A Bacillus velezensis and its application in controlling Sclerotinia sclerotiorum of plants

Abstract

The present invention discloses a Bacillus velezensis and its application in controlling Sclerotinia disease of plants. Its taxonomic name is Bacillus velezensis JD-3, which was registered and preserved at the China Center for Type Culture Collection on August 12, 2024, with the preservation number CCTCC M 20241775; the Bacillus velezensis JD-3 is applied to control Sclerotinia disease of plants. In the present invention, taking the stem segments of Morus atropurpurea Roxb. as materials, the Bacillus velezensis JD-3 was successfully isolated. It has an obvious inhibitory effect on the pathogen of Sclerotinia disease and has broad-spectrum antibacterial properties, providing a new strain resource for the biological control of Sclerotinia disease of plants and a practical basis for field application; the present invention provides data support for the research and development and application of biological agents, and at the same time opens up a new research path for the health management of plants such as Morus atropurpurea Roxb.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbiology. More specifically, the present invention relates to a Bacillus velezensis and its application in controlling Sclerotinia disease of plants. Background Art

[0002] Sclerotinia disease is the most serious disease in the development of the fruit mulberry industry, mainly damaging the fruits of fruit mulberry. It has the characteristics of rapid onset and wide range. It can infect mulberry flowers, leading to the occurrence of Sclerotinia disease in fruit mulberry. Generally, there are three symptoms, namely hypertrophic sclerotinia disease, small-grain sclerotinia disease, and dwarf sclerotinia disease. At present, chemical agents are commonly used to solve the problem of Sclerotinia disease in fruit mulberry. Thiophanate-methyl and procymidone are sprayed alternately, and the control effect is up to more than 90%. However, HPLC and GC analyses show that the pesticide residue in the fruits exceeds the international standard limit. Therefore, the irreversible decline in soil quality and serious health and environmental problems cannot be ignored. Overuse may also cause the pathogen to develop resistance. However, other methods, such as resistance breeding or traditional cultivation measures (crop rotation, soil management), etc., have no significant effect on the occurrence of Sclerotinia disease.

[0003] In recent years, biological control has attracted much attention in the control of Sclerotinia disease in fruit mulberry due to its advantages of being green, safe, efficient, and persistent. Most of the most common biocontrol bacteria are Bacillus, which is a class of aerobic or facultative anaerobic Gram-positive, chemoheterotrophic bacteria. There are 266 named species, which are widely present in soil, water, and air. The spores produced by them can have strong resistance to high temperature, ultraviolet rays, and ionizing radiation. The genus Bacillus is a producer of bioactive metabolites, and the prediction of biosynthetic gene clusters (BGCs) provides the possibility for the discovery and identification of secondary metabolite diversity. However, there are few reports on the inhibitory effect of Bacillus velezensis on Sclerotinia disease in fruit mulberry. Summary of the Invention

[0004] An object of the present invention is to solve at least the above problems and / or deficiencies and provide at least the advantages described hereinafter.

[0005] To achieve these objects and other advantages of the present invention, a Bacillus velezensis is provided, which is classified and named as Bacillus velezensis JD-3, and has been registered and preserved in the China Center for Type Culture Collection on August 12, 2024, with the preservation number of CCTCC NO: M 20241775.

[0006] Preferably, the Bacillus velezensis is derived from the stem segment of fruit mulberry.

[0007] Preferably, the biological characteristics of the Bacillus velezensis include: the colony is milky white, with an irregular edge, no viscosity, and is opaque.

[0008] Preferably, the 16S rDNA sequence of the Bacillus velezensis is as shown in SEQ ID NO.1.

[0009] Use of the Bacillus velezensis as described above in inhibiting fungal pathogens.

[0010] Preferably, the fungal pathogens include: the pathogen of hypertrophic sclerotinia disease, Epicoccum nigrum, Fusarium acuminatum, an endophytic fungus of Didymella, Alternaria alternata, and the blueberry fruit rot pathogen.

[0011] Use of the Bacillus velezensis as described above in inhibiting sclerotium germination.

[0012] Preferably, the inhibition rate of the Bacillus velezensis on sclerotium germination is 100%.

[0013] Use of the Bacillus velezensis as described above in promoting the growth of Morus atropurpurea Roxb.

[0014] Use of the Bacillus velezensis as described above in controlling sclerotinia disease of plants.

[0015] Preferably, the plant includes any one of Morus atropurpurea Roxb., Phaseolus vulgaris, Helianthus annuus, Apium graveolens, Lactuca sativa, flowers, Solanum lycopersicum, Capsicum annuum, Cucumis sativus, Sesamum indicum, and Foeniculum vulgare.

[0016] Preferably, when the plant is Morus atropurpurea Roxb., the specific application method is: spraying the fermented bacterial liquid of Bacillus velezensis during the flowering period of Morus atropurpurea Roxb. once a week for 3 consecutive times.

[0017] The present invention has at least the following beneficial effects: Using the stem segments of Morus atropurpurea Roxb. as materials, two strains of Bacillus velezensis (JT-3 and JD-3) with obvious inhibitory effects on the pathogen of sclerotinia disease are successfully isolated, and they have broad-spectrum antibacterial properties. Among them, JD-3 has a better effect, providing a new strain resource for the biological control of plant sclerotinia disease and a practical basis for field application. First, JD-3 can significantly inhibit the formation of sclerotia, and the inhibition rate of sclerotia germination in the soil reaches 100%, which provides a new idea for the prevention and control of sclerotinia disease in the future. Second, field control and growth promotion experiments show that the disease infection rate of Morus atropurpurea Roxb. after the action of JD-3 bacterial liquid can be reduced by up to 75.72% at most, and the plant height, stem diameter, number of leaves, leaf area, root length, root weight, and above-ground part weight are all significantly higher than those of the control. Third, the whole genome sequencing of the JD-3 strain is carried out using the ONT sequencing technology. The genome of JD-3 consists of a circular chromosome and a plasmid, and the genome size is 4,088,654 bp, of which the plasmid is 35,700 bp. Comparative genomic analysis shows that the specific gene families of JD-3 are related to functions such as cilium or flagellum-dependent cell motility, defense response, and hydrolase activity. 12 secondary metabolite gene clusters are predicted in JD-3, and plantazolicin is a specific product of JD-3. The present invention provides data support for the research and application of biological bactericides, and at the same time opens up a new research path for the health management of plants such as Morus atropurpurea Roxb.

[0018] Other advantages, objectives, and features of the present invention will be partially reflected by the following description, and partially will be understood by those skilled in the art through the research and practice of the present invention. Brief Description of the Drawings

[0019] Figure 1 For the inhibition of Sclerotinia sclerotiorum by JT-3 and JD-3 and the mycelial morphology of the pathogen in Example 2;

[0020] Figure 2 For the inhibition of sclerotia germination by JT-3 and JD-3 in Example 2;

[0021] Figure 3 For the antibacterial spectra of JT-3 and JD-3 in Example 2, a is the inhibitory effect of JT-3 on other pathogens, and b is the inhibitory effect of JD-3 on other pathogens;

[0022] Figure 4 For the colony morphology of JT-3 and JD-3 in Example 2;

[0023] Figure 5 Functional active substances and biofilm detection results of JT-3 and JD-3 in Example 3;

[0024] Figure 6 Effect of JD-3 on the growth of Morus atropurpurea Roxb. in Example 4; a, Effect of JD-3 on plant height; b, Effect of JD-3 on stem diameter; c, Effect of JD-3 on leaf number; d, Effect of JD-3 on leaf area; e, Effect of JD-3 on root length; f, Effect of JD-3 on root weight; g, Effect of JD-3 on the weight of the upper part;

[0025] Figure 7 Growth conditions of Morus atropurpurea Roxb. under different treatments of JD-3 in Example 4;

[0026] Figure 8 Effect of JD-3 on the incidence of sclerotinia of Morus atropurpurea Roxb. in Example 5;

[0027] Figure 9 Genome circle diagram of JD-3 in Example 6;

[0028] Figure 10 Phylogenetic tree of JD-3 in Example 6;

[0029] Figure 11 Nr function prediction diagram of JD-3 in Example 6;

[0030] Figure 12 Collinearity analysis of JD-3 in Example 6;

[0031] Figure 13 Venn diagram of JD-3 in Example 6;

[0032] Figure 14 GO analysis diagram of JD-3 in Example 6;

[0033] Figure 15 Predicted arrangement of gene clusters of JD-3 in Example 6;

[0034] Figure 16 Genes related to the synthesis of antibacterial active substances of JD-3 in Example 6. Detailed implementation mode

[0035] The present invention will be further described in detail below with reference to the accompanying drawings, so that those skilled in the art can implement it according to the description in the specification.

[0036] It should be understood that the terms such as "having", "comprising" and "including" used herein do not exclude the presence or addition of one or more other elements or their combinations.

[0037] Example 1

[0038] A Bacillus velezensis, classified and named Bacillus velezensis JD-3, was registered and preserved at the China Center for Type Culture Collection on August 12, 2024, with the preservation number CCTCC NO: M 20241775, and its 16S rDNA sequence is shown as SEQ ID NO.1.

[0039] Isolation and purification of Bacillus velezensis:

[0040] Take healthy branches from the fruit mulberry base of the Chongqing Academy of Agricultural Sciences, cut them into appropriate lengths, rinse them under running water for 1 h, transfer them to a laminar flow hood, soak them in 75% ethanol for 2 min, take them out with forceps, burn off the excess ethanol on an alcohol lamp, roll them on a sterile NB medium to detect whether there is contamination on the surface, and then use a sterile knife to layer and dissect the stem segments, place the cut surface down on PDA, WA, Gao's, LB, and NA media respectively, and culture them at 25°C. When colonies grow on the edges, pick different colonies according to colony color, edge shape, smoothness and other characteristics for streak purification culture.

[0041] Screening of Bacillus velezensis:

[0042] Primary screening: Using the pathogen of Sclerotinia sclerotiorum as the test target bacterium for primary screening. Specifically: inoculate a pathogen cake with a diameter of 5 mm in the center of a 90-mm-diameter PDA plate, use a sterilized pipette tip to cross-inoculate 4 different strains 25 mm away from the center of the bacterium cake, place it in an incubator at 25°C for 5 d, and screen for antagonistic bacteria that have obvious inhibitory effects on the pathogen for re-screening;

[0043] Secondary screening: Use the plate confrontation method to re-screen the strains with antagonistic effects in the primary screening. Specifically: inoculate a pathogen cake with a diameter of 5 mm in the center of a 90-mm-diameter PDA plate, and at the same time streak and inoculate the antagonistic bacteria symmetrically 25 mm away from the center. Use a plate without inoculating bacteria as a control, set 3 replicates, measure the colony diameter of the pathogen after culturing at 25°C for 5 d, calculate the inhibition rate, and finally screen out 2 strains with significant and stable antibacterial effects, numbered JT-3 and JD-3 respectively.

[0044] Example 2

[0045] Resistance evaluation of JT-3 and JD-3:

[0046] 1. Antibacterial test of JT-3 and JD-3 fermentation broth

[0047] First, inoculate the strain on NA medium and activate it at 33°C for 21 h. Then, inoculate a single colony into a triangular flask containing 20 mL of NB liquid medium and culture it at 180 r / min and 33°C for 21 h to obtain a seed solution. Pipette 1 mL of the seed solution into a triangular flask containing 50 mL of NB liquid medium and culture it at 180 r / min and 33°C for 21 h to obtain a fermentation broth with an OD 600 value of 1.7. Detect its antibacterial activity against pathogenic bacteria by the inhibition zone method. The specific method is as follows: Inoculate a pathogenic bacteria cake (Sclerotinia sclerotiorum) with a diameter of 5 mm in the center of a fresh PDA plate, and punch a hole with a 5 mm diameter puncher at a distance of 25 mm from the center of the bacteria cake. Inject 20 μL of the fermentation broth into the hole. The control is a blank plate without the fermentation broth. Set 3 replicates for each group and place them in an incubator at 25°C for 5 d, and then calculate the inhibition rate. The inhibition rate (%) = (control colony area - treated colony area) / control colony area × 100%.

[0048] 2. Effect on hyphal morphology

[0049] Pick the hyphae of pathogenic bacteria at the edge of the inhibition zone and the normally growing hyphae of the control group in the antibacterial test of the aforementioned fermentation broth. After treatment with 0.4% trypan blue staining solution, observe the effect of the strain on the hyphal morphology of pathogenic bacteria under an optical microscope.

[0050] 3. Effect on sclerotium germination

[0051] Collect the sclerotia of pathogenic bacteria that have grown on PDA medium for about 14 d, disinfect them with 75% ethanol for 2 min, and finally wash them with sterile water 3 - 5 times. Soak them in sterile water for 4 - 6 h, then use a dry filter paper to absorb the excess water, and embed them in a petri dish filled with sterile sand, 10 per dish. Pipette 200 μL of the fermentation broth into the position where the sclerotia are located, and add 200 μL of NB medium as the control. Set 3 treatments for the experiment, place the petri dishes in an incubator at 25°C for 1 week, and observe the sclerotium germination situation every day.

[0052] 4. Antibacterial spectrum test

[0053] The antibacterial zone method was used for broad-spectrum antibacterial determination. The fungi used were isolated from diseased mulberries, namely Periconia pseudobyssoides, Epicoccum nigrum, Fusarium acuminatum, Didymella segeticola, Alternaria alternata, and Diaporthe vaccinii. Specifically, the strain was first inoculated in NA medium at 33°C for activation for 21 hours, and then a single colony was inoculated into a triangular flask containing 20mL NB liquid medium, and cultured at 180r / min and 33°C for 21 hours to obtain seed liquid. 1mL of seed liquid was aspirated and inoculated into a triangular flask containing 50mL NB liquid medium, and cultured at 180r / min and 33°C for 21 hours to obtain OD 600 The value of the fermentation liquid was 1.7; the antibacterial activity against pathogens was detected by the inhibition zone method. The specific method was as follows: a pathogen cake with a diameter of 5 mm was inoculated in the center of a fresh PDA plate, and a hole was punched with a 5 mm diameter puncher 25 mm away from the center of the cake, and 20 μL of the fermentation liquid was injected into the hole. The control was a blank plate without fermentation liquid. Each group was set up with 3 replicates, and the inhibition rate was calculated after culturing in a 25°C incubator for 5 days.

[0054] 5. Morphological observation and physiological and biochemical determination

[0055] The strain was inoculated on NA medium and cultured at 33°C for 1 day before observing the morphological characteristics of the colonies. API 50CH was used for physiological and biochemical determination to understand the utilization of various carbon sources by the bacteria.

[0056] Figure 1 The antibacterial test and hyphae morphology of the control group, JT-3 group and JD-3 group, among which a and d are the control group, b and e are the JT-3 group, and c and f are the JD-3 group. The inhibition rates of JT-3 and JD-3 on Sclerotinia sclerotiorum were 56.06% and 75.42% respectively. It can be clearly seen that the edges of fungi and bacteria are light yellow, indicating that JT-3 and JD-3 biocontrol bacteria can have a significant destructive effect on the growth of pathogenic hyphae. In order to further confirm the effect, the edge hyphae were picked from the experimental plate and the hyphae morphology was observed under an optical microscope. The microscopic examination results showed that the cell wall and cell membrane of the pathogens acted on by the biocontrol bacteria had been destroyed, and had been invaded and stained by the dye. The hyphae showed morphological distortions such as enlargement, twisting, shrinkage, and breakage, while the control hyphae grew luxuriantly, smoothly and slenderly, and smooth and uniformly.

[0057] like Figure 2As shown, compared with the control group (a), JT-3 (b) and JD-3 (c) have a significant inhibitory effect on sclerotium formation, with the number of sclerotia produced being 0. The sclerotium soil germination experiment shows that the inhibitory rate of the fermented bacterial liquid on sclerotium germination is 100%.

[0058] As Figure 3 shown, JT-3 (a) and JD-3 (b) also have good inhibitory effects on other fungal pathogens (Periconia pseudobyssoides, Epicoccum nigrum, Fusarium acuminatum, Didymella segeticola, Alternaria alternata, Diaporthe vaccinii), showing broad-spectrum antibacterial properties, and can both be used to prepare products for inhibiting these fungal pathogens.

[0059] As Figure 4 shown, the colonies of JT-3 (a) and JD-3 (b) are both milky white, with irregular edges, no viscosity, opaque, and the surface of JT-3 is slightly wrinkled.

[0060] As shown in Table 1, there are no obvious differences in the physiological and biochemical characteristics of the two strains of bacteria. Both can utilize glycerol, D-ribose, inositol, a-methyl-D-glucoside, aesculin, D-lactose, D-xylose, mannitol, salicin, D-glucose, sorbitol, amygdalin, D-cellobiose, D-sucrose, D-raffinose, L-arabinose, D-fructose, arbutin, D-maltose, D-trehalose, but cannot utilize β-methyl-D-xyloside, inulin, D-lyxose, D-arabitol, 5-keto-gluconate, erythritol, D-galactose, L-sorbose, D-melezitose, xylitol, D-tagatose, L-arabitol, D-arabinose, L-xylose, L-rhamnose, D-gentiobiose, D-fucose, potassium gluconate, adonitol, dulcitol, a-methyl-D-mannoside, D-turanose, L-fucose, 2-keto-gluconate; there are only differences in the utilization of glycogen, N-acetylglucosamine, D-melibiose, and starch.

[0061] Table 1

[0062]

[0063] Example 3

[0064] Functional tests related to JT-3 and JD-3

[0065] The skim milk agar medium, starch medium, cellulose Congo red medium, and CAS medium were used to test the protease hydrolysis activity, amylase hydrolysis activity, cellulose decomposition ability, and siderophore secretion ability of the strains, respectively. The production of a transparent zone around the colony demonstrated that the strain had the corresponding biological activity. The nitrogen-free medium was used to test the nitrogen fixation ability. For the biofilm formation assay, a 48-well cell culture plate was used. The strain activated overnight was cultured with shaking in TSB medium. The bacterial solution in the logarithmic growth phase was diluted and used as the inoculum (OD 600 = 1.7). On this basis, 10-fold (EM10), 100-fold (EM100), and 500-fold (EM500) dilutions were performed; 1.5 mL of MSgg medium + 10 μL of the inoculum were added to the wells. Five replicates were set for each concentration, and sterile water was used as the control. The cultures were incubated statically at 30 °C for 72 h. The culture solution under the biofilm was slowly aspirated, and the biofilm was rinsed 3-4 times with PBS buffer. After standing at room temperature and air-drying naturally, the biofilm was stained with 1% crystal violet at 30 °C for 15 min, then rinsed repeatedly with PBS buffer until colorless. 2 mL of 33% glacial acetic acid was added and allowed to stand for 30 min. The biofilm amount was measured using a spectrophotometer at a wavelength of 570 nm.

[0066] As Figure 5 shown, both JT-3 and JD-3 had the ability to produce protease, amylase, siderophore, and IAA. The hydrolysis zone was not obvious on the cellulose Congo red medium, and they could grow well on the nitrogen-free medium. In terms of biofilm formation, the absorbance values at 570 nm of the biofilms produced by the 100-fold diluted bacterial solutions (EM100) of JT-3 and JD-3 were 1.32 ± 0.28 and 3.76 ± 0.05, respectively, indicating their strong biofilm-forming ability, and JD-3 showed a greater advantage, which was beneficial for the strain to adapt to the environment and improve its colonization and antibacterial activities in the host.

[0067] Example 4

[0068] Application of JD-3 in promoting the growth of Morus atropurpurea Roxb.:

[0069] Growth promotion test: Healthy, plump, and uniformly sized Morus atropurpurea Roxb. seeds were selected and sown in a seedling tray filled with sterile substrate. Morus atropurpurea Roxb. seedlings with consistent growth were selected and transplanted into pots with a diameter of 15 cm and a height of 25 cm for cultivation (peat soil: red soil = 1:1). All pots were placed in a greenhouse. After the seedlings grew 3-5 true leaves and had consistent growth, a root irrigation test was carried out. Specifically: the OD 600The fermented bacterial liquid with an OD of 1.7 was centrifuged (6000 rpm, 8 minutes). After discarding the supernatant, an equal volume of sterile water was added for resuspension, denoted as BM1. On this basis, 10-fold (BM10), 100-fold (BM100), and 500-fold (BM500) dilution treatments were carried out; 20 mL of BM1, BM10, BM100, and BM500 were respectively measured and poured on the roots of the seedlings. Using water as the control group, it was watered once every 2 days for 3 consecutive times. Each concentration was repeated for 10 plants. After 1 week, the number of leaves, plant height, stem diameter, and leaf area were counted, and after 1 month, the root weight, root length, and above-ground part weight were counted.

[0070] As Figure 6-7 shown, as time increased, obvious differences gradually emerged between the treatment group and the control group. Among them, in the BM10 and BM500 treatment groups, the growth advantage of the seedlings was particularly prominent, with significant increases in plant height, stem diameter, and the number of leaves. There were also obvious differences in leaf area, above-ground fresh weight, and root weight compared with the control. Based on the data analysis in the fifth week, compared with the control group, the plant height Figure 6 (a), stem diameter Figure 6 (b), number of leaves Figure 6 (c), root length Figure 6 (e), root weight Figure 6 (f), and above-ground part weight Figure 6 (g) increased by 75.38%, 42.91%, 32.35%, 140.84%, 204.09%, and 327.53% respectively after treatment with JD-3 (BM10); after treatment with JD-3 (BM500), the plant height, stem diameter, root length, root weight, and above-ground part weight increased by 91.87%, 35.76%, 42.65%, 155.09, 163.98%, and 275.40% respectively. The leaf area of the seedlings in the second week was randomly measured, and it was found that the BM10 and BM500 treatment groups still performed prominently. Among them, the leaf area of the JD-3 (BM10) treatment group increased by 89.62% Figure 6 (d) compared with the control.

[0071] Example 5

[0072] Application of JD-3 in controlling sclerotinia disease of fruit mulberry:

[0073] Field experiment: Two-year-old potted fruit mulberry was placed in a diseased fruit mulberry orchard. Referring to Example 2, the fermented bacterial liquid (OD 600 =1.7) was prepared, and 10-fold, 100-fold, and 500-fold dilution treatments were carried out on this basis; the bacterial liquid was sprayed during the flowering period of the fruit mulberry once a week for 3 consecutive times. After the fruits were set, bagging treatment was carried out, and the disease infection rate was counted after the fruits matured.

[0074] As Figure 8 shown, the incidence rate of sclerotinia disease of JD-3 was 2.44 - 8.17%, fromFigure 8 It can be clearly seen that the control effect of the 100-fold diluted bacterial solution is better than that of the undiluted solution, 10-fold dilution, and 500-fold dilution. The inhibitory effect of the 500-fold dilution is relatively poor. Compared with the control, the incidence rate of the 100-fold bacterial solution of JD-3 decreased by 75.72%, fully indicating that the field prevention and control result of JD-3 is relatively good, and the best spraying concentration is the 100-fold diluted bacterial solution.

[0075] Example 6

[0076] Whole-genome sequencing, assembly, prediction, and annotation:

[0077] Use the HiPure Bacterial DNA kit (Magen, Guangzhou, China) to extract the genomic DNA of the biocontrol bacterium according to the manufacturer's instructions. Execute according to the standard protocol provided by Oxford Nanopore Technologies (ONT), including sample quality detection, library construction, library quality detection, and library sequencing. Use the Canu v1.5 software to assemble the filtered reads, use the Racon v3.4.3 software to correct the assembly results using the third-generation reads, and use the Circlator v1.5.5 software for circularization and adjustment of the starting site. Use Infernal v1.1.3 to predict ribosomal RNA (rRNA) genes, use tRNAscan-SE v2.0 to predict transfer RNA (tRNA) genes, use the CRT v1.2 software to predict CRISPR in the genome, use IslandPath-DIMOB v0.2 to predict gene islands in the bacterial genome, use PhiSpy v2.3 to predict prophages in the bacterial genome, and perform gene prediction through the Prodigal v2.6.3 software, and compare and annotate with the Nr, Uniprot, COG, and KEGG databases.

[0078] Comparative genomic analysis:

[0079] To determine the taxonomic position of the biocontrol bacterium, 22 Bacillus strains were selected for genomic analysis with JD-3. Based on the protein sequences of the target strains, alignment was performed through the orthofinder (https: / / github.com / davidemms / OrthoFinder) software. The obtained alignment results were trimmed and aligned (mafft) (https: / / mafft.cbrc.jp / alignment / software), and a phylogenetic tree was constructed. Calculate the average nucleotide identity (ANIs) and in silico DNA-DNA hybridization (DDH), and then use the MAUVE comparison software for comparative genomic analysis of the selected related strains.

[0080] Analysis and verification of secondary metabolic gene clusters:

[0081] The software antiSMASH v5.0.0 was used to identify and analyze biosynthetic gene clusters (BGCs) in bacterial genomic sequences. Six functional genes related to the synthesis of secondary metabolites (PKSI, NRPS, Sfp, srfC, ItuD, FenD) were randomly selected for PCR amplification to verify the genes related to metabolic gene clusters. The PCR system (50 μL): 2×Taq MasterMix: 25 μL, Primer_F+R (each 10 μM): each 1 μL, gDNA: 1 μL, ddH 2 O: up to 50 μL; The PCR reaction program is shown in Table 2; The primers are shown in Table 3.

[0082] Table 2

[0083]

[0084] Table 3

[0085]

[0086] In this example, the ONT sequencing technology was used to efficiently and accurately complete the whole-genome sequencing of the target strain. Figure 9 This is the genomic circular map of JD-3; From the outer circle to the inner circle, it represents in turn: The outermost circle is the indication of the genome size, and each scale is 5 kb; The second and third circles are genes on the positive and negative strands of the genome respectively, and different colors represent different COG functional classifications; The fourth circle is repetitive sequences; The fifth circle is tRNA and rRNA, blue is tRNA, and purple is rRNA; The sixth circle is the GC content. The light yellow part indicates that the GC content in this region is higher than the average GC content of the genome, and the higher the peak, the greater the difference from the average GC content. The blue part indicates that the GC content in this region is lower than the average GC content of the genome; The innermost circle is GC-skew, dark gray represents the region where the G content is greater than C, and red represents the region where the C content is greater than G. The JD-3 genome consists of a circular chromosome and a plasmid. The chromosome length is 4,088,654 bp, the plasmid length is 35,700, the GC content is 46.48%, a total of 4018 protein-coding genes, 27 rRNAs, 87 tRNAs, 36 other non-coding RNA families, 4 CRISPR structures, 4 gene islands, 3 prophages, and 12 genomic clusters are predicted. JD-3 has 4011 annotated proteins, and the detailed information is shown in Table 3.

[0087] Table 3

[0088]

[0089] Phylogenetic tree ( Figure 10 ) showed that JD-3 clustered together with B. amyloliquefaciens-ZF57. To further confirm their phylogenetic relationship, the average nucleotide identity (ANI) value and in silico DNA-DNA hybridization (DDH) value were calculated. Generally, comparative strains with ANI value > 96% and DDH value ≥ 70% will be considered the same species. The ANI and DDH of JD-3 and B. amyloliquefaciens-ZF57 were 97.7% and 94.5% respectively; it is worth noting that the ANI and DDH indices of B. velezensis-PRO96 and B. amyloliquefaciens-ZF57 were both higher than 96%, further indicating the close phylogenetic relationship between the selected strains. Combining with the Nr functional prediction ( Figure 11 ), JD-3 was identified as B. velezensis, and its 16S rDNA sequence obtained by sequencing was shown as SEQ ID NO.1.

[0090] To clarify the functional characteristics of the target strain, based on the phylogenetic tree, we used Mauve to align the whole genome sequence of JD-3 with the closest relative strain B. amyloliquefaciens-ZF57 strain ( Figure 12 ). The collinearity results showed that the two groups of genomes were highly similar, with obvious collinearity, only gene rearrangements occurred, and no obvious insertions, deletions, or inversions were found. The Venn diagram ( Figure 13 ) results showed that there were 2035 gene families shared by JD-3 and B. amyloliquefaciens-ZF57, and the specific gene families were 1427 and 72 respectively. To further understand the functions of the specific genes of the target strain, GO functional analysis was performed on them. It is worth noting that the specific genes of JD-3 were annotated to functions such as cilium or flagellum-dependent cell motility, defense response, hydrolase activity, etc. ( Figure 14 ).

[0091] Secondary metabolites produced by bacteria are an important source of antibacterial agents and other bioactive compounds. The software antiSMASH v5.0.0 was used to accurately identify gene clusters in the strain that can encode secondary metabolites of known categories. Twelve gene clusters were retrieved in JD-3, including 4 encoding NRPS, 4 encoding transAT-PKS, 2 encoding T3PKS, 2 encoding terpene (no protein alignment), 1 encoding betalactone, 1 encoding NRP-metallophore, 1 encoding other, 1 encoding RRE-containing, LAP, 1 encoding PKS-like (with 7% similarity to predicted butirosin A / butirosin B), 1 encoding NRP-metallophore, and 1 encoding RiPP-like. Among them, the predicted products of 6 clusters were predicted in all strains, which are related to the formation of bacillaene, fengycin, difficidin, bacilysin, surfactin, and macrolacin H. Plantazolicin is specifically present in JD-3 (Table 5, Figure 15 ).

[0092] Table 5

[0093]

[0094]

[0095]

[0096] The amplification results of 6 genes related to the synthesis of antibacterial secondary metabolites showed that 5 genes related to the synthesis of antibacterial active substances, namely PKSI, NRPS, Sfp, ItuD, and Srfc, could be detected in the genome of JD-3 strain ( Figure 15 ), further verifying the existence of antibacterial bioactive substances in JD-3 strain.

[0097] Although the embodiments of the present invention have been disclosed as above, it is not limited to the applications listed in the specification and embodiments. It can be fully applied to various fields suitable for the present invention. For those familiar with the field, additional modifications can be easily made. Therefore, without departing from the general concept defined by the claims and the equivalent scope, the present invention is not limited to specific details and the examples shown and described herein.

Claims

1. A Bacillus Velezii ( Bacillus velezensis ) strain JD-3, characterized in that The Bacillus Velez strain JD-3 was registered and deposited in the China Center for Type Culture Collection on August 12, 2024, with the deposit number being CCTCC NO: M 20241775.

2. A use of the Bacillus Velez strain JD-3 according to claim 1 in inhibiting fungal pathogens, characterized in that: The fungal pathogen is selected from: Sclerotinia sclerotiorum pathogen Sclerotinia sclerotiorum Ephemerococcus nigromaculata Epicoccum nigrum Fusarium acuminate Fusarium acuminatum , Endophytic fungi of the genus Subsporus Didymella segeticola Alternaria Alternaria alternata and cyanobacterium rot Diaporthe vaccinii .

3. Use of the Bacillus Velez strain JD-3 as claimed in claim 1 in promoting the growth of mulberry.

4. Use of the Bacillus Velezii strain JD-3 as claimed in claim 1 in preventing and controlling plant sclerotinia disease.

5. The use of the Bacillus Velez strain JD-3 in preventing and treating plant sclerotinia disease as claimed in claim 4, characterized in that: The plant comprises any one of mulberry, kidney bean, celery, lettuce, tomato, pepper, cucumber, sesame and fennel.

6. Use of the Bacillus Velez strain JD-3 in preventing and treating plant sclerotinia disease as claimed in claim 5, characterized in that: When the plant is a mulberry, the specific application method is: using the fermented bacterial liquid of Bacillus Velez subtilis to spray during the flowering period of the mulberry, once a week, and spraying continuously for 3 times.

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