A preparation of beaaveria bassiana for prevention and control of western flower thrips and application thereof

Abstract

The present application relates to the field of agricultural microbial engineering and preparation processing, and discloses a Beauveria bassiana preparation for preventing and controlling Frankliniella occidentalis and application thereof.The steps are as follows: a crude drug dry powder is prepared by collecting the strain CGMCC 0877 through large-scale fermentation;5-15 parts of mixed emulsifiers, 2-3 parts of organic bentonite, 1-3 parts of humic acid coated nano titanium dioxide and 2-8 parts of trehalose are sequentially added in the remaining methyl oleate to preliminarily construct a three-dimensional thixotropic network;5-15 parts of the crude drug dry powder are connected for pre-dispersion, and the product biological oil suspension agent is obtained through temperature control continuous sanding refinement and homogenization curing;finally, the product biological oil suspension agent is diluted and sprayed.The present application improves the phenomena that the living preparation is prone to agglomeration, stratification and stress inactivation by constructing a thixotropic skeleton and a stress resistance protection barrier, and improves the physical stability of the preparation and the pest mortality rate.

Description

Technical Field

[0001] This invention relates to the fields of agricultural microbial engineering and biological pesticide formulation processing technology, specifically, to a Beauveria bassiana formulation for the control of western flower thrips and its application. Background Technology

[0002] With the development of modern green agriculture, the use of Beauveria bassiana to control western flower thrips has become an important biological control method. In the complex natural environment of the field, biological fungal preparations not only need to have high pathogenicity against the target pests, but also need to have excellent stress resistance and physical suspension stability to ensure long-term colonization and infection effects after application.

[0003] However, when traditional fungal oil suspensions are formulated with excipients such as UV protectants or dehydration resistant agents to enhance environmental tolerance, inorganic powders and live spores are prone to aggregation in the oil phase carrier, leading to microscopic stratification and flocculation of the system. This makes it difficult for the pesticide solution to effectively adhere to and spread on the hydrophobic body surface of pests. Furthermore, this multiphase live formulation has a narrow processing and application window: improper mechanical shearing and heat generation control during the sand milling and homogenization stages can easily damage and inactivate fragile live spores; during field spraying, if the formulation's rheological network is unstable, it is easily washed away by rainwater and subjected to strong light radiation, causing rapid attenuation of the strain. These problems—impaired biological activity, system degradation due to sedimentation, and unstable field efficacy—limit its application under harsh agricultural conditions. Summary of the Invention

[0004] In view of the aforementioned existing problems, the present invention is proposed.

[0005] Therefore, this invention provides a method for preparing a Beauveria bassiana formulation for the control of western flower thrips, solving a common technical problem faced in the fields of agricultural microbial engineering and biopesticide formulation processing.

[0006] To solve the above-mentioned technical problems, the present invention provides the following technical solution: This invention provides a method for preparing a Beauveria bassiana preparation for the control of western flower thrips, comprising the following steps: S1 extract was used to activate the highly virulent strain CGMCC 0877 of Beauveria bassiana selected from western flower thrips. The strain was then inoculated into a secondary seed culture medium and cultured for 40-55 hours until the late logarithmic growth stage. Subsequently, it was transferred to the main fermenter at a volume inoculation rate of 10%. The fermentation temperature and sterile air circulation were maintained, and large-scale fermentation was carried out at a main stirrer speed of 200-250 rpm. This ensured efficient gas-liquid dissolved oxygen mass transfer while controlling mechanical shearing damage to the mycelial skeleton. After fermentation, the strain was harvested, dehydrated, and dried at low temperature to obtain a high-activity technical powder. S2, by mass, adds the remaining methyl oleate as an oil phase carrier to the mixing vessel. While maintaining stirring, slowly add 5-15 parts of a mixed emulsifier composed of alkyl glycosides and castor oil polyoxyethylene ether to fully dissolve it and reduce the interfacial tension of the system. While maintaining the shear state, uniformly sieve in 2-3 parts of organic bentonite to initially construct a three-dimensional thixotropic anti-settling network. Then, sequentially add 1-3 parts of humic acid-coated nano-titanium dioxide and 2-8 parts of trehalose, relying on viscosity to support them to be uniformly suspended in the rheological network. S3. Add 5-15 parts of the high-activity active ingredient dry powder to the rheological system, and after pre-dispersion, pump it into a horizontal sand mill for continuous refining to control the thermal stress damage to the live spores caused by frictional heat generation of the grinding machinery. S4 introduces the finely ground material into a homogenizing device for homogenization and maturation, overcoming the van der Waals forces between powders, promoting the full development and shaping of the anti-settling rheological network structure, and producing a bio-oil suspension.

[0007] As a preferred embodiment of the preparation method of Beauveria bassiana preparation for the control of western flower thrips according to the present invention, in step S1, the fermentation temperature is 24-28℃ and the sterile air ventilation rate is 0.6-1 vvm.

[0008] As a preferred embodiment of the preparation method of Beauveria bassiana preparation for the control of western flower thrips according to the present invention, in step S1, the culture time in which the inoculated seed culture medium is 48 hours, and the main stirrer speed during the fermentation stage is 230 rpm.

[0009] In a preferred embodiment of the preparation method of the Beauveria bassiana preparation for the control of western flower thrips according to the present invention, in step S2, the amount of the mixed emulsifier added is 9 parts, the amount of the organic bentonite added is 2.5 parts, the amount of the humic acid-coated nano-titanium dioxide added is 2 parts, and the amount of the trehalose added is 5 parts; wherein, the mass ratio of the organic bentonite to the mixed emulsifier is 5:18.

[0010] In a preferred embodiment of the preparation method of Beauveria bassiana preparation for the control of western flower thrips according to the present invention, in step S3, the pre-dispersion speed is 2000-3000 rpm and the time is 15-25 minutes; preferably, the pre-dispersion speed is 2500 rpm and the time is 20 minutes.

[0011] As a preferred embodiment of the preparation method of Beauveria bassiana preparation for the control of western flower thrips according to the present invention, in step S3, the grinding speed of the horizontal sand mill is 1500-1800 rpm, and the cooling fluid circulation flow rate of the sand mill is 4-6 m³ / h.

[0012] In a preferred embodiment of the preparation method of Beauveria bassiana preparation for the control of western flower thrips according to the present invention, in step S3, the grinding speed of the horizontal sand mill is 1650 rpm, and the cooling fluid circulation flow rate of the sand mill is 5 m³ / h.

[0013] In a preferred embodiment of the preparation method of Beauveria bassiana preparation for the control of western flower thrips according to the present invention, in step S4, the rotation speed of the homogenizing equipment is 800-900 rpm and the time is 20-40 minutes.

[0014] In a preferred embodiment of the preparation method of Beauveria bassiana preparation for the control of western flower thrips according to the present invention, in step S4, the rotation speed of the homogenizing equipment is 850 rpm and the time is 30 minutes.

[0015] This invention also provides a Beauveria bassiana preparation for the control of western flower thrips, prepared by the process described in steps S1 to S4 above. The preparation has methyl oleate as the continuous phase and a three-dimensional thixotropic anti-settling network formed by fully swollen organic bentonite. The humic acid-coated nano-titanium dioxide is uniformly distributed in the system, and its surface humic acid molecules and nano-inorganic core work together to perform the dual functions of chemical photo-sieving and physical photo-shielding, constructing a photoprotective barrier that delays the ultraviolet aging of Beauveria bassiana spores. The trehalose and the flexible bentonite network structure work together to provide osmotic pressure physiological regulation and physical buffer space for live spores, constructing a stable and compatible microenvironment that reduces mechanical friction damage and effectively blocks the aggregation and stratification of multiphase particles.

[0016] The beneficial effects of this invention are as follows: A homogeneous suspension system constructed using humic acid-coated nano-titanium dioxide as the core for UV protection, supplemented by trehalose osmotic regulation and an organic bentonite thixotropic network, significantly improves the microscopic compatibility and overall environmental resistance of the formulation while maintaining the high virulence and infection characteristics of the strain. It overcomes the limitations of traditional multiphase live biological agents, which are prone to aggregation and stratification and rapid inactivation under environmental stress. This allows the bio-oil suspension to resist mechanical shear and thermal stress damage during high-intensity processing and grinding, and to exhibit suitable light shielding and physiological buffering capabilities when applied in the field to strong UV radiation or alternating drought conditions, thus broadening its application window in harsh agricultural environments. Furthermore, the three-dimensional thixotropic anti-settling network effectively slows down the aggregation and precipitation of powder particles, and the optimized gradient wetting pre-construction and steady-state homogenization process preserves the integrity of the microscopic rheological framework, resulting in better physical suspension stability and field target lethality of the control agent during long-term storage and practical application. Attached Figure Description

[0017] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the following description of the embodiments will be briefly introduced. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1a The graph shows the effect of the mass ratio of mixed emulsifier / organic bentonite on the suspension rate.

[0019] Figure 1b The graph shows the optimization effect of the mass ratio of mixed emulsifier / organic bentonite on the oil separation rate after standing.

[0020] Figure 1c The graph shows the effect of the mass ratio of mixed emulsifier / organo-bentonite on apparent viscosity.

[0021] Figure 2 Optimization diagram of the formulation for humic acid-coated nano-titanium dioxide as a UV protectant.

[0022] Figure 3 The diagram shows the optimized ratio of trehalose, a spore stress protectant.

[0023] Figure 4 This is a diagram showing the optimal culture time and process optimization for secondary seed culture.

[0024] Figure 5 This is a process optimization diagram for the agitator speed during the fermentation stage.

[0025] Figure 6 Optimization diagram of milling speed for processing bio-oil suspensions.

[0026] Figure 7 Optimization diagram for grinding cooling fluid temperature control system.

[0027] Figure 8 This is an optimized diagram of the homogenizer speed system.

[0028] Figure 9 This is a process flow diagram for Beauveria bassiana bio-oil suspension. Detailed Implementation

[0029] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings.

[0030] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0031] Secondly, the term "one embodiment" or "example" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the invention. The appearance of an embodiment in different places in this specification does not necessarily refer to the same embodiment, nor is it a single or selective embodiment that mutually excludes other embodiments.

[0032] Example 1 This embodiment aims to explore a screening system for highly virulent strains of Beauveria bassiana that combines efficient targeted killing and rapid lethality, in order to solve the technical bottlenecks of traditional biological control agents in dealing with western flower thrips, such as slow onset of action, large fluctuations in field control efficacy, and the difficulty for conventional wild strains to form effective infection in a short period of time.

[0033] 1.1 Screening and identification of basic fungal species To identify effective bioactive substances for controlling western flower thrips, a screening experiment of basic fungal species was first conducted.

[0034] Experimental groups: *Beauveria bassiana*; *Metarhizium anisopliae*; *Metarhizium chrysogenum*; *Verticillium chrysogenum*; *Cladosporium flavum*; *Cladosporium pulvinatum*; *Trichoderma harzianum*; *Trichoderma viride*; *Beauveria bassiana*; *Verticillium dahliae*; *Paecilomyces pulvinatum*; *Trichoderma thomsonii*; *Paecilomyces simonii*; *Bacillus lilacinus*; *Trichoderma harzianum*; *Beauveria bassiana*; *Verticillium dahliae*; *Paecilomyces pulvinatum*; *Trichoderma thomsonii*; *Bacillus niger*. According to NY / T 1156.4-2006 standard, tests were conducted in an artificial climate chamber at 25℃ and 90% relative humidity. The cumulative corrected mortality rate (%) on day 7 against second-instar western flower thrips nymphs was measured for each experimental group to assess their basic pathogenicity and direct killing ability against the target pest. According to GB / T 19526.1-2004 standard, after culturing on potato dextrose agar at 25℃ for 14 days, the sporulation rate per unit area (10^8 sporosomes / cm²) was measured to determine the industrial propagation and material supply capacity of the strains in large-scale fermentation production. According to the NY / T 2061.1-2011 standard, the environmental germination rate (%) of spores was measured 24 hours after inoculation under a stress environment with a relative humidity of 75% to evaluate the effectiveness, stability, and survival ability of the microbial material in low humidity field conditions. The raw test data were compiled and compared with the standard data as follows.

[0035] Among them, *Trichoderma harzianum*, *Trichoderma viride*, and *Verticillium dahliae* have high sporulation per unit area, providing a good foundation for industrial propagation. However, their cumulative corrected mortality rate against western flower thrips is extremely low, indicating insufficient pathogenicity and failing to meet the basic toxicity requirements for biocontrol agents as core insecticides. *Metarhizium anisopliae* and *Metarhizium chrysogenum* exhibit excellent lethality, but their environmental germination rate is extremely low, and they are highly sensitive to environmental humidity, posing a risk of spore inactivation and shedding before infection in common natural drying greenhouse applications. *Verticillium chrysogenum* and *Bifidobacterium tumefaciens* have good environmental resistance, but their low sporulation per unit area leads to low efficiency in subsequent technical preparation. *Paecilomyces lilacinus*, *Diplosporium globosum*, and *Metarhizium anisopliae* lack specific enzyme systems for degrading the western flower thrips' body wall, resulting in insufficient effective penetration and substandard cumulative mortality. *Trichoderma thomsonii* and *Paecilomyces cerevisiae* performed mediocrely across all evaluation dimensions. *Bryophytum rosifolium* has good pathogenicity, but its sporulation cycle is long and it is prone to degeneration, limiting its industrialization potential.

[0036] Beauveria bassiana exhibits excellent overall performance, demonstrating well-balanced characteristics without any obvious weaknesses. It possesses high lethal toxicity, high industrial propagation potential, and excellent adaptability to drought stress, thus it was chosen as a substrate species for development. Based on this, targeted screening experiments for high toxicity of single strains within the wild population of Beauveria bassiana will continue.

[0037] 1.2 Screening and identification of Beauveria bassiana strains to be tested To obtain specific production strains that exhibit high pathogenicity and rapid infectivity against western flower thrips, the next step is to conduct screening and identification experiments for the Beauveria bassiana test strains.

[0038] Experimental groups: Commonly available Beauveria bassiana strains and those published by relevant preservation centers were selected, namely GHA strain; ATCC 74040 strain; Z-77 strain; ACCC 30263 strain; Bb-208 strain; Vb-113 strain; Bb-102 strain; and CGMCC 0877 strain. A total of eight test groups were formed.

[0039] Indoor toxicity testing was conducted according to the NY / T 1156.4-2006 standard. Considering that ambient temperature and humidity are key factors determining whether fungal spores can successfully attach to and germinate on the surface of pests and penetrate their body walls, the entire experiment was conducted in an artificial climate chamber at 25℃ and 90% relative humidity. A uniform concentration (1.0 × 10^7 spores / mL) of spore suspension was sprayed evenly onto petri dishes lined with green bean leaves using a Porter spray tower. Healthy second-instar western flower thrips nymphs were then inoculated, and the cumulative corrected mortality rate (%) on day 7 was measured to assess its basic pathogenicity against the target pests. Daily observations were recorded, and the median lethal time (d) required to kill half of the test pests was calculated to determine its infection and mortality rates. According to GB / T 19526.1-2004 standard, after culturing on potato dextrose agar at 25℃ for 14 days, the sporulation rate per unit area (10^8 sporosomes / cm2) was scraped and measured to evaluate the material conversion rate of this strain in large-scale fermentation production. The raw test data were then compiled into standardized data comparison results as follows.

[0040] Among them, strains GHA and ACCC 30263 have high sporulation per unit area, indicating a good foundation for propagation and production. However, their cumulative corrected mortality rate is low, their half-lethal time is long, and their pathogenicity is weak, failing to meet the toxicity requirements for biocontrol agents as core insecticide active ingredients. Strains ATCC 74040 and Z-77 have high cumulative corrected mortality rates and good targeted lethality, but their sporulation per unit area is low, resulting in weak industrial fermentation efficiency and failing to meet the material supply requirements for large-scale pharmaceutical manufacturing. Strains Bb-208 and Vb-113 have short half-lethal times and good infection rates, but their cumulative corrected mortality rate is low, resulting in weak overall control efficacy and a risk of ineffective target control in many high insect population density application scenarios. Strain Bb-102 has a certain sporulation base, but its cumulative corrected mortality rate is also low.

[0041] The CGMCC 0877 strain exhibits excellent overall performance and demonstrates a balanced characteristic without any obvious weaknesses, thus it has been established as the core highly virulent Beauveria bassiana strain.

[0042] 1.3 Screening and Determination of Culture Medium Formulation After establishing Beauveria bassiana as the basic species for development, in order to ensure that the fungus can achieve good growth status and sporulation efficiency in subsequent experiments and large-scale propagation, screening experiments of different culture medium formulations were carried out.

[0043] Experimental groups: Potato dextrose agar; Sassirer's medium; Czapek's medium; Oat agar; Corn flour agar; Nutrient agar; Sorghum extract medium; Mixed potato medium. According to NY / T1736.5-2009 standard, under constant temperature and dark incubation at 25℃, 5 μL of Beauveria bassiana spore suspension of the same concentration (1.0 × 10^7 spores / mL) was inoculated into the center of each group's medium. The colony diameter (mm) was measured after 7 days of incubation to evaluate the medium's ability to promote mycelial vegetative growth. According to GB / T 19526.1-2004 standard, after 14 days of incubation at 25℃ on each medium, spore production per unit area (10^8 spores / cm2) was measured to determine the reproductive transformation efficiency of the medium in large-scale fermentation production. According to the NY / T 2061.1-2011 standard, newly generated spores on each culture medium were collected, prepared into suspensions, and dropped onto water agar plates. After incubation at 25℃ for 24 hours, the spore germination rate (%) was measured under a microscope to assess the physiological viability and initial infection potential of the progeny spores bred in the culture medium. The original test data were then compiled into standardized data comparison results, as shown below.

[0044] Among the tested media, nutrient agar and Czapek's agar showed small colony diameters and insufficient nutrient supply, failing to meet the basic growth requirements for the rapid propagation of *Beauveria bassiana*. Oat agar exhibited the largest colony diameter, significantly promoting mycelial vegetative growth; however, its sporulation yield per unit area was low, indicating that its nutrients more readily guide the fungus towards lateral spread rather than the reproductive stage to produce large quantities of spores, making it unsuitable as a carrier for obtaining large quantities of biological active ingredients. Sacchariform agar and corn flour agar showed mediocre performance in all tests, lacking significant propagation advantages.

[0045] The potato culture medium exhibits a balanced characteristic without any obvious shortcomings, and can take into account both mycelial expansion and efficient reproductive transformation. Therefore, it is established as the basic culture medium for subsequent targeted screening of highly virulent single plants and large-scale propagation in this invention.

[0046] 1.4 Screening and Determination of Green and Environmentally Friendly Oil Phase Carriers After establishing the basic culture medium and obtaining sufficient live Beauveria bassiana spores, in order to process them into an oil suspension that is easy to store for a long time and can adhere tightly to the surface of western flower thrips and crop leaves, a screening experiment for green and environmentally friendly oil-phase carriers was carried out.

[0047] Experimental groups: liquid paraffin; soybean oil; corn oil; sunflower oil; palm oil; rosin-based vegetable oil; soybean methyl ester; methyl oleate. According to GB / T 265-1988 standard, the kinematic viscosity (mm² / s) of each oil phase carrier was measured using a capillary viscometer at 25℃ to evaluate its flow resistance during sand milling and its atomization potential during field spraying. According to GB / T 30693-2014 standard, the wetting contact angle (°) of each oil phase on the surface of green bean leaves was measured using a contact angle meter to determine the rapid spreading and penetration ability of the pesticide solution after contacting the target plant or insect body wall. Live Beauveria bassiana spores were suspended in the above-mentioned sterile, dehydrated oil phases, sealed, and stored in a cool, dark place at 25℃. After 6 months, samples were taken and the spore survival rate (%) was measured on water agar plates to evaluate the long-term biosafety of the carrier to fragile microscopic organisms. The raw test data were compiled into standardized data comparison results as follows.

[0048] Common plant oils such as palm oil, soybean oil, corn oil, and sunflower oil showed good biocompatibility with the strains, but their high kinematic viscosity made them prone to overheating and killing the live bacteria in a sand mill due to excessive friction. Furthermore, they were difficult to break into fine droplets during spraying, and their large wetting contact angle prevented rapid film formation on the target surface. Liquid paraffin had good biostability and moderate viscosity, but its extremely large wetting contact angle caused the solution to easily condense into water droplets on leaves and slide off, lacking the ability to adhere and penetrate to pests in hidden areas. Pine resin-based plant oils had excellent spreading ability, but some of their volatile terpenes were toxic to Beauveria bassiana spores, resulting in extremely low spore survival rates and making them unsuitable as a long-term protective carrier for the live bacteria. Soybean oil methyl ester showed good overall performance, but there was room for improvement in all indicators.

[0049] Methyl oleate exhibits highly compatible physical and biological synergistic properties, thus establishing it as the core green and environmentally friendly oil phase carrier of the oil suspension of this invention.

[0050] 1.5 Screening and Determination of Emulsifiers To ensure that the formulation can be rapidly emulsified and dispersed after dilution with water, maintain physical stability during the storage period of the original drug without oil-water separation, and not cause chemical toxicity to the fragile live spores, single-agent screening and compound experiments of emulsifiers will be carried out next.

[0051] Experimental groups: Sodium dodecyl sulfate (SDS); Agricultural emulsion 500# (calcium dodecylbenzenesulfonate); Polyethylene glycol 400 (PEG-400); Fatty alcohol polyoxyethylene ether (AEO-7); Tween-80; Span-80; Agricultural emulsion 700#; Alkyl glycoside (APG); Castor oil polyoxyethylene ether (EL); Agricultural emulsion 500# and Tween-80 compound (mass ratio 1:1); Alkyl glycoside and Tween-80 compound (mass ratio 1:1); Alkyl glycoside and castor oil polyoxyethylene ether compound (mass ratio 1:1). A total of twelve test groups were established.

[0052] According to GB / T 14825-2006 standard, the oil suspensions prepared in each experimental group were diluted 200 times in standard hard water at 30℃. After standing for 1 hour, the suspension rate (%) of the active ingredient was measured to evaluate the dispersion stability of the solution in the field spraying container and avoid uneven spraying concentration due to sedimentation. Considering that the high temperature in conventional heat storage experiments would directly lead to the death of viable bacteria, this experiment adopted the centrifugation accelerated sedimentation method. The oil suspension was placed in a centrifuge and centrifuged at 3000 r / min for 15 minutes, and its centrifugal oil separation rate (%) was measured to determine the physical anti-stratification ability of the product during long-term static storage. According to NY / T 2061.1-2011 standard, each emulsifier was prepared into an aqueous solution equivalent to the field application concentration. After adding Beauveria bassiana spores for suspension treatment for 24 hours, the spore germination rate (%) was measured under a microscope to evaluate the degree of chemical stress of surfactants on the microscopic cell wall and biocompatibility. After obtaining the original test data, the data comparison results were compiled into standard data as follows.

[0053] While polyethylene glycol 400 (PEG-400) exhibits excellent biocompatibility, its hydrophilic-lipophilic balance is not well-suited to current oil-phase systems, resulting in low suspension and oil separation. Alkyl glycosides (APGs), due to their green nature derived from natural plant extracts, demonstrate high biocompatibility, but their ability to construct emulsion systems independently is limited. Castor oil polyoxyethylene ethers (ELs) exhibit good physical dispersibility, but their biocompatibility is slightly insufficient. In the compound group, the compound containing agricultural emulsion 500# still exhibits high biotoxicity. The compound of alkyl glycosides and Tween-80 shows good performance across the board, but a small amount of oil still separates under extreme centrifugation.

[0054] The compound of alkyl glycoside and castor oil polyoxyethylene ether (mass ratio 1:1) exhibits balanced characteristics without any obvious shortcomings, and is therefore established as the core emulsifier of this invention.

[0055] 1.6 Screening and Determination of UV Protectants Considering that the strong ultraviolet radiation from sunlight in the field application environment can quickly penetrate and destroy the cellular nucleic acid of Beauveria bassiana spores, causing a large number of living particles to die and fall off before contacting and infecting pests, the next step is to conduct a screening and determination experiment for UV-protective agents.

[0056] Experimental groups: nano-titanium dioxide; nano-zinc oxide; nano-silica; benzophenone-based UV absorber (UV-531); benzotriazole-based UV absorber (UV-328); phenyl salicylate; lignin-modified nano-silica; polyethylene glycol-modified nano-zinc oxide; chitosan-coated nano-titanium dioxide; humic acid-coated nano-titanium dioxide. A total of ten test groups were established.

[0057] According to GB / T 31899-2015 standard, the average transmittance (%) of each UV-blocking agent in the UV region (280nm~400nm) of the oil-phase system was determined using a UV-Vis spectrophotometer to evaluate its direct physical blocking or absorption capacity against UV radiation. According to GB / T 23762-2009 standard, the relative value (%) of photocatalytically generated reactive oxygen species (ROS) in the system was determined under simulated strong light irradiation (using the blank group without added protective agents as a baseline) to determine whether inorganic nanoparticles will generate highly oxidizing free radicals after absorbing light energy, thereby causing secondary oxidative damage to the spore cell wall. According to GB / T2423.24-2013, the standard for simulated ground solar radiation testing, suspensions containing spores and various groups of UV-protective agents were coated onto glass slides and continuously irradiated in a UV radiation chamber for 72 hours. After elution, the spore survival rate (%) was measured under a microscope to comprehensively evaluate the actual protective efficacy of the agents against microscopic organisms under extreme light pressure. The results of the original test data comparison are as follows.

[0058] Among them, nano-titanium dioxide and nano-zinc oxide have low average transmittance in the ultraviolet region, providing good physical shielding ability. However, their photocatalytic reactive oxygen species (ROS) production is relatively high, easily causing oxidative damage and failing to meet the basic biosafety requirements for Beauveria bassiana spores as a core active ingredient. Nano-silica has low photocatalytic activity but high average transmittance in the ultraviolet region, resulting in weak UV shielding ability and failing to meet the requirements for protection against strong light radiation. Phenyl salicylate has low photocatalytic activity, but its biosafety is also low. Lignin-modified nano-silica and polyethylene glycol-modified nano-zinc oxide provide a certain organic protective layer, but high ROS leakage leads to oxidative damage to spores and a decrease in survival rate. Chitosan-coated nano-titanium dioxide has good physical shielding ability, but its coating layer has weak stability and is sensitive to strong light. Under prolonged irradiation, the coating is prone to degradation, leading to reduced protective performance.

[0059] Humic acid-coated nano-titanium dioxide exhibits excellent overall performance and demonstrates balanced characteristics without any obvious shortcomings, thus establishing it as the core UV-protective agent.

[0060] 1.7 Screening and Determination of UV Protectants In order to impart good physical anti-settling properties to oil suspensions and give materials ideal rheological flow characteristics, and to prevent solid-liquid separation during long-term standing, the following experiment was conducted to screen and determine thickening and anti-settling agents.

[0061] Experimental groups: Attapulgite; Fumed silica; Kaolin; Aluminum stearate; Sodium carboxymethyl cellulose (CMC-Na); Xanthan gum; Polyvinyl alcohol (PVA); Dodecyl dimethyl benzyl ammonium chloride modified bentonite; Hexadecyl trimethyl ammonium bromide modified bentonite; Organobentonite. A total of ten test groups were established.

[0062] According to GB / T 22427.7-2008, the apparent viscosity (mPa·s) of each component after addition to the oil phase system at a shear rate of 20 s⁻¹ was determined using a rotational viscometer to evaluate its initial thickening and viscousing ability. According to GB / T 13531.4-2013, the samples were left to stand at room temperature for 30 days, and the percentage of oil separation at rest was calculated by measuring the height of the top oil layer to evaluate the static physical stability of the product during its shelf life. According to NY / T 2061.1-2011, the viable cell counts of each suspension system before and after the fine grinding process in a sand mill were determined, and the spore mechanical damage inactivation rate (%) during processing was calculated to evaluate the impact of viscosity fluctuations caused by rheology modifiers on spore shear tolerance. The results of the standard data comparison after obtaining the original test data are as follows.

[0063] Xanthan gum and sodium carboxymethyl cellulose (CMC-Na) exhibit low spore mechanical damage inactivation rates and good processing safety, but their low apparent viscosity and weak thickening ability fail to meet the basic anti-settling requirements for oil suspensions as stable systems. Fumed silica has a high apparent viscosity, but its high spore mechanical damage inactivation rate does not meet its processing survival rate requirements. Kaolin has a low apparent viscosity and weak anti-settling properties, posing a risk of oil separation in many long-term storage applications. Polyvinyl alcohol (PVA) has good low-damage characteristics, but its apparent viscosity is also low. Dodecyl dimethyl benzyl ammonium chloride-modified bentonite has high apparent viscosity and anti-oil separation properties, but its high shear heat generation leads to localized temperature increases and a decrease in spore survival rate. Hexadecyl trimethyl ammonium bromide-modified bentonite has a high apparent viscosity, but its high damage inactivation rate makes it sensitive to high-intensity shear forces, leading to structural damage under prolonged grinding and reduced rheological properties.

[0064] Organic bentonite exhibits excellent overall performance and demonstrates balanced characteristics without any obvious weaknesses, thus it has been established as the core thickening and anti-settling agent.

[0065] 1.8 Screening and Determination of Spore Stress Protection Agents Considering the high temperature and heat generated during long-term storage of the formulation, and the fact that the drought and low humidity environment encountered after spraying in the field would cause a large amount of Beauveria bassiana live spores to lose water and become inactive, the next step is to conduct a screening and determination experiment for spore stress protection agents.

[0066] Experimental groups: Glycerin; Polyethylene Glycol 8000 (PEG-8000); Skim Milk Powder; Maltodextrin; Ascorbic Acid (Vitamin C); Sodium Alginate; Proline; Trehalose; Betaine; Trehalose and Betaine Complex (mass ratio 1:1). A total of ten test groups were established. According to GB / T 19136-2021, the formulations of each experimental group were sealed and placed in a constant temperature incubator at 54±2℃ for heat storage testing. After 14 days, the heat storage survival rate (%) was measured to evaluate the heat-resistant degradation ability of the protectant under high-temperature storage conditions in summer. According to NY / T 2061.1-2011, the spore suspensions of each group were inoculated onto water agar plates and placed in an artificial climate chamber with a relative humidity of only 50% for 24 hours under stress. The low-humidity germination rate (%) was measured to determine its water retention and osmotic pressure resistance under arid field conditions. Simultaneously, the initial germination rate (%) was measured under a standard suitable environment of 25℃ and 90% relative humidity to assess the physiological compatibility of the protectant with live spores and whether it would cause respiratory inhibition. The raw test data were then compiled into standardized data comparison results as follows.

[0067] Comparative analysis of the above data shows that glycerol has a high low-humidity germination rate and good water retention and drought resistance, but its heat storage survival rate is low and its heat resistance is insufficient, failing to meet the basic survival requirements of biological agents as live commodities in high-temperature storage. Polyethylene glycol 8000 (PEG-8000) has good heat resistance, but its low low-humidity germination rate does not meet its field drought resistance requirements. Ascorbic acid has some resistance to high-temperature oxidation, but its low low-humidity germination rate is also low. Sodium alginate has some physical film-forming and water-retaining properties, but its low initial germination rate leads to respiratory inhibition and reduced infection capacity on the target surface. Proline has a good initial germination rate, but its heat storage survival rate is low; it is sensitive to sustained high temperatures and undergoes structural degradation during long-term storage, resulting in reduced protective performance.

[0068] Trehalose exhibits excellent overall performance and demonstrates a balanced characteristic without any obvious weaknesses, thus establishing it as a spore stress protectant.

[0069] Example 2 Referring to the accompanying drawings, this is the second embodiment of the present invention. This embodiment aims to optimize the quantitative proportions of the components screened and determined in Example 1 through a systematic matrix experimental design. Although the functionality of each individual component has been confirmed, the final comprehensive performance of the bio-oil suspension is not a simple summation of the effects of the excipients and the active ingredient. Rather, it depends on the dynamic balance between the adsorption force at the oil-water-solid three-phase interface and the suspension state of the microscopic spore particles in the dispersion environment of sand milling and water dilution, as well as the synergistic regulatory effect of the mass ratio of each component on the long-term physical anti-settling ability of the formulation, the survival rate of live microparticles under stress, and the field targeted contact infection control efficacy.

[0070] 2.1 Quantitative optimization of the ratio of mixed emulsifier and organic bentonite. Using 100 parts by weight of the final oil suspension as a baseline, this study aimed to investigate the performance boundaries of interfacial active molecules and rheologically modified networks under extreme formulation conditions during water dilution and long-term static storage. It also aimed to provide comparative data for establishing the physical stability and processing rheological range of the formulation, demonstrating the synergistic regulatory effect of the mixed emulsifier reducing interfacial tension and the organobentonite constructing a spatial thixotropic network structure. Matrix experiments were conducted with mixed emulsifier (alkyl glycoside and castor oil polyoxyethylene ether compound) in 1.0% increments from 5.0% to 12.0% and organobentonite in 0.5% increments from 1.0% to 4.5%. The appropriate process formulation was determined by evaluating parameters such as suspension rate, oil separation rate during static storage, and apparent viscosity.

[0071] Test results are as follows Figure 1a As shown in -c, when the amount of mixed emulsifier is fixed, the suspension rate shows a continuous upward trend with the increase of organobentonite content. When the amount of organobentonite is fixed, the suspension rate also shows an overall upward trend with the increase of mixed emulsifier content, then tends to level off after the mixed emulsifier content exceeds 9.0% and the bentonite content exceeds 2.5%. Relatively high values ​​appear in the range of high emulsifier and high bentonite content. The oil separation rate after standing shows a non-linear decreasing trend with the increase of organobentonite content, showing a significant convergence and stabilization when the bentonite content reaches around 2.5%. With the increase of mixed emulsifier content, the oil separation rate after standing shows a gradual decreasing trend. Lower values ​​appear in the range of high bentonite and medium-high emulsifier content. When the amount of mixed emulsifier is fixed, the apparent viscosity shows an approximately linear steep increase with the increase of organobentonite content. With the increase of mixed emulsifier content, the apparent viscosity also shows a gradual slight increase. High-viscosity regions that are difficult to process due to a sharp increase in shear friction resistance caused by excessively strong rheological networks appear in the range of extremely high bentonite ratios.

[0072] By normalizing the data of the three key indicators mentioned above and then performing weighted calculations, the dispersion, anti-settling, and rheological equilibrium points under the interaction of the oil-water-solid three-phase interface were simulated and searched. The theoretical equilibrium point for the comprehensive physical properties of the formulation was found to be a mixture of emulsifier and organobentonite of 9.15% and 2.46%. Considering the accuracy of feed control and batch stability for industrial-scale liquid preparation, a fixed ratio close to the theoretical point was finally selected as the process formulation, namely, a mixture of emulsifier of 9.0% and organobentonite of 2.5%.

[0073] 2.2 Optimization of UV protection agent dosage ratio To investigate the dynamic balance between physical shielding efficacy and photocatalytic oxidative stress of UV protectants at different concentrations, and to provide data support for ensuring the survival rate of Beauveria bassiana spores under strong field light conditions, a single-factor gradient experiment was conducted with UV protectant concentrations ranging from 0.5% to 4.0% in 0.5% increments, resulting in a total of 8 experimental groups.

[0074] According to GB / T 31899-2015 standard, the average transmittance (%) of each group of samples in the ultraviolet region (280nm~400nm) of the oil phase system was determined using a UV-Vis spectrophotometer to evaluate the direct physical blocking ability of concentration changes on ultraviolet radiation. According to GB / T 23762-2009 standard, the relative value (%) of photocatalytically generated reactive oxygen species in the system was determined under simulated strong light continuous irradiation to monitor the potential oxidative stress risk associated with increasing nanomaterial concentration. According to GB / T2423.24-2013 standard, each group of samples was coated on a glass slide, placed in an ultraviolet radiation chamber for continuous irradiation for 72 hours, and then eluted and the spore survival rate (%) was determined to comprehensively evaluate the actual protective efficacy of different formulations under strong light conditions.

[0075] Test results are as follows Figure 2 As shown, the heat storage survival rate and low-humidity germination rate were both low in the low addition range, failing to form a complete physical and physiological protective layer against stress, and not meeting the basic survival requirements of biological agents under high-temperature storage and drought conditions. In the high addition range, with the continuous increase of trehalose concentration, the heat storage survival rate and low-humidity germination rate did not show a significant increase, and even showed a slight decline. Excessively high concentrations of sugars can lead to high local osmotic pressure in the system's microenvironment, easily causing additional physiological osmotic stress on spore cells, resulting in reverse water loss, and also unnecessarily increasing the material cost of the formulation.

[0076] By normalizing the three performance indicators and performing weighted fitting analysis, the theoretical comprehensive equilibrium point was finally calculated to be 2.08%. Considering the accuracy tolerance and batch stability of the weighing system during industrial-scale liquid preparation and feeding, a value that is close to the theoretical equilibrium point and easy to operate was finally selected, namely, the determined dosage of humic acid-coated nano-titanium dioxide is 2.0%.

[0077] 2.3 Optimization of the dosage ratio of spore stress protectant Using 100 parts by weight of the final oil suspension as a baseline, and under the premise of keeping the optimal ratio of the mixed emulsifier (9.0%), organobentonite (2.5%), and UV protectant (2.0%) obtained in the above experiments constant, a single-factor optimization experiment was conducted on the ratio of the spore stress protectant (trehalose). In order to study the protective efficacy of the stress protectant against high-temperature storage decay and field drought stress at different addition concentrations, as well as the potential osmotic pressure effect of its own concentration on spore physiological activity, a single-factor gradient experiment of trehalose was set up with a step size of 1.0% from 1.0% to 8.0%, for a total of 8 experimental groups.

[0078] According to GB / T 19136-2021 standard, the formulations of each experimental group were sealed and placed in a constant temperature incubator at 54±2℃ for heat storage testing. After 14 days, the formulations were removed and their heat storage survival rate (%) was measured to evaluate the high-temperature degradation resistance of the protectant at different concentrations. According to NY / T 2061.1-2011 standard, the spore suspensions of each group were inoculated on water agar plates and placed in an artificial climate chamber with a relative humidity of only 50% for 24 hours of stress culture. The low-humidity germination rate (%) was measured to determine its field water retention and osmotic pressure resistance under different ratios. At the same time, the formulations were cultured under a standard suitable environment of 25℃ and 90% relative humidity, and the initial germination rate (%) was measured to assess whether the concentration of the protectant itself would cause physiological osmotic stress and respiration inhibition in live spores.

[0079] Test results are as follows Figure 3 As shown, the experimental groups with low addition levels exhibited low survival rates during heat storage and low-humidity germination rates, failing to form a complete physical and physiological protective layer against stress, thus not meeting the basic protection requirements for biological agents under high-temperature storage and drought conditions. Excessively high concentrations of sugars can lead to excessively high local osmotic pressure within the system's microenvironment, easily causing additional physiological osmotic stress on spore cells, resulting in reverse water loss and inhibition of normal germination. Through normalization and weighted fitting analysis of the above three performance indicators, the theoretical comprehensive equilibrium point was finally calculated to be 4.86%. Considering the accuracy tolerance and batch stability of the weighing system during industrial-scale liquid preparation and feeding, a value close to this theoretical equilibrium point and easily operable was ultimately selected, namely, the determined dosage of trehalose was 5.0%.

[0080] Example 3 Referring to the accompanying drawings, this is the third embodiment of the present invention. This embodiment aims to extend the core strain identified in Example 1 and the optimal formulation ratio determined in Example 2 to a pilot-scale scale. Considering that in the dynamic processing of continuous industrial production, the limitations of dissolved oxygen mass transfer and nutrient gradient shear in large-volume fermenters, as well as the high-frequency physical collisions and heat generation effects inside the continuous sand milling chamber, will cause significant differences between the viable sporulation quality of Beauveria bassiana and the microscopic dispersion and rheological state of various adjuvants in the oil phase system and the static microscopic results in the laboratory, dynamic pilot-scale experiments are needed to finely optimize core conditions such as aeration rate and stirring speed during the fermentation stage, and to correct process windows such as sand milling time and temperature and flow rate during the formulation processing stage. This ensures that under large-scale production, both a high concentration and high viability of high-quality spores can be obtained, and a uniform and stable anti-settling spatial network can be stably constructed, thereby comprehensively guaranteeing the high efficacy reproducibility and high consistency of this biological agent in field application.

[0081] 3.1 Gradient wetting and rheological pre-dispersion process for feeding materials The components were pre-dispersed using a gradient wetting and rheological pre-dispersion method. The remaining methyl oleate was added to the mixing vessel, and stirring was started to maintain surface flow. 9.0 parts by weight of a mixed emulsifier composed of alkyl glycosides and castor oil polyoxyethylene ether was slowly added to dissolve it, reducing interfacial tension and improving the powder wetting environment. Maintaining shear conditions, 2.5 parts by weight of organobentonite were uniformly sieved in, allowing it to swell fully and initially construct a three-dimensional thixotropic network. Next, 2.0 parts by weight of humic acid-coated nano-titanium dioxide and 5.0 parts by weight of trehalose were added sequentially. The existing viscosity of the system prevented the modified powder and sugars from settling and ensured uniform suspension. Finally, an appropriate amount of Beauveria bassiana technical was added. The formed flexible network structure provided physical buffering, reducing mechanical friction damage to the live spores. After all materials were added, the rotation speed was increased to 2500 rpm for continuous premixing for 20 minutes to achieve a microscopically uniform dispersion, completing the pre-dispersion step.

[0082] 3.2 Optimal Culture Time and Process Optimization for Secondary Seed Culture To ensure that Beauveria bassiana (CGMCC 0877 strain) can quickly adapt to the environment and enter the logarithmic rapid propagation stage after being inoculated into an industrial-grade pilot fermenter, a single-factor process parameter optimization experiment was conducted to optimize the optimal culture time of the secondary seed culture.

[0083] Considering that the physiological age of the seed culture directly determines its proliferation potential and final sporulation efficiency in the submerged fermentation system, a gradient experiment was set up with secondary seed culture time ranging from 24 hours to 66 hours, with a step size of 6 hours, for a total of 8 experimental groups. The activated primary shake-flask inoculum was inoculated into a secondary fermenter containing a comprehensive potato medium at a fixed volume ratio and cultured with aeration and stirring. After the predetermined time, samples were taken to measure the mycelial dry weight (g / L) to assess the degree of biomass accumulation, and the spore concentration in the system (10^8 spores / mL) was measured to assess its reproductive and developmental stage. Subsequently, the seed culture from each group was transferred to the pilot-scale main fermenter at a uniform inoculation rate of 10%, and the lag time (h), i.e., the time required for the cell growth rate to reach half of its maximum value, was monitored and recorded to determine its adaptation to the new environment and its start-up speed.

[0084] Test results are as follows Figure 4 As shown, in the shorter culture time range, the mycelial dry weight and spore concentration were both low, resulting in insufficient overall biomass. After inoculation into large-volume fermenters, they failed to establish a dominant population, leading to a prolonged lag phase and failing to meet the continuous production requirements for rapid start-up at the pilot-scale level. In the longer culture time range, although the mycelial dry weight and spore concentration reached higher levels, the accumulation of secondary metabolites and excessive consumption of nutrients in the culture medium caused the cells to gradually enter a senescent phase. Cellular physiological activity significantly decreased, resulting in a rebound and significantly prolonged lag phase after readjustment and recovery to a new environment.

[0085] The experimental group with a secondary seed culture time of 48 hours showed good performance, so it was established as the core culture time process parameter.

[0086] 3.3 Optimization of Agitator Speed ​​During Fermentation Stage To ensure the final propagation yield and viability of Beauveria bassiana, the following process parameter optimization experiment was conducted on the stirrer speed during the main fermentation stage. Using the initial effective liquid volume of the pilot-scale fermenter as a baseline, and under the premise of fixing the previously selected secondary seed culture and inoculating it at a 10% inoculum, while maintaining a constant sterile air ventilation rate (e.g., 0.8 vvm) and fermentation temperature (26℃), a single-factor gradient experiment was set up with stirrer speeds ranging from 120 rpm to 330 rpm in 30 rpm increments, for a total of 8 experimental groups.

[0087] The average dissolved oxygen saturation (%) during the logarithmic growth peak was monitored and recorded in real time using an online dissolved oxygen electrode to evaluate the efficiency of gas-liquid phase mixing and mass transfer at different stirring speeds. Before discharge from the fermentation tank at the end of the predetermined cycle (120 hours), samples were taken and the final sporulation yield (10⁻⁶) was determined using a hemocytometer. 8The fermentation broth was analyzed to directly assess the actual production capacity conversion of large-scale fermentation. Simultaneously, a suitable amount of fermentation broth was examined under a microscope and subjected to fluorescence staining to determine the mycelial shear damage rate (%), in order to assess the extent of damage to the fragile mycelial skeleton caused by the high-frequency physical shear force generated by the mechanical agitator impeller.

[0088] Test results are as follows Figure 5 As shown in the comparison and analysis of the above measurement data, it can be seen that the experimental group in the low rotation speed range had a lower mycelial shear damage rate and maintained a relatively complete microstructure. However, its average dissolved oxygen saturation was low, and the gas-liquid mixing was insufficient. This resulted in insufficient dissolved oxygen affecting the normal development of the mycelium during the vigorous aerobic metabolic period, ultimately significantly limiting the sporulation yield. In the experimental group in the high rotation speed range, although the average dissolved oxygen saturation was high and the mass transfer efficiency was high, the increased mechanical shear force easily led to the physical breakage of a large number of mycelia. The mycelial shear damage rate increased significantly, destroying the physiological integrity of the sporulation structure and resulting in a substantial decrease in the final sporulation yield.

[0089] To achieve a balance between gas-liquid dissolved oxygen mass transfer efficiency during peak fermentation, final sporulation yield in large-scale production, and controlling physical damage to the mycelial skeleton caused by mechanical shearing, the three performance indicators were normalized and weighted fitting analysis was performed, yielding a theoretical equilibrium point of 228.5 rpm. Considering the control precision tolerance of the variable frequency motor in industrial-scale fermenters and the mechanical stability during long-term operation, an integer value close to this theoretical equilibrium point and easily operable was selected; that is, the rotation speed of the main agitator in the fermenter was determined to be 230 rpm.

[0090] 3.4 Optimization of Milling Speed ​​in Bio-oil Suspension Process To ensure that the Beauveria bassiana oil suspension achieves a suitable physical dispersion fineness during the sand milling process, while effectively controlling the damage to live spores caused by shearing and frictional heat generated by high-frequency mechanical collisions, the sand milling speed process was optimized.

[0091] The pre-dispersed mixture was pumped into a pilot-scale horizontal sand mill, using zirconia beads of suitable particle size as the grinding media. A single-factor gradient experiment was conducted with mill speeds ranging from 1000 rpm to 2400 rpm in 200 rpm increments, resulting in a total of eight experimental groups. Continuous grinding tests were performed while maintaining constant material feed flow and cooling water circulation parameters. The ground material was extracted, and its average particle size D50 (μm) was measured using a laser particle size analyzer to evaluate the effects of physical pulverization and fine dispersion. An online temperature sensor was used to record the stable outlet temperature (°C) of the material at the mill outlet to monitor heat accumulation caused by high-frequency mechanical friction. Simultaneously, the effluent liquid phase was extracted for viable bacterial plate counting, and the overall spore damage and inactivation rate (%) during the sand milling process was calculated to assess the negative impact of the synergistic effect of shear stress and thermal effects on biological activity.

[0092] Test results are as follows Figure 6 As shown, in the low-speed range, although the outlet temperature is low, avoiding thermal damage, and the overall spore inactivation rate remains at a low level, the average particle size D50 is relatively large, failing to reach the micron-level fineness required for suspension products. This easily leads to product aggregation and physical anti-settling failure during long-term storage. In the high-speed range, the pulverization and finening effect is obvious, and the average particle size is small. However, the high-frequency grinding brings intense mechanical friction, causing the outlet temperature to rise sharply, exceeding the heat tolerance range of Beauveria bassiana. Coupled with strong mechanical shearing, this significantly increases the overall spore inactivation rate, destroying the effective components of the biological agent.

[0093] The three performance indicators were normalized and weighted fitting analysis was performed to calculate the theoretical equilibrium point as 1645 rpm. Considering the control accuracy tolerance and long-term mechanical stability of the motor in industrial-scale sand mills, an integer value that is close to the theoretical equilibrium point and easy to operate was selected, namely, the determined speed of the sand mill is 1650 rpm.

[0094] 3.5 Optimization of Grinding Cooling Fluid Temperature Control System Considering that high-frequency mechanical friction continuously generates a large amount of heat during pilot-scale and industrial-scale continuous grinding processes, if this heat cannot be dissipated in time, the nonlinear increase in the core temperature of the material within the grinding chamber will cause irreversible thermal damage to the heat-sensitive Beauveria bassiana spores. Therefore, process parameter optimization experiments were conducted on the cooling fluid temperature control system of the sand mill.

[0095] Under the premise of fixed material feed flow rate and determined mill speed, the cooling circulating water inlet temperature was kept constant at 10℃. A single-factor gradient experiment was conducted with cooling fluid circulation flow rates ranging from 2.0 m³ / h to 9.0 m³ / h in 1.0 m³ / h increments, resulting in a total of 8 experimental groups for continuous grinding tests. After the equipment ran continuously for 2 hours to reach thermal equilibrium, the stable outlet temperature (℃) of the material was recorded using an online temperature sensor to evaluate the actual efficiency of the cooling system in removing mechanical heat. The relative value (%) of cooling energy consumption was recorded using a flow meter and a water pump power meter to evaluate the industrial operating cost of dynamic temperature control. Simultaneously, the effluent liquid phase was extracted for viable bacterial plate counting, and the spore survival rate (%) during the grinding stage was calculated to determine the impact of comprehensive thermal stress on biological activity.

[0096] Test results are as follows Figure 7As shown, in the low flow rate range, although the cooling energy consumption is relatively low, the system's heat transfer efficiency is insufficient, causing the stable outlet temperature to exceed the suitable tolerance threshold for spores. Live cells experience severe heat accumulation within the cavity, resulting in a significant decrease in spore survival rate and failure to meet the quality requirements of biopesticides. In the high flow rate range, the system's cooling efficiency is high, and the outlet temperature is controlled at a low level, maintaining a high spore survival rate. However, as the flow rate continues to increase, the improvement in survival rate plateaus, while the operating load of the cooling water pump and the relative value of cooling energy consumption show a significant non-linear increase, raising the operating costs of industrial production.

[0097] The three performance indicators were normalized and weighted fitting analysis was performed to calculate the theoretical equilibrium point as 5.15 m³ / h. Considering the control accuracy tolerance and long-term operational stability of the industrial-scale water pump frequency conversion system, an integer value that is close to the theoretical equilibrium point and easy to operate was selected, namely, the cooling fluid circulation flow rate of the sand mill was determined to be 5.0 m³ / h.

[0098] 3.6 Homogenizer Speed ​​System Optimization After completing the sand milling and cooling temperature control processes, in order to ensure that the organic bentonite in the system can be fully peeled and swollen in the oil phase carrier, and to construct a three-dimensional thixotropic anti-settling network with appropriate strength, while avoiding excessive mechanical action from causing secondary damage to the formed rheological skeleton and fragile live spores, a single-factor process parameter optimization experiment was conducted on the homogenizing stirring shear force (characterized by the homogenizer speed) of the material after sand milling.

[0099] Under the premise of a fixed homogenization and maturation time of 30 minutes and maintaining a suitable material temperature, a single-factor gradient experiment was conducted with the homogenizer speed ranging from 500 rpm to 1200 rpm in 100 rpm increments, resulting in a total of 8 experimental groups undergoing continuous testing. After homogenization, samples from each group were extracted and allowed to stand, and their suspension rate (%) was determined according to relevant standards to evaluate the macroscopic physical anti-settling stability of the final formulation. The thixotropic recovery rate (%) of the system after high shear stress was measured using a rotational rheometer to determine the structural toughness and molding quality of the internal spatial network structure. Simultaneously, viable cell plate counting was performed on the extracted liquid phase, and the spore survival rate (%) after the homogenization stage was determined to monitor the final impact of terminal high-frequency shear on biological activity.

[0100] Test results are as follows Figure 8As shown, in the low-speed range, insufficient homogeneous shear force fails to overcome the van der Waals forces between powder particles, resulting in incomplete separation and unfolding of the organic bentonite layers, incomplete development of the rheological network, and low suspension and thixotropic recovery rates. This makes the product prone to stratification during long-term storage. In the high-speed range, excessive mechanical shear force forcibly severs the established hydrogen bonds and intermolecular forces, causing irreversible structural damage to the rheological framework. Suspension and thixotropic recovery rates decline significantly. Simultaneously, the intense end-effector shearing also drastically reduces spore survival rates, affecting the quality requirements of biopesticides.

[0101] To achieve a balance between the microscopic development of the rheological framework, the macroscopic physical anti-settling properties of the product, and the control of damage to live spores caused by end-effector mechanical shearing, the three performance indicators were normalized and weighted fitting analysis was performed, resulting in a theoretical equilibrium point of 842 rpm. Considering the frequency conversion control accuracy and actual operating conditions of industrial-scale homogenizing equipment, an integer value that is close to this theoretical equilibrium point and easy to operate was selected, namely, a homogenizing stirring speed of 850 rpm.

[0102] Example 4 refer to Figure 9 This embodiment aims to fully demonstrate the entire process of preparing Beauveria bassiana bio-oil suspension under a series of suitable ratios and process parameters determined in the early stage, and to comprehensively test the physical stability of its dispersion system, the stress resistance of live spores, and the final lethality against the core target, western flower thrips. To reflect the synergistic effect of the optimized scheme, a comparative ratio was set up for parallel testing.

[0103] The preparation process was strictly carried out according to the optimized parameters. First, the strain underwent fermentation culture. Activated Beauveria bassiana (CGMCC 0877 strain) was inoculated into a secondary seed culture medium and cultured for 48 hours to reach the late logarithmic growth stage. Then, a 10% inoculum was transferred to the pilot-scale main fermenter. The fermentation temperature was controlled at 26℃, the sterile air ventilation rate was maintained at 0.8 vvm, and the main stirrer speed was strictly set at 230 rpm to ensure sufficient gas-liquid dissolved oxygen mass transfer while controlling the physical damage to the mycelial skeleton caused by high-intensity mechanical shearing. After the fermentation cycle, the strain was harvested, dehydrated, and dried at low temperature to obtain highly viable Beauveria bassiana technical powder.

[0104] The process then proceeds to the oil suspension stage. Gradient wetting and rheological pre-mixing methods are used for feeding. The remaining methyl oleate, acting as a carrier, is added to the mixing tank. After stirring, 9.0 parts by weight of a mixed emulsifier of alkyl glycoside and castor oil polyoxyethylene ether is slowly added to dissolve it and reduce interfacial tension. Maintaining shear conditions, 2.5 parts by weight of organobentonite are uniformly sieved in to fully swell, initially constructing a three-dimensional thixotropic network. Next, 2.0 parts by weight of humic acid-coated nano-titanium dioxide with a core-shell double-layer structure and 5.0 parts by weight of trehalose are added sequentially. Finally, 10.0 parts by weight of Beauveria bassiana technical grade powder is added, utilizing the already formed flexible network structure to provide physical buffering. The rotation speed is increased to 2500 rpm for continuous premixing for 20 minutes. After pre-dispersion, the material is pumped into a horizontal sand mill, set at 1650 rpm, and the cooling fluid circulation flow rate is controlled at 5.0 m³ / h to remove mechanical heat. After being finely ground by sand milling, the material is homogenized and matured at 850 rpm for 30 minutes to promote the full development and shaping of the rheological network structure, ultimately yielding the finished product.

[0105] To verify the comprehensive performance of the products prepared by this process, the sample from Example 4 was selected, and comparative tests were conducted using Comparative Example 1 (prepared using a traditional direct mixing and centralized feeding method and a conventional 2400 rpm high-speed strong shear sand milling process) and Comparative Example 2 (UV protectant and trehalose components were removed, and the remainder was supplemented with methyl oleate). The static suspension rate (%), survival rate after 72 hours of simulated continuous ultraviolet light irradiation (%), and high-temperature survival rate after 14 days of heat storage at 54℃ were determined according to relevant national standards. Simultaneously, live potted plant tests were conducted in a greenhouse, using western flower thrips (Frankliniella occidentalis) nymphs as the target. Each group of preparations was diluted with water at equal amounts of active ingredients and sprayed evenly. The corrected lethality rate of western flower thrips (%) was calculated after 7 days. The original test data were then compiled into standardized data comparison results as follows.

[0106] Example 4: Under a systematically optimized formulation and dynamic processing technology, the mixed emulsifier and organic bentonite synergistically constructed a dense and uniform anti-settling network, ensuring an extremely high physical suspension rate. The UV protectant and stress-resistant agent, at specific concentrations, fully utilized their physical shielding and physiological buffering functions, significantly improving the survival rate of live spores in extreme environments. Various physicochemical and biological indicators mutually reinforced each other, ensuring a high mortality rate against the target pest, western flower thrips, achieving a good balance between processing rheological smoothness, live component barrier protection, and actual field efficacy.

[0107] In summary, the homogeneous suspension system, constructed with humic acid-coated nano-titanium dioxide as the core for UV protection, supplemented by trehalose osmotic regulation and an organic bentonite thixotropic network, significantly improves the microscopic compatibility and overall environmental resistance of the formulation while maintaining the high virulence and infection characteristics of the strain. It overcomes the limitations of traditional multiphase live biological agents, such as easy aggregation and stratification and rapid inactivation under environmental stress. This allows the bio-oil suspension to resist mechanical shear and thermal stress damage during high-intensity processing and grinding, and to exhibit suitable light shielding and physiological buffering capabilities when applied in the field to strong UV radiation or alternating drought conditions, thus broadening its application window in harsh agricultural environments. Furthermore, the three-dimensional thixotropic anti-settling network effectively slows down the aggregation and precipitation of powder particles, and the optimized gradient wetting pre-construction and steady-state homogenization process preserves the integrity of the micro-rheological framework, resulting in better physical suspension stability and field target lethality of the control agent during long-term storage and practical application.

[0108] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A method for preparing a Beauveria bassiana preparation for the control of western flower thrips, characterized in that, Includes the following steps: S1 extract was used to activate the highly virulent strain CGMCC 0877 of Beauveria bassiana selected from western flower thrips. The strain was then inoculated into a secondary seed culture medium and cultured for 40-55 hours until the late logarithmic growth stage. Subsequently, it was transferred to the main fermenter at a volume inoculation rate of 10%. The fermentation temperature and sterile air circulation were maintained, and large-scale fermentation was carried out at a main stirrer speed of 200-250 rpm. This ensured efficient gas-liquid dissolved oxygen mass transfer while controlling mechanical shearing damage to the mycelial skeleton. After fermentation, the strain was harvested, dehydrated, and dried at low temperature to obtain a high-activity technical powder. S2, by mass, adds the remaining methyl oleate as an oil phase carrier to the mixing vessel. While maintaining stirring, slowly add 5-15 parts of a mixed emulsifier composed of alkyl glycosides and castor oil polyoxyethylene ether to fully dissolve it and reduce the interfacial tension of the system. While maintaining the shear state, uniformly sieve in 2-3 parts of organic bentonite to initially construct a three-dimensional thixotropic anti-settling network. Then, sequentially add 1-3 parts of humic acid-coated nano-titanium dioxide and 2-8 parts of trehalose, relying on viscosity to support them to be uniformly suspended in the rheological network. S3. Add 5-15 parts of the high-activity active ingredient dry powder to the rheological system, and after pre-dispersion, pump it into a horizontal sand mill for continuous refining to control the thermal stress damage to the live spores caused by frictional heat generation of the grinding machinery. S4 introduces the finely ground material into a homogenizing device for homogenization and maturation, overcoming the van der Waals forces between powders, promoting the full development and shaping of the anti-settling rheological network structure, and producing a bio-oil suspension.

2. The method for preparing the Beauveria bassiana preparation for controlling western flower thrips according to claim 1, characterized in that, In step S1, the fermentation temperature is 24-28℃, and the sterile air ventilation rate is 0.6-1 vvm.

3. The preparation method of the Beauveria bassiana preparation for the control of western flower thrips according to claim 2, characterized in that, In step S1, the inoculation time in the secondary seed culture medium is 48 hours, and the main stirrer speed during the fermentation stage is 230 rpm.

4. The preparation method of the Beauveria bassiana preparation for the control of western flower thrips according to claim 1, characterized in that, In step S2, the amount of the mixed emulsifier added is 9 parts, the amount of the organic bentonite added is 2.5 parts, the amount of the humic acid-coated nano-titanium dioxide added is 2 parts, and the amount of the trehalose added is 5 parts; wherein, the mass ratio of the organic bentonite to the mixed emulsifier is 5:

18.

5. The method for preparing the Beauveria bassiana preparation for controlling western flower thrips according to claim 1, characterized in that, In step S3, the pre-dispersion speed is 2000-3000 rpm and the time is 15-25 minutes; preferably, the pre-dispersion speed is 2500 rpm and the time is 20 minutes.

6. The method for preparing the Beauveria bassiana preparation for controlling western flower thrips according to claim 1, characterized in that, In step S3, the grinding speed of the horizontal sand mill is 1500-1800 rpm, and the cooling fluid circulation flow rate of the sand mill is 4-6 m³ / h.

7. The method for preparing the Beauveria bassiana preparation for controlling western flower thrips according to claim 6, characterized in that, In step S3, the grinding speed of the horizontal sand mill is 1650 rpm, and the cooling fluid circulation flow rate of the sand mill is 5 m³ / h.

8. The method for preparing the Beauveria bassiana preparation for controlling western flower thrips according to claim 1, characterized in that, In step S4, the rotation speed of the homogenizing device is 800-900 rpm, and the time is 20-40 minutes.

9. The method for preparing the Beauveria bassiana preparation for controlling western flower thrips according to claim 8, characterized in that, In step S4, the homogenizing device rotates at 850 rpm for 30 minutes.

10. A Beauveria bassiana preparation for the control of western flower thrips, characterized in that, Prepared by the preparation method according to any one of claims 1 to 9, the prepared formulation has methyl oleate as the continuous phase and a three-dimensional thixotropic anti-settling network formed by fully swollen organic bentonite. The humic acid-coated nano-titanium dioxide is uniformly distributed in the system, and its surface humic acid molecules and nano-inorganic core work together to play a dual role in chemical photo-sieving and physical photo-shielding, constructing a photoprotective barrier that delays the ultraviolet aging of Beauveria bassiana live spores. The trehalose and the flexible bentonite network structure work together to provide osmotic pressure physiological regulation and physical buffer space for live spores, constructing a stable and compatible microenvironment that reduces mechanical friction damage and effectively blocks the aggregation and stratification of multiphase particles.

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