Pseudomonas sp. antagonistic to bursaphelenchus xylophilus and application thereof
By isolating and identifying the dispersible pan-mycelium CM-21 with highly efficient pine wilt nematode activity, the problem of the lack of effective strains for the biological control of pine wilt disease has been solved, achieving a 100% pine wilt nematode mortality rate and providing new biological control materials.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- GUANGDONG ACAD OF FORESTRY
- Filing Date
- 2026-03-16
- Publication Date
- 2026-06-05
AI Technical Summary
Current research on biological control of pine wilt disease is relatively backward, with a lack of effective strains, a failure to establish sustainable control technologies, and existing strains not being applied in production practice.
An endophytic bacterium, Pantoea dispersa CM-21, was isolated and identified and named CM-21. It has 100% activity against pine wilt disease and can be used to prepare plant disease control agents for the prevention and control of pine wilt disease.
A highly effective antagonistic strain of pine wilt disease, CM-21, was provided. The corrected mortality rate of pine wilt disease reached 100% after 48 hours of treatment with the fermentation filtrate, providing a new biological control material for the prevention and control of pine wilt disease.
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Figure CN122146523A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of biological control technology, and more specifically to a dispersible pan-winter fungus antagonistic to pine wood nematode and its application. Background Technology
[0002] Pine wilt disease is caused by the pine wilt nematode (Pinus thunbergii var. Bursaphelenchus xylophilus Pine wilt disease is a devastating disease of pine trees that has caused severe ecological and economic losses in many provinces of my country. my country has invested significant human, financial, and material resources in the research and development of integrated management techniques for pine wilt disease, and has carried out fruitful scientific research and exploration. However, due to the complex pathogenesis, rapid spread, and extreme difficulty in controlling the disease, a sustainable control technology has yet to be established.
[0003] Biological control is a scientific and green control measure for plant diseases. However, research on biological control of pine wilt nematodes is relatively lagging. Firstly, very few effective strains have been identified for use against pine wilt nematodes. Secondly, technologies and processes for applying existing nematicidal microbial strains to production practices have not yet been established. Therefore, identifying and stockpiling more microbial strains and determining their role in pine wilt nematode control is a necessary and long-term task in pine wilt nematode control technology research.
[0004] Therefore, how to provide a strain that antagonizes pine wood nematode is a technical problem that urgently needs to be solved by those skilled in the art. Summary of the Invention
[0005] In view of this, the present invention provides a dispersible pan-wine strain antagonistic to pine wood nematode and its application.
[0006] Plant endophytic bacteria possess various biological functions, including nitrogen fixation, growth promotion, and disease and pest resistance. Endophytic bacteria of the genera *Bacillus* and *Pseudomonas* have been isolated from pine trees such as *Pinus massoniana*, *Pinus thunbergii*, and *Pinus slashii*, exhibiting activity against pine wilt nematodes. This invention describes the isolation of an endophytic bacterium, *Panthera dispersa*, from diseased branches of *Pinus massoniana*. Pantoea dispersa The results of indoor immersion testing showed that the corrected mortality rate of pine wilt nematodes was 100% after 48 hours of treatment with the fermentation filtrate of *Panthera dispersans* CM-21. This indicates that *Panthera dispersans* CM-21 can be considered a bacterial strain with high potential for killing pine wilt nematodes, laying the foundation for the future development of microbial agents for the prevention and control of pine wilt disease.
[0007] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:
[0008] A dispersible pan-yeast strain antagonistic to pine wood nematode, named CM-21, is classified as follows: Pantoea dispersaIt was deposited on November 18, 2025 at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCCNo:67318, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.
[0009] A plant disease control agent comprising the above-mentioned dispersible pantothecin, its fermentation broth and / or its metabolites.
[0010] A method for controlling plant diseases, comprising applying the aforementioned plant disease control agent.
[0011] Furthermore, the plant disease mentioned is pine wilt disease.
[0012] The application of the above-mentioned dispersible pan-fungi or the above-mentioned plant disease control agents in the control of pine wilt disease.
[0013] The application of the above-mentioned dispersible pan-yeast or the above-mentioned plant disease control agents in the suppression and killing of pine wilt nematodes.
[0014] As can be seen from the above technical solution, compared with the prior art, the present invention has the following beneficial effects: Currently reported bacterial strains with nematicidal activity against pine wilt disease mainly focus on Bacillus and Pseudomonas genera, while no reports have been found on Pantotheca. This invention has discovered a dispersible Pantotheca strain with pine wilt disease activity. Pantoea dispersa CM-21 is a bacterium discovered for the first time in pine trees that has the potential to control pine wilt disease, and can provide new materials for the prevention and control of pine wilt disease. 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 embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.
[0016] Figure 1 The results of morphological identification of strain CM-21 in Example 1 of the present invention are shown in Figure A, which is a colony morphology of strain CM-21 on LB solid medium (containing 10 g tryptone, 5 g yeast extract, 10 g NaCl, 15 g agar, pH 7.0~7.2 per 1000 mL) and Figure B is a Gram staining image of strain CM-21.
[0017] Figure 2 This is a phylogenetic tree of strain CM-21 in Example 1 of the present invention. The Latin scientific name in the figure is followed by the Genbank number of the corresponding strain.
[0018] Figure 3 The images show the morphology of pine wood nematodes treated with the fermentation filtrate of Pantotheca CM-21 for 48 hours in Example 3 of this invention. In the image, A represents the morphology of pine wood nematodes treated with the treatment group (fermentation filtrate) for 48 hours, and B represents the morphology of pine wood nematodes treated with the control group (LB liquid culture medium) for 48 hours. Detailed Implementation
[0019] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. 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.
[0020] Example 1 Isolation and identification of strain CM-21 1. Isolation of strain CM-21 The Masson pine branches of this invention were collected from pine forests infected with pine wilt disease in Raoping County, Chaozhou City, Guangdong Province. Bacterial strains were isolated from the infected Masson pine branches, purified by streak culture on LB medium, and named CM-21. The strains were stored in a refrigerator at 4°C for later use.
[0021] 2. Identification of strain CM-21 The strain CM-21 was identified using the methods described in "Manual of Systematic Identification of Common Bacteria" edited by Dong Xiuzhu et al. (Beijing: Science Press, 2001: 349-398) and "Microbiology Experiment Manual" edited by Zhou Deqing et al. (Shanghai: Science and Technology Press, 1986: 1-137) and 16S rDNA.
[0022] (1) Morphological identification After streaking culture, strain CM-21 produces single, round, yellow, opaque, viscous, uniform, and non-protruding colonies. Figure 1 A), Gram staining negative ( Figure 1 B).
[0023] (2) Physiological and biochemical identification Physiological and biochemical characteristics of strain CM-21 were determined according to Bergey's Manual of Archaea and Bacteria (BISMiS, 2015 edition), and the results are shown in Table 1.
[0024] Table 1. Physiological and biochemical characteristics of strain CM-21
[0025] Note: "+" indicates positive, and "-" indicates negative.
[0026] The results showed that strain CM-21 could grow on semi-solid agar medium but could not be cultured on peptone water medium; it could produce acid using mannitol, inositol and melibiose, but not using sorbitol, raffinose, ribitol and other substances; the amino acid control, VP reaction, hydrogen sulfide generation, urease and MR experiments were negative.
[0027] (3) Molecular biological identification Strawberry strain CM-21 was streaked in LB solid medium, and single colonies were picked and directly transferred into the PCR reaction solution. PCR amplification was performed using universal primers 27F and 1492R for 16S rDNA. The amplification reaction system is shown in Table 2, the amplification reaction procedure is shown in Table 3, and the primer sequences are as follows.
[0028] 27F: 5'-AGAGTTTGATCCTGGCTCAG-3', SEQ ID No. 1; 1492R: 5'-GGTTACCTTGTTACGACTT-3', SEQ ID No. 2.
[0029] Table 2. PCR amplification reaction system of strain CM-21 16S rDNA
[0030] Table 3 PCR amplification reaction procedure
[0031] The PCR products were detected by 1.5% agarose gel electrophoresis. The amplified product was a DNA fragment of approximately 1500 bp. After purification and recovery of the PCR products using a DNA recovery kit, they were sent to Qingke Biotechnology Co., Ltd. (Guangzhou) for sequencing. The sequencing results are as follows.
[0032]
[0033] The sequenced sequences were subjected to BLAST alignment analysis in the NCBI database (http: / / blast.ncbi.nlm.nih.gov / ). A phylogenetic tree was constructed using the Mega X software with the Neighbor-Joining method for phylogenetic analysis. The values on each branch represent the percentage of support from 1000 Bootstrap resampling analyses.
[0034] Blast analysis showed that strain CM-21 was similar to Pantotheca dispersalis. Pantoea dispersa The homology was highest, with 100% similarity. Further phylogenetic analysis (…) Figure 2 ), found that strain CM-21 and Pantoea dispersa (FJ756349) is most closely related, clustered in the same branch, and is more distantly related to other strains, indicating that this strain is a dispersible pantothenic bacterium.
[0035] Based on the morphological characteristics, physiological and biochemical properties, and 16S rDNA sequence characteristics of the strain, strain CM-21 was identified as *U. dispersans*. Pantoea dispersa ).
[0036] Example 2 Preservation of strain CM-21 Dispersible pantothecin CM-21, its classification name is Pantoea dispersa It was deposited on November 18, 2025 at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No:67318, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.
[0037] Example 3 Nematode activity of dispersible pan-yeast CM-21 against pine wood nematode 1. Activate the dispersed pantothenic acid CM-21 stored at 4℃, pick a single activated colony and inoculate it into a 250 mL Erlenmeyer flask containing 100 mL LB liquid medium. Incubate in a shaker at 28 ℃ and 180 r / min for 2 days. Collect the fermentation broth of the strain, centrifuge at 10000 r / min for 10 min at 4℃, take the supernatant and filter it through a 0.22 μm microporous membrane. The fermentation filtrate after filtration is used for later use.
[0038] 2. Nematode activity was determined using a 24-well cell culture plate immersion method. Artificially cultured pine wood nematodes were collected and a nematode suspension with a concentration of 100 nematodes / 100 μL was prepared. 100 μL of the nematode suspension was added to each well of a 24-well cell culture plate. The treatment group received 900 μL of fermentation filtrate of dispersed pantothecin CM-21, while the control group received 900 μL of sterile LB liquid medium. After adding all materials, the culture plate was gently shaken to ensure thorough mixing of the liquid and pine wood nematodes. The cell culture plates were placed in a 28℃ incubator. After 12h, 24h, and 48h, the pine wood nematodes were observed for rigidity and death. Under a stereomicroscope, the nematodes were punctured with a needle; those that remained completely still were considered dead. The number of dead pine wood nematodes was counted, and the mortality rate and corrected mortality rate were calculated. The calculation formula is as follows: Mortality rate = (Number of dead pine wilt nematodes / Total number of pine wilt nematodes) × 100% Corrected mortality rate = (treatment mortality rate - control mortality rate) / (1 - control mortality rate) × 100% 3. Results Table 4. Corrected mortality rates of pine wood nematodes at different time points after treatment with dispersible pantothecin CM-21
[0039] The results showed that the corrected mortality rate of pine wood nematodes treated with the fermentation filtrate of *Plasmodium dispersum* CM-21 was 19.98% after 12 hours, 86.21% after 24 hours, and 100% after 48 hours (Table 4). After 48 hours of treatment with the fermentation filtrate of *Plasmodium dispersum* CM-21, the pine wood nematodes were in a rigid state; when pricked with a needle, they remained stiff and immobile, while the pine wood nematodes in the control group exhibited a twisting and bending motion. Figure 3 ).
[0040] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.
[0041] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims
1. A dispersible pan-wine fungus antagonizing pine wood nematode, characterized in that, The dispersible pantothecin was named CM-21, and its classification was named... Pantoea dispersa It was deposited on November 18, 2025 at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No:67318, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.
2. A plant disease control agent, characterized in that, Includes the dispersed pantothecin as described in claim 1, its fermentation broth, and / or its metabolites.
3. A method for controlling plant diseases, characterized in that, Apply the plant disease control agent as described in claim 2.
4. The method as described in claim 3, characterized in that, The plant disease in question is pine wilt disease.
5. The application of the dispersible pantothecin according to claim 1 or the plant disease control agent according to claim 2 in the control of pine wilt disease.
6. The application of the dispersible pantothecin as described in claim 1 or the plant disease control agent as described in claim 2 in the suppression and killing of pine wilt nematodes.