A chemical attractant and method for promoting the colonization of biocontrol bacteria in the rhizosphere of tobacco

Abstract

The present invention belongs to the technical field of tobacco disease control, and discloses a chemical attractant and method for promoting the colonization of biocontrol bacteria in the tobacco rhizosphere. The biocontrol bacteria is Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens ) CH1. The chemical attractant takes cinnamic acid as one of the active ingredients, and may also contain active ingredients such as 4-ethylphenol, benzyl cinnamate, and protocatechuic acid. The cinnamic acid, 4-ethylphenol, benzyl cinnamate, and protocatechuic acid provided by the present invention can selectively and efficiently promote the enrichment and colonization of strain CH1 in the tobacco rhizosphere, and at the same time can increase the biofilm amount of strain CH1, thereby being beneficial to enhancing the field control effect of strain CH1 on tobacco bacterial wilt disease; this scheme is safe and harmless to the environment and has good application prospects.

Description

Technical Field

[0001] The present invention belongs to the technical field of tobacco disease control, and particularly relates to a chemical attractant and method for promoting the colonization of biocontrol bacteria in the tobacco rhizosphere, 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 devastating diseases in the process of tobacco cultivation, causing huge economic losses to tobacco leaf production. Tobacco bacterial wilt is a typical vascular bacterial disease, and all parts of the root, stem, and leaf can be affected (Dan Xiaopeng, 2021). The most typical symptom is wilting, which mostly occurs during the vigorous growth and maturity stages of tobacco. Once the pathogen invades from the damaged part of the tobacco, it will rapidly multiply in large numbers and secrete extracellular polysaccharide proteins, etc. to block vascular transport, resulting in difficulty in the normal transport of nutrients in the tobacco plant. This causes the tobacco roots to turn black and gradually rot, the stems to form black stripes and start to form cavities, and the leaves to wilt until finally die. Research shows that high temperature and high humidity, low-lying and sticky, neutral to slightly acidic soils are prone to bacterial wilt, and the epidemic areas of bacterial wilt show a trend of spreading (Kong Fanyu, 2003). In order not to affect the development of the tobacco agricultural economy, the prevention and control of tobacco bacterial wilt is urgent.

[0003] Biological control, as a green, environmentally friendly, sustainable development method that conforms to the concept of green agricultural production, has been concerned by more and more researchers and is a control method with broad application prospects. Biological control mainly refers to the treatment of tobacco soil with microbial agents, and inhibits 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 structure of soil microorganisms and protect the tobacco field ecological environment, etc. (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 medium in the process of plant-microbe interactions. On the one hand, root exudates, as nutrients, enrich a large number of microbial populations around plant roots. On the other hand, as signal molecules, they participate in highly specific chemical communication between roots and soil microorganisms, establishing an interaction 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). Existing studies have found that secondary metabolites secreted by roots, as signal molecules, attract rhizobia and mycorrhizal fungi. For example, flavonoids can be regulators for leguminous plants to utilize nitrogen fixation by rhizobia, strigolactones provide a communication medium for the formation of a mutually beneficial relationship between mycorrhizal fungi and hosts, and organic acids can be important chemotactic substances for rhizosphere Bacillus (Hassan and Mathesius, 2012; Shen et al, 2022); organic acids such as malic acid and citric acid can recruit Bacillus to rhizosphere soil (Wu Linkun et al, 2014); the exudates of rice can attract Bacillus; the exudates of Arabidopsis play an important role in attracting Bacillus subtilis and enhancing rhizosphere colonization (Badri et al, 2013); the root exudates of citrus promote the growth and colonization of beneficial rhizosphere bacteria Pseudomonas putida and Novosphingobium (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 relationship between tobacco and biocontrol bacteria.

[0006] The technical solution of the present invention is specifically as follows:

[0007] In the first aspect of the present invention, a chemical attractant for promoting the colonization of biocontrol bacteria in the tobacco rhizosphere is provided, wherein the biocontrol bacteria is Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens ) CH1, with the preservation number of CCTCC NO: M208127, the preservation time of September 5, 2008, and the preservation address of China Center for Type Culture Collection (Wuhan University, Wuhan, Hubei Province).

[0008] The CH1 strain has been proven to have the ability to promote the growth of tobacco plants and antagonistic activity against Ralstonia solanacearum. The enrichment and colonization of the CH1 strain in the tobacco roots are the key factors for its growth promotion and disease resistance. Through the analysis of tobacco root exudates, the inventors found that among a large number of root exudates, the CH1 strain has chemotaxis to 4-ethylphenol, cinnamic acid, benzyl cinnamate, and protocatechuic acid. Introducing 4-ethylphenol, cinnamic acid, benzyl cinnamate, and protocatechuic acid into the tobacco rhizosphere can significantly enhance the enrichment and colonization ability of CH1 in the tobacco rhizosphere. Moreover, the experimental data show that cinnamic acid has the best effect, followed by benzyl cinnamate and 4-ethylphenol, and the mixture 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.

[0009] Preferably, the active ingredient of the chemical attractant at least includes cinnamic acid, and may also include one or more of 4-ethylphenol, benzyl cinnamate, and protocatechuic acid.

[0010] 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.

[0011] The second aspect of the present invention provides a method for promoting the colonization of biocontrol bacteria in the tobacco rhizosphere, specifically: applying the chemical attractant to the tobacco rhizosphere soil, thereby utilizing the chemotaxis of the biocontrol bacterium CH1 to the chemical attractant to achieve the enrichment of the CH1 strain in the tobacco rhizosphere 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.

[0012] Preferably, in the above method, the active ingredient of the chemical attractant at least includes cinnamic acid; more preferably, it also includes one or more of 4-ethylphenol, benzyl cinnamate, and protocatechuic acid.

[0013] The third aspect of the present invention provides the application of the above chemical attractant, including at least the following:

[0014] a) Promote the colonization of the biocontrol bacterium CH1 in the tobacco rhizosphere;

[0015] b) Enhance the field control effect of the biocontrol bacterium CH1 on tobacco bacterial wilt;

[0016] c) Enhance the ability of the biocontrol bacterium CH1 to promote the growth of tobacco;

[0017] d) Increase the biofilm production of the biocontrol bacterium CH1.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0019] The present invention provides a chemical attractant suitable for the biocontrol bacterium Bacillus amyloliquefaciens CH1, which can selectively and efficiently promote the enrichment and colonization of the biocontrol bacterium in the rhizosphere of tobacco, thereby being beneficial to enhancing the biocontrol effect of the strain. Moreover, the chemical attractant provided by the present invention is safe and pollution-free to the environment. Therefore, the solution of the present invention is beneficial to more efficiently, simply and economically controlling tobacco bacterial wilt and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 It is the chemotaxis detection result of strain CH1 to protocatechuic acid, benzyl cinnamate, 4-ethylphenol and cinnamic acid in Example 1 of the present invention;

[0021] Figure 2 It is the fluorescence intensity map of the colonization situation of strain CH1 in the rhizosphere of tobacco in Example 2 of the present invention;

[0022] Figure 3 It is the influence of different concentrations of protocatechuic acid, benzyl cinnamate, 4-ethylphenol and cinnamic acid on the biofilm of strain CH1 in Example 3 of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0023] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the technical field to which the present invention belongs. The term "comprising" and any variation thereof in the specification and claims of the present invention are intended to cover non-exclusive inclusion.

[0024] The technical solutions of the present invention will be clearly and completely described 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.

[0025] For those not specified in the following examples in terms of specific technologies or conditions, they are all carried out according to the technologies or conditions described in the literature in this field or according to the product specifications; for the reagents or instruments not specified in terms of the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0026] Example 1

[0027] In this example, 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:

[0028] Standard products of 4-ethylphenol, cinnamic acid, benzyl cinnamate and protocatechuic acid were respectively prepared into test solutions with concentrations of 0.05, 0.10, 0.20, 0.40 and 0.80 mmol / L for standby.

[0029] Inoculate strain CH1 into 5 mL of LB medium and culture it overnight at 37°C and 180 r / min for activation; transfer it to 5 mL of LB medium at an inoculation amount of 1% and culture it at 37°C and 180 r / min for 5 - 6 h until the mid-logarithmic phase (OD600 ≈ 1.0). Take 2 mL of the fermentation broth, centrifuge it at 25°C and 5000 r / min for 5 min, and after concentration, resuspend it in an equal volume of chemotaxis buffer (containing 4.4 g of K2HPO4, 4.2 g of KH2PO4, and 0.00745 g of Na2EDTA, made up to 1 L with water, and the pH adjusted to neutral) to prepare a bacterial suspension, and use a 200 μL pipette tip to aspirate 100 μL of the bacterial suspension for use.

[0030] Use a 1 mL syringe as the capillary for the chemotaxis test, aspirate 100 μL of the test solution, and insert the syringe needle into the narrow end of the above pipette tip to allow the test solution to come into full contact with the bacterial suspension; after standing at room temperature under sterile conditions for 2 h, carefully remove the syringe needle from the bacterial suspension, and detect the bacterial content in the test solution in the syringe by the dilution plate coating method, that is, count the number of colonies (CFU) after culturing at 37°C for 24 h. Set 3 replicates for each treatment; use the chemotaxis buffer as a control and calculate the average value of the number of colonies on 3 replicated plates.

[0031] The chemotaxis index (relative chemotaxis index, RIC) value is the ratio of the average value of CFU in the treatment group to the CFU in the control group. When RIC ≥ 2, it is determined that the chemotaxis of the treatment group is significantly different from that of the control group. The results are as Figure 1 shown. Strain CH1 showed chemotaxis to 0.4 mM protocatechuic acid (1A), strain CH1 showed chemotaxis to 0.1 mM, 0.2 mM, and 0.8 mM benzyl cinnamate (1B), strain CH1 showed chemotaxis to 0.05 mM, 0.2 - 0.8 mM 4-ethylphenol (1C), and strain CH1 showed chemotaxis to 0.05 - 0.8 mM cinnamic acid (1D).

[0032] Example 2

[0033] In this example, 4-ethylphenol, cinnamic acid, benzyl cinnamate, and protocatechuic acid in Example 2 were used as the active ingredients of the attractant respectively to detect the actual effects of different substances on the colonization ability of strain CH1 in the tobacco rhizosphere, including the following steps:

[0034] (1) Label CH1 with green fluorescent protein.

[0035] The T2 plasmid containing the GFP protein expression cassette was transferred into the cells of Bacillus amyloliquefaciens CH1 by natural transformation method. After culturing at 28 °C until single colonies appeared, PCR verification and fluorescence microscopy observation were carried out to obtain the GFP-labeled CH1 strain (denoted as GFP-tagged CH1 strain).

[0036] The overnight-activated GFP-tagged CH1 strain was added to the LB liquid medium without antibiotics at an inoculation amount of 1%, and cultured at 37 °C and 180 r / min for 5 h. This was repeated 10 times. After culturing 10 times, the culture solution was diluted by a certain multiple and then spread on resistant and non-resistant plates respectively. After single colonies grew out, the number of colonies on the two types of plates was counted and compared. The results showed that the GFP-tagged CH1 strain had genetic stability.

[0037] (2) Pot experiment of tobacco seedlings.

[0038] Tobacco seedlings with consistent growth were selected to set up a pot experiment. According to the detection results of Example 2, different concentrations of solutions were prepared for different substances and added to the rhizosphere soil of tobacco seedlings. The following treatment groups were specifically set:

[0039] T1: GFP-tagged CH1 + 0.10 mmol / L cinnamic acid;

[0040] T2: GFP-tagged CH1 + 0.10 mmol / L benzyl cinnamate;

[0041] T3: GFP-tagged CH1 + 0.05 mmol / L 4-ethylphenol;

[0042] T4: GFP-tagged CH1 + 0.40 mmol / L protocatechuic acid.

[0043] After 5 replicates for each treatment, after watering 5 mL of attractant for each seedling, 5 mL of 1×10 7 CFU / mL of GFP-tagged CH1 strain was inoculated at a position 3 - 5 cm away from the root. The wild type was used as the negative control (WT, Wild type of CH1), and the treatment group with the bacterial solution without attractant was used as the positive control (CK, GFP-tagged CH1).

[0044] The root tissues of tobacco seedlings were taken on the 7th d, 14th d and 28th d after root irrigation with the bacterial solution, and rinsed with sterile water; the colonization of GFP-tagged CH1 in the tobacco rhizosphere was observed through a plant in vivo imaging system (NightSHADE L985).

[0045] The results are asFigure 2 As shown in the figure, the fluorescence intensity gradually weakened over time, indicating that the colonization of CH1 in the rhizosphere gradually degraded with the increase of time. However, a certain amount of bacteria could still survive and reproduce in the rhizosphere for a long time ( Figure 2 ).

[0046] Example 3

[0047] The biocontrol efficacy of biocontrol bacteria is closely related to their biofilms. In this example, for 4-ethylphenol, cinnamic acid, benzyl cinnamate, and protocatechuic acid that can promote the colonization of CH1 strain in tobacco roots, the effects of these substances on the biofilm of CH1 strain were further detected, including the following steps:

[0048] Inoculate the CH1 strain into 5 mL of LB medium and culture overnight at 37°C and 180 r / min. Transfer it to 5 mL of LB medium at an inoculation amount of 1% and culture at 37°C and 180 r / min for 5-6 h until OD600 ≈ 1.0.

[0049] Use the standards of 4-ethylphenol, cinnamic acid, benzyl cinnamate, and protocatechuic acid to prepare the stock solutions of the test solutions respectively.

[0050] Add the stock solutions of the test solutions to a 24-well plate containing 2 mL of MSgg medium and make their final concentrations 0.05, 0.10, 0.20, 0.40, 0.80 mmol / L. Then add the bacterial solution to the 24-well plate containing the test solution at a ratio of 1%. The control group is the MSgg medium without adding the stock solution of the test solution. Incubate statically at 28°C for 48 h, observe and take pictures.

[0051] Gently aspirate the 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, 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 the blank control and measure the absorbance at 595 nm.

[0052] The results are as Figure 3 shown: After treatment with different attractants, CH1 formed a stronger biofilm structure ( Figure 3 A), that is, it promoted the formation of biofilms. Among them, 0.05 mM protocatechuic acid, 0.2 mM benzyl cinnamate, 0.2 mM 4-ethylphenol, and 0.05 mM cinnamic acid could all promote the production of biofilms by the CH1 strain ( Figure 3 B-E).

[0053] In summary, 4-ethylphenol, cinnamic acid, benzyl cinnamate, and protocatechuic acid can promote the enrichment and colonization of strain CH1 in the tobacco rhizosphere (among which cinnamic acid has the best effect), and at the same time can promote the increase of the biofilm of strain CH1. Therefore, it is expected to further improve the field control effect of strain CH1 on bacterial wilt by using the above substances.

[0054] It should be noted that the above embodiments are only a part rather than all of the embodiments of the present invention, and are only used to illustrate the technical solutions of the present invention rather than to limit them; based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present invention.

Claims

1. A method for promoting the colonization of biocontrol bacteria in the rhizosphere of tobacco, characterized in that: A chemical attractant containing 4-ethylphenol is applied to the rhizosphere soil of tobacco to promote the colonization of biocontrol bacteria in the rhizosphere of tobacco, wherein the biocontrol bacteria is Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens ) CH1, and its deposit number is CCTCC NO: M208127.

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