Bacillus subtilis SY8 capable of generating myriocin and preventing and treating black spot of Chinese herbaceous peony and application of bacillus subtilis SY8

Abstract

The invention discloses a bacillus subtilis SY8 capable of generating myriocin and preventing and treating paeonia lactiflora black spot and application of the bacillus subtilis SY8, the bacillus subtilis SY8 is preserved in China General Microbiological Culture Collection Center (CGMCC), the preservation number is CGMCC No.33763, and the preservation date is March 10, 2025. The bacillus subtilis SY8 is a biocontrol bacterium, the biocontrol bacterium can generate a natural active substance myriocin (Myriocin) with great value, the bacillus subtilis SY8 has a very strong inhibition effect on the growth of a pathogenic bacterium alternaria alternata of the black spot of the Chinese herbaceous peony, and the bacillus subtilis SY8 is verified to have a very strong prevention and treatment effect on the black spot through living body tieback of the Chinese herbaceous peony, so that the bacillus subtilis SY8 can be used for preventing and treating the black spot of the Chinese herbaceous peony. The strain has good application and development prospects in biological control of black spot of Chinese herbaceous peony and biological synthesis of myriocin.

Description

Technical Field

[0001] The invention belongs to the field of microorganisms, and particularly relates to a Bacillus subtilis SY8 capable of producing myriocin and preventing and treating peony black spot disease, and application thereof. Background Art

[0002] Myriocin is a bioactive substance with multiple functions. Its molecular formula is C 21 H 39 NO6, with the chemical formula of 2-amino-3,4-dihydroxy-2-hydroxymethyl-1-4-oxo-6-eicosenoic acid. Initial research on the biological activity of myriocin focused on immunosuppressive activity. Studies have shown that myriocin inhibits the activity of serine palmitoyl-CoA transferase (SPT), thereby affecting the biosynthesis of sphingolipids, a component of the cell membrane of eukaryotic organisms. Sphingolipids, as an important class of cytoplasmic membrane components commonly found in eukaryotic organisms, are important intracellular signaling molecules that participate in the regulation of various basic biological processes such as cell growth, aging, and apoptosis. In recent years, the antifungal activity of myriocin and its related functions of inhibiting biofilm activity have also been continuously developed. However, a cheap and efficient method for synthesizing myriocin has not yet been developed, resulting in high costs for the use of myriocin.

[0003] Black spot disease of peony is caused by the fungus Alternaria alternata, which primarily affects peony leaves. Initially, small brown spots form with a yellow outer ring and a distinct outer border. As the disease progresses, the spots gradually expand to become nearly circular or irregular in shape, dark brown in color. In severe cases, they can become widespread, causing leaves to wither and yellow, ultimately leading to the death of the entire plant. This leads to a significant decrease in the number and quality of flowers produced the following year, severely impacting the healthy development of the peony industry.

[0004] Currently, chemical pesticides remain the primary means of controlling black spot disease in peony. However, their widespread use has led to increasingly serious environmental, ecological, food safety, and human health issues. Therefore, while using chemical pesticides rationally and scientifically, we must also actively seek safe and environmentally friendly methods for controlling black spot disease. Biological control methods that utilize microorganisms to reduce or prevent pathogen infection, enhance plant disease resistance, and replace chemical fertilizers and pesticides can significantly reduce reliance on chemical pesticides and make a significant contribution to promoting the protection of China's agricultural natural environment. Summary of the Invention

[0005] Purpose of the invention: In response to the problems existing in the prior art, the present invention provides a Bacillus subtilis SY8 that can produce myriocin and prevent and control peony black spot disease. The Bacillus subtilis SY8 of the present invention can synthesize myriocin with high efficiency, has potential application value in the synthesis and extraction of myriocin, and has a strong control effect on black spot disease. It has good application and development prospects in both the biological control of peony black spot disease and the biosynthesis of myriocin.

[0006] The present invention also provides a biological bacterial agent of Bacillus subtili SY8 and application thereof.

[0007] Technical solution: In order to achieve the above-mentioned purpose, the Bacillus subtili SY8 described in the present invention has been deposited in the General Microbiology Center of the China Culture Collection Administration of Microorganisms, with the deposit number CGMCC No. 33763 and the deposit date March 10, 2025.

[0008] The invention discloses an application of the Bacillus subtilis SY8 in synthesizing myriocin.

[0009] Among them, myriocin was extracted from Bacillus subtilis SY8.

[0010] The invention relates to the application of the Bacillus subtilis SY8 in preventing and treating plant black spot disease.

[0011] Among them, black spot disease caused by Alternaria alternata is prevented and controlled by inoculating plants with Bacillus subtilis.

[0012] Wherein, the plant is peony.

[0013] The biological bacterial agent prepared by Bacillus subtilis SY8 of the present invention.

[0014] Among them, a single SY8 colony was picked and cultured and inoculated into LB liquid medium, and cultured with shaking until the OD value was 0.25-0.5.

[0015] The invention relates to the application of the biological bacterial agent in the synthesis of myriocin.

[0016] Application of the biological bacterial agent of the present invention in preventing and treating plant black spot disease.

[0017] The present invention provides a Bacillus subtilis SY8 that can produce myriocin and prevent and treat peony black spot disease and its application. The Bacillus subtilis SY8 is a biocontrol bacterium, classified and named Bacillus subtilis (Bacillus subtili). The biocontrol bacterium can produce a very valuable natural active substance myriocin (Myriocin). It has been verified that this substance has a strong inhibitory effect on the growth of the pathogen of peony black spot disease, Alternaria alternata. The Bacillus subtilis SY8 has a strong control effect on black spot disease through live peony inoculation. It has good application and development prospects in both the biological control of peony black spot disease and the biosynthesis of myriocin. Beneficial effects: Compared with the existing technology, the present invention has the following advantages:

[0018] The present invention isolated and screened a strain of Bacillus subtilis SY8, which can synthesize myriocin. This strain can efficiently synthesize myriocin and, by inoculating plants with Bacillus subtilis, can control black spot disease caused by Alternaria alternata. Measurements showed that the strain had a plate pathogen inhibition rate of 67.98%, a leaf inoculation disease index of 18.1%, and a control efficacy of 77.53%.

[0019] At the same time, the biological agent prepared by the strain of the present invention can be produced using general fermentation equipment in the fermentation industry, has low production cost and is easy to use, and is suitable for the biological control of peony black spot disease and the biosynthesis of myriocin. Therefore, the strain SY8 has great potential to be developed into a commercial live bacterial agent. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 For the confrontation experiment between SY8 and Alternaria plate culture

[0021] Figure 2 Phylogenetic tree of SY8 gene sequence alignment

[0022] Figure 3 SY8 single colony morphology and SEM morphology observation

[0023] Figure 4 Statistics of differential metabolites in SY8 antagonistic culture fluid

[0024] Figure 5 The inhibition of myriocin at different concentration gradients on the growth of Alternaria alternata

[0025] Figure 6 The present invention is to inoculate the strain SY8 of the present invention to the leaves infected by the black spot pathogen of peony. DETAILED DESCRIPTION

[0026] The present invention will be further described below with reference to the accompanying drawings and examples.

[0027] Unless otherwise specified, the raw materials and reagents in the present invention are commercially available.

[0028] Example 1

[0029] Separation and purification: First, gently shake off the large pieces of soil near the roots of peony, then use a sterile brush to gently brush the soil attached to the rhizosphere and collect it in a sterile centrifuge tube. Weigh 1g of rhizosphere soil sample, place it in a sterile triangular flask, add 99mL of sterile water and shake thoroughly to mix, then place it on a shaker and shake at 28°C and 200rpm for 10 minutes. At this time, the bacterial concentration in the soil is diluted to 10 -2 Prepare enough centrifuge tubes and sterile water in advance, and use a pipette to draw 1 mL 10 -2 The soil solution was transferred to 9 mL of sterile water and mixed by suction and diluted to 10 -3 Repeat this process until the soil solution concentration reaches 10 -7 Prepare LB solid culture medium, use a pipette to draw 200uL 10 -7 Concentrated soil solution is evenly added to the surface of the culture medium and spread evenly with a sterile coating rod until the surface is dry. Repeat the coating on 3 plates for each group of samples. After coating, the culture dish is placed in a 37°C incubator to culture and observe the growth of bacteria. The grown bacteria are separated and purified using the dilution coating plate method. Pick as many pure bacteria or single colonies as possible and purify them using the repeated streaking method. Repeat the operation several times until a single colony is obtained. Use an inoculation loop to pick the purified single colony and inoculate it into LB liquid culture medium. Culture it overnight in a shaker at 32°C and 200 rpm. After the bacterial solution becomes turbid, move it to an EP tube containing 50% sterilized glycerol and store it in a -80°C refrigerator.

[0030] Screening: (1) Pick a single bacterial colony that has been cultured for 18-24 hours and inoculate it into LB liquid medium. Incubate overnight in a 32°C constant-shake incubator. Measure the OD value of the bacteria in a UV-visible spectrophotometer, adjust the wavelength to 600 nm, and zero it with LB liquid medium. Then adjust the OD value of the culture of the bacteria to 0.25 with LB liquid medium.

[0031] (2) Take the preserved Alternaria pathogen (a wild-type Alternaria pathogen strain isolated and purified from leaves infected with black spot of peony) and inoculate it into PDA solid culture medium. After culturing in a constant temperature incubator at 28°C for 14 days, use a 9mm sterile punch to punch out the bacterial mass from the outermost circle of the well-grown pathogen colony and place it on one end of a new PDA solid culture medium plate. At the same time, use a sterile inoculation loop to dip the test bacterial solution and streak it, then inoculate it on the other end of the plate, ensuring that there is a 3cm gap between the bacterial inoculation area and the fungal mass.

[0032] (3) The culture medium was cultured at 28°C for 14 days to screen out strains that can effectively inhibit the growth of Alternaria. By accurately measuring the inhibition rate of each strain to be tested, the strain with the highest inhibition rate was screened out and named SY8. The growth inhibition rate of SY8 on Alternaria was 67.98%. (Inhibition rate (%) = (Rr) / R×100, where r is the radius of the pathogenic fungus colony after inoculation with bacteria, and R is the maximum radius of the pathogenic fungus colony after not inoculating with bacteria). The growth status of Alternaria is shown in Figure 2. Figure 1 As shown, SY8 significantly inhibited the growth of Alternaria alternata on plate culture medium compared with the control group.

[0033] Morphological and molecular biological identification: Use an inoculation loop to take an appropriate amount of the preserved bacterial liquid and streak it on an LB plate. Invert the plate and culture it at 32°C overnight. Select a single colony from the LB plate and inoculate it into LB liquid culture medium. Culture the bacteria at 32°C and 200 rpm overnight to obtain the activated SY8 bacterial liquid. The 16s sequence obtained by sequencing was used to perform a DNA sequence BLAST in the GenBank database. The results showed that the closest relative to strain SY8 was Bacillus subtili. The 16s sequences of representative strains of the genus Bacillus were combined with the 16s sequence of strain SY8 to construct a phylogenetic tree. The results are as follows: Figure 2 The strain Bacillus subtiliSY8 was deposited in the General Microbiology Center of China Culture Collection Administration of Microorganisms in Beijing, China, with the deposit number CGMCC No. 33763 and the deposit date March 10, 2025.

[0034] The morphology of strain SY8 single colony and SEM electron microscopy observation is as follows Figure 3 The morphological characteristics and physiological and biochemical characteristics are shown in Table 1 below:

[0035] Table 1

[0036]

[0037]

[0038] Example 2

[0039] Determination of relative content of polychaetococcin in SY8 bacterial suspension

[0040] (1) Determination of the relative content of polysaccharin in SY8 bacterial culture using non-targeted metabolic LC-MS. Sample pretreatment method: Pipette 100 μL of bacterial culture (SY8 fermentation broth, SY8-Alternaria co-fermentation broth) cultured for 48 h under the same culture conditions into a 2 mL centrifuge tube; add 400 μL of pre-cooled methanol:acetonitrile (1:1, v / v), vortex and oscillate for 30 s; place in a -20 ° C refrigerator and freeze for 30 min; centrifuge at 12000 rpm and 4 ° C for 10 min, take 400 μL of supernatant and vacuum concentrate to dryness; add 150 μL of 50% methanol (containing 5 ppm 2-chlorophenylalanine) to re-dissolve, vortex and oscillate for 30 s; centrifuge at 12000 rpm and 4 ° C for 10 min, take the supernatant and filter through a 0.22 μm filter membrane, and add the filtrate to the detection bottle; take 10-20 μL of each sample filtrate and mix them into a QC sample for evaluating instrument stability and data reliability.

[0041] (2) Using ACQUITY UPLCHSS T3 Column ( 1.8μm, 2.1mm×100mm), flow rate 0.4mL / min, column temperature 40°C, autosampler 8°C, injection volume 2μL. Positive and negative mode mobile phase: mobile phase A is 0.1% formic acid in water, mobile phase B is acetonitrile (containing 0.1% formic acid), elution gradient is as shown in Table 2:

[0042] Table 2

[0043]

[0044] (3) Thermo Orbitrap Exploris 120 mass spectrometer was controlled by Xcalibur software (version: 4.7, Thermo) to acquire DDA mass spectrometric data in both positive and negative ion modes. A HESI source was used, with a spray voltage of 3.5 kV / -3.0 kV, a sheath gas of 40 arb, an auxiliary gas of 15 arb, a capillary temperature of 325°C, an auxiliary gas temperature of 300°C, a primary resolution of 60,000, a scan range of 100-1000 m / z, an AGC Target Standard, a Max IT of 100 ms, and the top 4 ions of response were screened for secondary fragmentation. The dynamic exclusion time was 8 s, the secondary resolution was 15,000, the HCD collision energy was 30%, the AGC Target Standard, and the Max IT Auto. All formal samples and QC samples were loaded into the instrument according to the above-mentioned chromatographic and mass spectrometric methods. Two to four QC samples were injected before the formal injection to equilibrate the system. During the injection process, one QC sample was injected every 5 to 10 samples for subsequent data evaluation and quality control.

[0045] (4) Import the .raw format data into the commercial software CompoundDiscovererTM 3.3 (version 3.3.2.31, Thermo, Waltham, USA) uses the software's new peak detection and peak quality scoring algorithms to perform peak extraction, alignment, and correction. Its unique peak quality rating calculation and filter significantly reduce interference from background and low-quality peaks. Peaks that were not detected in >50% of QC samples were filtered, and missing values ​​for undetected peaks were filled using the software's FillGaps algorithm and normalized to the total peak area. Metabolite identification was performed based on self-built libraries, mzCloud online library (https: / / www.mzcloud.org / ), LIPID MAPS (https: / / www.lipidmaps.org / ), HMDB (https: / / hmdb.ca / ), MoNA (https: / / mona.fiehnlab.ucdavis.edu / ) and NIST_2020_MSMS spectral library. The MS1 mass tolerance MassTolerance was set to 15 ppm and the MS2 MatchFactorThreshold was set to 50.

[0046] (5) The R software package Ropls was used to perform principal component analysis (PCA), partial least squares discriminant analysis (PLS-DA), and orthogonal partial least squares discriminant analysis (OPLS-DA) dimensionality reduction analysis on the sample data. Score plots, loading plots, and S-plots were drawn to show the differences in metabolite composition between samples. The model was tested for overfitting using the permutation test method. R2X and R2Y represent the explanatory rate of the constructed model for the X and Y matrices, respectively, and Q2 indicates the predictive ability of the model. The closer their values ​​are to 1, the better the fit of the model, and the more accurately the samples in the training set can be divided into their original categories. The pvalue value was calculated based on the statistical test, the variable projection importance (VIP) was calculated using the OPLS-DA dimensionality reduction method, and the fold change (FC) was calculated to calculate the difference between the two groups. The intensity and explanatory power of the content of each metabolite component on the sample classification and discrimination were measured to assist in the screening of marker metabolites. When the pvalue value is 1, the metabolite molecule is considered to be statistically significant. The Pheatmap package (V1.0.12) in R was used to perform cluster analysis on the abundance values ​​of differential metabolites, and heat maps and trend analysis diagrams were drawn; VennDiagram (V1.7.3) and UpSetR (V1.4.0) were used to draw Venn diagrams and Upset diagrams for the differential substances compared between two different groups; corrplot (V4.0.3) was used to perform correlation analysis on the differential metabolites; ggplot2 (V3.4.1) was used to draw box plots and violin plots of differential metabolites to show the abundance of each differential substance between different groups; the differential metabolite results were further analyzed by machine learning (mlr3verse, V0.2.7) and ROC curve drawing (pROC, V1.18.2) to obtain key product information in the differential set.

[0047] (6) According to the analysis results Figure 4 The proportions of differential metabolite species between SY8 fermentation broth and blank medium, as well as the relative content fold differences of key differential metabolites between SY8 fermentation broth, SY8-Alternaria co-fermentation broth and blank medium were obtained. The results showed that the differential expression fold of myriocin in SY8 fermentation broth and blank medium was 262.04, and the differential expression fold of myriocin in SY8-Alternaria co-fermentation broth was 311.46, which was higher than that in SY8 fermentation broth, indicating that under the stimulation of the pathogen Alternaria, the secretion of myriocin by Bacillus subtilis SY8 increased.

[0048] Example 3

[0049] Inhibitory effects of myriocin on the growth of Alternaria alternata at different concentrations

[0050] (1) Myriocin was added to the unsolidified PDA culture medium to obtain PDA solid culture medium with myriocin concentrations of 25 mg / L, 50 mg / L, and 100 mg / L.

[0051] (2) The preserved Alternaria pathogen (a wild-type Alternaria pathogen strain isolated and purified from leaves infected with black spot disease of peony) was inoculated into PDA solid culture medium. After culturing in a constant temperature incubator at 28°C for 14 days, a 9mm sterile punch was used to punch out bacterial blocks from the outermost circle of the well-grown pathogen colony. The blocks were then placed on PDA solid culture medium plates with different myriocin concentrations for culture.

[0052] (3) The culture medium was cultured at 28°C for 14 days. The growth of Alternaria was as follows: Figure 5 As shown in the results, compared with the control group, myriocin at different concentrations can effectively inhibit the growth of Alternaria alternata. The higher the concentration, the more obvious the inhibitory effect. This shows that myriocin is the main effective substance of Bacillus subtilis SY8 in antagonizing Alternaria alternata.

[0053] Example 4

[0054] Effect of SY8 on the prevention and treatment of black spot disease on peony leaves

[0055] (1) Select three-year-old peony plants with consistent growth conditions and gently wipe the healthy peony leaves with sterile water to remove dust and other impurities attached to the leaf surface. After preparation, use a sterile syringe to punch holes on each healthy leaf and quickly use a pipette to draw 10 μL of Alternaria spore suspension (1×10 6 ~1×10 7 / mL) and drip it on the wound on the leaf.

[0056] (2) After the wound was naturally dried, the experimental group was inoculated with 10 μL of SY8 bacterial solution (OD value 0.25) at the same wound site, and the control group was inoculated with the same amount of LB liquid culture medium in the same way.

[0057] (3) To simulate a suitable humidity environment, use sterile plastic bags to wrap the treated plant leaves and spray water inside the plastic bags to maintain humidity. Then, place the plants in a well-ventilated environment with a constant temperature of 28°C for cultivation. After 48 hours of cultivation, observe the pathological conditions of the leaves. Figure 6As shown, 48 hours after inoculation, the control group had already developed disease at the inoculated site, while SY8 effectively inhibited the growth and development of Alternaria on peony leaves. According to statistical data, the disease index for the control group reached 81%, while that for the SY8-inoculated group was only 18.1%, resulting in a control efficacy of 77.53%. (Disease index = Σ(number of diseased leaves at each level × relative disease level) / (total number of leaves surveyed × highest disease level) × 100; control efficacy (%) = (disease index in the control area - disease index in the treated area) / control disease index × 100).

Claims

1. A strain of Bacillus subtili SY8, deposited in the General Microbiology Center of China Culture Collection Administration, with the deposit number CGMCC No. 33763 and the deposit date March 10, 2025.

2. Use of the Bacillus subtilis SY8 according to claim 1 in synthesizing myriocin.

3. The use according to claim 2, characterized in that Preferably, myriocin is extracted from Bacillus subtilis SY8.

4. Use of the Bacillus subtilis SY8 according to claim 1 in preventing and treating plant black spot disease.

5. The use according to claim 4, characterized in that Control black spot disease caused by Alternaria by inoculating plants with Bacillus subtilis.

6. The use according to claim 4, characterized in that The plant is peony.

7. A biological agent prepared by the Bacillus subtilis SY8 according to claim 1.

8. The biological agent according to claim 7, characterized in that A single SY8 colony was picked and inoculated into LB liquid medium, shaken and cultured until the OD value was 0.25-0.

5.

9. Use of the biological agent according to claim 7 in the synthesis of myriocin.

10. Use of the biological agent according to claim 7 in preventing and controlling plant black spot disease.

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