Mesoxin-producing strain and application thereof

Abstract

This invention relates to a streptomycin-producing strain and its application, wherein the strain is Streptomyces. Streptomyces sp. ZS-2 has good activity in producing kasugamycin, and its fermentation products have higher antibacterial activity and a wider antibacterial range.

Description

Technical Field

[0001] This invention belongs to the field of agricultural technology, specifically relating to a streptomycin-producing strain and its application. Background Technology

[0002] Streptomycin is an agricultural antibiotic fungicide independently developed in my country. It kills pathogens by inhibiting their protein synthesis and has the advantages of low toxicity and low residue. It can prevent and control a variety of crop diseases.

[0003] Streptomycin is a multi-component mixture produced by the fermentation of Streptomyces. Its main active ingredients include streptomycin A, B, C, D, E, and F. Different strains produce different types of active ingredients and the proportions of each ingredient, which leads to variations in their antibacterial ability.

[0004] Currently, obtaining strains with high kasugamycin yield and strong antibacterial ability is a key measure to improve pesticide efficiency and reduce pesticide use. Summary of the Invention

[0005] To address the aforementioned technical problems, this application provides a streptomycin-producing strain, which not only possesses good streptomycin-producing activity but also exhibits higher antibacterial activity in its fermentation products.

[0006] According to one aspect of this application, a streptomycin-producing strain is provided, said strain being Streptomyces. Streptomyces sp. ZS-2, the strain is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 35483.

[0007] According to one aspect of this application, a streptomycin-producing strain is provided, said strain being Streptomyces. Streptomyces sp. ZS-2, satisfying any one or a combination of the following:

[0008] (1) Contains a molecular marker, the nucleotide sequence of which is shown in SEQ ID NO: 1;

[0009] (2) Contains a molecular marker, wherein the nucleotide sequence of the molecular marker has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher homology with the nucleotide sequence shown in SEQ ID NO: 1;

[0010] (3) The genomic DNA was amplified using the primers shown in SEQ ID NO: 2 and SEQ ID NO: 3. The amplification products were analyzed by agarose gel electrophoresis to obtain specific bands.

[0011] (4) The genomic DNA was amplified using the primers shown in SEQ ID NO: 2 and SEQ ID NO: 3. The amplified products were sequenced and analyzed to obtain the nucleotide sequence shown in SEQ ID NO: 1; or

[0012] The obtained nucleotide sequence has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher homology with the nucleotide sequence shown in SEQ ID NO: 1.

[0013] According to one aspect of this application, a molecular marker is provided, the nucleotide sequence of which is shown in SEQ ID NO: 1; or

[0014] The nucleotide sequence of the molecular marker has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher homology with the nucleotide sequence shown in SEQ ID NO: 1.

[0015] According to one aspect of this application, the use of the above-mentioned molecular markers in identifying the above-mentioned strains is provided.

[0016] According to one aspect of this application, a primer pair is provided, wherein the nucleotide sequence of the forward primer is shown in SEQ ID NO:2;

[0017] The nucleotide sequence of the reverse primer is shown in SEQ ID NO: 3.

[0018] According to one aspect of this application, the use of the above primer pairs in identifying the above strains is provided.

[0019] According to one aspect of this application, a method for identifying the above-mentioned strain is provided, comprising the following steps:

[0020] (1) Sequencing of the strain;

[0021] (2) Obtain a sequence containing a specific molecular marker, wherein the specific molecular marker satisfies any one or a combination of the following:

[0022] 1) The nucleotide sequence is as shown in SEQ ID NO: 1; and / or

[0023] 2) The nucleotide sequence has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher homology with the nucleotide sequence shown in SEQ ID NO: 1.

[0024] Optionally, the sequencing is selected from:

[0025] (1) Genome sequencing; and / or

[0026] (2) Sequencing of PCR products.

[0027] Optionally, the PCR product is obtained by amplification using the primers shown in SEQ ID NO: 2 and SEQ ID NO: 3.

[0028] According to one aspect of this application, the use of the above-mentioned strain in the preparation of pesticide products is provided.

[0029] According to one aspect of this application, a pesticide product is provided, which contains a fermentation product of the above-mentioned strain, wherein the active ingredient of the fermentation product contains kasugamycin.

[0030] Optionally, the pesticide product is selected from any one of fermentation stock solution, fermentation dilution solution, fermentation concentrate, technical grade, mother drug, and formulation.

[0031] Optionally, the content of kasugamycin in the fermentation stock solution, fermentation dilution solution, or fermentation concentrate solution is 10-300000 μg / mL;

[0032] The mass fraction of kasugamycin in the technical grade and parent drug is 5-99%;

[0033] The mass fraction of kasugamycin in the preparation is 0.1-30%.

[0034] Optionally, the dosage form of the formulation is selected from any one of powder, granules, oil, and seed treatment suspension.

[0035] Optionally, the dosage form of the formulation is selected from sustained-release formulations.

[0036] Optionally, the dosage form of the formulation is selected from suspensions.

[0037] Optionally, the formulation is selected from suspension seed coating agents.

[0038] Optionally, the dosage form of the formulation is selected from any one of seed treatment dispersible powder, seed treatment soluble powder, seed treatment liquid, seed treatment emulsion, and seed treatment microcapsule suspension.

[0039] Optionally, the dosage form of the formulation is selected from any one of the following: large granules, fine granules, microparticles, microcapsules, wettable powders, oil-dispersible powders, water-dispersible granules, emulsions, effervescent granules, dispersible tablets, effervescent tablets, sustained-release blocks, sustained-release tubes, sustained-release granules, soluble powders, soluble granules, soluble tablets, soluble solutions, aqueous solutions, soluble gels, spreading oils, ultra-low volume liquids, ultra-low volume microcapsule suspensions, emulsions, latexes, dispersible liquids, pastes, concentrated gels, water emulsions, oil emulsions, microemulsions, liposomes, microcapsule suspensions, oil suspensions, and suspension emulsions.

[0040] According to one aspect of this application, a method for preparing the above-mentioned pesticide product is provided, the method comprising the following steps:

[0041] Using Streptomyces Streptomyces The pesticide product was obtained by fermentation of sp. ZS-2.

[0042] Optionally, the preparation method includes the following steps:

[0043] (1) Utilizing Streptomyces Streptomyces Fermentation was carried out using sp. ZS-2 to obtain the fermentation stock solution, which is the pesticide product mentioned above; or

[0044] The preparation method further includes the following steps:

[0045] (2) Further processing of the fermentation stock solution to obtain any one of the following: fermentation dilution, fermentation concentrate, technical material, mother drug, or formulation, which is the pesticide product.

[0046] According to one aspect of this application, the use of the above-described strain, the above-described pesticide product, or the pesticide product obtained by the above-described method in the control of plant diseases is provided.

[0047] According to one aspect of this application, the use of the above-described strain, the above-described pesticide product, or the pesticide product obtained according to the above-described method in the preparation of plant disease control agents is provided.

[0048] The positive and beneficial effects achieved by this application are as follows:

[0049] 1. This application provides the streptomycin-producing strain Streptomyces. Streptomyces sp. ZS-2 has high activity in producing streptomycin, and the mass content and bioavailability of streptomycin in its fermentation broth are significantly higher than those of existing strains.

[0050] 2. This application provides the streptomycin-producing strain Streptomyces. Streptomyces sp. ZS-2, whose fermentation products exhibit higher antibacterial activity and a wider antibacterial spectrum. Compared with existing strains, the strain presented in this application demonstrates a stronger antibacterial effect.

[0051] 3. This application provides the streptomycin-producing strain Streptomyces. Streptomyces sp. ZS-2, in addition to inhibiting bacterial pathogens, can also inhibit fungal pathogens such as tea anthracnose fungus, and has a wider range of antibacterial activity.

[0052] Preservation instructions: Streptomyces Streptomyces sp. ZS-2 was deposited at the China General Microbiological Culture Collection Center on July 31, 2025, and classified as Streptomyces. Streptomycessp., with accession number CGMCC No.35483. Attached Figure Description

[0053] Figure 1 This is an image of the inhibition zones of 10 single colonies in dish 1 of Example 1;

[0054] Figure 2 Streptomyces Streptomyces Phylogenetic tree of 16S rDNA of sp. ZS-2;

[0055] Figure 3 Streptomyces Streptomyces Liquid chromatography chromatogram of ZS-2 fermentation broth;

[0056] Figure 4 It is the Hainan variant of Streptomyces lilacinus ( Streptomyces lavendulae Liquid chromatography-mass spectra of UV-11 fermentation broth (var.hainanensis);

[0057] Figure 5 This is the mass spectrum of component 1;

[0058] Figure 6 The H-NMR spectrum of component 1;

[0059] Figure 7 For component 1 13 C-NMR spectrum;

[0060] Figure 8 The mass spectrum of component 2 is shown below.

[0061] Figure 9 The H-NMR spectrum of component 2;

[0062] Figure 10 For component 2 13 C-NMR spectrum;

[0063] Figure 11 The graph shows the inhibitory effects of various fermentation products on anthracnose pathogens in tea leaves.

[0064] Figure 12 The agarose gel electrophoresis results are for the amplification products of SEQ ID NO: 2 and SEQ ID NO: 3. Detailed Implementation

[0065] For the purposes of this invention, unless otherwise stated, the terms used in this application have the following meanings:

[0066] The term "active ingredient" refers to the component in pesticide products that has toxic and killing activity against diseases, insects, and weeds.

[0067] The term "raw material" refers to the product obtained during the production process, which consists of active ingredients and related impurities, and may include a small amount of additives if necessary.

[0068] The term "mother drug" refers to the product obtained during the production process, which consists of the active ingredient and related impurities, and may contain small amounts of necessary additives and appropriate diluents.

[0069] The term "formulation" refers to a stable product made from pesticide technical (mother drug) and suitable adjuvants, or processed by methods such as bio-fermentation and plant extraction.

[0070] The term "plant disease" refers to the phenomenon where, during the growth and development of a plant, its growth and development are significantly hindered due to infection by other organisms and adverse abiotic factors. This results in pathological changes both internally and externally, in terms of physiology and tissue structure, leading to illness or even death, and consequently, reduced yield and deteriorated quality. In this application, "plant disease" specifically refers to diseases caused by infection of plants by other organisms, including but not limited to fungi (such as *Anthracnose fungus* of the tea plantation) or bacteria (such as *Bacterium angularis* of the cucumber plantation).

[0071] The reagents and solvents used in the following examples are commercially available or prepared using conventional methods known to those skilled in the art. Experimental methods in the following examples, unless otherwise specified, were performed according to conventional methods and conditions.

[0072] Preparation of PDA medium: Weigh 200 g of potato, wash, peel and chop it, add 800 ml of distilled water and boil for half an hour, filter with gauze, then add 20 g of glucose and 20 g of agar, and make up to 1000 ml with distilled water, and sterilize at 121℃ for 30 minutes.

[0073] Preparation of LB medium: Weigh 10 g tryptone, 5 g yeast extract, 10 g NaCl and 20 g agar, dissolve in distilled water and bring the volume to 1000 ml, adjust the pH to 7.0, and sterilize at 121℃ for 20 min.

[0074] Seed culture medium preparation: Weigh the following materials according to the proportions: glucose 1.0%, corn starch 1.5%, corn flour 1.0%, soybean flour 2.0%, sodium chloride 0.3%, ammonium chloride 0.3%, calcium carbonate 0.3%, with the remainder being water. Adjust the pH to 6.6 and sterilize at 121℃ for 20 min.

[0075] Fermentation medium preparation: Weigh the following materials according to the proportions below: glucose 5.0%, corn starch 2.0%, corn flour 2.0%, soybean flour 4.0%, sodium chloride 0.6%, ammonium chloride 0.8%, calcium carbonate 0.7%, potassium dihydrogen phosphate 0.02%, magnesium sulfate 0.05%, with the remainder being water. Adjust the pH to 6.6 and sterilize at 121℃ for 20 min.

[0076] The liquid chromatography (HPLC) detection method and the biopotency detection method were performed in accordance with the enterprise standard Q / KLSW 043-2021 of Fujian Kaili Biological Products Co., Ltd., "80U / mg (6%) Viscumin Soluble".

[0077] Genomic DNA was extracted using a bacterial genomic DNA extraction kit from Tiangen Biotech Co., Ltd., following the instructions provided.

[0078] Specific amplification was performed using PrimeSTAR® Max DNA Polymerase, a rapid PCR polymerase from Takara Bio Inc., and the amplification system is as follows:

[0079]

[0080] The amplification procedure is as follows:

[0081]

[0082] The sources of the Streptomyces strains used in this application are described below:

[0083]

[0084] Example 1: Streptomyces Streptomyces Isolation and Identification of strain ZS-2

[0085] 1. Strains Isolation

[0086] Ten soil samples were collected from the Ulanhada Volcanic Geopark in Chahar Right Wing Rear Banner, Ulanqab City, Inner Mongolia Autonomous Region. 10 g of each sample was weighed and dissolved in 90 mL of sterile water. The samples were then serially diluted with sterile water to 100-fold and 1000-fold. An appropriate amount of the 1000-fold dilution was spread onto PDA agar plates and incubated at 28°C for 3 days. Eighteen single colonies were then further purified and cultured. These purified colonies were inoculated into Erlenmeyer flasks containing fermentation medium and fermented at 28°C and 180 rpm for 3 days to obtain the fermentation broth. The fermentation broth was filtered through a 0.22 μm bacterial filter to remove bacterial cells and was then used for further processing.

[0087] Take 200 mL of thawed and cooled LB medium to 48 ℃~50 ℃, and add 2 mL of 1×10 8 A CFU / mL suspension of *Cucumber angular leaf spot* was shaken well, and an appropriate amount was poured into a 15cm diameter petri dish. The mixture was allowed to solidify to obtain a bacterial layer. Two bacterial layers were prepared, designated dishes 1 and 2. Ten filter paper discs were evenly placed in dish 1, and eight filter paper discs were placed in dish 2. 20μL of the fermentation broth containing the 18 single colonies was added dropwise to each filter paper disc. The discs were then dried and incubated at 28℃ for one day. The size of the inhibition zone produced by the fermentation broth was observed. The strain with the largest inhibition zone (strain number 5 in dish 1) was selected; the inhibition effect is shown in [reference needed]. Figure 1 Further research will be conducted.

[0088] 2. Strain identification

[0089] The 16S rDNA identification method was used to identify strain 5 above. The specific identification steps are as follows:

[0090] (1) Genomic DNA extraction: After activating and culturing strain 5 on PDA medium plates, genomic DNA was extracted.

[0091] (2) 16S rDNA amplification: Using genomic DNA as a template, 16S rDNA fragments were amplified from genomic DNA using universal 16S rDNA primers (27F sequence as shown in SEQ ID NO: 4 and 1492R sequence as shown in SEQ ID NO: 5). The amplification products were sent to Beijing Liuhe BGI Genomics Co., Ltd. for analysis.

[0092] (3) 16S rDNA sequence analysis: The 16S rDNA sequence obtained from sequencing was compared with the 16S rDNA sequence in GenBank using BLAST. The results showed that it was consistent with... Streptomyces The highest similarity (>99%) was observed in sps 1-9. A phylogenetic tree was constructed using Mega 5.0 software, and this strain was related to... Streptomyces sp. 1-9 clustered on one branch (e.g.) Figure 2 (As shown).

[0093] Based on the above molecular identification results, the strain was identified as belonging to the genus Streptomyces. Streptomyces sp., named Streptomyces Streptomyces sp. ZS-2 is deposited at the China General Microbiological Culture Collection Center, Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 35483.

[0094] Example 2 Streptomyces Streptomyces sp. ZS-2 active substance identification

[0095] 1. Liquid chromatography detection

[0096] The components of Streptomyces were located using kasugamycin B, kasugamycin C, kasugamycin D, kasugamycin E, and kasugamycin F standards (these standards are owned by Yuanda Crop Science (Shaanxi) Co., Ltd.). Streptomyces The fermentation broth of sp. ZS-2 was analyzed by liquid chromatography, and the chromatogram is shown below. Figure 3 As shown.

[0097] Based on the positioning of the streptomycin standard, the results showed that Streptomyces Streptomyces In the fermentation broth of sp. ZS-2, the component with a peak time of 26.625 (component 1) is streptomycin B, and the component with a peak time of 17.821 (component 2) is streptomycin D.

[0098] The components were located using kasugamycin B, kasugamycin C, kasugamycin D, kasugamycin E, and kasugamycin F standards to locate the *Streptomyces hainanensis* var. *palmarginata*. Streptomyces lavendulae The fermentation broth of var. hainanensis (UV-11) was analyzed by liquid chromatography, and the chromatogram is shown below. Figure 4 As shown, the component with a peak time of 23.400 is kasugamycin B, the component with a peak time of 19.839 is kasugamycin C, the component with a peak time of 17.985 is kasugamycin D, the component with a peak time of 15.050 is kasugamycin E, and the component with a peak time of 12.155 is kasugamycin F.

[0099] The above results indicate that Streptomyces Streptomyces sp. ZS-2 and Streptomyces lilacinus Hainan variant ( Streptomyces lavendulae Both *var. hainanensis* and UV-11 can produce kasugamycin, but comparison shows that... Figure 3 and Figure 4 It was found that there were significant differences in the types of kasugamycin and the content and proportion of each component between the two strains.

[0100] 2. Structural identification

[0101] To further confirm the above-mentioned Streptomyces Streptomyces Whether components 1 and 2 of sp. ZS-2 are streptavidin B and D was determined by gel column chromatography, concentration, and drying from Streptomyces. StreptomycesThe above-mentioned components were obtained from the fermentation broth of sp. ZS-2. Components 1-2 were analyzed by mass spectrometry (MS) and nuclear magnetic resonance spectroscopy (NMR). The results showed that the molecular structures of components 1-2 corresponded to streptomycin B and streptomycin D, respectively, and their structural identification spectra are shown below:

[0102] The mass spectra of component 1 are as follows Figure 5 As shown in Figure 6, the H-NMR spectrum is as follows. 13 C-NMR such as Figure 7 As shown.

[0103] The mass spectra of component 2 are as follows Figure 8 As shown, H-NMR is as follows Figure 9 As shown, 13 C-NMR such as Figure 10 As shown.

[0104] The above results indicate that Streptomyces Streptomyces sp. ZS-2 can produce streptomycin, but the types of streptomycin components, as well as the content and ratio of each component, are different from those of *Streptomyces hainanensis* (var. *zinosa*). Streptomyces lavendulae There is a clear difference between var.hainanensis and UV-11.

[0105] Example 3 Streptomyces Streptomyces sp. ZS-2 fermentation level determination

[0106] Streptomyces Streptomyces sp. ZS-2, Streptomyces lilacinus Hainan variant ( Streptomyces lavendulae var. hainanensis) UV-11 and Streptomyces hainanensis ( Streptomyces lavendulae After being cultured in seed culture medium for 1 day, the 46# var.hainanensis were transferred to a 5L fermenter for 7 days of fermentation. Samples were then taken for testing of the content and bioavailability of the kasugamycin.

[0107] 1. Detection of the quality content of kasugamycin

[0108] Streptomyces were detected using high-performance liquid chromatography (HPLC). Streptomyces The fermentation broth of sp. ZS-2 was tested for streptomycin content, and the result was 15916 μg / mL.

[0109] The same method was used to treat *Streptomyces hainanensis* var. *lilacinus* (Hainan variety). Streptomyces lavendulae var. hainanensis) UV-11, Streptomyces hainanensis ( Streptomyces lavendulaeThe fermentation broth of var.hainanensis new var.) 46# was tested for streptomycin content, and the results were 13819 μg / mL and 3668 μg / mL, respectively.

[0110] The results above show that Streptomyces Streptomyces The mass content of streptomycin in the fermentation broth of sp. ZS-2 was higher than that of Streptomyces hainanensis (a strain of Streptomyces lilacinus). Streptomyces lavendulae The UV-11 concentration of *Streptomyces hainanensis* var. *hainanensis* was 15.2% higher than that of *Streptomyces hainanensis* var. *hainanensis*. Streptomyces lavendulae var.hainanensis newvar.) 46# is 333.9% higher.

[0111] 2. Bioactivity testing

[0112] Streptomyces Streptomyces The bioavailability of the fermentation broth of sp. ZS-2 was determined to be 22619 U / mL.

[0113] Streptomyces lilacinus Hainan variety ( Streptomyces lavendulae var. hainanensis) UV-11, Streptomyces hainanensis ( Streptomyces lavendulae The biopotency of the fermentation broth of var. hainanensis (new var.) 46# was tested, and the results were 18402 U / mL and 4564 U / mL, respectively.

[0114] The results above show that Streptomyces Streptomyces The bioefficiency of the fermentation broth of sp. ZS-2 compared to Streptomyces hainanense (a variant of Streptomyces lilacinus) Streptomyces lavendulae The UV-11 concentration of *Streptomyces hainanensis* var. *hainanensis* was 22.9% higher than that of *Streptomyces hainanensis* var. *hainanensis*. Streptomyces lavendulae var.hainanensis new var.) 46# higher than 395.6%.

[0115] The results above show that, regardless of whether the detection is based on the content of streptomycin or its biopotency, Streptomyces... Streptomyces The fermentation level of sp. ZS-2 was significantly higher than that of Streptomyces hainanensis (var. hainanensis). Streptomyces lavendulae var. hainanensis) UV-11 and Streptomyces hainanensis ( Streptomyces lavendulae var.hainanensis new var.) 46#.

[0116] Example 4 Streptomyces Streptomyces Antibacterial activity test of ZS-2 fermentation products

[0117] (1) Determination of antibacterial ability against bacterial angular leaf spot of cucumber

[0118] Take 1 mL of the Streptomyces prepared in Example 3 Streptomyces The fermentation broth of sp. ZS-2 was mixed with 317 mL of water to prepare a solution with a kasugamycin content of 50 μg / mL.

[0119] Take 1 mL of the *Streptomyces hainanense* var. *hainanense* prepared in Example 3 (… Streptomyces lavendulae The UV-11 fermentation broth of var. hainanensis was mixed with 275 mL of water to prepare a solution with a kasugamycin content of 50 μg / mL.

[0120] Take 1 mL of the *Streptomyces hainanense* var. *hainanense* prepared in Example 3 (… Streptomyces lavendulae (var.hainanensis new var.) 46# fermentation broth, add 72 mL of water to prepare a solution with a kasugamycin content of 50 μg / mL.

[0121] Take 200 mL of LB medium that has been melted and cooled to 48 ℃~50 ℃, and add 2 mL of 1×10 8 A suspension of *Cucumber angular leaf spot* at CFU / mL was shaken well, and an appropriate amount was poured into a 9cm diameter petri dish. The mixture was allowed to solidify, resulting in a bacterial layer. Three 6mm diameter filter paper discs were placed on each disc, and 10μL of the solution was added to each disc, repeating this process three times. After incubation at 28℃ for one day, the size of the inhibition zone produced by the fermentation broth was observed, and the results are as follows:

[0122]

[0123] (2) Determination of antibacterial ability against anthrax bacteria in tea leaves

[0124] Take 0.63 mL of the Streptomyces prepared in Example 3. Streptomyces The ZS-2 fermentation broth was added to 100 mL of melted PDA medium to prepare a solution with a kasugamycin content of 100 μg / mL, which was then poured into a petri dish to make a toxicity plate.

[0125] Take 0.72 mL of the *Streptomyces hainanense* var. *hainanense* prepared in Example 3 (…). Streptomyces lavendulae The UV-11 fermentation broth (var. hainanensis) was added to 100 mL of melted PDA medium to prepare a solution with a kasugamycin content of 100 μg / mL, which was then poured into a petri dish to make a toxicity plate.

[0126] Take 2.73 mL of the *Streptomyces hainanense* strain prepared in Example 3 (Hainan variety). Streptomyces lavendulaeAdd 100 mL of melted PDA medium to the fermentation broth of var.hainanensis (new var.) 46# to prepare a solution with a mesophytic bacteria content of 100 μg / mL, and pour it into a petri dish to make a toxicity plate.

[0127] Using a 6mm diameter punch, mycelial cakes were collected from the edge of activated tea anthracnose colonies (approximately 6 cm in diameter). These cakes were then inoculated onto the aforementioned toxic PDA plates, with a blank plate serving as a control. The inoculation was repeated three times. After incubation at 28℃ for 6 days, the inhibition rates of each fermentation product were investigated and calculated. The calculation formula is: Colony growth inhibition rate = (Control colony diameter - Treated colony diameter) / (Control colony diameter - Mycelial cake diameter) × 100%.

[0128] The growth status of each group of colonies is as follows: Figure 11 As shown, where A is Streptomyces. Streptomyces sp. ZS-2 fermentation broth, B is Streptomyces lilacinus Hainan variant ( Streptomyces lavendulae var. hainanensis) UV-11 fermentation broth, C is Streptomyces hainanensis (var. hainanensis) Streptomyces lavendulae (var. hainanensis new var.) 46# fermentation broth, D is the blank control. As can be seen from the figure, Streptomyces... Streptomyces sp. ZS-2 fermentation broth had the smallest colony diameter, and Streptomyces hainanensis (a type of Streptomyces purpureus) was the largest. Streptomyces lavendulae var. hainanensis new var.) 46# fermentation broth, Streptomyces hainanensis (var. hainanensis) Streptomyces lavendulae The diameters of the UV-11 fermentation broth (var. hainanensis) and the blank control were larger.

[0129] The specific colony diameter and antibacterial effect statistics are shown in the table below:

[0130]

[0131] The above results indicate that Streptomyces Streptomyces The fermentation product of sp. ZS-2 has a strong inhibitory effect on bacterial angular leaf spot of cucumber, and its inhibitory effect is better than that of Streptomyces hainanensis var. hainanensis. Streptomyces lavendulae var. hainanensis) UV-11 and Streptomyces hainanensis ( Streptomyces lavendulae (var.hainanensis new var.) 46#. Additionally, the applicant unexpectedly discovered Streptomyces. Streptomyces sp. ZS-2 also exhibits good inhibitory activity against *Streptomyces hainanensis*, the causal agent of anthracnose in tea, while *Streptomyces lilacinus* Hainan variant (… Streptomyces lavendulaevar. hainanensis) UV-11 and Streptomyces hainanensis ( Streptomyces lavendulae The antibacterial ability of *Streptomyces hainanensis* var. (new var.) 46# is relatively weak, which is significantly different from the existing *Streptomyces* strains that can produce streptomycin. This indicates that *Streptomyces*... Streptomyces sp ZS-2 has better antibacterial ability and a broader antibacterial spectrum.

[0132] Example 5 Streptomyces Streptomyces SCAR molecular marker analysis of sp. ZS-2

[0133] Streptomyces Streptomyces sp. ZS-2, Streptomyces lilacinus Hainan variant ( Streptomyces lavendulae var. hainanensis) UV-11, Streptomyces hainanensis ( Streptomyces lavendulae var.hainanensis new var.) 46#, Streptomyces Streptomyces sp. ZS-Ⅱ-D190515, Streptomyces MKL-2 ( Streptomyces microaureaus MKL-2), Streptomyces simonii ( Streptomyces caniferus NEAU6, Streptomyces Streptomyces Using genomic DNA from seven Streptomyces sp. 2356 strains as templates, random amplification was performed using 20 random primers (TGCCGAGCTG, AGTCAGCCAC, AATCGGGCTG, AGGGGTCTTG, GTTTCGCTCC, CATCCCCCCTG, CTGCTGGGAC, GTGAGGCGTC, CCGCATCTAC, GTGTGCCCCA, GGACCCAACC, TTGGCACGGG, AGCGCCATTG, GGTGCGGGAA, GTGACATGCC, TGCGCCCTTC, GGACTGCAGA, CTCACCGTCC, GGTGATCAGG, CTGAGACGGA). The amplified products were analyzed by agarose gel electrophoresis, revealing Streptomyces... Streptomyces sp. ZS-2 has unique bands that contain Streptomyces. Streptomyces The specific band of sp. ZS-2 was cut, recovered, and sequenced to obtain the sequence of the specific band.

[0134] Primers were designed based on the obtained unique band sequences (forward primer as shown in SEQ ID NO: 2; reverse primer as shown in SEQ ID NO: 3). Specific amplification was performed using the genomic DNA of the above 7 Streptomyces strains as templates. The amplification products were analyzed by agarose gel electrophoresis, and the results are as follows: Figure 12 As shown, lanes 1-7 are streptomyces in sequence. Streptomyces sp. ZS-2, Streptomyces lilacinus Hainan variant ( Streptomyces lavendulae var. hainanensis) UV-11, Streptomyces hainanensis ( Streptomyces lavendulae var.hainanensis new var.) 46#, Streptomyces Streptomyces sp. ZS-Ⅱ-D190515, Streptomyces MKL-2 ( Streptomyces microaureaus MKL-2), Streptomyces simonii ( Streptomyces caniferus NEAU6, Streptomyces Streptomyces sp.2356, the image shows only Streptomyces. Streptomyces sp. ZS-2 was able to amplify a specific band, while the other 6 strains were unable to amplify this specific band, verifying the specificity of the band sequence and confirming that the primers (SEQ ID NO: 2 and SEQ ID NO: 3) can specifically identify Streptomyces. Streptomyces sp. ZS-2.

[0135] SEQ ID NO: 2 and SEQ ID NO: 3 specifically amplified Streptomyces Streptomyces The specific band obtained from sp. ZS-2 was recovered and sequenced, and the sequence of the specific band is shown in SEQ ID NO: 1.

[0136] Blast alignment of the specific band sequence shown in SEQ ID NO: 1 in GenBank revealed no identical or similar sequences among all publicly available Streptomyces strains. This further demonstrates that the specific sequence shown in SEQ ID NO: 1 can be used to identify Streptomyces. Streptomyces sp. ZS-2.

[0137] The above results indicate that primers SEQ ID NO: 2 and SEQ ID NO: 3 are effective for identifying Streptomyces. Streptomyces The specific primers for strain ZS-2, and the PCR amplification product, as shown in SEQ ID NO: 1, are Streptomyces. StreptomycesSCAR molecular markers for sp. ZS-2. Primers SEQ ID NO: 2 and SEQ ID NO: 3, as well as the specific band sequence (SEQ ID NO: 1), can rapidly and effectively identify Streptomyces. Streptomyces sp. ZS-2.

[0138] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A streptomycin-producing strain, characterized in that, The strain is Streptomyces. Streptomyces sp. ZS-2, the strain is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 35483.

2. The streptomycin-producing strain according to claim 1, characterized in that, Satisfy any one or a combination of the following: (1) Contains a molecular marker, the nucleotide sequence of which is shown in SEQ ID NO: 1; (2) Genomic DNA was amplified using the primers shown in SEQ ID NO: 2 and SEQ ID NO:

3. The amplification products were analyzed by agarose gel electrophoresis to obtain specific bands. (3) The genomic DNA was amplified using the primers shown in SEQ ID NO: 2 and SEQ ID NO:

3. The amplified products were sequenced and analyzed, and the nucleotide sequence obtained was shown in SEQ ID NO:

1.

3. A molecular marker, characterized in that, The nucleotide sequence of the molecular marker is shown in SEQ ID NO:

1.

4. Use of the molecular marker of claim 3 in identifying the strains of any one of claims 1-2.

5. Primer pairs, characterized in that, The nucleotide sequence of the forward primer is shown in SEQ ID NO: 2; The nucleotide sequence of the reverse primer is shown in SEQ ID NO:

3.

6. Use of the primer pair of claim 5 in identifying the strain of any one of claims 1-2.

7. A method for identifying the strain according to any one of claims 1-2, characterized in that, Includes the following steps: (1) Sequencing of the strain; (2) Obtain a sequence containing a specific molecular marker, wherein the specific molecular marker satisfies the following: 1) The nucleotide sequence is shown in SEQ ID NO:

1.

8. The method according to claim 7, characterized in that, The sequencing data were selected from: (1) Genome sequencing; and / or (2) Sequencing of PCR products.

9. The method according to claim 8, characterized in that, The PCR product was obtained by amplification using the primers shown in SEQ ID NO: 2 and SEQ ID NO:

3.

10. Use of the strain according to any one of claims 1-2 in the preparation of pesticide products.

11. A pesticide product, characterized in that, It contains any one of the fermentation stock solution, fermentation dilution solution, and fermentation concentrate of the strain described in any one of claims 1-2.

12. The pesticide product according to claim 11, characterized in that, The content of kasugamycin in the fermentation stock solution, fermentation dilution solution, or fermentation concentrate solution is 10-300,000 μg / mL.

13. The pesticide product according to claim 11, characterized in that, The formulation of the pesticide product is selected from any one of powder, granule, oil, or seed treatment suspension.

14. The pesticide product according to claim 11, characterized in that, The formulation of the pesticide product is selected from slow-release formulations.

15. The pesticide product according to claim 11, characterized in that, The formulation of the pesticide product is selected from suspension concentrates.

16. The pesticide product according to claim 11, characterized in that, The formulation of the pesticide product is selected from suspension seed coating agents.

17. The pesticide product according to claim 11, characterized in that, The formulation of the pesticide product is selected from any one of seed treatment dispersible powder, seed treatment soluble powder, seed treatment liquid, seed treatment emulsion, and seed treatment microcapsule suspension.

18. The pesticide product according to claim 11, characterized in that, The formulation of the pesticide product is selected from any one of the following: large granules, fine granules, microparticles, microcapsule granules, wettable powders, oil-dispersible powders, water-dispersible granules, emulsion granules, effervescent granules, dispersible tablets, effervescent tablets, sustained-release blocks, sustained-release tubes, sustained-release granules, soluble powders, soluble granules, soluble tablets, soluble solutions, aqueous solutions, soluble gels, spreading oils, ultra-low volume liquids, ultra-low volume microcapsule suspensions, emulsifiable concentrates, latexes, dispersible liquids, pastes, concentrated gels, water emulsions, oil emulsions, microemulsions, greases, microcapsule suspensions, oil suspensions, and suspension emulsions.

19. A method for preparing the pesticide product according to any one of claims 11-18, characterized in that, The preparation method includes the following steps: Using Streptomyces Streptomyces The pesticide product was obtained by fermentation of sp. ZS-2.

20. The preparation method according to claim 19, characterized in that, The preparation method includes the following steps: (1) Utilizing Streptomyces Streptomyces Fermentation was carried out using sp. ZS-2 to obtain the fermentation stock solution, which is the pesticide product mentioned above; or The preparation method further includes the following steps: (2) Further processing of the fermentation stock solution to obtain either fermentation dilution or fermentation concentrate, which is the pesticide product.

21. Use of the strain according to any one of claims 1-2, the pesticide product according to any one of claims 11-18, or the pesticide product obtained by the method according to any one of claims 19-20 in the prevention and control of plant diseases.

22. Use of the strain according to any one of claims 1-2, the pesticide product according to any one of claims 11-18, or the pesticide product obtained by the method according to any one of claims 19-20 in the preparation of plant disease control agents.

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