WO2022076351A1 - Pseudomonas strains and their metabolites to control fish diseases - Google Patents
Pseudomonas strains and their metabolites to control fish diseases Download PDFInfo
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- WO2022076351A1 WO2022076351A1 PCT/US2021/053482 US2021053482W WO2022076351A1 WO 2022076351 A1 WO2022076351 A1 WO 2022076351A1 US 2021053482 W US2021053482 W US 2021053482W WO 2022076351 A1 WO2022076351 A1 WO 2022076351A1
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- 241000589516 Pseudomonas Species 0.000 title claims description 14
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N31/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic oxygen or sulfur compounds
- A01N31/02—Acyclic compounds
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
- A01N43/34—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one nitrogen atom as the only ring hetero atom
- A01N43/40—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one nitrogen atom as the only ring hetero atom six-membered rings
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
- A01N63/27—Pseudomonas
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
- A01P1/00—Disinfectants; Antimicrobial compounds or mixtures thereof
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
- A01P3/00—Fungicides
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
- C07D213/90—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having more than three double bonds between ring members or between ring members and non-ring members
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/38—Pseudomonas
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
- Y02A40/81—Aquaculture, e.g. of fish
Definitions
- This invention is in the field of biopesticides.
- the invention pertains to seven novel strains of Pseudomonas spp, 0617-T307, 0917-T305, 0917-T306, 0917-T307, 0118-T319, 0318-T327, and 0418-T328, the cell broth and novel metabolites produced from the bacterial strain that can inhibit the growth of a variety of fish pathogens.
- the Pseudomonas strains of 0617-T307, 0917-T305, 0917-T306, 0917-T307, 0118-T319, 0318- T327, and 0418-T328 have been deposited in the American Type Culture Collection (ATCC) and have ATCC accession number PTA-126796, PTA-126797, PTA-126798, PTA-126799, PTA-126800, PTA-126801, and PTA-126802, respectively.
- ATCC American Type Culture Collection
- Wild fish, farmed fish, and ornamental pet fish are susceptible to infectious diseases caused by a variety of bacterial pathogens.
- the most frequently described fish bacterial pathogens are Aeromonas, Edwardsiella, Pseudomonas, Shewanella, Mycobacterium, Streptococcus, Flavobacterium, and Vibrio.
- Aeromonas salmonicida causes furunculosis, haemorrhages, muscle lesions, inflammation of the lower intestine, spleen enlargement, and death in freshwater fish populations.
- Vibriosis caused by Vibrio species is responsible for mass mortality in marine ornamental fishes.
- Vibrio infected fishes include dark skin, pale gills, haemorrhages at the base of fins, exophthalmia, skin ulcers, corneal opacity, splenomegaly, and enteritis.
- V. parahaemolyticus is found to be the causative agent of tail rot disease in marine ornamental fish. The bacterium attacks the adipose fin, and progressive infection is always fatal.
- Two Gram-positive bacterial species, Streptococcus agalactiae and Streptococcus iniae also cause severe impact to the aquaculture industry. S.
- S. agalactiae causes meningitis in fish, and infected survival often shows neurological disorders, such as constant, aberrant swimming.
- S. agalactiae has become a major pathogen for tilapia.
- Freshwater and saltwater fish are susceptible to infection by S. iniae.
- S. iniae causes meningoencephalitis, skin lesions, and septicemia.
- S. iniae infections have been reported in at least 27 species of cultured or wild fish and, resulting in over US$100M in annual losses.
- S. iniae can cause disease in mammals, including humans. Fish handlers with hand injuries can become ill from a Strep infection after contacting S. iniae infected fish.
- Tenacibaculum maritimum is a bacterial pathogen that affects a large number of marine fish species in the world and is of considerable economic significance to aquaculture producers.
- the pathogen causes tenacibaculosis (an ulcerative disease), which is one of the most threatening of many species of commercial value.
- oomycete pathogen Saprolegnia parasitica is both a saprotroph and necrotroph.
- the pathogen causes saprolegniasis, a disease that is characterized by visible white or grey patches of filamentous mycelium on the body or fins of freshwater fish.
- Saprolegnia infections were kept under control with malachite green. However, banning the use of malachite green resulted in a dramatic re-emergence of Saprolegnia infections in aquaculture.
- S. parasitica is now causing economic impacts, especially on catfish, salmon, and trout species.
- a method of controlling a fish pathogen and associated disease of a fish includes several steps.
- One step includes producing an agricultural composition comprising Formula (I)
- Another step includes applying said agricultural composition to the fish to inhibit the growth of the fish pathogen and the associated disease on the fish.
- a method of controlling a fish pathogen disease includes a step of applying an agricultural composition comprising between about 1.0 x 10 5 and 1.0 x 10 9 cfu per mL Pseudomonas bacteria to fish to inhibit the growth of a fish pathogen and an associated disease.
- the present invention relates to a novel metabolite produced by seven Pseudomonas strains listed in this patent, such as 0617-T307, that exhibits antimicrobial activity against pathogenic microorganisms, including bacteria and fungi. From the 16S rRNA and other housekeeping gene sequences, the strain was identified as Pseudomonas soli 0617-T307 in the Pseudomonas putida group.
- the cell broth of the 7 bacterial strains, such as 0617-T307 contains a novel, potent 6-membered heterocycle natural product which is designated as RejuAgro A (Formula (I)) as depicted below:
- Biocontrol agents are a way of managing pests, such as pathogens, weeds and insects, safely, sustainably, and cost-effectively. These agents are introduced into the environment to target a pest species, with the aim of reducing the pest's population or abundance in the environment.
- Bios are preparations of living microorganisms (bacteria and yeasts) that produce colonies on the hosts. These microorganisms are applied mainly to slow the pathogen buildup during its infection (Soliman et al. (2019) and Tianna et al. (2016)).
- Biorational is a term applied to microbe-based biopesticides. These biopesticides are often made by fermenting microbial strains. Most of these products have both anti-bacterial and anti-fungal activity (Soliman et al. (2019) and Tianna et al. (2016)).
- Biopesticides is defined by The US Environmental Protection Agency (EP A) to be pesticides derived from natural materials and categorizes them as either biochemical pesticides, containing substances that control pests by nontoxic mechanisms, microbial pesticides, consisting of microorganisms that typically produce bioactive natural products (BNPs), or plant-incorporated-protectants with activity produced by plants because of added genetic materials (Gwinn K.D. (2018)).
- RejuAgro A corresponds to chemical compound having the formula (I), respectively, as illustrated below:
- a method of controlling a fish pathogen and associated disease of a fish includes several steps.
- One step includes producing an agricultural composition comprising Formula (I) (Formula (I)).
- Another step includes applying said agricultural composition to the fish to inhibit the growth of the fish pathogen and the associated disease on the fish.
- the method includes the fish pathogen being selected from the group consisting of Aeromonas salmonicida, Streptococcus agalactiae, Streptococcus iniae, Vibrio parahaemolyticus, Saprolegnia parasitica, and Tenacibaculum maritimum.
- the method includes the associated disease being selected from the group consisting of furunculosis, streptococcosis, streptococcosis, vibriosis, acute hepatopancreatic, saprolegniasis, and tenacibaculosis.
- the method includes the fish being selected from the group consisting of Salmon, Trout, Cyprinids, Pike, Perch, Bullheads, Turbot, Halibu, fresh-water fish, sea-water fish, wild fish, farm fish, fish, shrimp, squid, oyster, crab, and conch.
- a method of controlling a fish pathogen disease includes a step of applying an agricultural composition comprising between about 1.0 x 10 5 and 1.0 x 10 9 cfu per mL Pseudomonas bacteria to fish to inhibit the growth of a fish pathogen and an associated disease.
- the method includes the Pseudomonas bacteria being selected from the group consisting of Pseudomonas soli 0617-T307 (Accession No. PTA-126796), Pseudomonas soli 0917-T305 (Accession No.
- PTA-126797 Pseudomonas soli 0917-T306 (Accession No. PTA-126798), Pseudomonas soli 0917-T307 (Accession No. PTA-126799), Pseudomonas mosselii 0118-T319 (Accession No. PTA-126800), Pseudomonas mosselii 0318-T327 (Accession No. PTA-126801), and Pseudomonas mosselii 0418-T328 (Accession No. PTA-126802).
- the method includes the composition comprising between about 5.0 x 10 7 and 2.0 x 10 8 cfu per mL Pseudomonas bacteria.
- the method includes the fish pathogen being selected from the group consisting of Aeromonas salmonicida, Streptococcus agalactiae, Streptococcus iniae, Vibrio parahaemolyticus, Saprolegnia parasitica, and Tenacibaculum maritimum.
- the method includes the associated disease being selected from the group consisting of furunculosis, streptococcosis, streptococcosis, vibriosis, acute hepatopancreatic, saprolegniasis, and tenacibaculosis.
- the method includes the fish being selected from the group consisting of Salmon, Trout, Cyprinids, Pike, Perch, Bullheads, Turbot, Halibu, fresh-water fish, sea-water fish, wild fish, farm fish, fish, shrimp, squid, oyster, crab, and conch.
- ATCC Patent Depository On June 25, 2020, which were accorded unofficial ATCC patent numbers PTA-126796, PTA-126797, PTA-126798, PTA-126799, PTA-126800, PTA-126801, and PTA-126802, respectively. Following viability testing, the ATCC Patent Depository accorded these deposited bacterial strains the following Accession numbers, effective June 25, 2020: Pseudomonas soli 0617- T307 (Accession No. PTA-126796), Pseudomonas soli 0917-T305 (Accession No.
- PTA- 126797 Pseudomonas soli 0917-T306 (Accession No. PTA-126798), Pseudomonas soli 0917-T307 (Accession No. PTA-126799), Pseudomonas mosselii 0118-T319 (Accession No. PTA-126800), Pseudomonas mosselii 0318-T327 (Accession No. PTA-126801), and Pseudomonas mosselii 0418-T328 (Accession No. PTA-126802).
- Dr. Yang grants permission to Applicant to include this biological deposit disclosure in the present application.
- the MICs of RejuAgro A on Aeromonas salmonicida, Streptococcus agalactiae, Streptococcus iniae, Vibrio parahaemolyticus, and Tenacibaculum maritimum are 1.56, 1.56, 3.13, 0.39, and 12.5 pg/ml, respectively.
- the minimum lethal concentration (MLC) was determined in triplicate.
- the MLC of RejuAgro A on Saprolegnia parasitica is 100 pg/ml.
- RejuAgro A provides good inhibition on furunculosis, streptococcosis, saprolegniasis, and tenacibaculosis caused by Aeromonas salmonicida, Streptococcus agalactiae, Streptococcus iniae, Saprolegnia parasitica, and Tenacibaculum maritimum.
- RejuAgro A also shows inhibition on vibriosis, acute hepatopancreatic necrosis, and contamination in fish, shrimp, squid oyster, crab, and conch caused by Vibrio parahaemolyticus.
- RejuAgro A (ug/mL) RejuAgro A
- Aeromonas salmonicida (TSA/TSB)/20°C 1.56 (furunculosis in Salmon, Trout,
- Streptococcus agalactiae (TSAY/TSBY)/28°C 1.56 (streptococcosis in fresh and sea-water fish) Strain (related disease) Medium/Temperature MIC (pg/mL) MLC
- Example 2 Media culture compositions used in the Examples.
- Table 2 includes exemplary media compositions used in the Examples.
- Example 3 Bacterial strains, natural products, and references cited to same.
Abstract
The present disclosure concerns methods of using novel bacterial strains of 0617- T307, 0917-T305, 0917-T306, 0917-T307, 0118-T319, 0318-T327, and 0418-T328, the cell broth and novel metabolites produced from the bacterial strains, that can inhibit the growth of a variety of fish pathogens. The methods include use of novel, potent antimicrobial metabolites produced from the strains corresponding to a compound having Formula (I):
Description
PSEUDOMONAS STRAINS AND THEIR METABOLITES
TO CONTROL FISH DISEASES
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part application of International Patent Application No. PCT/US2020/54303, filed October 5, 2020 and International Patent Application No. PCT/US2021/53405, filed October 4, 2021, both entitled ‘‘‘‘PSEUDOMONAS STRAINS AND THEIR METABOLITES TO CONTROL PLANT DISEASES,” and claims priority to same, as well as to U.S. Patent Application Serial Number 17/063,540, filed October 5, 2020, U.S. Patent Application Serial Number 17/493,594, filed October 4, 2021, Argentina Patent Application Serial No. P 20 01 02757, filed October 5, 2020, and Taiwan Patent Application Serial No. 109134454, filed October 5, 2020, the contents of each application which are herein incorporated by reference in their entirety.
FIELD OF THE INVENTION
[0002] This invention is in the field of biopesticides. In particular, the invention pertains to seven novel strains of Pseudomonas spp, 0617-T307, 0917-T305, 0917-T306, 0917-T307, 0118-T319, 0318-T327, and 0418-T328, the cell broth and novel metabolites produced from the bacterial strain that can inhibit the growth of a variety of fish pathogens. The Pseudomonas strains of 0617-T307, 0917-T305, 0917-T306, 0917-T307, 0118-T319, 0318- T327, and 0418-T328 have been deposited in the American Type Culture Collection (ATCC) and have ATCC accession number PTA-126796, PTA-126797, PTA-126798, PTA-126799, PTA-126800, PTA-126801, and PTA-126802, respectively.
BACKGROUND OF THE INVENTION
[0003] Wild fish, farmed fish, and ornamental pet fish are susceptible to infectious diseases caused by a variety of bacterial pathogens. The most frequently described fish bacterial pathogens are Aeromonas, Edwardsiella, Pseudomonas, Shewanella, Mycobacterium, Streptococcus, Flavobacterium, and Vibrio. Aeromonas salmonicida causes furunculosis, haemorrhages, muscle lesions, inflammation of the lower intestine, spleen enlargement, and death in freshwater fish populations. Vibriosis caused by Vibrio species is
responsible for mass mortality in marine ornamental fishes. Common symptoms of Vibrio infected fishes include dark skin, pale gills, haemorrhages at the base of fins, exophthalmia, skin ulcers, corneal opacity, splenomegaly, and enteritis. V. parahaemolyticus is found to be the causative agent of tail rot disease in marine ornamental fish. The bacterium attacks the adipose fin, and progressive infection is always fatal. Two Gram-positive bacterial species, Streptococcus agalactiae and Streptococcus iniae, also cause severe impact to the aquaculture industry. S. agalactiae causes meningitis in fish, and infected survival often shows neurological disorders, such as constant, aberrant swimming. S. agalactiae has become a major pathogen for tilapia. Freshwater and saltwater fish are susceptible to infection by S. iniae. S. iniae causes meningoencephalitis, skin lesions, and septicemia. S. iniae infections have been reported in at least 27 species of cultured or wild fish and, resulting in over US$100M in annual losses. S. iniae can cause disease in mammals, including humans. Fish handlers with hand injuries can become ill from a Strep infection after contacting S. iniae infected fish. Tenacibaculum maritimum is a bacterial pathogen that affects a large number of marine fish species in the world and is of considerable economic significance to aquaculture producers. The pathogen causes tenacibaculosis (an ulcerative disease), which is one of the most threatening of many species of commercial value. Apart from bacterial pathogens, oomycete pathogen Saprolegnia parasitica is both a saprotroph and necrotroph. The pathogen causes saprolegniasis, a disease that is characterized by visible white or grey patches of filamentous mycelium on the body or fins of freshwater fish. Saprolegnia infections were kept under control with malachite green. However, banning the use of malachite green resulted in a dramatic re-emergence of Saprolegnia infections in aquaculture. S. parasitica is now causing economic impacts, especially on catfish, salmon, and trout species.
[0004] There is a need for new biopesticides derived from novel strains, cell broths and novel metabolites produced from such strains that can inhibit the growth of fish diseasecausing pathogens.
BRIEF SUMMARY OF THE INVENTION
[0005] In a first aspect, a method of controlling a fish pathogen and associated disease of a fish is provided. The method includes several steps. One step includes producing an agricultural composition comprising Formula (I)
Another step includes applying said agricultural composition to the fish to inhibit the growth of the fish pathogen and the associated disease on the fish.
[0006] In a second aspect, a method of controlling a fish pathogen disease is provided. The method includes a step of applying an agricultural composition comprising between about 1.0 x 105 and 1.0 x 109 cfu per mL Pseudomonas bacteria to fish to inhibit the growth of a fish pathogen and an associated disease.
DETAILED DESCRIPTION
[0007] The present invention relates to a novel metabolite produced by seven Pseudomonas strains listed in this patent, such as 0617-T307, that exhibits antimicrobial activity against pathogenic microorganisms, including bacteria and fungi. From the 16S rRNA and other housekeeping gene sequences, the strain was identified as Pseudomonas soli 0617-T307 in the Pseudomonas putida group. The cell broth of the 7 bacterial strains, such as 0617-T307, contains a novel, potent 6-membered heterocycle natural product which is designated as RejuAgro A (Formula (I)) as depicted below:
[0008] This compound, its method of production, and applications for inhibiting fish microbial pathogens is disclosed in greater detail herein.
Definitions
[0009] When introducing elements of aspects of the disclosure or particular embodiments, the articles “a,” “an,” “the,” and “said” are intended to mean that there are one
or more of the elements. The terms “comprising,” “including,” and “having” are intended to be inclusive and mean that there may be additional elements other than the listed elements. The term "or" means any one member of a particular list and also includes any combination of members of that list, unless otherwise specified.
[0010] As intended herein, the terms “substantially,” “approximately,” and “about” and similar terms are intended to have a broad meaning in harmony with the common and accepted usage in the art to which the subject matter of this disclosure pertains. It should be understood by those of skill in the art who review this disclosure that these terms are intended to allow a description of certain features described and claimed without restricting the scope of these features to precise numerical ranges provided. Accordingly, these terms should be interpreted as indicating that insubstantial or inconsequential modifications or alterations of the subject matter described and claimed are considered to be within the scope of the invention as recited in the appended claims.
[0011] “Biological control agents (or BCAs)” are a way of managing pests, such as pathogens, weeds and insects, safely, sustainably, and cost-effectively. These agents are introduced into the environment to target a pest species, with the aim of reducing the pest's population or abundance in the environment.
[0012] “Biologicals” are preparations of living microorganisms (bacteria and yeasts) that produce colonies on the hosts. These microorganisms are applied mainly to slow the pathogen buildup during its infection (Soliman et al. (2019) and Tianna et al. (2018)).
[0013] “Biorational” is a term applied to microbe-based biopesticides. These biopesticides are often made by fermenting microbial strains. Most of these products have both anti-bacterial and anti-fungal activity (Soliman et al. (2019) and Tianna et al. (2018)). [0014] “Biopesticides” is defined by The US Environmental Protection Agency (EP A) to be pesticides derived from natural materials and categorizes them as either biochemical pesticides, containing substances that control pests by nontoxic mechanisms, microbial pesticides, consisting of microorganisms that typically produce bioactive natural products (BNPs), or plant-incorporated-protectants with activity produced by plants because of added genetic materials (Gwinn K.D. (2018)).
[0015] The compound referred to as RejuAgro A corresponds to chemical compound having the formula (I), respectively, as illustrated below:
[0016] In a first aspect, a method of controlling a fish pathogen and associated disease of a fish is provided. The method includes several steps. One step includes producing an agricultural composition comprising Formula (I)
(Formula (I)).
Another step includes applying said agricultural composition to the fish to inhibit the growth of the fish pathogen and the associated disease on the fish.
[0017] In a first respect, the method includes the fish pathogen being selected from the group consisting of Aeromonas salmonicida, Streptococcus agalactiae, Streptococcus iniae, Vibrio parahaemolyticus, Saprolegnia parasitica, and Tenacibaculum maritimum. In a second respect, the method includes the associated disease being selected from the group consisting of furunculosis, streptococcosis, streptococcosis, vibriosis, acute hepatopancreatic, saprolegniasis, and tenacibaculosis. In a third respect, the method includes the fish being selected from the group consisting of Salmon, Trout, Cyprinids, Pike, Perch, Bullheads, Turbot, Halibu, fresh-water fish, sea-water fish, wild fish, farm fish, fish, shrimp, squid, oyster, crab, and conch.
[0018] In a second aspect, a method of controlling a fish pathogen disease is provided. The method includes a step of applying an agricultural composition comprising between about 1.0 x 105 and 1.0 x 109 cfu per mL Pseudomonas bacteria to fish to inhibit the growth of a fish pathogen and an associated disease.
[0019] In a first respect, the method includes the Pseudomonas bacteria being selected from the group consisting of Pseudomonas soli 0617-T307 (Accession No. PTA-126796), Pseudomonas soli 0917-T305 (Accession No. PTA-126797), Pseudomonas soli 0917-T306 (Accession No. PTA-126798), Pseudomonas soli 0917-T307 (Accession No. PTA-126799), Pseudomonas mosselii 0118-T319 (Accession No. PTA-126800), Pseudomonas mosselii 0318-T327 (Accession No. PTA-126801), and Pseudomonas mosselii 0418-T328 (Accession No. PTA-126802). In a second respect, the method includes the composition comprising between about 5.0 x 107 and 2.0 x 108 cfu per mL Pseudomonas bacteria. In a third respect, the method includes the fish pathogen being selected from the group consisting of Aeromonas salmonicida, Streptococcus agalactiae, Streptococcus iniae, Vibrio parahaemolyticus, Saprolegnia parasitica, and Tenacibaculum maritimum. In a fourth respect, the method includes the associated disease being selected from the group consisting of furunculosis, streptococcosis, streptococcosis, vibriosis, acute hepatopancreatic, saprolegniasis, and tenacibaculosis. In a fifth respect, the method includes the fish being selected from the group consisting of Salmon, Trout, Cyprinids, Pike, Perch, Bullheads, Turbot, Halibu, fresh-water fish, sea-water fish, wild fish, farm fish, fish, shrimp, squid, oyster, crab, and conch.
Biological Deposit Information
[0020] One of the inventors, Dr. Ching-Hong Yang, submitted the bacterial strains Pseudomonas soli 0617-T307, Pseudomonas soli 0917-T305, Pseudomonas soli 0917-T306, Pseudomonas soli 0917-T307, Pseudomonas mosselii 0118-T319, Pseudomonas mosselii 0318-T327, and Pseudomonas mosselii 0418-T328 to the American Type Culture Collection (ATCC®), P.O. Box 1549, Manassas, VA 20110 USA (“ATCC Patent Depository”) on June 25, 2020, which were accorded unofficial ATCC patent numbers PTA-126796, PTA-126797, PTA-126798, PTA-126799, PTA-126800, PTA-126801, and PTA-126802, respectively. Following viability testing, the ATCC Patent Depository accorded these deposited bacterial strains the following Accession numbers, effective June 25, 2020: Pseudomonas soli 0617- T307 (Accession No. PTA-126796), Pseudomonas soli 0917-T305 (Accession No. PTA- 126797), Pseudomonas soli 0917-T306 (Accession No. PTA-126798), Pseudomonas soli 0917-T307 (Accession No. PTA-126799), Pseudomonas mosselii 0118-T319 (Accession No. PTA-126800), Pseudomonas mosselii 0318-T327 (Accession No. PTA-126801), and
Pseudomonas mosselii 0418-T328 (Accession No. PTA-126802). Dr. Yang grants permission to Applicant to include this biological deposit disclosure in the present application.
EXAMPLES
[0021] Example 1. Use of RejuAgro A for inhibiting fish pathogens
[0022] Fish pathogens were grown in their respective broth and temperature (Table 1) to exponential phase. After incubation, broth cultures were diluted 1 : 10 and pipetted into individual well of 96 well plates containing the compound per concentration (0.39, 0.78, 1.56, 3.13, 6.25, 12.5, 25, 50, 100, 200pg/ml). In addition, pathogen control and blank control (media only) were included. The optical density (OD600) readings from each well were captured using a spectrophotometer to determine MIC. The pathogen control and technical replicates of each concentration were combined, and lOOpl plated on respective agar in triplicate to determine the final MIC. The MICs of RejuAgro A on Aeromonas salmonicida, Streptococcus agalactiae, Streptococcus iniae, Vibrio parahaemolyticus, and Tenacibaculum maritimum are 1.56, 1.56, 3.13, 0.39, and 12.5 pg/ml, respectively. The minimum lethal concentration (MLC) was determined in triplicate. The MLC of RejuAgro A on Saprolegnia parasitica is 100 pg/ml. These results demonstrate that RejuAgro A provides good inhibition on furunculosis, streptococcosis, saprolegniasis, and tenacibaculosis caused by Aeromonas salmonicida, Streptococcus agalactiae, Streptococcus iniae, Saprolegnia parasitica, and Tenacibaculum maritimum. RejuAgro A also shows inhibition on vibriosis, acute hepatopancreatic necrosis, and contamination in fish, shrimp, squid oyster, crab, and conch caused by Vibrio parahaemolyticus.
[0023] Table 1. Summary of the antimicrobial effect of RejuAgro A on different fish pathogens
Strain (related disease) Medium/Temperature MIC (jig/mL) MLC
RejuAgro A (ug/mL) RejuAgro A
Aeromonas salmonicida (TSA/TSB)/20°C 1.56 (furunculosis in Salmon, Trout,
Cyprinids, Pike, Perch, Bullheads, Turbot, and Halibu)
Streptococcus agalactiae (TSAY/TSBY)/28°C 1.56 (streptococcosis in fresh and sea-water fish)
Strain (related disease) Medium/Temperature MIC (pg/mL) MLC
RejuAgro A (jig/mL)
RejuAgro A
Streptococcus iniae (TSAY/TSBY)/28°C 3.13
(streptococcosis in fresh and sea-water fish)
Vibrio par ahaemolyticus (TSA2/TSB2)/37°C 0.39
(vibriosis, acute hepatopancreatic necrosis and contamination in fish, shrimp, squid oyster, crab, and conch)
Tenacibaculum maritimum (MA/MB)/15°C 12.5
(tenacibaculosis in marine fish)
Saprolegnia parasitica GY/20°C NA 100
(saprolegniasis in wild and farm fish)
NA: Not applicable for isolate
Not available
Example 2. Media culture compositions used in the Examples.
[0024] Table 2 includes exemplary media compositions used in the Examples.
[0025] Table 2. Media compositions.
No. Medium Name Composition g per liter Reference
Ml YME Yeast extract 4.0 g (Hamamoto, H., et. al. (2015)) Malt extract 10 g
Glucose 4.0 g
Tap water 1.0 L
M6 DAPG medium Malt extract 15.0 g (Gnanamanickam,
Samuel S. (2008))
Water
M7 PRN medium Glycerol 30.0 g (Gnanamanickam,
Samuel S. (2008))
K2HPO4 3.0 g
NaCl 5.0 g
MgSO4-7H2O 0.5 g
D-tryptophan 0.61 g
M8 IAA medium D-glucose 5.0 g (Gnanamanickam,
Samuel S. (2008))
Casamino acids 25.0 g
MgSO4-7H2O 0.3 g
K2HPO4 1.7 g
No. Medium Name Composition g per liter Reference
M12 TSA2/TSB2 Tryptic soy broth 30.0g (Onarinde, B., &
Dixon, R. (2018))
NaCl 15.0g
Agar (T S A2 only) 15.0g
M13 MA/MB Marine broth 37.4g (Mabrok M., et al.
(2016))
Agar (MA only) 15.0g
M14 GY Glucose 10.9g (Eszterbauer, et al.
(2020)
Yeast extract 2.5g
Agar 15.0g
Example 3. Bacterial strains, natural products, and references cited to same.
[0026] The bacterial strains and natural products described in this application and presented in the appended claims are well-known in the microbiology literature. These references are presented below in Table 3 for each of the cited bacterial strains and natural products disclosed herein, the contents of which are hereby incorporated by reference in their entirety.
[0027] Table 3. Bacterial strains, natural products and references cited in support as evidence of their availability.
Bacterial Strains Reference citation
0617-T307, 0917-T305, Pascual, J., Garcia-Lopez, M., Carmona, C., Sousa, T. da S., de 0917-T306, and Pedro, N., Cautain, B., Martin, J., Vicente, F., Reyes, F., Bills,
0917-T307 G. F., & Genilloud, O. (2014). Pseudomonas soli sp. nov., a novel producer of xantholysin congeners. Syst Appl Microbiol, . 412-416.
Bacterial Strains Reference citation
0118-T319, 0318-T327, Dabboussi, F., Hamze, M., Singer, E., Geoffroy, V., Meyer, J., and 0418-T328 & Izard, D. (2002). Pseudomonas mosselii sp . nov ., a novel species. Int J Syst Bacterio!, 52: 363-376.
Natural Products Reference citation
RejuAgro B Knackmuss, H., Medizinische, M., & Chemie, I. (1968). Methyl-substituted 2,3,6-trihydroxypyridines and their oxidation products. Eur. J. Inorg. Chem. 2689: 2679-2689.
Rt22.9 and Rt25.0 Loots, D. T., Erasmus, E., & Mienie, L. J. (2005).
Identification of 19 new metabolites induced by abnormal amino acid conjugation in isovaleric acidemia. Clin Chem, 51 : 1510-1512.
Rtl8.9 Osipov, A. M., Metlova, L. P., Baranova, N. V, & Rudakov, E. S. (1978). New derivatives of difuryl: 2,2’-difuryl-5,5’- dicarbinol and 2,2’-difuryl-5,5’-dicarboxylic acid. Ukrainskii
Khimicheskii Zhurnal (Russian Edition), 44: 398.
Citations
Banu, L., Conrads, G., Rehrauer, H., Hussain, H., Allan, E., & van der Ploeg, J. R. (2010). The Streptococcus mutans serine/threonine kinase, PknB, regulates competence development, bacteriocin production, and cell wall metabolism. Infect Im mun. 78: 2209-2220.
Dabboussi, F., Hamze, M., Singer, E., Geoffroy, V., Meyer, J., & Izard, D. (2002). Pseudomonas mosselii sp . nov ., a novel species. Int J Syst Bacterio!. 52: 363-376.
Eszterbauer, E., Hardy, T., Ronai, Z, Sipos, D., Zsigmond, G. (2020) Cryopreservation of three Saprolegnia species (Oomycota): Preliminary evidence for the long-term archiving of water mould species, Fungal Biology, 124: 682-687.
Gavrish, E., Bollmann, A., Epstein, S., & Lewis, K. (2008). A trap for in situ cultivation of filamentous actinobacteria. J Microbiol Methods 72:257-262.
Gnanamanickam, Samuel S. (Roanoke, VA, U. (2010). Pseudomonas bacterium (Patent No. 20100093538)
Gwinn K.D. (2018) Chapter 7 - Bioactive natural products in plant disease control, in: R. Atta ur (Ed.), Studies in Natural Products Chemistry, Elsevier, pp. 229-246.
Hamamoto, H., Urai, M., Ishii, K., Yasukawa, J., Paudel, A., Murai, M., Kaji, T., Kuranaga, T., Hamase, K., Katsu, T., Su, J., Adachi, T., Uchida, R., Tomoda, H., Yamada, M., Souma, M., Kurihara, H., Inoue, M., & Sekimizu, K. (2015). Lysocin e is a new antibiotic that targets menaquinone in the bacterial membrane. Nat Chem Biol 11 : 127-133.
Ishiguro, E., Kay, W., Ainsworth, T., Chamberlain, J., Austen, R., Buckley, J., Trust, T. (1981) Loss of virulence during culture Aeromonas salmonicida at high temperature. J Bacteriol. 148(l):333-40.
Knackmuss, H., Medizinische, M., & Chemie, I. (1968). Methyl-substituted 2,3,6- trihydroxypyridines and their oxidation products. Eur. J. Inorg. Chem. 2689: 2679-2689.
Loots, D. T., Erasmus, E., & Mienie, L. J. (2005). Identification of 19 new metabolites induced by abnormal amino acid conjugation in isovaleric acidemia. Clin Chem, 51 : 1510-1512.
Mabrok, M., Machado, M., Serra, C.R., Afonso, A., Valente, L.M.P. and Costas, B. (2016), Tenacibaculosis induction in the Senegalese sole (Solea senegalensis) and studies of Tenacibaculum maritimum survival against host mucus and plasma. J Fish Dis, 39: 1445- 1455.
Onarinde, B., & Dixon, R. (2018). Prospects for Biocontrol of Vibrio parahaemolyticus Contamination in Blue Mussels (Mytilus edulus)-N Year-Long Study. Frontiers in microbiology, 9, 1043.
Osipov, A. M., Metlova, L. P., Baranova, N. V, & Rudakov, E. S. (1978). New derivatives of difuryl: 2,2’-difuryl-5,5’-dicarbinol and 2,2’-difuryl-5,5’-dicarboxylic acid. Ukrainskii Khimicheskii Zhurnal (Russian Edition), 44: 398.
Pascual, J., Garcia-Lopez, M., Carmona, C., Sousa, T. da S., de Pedro, N., Cautain, B., Martin, J., Vicente, F., Reyes, F., Bills, G. F., & Genilloud, O. (2014). Pseudomonas soli sp. nov., a novel producer of xantholysin congeners. Sy st Appl Microbiol, . 412-416.
Soliman WS, Shaapan RM, Mohamed LA, Gayed SSR. (2019) Recent biocontrol measures for fish bacterial diseases, in particular to probiotics, bio-encapsulated vaccines, and phage therapy. Open Vet J. 9(3): 190-195.
Tianna D.K., Johnson; Rachel, Elkins; Tim, Smith; David, Granatstein. (2018) Organic Fire Blight Management in the Western U.S. - extension, Organic agriculture.
Incorporation by Reference
[0028] All literature, publications, patents, patent applications, and related material cited here are incorporated by reference as if fully set forth herein.
Claims
1. A method of controlling a fish pathogen and associated disease of a fish, comprising the steps of i. producing an agricultural composition comprising Formula (I)
(Formula (I)); and ii. applying said agricultural composition to the fish to inhibit the growth of the fish pathogen and the associated disease on the fish.
2. The method of claim 1, wherein the fish pathogen is selected from the group consisting of Aeromonas salmonicida, Streptococcus agalactiae, Streptococcus iniae, Vibrio parahaemolyticus, Saprolegnia parasitica, and Tenacibaculum maritimum.
3. The method according to claim 1, wherein the associated disease is selected from the group consisting of furunculosis, streptococcosis, streptococcosis, vibriosis, acute hepatopancreatic, saprolegniasis, and tenacibaculosis.
4. The method according to claim 1, wherein the fish is selected from the group consisting of Salmon, Trout, Cyprinids, Pike, Perch, Bullheads, Turbot, Halibu, fresh-water fish, sea-water fish, wild fish, farm fish, fish, shrimp, squid, oyster, crab, and conch.
5. A method of controlling a fish pathogen disease, comprising: applying an agricultural composition comprising between about 1.0 x 105 and 1.0 x 109 cfu per mL Pseudomonas bacteria to fish to inhibit the growth of a fish pathogen and an associated disease.
6. The method of claim 5, wherein the Pseudomonas bacteria is selected from the group consisting of Pseudomonas soli 0617-T307 (Accession No. PTA-126796), Pseudomonas soli 0917-T305 (Accession No. PTA-126797), Pseudomonas soli 0917-T306 (Accession No. PTA-126798), Pseudomonas soli 0917-T307 (Accession No. PTA-126799), Pseudomonas mosselii 0118-T319 (Accession No. PTA-126800), Pseudomonas mosselii 0318-T327 (Accession No. PTA-126801), and Pseudomonas mosselii 0418-T328 (Accession No. PTA- 126802).
7. The method according to claim 5, wherein the composition comprises between about 5.0 x 107 and 2.0 x 108 cfu per mL Pseudomonas bacteria.
8. The method of claim 5, wherein the fish pathogen is selected from the group consisting of Aeromonas salmonicida, Streptococcus agalactiae, Streptococcus iniae, Vibrio parahaemolyticus, Saprolegnia parasitica, and Tenacibaculum maritimum.
9. The method according to claim 5, wherein the associated disease is selected from the group consisting of furunculosis, streptococcosis, streptococcosis, vibriosis, acute hepatopancreatic, saprolegniasis, and tenacibaculosis.
10. The method according to claim 5, wherein the fish is selected from the group consisting of Salmon, Trout, Cyprinids, Pike, Perch, Bullheads, Turbot, Halibu, fresh-water fish, sea-water fish, wild fish, farm fish, fish, shrimp, squid, oyster, crab, and conch.
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| EP21794692.0A EP4225895A1 (en) | 2020-10-05 | 2021-10-05 | Pseudomonas strains and their metabolites to control fish diseases |
| CA3195920A CA3195920A1 (en) | 2020-10-05 | 2021-10-05 | Pseudomonas strains and their metabolites to control fish diseases |
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| ARP200102757 | 2020-10-05 | ||
| ARP200102757A AR120159A1 (en) | 2020-10-05 | 2020-10-05 | PSEUDOMONAS STRAINS AND THEIR METABOLITES TO CONTROL PLANT DISEASES |
| USPCT/US2020/054303 | 2020-10-05 | ||
| TW109134454 | 2020-10-05 | ||
| US17/063,540 US11582973B2 (en) | 2020-10-05 | 2020-10-05 | Pseudomonas strains and their metabolites to control plant diseases |
| TW109134454A TW202214837A (en) | 2020-10-05 | 2020-10-05 | Pseudomonas strains and their metabolites to control plant diseases |
| US17/063,540 | 2020-10-05 | ||
| PCT/US2020/054303 WO2022075969A1 (en) | 2020-10-05 | 2020-10-05 | Pseudomonas strains and their metabolites to control plant diseases |
| US17/493,594 US20220104487A1 (en) | 2020-10-05 | 2021-10-04 | Pseudomonas strains and their metabolites to control plant diseases |
| US17/493,594 | 2021-10-04 | ||
| USPCT/US2021/053405 | 2021-10-04 | ||
| PCT/US2021/053405 WO2022076323A1 (en) | 2020-10-05 | 2021-10-04 | Pseudomonas strains and their metabolites to control plant diseases |
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| CA3197854A1 (en) | 2022-04-14 |
| CL2023000979A1 (en) | 2023-09-22 |
| CN116634877A (en) | 2023-08-22 |
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