Streptomyces TRM 76323T and application thereof

Abstract

The invention discloses streptomyces TRM 76323T, and the preservation number of the streptomyces TRM is CCTCC (China Center for Type Culture Collection) NO: M2023092. The strain has broad-spectrum antifungal activity, has an obvious inhibition effect on pear black spot, enhances the growth of pears, and has the potential of developing a biocontrol inoculant for pear black spot, and the fermentation liquor of the strain and the active ingredient streptochlorin of the fermentation liquor have an obvious antagonistic effect on alternaria alternata. Therefore, the characteristics of biological prevention and control and plant pathogen antagonism of the streptomyces TRM 76323T have important significance on agricultural development.

Description

Technical Field

[0001] This invention relates to the field of microbial technology. Specifically, it relates to Streptomyces TRM 76323. T And its applications. Background Technology

[0002] Alternaria species are a class of fungi that can cause plant diseases. These fungi, which are usually found in plant residues and in plants, are very harmful to crops. Specifically, they can cause many pre- and post-harvest diseases in crops, resulting in considerable crop losses. Currently, the main methods for controlling Alternaria involve the application of chemical reagents.

[0003] Pear black spot disease is widespread, primarily caused by the fungus *Alternaria alternate (Fr.) Keissl*. It mainly affects pear leaves and fruits, often leading to premature leaf drop and extensive fruit rot in severe cases, causing significant economic losses to the pear industry and hindering its development. Biological control methods offer significant advantages over traditional chemical pest control, effectively reducing pesticide residues and avoiding harm to humans. From a biocontrol perspective, utilizing beneficial microorganisms is the best choice for managing fungal diseases.

[0004] The vast majority of biocontrol bacteria are isolated and screened from plants or soil. Xinjiang, located in the northwest border region of China, encompasses diverse landforms such as deserts (e.g., Gobi), lakes, basins, grasslands, mountains, rivers, and swamps. Due to its unique geographical location, microorganisms face various environmental stresses, including drought, high temperatures, and salinity, which may lead to the production of specific antibiotics. Microbial secondary metabolites (such as antibiotics) have long been an important source of many new drugs. They have a wide range of effects, including bactericidal, bacteriostatic, anti-infective, insecticidal, antitumor, herbicide, and immunosuppressant effects. They are extremely important for various industries, including pharmaceuticals, health, and agriculture, and are also applicable to environmental protection. These unknown, untapped resources have great application potential in medicine and health, biological control, and plant growth promotion, providing new biocontrol resources for the control of pear black spot disease. Summary of the Invention

[0005] Therefore, the technical problem to be solved by the present invention is to provide a Streptomyces TRM 76323. T It possesses broad-spectrum antifungal activity and exhibits high stable heritability. In vitro experiments show that TRM 76323 T The fermentation broth of this strain significantly inhibited the incidence of pear black spot disease. Furthermore, through isolation and purification, the antibacterial active substance streptochlor was obtained, which significantly inhibited the mycelial growth of *Alternaria alternata*. Therefore, strain TRM 76323... TIt has the potential to act as a biocontrol agent against pear black spot disease.

[0006] To solve the above-mentioned technical problems, the present invention provides the following technical solution:

[0007] A Streptomyces TRM 76323 T (Streptomyces sp. TRM 76323) with accession number CCTCC NO: M2023092 was deposited on February 6, 2023 at the China Center for Type Culture Collection, located at Wuhan University, Wuhan, China, on Luojia Mountain, Bayi Road, Wuchang District, Wuhan, Hubei Province.

[0008] The technical solution of the present invention achieves the following beneficial technical effects:

[0009] Streptomyces strain (TRM 76323) was isolated from soil. T It possesses broad-spectrum antifungal activity, and in genetic classification and identification, TRM 76323 has been identified. T The genetic taxonomic position of strain TRM76323 was verified. T The inhibitory activity of TRM 76323 against Alternaria pyriformis was investigated, and its active ingredient was identified. Scanning electron microscopy and in vitro experiments confirmed the activity of TRM 76323. T The inhibitory effects of this study on *Streptomyces oryzae* TRM 76323 at both the microscopic and macroscopic levels form the basis for future research on this fungus, which possesses biocontrol and pear growth-promoting properties. T It has important effects in the prevention and control of plant diseases in agriculture. Attached Figure Description

[0010] The Streptomyces sp. TRM 76323T shown in the attached figure has the accession number CCTCC NO: M2023092 and was deposited at the China Center for Type Culture Collection on February 6, 2023.

[0011] Figure 1 strain TRM 76323 T Schematic diagram of the morphology of strains cultured for 7 days in different culture media.

[0012] Figure 2 strain TRM 76323 T Schematic diagram of the morphology of strains cultured at different temperatures for 7 days.

[0013] Figure 3 strain TRM 76323 T Schematic diagram of the morphology of strains cultured for 7 days at different salt concentrations.

[0014] Figure 4strain TRM 76323 T A schematic diagram illustrating the morphological characteristics of mature colonies;

[0015] In the figure: A represents strain TRM 76323 T Colony morphology after 7 days of incubation in ISP4 medium; B represents strain TRM76323. T Scanning electron microscope images.

[0016] Figure 5 strain TRM 76323 T A schematic diagram of the colorimetric results of thin-layer chromatography plates for polar lipid types;

[0017] In the diagram: A is ninhydrin colorimetric reagent, B is anisaldehyde colorimetric reagent, and C is molybdenum phosphate colorimetric reagent.

[0018] Figure 6 strain TRM 76323 T A schematic diagram of the colorimetric results of thin-layer chromatography plates containing hydrolyzed sugars and amino acid components;

[0019] In the figure: A represents strain TRM 76323 T Thin-layer chromatography analysis of cell wall diaminopimelic acid type, B represents strain TRM 76323. T Results of thin-layer chromatography analysis of whole-cell glycosides.

[0020] Figure 7 strain TRM 76323 T Schematic diagram of a phylogenetic tree based on the 16S rDNA gene neighbor-joining method.

[0021] Figure 8 strain TRM 76323 T A schematic diagram of a phylogenetic tree based on the neighbor-joining method using housekeeper genes.

[0022] Figure 9 strain TRM 76323 T A schematic diagram of a phylogenetic tree based on the maximum likelihood method using housekeeper genes.

[0023] Figure 10 strain TRM 76323 T A schematic diagram of a phylogenetic tree based on the maximum parsimony method of housekeeper genes.

[0024] Figure 11 strain TRM 76323 T Schematic diagram of liquid phase and semi-preparative separation and purification process;

[0025] In the diagram: A represents TRM76323 TThe HPLC conditions for the crude extract were as follows: mobile phase: methanol and water; flow rate: 1 mL / min; column temperature: 40℃; wavelength: 190-800 nm; detection conditions: 10%-100% methanol for 40 min, 100% methanol for 10 min; injection volume: 10 μL.

[0026] B is the liquid phase chromatogram after extracting the sample with methanol of different concentrations. The HPLC conditions are as follows: mobile phase is methanol and water, flow rate is 1 mL / min, column temperature is 40℃, wavelength is 190-800 nm; detection conditions are: 10%-100% methanol for 40 min, 100% methanol for 10 min, and injection volume is 10 μL.

[0027] C is the liquid chromatogram of the semi-preparative conditions explored by changing the solvent. The HPLC conditions are: mobile phase is methanol and water, flow rate is 1 mL / min, column temperature is 40℃, wavelength is 190-800 nm; detection conditions are: 40%-100% acetonitrile for 30 min, 100-10% acetonitrile for 10 min, injection volume is 10 μL.

[0028] D is a liquid phase diagram of semi-preparative separation and purification;

[0029] E is the liquid phase chromatogram after semi-preparation. The HPLC conditions are as follows: mobile phase is methanol and water, flow rate is 1 mL / min, column temperature is 40℃, wavelength is 190-800 nm; detection conditions are: 10%-100% methanol for 40 min, 100% methanol for 10 min, and injection volume is 10 μL.

[0030] Figure 12 strain TRM 76323 T A schematic diagram comparing the NMR data of the active ingredient and streptochlor.

[0031] In the diagram: A represents the active ingredient. 1 H and 13 C is a chemical shift, and B is the chemical structure of the chain chlorine.

[0032] Figure 13 TRM 76323 prepared using different solvents T A diagram illustrating the antibacterial effect of the active ingredient.

[0033] Figure 14 TRM 76323 T A schematic diagram illustrating the activity and antibacterial effect of the active ingredient, streptochlor.

[0034] In the figure: A shows the effect of streptomycin on Alternaria, and B shows the antibacterial activity of streptomycin at different concentrations at the MIC (minimum inhibitory concentration).

[0035] Figure 15 TRM 76323 TA schematic diagram of the results of the plate confrontation experiment between the mycelium cake and the fermentation broth;

[0036] In the diagram: A represents TRM 76323 T Stipe inhibition diameter, B is TRM 76323 T The diameter of the antibacterial agent in the fermentation broth.

[0037] Figure 16 TRM 76323 under scanning electron microscope T A schematic diagram illustrating the effect of the active ingredient streptomycin on Alternaria alterniflora.

[0038] In the figure: A shows the morphology of Alternaria alternate (Fr.) Keissl under normal conditions, and B shows the morphology of Alternaria alternate (Fr.) Keissl after treatment with streptomycin.

[0039] Figure 17 strain TRM 76323 T A comparative diagram showing the control effect of fermentation broth on Alternaria. Detailed Implementation

[0040] This invention provides a Streptomyces TRM 76323 T Streptomyces sp. TRM 76323, with accession number CCTCC NO: M 2023092, was deposited on February 6, 2023, at the China Center for Type Culture Collection, located at Wuhan University, Wuhan, Hubei Province, China. This invention pertains to Streptomyces sp. TRM 76323. T The bacteria were isolated and screened from the rhizosphere soil samples of Tamarix chinensis collected from the Ahe Expressway in Xinjiang.

[0041] Example 1: Isolation and physiological and biochemical characteristics analysis of the strain

[0042] 1. Strain TRM 76323 T separation

[0043] Strains TRM 76323 were isolated from rhizosphere soil samples of Tamarix chinensis collected from the Ahe Expressway in Xinjiang (36.888E, 82.553N; altitude 1664.8m). T The strain was isolated using glycerol-arginine medium, which consisted of 2.00 g arginine, 12.00 g glycerol, 0.50 g MgSO4, 1.00 g K2HPO4, and 17.00 g agar. The pH of the glycerol-arginine medium was adjusted to 7.5 using Gao's medium to allow strain TRM 76323 to isolate. TGrow at 30℃ for 1 week.

[0044] 2. Analysis of culture characteristics and physiological and biochemical characteristics

[0045] To determine the optimal culture medium, strain TRM 76323 was used. T The strain was cultured on a series of international Streptomyces (ISP) media (ISP1, ISP2, ISP3, ISP4, ISP5, ISP6, and ISP7), as well as Gauze's medium, potato dextrose agar, and nutrient agar. The pH of all media was adjusted to 7.0 to allow the strain to grow and be maintained on ISP4 synthetic medium. Cell morphology of spores and mycelia on ISP4 medium plates incubated at 28°C for one week was examined using a JSM-6360 scanning electron microscope. Carbon source utilization was performed using basal medium. The strain was TRM 76323. T The ability to grow was tested at 4–45 °C (4, 12, 15, 20, 25, 28, 30, 37, 45) and pH 4–11 (pH 4, 5, 6, 7, 8, 9, 10, and 11). Furthermore, its tolerance to 0%–25% NaCl concentrations (0%, 5%, 10%, 15%, 20%, and 25% w / v) was determined, and related experiments were conducted on the production of peroxidase, urease, esterase, and catalase. Cellulose decomposition, starch hydrolysis, milk peptone formation and coagulation, nitrate reduction, and melanin production were investigated using a single carbon source (0.5% w / v).

[0046] strain TRM 76323 T The morphology of strains cultured for 7 days in different culture media is as follows Figure 1 As shown, strain TRM 76323 T It grows well on ISP4, GS, ISP1, and ISP5, with the best growth on ISP4. Growth is relatively poor on ISP6 and NA, and it cannot grow on ISP3. Therefore, ISP4 is the culture medium for strain TRM 76323. T The optimal culture medium.

[0047] strain TRM 76323 T The morphology of strains cultured at different temperatures for 7 days is as follows Figure 2 As shown, the suitable temperature for growth was observed to be 16–37℃, and optimal growth was observed at 28℃ and pH 7.0.

[0048] strain TRM 76323 T The morphology of the strain cultured at salt concentration for 7 days is as follows: Figure 3 As shown, the strain can grow at NaCl concentrations up to 5% (w / v).

[0049] TRM 76323 cultured in ISP 4 medium T The morphology of the strain is as follows Figure 4 As shown, TRM 76323 cultured for 7 days was observed using a scanning electron microscope. T Branched aerial hyphae and long, straight spores can be observed in the culture (when mature).

[0050] strain TRM 76323 T The physiological characteristics and the characteristics of the most closely related Streptomyces species are compared in Table 1 below. Nitrate reduction, catalase, milk peptone and coagulation, starch hydrolysis, cellulose hydrolysis and urease production all yielded positive results, but gelatin hydrolysis, oxidase production and melanin production all yielded negative results.

[0051] Table 1. Strain TRM 76323 T Comparison of characteristics with the most closely related Streptomyces species

[0052]

[0053] In the table: +: positive; -: negative; (+): weak positive; ND: not detected.

[0054] 3. Chemical taxonomic characteristics of the strain

[0055] 1) Analysis of the components of total cellular hydrolysate

[0056] (1) Preparation of cell-to-cell hydrolysate samples: Place the bacterial cells into an ampoule and add 250 μL of 0.5 mol / L hydrochloric acid. Treat at 121℃ for 15 min. After treatment, centrifuge and collect the supernatant for later use.

[0057] (2) Thin-layer chromatography treatment: Using the microcrystalline cellulose thin-layer chromatography method, weigh 10.0g of microcrystalline cellulose and 60mL of distilled water, grind them thoroughly for 30min, spread them evenly on a 10*20 glass plate, and let them dry.

[0058] (3) Spotting: Add 5 μL of cell total sugar hydrolysis sample and standard respectively, and spot the sample multiple times. Add sugar standard S1 (rhamnose, xylose, mannitol, galactose) and sugar standard S2 (glucose, ribose, arabinose);

[0059] (4) Development: Prepare the developing solvent (n-butanol:pyridine:toluene:water = 10:6:1:6) and develop the layers three times, successively running the developing solvent to the top 3cm, 2cm and 1cm;

[0060] (5) Color development: Spray the color development agent (1.66 g phthalic acid, 0.93 g aniline, 100 mL water-saturated n-butanol) onto the thin-layer plate after it has been developed, and observe and compare the hydrolyzed sugar components of the test strain with those of the sugar standard.

[0061] 2) Whole-cell amino acid composition analysis

[0062] (1) Preparation of whole cell amino acid samples: Place the bacterial cells into an ampoule and add 250 μL of 2 mol / L hydrochloric acid. Seal the ampoule with an alcohol burner and treat it at 121℃ for 6 h. After treatment, centrifuge and collect the supernatant for later use.

[0063] (2) Thin-layer chromatography treatment: Microcrystalline cellulose thin-layer chromatography method was adopted. 10.0g of microcrystalline cellulose and 60mL of distilled water were weighed, and 2.0×2.5cm filter paper was cut into pieces and added. The mixture was ground thoroughly for 30min and evenly spread on a 10*20 glass plate and then dried.

[0064] (3) Spotting: Multiple spotting was used to add 5 μL of whole-cell amino acid sample and amino acid standard DAP (diaminopimelic acid) respectively. The spotting position was 1.5 cm from the bottom of the chromatography plate.

[0065] (4) Development: Prepare the developing solvent (methanol:pyridine:glacial acetic acid:water = 10:1:0.25:5) and develop the layers twice. In the first development, the developing solvent is moved to 1 cm from the top of the chromatographic plate, and in the second development, the developing solvent is moved to the top of the chromatographic plate.

[0066] (5) Color development: Spray the color development agent (0.4g ninhydrin, water-saturated n-butanol, 100mL) onto the thin-layer plate after it has been spread, and observe and compare the cellular amino acids of the test strain with those of the amino acid standard.

[0067] 3) Polar lipid component analysis

[0068] (1) Preparation of polar lipid components: The liquid fermentation broth was dispensed into 50 mL centrifuge tubes and centrifuged at 4 °C and 12000 rpm for 10 min in a centrifuge. The supernatant was removed after centrifugation.

[0069] Add 15.0 mL of methanol to the collected bacterial cells, boil in water for 10 minutes, wait for cooling, add 10.0 mL of chloroform, and shake well.

[0070] Continuously add a 2.0% sodium chloride solution. After the layers separate, shake and mix for 10 minutes. Centrifuge at 12,000 rpm for 10 minutes. Take the lower layer liquid and evaporate it in a rotary evaporator flask.

[0071] Finally, dissolve the sample in a rotary evaporator flask using a chloroform:methanol mixture of 2:1. Place the sample in a 2mL centrifuge tube and centrifuge at 12000rpm for 10min. Collect the supernatant and store it at low temperature for use.

[0072] (2) Preparation of thin-layer silicone plates: Place the pre-purchased silicone plates in a 60℃ oven for 30 min to activate them;

[0073] (3) Spotting: Take 10.0 μL of the prepared polar lipid component sample and spot it 1.5 cm below the edge of the silica gel plate;

[0074] (4) Development: Polar lipid components were developed using biphase thin-layer chromatography. A biphase developing solution was prepared, wherein the first phase was chloroform:methanol:water = 65:25:4 and the second phase was chloroform:glacial acetic acid:methanol:water = 80:15:12:4. After spotting the sample on the silica gel plate, the first phase developing solution was developed. The developing solution was placed 1 cm away from the top of the silica gel plate. The silica gel plate was then removed and dried.

[0075] Rotate the silica gel plate 90° and place it into the developing solution containing the second phase for developing. When the developing solution reaches 1 cm from the silica gel plate, remove the silica gel plate and dry it.

[0076] (5) Color development: The color development of polar lipid components is slightly different. The No. 1 silica gel plate is first developed with ninhydrin color developer and placed in an 80℃ oven for 5 minutes. At this time, the presence of polar lipids with free amino groups in the sample is judged based on whether red appears and the position.

[0077] The No. 2 silica gel plate was developed with anisaldehyde colorimetric reagent and placed in an oven at 110℃ for 5 minutes. A yellow-green color indicates the presence of polar lipids containing sugar, while a light blue color indicates the presence of DPG, PE, PC, or PI in the sample.

[0078] The No. 3 silica gel plate was developed with molybdenum phosphate colorimetric reagent and placed in an oven at 110℃ for 5 minutes. All lipids were developed.

[0079] Colorimetric analysis results Figure 5 and Figure 6 As shown, where Figure 5 strain TRM 76323 T TLC thin-layer chromatography results of polar lipid components, strain TRM 76323 T Polar lipids include: phosphatidylinositol (PI), phosphatidylinositol mannoside (PIM), phosphatidylinositol dimannoside (PIDM), and phospholipids (PLS).

[0080] Figure 6 strain TRM 76323 T TLC (thin-layer chromatography) results of hydrolyzed sugar and amino acid fractions, with strain TRM 76323 observed in sample A. T The cell wall diaminopimelic acid is a cell wall diamino acid, and mannose was observed in the whole-cell hydrolysate group from B.

[0081] Based on strain TRM 76323 T The colony, cell morphology, and physiological and biochemical characteristics of the bacteria were determined to identify strain TRM76323. T It belongs to the genus Streptomyces.

[0082] Example 2: 16S rRNA gene and multisite sequence analysis of the strain

[0083] The 16S rRNA gene sequence was amplified by polymerase chain reaction using universal primers 27F (5'AGTTTGATCMTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3') for molecular identification of the strain.

[0084] The following housekeeping genes were selected for use in the Streptomyces multisite sequence analysis: atpD (ATP synthase, β subunit), gyrB (DNA gyrase B subunit), recA (recombinase a), rpoB (RNA polymerase, β subunit), and trpB (tryptophan synthase, β subunit). The average nucleotide identity (ANI) value was calculated to be 86.09% and the DNA-DNA hybridization value was 30.60% based on the genome sequence.

[0085] strain TRM 76323 T In contact with strain TRM 49605 T Based on comparison, TRM 76323 T The ANI value was 86.09%. Phylogenetic analysis of its 16S rRNA sequence was performed using the neighbor-joining method, and the results are as follows: Figure 7 As shown; furthermore, the sequences atpD, gyrB, recA, rpoB, and trpB are concatenated for neighbor-based joins, with the following results: Figure 8 As shown; the analyses performed by the maximum likelihood and maximum parsimony algorithms are respectively as follows: Figure 9 and Figure 10 As shown.

[0086] Example 3 TRM 76323 T Fermentation and analysis of active ingredients

[0087] 1. TRM 76323 T Fermentation and purification

[0088] ISP4 was selected as the seed culture medium, and 3L of ISP4 medium was prepared and inoculated and cultured at 30℃ and 150rpm for 3 days. Millet medium was selected as the fermentation medium, and the bacterial blocks were inoculated onto the millet medium. The required 50L of millet medium (15g / L millet, 5g / L glucose, 4g / L peptone and 3g / L sodium chloride, pH 7) was placed in the fermenter. At the same time, 3mol / L NaOH solution and HCl solution were prepared to adjust the pH value. Before fermentation, the fermenter was sterilized at 121℃ for 30min. After sterilization, the temperature was cooled to 30℃. The seed culture was inoculated and cultured in the fermenter for 7 days. Samples were taken every two days for observation, and the samples were spray-dried and stored.

[0089] The purification process is as follows: First, the crude extract is subjected to preliminary liquid chromatography analysis to determine the reagent extraction concentration based on the sample elution time. Second, the extracted sample is subjected to liquid chromatography analysis to determine the separation and purification effect. Finally, the reagent used for the sample is replaced with the reagent used for the semi-preparative liquid chromatography to further explore the conditions. Subsequently, the semi-preparative liquid chromatography is used for final condition exploration. Samples are collected according to the elution time, and the obtained samples are subjected to liquid chromatography analysis and NMR spectroscopy. Finally, the results are compared with publicly available microspectral data.

[0090] Figure 11 For liquid phase and semi-preparative separation and purification processes, in Figure 11 As observed in Figure A, a high concentration peak appeared at 28 min using liquid chromatography. In Figure B, repeated extraction with methanol at concentrations of 66.25% and 73% yielded a relatively pure peak. In Figure C, subsequent liquid chromatography under semi-preparative exploratory conditions showed a peak time of approximately 10 min. In Figure D, using semi-preparative exploratory conditions, the peak time was 14.4 min. In Figure E, a relatively pure sample was obtained at approximately 28 min.

[0091] The NMR data obtained from the purified sample from the fermentation broth were consistent with those obtained from the antibiotic, such as... Figure 12 As shown, the CH data of both are consistent. According to the dictionary of combinatorial chemistry, the antibiotic was identified as an anti-nematode compound. Furthermore, the NMR data analysis of this compound and streptomycin produced very similar results. Therefore, from TRM 76323... T A compound with tyrosinase inhibitory activity, namely streptochlor, was isolated from the methanol extract of strain TRM 76323. T The active ingredient is streptochlor, and a purified sample containing 12 mg of streptochlor can be obtained from 50 L of fermentation broth.

[0092] 2. TRM 76323 T Analysis of the active ingredient streptochlor in the fermentation broth

[0093] Proteins in the fermentation broth were precipitated in 75% ethanol to determine if they were active ingredients. The crude extract was added to pre-cooled ethanol (final 75%), and the resulting solution was incubated at 4°C for 4 hours. Then, it was centrifuged (10,000 rpm, 4°C, 20 minutes). The crude extract was collected and added to 20 mM Tris buffer (pH 6.8).

[0094] To verify TRM 76323 T The active ingredient, TRM76323 T The freeze-dried samples were treated with dichloromethane, ethyl acetate, methanol, and water to produce extracts. 100 μL aliquots of *Alternaria alternata* suspension were spread onto PDA medium. The inhibitory effect of the fermentation broth on *Alternaria alternata* was analyzed using the perforation antagonism method. Sterile blue pipette tips were used to punch holes in the solid medium, and 100 μL of fermentation broth was filled into each hole. The medium was then incubated at 28 °C. The inhibitory effect was verified using the perforation antagonism method. The results are as follows: Figure 13 As shown, TRM 76323 was prepared using different solvents. T Extract, TRM 76323 T The active ingredient, streptochlorine, is mainly extracted from ethyl acetate.

[0095] The effect of streptomycin (2 mg / mL) on Alternaria alternate was determined using the filter paper disc method. 5 μL aliquots of streptomycin or chloroform (control) were added to individual filter paper discs. This experiment was repeated three times. The effect of streptomycin on Alternaria alternate and its antibacterial activity at different concentrations were tested at a concentration of 2 mg / mL. The results are as follows: Figure 14 As shown, the inhibitory production has an active inhibition diameter of approximately 1.5 cm, and the minimum inhibitory concentration determined using the MIC method is 512 μg / mL.

[0096] Strain TRM76323 was cultured for 7 days in both ISP4 solid and liquid media at 30℃. Plate confrontation experiments were conducted using mycelial cake and fermentation broth, and the results are as follows: Figure 15 As shown, the antibacterial diameters were found to be 1.6 cm and 1.4 cm, respectively. The antibacterial diameters were basically the same as those of the monomer compound streptochlor, and the antibacterial effects of the two were similar. Therefore, the antibacterial effect of the fermentation broth was actually due to the active ingredient streptochlor.

[0097] The fermentation broth was divided into three portions for testing. Alternaria alternata (Alternate) was activated and cultured on PDA medium for 48 hours. PDA medium was used to culture Alternaria alternata as a control. Using the filter paper method, one plate and three plates were inoculated with 5 μL of a compound at a concentration of 1 mg / mL. After 7 days of culture, scanning electron microscopy was performed to observe the effects of the compound. The effects of the compound under scanning electron microscopy were as follows: Figure 16As shown in Figure A, the untreated sample has plump spores and rounded mycelia; Figure B shows the compound Streptochlorin (chemical formula C) extracted using TRM 76323T. 11 The mycelium of Alternaria treated with H7ClN2O (English name: 4-chloro-S-(1H-indol-3-yl)oxazole; Chinese name: 4-chloro-S(1H-indol-3-yl)oxazole) is coarse, irregular in shape, and has collapsed cell walls.

[0098] To further explore TRM 76323 T To investigate how fermentation broth can prevent and inhibit the growth and development of Alternaria alternata, an in vitro experiment was conducted using pears washed three times with 75% ethanol and 5% sodium hypochlorite. A damaged inoculation method and sterile water treatment were used as blanks. Samples were inoculated solely with Alternaria alternata, and then sprayed with 100 μL of fermentation broth after 24 hours. The samples were placed in sterile bags and incubated at 30°C for 7 days. Figure 17 As shown, pear fruits inoculated only with Alternaria exhibited black spot symptoms after 7 days of culture. Similarly, using TRM 76323... T Treating infected pear fruits with fermentation broth for 7 days reduced the incidence of pear black spot disease.

[0099] In summary, genetic classification and identification verified that strain TRM 76323... T The activity of TRM 76323 against Alternaria pyriformis was inhibited, and its active ingredient was identified; scanning electron microscopy and in vitro experiments confirmed the activity of TRM 76323. T It exhibits inhibitory effects against *Alternaria pyrifolia* at both microscopic and macroscopic levels; furthermore, the purified compound streptochlor has a minimum inhibitory concentration (MIC) of 512 μg / mL. These results indicate that strain TRM 76323... T Its fermentation broth or the active ingredient streptomycin in the fermentation broth has a preventive effect against Alternaria infection in Korla pear, and has an important effect in the prevention and control of plant diseases in agriculture.

[0100] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of the claims of this patent application.

Claims

1. Streptomyces TRM 76323 T Its characteristics are, The accession number is CCTCC NO: M2023092.

2. Claim 1, Streptomyces TRM 76323 T Its application in antagonizing plant pathogens to cause plant diseases is characterized by... The pathogens of the plant pathogens are Alternaria and / or Pear Black Spot Fungus.

3. The Streptomyces TRM 76323 as described in claim 1 T Its application in the preparation of plant pathogen inhibitors is characterized by, The pathogens of the plant pathogens are Alternaria and / or Pear Black Spot Fungus.

4. The Streptomyces TRM 76323 as described in claim 1 T Application in growth promoters for wheat, corn, celery, pepper, Aksu apples, jujubes, and / or Korean pears.

5. The Streptomyces TRM 76323 as described in claim 1 T Fermentation broth or from Streptomyces TRM 76323 T The application of active ingredients isolated from fermentation broth in the preparation of plant pathogen inhibitors, characterized in that, The pathogens of the plant pathogens are Alternaria and / or pear black spot fungus, derived from the Streptomyces TRM 76323. T The active ingredient isolated from the fermentation broth is streptochlorine.

6. The Streptomyces TRM 76323 as described in claim 1 T Fermentation broth or from Streptomyces TRM 76323 T The application of the active ingredient isolated from the fermentation broth in a biocontrol agent antagonizing Alternaria alternata and / or pear black spot fungus, characterized in that... From the Streptomyces TRM 76323 T The active ingredient isolated from the fermentation broth is streptochlorine.

7. The Streptomyces TRM 76323 as described in claim 1 T Fermentation broth or from Streptomyces TRM 76323 T The application of the active ingredients isolated from the fermentation broth in promoting the growth of wheat, corn, celery, pepper, Aksu apple, jujube, and / or Korean pear is characterized by the following: From the Streptomyces TRM 76323 T The active ingredient isolated from the fermentation broth is streptochlorine.

8. A bactericide, characterized in that, Including Streptomyces TRM 76323 T Bacterial cells, Streptomyces TRM 76323 T Fermentation broth or from Streptomyces TRM 76323 T The active ingredients isolated from the fermentation broth.

9. The bactericide according to claim 9, characterized in that, From the Streptomyces TRM 76323 T The active ingredient isolated from the fermentation broth is streptochlorine.

10. Streptomyces TRM 76323 according to any one of claims 5-9 T The application of the fermentation broth is characterized by, The fermentation broth is prepared as follows: Streptomyces is inoculated onto seed culture medium ISP4 and cultured. Millet culture medium is used as the fermentation medium. The bacterial blocks are then inoculated onto the millet culture medium and placed in a fermenter. The mixture is cultured at 30°C for 7 days, and the fermentation broth is collected.

Images