Application of streptomyces roseosporus in plant disease control

Abstract

The application belongs to the technical field of microbial application, and specifically discloses application of roseofungin Streptomyces roseofunginus in plant disease prevention and treatment. The application finds that after R&M synthesis genes in roseofungin Streptomyces roseofunginus are knocked out, the strain can still synthesize oxazolomycin A. Further finding is that oxazolomycin A shows good inhibitory effect on pepper Phytophthora capsici and Phytophthora sansomei, wherein the EC 50 value of pepper Phytophthora capsici PC35 is 2.95 μg / mL, and the EC 50 value of atractylodes Phytophthora sansomei AMPH-1 is 2.39 μg / mL. The oxazolomycin A prepared by the application can be developed into a biological pesticide that can effectively meet the agricultural development demand of "green ecology", and provides key biocontrol resource support for green prevention and control of plant diseases.

Description

Technical Field

[0001] This invention belongs to the field of microbial application technology and relates to the application of Streptomyces roseum in the prevention and control of plant blight. Background Technology

[0002] Phytophthora capsici ( Phytophthora capsicum ) belongs to the genus *Phytophthora* of the phylum Oomycetes, and is a plant of the genus *Capsicum*. Capsicum annuum Phytophthora capsici is a typical oomycete pathogen that exhibits significant harmful characteristics in agricultural production: First, it has a wide range of transmission and infection, spreading through the soil and being difficult to effectively block, leading to easy spread of the disease in the field; second, its infection mechanism is highly destructive. This pathogen mainly invades the plant through the roots, releasing vector effector proteins that directly attack the plant's own defense system, rendering the plant unable to resist the pathogen. After infection with Phytophthora capsici, chili plants will exhibit a series of typical disease symptoms, including root, stem, and fruit rot, accompanied by chili leaf blight. As the disease progresses, the pathogen's infection rate accelerates, eventually causing the entire chili plant to wilt and die. In severe outbreaks, the mortality rate of chili plants in the field is extremely high, even leading to total crop failure and significant economic losses to the chili planting industry.

[0003] As an important traditional Chinese medicine, Atractylodes macrocephala is susceptible to Atractylodes macrocephala blight pathogens during its production process (… Phytophthora sansomeana AMPH-1 infection. This pathogen primarily damages the stem base of Atractylodes macrocephala and has a wide infection period coverage—both seedlings and mature plants can be infected, with the seedling stage being the most severely affected. Seedlings are less resistant to pathogens and are prone to rapid symptoms such as stem base rot and lodging after infection, leading to a significant increase in seedling mortality. Even in mature plants, infection affects the quality and yield of the rhizomes, thus negatively impacting the industrial production of Atractylodes macrocephala as a traditional Chinese medicine.

[0004] Currently, the industry mainly employs two technical approaches for the prevention and control of Phytophthora blight in peppers and Atractylodes macrocephala: chemical control and biological control. Chemical control, due to its ability to rapidly suppress the spread of pathogens and achieve good disease control, has been the commonly used method in the past. However, with the long-term and large-scale use of chemical pesticides, their adverse effects have become increasingly apparent. These include pesticide residues in soil, fruits, and medicinal materials; damage to beneficial organisms in the farmland ecosystem; and the development of pesticide resistance in pathogens due to long-term use, leading to a gradual decline in control effectiveness. As the concept of "green ecology" gradually penetrates into agricultural production, reducing reliance on chemical pesticides and developing and producing pollution-free biological pesticides have become urgent priorities in plant disease prevention and control. Especially in crop cultivation, the requirements for the safety and environmental friendliness of biocontrol agents are even higher, necessitating technical solutions that meet the needs of green prevention and control. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides the application of *Streptomyces roseum* in the prevention and control of plant blight. This invention creatively discovers that strain 0116 of *Streptomyces roseum* can still produce oxazolidin A even after the R&M synthesis gene is knocked out. It has a significant inhibitory effect on *Phytophthora capsici* and *Phytophthora blight*, the pathogen of *Phytophthora* blight of *Atractylodes macrocephala*, and can be used for the prevention and control of plant blight such as *Phytophthora capsici* and *Phytophthora* blight of *Atractylodes macrocephala*.

[0006] To achieve the above-mentioned objectives, the embodiments of the present invention employ the following technical solutions:

[0007] In a first aspect, the present invention provides *Streptomyces roseum* ( Streptomyces roseoflavus Application of ) in the prevention and control of plant diseases.

[0008] Preferably, the *Streptomyces roseum* is *Streptomyces roseum* strain 0116.

[0009] The preservation number of Streptomyces roseum strain 0116 is CGMCC No.1471, and it was first disclosed in Chinese patent document CN200510117422.8 (Patent title: Application of fermentation broth residue of Streptomyces roseum strain 0116 as fertilizer); this strain is also named Streptomyces roseum Men-myco-93-63.

[0010] Preferably, the plant disease includes at least one of pepper blight or Atractylodes blight.

[0011] Streptomyces roseum strain 0116 is an antagonistic bacterium screened from soil naturally decaying with potato scab. Studies have found that this strain and its fermentation broth exhibit strong inhibitory effects against several important plant pathogens, including Verticillium wilt of cotton, powdery mildew of cucurbits, and scab of potatoes, demonstrating good potential for biocontrol applications. Previous studies have shown that a group of structurally similar pentaene macrocyclic lactone antibiotics were isolated from the fermentation broth of this bacterium, with two components present in high amounts: roflamycoin and men-myco-A, abbreviated as R&M. This group of antibiotics exhibits broad-spectrum and highly effective antifungal activity, showing potential for development into agricultural antibiotics. This invention creatively discovers the R&M synthesis gene in Streptomyces roseum strain 0116. rfmA The mutant strain obtained after knocking out (accession number MT968841) can still synthesize oxazolomycin A.

[0012] Secondly, the present invention provides *Streptomyces roseum* ( Streptomyces roseoflavus Application of strain 0116 in the preparation of oxazolidin A.

[0013] Thirdly, the present invention provides a method for preparing oxazolidin A, the method comprising the following steps: using *Streptomyces roseum* strain 0116 as the starting strain, knocking out the R&M synthesis gene— rfmA Genes were used to construct a mutant strain ∆rfmA; the mutant strain ∆rfmA was fermented, extracted, and isolated to obtain oxazolidin A.

[0014] Preferably, the fermentation temperature is 28℃-32℃, the rotation speed is 150rpm-250rpm, and the time is 96h-144h.

[0015] Preferably, the fermentation medium comprises the following components: 20g-30g glucose, 5g-10g soluble starch, 10g-20g peanut cake powder, 5g-10g corn steep liquor, 3g-5g sodium chloride, 1g-5g calcium carbonate, and 0.1g-0.3g potassium dihydrogen phosphate per 1L, with the balance being water, and the pH being 7.2-7.8.

[0016] Preferably, the extraction and separation includes the following steps: separating the fermentation broth obtained from fermentation into a solid-liquid mixture to obtain a solution;

[0017] The solution was extracted with a short-chain ester, and after removing the solvent from the resulting extract, a short-chain alcohol was added for redissolution. The extract was then purified by preparative liquid chromatography to obtain oxazolidin A.

[0018] More preferably, the short-chain esters include ethyl acetate.

[0019] More preferably, the short-chain alcohol includes methanol.

[0020] Preferably, the conditions for the preparation of liquid phase purification are: chromatographic column: octadecyl bonded silica gel column;

[0021] Detection wavelength: 274nm;

[0022] Mobile phase: Water is mobile phase A, and methanol is mobile phase B;

[0023] The elution procedure is gradient elution; the gradient elution procedure is as follows:

[0024] .

[0025] Fourthly, the present invention provides the application of oxazolidin A prepared by the method described in the third aspect in inhibiting plant pathogens, wherein the plant pathogens include Phytophthora capsici. Phytophthora capsicum Or Phytophthora infestans, the pathogen of Atractylodes macrocephala blight Phytophthora sansomeana At least one of them.

[0026] Currently, there is a shortage of agricultural antibiotic biocontrol resources that exhibit good inhibitory effects against both *Phytophthora capsici* and *Phytophthora sansembryophylla* pathogens, necessitating the discovery of new, highly effective biocontrol strains and derived active substances. The oxazolidin A provided by this invention can effectively inhibit the growth of *Phytophthora capsici* and *Phytophthora sansembryophylla* mycelia, demonstrating good inhibitory effects against these two pathogens. Specifically, the EC50 of *Phytophthora capsici* PC35... 50 The value was 2.95 μg / mL, the EC50 value of *Phytophthora sansemii* AMPH-1 in *Atractylodes macrocephala* blight control. 50 The value was 2.39 μg / mL.

[0027] Fifthly, the present invention provides the application of oxazolidin A prepared by the method of preparation of oxazolidin A provided in the third aspect in the preparation of plant blight inhibitors, wherein the plant blight includes at least one of pepper blight or Atractylodes blight.

[0028] Oxazolidin A can be used as an agricultural antibiotic, possessing core advantages such as no residue, easy decomposition in the environment, and ecosystem friendliness. The oxazolidin A prepared in this application can be developed into a biopesticide that effectively meets the needs of "green and ecological" agricultural development, providing crucial biocontrol bacterial resource support for the green control of plant diseases. Simultaneously, this invention provides important technical basis for the development of specialized biocontrol agents targeting blight in traditional Chinese medicinal materials or peppers, possessing significant technological research and application value. Attached Figure Description

[0029] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0030] Figure 1 This is a high-resolution mass spectrum of the target compound obtained in Example 1 of the present invention;

[0031] Figure 2 The 1H NMR spectrum of the target compound obtained in Example 1 of this invention;

[0032] Figure 3 The image shows the carbon NMR spectrum of the target compound obtained in Example 1 of this invention.

[0033] Figure 4 The results of the toxicity test of oxazolidin A against Phytophthora sansemella AMPH-1 obtained in Example 1 of the present invention are shown.

[0034] Figure 5 The results of the toxicity test of oxazolidin A against Phytophthora capsici PC35 obtained in Example 1 of the present invention are shown. Detailed Implementation

[0035] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0036] Unless otherwise specified, the raw materials and reagents used in this invention are all conventional commercially available products; unless otherwise specified, the methods used in this invention are all conventional methods in the field.

[0037] Example 1

[0038] This embodiment provides a method for preparing oxazolidin A, which includes the following steps:

[0039] S1. Mutant ∆ rfmA Construction

[0040] Using *Streptomyces roseum* strain 0116 (CGMCC No. 1471) as the starting strain, knockout was performed. rfmA Gene (NCBI accession number MT968841), construct mutant strain ∆rfmA; specific method is as follows:

[0041] Using the genomic DNA of Streptomyces roseum strain 0116 as a template, and primers K7392U-F / R and K7392D-F / R as primers, the amplification was performed. rfmA The homologous left and right arms, measuring 521bp and 575bp respectively, were introduced into the 5' ends of primers K7392U-F and K7392D-R. EcoRV and EcoR The restriction endonuclease site I was identified. Simultaneously, using plasmid pVB as a template and KanF-7392 and KanR-7392 as primers, the kanamycin resistance fragment was amplified. neo The size is 1057bp.

[0042] The purity and size of the products were determined by 1% agarose gel electrophoresis and the gel was excised and recovered. Then, overlap PCR was performed according to the left arm- neo The three fragments were ligated in the order of the right arm (for knockout vectors without antibiotic markers, the two fragments were ligated in the order of the left arm to the right arm), and the purity and size of the products were determined by 1% agarose gel electrophoresis and recovered. The obtained fragment ligation products and pKC1139 plasmid were then subjected to... EcoRV and EcoRI Enzyme digestion, followed by ligation with T4 ligase to obtain the gene-blocking plasmid pKO7392, was transformed into E. coli DH5α competent cells. Positive clones were picked, and the plasmid was extracted by shaking. EcoRV and EcoRI Double enzyme digestion and sequencing verification were performed. The verified plasmid was transformed into methylation-deficient *E. coli* ET12567 / puz8002, and then conjugated to *Streptomyces roseum* strain 0116 to obtain a mutant strain. ∆rfmA .

[0043] The specific sequences of the primers are shown in Table 1 below.

[0044] Table 1

[0045]

[0046] S2. Fermentation

[0047] mutant strain ∆rfmA The culture was spread on PDA medium and cultured for 5 days. A bacterial cake with a diameter of 8 mm was taken and inoculated into a shake flask (capacity 500 mL) containing 100 mL of fermentation medium. A total of 30 flasks were cultured at 30℃ and 200 rpm for 120 h to obtain the fermentation broth.

[0048] The fermentation medium includes the following components: 24g glucose, 8g soluble starch, 15g peanut cake powder (boiled for 15 minutes, filtered through eight layers of gauze, and the filtrate is collected), 8g corn steep liquor, 4g sodium chloride, 3g calcium carbonate, 0.2g potassium dihydrogen phosphate, and distilled water to make up to 1L. The pH is adjusted to 7.5 with 3M sodium hydroxide solution.

[0049] S3. Extraction of fermentation broth and purification of target product

[0050] The above fermentation broth was centrifuged at 4000 rpm for 10 min to obtain the supernatant of the fermentation broth;

[0051] Take the supernatant of the fermentation broth obtained above, add an equal volume of ethyl acetate for extraction, and evaporate the extract phase by rotary evaporation at 50°C to obtain a brown oily substance.

[0052] The above-mentioned brown oily substance was dissolved in methanol by chromatography, analyzed by liquid chromatography, and purified and recovered by preparative liquid chromatography. The collected liquid was concentrated by rotary evaporation at 50°C to obtain a yellow oily substance. The solvent was evaporated to obtain the target compound.

[0053] The chromatographic conditions for preparing the liquid phase are as follows:

[0054] Column: Waters SunFire® Prep C18 OBD TM The column was prepared with a packing particle size of 5μm and 19mm*250mm.

[0055] Detection wavelength: 274nm;

[0056] Injection volume: 1 mL; Flow rate: 10 mL / min;

[0057] Mobile phase: Water is mobile phase A, and methanol is mobile phase B;

[0058] The elution procedure is gradient elution; the gradient elution procedure is as follows:

[0059]

[0060] S4. Structural Identification

[0061] The collected liquid obtained from the above-mentioned liquid-phase purification was analyzed by high-resolution mass spectrometry to determine its molecular weight; the target compound was dissolved in deuterated methanol (Methanol-d4) and subjected to nuclear magnetic resonance (NMR). 1 H (600 MHz) and 13 The chemical structure of the compound was identified by C10 NMR (150 MHz). The high-resolution mass spectrum of the target compound is shown below. Figure 1 As shown, the proton NMR spectrum of the obtained target compound is as follows. Figure 2 As shown, the carbon NMR spectrum of the obtained target compound is as follows. Figure 3 As shown.

[0062] High-resolution mass spectrometry analysis revealed that the mass-to-charge ratio of this compound is [M+H]. + m / z The molecular weight of the compound is predicted to be 655, given the value of 656.3543. The molecular formula is: C 35 H 49 N3O9 has an unsaturation degree of 13.

[0063] The 1H NMR spectrum information is as follows: 1 H NMR (600 MHz, Methanol- d 4) δ 8.11 (1H, s, H-13'), 6.88 (1H, s, H-12'), 6.75 (1H, dd, J = 15.0, 11.3 Hz, H-8'), 6.48 (1H, d, J = 11.9 Hz, H-5'), 6.30 (1H, t, J = 11.6 Hz, H-6'), 6.27 - 6.22 (2H, m, H-9,10), 5.99 (1H, t, J = 11.1 Hz, H-7'), 5.81 (1H, dt, J= 14.4, 6.9 Hz, H-9'), 5.75- 5.67 (2H, m, H-8, 11), 4.86 (1H, overlapped, H-16a), 4.73 (1H, s, H-3'), 4.53 (1H, d, J = 6.4 Hz, H-16b), 4.00 (1H, t, J = 6.3 Hz, H-7), 3.85 (2H, d, J =6.0 Hz, H-12), 3.58 (2H, d, J = 6.9 Hz, H-10'), 3.54 (1H, t, J = 5.0 Hz, H-4),3.35 (s, 3H, 4-OCH3), 2.92 (3H, s, N-CH3), 2.52 (1H, q, J = 7.3 Hz, H-2), 2.08(1H, dt, J = 15.4, 5.4 Hz, H-6), 1.81 (3H s, 4'-CH3), 1.79 - 1.74 (2H, m, H-5), 1.28 (s, 3H, 2'-CH3), 1.20 (3H, d, J = 7.3 Hz, H-13), 1.09 (3H, s, 2'-CH3), 1.02(3H, d, J = 6.8 Hz, H-14);

[0064] The carbon NMR spectrum information is as follows: 13 C NMR (150 MHz, Methanol- d4) δ 179.8 (C-1'),177.6 (C-1), 171.9 (C-17), 153.1 (C-13'), 152.8 (C-11'), 140.0 (C-4'), 134.8(C-8), 132.4 (C-10), 132.4 (C-11),130.6 (C-9), 129.7 (C-9'), 129.5 (C-8'),129.0 (C-7'), 125.7 (C-6'), 125.2 (C-5'), 122.7 (C-12'), 87.5 (C-15), 84.4(C-4), 80.7 (C-3), 77.5 (C-7), 75.6 (C-3'), 66.7 (C-16), 57.4 (4-OCH3), 46.9(C-2'), 44.2 (C-2), 42.1 (C-12), 38.3 (C-6), 33.4 (C-5), 29.6 (C-10'), 26.3(CN-CH3), 26.0 (4'-CH3), 22.1 (2'-CH3), 20.1 (2'-CH3), 16.7 (C-14), 10.2 (C-13).

[0065] The 1H and 1C NMR spectra of the target compound indicate that it has a complex polycyclic or long-chain conjugated structure and contains multiple functional groups. 1 H NMR showed the presence of multiple olefin proton signals (δ). H 6.75–5.67), where δ H 6.75 (dd, J=15.0, 11.3Hz) and δ H 5.81 (dt, J=14.4, 6.9 Hz) indicates a trans double bond configuration; aromatic proton signal δ H 8.11 (s) and δ H 6.88 (s) belongs to the heterocyclic system. Furthermore, the oxymethylene signal (H-16a,b) at δ 4.86–4.53 and δ H 4.00 (t) and δ H The presence of an oxygen-containing methine proton at 3.54 (t) indicates the presence of an ether or hydroxyl substitution in the molecule. Methoxy (δ) H 3.35, s) and N-methyl (δ) H The 2.92, s) signal further confirmed the presence of ether and tertiary amine functional groups. 13 C NMR data conjugated carbonyl carbon signal (δ) C(179.8, 177.6, 171.9) are consistent with the structures of lactones or carboxylic esters; olefin carbon signals (δ) C (153.1–122.7) supports the existence of a conjugated framework, with multiple quaternary carbon signals (such as δ) C (87.5, 84.4, 80.7) indicates the presence of multiple chiral centers or ether rings in the molecular skeleton. The methyl carbon signal (δ) C 26.0, 22.1, 20.1, 16.7, 10.2) and 1 Multiple methyl peaks (δ) in H NMR H The values ​​of 1.81–1.02 indicate the presence of multiple methyl substituents. Based on the above analysis and comparison with literature, this compound is preliminarily identified as a member of the oxazolomycin family (C). 35 H 49 N3O9).

[0066] The oxazolomycin family compounds oxazolomycin A, B, and C differ only in the conjugated triene moiety at the 4′, 6′, and 8′ positions, which are (4'Z, 6'Z, 8'E), (4'E, 6'E, 8'E), and (4'Z, 6'E, 8'E), respectively. Furthermore, existing literature indicates that the chemical shifts of H-3′ in oxazolomycin A, B, and C are 4.65 ppm, 4.12 ppm, and 4.73 ppm, respectively, corresponding to Z, E, and Z double bond configurations at the 4′ position. The H-3′ in this compound exhibits a relatively large chemical shift (ẟ). H-3 The 4′ position (J′ 4.73) indicates that the compound has a Z configuration. The coupling constants between H-6′ and H-7′ (J6′-7′=11.6 Hz) and between H-8′ and H-9′ (J8′-9′=15.0 Hz) indicate that the double bond configuration of this compound is 6′Z, 8′E. Therefore, the geometry of the conjugated triene moiety of this compound is determined to be 4′Z, 6′Z, 8′E, thus ultimately identifying the compound as oxazolomycin A.

[0067] Example 2

[0068] This embodiment provides a method for preparing oxazolidin A, which is basically the same as that in Example 1, except that in step S2, the fermentation conditions are: 28℃, 220 rpm, and a fermentation time of 144 h. The fermentation medium includes the following components per 1 L: 20 g glucose, 10 g soluble starch, 10 g peanut cake powder (boiled for 15 min, filtered through eight layers of gauze, and the filtrate is collected), 10 g corn steep liquor, 5 g sodium chloride, 5 g calcium carbonate, 0.3 g potassium dihydrogen phosphate, and distilled water to a final volume of 1 L. The pH is adjusted to 7.6 using 3M sodium hydroxide solution. The remaining preparation methods are the same as in Example 1.

[0069] Example 3

[0070] This embodiment provides a method for preparing oxazolidin A, which is basically the same as that in Example 1, except that in step S2, the fermentation conditions are 32℃, 220 rpm, and the fermentation time is 96 h. The fermentation medium includes the following components per 1 L: 30 g glucose, 5 g soluble starch, 20 g peanut cake powder (boiled for 15 min, filtered through eight layers of gauze, and the filtrate is collected), 5 g corn steep liquor, 3 g sodium chloride, 2 g calcium carbonate, 0.15 g potassium dihydrogen phosphate, and distilled water to a final volume of 1 L. The pH is adjusted to 7.4 using 3M sodium hydroxide solution. The remaining preparation methods are the same as in Example 1.

[0071] Example of effect

[0072] This invention takes the preparation of oxazolidin A in Example 1 as an example to investigate its effect on Phytophthora capsici (… P. capsicum PC35 and Phytophthora sansemii, the pathogen of Atractylodes macrocephala blight ( P. sansomeana The inhibitory effect of AMPH-1 is detailed below:

[0073] The collected liquid obtained from the preparative liquid chromatography purification and recovery in Example 1 was concentrated by rotary evaporation at 50°C. After evaporating the solvent, pure oxazolidin A was obtained. The pure oxazolidin A was dissolved in different volumes of chromatographic methanol, filtered through a 0.22 μm organic filter membrane, and then subjected to the reaction with Phytophthora capsici (…). P. capsicum PC35 and Phytophthora sansemii, the pathogen of Atractylodes macrocephala blight ( P. sansomeana ) The toxicity of AMPH-1.

[0074] To investigate mycelial growth inhibition, oxazolomycin A concentration gradients of 10 μg / mL, 5 μg / mL, 2.5 μg / mL, 1 μg / mL, and 0.2 μg / mL were used in three replicates. A blank control was included, and the plates were incubated at 25°C for 3 days. The mycelial diameter of *Phytophthora indicum* was measured using the cross-multiplication method, and the growth inhibition rate of the fungicide against *Phytophthora indicum* was calculated using the following formula.

[0075] Mycelial growth inhibition rate (%) = (mycelial diameter of pathogen in control group - mycelial diameter of pathogen in treatment group) / mycelial diameter of pathogen in control group × 100%.

[0076] The virulence test results of oxazolidinone against the two Phytophthora species are shown in Table 2. The virulence test results of oxazolidinone A against Phytophthora sansemella AMPH-1 are shown in Table 2. Figure 4 As shown; Figure 4 In the table, A is the blank control, B is the methanol control; C is 10 μg / mL, D is 5 μg / mL, E is 2.5 μg / mL, F is 1 μg / mL, and G is 0.2 μg / mL. The results of the toxicity assay of oxazolidin A against Phytophthora capsici are as follows: Figure 5 As shown; Figure 5 In the table, A is the blank control, B is the methanol control; C is 10 μg / mL, D is 5 μg / mL, E is 2.5 μg / mL, F is 1 μg / mL, and G is 0.2 μg / mL.

[0077] Table 2

[0078]

[0079] Based on the toxicity regression equation, the effective inhibitory concentration (EC50) of the pure oxazolidinyl A prepared in Example 1 of this invention was calculated. 50 (μg / mL) and correlation coefficient (R) 2 EC of Phytophthora capsici PC35 50 The value was 2.95 μg / mL, EC 90 The value was 14.24 μg / mL; EC50 of Phytophthora sansemii AMPH-1 in Atractylodes macrocephala blight 50 The value was 2.39 μg / mL, EC 90 The value was 8.73 μg / mL.

[0080] As can be seen from the above, using *Streptomyces roseum* strain 0116 as the starting strain, knockout... rfmA Post-genetic mutant strain ∆rfmA The produced oxazolidinone A exhibits significant inhibitory effects against *Phytophthora capsici* PC35 and *Phytophthora sansem* AMPH-1, the pathogen causing *Atractylodes macrocephala* blight. Therefore, *Streptomyces roseum* strain 0116 also possesses the ability to synthesize oxazolidinone A. Thus, *Streptomyces roseum* strain 0116 and its mutant strain ∆rfmA can be used to prepare oxazolidinone A, which can then be further applied to the control of plant blight diseases such as *Phytophthora capsici* and *Atractylodes macrocephala* blight.

[0081] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions or improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. Streptomyces roseum ( Streptomyces roseoflavus The application of strain 0116 in the preparation of oxazolidin A is characterized by: With rose yellow Streptomyces strain 0116 as a starting strain, the rfmA gene is knocked out to construct a mutant strain rfmA; the mutant strain rfmA is fermented, extracted and separated to obtain oxazolomycin A.

2. A process for the preparation of oxazolmycin A, characterized by: The preparation method comprises the following steps: taking roseofinus muscorum strain 0116 as a starting strain, knocking out rfmA a gene, constructing a mutant strain rfmA ; fermenting, extracting and separating the mutant strain rfmA to obtain oxazolomycin A.

3. The process for the preparation of oxazolmycin A according to claim 2, characterized in that: The fermentation is carried out at a temperature of 28-32℃, a rotation speed of 150-250rpm, and a time of 96-144h; and / or The fermentation medium comprises the following components: 20-30g of glucose, 5-10g of soluble starch, 10-20g of peanut cake powder, 5-10g of corn syrup, 3-5g of sodium chloride, 1-5g of calcium carbonate, and 0.1-0.3g of potassium dihydrogen phosphate, with the balance being water, per 1L, and has a pH of 7.2-7.

8.

4. The process for preparing oxazolmycin A according to claim 2, wherein: The extraction and separation comprises the following steps: subjecting the fermentation broth obtained from the fermentation to solid-liquid separation to obtain a solution; The solution is extracted with short-chain esters, the obtained extraction phase is desolventized, a short-chain alcohol is added for redissolution, and preparative liquid phase purification is performed to obtain oxazolomycin A.

5. The process for the preparation of oxazolmycin A according to claim 4, characterized in that: The short-chain esters comprise ethyl acetate; and / or The short-chain alcohol comprises methanol.

6. The process for the preparation of oxazolmycin A according to claim 4, characterized in that: The preparative liquid phase purification is performed under the following conditions: a chromatographic column: a carbon octadecyl-bonded silica gel filler chromatographic column; a detection wavelength: 274nm; a mobile phase: water as mobile phase A and methanol as mobile phase B; an elution program: gradient elution; and the gradient elution program is as follows: 。 7. Use of oxazolmycin A produced according to the process of any one of claims 3 to 6 for inhibiting phytopathogenic fungi, characterized in that: The plant pathogen includes at least one of Phytophthora capsici Phytophthora capsicum or the white turmeric blight pathogen, Pythium Phytophthora sansomeana ​ 8. Use of oxazolmycin A produced by the process according to any one of claims 3 to 6 for the preparation of a plant disease inhibitor, characterized in that: The plant diseases comprise at least one of pepper blight or atractylodes blight.

Images