Streptomyces sp. HN-149F and application thereof

Abstract

The invention relates to the technical field of agricultural biological prevention and control, in particular to streptomyces cellostatus HN-149F and application of the streptomyces cellostatus HN-149F. The preservation number of Streptomyces cellostatus HN-149F is CGMCC (China General Microbiological Culture Collection Center) NO.36380, the Streptomyces cellostatus HN-149F is preserved in the China General Microbiological Culture Collection Center, the preservation date is October 28, 2025, and the preservation address is No.3, Yard 1, Beichen West Road, Chaoyang District, Beijing. Field pharmacodynamic test results show that the control effect of the streptomyces kasugamycin HN-149F on the magnaporthe oryzae reaches up to 72.73%, the streptomyces kasugamycin HN-149F has no obvious difference from chemical agents kasugamycin and tricyclazole, has no phytotoxicity to rice, effectively solves the problems of enhanced resistance to the magnaporthe oryzae, pesticide residues and environmental pollution caused by excessive dependence on chemical bactericides in the control process, and has a broad application prospect. Theoretical basis and material basis are provided for research and development of novel biopesticides, and technical progress in the field of agricultural pest control is promoted.

Description

Technical Field

[0001] This invention relates to the field of agricultural biological control, and more specifically, to a Streptomyces cytotoxicum HN-149F and its application, particularly the application of Streptomyces cytotoxicum HN-149F in the control of rice blast fungus. Background Technology

[0002] Rice blast is caused by the rice blast fungus ( ). Pyricularia oryzae Rice blast is one of the major diseases caused by pests and diseases. It is widely distributed and can cause a 10%–20% reduction in rice yield globally each year, with severe cases resulting in a 40%–50% reduction. Currently, the main control methods for rice blast include agricultural control, chemical control, and biological control. Among these, chemical control has become the primary method due to its simplicity, convenience, rapid effectiveness, and high efficiency. However, the long-term use of chemical pesticides has led to increasingly prominent problems such as increased pesticide resistance in pests and diseases, environmental pollution, and excessive pesticide residues. Therefore, developing a highly effective and environmentally friendly fungicide for controlling rice blast is crucial. Summary of the Invention

[0003] To address the aforementioned problems, this invention provides a Streptomyces cytotoxicum HN-149F and its applications.

[0004] To achieve the above objectives, the present invention is implemented through the following technical solution: In a first aspect, the present invention provides a Streptomyces cytotoxicum (Synthia spp.) Streptomyces cellostaticus HN-149F, with accession number CGMCC NO.36380, is deposited at the China General Microbiological Culture Collection Center on October 28, 2025, at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0005] In a second aspect, the present invention provides a *Streptomyces* strain described above (…). Streptomyces cellostaticus HN-149F microbial inoculant.

[0006] A third aspect of the present invention provides a *Streptomyces* strain described above (…). Streptomyces cellostaticus Application of HN-149F.

[0007] Under the preferred embodiment, the above-mentioned Streptomyces cytotoxicum (Streptomyces cytotoxicum) Streptomyces cellostaticus HN-149F is used to control rice blast fungus ( Pyricularia oryzae ) and / or tomato gray mold ( Botrytis cinerea ).

[0008] Under the preferred embodiment, the above-mentioned Streptomyces cytotoxicum (Streptomyces cytotoxicum) Streptomyces cellostaticus HN-149F is used to control rice blast fungus ( Pyricularia oryzae ).

[0009] Advantages of the present application The rice blast pathogenic bacteria biocontrol bacteria effectively solve the problems of enhanced resistance of rice blast pathogenic bacteria, pesticide residues and environmental pollution caused by excessive reliance on chemical fungicides. The present application provides an environmentally friendly alternative solution, which can not only continuously and specifically inhibit pathogenic bacteria, but also reduce the use of chemical pesticides, ensure the quality and safety of rice, and help maintain the balance of paddy microecosystem, which is a key way to realize green and sustainable production of rice.

[0010] The present application avoids the large use of chemical fungicides, reduces the pollution of pesticide residues to soil and water from the source, maintains the balance of paddy microecosystem, helps the green and sustainable development of agriculture, and realizes the continuous and specific inhibition of pathogenic bacteria through biological control means, prolongs the effective period of prevention and control measures. Reduce the potential harm of chemical pesticides to rice plants, ensure the safety of rice growth, and then help to stabilize or even improve the yield of rice, ensure food security, and provide a theoretical basis and material basis for the optimization of subsequent biological control technology and the research and development of new biological pesticides, promote the technological progress in the field of agricultural pest control. BRIEF DESCRIPTION OF DRAWINGS

[0011] Figure 1 is the inhibition effect of biocontrol bacteria HN-149F on rice blast pathogenic bacteria; Figure 2 is the inhibition effect of biocontrol bacteria HN-149F fermentation liquid on rice blast pathogenic bacteria; Figure 3 is the morphological characteristic identification result of biocontrol bacteria HN-149F; Figure 4 is the 16S rDNA gene sequence phylogenetic tree of biocontrol bacteria HN-149F; Figure 5 is the antibacterial spectrum determination result of biocontrol bacteria HN-149F; Figure 6 is the chemical detection result of biocontrol bacteria HN-149F; Figure 7 is the inhibition result of biocontrol bacteria HN-149F fermentation crude extract on rice blast pathogenic bacteria; Figure 8 is the inhibition result of biocontrol bacteria HN-149F fermentation crude extract separation component C3 on rice blast pathogenic bacteria. DETAILED DESCRIPTION

[0012] The technical solutions in the embodiments of the present application will be described below in a clear and complete manner. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor should belong to the scope of protection of the present application.

[0013] Unless otherwise indicated, the scientific and technical terms used in this disclosure have the meanings that are commonly understood by one of ordinary skill in the art. The experimental methods in the examples are routine methods unless specifically indicated otherwise. The examples are carried out under conventional conditions or under conditions recommended by the manufacturer, unless otherwise indicated. The reagents or instruments used are conventional products that can be obtained commercially, unless otherwise indicated.

[0014] The present application adopts dilution coating method and plate streaking method to isolate a strain of biocontrol bacteria from soil samples collected from Shenbei New District of Shenyang City, Liaoning Province, which has good antagonistic effect on Magnaporthe oryzae. The strain is a strain of Actinomycete, belongs to Streptomyces, and its classification name is Streptomyces coelicolor Streptomyces cellostaticus , named HN-149F. The strain has been preserved in China General Microbiological Culture Collection Center on October 28, 2025, with the preservation number of CGMCC NO. 36380 and the preservation address of No. 3, Beichen West Road, Chaoyang District, Beijing.

[0015] ①PDA culture medium: potato 200 g, glucose 20 g, agar 20 g, distilled water 1000 mL.

[0016] ②Citric acid culture medium: citric acid 0.12 g, sodium nitrate 1.5 g, K2HPO4·3H2O 0.1 g, anhydrous magnesium sulfate 0.1 g, calcium chloride 0.05 g, EDTA (ethylenediaminetetraacetic acid) 0.02 g, sodium carbonate 0.2 g, agar 15 g, distilled water 1000 mL.

[0017] ③ISP-2 culture medium: yeast extract 4 g, malt extract powder 10 g, glucose 4 g, distilled water 1000 mL.

[0018] ④Tomato oat culture medium: tomato juice 200 mL, oat 30 g, agar 30 g, distilled water 1000 mL.

[0019] Example 1: Isolation, purification, screening and identification of the strain 1. Isolation and purification of the strain Weigh 5 g of soil sample into a 50 mL centrifuge tube, add 45 mL of sterile water to the centrifuge tube, shake until there are no obvious soil clumps in the solution, and obtain the bacterial solution; the bacterial solution is sequentially diluted by 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 and 10 -6The 33 strains were obtained from soil samples collected from Shenbei New District of Shenyang City, Liaoning Province, including 1 strain of fungi, 10 strains of bacteria and 22 strains of actinomycetes.

[0020] 2. Screening of strains 2.1 Test pathogenic fungus strains Magnaporthe oryzae (Guy11) was provided and preserved by the Rice Disease Research Room of Shenyang Agricultural University. Pyricularia oryzae

[0021] 2.2 Plate confrontation method The 33 strains obtained by isolation and purification were screened for strains with high inhibitory effect on Magnaporthe oryzae by plate confrontation method using Magnaporthe oryzae as the indicator (target). The specific process is as follows: A 5mm-diameter Magnaporthe oryzae mycelium cake was taken and placed at the center of a PDA medium plate, and the strains for screening were inoculated symmetrically at three points 30mm away from the center of the Magnaporthe oryzae mycelium cake, and then placed in a 28℃ incubator; a PDA medium plate inoculated only with the Magnaporthe oryzae mycelium cake was used as a control group (CK), each treatment was repeated 3 times, and when the mycelium of the control group approached (7d) to the edge of the plate by 10mm, the diameter (mm) of the inhibition zone was recorded.

[0022] The present application selects one strain with high inhibitory effect on Magnaporthe oryzae and stable and persistent antagonistic effect by plate confrontation method, which is strain HN-149F, and is used for subsequent research. The inhibitory effect of biocontrol agent HN-149F on Magnaporthe oryzae is shown in Figure 1 and Table 1. The plate confrontation test results show that the inhibition zone diameter of biocontrol agent HN-149F on Magnaporthe oryzae Guy11 is 17.25±0.27mm.

[0023] Table 1 Inhibitory effect of biocontrol agent HN-149F on Magnaporthe oryzae

[0024] 2.3 Mycelial growth rate method ​The biocontrol bacteria HN-149F was inoculated into ISP-2 medium and cultured at 28°C and 180 rpm for 48 h to obtain a bacterial suspension; the bacterial suspension was divided into 50 mL centrifuge tubes and centrifuged at 10,000 rpm for 15 min at 20°C, and then filtered with a filter (φ=0.22 μm); the supernatant was mixed with streptomycin at a ratio of 100:1 to obtain a fermentation liquor of the biocontrol bacteria HN-149F; the fermentation liquor of the biocontrol bacteria HN-149F and the PDA medium were mixed at ratios of 1:9, 2:8 and 3:7 to prepare medium plates, and the plates prepared with sterile water medium and the PDA medium were used as blank controls (CK); 5 mm activated rice blast fungus cakes were punched with a puncher and placed in the center of the plates, and the colony diameters were measured after the plates were cultured at 28°C for 7 d; the inhibition rate (I) was calculated, each treatment was repeated three times, and the results are shown in Table 2. Figure 2 and Table 2.

[0025] I / % = [(D CK - D PT ) / (D CK - 5 mm)]×100% (1) D CK is the average mycelium diameter on the blank control PDA medium, mm; and D PT is the average mycelium diameter on the PDA medium after treatment, mm.

[0026] Table 2 Inhibition effect of the fermentation liquor of the biocontrol bacteria HN-149F on rice blast fungus

[0027] Note: The superscript letters indicate significant differences from CK (P<0.001).

[0028] Figure 2 Table 2 and Table 2 are the test results of the inhibition effect of the fermentation liquor of the biocontrol bacteria HN-149F on rice blast fungus by the mycelium growth rate method. The results show that when the content of the fermentation liquor of the biocontrol bacteria HN-149F is 30% (i.e., 3:7), the biocontrol bacteria can significantly inhibit rice blast fungus, and the inhibition rate is as high as 63.12%.

[0029] 3. Identification of the biocontrol bacteria HN-149F 3.1 Morphological identification and physiological and biochemical identification of the biocontrol bacteria HN-149F The morphological characteristics and physiological and biochemical properties of the biocontrol bacteria HN-149F were identified according to the methods in the Common Bacterial System Identification Manual (Buchanan, 1984) and the Bergey's Manual of Determinative Bacteriology (Dong Xi-zhu et al., 2001), and the results are shown in Table 3. Figure 3 and Table 3.

[0030] Figure 3For the morphological identification results of biocontrol bacteria HN-149F, the spore of biocontrol bacteria HN-149F is spherical to oval, the surface is raised and dry, and it is opaque.

[0031] Table 3 Identification results of physiological and biochemical characteristics of biocontrol bacteria HN-149F

[0032] Note: "+" indicates that the reaction is positive or can grow and utilize; "-" indicates that the reaction is negative or cannot grow and utilize.

[0033] 3.2 Molecular biological identification of biocontrol bacteria HN-149F The genomic DNA of biocontrol bacteria strain HN-149F was extracted, and PCR amplification was performed using bacterial 16S rDNA gene universal primers 27F and 1492R. The 16S rDNA reaction system was as follows:

[0034] The amplification conditions were performed according to the method of Yu Guohui et al. (2010).

[0035] The 16S rDNA sequence of biocontrol bacteria HN-149F was subjected to BLAST comparison on NCBI, and the 16S gene sequence was downloaded. The sequencing sequence and the sequence with high homology were analyzed using MEGA11 software, and the phylogenetic tree was constructed using the Neighbor-Joining method ( Figure 4 ). Combined with the physiological and biochemical identification results and morphological characteristics analysis, the biocontrol bacteria HN-149F was identified as Streptomyces coelicolor ( Streptomyces cellostaticus ).

[0036] The 16S sequencing spliced sequence of biocontrol bacteria HN-149F is as follows: Example 2: Inhibition spectrum determination of Streptomyces hyalurogenise HN-149F 1. Test pathogen strains The pathogen strains used in this example were provided and preserved by the Rice Disease Research Lab of Shenyang Agricultural University.

[0037] 2. Test method and results The inhibition spectrum of Streptomyces hyalurogenise HN-149F was determined by taking 5mm diameter pathogen mycelium disks (Table 4) and using the plate confrontation method provided in Reference Example 1 to determine the inhibitory effect of Streptomyces hyalurogenise HN-149F on different pathogen strains. The control group was inoculated with only pathogen mycelium disks, and each treatment was repeated three times. When the mycelium in the control group reached 1cm from the edge of the plate (7th day), the diameter of the inhibition zone (mm) was recorded. The inhibitory effect of Streptomyces hyalurogenise HN-149F on different pathogen strains is shown in Table 4 and Figure 5 .

[0038] Table 4 Inhibitory effect of Streptomyces hyalurogenise HN-149F on different pathogens

[0039] Table 4 and Figure 5 Table 4 and

[0040] Example 3: Preparation of biocontrol agent HN-149F fermentation crude extract and testing of its inhibitory activity 1. Preparation of biocontrol agent HN-149F fermentation crude extract The preparation method of biocontrol agent HN-149F fermentation crude extract includes the following steps: S1. Preparation of primary seed liquid: 5mL of ISP-2 medium was added to a 25mL test tube and sterilized at 121℃ for 30min. The edge of the biocontrol agent HN-149F colony was punched with a 5mm punch, and the obtained biocontrol agent HN-149F cake was inoculated into the sterilized ISP-2 medium. The test tube was placed in a shaker and cultured at 28℃, 180rpm for 48h to obtain the primary seed liquid.

[0041] S2. Preparation of secondary seed liquid: The primary seed liquid was transferred to a 250mL flask containing 50mL of ISP-2 medium and cultured at 28℃, 180r / min for 3d on a shaker. The growth state of the strain was observed during the fermentation period, and pure and uniform bacterial cells without impurities were preferred. The secondary seed liquid was obtained.

[0042] S3. Preparation of dichloromethane extract: The secondary seed liquid was poured into a 250 mL flask containing 100 mL of ISP-2 medium and adding 4% by volume of XAD-16 macroporous adsorption resin, and cultured at 28°C, 180 r / min for 7 days. After the fermentation was completed, the macroporous resin in the flask was collected and repeatedly soaked with clean water until there was no mycelium and medium residue. After washing, the macroporous adsorption resin was placed in a 30°C oven for drying until there was no moisture. Then the macroporous adsorption resin was completely soaked with methanol for 4 times, each time for 4 hours, one of which needed to be soaked overnight, and the methanol soaking liquid was collected. After the methanol soaking liquid was extracted with an equal amount of dichloromethane, the dichloromethane phase was collected, and the dichloromethane phase was concentrated using a rotary evaporator to obtain the dichloromethane extract, which was the crude extract of the biocontrol fungus HN-149F fermentation.

[0043] 2. Mycelial growth rate method for determining the antibacterial activity of the biocontrol fungus HN-149F fermentation crude extract Reference method (NY / T1156.2-2006; Yu Huiyuan, 2023), mycelial growth rate method was used to detect the antibacterial activity of the crude extract on plant pathogenic fungi.

[0044] The biocontrol fungus HN-149F fermentation crude extract was dissolved with dimethyl sulfoxide (DMSO) and 0.25% Tween-80, and the final concentration of the biocontrol fungus HN-149F fermentation crude extract was 100 μg / mL. An equal amount of PDA medium was added to prepare a drug-containing plate (Φ = 90 mm). The final mass fraction of DMSO was 0.5%, and the final mass fraction of Tween-80 was 0.25%. The treatment containing only DMSO and Tween-80 was used as the negative control. The pathogenic fungi (Φ = 5 mm) with consistent growth were inoculated into the plate with the mycelium facing down, and incubated at 25°C. Each group was repeated three times. When the control colony grew to 2 / 3 or 3 / 4 of the diameter of the plate, the colony diameter was measured by cross method, and the mycelial growth inhibition rate (I) was calculated according to formula (1).

[0045] Table 5 Inhibition results of biocontrol fungus HN-149F fermentation crude extract on Magnaporthe oryzae

[0046] Table 5 is the determination result of the antibacterial activity of the biocontrol fungus HN-149F fermentation crude extract on Magnaporthe oryzae. The mycelial growth rate method was used to determine the antibacterial activity of the biocontrol fungus HN-149F crude extract on Magnaporthe oryzae, and a preliminary screening test was conducted. The test results (Table 5) showed that at a final concentration of 100 μg / mL, the biocontrol fungus HN-149F fermentation crude extract had antibacterial activity on Magnaporthe oryzae, and the inhibition rate was 63.23%. Figure 6

[0047] ​3. Preparation of dichloromethane extract of biocontrol agent HN-149F The method for preparing the dichloromethane extract of biocontrol agent HN-149F includes the following steps: When the colony diameter of the biocontrol agent HN-149F reaches about 70 mm, punch a hole at the edge of the colony with a 5 mm puncher, and inoculate the bacterial cake into a 25 mL test tube as a primary liquid seed. Add 5 mL of ISP-2 medium to each test tube, sterilize at 121°C for 30 min, and use 3 test tubes without inoculation as blank controls. After inoculation, place all the test tubes in a shaker and incubate at 180 r / min and 28°C for 48 h. When the bacterial solution is turbid, remove it.

[0048] Transfer all the fermented primary seeds to 250 mL Erlenmeyer flasks as secondary liquid seeds. Add 50 mL of ISP-2 medium to each Erlenmeyer flask, sterilize at 121°C for 30 min, and use 3 Erlenmeyer flasks without inoculation as blank controls. Incubate at 180 r / min and 28°C for 48 h. When the bacterial solution is turbid and bacterial clumps are formed, remove it.

[0049] Transfer all the fermented secondary seeds to 2 L Erlenmeyer flasks. Add 400 mL of ISP-2 medium and 16 g of macroporous adsorption resin (XAD-16) to each Erlenmeyer flask, sterilize at 121°C for 30 min, and use 3 Erlenmeyer flasks without inoculation as blank controls. Incubate at 180 r / min and 28°C for 7 d, and a total of 24 L of fermentation is performed. (Large-scale fermentation) After the overall fermentation is completed, a total of 24 L of fermentation solution is obtained. The fermentation solution is washed multiple times with distilled water to remove the bacterial cells and medium. Collect the macroporous adsorption resin, gather all the resin in the flasks onto a tray, and dry it in an oven at 30°C. After the resin is dried, pour an appropriate amount of methanol into a 2 L separatory funnel to elute the resin. Replace the methanol every 4 hours, and a total of 4 times of soaking and elution are performed, with one overnight soaking. Collect and concentrate the eluate under reduced pressure to obtain the fermentation crude extract. Dissolve the fermentation crude extract in an appropriate amount of methanol, add an equal amount of distilled water and dichloromethane (V 甲醇 :V 水 =1:1, V 总 =600 mL), and perform a total of 4 times of extraction. Collect the dichloromethane phase, and concentrate it under reduced pressure to obtain the dichloromethane extract of the biocontrol agent HN-149F.

[0050] 4. Detection of chemical components in the dichloromethane extract of biocontrol agent HN-149F According to the reference method (Chen Mingqin, 2021), the thin layer chromatography (TLC) is used to detect the chemical components in the fermentation crude extract. According to the detection results, measure the solvent line and the distance from the center of different spots to the baseline, and calculate the relative migration rate Rf value according to formula (2) to determine the type and proportion of the eluent for the primary silica gel column chromatography.

[0051] (2) 4.1 Silica gel column chromatography test method: (1) Sample mixing: Dissolve the crude dichloromethane extract of the biocontrol bacteria in a small amount of dichloromethane, weigh an equal amount of silica gel [pore size 0.150-0.075 mm (100-200 mesh)] using an evaporating dish, and add the dissolved crude dichloromethane extract to the silica gel using a rubber bulb dropper. Stir the mixture with a spatula until the crude dichloromethane extract is evenly distributed in the silica gel. Place the evaporating dish in a fume hood until the organic solvent evaporates completely.

[0052] (2) Column loading: Use the dry method to load the column. Select a silica gel column based on the weight of the crude dichloromethane extract. Fix the silica gel column on an iron stand and insert a suitable amount of absorbent cotton into the top of the column and push it to the bottom to compact it. Fill the silica gel to the top of the column by 3-5 cm, and gently tap the column to make the silica gel surface flat. After filling, use a vacuum pump to perform vacuum treatment for more than 30 minutes until there are no bubbles in the silica gel column.

[0053] (3) Sample loading: Use the dry method to load the sample into the prepared silica gel column. Gently tap the outer wall of the silica gel column with a suction bulb to make the sample surface flat. After transferring the sample, cover the top of the column with a layer of absorbent cotton.

[0054] (4) Collection: Based on the TLC detection results of the crude dichloromethane extract, select a system to design gradient eluent. Use a 500 mL triangular flask to collect 150 mL per bottle. After reducing pressure concentration, transfer the eluent to a test tube using a rubber bulb dropper, and number them sequentially as a fraction. Each gradient collects 4 fractions, and 9 gradients collect 36 fractions.

[0055] (5) Sample combination: Use thin layer chromatography (TLC) to detect all fractions. According to the Rf value, 254 nm ultraviolet absorption results, and vanillin-concentrated sulfuric acid staining results, combine components with similar polarity and color, and then group them again. After grouping, each component is detected by TLC again, and then reduced pressure distillation and concentration are performed, and the weight and record of each component are recorded.

[0056] Table 6 Silica gel column chromatography eluent of biocontrol bacteria HN-149F dichloromethane extract

[0057] 4.2 Separation and purification of components: According to the TLC detection results ( Figure 6), the appropriate eluent system and proportion are selected, and each component is separated by silica gel column chromatography using silica gel with a mesh size of 0.075-0.050 mm (200-300 mesh), and the silica gel column chromatography operation steps are the same as those in 4.1. After silica gel column chromatography, the fractions containing the target compounds are combined by TLC detection, and then concentrated by vacuum distillation. The concentrated sample is subjected to silica gel column chromatography and gel column chromatography according to the TLC detection results. The operation steps of gel column chromatography are as follows: (1) Column loading: wet column loading is used. The appropriate length of the gel column and the corresponding eluent are selected according to the weight, polarity and solubility of the sample. An appropriate amount of eluent is added to a glass beaker containing the gel, and the gel is stirred with a glass rod to ensure complete swelling. Ultrasonic treatment is performed for 2-3 min to remove air bubbles. The gel column is fixed on an iron stand, and an appropriate amount of defatted cotton is inserted into the upper end of the column and pushed to the bottom to compact it. A 500 mL reservoir ball is connected to the upper end of the column, and the lower end valve is closed. Slowly pour the gel into the column to 1 / 3 of the column volume, stirring while gently tapping the outer wall of the gel column with an ear bulb to remove air bubbles. Then open the lower end valve and continue to pour the gel to 3-5 cm above the upper end of the column.

[0058] (2) Sample loading: wet sample loading is used. The sample is dissolved in the same eluent as the selected gel column. The gel column is allowed to flow at a constant speed. When the eluent reaches the surface of the gel, the completely dissolved sample is slowly added along the wall of the column. Pay attention to uniform and smooth dripping to avoid impacting the surface of the gel. When the sample solution reaches the surface of the gel, add an appropriate amount of eluent. After the sample layer moves a certain distance, add a large amount of eluent for elution.

[0059] (3) Collection: when the sample moves to 1 / 3 of the height of the column, collect the fractions using test tubes. According to the amount of sample and the impurity content of the sample, set the fraction collection time to make the volume of each fraction the same.

[0060] (4) Sample combination: combine the fractions containing the same compound according to the TLC results. After the combination of the components, perform TLC detection again. Each component is concentrated by vacuum distillation and weighed and recorded.

[0061] After multiple silica gel column chromatography, gel column chromatography separation and TLC detection, the thin layer chromatography silica gel plate has only one color spot according to the results of 254 nm ultraviolet absorption and vanillin-concentrated sulfuric acid staining. It is considered that the compound is separated and purified. The compound is stored in a refrigerator at 4°C.

[0062] The identified biocontrol strain HN-149F is subjected to macroporous resin adsorption and solvent extraction to obtain a dichloromethane crude extract of the secondary metabolites of the strain, and the chemical components of the crude extract are detected by thin layer chromatography. According to the results of ultraviolet irradiation at 254 nm and the results of vanillin-concentrated sulfuric acid staining, the HN-149F strain is selected as a subsequent test strain. Figure 7 It is shown by thin layer chromatography that the dichloromethane crude extract of the biocontrol strain is rich in components in a petroleum ether-ethyl acetate system, and can be used as a strain for the next step of testing.

[0063] After column chromatography, 40 fractions are obtained, and after sample combination, 8 components are obtained. Among them, component C3 (numbered C3) is rich in components (4 points) and has an obvious main point. The C3 is subjected to antibacterial activity test, and the test method is the same as that in Example 3. The results show that at a final concentration of 100 μg / mL, the inhibition rate of compound C3 on Magnaporthe grisea is 100% ( Figure 8 ), indicating that it has a very good inhibitory effect on Magnaporthe grisea.

[0064] Table 7 Inhibition results of biocontrol strain HN-149F crude extract separation component C3 on Magnaporthe grisea

[0065] Example 4: Field efficacy test of biocontrol strain HN-149F fermentation broth The preparation method of the biocontrol strain HN-149F fermentation broth comprises the following steps: S1. Solid culture: inoculate the biocontrol strain HN-149F into a lemon culture medium and culture at 28°C for 7 days. The formula of the lemon culture medium is: 1000 mL of distilled water, 0.12 g of citric acid, 1.5 g of sodium nitrate, 0.1 g of K2HPO4·3H2O, 0.1 g of anhydrous magnesium sulfate, 0.05 g of calcium chloride dihydrate, 0.02 g of EDTA (ethylene diamine tetraacetic acid), 0.2 g of sodium carbonate, 15 g of agar, and pH 7.2. S2. Liquid culture: punch out the fungus cake of the biocontrol strain HN-149F after solid culture with a 5 mm puncher, transfer it to a 250 mL triangular flask containing 100 mL of ISP-2 culture medium (the fungus cake is a single colony fungus cake of the biocontrol strain, and there are 5 fungus cakes in each flask), and culture at 28°C and 180 rpm for 7 days to obtain the biocontrol strain HN-149F fermentation broth.

[0066] Tested rice: Yanyong 47; Target organism: Magnaporthe grisea; The preparation method of the Magnaporthe grisea spore suspension comprises the following steps: Pyricularia grisea was inoculated on tomato oat medium at 28℃ for 7 days, and the cultured Pyricularia grisea was treated alternately under black light, i.e. treated under black light for 24 hours and normally cultured for 24 hours. The tomato oat medium formula is as follows: tomato juice 200 mL, oat 30 g, agar 30 g, distilled water 1000 mL.

[0067] The test rice field: field efficacy test was carried out in the rice test field of Shenyang Agricultural University in 2025.

[0068] Test grouping: the test was divided into 4 groups, including 1 test group, 2 positive control groups and 1 blank control group. The test group was sprayed with biocontrol bacteria HN-149F fermentation liquor, the positive control group was sprayed with tricyclazole and kasugamycin respectively, and the blank control group was sprayed with water.

[0069] Test method: protective measures and treatment measures were taken for each test group. The protective measures were to spray water, chemical agents or biocontrol bacteria HN-149F fermentation liquor first and then inoculate Pyricularia grisea, and the treatment measures were the opposite.

[0070] Protective measures: spray method was used for Pyricularia grisea inoculation. First, the mycelium of the above cultured Pyricularia grisea was washed with sterile water, filtered, and the filtrate was collected as spore suspension. The concentration was adjusted to 5×10 5 / mL (about 40-50 under 10 times microscope), 0.05% volume of Tween 20 was added, and mixed thoroughly. Then the spore suspension was uniformly sprayed on the rice plants to be inoculated with a spray pot, 3 times for each treatment, 15 holes of rice in each plot. According to the above test grouping, water, tricyclazole, kasugamycin and biocontrol bacteria HN-149F fermentation liquor were used for protective treatment, and the incidence of Pyricularia grisea was investigated about 7-14 days after heading.

[0071] Treatment measures: spray method was used for Pyricularia grisea inoculation. First, the mycelium of the above cultured Pyricularia grisea was washed with sterile water, filtered, and the filtrate was collected as spore suspension. The concentration was adjusted to 5×10 5 / mL (about 40-50 under 10 times microscope), 0.05% volume of Tween 20 was added, and mixed thoroughly. Then the spore suspension was uniformly sprayed on the rice plants to be inoculated with a spray pot, 3 times for each treatment, 15 holes of rice in each plot. According to the above test grouping, water, tricyclazole, kasugamycin and biocontrol bacteria HN-149F fermentation liquor were used for protective treatment, and the incidence of Pyricularia grisea was investigated about 7-14 days after heading.

[0072] Table 8 Field test control effect

[0073] As shown in Table 8, the test results show that in the protection treatment, the control effect of the biocontrol bacteria fermentation filtrate is not obvious in the field efficacy test; in the treatment, the control effect of the biocontrol bacteria fermentation liquid on rice blast is 72.73%, and there is no significant difference with the chemical agent proleukin (80.58%) and tricyclozole (79.41%), and the biocontrol bacteria fermentation liquid has no phytotoxicity to the rice plants and no adverse effects on the beneficial organisms in the field.

[0074] The above merely describes the preferred embodiments of the present application, but the protection scope of the present application is not limited to this. Any person skilled in the art can make equivalent replacements or changes according to the technical scheme and the inventive concept of the present application within the technical range disclosed by the present application, and all of them should be covered in the protection scope of the present application.

Claims

1. A type of Streptomyces ( Streptomyces cellostaticus HN-149F, characterized in that, Its accession number is CGMCC NO.36380, it is deposited at the China General Microbiological Culture Collection Center, the deposit date is October 28, 2025, and the deposit address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

2. A strain containing the Streptomyces cytotoxicum as described in claim 1 ( Streptomyces cellostaticus HN-149F microbial inoculant.

3. A Streptomyces cytotoxicum strain according to claim 1 ( Streptomyces cellostaticus Application of HN-149F.

4. The Streptomyces cytotoxicum according to claim 1 ( Streptomyces cellostaticus The application of HN-149F is characterized by, Used to control rice blast fungus ( Pyricularia oryzae ) and / or tomato gray mold ( Botrytis cinerea ).

5. The Streptomyces cytotoxicum according to claim 1 ( Streptomyces cellostaticus The application of HN-149F is characterized by, Used to control rice blast fungus ( Pyricularia oryzae ).

Images