DSE chlamydospore aqueous solution, its preparation method and application

Abstract

This invention provides a DSE (dichloropyrophyll-forming element) aqueous solution, its preparation method, and its application, belonging to the field of microbial preparation technology. It comprises chlamydospores from at least one dark-colored septate endophytic fungus, and also includes 5 g / mL to 10 g / mL of preservative and 5 g / mL to 10 g / mL of protective agent, wherein the chlamydospore content is greater than 2.5 × 10⁻⁶. 8 CFU / mL. This invention improves the properties of aqueous solutions by rationally optimizing the dosage of DSE chlamydospores and excipients, resulting in an aqueous solution with a chlamydospore content of 2.54 × 10⁻⁶ CFU / mL. 8 The concentration of CFU / mL meets national standards; it can be stored at room temperature for more than 12 months, extending its shelf life by more than 9 months compared to conventional conidial aqueous solutions. This DSE chlamydospore aqueous solution effectively controls bacterial wilt in tomatoes and black-streaked dwarf disease in southern rice, improving the growth of tomato and rice plants, and achieving control effects comparable to or even superior to the positive control *Trichoderma harzianum*. The DSE chlamydospore aqueous solution provided by this invention is simple to prepare, cost-effective, and has excellent performance, making it suitable for large-scale promotion and use as a novel biological pesticide product.

Description

Technical Field

[0001] This invention belongs to the field of microbial preparation technology, and specifically relates to a DSE thick-walled spore aqueous solution, its preparation method, and its application. Background Technology

[0002] Tomatoes and rice are important economic and food crops in my country and globally, and their yield and quality directly affect agricultural production security, food supply stability, and farmers' economic income. However, under intensive and large-scale planting models, the frequent occurrence and spread of soil-borne and viral diseases have become key issues restricting the high-quality and high-yield of these two crops. Among them, bacterial wilt of tomatoes is caused by Ralstonia solanacearum (Ralstonia solanacearum). Ralstonia solanacearum This disease, caused by the white-backed planthopper, is a typical soil-borne vascular disease characterized by rapid spread, a wide host range, and difficulty in control. After infection, it often leads to the rapid wilting and death of tomato plants. The incidence rate in general fields can reach 20%-30%, and in severely affected areas, it can even result in total crop failure, causing significant economic losses to the tomato industry. Southern rice black-streaked dwarf disease, on the other hand, is a viral disease caused by Southern rice black-streaked dwarf virus (SRBSDV), primarily transmitted by the white-backed planthopper (Synthia spp.). Sogatella furcifera The disease spreads and causes rice plants to become stunted, with dark green leaves, thick stems, and failure to head or produce empty heads. In recent years, it has broken out and spread multiple times in the main rice-producing areas of southern my country, posing a serious threat to rice yields and exacerbating the pressure on grain production.

[0003] Currently, biocontrol microorganisms targeting bacterial wilt of tomatoes are mainly Bacillus species. For example, Liu et al. (2024) isolated Bacillus belyss SB10, and both its bacterial suspension and fermentation broth showed preventive and curative effects against Ralstonia solanacearum. Mekonnen et al. (2022) found that Bacillus isolate BDUA1 could significantly reduce the incidence of bacterial wilt in tomatoes under greenhouse conditions. Due to the complex soil environment in the field and the high differentiation of physiological races of bacterial wilt pathogens, these biocontrol bacteria generally suffer from weak colonization ability and unstable control efficacy. Meanwhile, the control of rice black-streaked dwarf disease in southern China mainly relies on chemical agents such as imidacloprid, phosmet, and pymetrozine-thiamethoxam, with relatively little research on biological agents. Therefore, finding efficient, safe, and environmentally friendly biological control resources and developing new biological pesticides has become a research hotspot and urgent need in the field of green control of agricultural diseases.

[0004] Dark septate endophytic fungi (DSE) are a group of endophytic fungi that widely colonize the interior or surface of plant roots, forming dark septate hyphae and microsclerotia, and can form symbiotic relationships with a variety of plants. Studies have shown that DSE can enhance plant tolerance to biotic and abiotic stresses by promoting nutrient absorption, secreting growth regulators, inducing plant resistance, and inhibiting pathogen growth, making them highly promising for the biological control of crop diseases. DSE belongs to the "low abundance, high function" group. Its dark septate hyphae and microsclerotia can penetrate and colonize plant tissues, exhibiting advantages such as strong colonization ability, long-lasting activity, and broad-spectrum resistance, making its development and utilization promising. In existing research, the use of fungi often involves directly inoculating plants with mycelium or preparing inoculants using conidia. However, mycelium is easily inactivated during practical applications, with a very short shelf life (only 3-6 months), making it difficult to meet commercial requirements; while conventional conidial inoculants also have limited shelf life, making large-scale application difficult. Thick-walled spores of fungi have become the preferred ingredient for the development of fungal agents due to their strong resistance and stability. However, when the inventors tried to use spores to prepare aqueous solutions, they found the following problems: low spore germination rate or low number of effective viable cells made it difficult to exert their effects, which brought great challenges to the product development of DSE. Summary of the Invention

[0005] To address the above technical problems, this invention provides a DSE thick-walled spore aqueous solution, its preparation method, and its application.

[0006] This invention provides a DSE chlamydospore aqueous solution, comprising chlamydospores from at least one dark-colored septate endophytic fungus, and further comprising a preservative at a concentration of 0.5 mg / mL to 2.5 mg / mL and a protective agent at a concentration of 0.5 mg / mL to 1.0 mg / mL, wherein the content of the chlamydospores in the DSE chlamydospore aqueous solution is greater than 2.5 × 10⁻⁶. 8 CFU / mL.

[0007] Further, the dark-colored septate endophytic fungus is *Cladosporium*; preferably, the dark-colored septate endophytic fungus is *Cladosporium guangxiense* (…). Cladophialophora guangxiense The strain HX2, with accession number CGMCC NO. 41498.

[0008] Guangxi Cladosporium ( Cladophialophora guangxiense HX2 is a DSE strain isolated and screened from sugarcane fields. It is currently deposited at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing; deposit date: September 5, 2024; classification and nomenclature: Cladophialophora guangxiense Accession number: CGMCC NO. 41498.

[0009] Furthermore, the preservative is selected from at least one of calcium propionate, sodium benzoate, and potassium sorbate.

[0010] Further, the preservative is potassium sorbate, and the amount of potassium sorbate added to the DSE thick-walled spore aqueous solution is 0.5 mg / mL to 1.0 mg / mL, optionally 0.5 mg / mL to 0.75 mg / mL, optionally 0.5 mg / mL to 0.6 mg / mL, optionally 0.5 mg / mL.

[0011] Furthermore, the protective agent is selected from at least one of dextrin, humic acid, carboxymethyl cellulose, sodium acetate, and sodium citrate.

[0012] Further, the protectant is dextrin, and the amount of dextrin added to the DSE thick-walled spore aqueous solution is 0.5 mg / mL to 1 mg / mL, optionally 0.75 mg / mL to 1.0 g / mL, optionally 0.9 mg / mL to 1.0 mg / mL, optionally 1.0 mg / mL.

[0013] Another aspect of the present invention provides a method for preparing the above-mentioned DSE thick-walled spore aqueous solution, comprising the following steps:

[0014] (1) Preparation of thick-walled spore slurry: The dark-colored septate endophytic fungal strain is cultured in liquid basal medium, and the thick-walled spores in the fermentation broth are collected to obtain thick-walled spore slurry; Optionally, the method for preparing thick-walled spore slurry includes: inoculating on PDA medium plates, activating culture at 28°C, then transferring to basal medium, and culturing in a constant temperature shaker at 28°C for 14 days; the obtained thick-walled spore fermentation broth is filtered through three layers of sterile gauze to obtain thick-walled spore slurry;

[0015] (2) Preparation of finished aqueous solution: add preservative and protective agent to the thick-walled spore slurry in proportion to obtain the finished DSE thick-walled spore aqueous solution.

[0016] The third aspect of this invention provides the application of the above-mentioned DSE thick-walled spore aqueous solution in promoting the expression of plant defense enzymes, wherein the plant is tomato or rice, and the plant defense enzymes include PAL, PPO, POD, CAT and SOD.

[0017] The fourth aspect of this invention provides the application of the above-mentioned DSE thick-walled spore aqueous solution in the control of rice black-streaked dwarf disease in southern China.

[0018] Furthermore, the method of using the DSE chlamydospore aqueous solution is as follows: dilute the DSE chlamydospore aqueous solution to a concentration of 1×10⁻⁶ chlamydospores. 5 ~1×10 8 CFU / mL; optionally, the concentration of the chlamydospores is 1×10⁻⁶.5 ~1×10 6 CFU / mL; optionally, the concentration of the chlamydospores is 1×10⁻⁶. 6 CFU / mL.

[0019] The fifth aspect of this invention provides the application of the above-mentioned DSE thick-walled spore aqueous solution in the control of bacterial wilt of tomatoes.

[0020] Furthermore, the method of using the DSE chlamydospore aqueous solution is as follows: dilute the DSE chlamydospore aqueous solution to a concentration of 1×10⁻⁶ chlamydospores. 5 ~1×10 7 CFU / mL, optionally 1×10 6 ~1×10 7 CFU / mL, optionally 1×10 7 CFU / mL.

[0021] The sixth aspect of the present invention provides the application of the above-mentioned DSE thick-walled spore aqueous solution in promoting plant growth, wherein the plant includes at least one of rice and tomato.

[0022] In some embodiments, the plant is tomato, and the method of using the DSE chlamydospore aqueous solution is as follows: dilute the DSE chlamydospore aqueous solution to a concentration of 1×10⁻⁶ chlamydospores. 5 ~1×10 7 CFU / mL, optionally 1×10 6 ~1×10 7 CFU / mL, optionally 1×10 7 CFU / mL.

[0023] In other embodiments, the plant is rice, and the method of using the DSE chlamydospore aqueous solution is as follows: dilute the DSE chlamydospore aqueous solution to a concentration of 1×10⁻⁶ chlamydospores. 5 ~1×10 8 CFU / mL, optionally 1×10 5 ~1×10 6 CFU / mL, optionally 1×10 6 CFU / mL.

[0024] The beneficial effects of this invention are as follows: This invention provides a DSE chlamydospore aqueous solution. By rationally optimizing the dosage of DSE chlamydospores and excipients, the product properties of the aqueous solution are improved, and the chlamydospore content in the obtained aqueous solution can reach 2.54 × 10⁻⁶. 8With a concentration of CFU / mL and a pH of 7.48, all indicators meet national standards. It can be stored at room temperature for over 12 months, extending its shelf life by more than 9 months compared to conventional conidial fungal agents. Experiments show that this DSE chlamydospore aqueous solution effectively controls bacterial wilt in tomatoes and black-streaked dwarf disease in southern rice, improves the growth of both tomato and rice plants, and significantly increases the expression of five defensive enzymes (PAL, PPO, POD, CAT, and SOD) in both plants, achieving control effects comparable to or even superior to the positive control *Trichoderma harzianum*. The DSE chlamydospore aqueous solution provided by this invention is simple to prepare, cost-effective, and exhibits excellent performance, making it suitable for large-scale promotion and use as a novel biological pesticide product. Attached Figure Description

[0025] One or more embodiments are illustrated by way of example with reference to the accompanying drawings, and these illustrative examples are not intended to limit the embodiments. The term "illustrative" as used herein means "serving as an example, embodiment, or illustration." Any embodiment illustrated herein as "illustrative" is not necessarily to be construed as superior to or better than other embodiments.

[0026] Figure 1 The growth status of tomatoes after 15 days of seed soaking treatment in Example 5 is shown below: A is the comparison between tomatoes in group T1 and group CK3; B is the comparison between tomatoes in group T2 and group CK3; C is the comparison between tomatoes in group T3 and group CK3; and D is the comparison between tomatoes in group T4 and group CK3.

[0027] Figure 2 The figures show the growth status of tomatoes in Example 6 after 30 days of inoculation with Ralstonia solanacearum. Specifically, A represents the growth status of tomatoes in group CK4 after 30 days of inoculation with Ralstonia solanacearum; and B represents the growth status of tomatoes in group T6 after 30 days of inoculation with Ralstonia solanacearum.

[0028] Figure 3 The table shows the expression of defensive enzymes in tomato plants under different treatment groups in Example 7, where: A represents PAL expression; B represents PPO expression; C represents POD expression; D represents CAT expression; and E represents SOD expression.

[0029] Figure 4 This shows the growth status of rice seeds after 15 days of soaking treatment in Example 8.

[0030] Figure 5 This shows the growth status of rice 30 days after inoculation with the virus-carrying white-backed planthopper in Example 9.

[0031] Figure 6 The table shows the expression of defense enzymes in rice plants under different treatment groups in Example 10, where: A represents PAL expression; B represents PPO expression; C represents POD expression; D represents CAT expression; and E represents SOD expression.

[0032] Information on the preservation of biological materials

[0033] The strain information deposited in this application is as follows:

[0034] Guangxi Cladosporium ( Cladophialophora guangxiense HX2, its classification name is Cladophialophora guangxiense It is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, on September 5, 2024, with accession number CGMCC NO. 41498. Detailed Implementation

[0035] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise expressly stated, throughout the specification and claims, the term "comprising" or its variations such as "including" or "comprising of," etc., will be understood to include the stated elements or components, and does not exclude other elements or other components.

[0036] Furthermore, to better illustrate the present invention, numerous specific details are set forth in the following detailed embodiments. Those skilled in the art will understand that the present invention can still be practiced even without certain specific details. In some embodiments, materials, elements, methods, and means well known to those skilled in the art are not described in detail in order to highlight the spirit of the invention.

[0037] The preparation method provided by this invention is illustrated below using the *Cladosporium guangxiense* strain HX2 as an example. *Cladosporium guangxiense* ( Cladophialophora guangxiense HX2 is a DSE strain isolated and screened from sugarcane fields. It is currently deposited at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing; deposit date: September 5, 2024; classification and nomenclature: Cladophialophora guangxiense Accession number: CGMCC NO. 41498.

[0038] The following examples use *Ralstonia solanacearum* (tomato *Ralstonia solanacearum*). Ralstonia solanacearumGg24 was kindly provided by Professor Yuan Gaoqing's team from the College of Agriculture, Guangxi University. The tomato variety used for the trial was Yifeng Generation 1, purchased from Nanning Yifeng Agricultural Technology Co., Ltd. The rice variety used in the pot experiment was "Taichung Native 1 (TN1)," a variety susceptible to Southern Rice Black-Streaked Dwarf Disease. White-backed planthoppers were provided by the Virology Laboratory of the Plant Protection Institute, Guangxi Academy of Agricultural Sciences. All plants were reared using TN1 rice seedlings that had not been treated with any pesticides, under the following conditions: temperature 29℃, humidity 70-80%, and light:D = 16h:8h.

[0039] The culture medium formulation used in the following embodiments is as follows:

[0040] Potato glucose agar (PDA) medium: 200g potato, 20g glucose, 15g agar, 1000mL distilled water.

[0041] Basic culture medium: 84g molasses, 25g soybean flour, 26g corn flour, 1000mL distilled water.

[0042] LA agar medium: 10g tryptone, 5g yeast extract, 10g NaCl, 15g agar, 1000mL distilled water.

[0043] LB liquid medium: 10g tryptone, 5g yeast extract, 10g NaCl, 1000mL distilled water.

[0044] Calcium propionate, sodium benzoate, potassium sorbate, dextrin, humic acid, carboxymethyl cellulose, sodium acetate, and sodium citrate used in the following embodiments were all purchased from Guangxi Nanning Yikesong Biotechnology Co., Ltd. *Trichoderma harzianum* was purchased from Longwo Horticulture Luoyang Co., Ltd. Peroxidase (POD), polyphenol oxidase (PPO), phenylalanine ammonialyase (PAL), catalase (CAT), and superoxide dismutase (SOD) kits were all purchased from Suzhou Grace Biotechnology Co., Ltd.

[0045] The data obtained in the following examples were statistically analyzed using Microsoft Excel 2021 and DPS 7.05. The significance of differences was tested using Duncan's new multiple range method in one-way ANOVA, and Origin 2024 was used for graphing.

[0046] Example 1: Preparation of DSE thick-walled spore plasma

[0047] The preserved HX2 strain was inoculated onto PDA agar plates and activated at 28°C for 10 days. It was then transferred to basal medium and cultured in a constant-temperature shaker at 28°C and 170 rpm for 14 days. After calculating the chlamydospore content using the hemocytocyte count method, the chlamydospore fermentation broth was filtered through three layers of sterile gauze to obtain chlamydospore spore paste, containing 2.35 × 10⁻⁶ spores. 8 CFU / mL.

[0048] Example 2: Screening and Dosage Optimization of Preservatives

[0049] Under aseptic conditions, 30 mL of the thick-walled spore paste prepared in Example 1 was added to a sterilized 50 mL centrifuge tube, and DSE thick-walled spore aqueous solutions were prepared according to the following formulations:

[0050] Formula 1: Add potassium sorbate aqueous solution (concentration of 500 mg / mL) at volume fractions of 0.10%, 0.20%, 0.30%, 0.40%, and 0.50%, respectively, so that the mass concentration of potassium sorbate in DSE thick-walled spore aqueous solution is 0.5 mg / mL, 1.0 mg / mL, 1.5 mg / mL, 2.0 mg / mL, and 2.5 mg / mL, respectively;

[0051] Formula 2: Add calcium propionate aqueous solution (concentration of 500 mg / mL) at volume fractions of 0.10%, 0.20%, 0.30%, 0.40%, and 0.50%, respectively, so that the mass concentration of calcium propionate in DSE thick-walled spore aqueous solution is 0.5 mg / mL, 1.0 mg / mL, 1.5 mg / mL, 2.0 mg / mL, and 2.5 mg / mL, respectively;

[0052] Formula 3: Add sodium benzoate aqueous solution (concentration of 500 mg / mL) at volume fractions of 0.10%, 0.20%, 0.30%, 0.40%, and 0.50%, respectively, so that the mass concentration of sodium benzoate in DSE thick-walled spore aqueous solution is 0.5 mg / mL, 1.0 mg / mL, 1.5 mg / mL, 2.0 mg / mL, and 2.5 mg / mL, respectively.

[0053] An additional control group (CK1) was set up, which did not add any preservatives but only an equal volume of sterile water; each treatment was set up in triplicate. The centrifuge tubes were sealed and stored at room temperature in the dark. After 30 days, samples were taken, and the effective viable bacteria count and the number of contaminating bacteria were calculated using the dilution plating method. The contamination rate was also calculated, as was the spore germination rate.

[0054] Contamination rate = (Number of other non-target colonies / Total number of colonies) × 100%.

[0055] Method for determining the germination rate of chlamydospores: The test solution (preservative + spore paste) was diluted 10 times and spread on a PDA plate. After incubation at 28℃ for 30 h, the plate was placed under a microscope to count the number of germinating chlamydospores. Five replicates were set up for each treatment. Results with large differences were discarded, and the average value of three replicates was selected as the result for analysis. The germination rate was expressed as a percentage, with 100 spores per plate as the baseline.

[0056] The measurement results are shown in Table 1. When calcium propionate was used as a preservative, there were no significant differences in spore germination rate and effective viable cell count at different concentrations, and both were significantly lower than those in the CK1 treatment, while the number of miscellaneous bacteria was significantly higher than that in the CK1 treatment. Sodium benzoate at a concentration of 0.10-0.20% significantly improved the spore germination rate, but the number of effective viable bacteria was significantly lower than that in the CK1 treatment and the calcium propionate group, and the sodium benzoate concentration of 0.30% caused cell death. Potassium sorbate at a concentration of 0.30% also caused spore death, but a high spore germination rate could be obtained in the range of 0.10-0.20%. In particular, when the potassium sorbate concentration was 0.10%, the spore germination rate and the number of effective viable bacteria reached the highest level, significantly higher than those in the CK1 treatment and the calcium propionate group, indicating that potassium sorbate can effectively kill miscellaneous bacteria, and at an appropriate dosage, it is safe for HX2 cells and chlamydospores. Therefore, potassium sorbate can be used as the best preservative for this aqueous solution, and the optimal dosage is 0.10% (i.e., the mass concentration of potassium sorbate in DSE thick-walled spore aqueous solution is 0.5 mg / mL).

[0057] Table 1. Effects of preservative type and dosage on HX2 thick-walled spore aqueous solution

[0058]

[0059] *Different lowercase letters in the same column of the table indicate significant differences between treatments. P <0.05).

[0060] Example 3: Screening and Dosage Optimization of Protective Agents

[0061] Under aseptic conditions, 30 mL of the thick-walled spore paste prepared in Example 1 was added to a sterilized 50 mL centrifuge tube, and DSE thick-walled spore aqueous solutions were prepared according to the following formulations:

[0062] Formula 3: Add sodium carboxymethyl cellulose aqueous solution (concentration of 500 mg / mL) at volume fractions of 0.10% and 0.20% respectively, so that the mass concentration of sodium carboxymethyl cellulose in DSE thick-walled spore aqueous solution is 0.5 mg / mL and 1 mg / mL respectively;

[0063] Formula 4: Add mineral-derived sodium humate aqueous solution (concentration of 500 mg / mL) at volume fractions of 0.10% and 0.20% respectively, so that the mass concentration of mineral-derived sodium humate in DSE thick-walled spore aqueous solution is 0.5 mg / mL and 1 mg / mL respectively;

[0064] Formula 5: Add sodium citrate aqueous solution (concentration of 500 mg / mL) at volume fractions of 0.10% and 0.20% respectively, so that the mass concentration of sodium citrate in DSE thick-walled spore aqueous solution is 0.5 mg / mL and 1 mg / mL respectively;

[0065] Formula 6: Add dextrin aqueous solution (concentration of 500 mg / mL) at volume fractions of 0.10% and 0.20% respectively, so that the mass concentration of dextrin in DSE thick-walled spore aqueous solution is 0.5 mg / mL and 1 mg / mL respectively;

[0066] Formula 7: Add sodium acetate aqueous solution (concentration of 500 mg / mL) at volume fractions of 0.10% and 0.20% respectively, so that the mass concentration of sodium acetate in DSE thick-walled spore aqueous solution is 0.5 mg / mL and 1 mg / mL respectively.

[0067] An additional control group (CK2) was set up, which did not add any protectant but only added an equal volume of sterile water; each treatment was set up in triplicate. The centrifuge tubes were sealed and stored at room temperature in the dark. After 30 days, samples were taken, and the effective viable bacteria count and the number of contaminating bacteria were calculated using the dilution plating method provided in Example 2, and the contamination rate was calculated. At the same time, the spore germination rate was also calculated.

[0068] The experimental results are shown in Table 2. The spore germination rates of the groups with added sodium carboxymethyl cellulose and dextrin were comparable and significantly higher than the other groups. However, the effective viable cell count was lower in the group with added sodium carboxymethyl cellulose, while the effective viable cell count in the group with added dextrin was significantly higher than the other groups. In particular, when the dextrin dosage was 0.20%, not only was the spore germination rate highest, but the detected effective viable cell count also reached the highest level, exceeding the other groups by more than 6 times. This indicates that dextrin can effectively maintain the stability of the aqueous solution system, thereby effectively improving the spore germination rate and cell survival rate. Therefore, dextrin was selected as the best protectant for this aqueous solution, and the optimal dosage was 0.20% (i.e., the mass concentration of dextrin in the DSE thick-walled spore aqueous solution was 1.0 mg / mL).

[0069] Table 2. Effects of different protectants on HX2 thick-walled spore aqueous solution

[0070]

[0071] *Different lowercase letters in the same column of the table indicate significant differences between treatments. P<0.05).

[0072] Example 4: Preparation of DSE Thick-walled Spore Aqueous Solution

[0073] A DSE (dichlorocystis suis) aqueous solution is prepared by the following method:

[0074] (1) Preparation of chlamydospore slurry: The preserved HX2 strain was inoculated onto PDA medium plates and activated at 28°C for 10 days. Then it was transferred to basal medium and cultured in a constant temperature shaker at 28°C and 170 r / min for 14 days for later use. After calculating the chlamydospore content using the hemocytocyte count method, the chlamydospore fermentation broth was filtered through three layers of sterile gauze to obtain chlamydospore slurry for later use.

[0075] (2) Preparation of finished aqueous solution: Add 0.10% potassium sorbate aqueous solution (concentration of 500 mg / mL) and 0.20% dextrin aqueous solution (concentration of 500 mg / mL) to the above-mentioned thick-walled spore slurry, so that the mass concentration of potassium sorbate in the DSE thick-walled spore aqueous solution is 0.5 mg / mL and the mass concentration of dextrin is 1.0 mg / mL, stir evenly, and obtain the finished DSE thick-walled spore aqueous solution.

[0076] Referring to GB20287—2006 standard, the spore content, contamination rate, water content, fineness and pH of the prepared DSE thick-walled spore aqueous solution were determined, and the wetting time and suspension rate were determined using the method provided in Example 2.

[0077] Table 3. Quality Indicators of HX2 Thick-walled Spore Aqueous Solution

[0078]

[0079] The test results are shown in Table 3. The amount of chlamydospores in the finished DSE chlamydospore aqueous solution is 2.54 × 10⁻⁶. 8 The concentration of CFU / g is free of contaminants, the suspension rate is 71.5%, and the pH is 7.48, which meets the national standard for agricultural microbial agents (GB20287—2006).

[0080] Example 5: Effects of DSE Thick-walled Spore Aqueous Formulation on Tomato Growth

[0081] The effects of the DSE thick-walled spore aqueous solution prepared in Example 4 on tomato growth were verified. Tomato seeds were surface-sterilized with a 0.5% sodium hypochlorite solution for 15-20 minutes, followed by rinsing with sterile water 3-5 times to remove exogenous microorganisms. The seeds were then germinated in sterile water at a suitable temperature for 72 hours. Five treatment groups were set up: T1, T2, T3, and T4, each using a concentration of 1×10⁻⁶. 8 1×10 71×10 6 1×10 5 Seeds were soaked in HX2 thick-walled spore liquid inoculant at CFU / mL for 30 min; control group (CK3) seeds were soaked in water for 30 min and sown in seedling trays (55cm×25cm, 50 holes) containing an equal amount of sterilized seedling substrate, with 1 seed per tray, 15 seedlings per replicate, and 3 replicates per treatment. Root length, plant height, stem diameter, fresh weight, and dry weight were measured after 15 days.

[0082] The growth status of tomatoes after soaking seeds for 15 days is as follows Figure 1 As shown in the figure, the tomatoes treated with T2 to T4 all grew significantly better than the tomatoes treated with CK3, exhibiting taller plants, longer roots, more leaves, darker color, and larger area.

[0083] The measurement results are shown in Table 4. Statistical analysis of various growth indicators of tomato plants after 15 days of cultivation revealed that, among different concentrations of HX2 thick-walled spore liquid inoculant treatments, except for T1 (10... 8 Except for T2 (10 CFU / mL) treatment, which significantly inhibited tomato growth, all other treatments had varying degrees of growth-promoting effects. 7 The growth-promoting effect of the group with CFU / mL was the most significant, with all tomato indicators being significantly higher than those of the other groups. Among them, the root length, plant height, stem diameter, fresh weight, and dry weight increased by 50.91%, 51.52%, 25.12%, 75.44%, and 128.47% respectively compared to CK3.

[0084]

[0085] Table 4. Growth-promoting effects of different concentrations on tomatoes

[0086] *Different lowercase letters in the same column of the table indicate significant differences between treatments. P <0.05).

[0087] Based on the above information, it is evident that the DSE thick-walled spore aqueous solution provided in this application can effectively promote the growth of tomato plants, with T2 (10) being the most effective. 6 The CFU / mL ratio has the best reproductive effect.

[0088] Example 6: Control of Bacterial Wilt in Tomatoes by DSE Thick-walled Spore Aqueous Solution

[0089] The efficacy of the DSE thick-walled spore aqueous solution prepared in Example 4 in controlling bacterial wilt of tomato was verified. Five treatment groups (CK4 and T5~T8) were set up according to the method in Example 5, with the addition of treatment T9: spraying with a 2 g / L Trichoderma harzianum diluted solution after inoculation with the pathogen.

[0090] Preparation of *Ralstonia solanacearum* bacterial suspension: The pathogen Gg24 was inoculated onto LA medium for activation. After 2 days, single colonies were picked and inoculated onto LB medium. The medium was then incubated on a shaker at 180 rpm at 30°C for 2 days. The bacterial concentration (OD value) was measured using a UV-Vis spectrophotometer. OD 600 Prepare a bacterial solution with nm=1. After pricking the roots of tomato plants with a 0.4 mm diameter pin, inoculate each plant with 10 mL of Gg24 bacterial solution by root irrigation. Investigate disease incidence at 15 and 30 days. Calculate the disease index and control efficacy according to "Guidelines for Field Efficacy Trials of Pesticides, Part 32: Control of Bacterial Wilt in Tomato" NYT1464.32-2010.

[0091] Disease index = ∑ (number of plants at each disease level × representative value at each level) / (total number of plants surveyed × highest representative value) × 100;

[0092] Prevention efficacy (%) = (Control disease index - Treatment disease index) / Control disease index × 100%.

[0093] The growth status of tomatoes 30 days after inoculation with the pathogen is as follows Figure 2 As shown in the figure, the tomatoes treated with T6 grew significantly better than those treated with CK4, with taller, stronger plants and more leaves that were darker in color and larger in area. The disease index and control efficacy of tomatoes 15 and 30 days after pathogen inoculation are shown in Tables 5 and 6, respectively. At 15 and 30 days after inoculation, the disease index of tomatoes treated with T6 was comparable to that of the positive control T9 (Trichoderma harzianum). However, at 15 days, the disease index of the T7 treatment was significantly lower than that of the T9 treatment, while the control efficacy (53.50%) was significantly higher than that of the T9 treatment. At this time, the control efficacy of the T5 treatment was significantly lower than that of the T7 treatment. At 30 days, the control efficacy of the T6 treatment was significantly improved (reaching 48.18%), and significantly higher than that of the T7 and T9 treatments. This indicates that the DSE thick-walled spore aqueous solution of this application, at a specific spore concentration (e.g., a treatment concentration of 1×10⁻⁶), is effective. 7 The control efficacy against bacterial wilt of tomato (at CFU / mL) showed an increasing trend over time. After 15 days of treatment, the disease index of group T5 was significantly higher than that of CK4, and the control efficacy was negative. This may be because 15 days after treatment is the early stage of disease development, and some plants rapidly developed disease phenotypes due to the adverse conditions caused by pathogen invasion, even showing higher disease indices than the control. However, plants have their own regulatory capacity, and DSE thick-walled spore aqueous solution can also induce plant immune activation and improve plant resistance. Therefore, in the later stages (30 days after treatment), the disease index began to decrease significantly, and the control efficacy improved.

[0094] Table 5. Effects of different concentrations on the control of bacterial wilt in tomato after 15 days.

[0095]

[0096] *Different lowercase letters in the same column of the table indicate significant differences between treatments. P <0.05).

[0097] Table 6. Effects of different concentrations on the control of bacterial wilt in tomato after 30 days.

[0098]

[0099] *Different lowercase letters in the same column of the table indicate significant differences between treatments. P <0.05).

[0100] Based on the above information, it can be seen that the DSE thick-walled spore aqueous solution provided in this application can effectively control bacterial wilt of tomatoes, and its control effect is no less than or even higher than that of positively treated Trichoderma harzianum.

[0101] Example 7: Effect of DSE thick-walled spore aqueous solution on the activity of defensive enzymes in tomato plants

[0102] To determine whether the tomato plant's defense system is activated by HX2 chlamydospores, the activities of five defense enzymes (PAL, PPO, POD, CAT, and SOD) were tested at 1, 7, and 14 days after pathogen inoculation. Figure 3 The results showed that the activities of PAL, PPO, and POD in tomato plants treated with liquid inoculant T6 were highest 14 days after pathogen inoculation, increasing by 0.96, 0.89, and 0.18 times, respectively, compared to CK4. CAT activity peaked at 1 day, increasing by 0.84 times, while SOD activity peaked at 7 days, increasing by 0.39 times. These findings indicate that HX2 chlamydospores can significantly enhance the activity of defensive enzymes in tomato plants, thereby strengthening their resistance to Ralstonia solanacearum.

[0103] Example 8: Effects of DSE Thick-walled Spore Aqueous Formulation on Rice Growth

[0104] The effects of the DSE thick-walled spore aqueous solution prepared in Example 4 on rice growth were verified. Treatment groups (T10-T13) and a control group (CK5) were set up in the same manner as in Example 5. Rice seeds were treated with sterile water for 48 hours to promote germination, with 20 seeds / cup (16cm×8cm) per replicate, repeated 3 times. Plant height, stem diameter, root length, fresh weight, and dry weight were measured after 15 days.

[0105] The growth status of rice seeds after 15 days of soaking is as follows Figure 4As shown in the figure, the rice treated with T10 to T13 all grew significantly better than the rice treated with CK5, exhibiting longer roots, more tillers, and a more developed root system; among them, the rice treated with T12 grew the best.

[0106] The measurement results are shown in Table 7. Statistical analysis of various growth indicators of rice plants after 15 days of cultivation revealed that different concentrations of HX2 thick-walled spore liquid inoculant treatment all had varying degrees of growth-promoting effects on rice plants. Among them, T12 (10... 6 The growth-promoting effect of CFU / mL was the most significant. All indicators of rice were not lower than or even significantly higher than those of the other groups. Among them, root length, plant height, stem diameter, fresh weight and dry weight increased by 40.32%, 18.63%, 11.59%, 76.39% and 35.48% respectively compared with the control.

[0107] Table 7. Growth-promoting effects of different concentrations on rice

[0108]

[0109] *Different lowercase letters in the same column of the table indicate significant differences between treatments. P <0.05).

[0110] Example 9: Control of DSE Thick-walled Spore Aqueous Solution against Southern Rice Black-streaked Dwarf Disease

[0111] The efficacy of the DSE thick-walled spore aqueous solution prepared in Example 4 in controlling southern rice black-streaked dwarf disease was verified. Treatment groups (CK6 and T14~T18) were set up in the same manner as in Example 5, and the seeds were treated with sterile water for 48 hours to promote germination. Each replicate consisted of 20 seeds / cup (16cm×8cm) and was repeated 3 times.

[0112] Seven-day-old rice seedlings were inoculated with infected white-backed planthoppers at a rate of 0.5 planthoppers per seedling. The planthoppers were shaken off twice daily, once in the morning and once in the afternoon, to ensure even infection of the seedlings. Two days after infection, the planthoppers were removed and the rice seedlings were transplanted to large pots (65cm×25cm×20cm) with 15 seedlings per pot. This process was repeated three times. Disease incidence was assessed at 15 and 30 days, and the disease incidence and control efficacy were calculated using the following methods.

[0113] Incidence rate (%) = Number of infected plants / Total number of plants × 100%;

[0114] Prevention efficacy (%) = (control incidence rate - treatment incidence rate) / control incidence rate × 100%.

[0115] An investigation of rice plant disease incidence 15 and 30 days after virus inoculation revealed that, 15 days later, infected rice plants exhibited significantly slower growth, stunted growth, and a darker green color, symptoms that persisted throughout the plant's growth. The growth status of rice 30 days after virus inoculation was as follows... Figure 5 As shown, rice plants treated with T16 exhibited significantly better growth than those in the CK6 group. The number of roots, stem length, stem diameter, and leaf length and number in the T16 group were all significantly increased compared to the CK6 group. The disease incidence and control efficacy of rice 30 days after inoculation are shown in Table 8. The disease incidence rate of rice treated with T16 was significantly lower than that treated with T18, while the control efficacy was significantly higher. The disease incidence and control efficacy of T17 were second lowest, but comparable to those of T18. This indicates that both T16 and T17 treatments significantly reduced the disease incidence rate and significantly improved the control efficacy, with T16 showing the best control effect, reaching as high as 55.1%.

[0116] Based on the above information, it can be seen that the DSE thick-walled spore aqueous solution provided in this application can effectively control rice black-streaked dwarf disease in southern China, and its control effect is significantly higher than that of positively treated Trichoderma harzianum.

[0117] Table 8. Control effects of different treatments on black-streaked dwarf disease in southern rice.

[0118]

[0119] *Different lowercase letters in the same column of the table indicate significant differences between treatments. P <0.05).

[0120] This study comprehensively evaluated indicators such as spore germination rate and effective viable bacteria count, and screened potassium sorbate as the best preservative and dextrin as the best protective agent. The effects of each indicator were best when the addition amount was 0.10% and 0.20%, respectively. This can significantly optimize the physicochemical properties of the formulation and provide a guarantee for the long-term preservation of the formulation and its stable use in the field under ultraviolet environment.

[0121] Example 10: Effects of DSE thick-walled spore aqueous solution on the activity of defensive enzymes in rice plants

[0122] The activities of five defense enzymes (PAL, PPO, POD, CAT, and SOD) were measured in rice plants 1, 7, and 14 days after inoculation. Figure 6It was found that, compared with CK6, the T16 treatment significantly increased the activity of defensive enzymes. The activities of PPO, CAT, and SOD reached their highest levels 14 days after inoculation, increasing by 1.21 times, 0.78 times, and 1.5 times, respectively. PAL content peaked at 7 days after inoculation, increasing by 0.78 times, while POD reached its highest level 1 day after inoculation, increasing by 0.30 times. These findings indicate that different types of HX2 thick-walled spore inoculants can significantly increase the content of defensive enzymes in rice plants, thereby enhancing their resistance to Southern Rice Black-Streaked Dwarf Disease.

[0123] Example 11: Stability test of DSE thick-walled spore aqueous solution

[0124] The storage stability of the aqueous solution provided in this application was tested under normal temperature and 4°C storage conditions, namely, the concentration of viable bacteria (cells / g) and the spore germination rate were tested, and samples were taken for testing once a month.

[0125] The viable bacteria counting method is as follows: take 1 mL of aqueous solution and add it to sterile water, dilute it in a 10-fold serial manner, take 0.1 mL and spread it on a PDA plate, count the colonies after 7 days to calculate the viable bacteria count, and repeat three times.

[0126] Viable bacterial concentration (CFU / g) = number of colonies on plate × dilution factor / inoculation volume (mL).

[0127] Method for determining the germination rate of chlamydospores: PDA plates were incubated at 28℃ for 30 h, and the number of germinating chlamydospores was counted using a microscope (germination standard is that the length of the germ tube exceeds half of its own diameter). Five replicates were set for each treatment, and results with large differences were discarded. Three replicates were selected, and the average value was taken as the result for analysis. The germination rate was expressed as a percentage, with 100 spores per plate as the baseline.

[0128] Table 9. Results of the stability test of the aqueous solution

[0129]

[0130] As shown in the table above, the spore germination rate decreased with prolonged storage time. Under normal temperature storage conditions, as the storage time increased from 1 month to 11 months, the germination rate of chlamydospores continuously decreased from 99.51% to 81.67%, and the viable cell count was still 2.02 × 10⁻⁶ at the 12th month. 8 After 10 months of storage at 4℃, the germination rate of the chlamydospores remained at 82.12%, with a viable cell count of 2.02 × 10⁻⁶. 8Therefore, the shelf life of the DSE thick-walled spore aqueous solution provided in this application meets the relevant requirements of the national standard "Agricultural Microbial Agents" (GB 20287-2006) for aqueous formulations (shelf life ≥ 3 months), and can be stored at room temperature for more than 12 months, which is even more than 9 months longer than conventional conidial aqueous solutions.

[0131] This study confirms that HX2 chlamydospore aqueous solution has significant control effects on both diseases, but the optimal application concentration differs. For tomato bacterial wilt, at 15 and 30 days after inoculation, the disease index of treatment T6 was comparable to the positive control T9 (Trichoderma harzianum). However, at 15 days, the disease index of treatment T7 was significantly lower than that of treatment T9, while its control efficacy (53.50%) was significantly higher than that of treatment T9. At this time, the control efficacy of treatment T6 was significantly lower than that of treatment T7. However, at 30 days, the control efficacy of treatment T6 significantly improved (reaching 48.18%), and was significantly higher than that of treatments T7 and T9. This indicates that the control efficacy of the DSE chlamydospore aqueous solution against tomato bacterial wilt increases over time. It is noteworthy that the increasing control efficacy of treatment T6 over time may be related to the colonization ability of chlamydospores in the tomato rhizosphere. HX2 chlamydospores are highly resistant and can survive and gradually colonize in the root system for a long time. As the colonization amount increases, its biocontrol effect continues to strengthen. For Southern Rice Black-Streaked Dwarf Virus, the incidence rate of T16 treatment was significantly lower than that of T18 treatment, while its control efficacy was significantly higher. T17 treatment had the next lowest incidence rate and control efficacy, but was comparable to T18. This indicates that both T16 and T17 treatments significantly reduced rice disease incidence and significantly improved control efficacy, with T16 showing the best control effect, reaching up to 55.1%. This difference in concentration effect may be related to the pathogenesis of the two diseases and the crop growth characteristics. (Note: The last sentence about tomato bacterial wilt appears unrelated and likely refers to a different topic.) Ralstonia solanacearum The infection is caused by invasion of the vascular bundles, requiring high concentrations of biocontrol bacteria to form a dominant flora and inhibit pathogen infection. However, Southern Rice Black-Streaked Dwarf Disease is caused by a virus transmitted by the white-backed planthopper. HX2 may reduce viral replication and spread within the rice plant by regulating plant resistance, achieving ideal control efficacy at lower concentrations. Furthermore, the T16 treatment showed significantly better control efficacy against rice diseases than *Trichoderma harzianum*, further confirming the unique advantages of the HX2 strain in controlling viral diseases and compensating for the current lack of biocontrol agents for Southern Rice Black-Streaked Dwarf Disease.

[0132] 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 the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A DSE thick-walled spore aqueous solution, characterized in that, including sclerotia from dark septate endophytic fungi, also including preservatives and protectants, the content of the sclerotia in the DSE sclerotia water is 2.54 x 10 8 CFU / mL; The dark-colored septate endophytic fungus is *Cladosporium*, and *Cladosporium* is *Cladosporium guangxiense* (…). Cladophialophora guangxiense The strain HX2, with accession number CGMCC NO. 41498; The preservative is potassium sorbate, and the amount of potassium sorbate added to the DSE thick-walled spore aqueous solution is 0.5 mg / mL; The protectant is dextrin, and the amount of dextrin added to the DSE thick-walled spore aqueous solution is 1.0 mg / mL.

2. A method for preparing the DSE thick-walled spore aqueous solution according to claim 1, characterized in that, The steps include the following: (1) Preparation of thick-walled spore paste: Dark-colored septate endophytic fungal strains are cultured in liquid basal medium, and thick-walled spores are collected from the fermentation broth to obtain thick-walled spore paste; The method for preparing chlamydospore slurry includes: inoculating the chlamydospores onto PDA medium plates, activating them at 28°C, then transferring them to basal medium and culturing them in a constant temperature shaker at 28°C for 14 days; filtering the obtained chlamydospore fermentation broth through three layers of sterile gauze to obtain chlamydospore slurry. (2) Preparation of finished aqueous solution: Add preservatives and protectants to the thick-walled spore slurry in proportion to obtain the finished DSE thick-walled spore aqueous solution.

3. The application of the DSE thick-walled spore aqueous solution according to claim 1 in promoting the expression of plant defense enzymes, wherein the plant is tomato or rice, and the plant defense enzymes include PAL, PPO, POD, CAT and SOD.

4. The application of the DSE thick-walled spore aqueous solution according to claim 1 in the control of rice black-streaked dwarf disease in southern China, characterized in that, The method of using the DSE chlamydospore aqueous solution is as follows: Dilute the DSE chlamydospore aqueous solution to a concentration of 1×10⁻⁶ chlamydospores. 5 ~1×10 8 CFU / mL.

5. The application according to claim 4, characterized in that, The method of using the DSE chlamydospore aqueous solution is as follows: Dilute the DSE chlamydospore aqueous solution to a concentration of 1×10⁻⁶ chlamydospores. 5 ~1×10 6 CFU / mL.

6. The application of the DSE thick-walled spore aqueous solution according to claim 1 in the control of bacterial wilt of tomato, characterized in that, The method of using the DSE chlamydospore aqueous solution is as follows: Dilute the DSE chlamydospore aqueous solution to a concentration of 1×10⁻⁶ chlamydospores. 5 ~1×10 7 CFU / mL.

7. The application according to claim 6, characterized in that, The method of using the DSE chlamydospore aqueous solution is as follows: Dilute the DSE chlamydospore aqueous solution to a concentration of 1×10⁻⁶ chlamydospores. 6 ~1×10 7 CFU / mL.

8. The application of the DSE thick-walled spore aqueous solution according to claim 1 in promoting tomato growth, characterized in that, The method of using the DSE chlamydospore aqueous solution is as follows: Dilute the DSE chlamydospore aqueous solution to a concentration of 1×10⁻⁶ chlamydospores. 5 ~1×10 7 CFU / mL.

9. The application according to claim 8, characterized in that, The method of using the DSE chlamydospore aqueous solution is as follows: Dilute the DSE chlamydospore aqueous solution to a concentration of 1×10⁻⁶ chlamydospores. 6 ~1×10 7 CFU / mL.

10. The application of the DSE thick-walled spore aqueous solution according to claim 1 in promoting rice growth, characterized in that, The method of using the DSE chlamydospore aqueous solution is as follows: Dilute the DSE chlamydospore aqueous solution to a concentration of 1×10⁻⁶ chlamydospores. 5 ~1×10 8 CFU / mL.

11. The application according to claim 10, characterized in that, The method of using the DSE chlamydospore aqueous solution is as follows: Dilute the DSE chlamydospore aqueous solution to a concentration of 1×10⁻⁶ chlamydospores. 5 ~1×10 6 CFU / mL.

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