Biocontrol bacillus velezensis, biocontrol microbial inoculum and application of biocontrol bacillus velezensis and biocontrol microbial inoculum

Abstract

The invention discloses biocontrol bacillus velezensis, a biocontrol microbial agent and application of the biocontrol bacillus velezensis and the biocontrol microbial agent, and relates to the technical field of microbial control. The bacterial strain is bacillus velezensis DLYK-221, the bacterial strain is preserved in the China Center for Type Culture Collection on December 9, 2024, the preservation address is Wuhan University, Wuhan, China, and the preservation number is CCTCC NO: M20242761. The strain provided by the invention can prevent and treat tobacco spotted wilt caused by tomato spotted wilt virus, and the prevention and treatment effect reaches 99% or above, so that DLYK-221 can be used as a biocontrol strain for preventing and treating tobacco spotted wilt, and has a wide application prospect in the field of tobacco planting.

Description

Technical Field

[0001] The invention relates to the technical field of microbial control, and in particular to a biocontrol bacillus Velez subtilis, a biocontrol agent and applications thereof. Background Art

[0002] Yunnan Province is the region with the most Orthotospovirus species and the most serious disease incidence in my country. In recent years, tobacco spotted wilt caused by Tomato spotted wilt virus transmitted by thrips on tobacco has become increasingly serious, causing huge economic losses. Due to the diverse climate in Yunnan Province, the increase in the scope and severity of drought, the spread of Tomato spotted wilt virus diseases has accelerated. Tomato spotted wilt seriously restricts the safe production of tobacco leaves. At present, there is a lack of in-depth and systematic research on the occurrence characteristics and prevention and control of this disease in Dali tobacco-growing areas of Yunnan.

[0003] Microbial control is a new approach to pest control and has great application prospects in the field of disease control. Therefore, how to provide a biocontrol microorganism with the ability to induce plant disease resistance is a technical problem that those skilled in the art need to solve urgently. Summary of the invention

[0004] The invention aims to provide a method for biologically controlling tobacco spotted wilt caused by tomato spotted wilt virus, which can effectively control tobacco spotted wilt and has great application potential in the field of tobacco planting.

[0005] In order to achieve the above-mentioned purpose, the present invention provides a biocontrol Bacillus velezensis DLYK-221, which has been deposited in the China Center for Type Culture Collection on December 9, 2024, with the deposit address being Wuhan University, Wuhan, China, and the deposit number being CCTCC NO: M20242761.

[0006] The Bacillus Velezii DLYK-221 provided by the invention can be used to inhibit the expression of tomato spotted wilt virus and can be used to prevent and control tobacco spotted wilt.

[0007] The Bacillus Velezii DLYK-221 provided by the invention can also be used for preparing drugs for preventing and treating tobacco spotted wilt.

[0008] The present invention also provides a biocontrol agent for preventing and controlling tobacco spotted wilt, which comprises the above-mentioned Bacillus Velezii DLYK-221.

[0009] Preferably, the above-mentioned biocontrol agent is a bacterial suspension of Bacillus Velezii DLYK-221, and its spore concentration is preferably 1×10 7-10 CFU / mL.

[0010] The biocontrol agent provided by the invention can be used in the field of tobacco planting, and in particular can be used to prevent and control tobacco spotted wilt caused by tomato spotted wilt virus.

[0011] The present invention has the following advantages:

[0012] The present invention discovers a Bacillus velezensis DLYK-221 from the rhizosphere soil of healthy tobacco. Through verification, it is found that the strain can prevent and control tobacco spotted wilt caused by tomato spotted wilt virus, can be used as a biocontrol strain, and has application potential in the field of tobacco planting.

[0013] The Bacillus Velezii DLYK-221 provided by the invention can be prepared into a biocontrol fungicide, has better adaptability to the rhizosphere environment of tobacco, can effectively prevent and control tomato spotted wilt, and the prevention and control effect can reach more than 99%, is more conducive to the rapid colonization of biocontrol bacteria, improves the rhizosphere microecological environment of tobacco, and prevents and controls tomato spotted wilt. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 It is a colony characteristic diagram of the biocontrol strain DLYK-221 of the present invention, wherein A is a colony morphology diagram and B is a Gram staining microscopic examination diagram.

[0015] Figure 2 It is the phylogenetic tree of the biocontrol strain DLYK-221 of the present invention.

[0016] Figure 3 The control effect of the biocontrol agent on tomato spotted wilt is shown in the figure, wherein A is a control treated with sterile water root irrigation, and B is a control treated with DLYK-221 biocontrol agent root irrigation.

[0017] Figure 4 The present invention is to detect the relative content of viruses in tobaccos treated differently. DETAILED DESCRIPTION

[0018] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0019] Note: The experimental methods in the following examples are conventional methods unless otherwise specified, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples are all commercially available unless otherwise specified.

[0020] Part of the culture medium used in the present invention:

[0021] NA liquid culture medium: peptone 10 g, beef extract powder 3 g, sodium chloride 5 g, water 1000 mL, pH = 7.0, sterilize at 121 ° C for 30 min.

[0022] NA solid culture medium: Add 20 g of agar powder to every 1000 mL of NA liquid culture medium and sterilize at 121°C for 30 min.

[0023] Casein glucose agar CD medium: 5.0g yeast extract, 10.0g cheese acid hydrolyzate, 10.0mL 50% glucose sterile stock solution, 4.0g potassium dihydrogen phosphate, 17.0g agar powder, dilute to 1000mL. The 50% glucose sterile stock solution should be sterilized separately and added to the basic medium under aseptic conditions.

[0024] Example 1 Screening and identification of strains

[0025] Tobacco leaves infected with tomato spotted wilt and rhizosphere soil of healthy tobacco collected from tobacco fields in Dali, Yunnan were used as samples. Using the dilution plate method, 10 g of healthy tobacco rhizosphere soil sample was weighed and added to a 100 mL Erlenmeyer flask containing 10 mL of sterile water. The sample was taken out after shaking and incubating at 25°C and 180 rpm / min for 1 hour. After standing for 30 minutes and stratifying, 1 mL of the upper layer was taken and added to 9 mL of sterile water to obtain 10 -1 The gradient soil dilution was diluted to obtain 10 -2 to 10 -7 Soil dilutions of different concentration gradients. Use a pipette to draw 200 μL of soil suspensions of different concentrations and apply them on CD culture medium, and place them in a constant temperature incubator at 28°C for 24 to 48 hours. Select colonies of different morphologies, streak them on NA culture medium to obtain a single colony, which is named DLYK-221. The single colony DLYK-221 that has been purified multiple times is transferred to 60% glycerol and stored at -80°C for later use.

[0026] The selected strain DLYK-221 was inoculated on NA medium for 24 h activation, morphological observation, and Gram staining. The results were as follows: Figure 1 As shown in the figure, it can be seen that the colony edge of this strain is irregular, convex, milky white, and opaque; it is a Gram-positive bacterium with spores, rod-shaped, and 4 to 10 μm.

[0027] The universal primers 16S-27F and 16S-1492R were used as amplification primers. The specific primers are as follows. The 16s rRNA gene of strain DLYK-221 was amplified and identified. The sequencing results were compared with the BLAST homology in the NCBI database, and related sequences were screened out based on sequence similarity. The phylogenetic tree was constructed using the 16s rRNA gene sequence. The results are as follows Figure 2 As shown, the strain was further identified as Bacillus velezensis DLYK-221, and its taxonomic name was Bacillus velezensis. The strain was deposited in the China Center for Type Culture Collection on December 9, 2024, with the deposit address at Wuhan University, Wuhan, China, and the deposit number is CCTCC NO: M20242761.

[0028] The universal primers and amplification results are as follows:

[0029] 16S-27F (SEQ ID NO. 1):

[0030] 5′-AGAGTTTGATCTGGCTCAG-3′;

[0031] 16S-1492R (SEQ ID NO. 2):

[0032] 5'-TACGGCTACCTTGTTACGACTT-3'.

[0033] Amplified 16s rRNA gene sequence (SEQ ID NO.3):

[0034] CCGGGGGCTGCCTATACTGCAAGTCGAGCGGACAGATGGGAGCTTGCTCCCTGATGTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCTGTAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATGGTTGTCTGAACCGCATGGTTCAGACATAAAAGGTGGCTTCGGCTACCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTAGGGAAGAACAAGTGCCGTTCAAATAGGGCGGCACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGGGCTCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGGAACTTGAGTGCAGAAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGACTCTCTGGTCTGTAACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGGGGAACATGTGGTTTAATTCGAAGGAACGCGAAGAACCTTACAAGGGCTTGAA。

[0035] Example 2 Preparation of biocontrol agents and verification of biological control effects

[0036] 1. Preparation of biocontrol agents

[0037] The strain DLYK-221 activated on NA solid medium was inoculated into NA liquid medium and cultured at 28°C and 180 rpm for 12 h, then centrifuged at 8000 rpm for 5-8 min, and resuspended in sterile water to adjust the spore concentration to 1×10 7 CFU / mL, and obtain the biocontrol agent.

[0038] 2. Verification of biological control effectiveness

[0039] Weigh an appropriate amount of fresh tobacco leaves with tomato spotted wilt virus and grind them thoroughly in a clean mortar. Add 50mM pH=7.2 phosphate inoculation buffer to the leaves at a ratio of 1:5 to dilute and prepare the tomato spotted wilt virus inoculation solution. Use the friction method to inoculate the inoculation solution. Dip a cotton swab in the tomato spotted wilt virus inoculation solution and gently rub it on the surface of the tobacco leaves sprinkled with a small amount of quartz powder. Inoculate a total of 40 tobacco plants. After inoculation with tomato spotted wilt virus, place the plants in a plant growth room, control the temperature and humidity at 28°C and 70% respectively, and culture for 16 days.

[0040] Twenty-four susceptible tobacco plants with the same growth potential were selected and two treatments were set up, namely treatment A (root irrigation with clean water, recorded as CK) and treatment B (root irrigation with biocontrol agents, recorded as DLYK-221), with 12 plants in each treatment.

[0041] The test method is to start root irrigation when the diseased tobacco has four leaves and one heart, for a total of 3 times, 10 mL each time, with an interval of 5 days between each time.

[0042] The investigation method is to take photos and collect leaves 5 days after the last root irrigation, and perform RT-qPCR detection using tomato spotted wilt virus-specific primers (TSWV-F, TSWV-R). The specific primers are as follows:

[0043] TSWV-F (SEQ ID NO.4):

[0044] 5'-GTGAAGAAAGGGAAAGAGTATGCTGC-3';

[0045] TSWV-R (SEQ ID NO.5):

[0046] 5'-GGGGTTAAAAAACAGGCAAAACTCAC-3'.

[0047] The RT-qPCR detection method is as follows: Total RNA was extracted by using TRANZOL reagent (Invitrogen, catalog number 15596-026). cDNA was obtained using an RT kit. The reaction was performed on a fluorescence quantitative instrument using SYBR premix SuperMix (Vazyme TM , No. R133-01). The PCR reaction was carried out under the following conditions: 94°C for 10 min; 95°C for 15 s, 60°C for 1 min, 95°C for 15 s, 40 cycles; and finally 72°C for 5 min. Among them, the NbActin gene was selected as the internal reference.

[0048] Among them, the control results of the biocontrol agent on tomato spotted wilt are as follows: Figure 3 The relative content of tomato spotted wilt virus in tobacco treated with different methods is shown in Figure 4 As shown. It can be seen that after treatment with Bacillus Velez DLYK-221, the content of tomato spotted wilt virus in the bacterial agent group was almost undetectable compared with the water control group. Combined with the tobacco growth phenotype after bacterial agent treatment ( Figure 3 ), the results showed that Bacillus Velezii DLYK-221 had a good control effect on tobacco spotted wilt, had a strong inhibitory effect on the expression of tomato spotted wilt virus, and the control effect on tobacco spotted wilt caused by tomato spotted wilt virus was more than 99%.

[0049] The prepared biocontrol agent was diluted in a gradient manner to a spore concentration of 1×10 8 , 1×10 9 , 1×10 10 , 1×10 11 , 1×10 12 CFU / mL, and root irrigation was used to control the tobacco infected with tomato spotted wilt virus. At the same time, the growth phenotype of tobacco was observed and the expression of tomato spotted wilt virus was measured. The results showed that when the concentration was diluted to 1×10 10 When the spore concentration was as low as 1×10 11 When the fungal disease control effect is significantly reduced, it can be seen that the biocontrol agent provided by the present invention has a spore concentration of DLYK-221 of 1×107- 10 , can be used for the effective prevention and treatment of tobacco spotted wilt.

[0050] Therefore, the promotion and application of DLYK-221 is expected to replace chemical pesticides to control tobacco spotted wilt, protect the environment, reduce pesticide residues, and improve the safety of tobacco leaves. It can be used as a biocontrol strain for the prevention and control of tobacco spotted wilt, and has broad application prospects in the field of tobacco cultivation.

[0051] Although the content of the present invention has been described in detail through the above preferred embodiments, it should be appreciated that the above description should not be considered as a limitation of the present invention. After reading the above content, it will be apparent to those skilled in the art that various modifications and substitutions of the present invention will occur. Therefore, the protection scope of the present invention should be limited by the appended claims.

Claims

1. A biocontrol agent for Bacillus velezensis DLYK-221, characterized in that: This strain was deposited in the China Center for Type Culture Collection on December 9, 2024, with the deposit address being Wuhan University, Wuhan, China, and the deposit number being CCTCC NO:M20242761.

2. Use of Bacillus Velez DLYK-221 as claimed in claim 1 in inhibiting the expression of tomato spotted wilt virus.

3. Use of Bacillus Velezii DLYK-221 as claimed in claim 1 in preventing and treating tobacco spotted wilt.

4. Use of the Bacillus Velezii DLYK-221 as claimed in claim 1 in the preparation of a drug for preventing and treating tobacco spotted wilt.

5. A biocontrol agent for the prevention and treatment of tobacco spotted wilt, characterized in that: The biocontrol agent comprises the Bacillus Velezii DLYK-221 as claimed in claim 1.

6. The biocontrol agent according to claim 5, characterized in that The biocontrol agent is a bacterial suspension of Bacillus Velezii DLYK-221, and its spore concentration is 1×10 7-10 CFU / mL.

7. Use of the biocontrol agent according to claim 5 or 6 in the field of tobacco planting.

8. The use according to claim 7, characterized in that: The biocontrol fungicide can be used for preventing and controlling tobacco spotted wilt.

9. The use according to claim 8, characterized in that: The tobacco spotted wilt is caused by tomato spotted wilt virus.

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