Bacillus velezensis ts-6 and application thereof in prevention and treatment of grape white rot disease

Abstract

The application provides a bacillus velezensis Ts-6 and application thereof in prevention and treatment of grape botrytis blight, and belongs to the technical field of agricultural microorganisms. The bacillus velezensis Ts-6 has a preservation number of CGMCC No. 38544, contains a surfactin synthesis gene cluster, a shionin synthesis gene cluster and a bacillolysin synthesis gene cluster in a genome, can effectively inhibit botrytis cinerea, and has multiple plant growth promoting functions such as phosphorus solubilization, indole acetic acid production and extracellular enzyme production. The application further provides a microbial inoculum containing the strain, and application of the strain or the inoculum in prevention and treatment of grape botrytis blight and promotion of grape growth, so that the use of chemical pesticides can be reduced, and the application is suitable for green organic grape production.

Description

Technical Field

[0001] This invention relates to a Bacillus berberis Ts-6 strain and its application in the control of grape white rot, belonging to the field of agricultural microbial technology. Background Technology

[0002] Grape white rot, also known as grape canker, is a significant fungal disease in grape production. It primarily affects grape bunches, shoots, and leaves, and is particularly severe under hot and humid conditions, leading to substantial yield reductions or even crop failure. Currently, control of this disease mainly relies on chemical fungicides such as carbendazim and tebuconazole. However, the long-term and excessive use of chemical pesticides not only leads to increased pathogen resistance and decreased efficacy but also poses risks of pesticide residues, environmental pollution, and food safety. Therefore, developing safe, efficient, and environmentally friendly biological control methods has become an important direction for green grape production.

[0003] Bacillus belyssae is a recognized plant growth-promoting bacterium that produces a variety of antifungal metabolites and auxins, showing potential in the control of various crop diseases. However, the specific control efficacy of currently available Bacillus belyssae strains against grape white rot is not ideal, and they often lack systematic analysis of growth-promoting characteristics and clear elucidation of antimicrobial metabolite gene clusters. Summary of the Invention

[0004] The purpose of this invention is to provide a Bacillus berreatus Ts-6 strain and its application in the control of grape white rot, thereby solving the technical problems of existing chemical control methods that easily lead to drug resistance and environmental pollution, as well as the unsatisfactory control effect of existing biocontrol strains on grape white rot and the unclear growth-promoting function.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0006] In a first aspect, the present invention provides a Bacillus berleis Ts-6, which is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 38544.

[0007] In conjunction with the first aspect, the Bacillus belyssus Ts-6 genome further contains a surfactant synthesis gene cluster, a phytosalicylate synthesis gene cluster, and a bacilysin synthesis gene cluster.

[0008] In a second aspect, the present invention provides a microbial agent comprising Bacillus berberis Ts-6 as described in the first aspect.

[0009] In conjunction with the second aspect, the microbial agent is further described as a liquid agent, a solid agent, or a wettable powder.

[0010] Thirdly, the present invention provides the application of the microbial agent described in the second aspect in the prevention and control of grape white rot, wherein the pathogen causing grape white rot includes the pathogen Coniella diplodiella.

[0011] Fourthly, the present invention provides the application of Bacillus berberis Ts-6 described in the first aspect in the prevention and control of grape white rot, wherein the pathogen causing grape white rot includes the pathogen Coniella diplodiella.

[0012] In conjunction with the fourth aspect, further, the Bacillus berberis Ts-6 exhibits a plate confrontation inhibition rate of not less than 70% against the pathogen Coniella diplodiella.

[0013] Fifthly, the present invention provides the application of Bacillus berberis Ts-6 as described in the first aspect in promoting grape growth.

[0014] In conjunction with the fifth aspect, the Bacillus berberis Ts-6 further comprises the functions of phosphate solubilization, indoleacetic acid production, and / or extracellular enzyme production, wherein the extracellular enzymes include at least one of protease, amylase, and cellulase.

[0015] The present invention also provides a method for preventing and controlling grape white rot, the method comprising applying Bacillus berles Ts-6 as described in the first aspect or the microbial agent as described in the second aspect to grapevines and / or their growth environment.

[0016] Compared with the prior art, the beneficial effects achieved by the present invention are as follows:

[0017] This invention provides a Bacillus berberis Ts-6 and its application in the control of grape white rot, which has a plate confrontation inhibition rate of not less than 70% against grape white rot fungus and has strong specific inhibitory activity.

[0018] Bacillus berberis Ts-6 possesses multiple plant growth-promoting pathways, including phosphorus solubilization, indoleacetic acid production, and extracellular enzyme production. In practical applications, it can simultaneously achieve the dual functions of disease prevention and growth promotion, helping to reduce the use of chemical pesticides and fertilizers.

[0019] Bacillus berberis Ts-6 is environmentally friendly, poses no risk of chemical residues, and is suitable for organic or green grape production systems. Attached Figure Description

[0020] Figure 1 This is a colony morphology diagram of Bacillus berberis Ts-6 on LB medium provided in an embodiment of the present invention;

[0021] Figure 2 This is a graph showing the phosphorus solubilization effect of Bacillus berberis Ts-6 provided in an embodiment of the present invention;

[0022] Figure 3 A graph showing the ability of Bacillus berberis Ts-6 to produce indoleacetic acid provided in an embodiment of the present invention; Figure 4 This is an image showing the effect of Bacillus berberis Ts-6 producing extracellular enzymes according to an embodiment of the present invention;

[0023] Figure 5 This is a schematic diagram of the antimicrobial metabolite gene cluster structure in Bacillus belyssus Ts-6 provided in an embodiment of the present invention; Figure 6 This is a plate confrontation inhibition effect diagram of Bacillus berberis Ts-6 against Grape white rot fungus provided in an embodiment of the present invention;

[0024] Figure 7 The diagram shows the control effect of Bacillus berberis Ts-6 against grape white rot fungus, as provided in this embodiment of the invention. Detailed Implementation

[0025] The following examples are provided to further illustrate the present invention, but the scope of protection of the present invention is not limited thereto. Unless otherwise specified, the test methods used in the following examples are conventional test methods; unless otherwise specified, the reagents and materials used are commercially available.

[0026] This invention provides a Bacillus velezensis Ts-6 strain and its application in the control of grape white rot. The Bacillus velezensis Ts-6 strain has been deposited at the China General Microbiological Culture Collection Center (CGMCC), located at the Institute of Microbiology, Chinese Academy of Sciences, Beijing, China, with accession number CGMCC No. 38544, classified as Bacillus velezensis Ts-6, and deposited on May 6, 2026.

[0027] Example 1:

[0028] This embodiment describes the isolation and identification of a bacterial strain, Ts-6, isolated from the foliage of grapevines in the Yongbin Farm vineyard in Jurong City, Jiangsu Province. The process is as follows:

[0029] Microbial samples were obtained from the surface of the tested grape leaves and prepared into serially diluted bacterial suspensions. Isolation was performed using a dilution-spreading method, where the bacterial suspensions were spread onto LB solid medium and incubated at 30°C for 24–48 h. Single colonies were then picked and purified.

[0030] The colony morphology of the purified strain on LB medium is as follows: Figure 1As shown, the colonies on LB medium appear milky white, with a dry surface and irregular edges. The strain identification process is as follows: Genomic DNA of strain Ts-6 was extracted, and its 16S rDNA sequence was amplified using primers 27F / 1492R. Sequencing results were compared using BLAST, and highly similar sequences were selected. The sequence with the highest similarity to the 16S rDNA sequence of *Bacillus belyssiensis* was identified as *Bacillus belyssiensis* Ts-6.

[0031] Example 2:

[0032] This embodiment includes physiological and biochemical tests and life-promoting assays, specifically phosphate solubilization capacity tests, indoleacetic acid production capacity tests, and extracellular enzyme activity tests. Phosphate solubilization capacity test: Using inorganic and organic phosphorus media, *Bacillus belye* Ts-6 obtained in Example 1 was inoculated and cultured for 5 days. Clear zones appeared on all plates. Figure 2 As shown in the figure, a represents the effect of organic phosphorus solubilization, and b represents the effect of inorganic phosphorus solubilization. Both figures show a clear and distinct transparent zone around the colony, indicating that this strain has the ability to solubilize both inorganic and organic phosphorus. Indoleacetic acid (IAA) production test: *Bacillus belyeis* Ts-6 was cultured in LB medium containing L-tryptophan for 48 h. The Salkowski colorimetric method was used to determine the IAA production in the fermentation broth. Its IAA production capacity is shown in the figure below. Figure 3 As shown. Extracellular enzyme activity tests: Protease, cellulase, and amylase were detected separately. Protease activity test: The strain was spotted onto a skim milk plate, and a clear zone formed after incubation. Cellulase activity test: The strain was spotted onto a CMC-Na plate, and after incubation, Congo red staining showed a clear hydrolysis zone. Amylase activity test: The strain was spotted onto a starch plate, and after incubation, iodine staining showed a clear zone. All the above extracellular enzyme activity test results showed clear zones.

[0033] like Figure 4 As shown, Figure 4 Image a shows the clear zone of the plate containing the protease, image b shows the clear zone of the plate containing the cellulase, and image c shows the clear zone of the plate containing the amylase. All images show a clear zone of hydrolysis around the colony, proving that this strain can produce active protease, cellulase, and amylase.

[0034] Example 3:

[0035] In this embodiment, antimicrobial metabolite gene cluster analysis was performed. The whole genome of Bacillus belyssus Ts-6 was sequenced, and the secondary metabolite gene clusters were analyzed using antiSMASH software.

[0036] The results are as follows Figure 5As shown, the genome of Bacillus belyssus Ts-6 contains at least the surfactantin synthesis gene cluster, the fengycin synthesis gene cluster, and the bacilysin synthesis gene cluster.

[0037] Example 4:

[0038] In this embodiment, an antibacterial test was conducted. The tested pathogen was Coniella diplodiella, the causal agent of grape white rot, which was isolated and preserved in the laboratory.

[0039] The plate confrontation method was used. A 7 mm diameter pathogen colony disc was inoculated in the center of a PDA plate, and activated Bacillus belychnophora Ts-6 was symmetrically inoculated at a distance of 2.5 cm. The plates were incubated at 25°C for 3-5 days, and the pathogen growth radius was measured. Each treatment was repeated three times. The inhibition rate was calculated using the following formula. The average net growth radius of the pathogen and the inhibition rate are shown in Table 1. Inhibition rate (%) = (Control colony radius - Treatment colony radius) / Control colony radius × 100%

[0040] Table 1: Inhibition rate of Bacillus belyceta ts-6 against Grape white rot pathogen in plate confrontation

[0041]

[0042] Note: Since the inoculated mycelium cake itself has a diameter of 7 mm (radius 3.5 mm), the net growth radius = total measured radius - 3.5 mm.

[0043] like Figure 6 The figure shows the plate confrontation inhibition effect of Bacillus vesiculosus Ts-6 provided in the example against Grape white rot pathogen. In the figure, a is the control group inoculated only with Grape white rot pathogen without the addition of antagonistic bacteria. The pathogen colonies grow radially in a uniform manner, with net growth radii of 28.2 mm, 27.5 mm, and 28.4 mm, respectively. b is the treatment group inoculated with Bacillus vesiculosus Ts-6. Bacillus vesiculosus Ts-6 showed a significant inhibitory effect on Grape white rot pathogen, with net growth radii of 8.0 mm, 7.8 mm, and 8.4 mm, respectively. Its average plate confrontation inhibition rate was 71.2%.

[0044] Example 5:

[0045] This embodiment conducts a greenhouse control efficacy verification test of Bacillus vesicles Ts-6 against grape white rot, including applying Bacillus vesicles Ts-6 or microbial agents containing Bacillus vesicles Ts-6 to grapevines and / or their growth environment by spraying, drenching or broadcasting. The microbial agent can be a liquid agent, a solid agent or a wettable powder.

[0046] In vitro leaf experiment: Mature leaves from the middle part of healthy grapevines were collected, disinfected, and placed in petri dishes lined with moistened filter paper. Treatment groups were sprayed with Bacillus berghei Ts-6 fermentation broth (1×10⁻⁶). 8 (CFU / mL), the control group was sprayed with an equal volume of sterile water, and after drying, each group of leaves was inoculated with a 5mm diameter pathogen cake, and cultured at 25℃ for 3-5 days. The diameter of the lesions was measured, and the results are shown in Table 2 and 3. Figure 7 As shown.

[0047] Table 2: Control efficacy of Bacillus vesicle Ts-6 against white rot in Shine Muscat grape leaves

[0048]

[0049] Note: Different lowercase letters after the diseased area data in the same column indicate significant differences at the P<0.05 level. The control effect is calculated based on the lesion area.

[0050] In vitro fruit test: Healthy, uniformly sized grapes were selected, and after surface sterilization, they were fermented in Bacillus baijis Ts-6 broth (1×10⁻⁶). 8 Soak the fruit in a solution of CFU / mL for 30 minutes and then air-dry it. The control group was treated with sterile water. After 24 hours, two inoculation points were gently punctured at the top of each fruit using a sterile syringe (0.5 mm × 20 mm, 1 mL). Each inoculation point was then inoculated with 10 μL of a suspension of conidia of *Coniella diplodiella*, the causal agent of grape white rot. After inoculation, the fruit was wrapped in sterile, water-moistened cotton to retain moisture and then incubated at 30 ℃. Disease occurrence was observed and recorded regularly, and the diameter of lesions was measured. The results are shown in Table 3. Figure 7 As shown.

[0051] Table 3: Control efficacy of Bacillus vesicle B. ts-6 against white rot in Shine Muscat grapes

[0052]

[0053] Note: Different lowercase letters after the diseased area data in the same column indicate significant differences at the P<0.05 level. The control effect is calculated based on the lesion area.

[0054] The results showed that the incidence and disease index of white rot in grapes treated with Bacillus vesicles Ts-6 fermentation broth were significantly lower than those in the control group, where the area of ​​lesions on detached leaves was 20.3 cm². 2 16.4 cm 2 23.0 cm 2 The area of ​​lesions on detached leaves in the treatment group was 3.4 cm². 2 3.1cm 2 7.1 cm2 The control efficacy reached 77.2%; the lesion area on the fruit of the control group treated with Sunshine Rose fruit was 0.28 cm². 2 0.42 cm 2 0.56 cm 2 The lesion area of ​​the Sunshine Rose fruit in the treatment group was 0.07 cm². 2 0.12 cm 2 0.17 cm 2 The preventive efficacy was 71.4%.

[0055] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the technical principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A type of Bacillus belye Ts-6, characterized in that, The Bacillus berberis Ts-6 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 38544.

2. The Bacillus belyssus Ts-6 according to claim 1, characterized in that, The genome of Bacillus berreatus Ts-6 contains a surfactant synthesis gene cluster, a fertility gene cluster, and a bacilysin synthesis gene cluster.

3. A microbial inoculant, characterized in that, The microbial agent comprises Bacillus berberis Ts-6 as described in claim 1.

4. The microbial agent according to claim 3, characterized in that, The microbial agent is a liquid agent, a solid agent, or a wettable powder.

5. The application of the microbial agent according to claim 3 in the control of grape white rot, characterized in that, The pathogens causing grape white rot include pathogenic bacteria. Coniella diplodiella .

6. The application of Bacillus berberis Ts-6 as described in claim 1 in the control of grape white rot, characterized in that, The pathogens causing grape white rot include pathogenic bacteria. Coniella diplodiella .

7. The application according to claim 6, characterized in that, The Bacillus berberis Ts-6 is effective against pathogens. Coniella diplodiella The inhibition rate of the flat plate confrontation is no less than 70%.

8. The application of Bacillus berberis Ts-6 as described in claim 1 in promoting grape growth.

9. The application according to claim 8, characterized in that, The Bacillus belyssus Ts-6 has the functions of phosphate solubilization, indoleacetic acid production, and / or extracellular enzyme production, wherein the extracellular enzyme includes at least one of protease, amylase, and cellulase.

10. A method for controlling grape white rot, characterized in that, This includes applying the Bacillus berberis Ts-6 of claim 1 or the microbial agent of claim 3 to grapevines and / or their growth environment.

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