Delftia delbrueckii Y96 and application of Delftia delbrueckii Y96 in prevention and treatment of tobacco bacterial wilt
By providing Dellford fungi Y96 and its biodefense preparations, the problems of long breeding cycle of disease-resistant varieties in the prevention and control of tobacco bacterium wilt have been solved, and the problems of long breeding cycles of disease-resistant varieties, high environmental harm to the environment, fewer biodefense species and limited prevention and control effects have been achieved, and safe, non-toxic and residue-free tobacco bacterium wilt have been achieved, reducing the incidence rate and promoting the development of tobacco cultivation industry.
Patent Information
- Application Number
- CN202411983004.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-31
- Publication Date
- 2025-04-11
AI Technical Summary
The prior art has problems in the prevention and control of tobacco green wilt, with long breeding cycles of disease-resistant varieties, great harm to the environment, fewer types of bio-drugs, and limited prevention and control effects.
A strain of Delford fungi Y96 and its biocontrol agent were provided. By inoculating Delford fungi Y96 into culture medium, a biocontrol agent for application to the soil was prepared to prevent and treat tobacco green wilt.
Dellford fungus Y96 has an inhibitory effect on tobacco vermicelli. Its fermented substances have a good prevention and control effect on tobacco vermicellili. It has achieved safe, non-toxic and residual prevention and control of tobacco vermicellili, reduced the incidence rate and promoted the development of tobacco planting industry.
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Figure CN119799567A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of biocontrol bacteria, in particular to a strain of Delftia Y96 and application thereof in preventing and controlling tobacco bacterial wilt. Background Art
[0002] Bacterial wilt is a bacterial wilt disease caused by Ralstonia solanacearum, which can infect more than 200 plant species from 50 families, including economic crops such as tobacco, tomatoes and potatoes. The outbreak of this disease can cause a large-scale reduction in crop yields or even a total crop failure.
[0003] At present, the main means of prevention and control of tobacco bacterial wilt are agricultural prevention, chemical prevention and biological prevention. Among them, the breeding of disease-resistant varieties is an important measure of agricultural prevention and control, but the breeding cycle of disease-resistant varieties is long and the effect is slow, especially the pathogens mutate frequently, and highly resistant varieties cannot fully adapt to the rapid mutation of pathogens. Chemical control is currently the most commonly used prevention and control method. It has the advantages of quick effect, convenience and low loss, but the long-term use of chemical pesticides endangers environmental safety and causes irreversible damage to the soil. Biological control has the advantages of economy, pollution-free and long-lasting effect, which meets the requirements of sustainable agricultural development. However, there are few types of biocontrol bacteria at present, and the prevention and control effect is limited. In order to enrich the biocontrol microorganisms and microbial products of tobacco bacterial wilt, it is necessary to find a new biocontrol microorganism for tobacco bacterial wilt and prepare corresponding derivative products to promote the development of tobacco planting industry. Summary of the invention
[0004] In order to solve the above problems, the present invention provides a strain of Delftia Y96 and its application in preventing and controlling tobacco bacterial wilt. The Delftia Y96 provided by the present invention has an inhibitory effect on tobacco bacterial wilt pathogens, and its fermentation product also has a good prevention and control effect on tobacco bacterial wilt, which is conducive to promoting the development of tobacco planting industry.
[0005] In order to achieve the above object, the present invention provides the following technical solutions:
[0006] The invention provides a strain of Delftia sp. Y96, whose deposit number is CGMCCNO.31157.
[0007] The present invention provides a biocontrol agent, comprising Delftia Y96 and / or extracellular secretions of Delftia Y96; the Delftia Y96 is the Delftia Y96 described in the above technical solution.
[0008] Preferably, when the biocontrol agent contains Delftia Y96, the OD of Delftia Y96 in the biocontrol agent is 600 ≥1.
[0009] The present invention provides a method for preparing the biocontrol agent described in the above technical solution, including the first method, the second method or the third method;
[0010] The first method comprises: inoculating the Delftia Y96 into a culture medium, culturing to obtain a culture solution, which is the biocontrol agent; the culture medium comprises a TSB liquid culture medium, an LB liquid culture medium or a NB liquid culture medium;
[0011] The second method comprises: centrifuging the culture solution obtained by the first method to obtain a supernatant, which is the biocontrol agent;
[0012] The third method comprises: centrifuging the culture solution obtained by the first method to obtain bacterial precipitation; and resuspending the bacterial precipitation with sterile water to obtain the biocontrol agent.
[0013] Preferably, the culture temperature is 28-30°C, the rotation speed is 150-200 r / min, and the time is 1-2 days.
[0014] The present invention provides the use of Delftia Y96 described in the above technical solution, or the biocontrol agent described in the above technical solution, or the biocontrol agent prepared by the preparation method described in the above technical solution in preventing and controlling plant diseases caused by Ralstonia solanacearum.
[0015] Preferably, the plant disease includes tobacco bacterial wilt.
[0016] The present invention provides a method for preventing and treating tobacco bacterial wilt, comprising the following steps:
[0017] The biocontrol agent is applied to soil where tobacco is planted; the biocontrol agent is the biocontrol agent described in the above technical solution or the biocontrol agent prepared by the preparation method described in the above technical solution.
[0018] Preferably, the application method is root irrigation, and the application amount per tobacco plant is 10 mL.
[0019] Preferably, the timing of application is 1 to 3 days after the tobacco seedlings are transplanted.
[0020] Beneficial effects:
[0021] The present invention provides a strain of Delftia Y96, with a deposit number of CGMCC NO.31157. The Delftia Y96 provided by the present invention is a biocontrol bacterium screened from healthy tobacco root soil, has a good inhibitory effect on tobacco bacterial wilt, and has a good preventive effect on tobacco bacterial wilt. After testing, Delftia Y96 has an inhibitory effect on tobacco bacterial wilt on a plate, and its fermentation product has a good preventive and control effect on tobacco bacterial wilt, and the bacterium is isolated from tobacco rhizosphere soil, indicating that the bacterium can be colonized in the tobacco root system. Compared with chemical pesticides, the strain has no toxic side effects and no residue on tobacco plants and soil ecology, has good environmental safety, has good development prospects and application potential, and is particularly important for the prevention and control of tobacco bacterial wilt. BRIEF DESCRIPTION OF THE DRAWINGS
[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.
[0023] Figure 1 Delftia Y96 purified by plate streak method;
[0024] Figure 2 is the 16S rRNA phylogenetic tree of Delftia Y96;
[0025] Figure 3 This is a plate inhibition effect diagram of Delftia Y96 on tobacco bacterial wilt pathogen;
[0026] Figure 4 This is a diagram showing the plate inhibition effect of the supernatant culture solution of Delftia Y96 on tobacco bacterial wilt pathogen;
[0027] Figure 5 This is a graph showing the control effect of biocontrol agent Y96 on tobacco bacterial wilt.
[0028] Biological Deposit Description
[0029] Delftia sp. Y96, classified and named as Delftia sp., was deposited at the General Microbiology Center of China Culture Collection Administration on July 3, 2024. The deposit address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCCNO.31157. DETAILED DESCRIPTION
[0030] The invention provides a strain of Delftia sp. Y96, whose deposit number is CGMCCNO.31157.
[0031] The Delftia Y96 provided by the present invention is a biocontrol bacterium screened from healthy tobacco root soil, can colonize in the tobacco root system, has a good inhibitory effect on tobacco solanacearum, and has a good preventive effect on tobacco bacterial wilt, can achieve safe, non-toxic and residue-free prevention and control of tobacco bacterial wilt, and is beneficial to promoting the development of the tobacco planting industry.
[0032] The present invention provides a biocontrol agent, comprising Delftia Y96 and / or extracellular secretions of Delftia Y96; the Delftia Y96 is the Delftia Y96 described in the above technical solution.
[0033] As an embodiment, the OD of Delftia Y96 in the biocontrol agent is 600 ≥1.
[0034] The Delftia Y96 and the extracellular secretions thereof provided by the invention can inhibit the growth of tobacco solanacearum and can be used for preparing a biocontrol agent for preventing and controlling tobacco solanacearum.
[0035] The present invention provides a method for preparing the biocontrol agent described in the above technical solution, including the first method, the second method or the third method;
[0036] The first method comprises: inoculating the Delftia Y96 into a culture medium, culturing to obtain a culture solution, which is the biocontrol agent; the culture medium comprises a TSB liquid culture medium, an LB liquid culture medium or a NB liquid culture medium;
[0037] The second method comprises: centrifuging the culture solution obtained by the first method to obtain a supernatant, which is the biocontrol agent;
[0038] The third method comprises: centrifuging the culture solution obtained by the first method to obtain bacterial precipitation; and resuspending the bacterial precipitation with sterile water to obtain the biocontrol agent.
[0039] In one embodiment, the culture temperature is 28-30°C, the rotation speed is 150-200 r / min, and the time is 1-2 days. In another embodiment, the culture temperature is 30°C, the rotation speed is 150-180 r / min, and the time is 2 days.
[0040] Based on the above advantages, the present invention provides the use of Delftia Y96 described in the above technical solution or the biocontrol agent described in the above technical solution or the biocontrol agent prepared by the preparation method described in the above technical solution in preventing and controlling plant diseases caused by Ralstonia solanacearum. As an embodiment, the plant disease includes tobacco bacterial wilt.
[0041] Based on the above advantages, the present invention provides a method for preventing and controlling tobacco bacterial wilt, comprising the following steps:
[0042] The biocontrol agent is applied to soil where tobacco is planted; the biocontrol agent is the biocontrol agent described in the above technical solution or the biocontrol agent prepared by the preparation method described in the above technical solution.
[0043] As an implementation mode, the application method is root irrigation, and the application amount per tobacco plant is 10 mL.
[0044] As an embodiment, the timing of application is 1 to 3 days after the tobacco seedlings are transplanted. As an embodiment, the timing of transplanting is when the tobacco seedlings have 5 to 6 leaves.
[0045] The biocontrol agent provided by the present invention can effectively reduce the incidence of tobacco bacterial wilt (can reduce the incidence by 40%), can achieve safe, non-toxic and residue-free prevention and control of tobacco bacterial wilt, and is beneficial to promoting the development of tobacco planting industry.
[0046] To further illustrate the present invention, a strain of Delftia Y96 provided by the present invention and its application in preventing and controlling tobacco bacterial wilt are described in detail below in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.
[0047] The tobacco solanacearum used in the embodiment of the present invention is Ralstonia solanacearum, which is disclosed in the document [Tian Xuehui. Development of colloidal gold immunochromatographic test strips for tobacco solanacearum [D]. Nanjing Agricultural University, 2022.].
[0048] Example 1 Isolation and identification of Delftia Y96
[0049] The Delftia Y96 of the present invention is isolated and purified from healthy tobacco rhizosphere soil by dilution plating method and plate streak method, as follows:
[0050] 1. Isolation and purification of strains.
[0051] The separation method is as follows: 1 g of rhizosphere soil is placed in a 50 mL centrifuge tube, 9 mL of sterile water is added, and the tube is shaken at 180 r / min and 28 °C for 30 min to make 10 -1 Dilution. Pipette 100 μL 10 -1 Dilute the solution in 900 μL sterile water to obtain 10 -2 Dilution, dilute in this way to obtain 10% of the soil -3 and 10 -4 Dilution. Pipette 100 μL 10 -3 , 10 -4The 100-fold dilution was applied to the NA solid medium plate and evenly spread with a sterilized applicator. After constant temperature culture at 30°C for 72 hours, the colonies of different forms on the NA solid medium were picked up by an inoculation loop and streaked on a new NA solid medium plate. The strains were then purified by the plate streaking method, numbered and preserved, and antagonistic bacteria were screened ( Figure 1 ), a strain antagonistic to tobacco solanacearum was obtained, numbered as Y96. The NA solid medium was prepared as follows: 10.0 g glucose, 5.0 g peptone, 3.0 g beef extract, 1 g yeast extract, 18.0 g agar powder, 1000 mL distilled water, pH adjusted to 7.0, and sterilized at 121° C. with high pressure steam for 30 min.
[0052] 2. 16S rDNA sequence analysis.
[0053] After the DNA of strain Y96 was extracted using an extraction kit, PCR amplification was performed using 16S universal primers 27F and 1492R. The primer sequences are as follows:
[0054] 27F: 5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID NO: 1).
[0055] 1492R: 5'-TACGGCTACCTTGTTACGACTT-3' (SEQ ID NO: 2).
[0056] The amplified PCR stock solution was sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing. The 16S rDNA sequence of strain Y96 is shown in SEQ ID NO: 3, as follows:
[0057] 5'-CCGACTCTGCCTTAACATGCAAGTCGAACGGTAACAGGTCTTCGG ACGCTGACGAGTGGCGAACGGGTGAGTAATACATCGGAACGTGCCCAGTCGTGGGGGATAACTACTCGAAAGAGTAGCTAATACCGCATACGATCTGAGGATGAAAGCGGGGGACCTTCGGGCCTCGCGCGATTGGAGCGGCCGATGGCAGATTAGGTAGTTGGTGGGATAAAAGCTTACCAAGCCGACGATCTGTAGCTGGTCTGAGAGGACGACCAGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATTTTGGACAATGGGCGAAAGCCTGATCCAGCAATGCCGCGTGCAGGATGAAGGCCTTCGGGTTGTAAACTGCTTTTGTACGGAACGAAAAAGCTCCTTCTAATACAGGGGGCCCATGACGGTACCGTAAGAATAAGCACCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGTGCGAGCGTTAATCGGAATTACTGGGCGTAAAGCGTGCGCAGGCGGTTATGTAAGACAGATGTGAAATCCCCGGGCTCAACCTGGGAACTGCATTTGTGACTGCATGGCTAGAGTACGGTAGAGGGGGATGGAATTCCGCGTGTAGCAGTGAAATGCGTAGATATGCGGAGGAACACCGATGGCGAAGGCAATCCCCTGGACCTGTACTGACGCTCATGCACGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCCTAAACGATGTCAACTGGTTGTTGGGAATTAGTTTTCTCAGTAACGAAGCTAACGCGTGAAGTTGACCGCCTGGGGAGTACGGCCGCAAGGTTGAAACTCAAAGGAATTGACGGGGACCCGCACAAGCGGTGGATGATGTGGTTTAATTCGATGCAACGCGAAAAACCTTACCCACCTTTGACATGGCAGGAAGTTT-3'。
[0058] The sequence obtained was submitted to the GenBank database for BLAST analysis and comparison, and it was found that the homology between strain Delftialacustris and strain Y96 could reach 99.90%. The 16S rDNA sequence with higher homology was selected as the reference object to construct the phylogenetic tree. The results are shown in Figure 2 According to the phylogenetic tree, strain Y96 was identified as Delftia sp. and deposited in the General Microbiological Center of China Microbiological Culture Collection Administration with the deposit number CGMCC NO.31157.
[0059] Example 2 Plate antibacterial assay of strain Y96 against tobacco bacterial wilt pathogen
[0060] 1. Activation of tobacco solanacearum.
[0061] Take out the R. solanacearum strains stored in a -80℃ refrigerator, thaw naturally, pick up the bacterial liquid with an inoculation loop, streak it on the NA solid medium, and incubate it upside down in a 37℃ constant temperature incubator for 24 hours to grow a single colony for use in the antibacterial test.
[0062] 2. Determination of the antibacterial effect of strain Y96.
[0063] Take out strain Y96 stored in a -80℃ refrigerator, thaw naturally, use an inoculation loop to pick up the bacterial solution and streak it on NA solid culture medium, and invert and culture it in a 37℃ constant temperature incubator for 24 hours. Allow it to grow into a single colony, pick up a single colony and inoculate it into NA liquid culture medium, and culture it in a shaking incubator at 30℃ and 180r / min for 48 hours. Take 2μL of the bacterial solution and inoculate it on a NA plate, and culture it at 30℃ and statically for 48 hours. Pick up a single colony from the activated tobacco solanacearum plate and inoculate it into NA liquid culture medium, and culture it in a shaking incubator at 30℃ and 180r / min for 48 hours. Resuspend the bacterial solution with sterile water. Dilute with water to OD 600 =1, get OD 600 = 1 of the bacterial solution of tobacco solanacearum. The bacterial solution was evenly sprayed on the plate with Y96 colonies using a spray bottle method, and the plates were confronted. The results are shown in Figure 3 .
[0064] Depend on Figure 3 It can be seen that the strain Y96 isolated by the present invention has a good antibacterial effect on tobacco solanacearum.
[0065] 3. Determination of the antibacterial effect of extracellular secretions of strain Y96.
[0066] Strain Y96 was selected and placed in NB liquid culture medium, and cultured in a shaking incubator at 30°C and 150 r / min. The obtained bacterial suspension was centrifuged at 12000 rpm for 6 min, and the supernatant culture of strain Y96 was taken. The inhibitory activity against Ralstonia solanacearum was detected by the punching method. The specific operation was as follows: a 11 mm diameter punch was used to punch holes on the NA culture plate mixed with Ralstonia solanacearum, and the supernatant culture of strain Y96 was added to the holes respectively, and sterile NB liquid culture medium was used as a control. The experiment was repeated 3 times, and the diameter of the inhibition zone was measured after culturing at 37°C for 48 h. The test results are shown in Figure 4 The diameter of the inhibition zone was determined to be 9.0 mm.
[0067] Example 3 Greenhouse control test of strain Y96 on tobacco bacterial wilt
[0068] 1. Preparation of pathogens and biocontrol agents.
[0069] Take out the tobacco solanacearum strain stored in a -80℃ refrigerator, thaw naturally, pick up the bacterial liquid with an inoculation loop and streak it on the NA solid medium, incubate it upside down in a 37℃ constant temperature incubator for 16 hours, pick a single colony and inoculate it on the NA liquid medium, and incubate it on a shaking table at 37℃, 180r / min for 48 hours. Centrifuge to obtain the bacterial cells, resuspend the bacterial cells with sterile water to obtain a bacterial suspension. Use sterile water to adjust the OD 600 =1, obtain tobacco solanacearum bacterial liquid.
[0070] 2. Preparation of biocontrol agent Y96.
[0071] Take out strain Y96 stored in a -80℃ refrigerator, thaw naturally, pick up the bacterial liquid with an inoculation loop and streak it on NA solid medium, incubate it upside down in a 30℃ constant temperature incubator for 48h, pick up a single colony and inoculate it on NA liquid medium, incubate it on a shaking table at 30℃, 180r / min for 48h. Centrifuge to obtain the bacterial cells, resuspend them with sterile water to obtain a bacterial suspension. Use sterile water to adjust the OD 600 =1(about 1×10 8 CFU / mL), and obtained the biocontrol agent Y96.
[0072] 3. Experimental method.
[0073] The experiment was designed with two treatments, each with 15 tobacco plants, namely the biocontrol agent Y96 treatment group (denoted as RS+Y96) and the control group (denoted as RS+CK). The treatment methods are as follows:
[0074] Tobacco seedlings at the 5-6 leaf stage were selected and transplanted into pots. Three days after transplanting, each tobacco plant in the biocontrol agent Y96 treatment group was watered with 10 mL of biocontrol agent Y96, and each tobacco plant in the control group was watered with 10 mL of sterile water. Five days later, the roots were inoculated with 20 mL of tobacco solanacearum solution. The disease survey time was 21 days after the pathogen was watered, and the incidence rate and disease index were calculated. The tobacco disease classification and survey method refer to the national standard GB / T23222-2008. The results are shown in Table 1 and Figure 5 .
[0075] Disease index = [∑ disease level × number of plants at that disease level) / (highest disease level × total number of plants)] × 100%;
[0076] Control effect = [(control disease index - treatment disease index) / control disease index] × 100%.
[0077] Table 1 The potted control effect of Y96 fungicide on tobacco bacterial wilt
[0078] deal with Incidence % Disease index Control effect% Y96 13.3％* 0.12 94.2％ CK 53.3％ 2.08 /
[0079] *Indicates that there is a significant difference between the Y96-treated group and the control group (P<0.05).
[0080] The results show that the biocontrol agent provided by the present invention can significantly reduce the incidence of tobacco, and can reduce the incidence by 40%.
[0081] In summary, the Delftia Y96 provided by the present invention can colonize in the root system of tobacco, has a strong inhibitory effect on the pathogenic bacteria Ralstonia solanacearum of tobacco bacterial wilt, can achieve safe, non-toxic and residue-free prevention and control of tobacco bacterial wilt, and is conducive to promoting the development of tobacco planting industry.
[0082] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.
Claims
1. A strain of Delftia sp. Y96, characterized in that: The deposit number is CGMCCNO.31157.
2. A biocontrol agent, characterized in that: It comprises Delftia Y96 and / or the extracellular secretions of Delftia Y96; the Delftia Y96 is the Delftia Y96 described in claim 1.
3. The biocontrol agent according to claim 2, characterized in that When the biocontrol agent contains Delftia Y96, the OD of Delftia Y96 in the biocontrol agent is 600 ≥1.
4. The method for preparing the biocontrol agent according to claim 2 or 3, characterized in that: including the first method, the second method or the third method; The first method comprises: inoculating the Delftia Y96 into a culture medium, culturing to obtain a culture solution, which is the biocontrol agent; the culture medium comprises a TSB liquid culture medium, an LB liquid culture medium or a NB liquid culture medium; The second method comprises: centrifuging the culture solution obtained by the first method to obtain a supernatant, which is the biocontrol agent; The third method comprises: centrifuging the culture solution obtained by the first method to obtain bacterial precipitation; and resuspending the bacterial precipitation with sterile water to obtain the biocontrol agent.
5. The preparation method according to claim 4, characterized in that: The culture temperature is 28-30° C., the rotation speed is 150-200 r / min, and the culture time is 1-2 days.
6. Use of the Delftia Y96 according to claim 1, the biocontrol agent according to claim 2 or 3, or the biocontrol agent prepared by the preparation method according to claim 4 or 5 in preventing and controlling plant diseases caused by Ralstonia solanacearum.
7. The use according to claim 6, characterized in that: The plant diseases include tobacco bacterial wilt.
8. A method for preventing and treating tobacco bacterial wilt, characterized in that: The following steps are involved: The biocontrol agent is applied to soil where tobacco is planted; the biocontrol agent is the biocontrol agent according to claim 2 or 3 or the biocontrol agent prepared by the preparation method according to claim 4 or 5.
9. The method according to claim 8, characterized in that The application method is root irrigation, and the application amount for each tobacco plant is 10 mL.
10. The method according to claim 8 or 9, characterized in that: The timing of application is 1 to 3 days after the tobacco seedlings are transplanted.