A strain of streptomyces spheroides jiq021 and application thereof

Abstract

The present application relates to the field of microbiological technology, and specifically discloses a strain of Streptomyces rutilocolore JQ021 and its application. The strain has been preserved in the China Center for Type Culture Collection on October 9, 2025, and the preservation number is CCTCC NO: M 20252186. The strain JQ021 and its culture, metabolic extract produced therefrom all exhibit high and broad-spectrum inhibitory activity against various aquatic pathogenic Vibrio, especially Vibrio parahaemolyticus, Vibrio alginolyticus and Vibrio owensii. The present application also relates to a microbial preparation and an antibacterial composition comprising the strain or the metabolic extract, and the application thereof in preparing a preparation for preventing and treating aquatic vibrio disease. The strain provides a valuable microbial resource for developing safe and efficient antibiotic substitutes.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a Streptomyces rustii strain JQ021 and its applications. Background Technology

[0002] Vibrio are common pathogens widely distributed in marine environments. Among them, Vibrio parahaemolyticus, Vibrio alginolyticus, and Vibrio erwens pose particularly serious threats to aquaculture, often causing large-scale mortality of fish, shrimp, and shellfish, resulting in significant economic losses. Currently, the control of this disease still heavily relies on antibiotics, but this has led to increasingly prominent problems such as increased vibrio resistance, drug residues, and environmental pollution. Therefore, finding safe and effective antibiotic alternatives has become an urgent need for industry development.

[0003] Biocontrol using antagonistic bacteria or their metabolites is considered a promising green solution. Actinomycetes, as an important source of bioactive secondary metabolites, show great promise in this field. However, current research and applications mostly focus on strains such as Bacillus and Lactobacillus, or only on the broad-spectrum antibacterial activity of actinomycete metabolites. For example, although some studies have reported the inhibitory effects of Streptomyces coccidioides and Streptomyces freundii on single or a few species of Vibrio harveyi and Vibrio anguillarum, the existing publicly available antagonistic strains generally have a narrow inhibitory spectrum, lacking clear records and verification of highly efficient and synergistic inhibitory activity against the three key pathogenic Vibrio species: Vibrio parahaemolyticus, Vibrio alginolyticus, and Vibrio erwens.

[0004] Therefore, screening out a novel marine actinomycete strain that can broadly and efficiently inhibit a variety of important aquatic pathogenic Vibrio bacteria and is adapted to the aquaculture environment has important practical application value for developing a new generation of microecological preparations and promoting the healthy development of aquaculture. Summary of the Invention

[0005] The purpose of this invention is to overcome the problems of existing antagonistic strains used in aquaculture having narrow antibacterial spectra and difficulty in synergistically inhibiting multiple important Vibrio species. This invention provides a Streptomyces rustii strain JQ021 and its applications. By culturing this strain and obtaining its cells, fermentation broth, or metabolic extracts (such as crude ethyl acetate extract), and preparing them into microbial preparations or antibacterial compositions, highly efficient and broad-spectrum inhibition of three key aquatic pathogenic Vibrio species—Vibrio parahaemolyticus, Vibrio alginolyticus, and Vibrio erwens—has been successfully achieved. This technical solution provides a novel microorganism with a broader spectrum of action, superior efficacy, and low likelihood of inducing drug resistance for the green prevention and control of Vibrio diseases in aquaculture, along with its applications.

[0006] The first aspect of this invention provides a Streptomyces rubiginosohelvolus strain JQ021, which was deposited at the China Center for Type Culture Collection on October 9, 2025, with accession number CCTCC NO: M 20252186 and classified as Streptomyces rubiginosohelvolus.

[0007] A second aspect of the present invention provides a culture of the above-mentioned Streptomyces rustae-yellow strain JQ021, the culture comprising the strain and / or its fermentation broth.

[0008] A third aspect of the present invention provides a microbial preparation comprising the above-described Streptomyces rusticae strain JQ021 and / or the above-described culture, and an agriculturally or aquaculture-acceptable carrier.

[0009] A fourth aspect of the present invention provides the use of the above-described Streptomyces rustae strain JQ021 or the above-described culture in the preparation of a composition for inhibiting Vibrio.

[0010] Furthermore, the Vibrio is at least one of Vibrio parahaemolyticus, Vibrio alginolyticus, and Vibrio erwens.

[0011] Furthermore, the composition is used to prevent and treat vibriosis in aquatic animals.

[0012] A fifth aspect of the present invention provides a metabolic extract produced from the above-described Streptomyces rusticae strain JQ021 or the above-described culture.

[0013] Furthermore, the metabolic extract is a crude ethyl acetate extract prepared by the following method:

[0014] The strain JQ021 was inoculated into a liquid culture medium and cultured under constant temperature and shaking to obtain a fermentation broth;

[0015] Ethyl acetate was added to the fermentation broth for ultrasonic extraction, and the upper ethyl acetate phase was collected, concentrated, and dried to obtain the final product.

[0016] A sixth aspect of the present invention provides an antibacterial composition comprising the above-described metabolic extract and an adjuvant acceptable for agricultural or aquaculture purposes.

[0017] A seventh aspect of the present invention provides the use of the metabolic extract or the antimicrobial composition described above in the preparation of an agent for inhibiting Vibrio, characterized in that the Vibrio is at least one of Vibrio parahaemolyticus, Vibrio alginolyticus, and Vibrio owens.

[0018] The eighth aspect of the present invention provides a culture medium for selectively isolating the above-mentioned Streptomyces rustis strain JQ021, which is based on 2216E medium and contains antibiotics at the following final concentrations: nystatin 80-120 mg / L, nalidixic acid 20-40 mg / L, streptomycin 10-20 mg / L, and gentamicin 3-5 mg / L.

[0019] The 2216E medium consisted of 5.0 g / L tryptone, 1.0 g / L yeast extract, and 0.1 g / L ferric phosphate, dissolved in aged seawater, at a pH of 7.6 ± 0.2.

[0020] The ninth aspect of the present invention provides a method for isolating Streptomyces rustii strain JQ021, characterized by comprising the following steps: culturing and isolating samples from a marine environment using the above-described selective culture medium.

[0021] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0022] This invention discloses a Streptomyces rubiginosohelvolus strain JQ021 and its applications. This strain was deposited at the China Center for Type Culture Collection (CCTCC) on October 9, 2025, with accession number CCTCC NO: M 20252186, and is classified as *Streptomyces rubiginosohelvolus*. Strain JQ021, its culture, and its metabolic extracts (such as crude ethyl acetate extract) exhibit highly efficient and broad-spectrum inhibitory activity against various pathogenic Vibrio species in aquaculture, particularly *Vibrio parahaemolyticus*, *Vibrio alginolyticus*, and *Vibrio erwens*, successfully overcoming the technical shortcomings of existing antagonistic strains, such as narrow antibacterial spectrum and difficulty in synergistic inhibition of multiple Vibrio species. This invention also relates to microbial preparations and antimicrobial compositions containing the aforementioned strain or its metabolic extracts, and their application in the preparation of formulations for the prevention and control of vibrio diseases in aquaculture. This strain provides a clearly defined, highly efficient, and promising microbial resource for the development of antibiotic-alternative biocontrol agents for vibrio diseases in aquaculture. Attached Figure Description

[0023] Figure 1 This is a morphological observation data image of strain JQ021; among which, Figure 1 a. in the image shows the colony morphology of JQ021. Figure 1 b, c, and d in the figure show the morphology of JQ021 as observed by transmission electron microscopy (TEM).

[0024] Figure 2 Phylogenetic tree of strain JQ021.

[0025] Figure 3The results of inhibition zone experiments on three pathogenic Vibrio species are shown for JQ021 bacterial suspension and crude ethyl acetate extract.

[0026] Figure 4 The results of inhibition zone experiments on three pathogenic Vibrio species for JQ021 preliminary isolated extracts a and b are presented. Detailed Implementation

[0027] The present invention will now be described in further detail with reference to specific embodiments. However, this should not be construed as limiting the scope of the present invention to the following embodiments; all technologies implemented based on the content of the present invention fall within the scope of the present invention.

[0028] The first aspect of this embodiment provides a Streptomyces rubiginosohelvolus strain JQ021, which was deposited at the China Center for Type Culture Collection on October 9, 2025, with accession number CCTCC NO: M 20252186 and classified as Streptomyces rubiginosohelvolus.

[0029] The second aspect of this embodiment provides a culture of the above-mentioned Streptomyces rustae-yellow strain JQ021, the culture comprising the strain and / or its fermentation broth.

[0030] The third aspect of this embodiment provides a microbial preparation comprising the above-mentioned Streptomyces rusticae strain JQ021 and / or the above-mentioned culture, and an agriculturally or aquaculture-acceptable carrier.

[0031] The fourth aspect of this embodiment provides the use of the above-described Streptomyces rustae-yellow strain JQ021 or the above-described culture in the preparation of a composition for inhibiting Vibrio.

[0032] In one or more embodiments, the Vibrio is at least one of Vibrio parahaemolyticus, Vibrio alginolyticus, and Vibrio erwinis.

[0033] In one or more embodiments, the composition is used to prevent and treat vibrio disease in aquatic animals.

[0034] The fifth aspect of this embodiment provides a metabolic extract produced from the above-described Streptomyces rustae yellow strain JQ021 or the above-described culture.

[0035] In one or more embodiments, the metabolic extract is an ethyl acetate crude extract prepared by the following method:

[0036] The strain JQ021 was inoculated into a liquid culture medium and cultured under constant temperature and shaking to obtain a fermentation broth;

[0037] Ethyl acetate was added to the fermentation broth for ultrasonic extraction, and the upper ethyl acetate phase was collected, concentrated, and dried to obtain the final product.

[0038] The sixth aspect of this embodiment provides an antibacterial composition comprising the above-described metabolic extract and agriculturally or aquaculture-acceptable excipients.

[0039] The seventh aspect of this embodiment provides the use of the metabolic extract or the antimicrobial composition described above in the preparation of an agent for inhibiting Vibrio, characterized in that the Vibrio is at least one of Vibrio parahaemolyticus, Vibrio alginolyticus, and Vibrio owens.

[0040] The eighth aspect of this embodiment provides a culture medium for selectively isolating the above-mentioned Streptomyces rustis strain JQ021, which is based on 2216E medium and contains antibiotics at the following final concentrations: nystatin 80-120 mg / L, nalidixic acid 20-40 mg / L, streptomycin 10-20 mg / L, and gentamicin 3-5 mg / L.

[0041] The 2216E medium consisted of 5.0 g / L tryptone, 1.0 g / L yeast extract, and 0.1 g / L ferric phosphate, dissolved in aged seawater, at a pH of 7.6 ± 0.2.

[0042] The ninth aspect of this embodiment provides a method for isolating Streptomyces rusticae var. flavomarginata strain JQ021, characterized by comprising the following steps: culturing and isolating samples from a marine environment using the above-described selective culture medium.

[0043] To better understand the above embodiments, the following more detailed experimental examples are provided for further explanation.

[0044] Example 1: This example aims to provide a marine biofilm actinomycete strain that targets three common pathogenic Vibrio species: Vibrio parahaemolyticus, Vibrio alginolyticus, and Vibrio erwens. Both the strain itself and its metabolic extracts can inhibit the production of these three Vibrio species at 25°C. This strain exhibits characteristics of a highly antagonistic bacterial strain and can serve as a candidate resource for the biological control of pathogenic Vibrio species.

[0045] Actinomycete isolation and culture

[0046] Biofilms collected from rocks at a depth of approximately 1 m in the subtidal zone of Maidong, Qingdao, Shandong Province, China, were inoculated onto different agar media containing different substances and incubated at 24°C for 7 days. Single colonies were then selected for subculturing. After approximately 5 generations, single colonies appeared, and their 16-sigma RNA was sequenced (sequencing method as shown in section 1.2, Species Identification). Sequence alignment was performed on the NCBI website, and the phylum-level classification of strains isolated from different media was analyzed. Finally, the total number of strains isolated from different media, the total number of actinomycetes isolated, and the efficiency of actinomycete isolation were compared (Table 1) to obtain a relatively efficient method for isolating actinomycetes. The obtained actinomycetes were subjected to redundancy removal analysis using cd-hit, and strains with a similarity >99.99% were preserved. A total of 123 actinomycete strains were obtained, among which JQ021 was isolated using the ZLONA method.

[0047] Table 1. Methods for Isolation and Culture of Actinomycetes

[0048]

[0049] Letter code annotation

[0050] 1) ZQLNA / E / L medium composition: The 2216 medium composition is supplemented with 100 mg / L nystatin, 20 mg / L nalidixic acid, 15 mg / L streptomycin, and 4 mg / L gentamicin.

[0051] 2) ZQNA / E / L medium composition: 2216 e agar medium, with 100 mg / L nystatin, 20 mg / L nalidixic acid, and 4 mg / L gentamicin added.

[0052] 3) ZQLN medium composition: 2216 e agar medium, with 100 mg / L nystatin, 30 mg / L nalidixic acid, 15 mg / L streptomycin, and 4 mg / L gentamicin added.

[0053] 4) ZQCN medium composition: 2216e agar medium, with the addition of 100 mg / L nystatin, 30 mg / L naridinone acid, 1 ml / 300 ml 3% potassium dichromate, and 4 mg / L gentamicin.

[0054] JQ021 Morphological Observation and Species Identification

[0055] JQ021 Colony Morphology Observation

[0056] Inoculate JQ021 onto 2216e agar medium and incubate for 15 days, then observe its colony morphology. Figure 1As can be seen from 'a', the colonies of this bacterium are relatively regular concave circular shapes with powdery edges, white and opaque in color, rough in surface, and dry in texture.

[0057] 1.2 Morphological observation of JQ021 by transmission electron microscopy (TEM)

[0058] In this embodiment, the morphology of JQ021 was examined using transmission electron microscopy (TEM). JQ021 cells were inoculated into sterile 2216E liquid medium and cultured overnight at 25°C with shaking. Bacterial cells were collected by centrifugation at 3500 rpm for 5 minutes at room temperature, and resuspended in 1 mL of 2.5% glutaraldehyde solution (glutaraldehyde dissolved in 0.1 M PBS). Cells were fixed at room temperature for 20 minutes. After centrifugation at 3500 rpm for 5 minutes at room temperature, the supernatant was discarded, and cells were collected and resuspended in 50 μL of glutaraldehyde solution for negative staining. A copper grid coated with 300-mesh carbon was glow-discharged for 30 seconds, and then incubated on 50 μL of bacterial suspension for 1 minute. The liquid on the grid was blotted dry with filter paper. Each grid was immersed in 5 μL of ddH2O for 1 minute, and the liquid on the grid was blotted dry with filter paper. Each grid was incubated in 5 μL of 0.05% uranyl acetate for 30 seconds and allowed to dry before image acquisition. The morphology of JQ021 was observed using a JEM-1400 electron microscope (JEOL, Japan), and the resulting morphological images are shown below. Figure 1 As shown in b, c, and d.

[0059] 1.3 Species Identification

[0060] 16S rDNA sequencing

[0061] First, DNA was extracted from the strain. 400 μl of JQ021 bacterial culture in the logarithmic phase (cultured in 2216E liquid medium at 25℃ for 36 h) was placed in a 1.5 ml centrifuge tube, balanced, and centrifuged at 10000 rpm / min for 1 minute. The supernatant was collected in a clean bench, and 50 μl of distilled water was added. The mixture was heated in a 100℃ water bath for 5 min, then centrifuged for another 1 min. The supernatant was then used as the DNA. PCR amplification was then performed. The PCR reaction system was prepared according to Table 2, and the procedure in Table 3 was followed for PCR amplification in a gradient PCR instrument. The amplified sequence was obtained and then sequenced to obtain the 16S rDNA of JQ021, which was preliminarily identified as a Streptomyces bacterium.

[0062] Depository: China Center for Type Culture Collection; Deposit date: October 9, 2025; Accession number: CCTCC M 20252186; Classification name: JQ021 Streptomyces rubiginosohelvolus.

[0063] Table 2 PCR reaction system (20 μl)

[0064]

[0065] Table 3 PCR Procedure

[0066]

[0067] Genome draft sequencing

[0068] DNA was extracted from JQ021 using the Vazyme DNA Extraction Kit and sent to Novogene for genome draft sequencing using the Novaseq-PE150 sequencing platform to obtain genomic data. The genome was assembled using SPAdes genome assembler v4.0.0, and the contamination level (0) and integrity (100%) were checked using CheckM2 v1.0.2. Species annotation was performed using gtdbtk version 2.3.2. The taxonomic rank of JQ021 is:

[0069] JQ021 d-Bacteria p-Actinomycetota c-Actinomycetia o-Streptomycetalesf-Streptomycetaceae g-Streptomyces s-Streptomyces rubiginosohelvolus.

[0070] Constructing a phylogenetic tree

[0071] Using the assembled genomes of JQ021 and 11 other actinomycetes, a rootless tree was constructed using GTDBTK (gtdbtk:version 2.3.2), and then beautified using chiplot (Xie et al, (2023) Nucleic AcidsRes doi: 10.1093 / nar / gkad359), finally obtaining the phylogenetic tree of JQ021. Figure 2 .

[0072] Example 2: Fermentation culture of strain JQ021 and acquisition of crude extract

[0073] Based on the genome draft sequence (100% integrity, 0% contamination), strain JQ021 of this invention was annotated using GTDB-Tk2 (a genome classification database toolkit) and classified as *Streptomyces rubiginosohelvolus*. Its taxonomic hierarchy is as follows: Domain: Bacteria; Phylum: Actinobacteriota; Class: Actinomycetia; Order: Streptomycetales; Family: Streptomycetaceae; Genus: *Streptomyces*; Species: *Streptomyces rubiginosohelvolus*.

[0074] Strain JQ021 was inoculated at a rate of 1% (v / v) into 100 mL of 2216E liquid medium and cultured at 25°C with constant temperature shaking at 180 rpm for 10 days to obtain the fermentation broth. Subsequently, twice the volume of ethyl acetate was added to the fermentation broth, and ultrasonic extraction was performed for 50 minutes using an ultrasonic cleaning tank (640 W). After standing and separation, the upper ethyl acetate phase was collected, concentrated using a rotary evaporator, and dried to obtain crude ethyl acetate extract powder.

[0075] Under the same culture conditions, 150 ml of the JQ021 fermentation broth cultured for 10 days was concentrated and dried using a rotary evaporator to obtain crude fermentation extract powder. Subsequently, using an Agilent 1290 ultra-high performance liquid chromatography system, fractions were collected based on the chromatographic peak times of the target compounds, thus obtaining preliminary separated extracts a and b of JQ021. The results are shown in Table 5.

[0076] Liquid phase conditions:

[0077] Mobile phase A: Water + 1% formic acid;

[0078] Mobile phase B: Acetonitrile;

[0079] The gradient elution conditions are shown in Table 4.

[0080] Table 4 Gradient elution conditions

[0081]

[0082] Table 5 Collection time of preliminary separated extracts

[0083]

[0084] Example 3: Determination of the antagonistic effect of JQ021 bacterial suspension and related extracts against Vibrio.

[0085] The pathogenic Vibrio used in the experiment was isolated from the intestines of diseased shrimp. The assay method was based on GB / T 38483-2020 (Determination of antibacterial activity of secondary metabolites of microbial antibiotics using the inhibition zone method).

[0086] Pathogenic Vibrio strains EMS001, XV22, and VP were inoculated at a rate of 1% (v / v) into 4 mL of 2216E liquid medium and cultured overnight at 25°C and 180 rpm for antagonistic Vibrio experiments.

[0087] 2.1 Determination of antagonistic Vibrio antagonism in JQ021 bacterial suspension

[0088] Three types of 2216e solid culture media containing 1% of different pathogenic Vibrio species were obtained using the pre-added bacterial suspension pour plate method. Then, 0.6 cm diameter holes were punched in the solid culture media, and 100 μL of JQ021 bacterial suspension was added to each hole (with sterile 2216e liquid culture medium added as a negative control). The media were sealed and incubated upright at 25°C until inhibition zones appeared. Figure 3 It can be seen that JQ021 bacterial solution has an inhibitory effect on three types of Vibrio: EMS, XV22, and VP.

[0089] 2.2 Determination of antagonistic Vibrio activity in JQ021 bacterial extract

[0090] Three solid culture media containing different pathogenic Vibrio species were obtained using the spread plate method. Then, 0.6 cm sterile antimicrobial susceptibility testing slides were placed on the plates, and 20 μL of bacterial extract (using 50% methanol as a negative control) was added to each slide. The plates were sealed and incubated upright at 25°C until inhibition zones appeared. Figure 3 and Figure 4 It can be seen that the bacterial extract of JQ021 and the preliminary isolated extracts a and b all have good antibacterial effects against three types of Vibrio: EMS XV22 VP.

[0091] The 16S rDNA sequence of strain JQ021:

[0092]

[0093] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A Streptomyces sp. JQ021 strain, characterized in that, The strain was deposited in China Center for Type Culture Collection on October 9, 2025, and the deposit number is CCTCC NO: M 20252186, and the classification name is Streptomyces rubiginosohelvolus .

2. A culture of Streptomyces sp. JQ021 according to claim 1, characterized in that, The culture comprises the strain and / or a fermentation broth thereof.

3. A microbial preparation, characterized in that, A composition for use in inhibiting Vibrio, comprising the Streptomyces rubiginosohelvolus strain JQ021 of claim 1 and / or the culture of claim 2, and an agriculturally or aquaculturally acceptable carrier.

4. Use of the Streptomyces rubiginosohelvolus strain JQ021 of claim 1 or the culture of claim 2 in the preparation of a composition for inhibiting Vibrio; the Vibrio being at least one of Vibrio parahaemolyticus, Vibrio alginolyticus and Vibrio owensii.

5. The use according to claim 4, wherein the compound is ###0002### The composition is used for preventing and treating Vibrio disease of aquaculture animals.

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