Bacillus subtilis and application thereof in prevention and treatment of brown wrinkled fruit virus

Abstract

The invention discloses a bacillus subtilis strain and application thereof in prevention and treatment of tomato brown wrinkled fruit virus diseases. According to the bacillus subtilis strain, the bacillus subtilis strain is NA25, the preservation name is bacillus subtilis, the bacillus subtilis strain is preserved in the China General Microbiological Culture Collection Center (CGMCC), the address of the preservation unit is No.3, No.1 Yard, Beichen West Road, Chaoyang District, Beijing, and the preservation date is April 27, 2025; the preservation number of the strain is CGMCC No. 34380. The bacillus subtilis provided by the invention is separated from healthy tomato endophyte, and the strain fermentation liquor and cell suspension both have a good inhibition effect on the brown wrinkled fruit virus, and can be used for preventing and treating the tomato brown wrinkled fruit virus.

Description

Technical Field

[0001] This invention relates to the field of microbial application technology, specifically to a Bacillus subtilis strain and its application in the prevention and control of brown wrinkled fruit virus. Background Technology

[0002] Tomato brown rugose fruit virus (ToBRFV) is a novel virus that affects the health and yield of solanaceous crops such as tomatoes and peppers. This virus causes decreased plant vigor, shortened harvest cycles, and even premature death. It produces brown patches and stunted fruit on tomatoes, severely impacting yield and quality. It has already caused significant outbreaks in major tomato-producing areas of China, such as Shandong and Yunnan. Currently, there are no commercially available varieties resistant to ToBRFV, nor are there any specific chemical agents for its control. Therefore, control methods are very limited, making the development of green and environmentally friendly biological control technologies essential.

[0003] Bacillus is an important group of biocontrol bacteria, commercially developed into various biocontrol agents. Its active substances can inhibit the growth of various plant pathogenic fungi and bacteria. Among them, *Bacillus subtilis* is the most widely used and studied group of Bacillus. Its mechanism of action mainly involves the effective inhibition of pathogen growth by secreted lipopeptides, subtilisin, mycelial peptides, and cell wall enzyme-degrading substances. Simultaneously, certain substances secreted by *Bacillus subtilis* can induce systemic resistance in plants, activating plant defense mechanisms. Currently, reported applications of *Bacillus subtilis* are mainly for the control of plant pathogenic fungi and bacterial diseases. A very small number of Bacillus strains have also been reported for the control of viral diseases, including Tobacco Mosaic Virus (TMV), Potato Virus Y (PVY), and Tomato Spotted Wilt Virus (TSWV). No biocontrol strains targeting ToBRFV have been reported yet. Summary of the Invention

[0004] In response to the current serious problem of brown wrinkled fruit virus disease in tomatoes, the lack of resistant varieties, and the limited control methods, this paper presents the application of a strain of Bacillus subtilis in the control of brown wrinkled fruit virus disease.

[0005] To achieve the above objectives, the present invention adopts the following technical solutions:

[0006] In a first aspect, the present invention provides a Bacillus subtilis strain, wherein the Bacillus subtilis strain is NA25, the depositary name is Bacillus subtilis, and it is deposited at the China General Microbiological Culture Collection Center (CGMCC), the address of which is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, the deposit date is April 27, 2025, and the deposit number is CGMCC No. 34380.

[0007] The gene sequence of the Bacillus subtilis strain is the 16S rRNA nucleotide sequence and gumG gene sequence shown in SEQ ID No. 1.

[0008] The Bacillus subtilis inoculant prepared by the Bacillus subtilis strain of the present invention comprises at least one of the following (a) and (b):

[0009] (a) The aseptic fermentation broth of Bacillus subtilis as described in claim 1;

[0010] (b) The Bacillus subtilis cell suspension obtained according to claim 1;

[0011] Preparation of sterile fermentation broth of Bacillus subtilis: Bacillus subtilis strain NA25 was inoculated onto NA solid medium plates and incubated upside down at 28°C for 24 hours to obtain single colonies of Bacillus subtilis. These single colonies were then inoculated into 50 mL of NB liquid medium and cultured on a shaker at 28°C and 180 r / min for 12–24 hours to obtain a seed culture of Bacillus subtilis. The seed culture was inoculated at a ratio of 2%–5% (v / v) into 200 mL of NB liquid medium and cultured at 28°C and 180 r / min for 48–90 hours. The culture was then centrifuged at 4°C, 8000 rpm, and 15 min, and the supernatant was collected. The supernatant was filtered using a bacterial filter to obtain a sterile fermentation broth (diluted 50–100 times).

[0012] Preparation of Bacillus subtilis cell suspension: Bacillus subtilis strain NA25 was inoculated onto NA solid medium plates and incubated upside down at 28°C for 24 hours to obtain single colonies of Bacillus subtilis. These single colonies were then inoculated into 50 mL of NB liquid medium and cultured on a shaker at 28°C and 180 r / min for 12–24 hours to obtain a seed culture of Bacillus subtilis. The seed culture was inoculated at a ratio of 2%–5% (v / v) into 200 mL of NB liquid medium and cultured on a shaker at 28°C and 180 r / min for 48–90 hours. The cells were collected at 4°C, 8000 rpm, and 15 min, washed with sterile water, and diluted with sterile water to a concentration of 1×10⁻⁶. 8Cell suspension of cfu / mL.

[0013] Secondly, this invention provides an application of Bacillus subtilis as described in the first aspect in the prevention and control of brown wrinkled fruit virus disease: NA25 sterile fermentation broth, NA25 cell suspension, or a mixture of fermented NA25 can be used for foliar spraying of crops; the main targets of control include crops sensitive to brown wrinkled fruit virus such as tomatoes and peppers; when using foliar spraying, the fermentation stock solution is diluted 50-100 times and sprayed once every 3-5 days.

[0014] Compared with existing technologies, the advantages of this invention are: This invention discovers an endophytic strain isolated from tomatoes, Bacillus subtilis NA25, which exhibits high activity and stability. The aseptic fermentation broth of this strain shows a control efficiency of 41% against ToBRFV disease, and the cell suspension of the strain shows a control efficiency of 39% against ToBRFV, significantly inhibiting the replication of the virus in the host plant. It possesses characteristics of high efficiency, safety, and environmental friendliness, and has broad prospects for biocontrol applications. Attached Figure Description

[0015] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0016] Figure 1 This is a colony morphology diagram of Bacillus subtilis NA25 of the present invention;

[0017] Figure 2 The microscopic morphological characteristics of Bacillus subtilis NA25 of the present invention;

[0018] Figure 3 Phylogenetic tree analysis of Bacillus subtilis NA25 for the present invention;

[0019] Figure 4 The aseptic fermentation broth of Bacillus subtilis NA25 of the present invention Enzyme-linked immunosorbent assay Initial screening image;

[0020] Figure 5 The Bacillus subtilis NA25 cell suspension of the present invention Enzyme-linked immunosorbent assay Initial screening image;

[0021] Figure 6 The diagram shows the effect of the Bacillus subtilis NA25 sterile fermentation broth of the present invention on the prevention and control of brown wrinkled fruit virus disease;

[0022] Figure 7This image shows the control effect of the Bacillus subtilis NA25 cell suspension of the present invention on plants infected with brown wrinkled fruit virus. Detailed Implementation

[0023] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0024] Example 1

[0025] Isolation of Bacillus subtilis NA25

[0026] Bacillus subtilis NA25, preservation number CGMCC No. 34380, was collected from healthy tomato leaves in Yunnan Province. Under standard aseptic conditions, the leaves were immersed in 75% ethanol for 1 min, rinsed three times with sterile water, and blotted dry with sterile filter paper. Next, they were immersed in 0.2% HgCl2 for 3 min, rinsed ten times with sterile water, and blotted dry with sterile filter paper. The sterilized leaves were then ground with sterile water, and the resulting juice was placed on NA agar plates and incubated at 28°C for 2 days. After bacterial growth, the plates were streaked 3–5 times for purification. The purified strain was then identified by 16S rRNA sequence amplification.

[0027] Example 2

[0028] Identification of Bacillus subtilis NA25 strain

[0029] 1. Observation of morphological characteristics

[0030] After culturing on NA medium for 24 hours, the colony morphology of this strain (NA25) was observed. The colony surface was rough, opaque, and milky white. When growing on solid medium, it often formed wrinkled, opaque, and glossy colonies. A colony morphology diagram of Bacillus subtilis NA25 is attached. Figure 1 As shown. Electron microscopy with negative staining revealed that individual cells were 0.7–0.8 × 2–3 μm in size and uniformly stained. (No further information provided.) capsule Zhou Sheng flagellum It can move, as shown in the attached document. Figure 2 As shown.

[0031] 2.16S rRNA sequence analysis

[0032] The 16S rRNA gene sequence of strain NA25 was amplified by PCR using universal bacterial primers 27F (5'-AGAGTTTGATCTGGCTCG-3) and 1492R (5'-GGTTACCTTGTTACGACTT-3). The reaction mixture consisted of 12.5 μL of 2×TaqPCRMaster Mix, 1 μL each of forward and reverse primers, one single colony of sample, and 9.5 μL of ddH2O. The reaction program was: 94℃ for 3 min; 94℃ for 1 s, 55-58℃ for 30 s, 72℃ for 30 s, for 35 cycles; 72℃ for 5 min. After gel electrophoresis, the sequence was sent to Shanghai Bioengineering Co., Ltd. for sequencing. The full-length sequence was 1404 bp (see SEQ). The obtained sequences were submitted to the GenBank database for BLAST analysis and comparison. The strains with high homology to NA25 belonged to *Bacillus subtilis* (NCBI ID: CP103351.1). A phylogenetic tree was constructed using MEGA 7.0 software. NA25 and *Bacillus subtilis* are in the same branch (see attached). Figure 3 ).

[0033] Based on the above morphological characteristics analysis, 16S rRNA analysis and NJ phylogenetic tree, the strain was preliminarily identified as Bacillus subtilis.

[0034] Example 3

[0035] Preparation of aseptic fermentation broth of Bacillus subtilis strain NA25

[0036] Bacillus subtilis strain NA25 was inoculated onto NA solid medium plates and incubated upside down at 28°C for 24 hours to obtain single colonies of Bacillus subtilis. These single colonies were then inoculated into 50 mL of NB liquid medium and cultured on a shaker at 28°C and 180 r / min for 12–24 hours to obtain a seed culture of Bacillus subtilis. The seed culture was then inoculated at a ratio of 2%–5% (v / v) into 200 mL of NB liquid medium and cultured at 28°C and 180 r / min for 48–90 hours. The culture was then centrifuged at 4°C, 8000 rpm, and 15 min, and the supernatant was collected. The supernatant was filtered using a bacterial filter to obtain a sterile fermentation broth.

[0037] Example 4

[0038] Inhibitory activity of Bacillus subtilis NA25 strain sterile fermentation broth against ToBRFV

[0039] Three control groups were set up as follows:

[0040] Blank control: 5 mL of NB liquid culture medium;

[0041] Negative Control: 100 μL of purified brown wrinkled fruit virus (final virus concentration 0.1–0.2 mg / mL) was added to 5 mL of NB liquid culture medium.

[0042] Positive Control: 100 μL of purified brown wrinkled fruit virus and ningnanmycin (final concentration 0.16 g / mL) were added to 5 mL of NB liquid culture medium.

[0043] The experimental group (NA25) was set as follows: 100 μL of purified brown wrinkled fruit virus and 100 μL of activated Bacillus subtilis NA25 sterile fermentation broth were added to 5 mL of NB liquid culture medium.

[0044] Each group was divided into three replicates. Samples were incubated on a constant-temperature shaker at 28 ℃ and 180 r / min for 0–3 days. Samples were collected every 24 hours and frozen at -80℃. After all samples were collected, their antiviral activity was detected by ELISA. The results are attached. Figure 4 As shown, co-incubation of NA25 fermentation broth significantly reduced viral load compared to the negative control (see attached figure). Figure 4 ).

[0045] Example 5

[0046] Preparation of Bacillus subtilis NA25 strain cell suspension

[0047] Bacillus subtilis strain NA25 was inoculated onto NA solid medium plates and incubated upside down at 28°C for 24 hours to obtain single colonies. These single colonies were then inoculated into 50 mL of NB liquid medium and cultured on a shaker at 28°C and 180 rpm for 12–24 hours to obtain a seed culture of Bacillus subtilis. The seed culture was then inoculated at a ratio of 2%–5% (v / v) into 200 mL of NB liquid medium and cultured on a shaker at 28°C and 180 rpm for 48–90 hours. The bacterial cells were collected at 4°C, 8000 rpm, and 15 min, washed with sterile water, and diluted with sterile water to a concentration of 1×10⁻⁶. 8 Cell suspension of cfu / mL.

[0048] Example 6

[0049] Inhibitory activity of Bacillus subtilis NA25 strain cell suspension against ToBRFV

[0050] Three control groups were set up as follows:

[0051] Blank control: 5 mL of NB liquid culture medium;

[0052] Negative Control: 100 μL of purified brown wrinkled fruit virus (final virus concentration 0.1–0.2 mg / mL) was added to 5 mL of NB liquid medium.

[0053] Positive control: 100 μL of purified brown wrinkled fruit virus and ningnanmycin (final concentration 0.16 g / mL) were added to 5 mL of NB liquid culture medium.

[0054] The experimental group (NA25) was set as follows: 100 μL of purified brown wrinkled fruit virus and 100 μL of activated Bacillus subtilis NA25 cell suspension were added to 5 mL of NB liquid culture medium.

[0055] Each group was divided into three replicates. Samples were incubated on a constant-temperature shaker at 28 ℃ and 180 r / min for 0–3 days. Samples were collected every 24 hours and frozen at -80℃. After all samples were collected, their antiviral activity was detected by ELISA. The results are attached. Figure 5 As shown, co-incubation of NA25 cell suspension with the negative control significantly reduced viral load. Figure 4 As shown.

[0056] Example 7

[0057] The efficacy of aseptic fermentation broth of Bacillus subtilis strain NA25 against ToBRFV

[0058] A spraying experiment was conducted on tomato seedlings. The experiment included: CK group: healthy pepper seedlings; Water group: inoculated with brown wrinkled fruit virus; NA25 group: simultaneously inoculated with the virus and sterile fermentation broth of Bacillus subtilis (NA25) (diluted 50-100 times); each group had 5 replicates. Spraying was performed every three days, and the disease index and control rate were recorded after 14 days.

[0059] The disease index and prevention efficacy are calculated according to the following formulas.

[0060] Disease index = Σ(Number of diseased plants at each level × Disease level value) / (Total number of plants surveyed × Highest level value) × 100

[0061] Severity grading criteria: The disease grading criteria and formula are as follows: Grade 0: The entire plant is healthy and asymptomatic;

[0062] Grade 1: Slight variegation, no obvious dwarfing.

[0063] Level 3: The top 1 / 3 of the leaves show signs of variegation but are not deformed, or the plant is dwarfed to more than 3 / 4 of its normal height.

[0064] Level 5: One-third to one-half of the plant has variegated leaves, or a few leaves are deformed and the main veins are black, or the plant is stunted to two-thirds to three-quarters of its normal height.

[0065] Level 7: 1 / 2 to 2 / 3 of the plant has mosaic leaves, or the leaves are deformed and the main veins are necrotic, or the plant is stunted to 1 / 2 to 2 / 3 of its normal height. Level 9: The entire plant has mosaic leaves, or the leaves are severely deformed and necrotic, or the plant is stunted to more than 1 / 2 of its normal height.

[0066] Prevention and control rate (%) = (Control disease index - Treatment disease index) / Control disease index × 100

[0067] The control effect of NA25 fermentation broth on brown wrinkled fruit virus disease is shown in the attached figure. Figure 6 As shown, spraying with sterile fermentation broth significantly reduced viral load. According to the disease index statistics, the control efficiency of NA25 sterile fermentation broth spraying against ToBRFV was 41% (Table 1). Table 1

[0068]

[0069] Example 8

[0070] The efficacy of Bacillus subtilis NA25 strain cell suspension against ToBRFV

[0071] A spraying experiment was conducted on tomato seedlings. The experiment included: CK group (healthy pepper seedlings); Water group (inoculated with brown wrinkled fruit virus); and NA25 group (inoculated with both the virus and Bacillus subtilis (NA25) cell suspension (OD600 = 0.6-1)). Each group had 5 replicates. Disease index and control rate were recorded after 14 days.

[0072] The disease index and prevention efficacy are calculated according to the following formulas.

[0073] Disease index = Σ(Number of diseased plants at each level × Disease level value) / (Total number of plants surveyed × Highest level value) × 100

[0074] Severity grading criteria: The disease grading criteria and formula are as follows: Grade 0: The entire plant is healthy and disease-free;

[0075] Grade 1: Slight mosaic pattern, no obvious dwarfing. Grade 3: Top 1 / 3 of the leaves show mosaic pattern but are not deformed, or the plant is dwarfed to more than 3 / 4 of its normal height. Grade 5: 1 / 3 to 1 / 2 of the entire plant has mosaic pattern, or a few leaves are deformed and the main vein is blackened, or the plant is dwarfed to 2 / 3 to 3 / 4 of its normal height. Grade 7: 1 / 2 to 2 / 3 of the entire plant has mosaic pattern, or the leaves are deformed and the main vein is necrotic, or the plant is dwarfed to 1 / 2 to 2 / 3 of its normal height. Grade 9: The entire plant has mosaic pattern, or the leaves are severely deformed and necrotic, or the plant is dwarfed to more than 1 / 2 of its normal height.

[0076] Prevention and control rate (%) = (Control disease index - Treatment disease index) / Control disease index × 100

[0077] The control efficacy of NA25 cell suspension against brown wrinkled fruit virus disease is shown in the attached figure. Figure 7 As shown, spraying with cell suspension can significantly reduce viral load. According to the disease index, the control efficiency of NA25 cell suspension spraying against ToBRFV is 39% (Table 1).

[0078] Table 2

[0079]

[0080] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope. The scope of protection of the present invention is defined by the appended claims, specification, and their equivalents.

Claims

1. A Bacillus subtilis strain, characterized in that: The Bacillus subtilis strain is NA25, and the deposit name is Bacillus subtilis (Bacillus subtilis) Bacillus subtilis , which is preserved in the China General Microbiological Culture Collection Center (CGMCC) at No. 1, Xibanchenli, Chaoyang District, Beijing, on April 27, 2025, and the preservation number is CGMCC No. 34380.

2. The Bacillus subtilis strain according to claim 1, characterized in that: The gene sequence of the Bacillus subtilis strain is the 16S rRNA nucleotide sequence shown in SEQ ID No. 1 and gumG the gene sequence.

3. A Bacillus subtilis inoculum prepared from the Bacillus subtilis strain of claim 1.

4. A Bacillus subtilis inoculum prepared from the Bacillus subtilis strain of the present application, wherein the active ingredient comprises at least one of the following (a) and (b): (a) the sterile fermentation broth of the Bacillus subtilis of claim 1; (b) the cell suspension of the Bacillus subtilis of claim 1.

5. The process for the preparation of a sterile fermentation broth of Bacillus subtilis according to claim 4, characterized in that The Bacillus subtilis strain NA25 is inoculated on NA solid medium plate and incubated at 28°C for 24 hours to obtain single colonies of Bacillus subtilis. The single colony of Bacillus subtilis NA25 is inoculated in 50 mL NB liquid medium and incubated at 28°C and 180 r / min for 12-24 hours to obtain seed liquid of Bacillus subtilis. The seed liquid is inoculated in 200 mL NB liquid medium at a ratio of 2%-5% (v / v) and incubated at 28°C and 180 r / min for 48-90 hours. The supernatant is obtained by centrifugation at 4°C, 8000 rpm and 15 min. The supernatant is filtered by a bacterial filter to obtain sterile fermentation broth, which is diluted 50-100 times.

6. The method of claim 4, wherein the cell suspension of Bacillus subtilis is prepared by the steps of: Bacillus subtilis strain NA25 is inoculated on NA solid medium plate, and is cultured at 28°C for 24 hours to obtain a single colony of Bacillus subtilis. The single colony of Bacillus subtilis NA25 is inoculated in 50 mL NB liquid medium, and is cultured at 28°C and 180 r / min for 12-24 hours to obtain a seed liquid of Bacillus subtilis. The seed liquid is inoculated in 200 mL NB liquid medium at a ratio of 2%-5% (v / v), and is cultured at 28°C and 180 r / min for 48-90 hours. The bacterial cells are collected at 4°C, 8000 rpm, and 15 min, and are prepared into a cell suspension with a concentration of 1×10 8 cfu / mL by using sterile water.

7. The method for preparing aseptic fermentation broth and cell suspension of Bacillus subtilis according to claim 4, characterized in that: The NA solid medium comprises the following components: 10 g / L of proteose peptone, 3 g / L of beef extract powder, 5 g / L of sodium chloride, 15 g / L of agar, and 1 L of distilled water, with pH being 7.3±0.1, and sterilized at 121°C for 15 min.

8. The process for the preparation of a sterile fermentation broth and cell suspension of Bacillus subtilis according to claim 4, characterized in that The NB liquid medium comprises the following components: 10 g / L of proteose peptone, 3 g / L of beef extract powder, 5 g / L of sodium chloride, and 1 L of distilled water, with pH being 7.4±0.2, and sterilized at 121°C for 15 min.

9. The sterile fermentation broth and cell suspension of Bacillus subtilis of any one of claims 1-7 for use in the prevention and treatment of tomato brown rugose fruit virus disease.

10. Use according to claim 9, characterized in that: The sterile fermentation broth or cell suspension of Bacillus subtilis NA25 is used for spraying treatment of plant leaves, and is used for tomato brown rugose fruit virus disease on tomatoes and peppers.

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