CN116634877A - Pseudomonas strains and their metabolites for controlling fish diseases - Google Patents

Abstract

The present disclosure relates to methods of using novel bacterial strains 0617-T307, 0917-T305, 0917-T306, 0917-T307, 0118-T319, 0318-T327, and 0418-T328, cell broth produced by the bacterial strains, and novel metabolites that may inhibit the growth of a variety of fish pathogens. The method comprises the use of the strain to produce novel, potent antimicrobial metabolites corresponding to compounds having the formula (I):

Description

Pseudomonas strains and their metabolites for controlling fish diseases

Cross References to Related Applications

This application is a continuation-in-part 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 titled "Pseudomonas strains and metabolites thereof for use in the control of plant diseases," and claimed, together with U.S. Patent Application Serial No. 17/063,540, filed October 5, 2020, and U.S. Patent Application Serial No. 17/063,540, filed October 4, 2021 Priority of Application Serial No. 17/493,594, Argentine Patent Application Serial No. P 20 01 02757 filed October 5, 2020, and Taiwanese Patent Application Serial No. 109134454 filed October 5, 2020, the entire contents of each application Incorporated herein by reference.

field of invention

The invention belongs to the field of biological pesticides. In particular, the present invention relates to seven novel strains of Pseudomonas spp, 0617-T307, 0917-T305, 0917-T306, 0917-T307, 0118-T319, 0318, which can inhibit the growth of various fish pathogens -T327 and 0418-T328, cell broth and new metabolites produced by this bacterial strain. Pseudomonas strains 0617-T307, 0917-T305, 0917-T306, 0917-T307, 0118-T319, 0318-T327, and 0418-T328 have been deposited with the American Type Culture Collection (ATCC) and have ATCC Accession numbers PTA-126796, PTA-126797, PTA-126798, PTA-126799, PTA-126800, PTA-126801 and PTA-126802.

Background of the invention

Wild, farmed, and ornamental fish are susceptible to infectious diseases caused by a variety of bacterial pathogens. The most commonly described fish bacterial pathogens are Aeromonas, Edwardsiella, Pseudomonas, Shewanella, Mycobacterium , Streptococcus, Flavobacterium and Vibrio. Aeromonas salmonicida causes furunculosis, haemorrhage, muscle lesions, lower intestinal inflammation, splenomegaly, and death in freshwater fish. Vibriosis, caused by species of the genus Vibrio, is responsible for massive mortality in marine ornamental fish. Common symptoms in fish infected with Vibrio spp include dull skin, pale gills, hemorrhages at the base of the fins, exophthalmos, skin ulcers, corneal clouding, splenomegaly, and enteritis. Vibrio parahaemolyticus (V.parahaemolyticus) was found to be the causative agent of tail rot in marine ornamental fish. The bacterium attacks the adipose fins, and progressive infections are always fatal. Two species of Gram-positive bacteria, Streptococcus agalactiae and Streptococcus iniae, also have a serious impact on the aquaculture industry. S. agalactiae causes meningitis in fish, and survival of the infection often shows neurological disturbances, such as constant abnormal swimming. Streptococcus agalactiae has become the main pathogen of tilapia. Freshwater and saltwater fish are susceptible to S. iniae infection. Streptococcus iniae causes meningoencephalitis, skin lesions, and sepsis. S. iniae infections have been reported in at least 27 species of farmed or wild fish, resulting in annual losses of more than $100 million. S. iniae can cause disease in mammals, including humans. Fish handlers with hand injuries may become ill from strep throat after coming into contact with fish infected with S. iniae. Tenacibaculum maritimum is a bacterial pathogen that affects a large number of marine fish species worldwide and is of considerable economic importance to aquaculture producers. The pathogen causes tenacibaculosis, an ulcer disease, one of the most threatening to many commercially important species. In addition to bacterial pathogens, the oomycete pathogen Saprolegnia parasitica is both humic and necrotrophic. The pathogen causes saprolegniasis, a disease characterized by white or gray patches of filamentous mycelium visible on the body or fins of freshwater fish. Saprolegnia infection is controlled by malachite green. However, the ban on the use of malachite green led to a significant re-emergence of Saprolegnia infection in aquaculture. Parasitic Saprolegniasis is now causing economic impacts, especially on catfish, salmon and trout species.

There is a need for new biopesticides derived from new strains, cell broths and new metabolites produced by such strains, which can inhibit the growth of pathogens of fish diseases.

Brief description of the invention

In a first aspect, a method of controlling fish pathogens and associated diseases of fish is provided. The method includes several steps. One step comprises producing an agricultural composition comprising formula (I):

Another step involves applying the agricultural composition to fish to inhibit the growth of fish pathogens and related diseases on fish.

In a second aspect, a method for controlling fish pathogenic diseases is provided. The method includes the step of administering to the fish an agricultural composition comprising between about 1.0 x ¹⁰⁵ to 1.0 x ¹⁰⁹ cfu per mL of Pseudomonas bacteria to inhibit growth of fish pathogens and associated diseases.

Detailed description

The present invention relates to a novel metabolite produced by 7 Pseudomonas strains listed in this patent, such as 0617-T307, which exhibits antimicrobial activity against pathogenic microorganisms, including bacteria and fungi. From the 16S rRNA and other housekeeping gene sequences, the strain could be identified as Pseudomonas Soli 0617-T307 in the group Pseudomonas putida. Seven bacterial strains (eg 0617-T307) Cell Broth contains a novel, potent 6-membered heterocyclic natural product, named Rejuagro A (Formula (I)), as follows:

The compound, its method of preparation and use for the inhibition of fish microbial pathogens are disclosed in more detail herein.

definition

When introducing elements of various aspects of the disclosure or a particular embodiment, the articles "a," "an," "the," and "said" are intended to mean that there are one or more of the elements. The terms "comprising", "comprising" and "having" are intended to be inclusive and mean that there may be additional elements other than the listed elements. Unless stated otherwise, the term "or" means any one member of a particular list and also includes any combination of members of that list.

As contemplated herein, the terms "substantially," "about," and "about," and similar terms, are intended to have a broad meaning consistent with 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 the precise numerical ranges provided. Accordingly, these terms should be interpreted as indicating that insubstantial or inconsequential modifications or variations of the subject matter described and claimed are considered to be within the scope of the invention as set forth in the appended claims.

A "biological control agent (or BCA)" is a safe, sustainable and cost-effective way to manage pests such as pathogens, weeds and insects. These agents are introduced into the environment to target pest species with the goal of reducing the population or abundance of the pest in the environment.

A "biologic" is a preparation of live microorganisms (bacteria and yeast) that produces colonies on a host. These microorganisms are mainly used to delay pathogen accumulation during the epiphytic phase (Soliman et al. (2019) and Tianna et al. (2018)).

"Bioreasonable" is a term applied to microbial-based biopesticides. These biopesticides are usually prepared by fermenting microbial strains. Most of these products have both antibacterial and antifungal activity (Soliman et al. (2019) and Tianna et al. (2018)).

"Biopesticides" are defined by the U.S. Environmental Protection Agency (EPA) as pesticides derived from natural materials, and are classified as biochemical pesticides containing substances that control pests through non-toxic mechanisms, usually produced by biologically active natural products (BNP) Microbial pesticides composed of microorganisms of the phytochemicals, or plant incorporation protectants with activity produced by the plant due to added genetic material (Gwinn K.D. (2018)).

The compound called RejuAgro A corresponds to the compound having formula (I), as shown below:

In a first aspect, a method of controlling fish pathogens and associated diseases of fish is provided. The method includes several steps. One step comprises producing an agricultural composition comprising formula (I):

Another step involves applying the agricultural composition to fish to inhibit the growth of fish pathogens and related diseases on fish.

In a first aspect, the method comprises a fish pathogen selected from the group consisting of Aeromonas salmonicida, Streptococcus agalactiae, Streptococcus iniae, Vibrio parahaemolyticus, Saprolegnia parasitica and Adhesiobacterium marinum. In a second aspect, the method comprises a relevant disease selected from the group consisting of furunculosis, streptococcal disease, streptococcal disease, vibriosis, acute hepatopancreatic disease (acute hepatopancreatic), saprolegniasis (saprolegniasis) and adhesion bacillosis (tenacibaculosis). In a third aspect, the method includes fish selected from the group consisting of salmon, trout, carp, pike, perch, cod, turbot, plaice, freshwater fish, marine fish, wild fish, farmed Fish, fish, shrimp, squid, oysters, crab and conch.

In a second aspect, a method for controlling fish pathogenic diseases is provided. The method includes the step of administering to the fish an agricultural composition comprising between about 1.0 x ¹⁰⁵ to 1.0 x ¹⁰⁹ cfu per mL of Pseudomonas bacteria to inhibit growth of fish pathogens and associated diseases.

In a first aspect, the method comprises a Pseudomonas bacterium selected from the group consisting of: Pseudomonas agroides 0617-T307 (Accession No. PTA-126797), Pseudomonas soil 0917-T306 (Accession No. PTA-126798), Pseudomonas soil 0917-T307 (Accession No. -126800), Pseudomonas moldii 0318-T327 (Accession No. PTA-126801 ) and Pseudomonas moldei 0418-T328 (Accession No. PTA-126802). In a second aspect, the method comprises a composition comprising between about 5.0×10 ⁷ to 2.0×10 ⁸ cfu per mL of Pseudomonas bacteria. In a third aspect, the method includes a fish pathogen selected from the group consisting of Aeromonas salmonicida, Streptococcus agalactiae, Streptococcus iniae, Vibrio parahaemolyticus, Saprolegnia parasitica and Adhesiobacterium marinum. In a fourth aspect, the method comprises a related disease furunculosis, streptococcosis, streptococcosis, vibriosis, acute hepatopancreatic disease, saprolegniasis and sticky bacillosis selected from the group consisting of. In a fifth aspect, the method includes fish selected from the group consisting of salmon, trout, carp, pike, perch, cod, turbot, plaice, freshwater fish, marine fish, wild fish, farmed fish, Fish, shrimp, squid, oysters, crab and conch.

Biological deposit information

One of the inventors, Dr. Yang Qinghong, bacterial strains Pseudomonas soil 0617-T307, Pseudomonas soil 0917-T305, Pseudomonas soil 0917-T306, Pseudomonas soil 0917-T307, Morse Submitted to the American Type Culture Collection on June 25, 2020 for P. POBox 1549, Manassas, VA 20110 USA ("ATCC Patent Depository"), which has been assigned unofficial ATCC Patent Nos. PTA-126796, PTA-126797, PTA-126798, PTA-126799, PTA-126800, PTA-126801, and PTA-126802, respectively . Following survival testing, the ATCC Patent Depositary assigned the following accession numbers to these deposited bacterial strains, effective June 25, 2020: T305 (Accession No. PTA-126797), Pseudomonas soil 0917-T306 (Accession No. PTA-126798), Pseudomonas soil 0917-T307 (Accession No. PTA-126799), Pseudomonas mosei 0118-T319 (Accession No. PTA-126800), Pseudomonas morgii 0318-T327 (Accession No. PTA-126801 ) and Pseudomonas morgii 0418-T328 (Accession No. PTA-126802). Dr. Yang permits the applicant to include the disclosure of the biological deposit in this application.

Example

Example 1. Use of RejuAgro A for inhibiting fish pathogens

Fish pathogens were grown to exponential phase in their respective broths and temperatures (Table 1). After incubation, the broth culture was diluted 1:10 and pipetted into 96-well plates containing each concentration (0.39, 0.78, 1.56, 3.13, 6.25, 12.5, 25, 50, 100, 200 μg/mL) of the compound in a separate hole. Additionally, pathogen controls and blank controls (medium only) were included. Optical density (OD600) readings of each well were captured using a spectrophotometer to determine the MIC. Pathogen controls and technical replicates at each concentration were combined and 100 μl were plated on the corresponding agar in triplicate to determine the final MIC. The MICs of RejuAgro A against Aeromonas salmonicida, Streptococcus agalactiae, Streptococcus iniae, Vibrio parahaemolyticus, and marine adhesive bacteria were 1.56, 1.56, 3.13, 0.39, and 12.5 μg/ml, respectively. The minimum lethal concentration (MLC) was determined in triplicate. The MLC of RejuAgro A against Saprolegnia parasitica is 100μg/ml. These results confirm that RejuAgro A provides good protection against furunculosis, streptococcosis, saprolegniasis and adhesive bacillosis caused by A. inhibitory effect. RejuAgro A also showed inhibitory effects on vibriosis, acute hepatopancreatic necrosis, and contamination in fish, shrimp, squid, oysters, crabs, and sea snails caused by Vibrio parahaemolyticus.

Table 1. Summary of antimicrobial effects of RejuAgro A against different fish pathogens

NA: not applicable to isolates

"-" not available

The culture medium composition used in embodiment 2. embodiment

Table 2 includes exemplary media compositions in the Examples.

Table 2. Media composition

Example 3. Bacterial strains, natural products and references cited therein.

The bacterial strains and natural products described in this application and present in the appended claims are well known in the microbiological literature. For the various cited bacterial strains and natural products disclosed herein, these references are presented in Table 3 below, the contents of which are incorporated herein by reference in their entirety.

Table 3. Bacterial strains, natural products and supporting references cited as evidence of their availability

Citation

Banu, L., Conrads, G., Rehrauer, H., Hussain, H., Allan, E., & van der Ploeg, J.R. (2010). The Streptococcus mutans serine/threonine kinase, PknB, regulatescompetence development, bacteriocin production, and cell wall metabolism. Infect Immun, 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 Bacteriol, 52 :363–376.

Eszterbauer, E., Hardy, T., Rónai, Z, Sipos, D., Zsigmond, G. (2020) Cryopreservation of three Saprolegnia species (Oomycota): Preliminary evidence for the long-term archiving of water mold species, Fungal Biology, 124:682-687.

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Gwinn K.D.(2018)Chapter 7-Bioactive natural products in plant diseasecontrol,in:R.Attaur(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 virus during culture of Aeromonas salmonicida athigh temperature. J Bacteriol .148(1):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 Vibrioparahaemolyticus Contamination in Blue Mussels(Mytilus edulus)-A Year-Long Study. Frontiers in microbiology, 9, 1043.

Osipov, A.M., Metlova, L.P., Baranova, N.V, & Rudakov, E.S. (1978). Newderivatives 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., García-López, M., Carmona, C., Sousa, T. da S., de Pedro, N., Cautain, B., Martín, J., Vicente, F., Reyes, F. , Bills, G.F., & Genilloud, O. (2014). Pseudomonas soli sp.nov., a novel producer of xantholysin congeners. Syst ApplMicrobiol, 37:412–416.

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incorporated by reference

All literature, publications, patents, patent applications, and related materials cited herein are incorporated by reference as if fully set forth herein.

Claims (10)

1. A method of controlling fish pathogens and related diseases of fish comprising the steps of:

i. producing an agricultural composition comprising formula (I):

and

applying the agricultural composition to fish to inhibit growth of fish pathogens and related diseases on 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, hydromyces parasiticus and marine adhesion bacillus.

3. The method according to claim 1, wherein the related disease is selected from the group consisting of: furunculosis, streptococcosis, vibriosis, acute hepatopancreatic disease, saprolegniasis and colibacillosis.

4. The method of claim 1, wherein the fish is selected from the group consisting of: salmon, trout, carp, pike, weever, kohlrabi, turbot, plaice, freshwater fish, seawater fish, wild fish, farmed fish, shrimp, squid, oyster, crab and conch.

5. A method of controlling fish pathogen disease comprising:

administering a composition comprising at least about 1.0X10 to fish ⁵ Up to 1.0X10 ⁹ Agricultural compositions of Pseudomonas bacteria between cfu per mL to inhibit growth of fish pathogens and related diseases.

6. The method of claim 5, wherein the pseudomonas bacteria are selected from the group consisting of: pseudomonas soil 0617-T307 (accession number PTA-126796), pseudomonas soil 0917-T305 (accession number PTA-126797), pseudomonas soil 0917-T306 (accession number PTA-126798), pseudomonas soil 0917-T307 (accession number PTA-126799), pseudomonas moellensis 0118-T319 (accession number PTA-126800), pseudomonas moellensis 0318-T327 (accession number PTA-126801) and Pseudomonas moellensis 0418-T328 (accession number PTA-126802).

7. The method of claim 5, wherein the composition comprises at least about 5.0 x 10 ⁷ Up to 2.0X10 ⁸ Pseudomonas bacteria between cfu per mL.

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, hydromyces parasiticus and marine adhesion bacillus.

9. The method according to claim 5, wherein the related disease is selected from the group consisting of: furunculosis, streptococcosis, vibriosis, acute hepatopancreatic disease, saprolegniasis and colibacillosis.

10. The method of claim 5, wherein the fish is selected from the group consisting of: salmon, trout, carp, pike, weever, kohlrabi, turbot, plaice, freshwater fish, seawater fish, wild fish, farmed fish, shrimp, squid, oyster, crab and conch.