Bacillus velezensis and application thereof in prevention and treatment of sclerotinia rot of plants

Abstract

The invention discloses bacillus velezensis and application thereof in prevention and treatment of plant sclerotiniose, the classification name of the bacillus velezensis is bacillus velezensis JD-3, the bacillus velezensis is registered and preserved in China Center for Type Culture Collection on August 12, 2024, and the preservation number is CCTCC M 20241775; the bacillus velezensis JD-3 is applied to prevention and treatment of plant sclerotiniose. The bacillus velezensis JD-3 is successfully separated by taking fruit mulberry stems as materials, has an obvious inhibition effect on sclerotiniose pathogenic bacteria, has broad-spectrum antibacterial activity, provides a new strain resource for biological control of plant sclerotiniose, and provides a practical basis for field application; data support is provided for research, development and application of biological agents, and meanwhile, a new research approach is opened up for health management of plants such as fruit mulberries and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and more specifically, relates to Bacillus Velezii and application thereof in preventing and controlling plant sclerotinia disease. Background Art

[0002] Sclerotinia is the most serious disease in the development of the fruit mulberry industry. It mainly harms the fruit of the fruit mulberry, has the characteristics of rapid onset and wide range, can infect mulberry flowers, and cause the occurrence of fruit mulberry sclerotinia. There are generally three symptoms, namely, hypertrophic sclerotinia, small-grain sclerotinia and reduced sclerotinia. At present, chemical agents are a common method to solve the problem of fruit mulberry sclerotinia. The control effect of alternate spraying of methyl thiophanate and procymidone is as high as more than 90%, but HPLC and GC analysis show that the pesticide residue in the fruit exceeds the international standard limit. The irreversible decline in soil quality and serious health and environmental problems caused by this cannot be ignored. Excessive use may also cause pathogens to develop resistance, but other methods, such as resistance breeding or traditional cultivation measures (crop rotation, soil management) and other means have no significant effect on the occurrence of sclerotinia.

[0003] In recent years, biological control has attracted much attention in the prevention and control of mulberry sclerotinia due to its advantages of being green, safe, efficient and durable. The most common biocontrol bacteria are mostly Bacillus, which is a type of aerobic or facultative anaerobic Gram-positive, chemoheterotrophic bacteria with 266 named species. It is widely present in soil, water and air, and the spores it produces are highly resistant to high temperature, ultraviolet rays and ionizing radiation. Bacillus is a producer of bioactive metabolites, and the prediction of biosynthetic gene clusters (BGCs) provides the possibility for the discovery and identification of the diversity of secondary metabolites. However, there are few reports on the inhibitory effect of Bacillus Velez on mulberry sclerotinia. Summary of the invention

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

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

[0006] Preferably, the Bacillus Velezii is derived from mulberry stem segments.

[0007] Preferably, the biological characteristics of the Bacillus Velezii include: the colonies are milky white, have irregular edges, are non-sticky, and are opaque.

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

[0009] A use of the Bacillus Velezii as described above in inhibiting fungal pathogens.

[0010] Preferably, the fungal pathogens include: Sclerotinia sclerotiorum pathogen, Epenthes nigrosinus, Fusarium acuminate, endophytic fungi of the genus Subsporus, Alternaria alternata and blueberry fruit rot pathogen.

[0011] A use of the Bacillus Velezii as described above in inhibiting sclerotium germination.

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

[0013] A use of the Bacillus Velez subtilis as described above in promoting the growth of mulberry trees.

[0014] A use of the Bacillus Velezii as described above in preventing and controlling plant sclerotinia disease.

[0015] Preferably, the plant comprises any one of mulberry, kidney bean, sunflower, celery, lettuce, flower, tomato, pepper, cucumber, sesame and fennel.

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

[0017] The present invention includes at least the following beneficial effects: the present invention uses mulberry stem segments as materials, and successfully isolates two strains (JT-3, JD-3) of Bacillus Velezii that have obvious inhibitory effects on sclerotinia pathogens, and have broad-spectrum antibacterial properties, of which JD-3 has a better effect, providing a new bacterial resource for the biological control of plant sclerotinia, and providing a practical basis for field application. First, JD-3 can significantly inhibit the formation of sclerotia, and the germination inhibition rate of sclerotia in the soil is 100%, which provides a new idea for the prevention and control of sclerotinia in the future. Secondly, field control and growth promotion experiments show that the disease rate of mulberry after the action of JD-3 bacterial solution can be reduced by up to 75.72%, and the plant height, stem thickness, number of leaves, leaf area, root length, root weight, and aboveground weight are significantly higher than the control. Third, the JD-3 strain was sequenced using ONT sequencing technology. The JD-3 genome consists of a circular chromosome and a plasmid, with a genome size of 4,088,654bp, of which the plasmid is 35,700bp. Comparative genome analysis found that the JD-3-specific gene family is related to cilium or flagellum-dependent cell motility, defense response, hydrolase activity and other functions. JD-3 predicted 12 secondary metabolite gene clusters, of which plantazolicin is a unique product of JD-3. The present invention provides data support for the development and application of biological microbial agents, and opens up new research avenues for the health management of fruit mulberry and other plants.

[0018] Other advantages, objectives and features of the present invention will be embodied in part through the following description, and in part will be understood by those skilled in the art through study and practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The inhibition of JT-3 and JD-3 on Sclerotinia sclerotiorum and the hyphae morphology of the pathogen in Example 2;

[0020] Figure 2 The inhibition of sclerotium germination by JT-3 and JD-3 in Example 2;

[0021] Figure 3 is the antibacterial spectrum 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 The colony morphology of JT-3 and JD-3 in Example 2;

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

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

[0025] Figure 7 The growth conditions of mulberry trees under various treatments of JD-3 in Example 4;

[0026] Figure 8 The effect of JD-3 on the incidence of sclerotinia sclerotiorum in Example 5;

[0027] Fig. 9 is the genome circle map of JD-3 in Example 6;

[0028] Fig.10 is the phylogenetic tree of JD-3 in Example 6;

[0029] Fig.11 This is the Nr function prediction diagram of JD-3 in Example 6;

[0030] Fig.12 is the collinearity analysis of JD-3 in Example 6;

[0031] Fig.13 is the Venn diagram of JD-3 in Example 6;

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

[0033] Fig.15 The predicted arrangement of the gene cluster of JD-3 in Example 6;

[0034] Fig.16 It is a gene related to the synthesis of antibacterial active substances of JD-3 in Example 6. DETAILED DESCRIPTION

[0035] The present invention is further described in detail below in conjunction with the accompanying drawings so that those skilled in the art can implement the invention with reference to the description.

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

[0037] Example 1

[0038] A Bacillus velezensis, whose classification name is Bacillus velezensis JD-3, was registered and deposited in the China Center for Type Culture Collection on August 12, 2024, with a deposit number of CCTCC NO: M 20241775, and its 16SrDNA sequence is shown in SEQ ID NO.1.

[0039] Isolation and purification of Bacillus Velez:

[0040] Healthy branches were obtained from the fruit mulberry base of Chongqing Academy of Agricultural Sciences, cut into appropriate lengths, rinsed under running water for 1 h, transferred to a clean bench, immersed in 75% ethanol for 2 min, taken out with tweezers, and the excess ethanol was burned on an alcohol lamp. The branches were rolled on a sterile NB medium to detect whether the surface was contaminated. The stem segments were then cut into layers with a sterile knife and placed with the cut surface facing down on PDA, WA, Gao's, LB, and NA medium, respectively, and cultured at 25°C. When colonies grew on the edges, different colonies were selected for streak purification culture according to their color, edge shape, smoothness and other characteristics.

[0041] Screening of Bacillus Velezii:

[0042] Initial screening: The pathogen of Sclerotinia sclerotiorum was used as the target bacteria for preliminary screening. Specifically, a 5 mm diameter pathogen cake was inoculated in the center of a 90 mm diameter PDA plate. Four different strains were cross-inoculated at a distance of 25 mm from the center of the cake using a sterilized pipette tip. After culturing in a 25°C incubator for 5 days, antagonistic bacteria with a significant inhibitory effect on the pathogen were screened for rescreening.

[0043] Secondary screening: The plate confrontation method was used to rescreen the strains with antagonistic effects in the initial screening. Specifically, a pathogen cake with a diameter of 5 mm was inoculated in the center of a PDA plate with a diameter of 90 mm, and antagonistic bacteria were inoculated symmetrically 25 mm from the center. The plate without bacteria inoculation was used as the control. Three replicates were set up, and the colony diameters of the pathogens were measured after culturing at 25°C for 5 days. The inhibition rate was calculated, and finally two strains with significant and stable antibacterial effects were screened, numbered JT-3 and JD-3 respectively.

[0044] Example 2

[0045] JT-3 and JD-3 resistance evaluation:

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

[0047] The strain was first inoculated in NA medium at 33°C for activation for 21 h, and then a single colony was inoculated into a flask containing 20 mL of NB liquid medium, and cultured at 180 r / min and 33°C for 21 h to obtain seed solution. 1 mL of seed solution was inoculated into a flask containing 50 mL of NB liquid medium, and cultured at 180 r / min and 33°C for 21 h to obtain OD 600 The value of the fermentation bacteria liquid was 1.7; the antibacterial activity against pathogens was detected by the inhibition zone method, the specific method was: a pathogen cake (Sclerotinia sclerotiorum) 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 at a distance of 25 mm from the center of the bacteria cake, 20 μL of the fermentation bacteria liquid was injected into the hole, and the control was a blank plate without fermentation bacteria 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, and the inhibition rate (%) = (control colony area-treated colony area) / control colony area × 100%.

[0048] 2. Effect on mycelium morphology

[0049] The pathogen hyphae at the edge of the inhibition zone in the aforementioned fermentation broth inhibition test and the normal growth hyphae of the control group were selected, and after being treated with 0.4% trypan blue staining solution, the effect of the strain on the morphology of pathogen hyphae was observed under an optical microscope.

[0050] 3. Effect on sclerotium germination

[0051] The sclerotia of pathogens grown on PDA medium for about 14 days were collected, disinfected with 75% ethanol for 2 minutes, and finally washed with sterile water for 3-5 times. After soaking in sterile water for 4-6 hours, excess water was absorbed with dry filter paper, and embedded in a culture dish filled with sterile sand, 10 in each dish. 200 μL of fermentation solution was drawn and added to the position of the sclerotia. 200 μL of NB culture medium was added to the control. There were 3 treatments in the experiment. The culture dishes were placed in a 25°C incubator for 1 week, and the germination of sclerotia was observed 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 bacteria 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 bacterial cake, and 20 μL of the fermentation bacteria liquid was injected into the hole. The control was a blank plate without fermentation bacteria 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) had a significant inhibitory effect on sclerotium formation, and the number of sclerotia produced was 0. The sclerotium soil germination experiment showed that the inhibition rate of fermented bacterial liquid on sclerotium germination was 100%.

[0058] like Figure 3 As shown in the results, JT-3(a) and JD-3(b) also have good inhibitory effects on other fungal pathogens (Periconia pseudobyssoides, Epicoccum nigrum, Fusarium acuminatum, Didymella segeticol a, Alternaria alternata, Diaporthe vaccinii), have a broad-spectrum antibacterial activity, and can be used to prepare products that inhibit these fungal pathogens.

[0059] like Figure 4 As shown, the colonies of JT-3 (a) and JD-3 (b) were milky white with irregular edges, no stickiness, and opaque, and the surface of JT-3 was slightly wrinkled.

[0060] As shown in Table 1, there were no significant differences in the physiological and biochemical characteristics of the two strains. Both strains could utilize glycerol, D-ribose, inositol, α-methyl-D-glucoside, esculin, D-lactose, D-xylose, mannitol, salicin, D-glucose, sorbitol, amygdalin, D-cellobiose, D-sucrose, D-raffinose, L-arabinose, D-fructose, arbutin, D-maltose, and D-trehalose, but could not utilize β-methyl-D-xyloside, inulin, D-lyxose, and D-arabinose. Primary alcohol, 5-keto-gluconate, erythritol, D-galactose, L-sorbose, D-melezitose, xylitol, D-tagatose, L-arabinol, D-arabinose, L-xylose, L-rhamnose, D-gentiobiose, D-fucose, potassium gluconate, adonol dulcitol, a-methyl-D-mannoside, D-toulonose, L-fucose, 2-keto-gluconate; only differences were found in the utilization of glycogen, N-acetylglucosamine, D-melibiose, and starch.

[0061] Table 1

[0062]

[0063] Example 3

[0064] JT-3 and JD-3 related functional tests:

[0065] 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 iron carrier secretion ability of the strain. The transparent area around the colony proved that the strain had the corresponding biological activity. Nitrogen-free medium was used to test the nitrogen fixation ability. The biofilm formation test used a 48-well cell culture plate, and the strain activated overnight was cultured in TSB medium by shaking. The bacterial solution in the logarithmic growth phase was diluted and used as the bacterial culture solution (OD 600 =1.7), and on this basis, 10-fold (EM10), 100-fold (EM100), and 500-fold (EM500) dilutions were performed; 1.5 mL of MSgg culture medium + 10 μL of bacterial culture solution were added to the wells, 5 replicates were set for each concentration, sterile water was used as the control, and the cells were cultured at 30°C for 72 h, the culture solution under the biofilm was slowly aspirated, and the cells were repeatedly rinsed with PBS buffer for 3-4 times, allowed to stand at room temperature, and naturally air-dried. After staining with 1% crystal violet at 30°C for 15 min, the cells were repeatedly rinsed with PBS buffer until colorless, 2 mL of 33% glacial acetic acid was added, and the cells were allowed to stand for 30 min. The amount of biofilm was determined using a spectrophotometer at a wavelength of 570 nm.

[0066] like Figure 5 As shown, both JT-3 and JD-3 have the ability to produce proteases, amylases, siderophores, and IAA, and the hydrolysis zone is not obvious on the cellulose Congo red medium, and they can grow well on the nitrogen-free medium. In terms of biofilm formation, the biofilms produced by the 100-fold diluted bacterial solution (EM100) of JT-3 and JD-3 have absorbance values ​​of 1.32±0.28 and 3.76±0.05 at 570nm, respectively, indicating that they have strong film-forming ability, and JD-3 shows a greater advantage, which is beneficial for the strain to adapt to the environment and improve the colonization and antibacterial activity in the host.

[0067] Example 4

[0068] Application of JD-3 in promoting the growth of fruit mulberry:

[0069] Growth promotion test: Select healthy, plump, uniform-sized four-season mulberry seeds, sow them in a seedling tray filled with sterile substrate, select mulberry seedlings with uniform growth and transplant them into pots with a diameter of 15 cm and a height of 25 cm for cultivation (peat soil: red soil = 1:1), place all pots in a greenhouse, and conduct root irrigation test after the seedlings grow 3-5 true leaves and grow uniformly. Specifically, adjust the OD 600=1.7 was centrifuged (6000rpm, 8 minutes), the supernatant was discarded and an equal amount of sterile water was added to resuspend, recorded as BM1, and on this basis, 10 times (BM10), 100 times (BM100), and 500 times (BM500) were diluted; 20mL BM1, BM10, BM100, and BM500 were measured and irrigated on the roots of the seedlings respectively, with water as the control group, irrigated once every 2 days for 3 times in a row, and 10 plants were treated with each concentration repeatedly. The number of leaves, plant height, stem diameter, and leaf area were counted after 1 week, and the root weight, root length, and aboveground weight were counted after 1 month.

[0070] like Figure 6-7 As shown in the figure, with the increase of time, the treatment groups and the control group gradually showed obvious differences. Among them, the BM10 and BM500 treatment groups had particularly prominent advantages in seedling growth, with significant increases in plant height, stem thickness, and number of leaves. The leaf area, aboveground fresh weight, and root weight also showed significant differences from the control group. Taking the fifth week data as the benchmark for analysis, compared with the control group, the plant height ( Figure 6 a) Stem thickness ( Figure 6 b) Number of blades ( Figure 6 c) Root length ( Figure 6 e), root weight ( Figure 6 f) Weight of above-ground part ( Figure 6 g) increased by 75.38%, 42.91%, 32.35%, 140.84%, 204.09% and 327.53% respectively; plant height, stem diameter, root length, root weight and aboveground weight after JD-3 (BM500) treatment increased by 91.87%, 35.76%, 42.65%, 155.09%, 163.98% and 275.40% respectively. The leaf area of ​​seedlings in the second week was randomly measured, and it was found that BM10 and BM500 treatment groups still performed outstandingly, among which the leaf area of ​​JD-3 (BM10) treatment group increased by 89.62% ( Figure 6 d).

[0071] Example 5

[0072] Application of JD-3 in the prevention and treatment of sclerotinia rot in fruit mulberry:

[0073] Field test: Two-year-old potted mulberry trees were placed in a diseased mulberry garden, and fermentation liquid (OD 600 =1.7), and on this basis, 10-fold, 100-fold, and 500-fold dilutions were performed; the bacterial solution was sprayed during the flowering period of mulberry, once a week, for 3 consecutive times, and bagging was performed after the fruits were formed. The susceptibility rate was calculated after the fruits were ripe.

[0074] like Figure 8 As shown in Figure 2, the incidence of sclerotinia in JD-3 was 2.44-8.17%. Figure 8 It is obvious that the control effect of 100 times diluted bacterial solution is better than that of original solution, 10 times and 500 times, and the inhibition effect of 500 times is relatively poor. Compared with the control, the incidence rate of JD-3 100 times bacterial solution decreased by 75.72%, which fully shows that the field control result of JD-3 is relatively good, and the best spraying concentration is 100 times diluted bacterial solution.

[0075] Example 6

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

[0077] The HiPure Bacterial DNA Kit (Magen, Guangzhou, China) was used to extract the genomic DNA of biocontrol bacteria according to the manufacturer's instructions. The standard protocol provided by Oxford Nanopore Technologies (ONT) was followed, including sample quality detection, library construction, library quality detection, and library sequencing. The filtered reads were assembled using Canu v1.5 software, and the assembly results were corrected using the third-generation reads using Racon v3.4.3 software. Circularization and adjustment of the start site were performed using Circlator v1.5.5 software. Infernal v1.1.3 was used to predict ribosomal RNA (rRNA) genes, tRNAscan-SE v2.0 was used to predict transfer RNA (tRNA) genes, CRT v1.2 software was used to predict CRISPR in the genome, IslandPath-DIMOB v0.2 was used to predict gene islands in bacterial genomes, PhiSpyv2.3 was used to predict prophages in bacterial genomes, and Prodigal v2.6.3 software was used to predict genes, and compared and annotated with the Nr, Uniprot, COG, and KEGG databases.

[0078] Comparative genomic analysis:

[0079] In order to determine the taxonomic position of the biocontrol bacteria, 22 Bacillus strains were selected for genome analysis with JD-3. Based on the protein sequences of the target strains, the orthofinder (https: / / github.com / da videmms / OrthoFinder) software was used for alignment, and the alignment results were trimmed and aligned (mafft) (https: / / mafft.cbrc.jp / alignment / software), and the evolutionary tree was constructed, and the average nucleotide identifications (ANIs) and in silico DNA-DNA hybridization (DDH) were calculated. Then, the MAUVE comparison software was used to perform comparative genome analysis on the selected closely related strains.

[0080] Secondary metabolism gene cluster analysis and verification:

[0081] The software antiSMASH v5.0.0 was used to identify and analyze the biosynthetic gene clusters (BGCs) in the bacterial genome sequences. Six functional genes related to the synthesis of secondary metabolites (PKSI, NRPS, Sfp, srfC, ItuD, and FenD) were randomly selected for PCR amplification, and the genes related to the metabolic gene clusters were verified. The PCR system (50 μL) was as follows: 2×Taq MasterMix: 25 μL, Primer_F+R (each 10 μM): each 1 μL, gDNA: 1 μL, ddH2O: 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] This example uses ONT sequencing technology to efficiently and accurately complete the whole genome sequencing of the target strain. Fig. 9 The figure shows the circle diagram of JD-3 genome. From the outer circle to the inner circle, they represent: the outermost circle indicates the size of the genome, with each scale being 5kb; 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 GC content, the light yellow part indicates that the GC content of this region is higher than the average GC content of the genome, the higher the peak value, the greater the difference from the average GC content, and the blue part indicates that the GC content of this region is lower than the average GC content of the genome; the innermost circle is GC-skew, dark gray represents regions where the G content is greater than C, and red represents regions 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%, and there are 4018 predicted 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. JD-3 has 4011 annotated proteins, and detailed information is shown in Table 3.

[0087] Table 3

[0088]

[0089] Phylogenetic tree ( Fig.10 ) showed that JD-3 and B.amyloliquefaciens-ZF57 were clustered together. In order to further confirm their developmental relationship, the average nucleotide identity (ANI) value and computer DNA-DNA hybridization (DDH) value were calculated. Generally, the comparison strains with ANI values ​​>96% and DDH values ​​≥70% will be regarded as 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 indexes of B.velezensis-PRO96 and B.amyloliquefaciens-ZF57 were both higher than 96%, further indicating that the selected strains were closely related. Combined with Nr function prediction ( Fig.11 ), JD-3 was determined to be B. velezensis, and its 16S rDNA sequence was obtained by sequencing as shown in SEQ ID NO.1.

[0090] In order to clarify the functional characteristics of the target strain, we used Mauve to align the whole genome sequence of JD-3 with the closest relative strain B. amyloliquefaciens-ZF57 based on the phylogenetic tree ( Fig.12 ), the colinearity results showed that the two groups of genomes were highly similar, with obvious colinearity, only gene rearrangement occurred, and no obvious insertion, deletion, or inversion was found. Venn diagram ( Fig.13 ) The results showed that JD-3 and B. amyloliquefaciens-ZF57 had 2035 common gene families, and 1427 and 72 unique gene families, respectively. In order to further understand the functions of the unique genes of the target strain, GO functional analysis was performed. It is worth noting that the unique genes of JD-3 were annotated with cilium or flagellum-dependent cell motility, defense response, hydrolase activity and other functions ( Fig.14 ).

[0091] Secondary metabolites produced by bacteria are an important source of antimicrobial agents and other bioactive compounds. The software antiSMASH v5.0.0 was used to accurately identify gene clusters in strains that can encode known categories of secondary metabolites. JD-3 retrieved 12 gene clusters, 4 encoding NRPS, 4 encoding transAT-PKS, 2 encoding T3PKS, 2 encoding terpene (no protein was matched), 1 encoding betaact one, 1 encoding NRP-metallophore, 1 encoding other, 1 encoding RRE-containing, LAP, 1 encoding PKS-like (with a similarity of 7% to the predicted butirosin A / butirosin B), 1 encoding NRP-metallophore, and 1 encoding RiPP-like. Among them, 6 clusters of predicted products were predicted by all strains and were related to the formation of bacillaene, fengycin, difficidin, bacilysin, surfactin, and macrolacinH. Plantazolicin was specifically present in JD-3 (Table 5, Fig.15 ).

[0092] Table 5

[0093]

[0094]

[0095]

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

[0097] Although the embodiments of the present invention have been disclosed as above, they are not limited to the applications listed in the specification and the implementation modes, and they can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to the specific details and the illustrations shown and described herein.

Claims

1. A Bacillus Velezii, characterized in that Its classification name is Bacillus velezensis JD-3, and it was registered and preserved in the China Center for Type Culture Collection on August 12, 2024, with the preservation number CCTCC NO: M 20241775.

2. The Bacillus Velezii according to claim 1, characterized in that The 16SrDNA sequence of the Bacillus Velezii is shown as SEQ ID NO.

1.

3. The Bacillus Velezii according to claim 1, characterized in that The Velez Bacillus is derived from mulberry stem segments; the biological characteristics of the Velez Bacillus include: the colony is milky white, has irregular edges, is non-sticky, and is opaque.

4. A use of the Bacillus Velezii as claimed in claim 1 in inhibiting fungal pathogens, characterized in that: The fungal pathogens include: Sclerotinia sclerotiorum pathogen, Black Echinococcus, Fusarium acuminate, endophytic fungi of the genus Subsporus, Alternaria alternata and blueberry fruit rot pathogen.

5. Use of the Bacillus Velezii as claimed in claim 1 in inhibiting sclerotium germination.

6. The use of Bacillus Velezii in inhibiting sclerotium germination as claimed in claim 5, characterized in that: The inhibition rate of the Velez Bacillus on sclerotium germination is 100%.

7. Use of the Bacillus Velez subtilis as claimed in claim 1 in promoting the growth of mulberry.

8. Use of the Bacillus Velezii as claimed in claim 1 in preventing and treating plant sclerotinia disease.

9. The use of Bacillus Velezii in preventing and treating plant sclerotinia disease as claimed in claim 8, characterized in that: The plant comprises any one of mulberry, kidney bean, sunflower, celery, lettuce, flower, tomato, pepper, cucumber, sesame and fennel.

10. The use of Bacillus Velezii in preventing and treating plant sclerotinia disease according to claim 9, 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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