CN114292785A - Marine streptomyces and application thereof in preventing and controlling aeromonas hydrophila from infecting aquatic animals - Google Patents
Marine streptomyces and application thereof in preventing and controlling aeromonas hydrophila from infecting aquatic animals Download PDFInfo
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Abstract
The invention discloses a marine streptomyces and application thereof in preventing and controlling aeromonas hydrophila infection of aquatic animals, belonging to the technical field of biological prevention and control. The name of the Streptomyces is Streptomyces sp.SH5, and the accession number is as follows: GDMCC No: 62057. the SH5 bacterial strain is separated from the water environment, and is applied to the culture water body to reduce the ecological safety risk to a great extent. The SH5 bacterial strain is planted in intestinal tracts of zebra fish, a host immune system is activated, inflammatory reaction is inhibited, production of toxicity factors of aeromonas hydrophila and expression of virulence genes are reduced, survival rate of the zebra fish stressed by the aeromonas hydrophila can be effectively improved, and accordingly prevention and control of aeromonas hydrophila infection are achieved in a green and environment-friendly manner without influencing host micro-ecological balance.
Description
Technical Field
The invention belongs to the technical field of biological control, relates to marine streptomyces and application thereof in the field of aquatic disease control, and particularly relates to marine streptomyces SH5 capable of being planted in intestinal tracts of aquatic animals and application thereof in controlling aeromonas hydrophila to infect aquatic animals (such as zebrafish).
Background
Aeromonas hydrophila (Aeromonas hydrophila) is a common pathogenic bacterium in water environment, can infect various aquatic animals such as fish, shrimp, shellfish and the like to induce septicemia of Aeromonas mobilis, and the direct economic loss of China caused by outbreak of the disease reaches billions every year. Antibiotic pharmacotherapy is always a traditional means for preventing and treating aquatic diseases commonly used at home and abroad, but the problems of drug resistance, environmental pollution, ecological balance damage and the like caused by the large use of broad-spectrum antibiotics force people to develop safer, environment-friendly and reliable novel disease prevention and control medicines and methods as soon as possible so as to meet the strategic demands of national 'green development'. Biological control is considered to be a main technical means for replacing antibiotics due to the advantages of environmental protection, safety, health, economy, low price, convenient use and the like.
The streptomyces has attracted extensive attention in the field of biological prevention and control because of the advantages of good production of antagonistic active substances, degradation of enzymes, good stress resistance and the like. However, the aerobic life characteristics of streptomycete make the streptomycete have no relevant findings and basis for the streptomycete to exert biological prevention and control effects in aquatic organisms in a permanent planting way.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention aims to provide a marine streptomyces. The strain is a streptomycete strain which can be fixedly planted in intestinal tracts of zebra fish, and the strain fermentation liquor is applied to a culture water body, so that the survival rate of the zebra fish under the stress of aeromonas hydrophila can be effectively improved, the inflammatory reaction of the zebra fish is inhibited, and the production of aeromonas hydrophila virulence factors and the expression of related virulence genes are reduced.
The invention also aims to provide application of the marine streptomyces.
The purpose of the invention is realized by the following technical scheme:
the invention provides a strain of Streptomyces marinus, which is named as Streptomyces sp SH5 and is obtained by separating and purifying ocean sediment of Bay of Xinjiang province in Dalian city.
The preservation information of the marine Streptomyces (Streptomyces sp.) SH5 is as follows: the preservation unit: guangdong province microbial culture Collection (GDMCC), accession number: GDMCC No: 62057, deposit address: the microbiological research institute of Guangdong province, No. 59 building, No. 5 building, Guangdong province, of the Fuli Zhonglu 100, Guangzhou city, the preservation date: 11/12/2021.
The 16S rDNA sequence characteristics of the marine Streptomyces (Streptomyces sp.) SH5 are as follows: the 16SrDNA sequence has a nucleotide sequence shown as SEQ ID No.1, and the sequence length is 1374 bp.
A biological agent is prepared based on the marine streptomyces.
The biological agent is prepared by liquid culture of the marine streptomyces, and preferably comprises the following steps: the marine streptomyces is inoculated into an ISP1 liquid culture medium for culture, and then the biological agent can be obtained.
The culture is carried out at the temperature of 28-30 ℃ and the rpm of 180-200 for 48-72 h.
The marine streptomyces SH5 is applied to prevention and control of aeromonas hydrophila infection of aquatic animals.
The application of the marine streptomycete SH5 in the analysis of the intestinal tract colonization ability of aquatic animals;
further, the application of marine streptomyces SH5 in improving the immune response of aquatic animals.
The marine streptomyces SH5 is applied to the regulation of aeromonas hydrophila in the process of inflammatory reaction of aquatic animals.
Further, the marine streptomyces SH5 is applied to inhibiting the intestinal colonization of aquatic animals by aeromonas hydrophila.
Further, the marine streptomyces SH5 is applied to reducing inflammatory reaction of low-aquatic animals under the stress of pathogenic bacteria; the pathogenic bacteria are aeromonas hydrophila.
Further, the aquatic animal is zebra fish.
The marine streptomycete SH5 is applied to inhibiting the production of aeromonas hydrophila virulence factors and the expression of virulence genes.
The virulence genes are aerA, act, ast, hlyA, alt, ahp, ahyR, ompA and fur.
The principle of preventing and controlling the aeromonas hydrophila to infect the zebra fish by the SH5 strain is that SH5 is fixedly planted in intestinal tracts of the zebra fish to activate a host immune system, inhibit inflammatory reaction and reduce the virulence gene expression of the aeromonas hydrophila, so that the prevention and control of the aeromonas hydrophila infection are realized in a green and environment-friendly way without influencing the microecological balance of the host.
Compared with the prior art, the invention has the following advantages and effects:
(1) the invention provides a marine Streptomyces sp SH5 strain. The SH5 bacterial strain is separated from the water environment, and is applied to the culture water body to reduce the ecological safety risk to a great extent.
(2) The SH5 strain disclosed by the invention is capable of effectively improving the survival rate of the zebra fish under the stress of aeromonas hydrophila.
(3) The SH5 bacterial strain is an aerobic bacterial strain, but can be planted in intestinal tracts of zebra fish.
(4) The SH5 bacterial strain disclosed by the invention can effectively reduce the colonization amount of aeromonas hydrophila in the intestinal tracts of zebra fish.
(5) The SH5 strain of the invention can effectively inhibit the expression of virulence genes of pathogenic bacteria aeromonas hydrophila.
(6) The SH5 strain disclosed by the invention effectively improves the immune response of the zebra fish and obviously reduces the inflammatory reaction of the zebra fish under the stress of pathogenic bacteria.
Drawings
FIG. 1 is a graph showing the actual effect of the target strain SH5 on improving the survival rate of zebra fish under the stress of Aeromonas hydrophila; where SH5(L) denotes the low concentration treatment group, i.e. SH5 is calculated at 1: 1000 dilution fermentation liquor is added into culture water of the zebra fish juvenile fish; SH5(H) indicates the high concentration treatment group, i.e. SH5 was treated at a rate of 1: 100 dilution fermentation liquor is added into culture water of the zebra fish juvenile fish.
FIG. 2 shows the SH5 strain 16S rDNA sequence and the evolutionary tree.
FIG. 3 is a test for the specificity of primer SH 5F/R; wherein M is marker of DL2000, Lane 1 is SH5, Lane 2 is Streptomyces TK24, Lane 3 is Escherichia coli, Lane 4 is Aeromonas hydrophila, Lane 5 is Vibrio parahaemolyticus, and Lane 6 is Zebra fish.
FIG. 4 shows the colonization effect (A) of the fluorescent-labeled SH5 strain in the intestinal tract of zebra fish and the quantitative analysis (B) of the colonized SH 5.
FIG. 5 is the effect of SH5 strain on reducing colonization of Aeromonas hydrophila in the intestinal tract of zebra fish; wherein A is the number of aeromonas hydrophila of each zebra fish at different time points; b is the number of SH5 per zebra fish at different time points.
FIG. 6 shows the effect of SH5 strain on the expression of genes associated with the immune response and inflammatory response of zebrafish; wherein A is the result of 6h of toxic attack of aeromonas hydrophila; b is the result of 12h of toxic attack by Aeromonas hydrophila.
FIG. 7 is the effect of SH5 strain on inhibiting the expression of virulence genes of Aeromonas hydrophila; wherein A is the result of the SH5 and aeromonas hydrophila co-culture for 6 h; b is the result of the SH5 and aeromonas hydrophila co-culture for 12 h; a is the result of 24h of SH5 and Aeromonas hydrophila co-culture.
FIG. 8 is the antagonistic action of SH5 strain on Aeromonas hydrophila.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.
The test methods in the following examples, in which specific experimental conditions are not specified, are generally performed according to conventional experimental conditions or according to the experimental conditions recommended by the manufacturer. The materials, reagents and the like used are, unless otherwise specified, reagents and materials obtained from commercial sources.
Escherichia coli (Escherichia coli) GDMCC 1.215 and Aeromonas hydrophila (Aeromonas hydrophila) GDMCC 1.2306 were used in example 2, and were obtained from the microorganism culture Collection of Guangdong province;
vibrio parahaemolyticus is Vibrio parahaemolyticus (Vibrio parahaemolyticus) ATCC 17802 available from Kyoto Biotech, Inc.;
streptomyces lividans TK24 Streptomyces lividans TK24 is disclosed in "Xu D, Seghezzi N, Eshenult C, et al.reproduction of an antibacterial Production and transformation in Streptomyces coelicolor by Overexpression of a TetR Family Transmission promoter [ J ] Applied and Environmental Microbiology,2010,76(23): 1-.
Example 1
The method for screening and identifying the marine Streptomyces (Streptomyces sp.) SH5 comprises the following steps and results:
(1) weighing 1g of Dalianxing gulf sea mud sample, adding 10mL of sodium cholate (0.1% w/v) at 200r/min, shaking for 30min, centrifuging (500g, 1min), and storing the supernatant. 10mL sodium cholate (0.1% w/v) was added to the lower sea mud precipitate, gently treated in a low power ultrasonic water bath for 1min, centrifuged (500g, 1min), and the supernatant was retained. Mixing the two supernatants, and performing gradient dilution with sterile seawater to obtain dilution 103And (3) doubling the supernatant, uniformly coating 0.2mL of the diluent on an HV plate containing 15 mu g/mL nalidixic acid and 50 mu g/mL potassium dichromate, placing the HV plate in an incubator at 28 ℃ for 20 days, observing streptomycete colonies, picking single colonies, performing streak culture on a Bennett culture medium, and observing the growth form of the streptomycete strains after 7 days to confirm that no bacteria or fungi are polluted. The streptomycete agar block is taken from the Bennett culture medium and inoculated into the TSB liquid culture medium, the culture is carried out for 7 days at the temperature of 28 ℃ and the rpm of 200, 500 mu L of bacterial suspension is taken and is fully mixed with 40 percent glycerol with the same volume, and then the mixture is placed into a 1.5mL centrifuge tube and is stored in a refrigerator at the temperature of 70 ℃ below zero.
(2) And (5) activating the strain. Firstly, taking out the target streptomyces strain SH5 from a refrigerator at-70 ℃, coating 100 mu L of the target streptomyces strain SH5 in an ISP1 solid culture medium, culturing for 3 days at 28 ℃, and then inoculating strain agar blocksCultured in 50mL of ISP1 liquid medium at 28 ℃ and 200rpm for 3 days for use. Tu type zebrafish embryos (fertilized day 0) (purchased from Shanghai Fixi Biotech Co., Ltd.) were washed 3 times with sterile water, placed in 6-well plates, placed in a 28 ℃ incubator, and the photoperiod was controlled with 14h light and 10h dark to provide fertilization conditions. On the 3 rd day after fertilization (3dpf), adding the mixture into the zebra fish culture water body according to the ratio of 1: 100(SH5(H)), 1: 1000(SH5(L)) and the same volume of ISP1 liquid medium was added to the Control group (Control), and the original streptomyces broth concentration was maintained after daily water change. After 3 days of fermentation broth treatment, the broth is cultured in sterile water and added to a final concentration of 1X 108CFU/mL aeromonas hydrophila was challenged, and survival rate was recorded every 12 h. Each set was set up with 3 parallel experiments. The survival results are detailed in figure 1. As can be seen from fig. 1, in the case where SH5(Control) was not added, the survival rate of the zebra fish juvenile fish decreased 24 hours after challenge, sharply decreased 36 hours later, and at 48 hours, the survival rate of the zebra fish juvenile fish was 0%; in the SH5 treated group, the survival rate of the zebra fish juvenile fish after being attacked by poison is 100% at 24h, and exceeds 80% at 36h, which is 3 times that of the Control group (Control). The result shows that the survival rate of the zebra fish under the toxic attack of the aeromonas hydrophila can be effectively improved under the SH5 treatment.
The ISP1 liquid culture medium comprises the following components in percentage by weight: 3g of yeast extract powder, 5g of tryptone and sterile water with constant volume of 1L and pH of 7.2 +/-0.2; 20g of agar was also added to ISP1 solid medium.
(3) And (3) identifying the 16S rDNA sequence of the target strain SH 5. 200 mu L of SH5 strain fermentation liquor for 24h is taken, centrifuged and supernatant is discarded, collected hyphae are suspended in 100 mu L of sterile water and heated to 100 ℃ for 10min, and the obtained solution is directly used as a template for PCR. The general primers are adopted: 27F: 5'-AGAGTTTGATCCTGGCTCAG-3', 1492R: 5'-TACGGTTACCTTGTTACGACTT-3' are provided.
The PCR reaction system is as follows: ddH2O15.00. mu.L, KOD-Plus buffer 2.5. mu.L, dNTP mix (200mmol/L) 2.5. mu.L, primer 27F 1.0. mu.L, primer 1492R 1.0. mu.L, bacterial suspension template 1.0. mu.L, KOD-Plus enzyme 0.5. mu.L, MgSO 24(25mmol/L) 1.5. mu.L. The reaction program was set up as follows: pre-denaturation at 95 ℃ for 5min before entering the thermal cycle (x 30): 94 ℃ for 1 min; 54 ℃ 1min; 72 ℃, 2min, and finally 72 ℃ extension for 10 min. Carrying out 1.2% agarose gel electrophoresis (100V, 30min) on the obtained PCR amplification product, carrying out gel cutting recovery on the PCR product (the size is about 1.5kb), cloning the purified DNA fragment to a pMD18-T vector, transforming the obtained T-A connection product to Escherichia coli DH5 alpha competent cells, detecting the clone by adopting PCR, and carrying out gene sequencing on positive clones.
(4) And (3) establishing an evolutionary tree according to the 16S rDNA sequence of the SH5 strain. Firstly, introducing a measured 16S rDNA sequence into an EzTaxon professional database, performing homologous sequence comparison analysis on line, introducing a result into Cluxtalx 1.83 software for multi-sequence matching arrangement, applying the obtained result to Mega5 software, and establishing a phylogenetic tree by a Neighbor-joining method. The results are shown in FIG. 2, where SH5 strain was integrated with S.badius NRRL B-2567(AY 999783). The strain SH5 is identified as Streptomyces sp and named as marine Streptomyces sp 5.
The preservation information of the marine Streptomyces (Streptomyces sp.) SH5 is as follows: the preservation unit: guangdong province microbial culture Collection (GDMCC), accession number: GDMCC No: 62057, deposit address: the microbiological research institute of Guangdong province, No. 59 building, No. 5 building, Guangdong province, of the Fuli Zhonglu 100, Guangzhou city, the preservation date: 11/12/2021.
The 16S rDNA sequence of the marine Streptomyces (Streptomyces sp.) SH5 is shown as SEQ ID No.1 (1374 bp).
Example 2
The SH5 bacterial strain is fixedly planted in the intestinal tract of the zebra fish and reduces the effect of fixedly planting aeromonas hydrophila in the intestinal tract of the zebra fish.
(1) And directly observing the colonization condition of SH5 in the intestinal tract of the zebra fish by using a fluorescence microscope. First, SH5 was fluorescently labeled with the green fluorescent tracer dye CFDA-SE. Then, the dyed SH5 was applied at 1: 100 concentrations were added to zebra fish aquaculture water treated with PTU (phenylthiourea) to inhibit melanin production in the zebra fish larvae, and the initial concentration was maintained by supplementing fluorescent dye after daily water changes. After 3 days, the zebra fish are continuously raised by using normal culture water (without adding SH5), 5 zebra fish juvenile fishes are randomly selected from each group every 24 hours, and after MS-22 anesthesia, the permanent planting condition of SH5 is observed under a fluorescent microscope by utilizing the optical permeability of the zebra fish juvenile fishes. The results show that SH5 can stay in the zebrafish gut for more than 7 days and is present in greater numbers in the zebrafish foregut and midgut (fig. 4A).
(2) Analysis of SH5 colonization amount in intestinal tract of zebra fish. The RT-PCR technology is adopted to identify the specific number of SH5 colonized in intestinal tracts of zebra fishes at different time points, and the specific steps are as follows: firstly, performing polynucleotide sequence comparison on the 16S rDNA of streptomyces by using ClustalW, designing a specific primer (SH 5-F: 5'-TAACACTCTGTCCCGCATGG-3'; SH 5-R: 5'-TTACCCCACCAACAAGCTGA-3') of SH5 according to conserved and variable regions of the sequence, wherein an amplification region comprises a gamma hypervariable region of the 16S rDNA, and verifying the specificity of the primer SH5-F/R by using escherichia coli, aeromonas hydrophila, vibrio parahaemolyticus and other streptomyces (Streptomyces lividans TK24) and zebra fish, and the result shows that only SH5 is amplified to form a band and no band exists in other regions; as can be seen, primer SH5-F/R has good specificity (FIG. 3). Cloning the specific primer amplified fragment to pMD18-T vector, identifying the concentration of the constructed plasmid, according to the formula Copies/mu L ═ 6.02X 1023XDNA concentration (. mu.L) × 10-9The DNA length was multiplied by 660, and the number of copies of the standard sample was determined. The standard sample is diluted by 10 times of gradient to be used as a template, and a standard curve is drawn. And then, extracting total DNA of the intestinal bacteria of each group of zebra fish by adopting an OMEGA DNA kit, homogenizing the initial concentration, and identifying the specific amount of SH5 permanent planting in intestinal tracts of the zebra fish by using the total DNA as a template and SH5-F/SH5-R as specific primers through an RT-PCR (reverse transcription-polymerase chain reaction) experiment. The reaction system of RT-PCR is as follows: SYBR Green Realtime PCR Master Mix 10. mu.L, primer SH 5-F0.5. mu.L, primer SH 5-R0.5. mu.L, template DNA 1. mu.L, ddH2O8. mu.L. The RT-PCR reaction program was set up as follows: pre-denaturation at 95 ℃ for 5min before entering the thermal cycle (x 40): 30s at 94 ℃; at 58 ℃ for 30 s; 72 ℃ for 20 s. The melting signal collection conditions were set as follows: 65 ℃ to 95 ℃, 0.5 ℃/10 s. As a result, SH5 can stay in intestinal tracts of zebra fish for about 4 days, and the fixed planting quantity is stabilized at about 1X 10 times per fish2.5CFU (fig. 4B).
(3) SH5 inhibiting effect of aeromonas hydrophila on colonizing zebrafish. The RT-PCR technology is adopted to identify the specific effect of SH5 on the intestinal tract of the zebra fish colonized by the aeromonas hydrophila at different time points, and the related steps are as follows: first, DNA fragments were amplified by PCR using Aeromonas hydrophila specific primers (aerA 2-F: 5'-CGCCAGCTGGTCAAGACTGT-3', aerA 2-R: 5'-CCAGTTGGTGGCTGTGTCGT-3') and cloned into pMD18-T vector, and the number of copies of the standard sample was determined according to the method described above to draw a standard curve. And calculating the quantity of aeromonas hydrophila and SH5 of each zebra fish at different time points according to the standard curve. As a result, SH5 can effectively inhibit the colonization of aeromonas hydrophila in the intestinal tract of zebra fish (figure 5).
Example 3
The effect of the SH5 strain on zebrafish immune gene expression.
(1) SH5 pretreats zebra fish. After three days of SH5 culture, according to SH5 fermentation liquor: 1, 1: adding SH5 fermentation liquid into culture water of zebra fish juvenile fish (3dfp) at a ratio of 100, changing water every day, maintaining the original concentration of SH5 fermentation liquid, and treating for 3 d. The control group was supplemented with the same amount of ISP1 liquid medium as the experimental group. Each set was set to 3 replicates.
(2) Challenge experiment of Aeromonas hydrophila. After treating zebrafish for 3d with SH5 fermentation broth, adding Aeromonas hydrophila 12h TSB culture solution for counteracting toxic substance to final concentration of 1 × 108CFU/mL. 50 zebra fish juvenile fishes are taken to extract total RNA in 0h, 6h and 12h after the challenge.
(3) Extracting total RNA of zebra fish.
Sample treatment: zebrafish were washed 3 times with PBS, the PBS was aspirated off, and the zebrafish were mashed with a grinding bar. To the triturated sample was added 1mL Trizol, mixed well and left to stand for 5 min.
Chloroform extraction: to the sample was added 400. mu.L of chloroform, vortexed and vortexed for 30s to mix the solution uniformly, and centrifuged (12000rpm, 10min, 4 ℃). Carefully pipette approximately 400. mu.L of the supernatant into another clean centrifuge tube.
And (3) isopropanol precipitation: add 400. mu.L of isopropanol, mix by inversion, stand for 10min, centrifuge (12000rpm, 10min, 4 ℃ C.), remove supernatant.
Washing with ethanol: the precipitate was washed twice with 75% ethanol and centrifuged to remove the supernatant. The precipitate was placed in the air to allow sufficient evaporation of the ethanol.
Adding a proper amount of RNase-free water into the precipitate for dissolving, detecting the quality of RNA by agarose gel electrophoresis, and identifying the concentration by a nucleic acid quantitative analyzer.
(4) And qRT-PCR is used for detecting the immune gene expression quantity. cDNA was amplified using total RNA as a template and Reverse Transcriptase M-MLV Reverse Transcriptase (Promega). Total RNA was first: 2 μ g, oligo (dT) 18: 0.5 μ g of the mixture was mixed, DEPC-treated water was added to 15 μ L, and then the temperature was raised to 70 ℃ for 5 min. After cooling on ice, M-MLV 5 × Reaction Buffer 5. mu.L, dNTPs mix (10mM) 1. mu.L, M-MLV Reverse Transcriptase 1. mu.L, RNase Inhibitor 1. mu.L, DEPC treated water to 25. mu.L were added to the Reaction system. The reaction procedure was as follows: 42 ℃ of: 1h, 72 ℃: 10min, stop at 4 ℃. The samples were stored at-20 ℃.
The cDNA synthesized by reverse transcription was used as a template, and the expression level of the immune gene such as the level of expression was detected by RT-PCR using specific primers (Table 1). The housekeeping gene EF-1 alpha is used as an internal reference gene, 5 biological repeats are set for each sample, an experimental group without an amplification template is set as a blank control group at the same time, whether the sample is polluted by genome DNA is detected, and a relative fluorescence quantitative PCR experimental system and reaction conditions are the same as above. Experimental results show that after SH5 treatment, after Aeromonas hydrophila is subjected to toxicity attack, the expression levels of immune-related genes such as TLR3, Lysozyme and iNOs of zebra fish are remarkably increased, and inflammatory genes such as 1L-1 beta, 1L-6, MyD88 and TLR4a of zebra fish are remarkably reduced, so that the probiotic strain SH5 has the effects of activating host immunity and reducing inflammatory response (figure 6).
Example 4
The effect of the SH5 strain on the expression of virulence genes of Aeromonas hydrophila.
(1) Establishment of SH5 and aeromonas hydrophila co-culture system
SH5 and Aeromonas hydrophila were cultured by fermentation in ISP1 and TSB liquid media, respectively, to plateau. Then, 1mL of each of the cell suspension was placed in a baffled flask containing 100mL of TSB, incubated at 28 ℃ and 180rpm for 48 hours, and the same volume of ISP1 medium was used in place of the SH5 fermentation broth in the control group. Sampling is carried out at different time points of 0h, 6h, 12h and 24h in the culture process for extracting total RNA.
(2) Total RNA extraction
The specific steps refer to the operation provided by an RNA extraction kit (Beijing Kulaibo Biotech Co.):
and (3) cracking thalli: 1mL of the bacterial suspension sample was centrifuged (5000rpm, 10min) in a 1.5mL EP tube, and the supernatant was discarded. To the resulting precipitate, 0.5mL Trizol was added, mixed well and left at room temperature for about 3min until the bacteria were completely lysed.
Chloroform extraction: to the lysate was added 0.1mL of chloroform, vortexed and mixed for 30 s. Centrifuge at 12000rpm for 3min at room temperature, carefully aspirate the supernatant into a new centrifuge tube.
Ethanol precipitation: adding 200. mu.L of absolute ethyl alcohol into the supernatant, mixing uniformly, and loading into an RNA purification column. Centrifuge at 12000rpm for 1min, discard the permeate. 0.5mL of 75% ethanol was added to the purification column, followed by centrifugation at 12000rpm for 30s at room temperature, the permeate was discarded, and the washing step was repeated twice.
Collecting RNA: the column bound with RNA was centrifuged at 12000rpm for 1min, the core column was transferred to a 1.5mL RNase-free centrifuge tube, 30. mu.L of an RNA lysate was added, and the mixture was left at room temperature for 2min and centrifuged at 12000rpm for 1min to elute RNA. The obtained RNA is subjected to nucleic acid electrophoresis and nucleic acid quantitative analyzer to determine concentration and purity, and then stored at-80 ℃.
(3) And (3) detecting the expression quantity of virulence genes by qRT-PCR. The reverse transcription amplification of cDNA and the qRT-PCR method are as above. The specific primers used are shown in Table 1. The results showed that the expression levels of nine virulence genes, Aeromonas hydrophila aerolysin (aerA), cytoxic enterotoxin (act), heat-stable cytotoxin (ast), hemolysin A (hly), heat lipid cytotoxin (alt), serine protease (ahp), LuxI/R-type virulence regulator (ahyR), outer membrane protein A (ompA), and ferric upper regulator (fur) were significantly reduced when co-cultured with the SH5 strain (FIG. 7).
TABLE 1 primer List
Example 5
The SH5 strain has antagonistic effect on Aeromonas hydrophila.
A7 d SH5 agar block (diameter 8mm) was cultured with A1 solid medium by an agar diffusion method using a puncher with the side with streptomyces growing facing downward, placed on TSB solid medium coated with 100. mu.L of Aeromonas hydrophila, subjected to static culture at 28 ℃ for 24h, and the diameter of the zone of inhibition was measured and recorded, and each experiment was repeated three times. As can be seen from FIG. 8, no zone of inhibition was observed on the culture medium for Aeromonas hydrophila, indicating that SH5 has no antagonistic effect on Aeromonas hydrophila.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Sequence listing
<110> river-south university
<120> strain of marine streptomyces and application thereof in preventing and controlling aeromonas hydrophila infection of aquatic animals
<160> 59
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1374
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> 16S rDNA sequence of Streptomyces sp. SH5 strain
<400> 1
cgaacgatga agccgcttcg gtggtggatt agtggcgaac gggtgagtaa cacgtgggca 60
atctgccctt cactctggga caagccctgg aaacggggtc taataccgga taacactctg 120
tcccgcatgg gacggggttg aaagctccgg cggtgaagga tgagcccgcg gcctatcagc 180
ttgttggtgg ggtaatggcc taccaaggcg acgacgggta gccggcctga gagggcgacc 240
ggccacactg ggactgagac acggcccaga ctcctacggg aggcagcagt ggggaatatt 300
gcacaatggg cgaaagcctg atgcagcgac gccgcgtgag ggatgacggc cttcgggttg 360
taaacctctt tcagcaggga agaagcgaaa gtgacggtac ctgcagaaga agcgccggct 420
aactacgtgc cagcagccgc ggtaatacgt agggcgcaag cgttgtccgg aattattggg 480
cgtaaagagc tcgtaggcgg cttgtcacgt cggatgtgaa agcccggggc ttaaccccgg 540
gtctgcattc gatacgggct agctagagtg tggtagggga gatcggaatt cctggtgtag 600
cggtgaaatg cgcagatatc aggaggaaca ccggtggcga aggcggatct ctgggccatt 660
actgacgctg aggagcgaaa gcgtggggag cgaacaggat tagataccct ggtagtccac 720
gccgtaaacg ttgggaacta ggtgttggcg acattccacg tcgtcggtgc cgcagctaac 780
gcattaagtt ccccgcctgg ggagtacggc cgcaaggcta aaactcaaag gaattgacgg 840
gggcccgcac aagcagcgga gcatgtggct taattcgacg caacgcgaag aaccttacca 900
aggcttgaca tataccggaa agcatcagag atggtgcccc ccttgtggtc ggtatacagg 960
tggtgcatgg ctgtcgtcag ctcgtgtcgt gagatgttgg gttaagtccc gcaacgagcg 1020
caacccttgt tctgtgttgc cagcatgccc ttcggggtga tggggactca caggagactg 1080
ccggggtcaa ctcggaggaa ggtggggacg acgtcaagtc atcatgcccc ttatgtcttg 1140
ggctgcacac gtgctacaat ggccggtaca atgagctgcg atgccgcgag gcggagcgaa 1200
tctcaaaaag ccggtctcag ttcggattgg ggtctgcaac tcgaccccat gaagtcggag 1260
ttgctagtaa tcgcagatca gcattgctgc ggtgaatacg ttcccgggcc ttgtacacac 1320
cgcccgtcac gtcacgaaag tcggtaacac ccgaagccgg tggcccaacc cctt 1374
<210> 2
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> 27F
<400> 2
<210> 3
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> 1492R
<400> 3
tacggttacc ttgttacgac tt 22
<210> 4
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> aerA-F
<400> 4
gagaaggtga ccaccaagaa ca 22
<210> 5
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> aerA-R
<400> 5
arctgacatc ggccttgaac tc 22
<210> 6
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> ast-F
<400> 6
<210> 7
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> ast-R
<400> 7
<210> 8
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> ahp-F
<400> 8
tctatgcgct ggagtcgttc 20
<210> 9
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> ahp-R
<400> 9
<210> 10
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> act-F
<400> 10
<210> 11
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> act-R
<400> 11
<210> 12
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> hly-F
<400> 12
<210> 13
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> hly-R
<400> 13
<210> 14
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> ompA-F
<400> 14
<210> 15
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> ompA-R
<400> 15
<210> 16
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> ompW-F
<400> 16
<210> 17
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> ompW-R
<400> 17
<210> 18
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> fur-F
<400> 18
<210> 19
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> fur-R
<400> 19
cggagaactc gatcaccttg 20
<210> 20
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> ahyR-F
<400> 20
<210> 21
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> ahyR-R
<400> 21
<210> 22
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> alt-F
<400> 22
tggatgccga gcagaacat 19
<210> 23
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> alt-R
<400> 23
<210> 24
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> ela-F
<400> 24
<210> 25
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> ela-R
<400> 25
<210> 26
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> EF1α-F
<400> 26
aacagctgat cgttggagtc aa 22
<210> 27
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> EF1α-R
<400> 27
ttgatgtatg cgctgacttc ct 22
<210> 28
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> IL-1β-F
<400> 28
tggacttcgc agcacaaaat g 21
<210> 29
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> IL-1β-R
<400> 29
<210> 30
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> IL-6-F
<400> 30
tcaacttctc cagcgtgatg 20
<210> 31
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> IL-6-R
<400> 31
tctttccctc ttttcctcct g 21
<210> 32
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Lysozyme-F
<400> 32
cgtggatgtc ctcgtgtgaa g 21
<210> 33
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Lysozyme-R
<400> 33
<210> 34
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> TLR1-F
<400> 34
cagagcgaat ggtgccacta t 21
<210> 35
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> TLR1-R
<400> 35
<210> 36
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> TLR3-F
<400> 36
tggagcatca cagggataaa ga 22
<210> 37
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> TLR3-R
<400> 37
<210> 38
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> SOD1-F
<400> 38
<210> 39
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> SOD1-R
<400> 39
tgtcagcggg ctagtgctt 19
<210> 40
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Gpx1a-F
<400> 40
<210> 41
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Gpx1a-R
<400> 41
<210> 42
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> HSP70-F
<400> 42
aagcgacgaa ggatgcagga g 21
<210> 43
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> HSP70-R
<400> 43
<210> 44
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> TLR4a-F
<400> 44
tttcacaaga acaagccttt g 21
<210> 45
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> TLR4a-R
<400> 45
<210> 46
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> IAP-F
<400> 46
atgggagtgc cacggtttca g 21
<210> 47
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> IAP-R
<400> 47
cgatgccaac agactttcct tg 22
<210> 48
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Bcl-2-F
<400> 48
aggaaaatgg aggttgggat g 21
<210> 49
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Bcl-2-R
<400> 49
tgttaggtat gaaaacgggt gga 23
<210> 50
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> TNFα-F
<400> 50
<210> 51
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> TNFα-R
<400> 51
<210> 52
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> iNOs-F
<400> 52
ggagatgcaa ggtcagcttc 20
<210> 53
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> iNOs-R
<400> 53
ggcaaagctc agtgacttcc 20
<210> 54
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> MyD88-F
<400> 54
<210> 55
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> MyD88-R
<400> 55
<210> 56
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> aerA2-F
<400> 56
<210> 57
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> aerA2-R
<400> 57
<210> 58
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> SH5-F
<400> 58
<210> 59
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> SH5-R
<400> 59
Claims (9)
1. A strain of marine streptomyces, which is characterized in that: the strain is named as marine Streptomyces sp SH5, and is preserved in Guangdong province microorganism strain preservation center of Guangdong province microorganism research institute No. 59 building, No. 5 building, Guangdong province, No. 100 institute of Mieli Zhou, Guangzhou, Guangdong province, 11.12 days in 2021, with the preservation number: GDMCC No: 62057.
2. a biological agent prepared based on the marine Streptomyces according to claim 1.
3. The biological agent according to claim 2, characterized in that:
the biological agent is prepared by liquid culture of the marine streptomyces as claimed in claim 1.
4. The biological agent according to claim 3, characterized in that:
inoculating the marine streptomyces of claim 1 into ISP1 liquid culture medium for culture to obtain the biological preparation.
5. The biological agent according to claim 4, characterized in that:
the culture is carried out at the temperature of 28-30 ℃ and the rpm of 180-200 for 48-72 h.
6. Use of a marine streptomyces as claimed in claim 1 or a biological agent as claimed in any one of claims 2 to 5, wherein: the application is one of the following applications:
1) the marine streptomyces or the biological preparation are applied to preventing and controlling aeromonas hydrophila from infecting aquatic animals;
2) the marine streptomyces or the biological preparation are applied to the analysis of the intestinal colonization ability of aquatic animals;
3) the marine streptomyces or the biological agent are applied to improving the immune response of aquatic animals;
4) the marine streptomyces or the biological agent is applied to the regulation of aeromonas hydrophila in the process of inflammatory reaction of aquatic animals;
5) the marine streptomyces or the biological preparation is applied to inhibiting the intestinal colonization of aquatic animals by aeromonas hydrophila;
6) the marine streptomycete or the biological preparation is applied to the reduction of inflammatory reaction of low aquatic animals under the stress of pathogenic bacteria;
7) the marine streptomycete or the biological preparation is applied to inhibiting the production of aeromonas hydrophila virulence factors and the expression of virulence genes.
7. Use according to claim 6, characterized in that:
the aquatic animal is zebra fish.
8. Use according to claim 6 or 7, characterized in that:
the pathogenic bacteria are aeromonas hydrophila.
9. Use according to claim 6 or 7, characterized in that:
the virulence genes are aerA, act, ast, hlyA, alt, ahp, ahyR, ompA and fur.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111640257.XA CN114292785B (en) | 2021-12-29 | 2021-12-29 | Marine streptomycete and application thereof in preventing and controlling aeromonas hydrophila from infecting aquatic animals |
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| Application Number | Priority Date | Filing Date | Title |
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| CN202111640257.XA CN114292785B (en) | 2021-12-29 | 2021-12-29 | Marine streptomycete and application thereof in preventing and controlling aeromonas hydrophila from infecting aquatic animals |
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| CN114292785B CN114292785B (en) | 2023-07-28 |
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Citations (6)
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| CN101497867A (en) * | 2009-02-18 | 2009-08-05 | 淮海工学院 | Isolated culture method and use of Streptomycete from sea |
| CN104087523A (en) * | 2014-01-20 | 2014-10-08 | 中国科学院南海海洋研究所 | Marine Streptomyces, method for preparation of Enterocin therefrom and application of the same |
| CN106472932A (en) * | 2016-10-18 | 2017-03-08 | 李德田 | A kind of complex microorganism beverage and preparation method thereof |
| CN111979150A (en) * | 2020-08-21 | 2020-11-24 | 华南农业大学 | Marine streptomyces and isolated culture method and application thereof |
| WO2021209820A1 (en) * | 2020-04-15 | 2021-10-21 | Institut Pasteur | Bacterial strains for use as probiotics, compositions thereof, deposited strains and method to identify probiotic bacterial strains |
| CN113621533A (en) * | 2021-07-09 | 2021-11-09 | 湖南师范大学 | Streptomyces rubiginosus Z1-26, microecological preparation and preparation method thereof |
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2021
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| CN101497867A (en) * | 2009-02-18 | 2009-08-05 | 淮海工学院 | Isolated culture method and use of Streptomycete from sea |
| CN104087523A (en) * | 2014-01-20 | 2014-10-08 | 中国科学院南海海洋研究所 | Marine Streptomyces, method for preparation of Enterocin therefrom and application of the same |
| CN106472932A (en) * | 2016-10-18 | 2017-03-08 | 李德田 | A kind of complex microorganism beverage and preparation method thereof |
| WO2021209820A1 (en) * | 2020-04-15 | 2021-10-21 | Institut Pasteur | Bacterial strains for use as probiotics, compositions thereof, deposited strains and method to identify probiotic bacterial strains |
| CN111979150A (en) * | 2020-08-21 | 2020-11-24 | 华南农业大学 | Marine streptomyces and isolated culture method and application thereof |
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| CN114292785B (en) | 2023-07-28 |
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