A biocontrol agent and its application

Abstract

This invention belongs to the field of biological control of plant diseases, specifically relating to a biocontrol agent and its application. The concentration of *Urspora oryzae* in this water-in-oil emulsion is 2.88 × 10⁻⁶. 8 The emulsion exhibits satisfactory stability and excellent dispersibility; both storage and low-temperature stability are satisfactory, with a foam volume of 0 mL in 1 minute and a residue of 0.15 mL after pouring. Its sustained foaming and pourability meet standards. The pH of the emulsion is 7.86. The average particle size before heat storage is 1.216 μm, and the emulsion quality test is satisfactory. When sprayed on *Solanum nigrum* plants, *Ustilago maydis* fungicide appeared on leaves after 3 days; from 7 to 14 days, the leaf lesions gradually expanded; from 14 to 28 days, a small number of leaves withered after spraying the undiluted fungicide, and some plants even died. The fresh weight control efficacy was 62.79% on day 28 after spraying. This emulsion is readily available, unlikely to induce resistant weeds during use, safe for non-target crops, and environmentally friendly, with low residue and high safety, aligning with the development direction of green weed control technology.

Description

Technical Field

[0001] This invention belongs to the field of biological control of plant diseases, specifically relating to a biocontrol agent and its application. Background Technology

[0002] Solanum rostratum, also known as yellow thorny nightshade, is an annual herbaceous plant belonging to the genus Solanum in the family Solanaceae. It possesses rich genetic diversity, strong reproductive capacity, and high adaptability. It can survive and expand its population in adverse environments, competing with native plants for natural resources such as light, nutrients, and growing space, thereby occupying ecological niches and seriously affecting the growth of native plants (Zhang Ruihai, Song Zhen, Zhang Guoliang, et al., 2019. Characteristics of the soil seed bank of Solanum rostratum Dunal and its response to substitution control [J]. Chinese Journal of Eco-Agriculture (Chinese and English), 27(3):337-347.). It can also affect the growth of other plants through allelopathic effects (Shao Q, Chang L, Wei ZY, et al., 2018. Separation of four flavonolglycosides from Solanum rostratum Dunal using solvent sublation followed by HSCCC and low column temperature preparative HPLC [J]. Journal of Chromatographic Science, 56(8):695-701.). The rapid spread of Solanum rostratum can crowd out the growth of crops and increase the risk of the spread of crop diseases and pests. Besides directly harming crop growth, *Solanum rosifolium* is also an intermediate host for some crop pests and diseases, increasing the chances of crop infection. Its thorns contain toxins that produce a neurotoxin. When these toxins penetrate the fur of livestock or are ingested, they reduce livestock productivity (Zhou SX, Zhu XZ, Shi K, et al., 2021. Chemical composition and allelopathic potential of the invasive plant *Solanum rosifolium* Dunal essential oil[J]. Flora, 274:151730.), and can even scratch human skin, causing allergic reactions.

[0003] Solanum rostratum, native to North America, is a globally prevalent noxious weed. It has been reported in 21 countries and regions worldwide, and its spread continues. This weed thrives in sandy soils, alkaline fertile soils, or mixed clay soils, and can grow in various habitats such as fields, riverbanks, roadsides, wastelands, farmlands, courtyards, and pastures, causing serious harm to agriculture, animal husbandry, and the ecological environment (Zhao JL, Solis-Montero L, Lou AR, et al, 2013. Population structure and genetic diversity of native and invasive populations of Solanum rostratum (Solanaceae) [J]. PLoS ONE, 8(11):e79807.). Solanum spinosa is listed as a quarantine plant in the United States, Canada, Russia, Ukraine and China, and is prohibited from import in Romania, Czech Republic and Australia (Liu Yong, Liao Fang, Yang Xiuli, et al., 2011. Study on molecular biological detection of Solanum spinosa, an important quarantine weed [J]. Plant Quarantine, 25(2):51-54.).

[0004] Currently, the main methods for controlling *Solanum nigrum* are physical, chemical, and ecological control. Among these, the efficacy of chemical control decreases with increasing seedling age, and the use of herbicides is limited by the plant's growth period. Long-term overuse of chemical agents can induce or enhance the development of drug resistance in pathogens, leading to a decrease in control effectiveness. At the same time, pesticide residues can also cause environmental pollution, poisoning of humans and animals, and the ban on highly toxic pesticides has increased the difficulty of selecting chemical control methods. Physical control requires a lot of manpower and time, has low work efficiency, a small applicable area, and a high leakage rate (Pang Lidong, Sun Yuzhuo, 2016. Research progress on invasion mechanism and control strategy of *Solanum nigrum*. China Plant Protection Guide, 36(8):20-25.). In recent years, with the strengthening of green control and sustainable development concepts, the demand for more environmentally compatible biological control resources has increased. The use of pathogenic microorganisms to control target weeds has advantages such as high safety, environmental friendliness, and sustained efficacy, and has gradually become a hot topic in control research (Yan Wenfeng, Wang Jue, Zheng Yanan, 2022. Current status of damage and research progress of control technology of the major invasive plant *Solanum nigrum* [J]. Journal of Terrestrial Ecosystems and Conservation, 2(03):73-79.). In biological control, plant pathogenic microorganisms are an important source for developing pathogenic agents, among which pathogenic fungi are the most widely used.

[0005] Microbial herbicides are pesticides that utilize microorganisms themselves or their metabolites as precursors through artificial modification or synthesis. They are a major component of biopesticides (Radhakrishnan R, Alqarawi AA, Allah FE, 2018. Bioherbicides: Current knowledge on weed control mechanism[J]. Ecotoxicology and Environmental Safety, 2018, 158: 131-138.). They are characterized by abundant resources, low susceptibility to resistant weeds, safety for non-target crops, environmental friendliness, low residue, and high safety, making them the current international trend in green weed control technology (Frumi AC, Charline B, Thammys S, et al, 2023. Fungus-based bioherbicides on circular economy[J]. Bioprocess and biosystems engineering, 46(12): 1729-1754.). Currently, fungi are the most widely used microorganisms in the research and application of microbial herbicides.

[0006] The formulations of fungal herbicides used in the market are mainly divided into two categories: liquid and solid. Among them, the most common are spore suspensions, wettable powders, and water-dispersible granules. Since the active ingredient of microbial herbicides is live bacteria, its vitality is easily affected by the external environment. Therefore, such formulations are prone to problems such as short shelf life and unsatisfactory field control (Zhang Jingjing, Huang Yali, Ma Hong et al., 2016. Development of wettable powder of Trichoderma chlamydospores [J]. Plant Protection, 42(05):103-109.). In contrast, most of the components of water-in-oil emulsions are water, which has the characteristics of high efficiency and greenness. It is conducive to the moisturization of mycelia or spores and prolongs their survival time, which provides the possibility of improving control efficacy and has broad development prospects. In patent CN 117356586A, it was found that *Ulva oryzae* has strong pathogenicity to Solanaceae plants and can be used as a screening object for biocontrol agents to inhibit the growth of Solanaceae plants. At present, there is no relevant research on its use as a biocontrol agent formulation. Summary of the Invention

[0007] This invention uses *Ustilago maydis* isolated and purified from the leaves of naturally infected *Solanum lyratum* as the research object, and its mycelium as the active ingredient to develop a water-in-oil emulsion of *Ustilago maydis* for the control of *Solanum lyratum*. Based on this, the invention was completed.

[0008] In a first aspect, the present invention provides a biocontrol agent for controlling the growth of Solanaceae plants, wherein the biocontrol agent is an emulsion comprising a thickener, an antifoaming agent, an ultraviolet protectant, an emulsifier, a co-emulsifier, a solvent, and Mycelium of Ulva oryzae.

[0009] Furthermore, the thickener includes, but is not limited to, one or more of polyvinyl alcohol, gum arabic, polyethylene glycol and / or silica, preferably polyethylene glycol.

[0010] Furthermore, the content of the thickener is 0.01-1.00%, preferably 0.02-0.03%.

[0011] Furthermore, the defoamer includes, but is not limited to, xanthan gum and / or silicone, with silicone defoamers being preferred.

[0012] Furthermore, the defoamer content is 0.05-0.6%, preferably 0.3-0.5%.

[0013] Furthermore, the ultraviolet protectant includes, but is not limited to, one or more of dextrin, riboflavin and / or ascorbic acid, preferably dextrin.

[0014] Furthermore, the content of the ultraviolet protectant is 0.2-3.0%, preferably 0.4-0.5%.

[0015] Furthermore, the emulsifier is prepared by mixing a nonionic emulsifier and anionic emulsifier.

[0016] Furthermore, the nonionic emulsifier includes, but is not limited to, one or more of Tween-60, Tween-80 and / or castor oil polyoxyethylene ether, preferably castor oil polyoxyethylene ether.

[0017] Furthermore, the anionic emulsifier includes, but is not limited to, sodium dodecylbenzenesulfonate and / or dodecylbenzenesulfonic acid, preferably sodium dodecylbenzenesulfonate.

[0018] Furthermore, the mass ratio of the nonionic emulsifier to the anionic emulsifier includes, but is not limited to, one or more of 1:1, 1:2, 1:3, 1:4, 2:1, 2:3, 3:1, 3:2 and / or 4:1, preferably 3:2.

[0019] Furthermore, the co-emulsifier includes, but is not limited to, isopropanol and / or glycerin, preferably isopropanol.

[0020] Furthermore, the content of the co-emulsifier is 0.5%-8.0%, preferably 2.0%-5.0%.

[0021] Furthermore, the solvent includes, but is not limited to, one or more of xylene, sterile water, and / or methanol, with methanol being preferred.

[0022] Furthermore, the solvent content is 0.5%-8.0%, preferably 2.0%-5.0%.

[0023] Secondly, the present invention provides an application of the biocontrol agent as described in the first aspect in the control of Solanaceae plants, wherein the biocontrol agent has at least one of the following functions:

[0024] Inhibit plant growth;

[0025] Promotes leaf necrosis and abscission;

[0026] Kill the plant.

[0027] Furthermore, the Solanaceae plants include, but are not limited to, one or more of the following: Solanum kaxiense, Solanum semperflorens, Solanum sparsely thorny, Solanum nigrum, Solanum nigrum and / or Solanum nigrum, with Solanum nigrum being the preferred species.

[0028] Beneficial effects

[0029] The concentration of the rice black spore emulsion was 2.88 × 10⁻⁶. 8 The emulsion exhibits satisfactory stability and excellent dispersibility; both storage and low-temperature stability are satisfactory, with a foam volume of 0 mL in 1 minute and a residue of 0.15 mL after pouring. Its sustained foaming and pourability meet standards. The pH of the emulsion is 7.86. The average particle size before heat storage is 1.216 μm, and the emulsion quality test is satisfactory. When sprayed on *Solanum nigrum* plants, *Ustilago maydis* fungicide appeared on leaves after 3 days; from 7 to 14 days, the leaf lesions gradually expanded; from 14 to 28 days, a small number of leaves withered after spraying the undiluted fungicide, and some plants even died. The fresh weight control efficacy was 62.79% on day 28 after spraying. This emulsion is readily available, unlikely to induce resistant weeds during use, safe for non-target crops, and environmentally friendly, with low residue and high safety, aligning with the development direction of green weed control technology. Attached Figure Description

[0030] Figure 1 The effect of thickeners on the mycelial growth of *Urspora oryzae*.

[0031] Figure 2 Solubility of different thickeners in fermentation broth.

[0032] Figure 3 The effect of defoamers on the mycelial growth of *Ustilago maydis*.

[0033] Figure 4 The effect of ultraviolet protectants on the mycelial growth of *Urspora oryzae*.

[0034] Figure 5 Solubility of different emulsifiers in fermentation broth of *Ustilago maydis*.

[0035] Note: A1: Tween-60; A2: Tween-80; A3: Castor oil polyoxyethylene ether; A4: Dodecylbenzene sulfonic acid; A5: Sodium dodecylbenzene sulfonate

[0036] Figure 6 Solubility of different emulsifier mixtures in fermentation broth of *Ustilago maydis*.

[0037] Note: A1: Tween-60; A2: Tween-80; A3: Castor oil polyoxyethylene ether; A4: Dodecylbenzene sulfonic acid; A5: Sodium dodecylbenzene sulfonate.

[0038] Figure 7 Solutions of different ratios of A3:A5 stored at different temperatures for 14 days.

[0039] Note: Figure A: Solution before storage; Figure B: Solution after 14 days of storage at 54℃; Figure C: Solution after 14 days of storage at 25℃; Figure D: Solution after 14 days of storage at 0℃.

[0040] Figure 8 Solution states of different additives stored at different temperatures for 14 days.

[0041] Note: Figure A: Solution before storage; Figure B: Solution after 14 days of storage at 54℃; Figure C: Solution after 14 days of storage at 25℃; Figure D: Solution after 14 days of storage at 0℃.

[0042] Figure 9 Finished rice black spore mold water emulsion.

[0043] Figure 10 Plant growth status after spraying with rice black spore fungicide.

[0044] Note: A-1: ​​Plant growth status before application; A-2: Plant leaves before application; A-3: 3 days after spraying the undiluted fungicide; A-4: Leaves on the 3rd day after spraying the undiluted fungicide; A-5: 7 days after spraying the undiluted fungicide; A-6: Leaves on the 7th day after spraying the undiluted fungicide; A-7: 14 days after spraying the undiluted fungicide; A-8: Leaves on the 14th day after spraying the undiluted fungicide; A-9: 21 days after spraying the undiluted fungicide; A-10: Leaves on the 21st day after spraying the undiluted fungicide; A-11: 28 days after spraying the undiluted fungicide; A-12: ... Leaf of the inoculant stock solution on day 28; B-1: Plant growth status before application; B-2: Plant leaves before application; B-3: Leaf of the plant on day 3 after spraying with water; B-4: Leaf of the plant on day 3 after spraying with water; B-5: Leaf of the plant on day 7 after spraying with water; B-6: Leaf of the plant on day 7 after spraying with water; B-7: Leaf of the plant on day 14 after spraying with water; B-8: Leaf of the plant on day 14 after spraying with water; B-9: Leaf of the plant on day 21 after spraying with water; B-10: Leaf of the plant on day 21 after spraying with water; B-11: Leaf of the plant on day 28 after spraying with water; B-12: Leaf of the plant on day 28 after spraying with water. Detailed Implementation

[0045] The specific embodiments of the present invention will be further described below. It should be noted that these descriptions are for the purpose of aiding understanding the present invention, but do not constitute a limitation thereof. Furthermore, the technical features involved in the embodiments described below can be combined with each other as long as they do not conflict with each other.

[0046] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the experimental materials used in the following embodiments are all available through conventional commercial channels.

[0047] the term

[0048] HLB: is the balance value between lipophilic and hydrophilic. The higher the HLB value, the stronger the hydrophilicity, and the lower the HLB value, the stronger the lipophilicity. Generally speaking, the HLB value is between 1 and 40. When preparing hydrophilic water emulsions, the suitable HLB value for emulsifiers is usually 8-18, which has good stability for the emulsion.

[0049] Example 1 Test Materials

[0050] 1.1 Test materials

[0051] Thickeners: polyvinyl alcohol, gum arabic, polyethylene glycol, silica;

[0052] Defoamers: xanthan gum, silicone;

[0053] UV protectants: dextrin, riboflavin, ascorbic acid;

[0054] Emulsifiers: Tween-60 (A1), Tween-80 (A2), castor oil polyoxyethylene ether (A3), dodecylbenzene sulfonic acid (A4), sodium dodecylbenzene sulfonate (A5);

[0055] Co-emulsifiers: isopropanol, glycerin;

[0056] Solvents: xylene, methanol;

[0057] Sterile water.

[0058] 1.2 Preparation of fermentation broth of *Urspora oryzae*

[0059] (1) Weigh lactose, yeast and ammonium sulfate into sterile water;

[0060] (2) Add glycerol and adjust the pH to 5;

[0061] (3) Sterilize by adding the mother liquor of *Ustilago maydis*;

[0062] (4) After cultivation, the mycelium was filtered out and used as the test fermentation broth.

[0063] Example 2: Screening of thickeners for rice nigricans water-in-oil emulsion

[0064] 2.1 Thickener and bacterial strain compatibility test

[0065] Following the method of Liang Dingding (2020) (Liang Dingding. Optimization of liquid fermentation process and study on water-in-oil emulsion of Pseudomonas sylvestris strain SYAU-06 [D]. Shenyang Agricultural University, 2020.), the test strain was inoculated onto PDA medium for culture.

[0066] (1) PDA was used as the basic culture medium. Four thickeners, namely polyvinyl alcohol, gum arabic, polyethylene glycol and silica, were added to the medium in Table 1 and poured into the petri dish.

[0067] (2) Take the mycelium cake cultured on the plate and place it upside down in the plate;

[0068] (3) Prioritize inoculating the mycelium at the outer edge of the culture medium, and inoculate the mycelium block in the center of the plate and culture it.

[0069] (4) Using sterile water as a control, the diameter of the colonies was measured by the cross-sectional method.

[0070] Table 1. Thickener Formulation Ratio

[0071]

[0072] Note: The ratio in the table is: thickener / culture medium.

[0073] 2.2 Test Results

[0074] Compared with the control group, the four thickeners—silica, polyethylene glycol, gum arabic, and polyvinyl alcohol—had no significant effect on the mycelial growth of *Ulva oryzae*. Figure 1 ).

[0075] Solubility tests revealed that polyvinyl alcohol was insoluble in the fermentation broth, while silica, polyethylene glycol, and gum arabic were soluble in the fermentation broth. Figure 2 However, adding silica will make the solution cloudy.

[0076] Therefore, considering both mycelial growth and economic factors, 0.02% polyethylene glycol was selected as the thickener for the *Ustilago maydis* inoculant.

[0077] Example 3: Screening of antifoaming agents for *Urspora oryzae* water-based emulsions

[0078] 3.1 Determination of compatibility between defoamer and bacterial strain

[0079] Following the method of Chen Huaixiang (2020) (Chen Huaixiang. Development of 450g / L imazalil water-in-oil formulation [D]. Shanghai Normal University, 2020.), the test strains were inoculated onto PDA medium for culture.

[0080] (1) PDA was used as the basic culture medium. Xanthan gum and organosilicon were added to the contents shown in Table 2 and poured into the petri dish.

[0081] (2) Take the mycelium cake cultured on the plate and place it upside down in the plate;

[0082] (3) Prioritize inoculating the mycelium at the outer edge of the culture medium, and inoculate the mycelium block in the center of the plate and culture it.

[0083] (4) Using sterile water as a control, the diameter of the colonies was measured by the cross-sectional method.

[0084] Table 2. Defoamer Formulation Ratio

[0085]

[0086] Note: The ratio in the table is: defoamer / culture medium.

[0087] 3.2 Test Results

[0088] Compared with the control group, the two defoamers had no significant effect on the mycelial growth of *Urspora oryzae*. Figure 3 ).

[0089] In the persistent foaming test, it was found that xanthan gum with a concentration of 0.05–0.3% produced more than 25 mL of foam after 1 minute, making it unqualified as a bacterial defoamer. With silicone defoamers with a concentration of 0.05–0.3%, the foam volume gradually decreased after 1 minute as the concentration of silicone defoamer increased. Specifically, when the concentration of silicone defoamer was 0.3%, the foam volume after 1 minute was less than 25 mL, which was considered acceptable.

[0090] Therefore, 0.3% organosilicon defoamer was selected as the defoamer for the *Ustilago maydis* fungicide (Table 3).

[0091] Table 3. Screening Results of Defoamers

[0092]

[0093]

[0094] Example 4: Screening of UV Protectors for *Urspora oryzae* Water-in-Emulsion

[0095] 4.1 Determination of the compatibility between ultraviolet protectant and bacterial strain

[0096] Following the method of Li Yafang (2014) (Li Yafang. Study on stress resistance and physiological and biochemical effects of *Bryum flavomarginata* Pf9606 on cucumber [D]. Shanxi Agricultural University, 2014.), the test strain was inoculated onto PDA medium for culture.

[0097] (1) PDA culture was used as the basic culture medium. Three ultraviolet protectants, namely dextrin, riboflavin and ascorbic acid, were added to the contents shown in Table 4 and poured into the culture dish.

[0098] (2) Take the mycelium cake cultured on the plate and place it upside down in the plate;

[0099] (3) Prioritize inoculating the mycelium at the outer edge of the culture medium, inoculate the mycelium block in the center of the plate, and then incubate after UV irradiation;

[0100] (4) Using sterile water as a control, the diameter of the colonies was measured by the cross-sectional method.

[0101] Table 4. UV Protectant Formulation Ratio

[0102]

[0103] Note: The ratio in the table is: UV protectant / culture medium.

[0104] 4.2 Test Results

[0105] In the screening of ultraviolet protectants, it was found that *Ustilago maylis* could grow normally in low concentrations of ascorbic acid, with no significant difference from the control group, while *Ustilago maylis* mycelia did not grow in high concentrations of ascorbic acid.

[0106] Low concentrations of riboflavin inhibited the growth of *Ustilago maydis* mycelium, while high concentrations of riboflavin had no significant effect on the growth of *Ustilago maydis*.

[0107] Compared with the control group, different concentrations of dextrin had no significant effect on the growth of *Urspora oryzae* (e.g., ...). Figure 4 (as shown);

[0108] Therefore, 0.5% dextrin was selected as the ultraviolet protectant for *Ichthyophthirius multifiliis*.

[0109] Example 5: Screening of emulsifiers for *Urspora oryzae* water-in-oil emulsion

[0110] 5.1 Screening of emulsifiers for *Urspora oryzae*

[0111] 5.1.1 Test Procedure

[0112] (1) Weigh the emulsifier described in the experimental materials into sterile water and add the fermentation broth of *Ustilago maydis*.

[0113] (2) Mix well and observe whether the solution becomes transparent;

[0114] (3) Place it in a water bath at 30℃, 35℃, 40℃ and 45℃ and record the transparent temperature range when the emulsion forms (Feng Xiaolin. Preparation of Geraniol Nanoemulsion and Study on its Antibacterial Activity [D]. Jilin University, 2023.).

[0115] 5.1.2 Test Results

[0116] Depend on Figure 5 It is known that water-based emulsions prepared by different emulsifiers vary greatly. Among them, Tween-60 and sodium dodecylbenzenesulfonate cannot be prepared into emulsions; while Tween-80, castor oil polyoxyethylene ether, and dodecylbenzenesulfonic acid can be prepared into emulsions.

[0117] Spontaneous emulsification and stability tests revealed that Tween-80 and castor oil polyoxyethylene ether exhibited good spontaneous emulsification and excellent stability (as shown in Table 5).

[0118] Therefore, Tween-80 and castor oil polyoxyethylene ether have the potential to be used as emulsifiers for *Ustilago maydis*, but further research is needed on the compounding of emulsifiers to screen suitable emulsifier combinations to achieve product stability.

[0119] Table 5. Emulsifier Screening Results

[0120]

[0121] 5.2 Screening of Emulsifier Mixtures for *Urspora oryzae* Inoculant

[0122] 5.2.1 Test Procedure

[0123] Following the principle of mixing nonionic and anionic emulsifiers, the two emulsifiers were mixed at a mass ratio of 1:1 (0.75g of each) and then used (as shown in Table 6). The solution was then prepared according to the method in Example 5.1.1, and the transparent temperature range when the emulsion was formed was recorded. One optimal emulsifier mixture was selected (Wang Xinwei et al., 2004).

[0124] Table 6. Combinations of nonionic and anionic emulsifiers

[0125]

[0126] 5.2.2 Test Results

[0127] Depend on Figure 6 It can be seen that the solution after mixing Tween-60:sodium dodecylbenzenesulfonate has floating oil on the upper layer and sediment in the lower layer, and the emulsion is unqualified; the water emulsion prepared by the combination of Tween-80:dodecylbenzenesulfonic acid, Tween-60:dodecylbenzenesulfonic acid, Tween-80:sodium dodecylbenzenesulfonate, castor oil polyoxyethylene ether:dodecylbenzenesulfonic acid, and castor oil polyoxyethylene ether:sodium dodecylbenzenesulfonate has qualified self-emulsification and stability.

[0128] Among them, castor oil polyoxyethylene ether: sodium dodecylbenzene sulfonate emulsion exhibits the best self-emulsification and stability (as shown in Table 7).

[0129] Therefore, castor oil polyoxyethylene ether and sodium dodecylbenzene sulfonate were selected as emulsifiers for the *Ustilago maydis* fungicide.

[0130] Table 7. Screening Results of Emulsifier Mixtures

[0131]

[0132]

[0133] 5.3 Screening of the optimal ratio of *Ichthyophthirius multifiliis* emulsifier mixture

[0134] 5.3.1 Test Procedure

[0135] The two emulsifiers screened in Example 5.2 were mixed evenly according to the proportions in Table 8 and then used. The solution was prepared according to the method in Example 5.1.1. The phenomenon of the emulsion after being stored at high temperature (54°C), low temperature (0°C) and room temperature (25°C) for 2 weeks was observed, and the optimal ratio was selected (Zan Y, Wu Y, Ran D, et al, 2024. Nano-emulsion stabilized by multiple surfactants: An effective alternative for enhancing coal seam water injection effect[J]. Journal of Molecular Liquids, 398: 124150-124151.).

[0136] Table 8. Mixed content of emulsifiers at various ratios

[0137]

[0138] 5.3.2 Test Results

[0139] Two emulsifiers were mixed in different proportions and stored at 54℃, 25℃, and 0℃ for 14 days. The solution state was observed. Emulsions with proportions of 1:1, 1:2, 1:3, 1:4, 2:1, 2:3, 3:1, and 4:1 showed discoloration, trace precipitation, and turbidity. Therefore, the emulsions were substandard after storage. Figure 7 (as shown);

[0140] When the ratio is 3:2, the emulsion is a yellow transparent liquid without oil droplets, crystals, layering, precipitation, etc., and the emulsion is qualified after storage (as shown in Table 9).

[0141] Therefore, the ratio of castor oil polyoxyethylene ether to sodium dodecylbenzene sulfonate of 3:2 was chosen as the emulsifier mixture ratio.

[0142] Table 9. Screening of the optimal ratio of emulsifier mixtures

[0143]

[0144] Example 6: Screening of co-emulsifiers for *Urspora oryzae* water-based emulsions

[0145] 6.1 Test procedure (1) Weigh 15g of emulsifier mixture and 2g of co-emulsifier, take 15mL of rice black mold and add it to 68mL of sterile water; (2) Mix well and observe whether an emulsion can be formed;

[0146] (3) The prepared solutions were placed in a 54°C water bath and a 0°C refrigerator and stored for 14 days respectively.

[0147] (4) Determine the thermal storage (54℃) and cold storage (0℃) stability of the emulsion, and select a suitable co-emulsifier (Parveen R, Baboota S, Ali J, et al., 2015. Stability studies of silymarinnanoemulsion containing Tween

[0148] 80 as a surfactant[J]. Journal of Pharmacy and Bioallied Sciences, 7(4):321-324.).

[0149] 6.2 Test Results

[0150] Different co-emulsifiers have significantly different effects on the appearance of bacterial agents;

[0151] Isopropanol showed the best emulsification effect when used as a co-emulsifier. After storage at 54℃, 25℃, and 0℃ for 14 days, the solution was clear and transparent, without coagulation or stratification. Figure 8 (as shown);

[0152] Emulsions prepared using glycerol as a co-emulsifier became turbid and showed poor stability after being stored at 54°C for 14 days.

[0153] Therefore, isopropanol was selected as a co-emulsifier for *Ustilago maydis* inoculant (as shown in Table 10).

[0154] Table 10. Screening Results of Co-emulsifiers

[0155]

[0156] Example 7: Screening of solvents for water-based emulsions of *Urspora oryzae*

[0157] 7.1 Test Procedure

[0158] (1) Weigh the mixed emulsifier and co-emulsifier, mix the solvent and emulsifier in a ratio of 1:9, and then add *Ustilago maydis*.

[0159] Fermentation broth;

[0160] (2) Mix well and observe whether the solution becomes transparent;

[0161] (3) Place it in a water bath at 30℃, 35℃, 40℃ and 45℃ and record the transparent temperature range when the emulsion forms (Feng Xiaolin. Preparation of Geraniol Nanoemulsion and Study on its Antibacterial Activity [D]. Jilin University, 2023).

[0162] 7.2 Test Results

[0163] When xylene and methanol were added to the bacterial agent as solvents, it was found that the bacterial agent with xylene added had an oil floating layer on the top at 25-45℃, indicating that the bacterial agent was unqualified.

[0164] When methanol is added, the bacterial agent is a yellow transparent liquid, indicating that the bacterial agent is qualified (as shown in Table 11).

[0165] Therefore, methanol can be used as a solvent for *Ustilago maydis* inoculant.

[0166] Table 11. Solvent Screening Results for *Ustilago maydis* Inoculant Inoculant

[0167]

[0168] Example 8: Preparation of a water-based emulsion of *Urspora oryzae*

[0169] 8.1 Test Procedure

[0170] (1) Preparation of water-in-oil emulsion of *Urspora oryzae* using phase inversion method (Liu Zhenbang, Dong Lifeng, Wang Zhi, et al., 10% bifenthrin water-in-oil emulsion).

[0171] Formula research [J]. Pesticide Science and Management, 2012, 33(03):19-22.

[0172] (2) Determine the composition of the rice nitric acid emulsion based on the emulsion, drug content, and stability;

[0173] (3) Prepare according to the thickener, defoamer, UV protectant, emulsifier, co-emulsifier, solvent and their optimal ratio selected in Examples 2-7;

[0174] (4) Add mycelium, solvent, emulsifier and co-emulsifier respectively, mix well in a water bath environment, and use as oil phase;

[0175] (5) Add the adjuvant to the fermentation broth of *Ustilago maydis* in the optimal ratio, mix well, and the aqueous phase is obtained.

[0176] (6) Pour the oil phase into a beaker, stir, add the aqueous phase dropwise to the oil phase, mix well, and let it cool naturally to room temperature to obtain the finished product rice black spore mold water emulsion (Wang Yubo. Study on the control potential and environmental biosafety evaluation of Alternaria SC-018 against Sagittaria trifolia [D]. Shenyang Agricultural University, 2019.).

[0177] 8.2 Test Results

[0178] like Figure 9 The image shows a finished product of rice black spore mold water emulsion. This water emulsion is a yellow, transparent liquid with good fluidity and uniformity. It is stable and does not produce any floating paste, water separation, or other phenomena.

[0179] Example 9: Quality Testing of *Urspora oryzae* Water-in-Emulsion

[0180] 9.1 Active Ingredients

[0181] The active ingredients were determined using the "Standard Specifications for Pesticide Products (Current)" (National Standard HG / T2467.2-2003).

[0182] (1) Inject the sample into a vial, cool it, seal it, and weigh it;

[0183] (2) Place it in a metal container and keep it at 54℃±2℃ for 14 days;

[0184] (3) After cooling to room temperature, weigh the contents as shown in the following formula to determine the active ingredients:

[0185] Determination of active ingredient: X = (Original content of active ingredient - Content of active ingredient after heat storage) / Original content of active ingredient 9.2 pH value

[0186] pH value was determined using the "Method for Determination of pH Value of Pesticides (Current)" (National Standard GB / T1601-2023).

[0187] Weigh 1.0 g of the sample into a 100 mL stoppered graduated cylinder, fill to the mark, and mix well; transfer to a beaker and let stand; immerse the electrode in the sample solution and record the pH value of the sample solution.

[0188] 9.3 Dispersibility determination

[0189] The water-in-water emulsion sample was added to standard hard water, and its ability to form an emulsion in the hard water was observed:

[0190] A good quality product will immediately form a cloud-like mist when dropped into water.

[0191] Good quality is characterized by particles settling but being largely dispersed.

[0192] It needs to be shaken to disperse the particles and is considered neutral.

[0193] If the flocculent material cannot disperse automatically and requires strong shaking, it is considered a poor sign.

[0194] 9.4 Emulsion stability

[0195] The test was conducted in accordance with the "Determination of Stability of Pesticide Emulsions (Current)" (National Standard GB / T1603-2001).

[0196] Pipette 10 mL of the emulsion sample into standard hard water to prepare 100 mL of emulsion; after adding the emulsion, stir; transfer the emulsion to a graduated cylinder and let it stand in a constant temperature water bath; remove it and observe the separation of the emulsion: if there is no floating oil (paste), settled oil, or precipitate in the graduated cylinder, the emulsion stability is deemed to be qualified.

[0197] 9.5 Long-lasting foaming properties

[0198] The standard adopted is the "Standard Specifications for Pesticide Products (Current)" (National Standard HG / T2467.2-2003).

[0199] Add standard hard water to the graduated cylinder to the 180mL mark and place the graduated cylinder on the balance; weigh in 1.0g of sample, add hard water to the mark 9cm from the bottom of the graduated cylinder stopper, and stopper the cylinder; invert the graduated cylinder 30 times with the bottom as the center; let it stand for 1min, and record the foam volume. A sustained foam volume (after 1min) ≤25mL is acceptable.

[0200] 9.6 Low temperature stability

[0201] The standard adopted is the "Standard Specifications for Pesticide Products (Current)" (National Standard HG / T2467.2-2003).

[0202] Transfer 100 mL of sample into a centrifuge tube and keep it in a refrigerator at (0±2)℃ for 1 hour. Check and record the precipitation of solids or oily substances every 15 minutes for 15 seconds. Return the centrifuge tube to the refrigerator and continue to be placed at (0±2)℃. After 7 days, remove the centrifuge tube, let it stand at room temperature, centrifuge, and record the volume of the precipitate at the bottom of the tube.

[0203] 9.7 Thermal storage stability

[0204] The standard adopted is the "Standard Specification for Pesticide Products" (National Standard HG / T2467.2-2003).

[0205] Take 30 mL of sample into an ampoule, cool, seal, and weigh; place the sealed ampoule in a metal container and keep it at a constant temperature of 54℃±2℃ for 14 days; take it out, cool it to room temperature, weigh it, and calculate the decomposition rate within 24 hours for samples whose mass has not changed. A decomposition rate ≤5% is considered qualified.

[0206] 9.8 Particle size determination

[0207] Weigh 1g of water-in-oil emulsion sample and dilute it 100 times. Use a laser particle size analyzer to determine the particle size distribution of the water-in-oil emulsion sample before and after heat storage. The sample is considered qualified if the volume average particle size is less than 2μm.

[0208] 9.9 Test Results

[0209] The results of the tests on various quality indicators of the rice black spore emulsion are shown in Table 12.

[0210] The concentration of the water-in-oil emulsion is 2.88 × 10⁻⁶. 8 The emulsion exhibits satisfactory stability and excellent dispersibility.

[0211] Both thermal storage stability and low temperature stability are qualified. The foam volume is 0 mL in 1 minute and the residue after pouring is 0.15 mL. The sustained foaming and pouring properties both meet the standards.

[0212] The pH value of the emulsion is 7.86.

[0213] The average particle size before heat storage was 1.216 μm, and the water emulsion passed the quality test.

[0214] Table 12. Quality Control Indicators for *Ustilago maydis* Water Emulsion

[0215]

[0216] Example 10: Control results of *Urspora oryzae* inoculant on *Solanum nigrum* in the field.

[0217] Solanum nigrum after spraying with rice black spore fungicide Figure 10 As shown, leaf spots appeared on the 3rd day after spraying the fungicide. From the 7th to the 14th day after spraying, the leaf spots gradually enlarged, and some leaf tissue necrosis and abscission occurred. The fresh weight control efficacy was 60.26% on the 14th day after spraying. From the 14th to the 28th day after spraying, a small number of leaves withered after applying the undiluted fungicide, and some plants even died. The fresh weight control efficacy was 62.79% on the 28th day after spraying.

[0218] In summary, *Ustilago maydis* inoculants can be used to control *Solanum nigrum* (as shown in Table 13).

[0219] Table 13. Control efficacy of *Ustilago maydis* inoculant against *Solanum lyratum*.

[0220]

Claims

1. A biocontrol agent, wherein the biocontrol agent is an aqueous emulsion comprising 0.02% polyethylene glycol thickener, 0.3% silicone defoamer, 0.5% dextrin UV protectant, an emulsifier consisting of castor oil polyoxyethylene ether and sodium dodecylbenzene sulfonate in a mass ratio of 3:2, an isopropanol co-emulsifier, methanol solvent, and *Ustilago maydis*.

2. The application of the biocontrol agent as described in claim 1 in the control of Solanum nigrum, wherein the biocontrol agent has at least one of the following functions: Inhibit plant growth; Promotes leaf necrosis and abscission; Kill the plant.

Images