A Streptomyces mangrovicus strain B4503 and its application in controlling banana wilt

Abstract

The present invention belongs to the field of microbial technology, and specifically discloses a strain of Streptomyces sp. B4503 and its application in controlling banana wilt. The strain of Streptomyces sp. B4503 was deposited at the China Center for Type Culture Collection (CCTCC) on August 19, 2019, and the deposit number is CCTCC NO: M 2019647. In the present invention, the secondary metabolites produced by the strain of Streptomyces sp. B4503 have a significant inhibitory effect on the pathogenic fungus of banana wilt, physiological race 4 (Foc TR4), can inhibit the mycelial growth of Foc TR4, and change the typical growth morphology of the mycelium. The strain of Streptomyces sp. B4503 can be applied to control banana wilt and has the development and application value of a biological control agent.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbiology, and more specifically, relates to a Streptomyces sp. strain B4503 and its application in controlling banana wilt disease. Background Art

[0002] Banana (Musa spp.) is the fourth largest food crop in the world after rice, wheat, and corn, the largest bulk fruit in world trade, and one of the main agricultural pillar products in tropical and subtropical regions of China. However, banana cultivation has been threatened by the pathogen Fusarium oxysporum f. sp. cubense (Foc), especially its race 4 (Foc TR4), which can infect almost all cultivation systems (Siddhesh, International Journal of Pest Management, 2015, 61(3): 250-263). Banana wilt disease has the characteristics of soil-borne, can be transmitted through various media, and has a long survival period in the soil. Currently, no effective control method has been found.

[0003] Chemical bactericides, due to their direct and efficient characteristics, are one of the main methods for controlling banana wilt disease. However, long-term use is prone to drug resistance and environmental pollution, which makes pollution-free and bio-friendly microbial control technologies highly concerned. Screening microorganisms with the potential to inhibit plant diseases and pests, and exploring the control effects and action mechanisms of these microorganisms on plant diseases and pests are the basis for applying microbial control of plant diseases and pests.

[0004] Bacteria of the genus Streptomyces are widely distributed in soils of different habitats and can produce secondary metabolites with various structures and diverse biological activities. Currently, more than 75% of antibiotics are derived from Streptomyces, and some of them have been used as agricultural antibiotics in the control of plant diseases and pests. The genus Streptomyces has contained more than 1000 species of bacteria so far. Genomic analysis shows that the Streptomyces genome contains a large number of biosynthetic gene clusters of secondary metabolites. However, there are few reports on the active secondary metabolites from Streptomyces with inhibitory effects on the pathogen of banana wilt disease, and the biological activities of a large number of secondary metabolites have not been deeply explored. Therefore, excavating Streptomyces with significant biocontrol potential and studying its antibacterial mechanism are helpful for discovering active secondary metabolites and promoting their application in the biological control of plant diseases and pests. Summary of the Invention

[0005] Aiming at the problems existing in the prior art, the object of the present invention is a Streptomyces sp. B4503 strain and its application in controlling banana wilt. The Streptomyces sp. B4503 strain of the present invention can produce secondary metabolites that inhibit the activity of FocTR4, and this Streptomyces sp. B4503 strain has a good control effect on banana wilt.

[0006] The object of the present invention is achieved by the following technical solutions:

[0007] The present invention provides an application of a Streptomyces sp. B4503 strain in controlling banana wilt. The Streptomyces sp. B4503 strain was deposited at the China Center for Type Culture Collection (CCTCC) on August 19, 2019. Address: China Center for Type Culture Collection, Wuhan University, Wuhan, China, 430072. The deposit number is CCTCC NO: M2019647.

[0008] Preferably, the above Streptomyces sp. B4503 strain is subjected to conventional fermentation, and the supernatant after fermentation contains secondary metabolites for controlling banana wilt.

[0009] The Streptomyces sp. B4503 strain of the present invention was isolated from the soil of the mangrove forest in Beibu Gulf, Qinzhou, Guangxi. After culturing the Streptomyces sp. B4503 on ISP2 medium for 3 days, the colony is grayish-black, powdery, and has spores formed. By using ISP2 medium and changing the culture temperature, pH, and NaCl content in the medium of the bacteria, it is measured that the optimum growth temperature of the Streptomyces sp. B4503 is 28 °C, the optimal pH value is 7.0, and it can tolerate 0-7% NaCl.

[0010] Another object of the present invention is to provide an application of the Streptomyces sp. B4503 strain in the production and preparation of FocTR4 inhibitors. The specific technical solutions are as follows:

[0011] An application of the Streptomyces sp. B4503 strain in the production and preparation of FocTR4 inhibitors. The Streptomyces sp. B4503 strain was deposited at the China Center for Type Culture Collection on August 19, 2019, and the deposit number is CCTCC NO: M2019647.

[0012] Preferably, the method for producing and preparing the FocTR4 inhibitor by Streptomyces sp. B4503 strain comprises the following steps:

[0013] S1 Ferment the Streptomyces sp. B4503 strain with the preservation number of CCTCC NO: M2019647 to obtain a fermentation broth;

[0014] S2 Remove the thalli from the fermentation broth obtained in step S1 by centrifugation, filter the fermentation supernatant by suction, concentrate the suction filtrate under reduced pressure to obtain a crude extract of the fermentation broth, and through purification and separation, the FocTR4 inhibitor can be obtained.

[0015] Preferably, the fermentation conditions in step S1 are as follows: the medium is an oatmeal liquid medium, and the fermentation is carried out with shaking at 28 °C for 5 days.

[0016] Preferably, the suction filtration operation in step S2 is to add macroporous adsorption resin particles XAD-16N to the fermentation supernatant, filter by suction to enrich XAD-16N, first rinse the resin with deionized water, then rinse the resin with methanol, and collect the methanol phase.

[0017] Preferably, the purification and separation method in step S2 is to dissolve silica gel with a weight 100 times that of the crude extract in 20% methanol aqueous solution, load it into a glass chromatography column, perform gradient elution with a methanol / water system, and collect each component. Then dissolve it with methanol, filter, and place it for semi-preparative high performance liquid chromatography separation.

[0018] The present invention also provides the application of the FocTR4 inhibitor produced and prepared by the above-mentioned Streptomyces sp. B4503 strain in the preparation of a drug for preventing and treating the banana wilt pathogen race 4.

[0019] The present invention first uses Streptomyces sp. B4503 isolated from mangrove soil to ferment and isolate niphomycin C. Experiments show that this kind of compound can effectively inhibit the banana wilt pathogen race 4, and the minimum inhibitory concentration for spore germination is 32 μg / mL. It can destroy the hyphal structure, change the typical hyphal morphology, inhibit hyphal growth and spore germination, and cause the expression of related genes such as amino acid metabolism, cell signal transduction, and sugar metabolism pathways in race 4 cells, change the hyphal morphology, and make the hyphae change from smooth cylindrical to shriveled, curved and with obvious shrinkage, providing a good application prospect for the development of drugs for preventing and treating banana wilt.

[0020] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0021] The Streptomyces sp. strain B4503 provided by the present invention was isolated from the mangrove soil in Beibu Gulf, Qinzhou, Guangxi. It can significantly inhibit the growth of the 4th physiological race of the banana fusarium wilt pathogen, and the inhibition rate is 73.3%. The Streptomyces sp. strain B4503 of the present invention can produce active secondary metabolites that inhibit FocTR4. The secondary metabolite niphimycin C produced can cause the hyphae of FocTR4 to bend and shrivel, and wrinkles appear. The minimum inhibitory concentration for spores is 32 μg / mL. This Streptomyces sp. strain B4503 has a good control effect on banana fusarium wilt and has the development and application value of a biocontrol agent. Brief Description of the Drawings

[0022] Figure 1 It is the bacterial growth morphology (A) of Streptomyces sp. strain B4503 in Guangxi mangrove and its plate confrontation experiment (B) with FocTR4.

[0023] Figure 2 It is the phylogenetic analysis of Streptomyces sp. strain B4503 based on the 16S rRNA gene sequence.

[0024] Figure 3 It is a comparison chart of the control effect of Streptomyces sp. strain B4503 on FocTR4.

[0025] Figure 4 It is the chemical structure of the compound niphimycin C.

[0026] Figure 5 It is the flow chart of the isolation and purification of niphimycin C.

[0027] Figure 6 It is a scanning electron micrograph of the effect of niphimycin C on the growth morphology of FocTR4 hyphae. Figure 6 On the left is the hyphal morphology of FocTR4 under normal growth conditions, and on the right is the hyphal morphology of FocTR4 after the action of niphimycin C. Detailed Description of the Invention

[0028] The present invention will be further described below in conjunction with specific embodiments and the accompanying drawings. However, the embodiments do not impose any form of limitation on the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts belong to the scope of protection of the present invention. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in the technical field. Unless otherwise specified, the reagents and materials used in the present invention are commercially available.

[0029] Example 1 Isolation, Cultivation, and Identification of Streptomyces sp. Strain B4503

[0030] 1. Experimental Materials

[0031] 1.1 Isolation and Cultivation of Strains

[0032] The soil collected from the mangrove forest in Beibu Gulf, Qinzhou, Guangxi was dried in an oven at 60°C, diluted with sterile water to prepare sample solutions of 10-2 and 10-3 respectively, and 250 μL of each sample solution was pipetted and spread on ISP2 and 2216E agar media. After culturing in a constant temperature incubator at 28°C for several days, colonies were picked with a sterile inoculation needle, and the continuous streak culture method was used to streak culture in the medium until single colonies appeared.

[0033] 1.2 Test Media

[0034] The media used in this study are shown in Table 1.

[0035] Table 1 Formulation of Bacterial Media

[0036]

[0037] 2. Classification and Identification of Strains

[0038] 2.1 Determination of the 16S rDNA Gene Sequence of Streptomyces sp. B4503

[0039] The bacterial cells were scraped from the ISP2 agar medium. After extracting the total genomic DNA using a bio-Teke DNA extraction kit, the DNA was amplified using the universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3'). The total volume of the PCR reaction system was 25 μl, and the amplification conditions were 95°C for 8 min; 58°C for 45 s; 72°C for 90 s. After sequencing, a sequence with a length of 1516 bp (SEQ.ID.NO.1) was obtained.

[0040] 2.2 Identification of Streptomyces sp. B4503

[0041] The obtained sequence was submitted to the NCBI website for alignment with the published 16S rRNA gene sequences stored in the GenBank database, and it was determined that the homologous related strains of Streptomyces sp. B4503 were S. yatensis NBRC101000T (99.8%), S. antimycoticus NBRC12839T (99.4%), S. melanosporofaciens DSM40318T (99.3%), S. geldanamycininus DSM40318T (99.3%), and S. mordarskii NRRLB-1346T (99.0%).

[0042] As Figure 2 shown, based on the 16S rRNA gene sequence analysis, it was found that strain B4503 belongs to the genus Streptomyces and has the closest genetic relationship with S. yatensis NBRC101000T.

[0043] 2.3 Optimization of the growth conditions of Streptomyces sp. B4503

[0044] After Streptomyces sp. B4503 was cultured on ISP2 medium for 3 days, the colonies were grayish-black, powdery, and spore formation occurred ( Figure 1 -A). Using ISP2 medium, by changing the culture temperature, pH, and NaCl content in the medium of the bacteria, it was measured that the optimal growth temperature of Streptomyces sp. B4503 was 28 °C, the optimal pH value was 7.0, and it could tolerate 0 - 7% NaCl.

[0045] Example 2 Control effect of Streptomyces sp. B4503 strain on banana wilt

[0046] 1. Tested pathogenic bacteria

[0047] The 4th physiological race of the banana wilt pathogen, F. oxysporum f. sp. cubense Race4, was provided by the Guangxi Academy of Agricultural Sciences, and the strain medium is shown in Table 2.

[0048] Table 2 Formulation of the strain medium

[0049]

[0050] 2. Plate inhibitory activity of Streptomyces sp. B4503 against Foc TR4

[0051] The race 4 of Fusarium oxysporum f. sp. cubense (FocTR4) was inoculated at the center of a PDA medium, and Streptomyces sp. B4503 was inoculated 2 - 3 cm away from the pathogen block. A PDA agar plate inoculated only with FocTR4 was used as a control. There were three replicates in each group. After culturing at a constant temperature of 28°C for 7 days, the growth diameter of the FocTR4 colonies in the control group and the treatment group was measured, and the inhibition rate of Streptomyces sp. B4503 against FocTR4 was calculated.

[0052]

[0053] The inhibitory effect of Streptomyces sp. B4503 against FocTR4 was as Figure 1 shown in -B, and the inhibition rate was 73.3%.

[0054] 3. Control effect of Streptomyces sp. B4503 against FocTR4

[0055] FocTR4 was inoculated into a sterile mung bean medium and cultured with shaking at 30°C for 7 days to obtain a bacterial solution containing Foc TR4 spores. Banana seedlings with consistent growth were selected and immersed in the bacterial solution, and then transplanted into flower pots filled with 500 g of soil. Two treatments were set in the experiment: 1) The control group was watered with clear water daily; 2) In the experimental group, the cells of Streptomyces sp. B4503 were mixed with the soil in the flower pot and watered with clear water daily. The growth of bananas was observed every day, and the plant height, stem girth, number of leaves, and diameter were recorded. After 30 days of cultivation, the banana seedlings were carefully pulled out with their roots, and the rhizome part was cut with a sterile blade to observe the infection of FocTR4 on the banana plants.

[0056] The control effect of Streptomyces sp. B4503 against FocTR4 was as Figure 3 shown. As Figure 3 can be seen, the rhizome part of the banana seedlings without Streptomyces sp. B4503 was dark purple and slightly black in the center, and had been infected by FocTR4. While the rhizome part of the banana seedlings with Streptomyces sp. B4503 was white, and only a few showed light purple after infection, with less and lighter disease, which confirmed that Streptomyces sp. B4503 had a certain control effect against FocTR4.

[0057] Example 3 Isolation and purification of the antibacterial active secondary metabolites produced by Streptomyces sp. B4503

[0058] 1. The main reagents and instruments used in this study are shown in Tables 3 and 4

[0059] Table 3 Main common reagents and their sources

[0060]

[0061]

[0062] Table 4 Main instruments

[0063]

[0064] 2. Fermentation culture of Streptomyces sp. strain B4503

[0065] Scrape the mycelium of Streptomyces sp. B4503 growing on the ISP2 agar plate with a sterile inoculation needle and inoculate it into a triangular flask containing 100 mL of oat liquid medium. After shaking culture at 28 °C for 5 d, a fermentation seed liquid is obtained. Use a sterile pipette to aspirate 1 mL of the seed liquid into a triangular flask containing 200 mL of oat liquid medium, and inoculate a total of 50 L. Shake culture at 28 °C for 5 d.

[0066] Formula of oat liquid medium: 60.0 g of oat flour, 1000 mL of distilled water, pH 7.2.

[0067] 3. Isolation and purification of active secondary metabolites of Streptomyces sp. strain B4503

[0068] The fermentation broth is centrifuged (5000 rpm, 10 min) to remove the thalli. 10% (w / v) macroporous adsorption resin particles XAD-16N are added to the fermentation supernatant, and XAD-16N is enriched by suction filtration. First, wash the resin 3 times with deionized water, 500 mL each time, and then wash the resin 3 times with methanol, 500 mL each time. Collect the methanol phase and obtain the crude extract of the fermentation broth after concentration under reduced pressure. After activity testing, this crude extract has obvious inhibitory activity against the pathogen Foc TR4.

[0069] Silica gel weighing 100 times the weight of the crude extract is dissolved in 20% methanol aqueous solution and loaded into a glass chromatography column. Gradient elution is carried out with a methanol / water system (300 mL, v / v, 20%, 40%, 60%, 80% and 100%), and each fraction is collected. After concentration, the fraction Fr.4 containing the active compound is determined by activity testing.

[0070] The fraction containing the active compound is dissolved in 5 mL of methanol, filtered through a 0.22 μm filter head, and placed for semi-preparative high performance liquid chromatography separation. The separation conditions are: the column temperature is room temperature; the chromatographic column is XBridge, BEH, C18 , OBD (250X10 mm, 5 μm); the mobile phase is acetonitrile / water, 0 - 20 min, 40% - 60% acetonitrile, 20 - 30 min, 60% - 80% acetonitrile, 30 - 50 min, 80% - 100% acetonitrile; the injection volume is 80 μL; the flow rate is 1.0 mL / min.

[0071] After semi-preparative high performance liquid chromatography separation, a total of 5 monomeric compounds were collected. Among them, compound 3 was collected at 16 min (12 mg) and was verified as the target compound through activity verification.

[0072] The active compound 3 is a colorless solid. Its molecular weight was detected by high-resolution mass spectrometry to be 1142.73005 [M+H] + , and the speculated molecular formula is C 59 H 104 N3O 18 . According to the 1 H-NMR and COSY spectra of the compound, and by comparing with the literature, it was found that the spectral data of compound 3 was consistent with that reported in the literature. Compound 3 was determined to be niphimycin C (Hu et al. Journal of Natural Products, 2018, 81(1): 178 - 187). The planar structure of compound 3 was deduced from the NMR spectra, as Figure 4 shown. The NMR data of this compound are shown in Table 5.

[0073] Table 5 NMR data of compound 3 (deuterated reagent: CD3OD)

[0074]

[0075]

[0076] Example 4 Inhibitory effect of antibacterial active secondary metabolites produced by Streptomyces sp. B4503 on FocTR4

[0077] 1. Inhibitory effect of niphimycin C on the spore germination of FocTR4

[0078] Use a pipette to aspirate 100 μL of FocTR4 spore solution with a concentration of 1x10 5 CFU / mL into a 96-well plate, and then add another 100 μL of the mixture of RPMI 1640 medium and niphimycin C. After incubation at 28 °C for 12 h, measure the absorbance at 620 nm using an enzyme-linked immunosorbent assay reader. Use the sample without niphimycin C as the negative control. By comparison, the minimum inhibitory concentration of niphimycin C on FocTR4 spores was determined to be 32 μg / mL.

[0079] 2. Effects of Niphimycin C on the Mycelial Growth Morphology of the Pathogen FocTR4

[0080] Dissolve niphimycin C in DMSO to prepare a 32 μg / mL solution. Mix 0.5 mL of the solution, 0.5 mL of the spore suspension, and 19 mL of PDA agar medium, then prepare a plate. Cover it with a sterile glass paper and seal it, then place it in a constant temperature incubator at 28 °C for 7 days of incubation.

[0081] Use a sterile spreader to collect the FocTR4 mycelia on the glass paper. Fix the mycelia overnight at 4 °C with 2.5% (v / v) glutaraldehyde solution, then rinse it multiple times with 0.1 mol / L phosphate buffer solution (PBS, pH 7.4). Then, dehydrate it successively with gradient elution using 30%, 50%, 70%, 80%, 90%, 95%, and 100% ethanol solutions for 20 minutes each time. Finally, after vacuum drying and gold sputtering treatment, observe the mycelial morphology of FocTR4 with a scanning electron microscope. The results are as Figure 6 shown.

[0082] Figure 6 shown that niphimycin C can inhibit the mycelial growth of FocTR4, change the mycelial morphology, and make the mycelia change from smooth cylindrical to shriveled, curved, and with obvious shrinkage.

[0083] The specific embodiments of the present invention have been described in detail above, but they are only examples, and the present invention is not limited to the specific embodiments described above. For those skilled in the art, any equivalent modifications and substitutions made to the present invention are also within the scope of the present invention. Therefore, all equivalent transformations and modifications made without departing from the spirit and scope of the present invention should be covered within the scope of the present invention.

Claims

1. Application of Streptomyces mangroviensis ([ Streptomyces Streptomyces sp.) strain B4503 in preventing and controlling banana wilt, characterized in that, The Streptomyces mangrovicus( Streptomyces sp.) strain B4503 was deposited at the China Center for Type Culture Collection on August 19, 2019, with the deposit number CCTCC NO: M 2019647.

2. Use of Streptomyces mangrovicus ([ Streptomyces Streptomyces sp.) strain B4503 in controlling banana wilt, characterized in that, Ferment the Streptomyces mangrovicus ([ Streptomyces Streptomyces sp.) strain B4503 routinely. The supernatant after fermentation contains secondary metabolites for controlling banana wilt disease.

3. Application of Streptomyces mangrovicus ([ Streptomyces Streptomyces sp.) strain B4503 in the production and preparation of Foc TR4 inhibitor, characterized in that The Streptomyces mangrovicus( Streptomyces sp.) strain B4503 was deposited at the China Center for Type Culture Collection on August 19, 2019, with the deposit number CCTCC NO: M 2019647.

4. Use of Streptomyces mangrovicus ([ Streptomyces Streptomyces sp.) strain B4503 in the production and preparation of Foc TR4 inhibitor, characterized in that Streptomyces mangrovisoli ([ Streptomyces [ sp.) Method for producing and preparing strain B4503 Foc [ Method for producing a TR4 inhibitor, comprising the following steps: S1 ferment the Streptomyces mangrovicus ([ Streptomyces Streptomyces sp.) strain B4503 to obtain a fermentation broth; S2 removes the bacteria from the fermentation broth obtained in step S1 by centrifugation, the fermentation supernatant is filtered by suction, and the suction filtrate is concentrated under reduced pressure to obtain a crude extract of the fermentation broth. After purification and separation, Foc TR4 inhibitor can be obtained.

5. Use of Streptomyces mangrovicus ([ Streptomyces Streptomyces sp.) strain B4503 in the production and preparation of Foc TR4 inhibitor, characterized in that The fermentation conditions described in step S1 are as follows: the medium is an oat liquid medium, and the fermentation is carried out with shaking at 28 °C for 5 days.

6. Use of Streptomyces mangrovicus ([ Streptomyces Streptomyces sp.) strain B4503 in the production and preparation of Foc TR4 inhibitor, characterized in that The filtration method described in step S2 is that the fermentation supernatant is added with macroporous adsorption resin particles XAD-16N, and the XAD-16N is enriched by filtration. First, the resin is rinsed with deionized water, and then the resin is rinsed with methanol, and the methanol phase is collected.

7. Use of Streptomyces mangrovicus ([ Streptomyces Streptomyces sp.) strain B4503 in the production and preparation of Foc TR4 inhibitor, characterized in that The purification and separation method described in step S2 is to dissolve silica gel with a weight 100 times that of the crude extract in a 20% methanol aqueous solution, load it into a glass chromatography column, perform gradient elution with a methanol / water system, collect each component, then dissolve it with methanol, filter it, and place it for semi-preparative high performance liquid chromatography separation.

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