Identification and application of bacillus velezensis strain 913

Abstract

The invention discloses identification and application of a bacillus strain. The bacillus velezensis is a bacillus velezensis 913 bacterial strain, the bacterial strain is preserved in the China General Microbiological Culture Collection Center (CGMCC) in July 2024, and the preservation number of the bacterial strain is CGMCC No.31170. The bacillus velezensis is a bacillus velezensis strain, and the bacillus velezensis strain has been preserved in the China General Microbiological Culture Collection Center (CGMCC) in July 2024. The bacterial strain can be used for inhibiting Xanthomonas fragariae, so that the bacterial strain can be used for preventing and treating bacterial angular leaf spot and void disease of strawberries.

Description

Technical Field

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

[0002] Strawberry has a unique flavor and high nutritional value. It is rated as one of the best fruits by the World Health Organization and is known as the "Queen of Berries". Its cultivation area ranks first among small berries in the world. In recent years, my country's strawberry industry has developed rapidly, with both the planting area and output ranking first in the world, making it the world's largest strawberry producer and exporter. At present, my country's strawberries are mainly cultivated in facilities. The special environment of high temperature and high humidity in the facilities, coupled with the use of continuous crops, has led to the occurrence and spread of various diseases, especially the "cavity disease" (also known as hollow disease, decapitation disease, and medullary hollow disease, etc.) that has emerged in recent years. It is a new type of devastating bacterial disease. The pathogen of this disease is Xanthomonas fragariae. The pathogen is highly infectious, spreads quickly, and has a late onset. Generally, symptoms will appear one month after cultivation in autumn, and seedlings cannot be replenished in time, which seriously restricts 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 and isolated and purified a Bacillus velezensis strain from the rhizosphere soil of healthy strawberry plants, and its name is Bacillus velezensis strain 913. It was found that the Bacillus velezensis can effectively inhibit Xanthomonas fragariae and prevent and treat the strawberry "cavity disease" caused by Xanthomonas fragariae, and the 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 Bacillus velezensis strain, named Bacillus velezensis 913, having growth characteristics such as Figure 1 As shown: the colony is milky white, translucent, with smooth and regular edges, the bacterial secretions can spread in a cloud-like manner, and the colony has wrinkled protrusions after 24 hours of cultivation. It has been deposited in the China General Microbiological Culture Collection Administration with the deposit number CGMCC No.31170.

[0006] The present invention also relates to an antibacterial agent, which comprises the Velez subtilis strain 913 as an active ingredient or a fermentation filtrate obtained by aseptically filtering a bacterial solution obtained after fermentation of the Velez subtilis strain 913.

[0007] The present invention also relates to the use of the Velez Bacillus 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" 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 comprising the fermentation liquid of the Bacillus Velez strain 913 described in the present application has good stability at different temperatures, light treatments, ultraviolet treatments, and pH values, that is, after being treated 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 controlling 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 "cavity disease", which has good application prospects in strawberry production;

[0012] 2. The antibacterial agent containing the fermentation broth of Bacillus Velez 913 not only has 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 This is the colony morphology of Bacillus Velez strain 913 grown on LB solid medium for 24 hours.

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

[0016] Figure 3 This is the phylogenetic tree of the glpF, ilvD, tpiA, purH, rpoD, pycA, and ptA gene sequences of Bacillus velez 913.

[0017] Figure 4 This is the growth curve of Bacillus Velez strain 913 at different temperatures of 16℃, 25℃, 30℃, 37℃ and 40℃.

[0018] Figure 5 The inhibition zone diagram of the fermentation filtrate of Bacillus Velez strain 913 against Xanthomonas fragariae, with the LB control on the left and the inhibition zone of strain 913 on the right.

[0019] Figure 6 is the diameter of the inhibition zone of the fermentation filtrate of Bacillus Velez strain 913 against Xanthomonas fragariae.

[0020] Figure 7 The following are the sizes of inhibition zones of fermentation broth of Bacillus Velez strain 913 after treatment in different environments. 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 The control effect of Bacillus Velez 913 on the symptoms of angular leaf spot of strawberry "cavity disease".

[0022] Fig. 9 The incidence of angular spot disease on strawberry leaves after treatment with Bacillus Velez strain 913 for 15 days, where * indicates P < 0.05% and ** indicates P < 0.01%.

[0023] Fig.10 is the disease index of angular spot disease on strawberry leaves after treatment with Bacillus Velez strain 913 for 30 days, where * indicates P < 0.05% and ** indicates P < 0.01%.

[0024] Fig.11 This is the hollowness in the strawberry stem after being treated with Bacillus Velez strain 913 for 90 days.

[0025] Fig.12 This is the hollowing rate of strawberry stems after being treated with Bacillus Velez strain 913 for 90 days, where ** indicates P < 0.05% and **** indicates P < 0.01%.

[0026] Fig.13 This is the size of the inhibition zone after 4 days of confrontation culture between the fermentation filtrate of Bacillus Velezii strains 913 and Rf21 and Xanthomonas fragariae.

[0027] Fig.14 These are the hollowing rates of strawberry stems after treatment with Bacillus Velez strains 913 and Rf21 for 90 days, respectively. a, b, and c indicate statistical significance. DETAILED DESCRIPTION

[0028] The following are specific embodiments of the present invention, and the technical scheme of the present invention is described in detail. The examples given are only for illustrating the present invention, and are not intended to limit the scope of the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The materials, reagents, instruments, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial sources.

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

[0030] LB solid medium: yeast powder 5g / L, peptone 10g / L, NaCl 10g / L, agar powder 7g / L,

[0031] Sterilize at 121℃ for 25min.

[0032] LB liquid medium: yeast powder 5g / L, peptone 10g / L, NaCl 10g / L, sterilize at 121℃ for 25min.

[0033] NB solid culture medium: peptone 5g / L, sucrose 10g / L, yeast powder 1g / L, beef extract 3g / L, agar powder, adjust the pH to 7.0, and sterilize at 121℃ for 25min.

[0034] NB liquid culture medium: peptone 5g / L, sucrose 10g / L, yeast powder 1g / L, beef extract 3g / L, adjust the pH to 7.0, and sterilize at 121℃ for 25min.

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

[0036] After strain 913 was streaked on LB solid medium, a single colony was picked into a triangular flask filled with LB liquid medium and cultured at 37°C and 200r / min for 24h to obtain 913 bacterial solution. Using 913 bacterial solution as a template, 16s rRNA primers (27F5'-AGAGTTTGATCCTGGCTCAG-3'; 1492R5'-TACGGCTACCTTGTTACGACTT-3') were used for PCR amplification. The PCR reaction conditions were: 95°C for 3min; 95°C for 15s, 58°C for 15s, 72°C for 1min, 35 cycles; 72°C for extension for 5min, and stop at 4°C. The PCR product was sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing, and the DNA sequence corresponding to its 16s rRNA is shown in SEQ ID NO: 1. The sequencing results were compared with the existing bacterial 16s rRNA sequences in NCBI by BLAST analysis, and the results are shown in Figure 2 .comprehensive Figure 1 The comparison of colony morphology and 16s rRNA showed that strain 913 belonged to the genus Bacillus.

[0037] In the classification and identification of bacteria, although the molecular identification using 16s rRNA is widely used and the technology is relatively mature, it still has some limitations for some closely related bacterial species. Therefore, the classification of genus using 16s rRNA is more accurate, but not suitable for the classification and identification of species. The gene similarity between various species of Bacillus is very high, and it is particularly important to accurately identify bacterial species using the method of multi-locus sequence typing (MLST). Therefore, the present invention uses seven housekeeping genes, glpF, ilvD, tpiA, purH, rpoD, pycA, and ptA, to construct a phylogenetic tree of strain 913 and other 10 closely related bacterial species to accurately identify the species.

[0038] The bacterial suspension of strain 913 was used as template and glpF primers (glpF-F 5'-WTGACAGCATTTTGGGG-3'; glpF-R 5'-GTAAAATACRCCGCCGA-3') and ilvD primers (ilvD-F 5'-ATGAGATATTCGCTGCC-3'; ilvD-R5'-CTTCGTTAATGCGTTCTAAAGAG-3'), tpiA primer (tpiA-F5'-TCAGCTTCGTTGAAGAAGTGAAA-3'; tpiA-R5'-GGACTCTGCCATATATTCTTTA-3'), purH primer (purH-F5'-TTTGAGAAAAAA CAATCGCT-3'; purH-R5'-TCGGCTCCCTTTTCGTCGG-3'), rpoD primer (rpoD-F5'-GCCGAAGAAGAATTTGACCTTAA-3'; rpoD-R5'-CGTTTRCTTCTGCTHGGATGTCT-3'), pycA primer (pycA-F5'-AAATCAGARGCGAAAGC-3'; pycA-R 5'-CCTGAGCGGTAAGCCAT-3'), ptA primer (ptA-F5'-ATACATATGAAGGSATGGAAGA-3'; ptA-R5'-TAGCCGATGTTTCCTGCT-3') for PCR amplification. The PCR system is shown in Table 1.

[0039] Table 1 PCR system for gene amplification of strain 913

[0040]

[0041] The PCR reaction conditions were: 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 extension for 5 min, and 4°C for stop. The PCR product was purified and connected to the pMD19-T vector, and sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing. The sequencing results were: 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; tpiA: SEQ ID NO: 8. The above gene sequences of the other 10 closely related strains were derived from NCBI. After the gene sequences of each strain were used to establish MLST typing, the phylogenetic tree was constructed using MEGA7.0 software and the K2 model. The results are shown in Figure 3. Comprehensive comparison found that strain 913 was in the same branch as Bacillus velezensis, and had the highest similarity with Bacillus velezensis strain QST713. Strain 913 was identified as Bacillus velezensis and was officially named Bacillus velezensis strain 913. The strain was deposited in the General Microbiology Center of China Culture Collection Administration (CGMCC, Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, 100101) in July 2024, and its deposit 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 culture in a 37℃ constant temperature incubator for 3 days. After a single colony grows on the plate, pick a single colony and inoculate it into a 2mL centrifuge tube containing 1mL LB liquid medium, and set the shaking speed to 200r / min. Shake and culture for 2 days.

[0044] 2. After the strain activation is completed, the bacterial solution in the centrifuge tube was inoculated into a 250mL Erlenmeyer flask containing 100mL LB liquid culture medium at an inoculum size of 1:100. The shaking incubator temperature was set to 16℃, 25℃, 30℃, 37℃, and 40℃, respectively, and the shaking culture was performed at 200r / min.

[0045] 3. Take the bacterial solution at 0h, 2h, 4h, 6h, 8h, 10h, 12h, 18h, and 24h, and measure its absorbance at a wavelength of 600nm. Stop the measurement when the OD value stabilizes. Repeat each treatment three times and take the average value. Draw a growth curve of the strain.

[0046] 4. Results: The growth curves of strain 913 at different temperatures are as follows Figure 4 As shown, strain 913 was grown at 25-40℃

[0047] The growth rate was significantly inhibited at 16℃, and the proliferation was basically non-existent. At 25℃, there was basically no growth before 8h, and it began to grow rapidly after 8h and entered the logarithmic growth phase. At 30℃, it entered the logarithmic growth phase at 2-10h, and then entered the stable phase. At 37℃, it entered the logarithmic growth phase at 0-8h, and then the growth rate slowed down and entered the stable phase. The growth rate was the fastest at 40℃, and it basically reached the stable phase after 6h. The bacterial liquid concentration began to decrease after 12h.

[0048] Example 3: Detection of the antagonistic effect of the fermentation filtrate of strain 913 on Xanthomonas fragariae.

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

[0050] 1. Preparation of fermentation filtrate of strain 913: After streaking strain 913 on LB solid medium, pick a single colony into a 2mL centrifuge tube containing 1ml LB liquid medium, and culture overnight at 37°C and 200r / min to obtain culture solution; take 100μL of the culture solution into a 250mL Erlenmeyer flask containing 100mL LB liquid medium, and culture for 60h at 37°C and 200r / min. Centrifuge the fermented bacterial solution at 8000r / min for 25min, take the supernatant and filter it with a 0.45μm filter head, and then filter it with a sterile 0.22μm filter head to obtain sterile fermentation solution.

[0051] 2. Streak the strawberry Xanthomonas YL19 on NB medium, pick a single colony and put it into a triangular flask filled with 50mL NB liquid medium, and culture it at 25℃ and 200r / min until the OD value of the bacterial solution reaches 600 ≈1.0. Pipette 1 mL of bacterial solution into 50 mL of melted NB solid medium (temperature about 45°C), mix well, and then immediately pour into the plate to solidify.

[0052] 3. Use a sterile punch ring with a pore size of 5 mm to punch a hole in the center of the mixed NB plate, put a sterile absorbent cotton ball in the hole, and inject 300 μL of the sterile fermentation liquid of the tested Bacillus into the cotton ball, and inject liquid LB culture medium as a control. Place the prepared confrontation plate in a 28℃ constant temperature incubator for 4 days, observe the size of the inhibition zone and calculate the inhibition rate.

[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 × 100%.

[0055] 4. Results: The fermentation filtrate of strain 913 was plated against Xanthomonas fragariae. It can be seen that the control group ( Figure 5 Left) compared to strain 913 ( Figure 5 Right) The fermentation filtrate showed a significant inhibitory effect on the growth of Xanthomonas fragariae. The diameter of the inhibition zone between the 913 fermentation filtrate and Xanthomonas fragariae YL19 was measured ( Figure 6 The calculated inhibition rate of strain 913 was 36.2%, which was significantly different from the control group injected with liquid LB medium. The results of the plate confrontation experiment showed that the fermentation filtrate of strain 913 had a good inhibitory effect on Xanthomonas fragariae YL19.

[0056] Example 4: Stability test of the antibacterial effect of the fermentation filtrate of strain 913.

[0057] The stability test of the antibacterial effect of the fermentation liquid of strain 913 was achieved by the size of the inhibition zone on the agar plate after it was treated in different environments. All experiments used the controlled variable method, that is, when testing the stability of one environmental condition, the other environmental conditions were the same. The experiment was repeated three times and the average value was taken. The specific implementation steps of each experiment are as follows:

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

[0059] 2. Light stability test: After the fermentation liquid of the strain is placed under 10000Lx light for 2h, 4h, 6h, 8h, and 10h, a hole is punched in the center of the mixed NB plate with a sterile punch ring with a pore size of 5mm, a sterile absorbent cotton ball is placed in the hole, and 300μL of the light-treated Bacillus sterile fermentation liquid is injected into the cotton ball. The prepared confrontation plate is placed in a 28℃ constant temperature incubator for 4 days, and the size of the inhibition zone is recorded to verify its antibacterial effect. The fermentation liquid without light treatment is used as a control.

[0060] 3. UV stability test: The fermentation broth of the strain was placed in an ultraviolet light with an intensity of 75 μW / cm 2 After irradiation under ultraviolet light for 15min, 30min, 45min, 60min, and 75min, a hole was punched in the center of the mixed NB plate with a sterile punch ring with a pore size of 5mm, a sterile absorbent cotton ball was placed in the hole, and 300μL of ultraviolet-treated Bacillus sterile fermentation liquid was injected into the cotton ball. The prepared confrontation plate was placed in a 28℃ constant temperature incubator for 4 days, and the size of the inhibition zone was observed and recorded to verify its antibacterial effect. The fermentation liquid without ultraviolet irradiation was used as a control.

[0061] 4. Detection of acid-base stability: Adjust the pH value of the fermentation broth to 2, 4, 6, 8, 10, and 12 respectively, use a sterile punch ring with a pore size of 5 mm to punch a hole in the center of the mixed NB plate, put a sterile absorbent cotton ball in the hole, and inject 300 μL of the Bacillus sterile fermentation broth with adjusted pH into the cotton ball. Place the prepared confrontation plate in a 28°C constant temperature incubator for 4 days, observe and record the size of the inhibition zone, and verify its antibacterial effect. Accordingly, set the corresponding hydrochloric acid or KOH solution for each pH value as a control.

[0062] 5. Detection of metal ion stability: Cu 2+ and Cu 2+ were added to the fermentation broth of the strain. 2+ 、Zn 2+ , Fe 3+ , K + , Ca 2+ 、Na+ Mg 2+ Make the final concentration 0.1mol / L, use a sterile punch ring with a pore size of 5mm to punch a hole in the center of the mixed NB plate, put a sterile absorbent cotton ball in the hole, and inject 300μL of Bacillus sterile fermentation liquid with metal ions added into the cotton ball. Place the prepared confrontation plate in a 28℃ constant temperature incubator for 4 days and observe and record the size of the inhibition zone to verify its antibacterial effect. Accordingly, set the corresponding salt solution (concentration of 0.1mol / L) for each metal ion as a control.

[0063] 6. Temperature stability test: After the fermentation liquid of the strain is placed at -20℃, 4℃, 15℃, 28℃, 40℃, 60℃, 80℃, and 100℃ for 1h, a hole is punched in the center of the mixed NB plate with a sterile punch ring with a pore size of 5mm, a sterile absorbent cotton ball is placed in the hole, and 300μL of the sterile fermentation liquid of Bacillus treated at different temperatures is injected into the cotton ball. The prepared confrontation plate is placed in a 28℃ constant temperature incubator for 4 days, and the size of the antibacterial circle is observed and recorded to verify its antibacterial effect. 28℃ is used as the control.

[0064] 7. Results: The fermentation broth of strain 913 had good stability under different temperatures, light treatments, ultraviolet treatments, and pH. Even after being treated under different conditions, it could still significantly inhibit the growth of Xanthomonas fragariae on agar plates. Figure 7 .

[0065] Example 5: Potted experiment on controlling strawberry "cavity disease" with strain 913.

[0066] 1. Set three concentration gradients of strain 913 bacterial solution, OD 600 =0.4, 0.8, 1.2, a total of 3 treatments, namely 913-0.4, 913-0.8, 913-1.2, 10 plants in each treatment, repeated three times, with sterile water treatment as CK blank control. Except for the different experimental treatments, the cultivation management measures such as irrigation and fertilization were kept consistent.

[0067] 2. Select healthy strawberries with consistent growth potential, the variety is 'Tianxianzui', plant them in a sterile substrate, and carry out scientific water and fertilizer management for the strawberry plants. Wait until the strawberry plants have finished acclimatization and are growing vigorously before conducting test treatments.

[0068] 3. Strain activation and bacterial liquid preparation: After streaking strain 913 on LB solid medium, pick a single colony and place it in a 2mL centrifuge tube containing 1mL LB liquid medium. Culture it at 37°C and 200r / min overnight to obtain culture liquid. Take 100μL of the culture liquid and place it in a 250mL Erlenmeyer flask containing 100mL LB liquid medium. Culture it at 37°C and 200r / min for 12h to obtain bacterial liquid. Centrifuge the bacterial liquid at 5000r / min for 10min to collect the cells, remove the supernatant, and adjust the OD with sterile distilled water. 600 Are 0.4, 0.8, and 1.2.

[0069] 4. Streak the strawberry Xanthomonas YL19 on NB medium, pick a single colony and put it into a 2mL centrifuge tube containing 1mL NB liquid medium, culture it at 25℃ and 200r / min for 3 days, get the culture solution, take 100uL of the culture solution into a triangular flask containing 50ml NB liquid medium, shake and culture it at 25℃ and 200r / min for 3 days, centrifuge the bacterial solution at 5000r / min for 10min 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.

[0070] 5. The strawberry plants were treated with 913 bacterial solution for root irrigation and leaf spraying, and then the strawberry Xanthomonas suspension was mixed and sprayed on the whole strawberry seedlings, and the stems were inoculated. The method of stem inoculation is: simulate field agronomic operations, split the leaves, then take a sterile cotton ball and place it on the wound, use a pipette to absorb 1mL of strawberry Xanthomonas suspension and gently inject it into the sterile cotton ball, and the cotton ball is in contact with the split leaf wound to facilitate infection. The control group used sterile water instead of Bacillus bacterial solution.

[0071] 6. Leaf disease condition: Take photos of the leaves on the 15th and 30th days after treatment, and calculate the leaf disease incidence rate 15th day after treatment and the disease index 30th day after treatment. The disease grading standards are shown in Table 2. Observe the stem hollowing condition 90th day after treatment: Cut the strawberry stems, take photos, record and calculate the stem disease incidence rate and stem hollowing rate.

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

[0073]

[0074]

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

[0076] (2) Incidence rate (%) = number of diseased plants / number of investigated plants × 100

[0077] (3) Hollow rate (%) = number of plants with hollow stems / number of plants surveyed × 100

[0078] 7. Results:

[0079] (1) The leaf disease status at 15 and 30 days after treatment can be seen from Figure 8 It can be seen that 15 days after the inoculation, a small amount of angular spots appeared in the CK control group and the 913 treatment group. 30 days after the inoculation, the angular spots on the leaves of the strawberries in the control group and each treatment group were obvious. The angular spots on the leaves of the CK control group continued to spread, and the leaves appeared water-soaked and gradually lost their green color. The severity of the disease in the 913 treatment group was significantly alleviated, and the spread of angular spots on the leaves slowed down, and no water-soaked patches appeared. In particular, only sporadic angular spots appeared on the leaves of the 913-0.4 treatment group, and the symptoms were significantly alleviated.

[0080] (2) Plants with angular spots on their leaves 15 days after treatment were considered diseased plants. Fig. 9 As shown: 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 30 days of treatment is as follows Fig.10 As shown, compared with the control group, the treatment groups 913-0.4 and 913-0.8 could significantly reduce the disease index after 30 days of treatment.

[0082] (4) 90 days after inoculation, Fig.11 It can be seen that: the CK control group had serious cavitation in the stems, with obvious brown collapse and severe necrosis; among all the 913 treatment groups, the 913-0.4 treatment group had no obvious symptoms in the stems, and had the best effect in inhibiting the occurrence of cavitation; the 913-0.8 treatment group had brown stems and began to show a trend of cavitation; the 913-1.2 treatment group had cavitation, but the degree of cavitation was lighter than that of the CK control group.

[0083] (5) Effects of different treatments on stem hollowing rate Fig.12 As shown: Compared with the control, the void rate of the 913 treatment group decreased significantly, especially the 913-0.4 treatment group had the best effect.

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

[0085] 1. Preparation of fermentation filtrate of strains 913 and Rf21, and cultivation of Xanthomonas fragariae YL19: Same as Example 3.

[0086] 2. Use a sterile punch ring with a pore size of 5 mm to punch a hole in the center of the mixed NB plate, put a sterile absorbent cotton ball in the hole, and inject 300 μL of the sterile fermentation liquid of the tested Bacillus into the cotton ball, and inject liquid LB culture medium as a control. Place the prepared confrontation plate in a constant temperature incubator at 28℃ for 4 days and observe the size of its inhibition zone.

[0087] 3. Results: The fermentation filtrates of strains 913 and Rf21 were placed on a plate against Xanthomonas fragariae, and it can be seen that ( Fig.13 ), the diameter of the inhibition zone of the fermentation filtrate of strain 913 against Xanthomonas fragariae was significantly larger than that of Rf21, indicating that the inhibitory effect of the fermentation filtrate of strain 913 on Xanthomonas fragariae YL19 was better than that of Rf21.

[0088] Example 7: Comparative test on the effects of strains 913 and Rf21 in preventing and controlling strawberry "cavity disease".

[0089] 1. The experiment set up three treatments: 913 (913 bacterial solution, OD 600 =0.4, as confirmed in Example 5, OD 600 =0.4 is the best concentration for inhibiting cavitation disease), Rf21 (Rf21 bacterial solution, OD 600 =0.8, the applicant previously confirmed OD 600 =0.8 is the best concentration of Rf21 for inhibiting cavitation disease), 10 plants were treated in each experiment, and the experiment was repeated three times. Sterile water treatment was used as the CK blank control. Except for the different experimental treatments, the cultivation management measures such as irrigation and fertilization were kept consistent.

[0090] 2. Select healthy strawberries with consistent growth potential, the variety is 'Tianxianzui', plant them in a sterile substrate, and carry out scientific water and fertilizer management for the strawberry plants. Wait until the strawberry plants have finished acclimatization and are growing vigorously before conducting test treatments.

[0091] 3. Strain activation and bacterial liquid preparation: After streaking strains 913 and Rf21 on LB solid medium, pick a single colony and place it in a 2mL centrifuge tube containing 1mL LB liquid medium. Culture it at 37°C and 200r / min overnight to obtain culture liquid. Take 100μL of the culture liquid and place it in a 250mL Erlenmeyer flask containing 100mL LB liquid medium. Culture it at 37°C and 200r / min for 12h to obtain bacterial liquid. Centrifuge the bacterial liquid at 5000r / min for 10min to collect the cells, remove the supernatant, and adjust the concentration of strain 913 bacterial liquid to OD with sterile distilled water. 600 =0.4, the concentration of strain Rf21 was adjusted with sterile distilled water to OD 600 =0.8.

[0092] 4. Streak the strawberry Xanthomonas YL19 on NB medium, pick a single colony and put it into a 2mL centrifuge tube containing 1mL NB liquid medium, culture it at 25℃ and 200r / min for 3 days, get the culture solution, take 100uL of the culture solution into a triangular flask containing 50ml NB liquid medium, shake and culture it at 25℃ and 200r / min for 3 days, centrifuge the bacterial solution at 5000r / min for 10min 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. The strawberry plants were treated with 913 and Rf21 bacterial solutions by root irrigation and foliar spraying, and then the strawberry Xanthomonas suspension was mixed and sprayed on the whole strawberry seedlings, and the stems were inoculated. The method of stem inoculation is: simulate field agronomic operations, split the leaves, then take a sterile cotton ball and place it on the wound, use a pipette to absorb 1mL of YL19 bacterial suspension and gently inject it into the sterile cotton ball, the cotton ball is in contact with the split leaf wound, which is conducive to infection. The control group used sterile water instead of Bacillus bacterial solution. After 90 days of treatment, the strawberry stems were cut open and the incidence of stem cavities was counted.

[0094] 6. Results: 90 days after inoculation, the effects of different treatments on stem hollowing rate were as follows: Fig.14 As shown: Compared with the control, the hollowing 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 "hollow disease".

Claims

1. A Bacillus velezensis strain, named Bacillus velezensis 913, which has been deposited with China General Microbiological Culture Collection Committee under the deposit number of CGMCC No.31170.

2. An antibacterial agent comprising the Bacillus Velez strain according to claim 1 or a culture supernatant obtained after fermentation of the Bacillus Velez strain as an active ingredient.

3. Use of the Bacillus Velez strain according to claim 1 or the antibacterial agent according to claim 2 in preventing and treating strawberry bacterial angular spot disease and strawberry "cavity disease" disease caused by Xanthomonas fragariae.

4. Use of the Bacillus Velez strain according to claim 1 or the antibacterial agent according to claim 2 in the preparation of a biological preparation for inhibiting Xanthomonas fragariae.

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