Use of a new strain of streptomyces D67 as a bacteriostatic agent

Abstract

The application discloses application of Streptomyces sp. D67 as a bacteriostatic agent. A new actinomycete Streptomyces sp. D67 is obtained by separation, screening and identification from Kumutage desert soil in Xinjiang, and the bacterium has significant bacteriostatic properties, and has good bacteriostatic activity on Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli and Erwinia. The bacteriostatic agent of Streptomyces sp. D67 is diluted to 5 times the volume, and the inhibition rate of the bacteriostatic agent on the growth of Chromobacterium violaceum CV026 reaches 85.6%; the bacteriostatic agent is diluted to 2 times the volume, and the inhibition rate of the bacteriostatic agent on the growth of Pseudomonas aeruginosa reaches 84%; the bacteriostatic agent is diluted to 5 times the volume, and the inhibition rate of the bacteriostatic agent on the growth of Erwinia reaches 74.3%; and it is shown that the Streptomyces sp. D67 provided by the application as the bacteriostatic agent has good development and application prospects in the fields of biological pesticides, microbial preparations and other antibacterial related fields.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of microbial strains and their applications, and in particular to the application of a new strain Streptomyces D67 as a bacteriostatic agent. BACKGROUND

[0002] Plant diseases have always been a serious threat in agricultural production. Global climate change has brought serious epidemics to food crops, posing a huge threat to future food security. For example, Pseudomonas aeruginosa and Staphylococcus aureus are widely present in soil, crops, drinking water, and livestock, and are extremely easy to infect humans; Erwinia amylovora can cause pear fire blight and has a wide host range, which can harm more than 220 species of plants in more than 40 genera, such as pear, apple, hawthorn, wood jujube, and plum; Escherichia coli directly used can lead to the spread of plant diseases and pests, and produce toxic gases to cause plant root rot and seedling burn, etc. Crop protection is an important means to ensure the sustainable development of agriculture. The research of agricultural antibiotics in China began in the 1950s, and more than ten kinds of agricultural antibiotics such as Jingangmycin, Qingliangmycin, Difangmycin, Gongzhulingmycin, Jingfengmycin, and Zhongshengmycin were successfully developed, providing a strong guarantee for the prevention and control of crop diseases and pests and the high yield of crops. However, with the widespread use of the above-mentioned agricultural antibiotics, the drug resistance of related pathogens has increased, and it is increasingly important to find new agricultural bacteriostatic agents and biologically control plant diseases.

[0003] Actinomycetes have always been an important source of natural active metabolites. Since the British Fleming discovered penicillin in 1929, according to incomplete statistics, there are 33,500 natural active products from microorganisms, of which 13,700 are from actinomycetes, accounting for about 41%, and about 80% of them are from Streptomyces. The desert extreme environment has characteristics such as extreme drought, strong solar ultraviolet radiation, and extreme temperature changes in day and night cycles, which breeds a wealth of extreme environment microorganisms, among which actinomycetes are an important part. Related studies have shown that the metabolites of actinomycetes in arid regions exhibit high-temperature tolerance and easy solubility, and have broad application prospects in the fields of medicine and health, agricultural and pastoral production, and food processing. The Kumtag Desert is located in the eastern part of Xinjiang, south of Shanshan County, and is part of the Taklimakan Desert. It is hot and dry in summer, with a maximum temperature of 49.6°C, and the surface temperature can reach 82.5°C. In winter, the minimum temperature is generally -10°C, and the extreme minimum temperature can reach -29°C. There is occasional snow in the whole year, and its ecological environment is extremely special, providing a theoretical basis for mining related actinomycetes and developing related bacteriostatic agents. However, there are few reports on actinomycetes in this region and the development of related bacteriostatic agents in China, and it is necessary to continuously explore and find better strains and applications. SUMMARY

[0004] Aiming at the major demand of plant disease control and the advantage of actinomycete strains in extreme environment in natural product mining, whether new strains with high efficient bacteriostatic property and safe and reliable can be screened from extreme environment to obtain high efficient bacteriostatic agent needs to be researched, and the application of the new bacterium Streptomyces sp.D67 as a bacteriostatic agent is provided, the new bacterium Streptomyces sp.D67 with high yield of bacteriostatic activity is obtained by separation and screening from the soil of Kumtag Desert in Xinjiang, the bacterium has significant bacteriostasis, and has good bacteriostatic activity on Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli and Erwinia. The inhibition rate of Streptomyces sp.D67 bacteriostatic agent on the growth of Chromobacterium violaceum CV026 reaches 85.6% when diluted to 5 times the volume, the inhibition rate on the growth of Pseudomonas aeruginosa reaches 84% when diluted to 2 times the volume, and the inhibition rate on the growth of Erwinia reaches 74.3% when diluted to 5 times the volume, which shows that the Streptomyces sp.D67 bacteriostatic agent provided in the application has good development and application prospect in the fields of biological pesticides, microbial preparations and other antibacterial related fields.

[0005] In order to achieve the above technical effects, the application realizes the technical scheme as follows.

[0006] The application provides the application of the new bacterium Streptomyces sp.D67 as a bacteriostatic agent.

[0007] Specifically, the application provides the application of the new bacterium Streptomyces sp.D67 as a bacteriostatic agent, and the application of the new bacterium Streptomyces sp.D67 as a bacteriostatic agent in medicine and agriculture, which has good application value.

[0008] The application provides the application of the new bacterium Streptomyces sp.D67 as a bacteriostatic agent, and the application of the new bacterium Streptomyces sp.D67 as a bacteriostatic agent, which has good application value.

[0009] The new bacterium Streptomyces sp.D67 has been preserved in China Center for Type Culture Collection (CCTCC), and the strain preservation number is CCTCC NO: M 20221542; the gene sequence of Streptomyces sp.D67 is shown as SEQ ID NO:1.

[0010] The preparation process of the new bacteriostatic agent of Streptomyces sp. D67 is as follows: the preserved single colony of actinomycete new strain Streptomyces sp. D67 is inoculated into ISP2 liquid culture medium, and cultured at 30 DEG C and 150 rpm for 2 days to obtain Streptomyces sp. D67 seed liquid; the Streptomyces sp. D67 seed liquid is inoculated into Gao's No. 1 liquid culture medium at a volume ratio of 1%, and fermented and cultured at 30 DEG C and 150 rpm for 7 days to obtain Streptomyces sp. D67 fermentation liquid; after centrifugation and filtration to remove bacteria, the Streptomyces sp. D67 bacteriostatic agent is obtained, and stored at -20 DEG C for standby.

[0011] The preserved strain Streptomyces sp. D67 adopted in the application is screened and separated from a soil sample in the Kumtag region of Xinjiang, belongs to the genus Streptomyces through 16S rRNA gene sequence phylogenetic analysis and morphological analysis, and is determined as a new strain of the genus Streptomyces through gene sequencing of the strain, BLAST comparison analysis of the obtained sequence on the NCBI website, and the highest homology of the 16S rRNA gene sequence of Streptomyces sp. D67 with the standard model bacteria Streptomyces ardesiacus NBRC 15402 T , Streptomyces heliomycini NBRC 15899 T and Streptomyces pluripotens MUSC 135 T , and the similarity is 98.39%, 98.11% and 98.03% respectively. The 16S rRNA phylogenetic tree is constructed by selecting the sequences with higher homology, Streptomyces sp. D67 and Streptomyces pluripotens MUSC 135 T (CP021080) are more close in a branch. After a series of polyphasic classification and identification, Streptomyces sp. D67 is obviously different from Streptomyces ardesiacus NBRC 15402 T , Streptomyces heliomycini NBRC 15899 T and Streptomyces pluripotens MUSC 135 T , and can be determined as a new strain of the genus Streptomyces, and has the typical characteristics of a new strain.

[0012] The Streptomyces D67 adopted in the present application is identified by the above-mentioned commonly recognized strain system molecular level identification in the art, combined with morphological identification and physiological and biochemical characteristic identification analysis, and the present application relates to Streptomyces D67 which is gram-stained positive, the colony size is 0.2 cm-0.6 cm x 0.2 cm-0.6 cm, it is combined with culture medium tightly, the texture is dense, the aerial hypha is white and fluffy, the spore is white, and the intracellular hypha is light yellow.

[0013] The protease, amylase and cellulase activities of the Streptomyces D67 adopted in the present application are all positive; the Streptomyces D67 adopted in the present application can utilize the following sugar carbon sources: dextrin, xylose, pine disaccharide, sucrose, inositol, L-arabinose, D-arabitol, D-fructose, D-galactose, D-glucose and L-rhamnose. Through strain physiological and biochemical characteristic analysis, it is obviously different from the adjacent strains Streptomyces ardesiacus NBRC 15402 T , Streptomyces heliomycini NBRC 15899 T and Streptomycespluripotens MUSC 135 T .

[0014] The above-mentioned strain identification, as well as the commonly recognized strain system molecular level identification and strain physiological and biochemical system test verification in the art, confirm that the strain number D67 obtained is a typical new strain Streptomyces sp. D67 in the genus Streptomyces, and the strain has been preserved in the International Microorganism Preservation Unit of the Budapest Treaty: China Typical Culture Collection Center (CCTCC), address: Wuhan, Wuhan University, China, postcode: 430072, preservation number: CCTCC NO: M 20221542, and the preservation date is October 7, 2022.

[0015] The separation culture medium of the Streptomyces D67 adopted in the present application is: soluble starch 20 g / L, KNO3 1 g / L, K2HPO4 0.5 g / L, MgSO4·7H2O 0.5 g / L, NaCl 0.5 g / L, FeSO4·7H2O 0.01 g / L, agar 18 g / L, 10% soil extract 1000 mL, pH 7.5.

[0016] The purified culture medium of the Streptomyces D67 used in the application is: soluble starch 10.0 g / L, K2HPO4 1.0 g / L, MgSO4·7H2O 1.0 g / L, NaCl 1.0 g / L, (NH4)2SO4 2.0 g / L, CaCl2 2.0 g / L, FeSO4·7H2O 0.001 g / L, MnSO4 0.001 g / L, ZnSO4·7H2O 0.001 g / L, agar 15.0 g / L, distilled water 1.0 L, pH 7.2.

[0017] The following beneficial effects can be obtained by implementing the above specific technical solutions of the application.

[0018] The application provides the application of the new bacteria Streptomyces sp. D67 as an antibacterial agent. The new bacteria Streptomyces sp. D67 provided has significant antibacterial activity, and has significant antibacterial activity on Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli, and Erwinia. When the concentration of the antibacterial agent prepared by using the new bacteria Streptomyces sp. D67 is 10 mg / mL, the inhibition rate of the antibacterial agent on the growth of Chromobacterium violaceum CV026 reaches 85.6%; when the concentration of the antibacterial agent is 20 mg / mL, the inhibition rate of the antibacterial agent on the growth of Pseudomonas aeruginosa reaches 84%; and when the concentration of the antibacterial agent is 2 mg / mL, the inhibition rate of the antibacterial agent on the growth of Erwinia reaches 74.3%. It is shown that the Streptomyces sp. D67 provided as an antibacterial agent has good antibacterial capacity, and has good development and application prospects in the fields of biological pesticides, microbial preparations and other antibacterial related fields. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 A phylogenetic tree based on 16S rRNA sequences of Streptomyces sp. D67 is shown.

[0020] Figure 2 A colony morphology of Streptomyces sp. D67 is shown.

[0021] Figure 3 Effects of the antibacterial agent of Streptomyces sp. D67 on Escherichia coli, Staphylococcus aureus, Erwinia and Pseudomonas aeruginosa are shown.

[0022] Figure 4 Effects of antibacterial agents of Streptomyces sp. D67 with different concentrations on Chromobacterium violaceum CV026 are shown.

[0023] Figure 5The effect of Streptomyces sp. D67 bacteriostatic agent at different concentrations on Pseudomonas aeruginosa is shown.

[0024] Figure 6 The effect of Streptomyces sp. D67 bacteriostatic agent at different concentrations on Erwinia is shown. DETAILED DESCRIPTION

[0025] Hereinafter, the present application is illustrated by examples, but the present application is not limited to the following examples. All raw and auxiliary materials selected in the present application, and the selected strain culture method are well known in the art, and the % involved in the present application is mass percentage, unless otherwise specified.

[0026] In order to better explain the present application, the main content of the present application is further illustrated by combining specific examples, but the content of the present application is not limited to the following examples. If not specifically indicated, the technical means used in the examples is the conventional means familiar to those skilled in the art, and the raw materials used are commercial products.

[0027] The Gao's No. 1 medium of the present application: soluble starch 20 g / L, KNO3 1.0 g / L, K2HPO4 0.5 g / L, MgSO4·7H2O 0.5 g / L, NaCl 0.5 g / L, FeSO4·7H2O 0.01 g / L, agar 15 g / L, pure water 1000 mL, pH 7.4-7.6.

[0028] The LB medium (g / L) of the present application: tryptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, agar 15 g / L, pH 7.0.

[0029] Example 1: Preparation of Streptomyces sp. D67 bacteriostatic agent

[0030] I. Isolation, purification and identification of Streptomyces sp. D67

[0031] The actinomycetes were isolated by plate dilution method. The collected Xinjiang Kumutage desert soil sample was subjected to microbial isolation and purification by gradient dilution method. 5 g of soil sample was weighed and placed in 45 mL of sterile water, incubated at 150 r / min and 30℃ for 12 h; standard gradient dilution was used, and the dilution was prepared into 10 -1 -10 -3 Concentration of dilution; 100 μL of each concentration of dilution was taken and coated on the isolation medium plate by conventional coating method, and placed in a 30℃ constant temperature incubator for culture; after one week, the single colonies on the plate were picked and purified for culture; the purified single colonies were transferred to Gao's No. 1 medium slant for preservation.

[0032] (ii) 16S rRNA gene identification

[0033] 1. Extraction of PCR template DNA

[0034] The purified Streptomyces sp. D67 was inoculated into Gao's No. 1 liquid medium and cultured at 37°C for 2d. The bacterial cells were collected and the total genomic DNA was extracted using a DNA extraction kit.

[0035] 2. PCR amplification

[0036] 16S rRNA gene sequence primers:

[0037] 27F: 5'-AGAGTTTGATCCTGGCTCAG-3';

[0038] 1492R: 5'-GGTTACCTTGTTACGACTT-3';

[0039] The total volume of the PCR reaction system was 25μL. The PCR amplification conditions were as follows: pre-denaturation at 94°C for 5min; denaturation at 94°C for 30s, annealing at 54°C for 30s, extension at 72°C for 1min 30s, 30 cycles; and extension at 72°C for 7min.

[0040] 3. Sequence determination

[0041] The PCR amplification product was sequenced after electrophoretic detection and purification. The sequence length was 1421bp. The test results are shown in SEQ ID No: 1. BLAST homologous sequence retrieval was performed on NCBI. A phylogenetic tree was established by the Neighbor-Joining method using the commonly used MEGA 7.0 software in the art (1000 times of repeated sampling). The results are shown in the accompanying Figure 1 The sequence obtained was analyzed by comparison on the NCBI website. It was found that Streptomyces sp. D67 had the highest homology with Streptomyces ardesiacus NBRC15402 T , Streptomyces heliomycini NBRC15899 T and Streptomycespluripotens MUSC 135 T , with similarities of 98.39%, 98.11% and 98.03%, respectively. The 16S rRNA phylogenetic tree was constructed by selecting the sequences with higher homology. Streptomyces sp. D67 was more closely related to Streptomycespluripotens MUSC 135 T(CP021080) are more closely related to each other. After a series of polyphasic taxonomic identification, Streptomyces sp. D67 is more closely related to Streptomyces ardesiacus NBRC 15402 T , Streptomyces heliomycini NBRC 15899 T and Streptomyces pluripotens MUSC 135 T There are obvious differences, which prove that the obtained Streptomyces sp. D67 belongs to a typical new species within the genus Streptomyces.

[0042] (III) Colony morphological characteristics of Streptomyces sp. D67

[0043] Streptomyces D67 can grow well on ISP2, Gause I, Bennett agar and other culture media. Streptomyces D67 to be observed is inoculated on Gause I plate and cultured at 30℃ for 4 days. The colony characteristics are observed, recorded and photographed, and the scanning electron microscope photos of the colony are recorded. The results are shown in the following table and the photos in the accompanying drawings. Figure 2

[0044] From the accompanying Figure 2 results, it can be seen that the Streptomyces D67 provided by the present application is a gram-positive bacterium, and the colony size is 0.2cm-0.6cm x 0.2cm-0.6cm, which is tightly combined with the culture medium, and the texture is dense, the aerial hyphae are white and fluffy, the spores are white, and the subterranean hyphae are light yellow.

[0045] Based on the above biological characteristics, the above-mentioned Streptomyces D67 is identified as Streptomyces sp. belonging to a new strain of the genus Streptomyces. The strain has been preserved in the International Depository Authority of the Budapest Treaty on Microorganisms: China Center for Type Culture Collection (CCTCC), address: Wuhan University, Wuhan, China, postcode: 430072, preservation date: October 7, 2022, preservation number: CCTCC NO: M 20221542.

[0046] (IV) Physiological and biochemical characteristics of Streptomyces sp. D67

[0047] ​Streptomyces D67 was inoculated onto Gao's No. 1 medium, and its physiological and biochemical characteristics were tested. Analysis of growth temperature, salinity, pH range, gelatin liquefaction, nitrate reduction, hydrogen peroxide production, urease, amylase, esterase, cellulose decomposition, and carbon source utilization was mainly based on the *Handbook of Systematic Identification of Common Bacteria*. Streptomyces D67 showed positive results for protease, amylase, and cellulase activities. The carbohydrate carbon sources that Streptomyces D67 can utilize are dextrin, xylose, malinosate, sucrose, inositol, L-arabinose, D-arabinitol, D-fructose, D-galactose, D-glucose, and L-rhamnose. Antimicrobial assays showed that Streptomyces D67 exhibited some resistance to *Escherichia coli*, *Pseudomonas aeruginosa*, *Erwinia*, and *Staphylococcus aureus*.

[0048] Based on comprehensive analysis of 16S rRNA gene sequence homology, morphological identification, and comparison of physiological and biochemical characteristics, this Streptomyces D67 is clearly different from the standard type strain, thus identifying it as a new Streptomyces species with the characteristics of a new species within the same genus of Streptomyces.

[0049] (V) Preparation of Streptomyces sp. D67 antibacterial agent

[0050] A single colony of the preserved new actinomycete *Streptomyces* sp. D67 was inoculated into ISP2 liquid medium and cultured at 30°C and 150 rpm for 2 days to obtain *Streptomyces* sp. D67 seed culture. The *Streptomyces* sp. D67 seed culture was then inoculated into Gao's No. 1 liquid medium at a volume ratio of 1% and fermented at 30°C and 150 rpm for 7 days to obtain *Streptomyces* sp. D67 fermentation broth. After centrifugation and filtration to remove bacteria, the *Streptomyces* sp. D67 antibacterial agent was obtained and stored at -20°C for later use.

[0051] Example 2: Antibacterial activity of Streptomyces sp. D67 antibacterial agent

[0052] Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli, and Candida albicans were inoculated into 100 mL Erlenmeyer flasks containing 50 mL of LB liquid medium and cultured at 30 °C until the mid-to-late logarithmic phase. 1 mL of indicator bacteria was added to a Petri dish, followed by 25 mL of LB solid medium (approximately 50 °C), and the mixture was quickly mixed. After the medium solidified, it was placed in an 8 mm outer diameter Oxford cup. The prepared Streptomyces sp. D67 antibacterial agent was added to the cup, and the mixture was incubated at 30 °C for 12 h. The inhibition diameter was calculated to determine its antibacterial activity. Specific results are shown in the appendix. Figure 3 As shown.

[0053] From the appendix Figure 3The results show that the Streptomyces sp. D67 bacteriostatic agent provided by the application has significant inhibitory effect on Escherichia coli, Erwinia, Staphylococcus aureus and Pseudomonas aeruginosa, and the inhibitory effect on Escherichia coli is relatively strong, the bacteriostatic diameter is 2.52 cm, the bacteriostatic diameter of Erwinia is 2.21 cm, the bacteriostatic diameter of Staphylococcus aureus is 1.69 cm, and the bacteriostatic diameter of Pseudomonas aeruginosa is 0.8 cm, but the Streptomyces sp. D67 bacteriostatic agent has no inhibitory effect on fungi in the experiment, which shows that the Streptomyces sp. D67 bacteriostatic agent provided by the application has wide inhibitory bacterial activity.

[0054] Example 3: Antimicrobial activity of Streptomyces sp. D67 as a bacteriostatic agent

[0055] (1) Effect of Streptomyces sp. D67 bacteriostatic agent on the growth of purple bacillus CV026

[0056] The 96-well plate method was used, and the logarithmic middle and late stage of purple bacillus CV026 was inoculated, and the bacteriostatic agent of Streptomyces sp. D67 was inoculated into LB liquid medium at a mass volume ratio of 2% inoculation amount at different concentrations, wherein the bacteriostatic agent of Streptomyces sp. D67 was diluted to 10 times, 5 times, 2 times and 1 time, and the same volume of Gao's first medium was added as a control, and the same volume of sterile water was added as a positive control, and 3 parallel samples were set, and placed in an enzyme marker for 24h of 30℃ shaking culture, and the OD590 value was measured every 2h to determine the bacteriostatic effect of different concentrations of Streptomyces sp. D67 bacteriostatic agent, and the specific results are shown in the accompanying Figure 4 .

[0057] From the accompanying Figure 4 data, it can be seen that the high concentration of Streptomyces sp. D67 bacteriostatic agent has obvious inhibitory effect on purple bacillus, but the inhibitory effect on the bacteria is weakened at low concentration, when the Streptomyces sp. D67 bacteriostatic agent is diluted 2 times, the growth of purple bacillus CV026 is significantly inhibited, and the inhibition rate reaches 85.6%, and the growth of purple bacillus CV026 is still inhibited when the Streptomyces sp. D67 bacteriostatic agent is diluted to 5 times, which shows that the Streptomyces sp. D67 bacteriostatic agent provided by the application has significant inhibitory effect on purple bacillus CV026.

[0058] (2) Effect of Streptomyces sp. D67 bacteriostatic agent on the growth of Pseudomonas aeruginosa

[0059] The 96-well plate method was used to inoculate the logarithmic phase of Pseudomonas aeruginosa, and the inoculation amount was 2% (mass / volume). Different concentrations of Streptomyces sp. D67 bacteriostatic agent were added to the LB liquid medium, and the bacteriostatic agent was diluted to 10 times, 5 times, 2 times, and 1 time, respectively. The same volume of Gao's No. 1 medium was added as a control, and the same volume of sterile water was added as a positive control. Each group had 3 parallel samples, which were placed in the enzyme marker and shaken at 30°C for 24 hours. The OD value was measured every 2 hours to determine the bacteriostatic effect of different concentrations of Streptomyces sp. D67 bacteriostatic agent. The specific results are shown in the accompanying 590 . Figure 5

[0060] From the accompanying Figure 5 data, it can be seen that high concentrations of Streptomyces sp. D67 bacteriostatic agent have a significant inhibitory effect on the growth of Pseudomonas aeruginosa, and low concentrations of Streptomyces sp. D67 bacteriostatic agent have a weak inhibitory effect on the bacteria. Streptomyces sp. D67 bacteriostatic agent was diluted 2 times, which significantly inhibited the growth of Pseudomonas aeruginosa, with an inhibition rate of 84%. Streptomyces sp. D67 bacteriostatic agent was diluted 5 times, which weakly inhibited the growth of Pseudomonas aeruginosa, and as the concentration of Streptomyces sp. D67 bacteriostatic agent decreased, the inhibition rate of Pseudomonas aeruginosa also gradually decreased.

[0061] (3) Effect of Streptomyces sp. D67 bacteriostatic agent on the growth of Escherichia coli

[0062] The 96-well plate method was used to inoculate the logarithmic phase of Pseudomonas aeruginosa, and the inoculation amount was 2% (mass / volume). Different concentrations of Streptomyces sp. D67 bacteriostatic agent were added to the LB liquid medium, and the bacteriostatic agent was diluted to 10 times, 5 times, 2 times, and 1 time, respectively. The same volume of Gao's No. 1 medium was added as a control, and the same volume of sterile water was added as a positive control. Each group had 3 parallel samples, which were placed in the enzyme marker and shaken at 30°C for 24 hours. The OD value was measured every 2 hours to determine the bacteriostatic effect of different concentrations of Streptomyces sp. D67 bacteriostatic agent. The specific results are shown in the accompanying Figure 6 .

[0063] From the accompanying Figure 6 ​The results show that the high concentration of Streptomyces sp. D67 bacteriostatic agent has a significant inhibitory effect on the growth of E. coli, the low concentration of Streptomyces sp. D67 bacteriostatic agent has no obvious inhibitory effect on the growth of E. coli, the inhibitory rate of Streptomyces sp. D67 bacteriostatic agent on the growth of E. coli reaches 74.3% when the Streptomyces sp. D67 bacteriostatic agent is diluted by 5 times, and the inhibitory effect of Streptomyces sp. D67 bacteriostatic agent on the growth of E. coli is weak when the Streptomyces sp. D67 bacteriostatic agent is diluted by 10 times.

[0064] The data show that the Streptomyces sp. D67 provided by the application has a significant bacteriostatic effect, and has good bacteriostatic activity on P. aeruginosa, S. aureus, E. coli and E. coli. When the Streptomyces sp. D67 is used as a bacteriostatic agent and diluted to 5 times the volume, the inhibitory rate of Streptomyces sp. D67 on the growth of P. aeruginosa reaches 85.6%; when the Streptomyces sp. D67 bacteriostatic agent is diluted to 2 times the volume, the inhibitory rate of Streptomyces sp. D67 on the growth of P. aeruginosa reaches 84%; when the Streptomyces sp. D67 is used as a bacteriostatic agent and the prepared Streptomyces sp. D67 bacteriostatic agent is diluted to 5 times the volume, the inhibitory rate of Streptomyces sp. D67 on the growth of E. coli reaches 74.3%, which shows that the application of Streptomyces sp. D67 as a bacteriostatic agent has very good application value, and has good development and application prospect in the fields of biological pesticides, microbial preparations and other antibacterial related fields.

[0065] In summary, the above-described embodiments only describe the preferred embodiments of the present application, and do not limit the scope of the present application. Without departing from the spirit of the present application, various modifications and improvements of the technical solutions of the present application made by those skilled in the art shall fall within the protection scope of the present application.

Claims

1. Use of Streptomyces sp. D67 as a bacteriostatic agent, characterized in that, The new Streptomyces sp. D67 is preserved in China Center for Type Culture Collection (CCTCC), and the strain preservation number is CCTCC NO: M 20221542; the gene sequence of the new Streptomyces sp. D67 is shown in SEQ ID NO: 1; the bacteriostatic agent inhibits the activity of Pseudomonas aeruginosa, Chromobacterium violaceum and Erwinia; and the application is a non-therapeutic and diagnostic application.

2. Use of Streptomyces sp. D67 according to claim 1 as a bacteriostatic agent, characterized in that, The application of the new Streptomyces sp. D67 as a bacteriostatic agent in biological pesticides and microbial agents.

3. Use of Streptomyces sp. D67 according to claim 1 as a bacteriostatic agent, characterized in that, The bacteriostatic agent is obtained by fermentation of the new Streptomyces sp. D67.

4. Use of the novel Streptomyces sp. D67 according to any one of claims 1 or 2 as a bacteriostatic agent, characterized in that, The preparation process of the bacteriostatic agent is as follows: the preserved single colony of the new Streptomyces sp. D67 is inoculated into IPS2 liquid medium, and cultured at 30 DEG C and 150 rpm for 2 days to obtain the seed liquid of the new Streptomyces sp. D67; the seed liquid of the new Streptomyces sp. D67 is inoculated into Gao's No. 1 liquid medium at a volume ratio of 1% to be inoculated, and fermented and cultured at 30 DEG C and 150 rpm for 7 days to obtain the fermentation liquid of the new Streptomyces sp. D67; the fermentation liquid is centrifuged and filtered to remove bacteria, and the new Streptomyces sp. D67 bacteriostatic agent is obtained, which is stored at -20 DEG C for standby.

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