DSE chlamydospore wettable powder, preparation method and application thereof

Abstract

This invention provides a wettable powder of DSE (dichloropyrophylloides) spores, 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, a carrier, and excipients, wherein the chlamydospore content is greater than 2.35 × 10⁻⁶. 8 CFU / g. This invention improves the properties of wettable powder by rationally optimizing the dosage of DSE chlamydospores and excipients, achieving a chlamydospore content of 2.35 × 10⁻⁶ CFU / g in the finished product. 8 With a concentration of CFU / g, a wetting time of 24.25s, and a suspension rate of 73.8%, all indicators meet national standards. It can be stored at room temperature for over 12 months, extending its shelf life by more than 6 months compared to conventional conidiophore agents. This powder significantly increases the expression of five defense enzymes—PAL, PPO, POD, CAT, and SOD—in tomato and rice plants, thereby activating the plant's defense system. It effectively controls bacterial wilt in tomatoes and black-streaked dwarf disease in southern rice, improving the growth of tomatoes infected with bacterial wilt and rice infected with black-streaked dwarf disease. It is suitable for 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 wettable powder of DSE thick-walled spores, 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 endophytes (DSEs) 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 DSEs 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. Current research often involves directly inoculating plants with mycelium or preparing inoculants using conidia. However, mycelium is prone to inactivation during practical applications, resulting in a very short shelf life (only 3-6 months), making it difficult to meet commercial requirements; and conventional conidial inoculants also have limited shelf life, hindering large-scale application. Summary of the Invention

[0005] To address the above technical problems, this invention provides a wettable powder of DSE thick-walled spores, its preparation method, and its application.

[0006] This invention provides a DSE (dark spore) wettable powder, comprising chlamydospores from at least one dark-colored septate endophytic fungus and a carrier, further comprising a dispersant and a wetting agent with a total mass fraction of 1-10%, and a UV protectant with a mass fraction of 0.5-2.0%, wherein the content of the chlamydospores in the DSE wettable powder is greater than 2.35 × 10⁻⁶. 8 CFU / g.

[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] Further, the carrier is selected from at least one of diatomaceous earth, silica, talc, calcium carbonate, kaolin, and wood ash; optionally, the carrier is selected from at least one of diatomaceous earth, silica, and talc.

[0010] Preferably, the carrier is silica.

[0011] Further, the dispersant is selected from at least one of sodium lignosulfonate, sodium hexametaphosphate, sodium pyrophosphate, PEG8000 and sodium carboxymethyl cellulose, and the wetting agent is selected from at least one of Tween 60, saponin, sodium dodecyl sulfonate and sodium tripolyphosphate, and the mass ratio of the dispersant to the wetting agent is 5:1 to 1:5;

[0012] Preferably, the dispersant is PEG8000, the wetting agent is Tween 60, and the mass ratio of the dispersant to the wetting agent is 1:2 to 1:4, optionally 1:3.

[0013] Furthermore, the total mass fraction of the dispersant and the wetting agent in the DSE thick-walled spore wettable powder is 5-8%, optionally 8%.

[0014] Furthermore, the ultraviolet protectant is selected from at least one of dextrin, sodium alginate, xanthan gum, mineral-derived sodium humate, and ascorbic acid;

[0015] Preferably, the ultraviolet protectant is ascorbic acid, and the mass fraction of ascorbic acid in the DSE chlamydospore wettable powder is 0.5-1.5%, optionally 1.0%.

[0016] Another aspect of the present invention provides a method for preparing the above-mentioned DSE thick-walled spore wettable powder, comprising the following steps:

[0017] (1) Preparation of thick-walled spore paste: Dark-colored septate endophytic fungal strains are cultured in liquid basal medium, and thick-walled spores in the fermentation broth are collected to obtain thick-walled spore paste;

[0018] Preferably, the method for preparing the thick-walled spore slurry includes: inoculating a dark-colored septate endophytic fungal strain onto a PDA medium plate, activating it at 28°C, then transferring it to a basal medium and culturing it in a constant temperature shaker at 28°C for 14 days; filtering the obtained thick-walled spore fermentation broth through three layers of sterile gauze to obtain the thick-walled spore slurry.

[0019] (2) Preparation of mother powder: Add a carrier that has been sterilized at high temperature to the thick-walled spore slurry, so that the mass ratio of the thick-walled spore slurry to the carrier is 1:3; put the thick-walled spore slurry with the carrier into an oven at 40±1℃ and dry it for 6 hours, then grind it into powder to obtain the mother powder;

[0020] (3) Preparation of finished powder: Add dispersant and wetting agent according to proportion, and add ultraviolet protectant according to proportion, mix evenly to obtain finished DSE thick-walled spore wettable powder.

[0021] The third aspect of this invention provides the application of the above-mentioned DSE thallus wettable powder 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.

[0022] The fourth aspect of this invention provides the application of the above-mentioned DSE chlamydospore wettable powder in the control of rice black-streaked dwarf disease in southern China.

[0023] Furthermore, the method of using the DSE chlamydospore wettable powder is as follows: dilute the DSE chlamydospore wettable powder to a concentration of 1×10⁻⁶ chlamydospores. 5 ~1×10 6 CFU / mL, optionally 1×10 6 CFU / mL.

[0024] The fifth aspect of this invention provides the application of the above-mentioned DSE chlamydospore wettable powder in the control of bacterial wilt of tomatoes.

[0025] Furthermore, the method of using the DSE chlamydospore wettable powder is as follows: dilute the DSE chlamydospore wettable powder to a concentration of 1×10⁻⁶ chlamydospores. 5 ~1×10 8 CFU / mL, optionally 1×10 5 ~1×10 7 CFU / mL, optionally 1×10 7 CFU / mL.

[0026] The sixth aspect of the present invention provides the application of the above-mentioned DSE chlamydospore wettable powder in promoting the growth of diseased plants, said diseased plants including at least one of rice infected with Southern Rice Black-Streaked Dwarf Virus and tomato infected with Ralstonia solanacearum.

[0027] In some embodiments, the plant is tomato, and the method of using the DSE chlamydospore wettable powder is as follows: dilute the DSE chlamydospore wettable powder to a concentration of 1×10⁻⁶ chlamydospores. 5 ~1×10 8 CFU / mL, optionally 1×10 5 ~1×10 7 CFU / mL, optionally 1×10 7 CFU / mL.

[0028] In other embodiments, the plant is rice, and the method of using the DSE chlamydospore wettable powder is as follows: dilute the DSE chlamydospore wettable powder to a concentration of 1×10⁻⁶ chlamydospores. 5 ~1×10 6CFU / mL, optionally 1×10 6 CFU / mL.

[0029] The beneficial effects of this invention are as follows: This invention provides a wettable powder of DSE chlamydospores. By rationally optimizing the dosage of DSE chlamydospores and excipients, the product properties of the wettable powder are improved, and the chlamydospore content in the obtained wettable powder product can reach 2.35 × 10⁻⁶. 8 The DSE (dichlorodiphenyl spore) wettable powder exhibits the following properties: CFU / g, wetting time 24.25s, suspension rate 73.8%, pH 5.71, water content 16.67%, and fineness 98.81%, all meeting national standards. It can be stored at room temperature for over 12 months, extending its shelf life by more than 6 months compared to conventional conidial fungal agents. Experiments show that this DSE significantly increases the expression of five defense enzymes (PAL, PPO, POD, CAT, and SOD) in tomato and rice plants, thereby activating the plant's defense system. It effectively controls bacterial wilt in tomatoes and black-streaked dwarf disease in southern rice, improving the growth of tomato plants infected with bacterial wilt and rice plants infected with black-streaked dwarf disease, achieving control effects comparable to or even superior to the positive control *Trichoderma harzianum*. The DSE wettable powder 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. Attached Figure Description

[0030] 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.

[0031] Figure 1 The effects of dispersant and wetting agent on the properties of DSE chlamydospore wettable powder in Example 3 are shown in the figures: A represents the wetting time and suspension rate of the powder when different ratios of dispersant and wetting agent are added; B represents the chlamydospore germination rate of the powder when different ratios of dispersant and wetting agent are added; C represents the wetting time and suspension rate of the powder when different doses of dispersant and wetting agent are added; and D represents the chlamydospore germination rate of the powder when different doses of dispersant and wetting agent are added.

[0032] Figure 2 The image shows the growth status of tomatoes 30 days after inoculation with Ralstonia solanacearum in Example 6. In this image, A and B are tomato plants in the T2 treatment group, and C and D are tomato plants in the CK4 group.

[0033] Figure 3 The control effects of different treatments on bacterial wilt of tomatoes in Example 6 are shown, where: A is the disease index of tomato plants in different treatment groups; B is the control efficacy of different treatments.

[0034] Figure 4 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.

[0035] Figure 5 The image shows the growth status of rice plants in Example 8 30 days after inoculation with the virus-carrying white-backed planthopper. In this image, A represents rice plants in the T8 treatment group, and B represents rice plants in the CK5 group.

[0036] Figure 6 The table shows the expression of defense enzymes in rice plants under different treatment groups in Example 9, where: A represents PAL expression; B represents PPO expression; C represents POD expression; D represents CAT expression; and E represents SOD expression.

[0037] Information on the preservation of biological materials

[0038] The strain information deposited in this application is as follows:

[0039] 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

[0040] 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.

[0041] 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.

[0042] 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.

[0043] The following examples use *Ralstonia solanacearum* (tomato *Ralstonia solanacearum*). Ralstonia solanacearum Gg24 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.

[0044] The culture medium formulation used in the following embodiments is as follows:

[0045] Potato glucose agar (PDA) medium: 200g potato, 20g glucose, 15g agar, 1000mL distilled water.

[0046] Basic culture medium: 84g molasses, 25g soybean flour, 26g corn flour, 1000mL distilled water.

[0047] LA agar medium: 10g tryptone, 5g yeast extract, 10g NaCl, 15g agar, 1000mL distilled water.

[0048] LB liquid medium: 10g tryptone, 5g yeast extract, 10g NaCl, 1000mL distilled water.

[0049] The diatomaceous earth, silica, talc, calcium carbonate, kaolin, wood ash, sodium lignosulfonate (SLS), sodium hexametaphosphate (SHP), sodium pyrophosphate (SPP), PEG8000, Tween 60, saponin (SP), sodium dodecyl sulfate (SDS), sodium tripolyphosphate (STPP), sodium carboxymethyl cellulose (CMC), dextrin, sodium alginate, xanthan gum, mineral-derived sodium humate, and ascorbic acid (Vc) 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. The peroxidase (POD), polyphenol oxidase (PPO), phenylalanine ammonialyase (PAL), catalase (CAT), and superoxide dismutase (SOD) kits were all purchased from Suzhou Grace Biotechnology Co., Ltd.

[0050] 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.

[0051] Example 1: Preparation of DSE thick-walled spore plasma

[0052] 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.

[0053] Example 2: Screening of Carriers

[0054] Different carriers (diatomaceous earth, silica, talc, calcium carbonate, kaolin, and wood ash) were added to the chlamydospore slurry prepared in Example 1 at a mass ratio of 3:1. The mixture was then sterilized at 121°C for 30 min. After mixing, the mixture was placed in an oven at 40±1°C for 6 h and ground into powder to obtain the formulation master powder. The chlamydospore slurry without added carriers was used as a control (CK1), and the germination rate, suspension rate, wetting time, and adsorption rate of chlamydospores were determined.

[0055] Method for determining the germination rate of chlamydospores: The mother powder was diluted 10 times and spread on PDA plates. The plates were incubated at 28℃ for 30 h. The number of germinating chlamydospores was counted under a microscope. Five replicates were set up for each treatment. Results with large differences were discarded, and three replicates were selected. The average value was used as the result for analysis. The germination rate was expressed as a percentage, with 100 spores per plate as the baseline.

[0056] Suspension rate determination method: Refer to GB / T14825—2006 for determination.

[0057] Wetting time determination method: Refer to GB / T5451—2001 for determination.

[0058] Adsorption rate determination method: Weigh 2g of each carrier and place it in a clean cup. Use a dropper to add the 1.0×10⁻⁶ g of each carrier. 8 CFU / mL HX2 thick-walled spore slurry was added dropwise to different carriers, and stirred with a glass rod until the carriers clumped together. The total mass after adsorption of the thick-walled spore slurry was then weighed, and the adsorption rate of the carriers on the HX2 thick-walled spore slurry was calculated. Carrier adsorption rate (%) = (mass of carriers after adsorption of thick-walled spore slurry - mass of carriers before adsorption) / mass of carriers before adsorption × 100%.

[0059] The measurement results are shown in Table 1. When silica was used as the carrier, the HX2 chlamydospore wettable powder had the highest suspension rate (18.13%), chlamydospore germination rate (78.71%), and adsorption rate (2.52%) among all groups. Furthermore, the suspension rate and adsorption rate were significantly higher than the other groups, while the wetting time (24.97 s) was at a moderate level among the six carriers. The carrier of microbial preparations may inhibit spore germination to some extent. Therefore, the key to formulation lies in the selection of the carrier and the design of the formulation. Inappropriate selection will significantly reduce spore viability, while a suitable carrier can improve its stability and germination potential. This depends on the biocompatibility between the carrier material and the specific microbial spores. Therefore, through comprehensive comparison, silica was identified as the best carrier for the wettable powder.

[0060] Table 1. Effects of different carriers on HX2 chlamydospore wettable powder

[0061]

[0062] *Different lowercase letters in the same column of the table indicate significant differences between treatments. P <0.05).

[0063] Example 3: Screening and Dosage Optimization of Dispersants and Wetting Agents

[0064] Dispersants sodium lignosulfonate (SLS), sodium hexametaphosphate (SHP), sodium pyrophosphate (SPP), PEG8000, sodium carboxymethyl cellulose (CMC), and wetting agents Tween 60, saponins (SP), sodium dodecyl sulfate (SDS), and sodium tripolyphosphate (STPP) were added to the masterbatch (silica + spore paste) at a weight ratio of 1%. No dispersants or wetting agents were added to CK2. The germination rate, wetting time, and suspension rate of chlamydospores were determined according to the method provided in Example 2, and the optimal dispersants and wetting agents were screened.

[0065] The experimental results are shown in Table 2. Except for CK2, the group with added Tween 60 had the shortest wetting time, lowest suspension rate, and highest spore germination rate, showing significant differences from the other groups. The group with added PEG8000 had a spore germination rate comparable to the Tween 60 group, both exceeding 70%, and a wetting time second only to the Tween 60 group, also significantly shorter than the other groups. The suspension rate showed no significant difference from the other groups. Therefore, through comprehensive comparison, PEG8000 and Tween 60 were identified as the optimal dispersant and wetting agent for wettable powders.

[0066] Table 2. Effects of different wetting agents and dispersants on HX2 thick-walled spore wettable powder

[0067]

[0068] *Different lowercase letters in the same column of the table indicate significant differences between treatments. P <0.05).

[0069] The selected optimal dispersant and wetting agent (PEG8000 and Tween 60) were mixed with the masterbatch at mass ratios of 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, and 1:5, respectively. Various indicators were measured to determine the optimal mass ratio. Then, the total amount of dispersant and wetting agent was mixed with the masterbatch at mass fractions of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, and 10%. After drying to form a powder, its various indicators were measured to determine the optimal dosage of wetting agent and dispersant.

[0070] Experimental results for different mass ratios are as follows: Figure 1As shown in A and B in the figure. When the ratio of PEG8000 to Tween 60 is 1:3, the germination rate of thick-walled spores is the highest, the suspension rate is at the highest level, and the wetting time is at the lowest level. Therefore, through comprehensive comparison, 1:3 is identified as the optimal mass ratio of dispersant to wetting agent.

[0071] Experimental results for different addition amounts are as follows Figure 1 As shown in C and D, when the total addition of PEG8000 and Tween 60 was 8%, the germination rate of thick-walled spores was the highest (81.56%), with a relatively high suspension rate and a relatively low wetting time. Similar to the carrier, the addition of wetting agents and dispersants may also affect the consistency of spore germination, and the suitability of the additive components depends on the biocompatibility between the specific components and the specific microbial spores. Therefore, through comprehensive comparison, 8% was determined to be the optimal addition amount for dispersants and wetting agents.

[0072] Example 4: Screening of UV Protectants

[0073] According to the experimental results of Example 3, PEG8000 and Tween 60 (ratio 1:3) were added to the mother powder at a ratio of 8%. Dextrin, sodium alginate, xanthan gum, mineral-derived sodium humate, and ascorbic acid (Vc) were selected as UV protectants for the wettable powder and mixed with the mother powder (fumed silica + PEG8000 + Tween 60 + spore paste) at mass fractions of 0.5%, 1.0%, 1.5%, and 2.0%, respectively. After dilution to an appropriate multiple, 0.2 mL was taken and spread on a PDA plate. The petri dish lid was opened, and the plate was placed at 30 cm under a sterile 20W UV lamp (120 lx) with CK3 (fumed silica + PEG8000 + Tween 60 + spore paste, without UV protectant) for 3 min. The germination rate of chlamydospores was measured using the method provided in Example 2.

[0074] The experimental results are shown in Table 3. After UV irradiation, the spore germination rate of group CK3 was significantly lower than that of groups Tween-60 and PEG8000 in Example 3, indicating that UV irradiation significantly affects spore activity. The spore germination rates of all groups with added UV protectants were higher than those of group CK3 to varying degrees. In particular, the addition of ascorbic acid resulted in the highest spore germination rates at different concentrations, significantly higher than CK3, indicating that ascorbic acid has excellent protective effects on spore germination. When the addition amount was 0.5-1.5%, the spore germination rate was significantly higher than all other groups, with the 1% group showing the highest spore germination rate among all groups. Therefore, ascorbic acid was identified as the optimal UV protectant for wettable powders, and the optimal addition amount was 1%.

[0075] Table 3. Screening of UV Protectants for HX2 Thick-walled Spore Wettable Powder

[0076]

[0077] *Different lowercase letters in the same column of the table indicate significant differences between treatments. P <0.05).

[0078] Example 5: Preparation of DSE Thick-walled Spores Wettable Powder

[0079] A wettable powder of DSE (dichlorocystis suis) is prepared by the following method:

[0080] (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.

[0081] (2) Preparation of master powder: 25% of thick-walled spore slurry was added to 100% of fumed silica (sterilized at 121℃ for 30 min), and placed in an oven at 40±1℃ for 6 h. Thick-walled spore slurry without carrier was used as a control. The mixture was then ground into powder to obtain the preparation master powder.

[0082] (3) Preparation of finished powder: PEG8000 and Tween 60 are added in a ratio of 1:3 (the total mass of the two accounts for 8% of the mother powder), and ascorbic acid is added in a ratio of 1.0%. The mixture is mixed evenly to obtain the finished DSE thallus wettable powder.

[0083] Referring to GB20287—2006 standard, the spore content, contamination rate, moisture content, fineness and pH of the prepared DSE thick-walled spore wettable powder were determined, and the wetting time and suspension rate were determined using the method provided in Example 2.

[0084] The test results are shown in Table 4. The amount of chlamydospores in the finished DSE chlamydospore wettable powder is 2.35 × 10⁻⁶. 8 The concentration of CFU / g is free of contaminants, with a suspension rate of 73.8%, a wetting time of 24.25s, a pH of 5.71, a fineness of 98.81%, and a moisture content of 16.67%, which meets the national standard for agricultural microbial agents (GB20287—2006).

[0085] Table 4. Quality Indicators of HX2 Thick-walled Spores Wettable Powder

[0086]

[0087] Example 6: Control of Bacterial Wilt in Tomatoes by DSE Thick-walled Spore Wettable Powder

[0088] The efficacy of the DSE (thick-walled spores) wettable powder prepared in Example 5 in controlling bacterial wilt of tomato was verified. Tomato seeds were disinfected with a 0.5% sodium hypochlorite solution for 15-20 minutes, followed by rinsing with sterile water 3-5 times to remove surface microorganisms. Germination was then initiated with sterile water for 72 hours. Six treatment groups were established: T1, T2, T3, and T4, each treated with 1×10⁻⁶ sodium hypochlorite solution. 8 1×10 7 1×10 6 1×10 5 Seeds were soaked in HX2 chlamydospore wettable powder (CFU / mL) for 30 min; T5: sprayed with 2 g / L Trichoderma harzianum diluted solution after disease onset; CK4: seeds were soaked in water for 30 min. Seeds from each group were sown in seedling trays (55cm×25cm, 50 holes) containing an equal amount of sterilized seedling substrate, with one seed per tray, 15 seedlings per replicate, and 3 replicates per treatment.

[0089] 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.

[0090] Disease index = ∑ (number of plants at each disease level × representative value at each level) / (total number of plants surveyed × highest representative value) × 100;

[0091] Prevention efficacy (%) = (Control disease index - Treatment disease index) / Control disease index × 100%.

[0092] The growth status of tomatoes 30 days after inoculation with the pathogen is as follows Figure 2 As shown in the figure, A and B represent tomatoes treated with T2, while C and D represent tomatoes treated with CK4. The figure clearly shows that tomatoes treated with T2 grew significantly better than those treated with CK4, with taller, stronger plants and more numerous, darker, and larger leaves. The disease index of tomatoes is shown in the figure. Figure 3As shown in Figure A, at 15 and 30 days after inoculation with the pathogen, the disease index of tomatoes treated with T1-T4 was lower than that of CK4. At 15 days, the disease index of T4 treatment was the lowest, comparable to the positive control T5. At 30 days, the disease index of T1-T4 treatments was significantly lower than that at 15 days, even significantly lower than that of T5 treatment. The control efficacy of DSE chlamydospore wettable powder is as follows... Figure 3 As shown in B, the T4 treatment had the highest efficacy at 15 days, even significantly higher than the T5 treatment; at 30 days, the efficacy of both the T2 and T3 treatments increased significantly, with the T2 treatment showing a significantly higher efficacy than the T5 treatment and an increasing trend over time, while the T3 treatment also achieved a level of efficacy comparable to that of the T5 treatment.

[0093] Based on the above information, it can be seen that the DSE chrysophagus wettable powder product 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.

[0094] Example 7: Determination of the expression of defense enzymes in tomato plants

[0095] To determine whether the tomato plant's defense system would be activated by HX2 chlamydospores, leaf samples from the same parts of tomato plants in groups T2, T5, and CK4 were collected every 1, 7, and 14 days after the plants were inoculated with Ralstonia solanacearum in Example 6. The contents of their defense enzymes (POD, PPO, PAL, CAT, SOD) were determined using a detection kit from Suzhou Grace Biotechnology Co., Ltd.

[0096] The detection results of the five enzymes are as follows: Figure 4 As shown, A represents PAL expression; B represents PPO expression; C represents POD expression; D represents CAT expression; and E represents SOD expression. Compared with CK4, the tomato plants in the T2 treatment group showed significantly increased levels of defensive enzymes, and the levels of all five enzymes were higher than those in T5 (Trichoderma harzianum) on day 7. Among them, PPO (… Figure 4 B in ( ), POD ( Figure 4 C) and CAT Figure 4 The D content in PAL reached its peak at 7 days, increasing by 1.69 times, 3.39 times, and 0.25 times compared to CK4, respectively; while PAL ( Figure 4 A) and SOD ( Figure 4 The content of E) in the tomato plants reached its highest level at 14 days, increasing by 0.44 times and 1.17 times, respectively. These findings indicate that the DSE chlamydospores provided in this application can significantly increase the content of defense enzymes in tomato plants, effectively activate the plant's defense system, and thus enhance its resistance to Ralstonia solanacearum.

[0097] Example 8: Control of DSE Thick-walled Spores Wettable Powder against Southern Rice Black-streaked Dwarf Disease

[0098] The efficacy of the DSE chlamydospore wettable powder prepared in Example 5 in controlling rice black-streaked dwarf disease in southern China was verified.

[0099] The treatment groups (T6~T10) and the control group (CK5) were set up in the same manner as in Example 6. The rice seeds were treated and germinated with sterile water for 48 hours. Each replicate consisted of 20 seeds / cup (16cm×8cm) and was repeated 3 times.

[0100] Rice seedlings planted for 7 days were inoculated with white-backed planthoppers carrying the black-streaked dwarf fungus at a rate of 0.5 planthoppers per seedling. The planthoppers were shaken off twice a day, once in the morning and once in the afternoon, to ensure uniform infection of the rice 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 investigated at 15 days and 30 days, and the disease incidence and control efficacy were calculated using the following methods.

[0101] Incidence rate (%) = Number of infected plants / Total number of plants × 100%;

[0102] Prevention efficacy (%) = (control incidence rate - treatment incidence rate) / control incidence rate × 100%.

[0103] The growth status of rice 30 days after inoculation is as follows Figure 5 As shown in the figure, A represents rice treated with T8, and B represents rice treated with CK5. The figure shows that after 30 days, diseased rice plants exhibited typical symptoms such as slow growth, stunted growth, and a dark green color, which persisted throughout the rice's growth. Rice treated with T8 showed significantly better growth than CK5 rice, with taller, stronger plants and more, longer, and darker-colored leaves. The disease trend was consistent after 15 and 30 days of inoculation. Table 5 shows the incidence rate of rice and the control efficacy of DSE chlamydospore wettable powder. Compared to the positive control T10, both T8 and T9 treatments significantly reduced the incidence rate and significantly improved the control efficacy, with T8 showing the best control effect, reaching 63.64%.

[0104] Based on the above information, it can be seen that the DSE thick-walled spore wettable powder product 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.

[0105] Table 5. Control effects of different treatments on black-streaked dwarf disease in southern rice.

[0106]

[0107] *Different lowercase letters in the same column of the table indicate significant differences between treatments. P <0.05).

[0108] Example 9: Determination of the expression of defense enzymes in rice plants

[0109] To determine whether the defense system of rice plants would be activated by HX2 chlamydospores, leaf samples from the same parts of tomato plants in the T8, T10, and CK5 groups were collected every 1, 7, and 14 days after the plants were inoculated in Example 8. The contents of their defense enzymes (POD, PPO, PAL, CAT, SOD) were determined using a detection kit from Suzhou Grace Biotechnology Co., Ltd.

[0110] The detection results of the five enzymes are as follows: Figure 6 As shown, A represents PAL expression; B represents PPO expression; C represents POD expression; D represents CAT expression; and E represents SOD expression. Compared with CK5, the content of defense enzymes in rice plants treated with T8 was significantly increased, among which PPO (… Figure 6 B in ( ), CAT ( Figure 6 D) and SOD ( Figure 6 The levels of E in the samples reached their highest point 14 days after exposure, increasing by 0.12 times, 0.53 times, and 1.91 times respectively; PAL ( Figure 6 The content of A in the virus peaked at 7 days after exposure, increasing by 0.64 times; while the content of POD ( Figure 6 C) reached its peak 1 day after inoculation, increasing by 0.26 times. These findings indicate that the thick-walled spores provided in this application can significantly increase the content of defensive enzymes in rice plants, effectively activate the plant's defense system, and thus enhance its resistance to southern rice black-streaked dwarf disease.

[0111] Example 10: Stability test of DSE thick-walled spore wettable powder finished product

[0112] The storage stability of the wettable powder provided in this application under normal temperature and 4℃ storage conditions was tested, namely, the viable bacterial concentration (cells / g) and spore germination rate were tested, and samples were taken for testing once a month.

[0113] The method for determining the viable cell count is as follows: Add 1g of powder to sterile water, dilute it 10-fold, and spread 0.1mL onto a PDA plate. After 7 days, count the colonies to calculate the viable cell count. Repeat three times. Viable cell count (CFU / g) = Plate colony count × Dilution factor / Inoculation volume (mL).

[0114] Method for determining the germination rate of chlamydospores: After coating, PDA plates were incubated at 28℃ for 30 h. 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. Results with large differences were discarded, and three replicates were selected. The average value was used as the result for analysis. The germination rate was expressed as a percentage, with 100 spores per plate as the baseline.

[0115] Table 6. Results of stability tests on wettable powders

[0116]

[0117] 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 12 months, the germination rate of chlamydospores continuously decreased from 99.26% to 80.11%, and the viable cell count was still 2.08 × 10⁻⁶ at the 12th month. 8 After 11 months of storage at 4℃, the germination rate of the chlamydospores remained at 80.56%, with a viable cell count of 2.05 × 10⁻⁶. 8 Therefore, the shelf life of the DSE thick-walled spore wettable powder provided in this application meets the relevant requirements of the national standard "Agricultural Microbial Agents" (GB 20287-2006) for the shelf life of powder formulations (shelf life ≥ 6 months), and can be stored at room temperature for more than 12 months, which is even more than 6 months longer than conventional conidial agents.

[0118] This study comprehensively evaluated indicators such as adsorption rate, suspension rate, wetting time, and chlamydospore germination rate, and screened silica as the optimal carrier. The optimal dispersant and wetting agent ratio was PEG8000 and Tween 60, mixed at 1:3 with a total addition of 8%, resulting in the best performance across all indicators. At this ratio, the suspension rate increased to 63.33%, the wetting time was shortened to 11.07 s, and the chlamydospore germination rate reached 81.49%. This is closely related to the porous structure of silica; its high specific surface area not only provides stable attachment sites for chlamydospores, reducing spore damage, but also improves the dispersion performance of the formulation. Furthermore, the synergistic effect of the dispersant PEG8000 and the wetting agent Tween 60 effectively prevents spore aggregation, rapidly reduces the surface tension of the formulation, and enhances its wetting and spreading ability. The synergistic effect of these two agents significantly optimizes the physicochemical properties of the formulation. In addition, the results of UV protectant screening showed that 1.0% ascorbic acid (Vc) could maintain the germination rate of chlamydospores at 75.74%, which was significantly higher than the control (56.91%). This is because Vc has strong antioxidant properties, which can effectively remove reactive oxygen species generated by UV irradiation, reduce spore DNA damage, and ensure the stability of the formulation in the field under UV environment.

[0119] This study confirmed that HX2 chlamydospore wettable powder has significant control effects on both diseases, but the optimal application concentration differs. For tomato bacterial wilt, T2 (1×10⁻⁶) is the optimal concentration. 7 The efficacy of the (CFU / mL) treatment increased over time, reaching 50.9% at 30 days, significantly better than the low-concentration treatment T4 (1×10⁻⁶ CFU / mL). 5 CFU / mL), and the later control efficacy was close to or even better than T5 (Trichoderma harzianum treatment). It is worth noting that the increasing control efficacy of T2 treatment 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 disease, T8 (1×10⁻⁶ CFU / mL) was effective. 6 The concentration-controlled treatment (CFU / mL) showed the best efficacy, reaching 63.64%, significantly higher than other concentrations and the *Trichoderma harzianum* treatment. This difference in concentration effect may be related to the pathogenesis of the two diseases and the crop growth characteristics. Tomato bacterial wilt is caused by... 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 T8 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 addressing the current lack of biocontrol agents for Southern Rice Black-Streaked Dwarf Disease.

[0120] 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 wettable powder of DSE (dichloropyrophylloides) spores, characterized in that, The product comprises chlamydospores from dark-colored septate endophytic fungi and a carrier, as well as a dispersant, a wetting agent, and a UV protectant. The DSE chlamydospore wettable powder contains 2.35 × 10⁻⁶ chlamydospores. 8 CFU / g; The dark-colored septate endophytic fungus is *Cladosporium*, and *Cladosporium* is *Cladosporium guangxiense* (…). Cladophialophora guangxiense HX2 strain, preservation number CGMCC NO.41498; The carrier is silica; The dispersant is PEG8000, the wetting agent is Tween 60, and the mass ratio of the dispersant to the wetting agent is 1:

3. The total mass fraction of the dispersant and the wetting agent in the DSE thick-walled spore wettable powder is 8%; The ultraviolet protectant is ascorbic acid, and the mass fraction of ascorbic acid in the DSE chlamydospore wettable powder is 1.0%.

2. A method for preparing the DSE thick-walled spore wettable powder according to claim 1, characterized in that, Includes the following steps: (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 the thick-walled spore slurry includes: inoculating a dark-colored septate endophytic fungal strain onto a PDA medium plate, activating it at 28°C, then transferring it to a basal medium and culturing it in a constant temperature shaker at 28°C for 14 days; filtering the obtained thick-walled spore fermentation broth through three layers of sterile gauze to obtain the thick-walled spore slurry. (2) Preparation of mother powder: Add a carrier that has been sterilized at high temperature to the thick-walled spore slurry, so that the mass ratio of the thick-walled spore slurry to the carrier is 1:3; put the thick-walled spore slurry with the carrier into an oven at 40±1℃ and dry it for 6 hours, then grind it into powder to obtain the mother powder; (3) Preparation of finished powder: Add dispersant and wetting agent according to proportion, and add ultraviolet protectant according to proportion, mix evenly to obtain finished DSE thick-walled spore wettable powder.

3. The application of the DSE thallus wettable powder of 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 chlamydospore wettable powder according to claim 1 in the control of southern rice black-streaked dwarf disease, characterized in that, The method of using the DSE chlamydospore wettable powder is as follows: dilute the DSE chlamydospore wettable powder to a concentration of 1×10⁻⁶ chlamydospores. 5 ~1×10 6 CFU / mL.

5. The application of the DSE chlamydospore wettable powder according to claim 1 in the control of bacterial wilt of tomato, characterized in that, The method of using the DSE chlamydospore wettable powder is as follows: dilute the DSE chlamydospore wettable powder to a concentration of 1×10⁻⁶ chlamydospores. 5 ~1×10 8 CFU / mL.

6. The application according to claim 5, characterized in that, The method of using the DSE chlamydospore wettable powder is as follows: dilute the DSE chlamydospore wettable powder to a concentration of 1×10⁻⁶ chlamydospores. 5 ~1×10 7 CFU / mL.

7. The application of the DSE chlamydospore wettable powder according to claim 1 in promoting the growth of diseased plants, wherein the diseased plants are rice infected with Southern Rice Black-Streaked Dwarf Virus or tomatoes infected with Ralstonia solanacearum.

8. The application according to claim 7, characterized in that, The plant is tomato, and the method of using the DSE chlamydospore wettable powder is as follows: dilute the DSE chlamydospore wettable powder to a concentration of 1×10⁻⁶ chlamydospores. 5 ~1×10 8 CFU / mL.

9. The application according to claim 8, characterized in that, The method of using the DSE chlamydospore wettable powder is as follows: dilute the DSE chlamydospore wettable powder to a concentration of 1×10⁻⁶ chlamydospores. 5 ~1×10 7 CFU / mL.

10. The application according to claim 7, characterized in that, The plant is rice, and the method of using the DSE chlamydospore wettable powder is as follows: dilute the DSE chlamydospore wettable powder to a concentration of 1×10⁻⁶ chlamydospores. 5 ~1×10 6 CFU / mL.

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