Use of eugenol in preparation of bacteriostatic agent for enhancing efficiency of symbiotic nitrogen fixation of legume-rhizobium

Abstract

The application discloses application of eugenol in preparation of a bacteriostatic agent for enhancing symbiotic nitrogen fixation efficiency of legume-rhizobium, and relates to the field of agricultural microorganisms. ‑1 The application is safe and non-inhibiting to seed germination of Medicago polymorpha and target rhizobium LL2 in a concentration range of 500-1000 muL / L ‑1 of eugenol, can significantly reshape rhizosphere microbial community structure by selectively inhibiting competitive microorganisms such as bacillus and streptomyces in soil, and is environment-friendly and free of antibiotic residue risk, thereby providing a green and efficient new approach for improving symbiotic nitrogen fixation efficiency of legume-rhizobium.

Description

Technical Field

[0001] This invention relates to agricultural microbial technology, specifically to the application of eugenol in the preparation of antimicrobial agents for enhancing the nitrogen fixation efficiency of leguminous plants in rhizobia symbiotic relationships. Background Technology

[0002] Excessive application of chemical fertilizers not only reduces nitrogen use efficiency but also accelerates greenhouse gas emissions and groundwater pollution, threatening sustainable agricultural development. Against this backdrop, biological nitrogen fixation technology has become a crucial approach to addressing nitrogen fertilizer dependence and promoting green agriculture. A nitrogen fixation system jointly constructed by alfalfa and rhizobia possesses highly efficient and environmentally friendly nitrogen fixation capabilities, significantly reducing nitrogen fertilizer usage, improving soil quality, and increasing crop yield and quality. Therefore, it has significant application value in ecological agriculture and sustainable forage-livestock systems.

[0003] However, rhizobia often face strong competition from native microorganisms during actual field inoculation, leading to low colonization efficiency and weakened nodulation ability, thus limiting their widespread application. The survival and function of rhizobia in the soil are hindered by numerous native bacteria and fungi, especially some dominant species such as Bacillus and Streptomyces. These species significantly reduce the competitiveness of exogenous rhizobia through nutrient competition, space occupation, or the production of antimicrobial substances. Traditional research often uses antibiotic-based antimicrobial agents to suppress competing microorganisms to address this problem, but these methods easily lead to soil microbial imbalance, increased drug resistance, and environmental pollution, which is inconsistent with the development direction of green agriculture. Therefore, developing efficient, safe, and environmentally friendly green antimicrobial agents to selectively regulate the soil microbial community structure and enhance the field competitiveness and colonization ability of rhizobia has become an important research direction for optimizing rhizobia inoculation technology.

[0004] Eugenol is a plant-derived natural product with broad-spectrum antibacterial activity, but its application in regulating soil microbial communities and promoting nitrogen fixation by rhizobia has not been reported. Summary of the Invention

[0005] The purpose of this invention is to provide the application of eugenol in the preparation of antibacterial agents for enhancing the nitrogen fixation efficiency of leguminous plants-rhizobium symbiotic relationships. By selectively regulating the soil microbial community, the competitive advantage of exogenous rhizobia is enhanced, thereby improving the nitrogen fixation efficiency of leguminous plants-rhizobia symbiotic relationships and solving the problem of low colonization efficiency caused by competition from native microorganisms when rhizobia are intercropped in the field.

[0006] To achieve the above objectives, the present invention provides the following technical solution: the application of eugenol in the preparation of an antibacterial agent for enhancing the nitrogen fixation efficiency of leguminous plant-rhizobium symbiotic relationships, wherein the final concentration of eugenol in the antibacterial agent is 500-1000 μL·L⁻¹. -1 .

[0007] Furthermore, the final concentration of eugenol is 1000 μL·L⁻¹. -1 .

[0008] Furthermore, the legume is alfalfa.

[0009] Furthermore, the alfalfa mentioned is the Gannong No. 9 variety.

[0010] Furthermore, the rhizobium is *Rhizobium sinense*.

[0011] Furthermore, the *Rhizobium sinense* strain is LL2.

[0012] Furthermore, the antibacterial agent enhances the colonization advantage of rhizobia by selectively inhibiting competitive microorganisms in the soil, including at least one of Bacillus and Streptomyces.

[0013] Furthermore, the antibacterial agent can be applied in any one or more combinations of the following methods:

[0014] The antibacterial agent was used to soak legume seeds.

[0015] The antibacterial agent is pre-treated by applying it to the soil in which legumes are planted.

[0016] The antibacterial agent is mixed with the rhizobium bacterial solution and then applied simultaneously.

[0017] Furthermore, when the antibacterial agent is mixed with the rhizobium inoculum solution and applied simultaneously, the concentration of the rhizobium inoculum solution is 1×10⁻⁶. 9 CFU / mL, the inoculation amount is 0.5-2 mL per legume seedling.

[0018] Compared with the prior art, the application of eugenol provided by this invention in the preparation of antibacterial agents for enhancing the nitrogen fixation efficiency of leguminous plants-rhizobium symbiotic relationships involves distributing eugenol at 500-1000 μL·L⁻¹. -1 Within the specified concentration range, it is safe and does not inhibit alfalfa seed germination or the target rhizobium LL2. Furthermore, it can significantly reshape the rhizosphere microbial community structure by selectively inhibiting competing microorganisms such as Bacillus and Streptomyces in the soil.

[0019] The plant-derived antibacterial agent provided by this invention is environmentally friendly and has no risk of antibiotic residues, providing a green and efficient new approach to improve the nitrogen fixation efficiency of leguminous plants and rhizobia symbiotic relationships. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in this invention. For those skilled in the art, other drawings can be obtained based on these drawings.

[0021] Figure 1 This is a schematic diagram illustrating the effect of different concentrations of eugenol on alfalfa seed germination, provided in an embodiment of the present invention.

[0022] Figure 2 This is a schematic diagram illustrating the inhibitory effect of different concentrations of antibacterial agents on rhizobium LL2 provided in the embodiments of the present invention;

[0023] Figure 3 This is a schematic diagram of the community structure of soil bacteria at the phylum level under different treatments provided in the embodiments of the present invention;

[0024] Figure 4 This is a schematic diagram of the community structure of fungi at the phylum level under different treatments provided in the embodiments of the present invention;

[0025] Figure 5 This is a schematic diagram illustrating the relative abundance of soil Gram bacteria at the phylum level under different treatments provided in this embodiment of the invention.

[0026] Figure 6 A schematic diagram of nodulation indices of alfalfa under different antibacterial agent treatments provided in embodiments of the present invention;

[0027] Figure 7 This is a schematic diagram illustrating the activity of nitrogen-metabolizing enzymes in alfalfa roots under different antibacterial agent treatments, as provided in an embodiment of the present invention.

[0028] Figure 8 This is a schematic diagram illustrating the growth indicators of alfalfa under different antibacterial agent treatments provided in an embodiment of the present invention.

[0029] Figure 9 This is a schematic diagram illustrating the relative abundance of rhizobia and Gram bacteria in rhizosphere soil under different antibacterial agent treatments provided in this embodiment of the invention. Detailed Implementation

[0030] To enable those skilled in the art to better understand the technical solution of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings.

[0031] As attached Figure 1 To be continued Figure 9 As shown:

[0032] Materials and Methods

[0033] 1. Test materials

[0034] Eugenol: Purchased from Jiangxi Xuesong Natural Medicinal Oil Co., Ltd.

[0035] The Rhizobium sinense LL2 strain was provided by the Key Laboratory of Grassland Ecosystems of the Ministry of Education at Gansu Agricultural University. It was isolated from alfalfa root nodules in Longzhong and has a good affinity with Gansu Agricultural University No. 9 alfalfa.

[0036] Alfalfa seeds: Gannong No. 9, provided by the Key Laboratory of Grassland Ecosystems of the Ministry of Education, Gansu Agricultural University.

[0037] YMA liquid medium: K2HPO4·3H2O 0.5 g·L -1 ; MgSO4·7H2O 0.2 g·L -1 NaCl 0.1 g·L -1 Mannitol 10 g·L -1 1 g / L of yeast powder -1 1000 mL distilled water; 15 g / L agar -1 ;CaCO3 0.01g·L -1 .

[0038] TY medium: tryptone 5 g / L -1 5g / L yeast powder -1 ;CaCl2·6H2O0.1g·L -1 pH 7.0; Agar 15 g·L -1 (Solid), used for rhizobium activation.

[0039] 2. Data Processing and Analysis

[0040] SPSS 25 software was used for statistical analysis, Prism 8.0.2 software was used for plotting, and Qiime 2 software was used for microbial community data analysis.

[0041] Example 1:

[0042] This invention provides the effect of eugenol on alfalfa seed germination.

[0043] Germination tests were conducted according to the "Test Procedures for Forage Seeds" (GB / T2930.4-2017). Eugenol was prepared with sterile water to obtain concentrations of 0, 500, 1000, 2000, 5000, and 10000 μL·L⁻¹. -1 Six concentration gradients were used. Plump and uniform Gannong No. 9 alfalfa seeds were selected and soaked in solutions of various concentrations for 10 hours. After soaking, the seeds were blotted dry with filter paper. 100 seeds from each treatment were evenly placed in a petri dish (15 cm in diameter) lined with double-layered filter paper, and 30 mL of distilled water was added. Each treatment was repeated in triplicate. The seeds were weighed daily and water was added as needed. Germination potential was recorded on day 4, and germination rate was recorded on day 15. The seed hardness rate and imbibition rate were also recorded.

[0044] The results are as follows Figure 1 As shown. In the range of 0-1000 μL·L -1 Within the specified concentration range, both germination potential and germination rate were significantly higher than the control group (p<0.05), and the two values ​​largely overlapped, indicating uniform seed germination. (1000 μL·L⁻¹) -1 The best results are achieved when the concentration is increased to 2000 μL·L⁻¹, with a germination rate exceeding 95%. -1 At concentrations of 500-1000 μL·L, germination potential begins to decline, and the gap between germination rate and germination potential widens, indicating that high concentrations inhibit seed germination and lead to delayed germination. The hard seed rate is 500-1000 μL·L. -1 The concentration of eugenol was significantly lower than that of the control, while the imbibition rate increased with increasing concentration, indicating that high concentrations may be toxic to seeds. Therefore, the safe and effective concentration of eugenol was determined to be 500-1000 μL·L⁻¹. -1 The optimal value is 1000 μL·L. -1 .

[0045] Example 2:

[0046] This embodiment provides tolerance of eugenol to rhizobium LL2.

[0047] The Oxford cup method was used to determine the tolerance of rhizobium LL2 to eugenol. Activated LL2 bacterial culture (approximately 10 μL) was then used to determine the eugenol tolerance. 6 -10 8 A paste of eugenol (CFU / mL) is evenly spread on the surface of YMA solid medium. A sterile Oxford cup is placed in the center of the plate and gently pressed to ensure close contact with the medium. 200 μL of eugenol solution of different concentrations (500, 1000, 2000, 5000, 10000 μL·L⁻¹) is added to the Oxford cup. -1 After incubating at 28℃ for 18 hours, the diameter of the inhibition zone was observed.

[0048] The results are shown in Table 1 and Figure 2 As shown. In the range of 500-5000 μL·L -1 No inhibition zones were observed at any of the concentrations except at 10000 μL·L⁻¹. -1 A weak inhibition zone with a diameter of 9 mm was observed. According to the criteria for determining the inhibition zone (<10 mm indicates weak or no antibacterial effect), this indicates that eugenol has an inhibitory effect at ≤5000 μL·L⁻¹. -1 It had no inhibitory effect on rhizobium LL2. Based on seed germination experiments, a concentration of 1000 μL·L⁻¹ was selected. -1 This will be used as the concentration for subsequent tests.

[0049] Table 1. Tolerance of Rhizobium LL2 to Eugenol

[0050] <![CDATA[Eugenol concentration (μL·L -1 ).]]> 500 1000 2000 5000 10000 Diameter of the inhibition zone (mm) 0 0 0 0 9

[0051] Example 3:

[0052] This embodiment provides the effects of eugenol on soil microbial communities.

[0053] Soil samples were collected from the 0-5cm soil layer at the Lanzhou Pasture Experimental Station of Gansu Agricultural University, mixed thoroughly, and then dispensed. 10 mL of 1000 μL·L⁻¹ soil was added to each treatment group. -1 Eugenol solution was added to 1 L of soil and mixed thoroughly; an equal volume of sterile water was added to the control group. The mixture was placed in a light incubator (60% humidity, 25±2℃, natural light cycle) for 7 days. After treatment, genomic DNA was extracted from the soil and high-throughput sequencing was performed using the Illumina MiSeq platform to analyze the bacterial 16S rRNA gene and fungal ITS sequence.

[0054] α-diversity analysis

[0055] The results are shown in Table 2. After eugenol treatment, the Ace index and Chao index of soil bacteria and fungi were significantly lower than those of the control (p<0.05), indicating a decrease in species richness; the Shannon index decreased and the Simpson index increased, indicating a decrease in community evenness, an increase in dominant species, and an alteration in community structure.

[0056] Table 2. Soil bacterial and fungal α-diversity under different treatments

[0057]

[0058] Note: Different lowercase letters indicate significant differences (p<0.05).

[0059] Horizontal community structure

[0060] like Figure 3 and Figure 4 As shown, the dominant bacterial phyla in the control soil were Proteobacteria, Bacteroidetes, Actinobacteriota, and Firmicutes. After eugenol treatment, the relative abundance of Proteobacteria increased by 24.89%, Actinobacteria decreased by 50.62%, and Firmicutes decreased by 65.85%. At the fungal level, the relative abundance of Ascomycota and Basidiomycota decreased significantly.

[0061] Gram-related changes

[0062] Based on phylum-level classification, Actinobacteria and Firmicutes are classified as Gram-positive bacteria (G). + The remaining major categories are classified as Gram-negative bacteria (G). - ).like Figure 5As shown, after eugenol treatment, the relative abundance of Gram-negative bacteria increased by 6.05% compared with the control, while the abundance of Gram-positive bacteria decreased by 62.98%, indicating that eugenol selectively inhibits Gram-positive bacteria and is beneficial to the growth of Gram-negative rhizobia.

[0063] Changes in key bacterial communities at the genus level

[0064] As shown in Table 3, the dominant bacterial genera in the control soil were Bacillus (6.58%) and Streptomyces (3.75%). After eugenol treatment, the abundance of Bacillus decreased to below 1%, and Streptomyces decreased to 2.67%, while the relative abundance of Rhizobium-Ensifer increased from 1.01% to 3.98%, an increase of 294.1%. This indicates that eugenol effectively inhibited competing microorganisms and promoted the enrichment of Rhizobium.

[0065] Table 3. Dominant genera of bacteria with abundance >1% under different treatments

[0066] CK Abundance% Eugenol Abundance% Bacillus 6.58 spp. of Bacillus 6.38 spp. of Bacillus 5.87 Rhizobium 3.98 Streptomyces 3.75 Pseudomonas 3.67 Pseudomonas 3.67 Streptomyces 2.67 Bifidobacterium 3.31 Bifidobacterium 2.43 ... ... ... ... Rhizobium 1.01

[0067] Example 4:

[0068] This embodiment provides a potted plant inoculation verification test.

[0069] Experimental Design

[0070] Soil culture was used, and the process was carried out in a culture chamber (14h light / 25℃, 10h darkness / 20℃, light intensity 260-350 μmol·m). -2 ·s -1 (Relative humidity 60%-70%). Sterilized Gannong No. 9 seeds were sown in plastic cups (9cm in diameter, 12cm in height) filled with soil, 20 seeds per cup. After germination, 15 seedlings were retained. Three treatments were set up:

[0071] Control group (CK): Inoculated with rhizobium solution without antibacterial agent;

[0072] Eugenol treatment group (D): Inoculated with eugenol (final concentration 1000 μL·L⁻¹) -1 Rhizobium solution.

[0073] Each treatment was repeated 6 times. Rhizobium culture preparation: Activated LL2 bacterial culture was cultured in YMA medium with shaking until it reached 1×10⁻⁶. 9 CFU / mL, collect bacterial cells by centrifugation, resuspend in sterile water, and adjust to the target concentration with eugenol stock solution. Inoculate 1 mL per seedling when the first true leaf unfolds. During the growth period, alternately irrigate with nitrogen-free nutrient solution and distilled water to maintain moisture. Samples are taken for analysis after 40 days of culture.

[0074] Tumor index measurement

[0075] Carefully dig up the plant's root system, rinse it clean, count the number of effective root nodules per plant, and weigh each effective root nodule. Results are as follows: Figure 6 As shown in the figure, compared with the control (CK), the number of effective root nodules per plant increased by 41.87% and the weight of a single root nodule increased by 34.27% in the eugenol-treated group, with significant differences (p<0.05). This indicates that eugenol treatment significantly enhances the nodulation ability of rhizobia.

[0076] Assay of key enzyme activities in nitrogen metabolism

[0077] Fresh root samples were taken, and the activities of nitrate reductase (NR), glutamine synthase (GS), nitrite reductase (NiR), and glutamate synthase (GOGAT) were measured according to the kit instructions. Results are as follows: Figure 7 As shown, the activities of all four enzymes in the eugenol-treated group were significantly higher than those in the control group (p<0.05), with the most significant increases in GS and GOGAT activities (68.90% and 76.30%, respectively). This indicates that enhanced nitrogen fixation capacity in root nodules activates nitrogen assimilation metabolism in plants.

[0078] Growth index measurement

[0079] Ten plants were randomly selected from each treatment, and their height and root length were measured. The plants were then blanched at 105℃ for 15 minutes, dried at 75℃, and weighed. The results are as follows: Figure 8 As shown, the plant height in the eugenol-treated group increased by 16.73% and the root length increased by 19.84% compared to the control (CK), with significant differences (p<0.05). This indicates that the overall plant growth was promoted.

[0080] Rhizosphere microbial community analysis

[0081] Rhizosphere soil samples were collected and subjected to high-throughput sequencing to analyze the relative abundance of rhizobia and Gram-negative bacteria. Results are as follows: Figure 9 As shown, after eugenol treatment, the relative abundance of Rhizobium in the rhizosphere soil increased from 1.01% in the control (CK) to 3.98%, an increase of 294.1%; the relative abundance of Gram-negative bacteria increased by 6.05%, while the relative abundance of Gram-positive bacteria decreased by 62.98%, consistent with the trend in the overall soil sample. This confirms that eugenol also plays a selective regulatory role in the rhizosphere.

[0082] Example 5:

[0083] This embodiment provides the effects of different application methods.

[0084] Following the method in Example 4, different eugenol application methods were set up:

[0085] Method A: Seed soaking treatment. Soak the seeds in 1000 μL·L⁻¹ water. -1Soak in eugenol solution for 10 hours before sowing, followed by normal inoculation with rhizobium.

[0086] Method B: Soil pretreatment. Seven days before sowing, apply eugenol solution to the soil (10 mL of 1000 μL·L⁻¹ per liter of soil). -1 (Solution), then sow and inoculate with rhizobia.

[0087] Method C: Inoculate with rhizobia. Same as Example 4.

[0088] The results showed that all three methods significantly increased nodule number and plant growth, with method C showing the best effect, but methods A and B also exhibited significant promoting effects. This indicates that eugenol can exert its regulatory effects through multiple pathways.

[0089] The foregoing has only described certain exemplary embodiments of the present invention by way of illustration. Undoubtedly, those skilled in the art can modify the described embodiments in various ways without departing from the spirit and scope of the present invention. Therefore, the foregoing drawings and descriptions are illustrative in nature and should not be construed as limiting the scope of protection of the claims of the present invention.

Claims

1. The application of eugenol in the preparation of antibacterial agents for enhancing the nitrogen fixation efficiency of leguminous plant-rhizobium symbiotic relationships, characterized in that, The final concentration of eugenol in the antibacterial agent is 500-1000 μL·L. -1 .

2. The application of eugenol according to claim 1 in the preparation of an antibacterial agent for enhancing the nitrogen fixation efficiency of leguminous plant-rhizobium symbiotic relationships, characterized in that, The final concentration of eugenol is 1000 μL·L. -1 .

3. The application of eugenol according to claim 2 in the preparation of an antibacterial agent for enhancing the nitrogen fixation efficiency of leguminous plant-rhizobium symbiotic relationships, characterized in that, The legume in question is alfalfa.

4. The application of eugenol according to claim 3 in the preparation of an antibacterial agent for enhancing the nitrogen fixation efficiency of leguminous plant-rhizobium symbiotic relationships, characterized in that, The alfalfa mentioned is the Gannong No. 9 variety.

5. The application of eugenol according to claim 1 in the preparation of an antibacterial agent for enhancing the nitrogen fixation efficiency of leguminous plant-rhizobium symbiotic relationships, characterized in that, The rhizobium is *Rhizobium sinense*.

6. The application of eugenol according to claim 5 in the preparation of an antibacterial agent for enhancing the nitrogen fixation efficiency of leguminous plant-rhizobium symbiotic relationships, characterized in that, The Chinese rhizobium is strain LL2.

7. The application of eugenol according to claim 1 in the preparation of an antibacterial agent for enhancing the nitrogen fixation efficiency of leguminous plant-rhizobium symbiotic relationships, characterized in that, The antibacterial agent enhances the colonization advantage of rhizobia by selectively inhibiting competitive microorganisms in the soil, including at least one of Bacillus and Streptomyces.

8. The application of eugenol according to claim 1 in the preparation of an antibacterial agent for enhancing the nitrogen fixation efficiency of leguminous plant-rhizobium symbiotic relationships, characterized in that, The antibacterial agent is applied in any one or more combinations of the following methods: The antibacterial agent was used to soak legume seeds. The antibacterial agent is pre-treated by applying it to the soil in which legumes are planted. The antibacterial agent is mixed with the rhizobium bacterial solution and then applied simultaneously.

9. The application of eugenol according to claim 8 in the preparation of an antibacterial agent for enhancing the nitrogen fixation efficiency of leguminous plant-rhizobium symbiotic relationships, characterized in that, When the antibacterial agent is mixed with the rhizobium inoculum solution and applied simultaneously, the concentration of the rhizobium inoculum solution is 1×10⁻⁶. 9 CFU / mL, the inoculation amount is 0.5-2 mL per legume seedling.

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