Bacillus marini E7, biological preparation thereof and application of bacillus marini E7

Abstract

The invention relates to bacillus enteritidis E7 as well as a biological preparation and application thereof. The bacillus haynesii is preserved in the China Center for Type Culture Collection on July 28, 2025, and the preservation number of the bacillus haynesii is CCTCC (China Center for Type Culture Collection) M 20251704. According to the invention, a strain of bacillus enteritidis E7 is separated from corn rhizosphere soil and is comprehensively identified, and the bacillus enteritidis E7 is identified to be bacillus enteritidis. Experiments prove that the bacillus innamolii E7 has nitrogen fixation capacity, after the strain is fermented, the activity of nitrogenase is 1752.5 mu mol / mL / h, absorption of corn plants to nitrogen elements can be promoted, and dependence on exogenous chemical nitrogen fertilizer is reduced; plant growth is promoted; fusarium graminearum and colletotrichum gloeosporioides can be inhibited, and the bacteriostasis rate is greater than 60%, so that various plant fungal diseases are prevented and treated. The invention has important application value in agricultural production.

Description

Technical Field

[0001] This invention relates to the field of biological control technology, and in particular to a Bacillus hainanensis E7, its biological agents, and their applications. Background Technology

[0002] corn( Zeamays L. Corn is one of the world's most important crops, playing an irreplaceable role in food security, economic development, industrial applications, and energy production. As a high-yield crop, corn's growth and development are closely related to nitrogen. Nitrogen is one of the mineral nutrients corn requires in the largest quantity, directly affecting its yield and quality. Nitrogen is not only a core element in the formation of proteins and nucleic acids, but also a key factor in regulating crop growth and improving light energy utilization. To meet corn's high nitrogen requirements, the application of nitrogen fertilizer has been continuously increasing. While this has increased corn yield to some extent, excessive application has also led to a series of serious environmental problems, such as soil structure damage, soil salinization and acidification, reduced biodiversity, and excessive nitrate levels in groundwater. These problems pose a significant threat to the ecological environment and sustainable development. At the same time, over-reliance on chemical fertilizers to maintain high yields depletes soil fertility and reduces agricultural profitability. Therefore, there is an urgent need to develop biological agents with nitrogen-fixing properties to reduce dependence on chemical nitrogen fertilizers, improve soil structure, enhance soil fertility, alleviate environmental pressure, and achieve green and sustainable development in agriculture and society.

[0003] Ear rot and anthracnose are two major fungal diseases affecting maize production. Ear rot primarily damages the maize ear, and the pathogen is *Fusarium graminearum* (grass spp.). Fusarium graminearum Anthracnose multiplies in the corn ear, causing kernel rot, manifested as softening and mold growth at the tip or middle of the ear, resulting in kernel deterioration. A white or pink mold layer is commonly seen on diseased ears. The pathogen can also produce toxic metabolites (such as gibbenone toxins), severely affecting the quality and safety of corn and leading to a significant reduction in yield. Anthracnose is mainly caused by *Colletotrichum gloeosporioides* (…). ColletotrichumgloeosporioidesAnthracnose, commonly found on corn leaves, stems, and ears, is caused by various pathogens. Typical symptoms include circular or irregularly shaped lesions on the leaves, with a brown or black center and a distinct yellow or red border. Severe cases can lead to the death of the corn plant. Anthracnose not only affects corn growth and development but also reduces ear yield, impacting both grain yield and quality. Controlling corn ear rot and anthracnose is crucial for ensuring corn yield and quality. Currently, both diseases are primarily controlled through chemical and biological methods. While chemical control is simple and efficient, overuse can cause severe environmental pollution, lead to pathogen resistance, disrupt ecological balance with long-term use, cause toxicity to non-target organisms, and increase agricultural production costs. Biological control, on the other hand, involves introducing or propagating microorganisms that antagonize pathogens or utilizing their metabolites to inhibit the disease. It is environmentally friendly and less prone to developing resistance. However, in the field of microbial technology, problems remain, such as single-function strains and unstable application effects. Therefore, there is an urgent need to screen for highly active, functionally diverse, and stable strains to promote the vigorous development of the microbial industry.

[0004] The information disclosed in this background section is intended only to enhance the understanding of the background technology of this disclosure and should not be construed as an admission or in any way implying that the information constitutes prior art known to those skilled in the art. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the inventors isolated and purified a strain of Bacillus hainanensis E7 from the rhizosphere soil of corn. Research has shown that it possesses multiple functions, including nitrogen fixation, growth promotion, and prevention of fungal diseases.

[0006] According to one aspect of this disclosure, a type of Bacillus hainanensis ( ) was screened and obtained. Bacillus haynesii E7 was deposited on July 28, 2025 at the China Center for Type Culture Collection (Wuhan University, Wuhan, China, 430072, China) with accession number CCTCC NO: M 20251704.

[0007] According to another aspect of this disclosure, a biological agent containing the said Bacillus hainanensis E7 and / or its metabolic enzyme products is provided.

[0008] According to another aspect of this disclosure, the Bacillus hainanensis E7 or the biological agent is used in at least one of the following or in the preparation of a product having at least one of the following functions: (1) Nitrogen fixation; (2) Promotes corn growth; (3) Inhibits Fusarium graminearum and / or Colletotrichum gloeosporioides; (4) Control of ear rot and / or anthracnose; (5) Improve soil microbial community structure and / or enhance soil fertility.

[0009] The promotion of maize growth includes at least one of the following: increased root length, increased root fresh weight, increased root dry weight, increased leaf length, increased leaf fresh weight, and increased leaf dry weight.

[0010] According to another aspect of this disclosure, a method for preparing the biological agent is provided, comprising the following steps: inoculating the Bacillus hainanensis E7 into TSB liquid culture medium and culturing it with shaking at 25-30°C for 8-10 h to obtain a seed culture; inoculating the obtained seed culture into nitrogen-free liquid culture medium at a volume ratio of 1-5% and culturing it with shaking at 25-30°C for 10-14 h to obtain the biocontrol agent.

[0011] In some of the disclosed embodiments, the TSB liquid culture medium is composed of: 17.0 g / L of tryptone, 5.0 g / L of sodium chloride (NaCl), 3.0 g / L of soybean papain hydrolysate, 2.5 g / L of dipotassium hydrogen phosphate (K2HPO4), and 2.5 g / L of glucose.

[0012] In some of the disclosed embodiments, the nitrogen-free liquid culture medium is composed of: 10.0 g / L glucose or mannitol, 0.2 g / L potassium dihydrogen phosphate (KH2PO4), 0.2 g / L magnesium sulfate (MgSO4), 0.2 g / L sodium chloride, 0.1 g / L calcium sulfate (CaSO4), 5.0 g / L calcium phenolate, and the liquid is pure water with a pH of 7.0.

[0013] One or more technical solutions provided in the embodiments of this application have at least one of the following technical effects or advantages: (1) Nitrogen fixation: Bacillus hainanensis E7 can use nitrogenase (enzyme activity of 1752.5 μmol / mL / h) and ATP as energy to fix nitrogen in the air into available nitrogen, increase the available nitrogen content in the soil, and reduce dependence on exogenous chemical nitrogen fertilizers; (2) Promotes growth: Significantly promotes the growth of corn roots, stems and leaves, enhances the absorption of nitrogen nutrition, thereby improving crop yield and quality; (3) Biological control: Bacillus hainanensis E7 has a significant antagonistic effect on the pathogenic fungi of corn ear rot and anthracnose, which can inhibit the growth of pathogens, induce plant resistance, and achieve effective control of corn fungal diseases; (4) Improve soil: This strain can improve soil microecology and enhance soil aggregate structure and fertility.

[0014] In summary, the Bacillus subtilis E7 strain and its biological agents provided by this invention have multiple functions, including nitrogen fixation, growth promotion, prevention and control of fungal diseases, and soil improvement, thus promoting the green and sustainable development of agricultural production. Attached Figure Description

[0015] Figure 1 This is a streak plate image of Bacillus hainanensis E7 in one embodiment of this application.

[0016] Figure 2 This is an image of Bacillus hainanensis E7 colony in one embodiment of this application.

[0017] Figure 3 Figure A shows the physiological and biochemical analysis results of Bacillus hainanensis E7 in one embodiment of this application; Figure B shows the pH tolerance test results of E7; and Figure C shows the salt tolerance test results of E7.

[0018] Figure 4 This invention illustrates the effect of Bacillus hainanensis E7 on maize growth in one embodiment of this application.

[0019] Figure 5 This invention demonstrates the inhibitory effect of strain E7 on Fusarium graminearum and Anthrax glomeratus in one embodiment of this application. Detailed Implementation

[0020] Unless otherwise specified, the instruments and equipment involved in the following embodiments are all conventional instruments and equipment; unless otherwise specified, the reagents and culture media involved are all commercially available conventional reagents and culture media; unless otherwise specified, the experimental methods involved are all conventional methods.

[0021] To better understand the technical solution of this application, the above technical solution will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0022] Example 1: Screening and Identification of Bacillus hainanensis E7 1. Strain screening: The strain was isolated by Li Ke from maize rhizosphere soil in Anyang, Henan Province, at the Dongchang Experimental Base of the Cotton Research Institute of the Chinese Academy of Agricultural Sciences in August 2023.

[0023] Add 1.0 g of corn rhizosphere soil sample to 9.0 mL of sterile water, add sterile preservative, and vortex for 5 min to obtain the bacterial suspension (the dilution at this point is 10). -1 Take 1.0 mL of this bacterial suspension and add it to 9.0 mL of sterile water and mix thoroughly (the dilution is now 10). -2 ), and prepare 10 in sequence -3 and 10 -4 Diluted bacterial suspensions. Take 10 μL of each... -1 10 -2 10 -3 and 10 -4100 μL of diluted bacterial suspension was sequentially spread onto nitrogen-fixing Assumption medium (Haibo Biotechnology; HB8540) and incubated at 28℃ for 2 days. Single colonies were then picked and transferred to aerobic TSB medium (Haibo Biotechnology; HB4114) and incubated at 28℃ for 2 days. After removing non-target strains based on MALDI-TOF-MS results, the target strain was transferred to 96-well plates. Bacterial suspensions from the 96-well plates were then streaked for purification on aerobic nitrogen-fixing Assumption medium. (See streaking diagram). Figure 1 The colonies were incubated at 28℃ for 2 days. After single colonies grew, the colony morphology was observed to be small in diameter, nearly round, with neat edges without irregular bumps or depressions, smooth and moist surface, slightly glossy, without obvious wrinkles or roughness, and grayish-white and translucent. Figure 2 As shown. Based on the colony morphology of the bacteria growing on the petri dish, single colonies from the third or fourth zone were picked and cultured in aerobic TSB medium for 2 days, and finally identified by PCR.

[0024] 2. Strain Identification: Using the strain's genomic DNA as a template, the 16S rDNA gene was amplified using universal primers 25F and 1492R. PCR and cloning sequencing were then performed on the strain. The obtained 16S rDNA sequence was entered into GenBank for comparison to preliminarily determine the species and genus position of the isolated strain in taxonomy. The results showed that the isolated E7 strain was similar to *Bacillus hainanensis*. Bacillus haynesii (GenBank: OK189716.1) The similarity is close to 99.5%.

[0025] Further physiological and biochemical analyses of the strain were conducted based on indicators such as growth, pH tolerance, and salt tolerance. The results are as follows: Figure 3 As shown.

[0026] First, the E7 strain was subjected to a Gram staining test. The E7 staining result was purple, indicating that it was a positive bacterium.

[0027] The growth pattern of strain E7 within 0–24 h was studied using the optical density method. Strain E7 was inoculated into aerobic TSB medium and cultured at 28℃ and 180 rpm. The growth curve is shown below. Figure 3 As shown in Figure A. The incubation period for strain E7 is 0–10 h, the logarithmic growth phase is 10–16 h, and the stationary phase is 16–24 h. OD 600 It remains around 1.50.

[0028] pH tolerance tests were conducted on strain E7. A 1% inoculum was added to TSB liquid medium at different pH values, and after culturing at 180 rpm for 10 h, the OD value was measured. A blank control (CK) without inoculation was also included. Growth curves were plotted, and the results showed that ( Figure 3B), the E7 strain can grow in the pH range of 4.8 to 11.5, with the optimal pH being between 6.0 and 8.5. That is, the best growth environment for the E7 strain is a neutral to slightly alkaline environment.

[0029] Salt tolerance of strain E7 was tested. 1% inoculum was added to TSB liquid medium with different salt concentrations, and after culturing at 180 rpm for 10 h, the OD value was measured. A blank control (CK) without inoculation was also included. Growth curves were plotted, and the results showed that ( Figure 3 C) The E7 strain exhibited optimal growth at NaCl concentrations < 2 wt%, maintained activity within the NaCl concentration range of 0–5 wt%, and demonstrated a certain degree of salt tolerance.

[0030] Based on the above morphological characteristics, 16S rDNA gene sequence analysis, and physiological and biochemical properties, strain E7 should be classified as *Bacillus hainanensis*. Bacillus haynesii ).

[0031] Example 2: Determination of E7 nitrogenase activity in Bacillus hainanensis Nitrogenase activity of *Bacillus hainanensis* E7 was determined using the acetylene reduction method: the strain was inoculated into nitrogen-free medium and cultured at 28°C for 48 h. Then, acetylene gas (10% of the system volume) was injected into the culture system, and the culture was continued for another 24 h. Ethylene production was then measured using gas chromatography. The results showed that the nitrogenase activity of strain E7 was 1752.5 μmol / mL / h. This indicates that *Bacillus hainanensis* E7 has the ability to fix atmospheric nitrogen into available nitrogen, and the high enzyme activity indicates a strong biochemical ability for nitrogen fixation, which helps plants absorb nitrogen nutrients and thus promotes plant growth.

[0032] Example 3: Experiment on the growth-promoting effect of Bacillus hainanensis E7 on maize The seed culture was inoculated into nitrogen-free medium and cultured at 28°C for 16 h. The OD was then adjusted. 600 =0.6. With 2 mL of the original bacterial suspension (OD... 600 =0.6) The bacterial cell volume after centrifugation was used as the baseline for dilution, 10 -0.7 Bacterial count (diluted 5 times); 10 -1.3 Bacterial count (diluted 20 times); 10 -1.7 The bacterial cell count was determined (diluted 50-fold). Nitrogen-free liquid culture medium was then prepared for use. 10 mL of the above bacterial resuspension at different dilutions and 100 mL of nitrogen-free liquid culture medium were added to each tissue culture flask to prepare a 110 mL mixture, labeled E7-5, E7-20, and E7-50 respectively. The control (CK) only required 110 mL of nitrogen-free liquid culture medium, with other conditions remaining unchanged.

[0033] After sterilization, corn seeds were germinated in sterile water. Experiments were conducted when the corn seedlings reached the stage of two leaves and a central bud. The seedlings were then transplanted into tissue culture bottles and cultured in a greenhouse for 10-15 days (15000 lx light intensity, 25℃ temperature, 70% humidity, 16h light:8h dark cycle). Plants were photographed on the first day after the culture period ended. Figure 4 The root length, fresh weight, dry weight, leaf length, fresh weight, and dry weight of corn seedlings were measured, and the comprehensive results are shown in Table 1.

[0034] Compared with the control group (CK) treated only with nitrogen-free liquid culture medium, maize plants treated with Bacillus hainanensis E7 suspension showed significant increases in root length, root fresh weight, root dry weight, leaf length, leaf fresh weight, and leaf dry weight, indicating that Bacillus hainanensis E7 has a significant growth-promoting effect on maize. Among them, E7-5 showed the best growth-promoting effect in most indicators, with root length increasing by 95.92%, root fresh weight by 50.0%, root dry weight by 66.67%, leaf length by 19.44%, leaf fresh weight by 100.0%, and leaf-root weight by 87.50% compared with the CK group, and all indicators showed significant differences (P<0.05).

[0035] .

[0036] Example 4: Antagonistic effect of Bacillus hainanensis E7 on pathogenic fungi The inhibitory effect of Bacillus hainanensis strain E7 on Fusarium graminearum and Colletotrichum gloeosporioides was determined using the plate confrontation method: pathogenic mycelial blocks were inoculated in the center of PDA medium, and E7 bacterial suspension was inoculated 2 cm from the edge. The medium was incubated at 28°C for 5 days, and the colony diameter was measured. Results are shown below. Figure 5 The results showed that strain E7 exhibited an inhibition rate greater than 60% against *Fusarium graminearum* and *Colletotrichum gloeosporioides* (inhibition rate = antagonistic fungal colony radius / non-antagonistic fungal colony radius), significantly inhibiting the growth of pathogens. These results indicate that *Bacillus hainanensis* E7 possesses strong antagonistic ability against *Fusarium graminearum* and *Colletotrichum gloeosporioides*, and can be used to control fungal diseases (such as those caused by *Fusarium graminearum* and *Colletotrichum gloeosporioides*).

[0037] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this application and are not intended to limit the scope of protection of this application. Although this application has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of this application without departing from the substance and scope of the technical solutions of this application.

Claims

1. A type of Bacillus hainanensis ( Bacillus haynesii E7, characterized in that, The preservation number is CCTCC NO: M20251704.

2. A biological agent, characterized in that, The Bacillus haynesii E7 or / and metabolic enzyme product thereof of claim 1.

3. The Bacillus haynesii E7 of claim 1 or the biological preparation of claim 2 is applied to at least one of the following or to prepare a product with at least one of the following functions: (1) nitrogen fixation; (2) promoting corn growth; (3) inhibiting Fusarium graminearum or / and Glomerella cingulata; (4) preventing ear rot or / and anthracnose; (5) improving soil microbial community structure or / and enhancing soil fertility.

4. Use according to claim 3, characterized in that: The promoting corn growth includes at least one of the following: root length growth, root fresh weight increase, root dry weight increase, leaf length growth, leaf fresh weight increase, and leaf dry weight increase.

5. A method for preparing the biological agent according to claim 2, characterized in that, The method comprises the following steps: S1. Preparing seed liquid: inoculating the Bacillus haynesii E7 of claim 1 into TSB liquid medium, and culturing at 25-30°C for 8-10 h with shaking; S2. Preparing biological preparation: inoculating the seed liquid obtained in S1 into nitrogen-free liquid medium at a volume ratio of 1-5%, and culturing at 25-30°C for 10-16 h with shaking, thereby obtaining the biological preparation.

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