Preparation method and application of compound microorganism full water-soluble powder

Abstract

The invention discloses a preparation method and application of compound microorganism full water-soluble powder. According to the present invention, the Metarhizium anisopliae (WXH-M001) Metarhizium anisopliae and the Bacillus subtilis (WXH-B001) Bacillus velezensis are obtained through the directional screening of the bacterial strain function, and the Metarhizium anisopliae and the Bacillus subtilis are obtained through the screening; furthermore, the two strains are reasonably combined, and a special auxiliary agent is screened, so that the compound microorganism full water-soluble powder is prepared. The compound microorganism full-water-soluble powder provided by the invention has an obvious control effect on northern leaf blight, southern leaf blight, corn borer, beet armyworm and the like of corn, is beneficial to improving the corn yield, is not easy to induce pest or pathogenic bacterium resistance, and can replace chemical pesticides or reduce the application amount of pesticides; and meanwhile, the pesticide composition has the characteristic of quick dissolution, is free of residue, can be used through flight prevention, micro-spraying, drip irrigation and conventional mist spraying, is friendly to environment and non-target organisms, and can provide important technical support for realizing green agriculture.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of microorganisms, and relates to a preparation method of a composite microbial full water-soluble powder and application thereof. BACKGROUND

[0003] Metarhizium is a broad-spectrum insect pathogenic fungus, and according to statistics, the host insects of Metarhizium reach more than 200 species, which can parasitize scarab beetles, weevils, wireworms, lepidopteran pest larvae and pentatomid stink bugs and the like. Metarhizium can induce insects to produce Metarhizium disease and form repeated infection in the population. In application, Metarhizium anisopliae is mainly used to control pests, and Metarhizium has developed into a fungal insecticide second only to Beauveria bassiana in terms of control scale. Some studies show that Metarhizium also has fungicidal function. Metarhizium is harmless to humans and animals, safe to natural enemy insects, and does not pollute the environment. However, different strains have significant differences in infection efficiency on specific pests, and need to be selected accordingly. Limitations: sensitive to ultraviolet light, spore half-life < 24 hours under direct sunlight; slow onset, lethal time 3-7 days; ordinary spray drying survival rate < 50%.

[0004] Bacillus is a kind of gram-positive bacteria capable of forming resistant spores, which is widely used in agriculture, environmental protection, food and medicine and other fields. Different strains (even different subtypes of the same species) have different effects. Bacillus is a multifunctional and highly safe microbial preparation. Although it has some shortcomings such as slow effect and environmental dependence, the effect can be significantly improved through strain optimization, intelligent dosage form design and scientific application.

[0005] Therefore, there is an urgent need for a microbial preparation that can effectively control the pests and diseases of corn. SUMMARY

[0006] The purpose of the present application is to provide a preparation method of a composite microbial full water-soluble powder and application thereof, which combines Metarhizium and Bacillus into a full water-soluble microbial powder, and can effectively improve the control effect of microbial agents, is safe to the environment, and can be widely applied in the control of main diseases and pests of corn.

[0007] The purpose of the present application can be achieved by the following scheme:

[0008] In a first aspect, the present application provides a strain for controlling corn diseases and pests, comprising Metarhizium anisopliae and Bacillus velezensis.

[0009] The Metarhizium anisopliae has a preservation number of CCTCC NO: M 2025720, a preservation name of Metarhizium anisopliae WXH-M001, is preserved in the China Center for Type Culture Collection, and has a preservation address of Wuhan University, Wuhan, China and a preservation time of April 8, 2025.

[0010] The preservation number of the Bacillus velezensis is CCTCC NO: M 2025182, the preservation name is Bacillus velezensis WXH-B001, the preservation center is China Center for Type Culture Collection, the preservation address is Wuhan University, Wuhan, China, and the preservation time is January 20, 2025.

[0011] In a second aspect, the present application provides a compound microbial full water-soluble powder, which comprises Metarrhizium anisopliae and Bacillus velezensis as active ingredients. The Metarrhizium anisopliae is a fungicidal bacterium, and the Bacillus velezensis is a bacteriostatic and growth-promoting bacterium.

[0012] As an embodiment of the present application, the compound microbial full water-soluble powder comprises Metarrhizium anisopliae and Bacillus velezensis in a ratio of 1:2-4.

[0013] As an embodiment of the present application, the compound microbial full water-soluble powder comprises Metarrhizium anisopliae and Bacillus velezensis in a ratio of 1:2-4. 8 -1.5×10 8 CFU / g of Metarrhizium anisopliae and 1.0×10 8 -6×10 8 CFU / g of Bacillus velezensis.

[0014] As an embodiment of the present application, the compound microbial full water-soluble powder further comprises a protective agent, a water dispersing agent, a water-soluble carrier, a humectant, and a spreading agent.

[0015] Further, the protective agent comprises carboxymethylated inulin, the water dispersing agent comprises sodium lignosulfonate, the water-soluble carrier comprises guar gum, the humectant comprises sodium dodecyl benzene sulfonate, and the spreading agent comprises tea dregs.

[0016] As an embodiment of the present application, the compound microbial full water-soluble powder comprises the following components in the following mass percentages: 5-15% of a compound spore mother liquor, 5-20% of a protective agent, 1-2% of a water dispersing agent, 60-85% of a water-soluble carrier, 1-3% of a humectant, and 0.5-1.5% of a spreading agent. The compound spore mother liquor comprises Metarrhizium anisopliae and Bacillus velezensis. The compound microbial full water-soluble powder of the present application is dissolved in water in less than 60 seconds.

[0017] wherein:

[0018] Inulin is a natural soluble dietary fiber, mainly extracted from plants such as chicory and Jerusalem artichoke. It consists of β-(2,1)-D-fructose chains, often ending with a glucose unit. It possesses high water solubility, prebiotic properties, and environmental friendliness. This invention utilizes carboxymethylated inulin, which is produced by reacting inulin with chloroacetic acid under alkaline conditions to introduce carboxymethyl groups (-CH2COOH). These negatively charged groups can electrostatically adsorb positively charged microorganisms (such as Bacillus), increasing solubility to >50% (25℃). This process also enhances the viable spore rate and wetting time of microorganisms. The microbial agent prepared using carboxymethylated inulin as a protectant exhibits stable performance, preventing damage to the microorganisms caused by changes in the external environment, thus ensuring the compound microbial agent effectively exerts its insecticidal, bactericidal, and yield-increasing effects.

[0019] Jiawei powder (a multivitamin functional powder) is not only a nutritional supplement but also a highly efficient water-soluble carrier for microbial preparations, functional fertilizers, or drug delivery systems. It has broad compatibility, being compatible with microorganisms (such as Bacillus), minerals, and plant extracts without causing flocculation or stratification. It also provides synergistic nutritional effects, promoting the metabolic activation of active bacterial strains and accelerating their recovery and colonization. Furthermore, it enhances the stability of the microbial agent, providing a degree of protection, improving the microorganisms' resistance to high temperatures and dryness, making them more adaptable to different ecological environments, and increasing the survival rate of the microbial agent.

[0020] Tea seed cake is the residue left after pressing oil from camellia seeds. It is rich in active ingredients such as tea saponins, proteins, and polysaccharides. In agriculture, it can be used as a natural spreading agent (adjuvant) to improve the adhesion, penetration, and covering effect of pesticides or fertilizers. It has strong adhesion and resistance to rain erosion. Additionally, it contains small amounts of nitrogen, phosphorus, potassium, and trace elements, also providing foliar fertilizer benefits. Tea seed cake decomposes naturally, posing no risk of soil residue.

[0021] As one embodiment of the present invention, the application method of the composite microbial water-soluble powder includes any one of aerial spraying, micro-spraying, drip irrigation, and conventional spraying.

[0022] Thirdly, the present invention provides a method for preparing a composite microbial fully water-soluble powder, comprising the following steps:

[0023] S1. Metarhizium anisopliae and Bacillus belladonna were cultured in liquid fermentation to obtain Metarhizium anisopliae spore suspension and Bacillus belladonna spore suspension, and then mixed to obtain a composite spore mother liquor.

[0024] S2. The composite spore mother liquor is combined with a protectant, a water dispersant, and a water-soluble carrier to obtain a composite spore mother powder;

[0025] S3. Add a wetting agent and a spreading agent to the compound spore powder to obtain the compound microbial water-soluble powder.

[0026] As one embodiment of the present invention, in step S1, Metarhizium anisopliae and Bacillus spp. are used as production strains. The strains are cultured and fermented in pure culture, and the cells are enriched by centrifugation or filtration. The cells are mixed with the supernatant in a certain proportion and stirred evenly to prepare a mother liquor.

[0027] Fourthly, the present invention provides the application of the aforementioned composite microbial water-soluble powder, or the composite microbial water-soluble powder obtained by the aforementioned preparation method, in the prevention / treatment of corn leaf blight, corn leaf spot, corn borer, and beet armyworm.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] 1. This invention obtains Metarhizium anisopliae (WXH-M001, an insecticidal and fungicidal fungus) and Bacillus velezensis (WXH-B001, an antibacterial and growth-promoting bacterium) through functional-directed screening of strains. The combination of strains has significant control effects on maize leaf spot, corn leaf blight, corn borer, and beet armyworm, which is beneficial to the biological control of maize and thus increases maize yield.

[0030] 2. This invention combines Metarhizium anisopliae (WXH-M001) and Bacillus velezensis (WXH-B001) to prepare a fully water-soluble microbial agent, achieving multiple synergistic effects of pest control, disease inhibition, and growth promotion. Metarhizium anisopliae, a broad-spectrum parasitic fungus, kills insects (such as grubs, various moths, and leaf beetles) by secreting chitinase to destroy their epidermis. Bacillus velezensis secretes lipopeptide antibiotics (such as Surfactin and Iturin) to inhibit pathogenic fungi (Fusarium, Botrytis) and bacteria (Ralstonia solanacearum), showing significant effects in controlling soil-borne diseases (root rot) and foliar diseases. Furthermore, this invention utilizes the diverse mechanisms of action of the combination of Metarhizium anisopliae and Bacillus spp., leveraging the dual effects of Metarhizium anisopliae and Bacillus spp. to synergistically enhance the control of diseases such as corn leaf blight and small leaf blight, as well as pests such as corn borer and beet armyworm, thereby significantly increasing the yield per acre and yield increase rate of corn. The combined application of Metarhizium anisopliae and Bacillus spp. can reduce the number of pesticide applications, and the dead insects infected by Metarhizium anisopliae can become a nutrient source for Bacillus spp., extending the field residual effect period.

[0031] 3. The compound microbial water-soluble powder of this invention can completely dissolve in room temperature water within 1 minute without residue, making it suitable for modern application methods such as drip irrigation, aerial spraying, and micro-spraying. Among these, aerial spraying and micro-spraying are efficient, water-saving, and precise spraying technologies, especially suitable for the field application of water-soluble microbial preparations, significantly improving cell survival rate and application efficiency. In the future, with the popularization of intelligent control (such as IoT linkage), micro-spraying will become one of the core tools for promoting microbial technology in corn and other large-scale crops.

[0032] 4. In this invention, the compatibility of various adjuvants must consider not only their impact on the physicochemical properties of the formulation but also their toxic effects on the bioactive active ingredients, thus narrowing the range of adjuvants to choose from. This invention utilizes adjuvants such as carboxymethylated inulin, which have no toxic side effects on the microorganisms. Combined with other dispersants and spreading agents, this improves the viable spore rate and wetting time, effectively protecting the safety of the microbial active ingredients while enhancing the preventative efficacy.

[0033] 5. The compound microbial water-soluble powder of the present invention is environmentally safe, degradable, green and environmentally friendly, safe and reliable; and it is not likely to induce resistance in pests or pathogens, thus enabling the management of pesticide resistance in pests and diseases; at the same time, it can replace chemical pesticides or reduce the application of pesticides, without producing pesticide residues, and is friendly to the environment and non-target organisms, providing important technical support for the realization of green agriculture. Attached Figure Description

[0034] Other features, objects, and advantages of the present invention will become more apparent from the following detailed description of non-limiting embodiments with reference to the accompanying drawings:

[0035] Figure 1 The image shows the morphology of Metarhizium anisopliae strain WXH-M001 in Example 1; the left image is the front view of the PDA plate of Metarhizium anisopliae, and the right image is the back view of the PDA plate of Metarhizium anisopliae.

[0036] Figure 2 The image shows the hyphae and spores of Metarhizium anisopliae WXH-M001 in Example 1.

[0037] Figure 3 The phylogenetic tree of Metarhizium anisopliae WXH-M001 in Example 1;

[0038] Figure 4 The image shows the morphology of the Bacillus velezensis strain WXH-B001 in Example 1; the left image is a front view of the LBA plate of Bacillus velezensis, and the right image is a back view of the LBA plate of Bacillus velezensis.

[0039] Figure 5 Image of Bacillus velezensis spores WXH-B001 in Example 1;

[0040] Figure 6 This is the phylogenetic tree of Bacillus velezensis WXH-B001 in Example 1. Detailed Implementation

[0041] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. The following examples are implemented under the premise of the technical solution of the present invention, providing detailed implementation methods and specific operating procedures, which will help those skilled in the art to further understand the present invention. It should be noted that the scope of protection of the present invention is not limited to the following embodiments; any adjustments and improvements made under the concept of the present invention are all within the scope of protection of the present invention.

[0042] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0043] The Metarhizium anisopliae WXH-M001 strain described in the following examples has been deposited at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO: M 2025720, on April 8, 2025.

[0044] The Bacillus velezensis WXH-B001 in the following examples has been deposited at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO: M 2025182, on January 20, 2025.

[0045] Example 1

[0046] Isolation and identification of Metarhizium anisopliae (WXH-M001) and Bacillus velezensis (WXH-B001)

[0047] On October 29, 2021, *Metarhizium anisopliae* was collected from corn at the experimental farm of Shanghai Jiao Tong University's Minhang campus. After isolation, re-inoculation, and identification, the *Metarhizium anisopliae* strain of this invention, WXH-M001, was screened out. On August 15, 2023, the *Bacillus velezensis* strain of this invention, WXH-B001, was screened and identified from corn soil in protected cultivated land in Kalaqin Banner, Chifeng City.

[0048] I. Isolation and Identification of Strain Metarhizium anisopliae WXH-M001

[0049] 1. Separation and purification

[0050] Surface disinfection was performed on stunted insects collected from corn at the experimental farm of Shanghai Jiao Tong University's Minhang campus. The insects were then cut into segments on a clean bench and placed on PDA plates containing kanamycin. These segments were incubated at 28°C and observed every 12 hours. The colonies were counted and numbered, and then purified until they were identical. Mycelial cakes were collected from the PDA medium used for propagation using a 5mm punch, transferred to fresh PDA medium, and propagated again to confirm purification. Spores were scraped from the plates, and DNA was extracted using a kit. ITS sequences (full sequences of ITS1 and ITS2, as shown in SEQ ID NO. 3-4) were amplified by PCR using primers ITS1F and ITS4-R (as shown in SEQ ID NO. 3-4, respectively), and then sequenced for identification.

[0051] 2. Identification

[0052] (1) Morphological identification

[0053] When cultured on nutrient agar medium (PDA) at 28℃, the hyphae of *Metarhizium anisopliae* WXH-M001 initially appeared as short, white, fluffy hairs, spreading radially towards the edge of the plate, and the colonies gradually enlarged. Figure 1 As shown, pale green conidia begin to appear in the center of the colony after 3-4 days, gradually expanding outwards. The conidial mass is yellowish-green, and the color gradually fades from the center of the colony outwards in concentric rings. When viewed from the back of the colony, the center of the colony is dark yellow, and the color gradually brightens outwards in concentric rings, showing a bright yellow and light yellow color.

[0054] After fermentation in a liquid fermenter, the fermentation broth was examined under a microscope, such as... Figure 2 As shown, the hyphae of *Metarhizium anisopliae* WXH-M001 are long rods, reaching hundreds of micrometers in length, with few branches. The terminal spores are spherical or rugby ball-shaped. As the hyphae grow to a certain extent, they age and form spores. By controlling fermentation parameters, *Metarhizium anisopliae* hyphae can break into numerous rugby ball-shaped, thick-walled spores.

[0055] To determine the phylogenetic position of this strain, ITS sequence PCR amplification was performed using primers ITS1F and ITS4-R, followed by sequencing alignment for identification. Sequences of multiple reference strains were obtained from the NCBI (GenBank) database. The ITS1 and ITS2 sequences of the isolated strain and the reference strains were analyzed using BioEdit and MEGA11 software. A phylogenetic tree of the isolated strain and reference strains was constructed (e.g., Figure 3 (As shown). Thus, the strain of the isolated strain was identified as Metarhizium anisopliae and named WXH-M001.

[0056] II. Isolation and Identification of Bacillus strain WXH-B001

[0057] 1. Separation and purification

[0058] Soil samples collected from protected cultivated land cornfields in Kalaqin Banner, Chifeng City, were diluted with 10 grams of sample and 100 ml of sterile physiological saline. The mixture was shaken at 220 rpm for 15 minutes and then diluted to a final volume of 10. -3 10 -4 10 -5 Spread 100 μL of the culture onto LBA plates and incubate at 37°C for 1-2 days. Pick single colonies at different phenotypes and incubate at 37°C for 1-2 days. Dilute each single colony with 1 ml of sterile saline and spread 100 μL of the culture onto LBA plates, incubating at 37°C for 1 day. Repeat this process of picking single colonies and transferring them to plates until a single colony is observed. Dilute with 1 ml of sterile saline and spread 100 μL onto LBA plates, incubating at 37°C for 1 day. Pick a single colony and place it in an Erlenmeyer flask containing LB broth, incubating at 37°C and 200 rpm for 2-3 days. Extract DNA from the bacterial culture using a kit and sequence it using 16S rDNA (as shown in SEQ ID NO. 2).

[0059] 2. Identification

[0060] After incubation at 37°C for 24 hours in LBA medium, the observed colonies exhibited a serrated, slightly rough, and opaque morphology with serrated edges. The colonies were pale yellow in color, roughly round in shape, and had a viscous or gelatinous consistency (e.g., ...). Figure 4 (As shown).

[0061] Preliminary identification: The colony morphology of the isolated and purified bacteria was similar to that of *Bacillus belyssae* strain, and they were examined and observed under a microscope. Figure 5 As shown, the bacterial cells are tightly stacked like short rods, often clustered together, resembling a "microbial coral reef".

[0062] Multiple reference strain sequences were obtained from the NCBI (GenBank) database. The full-length 16S rDNA sequences of the isolated strains and reference strains were analyzed using BioEdit and MEGA11 software, and a phylogenetic tree of the isolated strains and reference strains was constructed. Figure 6 The strain was thus identified as Bacillus velezensis and named WXH-B001.

[0063] Example 2

[0064] Preparation of compound microbial fully water-soluble powder

[0065] The compound microbial water-soluble powder is made from the following raw materials in the following weight ratio: 10% (w / w) compound spore mother liquor, 5-20% carboxymethylated inulin, 60-85% glutaraldehyde powder, 1-2% sodium lignosulfonate, 1-3% sodium dodecylbenzenesulfonate, and 1% tea seed cake.

[0066] The preparation method of the above-mentioned composite microbial water-soluble powder is as follows:

[0067] 1. Preparation of Metarhizium anisopliae inoculum

[0068] (1) Activation of strain: Take the slant culture of Metarhizium anisopliae and streak it onto a PDA plate. Incubate in a constant temperature incubator at 25-28℃ for 7-10 days until a large number of light green conidia are produced.

[0069] (2) Seed culture preparation: Culture medium (1L formula, the same below) contains 20g glucose, 10g peptone, 5g yeast extract, 1g KH2PO4, 0.5g MgSO4·7H2O, pH 6.5-7.0. Spores are scraped from the plate with sterile water to prepare a spore suspension (concentration ≥1×10⁻⁶). 6 (CFU / mL). Inoculate the seed culture at a 5% inoculum and culture in a constant temperature shaker at 25-28℃ and 150-200 rpm for 48 hours.

[0070] (3) Large-scale fermentation:

[0071] ① Fermentation tank parameters: Temperature: 25-28℃; Dissolved oxygen (DO): ≥30% (controlled by aeration or stirring); pH: automatically adjusted to 6.5-7.0 (using ammonia / low concentration HCl).

[0072] ②Fermentation endpoint: Spore concentration ≥ 1×10 9 CFU / mL (microscopic count).

[0073] ③ Post-treatment: Collect the spore suspension. Centrifuge (8000 rpm, 2 min) to prepare a high-concentration spore concentrate (≥4×10⁻⁶).10 (CFU / mL)

[0074] 2. Preparation of Bacillus bacterial culture

[0075] (1) Activation of bacterial strain: Take Bacillus slant culture and streak it onto LB medium. Incubate at 37℃ for 24-48 hours until light yellow colonies are formed.

[0076] (2) Seed culture preparation: Culture medium (1L formula, the same below) 10g tryptone, 5g yeast extract, 10g NaCl, pH 7.0-7.2. Inoculation amount 1-2%, culture at 37℃ and 180rpm for 12-24 hours with shaking.

[0077] (3) Large-scale fermentation:

[0078] ① Inoculate the seed culture into the fermentation medium at an inoculation rate of 0.5%. Incubate at 37℃ and 200rpm for 24-48 hours (spore formation rate >90%).

[0079] ② Centrifuge (12000 rpm, 1 min) to prepare a high-concentration spore concentrate (≥12×10⁻⁶). 10 (CFU / mL).

[0080] 3. Preparation of compound spore mother liquor

[0081] Prepare a composite stock solution by mixing the spore concentrates from steps 1 and 2 above at a volume ratio of 1:1 (i.e., a spore quantity ratio of 1:3), wherein the concentration of Metarhizium anisopliae is ≥1×10⁻⁶. 9 CFU / mL, Bacillus ≥3×10 9 CFU / mL.

[0082] 4. Preparation of compound spore powder

[0083] Carboxymethyl inulin is added as a protectant at 5-20% (w / w) to the composite spore mother liquor, followed by 1-2% sodium lignosulfonate water dispersant and 60-85% vinyl chloride water-soluble carrier. The mixture is stirred until homogeneous.

[0084] 5. Preparation of composite microbial fully water-soluble powder

[0085] Add 1-3% of a wetting agent such as sodium dodecylbenzenesulfonate and 1% of a spreading agent such as tea seed cake to the compound spore powder, stir well, and dry in a spore dryer at 42℃ to prepare a fully water-soluble powder of Metarhizium anisopliae and Bacillus subtilis compound microorganisms (Metarhizium anisopliae ≥ 0.5 × 10⁻⁶). 8 CFU / g, Bacillus ≥1.5×10 8 CFU / g).

[0086] The following samples were prepared according to the above preparation method.

[0087] Preparation Example 1

[0088] The weight ratio of the compound microbial water-soluble powder is as follows: compound spore mother liquor 10%, carboxymethylated inulin 5%, sodium lignosulfonate 1%, gibberellin powder 80%, sodium dodecylbenzenesulfonate 3%, and tea seed cake 1%.

[0089] Preparation Example 2

[0090] The weight ratio of the compound microbial water-soluble powder is as follows: compound spore mother liquor 10%, carboxymethylated inulin 20%, sodium lignosulfonate 2%, gibberellic acid powder 64%, sodium dodecylbenzenesulfonate 3%, and tea seed cake 1%.

[0091] Preparation Example 3

[0092] In the compound spore stock solution, *Metarhizium anisopliae* was replaced with *Metarhizium anisopliae* CGMCC NO.3.11962; and *Bacillus* was replaced with *Bacillus* SF248, CCTCC NO.M 20241355. The rest was the same as in Preparation Example 1.

[0093] Among them, Metarhizium anisopliae has the accession number CGMCC NO.3.11962, is deposited at the China General Microbiological Culture Collection Center, located at No.3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with the accession name Metarhizium anisopliae and the deposit date of September 24, 2008.

[0094] The accession number of Bacillus SF248 is CCTCC NO.M 20241355. It is deposited at the China Center for Type Culture Collection, Wuhan University, Wuhan, China. The accession name is Bacillus velezensis SF248. The deposit date is June 24, 2024.

[0095] Preparation Example 4

[0096] In the compound microbial water-soluble powder, carboxymethylated inulin was replaced with inulin. The rest was the same as in Preparation Example 1.

[0097] Preparation Example 5

[0098] The adjuvant of the compound microbial water-soluble powder was changed to a wettable powder. The specific composition is as follows: 10% of the compound spore mother liquor in Preparation Example 1, 8% of the wetting agent soapberry powder, 3% of the dispersant MF-5, and the remainder is the carrier diatomaceous earth.

[0099] Preparation Example 6

[0100] The composite spore stock solution in the composite microbial water-soluble powder contains only Metarhizium anisopliae WXH-M001. The rest is the same as in Preparation Example 1.

[0101] Preparation Example 7

[0102] The composite spore mother liquor in the composite microbial water-soluble powder contains only Bacillus WXH-B001. Everything else is the same as in Preparation Example 1.

[0103] Example 3

[0104] Performance testing of compound microbial water-soluble powder

[0105] I. Determination of biological compatibility (number of viable spores)

[0106] 1. Add the preparation examples 1-7 to sterile water containing 0.5% Tween-80, and dilute by a certain factor to a final concentration of 1×10⁻⁶. 6 CFU / ml, a certain amount of spore suspension was added to the spore germination solution, shaken thoroughly, and then incubated at (25±1)℃ and 120r / min for 24h. Samples were taken and the number of germinated and ungerminated spores in the germination solution was counted using a hemocytometer to calculate the spore germination rate. Each concentration was repeated 3 times, with a spore suspension without adjuvants (containing only the compound spore mother liquor) serving as a control.

[0107] 2. The experimental results are shown in Table 1:

[0108] Table 1

[0109] Sample Viable spore rate (%) Preparation Example 1 91.20 Preparation Example 2 93.46 Preparation Example 3 80.26 Preparation Example 4 77.37 Preparation Example 5 74.00 Preparation Example 6 89.66 Preparation Example 7 90.01 Control 89.37

[0110] The compatibility of various adjuvants must consider not only their impact on the physicochemical properties of the formulation but also their toxic effects on the active ingredient of the pesticide, thus narrowing the range of adjuvants to choose from. Viable spore rate is an important indicator for evaluating the shelf life of pesticide formulations with conidia as the active ingredient. As shown in the table above, preparations 1 and 2 achieved viable spore rates of 91.2%-93.46%, which are superior to other preparations.

[0111] II. Wetting Time

[0112] 1. Pour 100mL of water into a beaker and let it stand until there is no vortex (avoid water flow interference). Weigh 1.0g of powder using weighing paper and evenly sprinkle it on the water surface at a height of 1cm (avoid piling up). Immediately start a stopwatch and observe the powder wetting process. Endpoint determination: Complete wetting: The powder is completely soaked in water, with no floating particles or "dry core". Complete sinking: The powder sinks completely in the water (this can be confirmed by slightly tilting the beaker). Record the wetting time (in seconds), repeat 3 times and take the average value.

[0113] 2. The experimental results are shown in Table 2:

[0114] Table 2

[0115] Sample Wetting time (s) Preparation Example 1 25 Preparation Example 2 27 Preparation Example 3 61 Preparation Example 4 65 Preparation Example 5 79 Preparation Example 6 37 Preparation Example 7 41

[0116] Wetting time is a key indicator for measuring the ability of fully water-soluble powders to disperse rapidly in water. A wetting time ≤ 30s indicates excellent solubility, making it suitable for rapid application in scenarios such as aerial spraying, drip irrigation, and micro-spraying. Preparations 1 and 2 both achieved wetting times within 30s, demonstrating the best results.

[0117] Example 4

[0118] The experiment investigated the effects of using a compound microbial water-soluble powder sprayed twice during the corn tasseling and silking stages to replace and reduce the application of chemical pesticides, as well as to control pests and diseases in the middle and late stages.

[0119] I. Basic Information of the Experiment

[0120] 1. The location is Mengga Town, Mangshi City, Dehong Prefecture, Yunnan Province.

[0121] 2. According to the plan, each treatment should have no less than 10 rows (row length no less than 4m), and the spacing between plants and rows should be based on local planting habits. The planting density is 3200 plants / acre.

[0122] 3. Previous crop: Wheat. Variety: Kangnong No. 2. Sowing method: direct sowing; thinning and final seedling establishment should be completed 15 days after planting.

[0123] 4. Base fertilizer (type, quantity, quality, application time and method): Apply 40 kg of Meifeng Bilifu compound fertilizer (N-P2O5-K2O: 15-15-15) per mu, in strips on the sowing furrow before sowing. The first application is 20 kg of urea per mu in holes on June 20th; the second application is 30 kg of urea per mu in holes on July 17th.

[0124] 5. Weeding (time, frequency, method and quality): Weeding was carried out manually twice, on June 25 and July 27, in conjunction with topdressing.

[0125] II. Experiment Content and Field Design

[0126] Strictly follow the test protocol when spraying pesticides and record the application details.

[0127] 1. All plots of land in front of the large funnel-shaped opening were treated with conventional chemical agents.

[0128] 2. Early stage of the large trumpet mouth stage: Treatments 1-7: Preparation examples 1-7 of the above-mentioned examples were used respectively; Treatment 8: Chemical treatment method was used; Control: No agent was sprayed.

[0129] 3. Apply pesticide during the mid-silking stage, with additional application for treatments 1-7, and normal spraying for treatment 8 area;

[0130] 4. After applying pesticides during the silking stage, control and treatment 1-8 should be used according to the occurrence of pests and diseases. If further application is necessary, conventional chemical agents should be used.

[0131] 5. Preparation of Example 1-7: Application of the compound microbial water-soluble powder: Use 80 grams per mu (667 square meters), diluted and sprayed by airplane or sprayer.

[0132] 6. The chemical reagents and their usage methods involved in the above-mentioned tests are shown in Table 3.

[0133] Table 3 List of Common Chemical Reagents and Usage Methods

[0134]

[0135] III. Methods for Investigating Prevention Efficacy and Measuring Yield

[0136] 1. During the mature plant stage (after the milk stage), select 5 points diagonally in each treatment area, and randomly survey 20 plants at each point, for a total of 100 plants. Investigate the number of diseased (pest-infested) plants in each treatment, and calculate the disease index and disease control efficacy for large leaf spot and small leaf spot, as well as the plant infestation rate and pest control efficacy for corn borer and beet armyworm.

[0137] 2. Calculation formula:

[0138] (1) Disease index = [(number of disease grade plants × representative value of the disease grade) / (total number of plants × representative value of the most severe disease grade)] × 100.

[0139] (2) Prevention efficacy = (Control disease index - Treatment disease index) / Control disease index × 100%

[0140] (3) Infestation rate = [(Number of infested plants after pesticide application - Number of infested plants before pesticide application) ÷ Total number of plants surveyed] × 100%;

[0141] (4) Pest control efficacy = [(Number of infected plants in control - Number of infected plants in treatment) / (Number of infected plants in control - Average number of infected plants before application)] × 100%

[0142] 3. Yield Measurement Methods

[0143] (1) During the harvest period, 5 sampling points were taken from each treatment to investigate 10 square meters of plants, and the yield per mu and the yield increase rate were measured.

[0144] IV. Survey Results

[0145] 1. Statistics on the effectiveness of disease prevention

[0146] (1) The control effect on maize leaf spot disease is shown in Table 4.

[0147] Table 4. Statistics on the control efficacy of different treatments against maize leaf spot disease.

[0148] Treatment Disease index Control effect (%) Treatment 1 12.47 85.49 Treatment 2 11.39 86.75 Treatment 3 29.83 65.30 Treatment 4 38.76 54.91 Treatment 5 59.52 30.76 Treatment 6 18.47 78.51 Treatment 7 21.93 74.49 Treatment 8 46.30 46.14 Blank control 85.96 /

[0149] (2) The control effect on corn leaf blight is shown in Table 5.

[0150] Table 5. Statistics on the control efficacy of different treatments against maize leaf spot disease.

[0151]

[0152]

[0153] 2. Pest control efficacy

[0154] The control efficacy against corn borer is shown in Table 6.

[0155] Table 6

[0156] Treatment Disease rate (%) Disease control effect (%) Treatment 1 1 97.62 Treatment 2 2 95.24 Treatment 3 21 50.00 Treatment 4 25 40.48 Treatment 5 28 33.33 Treatment 6 14 66.67 Treatment 7 16 61.90 Treatment 8 32 23.81 Blank control 42 /

[0157] The control efficacy against beet armyworm is shown in Table 7.

[0158] Table 7

[0159]

[0160]

[0161] 3. Yield per mu and yield increase rate are shown in Table 8.

[0162] Table 8

[0163] Treatment Yield per mu (kg) Yield increase rate (%) Treatment 1 562.7 23.70 Treatment 2 584.3 28.45 Treatment 3 473.6 4.11 Treatment 4 406.5 -10.64 Treatment 5 449.2 -1.25 Treatment 6 451.9 -0.66 Treatment 7 403.6 -11.28 Treatment 8 452.2 -0.59 Blank control 454.9 /

[0164] The experimental results of Example 4 show that the samples prepared in Examples 1 and 2 of this invention have significant efficacy in preventing and controlling corn diseases and pests. Their control effects are significantly higher than those of Examples 3-7 and the control group treated with chemical pesticides, and they also show significant yield increases per acre. The compound microbial water-soluble powder of Metarhizium anisopliae and Bacillus spp. exhibits a synergistic effect, enhancing the control of diseases and pests, reducing the amount of chemical pesticides used, preventing the development of pesticide resistance, improving crop stress resistance, promoting crop growth, and being environmentally safe.

[0165] The specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the specific embodiments described above, and those skilled in the art can make various modifications or variations within the scope of the claims, which do not affect the essence of the present invention.

Claims

1. A strain for controlling corn diseases and pests, characterized in that, Including Metarhizium anisopliae and Bacillus belye; The preservation number of the Metarhizium anisopliae is CCTCC NO: M 2025720, and the preservation name is Metarhizium anisopliae WXH-M001. The Bacillus velezensis described is registered under CCTCC NO: M 2025182 and its name is Bacillus velezensis WXH-B001.

2. A composite microbial water-soluble powder comprising the strain described in claim 1, characterized in that, Its active ingredients include the aforementioned Metarhizium anisopliae and Bacillus vesiculosus.

3. The composite microbial water-soluble powder according to claim 2, characterized in that, The spore ratio of Metarhizium anisopliae and Bacillus belysae is 1:2-4.

4. The composite microbial water-soluble powder according to claim 2, characterized in that, The total number of viable spores of *Metarhizium anisopliae* in the composite microbial water-soluble powder is 0.5 × 10⁻⁶. 8 -1.5×10 8 CFU / g, viable spore count of Bacillus belysinii is 1.0 × 10⁻⁶. 8 -6.0×10 8 CFU / g.

5. The composite microbial water-soluble powder according to claim 2, characterized in that, The composite microbial water-soluble powder also includes a protectant, a water dispersant, a water-soluble carrier, a wetting agent, and a spreading agent.

6. The composite microbial water-soluble powder according to claim 5, characterized in that, The protective agent includes carboxymethylated inulin, the water dispersant includes sodium lignosulfonate, the water-soluble carrier includes gavage powder, the wetting agent includes sodium dodecylbenzenesulfonate, and the spreading agent includes tea seed cake.

7. The composite microbial water-soluble powder according to claim 2, characterized in that, The composite microbial water-soluble powder comprises the following components in the indicated weight percentages: 5-15% composite spore mother liquor, 5-20% protective agent, 1-2% water dispersant, 60-85% water-soluble carrier, 1-3% wetting agent, and 0.5-1.5% spreading agent; the composite spore mother liquor contains Metarhizium anisopliae and Bacillus belye.

8. The composite microbial water-soluble powder according to claim 2, characterized in that, The application methods for the compound microbial water-soluble powder include any one of aerial spraying, micro-spraying, drip irrigation, and spraying.

9. A method for preparing the composite microbial water-soluble powder as described in any one of claims 2-8, characterized in that, Includes the following steps: S1. Metarhizium anisopliae and Bacillus belladonna were cultured in liquid fermentation to obtain Metarhizium anisopliae spore suspension and Bacillus belladonna spore suspension, and then mixed to obtain a composite spore mother liquor. S2. The composite spore mother liquor is combined with a protectant, a water dispersant, and a water-soluble carrier to obtain a composite spore mother powder; S3. Add a wetting agent and a spreading agent to the compound spore powder to obtain the compound microbial water-soluble powder.

10. The application of a composite microbial water-soluble powder as described in any one of claims 2-8, or a composite microbial water-soluble powder prepared by the method described in claim 9, in the prevention / treatment of maize leaf spot, corn leaf blight, corn borer, and beet armyworm.

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