After incubation at 35°C for 1 day, single colonies grew. After colony growth, the bacterial strain was purified three times on LB agar plates using the streak method to obtain a single bacterial strain, numbered DXGZ2-6.

[0029] (2) Physiological and biochemical identification of colonies

[0030] Observe the morphology and color of the colonies grown on the surface of LB medium according to Bergey's Manual of Bacteriological Identification (9th Edition). The colony morphology diagram is shown below. Figure 1As shown; young cultures were selected, smeared, Gram-stained, and observed under a microscope for bacterial morphology, size, Gram staining reaction, and the presence, morphology, and attachment position of spores, etc. The microscopic images are shown below. Figure 2 As shown.

[0031] (3) Sequence analysis of the strain's 16S rRNA

[0032] The strain DXGZ2-6 was sent to the Sequencing and Identification Department of Shanghai Sangon Biotech for sequencing identification. The sequencing results were compared and analyzed in the NCBI ribosome database. The 16S rDNA sequence analysis results showed that the strain of this invention had 100% homology with Bacillushaynesi of the genus Bacillus, that is, this strain is Bacillushaynesi.

[0033] The microorganisms in this embodiment were preserved as follows:

[0034] Bacillus haynesii DXGZ2-6, classified as Bacillus haynesii, is deposited at the China General Microbiological Culture Collection Center (CGMCC) at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. The deposit date is March 3, 2025, and the accession number is CGMCC No. 33710.

[0035] Example 2

[0036] The amylase-producing capacity of Bacillus haynesii DXGZ2-6, isolated and identified in Example 1, was determined. The specific steps are as follows:

[0037] Scrape a loopful of Bacillus haynesii DXGZ2-6 bacterial growth from an LB solid plate, inoculate it onto an amylase-specific medium, and incubate at 37°C for 24 hours. Observe whether a lysis zone appears.

[0038] The results showed that Bacillus haynesii DXGZ2-6 can secrete amylase and has a strong ability to decompose starch, with a melting zone diameter of 19.4 mm.

[0039] Example 3

[0040] The cellulase-producing capacity of Bacillus haynesii DXGZ2-6, isolated and identified in Example 1, was determined. The specific steps are as follows:

[0041] Scrape a loopful of Bacillus haynesii DXGZ2-6 bacterial colony from an LB agar plate and inoculate it onto a cellulose detection medium. Incubate at 28°C for 7 days. After 7 days, add 5 mL of 0.2 mg / mL Congo red staining solution to the plate and stain for 1 hour. Discard the Congo red staining solution, then wash with 1 mol / L sodium chloride solution for 1 hour and discard the washing solution. Observe the formation of hydrolysis zones around the colonies; the appearance of hydrolysis zones indicates the production of cellulase.

[0042] The results showed that Bacillus haynesii DXGZ2-6 could secrete cellulase and had a good ability to hydrolyze cellulose, with a hydrolysis zone diameter of 23.3 mm.

[0043] Example 4

[0044] The lignin-producing ability of Bacillus haynesii DXGZ2-6, isolated and identified in Example 1, was determined. The specific steps are as follows:

[0045] Scrape a loopful of Bacillus haynesii DXGZ2-6 bacterial growth from an LB solid plate, inoculate it onto aniline blue medium, and incubate it statically at 35°C for 3 days. Observe whether the aniline blue medium shows any discoloration.

[0046] The results showed that Bacillus haynesii DXGZ2-6 can produce ligninase, and the ligninase activity is strong, with a decolorization zone diameter of up to 28.2 mm.

[0047] Example 5

[0048] The protease-producing ability of Bacillus haynesii DXGZ2-6, which was isolated and identified in Example 1, was determined. The specific steps are as follows:

[0049] Scrape a loopful of Bacillus haynesii DXGZ2-6 bacterial growth from an LB solid plate, inoculate it onto a protease detection medium, and incubate at 28°C for 48 hours. Observe whether a lysis zone appears.

[0050] The results showed that Bacillus haynesii DXGZ2-6 can secrete proteases and has a strong ability to decompose proteins, with a lysis zone diameter of 18.9 mm.

[0051] Example 6

[0052] This embodiment provides a microbial agent, and the specific preparation steps are as follows:

[0053] (1) Bacillus haynesii DXGZ2-6 was activated on LB agar plates, and single colonies were picked and cultured on LB slant agar plates at 28°C to obtain activated strains;

[0054] (2) The activated strain was inoculated into LB liquid medium and cultured at 35°C for 24 hours with stirring to obtain seed culture;

[0055] (3) The seed liquid was inoculated into the fermentation medium at a volume ratio of 1:45 and fermented at 35°C and 170 rpm to obtain the fermentation broth;

[0056] (4) After solid-liquid separation of the fermentation broth, cells, spores and fermentation broth can be obtained;

[0057] (5) Concentrate the fermentation broth to obtain a concentrated solution;

[0058] (6) The above-mentioned bacterial cells, spores and concentrated liquid are mixed to obtain the bacterial agent.

[0059] Example 7

[0060] This embodiment provides a method for using the microbial agent from Example 6 in chili pepper cultivation, as detailed below:

[0061] At the 4-5 leaf stage of the chili pepper, begin root application of a 500-fold dilution of microbial agent, once every 7 days, for a total of 3 treatments, with 200ml of water used per pot each time. The first root application treatment used a 1000-fold dilution of compound fertilizer (N+P2O5+K2O, 20-20-20, Nanning Hanhe Biotechnology Co., Ltd.) as the stock solution, while the second and third treatments used tap water as the stock solution.

[0062] Example 8

[0063] This embodiment provides a method for using the microbial agent from Example 6 in chili pepper cultivation, as detailed below:

[0064] At the 4-5 leaf stage of the chili pepper, begin root application of an 800-fold dilution of microbial agent, once every 7 days, for a total of 3 treatments, each using 200ml of water per pot. The first root application used a 1000-fold dilution of compound fertilizer (N+P2O5+K2O, 20-20-20, Nanning Hanhe Biotechnology Co., Ltd.) as the stock solution, while the second and third treatments used tap water as the stock solution.

[0065] Comparative Example 1

[0066] This comparative example provides a method for growing chili peppers. The difference between this comparative example and Example 7 is that the first treatment in this comparative example only involves root application of compound fertilizer, while the second and third treatments only involve root application of tap water. All other operations are exactly the same.

[0067] Seven days after the third treatment, chili peppers from Examples 7 and 8 and Comparative Example 1 were collected. The following indicators were investigated on the collected chili peppers. The investigation indicators and methods are shown in Table 1, and the investigation results are shown in Table 2.

[0068] Table 1 Survey Indicators and Methods

[0069]

[0070] Table 2. Effects of different treatments on various indicators of chili peppers

[0071]

[0072] As shown in Table 2, the treatments in Examples 7 and 8 significantly increased the plant height, stem diameter, and above-ground and below-ground dry and fresh weight of the chili peppers, indicating that the application of the microbial agent of the present invention can effectively promote the growth of chili peppers. Furthermore, the SPDA values ​​of Examples 7 and 8 were significantly higher than those of Comparative Example 1, indicating that the microbial agent treatment of the present invention can slow down the decomposition or transfer of chlorophyll in older leaves, improve photosynthesis in chili pepper leaves, and delay plant senescence.

[0073] Example 9

[0074] This embodiment provides a method for using the microbial agent from Example 6 in lettuce cultivation, as detailed below:

[0075] When lettuce has 5-6 leaves, begin root application of a 500-fold dilution of microbial agent, once every 7 days, for a total of 3 treatments, using 200ml of water per pot each time. The first root application uses a 1000-fold dilution of compound fertilizer (N+P2O5+K2O, 20-20-20, Nanning Hanhe Biotechnology Co., Ltd.) as the stock solution, while the second and third treatments use tap water as the stock solution.

[0076] Example 10

[0077] This embodiment provides a method for using the microbial agent from Example 6 in lettuce cultivation, as detailed below:

[0078] When lettuce has 5-6 leaves, begin root application of an 800-fold dilution of microbial agent, once every 7 days, for a total of 3 treatments, using 200ml of water per pot each time. The first root application uses a 1000-fold dilution of compound fertilizer (N+P2O5+K2O, 20-20-20, Nanning Hanhe Biotechnology Co., Ltd.) as the stock solution, while the second and third treatments use tap water as the stock solution.

[0079] Comparative Example 2

[0080] This comparative example provides a method for growing lettuce. The difference between this comparative example and Example 10 is that the first treatment in this comparative example only involves root application of compound fertilizer, while the second and third treatments only involve root application of tap water. All other operations are exactly the same.

[0081] Seven days after the third treatment, lettuce samples were collected from Examples 9 and 10 and Comparative Example 2. The collected lettuce was investigated for the following indicators. The investigation indicators and methods are shown in Table 3, and the investigation results are shown in Table 4.

[0082] Table 3 Survey Indicators and Methods

[0083]

[0084] Table 4. Effects of different treatments on various indicators of lettuce

[0085]

[0086] As shown in Table 4, the whole plant dry weight and fresh weight of Examples 9 and 10 were increased to varying degrees compared with Comparative Example 2, especially Example 9, which showed a significant increase. This indicates that the application of the microbial agent of the present invention can effectively promote lettuce growth and increase its yield.

[0087] Example 11

[0088] The ability of Bacillus hainanensis DXGZ2-6 from Example 1 to inhibit smut fungus, which causes corn smut, was determined. The specific steps are as follows:

[0089] Activated *Ustilago maydis*, the causal agent of corn smut, was inoculated in the center of PDA medium. Using a cross-hatching method, 2 μL of activated *Bacillus haynesii* DXGZ2-6 bacterial suspension was inoculated at equidistant points of 2 cm from the pathogen. A control group without *Bacillus haynesii* DXGZ2-6 was used. The medium was incubated at 25℃ for 72 h. After the fungal hyphae in the control group had fully colonized the plate, the colony diameter was measured using calipers (measured twice using the cross-hatching method, and the average value was taken). The inhibition rate was calculated using the following formula:

[0090] Inhibition rate (%) = [(control colony diameter - treated colony diameter) / control colony diameter] × 100%.

[0091] The test results showed that the Bacillus haynesii DXGZ2-6 bacterial suspension had an inhibition rate of 87.43% against the corn smut pathogen, Smut vulgaris.

Claims

1. A type of Bacillus hainanensis ( Bacillus haynesii The application of DXGZ2-6 in inhibiting plant pathogens is characterized by, The plant pathogen is *Smutella smut*, which causes corn smut; the *Bacillus hainanensis* (… Bacillus haynesii The accession number of DXGZ2-6 is CGMCC No.33710.

2. The Bacillus hainanensis according to claim 1 ( Bacillus haynesii The application of DXGZ2-6 in inhibiting plant pathogens is characterized by, The Bacillus hainanensis DXGZ2-6 has the function of producing amylase, cellulase, ligninase and protease.

3. An inoculum containing Bacillus hainanensis DXGZ2-6 as described in claim 1.

4. The application of *Bacillus hainanensis* DXGZ2-6 as described in claim 1 or the inoculum agent as described in claim 3 in promoting plant growth, characterized in that... The plants mentioned are chili peppers and lettuce.

5. The application as described in claim 4, characterized in that, The promotion of plant growth includes increasing plant height, stem diameter, dry weight and / or fresh weight.

6. The application of *Bacillus hainanensis* DXGZ2-6 as described in claim 1 or the bacterial agent as described in claim 3 in slowing down the decomposition or transfer of chlorophyll in plant leaves, characterized in that... The plants mentioned are chili peppers and lettuce.

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