Biocontrol inoculant and application thereof

Abstract

The invention belongs to the field of biological control of plant diseases, and particularly relates to a biocontrol fungicide and application thereof. The concentration of the nigrospora oryzae emulsion in water is 2.88 * 10 < 8 >, and the emulsion is qualified in stability and excellent in dispersity; the storage stability and the low-temperature stability are both qualified, the foam volume in 1 min is 0 mL, the residue after pouring is 0.15 mL, and the lasting foamability and the pouring property both meet the standard. The pH value of the emulsion in water is 7.86. The volume average particle size before heat storage is 1.216 microns, and the quality of the emulsion in water is detected to be qualified. Spraying a nightshade oryzae fungicide on plants of solanum rostratum, wherein scab appears on leaves after 3 days; after 7-14 days, leaf scab is gradually enlarged; after 14-28 days, a small amount of leaves are withered and even the plants are dead when the fungicide stock solution is sprayed, and the fresh weight control effect is 62.79% when the fungicide stock solution is sprayed on the 28th day. The emulsion in water is easy to obtain, does not easily generate resistant weeds in the use process, is safe to non-target crops, has the characteristics of environmental friendliness, few residues, high safety and the like, and conforms to the development direction of a green weed prevention and control technology.

Description

Technical Field

[0001] The invention belongs to the field of biological control of plant diseases, and specifically relates to a biocontrol fungicide and application thereof. Background Art

[0002] Solanum rostratum, also known as yellow flower nightshade, is an annual herbaceous plant of the genus Solanum in the family Solanaceae. It has rich genetic diversity, strong reproductive capacity and adaptability. It can survive and expand its population in adverse environments, compete with local plants for natural resources such as light, nutrients and growth space, thereby occupying ecological niches and seriously affecting the growth of local plants (Zhang Ruihai, Song Zhen, Zhang Guoliang, et al., 2019. Characteristics of the soil seed bank of Solanum rostratum and its response to alternative 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 will crowd out the growth of crops and increase the risk of the spread of crop diseases and pests. In addition to directly harming the growth of crops, Solanum rostratum is also an intermediate host for some crop diseases and insect pests, increasing the probability of crop infection. Its plants have thorns and toxins, producing a neurotoxin that can pierce livestock fur or be eaten, reducing livestock productivity (Zhou SX, Zhu XZ, Shi K, et al, 2021. Chemical composition and allelopathic potential of the invasive plant Solanumrostratum Dunal essential oil[J].Flora, 274:151730.), and even scratch human skin, causing allergic reactions.

[0003] Solanum rostratum is native to North America and is a global malignant weed. It has been reported to be distributed in 21 countries and regions around the world, and there is a trend of continued spread. The weed is suitable for growing in sandy soil, alkaline loam or mixed clay soil. It can grow in fields, river banks, roadsides, wastelands, farmlands, courtyards, pastures and other habitats, causing serious damage 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 rostratum is listed as a quarantine plant in the United States, Canada, Russia, Ukraine and China, and is prohibited from import in Romania, the Czech Republic and Australia (Liu Yong, Liao Fang, Yang Xiuli, et al., 2011. Research on molecular biological detection of important quarantine weed Solanum rostratum [J]. Plant Quarantine, 25(2):51-54.).

[0004] At present, physical control, chemical control and ecological control are mainly used for Solanum serratum. Among them, the efficacy of chemical control decreases with the increase of seedling age, and the use of herbicides is limited by the growth period of plants. Long-term excessive use of chemical agents will induce or enhance the resistance of pathogens, resulting in reduced control effect; at the same time, pesticide residues will also cause environmental pollution, human and animal poisoning and other problems. The prohibition of highly toxic pesticides has also increased the difficulty of choosing chemical control. 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 the invasion mechanism and control strategy of Solanum serratum. China Plant Protection Guide, 36(8):20-25.). In recent years, with the strengthening of green control and sustainable development concepts, the demand for biological control resources with better environmental compatibility has increased. The use of pathogenic microorganisms to control target weeds has the advantages of high safety, environmental friendliness and sustained control effect, and has gradually become a hot topic in control research (Yan Wenfeng, Wang Jue, Zheng Yanan, 2022. Current status of damage and research progress on control technology of major invasive plant Solanum serrata [J]. Journal of Terrestrial Ecosystems and Conservation, 2(03):73-79.). In biological control, plant pathogenic microorganisms are an important source for the development of pathogenic agents, among which pathogenic fungi are the most used.

[0005] Microbial herbicides are pesticides that are artificially modified or synthesized using microorganisms themselves or their metabolites as precursors. They are the main components of biological pesticides (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 rich in resources, not easy to produce resistant weeds, safe for non-target crops, environmentally friendly, low in residues, and highly safe. They are the current development direction of green weed control technology in the world (Frumi AC, Charline B, Thamarys S, et al, 2023. Fungus-based bioherbicides on circular economy [J]. Bioprocess and biosystems engineering, 46 (12): 1729-1754.). At present, fungi are the most widely used microorganisms in the research and development 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 which the most common ones are spore suspensions, wettable powders, and water-dispersible granules. Since the active ingredients of microbial herbicides are live bacteria, their vitality is easily affected by the external environment, so such preparations are prone to problems such as short shelf life and unsatisfactory field control effects (Zhang Jingjing, Huang Yali, Ma Hong, et al., 2016. Development of wettable powder of Trichoderma chlamydospores [J]. Plant Protection, 42(05): 103-109.). Most of the ingredients in aqueous emulsions are water, which has the characteristics of high efficiency and greenness, is conducive to the moisture retention of mycelium or spores, prolongs the survival time, and provides a possibility for improving control effects, and has broad development prospects. In patent CN 117356586A, it was found that the rice black spore mold has a strong pathogenicity to Solanaceae plants and can be used as a screening object for a biocontrol agent that inhibits the growth of Solanaceae plants. At present, there is no relevant research on it as a biocontrol agent preparation. Summary of the invention

[0007] The present invention takes the rice black spore mold isolated and purified from the naturally diseased leaves of Solanum spp. as the research object, uses its mycelium as the effective ingredient, and develops a rice black spore mold aqueous emulsion for preventing and controlling Solanum spp., based on which the present invention is completed.

[0008] In a first aspect, the present invention provides a biocontrol agent for controlling the growth of Solanaceae plants. The biocontrol agent is an aqueous emulsion comprising a thickener, a defoamer, an ultraviolet protectant, an emulsifier, an emulsifier aid, a solvent and mycelium of Nigrospora oryzae.

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

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

[0011] Furthermore, the defoaming agent includes but is not limited to xanthan gum and / or silicone, etc., preferably a silicone defoaming agent.

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

[0013] Furthermore, the UV protection agent 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 protection agent is 0.2-3.0%, preferably 0.4-0.5%.

[0015] Furthermore, the emulsifier is prepared by mixing a nonionic emulsifier and an 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 dodecylbenzene sulfonate and / or dodecylbenzene sulfonic acid, preferably sodium dodecylbenzene sulfonate.

[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 glycerol, preferably isopropanol.

[0020] Furthermore, the content of the auxiliary 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, preferably methanol.

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

[0023] In a second aspect, the present invention provides a use of the biocontrol agent according to the first aspect in the control of Solanaceae plants, wherein the biocontrol agent has at least one of the following effects:

[0024] Inhibit plant growth;

[0025] Promote leaf necrosis and shedding;

[0026] Kill plants.

[0027] Further, the Solanaceae plants include but are not limited to one or more of Solanum lycopersicum, Solanum ovata, Solanum sparsely spinulosum, Solanum nigrum, Solanum nigrum and / or Solanum spicatum, preferably Solanum nigrum.

[0028] Beneficial Effects

[0029] The concentration of the rice black spore emulsion was 2.88×10 8 , the emulsion stability is qualified, the dispersibility is excellent; the storage stability and low temperature stability are qualified, the foam volume of 1min is 0mL, the residue after pouring is 0.15mL, and the persistent foaming and pouring properties meet the standards. The pH value of the water emulsion is 7.86. The volume average particle size before hot storage is 1.216um, and the quality inspection of the water emulsion is qualified. Spraying the rice black spore fungus agent on the plants of Solanum nigrum, lesions appeared on the leaves on the 3d; 7-14d, the leaf lesions gradually expanded; 14-28d, a small number of leaves died after spraying the original solution of the fungus agent, and even some plants died. The fresh weight control effect was 62.79% on the 28th day of spraying. The water emulsion is easy to obtain, it is not easy to produce resistant weeds during use, it is safe for non-target crops, and it has the characteristics of environmental friendliness, less residue, and high safety, which is in line with the development direction of green weed control technology. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Effects of thickeners on the mycelial growth of Nigrospora oryzae.

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

[0032] Figure 3 Effects of defoamers on mycelial growth of Nigrosporium oryzae.

[0033] Figure 4 Effects of UV protectants on mycelial growth of Nigrosporium oryzae.

[0034] Figure 5 Solubility of different emulsifiers in fermentation broth of Nigrospora oryzae.

[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 Nigrospora oryzae.

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

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

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

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

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

[0042] Fig. 9 Finished product of rice black spore mold aqueous emulsion.

[0043] Fig.10 Plant growth status after spraying rice black spore fungus agent.

[0044] Note: A-1: ​​plant growth before spraying; A-2: plant leaves before spraying; A-3: 3 days after spraying the original solution of the fungicide; A-4: leaves on the 3rd day after spraying the original solution of the fungicide; A-5: 7 days after spraying the original solution of the fungicide; A-6: leaves on the 7th day after spraying the original solution of the fungicide; A-7: 14 days after spraying the original solution of the fungicide; A-8: leaves on the 14th day after spraying the original solution of the fungicide; A-9: 21 days after spraying the original solution of the fungicide; A-10: leaves on the 21st day after spraying the original solution of the fungicide; A-11: 28 days after spraying the original solution of the fungicide; A-12: Leaves on the 28th day after spraying with the original solution of the fungicide; B-1: plant growth status before application; B-2: leaves of plants before application; B-3: 3rd day after spraying with clean water; B-4: leaves on the 3rd day after spraying with clean water; B-5: 7th day after spraying with clean water; B-6: leaves on the 7th day after spraying with clean water; B-7: 14th day after spraying with clean water; B-8: leaves on the 14th day after spraying with clean water; B-9: 21st day after spraying with clean water; B-10: leaves on the 21st day after spraying with clean water; B-11: 28th day after spraying with clean water; B-12: leaves on the 28th day after spraying with clean water. DETAILED DESCRIPTION

[0045] The specific embodiments of the present invention are further described below. It should be noted that the description of these embodiments is used to help understand the present invention, but does not constitute a limitation of the present invention. In addition, 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] The experimental methods in the following examples are conventional methods unless otherwise specified, and the experimental materials used in the following examples are commercially available unless otherwise specified.

[0047] the term

[0048] HLB: It is the balance value of lipophilicity or hydrophilicity. The larger the HLB value, the stronger the hydrophilicity, and the smaller the HLB value, the stronger the lipophilicity. Generally speaking, the HLB value ranges from 1 to 40. The HLB value of the emulsifier suitable for preparing hydrophilic water emulsions is usually 8-18, which has better stability for the emulsion.

[0049] Example 1 Test materials

[0050] 1.1 Test materials

[0051] Thickener: polyvinyl alcohol, gum arabic, polyethylene glycol, white carbon black;

[0052] Defoaming agent: xanthan gum, silicone;

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

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

[0055] Co-emulsifier: isopropyl alcohol, glycerin;

[0056] Solvents: xylene, methanol;

[0057] Sterile water.

[0058] 1.2 Preparation of fermentation broth of Nigrosporium oryzae

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

[0060] (2) adding glycerol and adjusting the pH to 5;

[0061] (3) sterilizing by adding the mother liquor of black spore mold of rice;

[0062] (4) After cultivation, the mycelium is filtered out and used as the fermentation liquid for testing.

[0063] Example 2 Screening of Thickeners for Rice Black Sporophyte Emulsions

[0064] 2.1 Compatibility determination of thickener and strain

[0065] Referring to the method of Liang Dingding (2020) (Liang Dingding. Optimization of liquid fermentation process of Phyllostomia SYAU-06 strain and research on water emulsion [D]. Shenyang Agricultural University, 2020.), the test strain was inoculated on PDA medium for culture.

[0066] (1) PDA was used as the basic culture medium, and four thickeners, polyvinyl alcohol, gum arabic, polyethylene glycol and white carbon black, were added in the amounts shown in Table 1, and then poured into a culture dish;

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

[0068] (3) The mycelium at the outer edge of the culture medium is preferentially inoculated, and the bacterial mass is inoculated and cultured in the center of the plate;

[0069] (4) Add sterile water as a control and measure the diameter of the colony using the cross method.

[0070] Table 1. Thickener preparation 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, white carbon black, polyethylene glycol, gum arabic, and polyvinyl alcohol, had no significant effect on the mycelial growth of Nigrosporium oryzae ( Figure 1 ).

[0075] In the solubility test, it was found that polyvinyl alcohol was insoluble in the fermentation broth, while white carbon black, polyethylene glycol and gum arabic were soluble in the fermentation broth ( Figure 2 ), but adding white carbon black will make the solution turbid.

[0076] Therefore, from the perspective of mycelium growth and economy, 0.02% polyethylene glycol was selected as the thickener for the rice black spore mold inoculant.

[0077] Example 3 Screening of Rice Black Sporophyte Emulsion Defoamer

[0078] 3.1 Determination of compatibility between defoamers and strains

[0079] Referring to the method of Chen Huaixiang (2020) (Chen Huaixiang. Research and development of 450g / L prochloraz emulsion formula [D]. Shanghai Normal University, 2020.), the test strain was inoculated on PDA medium for culture.

[0080] (1) PDA was used as the basic culture medium, and two defoaming agents, xanthan gum and organosilicon, were added at the amounts shown in Table 2, and then poured into a culture dish;

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

[0082] (3) The mycelium at the outer edge of the culture medium is preferentially inoculated, and the bacterial mass is inoculated and cultured in the center of the plate;

[0083] (4) Add sterile water as a control and measure the diameter of the colony using the cross method.

[0084] Table 2. Defoamer preparation 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 Nigrosporium oryzae ( Figure 3 ).

[0089] In the persistent foaming test, it was found that the foam volume of 0.05-0.3% xanthan gum was greater than 25mL after 1 minute, which was unqualified as a bacterial defoamer. In the 0.05-0.3% organosilicon defoamer, as the concentration of the organosilicon defoamer increased, the foam volume gradually decreased after 1 minute. When the concentration of the organosilicon defoamer was 0.3%, the foam volume of the bacterial agent was less than 25mL after 1 minute, which was qualified.

[0090] Therefore, 0.3% organosilicon defoamer was selected as the defoamer for the rice black spore mold agent (Table 3).

[0091] Table 3. Defoamer screening results

[0092]

[0093]

[0094] Example 4 Screening of the UV Protective Agent of the Rice Black Sporoderm Emulsion

[0095] 4.1 Determination of compatibility between UV protectants and strains

[0096] Referring to the method of Li Yafang (2014) (Li Yafang. Research on the stress resistance of Pf9606 and its physiological and biochemical effects on cucumber [D]. Shanxi Agricultural University, 2014.), the test strain was inoculated on PDA medium for culture.

[0097] (1) PDA culture was used as the basic culture medium, and three UV protectants, dextrin, riboflavin and ascorbic acid, were added in the amounts shown in Table 4 and poured into a culture dish;

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

[0099] (3) The mycelium at the outer edge of the culture medium is preferentially inoculated, and the bacterial block is inoculated in the center of the plate, and cultured after irradiation with ultraviolet light;

[0100] (4) Add sterile water as a control and measure the diameter of the colony using the cross method.

[0101] Table 4. UV protection agent preparation ratio

[0102]

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

[0104] 4.2 Test results

[0105] In the screening of UV protectants, it was found that the rice black spore mold could grow normally in low concentrations of ascorbic acid, with no significant difference from the control group, while the mycelium of the rice black spore mold did not grow in high concentrations of ascorbic acid;

[0106] Low concentration of riboflavin inhibited the growth of mycelium of Nigrospora oryzae, while high concentration of riboflavin had no significant effect on the growth of Nigrospora oryzae.

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

[0108] Therefore, 0.5% dextrin was selected as the UV protectant for Nigrosporium oryzae.

[0109] Example 5 Screening of emulsifiers for rice black spore mold aqueous emulsion

[0110] 5.1 Screening of rice black spore mold emulsifier

[0111] 5.1.1 Test steps

[0112] (1) Weigh the emulsifier described in the experimental materials into sterile water, and add the fermentation liquid of Nigrosporium oryzae;

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

[0114] (3) Place it in a water bath at 30°C, 35°C, 40°C, and 45°C, and record the transparent temperature range when the emulsion is formed (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 can be seen that the aqueous emulsions prepared by different emulsifiers are very different. Among them, Tween-60 and sodium dodecylbenzene sulfonate cannot be prepared into emulsions, while Tween-80, castor oil polyoxyethylene ether, and dodecylbenzene sulfonic acid can be made into emulsions.

[0117] In the spontaneous emulsification and stability tests, it was found that Tween-80 and castor oil polyoxyethylene ether had 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 Nigrosporium oryzae, but further research is needed on the compounding of emulsifiers to screen the appropriate emulsifier combination to achieve product stability.

[0119] Table 5. Emulsifier screening results

[0120]

[0121] 5.2 Screening of mixtures of rice black spore fungus agent and emulsifier

[0122] 5.2.1 Test steps

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

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

[0125]

[0126] 5.2.2 Test results

[0127] Depend on Figure 6 It can be seen that after Tween-60 and sodium dodecylbenzene sulfonate are mixed, there is floating oil on the upper layer of the solution and precipitation in the lower layer, and the emulsion is unqualified; the aqueous emulsions prepared by the combinations of Tween-80: dodecylbenzene sulfonic acid, Tween-60: dodecylbenzene sulfonic acid, Tween-80: sodium dodecylbenzene sulfonate, castor oil polyoxyethylene ether: dodecylbenzene sulfonic acid, castor oil polyoxyethylene ether: sodium dodecylbenzene sulfonate, etc. have qualified spontaneous emulsification and stability.

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

[0129] Therefore, castor oil polyoxyethylene ether and sodium dodecylbenzene sulfonate were selected as the emulsifier for the rice black spore mold agent.

[0130] Table 7. Emulsifier mixture screening results

[0131]

[0132]

[0133] 5.3 Screening of the optimal ratio of rice black spore mold emulsifier mixture

[0134] 5.3.1 Test steps

[0135] The two emulsifiers screened out in Example 5.2 were mixed evenly according to the ratio in Table 8 and then prepared into a solution according to the method of Example 5.1.1. The phenomenon of the formed emulsion after being stored at high temperature (54°C), low temperature (0°C) and room temperature (25°C) for 2 weeks was observed to select the best ratio (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. Emulsifier content in different proportions

[0137]

[0138] 5.3.2 Test results

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

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

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

[0142] Table 9. Screening of the best ratio of emulsifier mixture

[0143]

[0144] Example 6 Screening of co-emulsifiers for the rice black spore mold aqueous emulsion

[0145] 6.1 Test steps (1) Weigh 15 g of emulsifier mixture, 2 g of co-emulsifier, and 15 mL of rice black spore mold, and add them to 68 mL 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 for 14 days;

[0147] (4) Determine the heat storage (54°C) and cold storage (0°C) stability of the emulsion and select the appropriate co-emulsifier (Parveen R, Baboota S, Ali J, et al, 2015. Stability studies of silymarin nanoemulsion containing Tween

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

[0149] 6.2 Test results

[0150] The effects of different emulsifiers on the appearance of bacterial agents vary greatly;

[0151] Among them, isopropanol had the best emulsification effect when used as an emulsifier. After storage at 54℃, 25℃, and 0℃ for 14 days, the solution was clear and transparent, without coagulation and stratification (such as Figure 8 shown);

[0152] However, the emulsion prepared with glycerol as the emulsifier became turbid after being stored at 54°C for 14 days, indicating poor stability.

[0153] Therefore, isopropanol was selected as the co-emulsifier for the rice black spore mold agent (as shown in Table 10).

[0154] Table 10. Screening results of emulsifiers

[0155]

[0156] Example 7 Screening of solvents for the aqueous emulsion of black spore mold of rice

[0157] 7.1 Test steps

[0158] (1) Weigh the mixed emulsifier and the co-emulsifier, mix the solvent and the emulsifier in a ratio of 1:9, and add the black spore mold

[0159] Bacterial fermentation broth;

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

[0161] (3) Place it in a water bath at 30°C, 35°C, 40°C, and 45°C, and record the transparent temperature range when the emulsion is formed (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 as solvents to the bacterial agent, it was found that the bacterial agent with xylene added had floating oil in the upper layer at 25-45°C, and the bacterial agent was unqualified;

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

[0165] Therefore, methanol can be selected as the solvent of the rice black spore mold agent.

[0166] Table 11. Screening results of solvents for the black spore mold of rice

[0167]

[0168] Example 8 Preparation of Black Sporophyte Emulsion in Water

[0169] 8.1 Test steps

[0170] (1) Preparation of an aqueous emulsion of Nigrosporium oryzae by phase inversion method (Liu Zhenbang, Dong Lifeng, Wang Zhi, et al. Bifenthrin 10% aqueous emulsion

[0171] Research on formulation of pesticides[J]. Pesticide Science and Management, 2012, 33(03):19-22.);

[0172] (2) Determine the composition of the Nigrosporium oryzae emulsion in water based on the emulsion in water, drug content and stability;

[0173] (3) preparing the thickener, defoamer, UV protectant, emulsifier, co-emulsifier, solvent and their optimal ratios selected from Examples 2-7;

[0174] (4) adding mycelium, solvent, emulsifier, and co-emulsifier respectively, and mixing them in a water bath to form the oil phase;

[0175] (5) adding the auxiliary agent to the fermentation liquid of the black spore mold of rice in an optimal ratio and mixing well to obtain an aqueous phase;

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

[0177] 8.2 Test results

[0178] like Fig. 9 As shown, the finished product of rice black spore mold aqueous emulsion is yellow and transparent, has good fluidity and is a uniform liquid. It is stable and has no floating paste, water precipitation and other phenomena.

[0179] Example 9 Quality Test of Rice Black Sporophyte 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 in a metal container at 54°C ± 2°C for 14 days;

[0184] (3) Take out and cool to room temperature, weigh them respectively, and determine the effective ingredient as described in the following formula:

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

[0186] The pH value was determined using the "Pesticide pH Determination Method (Current)" (National Standard GB / T1601-2023).

[0187] Weigh 1.0 g of the sample into a 100 mL stoppered measuring 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] Add the water emulsion sample into standard hard water to observe its ability to form an emulsion in hard water:

[0190] If it immediately turns into a cloud when dropped into water, it is considered excellent;

[0191] There are particles sinking but they are basically dispersed, which is considered good;

[0192] Needs shaking to disperse is judged as medium;

[0193] The flocculent sediment cannot disperse automatically and needs strong shaking, which is considered poor.

[0194] 9.4 Emulsion stability

[0195] Refer to the "Test Method for Stability of Pesticide Emulsion (Current)" (National Standard GB / T1603-2001).

[0196] Pipette 10mL of emulsion sample into standard hard water to make 100mL of emulsion; stir after adding the emulsion; transfer the emulsion to a measuring cylinder and let it stand in a constant temperature water bath; take it out and observe the separation of the emulsion: if there is no floating oil (ointment), sinking oil or precipitation in the measuring cylinder, the emulsion stability is judged to be qualified.

[0197] 9.5 Persistent foaming

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

[0199] Add standard hard water to the 180mL mark in the measuring cylinder and place it on a balance; weigh 1.0g of the sample and add hard water to the mark 9cm from the bottom of the measuring cylinder stopper and cover it with the stopper; invert the measuring cylinder 30 times with the bottom of the cylinder as the center; let it stand for 1min and record the foam volume. The tester shall be qualified if the sustained foaming volume (after 1min) is ≤25mL.

[0200] 9.6 Low temperature stability

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

[0202] Pipette 100 mL of the sample into a centrifuge tube and keep it in a (0±2)℃ refrigerator for 1 hour; check and record the precipitation of solid or oily matter every 15 minutes for 15 seconds each time; return the centrifuge tube to the refrigerator and continue to place it at (0±2)℃. After 7 days, take out the centrifuge tube, let it stand at room temperature, and after centrifugation, record the volume of the precipitate at the bottom of the tube.

[0203] 9.7 Hot Storage Stability

[0204] Adopt the "Standard Specifications for Pesticide Products" (National Standard HG / T2467.2-2003).

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

[0206] 9.8 Particle size determination

[0207] Weigh 1g of the aqueous emulsion sample and dilute it 100 times. Use a laser particle size distribution analyzer to measure the particle size distribution of the aqueous emulsion sample before and after hot storage. It is considered qualified if the volume average particle size is less than 2μm.

[0208] 9.9 Test results

[0209] Various quality indicators of the rice black spore mold aqueous emulsion were measured, and the test results are shown in Table 12.

[0210] The concentration of water emulsion is 2.88×10 8 , the emulsion stability is qualified and the dispersibility is excellent.

[0211] Both the hot storage stability and the low-temperature stability are qualified, the foam volume in 1 minute is 0 mL, the residue after pouring is 0.15 mL, and the persistent foaming and pouring properties are in compliance with the standards.

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

[0213] The volume average particle size before hot storage is 1.216um, and the water emulsion quality test is qualified.

[0214] Table 12. Quality control indexes of rice black spore emulsion

[0215]

[0216] Example 10 Control results of rice black spore mold agent on field Solanum nigrum

[0217] Solanum nigrum after spraying with rice black spore fungicide Fig.10 As shown in the figure, on the 3rd day after spraying the fungicide, lesions appeared on the leaves. On the 7th to 14th day after spraying, the lesions on the leaves gradually expanded, and some leaf tissues necrotized and fell off. On the 14th day after spraying, the fresh weight control efficiency was 60.26%. On the 14th to 28th day after spraying, a small number of leaves died after spraying the original solution of the fungicide, and some plants even died. On the 28th day after spraying, the fresh weight control efficiency was 62.79%.

[0218] In summary, the rice black spore fungicide can be used to control Solanum nigrum (as shown in Table 13).

[0219] Table 13. Control effect of rice black spore fungicide on Solanum nigrum

[0220]

Claims

1. A biocontrol agent for controlling the growth of Solanaceae plants, the biocontrol agent being an aqueous emulsion comprising a thickener, a defoamer, an ultraviolet protective agent, an emulsifier, an emulsifier aid, a solvent and mycelium of Nigrospora oryzae.

2. The biocontrol agent according to claim 1, in, The thickener includes but is not limited to one or more of polyvinyl alcohol, gum arabic, polyethylene glycol and / or white carbon black, preferably polyethylene glycol; the content of the thickener is 0.01-1.00%, preferably 0.02-0.03%.

3. The biocontrol agent according to claim 1, in, The defoaming agent includes but is not limited to xanthan gum and / or silicone, etc., preferably silicone defoaming agent; the content of the defoaming agent is 0.05-0.6%, preferably 0.3-0.5%.

4. The biocontrol agent according to claim 1, in, The ultraviolet protective agent includes but is not limited to one or more of dextrin, riboflavin and / or ascorbic acid, etc., preferably dextrin; the content of the ultraviolet protective agent is 0.2-3.0%, preferably 0.4-0.5%.

5. The biocontrol agent according to claim 1, in, The emulsifier is prepared by mixing a nonionic emulsifier and an anionic emulsifier; 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; the anionic emulsifier includes but is not limited to sodium dodecylbenzene sulfonate and / or dodecylbenzene sulfonic acid, preferably sodium dodecylbenzene sulfonate.

6. The biocontrol agent according to claim 1, in, 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, etc., preferably 3:

2.

7. The biocontrol agent according to claim 1, in, The auxiliary emulsifier includes but is not limited to isopropanol and / or glycerol, preferably isopropanol; the content of the auxiliary emulsifier is 0.5%-8.0%, preferably 2.0%-5.0%.

8. The biocontrol agent according to claim 1, in, The solvent includes but is not limited to one or more of xylene, sterile water and / or methanol, preferably methanol; the content of the solvent is 0.5%-8.0%, preferably 2.0%-5.0%.

9. Use of the biocontrol agent according to claim 1 in controlling Solanaceae plants, wherein the biocontrol agent has at least one of the following effects: Inhibit plant growth; Promote leaf necrosis and shedding; Kill plants.

10. The Solanaceae plant as claimed in claim 9 includes but is not limited to one or more of Solanum calycifolium, Solanum sibiricum, Solanum sparsely spinulosum, Solanum nigrum and / or Solanum spicatum, preferably Solanum nigrum.

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