Lipomycete capable of preventing and controlling bacterial wilt and application thereof
By preparing biocontrol agents using Leuconostoc melitica, the problems of decreased control efficacy and environmental pollution caused by chemical pesticides have been solved, achieving effective and green control of bacterial wilt in solanaceous crops.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- SOUTHWEST UNIV
- Filing Date
- 2026-03-25
- Publication Date
- 2026-06-23
Smart Images
Figure CN122256184A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of crop disease control, and more particularly to a Leuconostoc citrate-loving bacterium that can control bacterial wilt and its application. Background Technology
[0002] Solanaceae crops are important economic and vegetable crops globally, encompassing multiple varieties such as tomatoes, eggplants, peppers, and tobacco. They occupy a core position in my country's vegetable industry and are key crops for ensuring vegetable supply to urban and rural residents and increasing farmers' income. Therefore, research and control of diseases affecting solanaceae crops are of great importance. Bacterial wilt is one of the major diseases affecting solanaceae crops. This disease is characterized by rapid spread, a wide host range, and strong pathogenicity. Its pathogen can widely infect solanaceae crops such as tomatoes, eggplants, peppers, and tobacco. After infection, it easily leads to wilting, yellowing, and death of plants, significantly reducing crop yields. In severely affected areas, yield reductions can reach more than 50%, or even total crop failure, causing huge economic losses.
[0003] In the control of bacterial wilt in solanaceous crops, the long-term and large-scale use of chemical pesticides has not only led to the spread of drug-resistant pathogens and caused repeated outbreaks of the disease, resulting in a year-by-year decline in the control effect, but also left behind chemical residues that pose a potentially serious threat to the soil, water bodies and other ecosystems, as well as to food products such as vegetables and agricultural products, which does not meet the needs of sustainable agricultural development.
[0004] Biological control technologies, represented by biocontrol agents, offer new ideas for the green control of bacterial wilt. By screening candidate microbial strains with antagonistic effects against the pathogen causing bacterial wilt in solanaceous crops from the natural environment and optimizing their fermentation and culture conditions, biocontrol agents suitable for greenhouse and field control can be created, achieving green control of bacterial wilt in solanaceous crops such as tomatoes, eggplants, peppers, and tobacco. Since bacterial wilt is a soil-borne bacterial disease, a large number of pathogens reside in the rhizosphere of crops. Given the porous and loose physical structure of soil, fumigant pesticides have become an ideal means of controlling soil-borne diseases. However, the high toxicity of fumigants to non-target organisms limits their use. However, some environmental microorganisms can produce volatile antibacterial substances that have a similar effect to fumigants, inhibiting the growth of pathogens in their surrounding environment while avoiding damage to crops. These microorganisms show great promise for the control of soil-borne diseases such as bacterial wilt. Summary of the Invention
[0005] To address the above problems, this invention provides a Leuconostoc citreum strain that can prevent and control bacterial wilt, which was deposited on February 9, 2026, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 37755.
[0006] The present invention also provides the application of the above-mentioned Leuconostoc citrate in the prevention and control of bacterial wilt.
[0007] The present invention also provides a method for preparing a biocontrol agent, comprising the step of preparing the above-mentioned Leuconostoc citrate into an agent.
[0008] In one specific implementation, the method includes the following steps:
[0009] S1: The *Leuconostoc citrate* was inoculated into a culture medium to obtain fermentation raw material;
[0010] S2: Cultivate the fermentation raw materials to obtain Leuconostoc meliophilus fermentation broth, which is the biocontrol agent.
[0011] In one specific embodiment, the Leuconostoc citrate is inoculated and cultured in NB medium.
[0012] In one specific embodiment, the Leuconostoc citrate is cultured at 28°C and 110-180 rpm for 12-24 h.
[0013] The present invention also provides a biocontrol agent prepared by the above method.
[0014] The present invention also provides a method for preventing and controlling bacterial wilt, comprising the step of applying the above-mentioned biocontrol agent to the target plant.
[0015] In one specific implementation, the biocontrol agent is applied to the environment of the target plant by root irrigation.
[0016] In one specific implementation, the target plant is a plant belonging to the Solanaceae family.
[0017] The antagonistic strain Q2-1 provided by this invention is *Leuconostoc mellitus* Q2-1, a strain isolated from plant rhizosphere. This bacterium was identified as *Leuconostoc mellitus* citric acid lovage, belonging to the genus *Leuconostoc* of the family Streptococcus, and is a strain harmless to humans and animals. This strain exhibits significant antagonistic activity against *Ralstonia solanacearum*. Furthermore, plate inhibition analysis of the volatile metabolites of the antagonistic strain Q2-1 revealed that certain metabolites in the gas produced by the antagonistic strain Q2-1 have an inhibitory effect on the bacterial wilt pathogen CQPS-1. Therefore, the antagonistic strain Q2-1 of this invention has a control effect on bacterial wilt in solanaceous crops, providing a new and effective approach for the control of bacterial wilt in solanaceous crops and showing good application prospects.
[0018] Preservation of biological materials
[0019] The purified Leuconostoc meliticus Q2-1 of this invention was deposited on February 9, 2026, at the China General Microbiological Culture Collection Center (CGMCC) of the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 37755. Attached Figure Description
[0020] Figure 1 Scanning electron microscope image of Leuconostoc meliophilus Q2-1 cells.
[0021] Figure 2 The antibacterial effect of Q2-1 fermentation filtrate on three pathogens was measured.
[0022] Figure 3 This is a schematic diagram of the flat-plate fastening method.
[0023] Figure 4 The antagonistic effect of Q2-1 volatile metabolites on CQPS-1.
[0024] Figure 5 The incidence of bacterial wilt in tobacco after root drenching treatment Q2-1.
[0025] Figure 6 Comparison of tobacco bacterial wilt disease index among different treatment groups. Detailed Implementation
[0026] The principles and features of the present invention are described below with reference to the accompanying drawings. The examples given are only for explaining the present invention and are not intended to limit the scope of the present invention.
[0027] 1. Identification and preservation of Leuconostoc mesenteroides Q2-1
[0028] Rhizosphere samples were collected in April 2025 at Jingangbei, Beibei District, Chongqing, and placed in sterile plastic-sealed bags. 10g of each sample was weighed and placed in a 50mL centrifuge tube (pre-filled with 30mL of 1% sterile magnesium chloride). The tube was shaken at 28℃ and 180r / min for 2h30min to obtain a rhizosphere bacterial suspension. Each rhizosphere bacterial suspension was then diluted separately. , , Spread 200 μL of the sample onto NA plates and incubate upside down at 28°C for 24 h. Pick single colonies and spot them onto four NA plates. After 24 h, add *Ralstonia solanacearum*. 2.5 μL was dotted to the center of four points to screen for strains with antibacterial effects. Strawberry purification yielded strain Q2-1. The bacterial culture was placed in a 40% sterile glycerol tube and stored at -80℃ for later use.
[0029] Single colonies of the antagonistic strain Q2-1 were picked and inoculated into NB medium and cultured on a shaker at 28°C and 180 r / min for 24 h. Then, the morphological characteristics of single colonies of the antagonistic strain Q2-1 were observed after culturing on NA plates at 28°C for 24 h. On the plates, single colonies of this strain were round, white, and had smooth edges.
[0030] The cell morphology of this bacterium is as follows Figure 1 As shown, the bacterial cells are spherical or nearly spherical, with full outlines, irregular edges, and uneven surfaces. No obvious flagella, pili, or other accessory structures are observed.
[0031] After incubating the bacterial strain in NB medium at 28°C and 200 rpm for 24 h with shaking, the bacterial culture was aspirated for 16S rRNA sequencing (primer sequence). The amplification of 1492R (5'-CGGCTACCTTGTTACGAC-3') was performed using the following PCR reaction system: 12.5 μL 2×Taq PCR Mix, 1.0 μL 27F (10 μmol / L), 1.0 μL 1492R (10 μmol / L), 1.0 μL bacterial template, and 9.5 μL ddH2O. The PCR reaction conditions were: 95℃ pre-denaturation for 3 min; 94℃ denaturation for 30 s, 57℃ annealing for 30 s, 72℃ extension for 2 min, for 30 cycles; and a final extension at 72℃ for 5 min.
[0032] PCR amplification products were detected by electrophoresis on a 1% agarose gel. Successfully amplified products were sent to Qingke Biotechnology Co., Ltd. for purification and sequencing to obtain the 16S rRNA sequence of the antagonistic strain Q2-1, as shown in SEQ ID NO.3. This sequence was BLAST-aligned in the NCBI database and submitted to GenBank for further information. A phylogenetic tree was constructed using the neighbor-joining method with 1000 replicates using Mega 9.0 software to determine the taxonomic position of the strain. The results showed that it aggregated with the 16S rRNA sequence of *Leuconostoc citreum*.
[0033] Based on morphological characteristics, the strain was identified as Leuconostoc citreum.
[0034] The above-mentioned strain was deposited on February 9, 2026, at the China General Microbiological Culture Collection Center (CGMCC) of the Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 37755.
[0035] 2. Preparation of biocontrol agents
[0036] Preparation method of bacterial agent: Take out the Leuconostoc melitica Q2-1 strain from a 40% sterile glycerol tube stored at -80℃, and under aseptic conditions, use a sterile inoculation loop to pick up a small amount of bacterial solution and streak it onto a NA medium plate; place the inoculated plate upside down in a constant temperature incubator and incubate at 28℃ for 24 hours. When a round, white single colony with smooth edges grows on the plate, it is the activated Q2-1 strain.
[0037] Under aseptic conditions, a single colony of activated Q2-1 was picked from an NA plate and inoculated into an Erlenmeyer flask containing 100 mL of sterile NB medium. The flask was then placed in a constant-temperature shaker at 28°C and a rotation speed of 150 r / min (110-180 r / min) for 12-24 h to obtain the Q2-1 bacterial suspension. The OD value of the bacterial suspension was measured using a UV spectrophotometer. 600 Once the value reaches 0.3-0.5, it can be used as a biocontrol agent for root irrigation.
[0038] 3. Antibacterial effect of the strain
[0039] Antibacterial assay: Single colonies of antagonistic strains were picked using an inoculation loop, inoculated onto NB medium, and incubated at 28℃ and 120 rpm for 12 h. The Oxford cup method was used, with NA medium containing purified Ralstonia solanacearum CQPS-1, OD... 600 =0.1, 100 μL plate, punch a well in the center of NA medium, spot 50 μL of Q2-1 fermentation filtrate, remove the plate after 2 days, and observe the growth of CQPS-1 colonies. An inhibition zone will appear if there is an inhibitory effect. The size of the inhibition zone indicates the strength of the inhibitory effect. Figure 2 As shown in Figure A, the CQPS-1 plate containing the Q2-1 fermentation filtrate exhibited a 25% inhibition rate, indicating that the growth of CQPS-1 was inhibited. Figure 2 As shown in Figure B, the RS105 plate containing the fermentation filtrate of Q2-1 showed an inhibition rate of 12%, indicating that the growth of RS105 was inhibited. Figure 2 As shown in Figure C, the GMI1000 plate containing the fermentation filtrate of Q2-1 showed an inhibition rate of 36%, indicating that the growth of GMI1000 was inhibited.
[0040] The inhibition rate was calculated using the area method, with the formula: Inhibition rate = (effective area of inhibition zone / total area of plate) × 100%, where the plate diameter is 90 mm; the effective diameter of the inhibition zone of CQPS-1 plate is 45 mm, corresponding to an inhibition rate of 25%; the effective diameter of the inhibition zone of RS105 plate is 32 mm, corresponding to an inhibition rate of 12%; and the effective diameter of the inhibition zone of GMI1000 plate is 54 mm, corresponding to an inhibition rate of 36%.
[0041] Experimental results showed that the antagonistic strain Q2-1 had an inhibitory effect on Ralstonia solanacearum CQPS-1, GMI1000, and RS105, a pathogenic species of rice bacterial leaf streak.
[0042] 4. Determination of the antibacterial effect of volatile metabolites of antagonistic strain Q2-1
[0043] This embodiment measures the antibacterial effect of the volatile metabolites of the antagonistic strain Q2-1 on CQPS-1, and determines the antibacterial effect of the metabolites of the antagonistic strain Q2-1 on CQPS-1.
[0044] like Figure 3 As shown, the NA medium was used with purified CQPS-1, OD 600 =0.1, 100 μL plated, another NA medium was used with purified Q2-1, OD 600 =0.1, 100 μL was plated, and the control group was an empty plate. The two plates were inverted for 2 days without direct contact between the bacteria. After 2 days, the plates were removed and the growth of these pathogenic bacteria colonies was observed. If there is an inhibitory effect, the growth of CQPS-1 will be inhibited, and the color will be light white or transparent. The color of CQPS-1 indicates the strength of the inhibitory effect.
[0045] according to Figure 4 It can be seen that the CQPS-1 plate in 4B, which was inverted with the antagonistic bacteria Q2-1, was transparent and showed no pathogen growth; while the CQPS-1 plate in 4A, which was inverted with an empty plate, was white and showed good CQPS-1 growth. This indicates that the volatile metabolites of the antagonistic bacteria Q2-1 have an inhibitory effect on the pathogen, and the inhibitory effect is relatively strong.
[0046] 5. Control effect of antagonistic strain Q2-1 on tobacco bacterial wilt
[0047] Tobacco Treatment: Five tobacco seedlings (5-6 true leaves stage) were planted in each group. The control group was irrigated with 10 mL of pure water, while the experimental group was irrigated with 10 mL of OD245 solution. 600 Three days after root drenching with Q2-1 bacterial solution at a concentration of 0.3%, inoculate the OD at a dose of 10 ml per plant through root drenching. 600 =0.1 CQPS-1 bacterial culture
[0048] Pathogenicity test:
[0049] CQPS-1 cultured in NA medium at 28°C for 24 hours was prepared to obtain OD values. 600A bacterial solution with a concentration of 0.1 was prepared and poured into a sterile beaker. The solution was then slowly poured into the substrate around the base of the tobacco stems, circling the beaker wall, avoiding direct rinsing of the stem base. The first survey was conducted starting on day 3, followed by daily surveys at a fixed time (e.g., 9:00 AM) until all plants in the control group showed symptoms or died (the peak incidence of disease is usually 7-14 days after inoculation). The indoor grading standard for tobacco bacterial wilt was used.
[0050] Depend on Figure 5 It can be seen that, Figure 5 In control group A, there was obvious disease, with 3 plants wilting and dying completely, 1 plant with drooping leaves and slight browning at the stem base but no purulent discharge, and 1 plant showing no wilting symptoms and normal root system and stem base. Figure 5 In the group with antagonistic strain Q2-1, one plant showed wilting of the lower 3-5 leaves, drooping leaves, and slight browning at the stem base, without purulent discharge. Four plants showed no wilting symptoms, and their root systems and stem bases were normal. Antagonistic bacteria Q2-1 inhibited the development of tobacco bacterial wilt.
[0051] according to Figure 5 and 6 The indoor grading standard for tobacco bacterial wilt was established. In the control group, 1 plant was grade 0, 1 plant was grade 1, and 3 plants were grade 4. In the experimental group, after treatment with the antagonistic strain, 4 plants were grade 0 and 1 plant was grade 2. The disease index of the control group was 65, and that of the experimental group was 10. This experiment was performed in three biological replicates. In the other two replicates, the disease indices were 75 and 68 for the control group, and 16.7 and 15 for the experimental group. The relative efficacy of the biocontrol bacterium Q2-1 in the experimental group was 85%. Q2-1 conferred resistance to tobacco bacterial wilt. Disease index = ∑ (number of diseased plants × representative value of the disease level) / (total number of plants surveyed × highest representative value) × 100, relative control efficacy = (control disease index - treatment disease index) / control disease index × 100%, where level 0 means no disease in the whole plant, level 1 means 1%-25% of leaves wilting, level 2 means 26%-50% of leaves wilting, level 3 means 51%-75% of leaves wilting, and level 4 means the whole plant wilting.
[0052] The above experiments show that the strain Q2-1 of the present invention has an inhibitory effect on bacterial wilt pathogen CQPS-1 and can be used for the prevention and control of bacterial wilt.
[0053] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.
Claims
1. A Leuconostoc citreum strain that can prevent and control bacterial wilt, characterized in that, It was deposited on February 9, 2026 at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 37755.
2. The application of Leuconostoc citrate as described in claim 1 in the prevention and control of bacterial wilt.
3. A method for preparing a biocontrol agent, characterized in that, The step includes preparing the Leuconostoc citrate-loving bacteria as described in claim 1 into a bacterial agent.
4. The method according to claim 3, characterized in that, Includes the following steps: S1: The *Leuconostoc citrate* was inoculated into a culture medium to obtain fermentation raw material; S2: Cultivate the fermentation raw materials to obtain Leuconostoc meliophilus fermentation broth, which is the biocontrol agent.
5. The method according to claim 4, characterized in that, The Leuconostoc citrate was inoculated and cultured in NB medium.
6. The method according to claim 5, characterized in that, The *Leuconostoc melitica* strain was cultured at 28°C and 110-180 rpm for 12-24 h.
7. A biocontrol agent, characterized in that, It is prepared by the method according to any one of claims 3-6.
8. A method for preventing and controlling bacterial wilt, characterized in that, The step includes applying the biocontrol agent of claim 7 to the environment of the target plant.
9. The method according to claim 8, characterized in that, The biocontrol agent is applied to the environment of the target plant by root irrigation.
10. The method according to claim 8 or 9, characterized in that, The target plant is a plant of the Solanaceae family.