Biocontrol bacterium agent as well as preparation method and application thereof

Abstract

The invention discloses a biocontrol bacterium agent and a preparation method and application thereof.The biocontrol bacterium agent comprises a nano carrier and biocontrol bacteria, the carrier is halloysite nanotubes modified by coating the surfaces of the halloysite nanotubes with lecithin, sucrose ester or sophorolipid, and the biocontrol bacteria are bacillus subtilis; the preparation method comprises the following steps: (1) soaking halloysite nanotubes in a hydrochloric acid solution for pretreatment; (2) adding halloysite, lecithin, sucrose ester or sophorolipid into a solvent, uniformly mixing, reacting, and after the reaction is completed, washing and drying to obtain a modified halloysite nanotube; and (3) mixing a bacillus subtilis liquid with the modified halloysite nanotube, incubating, and drying to obtain the halloysite nanotube. According to the invention, lecithin, sucrose ester or sophorolipid is adopted to modify the halloysite nanotube and then is used as a carrier, so that the loading effect on bacillus subtilis is improved, and the fungicide has excellent prevention and treatment effects on tobacco brown spot, black shank, anthracnose and sweet leaf fungus damping-off.

Description

Technical Field

[0001] This invention relates to the field of biological control technology, and in particular to a biocontrol agent, its preparation method, and its application. Background Technology

[0002] Tobacco brown spot is primarily caused by *Alternaria alternata*. Tobacco black shank is mainly caused by *Phytophthora parasitica* var. *nicotianae*, a variant of *Phytophthora parasitica*, which severely damages tobacco growth. These diseases occur frequently in major tobacco-growing regions worldwide, causing significant economic losses to the tobacco industry. Currently, tobacco disease control still relies primarily on chemical control, but the long-term use of chemical pesticides has brought many drawbacks, such as environmental pollution, pesticide residues, and increased pathogen resistance. Therefore, the development of green, safe, efficient, and economical biocontrol agents has extremely broad application prospects.

[0003] In recent years, scholars at home and abroad have conducted research on the biological control of tobacco red spot disease, black shank disease and anthracnose (such as using Bacillus), but there are generally problems such as poor leaf wettability and poor control effect (Etesami, H., BR Jeong and BR Glick, Biocontrol of plant diseases by Bacillus spp. Physiological and Molecular Plant Pathology, 2023. 126: p. 102048; De O. Nunes, PS, et al., Bacillus subtilis and Bacillus licheniformis promote tomato growth. Brazilian Journal of Microbiology, 2023. 54(1): p. 397-406.). Yang Dezheng et al. from Guizhou University screened three antagonistic bacteria—Bacillus subtilis T11, Bacillus belyssus Y2, and Y6—that showed an inhibition rate of only about 50% against the causal agent of tobacco auricularia auricula-judae (Yang Dezheng, Sun Guangjun, and Sang Weijun, Screening, Identification, and Culture Medium Optimization of Antagonistic Bacteria for Tobacco Auricularia Auricula-judae. Hubei Agricultural Sciences, 2021, 60(S1): 173-177). Ye Aiping et al. screened a strain of Bacillus paralichrysiforme Q96, which inhibited the germination of auricularia auricula-judae spores and mycelial growth by competing for nutrients and squeezing out the pathogen's living space. The in vitro control efficacy was 60.04% (Ye Aiping et al., Screening and identification of antagonistic bacteria Q96 for tobacco red spot disease and its disease control effect. China Tobacco Science, 2022, 43(05): 56-60). Huang Zhihua et al. used nano-silver to coordinate the control of tobacco red spot disease, and the field control effect was 60.1%, but the price of nano-silver was relatively high (Yuxi Company of Yunnan Tobacco Company. A method for preparing a fungal agent for synergistic control of tobacco red spot disease with nano-silver: CN202310057831.1[P]. 2023-03-31). Summary of the Invention

[0004] Objectives of the invention: The first objective of this invention is to provide a biocontrol agent that enhances leaf adhesion and resistance to tobacco red spot disease; the second objective of this invention is to provide a method for preparing the biocontrol agent; and the third objective of this invention is to provide applications of the biocontrol agent.

[0005] Technical solution: The biocontrol agent of the present invention, wherein the carrier is halloysite nanotubes modified by coating the surface of halloysite nanotubes with lecithin, sucrose lipids or sophorolipids, and the biocontrol bacteria is Bacillus subtilis.

[0006] Lecithin, sucrose, or sophorolipid bind to the hydroxyl groups on the surface of halloysite nanotubes via hydrogen bonds through polar heads, while hydrophobic chains interact with the halloysite tube walls through van der Waals forces.

[0007] Preferably, the Bacillus subtilis is Bacillus subtilis YR08, preservation number: CGMCC No.29125 (patent publication number CN 118048271 A).

[0008] Preferably, the mass ratio of halloysite nanotubes to lecithin, sucrose lipids, or sophorolipids is (1:1) to (1:5). This ratio affects both the bactericidal effect of the final biocontrol agent and the wettability on tobacco leaves. If the proportion of lecithin, sucrose lipids, or sophorolipids is too high or too low, both the bactericidal effect and wettability decrease; a ratio of 1:2 is optimal.

[0009] Preferably, the biocontrol agent further includes a protectant and an adjuvant. The protectant is dextrin, monosodium glutamate, or modified starch, and the adjuvant is dextrin or plant polysaccharides. The core function of the protectant is to maximize the activity and stability of the biocontrol bacteria during processing, storage, transportation, and post-application. The core function of the adjuvant is to improve the physical properties of the formulation, optimize its application process, and enhance the colonization, reproduction, and efficacy of the biocontrol bacteria in the field.

[0010] The preparation method of the biocontrol agent of the present invention includes the following steps:

[0011] (1) Preprocessing of HNTs:

[0012] Halloysite nanotubes were soaked in hydrochloric acid solution and then washed with water until neutral.

[0013] (2) Preparation of HNTs-PC:

[0014] Halloysite nanotubes (HNTs) are added to a solvent with lecithin (PC), sucrose lipids or sophorolipids, mixed evenly, and then transferred to a reaction vessel for reaction. The solution changes from turbid to clear. After the reaction is completed, the modified halloysite nanotubes are obtained by washing and drying.

[0015] (3) Preparation of biocontrol agents:

[0016] The biocontrol agent is obtained by mixing Bacillus subtilis bacterial culture with modified halloysite nanotubes, incubating directly or after adding protectants and adjuvants, and then drying.

[0017] Preferably, in step (1), a natural halloysite ore source with uniform pipe diameter, appropriate length-to-diameter ratio, and good crystallinity is selected.

[0018] Preferably, in step (1), the concentration of hydrochloric acid is 1–5 mol / L. The purpose of using hydrochloric acid to pretreat halloysite is to remove impurities and improve reactivity. If the concentration of hydrochloric acid is too low, it will not have an activation effect, and if the concentration is too high, it will partially destroy the tubular structure. A concentration of 2 mol / L is optimal.

[0019] Preferably, in step (2), the solvent is ethanol; the washing is performed by washing with ethanol and deionized water respectively.

[0020] HNTs-PC were synthesized using a solvothermal method. During stirring, the hydrophobic chains of lecithin, sucrose lipids, or sophorolipids bind to the hydrophobic regions on the surface of halloysite nanotubes through hydrophobic interactions. At the same time, the polar head groups form hydrogen bonds with the hydroxyl groups on the surface of halloysite, further enhancing the adsorption stability.

[0021] Preferably, in step (2), the reaction temperature is 40–60°C. If the temperature is too low, the activity of lecithin molecules is limited, the modification is incomplete, and the improvement in nanotube dispersion is not significant. If the temperature is too high, the reaction rate accelerates, but it easily leads to partial destruction of the lecithin molecular structure, reducing the stability of the modification effect. The Heratus point of sucrose lipids (the temperature at which an aqueous solution of sucrose lipids changes from clear to turbid upon heating) is approximately 50°C. Above this point, the hydrophobic portion of sucrose lipids has a stronger effect, making it more conducive to its adsorption onto the surface of materials such as halloysite through hydrophobic interactions. If the temperature is too high, sucrose lipids may decompose or caramelize. Sophorolipids form micelles in aqueous solution, and their critical micelle concentration (CMC) decreases with increasing temperature. At higher temperatures, lower concentrations of sophorolipids can begin to form micelles, more effectively adsorbing onto the halloysite surface. Excessively high temperatures may cause the micelle structure to become too "active" or change, which is detrimental to the formation of a stable and ordered coating layer on the halloysite surface.

[0022] Preferably, in step (3), the incubation conditions are: at 15-37°C, preferably 25-30°C, in a constant temperature shaking box or after mixing, statically incubate for 0.5-5 hours, preferably 1-2 hours.

[0023] Preferably, in step (3), the drying is freeze drying or spray drying; the freeze drying temperature is -20 to -60°C and the time is 16 to 24 hours, preferably -30 to -40°C and the time is 18 to 20 hours; the spray drying inlet air temperature is 80 to 120°C and the outlet air temperature is 50 to 90°C.

[0024] The application of the biocontrol agent described in this invention in the control of tobacco red spot disease, black shank disease, anthracnose, or in the control of damping-off disease caused by Betalain.

[0025] Preferably, the biocontrol agent is used in the coating agent of tobacco seeds or stevia seeds.

[0026] Invention Mechanism:

[0027] Based on previous research, the inventors screened a strain of Bacillus subtilis (CGMCC No. 29125) with excellent biotransformation and pathogen inhibition properties. A series of patents have been applied for its application in biotransformation (Chen Yuru et al., A method for converting luteolin into four flavonoid compounds using biotransformation CN202510446366.X; A method for converting a series of aglycones in Scutellaria baicalensis into glycosyl or succinyl derivatives CN202410278362.0; A Bacillus subtilis strain). Subtilis and its application methods (CN202410278444.5). A method for one-step bioconversion of genistein into two flavonoid compounds (CN202410278765.5). A method for bioconverting quercetin into quercetin-7-O-succinyl glucoside (CN202410278351.2). A method for bioconverting isorhamnetin (CN202411616691.8). A method for bioconverting the active ingredient in Oroxylum indicum as a flavonoid derivative (CN202410278381.3). Bacillus has the advantages of rapid growth, low culture cost, green and safe production, and strong stress resistance, and has great potential for the biological control of plant diseases. Therefore, the team of this invention used this strain to study the control of tobacco red spot disease, black shank disease, anthracnose and stevia damping-off, and found that it has better control effect than traditional biological agents, such as Trichoderma preparations and Bacillus preparations, and has broad application prospects.

[0028] Bacillus subtilis secretes various antibacterial substances (such as lysozyme and ferritin) during its growth and metabolism. These substances play a crucial role in inhibiting the mycelial growth and spore germination of *Acer tumefaciens*, the causal agent of tobacco auricularia auricula-judae. In the rhizosphere and foliage environment of tobacco, Bacillus subtilis and *Acer tumefaciens* engage in fierce resource competition. Compared to *Acer tumefaciens*, Bacillus subtilis exhibits stronger adaptability and reproductive capacity, enabling it to rapidly seize dominant ecological niches. Furthermore, Bacillus subtilis secretes various plant hormones during its growth and metabolism, such as auxins and cytokinins, which play a vital regulatory role in the growth and development of tobacco.

[0029] Halloysite nanotubes (HNTs) are natural clay minerals with a unique hollow tubular morphology in their microstructure. This special hollow structure gives them a large specific surface area, providing ample attachment sites for other substances. When used as a carrier for Bacillus subtilis, this facilitates the adsorption and dispersion of the bacterial agent, ensuring sufficient contact between the agent and the external environment, thereby enabling it to exert its biocontrol function efficiently.

[0030] Meanwhile, the surface charge characteristics of halloysite nanotubes are closely related to the pH of the solution. When the pH of the solution is in the range of 2 to 8, the inner surface of the tube exhibits a positive charge, while the outer surface exhibits a negative charge. For Bacillus subtilis, in a near-neutral environment [such as soil solutions (pH often close to 7)], the bacterial cell surface usually carries a negative charge. The negative charge on the outer surface of the halloysite nanotube can prevent excessive aggregation of bacteria due to electrostatic attraction, thus preventing damage to bacterial activity. The positive charge on the inner surface of the tube can attract bacteria through electrostatic attraction, effectively loading Bacillus subtilis into the tube, providing a relatively stable attachment environment for the bacterial agent, reducing the impact of adverse external factors on the activity of the bacterial agent, ensuring its stability during storage and application, and laying the foundation for subsequent control of tobacco scab and other pathogens.

[0031] For Bacillus subtilis, modification with lecithin (PC), sucrose lipids, or sophorolipids significantly improves the affinity between nanotubes and the bacterial cells. The surface of Bacillus subtilis cells is rich in various proteins and polysaccharides, carrying certain charges and polar groups. Unmodified halloysite nanotubes, due to their relatively simple surface properties, may experience limitations in adsorption and survival of the bacterial agent upon contact with the bacterial cells due to factors such as charge mismatch and uncoordinated hydrophobic interactions. The amphipathic nature of lecithin, sucrose lipids, or sophorolipids allows them to not only bind stably to nanotubes but also interact with Bacillus subtilis surface components through weak interactions such as electrostatic attraction and hydrogen bonding. Nanotubes modified with lecithin, sucrose lipids, or sophorolipids can adsorb bacterial agents more efficiently, and the bacterial cells exhibit higher growth and metabolic activity on their surface, resulting in a corresponding increase in secreted antibacterial substances. This is of great significance for the synergistic control of Tobacco Red Spot Disease, allowing the entire system's biocontrol function to be fully realized.

[0032] Furthermore, lecithin, sucrose lipids, and sophorolipids, as zwitterionic surfactants, possess unique molecular structures, each containing both hydrophilic groups (sucrose in sucrose esters, sophorose in sophorolipids, and phosphocholine in lecithin) and lipophilic groups (fatty acid chains), which forms the basis of their surfactant properties. When modifying halloysite nanotubes, the hydrophobic chains bind tightly to hydroxyl groups and other groups on the nanotube surface through hydrophobic interactions, while the hydrophilic heads extend into the solution. From a molecular perspective, this process is similar to "molecular anchoring," with numerous lecithin, sucrose lipids, or sophorolipid molecules arranged in an orderly fashion on the nanotube surface, forming a protective film. On one hand, this film shields the direct interactions between nanotubes, preventing aggregation caused by van der Waals forces, allowing the nanotubes to exist as relatively independent entities in solution. On the other hand, the hydrophilic groups endow the nanotubes with better hydrophilicity, making them easier to disperse in aqueous environments.

[0033] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: (1) The present invention uses lecithin, sucrose lipid and sophorolipid to modify halloysite nanotubes as a carrier, which significantly improves the loading effect and biocompatibility of Bacillus subtilis, and the agent has excellent stability and leaf wettability, which improves the prevention effect of tobacco red spot disease, black shank disease, anthracnose or stem rot of Betula platyphylla; (2) The biocontrol agent has excellent characteristics such as environmental friendliness, long-lasting control effect and low production cost; (3) The preparation method is simple and easy to industrialize; (4) The biocontrol agent of the present invention has excellent effect on the prevention and control of tobacco red spot disease, black shank disease, anthracnose or stem rot of Betula platyphylla, and provides a new and effective way to prevent and control tobacco red spot disease, black shank disease, anthracnose or stem rot of Betula platyphylla. Attached Figure Description

[0034] Figure 1 Scanning electron microscope (SEM) images of the HNTs-PC nanocarrier and biocontrol agent prepared using Example 1;

[0035] Figure 2 Infrared spectra of HNTs-PC nanocarriers and halloysite nanotubes prepared using Example 1;

[0036] Figure 3 Thermogravimetric analysis (TGA) diagrams of the HNTs-PC nanocarrier and biocontrol agent prepared using Example 1;

[0037] Figure 4 X-ray diffraction patterns of the HNTs-PC nanocarrier and biocontrol agent prepared using Example 1;

[0038] Figure 5 A comparative diagram showing the antibacterial activity of HNTs-PC-Bacillus subtilis prepared in Example 1, HNTs-Bacillus subtilis prepared in Comparative Example 1, and Bacillus subtilis.

[0039] Figure 6 The bar chart shows the growth of the antibacterial rate of HNTs-PC-Bacillus subtilis prepared in Example 1, HNTs-Bacillus subtilis prepared in Comparative Example 1, and Bacillus subtilis.

[0040] Figure 7 Before and after scanning electron micrographs of the mycelia of pathogens treated with the biocontrol agent prepared in Example 1;

[0041] Figure 8 The diagram shows the water contact angle between the biocontrol agent prepared in Example 1 and the Bacillus subtilis bacterial solution.

[0042] Figure 9 The graph shows the water contact angle of the biocontrol agent prepared in Example 1 and Bacillus subtilis bacterial solution over time. Detailed Implementation

[0043] The technical solution of the present invention will be further described below with reference to the embodiments.

[0044] Example 1

[0045] The solid-phase antibacterial agent of the present invention is prepared by means of the following steps:

[0046] (1) Preprocessing of HNTs:

[0047] Halloysite nanotubes with an aspect ratio of 5:1 to 15:1 and a purity of 99% were selected and soaked in 2 mol / L hydrochloric acid solution for 6 hours, followed by thorough washing with water until neutral.

[0048] (2) Preparation of HNTs-PC:

[0049] HNTs-PC was synthesized using a solvothermal method. 100 mg of HNTs was added to 50 mL of a 98% ethanol solution, ultrasonically dispersed, and then 200 mg of PC was added. The mixture was transferred to a reaction vessel and stirred at 50 °C for 24 h. After the reaction was complete, the mixture was washed with ethanol and deionized water, respectively, and dried to obtain HNTs-PC.

[0050] (3) Preparation of biocontrol agents:

[0051] Preparation of Bacillus subtilis bacterial suspension: Bacillus subtilis [Bacillus subtilis YR08 preservation number: CGMCC No. 29125, patent publication number CN 118048271 A] was incubated in MRS medium for 24 hours to obtain a bacterial suspension with a concentration of 8.3 × 10⁻⁶. 8 cfu / ml.

[0052] 1 mL of a concentration of 8.3 × 10 8 A mixture of CFU / ml Bacillus subtilis bacterial suspension and 1000 mg HNTs-PC was incubated in a constant temperature shaking incubator at 28℃ for 2 hours. Electron microscopy showed that the number of bacteria on the carrier no longer increased. The suspension was then pre-frozen in a low-temperature freezer and freeze-dried at -60℃ for 20 hours to obtain HNTs-PC-Bacillus subtilis, which is the biocontrol agent.

[0053] Example 2

[0054] (1) Preprocessing of HNTs:

[0055] Halloysite nanotubes with an aspect ratio of 5:1 to 15:1 and a purity of 99% were selected and soaked in 1 mol / L hydrochloric acid solution for 6 hours, followed by thorough washing with water until neutral.

[0056] (2) Preparation of HNTs-PC:

[0057] HNTs-PC was synthesized using a solvothermal method. 100 mg of HNTs was added to 50 mL of a 98% ethanol solution, ultrasonically dispersed, and then 200 mg of PC was added. The mixture was transferred to a reaction vessel and stirred at 50 °C for 24 h. After the reaction was complete, the mixture was washed with ethanol and deionized water, respectively, and dried to obtain HNTs-PC.

[0058] (3) Preparation of biocontrol agents:

[0059] Bacillus subtilis bacterial suspension (prepared as in Example 1) was mixed with HNTs-PC and incubated in a constant temperature shaking incubator at 28°C for 1 hour. The suspension was then pre-frozen in a low-temperature freezer, and after pre-freezing, it was freeze-dried in a vacuum freeze dryer at -60°C for 20 hours to obtain HNTs-PC-Bacillus subtilis.

[0060] Example 3

[0061] The biocontrol agent of the present invention is prepared by means of the following steps:

[0062] (1) Preprocessing of HNTs:

[0063] Halloysite nanotubes with an aspect ratio of 5:1 to 15:1 and a purity of 99% were selected and soaked in 3 mol / L hydrochloric acid solution for 6 hours, followed by thorough washing with water until neutral.

[0064] (2) Preparation of HNTs-PC:

[0065] HNTs-PC was synthesized using a solvothermal method. 100 mg of HNTs was added to 50 mL of a 98% ethanol solution, ultrasonically dispersed, and then 200 mg of PC was added. The mixture was transferred to a reaction vessel and stirred at 50 °C for 24 h. After the reaction was complete, the mixture was washed with ethanol and deionized water, respectively, and dried to obtain HNTs-PC.

[0066] (3) Preparation of biocontrol agents:

[0067] Bacillus subtilis bacterial suspension (prepared as in Example 1) was mixed with HNTs-PC and incubated in a constant temperature shaking incubator at 28°C for 3 hours. The suspension was then pre-frozen in a low-temperature freezer, and after pre-freezing, it was freeze-dried in a vacuum freeze dryer at -60°C for 20 hours to obtain HNTs-PC-Bacillus subtilis.

[0068] Example 4

[0069] The biocontrol agent of the present invention is prepared by means of the following steps:

[0070] (1) Preprocessing of HNTs:

[0071] Halloysite nanotubes with an aspect ratio of 5:1 to 15:1 and a purity of 99% were selected and soaked in 4 mol / L hydrochloric acid solution for 6 hours, followed by thorough washing with water until neutral.

[0072] (2) Preparation of HNTs-PC:

[0073] HNTs-PC was synthesized using a solvothermal method. 100 mg of HNTs was added to 50 mL of a 98% ethanol solution, ultrasonically dispersed, and then 200 mg of PC was added. The mixture was transferred to a reaction vessel and stirred at 50 °C for 24 h. After the reaction was complete, the mixture was washed with ethanol and deionized water, respectively, and dried to obtain HNTs-PC.

[0074] (3) Preparation of biocontrol agents:

[0075] Bacillus subtilis bacterial suspension (prepared as in Example 1) was mixed with HNTs-PC and statically incubated at 28°C for 2 hours. The suspension was then pre-frozen in a low-temperature freezer, and subsequently freeze-dried at -60°C for 20 hours to obtain HNTs-PC-Bacillus subtilis.

[0076] Example 5

[0077] The biocontrol agent of the present invention is prepared by means of the following steps:

[0078] (1) Preprocessing of HNTs:

[0079] Halloysite nanotubes with an aspect ratio of 5:1 to 15:1 and a purity of 99% were selected and soaked in 5 mol / L hydrochloric acid solution for 6 hours, followed by thorough washing with water until neutral.

[0080] (2) Preparation of HNTs-PC:

[0081] HNTs-PC was synthesized using a solvothermal method. 100 mg of HNTs was added to 50 mL of a 98% ethanol solution, ultrasonically dispersed, and then 200 mg of PC was added. The mixture was transferred to a reaction vessel and stirred at 50 °C for 24 h. After the reaction was complete, the mixture was washed with ethanol and deionized water, respectively, and dried to obtain HNTs-PC.

[0082] (3) Preparation of biocontrol agents:

[0083] Bacillus subtilis bacterial suspension (prepared as in Example 1) was mixed with HNTs-PC and incubated in a constant temperature shaking incubator at 28°C for 2 hours. The suspension was then pre-frozen in a low-temperature freezer, and after pre-freezing, it was freeze-dried in a vacuum freeze dryer at -60°C for 20 hours to obtain HNTs-PC-Bacillus subtilis.

[0084] Example 6

[0085] Based on Example 1, the mass of PC was changed to 100 mg, while the other conditions remained the same.

[0086] Example 7

[0087] Based on Example 1, the mass of PC was changed to 300 mg, while the other conditions remained the same.

[0088] Example 8

[0089] Based on Example 1, the mass of PC was changed to 400 mg, while the other conditions remained unchanged.

[0090] Example 9

[0091] Based on Example 1, the mass of PC was changed to 500 mg, while the other conditions remained the same.

[0092] Example 10

[0093] The biocontrol agent of the present invention is prepared by means of the following steps:

[0094] (1) Preprocessing of HNTs:

[0095] Halloysite nanotubes with an aspect ratio of 5:1 to 15:1 and a purity of 99% were selected and soaked in 2 mol / L hydrochloric acid solution for 6 hours, followed by thorough washing with water until neutral.

[0096] (2) Preparation of HNTs-PC:

[0097] HNTs-PC was synthesized using a solvothermal method. 100 mg of HNTs was added to 50 mL of a 98% ethanol solution, ultrasonically dispersed, and then 200 mg of PC was added. The mixture was transferred to a reaction vessel and stirred at 40 °C for 24 h. After the reaction was complete, the mixture was washed with ethanol and deionized water, respectively, and dried to obtain HNTs-PC.

[0098] (3) Preparation of biocontrol agents:

[0099] Bacillus subtilis bacterial suspension (prepared as in Example 1) was mixed with HNTs-PC and incubated in a constant temperature shaking incubator at 28°C for 2 hours. The suspension was then spray-dried (inlet temperature 120°C, outlet temperature 70°C, solids content 20%) to obtain HNTs-PC-Bacillus subtilis.

[0100] Example 11

[0101] The biocontrol agent of the present invention is prepared by means of the following steps:

[0102] (1) Preprocessing of HNTs:

[0103] Halloysite nanotubes with an aspect ratio of 5:1 to 15:1 and a purity of 99% were selected and soaked in 2 mol / L hydrochloric acid solution for 6 hours, followed by thorough washing with water until neutral.

[0104] (2) Preparation of HNTs-PC:

[0105] HNTs-PC was synthesized using a solvothermal method. 100 mg of HNTs was added to 50 mL of a 98% ethanol solution, ultrasonically dispersed, and then 200 mg of PC was added. The mixture was transferred to a reaction vessel and stirred at 60 °C for 24 h. After the reaction was complete, the mixture was washed with ethanol and deionized water, respectively, and dried to obtain HNTs-PC.

[0106] (3) Preparation of biocontrol agents:

[0107] Bacillus subtilis bacterial suspension (prepared as in Example 1) was mixed with HNTs-PC and incubated in a constant temperature shaking incubator at 28°C for 2 hours. The suspension was then pre-frozen in a low-temperature freezer, and after pre-freezing, it was freeze-dried in a vacuum freeze dryer at -60°C for 20 hours to obtain HNTs-PC-Bacillus subtilis.

[0108] Example 12

[0109] The biocontrol agent of the present invention is prepared by means of the following steps:

[0110] (1) Preprocessing of HNTs:

[0111] Halloysite nanotubes with an aspect ratio of 5:1 to 15:1 and a purity of 99% were selected and soaked in 2 mol / L hydrochloric acid solution for 6 hours, followed by thorough washing with water until neutral.

[0112] (2) Preparation of HNTs-PC:

[0113] HNTs-PC was synthesized using a solvothermal method. 100 mg of HNTs was added to 50 mL of a 98% ethanol solution, ultrasonically dispersed, and then 200 mg of PC was added. The mixture was transferred to a reaction vessel and stirred at 50 °C for 24 h. After the reaction was complete, the mixture was washed with ethanol and deionized water, respectively, and dried to obtain HNTs-PC.

[0114] (3) Preparation of biocontrol agents:

[0115] Bacillus subtilis bacterial suspension (prepared as in Example 1) was mixed with HNTs-PC and incubated in a constant temperature shaking incubator at 28°C for 2 hours. The suspension was then pre-frozen in a low-temperature freezer, and after pre-freezing, it was freeze-dried in a vacuum freeze dryer at -60°C for 16 hours to obtain HNTs-PC-Bacillus subtilis.

[0116] Example 13

[0117] The biocontrol agent of the present invention is prepared by means of the following steps:

[0118] (1) Preprocessing of HNTs:

[0119] Halloysite nanotubes with an aspect ratio of 5:1 to 15:1 and a purity of 99% were selected and soaked in 2 mol / L hydrochloric acid solution for 6 hours, followed by thorough washing with water until neutral.

[0120] (2) Preparation of HNTs-PC:

[0121] HNTs-PC was synthesized using a solvothermal method. 100 mg of HNTs was added to 50 mL of a 98% ethanol solution, ultrasonically dispersed, and then 200 mg of PC was added. The mixture was transferred to a reaction vessel and stirred at 50 °C for 24 h. After the reaction was complete, the mixture was washed with ethanol and deionized water, respectively, and dried to obtain HNTs-PC.

[0122] (3) Preparation of biocontrol agents:

[0123] Bacillus subtilis bacterial suspension (prepared as in Example 1) was mixed with HNTs-PC and incubated in a constant temperature shaking incubator at 28°C for 2 hours. The suspension was then pre-frozen in a low-temperature freezer, and after pre-freezing, it was freeze-dried in a vacuum freeze dryer at -60°C for 18 hours to obtain HNTs-PC-Bacillus subtilis.

[0124] Example 14

[0125] The biocontrol agent of the present invention is prepared by means of the following steps:

[0126] (1) Preprocessing of HNTs:

[0127] Halloysite nanotubes with an aspect ratio of 5:1 to 15:1 and a purity of 99% were selected and soaked in 2 mol / L hydrochloric acid solution for 6 hours, followed by thorough washing with water until neutral.

[0128] (2) Preparation of HNTs-PC:

[0129] HNTs-PC was synthesized using a solvothermal method. 100 mg of HNTs was added to 50 mL of a 98% ethanol solution, ultrasonically dispersed, and then 200 mg of PC was added. The mixture was transferred to a reaction vessel and stirred at 50 °C for 24 h. After the reaction was complete, the mixture was washed with ethanol and deionized water, respectively, and dried to obtain HNTs-PC.

[0130] (3) Preparation of biocontrol agents:

[0131] Bacillus subtilis bacterial suspension (prepared as in Example 1) was mixed with HNTs-PC and incubated in a constant temperature shaking incubator at 28°C for 2 hours. The suspension was then pre-frozen in a low-temperature freezer, and after pre-freezing, it was freeze-dried in a vacuum freeze dryer at -60°C for 20 hours to obtain HNTs-PC-Bacillus subtilis.

[0132] Example 15

[0133] The biocontrol agent of the present invention is prepared by means of the following steps:

[0134] (1) Preprocessing of HNTs:

[0135] Halloysite nanotubes with an aspect ratio of 5:1 to 15:1 and a purity of 99% were selected and soaked in 2 mol / L hydrochloric acid solution for 6 hours, followed by thorough washing with water until neutral.

[0136] (2) Preparation of HNTs-PC:

[0137] HNTs-PC was synthesized using a solvothermal method. 100 mg of HNTs was added to 50 mL of a 98% ethanol solution, ultrasonically dispersed, and then 200 mg of PC was added. The mixture was transferred to a reaction vessel and stirred at 50 °C for 24 h. After the reaction was complete, the mixture was washed with ethanol and deionized water, respectively, and dried to obtain HNTs-PC.

[0138] (3) Preparation of biocontrol agents:

[0139] Bacillus subtilis bacterial suspension (prepared as in Example 1) was mixed with HNTs-PC and incubated in a constant temperature shaking incubator at 28°C for 2 hours. The suspension was then pre-frozen in a low-temperature freezer, and after pre-freezing, it was freeze-dried in a vacuum freeze dryer at -60°C for 22 hours to obtain HNTs-PC-Bacillus subtilis.

[0140] Example 16

[0141] The biocontrol agent of the present invention is prepared by means of the following steps:

[0142] (1) Preprocessing of HNTs:

[0143] Halloysite nanotubes with an aspect ratio of 5:1 to 15:1 and a purity of 99% were selected and soaked in 2 mol / L hydrochloric acid solution for 6 hours, followed by thorough washing with water until neutral.

[0144] (2) Preparation of HNTs-PC:

[0145] HNTs-PC was synthesized using a solvothermal method. 100 mg of HNTs was added to 50 mL of a 98% ethanol solution, ultrasonically dispersed, and then 200 mg of PC was added. The mixture was transferred to a reaction vessel and stirred at 50 °C for 24 h. After the reaction was complete, the mixture was washed with ethanol and deionized water, respectively, and dried to obtain HNTs-PC.

[0146] (3) Preparation of biocontrol agents:

[0147] Bacillus subtilis bacterial suspension (prepared as in Example 1) was mixed with HNTs-PC and incubated in a constant temperature shaking incubator at 28°C for 2 hours. The suspension was then pre-frozen in a low-temperature freezer, and after pre-freezing, it was freeze-dried in a vacuum freeze dryer at -60°C for 24 hours to obtain HNTs-PC-Bacillus subtilis (29125).

[0148] Example 17

[0149] Based on Example 1, the lecithin used was replaced with sucrose lipid, while the other conditions remained unchanged.

[0150] Example 18

[0151] Based on Example 1, the lecithin used was replaced with sophorolipid, while the other conditions remained unchanged.

[0152] Example 19

[0153] Based on Example 1, step (3) is as follows: 1 mL of Bacillus subtilis bacterial suspension and 1000 mg of HNTs-PC are mixed and incubated in a constant temperature shaking box at 28°C for 2 h. Then, 2% of sucrose lipid protectant and 1% of sodium glutamate adjuvant are added to the bacterial suspension. The suspension is placed in a low temperature freezer for pre-freezing. After pre-freezing, the suspension is placed in a vacuum freeze dryer and freeze-dried at -60°C for 20 h to obtain HNTs-PC-Bacillus subtilis, which is a biocontrol agent.

[0154] Example 20

[0155] The biocontrol agent described in this invention is the same as in Example 1, except that the protective agent used is replaced with modified starch, and all other conditions remain unchanged.

[0156] Example 21

[0157] Based on Example 1, the vacuum freeze-drying method used in step (3) is replaced with spray drying method, wherein the air inlet temperature is 120°C and the air outlet temperature is 50°C.

[0158] Example 22

[0159] The microbial agent prepared in Example 1 was used as a seed coating agent for tobacco seeds, with a dosage of 5% in the seed coating agent.

[0160] Example 23

[0161] The microbial agent prepared in Example 1 was used as a seed coating agent for stevia, with an amount of 3% in the seed coating agent.

[0162] Comparative Example 1

[0163] Based on Example 1, without performing step (2), Bacillus subtilis was directly fixed onto HNTs to obtain HNTs-Bacillus subtilis.

[0164] Structural characterization

[0165] The HNTs material, the HNTs-PC nanocarrier prepared in Example 1, and the biocontrol agent were characterized, and their structures are as follows: Figures 1-4 As shown.

[0166] Depend on Figure 1Scanning electron microscopy revealed that, compared to halloysite nanotubes (HNTs), halloysite nanotubes modified with lecithin (HNTs-PC) exhibited a uniformly dispersed tubular structure with smooth walls and no obvious aggregation when not loaded with Bacillus subtilis. This is attributed to the fact that lecithin modification enhanced the stability and dispersibility of the nanotubes. When loaded with Bacillus subtilis (HNTs-Bacillus subtilis), although a small number of bacteria were loaded on the surface of HNTs, they tended to aggregate due to their hydrophilicity and surface charge, resulting in localized accumulation and poor dispersibility. In contrast, HNTs-PC-Bacillus subtilis clearly showed a large number of rod-shaped bacterial cells attached to its surface. The size of the cells matched the morphological characteristics of Bacillus subtilis, indicating that Bacillus subtilis was successfully loaded onto HNTs-PC. The nanotubes provided attachment sites for the bacteria, allowing them to be distributed relatively evenly, preventing bacterial aggregation, and facilitating subsequent biocontrol functions.

[0167] Depend on Figure 2 Infrared spectroscopy reveals that the unmodified halloysite nanotubes exhibit FTIR spectra in the 3690-3620 cm⁻¹ range. -1 The sharp absorption peaks observed at this point are attributed to the stretching vibrations of the Al-OH groups on the inner surface of the tube, 1030-1000 cm⁻¹. -1 The nearby strong peaks correspond to the Si—O—Si antisymmetric stretching vibration. After lecithin modification, the vibration occurs in the 2920-2850 cm⁻¹ range. -1 A new absorption peak appears, originating from the C-H stretching vibration of the methyl and methylene groups in lecithin. A new absorption peak also appears at 1735 cm⁻¹, originating from the ester carbonyl group in lecithin. Simultaneously, a new absorption peak appears from 3690 to 3620 cm⁻¹. -1 and 3436cm -1 The peak broadened and its intensity decreased, indicating that lecithin chemically bonded to the nanotube surface groups via hydroxyl groups. Furthermore, a complex interaction system was formed between lecithin, nanotubes, and nanotubes, laying the chemical foundation for their synergistic antibacterial effect.

[0168] Depend on Figure 3Thermogravimetric analysis (TGA) revealed that unmodified halloysite, when heated to 150°C, primarily undergoes the removal of adsorbed water and surface hydroxyl groups, resulting in a weight loss of approximately 5–10%. At 600°C, structural water (interlayer water molecules in halloysite) is removed, leading to a weight loss of approximately 10–15%. Above 600°C, the halloysite structure begins to decompose, but the weight loss is relatively small, mainly due to dehydroxylation reactions, with the residue primarily consisting of silica-alumina oxides. Lecithin-modified halloysite exhibits slightly higher weight loss before 150°C than unmodified halloysite (due to the potential increase in surface adsorption capacity of lecithin). From 150°C to 600°C, the weight loss is significant (approximately 20–30%), primarily due to the decomposition of organic components in lecithin (such as fatty acid chains and phosphate groups). At 800°C, the residual mass is typically 50–70%, lower than that of unmodified halloysite, because the decomposition of lecithin leads to the complete loss of organic components. Thermogravimetric analysis (TGA) showed that lecithin modification significantly altered the thermal behavior of halloysite, primarily manifested in increased weight loss due to lecithin decomposition and a decrease in thermal stability. This result provides important thermal performance references for the application of lecithin-modified halloysite (such as drug carriers and sustained-release materials).

[0169] Depend on Figure 4 The X-ray diffraction patterns of the unmodified halloysite nanotubes revealed typical characteristic peaks, such as those at 20.3° and 24.9° corresponding to specific crystal planes, indicating good crystallinity. After lecithin modification, the positions and intensities of the characteristic peaks remained largely unchanged, but the peak shapes broadened slightly. This suggests that the lecithin modification did not disrupt the main framework of the nanotube crystal structure, only producing a weak effect on the surface. This further confirms the structural stability of the load system, ensuring its reliable performance in complex environments.

[0170] Performance testing

[0171] 1. Antibacterial activity test of Tobacco Star Disease Bacteria:

[0172] The antibacterial activity of the biocontrol agents prepared in Example 1 and Comparative Example 1 was tested.

[0173] Test method:

[0174] (1) Marking the culture dish: Take 3 PDA plates and mark five points on the bottom of the plates, located at the center of the plate and at four positions above, below, left and right 2.5 cm away from the center.

[0175] (2) Inoculation with pathogens: Inoculate the center of the plate with tobacco red star pathogen (the applicant provided wild type Alternaria alternata), pick up a 0.5 cm diameter mycelium with a sterile inoculation needle and gently press it onto the surface of the culture medium.

[0176] (3) Inoculation with biocontrol agents: Inoculate at the marked locations on the top, bottom, left and right sides of the plate. Inoculate 10 μL of sterile water into each of the three plates in sequence, and then inoculate 10 μL of Bacillus subtilis bacterial solution, 10 μL of biocontrol agent prepared in Comparative Example 1, and 10 μL of biocontrol agent prepared in Example 1 respectively. Repeat the above inoculation treatment three times.

[0177] Cultivation and Observation: Plates were incubated at 28℃ for 9–10 days. Colony growth was observed and recorded daily, particularly the inhibition of growth of *Tobacco Red Spot* pathogens. Test results are as follows: Figures 5-7 As shown.

[0178] Measure and record the colony diameter of *Tobacco Aster spp.* in each treatment group, and calculate the inhibition rate using the following formula:

[0179]

[0180] Depend on Figure 5 and Figure 6 It was found that after 9 days of incubation in a constant temperature incubator, the CK group had completely covered the plate, indicating that sterile water had no antibacterial effect, the experimental conditions were suitable for pathogen growth, and the experimental operation was correct, providing a reliable control basis for the experimental group's results. Compared with the CK group, the Bacillus subtilis group formed an inhibition zone of a certain size, with a maximum inhibition rate of 53%, indicating that it had a certain inhibitory effect on tobacco scab. Compared with the Bacillus subtilis group, the halloysite-loaded Bacillus subtilis group (HNTs-Bacillus subtilis) showed an improved inhibition rate, indicating that the presence of the carrier improved the stability and sustained-release effect of the bacteria, prolonged the action time, and enhanced the bactericidal effect. Compared with the halloysite-loaded Bacillus subtilis group, the lecithin-modified halloysite-loaded Bacillus subtilis group (HNTs-PC-Bacillus subtilis) showed a more significant increase in antibacterial rate on day 9, with a maximum antibacterial rate of 76%. This indicates that the modification of lecithin enhanced the interaction between halloysite nanotubes and pathogens, further improving the bactericidal effect.

[0181] Depend on Figure 7 Scanning electron microscopy (SEM) images show that before treatment with Bacillus subtilis, the hyphae have a smooth surface, intact structure, vigorous growth, and no abnormal attachments. After treatment with Bacillus subtilis, the hyphae of *Acer tumefaciens* become rough, with deformation or damage in some areas, and an increase in surface attachments. These attachments may be metabolic products or enzymes secreted by Bacillus subtilis, further disrupting the hyphae structure. SEM characterization revealed significant changes, including increased hyphae surface roughness, structural damage, and increased attachments, indicating that Bacillus subtilis has a significant bactericidal effect on *Acer tumefaciens*.

[0182] 2. Water contact angle test of biocontrol agents

[0183] The water contact angle of the biocontrol agent prepared in Example 1 and the Bacillus subtilis bacterial solution was tested.

[0184] Test method: Bacterial concentrations of 8.3*10⁻⁶ were prepared. 8 CFU / ml biocontrol agent and Bacillus subtilis bacterial solution were used. Freshly harvested tobacco leaves were rinsed, dried, and placed on a glass slide. The two sample solutions were then dropped onto the leaves, and the changes in contact angle at different times were observed and recorded. The test results are as follows: Figures 8-9 As shown.

[0185] Depend on Figures 8 to 9 It is evident that the angles of both the biocontrol agent and the Bacillus subtilis bacterial solution decreased rapidly in the first two minutes, initially attributed to the effect of gravity. After two minutes, the descent rate of the Bacillus subtilis bacterial solution slowed significantly, likely due to the interaction between droplet surface tension and leaf surface characteristics. However, the angle of the biocontrol agent still decreased more rapidly, indicating that it spreads more easily on the tobacco surface. At the initial contact between the droplets and the tobacco leaf surface, the water contact angles of the Bacillus subtilis bacterial solution and the biocontrol agent were 85.2° and 50.7°, respectively. After 8 minutes, the contact angle of the Bacillus subtilis bacterial solution was 50.3°, while that of the biocontrol agent was 33°. This is partly due to the amphiphilic (hydrophilic and hydrophobic) nature of lecithin; the modified halloysite may have facilitated a more uniform distribution of the bacterial solution on the tobacco leaf surface, reducing the water contact angle and improving wettability. On the other hand, the nanotube structure of halloysite aids in the adhesion and slow release of the bacterial solution, potentially further improving wettability.

Claims

1. A biocontrol agent, characterized in that, It includes a nanocarrier and a biocontrol bacterium. The carrier is halloysite nanotubes modified by coating the surface of halloysite nanotubes with lecithin, sucrose lipids or sophorolipids, and the biocontrol bacterium is Bacillus subtilis.

2. The biocontrol agent according to claim 1, characterized in that, The mass ratio of halloysite nanotubes to lecithin, sucrose lipids or sophorolipids is (1:1) to (1:5).

3. The biocontrol agent according to claim 1, characterized in that, The biocontrol agent also includes a protectant and an adjuvant. The protectant is dextrin, monosodium glutamate, or modified starch, and the adjuvant is dextrin or plant polysaccharide.

4. A method for preparing a biocontrol agent according to any one of claims 1 to 3, characterized in that, Includes the following steps: (1) Preprocessing of HNTs: Halloysite nanotubes were soaked in hydrochloric acid solution and then washed with water until neutral. (2) Preparation of HNTs-PC: Halloysite nanotubes were added to a solvent with lecithin, sucrose lipids or sophorolipids, mixed evenly, and then transferred to a reaction vessel for reaction. The solution changed from turbid to clear. After the reaction was completed, the modified halloysite nanotubes were obtained by washing and drying. (3) Preparation of biocontrol agents: The biocontrol agent is obtained by mixing Bacillus subtilis bacterial culture with modified halloysite nanotubes, incubating, and then drying directly or after adding protectants and adjuvants.

5. The method for preparing the biocontrol agent according to claim 4, characterized in that, In step (2), the reaction temperature is 40–60°C.

6. The method for preparing the biocontrol agent according to claim 4, characterized in that, In step (3), the incubation temperature is 15 to 37°C.

7. The method for preparing the biocontrol agent according to claim 4, characterized in that, In step (3), the drying is freeze drying or spray drying, and the freeze drying temperature is -20 to -60°C, and the time is 16 to 24 hours.

8. The method for preparing the biocontrol agent according to claim 4, characterized in that, In step (2), the solvent is ethanol, and the washing is performed by washing with ethanol and deionized water respectively.

9. The use of any one of the biocontrol agents according to claims 1 to 3 in the control of tobacco red spot disease, black shank disease, anthracnose, or in the control of damping-off disease caused by *Saccharomyces cerevisiae*.

10. The application according to claim 9, characterized in that, The biocontrol agent is used as a coating agent for tobacco seeds or stevia seeds.

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