Chemical attractant and method for promoting colonization of biocontrol bacteria in tobacco rhizosphere

Abstract

The invention belongs to the technical field of tobacco disease prevention and control, and discloses a chemical attractant and method for promoting colonization of biocontrol bacteria in tobacco rhizosphere, the biocontrol bacteria are bacillus amyloliquefaciens CH1, the chemical attractant takes cinnamic acid as one of active ingredients, and the chemical attractant can also contain 4-ethyl phenol, benzyl cinnamate, protocatechuic acid and other active ingredients. Cinnamic acid, 4-ethylphenol, benzyl cinnamate and protocatechuic acid provided by the invention can selectively and efficiently promote enrichment and colonization of the CH1 strain in tobacco rhizosphere, and can increase the biofilm quantity of the CH1 strain, thereby being beneficial to enhancing the field control effect of the CH1 strain on tobacco bacterial wilt; the scheme is safe and harmless to the environment and has a good application prospect.

Description

Technical Field

[0001] The present invention belongs to the technical field of tobacco disease prevention and control, and specifically relates to a chemical attractant and method for promoting the colonization of biocontrol bacteria in the rhizosphere of tobacco, wherein the biocontrol bacteria is Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens )CH1. Background Art

[0002] Bacterial wilt is a soil-borne bacterial disease and one of the main destructive diseases in tobacco cultivation, causing huge economic losses to tobacco leaf production. Tobacco bacterial wilt is a typical vascular bacterial disease that can affect roots, stems, and leaves (Shan Xiaopeng, 2021). The most typical symptom is wilting, which often occurs during the vigorous growth and maturity of tobacco. Once the pathogen invades the damaged part of tobacco, it will multiply rapidly and secrete extracellular polysaccharide proteins to block vascular transport, making it difficult for nutrients to be transported normally in tobacco plants, causing the roots of tobacco to turn black and gradually rot, black streaks to form on the stems and begin to form cavities, and the leaves to wilt until they eventually die. Studies have shown that high temperature and humidity, low-lying, heavy, neutral and acidic soils are prone to bacterial wilt, and the epidemic area of ​​​​bacterial wilt shows a trend of spreading (Kong Fanyu, 2003). In order not to affect the development of tobacco agricultural economy, the prevention and control of tobacco bacterial wilt is urgent.

[0003] As a green, environmentally friendly, sustainable and green agricultural production method, biological control has attracted the attention of more and more researchers and is a control method with broad application prospects. Biological control mainly refers to the use of microbial agents to treat tobacco soil, inhibiting the growth of pathogens through nutritional competition, niche competition and the production of antagonistic substances among microorganisms, thereby reducing the occurrence of diseases. This control method can not only inhibit tobacco bacterial wilt, but also change the composition and structure of soil microorganisms and protect the ecological environment of tobacco fields (Ding et al, 2021).

[0004] Studies have shown that plant root exudates play an important role in determining the structure of rhizosphere microbial communities and plant defense responses, and are considered to be the core media in the plant-microbe interaction process. On the one hand, root exudates act as nutrients to enrich a large number of microbial populations around plant roots. On the other hand, they act as signal molecules to participate in highly specific chemical communications between roots and soil microorganisms, establishing an interactive relationship between plants and microorganisms, and affecting the structure of rhizosphere microbial communities and their biocontrol effects by attracting (recruiting) specific microorganisms to the rhizosphere or repelling pathogenic microorganisms (Vives-Peris et al., 2020). Studies have found that secondary metabolites secreted by roots act as signal molecules to attract rhizobia and mycorrhizal fungi. For example, flavonoids can act as regulators of nitrogen fixation by rhizobia in leguminous plants, and strigolactones provide communication media for the formation of mutualistic relationships between mycorrhizal fungi and hosts, while organic acids can serve as important chemotactic substances for rhizospheric Bacillus (Hassan and Mathesius, 2012; Shen et al., 2022). Organic acids such as malic acid and citric acid can recruit Bacillus to the rhizosphere soil (Wu Linkun et al., 2014). Rice secretions can attract Bacillus. Arabidopsis secretions play an important role in attracting Bacillus subtilis and enhancing rhizosphere colonization (Badri et al., 2013). Citrus root exudates promote the growth and colonization of beneficial rhizosphere bacteria Pseudomonas putida and Neocotyledons (Vives-Peris et al., 2018). Summary of the invention

[0005] In view of this, the present invention aims to provide a new strategy for improving the field control effect of biocontrol bacteria by exploring the interaction between tobacco and biocontrol bacteria.

[0006] The technical solution of the present invention is as follows: The first aspect of the present invention provides a chemical attractant for promoting the colonization of biocontrol bacteria in the rhizosphere of tobacco, wherein the biocontrol bacteria is Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens )CH1, its deposit number is CCTCC NO:M208127, the deposit time is September 5, 2008, and the deposit address is China Center for Type Culture Collection (Wuhan University, Wuhan, Hubei Province).

[0007] The CH1 strain has been proven to have the ability to promote tobacco plant growth and antagonize Ralstonia solanacearum, and the enrichment and colonization of the CH1 strain in the tobacco root is the key factor in its growth promotion and disease resistance. The inventors analyzed the root secretions of tobacco and found that among a large number of root secretions, the CH1 strain has chemotaxis to 4-ethylphenol, cinnamic acid, benzyl cinnamate and protocatechuic acid, and the introduction of 4-ethylphenol, cinnamic acid, benzyl cinnamate and protocatechuic acid into the rhizosphere of tobacco can significantly enhance the enrichment and colonization ability of CH1 in the rhizosphere of tobacco; and the experimental data show that cinnamic acid has the best effect, followed by benzyl cinnamate and 4-ethylphenol, and the mixing of the four substances also has an effect. Therefore, in the chemical attractant of the present invention, the active ingredient can be one or more of 4-ethylphenol, cinnamic acid, benzyl cinnamate and protocatechuic acid.

[0008] Preferably, the active ingredient of the chemical attractant includes at least cinnamic acid, and may also include one or more of 4-ethylphenol, benzyl cinnamate and protocatechuic acid.

[0009] More preferably, in the above chemical attractant, the concentration of cinnamic acid is 0.04-0.15 mmol / L, the concentration of 4-ethylphenol is 0.05-1.0 mmol / L, the concentration of benzyl cinnamate is 0.1-1.0 mmol / L, and the concentration of protocatechuic acid is 0.4-0.5 mmol / L.

[0010] The second aspect of the present invention provides a method for promoting the colonization of biocontrol bacteria in the rhizosphere of tobacco, specifically: applying a chemical attractant to the rhizosphere soil of tobacco, thereby utilizing the chemotaxis of the biocontrol bacteria CH1 to the chemical attractant to achieve the enrichment of the CH1 strain in the rhizosphere of tobacco and enhance the colonization ability; wherein the active ingredient of the chemical attractant is at least one of 4-ethylphenol, cinnamic acid, benzyl cinnamate and protocatechuic acid.

[0011] Preferably, in the above method, the active ingredient of the chemical attractant includes at least cinnamic acid; more preferably, it also includes one or more of 4-ethylphenol, benzyl cinnamate and protocatechuic acid.

[0012] The third aspect of the present invention provides the use of the above-mentioned chemical attractant, which at least includes the following: a) Promote the colonization of biocontrol bacteria CH1 in the rhizosphere of tobacco; b) Enhance the field control effect of biocontrol bacteria CH1 on tobacco bacterial wilt; c) Enhance the ability of biocontrol bacteria CH1 to promote tobacco growth; d) Increase the biofilm production of biocontrol bacteria CH1.

[0013] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a kind of biocontrol bacteria Bacillus amyloliquefaciens The chemical attractant of CH1 can selectively and efficiently promote the enrichment and colonization of biocontrol bacteria in the rhizosphere of tobacco, thereby helping to enhance the biocontrol effect of the strain, and the chemical attractant provided by the present invention is safe and harmless to the environment. Therefore, the scheme of the present invention is conducive to more efficient, simple and economical prevention and control of tobacco bacterial wilt, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 The chemotaxis test results of the CH1 strain in Example 1 of the present invention to protocatechuic acid, benzyl cinnamate, 4-ethylphenol and cinnamic acid; Figure 2 This is a fluorescence intensity diagram of the colonization of the CH1 strain in the tobacco rhizosphere in Example 2 of the present invention; Figure 3 The figure shows the effects of different concentrations of protocatechuic acid, benzyl cinnamate, 4-ethylphenol and cinnamic acid on the biofilm of the CH1 strain in Example 3 of the present invention. DETAILED DESCRIPTION

[0015] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs. The term "comprises" and any variations thereof in the specification and claims of the present invention are intended to cover non-exclusive inclusions.

[0016] The technical solution of the present invention will be described clearly and completely below in conjunction with specific embodiments. It should be understood that the embodiments described herein are only used to illustrate and explain the present invention and are not used to limit the present invention.

[0017] If no specific techniques or conditions are specified in the following examples, all of them are carried out according to the techniques or conditions described in the literature in the field or according to the product instructions; if the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be obtained commercially.

[0018] Example 1 In this case, the capillary method was used to detect the chemotaxis of strain CH1 to 4-ethylphenol, cinnamic acid, benzyl cinnamate and protocatechuic acid, including the following steps: Standard solutions of 4-ethylphenol, cinnamic acid, benzyl cinnamate and protocatechuic acid were prepared at concentrations of 0.05, 0.10, 0.20, 0.40 and 0.80 mmol / L, respectively.

[0019] Inoculate CH1 strain in 5 mL LB medium, and culture overnight at 37℃, 180 r / min for activation; transfer to 5 mL LB medium at 1% inoculum, and culture at 37℃, 180 r / min for 5-6 h until mid-logarithmic phase (OD600 ≈ 1.0). Take 2 mL of fermentation broth, centrifuge at 25℃, 5000 r / min for 5 min, concentrate and resuspend in an equal volume of chemotaxis buffer (containing K2HPO44.4 g, KH2PO44.2 g and Na2EDTA 0.00745 g, dilute to 1 L with water, and adjust pH to neutral) to make bacterial suspension, and use a 200 μL pipette tip to draw 100 μL of bacterial suspension for use.

[0020] A 1 mL syringe was used as a capillary for the chemotaxis test. 100 μL of the test solution was drawn and the syringe needle was inserted into the narrow end of the above-mentioned pipette tip to allow the test solution to fully contact the bacterial suspension. After standing for 2 h under sterile conditions at room temperature, the syringe needle was carefully removed from the bacterial suspension, and the bacterial content of the test solution in the syringe was detected by the dilution plate coating method, that is, the colony count (CFU) was counted after culturing at 37°C for 24 h. Each treatment was repeated 3 times; the chemotaxis buffer was used as a control, and the average colony count of the 3 repeated plates was calculated.

[0021] The relative chemotaxis index (RIC) value is the ratio of the average CFU of the treatment group to the CFU of the control group. When RIC ≥ 2, the chemotaxis of the treatment group is judged to be significantly different from that of the control group. Figure 1 As shown, the CH1 strain showed chemotaxis to 0.4 mM protocatechuic acid (1A), the CH1 strain showed chemotaxis to 0.1 mM, 0.2 mM and 0.8 mM benzyl cinnamate (1B), the CH1 strain showed chemotaxis to 0.05 mM and 0.2-0.8 mM 4-ethylphenol (1C), and the CH1 strain showed chemotaxis to 0.05-0.8 mM cinnamic acid (1D).

[0022] Example 2 In this example, 4-ethylphenol, cinnamic acid, benzyl cinnamate and protocatechuic acid in Example 2 were used as attractant active ingredients to detect the actual effects of different substances on the colonization ability of the CH1 strain in the tobacco rhizosphere, including the following steps: (1) CH1 is labeled with green fluorescent protein.

[0023] The T2 plasmid containing the GFP protein expression cassette was transferred into the bacteria of Bacillus amyloliquefaciens CH1 by natural transformation and cultured at 28°C until single colonies appeared. PCR verification and fluorescence microscopy observation were performed to obtain the GFP-tagged CH1 strain (referred to as GFP-tagged CH1 strain).

[0024] The GFP-tagged CH1 strain activated overnight was added to the LB liquid medium without antibiotics at a 1% inoculum, and cultured at 37°C and 180 r / min for 5 h. This was repeated 10 times. The culture solution after 10 times of culture was diluted a certain multiple and spread on resistant and non-resistant plates respectively. After a single colony grew, the number of colonies on the two plates was counted and compared. The results showed that the GFP-tagged CH1 strain was genetically stable.

[0025] (2) Potted experiment of tobacco seedlings.

[0026] Tobacco seedlings with consistent growth were selected and potted experiments were set up. According to the test results of Example 2, solutions of different concentrations were prepared for different substances and added to the rhizosphere soil of the tobacco seedlings. Specifically, the following treatment groups were set up: T1: GFP-tagged CH1 + 0.10 mmol / L cinnamic acid; T2: GFP-tagged CH1 + 0.10 mmol / L benzyl cinnamate; T3: GFP-tagged CH1 + 0.05 mmol / L 4-ethylphenol; T4: GFP-tagged CH1+0.40 mmol / L protocatechuic acid.

[0027] Each treatment was repeated five times. After each seedling was watered with 5 mL of attractant, 5 mL of 1×10 7 CFU / mL of GFP-tagged CH1 strain was inoculated at a distance of 3-5 cm from the root. The wild type was used as the negative control (WT, Wild type of CH1), and the bacterial solution treatment group without attractant was used as the positive control (CK, GFP-tagged CH1).

[0028] Root tissues of tobacco seedlings were collected on the 7th, 14th and 28th days after root irrigation with the bacterial solution and rinsed with sterile water. The colonization of GFP-tagged CH1 in the rhizosphere of tobacco was observed using a plant living imaging system (NightSHADE L985).

[0029] The results are as follows Figure 2As shown in the figure, the fluorescence intensity gradually weakened with the passage of time, indicating that the colonization of CH1 in the rhizosphere gradually degraded with the increase of time, but there was still a certain amount of bacteria that could survive and reproduce in the rhizosphere for a long time ( Figure 2 ).

[0030] Example 3 The efficacy of biocontrol bacteria is closely related to their biofilm. In this case, 4-ethylphenol, cinnamic acid, benzyl cinnamate and protocatechuic acid, which can promote the colonization of CH1 strains in tobacco roots, were further tested for their effects on the biofilm of CH1 strains, including the following steps: Inoculate CH1 strain into 5 mL LB medium, culture at 37℃, 180 r / min overnight, transfer to 5 mL LB medium at 1% inoculum, culture at 37℃, 180 r / min for 5-6 h until OD600 ≈ 1.0.

[0031] Use the standards of 4-ethylphenol, cinnamic acid, benzyl cinnamate and protocatechuic acid to prepare the stock solutions of the test solutions.

[0032] The stock solution of the test solution was added to a 24-well plate containing 2 mL of MSgg medium, and the final concentration was 0.05, 0.10, 0.20, 0.40, and 0.80 mmol / L, and then the bacterial solution was added to the 24-well plate containing the test solution at a ratio of 1%. The control group was MSgg medium without the stock solution of the test solution, and was cultured at 28°C for 48 h, and observed and photographed. Gently aspirate the culture medium and free cells in each well, wash twice with distilled water, add 2 mL of 0.1% crystal violet staining solution to each well, let stand at room temperature for 30 min, carefully and slowly rinse the biofilm with distilled water until the washing solution is colorless, and then dry overnight. Add 1 mL of 33% acetic acid to dissolve the stained biofilm, shake at room temperature to fully dissolve the crystal violet, and appropriately dilute the sample with 33% acetic acid, use 33% acetic acid as a blank control, and measure the absorbance at 595 nm.

[0033] The results are as follows Figure 3 As shown: CH1 formed a more solid biofilm structure after treatment with different attractants ( Figure 3 A), that is, it promoted the formation of biofilm; among them, 0.05 mM protocatechuic acid, 0.2 mM benzyl cinnamate, 0.2 mM 4-ethylphenol and 0.05 mM cinnamic acid can promote the formation of biofilm of CH1 strain ( Figure 3 BE).

[0034] In summary, 4-ethylphenol, cinnamic acid, benzyl cinnamate and protocatechuic acid can promote the enrichment and colonization of CH1 strain in the tobacco rhizosphere (cinnamic acid has the best effect), and can also promote the increase of CH1 strain biofilm. Therefore, it is expected that the above substances can be used to further improve the field control effect of CH1 strain on bacterial wilt disease.

[0035] It should be noted that the above embodiments are only part of the embodiments of the present invention rather than all the embodiments, and are only used to illustrate the technical solution of the present invention rather than to limit it. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

Claims

1. A chemical attractant for promoting the colonization of biocontrol bacteria in the rhizosphere of tobacco, characterized in that: The biocontrol bacteria is Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens ) CH1, whose deposit number is CCTCC NO: M208127, and the chemical attractant has cinnamic acid as one of the active ingredients.

2. The chemical attractant according to claim 1, characterized in that In the chemical attractant, the concentration of cinnamic acid is 0.04-0.15 mmol / L.

3. The chemical attractant according to claim 1, characterized in that The active ingredients in the chemical attractant also include one or more of 4-ethylphenol, benzyl cinnamate and protocatechuic acid.

4. The chemical attractant according to claim 3, characterized in that The concentration of the 4-ethylphenol is 0.05-1.0 mmol / L, the concentration of the benzyl cinnamate is 0.1-1.0 mmol / L, and the concentration of the protocatechuic acid is 0.4-0.5 mmol / L.

5. A method for promoting the colonization of biocontrol bacteria in the rhizosphere of tobacco, characterized in that: A chemical attractant containing at least cinnamic acid is applied to the rhizosphere soil of tobacco, wherein the biocontrol bacteria is Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens ) CH1, and its deposit number is CCTCC NO: M208127.

6. The method according to claim 5, characterized in that The chemical attractant also includes at least one of 4-ethylphenol, benzyl cinnamate and protocatechuic acid.

7. Use of the chemical attractant according to any one of claims 1 to 4 or the method according to any one of claims 5 to 6 in enhancing the control effect of biocontrol bacteria on tobacco diseases in the field.

8. The use according to claim 7, characterized in that: The tobacco disease is tobacco bacterial wilt.

9. Use of the chemical attractant according to any one of claims 1 to 4 or the method according to any one of claims 5 to 6 in enhancing the ability of biocontrol bacteria to promote the growth of tobacco plants.

10. Use of the chemical attractant according to any one of claims 1 to 4 in increasing the biofilm yield of Bacillus amyloliquefaciens CH1.

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