Deep-sea bacillus oceanic strain and application thereof

Abstract

The invention discloses a deep-sea bacillus oceanic strain and application thereof, and belongs to the technical field of agricultural microorganisms and biological prevention and control. According to the invention, a deep-sea bacillus oceanic strain NT-3-5 is obtained through screening and identification, and experiments prove that the deep-sea bacillus oceanic strain NT-3-5 provided by the invention is resistant to salt and alkali, and can grow in an environment of 12% NaCl and pH 9; in addition, the strain NT-3-5 can obviously inhibit various plant fungal pathogenic bacteria and plant bacterial pathogenic bacteria; the potting prevention efficiency of the strain NH-3-5 on pepper southern blight reaches 72.09%. Therefore, the deep-sea bacillus oceanic strain NT-3-5 provided by the invention can be used for preparing products for inhibiting plant fungal pathogenic bacteria and plant bacterial pathogenic bacteria and preventing and treating plant diseases, 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 deep-sea oceanic Bacillus strain and its applications. Background Technology

[0002] Soil salinization is a core stressor problem hindering the sustainable development of global agriculture. As a typical extreme habitat, saline-alkali land suffers from the dual stress of high salt ion toxicity and high pH, ​​which directly disrupts the rhizosphere microecological balance, disrupts cell osmotic balance and ion homeostasis, inhibits root nutrient absorption and normal plant growth and development, and consequently leads to a sharp decline in crop yield. At the same time, saline-alkali stress reduces the plant's own disease resistance and stress tolerance, exacerbates the occurrence and spread of soil-borne fungal diseases such as Fusarium and Pythium, forming a dual hazard of "salt-alkali stress + disease infection". Compared with ordinary arable land, the large-scale development and utilization of saline-alkali land for crop cultivation has irreplaceable strategic significance: it can revitalize a large amount of idle and inefficient land resources, effectively expand arable land, and strengthen the national food security barrier, while also improving regional soil ecology and curbing further soil salinization and degradation, achieving a win-win situation for both ecological and economic benefits. The special extreme environment of saline-alkali land also places far greater demands on crop stress resistance and growth promotion, as well as green control of soil-borne fungal diseases than on ordinary soil. Traditional chemical soil conditioners and pesticides are not only prone to failure and reduced effectiveness in high-salt and high-alkali environments, but also cause secondary problems such as secondary soil pollution, increased pathogen resistance, and imbalance of rhizosphere microbial communities, making them unsuitable for the ecological improvement needs of saline-alkali land. In contrast, bio-improvement technology mediated by salt-tolerant functional microorganisms, with its eco-friendly, highly targeted, and sustainable characteristics, can actively adapt to extreme saline-alkali environments and directionally regulate the rhizosphere microecology, making it the core research and application direction in the current field of saline-alkali land agriculture.

[0003] As an important type of plant rhizosphere growth-promoting bacteria, Bacillus possesses the potential for salt and alkali tolerance, antibacterial activity, and growth promotion. It can alleviate plant salt stress through metabolic regulation, adapt to the extreme habitats of saline-alkali lands, and specifically inhibit the secretion of antibacterial substances by common soil-borne fungal pathogens in saline-alkali lands. Furthermore, it can promote crop growth through nitrogen fixation, phosphorus solubilization, and the production of growth hormones, meeting the specific needs of saline-alkali land agriculture. Therefore, screening for high-quality salt-tolerant microbial strains with outstanding salt and alkali tolerance, significant antibacterial activity against soil-borne fungal pathogens specific to saline-alkali lands, stable growth-promoting effects, and strong rhizosphere colonization ability, and developing their applications in saline-alkali soil ecological improvement, biological control of soil-borne fungal diseases in crops, and crop stress resistance, growth promotion, and yield increase, can not only fill the current gap in highly adaptable functional microbial resources for saline-alkali lands but also promote the development of large-scale, green agriculture in saline-alkali lands. This has significant practical implications and application value for achieving efficient utilization of saline-alkali land resources, developing ecologically sustainable agriculture, and ensuring food security. It is also a key research direction urgently needing breakthroughs in the fields of agricultural microbiology and saline-alkali land management. Summary of the Invention

[0004] The purpose of this invention is to provide a deep-sea oceanic Bacillus strain and its applications to solve the problems existing in the prior art. The deep-sea oceanic Bacillus strain NT-3-5 provided by this invention can be used to prepare products that inhibit plant fungal and bacterial pathogens and prevent and control plant diseases, which helps to promote sustainable agricultural development and can greatly reduce environmental pollution, thus having good application prospects.

[0005] To achieve the above objectives, the present invention provides the following solution: This invention provides a strain of deep-sea oceanic Bacillus (Bacillus) Oceanobacillus profundus The deep-sea oceanic Bacillus NH-3-5 was deposited on January 4, 2026 at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No: 67589, located at the Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0006] The present invention also provides the application of the aforementioned deep-sea oceanic Bacillus NH-3-5 in the preparation of microbial agents.

[0007] The present invention also provides a microbial agent comprising the aforementioned deep-sea oceanic Bacillus NH-3-5 or its fermentation broth.

[0008] The present invention also provides the application of the aforementioned deep-sea oceanic Bacillus NH-3-5 or its fermentation broth or the aforementioned microbial agent in inhibiting plant fungal pathogens and plant bacterial pathogens.

[0009] The present invention also provides the application of the aforementioned deep-sea oceanic Bacillus NH-3-5 or its fermentation broth or the aforementioned microbial agent in the preparation of products that inhibit plant fungal pathogens and plant bacterial pathogens.

[0010] Optionally, the plant fungal pathogens include *Sclerotium variegatum*, the causal agent of white mold on pepper (…). Sclerotium rolfsii ), Phytophthora capsici ( Phytophthora capsici Leonian Corn sheath blight fungus ( Rhizoctonia solani ) and / or pepper anthracnose ( Colletotrichum capsici ); The plant bacterial pathogens include *Radix black rot* (… Xanthomonas campestris pv. campestris ), Konjac soft rot bacteria (Erwinia carotovora subsp. carotovora)、 Rice bacterial blight pathogen ( Ralstonia solanacearum ) and / or Bacterium wilt of tomato ( Ralstonia solanacearum ).

[0011] The present invention also provides the application of the aforementioned deep-sea oceanic Bacillus NH-3-5 or its fermentation broth or the aforementioned microbial agent in the prevention and control of plant diseases, including pepper white mold disease.

[0012] The present invention also provides the application of the aforementioned deep-sea oceanic Bacillus NH-3-5 or its fermentation broth or the aforementioned microbial agent in the preparation of products for the prevention and control of plant diseases, including pepper white mold disease.

[0013] The present invention also provides a method for inhibiting plant fungal pathogens and plant bacterial pathogens, comprising the step of treating plant fungal pathogens and plant bacterial pathogens with the fermentation broth of the aforementioned deep-sea oceanic Bacillus NH-3-5; The plant fungal pathogens include *Sclerotium sclerotiorum*, *Phytophthora indicum*, *Phytophthora indicum*, and / or *Anthracnose indicum*. The plant bacterial pathogens include *Radish Black Rot* and *Konjac Soft Rot*. 、 Rice bacterial blight and / or tomato bacterial wilt.

[0014] 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 deep-sea oceanic Bacillus NH-3-5; The plant diseases mentioned include white mold disease of pepper.

[0015] The present invention discloses the following technical effects: This invention obtained a deep-sea oceanic Bacillus strain NT-3-5 through screening and identification. Experimental verification showed that the deep-sea oceanic Bacillus strain NT-3-5 provided by this invention is tolerant of saline-alkali environments and can grow in an environment with 12% NaCl and pH 9. Furthermore, strain NT-3-5 can significantly inhibit plant fungal pathogens (such as *Phytophthora indicum*, ... and *Phytophthora indicum*) and plant bacterial pathogens (such as *Phytophthora indicum*, *Phytophthora indicum*, and 、 The strain NH-3-5 (which inhibits bacterial wilt of rice and bacterial wilt of tomato) achieved a potted plant control efficacy of 72.09% against white mold disease of pepper. Therefore, the deep-sea Bacillus strain NT-3-5 provided by this invention can be used to prepare products that inhibit plant fungal and bacterial pathogens and control plant diseases, contributing to sustainable agricultural development while significantly reducing environmental pollution, and has promising application prospects. Attached Figure Description

[0016] 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.

[0017] Figure 1 The colony morphology of strain NT-3-5 after culturing for 24 h under saline-alkali conditions of 12% NaCl and pH 9.0; Figure 2 A phylogenetic tree for strain NT-3-5 constructed based on the 16S rRNA sequence; Figure 3 The plate confrontation effect of strain NT-3-5 against fungal pathogens: A: *Sclerotium solani* (the causal agent of white mold on pepper); B: *Phytophthora infestans* (the causal agent of Phytophthora infestans on pepper); C: *Rhizoctonia solani* (the causal agent of sheath blight on corn); D: *Anthracnose fungus* (the causal agent of anthracnose on pepper). Figure 4 The plate inhibition effect of strain NT-3-5 against bacterial pathogens; A: Radish black rot fungus; B: Konjac soft rot fungus; C: Rice bacterial blight fungus; D: Tomato bacterial wilt fungus; Figure 5 Pot experiment to control white mold disease of pepper with strain NT-3-5. Detailed Implementation

[0018] 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.

[0019] 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 within the scope of this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0020] 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.

[0021] 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.

[0022] 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.

[0023] Example 1: Isolation, identification and preservation of Bacillus subtilis strain NT-3-5 from the deep sea. (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.

[0024] 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.

[0025] 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.

[0026] LB solid medium (12% NaCl): 5 g yeast extract, 10 g peptone, 120 g sodium chloride, 15 g agar, 1000 mL deionized water, pH 9.0, sterilized at 121℃ for 20 min.

[0027] The inventors collected rhizosphere soil samples from crops in 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 samples were 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.

[0028] (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, were studied according to Bergey's Manual of Bacteriological Identification.

[0029] See results Figure 1 As shown in Table 1, the colonies on LB solid medium are pale yellow, opaque, with a raised center and neat edges; they are Gram-positive, facultative anaerobic, and can utilize various sugars such as starch, sucrose, glucose, and maltose. They can grow in an environment with 12% NaCl and pH 9.0.

[0030] Table 1. Physiological and biochemical characteristics of strain NT-3-5 (3) 16S rRNA sequence identification and phylogenetic tree alignment DNA extracted from the strain was extracted using a boiling method and used as a template. The 16S rRNA gene of strain NT-3-5 was amplified by PCR using the universal primer pair 27F / 1492R. The PCR products were sequenced, and a phylogenetic tree was constructed using MEGA 7.0. The results are as follows: Figure 2 As shown, strain NH-3-5 and Oceanobacillus profundus They gathered together as one.

[0031] Based on the 16S rRNA sequence analysis and considering the colony morphology and physiological and biochemical characteristics of the strain, strain NT-3-5 was identified as *Bacillus subtilis* (a deep-sea marine bacterium). Oceanobacillus profundus The strain, named NH-3-5, was deposited on January 4, 2026, at the Guangdong Provincial Center for Microbial Culture Collection (GDMCC No.: 67589). The deposit address is the Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0032] SEQ ID NO.1 (16S rRNA sequence):

[0033] Example 2: Control effect of deep-sea Bacillus strain NT-3-5 on plant pathogens The pathogens used in this embodiment were all provided by the microbiology laboratory of Xinxiang Jingu Biotechnology Co., Ltd.

[0034] 1. Preparation of fermentation broth (1) Slant seed culture: The cells of strain NT-3-5 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 inoculated into LB 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 medium at an inoculation rate of 1%, ferment at 30℃ and 180 r / min for 18h, and collect the fermentation mixture.

[0035] 2. Control efficacy of strain NT-3-5 against white mold scab 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.

[0036] 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 perimeter. 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 strain NT-3-5 against *Sclerotium oxysporum* var. *pepifolium* was 82.26%. Figure 3 (Table 2).

[0037] 3. Control efficacy of strain NT-3-5 against other crop pathogens. Simultaneously, the effects of strain NT-3-5 on other plant fungal pathogens (such as Phytophthora capsici) were tested. Phytophthora capsici Leonian Corn sheath blight fungus ( Rhizoctonia solani ) and pepper anthracnose bacteria ( Colletotrichum capsici The control effect of strain NT-3-5 on white spot on pepper is as follows: The specific operation and calculation method of inhibition rate are the same as in "2. Control effect of strain NT-3-5 on white spot on pepper". The inhibition effect is as follows: Figure 3 As shown in Table 2, strain NH-3-5 can also significantly inhibit white mold of pepper, late blight of potato, sheath blight of corn and anthracnose of pepper.

[0038] Table 2. The antagonistic effect of strain NT-3-5 against fungal pathogens. 4. Control efficacy of strain NT-3-5 against crop pathogenic bacteria Further testing was conducted to determine the control efficacy of strain NT-3-5 against plant bacterial pathogens. The test method involved introducing the bacterial pathogen (Radish Black Rot Bacterium) into the bacterial culture medium. Xanthomonas campestris pv. campestris ), Konjac soft rot bacteria (Erwinia carotovora subsp. carotovora)、 Rice bacterial blight pathogen ( Ralstonia solanacearum ) and tomato bacterial wilt ( Ralstonia solanacearum The antagonistic effects were observed when the culture was inoculated into LB liquid medium and cultured overnight at 180 r / min. The inoculated medium was then mixed with LB solid medium melted to 40℃ at a ratio of 1:100 to prepare a plate. A well was punched in the center of each LB solid plate, and 50 μL of fermentation broth was added. The plates were incubated at 30℃, and the size of the clear zone was measured. Figure 4 As shown in Table 3, strain NH-3-5 can significantly inhibit black rot of radish, soft rot of konjac, bacterial blight of rice, and bacterial wilt of tomato.

[0039] Table 3. Inhibition zones of strain NT-3-5 against bacterial pathogens. Example 3: Pot experiment on the control of white mold disease in peppers using fermentation broth of strain NT-3-5 The experiment consisted of two treatments (control group and treatment group), with 24 pepper seedlings in each treatment. The treatment group and control group were irrigated with 15 mL of NT-3-5 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 solani*, 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. The experimental results are as follows: Figure 5 See Table 4. The results show that strain NT-3-5 can control white mold disease in peppers, with a control efficacy of 72.09% in potted plants.

[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 = (disease index of CK group - disease index of treatment group) / disease index of CK group × 100%.

[0041] Table 4. Control efficacy of strain NT-3-5 against white mold disease in potted peppers. Note: Different lowercase letters in the table represent significant differences between treatments (P<0.05).

[0042] 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 deep-sea oceanic Bacillus ( Oceanobacillus profundus NH-3-5, characterized in that, The deep-sea oceanic Bacillus NH-3-5 was deposited on January 4, 2026 at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No: 67589, located at the Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

2. The application of the deep-sea oceanic Bacillus NH-3-5 as described in claim 1 in the preparation of microbial inoculants.

3. A microbial inoculant, characterized in that, The microbial agent comprises the deep-sea oceanic Bacillus NH-3-5 or its fermentation broth as described in claim 1.

4. The application of the deep-sea oceanic Bacillus NH-3-5 or its fermentation broth as described in claim 1, or the microbial agent as described in claim 3, in inhibiting plant fungal pathogens and plant bacterial pathogens.

5. The application of the deep-sea oceanic Bacillus NH-3-5 or its fermentation broth as described in claim 1, or the microbial agent as described in claim 3, in the preparation of products that inhibit plant fungal pathogens and plant bacterial pathogens.

6. The application as described in claim 4 or 5, characterized in that, The plant fungal pathogens include *Sclerotium thuringiensis* (pepper white mold). Sclerotium rolfsii ), Phytophthora capsici ( Phytophthora capsici Leonian ), corn sheath blight fungus ( Rhizoctonia solani ) and / or pepper anthracnose ( Colletotrichum capsici ); The plant bacterial pathogens include *Radix black rot* (… Xanthomonas campestris pv. campestris ), Konjac soft rot bacteria (Erwinia carotovora subsp. carotovora), Rice bacterial blight pathogen ( Ralstonia solanacearum ) and / or Bacterium wilt of tomato ( Ralstonia solanacearum ).

7. The application of the deep-sea oceanic Bacillus NH-3-5 or its fermentation broth as described in claim 1, or the microbial agent as described in claim 3, in the prevention and control of plant diseases, characterized in that, The plant diseases mentioned include white mold disease of pepper.

8. The application of the deep-sea oceanic Bacillus NH-3-5 or its fermentation broth as described in claim 1, or the microbial agent as described in claim 3, in the preparation of products for the prevention and control of plant diseases, characterized in that, The plant diseases mentioned include white mold disease of pepper.

9. A method for inhibiting plant fungal pathogens and plant bacterial pathogens, characterized in that, The steps include treating plant fungal pathogens and plant bacterial pathogens with the fermentation broth of Bacillus NH-3-5 as described in claim 1; The plant fungal pathogens include *Sclerotium sclerotiorum*, *Phytophthora indicum*, *Phytophthora indicum*, and / or *Anthracnose indicum*. The plant bacterial pathogens include *Radish Black Rot* and *Konjac Soft Rot*. 、 Rice bacterial blight and / or tomato bacterial wilt.

10. A method for preventing and controlling plant diseases, characterized in that, The step includes irrigating plants with the fermentation broth of Bacillus NH-3-5 from the deep sea as described in claim 1; The plant diseases mentioned include white mold disease of pepper.

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