Application of 3-phenylpropionic acid in improving the efficacy of Bacillus sp. in controlling plant diseases

Abstract

The present invention discloses the use of 3-phenylpropionic acid (3-phenylpropionic acid) in improving the ability of Bacillus to prevent and control plant diseases, wherein 3-phenylpropionic acid is used to induce the enrichment and colonization of Bacillus in the rhizosphere of plants. The present invention, by treating cucumber root exudates with shiitake mushroom waste, identifies for the first time that 3-phenylpropionic acid in the root exudates can serve as a signal substance for inducing the enrichment of Bacillus in the rhizosphere. Furthermore, by co-treating plants with Bacillus and 3-phenylpropionic acid, the plants' resistance to Fusarium wilt is significantly improved, providing a new approach for utilizing beneficial Bacillus to further enhance plant disease resistance.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant disease prevention and control, and specifically relates to the use of 3-phenylpropionic acid as a signal substance for regulating rhizosphere microbial communities, thereby enhancing the prevention and control effect of Bacillus on plant diseases (such as wilt) by recruiting Bacillus to colonize in the plant rhizosphere. Background Art

[0002] Root exudates serve as a medium for communication between plants and rhizosphere microorganisms. By adjusting the chemical composition of root exudates, plants can actively recruit beneficial soil bacteria to colonize the rhizosphere in response to various environmental stresses. Studies have found that infection with soil-borne pathogens alters the composition of plant rhizosphere bacterial communities and leads to an increase in the abundance of beneficial plant bacteria such as Bacillus, Pseudomonas, and Actinomycetes in the rhizosphere, thereby enhancing plant disease resistance. Therefore, researchers hope to enhance disease resistance by artificially manipulating plant root exudates to encourage the recruitment of beneficial microorganisms to plant roots.

[0003] At present, although some signal substances in root exudates that can regulate rhizospheric microbiota have been identified, people still lack understanding of the mechanism by which plants selectively recruit beneficial bacteria through root exudates.

[0004] Lentinus edodes waste is a soil conditioner. Studies have shown that treating soil with Lentinus edodes waste can effectively control crop diseases (such as Fusarium wilt), and that rhizosphere microbiota plays a key role in this disease prevention. Lentinus edodes waste soil treatment promotes the accumulation of plant-beneficial Bacillus spores in the rhizosphere, inhibiting root infection by pathogens such as Fusarium oxysporum and Verticillium dahliae. However, the signaling substances responsible for the recruitment of Bacillus spores by Lentinus edodes waste remain unknown. Summary of the Invention

[0005] In view of this, the present invention aims to explore signal substances for recruiting Bacillus through in-depth research on plant root secretions under conditions of shiitake mushroom husk soil treatment, and provide a new way to further enhance plant disease resistance by utilizing beneficial Bacillus.

[0006] The technical solutions of the present invention are as follows:

[0007] In a first aspect, the present invention provides the use of 3-phenylpropionic acid (C6H5CH2CH2COOH, CAS: 501-52-0) in improving the effectiveness of Bacillus in controlling plant diseases, wherein 3-phenylpropionic acid serves as a signal substance that induces Bacillus to enrich and colonize in the rhizosphere of plants.

[0008] The present invention uses shiitake mushroom waste as a soil treatment agent and simultaneously inoculates wilt pathogens. By collecting and analyzing plant root exudates, chemical components in the root exudates that are upregulated under shiitake mushroom waste treatment are obtained, and further analysis and identification show that 3-phenylpropionic acid can serve as a signal substance for recruiting beneficial Bacillus.

[0009] Preferably, in the above application, Bacillus and 3-phenylpropionic acid can be mixed to prepare an inoculum solution, which is then applied to plants. 3-phenylpropionic acid can actively promote the enrichment and colonization of Bacillus in the rhizosphere, thereby enhancing the disease resistance of the plants.

[0010] More preferably, in the above application, the concentration of 3-phenylpropionic acid in the inoculum is 1 to 70 μg / mL, with 10 μg / mL being optimal. Experimental results of the present invention indicate that 3-phenylpropionic acid has a strong chemotactic attraction to Bacillus at low concentrations, while high concentrations can inhibit the chemotaxis of Bacillus.

[0011] In a specific embodiment of the present invention, Bacillus Velez and an appropriate amount of 3-phenylpropionic acid were prepared into an inoculum and inoculated into cucumbers, which were then infected with the wilt pathogen Fusarium oxysporum. The results showed that compared with the case of inoculation with Bacillus Velez only, the co-treatment of Bacillus Velez and 3-phenylpropionic acid could significantly reduce the incidence rate and effectively increase the colonization of Bacillus Velez in the cucumber rhizosphere.

[0012] A second aspect of the present invention provides a preparation for preventing and treating cucumber wilt, comprising Bacillus velezensis and 3-phenylpropionic acid. It is understood that the preparation can be a mixed liquid preparation or a preparation in other forms.

[0013] A third aspect of the present invention provides an agent for enhancing the ability of Bacillus to colonize the cucumber rhizosphere, comprising 3-phenylpropionic acid as an active ingredient. Experiments in the present invention have shown that 3-phenylpropionic acid at specific concentrations has a strong chemotactic attraction for Bacillus velezensis, inducing the enrichment of Bacillus in the plant rhizosphere. Therefore, 3-phenylpropionic acid can be used to prepare an agent for enhancing the colonization of Bacillus in the plant rhizosphere.

[0014] The beneficial effects of the present invention are:

[0015] The present invention induces cucumber root exudates by treating shiitake mushroom waste, and further identifies for the first time that 3-phenylpropionic acid in the root exudates can serve as a signal substance that induces Bacillus rhizosphere enrichment; compared with treatment with Bacillus alone, co-treatment of Bacillus and 3-phenylpropionic acid can significantly increase the colonization of Bacillus in the plant rhizosphere and significantly improve the disease resistance of the plant.

[0016] The present invention also reveals the mechanism by which soil treatment with shiitake mushroom waste promotes the enrichment of plant-beneficial Bacillus in the rhizosphere. Specifically, treatment with shiitake mushroom waste induces an increase in the content of 3-phenylpropionic acid in plant root exudates, and 3-phenylpropionic acid has a strong chemotactic attraction for Bacillus. This invention helps deepen our understanding of the mechanisms by which plants recruit beneficial bacteria and provides a basis for enhancing plant disease resistance by altering the composition of plant rhizosphere bacterial communities through signaling substances. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The chemotaxis results of cucumber root exudates to Bacillus Velezii B31 strain in Example 1 are shown.

[0018] Figure 2 This is the result of non-targeted metabolomics analysis of cucumber root exudates in Example 1.

[0019] Figure 3 The chemotaxis results of different chemical standards in Example 1 on Bacillus velezensis B31 strain are shown.

[0020] Figure 4 This is the effect of 3-phenylpropionic acid in Example 2 on the control of cucumber wilt and rhizosphere colonization by Bacillus Velez B31 strain. DETAILED DESCRIPTION

[0021] The technical solution of the present invention will be described clearly and completely below in conjunction with the embodiments and drawings. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present invention and are not intended to limit the present invention.

[0022] 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 pertains. The term "comprise" and any variations thereof in the description and claims of the present invention are intended to cover non-exclusive inclusions.

[0023] If no specific techniques or conditions are specified in the following examples, the procedures were carried out in accordance with the techniques or conditions described in the literature in the field or in accordance with the product instructions; if no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0024] Example 1

[0025] In this study, we analyzed cucumber root exudates obtained by induction with Lentinus edodes waste and identified 3-phenylpropionic acid, which is found in increased levels in cucumber root exudates, as a signal substance for recruiting Bacillus spores. The experimental process is as follows:

[0026] (1) Preparation of inoculant and soil treatment.

[0027] Inoculant Preparation: Inoculate a PDA plate with Fusarium oxysporum (FOC) pathogen (stored in a refrigerator at 4°C) and incubate at 25°C for 5 days. A bacterial cake is then inoculated into PDB culture medium, shaking at 180 rpm and 25°C for 4 days. Filter the culture medium through four layers of gauze to remove mycelium. Centrifuge the filtrate at 8000 rpm at 4°C for 15 minutes, discard the supernatant, resuspend the conidia in water, and count them microscopically using a hemocytometer.

[0028] Soil treatment: FOC conidia suspension was inoculated into sterilized soil to prepare conidia soil, so that the final conidia concentration was 1*10 5 / g soil; 2% (w / w) shiitake mushroom residue powder was thoroughly mixed with the inoculated soil for cucumber seedling cultivation.

[0029] (2) Collection of cucumber root secretions.

[0030] Cucumber seeds were surface-sterilized and sown in seedling pots. Once two cotyledons had fully expanded, they were transplanted into plastic pots filled with inoculated soil. The treatment with Lentinus edodes spores was designated SMS, while the treatment without Lentinus edodes spores was designated CK. Each treatment consisted of three replicates, each containing 30 cucumber seedlings.

[0031] Ten days after transplanting, carefully remove the cucumber seedlings from the pots and rinse the roots with running water to remove any soil. Immerse the washed cucumber seedling roots in sterile deionized water at 25°C in the dark for 6 hours. Filter the collected solution through a 0.2 μm filter to sterilize it and freeze-dry it for storage.

[0032] (3) Chemotaxis of cucumber root secretions to Bacillus.

[0033] Chemotaxis was quantitatively determined by capillary tube method, specifically: Bacillus velezensis B31 strain was shaken and cultured to OD 600 The concentration of the culture medium was 0.8, and the bacteria were collected by centrifugation and resuspended in chemotaxis buffer (100 mM potassium phosphate (pH 7.0) + 20 μM EDTA); the bacterial suspension was aspirated into a 200 μL pipette tip; 200 μL of root exudate solution (concentration of 10 μg / mL, 50 μg / mL or 100 μg / mL) was aspirated into a 1 mL syringe, and the syringe needle was inserted into the pipette tip containing the bacterial suspension and allowed to stand for 2 hours; then, the liquid in the syringe was injected into the LB medium plate and spread evenly, incubated at 30°C for 24 hours, and the number of colonies was counted.

[0034] The chemotaxis was calculated using the chemotaxis buffer solution without root exudates as a control. Figure 1As shown in the results, cucumber root exudates had strong chemotaxis to B31 strain at the tested concentrations, and when the root exudate concentrations were 10 μg / mL, 50 μg / mL and 100 μg / mL, the chemotactic attraction to B31 strain increased by 2 times, 7.4 times and 15.1 times respectively compared with the chemotaxis buffer.

[0035] (4) Non-targeted metabolome analysis of cucumber root exudates.

[0036] In this study, an Agilent 8890B-5977B gas chromatograph-mass spectrometer (Agilent, USA) was used for untargeted metabolomic analysis of cucumber root exudates. Chromatographic conditions: After derivatization, the sample was injected into the GC-MS system in split mode for analysis, with an injection volume of 1 µL and a split ratio of 10:1. The sample was separated on a DB-5MS capillary column (40 m × 0.25 mm × 0.25 µm, Agilent 122-5532G) before being detected by mass spectrometry. The inlet temperature was 300°C, the carrier gas was high-purity helium at a flow rate of 1 mL / min, the septum purge flow rate was 3 mL / min, and the solvent delay was 5.5 min. The temperature program was as follows: initial temperature 60°C, equilibration for 0.5 min, then ramped to 310°C at a rate of 8°C / min and held for 6 min. Mass spectrometry conditions: electron impact ion source (EI), transfer line temperature 310°C, ion source temperature 280°C, quadrupole temperature 150°C, electron energy 70 eV. Scan mode: full scan (SCAN), mass scan range: m / z 50-500, scan frequency 3.2 scans / s.

[0037] The results of non-targeted metabolomics analysis were as follows Figure 2 As shown in the results: Through non-targeted metabolome analysis, it was found that the chemical components of cucumber root exudates of SMS were significantly different from those of CK. A total of 401 chemical components were identified in the cucumber root exudates, of which the contents of 231 components were significantly different between SMS and CK treatments. Compared with CK, there were 135 components significantly upregulated and 96 components significantly downregulated in SMS; the two treatments were clustered separately, indicating that there were significant differences in cucumber root exudates between SMS and CK treatments.

[0038] As shown in Table 1, 10 differential metabolites were screened based on the sample classification contribution (VIP value), significance analysis (p value) and difference fold (log2fold change). Among them, 3-phenylpropionic acid and (3R,4R)-(-)-D-erythroleucone lactone were strongly correlated with the chemotaxis of root exudates to B31 strain (R>0.8 and P <0.05).

[0039] Table 1 Difference analysis and correlation analysis of cucumber root exudates

[0040]

[0041] (4) Chemotaxis of B31 strain to chemical standards.

[0042] Chemical standards of 3-phenylpropionic acid and (3R,4R)-(-)-D-erythroleucone lactone were prepared in chemotaxis buffer (100 mM potassium phosphate (pH 7.0) + 20 μM EDTA) at concentrations of 1 μg / mL, 10 μg / mL, 50 μg / mL, and 100 μg / mL, respectively.

[0043] The above solutions were used as chemical attractants, and the chemotaxis of strain B31 to 3-phenylpropionic acid and (3R,4R)-(-)-D-erythrolide was determined by capillary tube method in step (3). Figure 3 As shown in the results, 3-phenylpropionic acid has a strong chemotactic attraction to B31 at low concentrations, but inhibits B31 chemotaxis at high concentrations, while (3R,4R)-(-)-D-erythroleucone lactone has no significant effect on B31.

[0044] Example 2

[0045] In this case, Bacillus and 3-phenylpropionic acid were co-treated to verify their effect on the resistance of cucumber to Fusarium wilt. The specific operation is as follows:

[0046] After culturing the B31 strain in LB liquid medium with shaking for 12 hours, centrifuge at 8000 rpm for 15 minutes, remove the supernatant, and resuspend the pellet in chemotaxis buffer to an OD600 value of 0.8. Add 3-phenylpropionic acid to the B31 suspension to a final concentration of 10 μg / mL to serve as the Bacillus inoculum.

[0047] After surface disinfection, cucumber seeds were sown in seedling trays containing sterilized substrate. Once two cotyledons had fully expanded, they were transplanted into pots containing sterilized substrate. Each cucumber seedling was inoculated with 5 mL of the aforementioned Bacillus inoculum. A B31 suspension without 3-phenylpropionic acid was used as a control. Each treatment consisted of three replicates, with 20 seedlings per replicate.

[0048] The cucumber wilt pathogen, Fusarium oxysporum, was activated on a PDA plate and inoculated into PDB liquid medium at 25°C and shaken at 180 rpm for 5 days. After filtering through four layers of gauze to remove mycelium, the mixture was centrifuged at 8000 rpm for 15 minutes, the supernatant was discarded, and the cells were resuspended in sterile water to a spore concentration of 1×10 7 spores / mL of pathogen inoculum.

[0049] Two days after inoculation with B31, cucumber seedlings were inoculated with 10 mL of the pathogen inoculum per seedling. Seven days later, the incidence of cucumber wilt was assessed, and rhizosphere soil was collected and the colonization of B31 in the rhizosphere soil was determined using a gradient dilution method.

[0050] The results of the detection of the incidence of cucumber wilt and the colonization of B31 strain in rhizosphere soil under different treatment conditions are as follows: Figure 4 The results showed that compared with the blank control (CK) without B31 inoculation, both B31 treatment and B31 + 3-phenylpropionic acid treatment (i.e., B31 + PA) significantly reduced the incidence of cucumber wilt, with the incidence rates reduced by 55.02% and 69.29%, respectively. The combined effect of B31 + PA was significantly better than that of B31 alone, with the incidence rate reduced by 31.72%. At the same time, the rhizosphere colonization of B31 was investigated, showing that the rhizosphere colonization of B31 alone was 3.7×10 5 The rhizosphere colonization rate of the B31+PA combination was 1.9×106 CFU / g soil, while the colonization rate in the rhizosphere was 1.9×106 CFU / g soil, a 4.14-fold increase. These results indicate that 3-phenylpropionic acid can significantly increase the colonization rate of the B31 strain in the cucumber rhizosphere and enhance the control effect of the B31 strain against cucumber wilt.

[0051] In summary, treatment with Lentinus edodes waste can induce an increase in the content of 3-phenylpropionic acid in cucumber root exudates. Co-treatment of 3-phenylpropionic acid with Bacillus can increase the rhizosphere colonization of Bacillus and enhance the control effect of cucumber against Fusarium wilt. It can be seen that 3-phenylpropionic acid plays a positive role in preventing and controlling plant diseases as a signal substance for recruiting beneficial bacteria in the soil.

[0052] 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 solutions of the present invention rather than to limit them. 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

The application of 1.3-phenylpropionic acid in improving the control of plant diseases by Bacillus sp. is characterized in that: The 3-phenylpropionic acid is used to induce Bacillus to enrich and colonize in the cucumber rhizosphere. The Bacillus and 3-phenylpropionic acid are mixed to prepare an inoculum solution, which is then applied to the cucumber. The plant disease is cucumber wilt, the Bacillus is the Bacillus velezensis B31 strain with a preservation number of CGMCC No. 24599, and the concentration of 3-phenylpropionic acid in the inoculum solution is 1-70 μg / mL.

2. The use according to claim 1, characterized in that The concentration of 3-phenylpropionic acid was 10 μg / mL.

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