Composite microbial agent for preventing and treating cotton verticillium wilt based on rare earth elements and preparation method of composite microbial agent
By using rare earth elements in combination with Bacillus polymyxa YUPP-8 and Bacillus subtilis YUPP-2 as a microbial agent, the problem of controlling cotton Verticillium wilt was solved, achieving highly efficient disease control, and significantly improving cotton yield and quality, especially under field conditions.
Patent Information
- Application Number
- CN202510886817.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-06-30
- Publication Date
- 2025-11-14
AI Technical Summary
There is a lack of effective fungicides in the current technology to inhibit or kill the pathogen that causes cotton Verticillium dahliae, and the control effect of a single endophytic fungus is not good at different plant growth stages.
A composite microbial agent using rare earth elements and Bacillus polymyxa YUPP-8 and Bacillus subtilis YUPP-2 was prepared. Through the enzyme regulation and photosynthesis of rare earth elements, combined with endogenous antagonism, the prepared composite microbial agent was stored at -80 to -4℃ to inhibit the growth of Verticillium dahliae.
It achieved highly efficient control of cotton Verticillium wilt, with an inhibition rate of ≥75% and a cure rate of 87.4-88.5% in field experiments, showing a significant synergistic antibacterial effect.
Smart Images
Figure CN120937866A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of compound microbial agents, and in particular relates to a compound microbial agent for the prevention and control of cotton Verticillium wilt based on rare earth elements and its preparation method. Background Technology
[0002] Verticillium dahliae Kleb is one of the most devastating diseases affecting cotton yield and quality in cotton-producing countries. It can spread through multiple pathways, including seeds, soil, and infected plants. Currently, few fungicides reach effective doses that inhibit or even kill the pathogen. While rice rotation can eliminate sclerotia in the soil, insufficient water is available for this control measure in major cotton-producing areas. Generally, biocontrol agents can colonize in niches similar to those of plant vascular pathogens, allowing endophytic microorganisms to play a crucial role in combating soil-borne fungal pathogens. Most biocontrol methods for plant diseases use single biocontrol agents, but in practice, the application of a single endophytic microorganism may be affected by a sharp decline in population at different stages of plant growth, potentially leading to poor control of Verticillium dahliae Kleb. Summary of the Invention
[0003] In view of this, the present invention aims to overcome the deficiencies in the prior art and proposes a compound microbial agent for the control of cotton Verticillium wilt based on rare earth elements and its preparation method. Combining the enzyme-regulating effect and photosynthetic advantages of rare earth elements with the endophytic antagonistic effect of the microbial agent, a multidimensional control of cotton Verticillium wilt is achieved. The present invention isolated antagonistic endophytic bacteria *Bacillus polymyxa* YUPP-8 (CCTCC:M209013) and *Bacillus subtilis* YUPP-2 (CCTCC:M209012) at the cotton seedling stage.
[0004] To achieve the above objectives, the technical solution of the present invention is implemented as follows:
[0005] In a first aspect, the present invention provides a method for preparing a compound microbial agent, comprising the following steps:
[0006] Rare earth compounds are dissolved in water, heated and stirred, and then catechin compounds are added to react. After standing and cooling, a mixed solution is obtained. Bacterial solution is added to the mixed solution for compounding to obtain a compound microbial agent for the prevention and control of cotton Verticillium wilt based on rare earth elements. The agent is stored at -80 to -4°C for later use. The bacterial solution is Bacillus polymyxa YUPP-8 bacterial solution and / or Bacillus subtilis YUPP-2 bacterial solution.
[0007] Preferably, the OD400 value of the polymyxin Bacillus YUPP-8 bacterial culture is 0.25-0.4.
[0008] Preferably, the OD400 value of Bacillus subtilis YUPP-2YUPP-8 in the Bacillus subtilis YUPP-2 bacterial solution is 0.25-0.4.
[0009] Preferably, the volume ratio of the bacterial solution to the mixed solution is 1:(7-8).
[0010] Preferably, the rare earth compounds include LaCl3·7H2O, CeCl3·6H2O, and YCl3·6H2O.
[0011] Preferably, the molar concentration of rare earth compounds in the mixed solution is 4-10 mmol / L, wherein the molar ratio of LaCl3·7H2O, CeCl3·6H2O and YCl3·6H2O is (6-12):1:1.
[0012] Preferably, the catechin polyphenol compound comprises one or more of catechin, epigallocatechin, and gallocatechin; more preferably, it is catechin.
[0013] Preferably, the concentration of catechin polyphenol compounds in the mixed solution is 5-15 g / L.
[0014] The polymyxin Bacillus YUPP-8 has the accession number CCTCC:M209013, and the Bacillus subtilis YUPP-2 has the accession number CCTCC:M209012. Both were purchased from the Shanghai Center for Microbial Preservation.
[0015] Secondly, the present invention provides a composite microbial agent prepared by the above preparation method.
[0016] Thirdly, the present invention also provides the application of the above-mentioned composite microbial agent in the preparation of a biocontrol agent for inhibiting Verticillium dahliae.
[0017] Fourthly, the present invention provides the application of the above-mentioned compound microbial agent in the preparation of a biological control agent for cotton Verticillium wilt.
[0018] This invention discovers that rare earth microbial agents have a combined control effect on cotton Verticillium wilt. Through the electrostatic effect of rare earth elements, they are adsorbed onto the cell wall of Verticillium dahliae, interfering with its physiological metabolism. At the same time, the microbial agents are released into the cell wall, inhibiting its proliferation through the antagonistic effect of Bacillus polymyxa YUPP-8 or Bacillus subtilis YUPP-2.
[0019] Compared with the prior art, the present invention has the following advantages:
[0020] (1) The compound microbial agent of the present invention has a highly effective antibacterial function against Verticillium dahliae, which causes Verticillium wilt of cotton, with an inhibition rate of ≥75%.
[0021] (2) In this invention, the bacterial solution of Bacillus polymyxa YUPP-8 or Bacillus subtilis YUPP-2 with antibacterial activity has a strong synergistic effect in the process of compounding with rare earth elements. Attached Figure Description
[0022] Figure 1 The coating effect of the microbial inoculants prepared in the examples and comparative examples after being reacted with Verticillium dahliae;
[0023] Figure 2 A statistical chart showing the antibacterial rate;
[0024] Figure 3 This represents the actual trial results from the field experiment. Detailed Implementation
[0025] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.
[0026] In this document, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.
[0027] In this document, when values are described as ranges, it should be understood that such disclosure includes disclosure of all possible subranges within that range, as well as the specific numerical values falling within that range, regardless of whether the specific numerical value or specific subrange is explicitly specified.
[0028] In this article, the terms "multiple" or "more than" are used unless otherwise specified, referring to a quantity greater than or equal to 2. For example, "one or more" means one or more types.
[0029] In this document, the terms "preferred" and "more preferred" are used only to describe implementation methods or embodiments with better effects, and should be understood as not constituting a limitation on the scope of protection of this invention.
[0030] In this document, terms such as "further" are used for descriptive purposes to indicate differences in content, but should not be construed as limiting the scope of protection of this invention.
[0031] In this article, the term "and / or" describes an association between objects, indicating that three relationships can exist. For example, A and / or B means: A or B, or A and B.
[0032] In this document, the term "about" means a specified value of + / - 10%, preferably + / - 5%, and more preferably + / - 1%.
[0033] In this article, the terms “include,” “including,” “have,” “contain,” etc., are all open-ended terms, meaning that they include but are not limited to.
[0034] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar to or equivalent to those described herein may be used in the implementation or testing of this invention.
[0035] The present invention will be described in detail below with reference to the embodiments.
[0036] Example 1
[0037] (1) In a 500 mL round-bottom flask, add 200 mL of double-distilled water, and add 1.6 mmol LaCl3·7H2O, 0.2 mmol CeCl3·6H2O, and 0.2 mmol YCl3·6H2O. After heating and stirring the above solution at 90 °C for 6 h, introduce 2 g of catechin (EC) into the above mixed solution and continue the reaction for 3 h. Let the mixed solution stand and cool to obtain rare earth material (abbreviated as RE-EC, the same below).
[0038] (2) Preparation of biocontrol bacteria culture medium: 1% sucrose, 0.5% peptone, 0.3% yeast extract, 0.1% KH2PO4 and 0.02% MgSO4 were mixed and the pH was adjusted to 7.0. The strain *Bacillus polymyxa* YUPP-8 purchased from the Shanghai Microbiological Culture Collection Center was transferred into the above culture medium for cultivation to obtain *Bacillus polymyxa* YUPP-8 bacterial suspension. The OD400 value of *Bacillus polymyxa* YUPP-8 in the bacterial suspension was 0.25.
[0039] (3) The polymyxin Bacillus YUPP-8 bacterial solution was introduced into the solution of rare earth material RE-EC at a volume ratio of 1:8 to obtain a compound microbial agent, which was then transferred to a 4°C refrigerator for storage.
[0040] Example 2
[0041] (1) In a 500 mL round-bottom flask, add 200 mL of double-distilled water, and add 1.2 mmol LaCl3·7H2O, 0.2 mmol CeCl3·6H2O, and 0.2 mmol YCl3·6H2O. After heating and stirring the above solution at 90 °C for 6 h, introduce 1 g of epigallocatechin into the above mixed solution and continue the reaction for 3 h. Let the mixed solution stand and cool to obtain rare earth material RE-EC.
[0042] (2) Preparation of biocontrol bacteria culture medium: 1% sucrose, 0.5% peptone, 0.3% yeast extract, 0.1% KH2PO4 and 0.02% MgSO4 were mixed and the pH was adjusted to 7.0. Bacillus subtilis YUPP-2 purchased from the Shanghai Microbiological Preservation Center was transferred into the above culture medium for cultivation to obtain Bacillus subtilis YUPP-2 bacterial suspension. The OD400 value of Bacillus subtilis YUPP-2 in the Bacillus subtilis YUPP-2 bacterial suspension was 0.4.
[0043] (3) The Bacillus subtilis YUPP-2 bacterial solution was introduced into the solution of rare earth material RE-EC at a volume ratio of 1:7 to obtain a compound microbial agent, which was then transferred to a refrigerator at 4°C for storage.
[0044] Example 3
[0045] (1) In a 500 mL round-bottom flask, add 200 mL of double-distilled water, and add 0.8 mmol LaCl3·7H2O, 0.2 mmol CeCl3·6H2O, and 0.2 mmol YCl3·6H2O. After heating and stirring the above solution at 90 °C for 6 h, introduce 3 g gallocatechin into the above mixed solution and continue the reaction for 3 h. Let the mixed solution stand and cool to obtain rare earth material RE-EC.
[0046] (2) Preparation of biocontrol bacteria culture medium: 1% sucrose, 0.5% peptone, 0.3% yeast extract, 0.1% KH2PO4 and 0.02% MgSO4 were mixed and the pH was adjusted to 7.0. Bacillus subtilis YUPP-2 purchased from the Shanghai Microbiological Preservation Center was transferred into the above culture medium for cultivation to obtain Bacillus subtilis YUPP-2 bacterial suspension. The OD400 value of Bacillus subtilis YUPP-2 in the Bacillus subtilis YUPP-2 bacterial suspension was 0.25.
[0047] (3) The Bacillus subtilis YUPP-2 bacterial solution was introduced into the solution of rare earth material RE-EC at a volume ratio of 1:7 to obtain a compound microbial agent, which was then transferred to a refrigerator at 4°C for storage.
[0048] Comparative Example 1
[0049] The microbial agent used in this comparative example is only the Bacillus polymyxa YUPP-8 culture obtained in Example 1 with an OD400 value of 0.25.
[0050] Comparative Example 2
[0051] The microbial agent used in this comparative example is only the Bacillus subtilis YUPP-2 bacterial culture obtained in Example 1 with an OD400 value of 0.4.
[0052] Comparative Example 3
[0053] The difference between this comparative example and Example 1 is that 1.6 mmol LaCl3·7H2O and 0.2 mmol CeCl3·6H2O were added during the preparation of the rare earth material RE-EC.
[0054] Comparative Example 4
[0055] The difference between this comparative example and Example 1 is that 1.6 mmol LaCl3·7H2O and 0.2 mmol YCl3·6H2O were added during the preparation of the rare earth material RE-EC.
[0056] Comparative Example 5
[0057] The difference between this comparative example and Example 1 is that 1.6 mmol of LaCl3·7H2O was added during the preparation of the rare earth material RE-EC.
[0058] Comparative Example 6
[0059] The difference between this comparative example and Example 1 is that 5g of catechin was added during the preparation of the rare earth material RE-EC.
[0060] Test Example 1
[0061] Verticillium dahliae was cultured for 5 days in liquid culture medium (a mixture of 0.3% NaNO3, 0.5% peptone, 0.2% yeast extract, 0.1% sucrose, and 0.3% chitin, adjusted to pH 7.0). The selected biocontrol culture medium (a mixture of 1% sucrose, 0.5% peptone, 0.3% yeast extract, 0.1% KH2PO4, and 0.02% MgSO4, adjusted to pH 7.0) was cooled to 45°C. 1 mL of Verticillium dahliae culture was added to the medium, shaken well, and incubated for 1–2 days. Then, 0.5 mL of the bacterial agents prepared in Examples 1–3 and Comparative Examples 1–5 were added, mixed thoroughly, and experimental plates were prepared. Sterile water was used as a blank control. The mixture was spread evenly with a spreading stick and transferred to a constant temperature incubator. The coverage area of Verticillium dahliae was observed periodically.
[0062] After 10 days of incubation, the results were filtered, and images with uniform distribution were selected for display. For example... Figure 1As shown, the Verticillium dahliae in the blank control group grew well, evenly distributed on the agar plate, covering an area of over 50%. By statistically analyzing the colony diameters of the three parallel experiments, we obtained an average colony diameter of 2.8 cm for the three plates. After incubation of Comparative Example 1 with Verticillium dahliae, the average colony diameter of the three parallel experiments was 2.8 cm, indicating that this strain alone did not inhibit the growth of Verticillium dahliae. After incubation of Comparative Example 2 with Verticillium dahliae, the colony diameter directly increased to 2.9 cm. However, the area of the Verticillium dahliae plaques after introducing the composite microbial agents of Examples 1, 2, and 3 did not increase further, indicating that the rare earth composite microbial agent has a strong synergistic antibacterial effect.
[0063] like Figure 2 As shown, we statistically analyzed the fungal coverage area in three parallel experiments, using the *Verticillium dahliae* control group as a baseline. The survival rate of *Verticillium dahliae* in the control group was 100%. After incubation with the compound microbial agents from Examples 1, 2, and 3, the survival rate of *Verticillium dahliae* was only 0.1-1%. At this point, the variance fluctuation of the three parallel experiments was small, indicating the stability of the material and resulting in minimal experimental bias. This single-variable method demonstrates that the combined application of microbial agents and rare earth elements has a good control effect on *Verticillium dahliae*, the cause of cotton verticillium wilt. This invention provides a new strategy for the control of soil-borne diseases caused by cotton verticillium wilt.
[0064] Test Example 2
[0065] The field experiment was conducted in cotton-growing areas of Xinjiang, specifically in regions where Verticillium wilt was prevalent, from June to August. The compound microbial inoculants described in Examples 1 and 2 were applied to the roots of diseased cotton plants via root drenching. A control group received no treatment. The experiment was conducted per mu (approximately 0.16 acres) of planted area, with an average of 2 tons of diluted inoculant applied per mu. Each experiment consisted of three parallel groups. Two months after application, external observations and statistics on average cotton yield and quality were performed to determine the pesticide's effectiveness against Verticillium wilt and to calculate the cure rate. Figure 3 As shown, experiments have revealed that in actual field use, the compound microbial agents of Examples 1 and 2, under conditions that completely simulate pesticide application, achieved cure rates of up to 87.4% and 88.5% against cotton Verticillium wilt, respectively, demonstrating remarkable control effects.
[0066] 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 method for preparing a compound microbial agent, characterized in that: Includes the following steps: Rare earth compounds are dissolved in water, heated and stirred, and then catechin compounds are added to react. After standing and cooling, a mixed solution is obtained. Bacterial solution is added to the mixed solution for compounding to obtain a compound microbial agent for the prevention and control of cotton Verticillium wilt based on rare earth elements. The agent is stored at -80 to -4°C for later use. The bacterial solution is Bacillus polymyxa YUPP-8 bacterial solution and / or Bacillus subtilis YUPP-2 bacterial solution.
2. The method for preparing the composite microbial agent according to claim 1, characterized in that: The OD400 value of the polymyxin Bacillus YUPP-8 bacterial culture was 0.25-0.
4.
3. The method for preparing the composite microbial agent according to claim 1, characterized in that: The OD400 value of Bacillus subtilis YUPP-2YUPP-8 in the Bacillus subtilis YUPP-2 bacterial solution was 0.25-0.
4.
4. The method for preparing the composite microbial agent according to claim 1, characterized in that: The volume ratio of the bacterial solution to the mixed solution is 1:(7-8).
5. The method for preparing the composite microbial agent according to claim 1, characterized in that: The rare earth compounds include LaCl3·7H2O, CeCl3·6H2O and YCl3·6H2O.
6. The method for preparing the composite microbial agent according to claim 1, characterized in that: The molar concentration of rare earth compounds in the mixed solution is 4-10 mmol / L, wherein the molar ratio of LaCl3·7H2O, CeCl3·6H2O and YCl3·6H2O is (6-12):1:
1.
7. The method for preparing the composite microbial agent according to claim 1, characterized in that: The catechin polyphenol compound comprises one or more of catechin, epigallocatechin, and gallocatechin; preferably, the concentration of the catechin polyphenol compound in the mixed solution is 5-15 g / L.
8. The composite microbial agent prepared by the preparation method according to any one of claims 1-7.
9. The application of the composite microbial agent according to claim 8 in the preparation of a biocontrol agent for inhibiting Verticillium dahliae.
10. The application of the compound microbial agent according to claim 8 in the preparation of a biological control agent for cotton Verticillium wilt.