Identification of a bacillus velezensis strain 913 and application thereof

Abstract

The application discloses identification of a bacillus strain and application thereof. The bacillus strain is a bacillus velezensis 913 strain, which has been preserved in the China General Microbiological Culture Collection Center (CGMCC) in July 2024, and the preservation number is CGMCC No. 31170. The strain can inhibit Xanthomonas fragariae, and thus can be used for preventing and treating strawberry bacterial angular spot and "hollow disease".

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and mainly relates to application of Bacillus Velez subtilis in preventing and controlling strawberry diseases. Background Art

[0002] With its unique flavor and high nutritional value, strawberries are recognized by the World Health Organization as one of the best fruits, earning them the nickname "Queen of Berries." Their cultivated area leads the world in small berries. In recent years, my country's strawberry industry has experienced rapid growth, ranking first in both cultivated area and yield, making it the world's largest producer, processor, and exporter of strawberries. Currently, strawberries in my country are primarily cultivated in greenhouses. The high-temperature, high-humidity environment within these facilities, coupled with continuous cropping, has led to the occurrence and prevalence of various diseases, most notably the recently emerging "cavity disease" (also known as hollow heart disease, head decapitation disease, and pith empty disease). This is a new and devastating bacterial disease caused by Xanthomonas fragariae. This pathogen is highly contagious, spreads rapidly, and develops late, typically not until a month after planting in the fall. This prevents timely replanting, severely hindering the sustainable and healthy development of the strawberry industry. As a new disease, strawberry "cavity disease" currently has no effective prevention and control method. Most farmers use chemical pesticides to control the disease, which has little effect and easily causes environmental pollution. Therefore, there is an urgent need to find an efficient and environmentally friendly prevention and control method for strawberry "cavity disease". Summary of the Invention

[0003] The technical problem to be solved by the present invention is to biologically control strawberry "cavity disease" caused by Xanthomonas fragariae.

[0004] The present invention discovered, isolated and purified a Bacillus velezensis strain from the rhizosphere soil of healthy strawberry plants, named Bacillus velezensis strain 913. It was found that this Bacillus velezensis strain can effectively inhibit Xanthomonas fragariae and prevent and treat strawberry "cavitation disease" caused by Xanthomonas fragariae, and its inhibitory effect is significantly better than that of Bacillus RF21 previously discovered by the applicant.

[0005] One aspect of the present invention relates to a strain of Bacillus velezensis, named Bacillus velezensis 913, which has growth characteristics such as Figure 1 As shown: The colonies are milky white, translucent, with smooth, regular edges. Secretions can diffuse in a cloud-like pattern. After 24 hours of incubation, the colonies develop wrinkled protrusions. It has been deposited with the China General Microbiological Culture Collection under the accession number CGMCC No. 31170.

[0006] The present invention also relates to an antibacterial agent, which comprises as an active ingredient the Bacillus Velez subtilis strain 913 or a fermentation filtrate obtained by sterile filtration of a bacterial liquid obtained by fermenting the Bacillus Velez subtilis strain 913.

[0007] The present invention also relates to the use of the Bacillus Velez strain 913 or an antibacterial agent containing the Bacillus strain 913 as an active ingredient in preventing and controlling strawberry bacterial angular spot disease and strawberry "cavity disease" caused by Xanthomonas fragariae.

[0008] The present invention also relates to use of the Bacillus Velez strain 913 or an antibacterial agent containing the Bacillus Velez strain 913 as an active ingredient in preparing a biological preparation for inhibiting Xanthomonas fragariae.

[0009] The antibacterial agent containing the fermentation liquid of the Bacillus Velez strain 913 described in the present application has good stability under different temperatures, light treatments, ultraviolet treatments, and pH values, that is, after treatment under different conditions, it can significantly inhibit the growth of strawberry Xanthomonas on agar plates; in potted plant experiments, spraying the bacterial liquid of strain 913 can effectively inhibit the incidence of strawberry "cavity disease" and reduce the disease index, showing good effects in preventing and treating strawberry "cavity disease".

[0010] Compared with the prior art, the advantages and progress of the present invention are:

[0011] 1. Bacillus Velez 913 and its fermentation liquid have a significant inhibitory effect on Xanthomonas fragariae, providing a biocontrol strain for the prevention and control of strawberry cavitation disease and has good application prospects in strawberry production.

[0012] 2. The antibacterial agent containing the fermentation broth of Bacillus velez 913 not only has an excellent antibacterial effect, but also has good stability, that is, it can exert its antibacterial effect in various temperature, acid, alkali, light and other environments, is resistant to processing, and is suitable for promotion and use.

[0013] 3. Compared with the previously discovered Bacillus Rf21, Bacillus Velez 913 has a significantly better inhibitory effect on strawberry "cavity disease" caused by Xanthomonas fragariae. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 The colony morphology of Bacillus Velez strain 913 grown on LB solid medium for 24 hours.

[0015] Figure 2 BLAST alignment results of the 16s rRNA sequence of Bacillus velez 913.

[0016] Figure 3 Phylogenetic tree of glpF, ilvD, tpiA, purH, rpoD, pycA, ptA gene sequences of Bacillus velezensis 913.

[0017] Figure 4 Growth curve of Bacillus velezensis strain 913 at different temperatures of 16℃, 25℃, 30℃, 37℃ and 40℃.

[0018] Figure 5 Inhibition zone diagram of Bacillus velezensis strain 913 fermentation filtrate on Pseudomonas tolicola. The left is LB control, and the right is the inhibition zone of strain 913.

[0019] Figure 6 Inhibition zone diameter of Bacillus velezensis strain 913 fermentation filtrate on Pseudomonas tolicola.

[0020] Figure 7 Inhibition zone size of Bacillus velezensis strain 913 fermentation broth after different environmental treatment. A is light treatment, B is ultraviolet treatment, C is different temperature treatment, D is different pH value treatment, and E is various metal ion treatment. The control is the fermentation broth without any treatment.

[0021] Figure 8 Preventive effect of Bacillus velezensis 913 on leaf angular spot symptoms of strawberry "hollow disease".

[0022] Figure 9 The incidence of angular spot of strawberry leaves treated with Bacillus velezensis strain 913 for 15 days, in which * represents P<0.05%, and ** represents P<0.01%.

[0023] Figure 10 The disease index of angular spot of strawberry leaves treated with Bacillus velezensis strain 913 for 30 days, in which * represents P<0.05%, and ** represents P<0.01%.

[0024] Figure 11 Strawberry stem hollow treated with Bacillus velezensis strain 913 for 90 days.

[0025] Figure 12 The hollow rate of strawberry stem treated with Bacillus velezensis strain 913 for 90 days, in which ** represents P<0.05%, and **** represents P<0.01%.

[0026] Figure 13 Inhibition zone size of Bacillus velezensis strain 913 and Rf21 fermentation filtrate against Pseudomonas tolicola after 4 days of confrontation culture.

[0027] Figure 14 The hollow rate of strawberry stem after 90 days of treatment with Bacillus velezensis strains 913 and Rf21, respectively, wherein a, b and c represent statistical significance. DETAILED DESCRIPTION

[0028] The following is a specific embodiment of the present application, which describes the technical solutions of the present application in detail. The embodiments given are only to illustrate the present application, and are not intended to limit the scope of the present application. The experimental methods in the following examples are all conventional methods unless otherwise specified. The materials, reagents, instruments, etc. used in the following examples can be obtained from commercial channels unless otherwise specified.

[0029] First, the culture medium used in the examples is:

[0030] LB solid medium: yeast powder 5 g / L, peptone 10 g / L, NaCl 10 g / L, agar powder 7 g / L, pH 7.0, 121°C sterilization for 25 min.

[0031] 121°C sterilization for 25 min.

[0032] LB liquid medium: yeast powder 5 g / L, peptone 10 g / L, NaCl 10 g / L, 121°C sterilization for 25 min.

[0033] NB solid medium: peptone 5 g / L, sucrose 10 g / L, yeast powder 1 g / L, beef extract 3 g / L, agar powder, pH 7.0, 121°C sterilization for 25 min.

[0034] NB liquid medium: peptone 5 g / L, sucrose 10 g / L, yeast powder 1 g / L, beef extract 3 g / L, pH 7.0, 121°C sterilization for 25 min.

[0035] Example 1: Rapid identification of Bacillus velezensis strain 913 based on 16s rRNA and accurate identification based on glpF, ilvD, tpiA, purH, rpoD, pycA, ptA genes.

[0036] After the strain 913 was streaked on LB solid medium, a single colony was picked into a flask containing LB liquid medium, and cultured at 37℃, 200r / min for 24h to obtain 913 bacterial liquid. The 913 bacterial liquid was used as a template, and 16s rRNA primers (27F 5'-AGAGTTTGATCCTGGCTCAG-3'; 1492R 5'-TACGGCTACCTTGTTACGACTT-3') were used for PCR amplification. The PCR reaction conditions were as follows: 95℃ for 3min; 95℃ for 15s, 58℃ for 15s, 72℃ for 1min, 35 cycles; 72℃ for 5min, 4℃ for stopping. The PCR product was sent to Beijing Qikang Biological Technology Co., Ltd. for sequencing, and the 16s rRNA corresponding DNA sequence is shown as SEQ ID NO:1. The sequencing results were compared with the existing bacterial 16s rRNA sequences in NCBI by BLAST alignment analysis, and the results are shown in Figure 2 . The colony morphology and 16s rRNA alignment results were combined Figure 1 It can be known that the strain 913 belongs to Bacillus.

[0037] In the classification and identification of bacteria, although 16s rRNA is widely used and the technology is relatively mature, there are some limitations for some bacteria species with close genetic relationship. Therefore, 16s rRNA is more accurate in the classification of genus, but not suitable for the classification and identification of species. The gene similarity between various Bacillus is very high, and it is particularly important to accurately identify the bacterial species by using the method of multilocus sequence typing (MLST). Therefore, the present application uses seven housekeeping genes glpF, ilvD, tpiA, purH, rpoD, pycA and ptA to construct a phylogenetic tree of the strain 913 and other 10 close bacterial species, so as to accurately identify the species.

[0038] The above strain 913 bacterial liquid was used as a template, and glpF primers (glpF-F 5'-WTGACAGCATTTTGGGG-3'; glpF-R 5'-GTAAAATACRCCGCCGA-3'), ilvD primers (ilvD-F 5'-ATGAGATATTCGCTGCC-3'; ilvD-R 5'-CTTCGTTAATGCGTTCTAAAGAG-3'), tpiA primers (tpiA-F 5'-TCAGCTTCGTTGAAGAAGTGAAA-3'; tpiA-R 5'-GGACTCTGCCATATATTCTTTA-3'), purH primers (purH-F 5'-TTTGAGAAAAAACAATCGCT-3'; purH-R 5'-TCGGCTCCCTTTTCGTCGG-3'), rpoD primers (rpoD-F 5'-GCCGAAGAAGAATTTGACCTTAA-3'; rpoD-R 5'-CGTTTRCTTCTGCTHGGATGTCT-3'), pycA primers (pycA-F 5'-AAATCAGARGCGAAAGC-3'; pycA-R 5'-CCTGAGCGGTAAGCCAT-3'), and ptA primers (ptA-F 5'-ATACATATGAAGGSATGGAAGA-3'; ptA-R 5'-TAGCCGATGTTTCCTGCT-3') were used for PCR amplification. The PCR system is shown in Table 1.

[0039] Table 1 PCR system for amplifying genes of strain 913

[0040]

[0041] The PCR reaction conditions were as follows: 95 °C for 3 min; 95 °C for 15 s, 55 °C for 15 s, 72 °C for 1 min, 35 cycles; 72 °C for 5 min, and 4 °C for stopping. The PCR product was purified and connected to a pMD19-T vector, and was sent to Beijing Genki Biotechnology Co., Ltd. for sequencing. The sequencing results were as follows: glpF: SEQ ID NO: 2; ilvD: SEQ ID NO: 3; ptA: SEQ ID NO: 4; purH: SEQ ID NO: 5; pycA: SEQ ID NO: 6; rpoD: SEQ ID NO: 7; and tpiA: SEQ ID NO: 8. The gene sequences of the above 10 closely related strains were obtained from NCBI. After the gene sequences of each strain were established for MLST typing, the MEGA7.0 software was used to construct a phylogenetic tree using the K2 model, and the results are shown in FIG. 1. Figure 3Strain 913 was found to be in the same branch as Bacillus velezensis by comprehensive comparison, and had the highest similarity with Bacillus velezensis strain QST713. It can be determined that strain 913 is Bacillus velezensis, and is officially named Bacillus velezensis strain 913. The strain has been preserved in the China General Microbiological Culture Collection Center (CGMCC) since July 2024 (address: No. 1, Huayuancun, Beijing, China, 100101), and the preservation number is CGMCC No. 31170.

[0042] Example 2: Determination of the growth curve of strain 913 at different temperatures.

[0043] 1. Activate the strain, streak strain 913 on LB solid medium, and incubate in a 37°C constant temperature incubator for 3 days. After single colonies grow on the plate, pick a single colony and inoculate into a 2 mL centrifuge tube containing 1 mL of LB liquid medium, and set the shaking speed to 200 r / min. Shake culture for 2 days.

[0044] 2. After the strain is activated, inoculate the shake-cultured bacterial solution in the centrifuge tube into a 250 mL flask containing 100 mL of LB liquid medium at a 1:100 inoculation amount, and set the shaking temperature to 16°C, 25°C, 30°C, 37°C, and 40°C, respectively, and shake at 200 r / min.

[0045] 3. Take the bacterial solution at 0 h, 2 h, 4 h, 6 h, 8 h, 10 h, 12 h, 18 h, and 24 h to measure the absorbance at 600 nm, and stop measuring when the OD value is stable. Each treatment is repeated three times, and the average value is taken. Draw the growth curve of the strain.

[0046] 4. Results: The growth curve of strain 913 at different temperatures is shown in Figure 4 913 strain can grow normally at 25-40°C

[0047] The growth at 16°C was significantly inhibited, and the strain basically did not proliferate. At 25°C, the strain basically did not grow before 8 h, and then started to grow rapidly, entering the logarithmic growth phase. At 30°C, the strain entered the logarithmic growth phase at 2-10 h, and then entered the stationary phase. At 37°C, the strain entered the logarithmic growth phase at 0-8 h, and then the growth rate slowed down, entering the stationary phase. At 40°C, the growth rate was the fastest, and the strain basically reached the stationary phase after 6 h, and the bacterial solution concentration started to decrease after 12 h.

[0048] Example 3: Detection of antagonistic effect of strain 913 fermentation filtrate on Pseudomonas syringae.

[0049] The experiment was repeated three times, and the average diameter of the inhibition zone was taken. The specific operation steps are as follows:

[0050] 1. Preparation of strain 913 fermentation filtrate: After streak culture of strain 913 on LB solid medium, single colonies were picked into 2 mL centrifuge tubes containing 1 mL of LB liquid medium and incubated at 37°C and 200 r / min overnight to obtain the culture solution. 100 μL of the culture solution was added to a 250 mL flask containing 100 mL of LB liquid medium, and incubated at 37°C and 200 r / min for 60 h. The fermented bacterial solution was centrifuged at 8000 r / min for 25 min, and the supernatant was filtered with a 0.45 μm filter head and then with a sterile 0.22 μm filter head to obtain sterile fermentation broth.

[0051] 2. Streak culture of Pseudomonas syringae YL19 on NB medium, and single colonies were picked into a flask containing 50 mL of NB liquid medium and incubated at 25°C and 200 r / min until the OD 600 ≈1.0. 1 mL of the bacterial solution was added to 50 mL of melted NB solid medium (temperature about 45°C) and mixed well, and then immediately poured into a flat plate to solidify.

[0052] 3. A sterile punch ring with a pore size of 5 mm was used to punch a hole in the center of the mixed NB plate, and a sterile cotton ball was placed in the hole, and 300 μL of the sterile fermentation broth of Bacillus sp. to be tested was injected into the cotton ball, with injection of liquid LB medium as a control. The prepared confrontation plate was placed in a 28°C constant temperature incubator for 4 d, and then the size of the inhibition zone was observed and the inhibition rate was calculated.

[0053] The formula for calculating the inhibition rate is as follows:

[0054] Inhibition rate (%) = (diameter of normally growing colonies - diameter of inhibited colonies) ÷ diameter of normally growing colonies x 100%.

[0055] 4. Results: Plate confrontation of strain 913 fermentation filtrate and Pseudomonas syringae showed that, compared with the injection of liquid LB medium control ( Figure 5 left), the fermentation filtrate of strain 913 ( Figure 5 right) had a significant inhibitory effect on the growth of Pseudomonas syringae. The diameter of the inhibition zone of the 913 fermentation filtrate and Pseudomonas syringae YL19 was obtained by measurement ( Figure 6 ). The inhibition rate of strain 913 was calculated to be 36.2%, which was significantly different from the injection of liquid LB medium control group. The plate confrontation experiment results showed that the fermentation filtrate of strain 913 had good inhibitory effect on Pseudomonas syringae YL19.

[0056] Example 4: Stability detection of the bacteriostatic effect of strain 913 fermentation filtrate.

[0057] The stability detection of the bacteriostatic effect of strain 913 fermentation filtrate was achieved by the size of the inhibition zone on the agar plate after treatment in different environments. All experiments used the controlled variable method, that is, when detecting the stability of one environmental condition, other environmental conditions were consistent. Each experiment was repeated three times, and the average value was taken. The specific implementation steps of each experiment were as follows:

[0058] 1. Preparation of strain 913 fermentation filtrate: same as Example 3.

[0059] 2. Detection of light stability: The strain fermentation broth was treated under 10000Lx light for 2h, 4h, 6h, 8h, 10h, respectively, and then a sterile punch ring with a pore size of 5mm was used to punch a hole in the center of the mixed NB plate. A sterile cotton ball was placed in the hole, and 300μL of the sterilized Bacillus fermentation broth treated by light was injected into the cotton ball. The prepared confrontation plate was placed in a 28℃ constant temperature incubator for 4d, and then the size of the inhibition zone was recorded to verify the bacteriostatic effect. The fermentation broth without light treatment was used as a control.

[0060] 3. Detection of ultraviolet stability: The strain fermentation broth was irradiated under a 75μW / cm 2 ultraviolet lamp for 15min, 30min, 45min, 60min, 75min, respectively, and then a sterile punch ring with a pore size of 5mm was used to punch a hole in the center of the mixed NB plate. A sterile cotton ball was placed in the hole, and 300μL of the sterilized Bacillus fermentation broth treated by ultraviolet irradiation was injected into the cotton ball. The prepared confrontation plate was placed in a 28℃ constant temperature incubator for 4d, and then the size of the inhibition zone was observed and recorded to verify the bacteriostatic effect. The fermentation broth without ultraviolet irradiation was used as a control.

[0061] 4. Detection of acid-base stability: The pH value of the strain fermentation broth was adjusted to 2, 4, 6, 8, 10, 12, respectively, and then a sterile punch ring with a pore size of 5mm was used to punch a hole in the center of the mixed NB plate. A sterile cotton ball was placed in the hole, and 300μL of the sterilized Bacillus fermentation broth with adjusted pH was injected into the cotton ball. The prepared confrontation plate was placed in a 28℃ constant temperature incubator for 4d, and then the size of the inhibition zone was observed and recorded to verify the bacteriostatic effect. Accordingly, hydrochloric acid or KOH solution corresponding to each pH value was set as a control.

[0062] 5. Detection of metal ion stability: Cu 2+ , Zn 2+ , Fe 3+ , K + , Ca 2+ , Na+ Mg 2+ The final concentration is 0.1 mol / L, and a sterile punch ring with a pore size of 5 mm is used to punch the center of the mixed NB plate, a sterile cotton ball is placed in the hole, and 300 μL of Bacillus sterile fermentation broth with metal ions is injected into the cotton ball. After placing the prepared confrontation plate in a constant temperature incubator at 28°C for 4d, observe and record the size of the inhibition zone, and verify the bacteriostatic effect. Correspondingly, set up the corresponding salt solution (concentration of 0.1 mol / L) of each metal ion as a control.

[0063] 6. Detection of temperature stability: The strain fermentation broth was treated at -20°C, 4°C, 15°C, 28°C, 40°C, 60°C, 80°C and 100°C for 1h, and then a sterile punch ring with a pore size of 5 mm was used to punch the center of the mixed NB plate, a sterile cotton ball was placed in the hole, and 300 μL of Bacillus sterile fermentation broth treated at different temperatures was injected into the cotton ball. After placing the prepared confrontation plate in a constant temperature incubator at 28°C for 4d, observe and record the size of the inhibition zone, and verify the bacteriostatic effect. Take 28°C as a control.

[0064] 7. Results: The fermentation broth of strain 913 has good stability under different temperature, light treatment, ultraviolet treatment and pH, and can still significantly inhibit the growth of Pseudomonas tolicola on agar plate after different conditions treatment, see Figure 7 .

[0065] Example 5: Pot experiment of strain 913 for preventing and treating strawberry "hollow disease".

[0066] 1. Set three concentration gradients of strain 913 broth, OD 600 =0.4, 0.8, 1.2, a total of 3 treatments, 913-0.4, 913-0.8, 913-1.2, 10 strains for each treatment, repeated three times, and the sterile water treatment is CK blank control. Except for different experimental treatments, irrigation, fertilization and other cultivation management measures are consistent.

[0067] 2. Select healthy strawberries with consistent growth, variety is 'Tianxianzui', and plant in sterile substrate. Scientific water and fertilizer management is carried out on strawberry plants, and the test treatment is carried out when the strawberry plants are recovered and the growth is vigorous.

[0068] 3. Strain activation and bacterial liquid preparation: After streaking culture of strain 913 on LB solid medium, single colonies were picked into 2 mL centrifuge tubes containing 1 mL of LB liquid medium, and cultured at 37°C and 200 r / min overnight to obtain the culture solution. 100 μL of the culture solution was added to a 250 mL flask containing 100 mL of LB liquid medium, and cultured at 37°C and 200 r / min for 12 h to obtain the bacterial liquid. The bacterial liquid was centrifuged at 5000 r / min for 10 min to collect the bacterial cells, and the supernatant was removed. The bacterial cells were resuspended in sterile distilled water to adjust the OD 600 to 0.4, 0.8, 1.2.

[0069] 4. Xanthomonas campestris YL19 was streaked on NB medium, and single colonies were picked into 2 mL centrifuge tubes containing 1 mL of NB liquid medium, and cultured at 25°C and 200 r / min for 3 d to obtain the culture solution. 100 μL of the culture solution was added to a flask containing 50 mL of NB liquid medium, and cultured at 25°C and 200 r / min for 3 d. The bacterial liquid was centrifuged at 5000 r / min for 10 min to collect the bacterial cells, and the supernatant was removed. The bacterial cells were resuspended in sterile distilled water and diluted to a concentration of about 1 x 10 8 cfu / mL.

[0070] 5. The bacterial liquid of 913 was used for root irrigation and foliar spraying treatment on strawberry plants, and then Xanthomonas campestris resuspension was mixed and sprayed on the whole strawberry seedlings, and stem inoculation was performed. The method of stem inoculation was as follows: simulating field agronomic operation, leaf splitting was performed, and then a sterile cotton ball was placed at the wound, 1 mL of Xanthomonas campestris bacterial suspension was gently injected into the sterile cotton ball using a pipette gun, and the cotton ball was in contact with the split leaf wound to facilitate infection. The control group used sterile water instead of Bacillus bacterial liquid.

[0071] 6. Leaf disease situation: on the 15th and 30th day after treatment, the leaves were photographed and recorded, the leaf disease rate after 15 d was counted, and the disease index after 30 d was counted. The disease grading standard is shown in Table 2. After 90 d of treatment, the stem cavity situation was observed: the strawberry stem was cut open, photographed and recorded, and the stem disease rate and stem cavity rate were counted.

[0072] Table 2 Disease grading of strawberry bacterial angular leaf spot and "cavity disease"

[0073]

[0074]

[0075] (1) Disease index = Σ (disease number of each level x representative value of each level) / (total number of leaves surveyed x highest representative value) x 100%

[0076] (2) Disease rate (%) = number of diseased plants / number of plants surveyed x 100

[0077] (3) Cavity rate (%) = number of stem cavity plants / number of investigated plants x 100

[0078] 7. Results:

[0079] (1) The leaf disease situation of 15d and 30d treatment can be seen from Figure 8 : After 15d of inoculation, a small amount of angular spots appeared in the CK control group and the 913 treatment group. After 30d of inoculation, the leaf angular spot symptoms of the control group and each treatment group were obvious. The angular spots of the CK control group continued to spread, the leaves appeared in patches of water spots, and gradually lost green, while the 913 treatment group significantly reduced the degree of disease, the spread of leaf angular spots slowed down, and there was no patchy water spots. Especially the 913-0.4 treatment group only had sporadic angular spots, and the disease was significantly reduced.

[0080] (2) After 15d of treatment, plants with angular spots on their leaves were considered to be diseased plants. The incidence rate after 15d of treatment is shown in Figure 9 : Compared with the CK control group, the incidence rate of the 913 control group was significantly reduced, especially the 913-0.4 treatment group.

[0081] (3) The disease index after 30d of treatment is shown in Figure 10 : Compared with the control group, the 913-0.4 and 913-0.8 treatment groups can significantly reduce the disease index after 30d of treatment.

[0082] (4) After 90d of inoculation, it can be seen from Figure 11 : The CK control group had severe cavity phenomenon on the stem, and the stem had obvious brown collapse and severe necrosis. In all 913 treatment groups, the 913-0.4 treatment group had no obvious disease symptoms on the stem, and the effect of inhibiting cavity was the best. The 913-0.8 treatment group showed brown on the stem and began to show a tendency to cavitate. The 913-1.2 treatment group showed cavitation, but the degree of cavitation was lighter than the CK control group.

[0083] (5) The effect of different treatments on the cavity rate of the stem is shown in Figure 12 : Compared with the control, the cavity rate of the 913 treatment group decreased significantly, especially the 913-0.4 treatment group had the best effect.

[0084] Example 6: Comparison test of the antagonistic effect of strain 913 and Rf21 fermentation filtrate on Pseudomonas tomlorii The experiment was repeated three times, and the average diameter of the inhibition zone was taken. The specific operation steps are as follows:

[0085] 1. Preparation of strain 913 and Rf21 fermentation filtrate, and culture of Pseudomonas tomlorii YL19: same as Example 3.

[0086] 2. Use a sterile punch ring with a 5mm aperture to punch a hole in the center of the mixed NB plate, place a sterile cotton ball in the hole, inject 300μL of the Bacillus sp. to be tested into the cotton ball, and inject LB liquid medium as a control. Place the prepared plate in a 28℃ incubator for 4 days, then observe the size of the inhibition zone.

[0087] 3. Results: Plate confrontation of strain 913 and Rf21 fermentation filtrate with P. fragariae showed that the inhibition zone diameter of strain 913 fermentation filtrate on P. fragariae was significantly larger than that of Rf21, indicating that the inhibition effect of strain 913 fermentation filtrate on P. fragariae YL19 was better than that of Rf21. Figure 13

[0088] Example 7: Comparison of the effects of strain 913 and Rf21 on the prevention and treatment of strawberry "cavity disease".

[0089] 1. Three treatments were set up: 913 (913 bacterial solution, OD 600 = 0.4, as confirmed in Example 5, OD 600 = 0.4 is the best applicable concentration for inhibiting cavity disease), Rf21 (Rf21 bacterial solution, OD 600 = 0.8, the applicant has previously confirmed that OD 600 = 0.8 is the best applicable concentration for Rf21 to inhibit cavity disease), 10 plants for each treatment, repeated three times, with sterile water treatment as the CK blank control. Except for the different treatments, the irrigation, fertilization and other cultivation management measures were consistent.

[0090] 2. Select healthy strawberries with consistent growth, variety 'Tianxianzui', and plant them in sterile substrate. Scientifically manage the water and fertilizer of the strawberry plants. When the strawberry plants have finished hardening off and are in good condition, proceed with the test treatment.

[0091] 3. Strain activation and bacterial solution preparation: After streaking culture of strains 913 and Rf21 on LB solid medium, single colonies were picked into 2mL centrifuge tubes containing 1mL of LB liquid medium, and cultured at 37℃, 200r / min overnight to obtain the culture solution. 100μL of the culture solution was added to a 250mL Erlenmeyer flask containing 100mL of LB liquid medium, and cultured at 37℃, 200r / min for 12h to obtain the bacterial solution. The bacterial solution was centrifuged at 5000r / min for 10min to collect the bacterial cells, and the supernatant was removed. The concentration of strain 913 bacterial solution was adjusted to OD 600 = 0.4 with sterile distilled water, and the concentration of strain Rf21 bacterial solution was adjusted to OD 600 = 0.8 with sterile distilled water.

[0092] ​4. Streak the strawberry Xanthomonas sp. YL19 on NB medium, pick a single colony and transfer it to a 2 mL centrifuge tube containing 1 mL of NB liquid medium. Cultivate it at 25°C and 200 rpm for 3 days to obtain the culture solution. Take 100 μL of the culture solution and transfer it to a flask containing 50 mL of NB liquid medium. Cultivate it at 25°C and 200 rpm for 3 days. Centrifuge the culture solution at 5000 rpm for 10 minutes to collect the bacteria, remove the supernatant, and resuspend and dilute it with sterile distilled water to a concentration of about 1 × 10 8 cfu / mL.

[0093] 5. Strawberry plants were treated with 913 and Rf21 bacterial suspensions, respectively, using root irrigation and foliar spraying. A resuspended suspension of Xanthomonas fragariae was then sprayed onto the entire strawberry plant, followed by stem inoculation. The stem inoculation method involved splitting the leaves, simulating field agronomic practices. A sterile cotton ball was then placed over the wound. Using a pipette, 1 mL of the YL19 suspension was gently pipetted into the sterile cotton ball, allowing contact between the cotton ball and the wound to facilitate infection. A control group received sterile water instead of the Bacillus spp. suspension. After 90 days of treatment, the strawberry stems were cut open, and the incidence of stem cavities was measured.

[0094] 6. Results: 90 days after inoculation, the effects of different treatments on the stem cavity rate were as follows: Figure 14 As shown: Compared with the control, the cavity rate of the 913 treatment group decreased most significantly, indicating that compared with Rf21, the 913 treatment can more significantly and effectively inhibit the occurrence of strawberry "cavity disease".

Claims

1. A Bacillus velezinoffii ( Bacillus velezensis ) strain, named Bacillus velez 913, which has been deposited in the China General Microbial Culture Collection Administration with the deposit number CGMCC No.31170.

2. A bacteriostatic agent comprising the Bacillus velezensis strain according to claim 1 as an active ingredient.

3. The Velez Bacillus strain as claimed in claim 1 or the antibacterial agent as claimed in claim 2 is effective in preventing and treating the bacterial infection caused by Xanthomonas fragariae ( Xanthomonas fragariae ) caused by bacterial angular spot of strawberry and strawberry "cavity disease".

4. The Velez Bacillus strain according to claim 1 or the antibacterial agent according to claim 2 is used in the preparation of a bacteriostatic agent for inhibiting Xanthomonas fragariae ( Xanthomonas fragariae ) in biological preparations.

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