Method for controlling fish pathogenic bacteria of phylum bacteroidota

Abstract

Provided is new predatory bacteria for controlling fish disease caused by bacteria of the phylum Bacteroidota, especially fish pathogenic bacteria of the genus Flavobacterium.　The present invention relates to: a method for controlling fish pathogenic bacteria in the phylum Bacteroidota present in fish or fish habitats, wherein the method is characterized in that predatory bacteria or variants thereof that prey upon the fish pathogenic bacteria are applied to the fish or fish habitats; predatory bacteria or variants thereof that prey upon fish bathogenic bacteria of the phylum Bacteroidota; and a composition containing the predatory bacteria or variants thereof.

Description

Methods for controlling fish pathogenic bacteria of the phylum Bacteroideta

[0001] The present invention relates to a method for controlling fish pathogenic bacteria of the phylum Bacteroidota present in fish or fish habitats, and a method for suppressing the occurrence of fish infectious diseases caused by the fish pathogenic bacteria.The present invention also relates to a predatory bacterium or a mutant thereof that preys on fish pathogenic bacteria of the phylum Bacteroidota.

[0002] Stable production through aquaculture is essential for a sustainable and stable supply of fish. In aquaculture ponds, farmed organisms live at high densities and there is little water exchange, so the water tends to become eutrophic due to the excrement and surplus feed of the farmed organisms. In such places, fish diseases occur due to the abnormal proliferation of microbial communities that have adapted to the environment. Once fish diseases occur, they can lead to mass deaths, so it is important to prevent them before they occur.

[0003] The main method of disease control has been the administration of antibiotics through feeding, but there is a strong demand both domestically and internationally for a reduction in the amount of antibiotics used due to concerns about the impact on ecosystems and the emergence of drug-resistant microorganisms. Furthermore, the administration of vaccines to young fish needs to be combined with effective adjuvants to enhance their effectiveness. Against this backdrop, research and development is underway in the fisheries industry, focusing on bacterivorous bacteria (Bdellovibrio and similar microorganisms, also known as BALOs) as an alternative disease control technique to the administration of antibiotics.

[0004] The genus Flavobacterium in the phylum Bacteroideta, along with the genera Vibrio and Aeromonas in the phylum Pseudomonadota (formerly Proteobacteria), are known as the three major pathogenic bacterial genera of fish. Diseases caused by pathogenic bacteria in each genera include bacterial coldwater disease (Flavobacterium psychrophilum), columnaris disease (Flavobacterium columnaris), bacterial gill disease (Fravobacterium branchiophilum), vibriosis (Vibrio anguillarum and Vibrio harveyi), and furunculosis (Aeromonas salmonicida). Most farmed freshwater fish, such as salmon, sweetfish, trout, eels, carp, and goldfish, are affected by at least one of bacterial coldwater disease, columnaris disease, and bacterial gill disease, particularly those caused by Flavobacterium bacteria.

[0005] Previous BALOs research has reported numerous bacteria that prey on fish-pathogenic bacteria in the phylum Pseudomonadota (Non-Patent Documents 1 to 4). For example, Bdellovibrio bacteria prey on Aeromonas hydrophila (Non-Patent Document 1), Bacteriovorax bacteria prey on Vibrio parahaemolyticus (Non-Patent Document 2), Bdellovibrio bacteria prey on Aeromonas hydrophila, Vibrio harveyi, and the like (Non-Patent Document 3), and Bdellovibrio bacteria prey on Aeromonas veronii (Non-Patent Document 4) are known.

[0006] Furthermore, Patent Document 1 also reports a method for treating vibrio infections in seafood using bacteria of the genus Bdellovibrio.

[0007] However, no bacteria have been reported to prey on fish pathogenic bacteria of the phylum Bacteroideta, especially on the genus Flavobacterium.

[0008] Patent No. 2583473

[0009] Cao, H. et al. (2012), Veterinary microbiology, 154(3-4), 413-418Kongrueng, J. et al. (2017), Diseases of Aquatic Organisms, 124(3), 223-232Chu, WH and Zhu, W. (2010), Zoonoses and public health, 57(4), 258-264Yang, H. et al. (2023), Aquaculture, 562, 738741

[0010] Therefore, there has been a need to search for novel predatory bacteria to control fish diseases caused by bacteria of the phylum Bacteroidetes, particularly fish pathogenic bacteria of the genus Flavobacterium, as an alternative to antibiotic administration.

[0011] As a result of extensive research to solve the above-mentioned problems, the present inventors have discovered bacteria that prey on bacteria of the phylum Bacteroideta in soil, and have completed the present invention. Specifically, they have discovered three strains of bacteria of the family Bacteriovoracaceae: strains HFL-1, HFL-10, and HFL-13. They have also found that these strains have the same feeding spectrum and can efficiently control fish-pathogenic bacteria of the phylum Bacteroideta present in fish or fish habitats, and have completed the present invention.

[0012] That is, the present invention provides the following: [1] A method for controlling fish pathogenic bacteria of the phylum Bacteroida present in fish or fish habitats, the method comprising applying to the fish or fish habitat a predatory bacterium or a mutant thereof that preys on the fish pathogenic bacteria. [2] A method for suppressing the occurrence of fish infectious diseases caused by fish pathogenic bacteria of the phylum Bacteroida present in fish or fish habitats, the method comprising applying to the fish or fish habitat a predatory bacterium or a mutant thereof that preys on the fish pathogenic bacteria. [3] The method according to [1] or [2], wherein the predatory bacterium or mutant thereof is a strain or mutant thereof belonging to the family Bacteriovoracaceae. [4] The method according to any one of [1] to [3], wherein the base sequence of the 16S rRNA gene contained in the predatory bacterium or its mutant has: (i) a sequence identity of more than 94.8% to the base sequence of the 16S rRNA gene represented by SEQ ID NO: 6; (ii) a sequence identity of more than 94.7% to the base sequence of the 16S rRNA gene represented by SEQ ID NO: 7; or (iii) a sequence identity of more than 96.1% to the base sequence of the 16S rRNA gene represented by SEQ ID NO: 8. [5] The method according to any one of [1] to [4], wherein the predatory bacterium or a mutant thereof is a Bacteriovoracaceae family bacterium strain HFL-1 (deposit number NITE ABP-03906), a Bacteriovoracaceae family bacterium strain HFL-10 (deposit number NITE ABP-03907), a Bacteriovoracaceae family bacterium strain HFL-13 (deposit number NITE ABP-03908), or a mutant thereof. [6] The method according to any one of [1] to [5], wherein the predatory bacterium or a mutant thereof comprises a 16S rRNA gene having a sequence identity of more than 97.9% to the consensus base sequence of the 16S rRNA gene represented by SEQ ID NO: 15. [7] The method according to any one of [1] to [6], wherein the fish pathogenic bacterium belongs to the class Flavobacteria.[8] The method according to any one of [1] to [7], wherein the fish pathogenic bacterium belongs to the family Flavobacteriaceae or Weeksellaceae. [9] The method according to any one of [1] to [8], wherein the fish pathogenic bacterium is selected from the group consisting of bacteria of the genus Flavobacterium, bacteria of the genus Tenacibaculum, bacteria of the genus Elizabethkingia, and bacteria of the genus Chryseobacterium.

[10] The method according to [9], wherein the bacterium of the genus Flavobacterium is Flavobacterium psychrophilum, Flavobacterium columnare, or Flavobacterium branchiophilum.

[11] The method according to any one of [2] and [3] to

[10] , wherein the fish infectious disease is bacterial coldwater disease caused by Flavobacterium psychrophilum, columnaris disease caused by Flavobacterium columnare, or bacterial gill disease caused by Flavobacterium branchiophilum.

[12] The method according to any one of [1] to

[11] , wherein the application is carried out at a temperature of 0 to 38° C.

[13] A predatory bacterium or a mutant thereof that preys on fish pathogenic bacteria of the phylum Bacteroidota.

[14] The predatory bacterium or a mutant thereof according to

[13] , wherein the predatory bacterium or a mutant thereof is a strain or a mutant thereof belonging to the family Bacteriovoracaceae.

[15] The predatory bacterium or mutant thereof according to

[13] or

[14] , wherein the predatory bacterium or mutant thereof comprises a 16S rRNA gene having greater than 97.9% sequence identity to the consensus nucleotide sequence of the 16S rRNA gene represented by SEQ ID NO: 15.

[16] The predatory bacterium or mutant thereof according to any one of

[13] to

[15] , wherein the nucleotide sequence of the 16S rRNA gene comprised by the predatory bacterium or mutant thereof (i) has greater than 94.8% sequence identity to the nucleotide sequence of the 16S rRNA gene represented by SEQ ID NO: 6, (ii) has greater than 94.7% sequence identity to the nucleotide sequence of the 16S rRNA gene represented by SEQ ID NO: 7, or (iii) has greater than 96.1% sequence identity to the nucleotide sequence of the 16S rRNA gene represented by SEQ ID NO: 8.

[17] The predatory bacterium or mutant thereof according to any one of

[13] to

[16] , wherein the predatory bacterium or mutant thereof is a Bacteriovoracaceae family bacterium HFL-1 strain (deposit number NITE ABP-03906), a Bacteriovoracaceae family bacterium HFL-10 strain (deposit number NITE ABP-03907), a Bacteriovoracaceae family bacterium HFL-13 strain (deposit number NITE ABP-03908), or a mutant thereof.

[18] The predatory bacterium or mutant thereof according to any one of

[13] to

[17] , wherein the fish pathogenic bacterium belongs to the class Flavobacteria.

[19] The predatory bacterium or mutant thereof according to any one of

[13] to

[18] , wherein the fish pathogenic bacterium belongs to the family Flavobacteriaceae or Weeksellaceae.

[20] The predatory bacterium or mutant thereof according to any one of

[13] to

[19] , wherein the fish pathogenic bacterium is selected from the group consisting of bacteria of the genus Flavobacterium, bacteria of the genus Tenacibaculum, bacteria of the genus Elizabethkingia, and bacteria of the genus Chryseobacterium.

[21] The predatory bacterium or mutant thereof according to

[20] , wherein the bacterium of the genus Flavobacterium is Flavobacterium psychrophilum, Flavobacterium columnare, or Flavobacterium branchiophilum.

[22] A composition for controlling fish pathogenic bacteria of the phylum Bacteroidota or for suppressing the occurrence of infectious diseases in fish caused by the fish pathogenic bacteria, comprising the predatory bacterium or mutant thereof according to any one of

[13] to

[21] .

[0013] According to the present invention, a strain of bacteria belonging to the Bacteriovoracaseaceae family or a mutant thereof can efficiently control fish-pathogenic bacteria of the phylum Bacteroidetes present in fish or fish habitats.

[0014] Figure 1 shows the predatory activity of strains HFL-1, HFL-10, and HFL-13 against Flavobacterium psychrophilum NBRC100250 (a bacterial coldwater disease bacterium) over time at 28°C (A), 18°C ​​(B), and 10°C (C). The control shows HEPES buffer without predatory bacteria. Figure 2 shows the predatory activity of strains HFL-1, HFL-10, and HFL-13 against Flavobacterium columnarum NBRC100251 (a columnaris disease bacterium) over time at 28°C (A), 18°C ​​(B), and 10°C (C). The control shows HEPES buffer without predatory bacteria. 3 shows the predatory activity of strains HFL-1, HFL-10, and HFL-13 against Flavobacterium branchiophyllum NBRC15030 (a bacterial gill disease bacterium) over time at temperatures of 28° C. (A), 18° C. (B), and 10° C. (C). The control is HEPES buffer without inoculation of predatory bacteria.

[0015] The present invention will be described in detail below with reference to specific embodiments. However, the present invention is not limited to the following embodiments and can be practiced with appropriate modifications.

[0016] A first embodiment of the present invention is a method for controlling fish pathogenic bacteria of the phylum Bacteroideta present in fish or fish habitats, characterized in that a predatory bacterium or a mutant thereof that preys on the fish pathogenic bacteria is applied to the fish or fish habitat. In the present invention, "present in fish" means present in the body of a fish via the fish's body surface or food. In the present invention, "predatory bacteria" refers to "obligate predatory bacteria" that, upon collision with a host bacterium of the phylum Bacteroideta, invade the cell and simultaneously cause the host cell to die. The invaded predatory bacteria grow sufficiently within the host cell, self-multiply, and then destroy the host cell and are released outside the cell. The "predatory bacteria" of the present invention may be any bacteria capable of preying on bacteria of the phylum Bacteroideta, and may further be any bacteria that belong to the family Bacteriovoracaseae and are capable of preying on bacteria of the phylum Bacteroideta. The predatory bacteria of the present invention do not prey on bacteria of the phylum Pseudomonadota, Actinomycetota (formerly Actinobacteriota), or Bacillota (formerly Firmicutes), as described in Example 2 below. Bacteria known as "facultative predatory bacteria" are also sometimes called predatory bacteria, but facultative predatory bacteria are nutritionally dependent and use lytic enzymes or the like to lyse other microorganisms and then absorb the decomposition products, and are therefore clearly distinguishable from the predatory bacteria of the present invention.

[0017] Specifically, three strains of predatory bacteria of the present invention, HFL-1, HFL-10, and HFL-13, which prey on Flavobacterium sp. GSB-24, were isolated from soil. As used herein, "soil" generally refers to a mixture of naturally occurring, solid inorganic matter such as minerals, rocks, and sand; organic matter such as biological products, excrement, corpses, and humic substances; natural liquids such as petroleum, petroleum precursors, petroleum spring water, mineral spring water, hot spring water, river water, lake water, and seawater; and living organisms (living organisms). Hereinafter, the predatory bacteria of the present invention will be referred to as "bacteria of the present invention" as appropriate, and when referring to a specific strain, it will be referred to as "strain of the present invention." Furthermore, the term "bacteria" of the present invention may sometimes mean "bacteria or a mutant thereof," and the term "strain" of the present invention may sometimes mean "strain or a mutant thereof."

[0018] The strain of the present invention can be isolated by a conventional method (see, for example, Jurkevitch, E. (2012). Current protocols in microbiology, 26(1), 7B-1). Specifically, soil is suspended in a washing solution, centrifuged, and the supernatant is collected. This supernatant is further centrifuged to collect a solid, and the solid suspension is used to detect plaques (bacterial plaques) by the double plate method.

[0019] Regarding the phylogenetic properties of the strains of the present invention, identity analysis of the nucleotide sequence of the 16S rRNA gene (EZBioCloud Microbiome Taxonomic Profiling, CJ Bioscience) was performed. As a result, it was found that the taxonomic type strain closely related to the HFL-1, HFL-10, and HFL-13 strains is the Perezibacter stari A3.12 strain (accession number AF084852) belonging to the Bacteriovoracaseaceae family, and that the nucleotide sequence of the 16S rRNA gene of the A3.12 strain (SEQ ID NO: 5) has 93.6%, 93.4%, and 95.7% identities to the 16S rRNA gene sequences of the HFL-1, HFL-10, and HFL-13 strains (SEQ ID NOs: 6, 7, and 8, respectively), respectively. Therefore, the HFL-1, HFL-10, and HFL-13 strains were all identified as novel strains belonging to the family Bacteriovoracaseaceae. In addition, identity analysis of the base sequence of the 16S rRNA gene using the BLAST (Basic Local Alignment Search Tool) program revealed that a known bacterial species closely related to the HFL-1, HFL-10, and HFL-13 strains is uncultured bacterium SSmCB08-70 (accession number AB176231), and the base sequence of the 16S rRNA gene of SSmCB08-70 (SEQ ID NO: 9) is the base sequence of the 16S rRNA gene of the HFL-1, HFL-10, and HFL-13 strains (represented by SEQ ID NOs: 6, 7, and 8, respectively). It was found to have at least 94.7% base sequence identity. Specifically, the base sequence of the 16S rRNA gene of SSmCB08-70 has 94.8%, 94.7%, and 96.1% base sequence identity with the base sequences of the 16S rRNA genes of the HFL-1, HFL-10, and HFL-13 strains, respectively. In the present invention, "sequence identity" refers to a value that can be calculated by appropriately aligning at least two sequences to be compared, determining the identical bases present in each sequence, determining the number of matching sites, and then dividing the number of matching sites by the total number of bases in the sequence region to be compared and multiplying the obtained value by 100.Specifically, such sequence identity can be calculated using, for example, phylogenetic analysis software (EZBioCloud Microbiome Taxonomic Profiling) or the BLAST (Basic Local Alignment Search Tool) program (Altschul et al., J. Mol. Biol., (1990), 215(3):403-10).

[0020] The present inventors applied to deposit the above three strains at the Patent Microorganism Deposit Center, Biotechnology Center, National Institute of Technology and Evaluation (Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture), and the above three strains were deposited on June 13, 2023 under the following accession numbers (notification date: July 27, 2023) and are currently preserved at the institution. (1) HFL-1 strain (Accession number NITE P-03906) (2) HFL-10 strain (Accession number NITE P-03907) (3) HFL-13 strain (Accession number NITE P-03908) Subsequently, the above three strains were submitted to the above-mentioned Patent Microorganisms Depositary Center, and an application for transfer from domestic deposit (original deposit date: June 13, 2023) to deposit under the Budapest Treaty was submitted. Viability was verified, and the application for transfer to deposit (international deposit) under the Budapest Treaty was received under the following receipt number (received date: August 23, 2024). (1) HFL-1 strain (received number NITE ABP-03906) (2) HFL-10 strain (received number NITE ABP-03907) (3) HFL-13 strain (received number NITE ABP-03908)

[0021] The bacterial strain of the present invention may be a viable bacterial cell, and a culture solution containing the viable bacterial cell may also be preferably used. The viable bacterial cell may be in a form that allows it to survive and be active on the surface of the fish body, inside the fish, or in the fish habitat to which it is applied.

[0022] The "mutant" of the HFL-1, HFL-10, or HFL-13 strain of the present invention may be any mutant derived from (a) the strain of the present invention, as long as it has the effect of preying on fish pathogenic bacteria of the phylum Bacteroides. Such mutants include strains generated by spontaneous mutations that occur in nature, including culturing and subculture of strains; strains created by artificial mutations induced by exposure to radiation, ultraviolet rays, X-rays, chemicals, etc.; strains created by genetic recombination; and strains created by genome editing. Furthermore, the "mutant" of the strain of the present invention also includes (b) any strain not derived from the strain of the present invention that is highly similar to the strain of the present invention and has predatory activity. The mutant of the present invention preferably has a 16S rRNA gene nucleotide sequence that has greater than 94.8%, greater than 94.7%, and greater than 96.1% sequence identity to the 16S rRNA gene nucleotide sequences represented by SEQ ID NOs: 6, 7, and 8, respectively. Specifically, the base sequence of the 16S rRNA gene of the mutant of the present invention has (i) greater than 94.8% sequence identity to the base sequence of the 16S rRNA gene represented by SEQ ID NO: 6, (ii) greater than 94.7% sequence identity to the base sequence of the 16S rRNA gene represented by SEQ ID NO: 7, or (iii) greater than 96.1% sequence identity to the base sequence of the 16S rRNA gene represented by SEQ ID NO: 8. The mutant of the present invention more preferably has greater than 97.9% sequence identity to the consensus base sequence (SEQ ID NO: 15) of the 16S rRNA genes of the HFL-1, HFL-10, and HFL-13 strains. As used herein, the term "consensus base sequence" refers to a calculated sequence representing the most frequently occurring nucleotide residue at each position in the base sequence of the 16S rRNA gene of each of the HFL-1, HFL-10, and HFL-13 strains. Typically, consensus sequences are determined by sequence alignment, where similar sequences are compared to each other and similar sequence motifs are calculated (see Example 3 below for details).

[0023] The base sequence of the genes in the entire genome of each of the mutants of the HFL-1 strain, HFL-10 strain, and HFL-13 strain of the present invention is preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 96.2%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably at least 99.1%, more preferably at least It has 99.2%, more preferably at least 99.3%, more preferably at least 99.4%, more preferably at least 99.5%, more preferably at least 99.6%, more preferably at least 99.7%, more preferably at least 99.9%, more preferably at least 99.91%, more preferably at least 99.92%, more preferably at least 99.93%, more preferably at least 99.94%, at least 99.95%, more preferably at least 99.96%, more preferably at least 99.97%, more preferably at least 99.98%, and even more preferably at least 99.99% sequence identity.

[0024] In the present invention, "fish" includes, for example, saltwater fish, freshwater fish, as well as crustaceans, shellfish, echinoderms, mollusks, amphibians, etc. Specific examples include saltwater fish such as salmon, trout, yellowtail, yellowtail, red sea bream, amberjack, pufferfish, flounder, bluefin tuna, striped jack, horse mackerel, yellowtail amberjack, stonefish, filefish, rockfish, scorpionfish, black porgy, rockfish, sea bass, crimson sea bream, grunt, mejina, black sea bream, chub mackerel, grouper, grouper, and coho salmon, and freshwater fish such as carp, rainbow trout, eel, sweetfish, dace, goldfish, and medaka. Crustaceans such as whiteleg shrimp, button shrimp, kuruma prawn, cherry shrimp, white shrimp, sweet shrimp, Nanban shrimp, red shrimp, grape shrimp, black shrimp, Pansian shrimp, spiny lobster, fan shrimp, slipper shrimp, elephant shrimp, mitten crab, snow crab, red snow crab, spider crab, king crab, blue crab, hairy crab, Hanasaki crab, and blue crab. Mega iawabi (sea balone), black abalone, scallops, clams, short-necked clams, blood shells, turban shells, Pacific oysters, rock oysters, Japanese oysters, Suminoe oysters, giant oysters, Portuguese oysters, European flat oysters, Shikamegaku oysters, pearl oysters, pea shells, cockles, stone clams, pearl oysters, pen shells, and echinoderms such as purple sea urchins, red sea urchins, Ezo bafun sea urchins, Chilean sea urchins, and mandarin sea urchins. Examples of mollusks include cuttlefish, Japanese common cuttlefish, Pacific common cuttlefish, Japanese pacific octopus, spear squid, common flathead squid, bigfin reef squid, common octopus, Pacific giant octopus, Japanese ...

[0025] Examples of fish pathogenic bacteria of the phylum Bacteroides include bacteria of the genus Flavobacterium, bacteria of the genus Tenacibacterium, bacteria of the genus Chryseobacterium, which belong to the family Flavobacteriaceae of the class Flavobacteria, or bacteria of the genus Elizabethkingia, which belong to the family Weechsellaceae of the class Flavobacteria.

[0026] Examples of bacteria of the genus Flavobacterium include Flavobacterium psychrophilum, Flavobacterium columnare, and Flavobacterium branchiophyllum. Examples of bacteria of the genus Tenacivaculum include Tenacivaculum maritimum (Sci Rep. 2021 May 4; 11(1):9453. doi: 10.1038 / s41598-021-88672-z). Examples of bacteria of the genus Chryseobacterium include Chryseobacterium balustinum, Chryseobacterium scophtalmum, Chryseobacterium joostei, Chryseobacterium piscicola, Chryseobacterium chaponense, Chryseobacterium aquaticum, Chryseobacterium daecheongense, Chryseobacterium indologenes, and Chryseobacterium biscerum. viscerum), Chryseobacterium gleum, Chryseobacterium oncorhynchi, Chryseobacterium aahli, Chryseobacterium siluri, etc. (BMC Veterinary Research 8, Article number: 77 (2012)).Examples of Elizabethkingia bacteria include Elizabethkingia miricola (Front. Cell. Infect. Microbiol., 14 March 2023; Fish & Shellfish Immunology Volume 130, November 2022, Pages 93-102).

[0027] The "fish infectious disease" in the present invention is not particularly limited as long as it is an infectious disease caused by a fish pathogenic bacterium of the phylum Bacteroides. Examples include bacterial coldwater disease caused by Flavobacterium psychrophilum and occurring in salmonid fish and sweetfish, columnaris disease caused by Flavobacterium columnare and occurring in carp, crucian carp, eel, and goldfish, bacterial gill disease caused by Flavobacterium branchiophilum and occurring in salmonid fish, and fish skin disease caused by Tenacibacillus maritimum.

[0028] In the present invention, "application" can be performed directly on fish or in fish habitats. Methods of application to fish include, for example, spraying on the surface of the fish's body or injecting into the fish's body. Methods of injection into the fish's body include mixing with feed and orally administering to the fish. Methods of application to fish habitats include spraying in aquariums, bodies of water, or oceans used as aquaculture sites, or attachment to underwater facilities at aquaculture sites. The application temperature refers to the water temperature at the time of application to fish (excluding injection into the fish's body) and is not particularly limited. For example, a range of about 0°C to about 38°C is preferred, a range of about 8°C to about 30°C is more preferred, and a range of about 10°C to about 28°C is particularly preferred. The optimal water temperature is about 28°C. Some strains of the present invention exhibit sufficient predatory activity even at temperatures as low as 8°C. The temperature at which the agent is injected into the fish's body may also be, for example, about 0°C to about 38°C.

[0029] The predatory bacteria of the present invention may be used in any form as long as they are viable, such as the bacterial cells themselves; a culture solution containing the bacterial cells; a suspension containing the bacterial cells; a concentrate, paste, frozen product, freeze-dried product, or diluted product thereof; etc.

[0030] The application concentration of the predatory bacteria of the present invention varies depending on the type of fish to be applied, the number of fish, the size of the fish, the size of the aquaculture tank, the application time, the amount of wastewater, etc. For example, when applied to fish, the concentration is 1 x 10 in terms of bacterial cell concentration. ２ ~1 x 10 １２ pfu / mL (plaque forming units), preferably 1 x 10 ４ ~1 x 10 １２ pfu / mL, preferably 1 x 10 ６ ~1 x 10 １２ pfu / mL.

[0031] The application amount of the composition of the present invention varies depending on the type of fish to be applied, the number of fish, the size of the fish, the size of the aquaculture tank, the application time, the amount of wastewater, etc. When the composition of the present invention is applied to fish by feeding, it is about 0.01 to 0.1% of the total weight of the feed, and it is applied for a certain period of time, for example, at weekly intervals, more preferably at 3 to 5 day intervals, more preferably at 1 to 2 day intervals.

[0032] In the present invention, "control" refers to the fact that the concentration of fish pathogenic bacteria when the predatory bacteria or mutant thereof of the present invention is applied is lower than the concentration of fish pathogenic bacteria when the predatory bacteria or mutant thereof of the present invention is not applied. Specifically, the predatory bacteria or mutant thereof of the present invention is said to have a control effect on fish pathogenic bacteria of the phylum Bacteroides when the concentration of fish pathogenic bacteria after treatment with the predatory bacteria or mutant thereof of the present invention is less than about 50%, preferably less than about 40%, preferably less than about 30%, preferably less than about 20%, and particularly preferably less than 10% of the concentration of fish pathogenic bacteria in the untreated (control) condition. The concentration of fish pathogenic bacteria after treatment can be measured by methods well known in the art. Examples include colony counting, absorbance using a spectrophotometer, and turbidity.

[0033] A second embodiment of the present invention is a method for suppressing the occurrence of fish infectious diseases caused by fish pathogenic bacteria of the phylum Bacteroides present in fish or fish habitats, the method comprising applying to the fish or fish habitat predatory bacteria or mutants thereof that prey on the fish pathogenic bacteria. In the present invention, "suppressing" the occurrence of fish infectious diseases means that the predatory bacteria or mutants thereof of the present invention exert a predatory action on the bacteria that cause the fish infectious disease, thereby preventing or curing the fish infectious disease caused by the bacteria. Specifically, this means that the incidence of fish infectious diseases in fish cultured in a growth environment containing fish pathogenic bacteria and to which the predatory bacteria or mutants thereof of the present invention have been applied is lower than the incidence of fish infectious diseases in fish cultured under the same conditions except that the predatory bacteria or mutants thereof of the present invention have not been used. Specifically, the predatory bacteria or their mutants of the present invention are said to have the effect of suppressing the occurrence of fish infections caused by fish pathogenic bacteria of the phylum Bacteroides if the incidence of fish infections in fish cultured in a growth environment containing fish pathogenic bacteria and treated with the predatory bacteria or their mutants of the present invention is less than about 50%, preferably less than about 40%, preferably less than about 30%, preferably less than about 20%, and particularly preferably less than 10%, of the incidence of fish infections in fish cultured under the same conditions but without the predatory bacteria or their mutants of the present invention. The degree of suppression of fish infections can be measured by methods well known in the art. Examples include colony counting, absorbance using a spectrophotometer, and turbidity.

[0034] As shown in Example 2 below, the strain of the present invention or its mutant has the effect of preying on fish pathogenic bacteria of the phylum Bacteroides. Therefore, for example, if the strain of the present invention or its mutant is applied to fish or fish habitats under the above-mentioned application conditions, it is expected that fish infectious diseases caused by the pathogenic bacteria will be effectively suppressed (Non-patent Document 4).

[0035] The predatory bacteria or their mutants, the fish or fish habitat, the fish pathogenic bacteria of the phylum Bacteroides, and the fish infectious disease are as described in the first embodiment.

[0036] A third embodiment of the present invention is a composition for controlling fish pathogenic bacteria of the phylum Bacteroides or for inhibiting the occurrence of fish infectious diseases caused by said fish pathogenic bacteria, comprising the predatory bacterium of the present invention or a mutant thereof.

[0037] The predatory bacteria or their mutants, fish pathogenic bacteria of the phylum Bacteroides, and fish infectious diseases, as well as "control" and "suppression" are as described in the first and second embodiments.

[0038] The composition of the present invention can be produced in the form of a solid preparation such as a dust, granule, wettable powder, or tablet; a liquid preparation such as an emulsion, flowable, or oil; or a dry preparation prepared by freeze-drying, by adding a carrier such as a solid carrier or liquid carrier to a culture solution containing the cells of the predatory bacteria, a suspension containing the cells, or a concentrate thereof, as needed.

[0039] The concentration of the predatory bacteria of the present invention contained in the composition of the present invention varies depending on the type of disease, the type of fish to which the composition is applied, the formulation of the composition, etc. For example, in the case of a liquid formulation, the concentration of the predatory bacteria in the liquid formulation is usually 1 x 10 ２ ~1 x 10 １１ pfu / mL (plaque forming units).

[0040] If necessary, the composition may contain carriers and auxiliary materials typically used in agricultural chemicals, such as surfactants, liquidity adjusters (such as pH adjusters), spreaders, wetting agents, stabilizers, drift inhibitors, etc., within the range that does not impair the effect of the predatory bacteria of the present invention in controlling fish pathogenic bacteria. When a carrier or auxiliary material is added to the composition of the present invention, the total concentration of the carrier or auxiliary material used is usually about 1 to 99.9% by weight, preferably about 10 to 99% by weight, based on the total weight of the composition of the present invention.

[0041] The compositions of the present invention can also be used in combination with vaccines derived from fish pathogenic bacteria (e.g., WO2009 / 080767, WO2009 / 056629).

[0042] The present invention will be described in more detail below with reference to examples. However, the present invention is not limited to the following examples and can be practiced with appropriate modifications.

[0043] Example 1 Isolation and cultivation of bacterivorous bacteria 2.5 g of soil (Hyogo Prefecture) was diluted with 22.5 ml of washing solution (6 mM CaCl ２ ・2H ２ 0, 4 mM MgCl ２ ・7H ２ The mixture was added to the washing solution and vigorously shaken for 5 minutes at room temperature. The mixture was centrifuged at 500 x g and 4°C for 5 minutes to obtain the supernatant, which was then centrifuged at approximately 30,000 x g and 4°C for 20 minutes to obtain a pellet. The pellet was suspended in the washing solution and centrifuged at approximately 30,000 x g and 4°C for 20 minutes to obtain a pellet. The pellet was suspended in the washing solution and filtered twice with a 1.0 μm pore size filter. Plaques were detected using the filtrate by the double plate method. In the double plate method, 0.1 mL of the filtrate, 0.25 mL of a suspension of Flavobacterium sp. GSB-24 strain, and 0.25 mL of a soft agar medium (25 mM HEPES, pH 7.4, 6 mM CaCl ２ ・2H ２ 0, 4 mM MgCl ２ ・7H ２ 0, 100-fold diluted tryptic soy broth (0.6% agar concentration) 3 mL, and this was plated on a medium (25 mM HEPES, pH 7.4, 6 mM CaCl ２ ・2H ２ 0, 4 mM MgCl ２ ・7H ２ The plaques were then cut out together with the surrounding food bacteria and placed in a buffer solution (25 mM HEPES, pH 7.4, 4 mM MgCl ). ２ ・7H ２ The suspension was serially diluted and plaque formation was repeated by duplicate plating to isolate the predatory bacteria.

[0044] Example 2 Identification of Isolated Predatory Bacteria and Their Relationship to Known Related Microorganisms The isolated predatory bacteria (strains HFL-1, HFL-10, and HFL-13) were identified as follows. First, genomic DNA was extracted from each strain using an InstaGene DNA Purification Matrix (BIO-RAD). The 16S rRNA gene of each strain was sequenced from 1378 bp to 1422 bp using the following specific amplification primers: Forward primer: 27f (AGAGTTTGATCMTGGCTCAG) (SEQ ID NO: 1) Forward primer: 515f (GTGCCAGCMGCCGCGGTAA) (SEQ ID NO: 2) Reverse primer: 806r (GGACTACHVGGGTWTCTAAT) (SEQ ID NO: 3) Reverse primer: 1492r (TACGGHTACCTTGTTACGACTT) (SEQ ID NO: 4) Phylogenetic analysis was performed by sequencing the 16S rRNA gene (EZBioCloud Microbiome Taxonomic Profiling, CJ Bioscience), and taxonomic type strains closely related to the HFL-1, HFL-10, and HFL-13 strains were investigated. As a result, the 16S sequence of the HFL-1 strain was The base sequence of the rRNA gene (SEQ ID NO: 6) of the 16S rRNA gene of Perezibacter stari A3.12 (accession number AF084852 (https: / / www.ezbiocloud.net)) belonging to the family Bacteriovoracaceae has 93.6% sequence identity with the base sequence of the 16S rRNA gene (SEQ ID NO: 5), the base sequence of the 16S rRNA gene of the HFL-10 strain (SEQ ID NO: 7) of Perezibacter stari A3.12 It had 93.4% sequence identity with the base sequence of the 16S rRNA gene of the HFL-13 strain (SEQ ID NO: 8) of Perezibacter stari A3.12 It had 95.7% sequence identity with the base sequence of the 16S rRNA gene (Table 1). Therefore, strains HFL-1, HFL-10, and HFL-13 were identified as novel strains belonging to the family Bacteriovoracaseaceae.

[0045]

[0046] In addition, the base sequences of the 16S rRNA genes of the HFL-1, HFL-10, and HFL-13 strains belonging to the Bacteriovoracaseaceae family (SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, respectively) were used as query sequences, and identity analysis was performed on all base sequences in the database using the BLAST (Basic Local Alignment Search Tool) program. The obtained results were sorted in descending order by Query cover (the percentage (%) of the result sequence that covers the query sequence), and the results for the five strains with the highest identity values ​​were obtained from among the result sequences whose result sequences were longer than the query sequence, i.e., among the result sequences with 100% Query cover, the results for the five strains with the highest identity values ​​were obtained. It was found that the known bacterial species closely related to the HFL-1, HFL-10, and HFL-13 strains is uncultured bacterium SSmCB08-70 (the 16S rRNA gene has been registered under accession number AB176231), and the base sequence of the 16S rRNA gene of SSmCB08-70 (SEQ ID NO: 9) is the same as the 16S rRNA gene of the HFL-1, HFL-10, and HFL-13 strains. It was found that they had 94.8%, 94.7%, and 96.1% sequence identity with the base sequence of the rRNA gene, respectively (Tables 2, 3, and 4).The second, third, fourth, and fifth most closely related known microorganisms to strain HFL-1 are uncultured delta proteobacterium clone 236 (16S rRNA gene registered under accession number EF188429; the nucleotide sequence of the 16S rRNA gene is shown in SEQ ID NO: 10), uncultured bacterium SSmCB08-8 (16S rRNA gene registered under accession number AB176175; the nucleotide sequence of the 16S rRNA gene is shown in SEQ ID NO: 11), Peredibacter stari A3.12 (16S rRNA gene registered under accession number CP139487; the nucleotide sequence of the 16S rRNA gene is shown in SEQ ID NO: 12), and uncultured bacterium SSmCB08-62 (16S rRNA gene registered under accession number CP139487; the nucleotide sequence of the 16S rRNA gene is shown in SEQ ID NO: 13), respectively. The rRNA gene has been registered under accession number AB176223; the base sequence of the 16S rRNA gene is shown in SEQ ID NO: 13), and the sequence identities to the base sequence of the 16S rRNA gene of the HFL-1 strain were 94.4%, 94.1%, 93.6%, and 93.5%, respectively (Table 2). The second, third, fourth, and fifth most closely related known microorganisms to strain HFL-10 are Uncultured Bacterium clone E18 (16S rRNA gene registered under accession number HG917723; the nucleotide sequence of the 16S rRNA gene is shown in SEQ ID NO: 14), Uncultured Deltaproteobacterium clone 236 (16S rRNA gene registered under accession number EF188429; the nucleotide sequence of the 16S rRNA gene is shown in SEQ ID NO: 10), Uncultured Bacterium SSmCB08-8 (16S rRNA gene registered under accession number AB176175; the nucleotide sequence of the 16S rRNA gene is shown in SEQ ID NO: 11), and Peredibacter salivarius A3.12 (16S rRNA gene registered under accession number AB176175; the nucleotide sequence of the 16S rRNA gene is shown in SEQ ID NO: 12). The rRNA gene has been registered under accession number CP139487; the nucleotide sequence of the 16S rRNA gene is shown in SEQ ID NO: 12), and the nucleotide sequence identities to the 16S rRNA gene sequence of the HFL-10 strain were 94.5%, 94.3%, 94.0%, and 93.4%, respectively (Table 3).The second, third, fourth, and fifth most closely related known microorganisms to strain HFL-13 are Peredibacter staphylococcus aureus A3.12 (the 16S rRNA gene has been registered under accession number CP139487; the nucleotide sequence of the 16S rRNA gene is shown in SEQ ID NO: 12), Uncultured Deltaproteobacterium clone 236 (the 16S rRNA gene has been registered under accession number EF188429; the nucleotide sequence of the 16S rRNA gene is shown in SEQ ID NO: 10), Uncultured Bacterium SSmCB08-8 (the 16S rRNA gene has been registered under accession number AB176175; the nucleotide sequence of the 16S rRNA gene is shown in SEQ ID NO: 11), and Uncultured Bacterium SSmCB08-62 (the 16S rRNA gene has been registered under accession number AB176175; the nucleotide sequence of the 16S rRNA gene is shown in SEQ ID NO: 12). The rRNA gene has been registered under accession number AB176223; the base sequence of the 16S rRNA gene is shown in SEQ ID NO: 13), and the base sequence identities to the base sequence of the 16S rRNA gene of the HFL-13 strain were 95.9%, 95.5%, 95.2%, and 94.7%, respectively (Table 4). Note that the term "uncultivated" means "not derived from cultured bacteria," and the strain has not been isolated as a bacterium; its base sequence is only known through metagenomic analysis or the like.

[0047]

[0048]

[0049]

[0050]

[0051]

[0052]

[0053]

[0054]

[0055]

[0056]

[0057]

[0058]

[0059]

[0060] Example 3: Obtaining a consensus sequence and the relationship between the consensus sequence and known closely related microorganisms As shown in Example 2, the three isolated predatory bacteria strains of the present invention belong to the Bacteriovoracaseaceae family and are closely related, but have been shown to be different bacteria. In order to clarify the relationship between these three strains, we attempted to obtain a sequence common to the 16S rRNA gene base sequences of each strain, i.e., a consensus sequence.

[0061] The 16S rRNA gene sequences of strains HFL-1, HFL-10, and HFL-13 belonging to the Bacteriovoracaseaceae family (SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, respectively) were extracted using a multiple alignment tool (SnapGene (registered trademark), ver. 5.3.3, GSL Biotech LLC) to extract only the nucleotide sequence common to all three 16S rRNA gene sequences. Nucleotide sequences and gap sequences that were not identical among the three strains were designated N, and Ns at both ends were deleted. The resulting nucleotide sequence is hereinafter referred to as the "16S rRNA gene consensus nucleotide sequence." The consensus nucleotide sequence of the 16S rRNA gene was 1,361 bases long and consisted of 1,289 unique bases and 72 Ns that allowed any base (SEQ ID NO: 15).

[0062]

[0063] Next, the obtained consensus base sequence of the 16S rRNA gene was used as the query sequence, and identity analysis was performed on all base sequences in the database using the BLAST (Basic Local Alignment Search Tool) program in the same manner as in Example 2. The obtained results were sorted in descending order by query cover (the percentage (%) of the result sequence that covers the query sequence), and the results for the five strains with the highest identity values ​​were obtained from the result sequences whose result sequences were longer than the query sequence, i.e., among the result sequences with a query cover of 100%, the results were obtained in descending order of identity. Here, in a typical BLAST search algorithm, N in the query sequence allows any base, so if any of the bases A, T, C, or G is present at the corresponding position in the result sequence, it should be determined to be identical, but N is considered to be a different base and the result is output. Therefore, the number representing the apparent identity will be lower. Therefore, in the case of N, a correction was made so that any of the bases A, T, C, and G is considered to be a match. The results are shown in Table 5. The closely related known microorganism having a 16S rRNA gene with the highest identity to the 16S rRNA gene consensus base sequence is uncultured bacterium SSmCB08-70 (the 16S rRNA gene has been registered under accession number AB176231), and the base sequence of the 16S rRNA gene of SSmCB08-70 (SEQ ID NO: 9) was found to have 97.9% base sequence identity to the 16S rRNA gene consensus base sequence.The second, third, fourth, and fifth most closely related known microorganisms to the HFL-1 strain were uncultured delta proteobacterium clone CK06-06_Mud_MAS4B-31n (the 16S rRNA gene has been registered under accession number AB369189; the nucleotide sequence of the 16S rRNA gene is shown in SEQ ID NO: 16), uncultured bacterium clone 236 (SEQ ID NO: 10), SSmCB08-8 (SEQ ID NO: 11), and Peredibacter stari A3.12 (SEQ ID NO: 12), and the nucleotide sequence identities to the consensus nucleotide sequence of the 16S rRNA gene were 97.9%, 97.5%, 97.1%, and 97.0%, respectively (Table 5).

[0064]

[0065] SEQ ID NO: 16 (nucleotide sequence of 16S rRNA gene of uncultured Deltaproteobacterium clone CK06-06_Mud_MAS4B-31 (accession number AB369189)): AACGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAACGTGAAAGTCCTTCGGGATGAGTAAAGTGGC GCACGGGTGAGTAACACGTAGGTAATCTGCCTTTCAGTGGGGAATAACTTCGGGAAACCGGAGCTAATGC CGCATACGTAAGTAATTAGAAAGAAGGCCTCTATTTATAAGCTTTCGCTGAGAGAAGAGCCTGCGTCCTA TTAGCTAGTTGGTAGGGTAAAAGCCTACCAAGGCAACGATAGGTATCCGGTCTGAGAGGATGATCGGACA CACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTAGGGAATATTGCGCAATGGGGGAAA CCCTGACGCAGCAACGCCGCGTGAGTGAGGAAGGTCTTAGGATTGTAAAACTCTTTTATTTGGGAAAAAT GACCATATGGCTAATATCCATGTGGAGTGATGGTACCAAAGGAATAAGCACCGGCTAACTTCGTGCCAGC AGCCGCGGTAATACGAAGGGTGCAAGCGTTGTTCGGAATCATTGGGCGTAAAGCGCGCGCAGGCGGATCA GCAAGTCAGATGTGAAATCTCGAAGCTCAACTTCGAAACTGCGTCTGAAACTGCTAGTCTAGAATGTCGG AGGGGGCAGGGGAATTTCACGTGTAGGGGTAAAATCCGTAGAGATGTGAAGGAACACCGGAGGCGAAGGC GCCTGCCTGGACGACTATTGACGCTGAGGCGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTA GTCCACGCCGTAAACGATGAGCACTAGTTATTGGGGGTATTGACTCCCTCAGTGACGCAGCTAACGCATTAAGTGCTCCGCCTGGGAAGTACGGTCGCAAGACTAAAACTCAAACAAATTGACGGGGGCCCGCACAAGCG GTGGACTATGTGGTTTAATTCGAAGCAACGCGCAGAACCTTACCTAGGCTTGAACTCCTACGAATCCGGG GTAATGCCTGGAGTGTCCGCAAGGAAATGTAGTGAGAGGTGCTGCAAGGCTGTCGTCAGCTCGTGTTGTG AAATGTTGGGTTAAGTCTCGCAACGAGCGCAACCCCTATCGTCTGTTGCCAGCATTAAGTTGGGCACTCT GACGAGACTGCCTGGGTTAACCAGGAGGAAGGTGGGGATGACGTCAAGTCCTCATGGCCCTTATGTCTAG GGCTACACACGTAATACAATGGCGCGTACAGAGGGAAGCGAACCAGCGATGGGGAGCAAATCTCAAAAAG CGCGCCTCAGTTCGGATTGGAGTCTGCAACTCGACTCCATGAAGTTGGAATCGCTAGTAATCGGAGATCA GCACGCTCCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTGGGCTGCAAA AGAAGTGGGTAGTTTAACCTTCGGAGGACGCTCACCACTTTGTGGTTCATGACTGGGGTG

[0066] Example 4 Cultivation of Predatory Bacteria The HFL-1, HFL-10, and HFL-13 strains were grown together with Flavobacterium sp. GSB-24 strain in a buffer solution at 200 rpm and 28°C. After the GSB-24 strain was removed by filtration through a 0.45 μm pore size filter, the resulting mixture was used as an inoculum of HFL-1 strain (approximately 2.8 × 10 ７ PFU / ml), HFL-10 strain inoculum (approximately 3.3 x 10 ７ PFU / ml), and HFL-13 strain inoculum (approximately 2.2 × 10 ７The GSB-24 strain was isolated from the roots of Dendrobium kokuensis using PS-DR2A medium (Nishioka, T and Tamaki, H. (2022), Microbiology Spectrum, 10(6), e02238-22; Nishioka et al. (2023), Microbiology Resource Announcements, 12(3), e01343-22).

[0067] Example 5 Verification of Predation Range The predation range of the HFL-1, HFL-10, and HFL-13 strains was verified using 13 test bacteria: Chitinophaga japonensis NBRC16041 strain, Flexibacter sp. GSC-23 strain, Flavobacterium pectinovorum NBRC15945 strain, Flavobacterium sp. GSB-24 strain, Pedobacter africanus NBRC100065 strain, Caulobacter segnis NBRC15250 strain, Sphingomonas herbicidvorans herbicidovorans strain NBRC16415, Rhizobium radiobacter strain NBRC15193, Paraburkholderia caledonica strain NBRC102488, Pectobacterium carotovorum subsp. carotovorum strain NBRC103133, Pseudomonas citronellolis strain NBRC103043, Rhodococcus erythropolis strain NBRC15567, Bacillus subtilis subsp. The following liquid culture method was carried out using the NBRC13719 strain of L. subtilis in a buffer solution (25 mM HEPES, pH 7.4, 6 mM CaCl ２ ・2H ２ 0, 4 mM MgCl ２ ・7H ２ 600 nm absorbance (OD ６００Each test bacterial strain was suspended to a pH of 0.8 to 1.2, and 4.75 ml of the inoculum containing HFL-1, HFL-10, or HFL-13 strains, or 0.25 ml of buffer solution containing no predatory bacteria (control), was mixed with the inoculum and incubated at 200 rpm and 28°C. After 24 hours of incubation, the absorbance at 600 nm of each inoculum and the control was measured. This experiment was repeated three times. GSC-23 strain was isolated from the roots of Dendrobium kokum using GG-DTS medium (Nishioka, T and Tamaki, H. (2022), Microbiology Spectrum, 10(6), e02238-22). The NBRC16041 strain, NBRC100065 strain, NBRC15250 strain, NBRC16415 strain, NBRC15193 strain, NBRC102488 strain, NBRC103133 strain, NBRC103043 strain, NBRC15567 strain, and NBRC13719 strain were obtained from the NITE Biotechnology Center (NBRC).

[0068] The growth inhibitory effect of the test bacterial strains was evaluated using the following three grades: ∘: The absorbance of the test bacteria after treatment with the strain of the present invention was less than 70% of the absorbance of the test bacteria not treated with predatory bacteria; Δ: The absorbance of the test bacteria after treatment with the strain of the present invention was 70% to 90% of the absorbance of the test bacteria not treated with predatory bacteria; ×: The absorbance of the test bacteria after treatment with the strain of the present invention was more than 90% of the absorbance of the test bacteria not treated with predatory bacteria. All test bacterial strains were graded as either "∘" or "×" (Table 2), and none were graded as "Δ".

[0069]

[0070] As shown in Table 6, strains HFL-1, HFL-10, and HFL-13 all exhibited predatory activity against Chitinophaga japonensis (a member of the Chitinophagia class of the Bacteroideta phylum), Flexibacter sp. (a member of the Cytophagia class), Flavobacterium pectinovorum and Flavobacterium sp. (a member of the Flavobacteria class), and Pedobacter africanus (a member of the Sphingobacteria class), demonstrating their ability to prey on bacteria belonging to the Bacteroideta phylum. On the other hand, they did not exhibit predatory activity against eight strains belonging to the Pseudomonadota, Actinomycetota, and Bacillota phyla. These results demonstrate that the HFL-1, HFL-10, and HFL-13 strains have the ability to prey on bacteria belonging to the phylum Bacteroidetes. In particular, these three strains were shown to have the ability to prey on bacteria belonging to the phylum Bacteroidetes.

[0071] Example 6 Verification of predatory activity against Flavobacterium species under different water temperature conditions The HFL-1, HFL-10, and HFL-13 strains were tested by liquid culture under different water temperature conditions to verify that they have predatory activity against three fish pathogenic bacteria: Flavobacterium psychrophilum NBRC100250, Flavobacterium columnare NBRC100251, and Flavobacterium branchiophyllum NBRC15030. The strains were cultured in a buffer solution (25 mM HEPES, pH 7.4, 6 mM CaCl ２ ・2H ２ 0, 4 mM MgCl ２ ・7H ２ 600 nm absorbance (OD ６００Each fish pathogenic bacterial strain was suspended in 4.75 ml of a solution to achieve a pH of 0.8 to 0.9, and 0.25 ml of the HFL-1 strain inoculum, HFL-10 strain inoculum, HFL-13 strain inoculum, or buffer solution (a control without predatory bacteria inoculated) was mixed with the suspension. The mixture was then cultured at three water temperatures (28°C, 18°C, and 10°C) while stirring at 200 rpm. The absorbance at 600 nm of each inoculated strain and the control was measured every 24 hours after the start of culture. This experiment was repeated three times.

[0072] The predatory activity of each strain against the three fish pathogenic bacteria is shown in Figures 1, 2, and 3. Strains HFL-1, HFL-10, and HFL-13 all exhibited high predatory activity against the three fish pathogenic bacteria at water temperatures of 28°C (A in each figure), 18°C ​​(B in each figure), and 10°C (C in each figure).

[0073] The predatory bacteria of the present invention can efficiently control fish pathogenic bacteria belonging to the phylum Bacteroides and can be used to ensure a stable supply of fish through aquaculture.

Claims

1. A method for controlling fish pathogenic bacteria of the phylum Bacteroidota present in fish or in a fish habitat, the method comprising applying to the fish or to the fish habitat a predatory bacterium or a mutant thereof that preys on the fish pathogenic bacteria.

2. A method for suppressing the occurrence of fish infectious diseases caused by fish pathogenic bacteria of the phylum Bacteroidota present in fish or in a fish habitat, the method being characterized in that a predatory bacterium or a mutant thereof that preys on the fish pathogenic bacteria is applied to the fish or in the fish habitat.

3. The method according to claim 1 or 2, wherein the predatory bacterium or a mutant thereof is a strain or a mutant thereof belonging to the Bacteriovoracaceae family of bacteria.

4. The method of claim 1 or 2, wherein the predatory bacterium or a mutant thereof comprises a 16S rRNA gene having a sequence identity of more than 97.9% to the consensus base sequence of the 16S rRNA gene represented by SEQ ID NO:

15.

5. The method according to claim 1 or 2, wherein the base sequence of the 16S rRNA gene contained in the predatory bacterium or its mutant has: (i) a sequence identity of more than 94.8% to the base sequence of the 16S rRNA gene represented by SEQ ID NO: 6; (ii) a sequence identity of more than 94.7% to the base sequence of the 16S rRNA gene represented by SEQ ID NO: 7; or (iii) a sequence identity of more than 96.1% to the base sequence of the 16S rRNA gene represented by SEQ ID NO:

8.

6. The method according to claim 1 or 2, wherein the predatory bacterium or a mutant thereof is a Bacteriovoracaceae family bacterium HFL-1 strain (deposit number NITE ABP-03906), a Bacteriovoracaceae family bacterium HFL-10 strain (deposit number NITE ABP-03907), a Bacteriovoracaceae family bacterium HFL-13 strain (deposit number NITE ABP-03908), or a mutant thereof.

7. The method according to claim 1 or 2, wherein the fish pathogenic bacterium belongs to the class Flavobacteriia.

8. The method according to claim 1 or 2, wherein the fish pathogenic bacterium belongs to the family Flavobacteriaceae or Weeksellaceae.

9. The method according to claim 1 or 2, wherein the fish pathogenic bacteria are selected from the group consisting of bacteria of the genus Flavobacterium, bacteria of the genus Tenacibaculum, bacteria of the genus Elizabethkingia, and bacteria of the genus Chryseobacterium.

10. The method according to claim 9, wherein the bacterium of the genus Flavobacterium is Flavobacterium psychrophilum, Flavobacterium columnare, or Flavobacterium branchiophilum.

11. The method of claim 2, wherein the fish infection is bacterial cold water disease caused by Flavobacterium psychrophilum, columnaris disease caused by Flavobacterium columnare, or bacterial gill disease caused by Flavobacterium branchiophilum.

12. The method according to claim 1 or 2, wherein the application is carried out at a temperature between 0 and 38°C.

13. A predatory bacterium or its mutant that preys on fish pathogenic bacteria of the phylum Bacteroidota.

14. The predatory bacterium or its mutant according to claim 13, wherein the predatory bacterium or its mutant is a strain or mutant thereof belonging to the Bacteriovoracaceae family of bacteria.

15. The predatory bacterium or its mutant described in claim 13, wherein the predatory bacterium or its mutant comprises a 16S rRNA gene having greater than 97.9% sequence identity to the consensus base sequence of the 16S rRNA gene represented by SEQ ID NO:

15.

16. The predatory bacterium or its mutant according to claim 13, wherein the base sequence of the 16S rRNA gene contained in the predatory bacterium or its mutant (i) has a sequence identity of more than 94.8% to the base sequence of the 16S rRNA gene represented by SEQ ID NO: 6, (ii) has a sequence identity of more than 94.7% to the base sequence of the 16S rRNA gene represented by SEQ ID NO: 7, or (iii) has a sequence identity of more than 96.1% to the base sequence of the 16S rRNA gene represented by SEQ ID NO:

8.

17. The predatory bacterium or a mutant thereof according to claim 13, wherein the predatory bacterium or a mutant thereof is a Bacteriovoracaceae family bacterium HFL-1 strain (deposit number NITE ABP-03906), a Bacteriovoracaceae family bacterium HFL-10 strain (deposit number NITE ABP-03907), a Bacteriovoracaceae family bacterium HFL-13 strain (deposit number NITE ABP-03908), or a mutant thereof.

18. The predatory bacterium or its mutant according to claim 13, wherein the fish pathogenic bacterium belongs to the class Flavobacteriia.

19. The predatory bacterium or a mutant thereof according to claim 13, wherein the fish pathogenic bacterium belongs to the family Flavobacteriaceae or Weeksellaceae.

20. The predatory bacterium or its mutant described in claim 13, wherein the fish pathogenic bacterium is selected from the group consisting of bacteria of the genus Flavobacterium, bacteria of the genus Tenacibaculum, bacteria of the genus Elizabethkingia, and bacteria of the genus Chryseobacterium.

21. The predatory bacterium or a mutant thereof according to claim 20, wherein the Flavobacterium bacterium is Flavobacterium psychrophilum, Flavobacterium columnare, or Flavobacterium branchiophilum.

22. A composition for controlling fish pathogenic bacteria of the phylum Bacteroidota or for suppressing the occurrence of infectious diseases in fish caused by said fish pathogenic bacteria, comprising the predatory bacterium or a mutant thereof according to any one of claims 13 to 21.

Images