Halophilous shorebone bacillus and application thereof

Abstract

The invention discloses halophilus shore and application thereof, and belongs to the technical field of agricultural microorganisms and biological control. According to the invention, the halophilous bacillus strain NH-1-2 is obtained through screening and identification, and experiments prove that the halophilous bacillus strain NH-1-2 provided by the invention is resistant to salt and alkali, and can grow in an environment of 10% NaCl and pH 10; the strain NH-1-2 can significantly inhibit pepper southern blight, phytophthora capsici and colletotrichum capsici, and the potting prevention efficiency of the pepper southern blight reaches 74.66%; meanwhile, the strain NH-1-2 has an excellent growth promoting effect on plants. Therefore, the halophilous bacillus strain NH-1-2 provided by the invention can be used for preparing products for inhibiting the growth of pathogenic bacteria, preventing and treating plant diseases or promoting the growth of plants, is beneficial to promoting the sustainable development of agriculture, can greatly reduce the pollution to the environment, and has a good application prospect.

Description

Technical Field

[0001] This invention relates to the field of agricultural microbiology and biological control technology, and in particular to a strain of Haloxylon ammodendron and its application. Background Technology

[0002] Soil salinization is a major bottleneck facing sustainable agricultural development globally. As an extreme habitat, its high salinity and alkalinity not only damage the rhizosphere microenvironment of plants and inhibit crop growth and development, but also lead to a significant decline in crop yields. Compared with ordinary soils, the development of agriculture on saline-alkali land has special and important significance. It can effectively revitalize idle saline-alkali land resources, expand arable land area, and ensure food security, while also improving the regional ecological environment and curbing soil degradation. At the same time, the special habitat of saline-alkali land has more urgent and specific needs for promoting crop growth and controlling fungal diseases. Traditional chemical control methods are not only difficult to adapt to the extreme environment of saline-alkali land, but also easily cause secondary soil pollution and increased drug resistance of pathogens. In contrast, microbial improvement technology, due to its eco-friendly nature, strong sustainability, and ability to be specifically adapted to the special habitat of saline-alkali land, has become a research hotspot for solving the above problems.

[0003] Existing *Halophilic Bacillus* strains have limitations such as limited salt and alkali tolerance, a narrow spectrum of inhibition (especially poor inhibition of fungal pathogens specific to saline-alkali land), and unstable growth-promoting effects. They are difficult to adapt to complex saline-alkali land environments and cannot meet the comprehensive needs of soil improvement, fungal disease control, and crop yield enhancement in saline-alkali land agriculture.

[0004] Therefore, screening for *Bacillus halophilus* strains with strong salt and alkali tolerance, significant antibacterial activity against fungal pathogens specific to saline-alkali land, and stable growth-promoting effects, and developing their application in saline-alkali land improvement, crop fungal disease control, and growth promotion, is of great practical significance for promoting the resource utilization of saline-alkali land, assisting the high-quality development of saline-alkali land planting industry, and developing green ecological agriculture. It is also an urgent need in the current field of microbial agriculture. Summary of the Invention

[0005] The purpose of this invention is to provide a strain of Halophilic Bacillus simonii and its applications to solve the problems existing in the prior art. The Halophilic Bacillus simonii strain NH-1-2 provided by this invention can be used to prepare products that inhibit the growth of pathogens, prevent and control plant diseases, or promote plant growth, which helps to promote sustainable agricultural development and can greatly reduce environmental pollution, thus having good application prospects.

[0006] To achieve the above objectives, the present invention provides the following solution: This invention provides a strain of Haloxylon ammodendron (Bacillus simulans) Halobacillus litoralisThe *Haloxylon ammodendron* NH-1-2 was deposited on January 4, 2026, at the Guangdong Provincial Center for Microbial Culture Collection (GDMCC No.: 67588), located at the Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0007] The present invention also provides the application of the aforementioned Halophilic Bacillus NH-1-2 in the preparation of microbial inoculants.

[0008] The present invention also provides a microbial agent comprising the aforementioned Halophilic Bacillus NH-1-2 or its fermentation broth.

[0009] This invention also provides the use of the aforementioned *Bacillus halophilus* NH-1-2 or its fermentation broth, or the aforementioned microbial agent, in any of the following: (1) Inhibits the growth of pathogens; (2) Prepare products that inhibit the growth of pathogens; (3) Prevention and control of plant diseases; (4) Prepare products for the prevention and control of plant diseases; (5) Promotes plant growth; (6) Prepare products that promote plant growth; The pathogens include *Sclerotium thuringiensis* (pepper white mold). Sclerotium rolfsii ), Phytophthora capsici ( Phytophthora capsici ) and / or pepper anthracnose ( Colletotrichum capsici ); The plant diseases mentioned include white mold disease of pepper.

[0010] Optionally, promoting plant growth includes increasing one or more of the following: stem width, root length, plant height, fresh weight, and dry weight.

[0011] Optionally, the plant includes chili peppers.

[0012] The present invention also provides a method for inhibiting the growth of pathogens, comprising the step of treating the pathogens with the fermentation broth of the aforementioned Halophilic Bacillus NH-1-2, thereby inhibiting the growth of the pathogens; The pathogens include *Sclerotium sclerotiorum*, *Phytophthora capsici*, and / or *Anthracnose capsici*.

[0013] The present invention also provides a method for preventing and controlling plant diseases, including the step of irrigating plants with the fermentation liquid of the aforementioned Bacillus halophilus NH-1-2; The plant diseases mentioned include white mold disease of pepper.

[0014] The present invention also provides a method for promoting plant growth, comprising the step of irrigating plants with the fermentation broth of the aforementioned Bacillus halophilus NH-1-2.

[0015] Optionally, promoting plant growth includes increasing one or more of the following: stem width, root length, plant height, fresh weight, and dry weight. The plant mentioned includes chili peppers.

[0016] The present invention discloses the following technical effects: This invention obtained a *Bacillus halophilus* strain NH-1-2 through screening and identification. Experimental verification showed that the *Bacillus halophilus* strain NH-1-2 provided by this invention is tolerant of saline-alkali environments and can grow in 10% NaCl and pH 10 environments. Strain NH-1-2 significantly inhibits *Sclerotium wiltii*, *Phytophthora indicum*, and *Anthracnose indicum* of pepper, with inhibition rates exceeding 80% in all cases. The potted plant control efficacy of strain NH-1-2 against *Sclerotium wiltii* of pepper reaches 74.66%. Simultaneously, strain NH-1-2 exhibits excellent growth-promoting effects on plants, increasing stem width, root length, plant height, fresh weight, and dry weight of pepper plants. Therefore, the *Bacillus halophilus* strain NH-1-2 provided by this invention can be used to prepare products that inhibit pathogen growth, control plant diseases, or promote plant growth, contributing to sustainable agricultural development while significantly reducing environmental pollution, and has promising application prospects. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 The colony morphology of strain NH-1-2 after culturing for 24 hours under saline-alkali conditions of 10% NaCl and pH 10.0; Figure 2 A phylogenetic tree for strain NH-1-2 constructed based on the 16S rRNA gene sequence; Figure 3 The plate confrontation effect of strain NH-1-2 against *Phytophthora capsici*, *Anthracnose capsici*, and *White rot capsici* was investigated. Figure 4 The results of a pot experiment on the control of white mold disease in peppers using strain NH-1-2; Figure 5 The results of a pot experiment on the growth-promoting effect of strain NH-1-2 on chili peppers. Detailed Implementation

[0019] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0020] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0021] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0022] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0023] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0024] Example 1: Isolation, identification and preservation of Bacillus halophilus strain NH-1-2 (1) Separation and purification Beef extract peptone medium: 3.0g beef extract, 10.0g peptone, 5.0g NaCl, 15g agar, 1000 mL deionized water, pH 7.0, sterilized at 121℃ for 20 min.

[0025] LB solid medium: 5 g yeast extract, 10 g peptone, 10 g sodium chloride, 15 g agar, 1000 mL deionized water, pH 7.0, sterilized at 121℃ for 20 min.

[0026] LB liquid medium: 5 g yeast extract, 10 g peptone, 10 g sodium chloride, 1000 mL deionized water, pH 7.0, sterilized at 121℃ for 20 min.

[0027] LB solid medium (10% NaCl): 5 g yeast extract, 10 g peptone, 100 g sodium chloride, 15 g agar, 1000 mL deionized water, pH 10.0, sterilized at 121℃ for 20 min.

[0028] The inventor collected plant rhizosphere soil samples from Tuoketuo County, Hohhot, Inner Mongolia Autonomous Region. After air-drying, 10 g of the sample was weighed and dissolved in 90 mL of sterile water to prepare a soil suspension. This suspension was then serially diluted 10-fold with sterile water, and 10 g of the sample was taken from each suspension. -3 and 10 -4 100 μL of soil suspension was evenly spread onto beef extract peptone agar plates. After incubating the plates at 30°C for 48 h, different colonies were picked according to their morphology, color, and size, and purified on new LB agar plates until pure colonies were obtained and numbered for storage.

[0029] (2) Morphological and physiological biochemical identification Pure colony strains were streaked onto LB agar plates and incubated at 30°C for 48 h. The morphology and color of single colonies were then observed under an optical microscope. Physiological and biochemical characteristics of the strains, including Gram staining and starch hydrolysis, were studied according to Bergey's Manual of Bacterial Identification and the Handbook of Common Bacteria.

[0030] The results are shown in Table 1 and... Figure 1 Colonies on LB solid medium are yellow and opaque; spores are short rod-shaped, with a slightly raised surface, neat edges, and a slightly moist appearance. Gram staining and methyl red reaction tests are positive. It can grow in 10% NaCl and pH 10 environments and can utilize maltose, trehalose, D-xylose, D-fructose, D-mannitol, D-glucose, etc.

[0031] Table 1. Physiological and biochemical characteristics of strain NH-1-2 (3) 16S rRNA sequence identification and phylogenetic tree DNA extracted from the strain was extracted using a boiling method and used as a template. The 16S rRNA gene of strain NH-1-2 was amplified by PCR using the universal primer pair 27F / 1492R. The PCR products were sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing, and a phylogenetic tree was constructed using MEGA 7.0. The results are as follows: Figure 2 As shown, strain NH-1-2 and Halobacillus litoralis They gathered together as one.

[0032] Based on the 16S rRNA sequence analysis and considering the colony morphology and physiological and biochemical characteristics of the strain, strain NH-1-2 was identified as *Bacillus halophilus* (Haloxylon ammodendron). Halobacillus litoralis The strain, named NH-1-2, was deposited on January 4, 2026, at the Guangdong Provincial Center for Microbial Culture Collection (GDMCC No.: 67588). The deposit address is the Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0033] SEQ ID NO.1 (16S rRNA sequence): GGAAGCGAGTGGATCCCTTCGGGGTGAAGCTCGTGGAACGAGCGGCGGACGGGTGAGTAACACGTGGGCAACCTACCTGTAAGATCGGAATAACCCCGGGAAACCGGGGCTAATGCCGGGTAATACTTTTCTTCGCATGAAGGAAAGTTGAAAGATGGCTTCTTGCTATCACTTACAGATGGGCCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAACGATGAAGGTCTTCGGATCGTAAAGTTCTGTTGTTAGGGAAGAACAAGTACCGTGCGAATAGAGCGGTACCTTGACGGTACCTAACGAGGAAGCCCCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGCGCGCGCAGGCGGTTCTTTAAGTCTGATGTGAAAGCCCACGGCTCAACCGTGGAAGGTCATTGGAAACTGGGGAACTTGAGGACAGAAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGATATGTGGAGGAACACCAGTGGCGAAGGCGACTCTCTGGTCTGTTTCTGACGCTGAGGTGCGAAAGCGTGGGTAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAGGTGTTAGGGGGCTTCCACCCCTTAGTGCTGAAGTTAACGCATTAAGCACTCCGCCTGGGGAGTACGGCCGCAAGGCTGAAACTCAAA。

[0034] Example 2 Control effect of Bacillus halophilus strain NH-1-2 on plant pathogens The pathogenic bacteria used in this example were all provided by the Microbiology Laboratory of Xinxiang Jingu Biotechnology Co., Ltd.

[0035] 1. Preparation of fermentation broth (1) Slant seed culture: The bacterial cells of strain NH-1-2 were aseptically streaked onto LB solid medium slant and cultured at 30℃ for 24 h to obtain slant seeds; (2) Liquid seed culture: single colonies activated from the slant were picked and inoculated into LB liquid medium and cultured at 30℃ and 180r / min for 24 h to obtain seed culture solution; (3) Fermentation culture: Inoculate the seed culture solution into LB liquid medium at an inoculation rate of 1%, ferment at 30℃ and 180 r / min for 18 h, and collect the fermentation mixture.

[0036] 2. Control effect of strain NH-1-2 on white mold disease of pepper. Potato glucose agar (PDA) medium: 200 g potato, 20 g glucose, 20 g agar, 1000 mL sterile water, pH 7.0±0.1, sterilized at 121℃ for 20 min.

[0037] Inoculate the center of the PDA medium with *Sclerotium variegatum* (the causal agent of pepper white mold). Sclerotium rolfsii Mycelial cakes (d=7 mm) were inoculated with 50 μL of fermentation broth at a distance of 2.5 cm around the cakes. Sterile LB broth plates were used as a negative control. The experiment was repeated three times. The inhibition rate was calculated when the negative control mycelia completely covered the plates. The relative inhibition rate (%) was calculated as: (control group colony diameter - treatment group colony diameter) / (control group colony diameter - 0.7) × 100%. The calculated control efficacy of this bacterium against *Sclerotium solani* (white mold scab) was 82.08%. Figure 3 (Table 2).

[0038] 3. Control efficacy of strain NH-1-2 against other crop pathogens. Simultaneously, the effects of strain NH-1-2 on other plant fungal pathogens (such as Phytophthora capsici) were tested. Phytophthora capsici ), pepper anthracnose bacteria ( Colletotrichum capsici The control effect of strain NH-1-2 on white mold disease of pepper is as follows: The specific operation and calculation method of inhibition rate are the same as in "2. Control effect of strain NH-1-2 on white mold disease of pepper". The inhibition effect is as follows: Figure 3 As shown in Table 2, strain NH-1-2 also significantly inhibited *Phytophthora capsici* and *Anthracnose capsici*.

[0039] Table 2. Antifungal effect of strain NH-1-2 against fungal pathogens. Example 3: Pot experiment on the control of white mold disease in peppers by fermentation broth of strain NH-1-2 The experiment consisted of two treatments (CK1 group and treatment group), each including 18 pepper seedlings of uniform growth. The treatment group and CK1 group were irrigated with 15 mL of NH-1-2 fermentation broth and sterile LB liquid medium, respectively. Five days later, the base of the pepper roots was punctured, and mycelial blocks (d=7 mm) of *Sclerotium oxysporum*, the pathogen of pepper white mold, were inoculated at the wounds and covered with sterile cotton balls to maintain moisture. Disease incidence was recorded 5 days after inoculation. Results are shown below. Figure 4 And Table 3.

[0040] Grading criteria for pepper white mold disease severity levels 0-4: Level 0: Asymptomatic; Level 1: Disease only occurs at the base of the stem; Level 2: The infected part accounts for less than 1 / 3 of the whole plant; Level 3: The infected part accounts for less than 2 / 3 of the whole plant; Level 4: The plant is nearing or has already died. Disease index = ∑ (number of diseased plants at each level × disease level) / (total number of plants investigated × highest disease level) × 100; Control effect = (CK1 disease index - treatment disease index) / CK1 disease index × 100%.

[0041] like Figure 4 As shown in Table 3, strain NH-1-2 has a control efficacy of 74.66% against white mold disease of pepper, indicating that strain NH-1-2 can effectively control white mold disease of pepper.

[0042] Table 3. Potted plant control efficacy of strain NH-1-2 against white mold disease of pepper. Note: Different lowercase letters in the table represent significant differences between treatments (P<0.05).

[0043] Example 4: Growth-promoting effect of fermentation broth of strain NH-1-2 on plants The experiment consisted of two treatments (CK2 group and treatment group), each including 30 pepper seedlings at the 4-leaf stage. The treatment group and CK2 group were irrigated with 10 mL of the bacterial fermentation broth and water every 3 days, respectively. After 25 days, plant morphology was observed. Simultaneously, the plants were removed from the pots, the root surface soil was washed off, and the stem width, root length, and plant height were measured. Fresh weight was measured, and the plants were dried at 50℃ to constant weight before being weighed dry. The growth rate of each growth index was calculated. The growth rate (%) was calculated as (treatment group - CK2 group) / CK2 group × 100%. Results are as follows: Figure 5 As shown in Table 4.

[0044] Table 4. Growth-promoting effects of strain NH-1-2 on plants like Figure 5As shown in Table 4, strain NH-1-2 has excellent growth-promoting effects on plants, significantly increasing stem width, root length, plant height, fresh weight, and dry weight.

[0045] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A strain of *Haloxylon ammodendron* ( Halobacillus litoralis NH-1-2, characterized in that, The *Halophilic Bacillus NH-1-2* was deposited on January 4, 2026, at the Guangdong Provincial Center for Microbial Culture Collection (GDMCC No.: 67588), located at the Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

2. The application of Bacillus halophilus NH-1-2 as described in claim 1 in the preparation of microbial inoculants.

3. A microbial inoculant, characterized in that, The microbial agent comprises the Haloxylon ammodendron NH-1-2 or its fermentation broth as described in claim 1.

4. The use of *Bacillus halophilus* NH-1-2 as described in claim 1 or its fermentation broth, or the microbial agent as described in claim 3, in any of the following: (1) Inhibits the growth of pathogens; (2) Prepare products that inhibit the growth of pathogens; (3) Prevention and control of plant diseases; (4) Prepare products for the prevention and control of plant diseases; (5) Promotes plant growth; (6) Prepare products that promote plant growth; The pathogens include *Sclerotium thuringiensis* (pepper white mold). Sclerotium rolfsii ), Phytophthora capsici ( Phytophthora capsici ) and / or pepper anthracnose ( Colletotrichum capsici ); The plant diseases mentioned include white mold disease of pepper.

5. The application as described in claim 4, characterized in that, The promotion of plant growth includes increasing one or more of the following: stem width, root length, plant height, fresh weight, and dry weight.

6. The application as described in claim 4, characterized in that, The plant mentioned includes chili peppers.

7. A method for inhibiting the growth of pathogenic bacteria, characterized in that, The method includes the step of treating the pathogen with the fermentation broth of Bacillus halophilus NH-1-2 as described in claim 1, thereby inhibiting the growth of the pathogen; The pathogens include *Sclerotium sclerotiorum*, *Phytophthora capsici*, and / or *Anthracnose capsici*.

8. A method for preventing and controlling plant diseases, characterized in that, The step includes irrigating plants with the fermentation broth of Bacillus halophilus NH-1-2 as described in claim 1; The plant diseases mentioned include white mold disease of pepper.

9. A method for promoting plant growth, characterized in that, The step includes irrigating plants with the fermentation broth of Bacillus halophilus NH-1-2 as described in claim 1.

10. The method as described in claim 9, characterized in that, The promotion of plant growth includes increasing one or more of the following: stem width, root length, plant height, fresh weight, and dry weight. The plant mentioned includes chili peppers.

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