CN115261422B - Preparation method and application of new species-derived thiolutin - Google Patents

Abstract

The present invention relates to a preparation method and application of thiolutin produced by Streptomyces populiradicis TRM70303. The present invention utilizes a new actinomycete species, Streptomyces populiradicis TRM70303, separated from rhizosphere soil of poplars in the Taklimakan Desert in Xinjiang, and obtains thiolutin through culturing and separation and purification of fermentation liquid, with a yield of 0.27 mg/mL. Compared with the thiolutin preparation method disclosed in the prior art, the yield is greatly improved, the preparation process is simple, the strain fermentation level is high, the cost is low, and the fermentation cycle is short; the antibacterial activity experimental test confirms that the thiolutin obtained by the present invention has a broad-spectrum and efficient antagonistic activity against cotton Verticillium wilt, cotton Fusarium wilt, fragrant pear rot, amylopectin, Shigella, pear black spot, and Acinetobacter baumannii, and has important application value and good development prospects for the prevention and control of plant diseases and bacterial infection.

Description

Preparation method and application of thiolutin from a new species

Technical Field

The present invention relates to the technical field of microbial strains and their applications, and in particular to a preparation method and application technical field of thiolutein produced by a new actinomycete species, Streptomyces populiradicis TRM70303.

Background technique

Agricultural pests and diseases are the main disasters in my country's agriculture. According to the statistics of the Food and Agriculture Organization of the United Nations (FAO), the average annual loss of production due to plant diseases is 10%-15% of the total output. Agricultural antibiotics have developed rapidly, and new products are constantly being put on the market. However, due to the low production level, high production cost, low prevention and control efficiency, etc., the efficient use of them on cash crops is largely limited.

As the human race calls for environmental protection, the development of chemical pesticides is becoming increasingly difficult. Agricultural antibiotics, as an important part of biological control, have become a hot topic in research and development. One way to solve this series of new problems is to find and discover a large number of new bioactive substances. The research on microbial antibiotics has become the focus of scientific research in my country. As the basis and source for obtaining lead compounds, the acquisition of strain resources is crucial, especially the synthesis of extreme environment microbial antibiotics, which is a hot area of research in the academic community.

As a special ecological environment, the desert has the characteristics of strong solar radiation, large water evaporation, extreme drought and heat, poor nutrition, and soil salinization. In order to adapt to the natural environment, microorganisms have formed their own unique metabolites in the long-term life and evolution process. These substances have high research and development value and are also important resources for finding new active secondary metabolites.

Thiolutin is a dithiopyrrolone antibiotic with broad-spectrum antibacterial activity. Although there are reports on the preparation of thioolutin by microbial fermentation, a Chinese patent ("Use of thioolutin dioxide and its derivatives in the preparation of drugs for treating CNS diseases and preparation method thereof", CN1279906C) discloses the use of Nocardia species ST100692, DSM 13834 produces thioluteolin dioxide, and the literature ("Screening of interleukin-4 receptor antagonists from microorganisms", Wang Ke et al., Chinese Journal of New Drugs, 2007, 16 (14): 1088-1090) discloses the use of Streptomyces 4849 fermentation broth to separate and purify three compounds containing aureusin, thioluteolin and 4,7-dimethoxyisoflavone, but the yield of thioluteolin is not high, the preparation process is complicated and the cost is high, and the fermentation level of the original strain is low; the chemical synthesis of thioluteolin is complicated, and there are relatively extreme chemical processes such as high temperature, high pressure, strong acid and strong alkali, the production safety is low and the environmental pollution is serious; at the same time, existing technical reports have shown that thioluteolin has good activity against Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa, tumor cells, etc., but there is no report on whether it has an inhibitory effect on cotton wilt, cotton verticillium wilt, fragrant pear rot, apricot fire blight, Shigella and Acinetobacter baumannii.

Summary of the invention

In view of the current technical status that the preparation process of thiolutin is complicated, costly, low in safety, has serious environmental pollution, and has low yield by microbial fermentation, and there is no literature or patent record on the preparation of thiolutin by fermentation of Streptomyces populiradicis, the present invention aims to provide a preparation method and application of thiolutin produced by a new actinomycete species, Streptomyces populiradicis TRM70303. TRM70303, fermentation, separation and purification to obtain the compound thioluteolin, with a yield of 0.27 mg/mL. Compared with the existing thioluteolin preparation method, the yield is greatly improved, the preparation process is simple, the strain fermentation level is high, the cost is low, the fermentation cycle is short, and the large-scale production of active substances can be achieved through large-scale fermentation, thereby fundamentally solving the shortage of drug sources; the antibacterial activity test shows that the thioluteolin obtained by the preparation method of the present invention has a broad-spectrum and highly effective antagonistic activity against cotton Verticillium wilt, cotton Fusarium wilt, fragrant pear rot, amylopectin fire blight, Shigella, pear black spot, and Acinetobacter baumannii, and has important application value and good development prospects for the prevention and control of plant diseases and bacterial infections. It is used in the preparation of drugs for preventing and controlling cotton Verticillium wilt, cotton Fusarium wilt, fragrant pear rot, amylopectin fire blight, Shigella, pear black spot and Acinetobacter baumannii, and has a wide range of biological pesticides and biomedicine values.

In order to achieve the above technical effects, the present invention is implemented through the following technical solutions.

A preparation method of thiolutin produced by Streptomyces populiradicis TRM70303 specifically comprises the following steps: inoculating a single colony of the strain Streptomyces populiradicis TRM70303 in a TSB culture medium, culturing at 33°C and 120rpm for 3 days to obtain a Streptomyces populiradicis TRM70303 seed solution; inoculating the Streptomyces populiradicis TRM70303 seed solution into an oat liquid culture medium at an inoculation volume ratio of 1%, fermenting and culturing at 33°C and 120rpm for 5 days to obtain a Streptomyces populiradicis TRM70303 fermentation liquid; collecting the fermentation liquid, filtering it, loading it onto a D101 macroporous resin, eluting it with 100% methanol, collecting the eluate, and concentrating it under reduced pressure at 50°C to obtain a flaky crystalline thiolutin.

In the method for preparing thiolutein of the present invention, the Streptomyces populiradicis TRM70303 strain is preserved in China Center for Type Culture Collection, and the strain preservation number is CCTCC No: M2021450.

In the method for preparing thiolutein of the present invention, the gene sequence of Streptomyces populiradicis TRM70303 is shown as SEQ ID NO: 1.

In the preparation method of thiolutein of the present invention, the separation culture medium of the new microbial species Streptomyces populiradicis TRM70303 is: soluble starch 20g/L, _{KNO3 1g} /L, K2HPO4 0.5g/L, MgSO4·7H2O _0.5g /L, NaCl 0.5g/ _L , _FeSO4 · _7H2O 0.01g/L, agar _18g /L, soil extract 1000mL, pH 7.5.

In the method for preparing thiolutein of the present invention, the purification culture medium of the new microbial species Streptomyces populiradicis TRM70303 is: _10.0g soluble starch, _{1.0g K2HPO4 ,} 1.0g _MgSO4 ·7H2O _{, 1.0g} NaCl _{, 2.0g} ( _NH4 )2SO4, _2.0g CaCl2, 0.001g FeSO4·7H2O, 0.001g MnSO4, 0.001g ZnSO4· _7H2O , 15.0g agar, _1.0L distilled water, and pH 7.2.

The actinomycete Streptomyces populiradicis TRM70303 provided by the present invention is screened and separated from the rhizosphere soil of Populus euphratica in the Taklimakan Desert of Xinjiang. After 16Sr RNA sequence phylogenetic analysis and morphological analysis, the strain TRM70303 belongs to Streptomyces. By sequencing the gene of the strain, the obtained sequence is subjected to BLAST comparison analysis on the NCBI website. The 16Sr RNA gene sequence of the strain TRM70303 has the highest similarity with that of Streptomyces lycii TRM 66187 ^T , which is 98.15%. Sequences with higher homology are selected to construct a 16Sr RNA phylogenetic tree. The strain TRM70303 is most closely related to Streptomyces lycii TRM 66187 ^T (MN567187). After a series of polyphasic classification identifications, it can be known that the strain TRM70303 is determined to be a new species of Streptomyces, which has the typical characteristics of a new species.

The actinomycete Streptomyces populiradicis TRM70303 provided by the present invention has been subjected to the above-mentioned molecular level identification of the strain system well known in the art, combined with multi-phase classification system identification analysis such as morphological identification, physiological and biochemical characteristics, and chemical characteristics. The present invention relates to the actinomycete TRM70303 and the three standard model strains with the closest homology to the genus. There are many differences, confirming that the actinomycete strain TRM70303 belongs to a new species of the genus Streptomyces. It is named Streptomyces populiradicis TRM70303, which has been deposited in the China Center for Type Culture Collection, with the strain deposit number of CCTCC No: M2021450, and the deposit date: April 25, 2021.

At the same time, the present invention provides an application of thioluteolin produced by Streptomyces populiradicis TRM70303 in the preparation of drugs for preventing and treating cotton wilt, cotton verticillium wilt, fragrant pear rot, amylovora, Shigella and Acinetobacter baumannii.

Through the above technical scheme, the present invention obtains the following technical effects:

(1) The present invention utilizes the obtained new actinomycete species Streptomyces populiradicis TRM70303 to ferment and produce thiolutein with a yield of 0.27 mg/mL. Compared with the thiolutein produced by fermentation metabolism and chemical synthesis of common microbial strains in the field, the yield is significantly improved, the preparation method is simple, the cost is low, the fermentation cycle is short, the safety is high, and the pollution is low. The active substance can be mass-produced by large-scale fermentation, thereby fundamentally solving the shortage of drug sources.

(2) The thiolutin prepared by the present invention using the main secondary metabolites of the new actinomycete species Streptomyces populiradicis TRM70303 has high application value in the prevention and control of cotton Verticillium dahliae (ACCC 36211), cotton wilt (Fusarium oxysporumfsp ACCC 31038), fragrant pear rot (Valsapyri WTZ-19), apricot fire blight (Erwinia amylovor TRM-03), Shigella Castellani TRM-11, pear black spot (Alternaria gaisen TRM-09), and Acinetobacter baumannii (Acinetobacter baumannii ATCC19606), and can be used to develop efficient biological pesticides and biomedicines.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the phylogenetic tree of strain TRM70303 based on 16S r RNA sequences.

FIG2 shows the morphological characteristics of strain TRM70303, wherein A is the colony morphology of strain TRM70303 on the culture medium; and B is a scanning electron microscope photograph of the colony.

FIG3 is a schematic diagram showing the molecular structure of thiolutin of the present invention.

FIG4 shows the ¹ H NMR spectrum of thiolutin of the present invention.

Figure 5 shows the ¹³ C NMR spectrum of thiolutin of the present invention.

FIG6 shows the HSQC chart of thiolutein of the present invention.

FIG. 7 shows the HMBC chart of thiolutin of the present invention.

FIG8 shows the HPLC chart of 0.5 mg/mL thiolutin in the present invention.

FIG. 9 shows a HPLC standard curve of thiolutein of the present invention.

FIG. 10 shows the HPLC chart of the crude extract of strain TRM 70303 of the present invention.

FIG11 shows the results of the antibacterial activity of thiolutein of the present invention determined by the filter paper method, wherein C.a is Candida albicans, E.c is Escherichia coli, and S.a is Staphylococcus aureus.

Detailed ways

The present invention is described below with reference to the following examples, but the present invention is not limited to the following examples. All raw and auxiliary materials used in the present invention, as well as the selected bacterial strain culture methods are well known in the art, and the % involved in the present invention is volume percentage unless otherwise specified.

The TSB culture medium used in the preparation of the Streptomyces populiradicis TRM70303 microbial agent of the present invention comprises (g/L): 15 g/L of trypticase, 5.0 g/L of soybean papain hydrolyzate, 5.0 g/L of NaCl, and a pH value of 7.3±0.2.

Example 1: Fermentation and metabolism of actinomycete Streptomyces populiradicis TRM70303 to produce thiolutein

(I) Separation and purification

Actinomycetes were isolated by the plate dilution method. 1 g of rhizosphere soil sample of Populus euphratica in the Taklimakan Desert was sampled and diluted to 10 ^-4 with sterile water. 0.2 mL of each sample was evenly spread on the separation plate culture medium. Ten plates were spread on each dilution gradient and inverted at 37°C. Single colonies were picked and streaked for purification. The separation culture medium was: soluble starch 20 g/L, KNO ₃ 1 g/L, K ₂ HPO ₄ 0.5 g/L, MgSO ₄ ·7H ₂ O 0.5 g/L, NaCl 0.5 g/L, FeSO ₄ ·7H ₂ O 0.01 g/L, agar 18 g/L, soil extract 1000 mL, pH 7.5; the purified culture medium was: soluble starch 10.0 g, K ₂ HPO ₄ 1.0 g, MgSO ₄ ·7H ₂ O1.0 g, NaCl 1.0 g, (NH ₄ ) ₂ SO ₄ 2.0 g, CaCl ₂ 2.0 g, FeSO ₄ ·7H ₂ O 0.001 g, MnSO ₄ 0.001 g, ZnSO ₄ ·7H ₂ O 0.001 g, agar 15.0 g, distilled water 1.0 L, pH 7.2.

(II) 16S rRNA gene identification

1. DNA Extraction

Streptomyces populiradicis TRM70303 was inoculated into purified liquid culture medium and cultured at 37°C for 7 days to obtain bacterial cells. Genomic DNA was extracted using a new plant genomic DNA rapid extraction kit.

2. PCR Amplification

16Sr RNA sequence primers:

27F: 5′-AGA GTT TGA TCC TGG CTC-3′;

1492R: 5′-CGG CTA CCT TGT TAC GAC TT-3′.

Reaction system and conditions:

3. Sequence Determination

The PCR amplification product was detected by electrophoresis and purified and then sequenced. The sequence length was 1407 bp. The sequencing result is shown in SEQ ID No: 1. BLAST homologous sequence search was performed on NCBI, and a phylogenetic tree was established by the Neighbor-Joining method using MEGA 7.0 software commonly used in the field (repeated sampling 1000 times). The result is shown in Figure 1. The obtained sequence was compared and analyzed on the NCBI website. It was found that the 16Sr RNA gene sequence of strain TRM70303 had the highest homology with Streptomyces lycii TRM 66187 ^T , with a similarity of 98.43%. In the phylogenetic tree constructed with the 16Sr RNA gene sequence, the 16Sr RNA sequence of strain TRM70303 had the highest homology with Streptomyces lycii TRM 66187 ^T. The strains of (MN567187) are the closest relatives, but they are not on the same branch, indicating that the support rate of the actinomycete TRM70303 as a new species is extremely high, and it has excellent stability in the evolutionary tree. Through the comprehensive judgment of the similarity and homology of the strains, it is confirmed that the strain number TRM70303 obtained within the genus Streptomyces is a typical new species of actinomycetes.

(III) Colony morphology characteristics of Streptomyces populiradicis TRM70303

The strain TRM70303 to be observed was inoculated onto Gao's No. 1 plate and cultured at 37°C for 5 days. When the colonies grew all over the plate, the characteristics of the colonies were observed and recorded and photographed for preservation. At the same time, scanning electron microscope photos of the colonies were recorded. The recording results are shown in Figure 2.

As shown in the results of Figure 2, strain TRM70303 was cultured in Gao's medium No. 1 at 37°C for 5 days, and a large number of bright yellow and white aerial hyphae were observed attached to its surface. The hyphae had few branches, no transverse septa, and were bright yellow; the aerial hyphae were abundant, forming spore chains of varying lengths. The spores were oval, smooth and thornless, growing in clusters, and the spore threads were spiral.

Based on the above biological characteristics, the strain TRM70303 was identified as Streptomyces. The strain has been deposited in the Budapest Treaty International Microbiological Depository: China Center for Type Culture Collection (CCTCC), address: Wuhan University, Wuhan, China, postal code: 430072, deposit date: April 25, 2021, deposit number: CCTCC No: M2021450.

(IV) Physiological and biochemical characteristics of Streptomyces populiradicis TRM70303

The strain TRM70303 was inoculated on Gao's medium No. 1, and the physiological and biochemical characteristics were detected. The growth temperature, salinity, pH range, gelatin liquefaction, nitrate reduction, hydrogen peroxide production, urease, amylase, esterase, cellulose decomposition, carbon source utilization and other characteristic analyses were mainly based on the "Common Bacterial System Identification Manual" and compared with the physiological and biochemical characteristics of similar strains S. lycii TRM66187 ^T , S. chumphonensis K1-2 ^T , and S. durbertens NEAU-S1GS20 ^T. The specific results are shown in Tables 1-3.

From the data in Table 1, it can be seen that the growth tolerance temperature range of strain TRM70303 is 15-45°C, the pH value is 6-10, the NaCl tolerance test range is 0-10%, and the optimal growth conditions are: 40°C, 1% NaCl, pH=7; strain TRM70303 can utilize carbon sources such as lactose, arabinose, D-sorbose, sucrose, mannose, glucose, D-fructose, and rhamnose, but cannot utilize raffinose, xylose, and inositol as the sole carbon source; the results of urease, nitrate reduction, cellulose hydrolase, starch hydrolase, and lipase are positive, and the results of catalase and gelatin liquefaction are negative.

Table 1: Comparative test of physiological and biochemical characteristics of strain TRM70303

(V) Chemical characteristics of Streptomyces populiradicis TRM70303

The whole cell hydrolyzed sugar components, whole cell hydrolyzed amino acid components, polar lipids, major quinone types, and fatty acid components of strain TRM70303 were detected (Lechevalier MP, Lechevalier HA (1980) The chemotaxono-my of actinomycetes. In: Dietz A, Thayer DW (eds) Actinomycete taxonomy special publication, vol 6. Society of Industrial Microbiology, Arlington, pp 227-291).

Tables 2 and 3 show the test results of whole cell hydrolyzed sugar components, whole cell hydrolyzed amino acid components, polar lipids, main quinone types, and fatty acid components of strain TRM70303. The whole cell hydrolyzed sugar types of strain TRM70303 are mainly glucose and ribose, which are consistent with other similar strains; the whole cell hydrolyzed amino acid DAP type is mainly LL-DAP; the main polar lipids are PE, PC, PG and PI, which are similar to other strains; the main quinone types are MK-9 (H ₆ ), MK-9 (H ₈ ), and MK-10 (H ₂ ), which are similar to other strains, but the content is different. The main fatty acid is anteiso-C _15:0 , with a content of 33.87%, which is similar to the three similar strains, but it also has two unique components, anteiso-C _17:1 and iso-C _16:1 .

Table 2: Differences in cellular chemical components between strain TRM70303 and similar strains

Table 3: Differences in fatty acid composition between strain TRM70303 and similar strains (>2%)

It can be seen that strain TRM70303 has similar physiological and chemical characteristics to its closest homologous strains S. lycii TRM66187 ^T , S. chumphonensis K1-2 ^T and S. durbertens NEAU-S1GS20 ^T , but has many differences, and is a potential new species of the genus Streptomyces.

Based on the comparative analysis of 16S rRNA gene sequence homology, morphological identification, comparison of physiological and biochemical characteristics, and chemical characteristic analysis, the strain TRM70303 was identified as a new species of actinomycete Streptomyces and was named Streptomyces populiradicis according to its isolation source, showing distinct differences from the three most homologous standard model strains.

Example 2: Preparation of Thiolutin Using Actinomycete Streptomyces populiradicis TRM70303

Based on Example 1, this example uses the obtained new actinomycete species Streptomyces populiradicis TRM70303 to produce thiolutin, which specifically includes the following steps: inoculating a single colony of the strain Streptomyces populiradicis TRM70303 in TSB culture medium, culturing at 33°C and 120rpm for 3 days to obtain Streptomyces populiradicis TRM70303 seed solution; inoculating the Streptomyces populiradicis TRM70303 seed solution into an oat liquid culture medium at an inoculum volume ratio of 1%, fermenting and culturing at 33°C and 120rpm for 5 days to obtain Streptomyces populiradicis TRM70303 fermentation liquid; collecting the fermentation liquid, filtering it, loading it on a D101 model macroporous resin, eluting it with 100% methanol, collecting the eluate, and concentrating it under reduced pressure at 50°C to remove the methanol-soluble part to obtain flaky crystalline thiolutin.

The oat culture medium is: 20 g oats, 1000 mL water, pH 7.5.

TSB culture medium is (g/L): trypticase 15 g/L, soybean papain hydrolysate 5.0 g/L, NaCl 5.0 g/L, pH 7.3±0.2.

Example 3: Identification of Thiolutin Producing from Streptomyces populiradicis TRM70303

In this example, based on Example 2, a single colony of the strain Streptomyces populiradicis TRM70303 was inoculated into TSB medium, and cultured at 37°C and 120rpm for 3 days to obtain a Streptomyces populiradicis TRM70303 seed solution; the Streptomyces populiradicis TRM70303 seed solution was inoculated into an oat liquid culture medium at an inoculum ratio of 1% by volume, and fermented and cultured at 37°C and 120rpm for 5 days to obtain a Streptomyces populiradicis TRM70303 fermentation liquid; the fermentation liquid was collected, filtered, and then loaded onto a D101 macroporous resin, eluted with 100% methanol, the eluate was collected, and concentrated under reduced pressure at 50°C to remove the methanol-soluble portion to obtain a pure product of thiolutein, the molecular structure of which is shown in FIG3 .

(1) Identification and purity testing

The pure product of thiolutin obtained in this experiment is a golden yellow flaky crystal, soluble in DMSO, and almost insoluble in methanol and water. Melting point: 273-276°C. The structure of thiolutin was identified by NMR. See Figure 4 for the nuclear magnetic resonance spectroscopy ¹ H NMR spectrum, see Figure 5 for the ¹³ C NMR spectrum, see Figure 6 for the HSQC (Heteronuclear Single Quantum Coherence) spectrum, and see Figure 7 for the HMBC (Heteronuclear Multiple-Bond Correlation) spectrum. Its spectral data is consistent with the thiolutin reported in the literature, indicating that the compound obtained by fermentation, separation and purification of strain TRM70303 in the present invention is thiolutin. The NMR data of the compound are shown in Table 4.

Table 4: NMR data of thiolutin (DMSO-d6, ¹ H:500MHz, ¹³ C:125MHz)

TLC thin layer chromatography (dichloromethane: methanol = 10: 1, petroleum ether: ethyl acetate = 3: 1, 3 color development methods: 254nm ultraviolet color development, iodine color development, ethanol-concentrated sulfuric acid color development, all into a single general point) was used, and high performance liquid chromatography HPLC was used for determination: by area rule method, 5 different wavelengths (254nm, 302nm, 365nm, 388nm, 440nm) were used for determination, and the time width of the chromatogram was recorded to be more than 2 times the peak time of thiolutin. As can be seen from the data in Figure 8, at the selected 5 wavelengths, the purity of thiolutin was greater than 98%, which met the purity requirements of the standard product.

(2) Drawing of standard curve

The above thiolutin with a purity greater than 98% was used as a standard product, and the standard curve was determined by the area normalization method using HPLC (mobile phase: 100% methanol-water solution, flow rate 1 mL/min, sample concentration 0.5 mg/mL, diluted to 0.4 mg/mL, 0.3 mg/mL, 0.2 mg/mL, 0.1 mg/mL in sequence, injection volume 10 μL, detection wavelength 388 nm). The specific results are shown in Figure 9. The standard curve of thiolutin is: y=(2E+07x)-111616, R2=0.9995, where x is the peak area, y is the content of thiolutin, and the value of R2 indicates that the linear relationship of the curve is good, and it can be used for the determination of the content of thiolutin.

(3) Quantitative analysis of thiolutin

The yield of thiolutin in the crude extract of 200 mL of fermentation broth was analyzed by HPLC. The chromatographic conditions were: 4.6 mm × 150 mm C18 chromatographic column (0.5 μm), 100% methanol-water as mobile phase, flow rate of 1 mL/min, detection wavelength of 388 nm, and the specific results are shown in Figure 10.

The preparation method of thiolutein provided by the present invention has a thiolutein yield of 0.27 mg/mL, which is greatly improved compared with the preparation method of thiolutein reported currently, has a simple preparation process, a high strain fermentation level, low cost, a short fermentation cycle, high safety, and reduced pollution, and can achieve mass production of active substances through large-scale fermentation, thereby fundamentally solving the shortage of drug sources.

Example 4: Antibacterial activity test of thiolutein produced by Streptomyces populiradicis TRM70303

This example is based on Examples 1-3, and the filter paper method is used to conduct a preliminary test of antagonistic activity. A fungus, a Gram-negative bacterium, and a Gram-positive pathogen were selected as representative target bacteria, respectively, to preliminarily investigate the antibacterial activity of the thiolutein produced by Streptomyces populiradicis TRM70303 of the present invention. The selected pathogens are Candida albicans (ATCC 64550), Escherichia coli (ATCC 25922), and Staphylococcus aureus (ATCC 6538).

Escherichia coli (ATCC 25922) and Staphylococcus aureus (ATCC 6538) were selected and inoculated into LB liquid culture medium, cultured overnight at 37°C to prepare a standby bacterial suspension, and 200 μL was evenly spread on an LB solid plate for later use; Candida albicans (ATCC 64550) was inoculated into PDA liquid culture medium and cultured overnight at 28°C to prepare a standby bacterial suspension, and 200 μL was evenly spread on a PDA solid plate for later use.

10 μL of 1 mg/mL thiolutin was taken on a filter paper (about 0.6 mm in diameter) and pasted on the culture medium coated with pathogens, 3 pieces per dish (DMSO and methanol were set as blank controls). After marking, the plates were cultured at 37°C and 28°C overnight, the size of the inhibition zone was measured and photographed for record, and the specific results are shown in Figure 11 and Table 5.

Table 5: Statistics of inhibition zone size

Target bacteria Diameter of inhibition zone Candida albicans (C.aATCC 64550) 26.1±0.05mm Escherichia coli (E.cATCC 25922) 24.0±0.15mm Staphylococcus aureus (S.aATCC 6538) 12.0±0.15mm

As shown in Figure 11 and the data in Table 5, thiolutin has good antibacterial activity against fungi (Candida albicans), Gram-negative bacteria (Escherichia coli), and Gram-positive bacteria (Staphylococcus aureus), and has broad application prospects.

Example 5: Biocontrol application test using Streptomyces populiradicis TRM70303 to produce thiolutein

Based on the 0.05 mg/mL concentration of thiolutin, a two-fold serial dilution was performed to make the final concentrations 50 μg/mL, 25 μg/mL, 12.5 μg/mL, 6.25 μg/mL, 3.125 μg/mL, 1.7 μg/mL, 0.8 μg/mL, and 0.4 μg/mL, respectively.

Pick out different pathogenic bacteria colonies and activate them in LB medium. After growth, the bacterial solution was prepared into 0.5 McFarland turbidimetric standard with physiological saline, and then diluted 1:10 with MH broth to make the bacterial content 10 ⁷ CFU/mL. When inoculating, use a micropipette to take 0.1mL of bacterial solution and add samples from low concentration to high concentration in sequence, so that the final bacterial solution concentration in each well is 5×10 ⁵ CFU/mL. After inoculation, incubate at 37℃ for 16-20h. The lowest drug concentration that completely inhibits bacterial growth in the small well is the MIC value of the antibacterial drug for the tested bacteria. Each treatment was repeated 3 times. The test results are shown in Table 6.

Table 6: Antibacterial effect of thiolutin

Pathogen strains Disease name Thiolutin MIC value Verticillium dahliae ACCC 36211 Verticillium dahliae 12.5 μg/mL Fusarium oxysporumfsp ACCC 31038 Fusarium wilt 12.5 μg/mL Valsa pyri WTZ-19 Fragrant pear rot 6.215μg/mL Erwinia amylovor TRM-03 Fire blight 12.5 μg/mL Shigella Castellani TRM-11 Shigella 12.5 μg/mL Alternaria gaisen TRM-09 Pear Black Spot 12.5 μg/mL Acinetobacter baumannii ATCC 19606 Acinetobacter baumannii 12.5 μg/mL

As shown in Table 6, the minimum inhibitory concentration of the new strain Streptomyces populiradicis TRM70303 provided by the present invention to produce sulfolutein against cotton Verticillium wilt, cotton Fusarium wilt, amylovora, Shigella, pear black spot and Acinetobacter baumannii is 12.5 μg/mL, and the minimum inhibitory concentration against fragrant pear rot is only 6.215 μg/mL, indicating that the new species Streptomyces populiradicis TRM70303 provided by the present invention can produce sulfolutein against cotton Verticillium wilt, cotton Fusarium wilt, amylovora, Shigella, pear black spot and Acinetobacter baumannii. The thioluteolin produced by TRM70303 has a significant inhibitory effect on cotton wilt, cotton verticillium wilt, fragrant pear rot, amylopectin, Shigella, and Acinetobacter baumannii, and has the strongest inhibitory effect on fragrant pear rot pathogens. This shows that the thioluteolin produced by the fermentation metabolism of this strain in the preparation of drugs for the prevention and treatment of cotton wilt, cotton verticillium wilt, fragrant pear rot, amylopectin, Shigella, and Acinetobacter baumannii has important application value and good development prospects for the prevention and control of plant diseases and bacterial infections.

From the above data, it can be seen that the preparation method of thioluteolin provided by the present invention has a yield of 0.27 mg/mL. Compared with the existing preparation method of thioluteolin, the yield is greatly improved, the preparation process is simple, the strain fermentation level is high, the cost is low, the fermentation cycle is short, and the mass production of active substances can be achieved through large-scale fermentation, thereby fundamentally solving the shortage of drug sources; the thioluteolin obtained by separating and purifying the main secondary metabolites of the new actinomycete species Streptomyces populiradicis TRM70303 has an effective effect on Verticillium dahliae (ACCC 36211), Fusarium oxysporum fsp ACCC 31038, Valsa pyri WTZ-19, Erwinia amylovor TRM-03, Shigella Castellani TRM-11, and Alternaria gaisen TRM-09) and Acinetobacter baumannii ATCC19606 have high application value and can be used to develop efficient biological pesticides and biomedicines.

The above embodiments are merely descriptions of the preferred implementation methods of this test, and are not intended to limit the scope of this test. Without departing from the spirit of the design of this test, various modifications and improvements made to the technical solutions of this test by ordinary technicians in this field should all fall within the protection scope determined by this test.

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<400> 1

cgcatgcaag tcgaacgatg aagccgcttc ggtggtggaa gagtggcgaa cgggtgagta 60

acacgtgggc aatctgccct gcactctggg acaagccctg gaaacggggt ctaataccgg 120

atacgactgc cggccgcatg gtctggtggt ggaaagctcc ggcggtgcag gatgagcccg 180

cggcctatca gcttgttggt ggggtgatgg cctaccaagg cgacgacggg tagccggcct 240

gagagggcga ccggccacac tgggactgag acacggccca gactcctacg ggaggcagca 300

gtggggaata ttgcacaatg ggcgcaagcc tgatgcagcg acgccgcgtg agggatgacg 360

gccttcgggt tgtaaacctc tttcagcagg gaagaagcgc aagtgacggt acctgcagaa 420

gaagcaccgg ctaactacgt gccagcagcc gcggtaatac gtagggtgcg agcgttgtcc 480

ggaattattg ggcgtaaaga gctcgtaggc ggcttgtcac gtcggatgtg aaagcccggg 540

gcttaacccc gggtctgcat tcgatacggg caggctagag ttcggtaggg gagatcggaa 600

ttcctggtgt agcggtgaaa tgcgcagata tcaggaggaa caccggtggc gaaggcggat 660

ctctgggccg atactgacgc tgaggagcga aagcgtgggg agcgaacagg attagatacc 720

ctggtagtcc acgccgtaaa cgttgggaac taggtgtggg cgacattcca cgtcgtccgt 780

gccgcagcta acgcattaag ttccccgcct ggggagtacg gccgcaaggc taaaactcaa 840

aggaattgac gggggcccgc acaagcggcg gagcatgtgg cttaattcga cgcaacgcga 900

agaaccttac caaggcttga catacaccgg aaaaccgtgg agacacggtc ccccttgtgg 960

tcggtgtaca ggtggtgcat ggctgtcgtc agctcgtgtc gtgagatgtt gggttaagtc 1020

ccgcaacgag cgcaaccctt attctgtgtt gccagcacgc ccttcggggt ggtggggact 1080

cacaggagac tgccggggtc aactcggagg aaggtgggga cgacgtcaag tcatcatgcc 1140

ccttatgtct tgggctgcac acgtgctaca atggccggta caatgagctg cgatgccgtg 1200

aggtggagcg aatctcaaaa agccggtctc agttcggatt ggggtctgca actcgacccc 1260

atgaagtcgg agtcgctagt aatcgcagat cagcattgct gcggtgaata cgttcccggg 1320

ccttgtacac accgcccgtc acgtcacgaa agtcggtaac acccgaagcc ggtggcctaa 1380

cccccttgtg gggagggaat cgtcgaa 1407

Claims (1)

1. A preparation method of Streptomyces populiradicis TRM70303 sulfur-producing gambogin is characterized in that single colony of a strain Streptomyces populiradicis TRM70303 is inoculated in a TSB culture medium and cultured for 3 days at 33 ℃ and 120rpm to obtain Streptomyces populiradicis TRM70303 seed liquid; inoculating Streptomyces populiradicis TRM70303 seed liquid into an oat liquid culture medium according to the inoculum size with the volume ratio of 1%, and fermenting and culturing at 33 ℃ and 120rpm for 5 days to obtain Streptomyces populiradicis TRM70303 fermentation liquid; collecting fermentation liquor, filtering, loading on D101 macroporous resin, eluting with 100% methanol, collecting eluate, and concentrating under reduced pressure at 50deg.C to obtain sheet crystalline thiolutin; the Streptomyces populiradicis TRM70303 strain is preserved in China Center for Type Culture Collection (CCTCC) with a strain preservation number of M2021450.