Halophilic bdellovibrio and application thereof

Abstract

The invention relates to the field of microbial technology and biological prevention and control of aquaculture diseases, in particular to halophilic bdellovibrio and application thereof.A preservation number of the halophilic bdellovibrio is GDMCC No.67013. The strain has a typical bdellovibrio form, the optimum growth salinity is 20-30% o, the growth temperature range is 4-40 DEG C, and the halophilic bdellovibrio can be used for preparing the halophilic bdellovibrio. The invention further discloses a high-density fermentation method of the strain, a microbial agent prepared from the strain and application of the strain or the microbial agent to prevention and control of bacterial diseases caused by the two types of pathogenic bacteria in mariculture. The invention has the outstanding advantages of double effect, broad spectrum and high adaptability, provides a brand new and efficient biological solution for green prevention and control of mariculture diseases, and has wide application prospects.

Description

Technical Field

[0001] This invention relates to the field of microbial technology and biological control of diseases in aquaculture, specifically to a halophilic leech vibrio that has the ability to prey on and lyse various marine aquaculture pathogens such as Vibrio and Aeromonas, and can grow in a wide temperature range of 4-40℃, and its applications. Background Technology

[0002] Marine aquaculture faces a serious threat from bacterial diseases caused by Gram-negative bacteria, with Vibrio spp. and Aeromonas spp. being the two most prevalent pathogens. Vibrio can cause early mortality syndrome / AHPND in shrimp, acute hepatopancreatic necrosis disease (AHPND), and vibrio disease in fish; while Aeromonas causes septicemia, ulcer disease, and furunculosis in marine fish. These two types of pathogens often cause mixed infections, resulting in complex and difficult-to-control diseases and leading to huge economic losses. Currently, control measures mainly rely on antibiotics and chemical disinfectants, but the resulting drug resistance, drug residues, and environmental pollution problems are becoming increasingly prominent, seriously restricting the sustainable development of aquaculture. Therefore, developing a highly efficient, environmentally friendly, and residue-free biological control technology is urgently needed.

[0003] Bdellovibrio is a group of tiny parasitic bacteria that obligately prey on Gram-negative bacteria, often referred to as "living antibiotics." They can invade the periplasmic space of host bacteria, utilize the host cell's resources for growth and reproduction, ultimately leading to the host cell's lysis and death. Utilizing the specific predatory characteristics of Bdellovibrio for biocontrol offers significant advantages, including strong targeting, low risk of developing drug resistance, and environmental friendliness. Halophilic Bdellovibrio is a group of Bdellovibrio species exhibiting halophilic growth characteristics and highly efficient predation capabilities. Therefore, isolating a halophilic Bdellovibrio strain that can adapt to the high-salt environment of seawater and possesses highly efficient predatory activity against major pathogenic Vibrio and Aeromonas bacteria, and developing it into a biocontrol agent suitable for marine aquaculture, has significant industrial application value and market potential. For example, patent document CN118685324A describes an efficient propagation method for halophilic Bdellovibrio and its application. The halophilic Bdellovibrio described in this prior art mainly preys on Vibrio alginolyticus, and its temperature range of adaptation is relatively narrow: the suitable conditions are 28℃, 20℃, and 15℃, which limits the application of halophilic Bdellovibrio in the aquaculture industry. Summary of the Invention

[0004] This invention addresses the shortcomings and deficiencies of existing technologies by proposing a novel halophilic bacteriophage, Bdellovibrio, which possesses highly efficient predation capabilities against pathogenic Vibrio and Aeromonas bacteria and can be used over a wide temperature range, along with its applications.

[0005] This invention achieves its purpose through the following measures:

[0006] A halophilic bacterophage, characterized in that the strain belongs to the genus Halobacteriovorax, named *Halobacteriovorax aquimaris* ZH4, and was deposited on September 22, 2025, at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), located at No. 100, Xianlie Middle Road, Yuexiu District, Guangzhou, Guangdong Province, with accession number GDMCC No. 67013.

[0007] The seawater halophilic bacteriophyte ZH4 described in this invention was obtained from sediments in the coastal waters of Weihai, China, using the seawater agar double-layer plate method with Vibrio alginolyticus as the bait host, and through multiple rounds of "purification-enrichment" cycle separation and purification.

[0008] The *Halobacteriovorax* ZH4 strain described in this invention exhibits the following morphological characteristics: Under transmission electron microscopy, the ZH4 strain displays a typical *Halobacteriovorax* morphology, being rod-shaped with polar flagella. The cell size is approximately 0.3 μm × 0.5–1.0 μm, and the flagella length is approximately 2.6–3.1 μm. Physiological and biochemical characteristics include: strict aerobicness, with an optimal salinity of 20–30‰ and an optimal temperature of 33–37℃, although it can grow in a temperature range of 4–40℃. The optimal pH is 7.0–8.0. These characteristics make it perfectly adapted to typical marine aquaculture environments. Sequencing of its 16S rRNA gene sequence and genome sequence, along with phylogenetic analysis, identified it as belonging to the genus *Halobacteriovorax*. However, its sequence shows significant differences from known type strains (similarity <98.5%), indicating that it is a novel species within the genus *Halobacteriovorax*.

[0009] The sequence listing of the ZH4 strain described in this invention is as follows:

[0010]

[0011] The present invention also provides a method for culturing the marine halophilic bacteriophage Bdellovibrio ZH4 as described above, characterized by comprising the following steps:

[0012] Fermentation was carried out using live bacterial prey as a nutrient source under aerobic conditions with a salinity of 20-30‰, a temperature of 33-37℃, a pH of 7.0-8.0, and the live bacterial prey consisted of Vibrio and Aeromonas.

[0013] The present invention also provides a high-density fermentation method for seawater halophilic bacteriophage Bdellovibrio ZH4 as described above, characterized by comprising the following steps:

[0014] Step 1: Use seawater as the basic culture medium for fermentation;

[0015] Step 2: Using Vibrio alginolyticus cells as a nutrient source, inoculate with ZH4 strain, and ferment continuously for 72 hours at 30℃, 200 rpm, and an aeration rate of 1.0 vvm, with nutrient source added every 24 hours.

[0016] Step 3: After fermentation, collect the cells by centrifugation and resuspend them in artificial seawater.

[0017] This invention also includes an application of the seawater halophilic bacterophage Bdellovibrio ZH4 as described above, characterized in that the seawater halophilic bacterophage Bdellovibrio ZH4 is applied to water for sterilization, wherein the water contains at least one of Vibrio or pathogenic Aeromonas, wherein the Vibrio includes Vibrio harveyi, Vibrio parahaemolyticus, Vibrio alginolyticus, and Vibrio vulnificus, and the pathogenic Aeromonas includes Aeromonas hydrophila and Aeromonas vesiculosus.

[0018] The present invention also proposes a microbial agent, characterized in that it contains the above-mentioned marine halophilic bacteriophage Bdellovibrio, and the dosage form of the microbial agent is liquid, lyophilized powder or microcapsule formulation.

[0019] When the microbial agent of the present invention is in liquid form, the agent contains the bacterial cells collected by centrifugation after fermentation and glycerol as a protectant. The glycerol accounts for 10% of the total mass of the microbial agent, and the number of effective viable bacteria per milliliter of the agent is ≥1×10^10 CFU.

[0020] Compared with existing technologies, this invention has the following beneficial effects: (1) Dual-effect broad spectrum: One strain targets both Vibrio and Aeromonas, the two most important aquatic pathogens, resulting in a wider spectrum of prevention and control and higher application value. (2) High efficiency and specificity: The ZH4 strain can specifically lyse pathogenic Vibrio and Aeromonas, without harming beneficial bacteria and farmed animals, and has high safety. (3) Environmentally friendly: It is a natural biological control method with no chemical residues, no environmental pollution, and no induction of drug resistance. (4) Strong environmental adaptability: Its halophilic characteristics make it particularly suitable for seawater and high-salinity brackish water aquaculture environments. Its wide temperature growth range adapts to multiple seawater aquaculture seasons, solving the key problem of limited application of common Bdellovibrio. (5) Good application effect: It can effectively reduce the number of Vibrio and Aeromonas in aquaculture water and aquatic animals, and significantly improve the survival rate of aquaculture. (6) Huge potential: One agent provides multiple preventions, reducing the cost of using multiple single bacterial agents, providing a new and effective solution to replace antibiotics, and conforming to the development direction of green aquaculture. Attached Figure Description

[0021] Figure 1 This is a photograph of the lysis plaque of the halophilic bacteriophage Bdellovibrio ZH4 in this invention, to show its predatory characteristics.

[0022] Figure 2 This is a transmission electron microscope image of the halophilic bacteriophage Bdellovibrio ZH4 in this invention to show its morphology and polar flagella.

[0023] Figure 3 This is a phylogenetic tree constructed based on the 16S rRNA gene sequence in this invention to show the taxonomic position of strain ZH4.

[0024] Figure 4 The lysate diameters of the halophilic bacteriophage ZH4 cultured at different temperatures for 5 days in this invention are shown to demonstrate its wide growth temperature range.

[0025] Figure 5 This is a lysis curve of strain ZH4 against common aquatic pathogenic Vibrio and Aeromonas in the embodiments of the present invention, to show its high predation efficiency.

[0026] Figure 6 This is a comparative graph showing the changes in the number of Vibrio and Bdellovibrio bacteria in shrimp farming ponds after using ZH4 bacterial agent in an embodiment of the present invention. Detailed Implementation

[0027] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0028] Example 1:

[0029] This example provides a method for culturing the marine halophilic bacteriophage strain ZH4, including the following steps:

[0030] The ZH4 strain was cultured using the seawater double-layer plate method. The lower layer consisted of 1.2% seawater agar, sterilized at 115°C for 30 minutes before being poured into plates for later use. The upper layer consisted of 0.6% seawater agar, sterilized at 115°C for 30 minutes before use. After cooling to approximately 50°C, 20 mL of OD200 was added per 100 mL of agar. 600 ≈1% of the prey bacteria (Vibrio alginolyticus or other prey bacteria). Pour onto a pre-prepared lower plate. Dilute the isolated ZH4 strain to different gradients of 10. -1 10 -2 10 -3 Take 100 μL of each of the different dilution ratios and spread it onto a seawater bilayer plate, or add the Bdellovibrio diluted to the required gradient directly when preparing the upper layer of the bilayer plate. After mixing evenly, pour it onto the prepared lower layer plate and incubate at a constant temperature of 30°C.

[0031] Example 2:

[0032] This example investigated the trapping diet of the marine halophilic bacteriophage Bdellovibrio ZH4 and determined its lysis rate:

[0033] The double-layer plate spotting method and the plate lysis plaque assay were used. Indicator pathogenic Vibrio (such as Vibrio alginolyticus and Aeromonas hydrophila) were mixed with soft agar and poured onto a plate. After solidification, a ZH4 bacterial suspension was spotted in the center of the plate. After incubation at 33°C for 24-48 hours, the diameter of the clear lysis plaques was observed and measured. Simultaneously, in a liquid co-culture system, ZH4 was mixed with the host pathogen at a specific ratio and incubated at 33°C with shaking at 180 rpm. Samples were taken at regular intervals, and the concentration change of the host bacteria was determined using the plate colony counting method.

[0034] The results showed that ZH4 could cause lysate plaques up to 15 mm in diameter against Vibrio alginolyticus. Figure 1 In liquid culture, it can reduce the concentration of various pathogenic host bacteria (Vibrio and Aeromonas) by 90% within 24 hours. Figure 5 ).

[0035] Example 3:

[0036] This example provides a method for high-density fermentation and inoculum preparation of strain ZH4, specifically including the following steps:

[0037] (1) Using seawater as the basic culture medium for fermentation;

[0038] (2) Using Vibrio alginolyticus cells as a nutrient source, inoculate with ZH4 strain, and ferment continuously for 72 hours at 30℃, 200 rpm, and 1.0 vvm aeration rate, with nutrient source added every 24 hours; (3) After fermentation, collect the cells by centrifugation and resuspend them in artificial seawater.

[0039] (4) Add glycerol to a final concentration of 10% as a preservative, mix well, and store at low temperature. The effective viable count per milliliter of bacterial agent is ≥1×10^10 CFU.

[0040] Example 4:

[0041] This example examines the application of the ZH4 bacterial agent obtained in Example 3 in shrimp farming (taking vibriosis as an example):

[0042] Water samples were collected from shrimp ponds infected with Vibrio disease and divided into experimental and control groups. The experimental group received ZH4 bacterial agent at a final concentration of 1×10^3 PFU / mL, while the control group received no agent. Water samples were collected before application and at 6, 12, 24, 30, and 36 hours after application to detect the total number of Vibrio bacteria in the water.

[0043] The results are attached. Figure 6 As shown, the number of Vibrio bacteria in the water of the experimental group decreased by more than 80% after 36 hours, which was significantly lower than that of the control group.

[0044] In summary, the strain provided by this invention has the following characteristics compared with the prior art: (1) The ZH4 strain is a new strain isolated from a high-salinity marine environment, and has genetic novelty. (2) Highly efficient predation diet: The unique feature of the ZH4 strain is that it exhibits highly efficient and specific predation ability against two major categories of aquatic pathogens (Vibrio and Aeromonas), including but not limited to: Vibrio harveyi, Vibrio parahaemolyticus, Vibrio alginolyticus, Vibrio vulnificus, Aeromonas hydrophila, and Aeromonas vesiculosus. Its lysis efficiency against Vibrio alginolyticus and Aeromonas hydrophila can reach more than 90% within 24 hours, which is something that ordinary freshwater Bdellovibrio cannot achieve in a high-salinity environment. (3) Environmental adaptability: The halophilic characteristics of the ZH4 strain (optimal salinity 20-30‰) and the wide growth temperature range (4-40℃) enable it to maintain high activity directly in seawater aquaculture without an adaptation process, overcoming the technical bottleneck of low activity and unstable effect of ordinary Bdellovibrio in seawater applications. (4) Preparation and application of the microbial agent: The ZH4 strain was fermented and cultured with continuous feeding to concentrate the bacterial cells. This was then mixed with an appropriate protectant to prepare a liquid biological control agent. This agent can be applied by whole-pond application, feed addition, or water circulation treatment to prevent and treat bacterial diseases in marine aquaculture animals such as shrimp, fish, and shellfish. It effectively reduces the concentration of pathogenic Vibrio and Aeromonas in the water, ensuring the health of the farmed animals.

Claims

1. A halophilic bacteriophage of Bdellovibrio saltans, characterized in that, The strain is a halophilic bacteriophage of the genus Halobacteriovorax, named Halobacteriovorax aquimaris ZH4, which has been preserved in the Guangdong Microbial Culture Collection Center (GDMCC) on September 22, 2025, at the address of 100, Martyrs Road, Yuexiu District, Guangzhou, Guangdong Province, with the preservation number of GDMCC No. 67013.

2. The culture method of the marine halophilic bacteriophage Bdellovibrio ZH4 according to claim 1, wherein, The method comprises the following steps: The fermentation culture is carried out under the conditions of salinity 20-30‰, temperature 33-37℃, pH 7.0-8.0 and aerobic condition, using live prey as a nutrient source, wherein the live prey is a Vibrio or Aeromonas.

3. A high-density fermentation method for seawater halophilic bacteriophage Bdellovibrio ZH4 as described in claim 1, characterized in that, After obtaining the strain, the following steps are performed: Step 1: using seawater as the fermentation base medium; Step 2: inoculating ZH4 strain with Vibrio alginolyticus as a nutrient source, and continuously culturing for 72 hours at 30℃, 200rpm, 1.0vvm of aeration, and adding the nutrient source every 24 hours; Step 3: after the fermentation is completed, the bacterial cells are collected by centrifugation and resuspended in artificial seawater.

4. The use of the marine halophilic bacteriophage Bdellovibrio ZH4 according to claim 1, characterized in that, The seawater halophilic bacteriophage Halobacteriovorax ZH4 described above is applied to water sterilization, and the water body contains at least any one of Vibrio or pathogenic Aeromonas, wherein the Vibrio includes Vibrio harveyi, Vibrio parahaemolyticus, Vibrio alginolyticus, Vibrio vulnificus, and the pathogenic Aeromonas includes Aeromonas hydrophila and Aeromonas veronii.

5. A microbial inoculant, characterized in that, The microbial agent contains the seawater halophilic bacteriophage Halobacteriovorax as described in claim 1, and the dosage form of the microbial agent is liquid, freeze-dried powder or microcapsule preparation.

6. The microbial inoculant of claim 6, wherein, When the microorganism is in liquid form, the microbial agent contains the bacterial cells collected by centrifugation after the fermentation is completed and glycerol as a protective agent, and the glycerol accounts for 10% of the total mass percentage of the microbial agent, and the effective viable bacterial count in each milliliter of the microbial agent is ≥1×10^10 CFU.

7. Use of the marine halophilic bacteriophage Bdellovibrio of claim 1, characterized in that, The seawater halophilic bacteriophage Halobacteriovorax is used for preparing a product for preventing and / or treating bacterial diseases of marine animals.

8. The use of the marine halophilic bacteriophage N. eurkinae according to claim 7, characterized in that, The marine animals include prawns, fish and shellfish.

9. The use of the marine halophilic bacteriophage N. euri according to claim 7, characterized in that, The application method is whole-pool spraying, feed addition or participation in water circulation treatment. The application method is whole-pool spraying, feed addition or participation in water circulation treatment.

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