CN114631544A - Streptomyces mangrove B4503 strain and application thereof in preventing and treating banana wilt - Google Patents

Abstract

The invention belongs to the technical field of microorganisms, and particularly discloses a Streptomyces mangrove sp B4503 strain and application thereof in preventing and treating banana wilt, wherein the Streptomyces mangrove sp B4503 strain is preserved in China Center for Type Culture Collection (CCTCC) in 2019, 8 and 19 months, and the preservation number is CCTCC NO: m2019647. The secondary metabolite produced by the B4503 strain has a remarkable inhibiting effect on the No. 4 physiological race of banana vascular wilt pathogenic bacteria (Foc TR4), can inhibit the hypha growth of Foc TR4 and change the typical growth morphology of the hypha, and the Streptomyces mangrove sp B4503 strain can be applied to preventing and treating banana vascular wilt and has development and application values of biocontrol microbial inoculum.

Description

A strain of Streptomyces mangrove B4503 and its application in the control of banana fusarium wilt

technical field

The invention belongs to the technical field of microorganisms, and more particularly, relates to a strain of Streptomycess p. B4503 and its application in the prevention and treatment of banana fusarium wilt.

Background technique

Banana (Musa spp.) is the fourth largest food crop in the world after rice, wheat and corn. However, banana cultivation has always been threatened by Fusarium oxysporum f.sp. cubenense (Foc), especially its physiological race 4 (Foc TR4), which can infect almost all cultivation systems ( Siddhesh, International Journal of Pest Management, 2015, 61(3):250-263). Banana fusarium wilt has soil-borne characteristics and can be transmitted through various media.

Chemical inoculants are one of the main methods for the control of banana fusarium wilt due to their direct and efficient characteristics, but long-term use is prone to produce drug resistance and pollute the environment, which makes pollution-free and bio-friendly microbial control technologies attract much attention. Screening of microorganisms with the potential to inhibit plant diseases and insect pests, and exploring the control effect and mechanism of these microorganisms on plant diseases and insect pests are the basis for the application of microorganisms to control plant diseases and insect pests.

Streptomyces bacteria are widely distributed in the soil of different habitats and can produce secondary metabolites with various structures and biological activities. At present, more than 75% of antibiotics are derived from Streptomyces, some of which have been used as agricultural products. Antibiotics are used in plant pest control. The genus Streptomyces has so far contained more than 1000 species of bacteria. Genome analysis shows that the genome of Streptomyces contains a large number of secondary metabolite biosynthesis gene clusters, while the active secondary metabolites derived from Streptomyces that inhibit the pathogenic bacteria of banana fusarium wilt are rarely reported. However, the biological activities of a large number of secondary metabolites have not been thoroughly explored. Therefore, exploring Streptomyces with significant biocontrol potential, and researching its antibacterial mechanism will help to discover active secondary metabolites and promote its application in biological control of plant diseases and insect pests.

SUMMARY OF THE INVENTION

For the problems existing in the prior art, the object of the present invention is a kind of mangrove Streptomyces (Streptomycessp.) B4503 bacterial strain and its application in the control of banana fusarium wilt. The Streptomycess p. B4503 strain of the present invention can produce secondary metabolites that inhibit the activity of Foc TR4, and the Streptomycess p. B4503 strain has a good control effect on banana fusarium wilt.

The object of the present invention is achieved through the following technical solutions:

The present invention provides an application of a mangrove Streptomyces sp. B4503 strain in the prevention and control of banana fusarium wilt, and the mangrove Streptomyces sp. B4503 strain was preserved in a Chinese typical on August 19, 2019 Culture Collection Center (CCTCC), address: China. Wuhan. Wuhan University Chinese Type Culture Collection, zip code 430072, deposit number CCTCC NO: M 2019647.

Preferably, the above-mentioned mangrove Streptomyces sp. B4503 strain is subjected to conventional fermentation, and the supernatant after fermentation contains secondary metabolites for preventing and treating banana fusarium wilt.

The mangrove Streptomyces sp. B4503 strain of the present invention is isolated from the soil of the mangroves in Beibu Gulf, Qinzhou, Guangxi. Spores are formed. Using ISP2 medium, by changing the bacterial culture temperature, pH and NaCl content in the medium, the optimum growth temperature of Streptomyces sp. B4503 was determined to be 28°C, the optimum pH value was 7.0, and it could tolerate 0-7 %NaCl.

Another object of the present invention is to provide the application of Streptomyces sp. B4503 strain in the production and preparation of Foc TR4 inhibitors. The specific technical solutions are as follows:

Application of Streptomyces sp. B4503 strain in the production and preparation of Foc TR4 inhibitors, the Streptomyces sp. B4503 strain was deposited in the China Center for Type Culture Collection on August 19, 2019 , the deposit number is CCTCC NO: M 2019647.

Preferably, the method for producing and preparing Foc TR4 inhibitor by Streptomyces sp. B4503 strain comprises the following steps:

S1 ferments the B4503 bacterial strain of mangrove Streptomyces sp. with preservation number CCTCC NO: M 2019647 to obtain fermentation broth;

S2 the fermentation broth obtained in step S1 is removed by centrifugation, the fermentation supernatant is suction filtered, and the suction filtrate is concentrated under reduced pressure to obtain a crude extract of the fermentation broth, which is purified and separated to obtain Foc TR4 inhibitors.

Preferably, the fermentation conditions described in step S1 are: the culture medium is an oat liquid culture medium, and the fermentation temperature is 28°C for 5 d of shaking fermentation.

Preferably, the suction filtration operation described in step S2 is to add macroporous adsorption resin particles XAD-16N to the fermentation supernatant, suction filtration to enrich XAD-16N, first use deionized water to rinse the resin, then use methanol to rinse the resin, and collect methanol Mutually.

Preferably, the purification and separation method described in step S2 is to take 100 times the weight of silica gel of the crude extract and dissolve it in a 20% methanol aqueous solution, then load it into a glass chromatography column, carry out gradient elution with a methanol/water system, and collect each components. After dissolving with methanol, it was filtered and placed in semi-preparative high performance liquid chromatography for separation.

The present invention also provides the application of the Foc TR4 inhibitor prepared by the production of the above-mentioned Streptomyces sp. B4503 strain in the preparation of a medicament for controlling the physiological race No. 4 of Fusarium wilt pathogen of banana.

The present invention uses mangrove soil Streptomyces sp. B4503 for the first time to ferment and isolate nifemycin C. The test shows that this compound can effectively inhibit the physiological race of banana fusarium wilt pathogen No. 4, and the minimum inhibitory concentration to spore germination is: 32μg/mL, can destroy the structure of mycelium, change the typical shape of mycelium, inhibit the growth of mycelium and spore germination, and cause the expression of amino acid metabolism, cell signal transduction, glucose metabolism pathway and other related genes in race 4 cells, and change the bacteria. The filament shape changes the mycelium from smooth cylindrical to shriveled, curved and obviously shrunken, which provides a good application prospect for the development and control of banana fusarium wilt.

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

The Streptomyces sp. B4503 strain provided by the present invention is isolated from the mangrove soil of Beibu Gulf, Qinzhou, Guangxi, and can significantly inhibit the growth of physiological race 4 of Fusarium wilt pathogen, with an inhibition rate of 73.3%. The Streptomyces sp. B4503 strain of the present invention can produce active secondary metabolites that inhibit Foc TR4, and the produced secondary metabolite niphimycin C can cause Foc TR4 hyphae to bend and shrivel , and folds appeared, and the minimum inhibitory concentration to spores was 32 μg/mL. The Streptomyces sp. B4503 strain had good control effect on banana fusarium wilt, and had the development and application value of biocontrol fungicides.

Description of drawings

Fig. 1 is the bacterial growth morphology of Guangxi mangrove Streptomyces sp. B4503 strain (A) and its plate confrontation experiment on Foc TR4 (B).

Figure 2 is a phylogenetic analysis of Streptomyces sp. B4503 based on the 16S rRNA gene sequence.

Figure 3 is a comparison diagram of the control effect of Streptomyces sp. B4503 on Foc TR4.

Figure 4 is the chemical structure of the compound niphimycin C.

Figure 5 is a flow chart of the separation and purification of niphimycin C.

Figure 6 is a scanning electron microscope image of the effect of niphimycin C on the growth morphology of Foc TR4 hyphae. The left side of Figure 6 is the morphology of Foc TR4 mycelium under normal growth conditions, and the right side is the morphology of Foc TR4 mycelium after the action of nifemycin C.

Detailed ways

The present invention is further described below in conjunction with specific embodiments and accompanying drawings, but the embodiments do not limit the present invention in any form. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts shall fall within the protection scope 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.

Example 1 Isolation, culture and identification of Streptomyces sp. B4503 strain

1. Test material

1.1 Isolation and culture of strains

The soil collected from the Beibu Gulf mangrove forest in Qinzhou, Guangxi was dried in a drying oven at 60 °C, diluted with sterile water to make 10-2 and 10-3 sample solutions, respectively, and 250 μL of the sample solutions were drawn and applied to ISP2 and 2216E. The agar medium was cultured in a constant temperature incubator at 28°C for several days, and the colonies were picked with a sterile inoculation needle, and were streaked in the medium by the continuous streak culture method until a single colony appeared.

1.2 Test medium

The culture medium used in this study is shown in Table 1.

Table 1 Bacteria culture medium formula

2. Classification and identification of strains

2.1 Determination of 16S rDNA gene sequence of Streptomyces sp. B4503

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

2.2 Identification of Streptomyces sp. B4503

The obtained sequence was submitted to the NCBI website and compared with the published 16S rRNA gene sequence stored in the GenBank database, and the homologous relative strain of Streptomyces sp. B4503 was determined to be S.yatensis NBRC101000T (99.8%), S.antimycoticus NBRC 12839T (99.4%), S. melanosporofaciens DSM40318T (99.3%), S. geldanamycininus DSM 40318T (99.3%) and S. mordarskii NRRL B-1346T (99.0%).

As shown in Figure 2, based on 16S rRNA gene sequence analysis, it was found that the strain B4503 belongs to the genus Streptomyces, and has the closest relationship with Streptomyces (S. yatensis) NBRC 101000T.

2.3 Optimization of growth conditions for Streptomyces sp. B4503

After Streptomyces sp. B4503 was cultured on ISP2 medium for 3 days, the colonies were gray-black, powdery, and sporulated (Fig. 1-A). Using ISP2 medium, by changing the bacterial culture temperature, pH and NaCl content in the medium, the optimum growth temperature of Streptomyces sp. B4503 was determined to be 28°C, the optimum pH value was 7.0, and it could tolerate 0-7 %NaCl.

Example 2 Control effect of Streptomyces sp. B4503 strain on banana fusarium wilt

1. Test pathogens

The physiological race F.oxysporum f.sp.cubense Race 4 of Fusarium wilt pathogen No. 4 was provided by Guangxi Academy of Agricultural Sciences, and the culture medium is shown in Table 2.

Table 2 strain culture medium formula

2. Plate inhibitory activity of Streptomyces sp. B4503 on Foc TR4

Banana wilt pathogen No. 4 race (Foc TR4) was inoculated in the center of the PDA medium, Streptomyces sp. B4503 was inoculated at 2-3 cm from the pathogenic bacteria block, and the PDA agar plate only inoculated with Foc TR4 was used as a control. , three replicates for each group, after 7 days of constant temperature incubation at 28°C, the growth diameter of Foc TR4 colonies in the control group and the treatment group was measured, and the inhibition rate of Streptomyces sp. B4503 strain on Foc TR4 was calculated.

The inhibitory effect of Streptomyces sp. B4503 on Foc TR4 is shown in Figure 1-B, and the inhibition rate is 73.3%.

3. The control effect of Streptomyces sp. B4503 on Foc TR4

Foc TR4 was inoculated into sterile mung bean medium, cultured with shaking at 30°C for 7 days to obtain a bacterial solution containing Foc TR4 spores, and banana seedlings with consistent growth were immersed in the bacterial solution, and then transplanted into flowerpots containing 500 g of soil. Two treatments were set up in the experiment: 1) the control group was watered with clean water every day; 2) the experimental group was mixed with the microbial cells of Streptomyces sp. B4503 and the soil in the flowerpot, and watered with clean water every day. The growth of bananas was observed every day, and the plant height, stem circumference, number and diameter of leaves were recorded. After cultivating 30 days, the banana seedlings were carefully pulled out by the roots, and the rhizome was cut with a sterile blade to observe the infection situation of Foc TR4 on the banana plants.

Figure 3 shows the control effect of Streptomyces sp. B4503 on Foc TR4. As can be seen from Figure 3, the rhizomes of banana seedlings without Streptomyces sp. B4503 were dark purple, and the center was slightly black, which had been infected by Foc TR4. However, the rhizomes of banana seedlings with Streptomyces sp. B4503 were white, and only a few showed light purple after infection, and the incidence was less and lighter. It was confirmed that Streptomyces sp. Foc TR4 has a certain preventive effect.

Example 3 Separation and purification of antibacterial activity secondary metabolites produced by Streptomyces sp. B4503

1. The main reagents and instruments used in this study are shown in Table 3 and Table 4

Table 3 Main commonly used reagents and sources

Table 4 Main instruments

2. Fermentation culture of Streptomyces sp. B4503 strain

The Streptomyces sp. B4503 grown on the ISP2 agar plate was inoculated into a conical flask containing 100 mL of oat liquid medium by scraping the mycelium with a sterile inoculating needle, and the fermented seed liquid was obtained after shaking at 28°C for 5 days. Use a sterile pipette to draw 1 mL of seed solution into a conical flask containing 200 mL of oat liquid medium, inoculate 50 L in total, and culture with shaking at 28 °C for 5 d.

Oat liquid culture medium formula: 60.0 g of oat flour, 1000 mL of distilled water, pH 7.2.

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

The fermentation broth was removed by centrifugation (5000rpm, 10min) to remove bacterial cells, and 10% (w/v) macroporous adsorption resin particles XAD-16N were added to the fermentation supernatant, and the XAD-16N was enriched by suction filtration, and the resin was rinsed with deionized water first. 3 times, 500 mL each time, and then use methanol to rinse the resin 3 times, 500 mL each time, collect the methanol phase, and concentrate under reduced pressure to obtain the crude extract of the fermentation broth. After the activity test, the crude extract has obvious inhibitory activity against Foc TR4 pathogenic bacteria.

100 times the weight of the silica gel of the crude extract was dissolved in 20% methanol aqueous solution, and then loaded into a glass chromatography column, followed by gradient elution with methanol/water system (300 mL, v/v, 20%, 40%, 60% , 80% and 100%), each fraction was collected, and after concentration, the fraction Fr.4 containing the active compound was determined by the activity test.

The active compound-containing components were dissolved in 5 mL of methanol, filtered through a 0.22 μm filter head, and separated by semi-preparative high performance liquid chromatography. The separation conditions are: the column temperature is room temperature; the chromatographic column is XBridge, BEH, C ₁₈ , OBD (250X 10mm, 5μm); the mobile phase is acetonitrile/water, 0-20min, 40%-60% acetonitrile, 20-30min, 60 %-80% acetonitrile, 30-50 min, 80%-100% acetonitrile; injection volume is 80 μL; flow rate is 1.0 mL/min.

After separation by semi-preparative high performance liquid chromatography, a total of 5 monomer compounds were collected, and compound 3 was collected in 16 min (12 mg) as the target compound after activity verification.

Active compound 3 is a colorless solid, and its molecular weight is 1142.73005[M+H] ⁺ detected by high-resolution mass spectrometry, and the molecular formula is presumed to be C ₅₉ H ₁₀₄ N ₃ O ₁₈ . According to the ¹ H-NMR and COSY spectra of compound 1, and the literature comparison, it is found that the spectral data of compound 3 is consistent with the report in the literature, and it is determined that compound 3 is nifemycin C (Hu et al. Journal of Natural Products, 2018, 81(1) :178-187). The planar structure of compound 3 was resolved by nuclear magnetic spectrum, as shown in FIG. 4 , and the nuclear magnetic data of this compound is shown in Table 5.

Table 5 NMR data of compound 3 (deuterated reagent: CD ₃ OD)

Example 4 Inhibitory effect of antibacterial activity secondary metabolites produced by Streptomyces sp. B4503 on FocTR4

1. Inhibitory effect of niphimycin C on spore germination of Foc TR4

Use a pipette to pipette 100 μL of Foc TR4 spore solution with a concentration of 1×10 ⁵ CFU/mL into a 96-well plate, add 100 μL of a mixture of RPML 1640 medium and nifemycin C, and incubate at 28°C for 12 h. The absorbance was measured at 620 nm with a meter, and the sample without niphamycin C was used as a negative control, and the lowest inhibitory concentration of nifemycin C on FocTR4 spores was determined by comparison to be 32 μg/mL.

2. The effect of niphimycin C on the growth morphology of Foc TR4 pathogenic bacteria

Dissolve nifemycin C in DMSO to prepare a solution of 32 μg/mL, mix 0.5 mL of solution, 0.5 mL of spore suspension and 19 mL of PDA agar medium to prepare a plate, cover it with a piece of sterile cellophane, and place it at 28. Cultivated in a constant temperature incubator for 7 days.

Foc TR4 hyphae on cellophane were collected using a sterile spreader bar, fixed with 2.5% (v/v) glutaraldehyde solution overnight at 4°C, and then 0.1 mol/L phosphate buffered saline (PBS, pH 7.4) After rinsing several times, dehydrate with gradient elution of 30%, 50%, 70%, 80%, 90%, 95% and 100% ethanol solution, 20min each time, and finally vacuum dry and spray gold. The hyphal morphology of Foc TR4 was observed by scanning electron microscope, and the results are shown in Figure 6.

Figure 6 shows that Nifemycin C can inhibit the growth of the hyphae of Foc TR4, change the shape of the hyphae, and make the hyphae from smooth cylindrical to shriveled, curved and significantly shrunken.

The specific embodiments of the present invention are described above in detail, but they are only used as 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, equivalent changes and modifications made without departing from the spirit and scope of the present invention should be included within the scope of the present invention.

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Claims (10)

1. the application of mangrove streptomyces (Streptomyces sp.) B4503 bacterial strain in the control of banana fusarium wilt, it is characterized in that, described mangrove streptomyces (Streptomyces sp.) B4503 bacterial strain is preserved in Chinese typical on August 19, 2019 Culture Collection Center, the deposit number is CCTCC NO: M 2019647. 2. the application of mangrove streptomyces (Streptomyces sp.) B4503 bacterial strain according to claim 1 in the control of banana fusarium wilt, it is characterized in that, the mangrove streptomyces (Streptomyces sp.) B4503 bacterial strain is carried out conventional fermentation, fermentation The latter supernatant contains secondary metabolites for controlling banana fusarium wilt. 3. the application of mangrove streptomyces (Streptomyces sp.) B4503 strain in the production and preparation of Foc TR4 inhibitor, it is characterized in that, described mangrove streptomyces (Streptomyces sp.) B4503 strain was preserved in August 19, 2019 in China Type Culture Collection Center, the deposit number is CCTCC NO: M 2019647. 4. the application of mangrove streptomyces (Streptomyces sp.) B4503 bacterial strain according to claim 3 in production and preparation of FocTR4 inhibitor, it is characterized in that, mangrove streptomyces (Streptomyces sp.) B4503 bacterial strain is produced to prepare Foc TR4 inhibitory The method of preparation, comprising the steps of: S1 ferments the mangrove Streptomyces sp. B4503 bacterial strain of claim 3 to obtain fermentation broth; S2 The fermentation broth obtained in step S1 is centrifuged to remove bacterial cells, the fermentation supernatant is suction filtered, and the suction filtrate is concentrated under reduced pressure to obtain a crude extract of the fermentation broth, which is purified and separated to obtain Foc TR4 inhibitors. 5. mangrove Streptomyces sp. B4503 bacterial strain according to claim 4 produces the method for preparing FocTR4 inhibitor, it is characterized in that, described in step S1, fermentation condition is: substratum is oat liquid medium, fermentation The temperature was 28°C for 5 days of shaking fermentation. 6. mangrove Streptomyces sp. B4503 bacterial strain according to claim 4 produces the method for preparing FocTR4 inhibitor, it is characterised in that the suction filtration method described in step S2 is that fermentation supernatant adds macroporous adsorption resin Particle XAD-16N, enrich XAD-16N by suction filtration, rinse the resin with deionized water first, then rinse the resin with methanol, and collect the methanol phase. 7. mangrove Streptomyces sp. B4503 bacterial strain according to claim 4 produces the method for preparing FocTR4 inhibitor, it is characterised in that the purification and separation method described in step S2 is to get 100 times of weight in the crude extract After the silica gel was dissolved in 20% methanol aqueous solution, it was loaded into a glass chromatography column, and gradient elution was carried out with methanol/water system. The components were collected, dissolved in methanol, filtered, and separated by semi-preparative high performance liquid chromatography. 8. The application of the Foc TR4 inhibitor prepared by the method of any one of claims 4-7 in the control of banana fusarium wilt. 9. The application of nifemycin C in the preparation of inhibitor of physiological race 4 of Fusarium wilt pathogen of banana. 10. The application of nifemycin C in the preparation and control of banana fusarium wilt pathogen.

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