Streptomyces rustproof strain JQ021 and application thereof

Abstract

The invention relates to the technical field of microorganisms, and particularly discloses a streptomyces rust red strain JQ021 and application thereof. The strain is preserved in the China Center for Type Culture Collection on October 9, 2025, and the preservation number of the strain is CCTCC NO: M 20252186. The strain JQ021, a culture of the strain JQ021 and a metabolic extract produced by the strain JQ021 show efficient and broad-spectrum inhibitory activity on various aquatic pathogenic vibrios, especially vibrio parahaemolyticus, vibrio alginolyticus and vibrio Ewana. The invention also relates to a microbial preparation and an antibacterial composition containing the strain or the metabolic extract, and an application of the microbial preparation and the antibacterial composition in preparation of a preparation for preventing and treating aquaculture vibriosis. The strain provides valuable microbial resources for developing safe and efficient antibiotic substitutes.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microbial technology, and in particular to a strain of Streptomyces rutilans JQ021 and its application. BACKGROUND

[0002] Vibrio is a common pathogenic bacteria widely distributed in marine environment, among which Vibrio parahaemolyticus, Vibrio alginolyticus and Vibrio owensii are particularly harmful to aquaculture industry, often leading to large-scale death of fish, shrimp and shellfish, and causing significant economic losses. At present, the prevention and control of the disease still highly depends on antibiotics, but the problems such as increased drug resistance of Vibrio, drug residues and environmental pollution are increasingly prominent. Therefore, it is an urgent need for the industry development to seek safe and efficient antibiotic substitutes.

[0003] Biological control using antagonistic bacteria or their metabolites is considered as a potential green solution. As an important source of active secondary metabolites, actinomycetes show great prospects in this field. However, existing researches and applications are mostly focused on strains of Bacillus, Lactobacillus and other bacteria, or only pay attention to the broad-spectrum antibacterial activity of actinomycete metabolites. For example, although some studies have reported that Streptomyces globosus and Streptomyces fradiae can inhibit single or a few species of Vibrio harveyi and Vibrio anguillarum, the existing antagonistic bacterial strain resources generally have the problem of narrow inhibition spectrum, and there is no clear record and verification of a strain that can simultaneously exhibit high and synergistic inhibitory activity against Vibrio parahaemolyticus, Vibrio alginolyticus and Vibrio owensii, which are three key pathogenic Vibrio.

[0004] Therefore, it is of great practical application value to screen a new marine actinomycete strain that can inhibit a variety of important aquatic pathogenic Vibrio in a broad and efficient manner and adapt to the breeding environment, for the development of a new generation of microecological preparations and the promotion of the healthy development of aquaculture industry. SUMMARY

[0005] The present application aims to overcome the problem of narrow inhibition spectrum and difficulty in synergistically inhibiting multiple important Vibrio of the existing antagonistic bacterial strains for aquaculture. A strain of Streptomyces rutilans JQ021 and its application are provided. By culturing the strain and obtaining its bacterial body, fermentation broth or metabolic extract (such as ethyl acetate crude extract), and preparing them into microbial preparations or antibacterial compositions, high and broad-spectrum inhibition of Vibrio parahaemolyticus, Vibrio alginolyticus and Vibrio owensii, which are three key aquatic pathogenic Vibrio, is successfully achieved. The technical solution provides a new microorganism with a wider action spectrum, better effect and less drug resistance for the green prevention and control of aquatic Vibrio disease and its application.

[0006] The first aspect of the present application provides a Streptomyces rubiginosohelvolus strain JQ021, which was preserved in the China Center for Type Culture Collection on October 9, 2025, and the preservation number is CCTCC NO: M 20252186, and the classification name is Streptomyces rubiginosohelvolus.

[0007] The second aspect of the present application provides a culture of the above-mentioned Streptomyces rubiginosohelvolus strain JQ021, wherein the culture comprises the strain and / or a fermentation liquor thereof.

[0008] The third aspect of the present application provides a microbial preparation, comprising the above-mentioned Streptomyces rubiginosohelvolus strain JQ021 and / or the above-mentioned culture, and an agriculturally or aquaculturally acceptable carrier.

[0009] The fourth aspect of the present application provides a use of the above-mentioned Streptomyces rubiginosohelvolus strain JQ021 or the above-mentioned culture in the preparation of a composition for inhibiting Vibrio.

[0010] Further, the Vibrio is at least one of Vibrio parahaemolyticus, Vibrio alginolyticus and Vibrio owensii.

[0011] Further, the composition is used for preventing and treating Vibrio disease of aquaculture animals.

[0012] The fifth aspect of the present application provides a metabolic extract produced by the above-mentioned Streptomyces rubiginosohelvolus strain JQ021 or the above-mentioned culture.

[0013] Further, the metabolic extract is an ethyl acetate crude extract prepared by the following method: The strain JQ021 is inoculated into a liquid culture medium, and incubated at a constant temperature with shaking to obtain a fermentation liquor; Ethyl acetate is added to the fermentation liquor for ultrasonic extraction, and the upper ethyl acetate phase is collected, and then concentrated and dried to obtain.

[0014] The sixth aspect of the present application provides an antibacterial composition, comprising the above-mentioned metabolic extract, and an agriculturally or aquaculturally acceptable excipient.

[0015] The seventh aspect of the present application provides a use of the above-mentioned metabolic extract or the above-mentioned antibacterial composition in the preparation of a preparation for inhibiting Vibrio, characterized in that the Vibrio is at least one of Vibrio parahaemolyticus, Vibrio alginolyticus and Vibrio owensii.

[0016] The eighth aspect of the present application provides a selective medium for separating the C. licheniformis strain JQ021, which is based on 2216E medium and supplemented with 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.

[0017] The 2216E medium comprises 5.0 g / L tryptone, 1.0 g / L yeast extract, 0.1 g / L ferric phosphate, dissolved in aged seawater, and has a pH of 7.6±0.2.

[0018] The ninth aspect of the present application provides a method for separating the C. licheniformis strain JQ021, which comprises the following steps: using the selective medium described above to culture and separate samples from a marine environment.

[0019] Compared with the prior art, the present application has the following beneficial effects: The present application discloses a C. licheniformis strain JQ021 and its application. The strain is preserved in the China Center for Type Culture Collection on October 9, 2025, has a preservation number of CCTCC NO: M 20252186, and is classified and named as Streptomyces rubiginosohelvolus. The strain JQ021 and its culture, metabolic extract (such as ethyl acetate crude extract) produced by the strain all exhibit high and broad-spectrum inhibitory activity on various aquatic pathogenic Vibrio, especially Vibrio parahaemolyticus, Vibrio alginolyticus and Vibrio owensii, and successfully overcome the technical defects of narrow inhibitory spectrum and difficulty in synergistically inhibiting multiple Vibrio of existing antagonistic strains. The present application also relates to a microbial preparation and an antibacterial composition containing the strain or the metabolic extract, and application of the microbial preparation and the antibacterial composition in preparation of a preparation for preventing and treating Vibrio disease in aquaculture. The strain provides a clear, efficient and widely applicable microbial resource for developing a biological prevention and treatment preparation for replacing antibiotics for Vibrio disease in aquaculture. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 Figure 1 is a morphological observation data graph of the JQ021 strain; wherein, Figure 1 Figure 1a shows the colony morphology of the JQ021 strain, Figure 1 Figures 1b, 1c and 1d show the morphology of the JQ021 strain observed by transmission electron microscopy (TEM).

[0021] Figure 2 Figure 2 is a phylogenetic tree of the JQ021 strain.

[0022] Figure 3 Figure 3 is the experimental results of the inhibition zone of the JQ021 strain liquid and ethyl acetate crude extract on three kinds of pathogenic Vibrio.

[0023] Figure 4 The results of the preliminary separation and extraction of JQ021 extracts a and b on the inhibition zone of three pathogenic Vibrio. DETAILED DESCRIPTION

[0024] The application will be described in further detail below with specific examples. However, it should not be understood that the scope of the above-mentioned subject matter of the application is limited to the following examples, and any technology achieved based on the content of the application falls within the scope of the application.

[0025] The first aspect of the present embodiment provides a Streptomyces rubiginosohelvolus strain JQ021, which was preserved in the China Center for Type Culture Collection on October 9, 2025, with a preservation number of CCTCC NO: M 20252186 and a classification name of Streptomyces rubiginosohelvolus.

[0026] The second aspect of the present embodiment provides a culture of the above-mentioned Streptomyces rubiginosohelvolus strain JQ021, which comprises the strain and / or a fermentation broth thereof.

[0027] The third aspect of the present embodiment provides a microbial preparation comprising the above-mentioned Streptomyces rubiginosohelvolus strain JQ021 and / or the above-mentioned culture, and an agriculturally or aquaculturally acceptable carrier.

[0028] The fourth aspect of the present embodiment provides a use of the above-mentioned Streptomyces rubiginosohelvolus strain JQ021 or the above-mentioned culture in the preparation of a composition for inhibiting Vibrio.

[0029] In one or more embodiments, the Vibrio is at least one of Vibrio parahaemolyticus, Vibrio alginolyticus, and Vibrio owensii.

[0030] In one or more embodiments, the composition is used for preventing and treating Vibrio disease of aquaculture animals.

[0031] The fifth aspect of the present embodiment provides a metabolic extract produced by the above-mentioned Streptomyces rubiginosohelvolus strain JQ021 or the above-mentioned culture.

[0032] In one or more embodiments, the metabolic extract is an ethyl acetate crude extract prepared by the following method: The strain JQ021 is inoculated into a liquid medium and incubated at a constant temperature with shaking to obtain a fermentation broth; Ethyl acetate is added to the fermentation broth for ultrasonic extraction, and the upper ethyl acetate phase is collected to obtain the ethyl acetate crude extract after concentration and drying.

[0033] The sixth aspect of the embodiment provides an antibacterial composition comprising the metabolic extract described above and an agriculturally or aquaculturally acceptable adjuvant.

[0034] The seventh aspect of the embodiment provides an application of the metabolic extract described above or the antibacterial composition described above in preparing a preparation for inhibiting Vibrio, characterized in that the Vibrio is at least one of Vibrio parahaemolyticus, Vibrio alginolyticus and Vibrio owensii.

[0035] The eighth aspect of the embodiment provides a culture medium for selectively isolating the strain Streptomyces rutilus JQ021 described above, wherein the culture medium is based on 2216E medium and further comprises 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.

[0036] The 2216E medium comprises 5.0 g / L tryptone, 1.0 g / L yeast extract, 0.1 g / L ferric phosphate, dissolved in aged seawater, and pH 7.6±0.2.

[0037] The ninth aspect of the embodiment provides a method for isolating the strain Streptomyces rutilus JQ021, characterized in that the method comprises the following steps: using the selective culture medium described above to culture and isolate samples from a marine environment.

[0038] In order to more clearly understand the above-mentioned embodiments, the following more detailed experimental examples are provided to make further explanations.

[0039] Example 1: The present embodiment aims to provide a marine biofilm actinomycete strain against three common pathogenic Vibrio, i.e. Vibrio parahaemolyticus, Vibrio alginolyticus and Vibrio owensii, which can inhibit the generation of the three Vibrio at 25℃ environment, and the strain itself and the metabolic extract thereof can have a very high antagonistic strain feature, and can become an alternative resource for the biological control of pathogenic Vibrio.

[0040] Actinomycete isolation culture The biofilm on the rock collected from the subtidal zone of Ma Dao, Qingdao City, Shandong Province, China at about 1 m was inoculated on different agar medium added with different substances, placed in a 24°C constant temperature incubator for culture for 7 days, and then single colonies were selected for subculture. Single colonies appeared after subculture for about 5 generations, then 16SIRNA was sequenced (the sequencing method is shown in 1.2 species identification), and sequence alignment was performed on the NCBI website. The classification of the strains isolated by different culture media at the door level was counted, and finally the total number of strains isolated by different culture media, the total number of actinomycetes isolated and the efficiency of isolating actinomycetes (Table 1) were compared to obtain a relatively efficient method for isolating actinomycetes. The obtained actinomycetes were subjected to redundancy analysis by cd-hit, and strains with similarity > 99.99% were preserved. Finally, 123 strains of actinomycetes were obtained, of which JQ021 was isolated by the method using ZLONA.

[0041] Table 1 Actinomycete isolation and culture method

[0042] Letter code note 1) ZQLNA / E / L medium composition: 2216 medium composition, added with 100 mg / L nystatin, 20 mg / L nalydic acid, 15 mg / L streptomycin, 4 mg / L gentamicin.

[0043] 2) ZQNA / E / L medium composition: 2216 e agar medium, added with 100 mg / L nystatin, 20 mg / L nalydic acid, 4 mg / L gentamicin.

[0044] 3) ZQLN medium composition: 2216 e agar medium, added with 100 mg / L nystatin, 30 mg / L nalydic acid, 15 mg / L streptomycin, 4 mg / L gentamicin.

[0045] 4) ZQCN medium composition: 2216 e agar medium, added with 100 mg / L nystatin, 30 mg / L nalydic acid, 1 ml / 300 ml 3% potassium dichromate, 4 mg / L gentamicin.

[0046] JQ021 morphological observation and species identification JQ021 colony morphology observation JQ021 was inoculated on 2216 e agar medium and cultured for 15 days to observe the colony morphology, as shown in Figure 1 a in which the colony of the bacteria is a relatively regular concave round, the edge is powdery, the color is white and opaque, the surface is rough, and the texture is dry.

[0047] 1.2 Transmission electron microscopy (TEM) observation of the morphology of JQ021 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.

[0048] 1.3 Species Identification 16S rDNA sequencing 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.

[0049] Depository: China Center for Type Culture Collection; Deposit date: October 9, 2025; Accession number: CCTCC M 20252186; Classification name: JQ021 Streptomyces rubiginosohelvolus.

[0050] Table 2 PCR reaction system (20 μl)

[0051] Table 3 PCR Procedure

[0052] Genome draft sequencing DNA of JQ021 was extracted using the vazyme DNA extraction kit, and sent to Novaseq-PE150 of Novogene for genome sketch sequencing to obtain genome data. SPAdes genome assembler v4.0.0 was used for assembly, CheckM2 v1.0.2 was used to check the degree of pollution (0) and the degree of integrity (100%), gtdbtk: version 2.3.2 was used for species annotation, and the taxonomic rank of JQ021 was: JQ021 d-Bacteria p-Actinomycetota c-Actinomycetia o-Streptomycetalesf-Streptomycetaceae g-Streptomyces s-Streptomyces rubiginosohelvolus.

[0053] Establishment of phylogenetic tree Using the assembled JQ021 and the genomes of the remaining 11 actinomycetes, GTDBTK (gtdbtk: version 2.3.2) was used to establish an unrooted tree, and chiplot was used for beautification (Xie et al, (2023) Nucleic Acids Res doi: 10.1093 / nar / gkad359), and finally the phylogenetic tree of JQ021 was obtained Figure 2 .

[0054] Example 2: Fermentation culture of JQ021 strain and obtaining of crude extract The strain JQ021 of the present application is annotated as Streptomyces rubiginosohelvolus by GTDB-Tk2 (Genome Taxonomy Database Toolkit) based on the genome sketch sequence (100% completeness, 0 contamination), and its taxonomic rank is as follows: domain: Bacteria; phylum: Actinobacteriota; class: Actinomycetia; order: Streptomycetales; family: Streptomycetaceae; genus: Streptomyces; species: Streptomyces rubiginosohelvolus.

[0055] JQ021 strain was inoculated in 100 mL 2216E liquid medium at an inoculation amount of 1% (v / v) and incubated at 25°C and 180 rpm for 10 days to obtain a fermentation broth. Then, twice the volume of ethyl acetate was added to the fermentation broth, and ultrasonic extraction was performed using an ultrasonic cleaning tank (640 W) for 50 minutes. After standing and layering, the upper ethyl acetate phase was collected, concentrated and dried by a rotary evaporator to obtain an ethyl acetate crude extract powder.

[0056] Under the same culture conditions, 150 ml of the JQ021 fermentation broth cultured for 10 days was concentrated and dried by a rotary evaporator to obtain a fermentation crude extract powder. Then, using an Agilent 1290 ultra-high performance liquid chromatography system, based on the chromatographic peak time of the target compound, fraction collection was performed to obtain preliminary separation extracts a and b of JQ021, and the results are shown in Table 5.

[0057] Liquid phase conditions: Mobile phase A: water + 1% formic acid; Mobile phase B: acetonitrile; The gradient elution conditions are shown in Table 4.

[0058] Table 4 Gradient elution conditions

[0059] Table 5 Collection time of preliminary separation extract Example 3: Determination of the effect of JQ021 bacterial liquid and related extracts on antagonizing Vibrio The pathogenic Vibrio used in the experiment was isolated from the intestinal tract of diseased shrimp. The determination method refers to GB / T 38483-2020 (Determination of antibacterial activity of secondary metabolites from microbial sources Antibacterial circle method).

[0060] The pathogenic Vibrio EMS001, XV22 and VP strains were inoculated in 4 mL 2216E liquid medium at an inoculation amount of 1% (v / v) and incubated at 25°C and 180 rpm for one night to be used for antagonistic Vibrio experiments.

[0061] 2.1 Determination of JQ021 bacterial liquid antagonizing Vibrio Three 2216e solid culture media containing 1% different pathogenic Vibrio were obtained by pouring the pre-inoculated liquid into the plates, and then punching holes with a diameter of 0.6 cm on the solid culture medium. Then, 100 ul of JQ021 bacterial liquid was added to the holes (with 2216e liquid medium without bacteria as the negative control), sealed and placed in a 25°C constant temperature incubator for culture until the appearance of the inhibition zone. The diameter of the inhibition zone was measured by Figure 3It can be seen that JQ021 bacterial solution has inhibitory effect on the three Vibrio of EMS XV22 VP.

[0062] 2.2 JQ021 bacterial solution extract antagonizes Vibrio determination Three 2216e solid culture media containing different pathogenic Vibrio were obtained by coating plate method, and then 0.6 cm sterile drug sensitive piece was placed on the plate, 20 ul of bacterial solution extract was added on the drug sensitive piece (50% methanol was added as negative control), and then it was sealed and placed in 25℃ constant temperature incubator for culture until inhibition ring appeared, and then the inhibition effect was observed. Figure 3 and Figure 4 It can be seen that JQ021 bacterial solution extract and preliminary separation extract a and b have good inhibitory effect on the three Vibrio of EMS XV22 VP.

[0063] The 16S rDNA sequence of JQ021 strain: The above description is only the preferred embodiment of the present application, and is not intended to limit the present application. Any modification, equivalent replacement and improvement made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

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

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

3. A medium for selectively isolating the strain of Streptomyces sp. JQ021 as claimed in claim 1, characterized in that, On the basis of 2216E medium, the following antibiotics are added at the final concentrations: nystatin 80-120 mg / L, nalidixic acid 20-40 mg / L, streptomycin 10-20 mg / L, and gentamicin 3-5 mg / L.

4. A method for isolating a Streptomyces rutilus strain JQ021, characterized in that, The method comprises the following steps: The selective medium as claimed in claim 3 is used to culture and isolate samples from marine environment.

5. A microbial preparation, characterized in that, The culture of the Streptomyces rubiginosohelvolus strain JQ021 of claim 1 and / or the culture of claim 2, and an agriculturally or aquaculturally acceptable carrier.

6. 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.

7. Use according to claim 6, wherein The composition is used for preventing and treating Vibrio disease of aquaculture animals.

8. A metabolic extract produced by the Streptomyces rubiginosohelvolus strain JQ021 of claim 1 or the culture of claim 2.

9. An antibacterial composition, characterized by, The metabolic extract of claim 8, and an agriculturally or aquaculturally acceptable adjuvant.

10. Use of a metabolic extract as claimed in claim 8 for the preparation of a preparation for inhibiting Vibrio, characterized in that, The Vibrio is at least one of Vibrio parahaemolyticus, Vibrio alginolyticus, and Vibrio owensii.

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