Nano-coated growth promoting bacterial agent and composite growth promoting bacterial agent, and preparation method and application thereof

Abstract

The application belongs to the technical field of microorganisms, and particularly relates to a nano-coated growth promoting microbial agent and a composite growth promoting microbial agent, and a preparation method and application thereof. The nano-coated growth promoting microbial agent comprises a growth promoting microbial agent and a nano-coating formed by self-assembly of plant-derived polyphenols and soluble multi-valence metal ions on the surface of the growth promoting microbial agent. The composite growth promoting microbial agent contains the nano-coated growth promoting microbial agent and a prebiotic. The key of the application is to form a nano-coating by self-assembly of plant-derived polyphenols and soluble multi-valence metal ions on the surface of the growth promoting microbial agent. Based on the interaction between the nano-coated growth promoting microbial agents in the interfoliar space, the leaf surface adhesion performance of the growth promoting microbial agents is significantly enhanced, the loss rate of the growth promoting microbial agents under rainwater scouring is effectively reduced, and the survival and colonization of the growth promoting microbial agents in the interfoliar space are enhanced.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a nano-coated growth promoter and a compound growth promoter, as well as their preparation methods and applications. Background Technology

[0002] Plant growth-promoting bacteria are a class of bacteria that promote plant growth and can colonize on or inside plants. They can promote nutrient absorption, induce the production of plant hormones, decompose harmful substances, or resist pathogen infection, thus achieving direct or indirect growth-promoting effects. In today's world of population growth and increasingly urgent food demands, the overuse of chemical fertilizers and pesticides in agricultural production has led to a series of serious environmental pollution problems, such as declining soil fertility and eutrophication of water bodies. Utilizing plant growth-promoting bacteria to prepare microbial inoculants is an important means of replacing chemical fertilizers and achieving healthy and sustainable agricultural development. Microbial inoculants need to survive and successfully colonize on the surface or inside of crops after application in order to interact with plants and exert their growth-promoting effects. Recent studies have gradually revealed that the abundant leaf area of ​​plants provides a good habitat for microorganisms. Compared to the soil environment, the foliar habitat and microbial community structure are relatively simple, with abundant leaf area and more flexible spraying. The key to using plant growth-promoting bacteria to prepare foliar microbial inoculants lies in exploring suitable inoculant systems that help them attach to the crop foliage and establish a symbiotic colonization relationship.

[0003] Current research on foliar microbial inoculation systems is still in its early stages, with limited application studies, mostly limited to simple spraying of growth-promoting bacterial suspensions. During foliar spraying, traditional bacterial formulations have insufficient effective residence time on crop leaves, and issues such as droplet dispersion, jumping, rolling, rain washing, and decomposition all reduce the effective colonization rate of growth-promoting bacteria. Furthermore, the strong hydrophobicity of the leaf surface wax layer and environmental stresses such as dryness and ultraviolet radiation also pose many challenges to the survival and colonization of growth-promoting bacteria. Therefore, there is an urgent need to develop novel foliar growth-promoting bacterial agents with strong adhesion to plant leaves, long foliar residence time, and favorable conditions for the survival and colonization of growth-promoting bacteria in the foliar region. Summary of the Invention

[0004] One of the objectives of this invention is to provide a novel nano-coated growth-promoting bacterial agent, which has strong adhesion to plant leaves and a long survival time, and can effectively enhance the colonization of growth-promoting bacteria in the leaf margin.

[0005] The second objective of this invention is to provide a method for preparing the above-mentioned nano-coated growth-promoting bacterial agent.

[0006] The third objective of this invention is to provide a compound growth-promoting bacterial agent.

[0007] The fourth objective of this invention is to provide applications based on the above-mentioned nano-coated growth promoters and / or composite growth promoters.

[0008] Specifically, the nano-coated growth promoter provided by the present invention includes a growth promoter and a nano-coating formed by the reaction of plant-derived polyphenols and soluble multivalent metal ions on the surface of the growth promoter.

[0009] In a preferred embodiment, the thickness of the nanocoating is 20–100 nm.

[0010] In a preferred embodiment, the mass ratio of the plant-derived polyphenols to the soluble multivalent metal ions is (1-10):1.

[0011] In a preferred embodiment, the growth-promoting agent is selected from at least one of Klebsiella pneumoniae, Bacillus polymyxa, Bacillus belye, and Pseudomonas.

[0012] In a preferred embodiment, the plant-derived polyphenols are selected from at least one of tannic acid, gallic acid, catechins, and apple polyphenols.

[0013] In a preferred embodiment, the soluble multivalent metal ion is selected from Fe. 3+ Al 3+ Mg 2+ Zn 2+ and Ti 4+ At least one of them.

[0014] The preparation method of the nano-coated growth-promoting agent provided by the present invention includes mixing the growth-promoting agent, plant-derived polyphenols, and soluble multivalent metal ions in a solution, so that the plant-derived polyphenols and soluble multivalent metal ions self-assemble on the surface of the growth-promoting agent to form a nano-coating.

[0015] In a preferred embodiment, the mixing is performed by including the following steps:

[0016] S1. Vortex mix the growth-promoting bacterial suspension with the plant-derived polyphenol solution to obtain the bacterial suspension / plant-derived polyphenol solution.

[0017] S2. Vortex mix the bacterial suspension / plant-derived polyphenol solution with the soluble multivalent metal ion solution, and then wash with PBS buffer (optionally) to obtain the nano-coated growth-promoting bacterial agent.

[0018] In a preferred embodiment, in step S1, the growth-promoting bacterial suspension is obtained by culturing the activated growth-promoting bacterial agent to the late logarithmic growth phase, centrifuging to collect the bacterial cells and washing them, and then resuspending the obtained growth-promoting bacterial agent in deionized water.

[0019] In a preferred embodiment, in step S1, the number of viable bacteria in the growth-promoting bacterial suspension is 1×10⁻⁶. 8 ~1×10 9CFU / mL.

[0020] In a preferred embodiment, in step S1, the concentration of the plant-derived polyphenol solution is 5–20 mg / mL.

[0021] In a preferred embodiment, in step S1, the ratio of the growth-promoting bacterial suspension to the plant-derived polyphenol solution is 600 μL:(25-100) μL.

[0022] In a preferred embodiment, in step S1, the vortex mixing time is 10 to 30 seconds.

[0023] In a preferred embodiment, in step S2, the concentration of the soluble multivalent metal ion solution is 0.5–5 mg / mL.

[0024] In a preferred embodiment, in step S2, the ratio of the soluble multivalent metal ion solution to the growth-promoting bacterial suspension is (25-100) μL: 600 μL.

[0025] In a preferred embodiment, the vortex mixing time in step S2 is 10 to 30 seconds.

[0026] In a preferred embodiment, in step S2, the pH value of the PBS buffer is 7.2 to 7.4.

[0027] The compound growth-promoting agent provided by this invention contains nano-coated growth-promoting bacteria and prebiotics.

[0028] In a preferred embodiment, the mass ratio of the nano-coated probiotics to the prebiotics is 100:(1-100).

[0029] In a preferred embodiment, the prebiotic is alginate.

[0030] In a preferred embodiment, the compound growth promoter is used in solution form.

[0031] The present invention also provides the application of the nano-coated growth-promoting bacteria agent and / or compound growth-promoting bacteria agent in promoting plant leaf growth.

[0032] The key to this invention lies in the self-assembly of plant-derived polyphenols and soluble multivalent metal ions on the surface of a growth-promoting bacterial agent to form a nanocoating. Based on the interaction between the nanocoated growth-promoting bacterial agent and the leaf surface, its adhesion performance is significantly enhanced, effectively reducing the loss rate of the growth-promoting bacterial agent under rainwater runoff, and increasing the survival rate and colonization of the growth-promoting bacteria on the leaf surface. Furthermore, the nanocoating formed by the reaction of plant-derived polyphenols and soluble multivalent metal ions on the surface of the growth-promoting bacteria is a single-cell coating, which helps the growth-promoting bacteria resist environmental stress. This nanocoating does not affect the metabolism and reproduction of the growth-promoting bacteria, exhibits good biocompatibility and storage stability, and can protect the growth-promoting bacteria from stresses such as reactive oxygen species and ultraviolet radiation. In addition, the self-assembly reaction of this invention is carried out in a pH-mild buffer solution and can be stably stored in an aqueous buffer solution for a long period. Attached Figure Description

[0033] Figure 1 Scanning electron microscope image of the nano-coated growth-promoting agent provided by the present invention;

[0034] Figure 2 Transmission electron microscopy image of the nano-coated growth-promoting agent provided by the present invention;

[0035] Figure 3 A laser confocal image of the nano-coated growth-promoting agent provided by this invention;

[0036] Figure 4 Metabolic activity diagram of the nano-coated growth-promoting agent provided by the present invention;

[0037] Figure 5 The storage stability results of the nano-coated growth-promoting agent provided by this invention are shown in the figure.

[0038] Figure 6 A graph showing the survival rate of the nano-coated growth promoter provided by this invention in hydrogen peroxide.

[0039] Figure 7 A graph showing the survival rate of the nano-coated growth-promoting bacterial agent provided by this invention under ultraviolet irradiation;

[0040] Figure 8 A comparison of the rolling angles of the nano-coated growth-promoting bacterial agent provided by this invention on the surface of rice leaves;

[0041] Figure 9 The figure shows the effect of the nano-coated growth-promoting bacterial agent provided by this invention on rice biomass in a greenhouse experiment. Detailed Implementation

[0042] The nano-coated growth-promoting bacterial agent provided by this invention comprises a growth-promoting bacterial agent and a nano-coating formed by the self-assembly of plant-derived polyphenols and soluble multivalent metal ions on the surface of the growth-promoting bacterial agent. This nano-coated growth-promoting bacterial agent possesses a supramolecular network structure, exhibits good microbial compatibility, protects the growth-promoting bacteria from stresses such as reactive oxygen species and ultraviolet radiation, and simultaneously interacts with the leaf stalk, enhancing the leaf stalk adhesion performance of the growth-promoting bacteria, effectively reducing the loss rate under rainwater runoff, and enhancing the leaf stalk colonization of the growth-promoting bacteria, thus significantly promoting crop growth. The thickness of the nano-coating is preferably 20–100 nm, such as 20 nm, 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, or any value between them. The mass ratio of the plant-derived polyphenols to the soluble multivalent metal ions is preferably (1–10):1, such as 1:1, 2:1, 4:1, 6:1, 8:1, 10:1, or any value between them.

[0043] In this invention, the growth-promoting bacterial agent can be any existing bacteria capable of promoting plant growth, particularly preferably at least one of Klebsiella pneumoniae, Bacillus polymyxa, Bacillus belyssus, and Pseudomonas. The inventors of this invention have discovered that selecting at least one of Klebsiella pneumoniae, Bacillus polymyxa, Bacillus belyssus, and Pseudomonas as the growth-promoting bacterial agent is more conducive to attachment to the leaf foliage and the establishment of a symbiotic colonization relationship, thereby better promoting plant growth.

[0044] In this invention, the plant-derived polyphenols are polyphenolic compounds found in plants, possessing a benzene ring conjugated structure and phenolic hydroxyl groups, endowing the inoculant with antioxidant and ultraviolet absorption capabilities, and protecting growth-promoting bacteria from stresses such as reactive oxygen species and ultraviolet radiation. The plant-derived polyphenols can originate from fruits, vegetables, nuts, tea, grains, legumes, etc. Common polyphenolic compounds include flavonoids, resveratrol, stilbene, lignans, etc. Specific examples include, but are not limited to, at least one of anthocyanins, flavanols, isoflavones, tannic acid, gallic acid, quercetin, kaempferol, catechins, and apple polyphenols, preferably selected from at least one of tannic acid, gallic acid, catechins, and apple polyphenols.

[0045] In this invention, the term "soluble multivalent metal ion" refers to a soluble metal ion with a valence of two or higher. The preferred valence of the soluble multivalent metal ion is +2, +3, or +4. Specific examples of +2 valent soluble metal ions include, but are not limited to, Mg. 2+ Zn 2+ Mn 2+ Examples of soluble metal ions with a positive trivalent valence include, but are not limited to: Fe. 3+ Al 3+ Cr 3+Examples of tetravalent soluble metal ions include, but are not limited to: Ti 4+ Zr 4+ Etc. Most preferably, the soluble multivalent metal ion is selected from Fe. 3+ Al 3+ Mg 2+ Zn 2+ and Ti 4+ At least one of them. In addition, the soluble multivalent metal ions may exist in the form of their salts, such as chlorides, sulfates, etc.

[0046] The inventors of this invention discovered that when tannic acid, gallic acid, catechins, and apple polyphenols are selected as plant-derived polyphenols, and Fe is also selected... 3+ Al 3+ Mg 2+ Zn 2+ and Ti 4+ When at least one of the components is a soluble multivalent metal ion, plant-derived polyphenols and soluble multivalent metal ions can play a better synergistic role. The nanocoating formed by the two can better adhere to the leaf margin, which can further enhance the survival rate and colonization of growth-promoting bacteria in the leaf margin.

[0047] The preparation method of the nano-coated growth-promoting agent provided by the present invention includes mixing the growth-promoting agent, plant-derived polyphenols, and soluble multivalent metal ions in a solution, so that the plant-derived polyphenols and soluble multivalent metal ions self-assemble on the surface of the growth-promoting agent to form a nano-coating.

[0048] This invention does not particularly limit the mixing form, as long as it enables the plant-derived polyphenols and soluble multivalent metal ions to react on the surface of the growth-promoting agent to form a nano-coating. In a preferred embodiment, the mixing is carried out by the following steps: S1, vortexing the growth-promoting agent suspension with the plant-derived polyphenol solution to obtain a suspension / plant-derived polyphenol solution; S2, vortexing the suspension / plant-derived polyphenol solution with a soluble multivalent metal ion solution, and then optionally washing with PBS buffer to obtain the nano-coated growth-promoting agent.

[0049] In the preparation process of the above-mentioned nano-coated growth-promoting bacterial agent, in step S1, the number of live bacteria in the growth-promoting bacterial suspension is preferably 1×10⁻⁶. 8 ~1×10 9 CFU / mL, such as 1×10 8 2×10 8 4×10 8 6×10 8 8×10 8 1×10 9The concentration of CFU / mL or any value between them may be used. The growth-promoting bacterial suspension can be commercially available or prepared using various existing methods. For example, it can be obtained by culturing the activated growth-promoting bacterial agent to the late logarithmic growth phase, centrifuging to collect the cells, washing them, and then resuspending the resulting growth-promoting bacterial agent in deionized water. The washing solvent can be deionized water or PBS buffer. The concentration of the plant-derived polyphenol solution is preferably 5–20 mg / mL, such as 5, 8, 10, 12, 15, 18, 20 mg / mL or any value between them. The preferred volume ratio of the growth-promoting bacterial suspension to the plant-derived polyphenol solution is 600 μL:(25–100) μL, such as 600 μL:25 μL, 600 μL:50 μL, 600 μL:75 μL, 600 μL:100 μL or any value between them. The preferred vortex mixing time is 10 to 30 seconds, such as 10 seconds, 15 seconds, 20 seconds, 25 seconds, 30 seconds, or any value between them.

[0050] In the preparation process of the above-mentioned nano-coated growth-promoting agent, in step S2, the concentration of the soluble multivalent metal ion solution is preferably 0.5–5 mg / mL, such as 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5 mg / mL or any value between them. The volume ratio of the soluble multivalent metal ion solution to the growth-promoting agent suspension is preferably (25–100) μL:600 μL, such as 25 μL:600 μL, 50 μL:600 μL, 75 μL:600 μL, 100 μL:600 μL or any value between them. The vortex mixing time is preferably 10–30 s, such as 10 s, 15 s, 20 s, 25 s, 30 s or any value between them.

[0051] In the preparation of the above-mentioned nano-coated growth-promoting bacterial agent, the pH value of the PBS buffer used is preferably 7.2 to 7.4, such as 7.20, 7.25, 7.30, 7.35, 7.40 or any value between them.

[0052] In one specific embodiment, the preparation method of the nano-coated growth-promoting agent includes:

[0053] S1ˋ Preparation of growth-promoting bacterial suspension: After culturing the activated growth-promoting bacteria to the late logarithmic growth phase, the bacterial cells are collected by centrifugation. The obtained bacterial cells are then washed 1–5 times with deionized water or PBS buffer. An appropriate amount of the growth-promoting bacteria is resuspended in deionized water to obtain a concentration of 1×10⁻⁶. 8 ~1×10 9 CFU / mL growth-promoting bacterial suspension;

[0054] S2ˋ Preparation of nano-coated growth-promoting bacterial agent: Add a plant-derived polyphenol solution with a concentration of 5-20 mg / mL to the growth-promoting bacterial agent suspension obtained in step S1ˋ, with a volume ratio of 600 μL:(25-100) μL. Vortex mix for 10-30 s. Then add a soluble multivalent metal ion solution with a concentration of 0.5-5 mg / mL to the resulting system, with a volume ratio of 600 μL:(25-100) μL. Vortex mix for 10-30 s. Finally, add 200-400 μL of the solution to the system. PBS buffer is used to self-assemble nano-coatings on the surface of growth-promoting bacteria. The mixture is then centrifuged at 6000–12000 rpm for 3–10 min. Unbound polyphenols or metal ions in the supernatant are discarded. The solid product is resuspended in PBS buffer and washed 1–3 times. Step S2 can be repeated 1–3 times to obtain nano-coating layers of different thicknesses, thus obtaining nano-coated growth-promoting agents.

[0055] In this invention, the nano-coated growth promoter can be transported and stored in solid form, and then prepared into a nano-coated growth promoter suspension using PBS buffer as a solvent when needed. Alternatively, it can be prepared directly into a nano-coated growth promoter suspension using PBS buffer as a solvent during the preparation process. There are no particular limitations on this.

[0056] The compound growth-promoting bacterial agent provided by this invention contains nano-coated growth-promoting bacteria and prebiotics. The nano-coated growth-promoting bacteria, when used independently, can protect the bacteria on leaves from environmental stresses such as ultraviolet radiation, enhance their leaf surface adhesion, and promote the survival rate and colonization of the bacteria in the foliage. When the nano-coated growth-promoting bacteria and prebiotics are used in combination, a more effective protective and adhesive microenvironment can be provided for the colonization of the bacteria in the foliage. The preferred mass ratio of the nano-coated growth-promoting bacteria to the prebiotics is 100:(1-100). The prebiotics are particularly preferably alginate, specifically selected from at least one of sodium alginate, potassium alginate, magnesium alginate, etc., with sodium alginate being particularly preferred. The inventors of this invention have found that when the natural polymer sodium alginate is used as a prebiotic, the corresponding compound growth-promoting bacterial agent can more effectively improve the effectiveness of microbial inoculation and is more conducive to promoting crop growth.

[0057] The composite growth-promoting bacterial agent provided by this invention can be obtained by adding an alginate solution to a nano-coated growth-promoting bacterial suspension and then vortexing to mix. The specific preparation process of the nano-coated growth-promoting bacterial suspension has been described above and will not be repeated here.

[0058] The present invention also provides the application of the nano-coated growth-promoting bacteria agent and / or compound growth-promoting bacteria agent in promoting plant leaf growth.

[0059] The nano-coated and compound growth-promoting bacterial agents provided by this invention exhibit good microbial compatibility, protecting the growth-promoting bacteria from stresses such as reactive oxygen species and ultraviolet radiation. They also enhance foliar adhesion through hydrogen bonding interactions, thereby improving foliar colonization and effectively reducing the loss rate of the growth-promoting bacterial agents due to rainwater runoff. These agents improve the effectiveness of microbial inoculation and significantly promote crop growth, demonstrating practical applicability. Furthermore, the preparation process of these agents is simple, low-cost, and highly practical, making them significant for the promotion and application of novel microbial agents.

[0060] The foliar growth-promoting bacteria agent based on nano-coating provided by this invention is not particularly limited to any particular type of plant leaf and is applicable to various existing crop leaves, such as rice leaves, wheat leaves, corn leaves, soybean leaves, potato leaves, tobacco leaves, tomato leaves, cotton leaves, peanut leaves, potato leaves, etc.

[0061] The application scope of the foliar growth-promoting bacterial agent based on nano-coating provided by this invention includes bio-fertilizer, increasing crop yield, plant disease resistance, and enhancing plant stress resistance.

[0062] The present invention will be described in detail below through embodiments.

[0063] Example 1

[0064] This embodiment uses the highly efficient nitrogen-fixing strain Klebsiella variicola W12 (hereinafter referred to as "W12", preservation number CGMCC NO.25294), tannic acid, and iron ions to prepare nano-coated growth-promoting bacteria. The specific steps are as follows:

[0065] S1. The activated growth-promoting bacteria were cultured to the late logarithmic growth phase, and then centrifuged at 8000 rpm / min for 10 min to collect W12 cells. The obtained cells were washed twice with PBS buffer, and the growth-promoting bacteria were resuspended in an appropriate amount of deionized water. The OD value was adjusted to 0.8 to obtain a concentration of 3 × 10⁻⁶. 8 CFU / mL growth-promoting bacterial suspension;

[0066] S2. Take 600 μL of the growth-promoting bacterial suspension obtained in step S1, add 50 μL of tannic acid solution with a concentration of 5 mg / mL, vortex and mix for 20 s, then add 25 μL of FeCl3 solution with a concentration of 1 mg / mL to the obtained system, vortex and mix for 20 s, then add 300 μL of PBS buffer (pH 7.4, the same below) to the system, then centrifuge at 10000 rpm / min for 4 min, discard the supernatant and wash the obtained solid product twice with PBS buffer, then resuspend in PBS buffer to obtain the nano-coated growth-promoting bacterial suspension, denoted as W12-TA-Fe.

[0067] Example 2

[0068] Add an appropriate amount of sodium alginate mother liquor with a concentration of 2% to the nano-coated growth-promoting bacterial suspension W12-TA-Fe obtained in Example 1, so that the final concentration of sodium alginate in the system is 0.05%, and vortex mix for 50s to obtain the nano-coated growth-promoting bacterial suspension, denoted as W12-TA-Fe-SA.

[0069] Example 3

[0070] In this embodiment, nano-coated growth-promoting bacteria were prepared using Paenibacillus polymyxa (hereinafter referred to as "Pae", strain number ACCC 10252), tannic acid, and iron ions. The specific steps are as follows:

[0071] S1. The activated growth-promoting bacteria were cultured to the late logarithmic growth phase, and then centrifuged at 8000 rpm / min for 10 min to collect Pae cells. The obtained cells were washed twice with PBS buffer, and the growth-promoting bacteria were resuspended in deionized water and the OD value was adjusted to 0.7.

[0072] S2. Take 8 mL of the growth-promoting bacterial suspension obtained in step S1 and add 700 μL of tannic acid solution with a concentration of 10 mg / mL. Vortex and mix for 20 s. Then add 500 μL of FeCl3 solution with a concentration of 2 mg / mL to the system and vortex and mix for 20 s. Next, add 4 mL of PBS buffer to the system and centrifuge at 10000 rpm / min for 4 min. Discard the supernatant and wash the obtained solid product twice with PBS buffer. Resuspend in PBS buffer to obtain the nano-coated growth-promoting bacterial suspension, denoted as Pae-TA-Fe.

[0073] S3. Add an appropriate amount of sodium alginate solution with a concentration of 2% to the nano-coated growth-promoting bacterial suspension Pae-TA-Fe obtained in step S2, so that the final concentration of sodium alginate in the system is 0.1%. Vortex mix for 50s to obtain the nano-coated growth-promoting bacterial suspension, denoted as Pae-TA-Fe-SA.

[0074] Example 4

[0075] In this embodiment, Bacillus velezensis (hereinafter referred to as "Bac", strain number ACCC 60428), tannic acid, and iron ions were used to prepare nano-coated growth-promoting bacteria. The specific steps are as follows:

[0076] S1. The activated growth-promoting bacteria were cultured to the late logarithmic growth phase, and then centrifuged at 8000 rpm / min for 10 min to collect Bac cells. The obtained cells were washed twice with PBS buffer, and the growth-promoting bacteria were resuspended in deionized water and the OD value was adjusted to 0.85.

[0077] S2. Add 1 mL of 4 mg / mL tannic acid solution to 10 mL of the growth-promoting bacterial suspension obtained in step S1, vortex and mix for 20 s. Then add 500 μL of 3.2 mg / mL FeCl3 solution to the system, vortex and mix for 20 s. Add 5 mL of PBS buffer to the system, centrifuge at 10000 rpm / min for 4 min, discard the supernatant, wash the obtained solid product twice with PBS buffer, and resuspend in PBS buffer to obtain the nano-coated growth-promoting bacterial suspension, denoted as Bac-TA-Fe.

[0078] S3. Add an appropriate amount of sodium alginate solution with a concentration of 2% to the nano-coated growth-promoting bacterial suspension Bac-TA-Fe obtained in step S2, so that the final concentration of sodium alginate in the system is 0.01%. Vortex mix for 50s to obtain the nano-coated growth-promoting bacterial suspension, denoted as Bac-TA-Fe-SA.

[0079] Test Example 1

[0080] Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were used to observe the untreated growth-promoting bacteria, nano-coated growth-promoting bacteria, and composite foliar growth-promoting bacteria agents. The SEM images of the untreated growth-promoting bacteria W12, the composite foliar growth-promoting bacteria agent W12-TA-Fe obtained in Example 1, and the composite foliar growth-promoting bacteria agent W12-TA-Fe-SA obtained in Example 2 are shown below. Figure 1 As shown, the transmission electron microscope image is as follows: Figure 2 As shown. By Figure 1 and Figure 2 It can be seen that growth-promoting bacteria based on nanocoating were successfully prepared, and there are significant morphological differences between them and untreated growth-promoting bacteria. Compared with untreated growth-promoting bacteria, the size of the nanocoated growth-promoting bacteria is increased, and the edges of W12-TA-Fe and W12-TA-Fe-SA are rougher, with single-cell nanocoatings of varying thickness visible on the surface.

[0081] Test Example 2

[0082] Laser confocal scanning was performed on the nano-coated growth-promoting bacterial suspensions obtained in Examples 1, 3, and 4. The results are shown in [Figure 1]. Figure 3 .like Figure 3 As shown, the nanocoating layer around the growth-promoting bacteria can be seen by fluorescent labeling, indicating that nanocoating was successfully prepared on the surface of different growth-promoting bacteria.

[0083] Test Example 3

[0084] The cell metabolic activity of untreated growth-promoting bacteria and the compound foliar growth-promoting bacteria agent obtained from the above examples was detected. The specific process is as follows:

[0085] After adding 5 μL of alpha blue assay reagent to 100 μL of bacterial suspension and incubating in the dark for 30 min, the solution color changed from indigo blue to pink. The relative fluorescence units (RFU) were then measured using a fluorescence microplate reader. The results are shown in Table 2. Figure 4 As shown in Table 2 and Figure 4 It is known that growth-promoting bacteria based on nanocoating do not affect their cellular metabolic activity and have good biocompatibility.

[0086] Table 1

[0087] project Growth-promoting bacteria Relative fluorescence unit (RFU) Untreated control W12 803571.3±6723.7 Example 1 W12-TA-Fe 1276059.3±101517.6 Example 2 W12-TA-Fe-SA 1092811.7±285991.5 Example 3 Pae-TA-Fe-SA 1223568.7±134415.6 Example 4 Bac-TA-Fe-SA 1062003.7±176224.2

[0088] Test Example 4

[0089] The storage viability of untreated growth-promoting bacteria and the compound foliar growth-promoting bacteria agent obtained from the above examples was tested. The specific process is as follows:

[0090] The four bacterial agents were adjusted to an OD of 0.8 and stored at 4°C to simulate the storage of conventional commercial liquid bacteria. Every week, 100 μL of the agent was serially diluted and plated onto selective solid culture plates. After incubating the plates at 30°C for 24 hours, the number of colonies was counted to obtain the effective viable bacterial count. Sampling was conducted continuously for four weeks, with three parallel experiments, and the average value was taken. The changes in the effective growth-promoting bacterial count over time for different agents are shown below. Figure 5 As shown. By Figure 5 It can be seen that the growth-promoting bacterial agent based on nano-coating can enhance its activity during long-term storage. Among them, after 4 weeks, the effective viable bacteria count of the bacterial agent W12-TA-Fe-SA exceeded 2×10⁻⁶. 7 CFU / mL, more than 2.5 times the amount of untreated W12 bacterial agent.

[0091] Test Example 5

[0092] The survival rates of untreated growth-promoting bacteria and the compound foliar growth-promoting bacteria agent obtained from the above examples were tested in hydrogen peroxide. The specific process is as follows:

[0093] 1 mL of bacterial agent was added to 8 mmol / L H2O2. After 90 min, the solution was removed, centrifuged, and washed. 100 μL of both the H2O2-treated and untreated bacterial agents were serially diluted and plated onto selective solid culture plates. The plates were incubated at 30℃ for 24 h, and the number of viable colonies was counted. This experiment was repeated three times, and the average value was taken. Survival ratio = number of surviving colonies / total number of colonies. The survival rates of different bacterial agents in hydrogen peroxide are shown in Table 2 and... Figure 6 As shown in Table 2 and Figure 6 It can be seen that the growth-promoting agent based on nano-coating can protect nitrogen-fixing bacteria in H2O2 and has a higher survival rate than the untreated growth-promoting bacteria W12. Both nano-coating and sodium alginate contribute certain antioxidant capacity.

[0094] Table 2

[0095] project Growth-promoting bacteria Survival rate (%) Untreated control W12 3.48±0.85 Example 1 W12-TA-Fe 12.55±1.34 Example 2 W12-TA-Fe-SA 16.95±0.40 Example 3 Pae-TA-Fe-SA 16.11±0.70 Example 4 Bac-TA-Fe-SA 17.49±0.34

[0096] Test Example 6

[0097] The survival rates of untreated growth-promoting bacteria and the compound foliar growth-promoting bacteria agent obtained from the above examples were tested under ultraviolet irradiation. The specific process is as follows:

[0098] 500 μL of bacterial agent was placed in a capped 24-well plate and irradiated at a fixed distance from the UV lamp in a clean bench for 5 min, 30 min, and 60 min. Samples were taken according to the time series, and 100 μL of the above bacterial agent and the untreated bacterial agent were serially diluted and spread onto selective solid culture plates. After incubating the plates at 30℃ for 24 h, the number of colonies was counted to determine the number of viable bacteria. This experiment was repeated three times, and the average value was taken. Survival ratio = number of surviving colonies / total number of colonies. The survival rates of different bacterial agents under UV irradiation at 5 min, 30 min, and 60 min are shown below. Figure 7 As shown in Table 3, the survival rates of different bacterial agents under UV irradiation for 5 minutes are as follows. From Table 3 and... Figure 7 It is known that nano-coated nitrogen-fixing bacteria inoculants can protect nitrogen-fixing bacteria from high-intensity UVC stress.

[0099] Table 3

[0100] project Growth-promoting bacteria Survival rate (%) Untreated control W12 35.32±1.76 Example 1 W12-TA-Fe 53.76±6.14 Example 2 W12-TA-Fe-SA 50.24±3.04 Example 3 Pae-TA-Fe-SA 54.59±0.85 Example 4 Bac-TA-Fe-SA 58.10±0.97

[0101] Test Example 7

[0102] Leaf roll-off angle tests were conducted on untreated growth-promoting bacteria and the compound foliar growth-promoting bacteria agent obtained from the above examples. The specific process is as follows:

[0103] Cut the rice leaves into 2cm pieces. 2 ×6cm2 The test platform was then fixed at a 30° angle. Using a microsyringe, 10 μL of bacterial solution was dropped onto the leaf surface. The test platform was slowly rotated; the critical tilt angle at which the droplet began to roll is the roll angle. A larger roll angle indicates stronger adhesion of the bacterial agent droplet to the leaf surface. The roll angles of different bacterial agents on rice leaves are shown in Table 4. Figure 8 As shown.

[0104] Table 4

[0105] project Growth-promoting bacteria Roll angle (°) Untreated control W12 8.83±1.04 Example 1 W12-TA-Fe 17.03±1.27 Example 2 W12-TA-Fe-SA 17.97±2.35 Example 3 Pae-TA-Fe-SA 20.63±0.85 Example 4 Bac-TA-Fe-SA 16.43±1.40

[0106] From Table 4 and Figure 8 It can be seen that the growth-promoting bacterial agent based on nano-coating improves the adhesion ability of rice leaves, and the rolling angle of the bacterial agent W12-TA-Fe-SA on the rice leaf surface is about 2.3 times higher than that of W12.

[0107] Test Example 8

[0108] The application effect of the growth-promoting bacteria obtained in the above embodiments on the leaf foliage of greenhouse rice was tested, and the process is as follows:

[0109] (1) Take Nipponbare rice seeds, disinfect them, germinate them, and then transfer them to a hydroponic box to continue growing.

[0110] (2) The experiment was divided into two groups: one group was the experimental group with the application of bacterial agents (untreated growth-promoting bacteria W12, and growth-promoting bacterial agents W12-TA-Fe, W12-TA-Fe-SA, Pae-TA-Fe-SA, and Bac-TA-Fe-SA obtained in Examples 1-4, respectively), and the other group was the control group with no bacterial agents applied by spraying water. Each group had 4 replicates.

[0111] (3) When the rice seedlings have grown to two true leaves, select seedlings with similar growth and spray them with bacteria. The inoculation suspension has an OD of 0.8 and a concentration of 0.5 mL per plant.

[0112] (4) Rice plants were harvested 37 days later, and the biomass of each group was measured. The results are shown in the table below. Figure 9 .

[0113] Depend on Figure 9 It can be seen that, 37 days after spraying, compared with the water spraying control group, the inoculation of each group of nano-coated growth-promoting bacteria significantly promoted rice growth.

[0114] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention without departing from the principles and spirit of the present invention.

Claims

1. A nano-coated growth promoter, characterized in that, The nano-coated growth-promoting agent comprises a growth-promoting agent and a nano-coating formed by the self-assembly of plant-derived polyphenols and soluble multivalent metal ions on the surface of the growth-promoting agent; the mass ratio of the plant-derived polyphenols to the soluble multivalent metal ions is (1~10):1; the growth-promoting agent is selected from at least one of Klebsiella pneumoniae, Bacillus polymyxa, Bacillus belye, and Pseudomonas; the plant-derived polyphenols are selected from at least one of tannic acid, gallic acid, catechin, and apple polyphenols; the soluble multivalent metal ions are selected from Fe 3+ Al 3+ Mg 2+ Zn 2+ and Ti 4+ At least one of them.

2. The nano-coated growth promoter according to claim 1, characterized in that, The thickness of the nanocoating is 20~100nm.

3. The method for preparing the nano-coated growth-promoting bacterial agent according to claim 1 or 2, characterized in that, The method involves mixing a growth-promoting agent, plant-derived polyphenols, and soluble multivalent metal ions in a solution to form a nanocoating on the surface of the growth-promoting agent through self-assembly of the plant-derived polyphenols and soluble multivalent metal ions.

4. The method for preparing the nano-coated growth-promoting agent according to claim 3, characterized in that, The mixing is performed using the following steps: S1. Vortex mix the growth-promoting bacterial suspension with the plant-derived polyphenol solution to obtain the bacterial suspension / plant-derived polyphenol solution. S2. Vortex mix the bacterial suspension / plant-derived polyphenol solution with the soluble multivalent metal ion solution, and then wash with PBS buffer (optionally) to obtain the nano-coated growth-promoting bacterial agent.

5. The method for preparing the nano-coated growth-promoting agent according to claim 4, characterized in that, In step S1, the growth-promoting bacterial suspension is obtained by culturing the activated growth-promoting bacterial agent to the late logarithmic growth phase, centrifuging to collect the bacterial cells and washing them, and then resuspending the obtained growth-promoting bacterial agent in deionized water.

6. The method for preparing the nano-coated growth-promoting agent according to claim 5, characterized in that, The number of viable bacteria in the growth-promoting bacterial suspension is 1×10⁻⁶. 8 ~1×10 9 CFU / mL.

7. The method for preparing the nano-coated growth-promoting agent according to claim 5, characterized in that, The concentration of the plant-derived polyphenol solution is 5~20 mg / mL.

8. The method for preparing the nano-coated growth-promoting agent according to claim 5, characterized in that, The ratio of the growth-promoting bacterial suspension to the plant-derived polyphenol solution is 600 μL:(25~100) μL.

9. The method for preparing the nano-coated growth-promoting agent according to claim 5, characterized in that, The vortex mixing time is 10~30s.

10. The method for preparing the nano-coated growth-promoting bacterial agent according to claim 4, characterized in that, In step S2, the concentration of the soluble multivalent metal ion solution is 0.5~5 mg / mL.

11. The method for preparing the nano-coated growth-promoting bacterial agent according to claim 10, characterized in that, The ratio of the soluble multivalent metal ion solution to the growth-promoting bacterial suspension is (25~100) μL: 600 μL.

12. The method for preparing the nano-coated growth-promoting bacterial agent according to claim 4, characterized in that, The vortex mixing time is 10~30s.

13. The method for preparing the nano-coated growth-promoting bacterial agent according to claim 4, characterized in that, The pH value of the PBS buffer is 7.2~7.

4.

14. A compound growth-promoting bacterial agent, characterized in that, The compound growth-promoting agent contains the nano-coated growth-promoting bacteria as described in claim 1 or 2, as well as prebiotics.

15. The compound growth-promoting bacterial agent according to claim 14, characterized in that, The mass ratio of the nano-coated probiotics to prebiotics is 100:(1~100).

16. The compound growth-promoting bacterial agent according to claim 14, characterized in that, The prebiotic is alginate.

17. The compound growth-promoting bacterial agent according to claim 14, characterized in that, The compound growth-promoting bacterial agent is used in solution form.

18. The application of the nano-coated growth-promoting bacterial agent according to claim 1 or 2 and / or the composite growth-promoting bacterial agent according to any one of claims 14 to 17 in promoting plant leaf growth.

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