Pseudomonas koreensis X02 and application thereof in preparation of low-pathogenicity ralstonia solanacearum attenuated strain
Through the Korean Pseudomonas X02, the strong strain of the blue whale bacteria is transformed into a low-pathogenic and insufficiency strain, the problem of difficulty in effectively preventing and controlling blue whale in the existing technology is solved, and the effect of significantly reducing the pathogenicity of blue whale is achieved, and an environmentally friendly and efficient anti-bacterial agent is provided.
Patent Information
- Application Number
- CN202411883775.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-19
- Publication Date
- 2025-04-11
AI Technical Summary
It is difficult to effectively prevent and control bacterium wilt in the existing technology. Traditional prevention and control measures such as methane bromide and streptomycin have environmental and food safety problems, biopesticides are costly and have a short life of effect, and there is a lack of efficient antibacterial agents to control the spread of bacterium wilt.
Through Pseudomonas Korea X02, the strong strain of the blue wax bacteria is transformed into a low-pathogenic and inadequate strain, thereby reducing the pathogenicity of the blue wax and achieving the bioprevention effect on the blue wax.
By converting the strong strain of Cyanobacteria into an inadequate strain, the pathogenicity and toxicity of Cyanobacteria is significantly reduced, the prevention and control effect of Cyanobacteria is improved, and environmental and food safety issues of traditional prevention and control measures are avoided.
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Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of agricultural microorganisms, and specifically relates to a Korean Pseudomonas X02 and an application thereof in preparing a low-pathogenicity solanacearum attenuated strain. Background Art
[0002] Ralstonia solanacearum is the world's second largest plant pathogen. Bacterial wilt caused by its infection is a devastating soil-borne disease, with general losses of 30% to 70%, and even total crop failure in severe cases. Bacterial wilt is widely distributed in tropical, subtropical and temperate regions of the world. Except for a few areas, it is distributed in most parts of my country, especially in the south of the Yangtze River. In recent years, the climate has warmed and the damage in the northern region has also increased year by year. The host of Ralstonia solanacearum is very wide, with as many as 45 families and more than 200 species of plants. Among them, Solanaceae crops with high economic value, including tomatoes, eggplants, peppers, tobacco, etc., and ginger are the most seriously affected, causing serious continuous cropping obstacles. Ralstonia solanacearum has a strong survival ability and can survive for up to ten years even in the absence of hosts. Therefore, conventional dry-dry rotation and short-term water-dry rotation cannot play a good prevention and control role. Methyl bromide fumigation is currently the most effective prevention and control measure, but it has been banned globally by the United Nations due to its damage to the ozone layer. Agricultural streptomycin was once the main control agent, but it is banned from use in agriculture due to cross-resistance, environmental and food safety issues. Disease-resistant varieties are the most economical and effective measure, but to date, no disease-resistant varieties have been used commercially. Biological pesticides, including Bacillus polymyxa and sinoprotinin, can all play a role in preventing and controlling bacterial wilt, but due to high costs and short duration of effectiveness, they have not been widely used in production. Developing new and efficient anti-disease agents to control the spread and damage of bacterial wilt is crucial to promoting sustainable agricultural development. Summary of the invention
[0003] The object of the present invention is to provide a Korean Pseudomonas X02 that can transform a strong strain of Ralstonia solanacearum into a weak strain with low pathogenicity, and the deposit number of the strain is CCTCC NO: 20242816.
[0004] Another object of the present invention is to provide a use of Korean Pseudomonas X02.
[0005] In order to achieve the above object, the present invention is implemented by the following technical solutions:
[0006] Korean Pseudomonas X02 was isolated from tomato-growing soil in Dongxihu District, Hubei Province, and belongs to Korean Pseudomonas (Pseudomonas koreensis). The fermentation broth and / or fermentation supernatant of the strain has no direct inhibitory effect on bacterial wilt, but it can achieve the biological control effect on bacterial wilt by converting the strong strain of bacterial wilt into a weak strain with low pathogenicity. The strain was submitted to the China Center for Type Culture Collection (CCTCC) for preservation on December 16, 2024, with a preservation number of CCTCCNO: 20242816, classification name: Pseudomonas koreensis X02, address: Wuhan University, Wuhan, China, and the Korean Pseudomonas X02 contains the polynucleotides shown in SEQ ID NO.1~18.
[0007] The protection scope of the present invention also includes:
[0008] The fermentation product of Korean Pseudomonas X02 contains live bacteria of Korean Pseudomonas X02.
[0009] A composition containing Pseudomonas koreana X02 or a fermentation product thereof.
[0010] Application of Korean Pseudomonas X02, a fermentation product of Korean Pseudomonas X02 or a composition containing Korean Pseudomonas X02 or its fermentation product in the preparation of a low pathogenicity solanacearum attenuated strain.
[0011] In the above-mentioned application, preferably, the application process comprises: mixing Korean Pseudomonas X02, a fermentation product of Korean Pseudomonas X02, or a composition containing Korean Pseudomonas X02 or its fermentation product with a strong strain of Ralstonia solanacearum.
[0012] Use of Korean Pseudomonas X02, a fermentation product of Korean Pseudomonas X02, or a composition containing Korean Pseudomonas X02 or a fermentation product thereof in biological control of Ralstonia solanacearum;
[0013] The application described above is to irrigate the roots with the above substances.
[0014] Application of Korean Pseudomonas X02, fermentation products of Korean Pseudomonas X02 or a composition containing Korean Pseudomonas X02 or its fermentation products in the preparation of a biological control agent for bacterial wilt.
[0015] Compared with the prior art, the present invention has the following significant features:
[0016] Different from the disease prevention mechanism of existing biological or chemical fungicides, the fermentation broth of Korean Pseudomonas X02 has no antibacterial activity against Ralstonia solanacearum. Instead, it prevents the occurrence of Ralstonia solanacearum by converting highly virulent strains of Ralstonia solanacearum into weak strains and significantly reducing the expression level of toxic genes, resulting in a significant decrease in the pathogenicity of Ralstonia solanacearum. BRIEF DESCRIPTION OF THE DRAWINGS
[0017] Figure 1 The colony morphology and optical microscope image of Korean Pseudomonas X02 of the present invention;
[0018] Among them: Figure A is the colony morphology of Korean Pseudomonas X02; Figure B is the optical microscope image of Korean Pseudomonas X02.
[0019] Figure 2 The antagonistic activity diagram of Korean Pseudomonas X02 and streptomycin against Ralstonia solanacearum of the present invention;
[0020] Among them: Figure A is the inhibition zone diagram of the Korean Pseudomonas X02 fermentation broth after filtration through a bacterial filter; Figure B is the antagonistic activity of streptomycin against Ralstonia solanacearum.
[0021] Figure 3 The effect diagram of the different components of Korean Pseudomonas X02 of the present invention on the prevention and control of tomato bacterial wilt;
[0022] Among them: Figure A is a picture showing the prevention and control effect of the original fermentation broth of Korean Pseudomonas X02; Figure B is a picture showing the prevention and control effect of the supernatant of the original fermentation broth of Korean Pseudomonas X02 after centrifugation at 8000rpm; CK is the control.
[0023] Figure 4 The disease pattern of tomato inoculated with the weak strain obtained by treating the strong strain of Ralstonia solanacearum with the Korean Pseudomonas X02 of the present invention;
[0024] Among them: Figure A shows the disease occurrence of tomatoes inoculated with a strong strain of Ralstonia solanacearum; Figure B shows the disease occurrence of tomatoes inoculated with a weak strain of Ralstonia solanacearum obtained by treating Korean Pseudomonas X02. DETAILED DESCRIPTION
[0025] The technical solutions described in the present invention, unless otherwise specified, are all conventional solutions in the art, and the reagents or materials, unless otherwise specified, are all from commercial channels.
[0026] Embodiment 1:
[0027] Screening and acquisition of Korean Pseudomonas X02:
[0028] (1) Isolation and purification: The strain was isolated from tomato planting soil in Dongxihu District, Wuhan City, Hubei Province, in October 2022. Take 10g of the above soil and add it to 90ml of sterile water for shaking and mixing, then dilute it by gradient dilution method, spread the dilution plate on LB solid culture medium, place it in a 28℃ incubator for 48 hours, pick a single colony and repeat the streaking 2 to 3 times to obtain a pure isolate. The purified single colony was inoculated into LB liquid culture medium, shaken and cultured at 28℃ and 150rpm for 48 hours, and the fermentation liquid was used for potted disease prevention effect screening.
[0029] (2) Screening of disease prevention effects:
[0030] Preparation of potting soil: Potting test is used for disease prevention effect screening. The matrix is peat soil: vermiculite: perlite in a volume ratio of 8:1:1, mixed evenly and put into a plastic pot of size 300g.
[0031] Preparation of Ralstonia solanacearum toxic soil: Tomato Ralstonia solanacearum pathogen (strong strain) was cultured in SPA medium for 48 h and OD was adjusted. 620 To 1.2, then dilute 10 times and pour 10 mL into each pot.
[0032] Transplantation and root irrigation: 48 hours after the solanacearum was injected into the potting soil, the 4-5 leaf stage tomato seedlings were transplanted into plastic pots, and the fermentation liquid of the isolated strain obtained in step (1) was diluted 5 times for root irrigation of tomatoes. The control group was injected with the same volume of sterile water. The occurrence standard of bacterial wilt disease was that the leaves of the tomato seedlings wilted for more than 2 days and could not be relieved by watering. The corrected control effect was calculated according to the following formula:
[0033] Incidence rate (%) = number of diseased plants / total number of plants ╳ 100;
[0034] Corrected control efficacy (%) = (control incidence - treatment incidence) / control incidence ╳ 100;
[0035] 30 days after transplanting, the incidence of the blank control was 100%, and the incidence of an isolated strain treated with fermentation liquid was 3%, with a corrected control efficiency of 97%, confirming that the strain has a significant effect in preventing and controlling bacterial wilt disease, and further identification is required.
[0036] (3) 16S rDNA sequence identification
[0037] The above-mentioned isolate was amplified by PCR using the prokaryotic 16S rDNA universal primers (forward primer) 27F (5'AGAGTTTGATCMTGGCTCAG3') and (reverse primer) 1492R (5'GGYTACCTTGTTACGACTT3'), and the obtained sequence was used to construct a phylogenetic evolutionary tree of the bacteria using CLUSTALW software (https: / / www.genome.jp / tools-bin / clustalw), and it was found that the isolate of the present invention can stably cluster with Korean Pseudomonas strains. The present invention identified the strain as Pseudomonas koreensis X02.
[0038] (4) Biological characteristics of Pseudomonas koreensis X02
[0039] The colonies of the Korean Pseudomonas X02 isolated in the LB solid culture medium are yellow, round, with a diameter of about 0.2-0.3 cm and smooth edges ( Figure 1 ).
[0040] (5) Korean Pseudomonas X02 genome
[0041] The whole genome shotgun (WGS) strategy was used to construct libraries with different inserts, and the libraries were sequenced using the second-generation sequencing technology (NGS) based on the Illumina NovaSeq sequencing platform, and the third-generation single-molecule sequencing technology based on the Oxford Nanopore ONT sequencing platform. The genome was assembled using Unicycler and Flye software, and the high-quality data of the second generation was corrected using pilon software, and the final Korean Pseudomonas X02 genome was 5.97M bp.
[0042] (6) Korean Pseudomonas X02 genome-specific DNA sequence
[0043] The BLASTN program was used to compare the Korean Pseudomonas X02 genome with the bacterial genome data in NCBI to find specific DNA sequences, and the PCR method was used for verification. Through the above method, a total of 18 specific DNA sequences were obtained, as shown in SEQ ID NO.1-18.
[0044] The strain was sent to the China Center for Type Culture Collection (CCTCC) for preservation on December 16, 2024, with the deposit number CCTCC NO:20242816, classification name: Pseudomonas koreensis X02, address: Wuhan University, Wuhan, China.
[0045] Embodiment 2:
[0046] Fermentation method of Korean Pseudomonas X02:
[0047] Korean Pseudomonas X02 stored at -80℃ was first activated by streaking on an LB plate. After culturing at 28℃ for 48h, a single colony was picked and placed in a shake flask containing 50mL LB liquid culture medium and cultured at 28℃ and 150rpm for 48h. The OD 600 When the pH value reaches 1.3-1.4, the fermentation liquid of Korean Pseudomonas X02 is obtained.
[0048] Embodiment 3:
[0049] Plate confrontation experiment between Korean Pseudomonas X02 and Ralstonia solanacearum
[0050] (1) Melt the sterilized 1.5% agar water culture medium and add 10 mL / plate to a 9 cm culture dish. After cooling, place an Oxford cup (6 mm inner diameter, 8 mm outer diameter, 10 mm high) in the center of the culture dish. Then add the tomato solanacearum described in Example 1 to the TTC culture medium cooled to 45°C at a volume ratio of 1:100, mix well, and add 10 mL / plate to a culture dish with a diameter of 9 cm until it is completely cooled.
[0051] (2) The fermentation broth of Pseudomonas koreaensis X02 was obtained according to the method described in Example 2, centrifuged at 8000 rpm for 10 minutes, the supernatant was filtered through a 0.22 μm bacterial filter, and 200 μL of the filtrate was added to an Oxford cup. 100 mg / L streptomycin was used as a positive control.
[0052] The results are as follows Figure 2 As shown, the Korean Pseudomonas X02 fermentation supernatant provided by the present invention has a zero inhibition zone against Ralstonia solanacearum, while the streptomycin inhibition zone is obvious, reaching 28 mm, which is significantly higher than the Korean Pseudomonas X02 fermentation supernatant. This indicates that the Korean Pseudomonas X02 fermentation supernatant of the present invention has no effect of inhibiting Ralstonia solanacearum.
[0053] Embodiment 4:
[0054] Application of Korean Pseudomonas X02 in the preparation of low pathogenicity Ralstonia solanacearum weak strains:
[0055] (1) Preparation of Korean Pseudomonas X02 supernatant
[0056] Korean Pseudomonas X02 fermentation used LB medium, the culture conditions were 28°C shaking culture for 48 hours, the fermentation liquid was centrifuged at 8000 rpm / min for 10 minutes, and the supernatant was filtered through a 0.22 μm bacterial filter for use.
[0057] (2) Preparation of highly virulent strains of Ralstonia solanacearum
[0058] The culture medium of tomato solanacearum (hereinafter referred to as solanacearum) described in Example 1 is SPA medium, and the culture conditions are shaking culture at 28°C for 48 hours, and the OD is adjusted to 600 is 1.2.
[0059] (3) Test of attenuated transformation of Ralstonia solanacearum and toxic gene expression
[0060] 1.5mL Korean Pseudomonas X02 supernatant, 0.5mL Ralstonia solanacearum and 8mL SPA medium were added to 50mL test tubes respectively; 1.5mL LB medium, 0.5mL Ralstonia solanacearum and 8mL SPA medium were used as controls. The above treatments were shaken and cultured at 28°C and 150rpm for 12h. 1mL of fermentation broth was taken from each tube, RNA was extracted using the RNA extraction kit produced by Nanjing Novozymes Biotech Co., Ltd., and then cDNA was synthesized using the RevertAid First Strand cDNA kit produced by Thermo Fisher Scientific. The iTaq Universal SYBRGreen supermix fluorescence quantitative kit produced by Bio-Rad Biomedical Products (Shanghai) Co., Ltd. was used for fluorescence quantitative PCR analysis. The 20μL reaction system: SYBR Greensupermix 10.0μL, 1.0μL each of forward and reverse primers, 3μL ddH2O, and 5μL DNA template. The reaction conditions were as follows: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 15 s, annealing at 60°C for 15 s, and extension at 72°C for 30 s, for a total of 40 cycles; and full extension at 72°C for 2 min.
[0061] Taking the highly virulent strain of Ralstonia solanacearum (i.e., the tomato Ralstonia solanacearum described in Example 1) as a reference, the relative expression of the toxic genes of the weakened strain of Pseudomonas koreana X02 was calculated, and the calculation formula is as follows:
[0062] ΔCt 弱毒 =Ct (target gene of weak strain) - Ct (reference gene)
[0063] ΔCt 强毒 =Ct (target gene of virulent strain) - Ct (reference gene)
[0064] ΔΔCt 弱毒 =ΔCt 弱毒 —ΔCt 强毒
[0065] Relative to the strong strain gene expression = 2^ΔΔCt 弱毒
[0066] Gene expression reduction (%) = (1-gene expression level) x 100
[0067] The results are as follows:
[0068] The primers for the internal reference gene and the six pathogenicity-related toxicity genes of Ralstonia solanacearum used in the fluorescence quantitative PCR reaction are as follows:
[0069] Toxicity Genes of Ralstonia solanacearum and Their Fluorescence Quantitative PCR Primers
[0070]
[0071]
[0072] The expression level of toxic genes of Ralstonia solanacearum after being treated with Korean Pseudomonas 02
[0073]
[0074] As shown in the table above: After mixed culture treatment with Korean Pseudomonas X02, the expression levels of 6 pathogenicity-related genes of Ralstonia solanacearum, including hrpB, hrpX, pehB, Rs5-4 819, Rs5-4 374 and epsC-2f, were significantly downregulated, indicating that the pathogenicity and toxicity of Ralstonia solanacearum were significantly reduced.
[0075] Embodiment 5:
[0076] Disease prevention effect of fermentation broth and supernatant of Korean Pseudomonas X02 on tomato bacterial wilt
[0077] (1) Preparation of Korean Pseudomonas X02 fermentation broth and supernatant
[0078] According to the method described in Example 2, Korean Pseudomonas X02 was fermented, and a portion of the fermentation broth was centrifuged at 8000 rpm / min for 10 mins. The supernatant was filtered through a 0.22 μm bacterial filter to obtain the fermentation broth supernatant of Korean Pseudomonas X02.
[0079] (2) Preparation of highly virulent strains of Ralstonia solanacearum
[0080] The culture medium of tomato solanacearum (hereinafter referred to as solanacearum) described in Example 1 is SPA medium, and the culture conditions are shaking culture at 28°C for 48 hours, and the OD is adjusted to 600 is 1.2.
[0081] (3) Potted plant test verification of Korean Pseudomonas X02 fermentation broth and supernatant for the prevention and control of tomato bacterial wilt
[0082] First, prepare potting medium, mix peat soil, vermiculite and perlite in a volume ratio of 8:1:1 and put them into a plastic pot with a size of 7.5cm×10cm×5cm, with a potting volume of 300g. Then, add the fermentation liquid of the strong strain of Ralstonia solanacearum obtained in step (2) to the OD 620 Adjust to 1.2, then dilute 10 times and pour 10 mL into each pot. 48 hours after the injection of Ralstonia solanacearum, transplant the 4-5 leaf stage tomato seedlings into the plastic pots, and at the same time, pour the Korean Pseudomonas X02 fermentation liquid and supernatant obtained in step (1) into the roots of the tomato seedlings, respectively, at a dosage of 30 mL / pot. Check the test results 30 days after transplanting the tomato seedlings. The occurrence of leaf wilt in tomato seedlings, which lasts for more than 2 days and cannot be alleviated by watering, is used as the criterion for the onset of bacterial wilt. The toxicity reduction rate and incidence rate are calculated according to the following formula:
[0083] Treatment incidence rate (%) = number of diseased plants / total number of treated plants ╳ 100
[0084] Toxicity reduction rate (%) = (incidence of tomato treated with strong strains - incidence of tomato treated with weak strains) / incidence of tomato treated with strong strains × 100. The results are as follows: Figure 3 As shown in A, the Korean Pseudomonas X02 fermentation liquid has a 90% efficacy in preventing tomato bacterial wilt. Figure 3 Figure B shows that the supernatant of the fermentation liquid of Korean Pseudomonas X02 has a 90% control effect on tomato bacterial wilt. The two methods have the same control effect on tomato bacterial wilt.
[0085] Embodiment 6:
[0086] The pathogenicity pot test of tomato solanacearum and Korean Pseudomonas X02 after attenuation provided in Example 1 verified:
[0087] First, prepare the potting medium. Mix peat soil, vermiculite and perlite in a volume ratio of 8:1:1 and use it as the medium. Put it into a plastic pot of 7.5cm╳10cm╳5cm in size, with a filling amount of 300g.
[0088] Then, the strong and weak strains of Ralstonia solanacearum described in Example 1 were cultured in SPA medium for 48 h, and the OD 620 Adjust to 1.2 respectively, then dilute 10 times and pour 10 mL into each pot.
[0089] 48 hours after the injection of bacterial wilt, the 4-5 leaf stage tomato seedlings were transplanted into plastic pots. 30 days after the transplanting of the tomato seedlings, the test results were checked. The occurrence of leaf wilt in the tomato seedlings, which lasted for more than 2 days and could not be relieved by watering, was regarded as the criterion for bacterial wilt. The toxicity reduction rate and incidence rate were calculated according to the following formula:
[0090] Treatment incidence rate (%) = number of diseased plants / total number of treated plants ╳ 100
[0091] Toxicity reduction rate (%) = (incidence of tomato treated with strong strains - incidence of tomato treated with weak strains) / incidence of tomato treated with strong strains × 100. The results are as follows: Figure 4 As shown in A, the tomato disease rate after inoculation with a highly virulent tomato strain is 90%. Figure 4 Figure B shows that the morbidity of tomatoes inoculated with a weak strain of Ralstonia solanacearum is 10%. Taking the strong strain of Ralstonia solanacearum as the control, the toxicity reduction rate of the weak strain of Ralstonia solanacearum is 88.89%. The morbidity of tomatoes treated with the original strong strain of Ralstonia solanacearum is more than 90%. After being treated with Korean Pseudomonas X02, the strong strain of Ralstonia solanacearum is transformed into a weak strain and the pathogenicity is significantly reduced.
Claims
1. An isolated Korean Pseudomonas X02 ( Pseudomonas koreensis X02), the deposit number of the Korean Pseudomonas is CCTCC NO: 20242816.
2. The Korean Pseudomonas according to claim 1, wherein the Korean Pseudomonas contains the polynucleotides shown in SEQ ID NOs. 1 to 18.
3. The fermentation product of Korean Pseudomonas X02 according to claim 1, wherein the fermentation product contains live Korean Pseudomonas X02 bacteria.
4. A composition containing Pseudomonas koreana X02 or the fermentation product of claim 3.
5. Use of the Korean Pseudomonas X02 according to claim 1, the fermentation product of the Korean Pseudomonas X02 according to claim 3, or the composition according to claim 4 in preparing a low pathogenicity attenuated strain of Ralstonia solanacearum.
6. The use according to claim 5, characterized in that: The method comprises mixing Korean Pseudomonas X02, a fermentation product of Korean Pseudomonas X02 or a composition containing Korean Pseudomonas X02 or a fermentation product thereof with a strong strain of Ralstonia solanacearum.
7. Use of the Korean Pseudomonas X02 according to claim 1, the fermentation product of the Korean Pseudomonas X02 according to claim 3, or the composition according to claim 4 in biological control of Ralstonia solanacearum.
8. The use according to claim 7, characterized in that: The method comprises irrigating the roots with the Korean Pseudomonas X02 according to claim 1, the fermentation product of the Korean Pseudomonas X02 according to claim 3 or the composition according to claim 4.
9. Use of the Korean Pseudomonas X02 according to claim 1, the fermentation product of the Korean Pseudomonas X02 according to claim 3, or the composition according to claim 4 in the preparation of a biological control agent for bacterial wilt.