A method for preparing and using a spore suspension of the strain Clitopilus passeckerianus.

Abstract

The application relates to a preparation method and application of a Clitopilus passeckerianus strain spore suspension, and the strain has a preservation number of CGMCC No.41407. Through antibacterial activity test on the Clitopilus passeckerianus spore suspension, it is proved that the Clitopilus passeckerianus spore suspension has excellent bacteriostatic activity on plant bacterial diseases, can be applied to preparation of a plant pathogenic bacterial prevention and treatment agent, and has simple preparation process and low production cost.

Description

Technical Field

[0001] This invention relates to the field of microbial and biopesticide technology, specifically to a method for preparing a suspension of Clitopilus passeckerianus spores and its application. Background Technology

[0002] Bacterial plant diseases are the second largest category of plant diseases after fungal diseases. According to incomplete statistics, bacterial diseases currently affect an area of ​​120 million mu (approximately 8 million hectares) in my country, characterized by rapid onset, wide distribution, and difficulty in control. In recent years, with changes in crop cultivation systems and environmental climate in my country, bacterial diseases of crops such as rice bacterial blight (Xoo), rice bacterial leaf streak (Xoc), and citrus canker (Xac) have shown a trend of increasing incidence. Currently, compared with fungal disease control agents, there is a significant lack of fungicides for bacterial diseases both domestically and internationally. With continuous cropping, bacterial diseases are increasingly showing a trend of mixed, multiple, and long-term outbreaks. Currently, chemical control of bacterial diseases is the main method, but the types of agents are limited and the varieties are limited, resulting in problems such as poor compatibility, poor safety, easy induction of spider mites, high resistance, and high reproductive toxicity. Compared with traditional chemical pesticides, biological pesticides have significant advantages. Biological pesticides originate from natural ecosystems, their active ingredients are easily decomposed, they have minimal environmental impact, are highly selective, safe for humans and animals, and do not cause pollution or residues. The search for rapid and effective biological control methods for plant bacterial diseases is receiving increasing attention.

[0003] Clitopilus passeckerianus spore suspension is a novel bactericidal biological pesticide with excellent antibacterial activity against pathogens such as rice bacterial blight (Xoo), rice bacterial leaf streak (Xoc), and citrus canker (Xac). It exhibits excellent control efficacy against bacterial diseases such as rice bacterial blight, rice bacterial leaf streak, and citrus canker. Summary of the Invention

[0004] To address the aforementioned technical problems in agricultural production and the shortcomings of existing technologies, this invention provides a strain of Clitopilus passeckerianus with good control efficacy against plant bacterial diseases, a spore suspension, its preparation method, and its application.

[0005] To achieve this objective, the present invention adopts the following technical solution.

[0006] In a first aspect, the present invention provides a strain of Clitopilus passeckerianus, which was collected from Yunnan and deposited on July 8, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 41407, at the Institute of Microbiology, Chinese Academy of Sciences.

[0007] In a second aspect, a method for preparing a suspension of *Cyclocarya cathartica* spores is provided, comprising the following steps:

[0008] (1) Inoculate the cat ear slant umbrella strain onto PDA solid medium and culture at 25℃ for 48-72h;

[0009] (2) The activated strain obtained in step (1) is inoculated onto the sporulation medium at a surface area ratio of 5-10% of the sporulation solid medium and fermented at 25℃-27℃ for 120-144h.

[0010] (3) Add 0.5% Tween-80 solution after autoclaving to the spore-producing medium obtained in step (2), gently scrape the surface of the medium to suspend all the spores in water, filter with 300 mesh gauze to obtain spore suspension, count the spores under a microscope using a hemocytometer, and then dilute to the required concentration.

[0011] Preferably, the sporulation culture medium in step (2) is prepared as follows:

[0012] Mix 20-25 parts oats, 20-25 parts oat bran, 5-8 parts glucose, ammonium molybdate, ferric sulfate, and 0.5-2 parts disodium EDTA-2Na with water, and sterilize at 121°C for 20 minutes to obtain the final product.

[0013] In a third aspect, the present invention is applied to the prevention and control of rice bacterial leaf blight (Xoo), rice bacterial leaf streak (Xoc), and citrus canker (Xac).

[0014] Pesticide adjuvants include, but are not limited to, one or more of the following: fillers, carriers, emulsifiers, dispersants, wetting agents, disintegrants, binders, or synergists.

[0015] The beneficial effects of this invention are as follows:

[0016] The cat's ear spore suspension provided by this invention has a good control effect on rice bacterial blight (Xoo).

[0017] The cat's ear spore suspension provided by this invention has good control effect on bacterial leaf streak (Xoc) of rice.

[0018] The cat's ear spore suspension provided by this invention has a good preventive effect against citrus canker (Xac).

[0019] The cat's ear spore suspension preparation conditions provided by this invention are simple, easy to industrialize and preserve, and have good development and application prospects.

[0020] Preservation Information: Clitopilus passeckerianus strain, which was deposited on July 8, 2024 at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 41407, at the Institute of Microbiology, Chinese Academy of Sciences. Attached Figure Description

[0021] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings:

[0022] Figure 1 Electron micrograph of spores of the strain Clitopilus passeckerianus;

[0023] Figure 2 This is a photograph of the cat-ear-shaped mushroom harvested from Yunnan in Example 1;

[0024] Figure 3 This is a photograph of the *Clitopilus passeckerianus* strain cultured on PDA in Example 1;

[0025] Figure 4 This is a microscope image of the *Cyclocarya cathartica* suspension from Example 2;

[0026] Figure 5 The protective activity of *Cyclocarya cathartica* suspension against rice bacterial blight at concentrations of 1 billion and 1.5 billion spores / mL in Example 3 was demonstrated.

[0027] Figure 6 This demonstrates the therapeutic activity of *Agrostis chinensis* suspension at a concentration of 1 billion spores / mL against rice bacterial blight in Example 3.

[0028] Figure 7 The protective activity of *Agrosporium argentea* suspension against bacterial leaf streak in rice at concentrations of 1 billion and 1.5 billion spores / mL in Example 4 was demonstrated.

[0029] Figure 8 The therapeutic activity of *Agrosporium argentea* suspension against bacterial leaf streak in rice at concentrations of 1 billion and 1.5 billion spores / mL in Example 4 was demonstrated.

[0030] Figure 9This study demonstrates the protective activity of *Cyclocarya cathartica* suspension against citrus canker at a concentration of 1 billion spores / mL, as described in Example 5. Example

[0031] Various exemplary embodiments of this application are now described in detail. This detailed description should not be considered as a limitation of this application, but rather as a more detailed description of certain aspects, features, and embodiments of this application. It should be understood that the terminology used in this application is merely for describing particular embodiments and is not intended to limit this application. Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art described herein. Although only preferred methods and materials are described in this application, any methods and materials similar to or equivalent to those described herein may be used in the implementation or testing of this application.

[0032] Example 1

[0033] Isolation, purification and identification of *Cercidiphyllum micranthum*:

[0034] (1) Collection and isolation of bacterial strains:

[0035] The cat-ear slanted umbrella cultivar was cultured using PDA plates. The culture medium consisted of 6 g / L potato starch, 20 g / L glucose, 20 g / L agar, and a natural pH.

[0036] After harvesting the fruiting bodies in Yunnan ( Figure 2 Spores were picked up with an inoculation needle and inoculated onto a PDA plate to grow colonies. The colonies were then isolated and purified using the single-spore isolation method and stored for later use.

[0037] (2) Strain identification:

[0038] Genomic DNA was extracted from the strain using a fungal genomic DNA extraction kit (Solarbio, Beijing). Amplification was performed using primers ITS1-F / ITS4. The PCR products were sequenced by Beijing Qingke Biotechnology Co., Ltd., and the results were compared with NCBI data. Electron micrographs of the spores were also taken. The spores were almond-shaped, a typical characteristic of spores in the genus *Pleurotus* of the family Phyllostachys. Based on comparisons of fruiting body morphology, ITS sequence, and spore morphology, this strain was identified as *Pleurotus* var. *mammalis*. Figure 3 It has been deposited at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 41407.

[0039] Example 2

[0040] Preparation of cat's ear cyperus suspension:

[0041] (1) Activation and sporulation of fungal strains

[0042] Sporulation of *Cereus uncinata* was carried out using a sporulation culture medium composed of: 20-25 parts oats, 20-25 parts oat bran, 5-8 parts glucose, ammonium molybdate, ferric sulfate, 0.5-2 parts disodium EDTA-2Na, and 50-60 parts water.

[0043] Activate *Corydalis edulis* on PDA solid medium and incubate at 25°C for 48-72 hours. After mycelium growth, inoculate the edge of the mycelial block onto the sporulation medium along with the PDA plate at a ratio of 5-10% of the surface area of ​​the sporulation solid medium, and ferment at 25°C-27°C for 120-144 hours.

[0044] (2) Preparation of spore suspension

[0045] Add 0.5% Tween-80 solution to the sporulation medium, gently scrape the surface of the medium to suspend all spores in the 0.5% Tween-80 solution, and filter through a Buchner funnel using 300-mesh gauze to obtain a spore suspension. The 0.5% Tween-80 solution, 300-mesh gauze, and Buchner funnel must all be autoclaved at 121°C for 20 minutes before use.

[0046] (3) Spore counting under a microscope in spore suspension

[0047] The spore suspension was diluted 50-fold, and 10 μL was placed on a hemocytometer. The spores were counted under a 22x eyepiece and a 40x objective microscope. The results showed that the spore concentration reached 2.095 × 10⁻⁶. 9 cells / mL ( Figure 4 ).

[0048] (4) Concentration dilution

[0049] The spore suspension can be diluted to the target concentration using a sterilized 0.5% Tween-80 solution before use.

[0050] Example 3

[0051] In vivo pot experiment on bacterial blight of rice using a suspension of *Cyclocarya cathartica* spores.

[0052] The protective activity of spore suspensions at a concentration of 1 billion spores / mL against rice bacterial blight was determined using the leaf-cutting method. Thiazole zinc and Bacillus subtilis were used as control agents. Thiazole zinc was prepared with 0.5% Tween-80 solution to a concentration of 200 μg / mL, and Bacillus subtilis was prepared with 0.5% Tween-80 solution to a concentration of 100 million spores / mL. The tips of rice leaves were cut off 2-3 cm. A control (CK) without any treatment (0.5% Tween-80 solution) was also included. Each treatment had at least 10 leaves, with three replicates. Fourteen days after application, the length of lesions on rice leaves was recorded. The spores were sprayed evenly onto the leaves until droplets fell. One day later, the protective activity was calculated based on the length of the lesions using a scissor containing logarithmic Xoo bacterial suspension. Figure 5 The therapeutic activity was determined by first cutting off 2-3 cm of the rice leaf tip with scissors containing logarithmic Xoo bacterial suspension. One day later, a spore suspension at a concentration of 10 or 1.5 billion / mL was evenly sprayed onto the rice leaves until droplets fell. A control group was established using a 0.5% Tween-80 solution without the pesticide. Each treatment involved at least 10 leaves, with three replicates. Fourteen days after application, the length of lesions on the rice leaves was recorded, and the therapeutic activity was calculated based on the lesion length. Figure 6 ).

[0053] Control efficacy (%) = (Leg length of control group - lesion length of treatment group) ÷ Lesion length of control group × 100%

[0054] Example 4: In vivo pot experiment on bacterial leaf streak of rice using *Cyclocarya cathartica* suspension.

[0055] The protective activity of spore suspensions against bacterial leaf streak of rice at concentrations of 1.5 billion and 1 billion / mL was determined using the osmotic pressure method. Thiazole zinc was used as a control. Thiazole zinc was prepared with 0.5% Tween-80 solution to a concentration of 200 μg / mL and sprayed evenly onto rice leaves until droplets fell. One day later, a syringe (without the needle tip) containing logarithmic Xoc bacteria suspension was used to osmotically penetrate the leaf tips to the 1 / 3 to 1 / 2 of the leaf surface. A control (CK) without 0.5% Tween-80 solution was also included. Each treatment involved at least 10 leaves, with three replicates. The length of lesions on rice leaves was measured 14 days after application, and the protective activity was calculated based on the lesion length. Figure 7 The therapeutic activity was determined by first injecting a syringe (without the needle tip) containing logarithmic-phase Xoc bacterial solution into the tip 1 / 3 to 1 / 2 of the rice leaf. One day later, a 200 μg / mL thiamethoxam zinc solution and 1 billion spores / mL Bacillus subtilis were evenly sprayed onto the rice leaves. A control group (0.5% Tween-80 solution without treatment) was also included. Each treatment involved at least 10 leaves, with three replicates. Fourteen days after treatment, the length of lesions on the rice leaves was recorded, and the therapeutic activity was calculated based on the lesion length. Figure 8 ).

[0056] Example 5: Experiment on live potted plants infected with Citrus canker using suspension of *Cyclocarya cathartica* spores.

[0057] A suspension of *Cyclocarya paliurus* spores was prepared to the required concentration of 1 billion spores / mL. The control agent *Bacillus subtilis* was prepared to the required concentration. The control agents thiamethoxam and thiamethoxam were prepared to the required concentration using a 0.5% Tween-80 solution. All were sprayed evenly onto citrus leaves until droplets appeared. One day later, 20 holes were pierced into each citrus leaf using a syringe. Paper soaked in a solution containing log-phase *Xac. var. pachycarpa* was placed over the holes for 24 hours. At least 15 leaves were treated per treatment. A water control and a control using 0.5% Tween-80 solution without any agents were also included. Thirty days after application, the length of lesions on the citrus leaves was measured, and the protective activity was calculated based on the number of lesions. Figure 9 ).

[0058] Table 1. Protective activity of *Cyclocarya paliurus* spore suspension against rice bacterial blight at 1 billion and 1.5 billion spores / mL.

[0059]

[0060] The protective activity of *Cyclocarya cathartica* suspension against rice bacterial blight at concentrations of 1.5 billion and 1 billion / mL is shown in Table 1. Figure 5 As shown, at concentrations of 1.5 billion and 1 billion / mL, the therapeutic activity of *Cyclocarya cateri* suspension was 75.0% and 61.4%, respectively, both superior to the control agents 200 μg / mL thiazoxazine (55.0%) and 1 billion / mL *Bacillus subtilis* (44.5%).

[0061] Table 2. The therapeutic activity of *Cyclocarya paliurus* spore suspension against rice bacterial blight at a concentration of 1 billion spores / mL.

[0062]

[0063] The therapeutic activity of *Cyclocarya cathartica* suspension against rice bacterial blight at a concentration of 1 billion / mL is shown in Table 2. Figure 6 As shown, at a concentration of 1 billion / mL, the therapeutic activity of *Cyclocarya cathartica* suspension against bacterial blight in rice was 72.9%, which was comparable to the control agent 200 μg / mL thiamethoxam zinc (79.8%), and superior to the control agent 100 million / mL *Bacillus subtilis* (5.1%).

[0064] Table 3. Protective activity of *Cyclocarya paliurus* spore suspension against bacterial leaf streak in rice at concentrations of 1.5 billion and 1 billion spores / mL.

[0065]

[0066] The protective activity of *Agrostis chinensis* suspension against bacterial leaf streak of rice at concentrations of 1.5 billion and 1 billion / mL is shown in Table 3. Figure 7 As shown, at concentrations of 1.5 billion and 1 billion / mL, the protective activity of *Cyclocarya cathartica* suspension was 86.3% and 81.5%, respectively, both significantly better than the control agent 200 μg / mL thiazoxazine zinc (29.2%).

[0067] Table 4. The therapeutic activity of *Cyclocarya paliurus* spore suspension against bacterial leaf streak in rice at 1 billion and 1.5 billion spores / mL.

[0068]

[0069]

[0070] The therapeutic activity of *Cyclocarya cathartica* suspension against bacterial leaf streak in rice at concentrations of 1.5 billion and 1 billion / mL is shown in Table 4. Figure 8 As shown, at concentrations of 1.5 billion and 1 billion / mL, the therapeutic activity of *Cyclocarya cateri* suspension was 79.3% and 75.4%, respectively, both significantly better than the control agent 200 μg / mL thiazoxazine zinc (43.8%).

[0071] Table 5. Protective activity of *Agrosporium argentea* suspension against citrus canker at 1 billion spores / mL.

[0072]

[0073] The therapeutic activity of *Cyclocarya cathartica* suspension against bacterial leaf streak in rice at concentrations of 1.5 billion and 1 billion / mL is shown in Table 5. Figure 9 As shown, at 1.5 billion CFU / mL, the suspension of *Cyclocarya cathartica* spores exhibited good protective activity against citrus canker, with a protective activity of 48.0%, comparable to that of *Bacillus subtilis* at 1 billion CFU / mL (44.6%), and significantly superior to the protective activities of 200 μg / mL thiamethoxam zinc (32.0%) and 200 μg / mL thiamethoxam copper (10.2%).

[0074] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A cat-ear slanted umbrella ( Clitopilus passeckerianus The strain is characterized by: The accession number is CGMCC No.41407.

2. A suspension of *Cyclocarya cathartica* spores, characterized in that: Derived from the cat-ear slanted umbrella of claim 1 ( Clitopilus passeckerianus ) strain.

3. The method for preparing the *Cyclocarya cathartica* spore suspension according to claim 2, characterized in that, Includes the following steps: (1) The strain of *Amanita muscaria* with accession number CGMCC No. 41407 was used to extract the *Amanita muscaria* strain. Clitopilus passeckerianus Inoculate onto PDA solid medium and incubate at 25℃ for 48-72 hours; (2) The activated strain obtained in step (1) is inoculated onto the sporulation medium at a surface area ratio of 5-10% of the sporulation solid medium and fermented at 25℃-27℃ for 120-144h. (3) Add 0.5% Tween-80 solution after autoclaving to the spore-producing medium obtained in step (2), gently scrape the surface of the medium to suspend all the spores in water, filter with 300 mesh gauze to obtain spore suspension, count the spores under a microscope using a hemocytometer, and then dilute to the required concentration.

4. The method for preparing the spore suspension according to claim 3, characterized in that, The spore-forming solid culture medium in step (2) is prepared as follows: 20-25 parts of oats, 20-25 parts of oat bran, 5-8 parts of glucose, ammonium molybdate, ferric sulfate, and 0.5-2 parts of disodium EDTA-2Na are mixed with water and sterilized at 121°C for 20 minutes.

5. A composition, characterized in that: Including the cat-ear slanted umbrella as described in claim 1 ( Clitopilus passeckerianus The strain or the cat's ear spore suspension as described in claim 2, and the adjuvants; the dosage form of the composition is selected from emulsifiable concentrates, powders, granules, aqueous solutions, suspensions, ultra-low volume sprays, microcapsules, and fumigants.

6. The cat-ear slanted umbrella as described in claim 1 ( Clitopilus passeckerianus The use of the strain, the *Agrostis cuspidatum* suspension of claim 2, or the composition of claim 5 in the control of plant bacterial diseases, characterized in that: The plant bacterial diseases mentioned are rice bacterial leaf blight, rice bacterial leaf streak, and citrus canker.

7. A method for preventing and controlling agricultural pests and diseases, characterized in that: The cat-ear slanted umbrella as described in claim 1 ( Clitopilus passeckerianus The strain, the *Cyclocarya cathartica* suspension of claim 2, or the composition of claim 5 are applied to harmful substances or the living environment. The agricultural pests and diseases are plant bacterial diseases, including rice bacterial blight, rice bacterial leaf streak, and citrus canker.

8. A method for protecting plants from agricultural pests and diseases, comprising using the cat-ear slanted umbrella (as described in claim 1) Clitopilus passeckerianus The method steps of contacting the strain, the *Cyclocarya cathartica* suspension of claim 2, or the composition of claim 5 with agricultural pests and diseases; wherein the agricultural pests and diseases are plant bacterial diseases, and the plant bacterial diseases are rice bacterial leaf blight, rice bacterial stripe disease, and citrus canker.

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