Trichoderma asperellum strain ZYC2023 and application thereof in preventing and treating anthrachnoe of panax notoginseng

Abstract

This invention belongs to the field of microbial technology, specifically relating to a biocontrol fungus, *Trichoderma echinocandii* ZYC2023, and its application in controlling *Aconitum carmichaelii* scabies disease. The *Trichoderma echinocandii* ZYC2023 provided by this invention can inhibit the mycelial growth and sclerotium formation of the pathogen causing *Aconitum carmichaelii* scabies, and shows excellent antifungal effects in both pot and field trials. The *Trichoderma echinocandii* ZYC2023 provided by this invention has a significant inhibitory effect on *Aconitum carmichaelii* scabies disease, and its inhibitory effect is superior to commercially available Bacillus subtilis agents and *Trichoderma harzianum* agents, making it suitable for the control of *Aconitum carmichaelii* scabies disease. The *Trichoderma echinocandii* ZYC2023 provided by this invention originates from the rhizosphere of *Aconitum carmichaelii*, has a high affinity for *Aconitum carmichaelii*, and will not affect its quality. It avoids the problems of environmental pollution, pathogen resistance, and disease resurgence caused by pesticide abuse, and has broad application prospects in the control of diseases in traditional Chinese medicinal materials.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a biocontrol bacterium Trichoderma echinocandes ZYC2023 and its application in the prevention and control of Aconitum carmichaelii white rot. Background Technology

[0002] White mold disease is a serious threat to the cultivation of root and rhizome medicinal herbs, leading to weakened growth, reduced yield, and poor quality, and in severe cases, even total crop failure. The white mold fungus has a wide host range, infecting more than 200 species of plants from over 100 families, including medicinal plants such as Atractylodes macrocephala, Pinellia ternata, Artemisia argyi, Pseudostellaria heterophylla, Sarcandra glabra, Aster tataricus, Scrophularia ningpoensis, Dendrobium officinale, Salvia miltiorrhiza, and Aconitum carmichaelii.

[0003] Currently, the main method for controlling white mold in production is chemical control. Song Min et al. (Song Min, Chen Xiaofeng, Wu Cuixia, et al. Field control efficacy of 21% thifluzamide·fludioxonil·pyraclostrobin suspension seed dressing agent against peanut white mold [J]. Pesticides, 2021, 60(09):691-693+702.) found that as a seed disinfection method, 21% thifluzamide·fludioxonil·pyraclostrobin suspension seed dressing agent has a good control effect on peanut white mold. Li Jinxin et al. (Li Jinxin, Chen Qiaohuan, Miao Yuhuan, et al. Identification, biological characteristics and effective fungicides of the pathogen of white rot of *Aconitum carmichaelii* [J / OL]. Chinese Journal of Traditional Chinese Medicine: 1-13 [2021-07-07]. 20210318.101.) screened agents for the control of white rot of *Aconitum carmichaelii* and found that the chemical fungicide lime sulfur, flusilazole and the plant-derived fungicide osthol had good inhibitory effects on *Aconitum carmichaelii* BQ-1. However, the efficacy of chemical agents is short-lived, and for medicinal materials with long growth cycles, they are often used multiple times, which easily leads to the "3R" problem of chemical pesticides. At present, many agents have reduced efficacy in controlling white rot of *Aconitum carmichaelii*. At the same time, the disease breaks out on a large scale, and there is a problem of "no medicine to prevent, and medicine that is difficult to prevent", which seriously restricts the healthy development of the *Aconitum carmichaelii* industry.

[0004] Biological control is safe, effective, and long-lasting, and in particular, it avoids a series of problems associated with chemical control. Therefore, screening out biocontrol bacteria for controlling white rot is of great practical significance in application. Summary of the Invention

[0005] The purpose of this invention is to provide a biocontrol fungus, Trichoderma echinocandii ZYC2023, and its application in the prevention and control of Aconitum carmichaelii white mold disease, which inhibits the mycelial growth and sclerotium formation of the pathogen causing Aconitum carmichaelii white mold disease, and effectively reduces the incidence of white mold disease.

[0006] This invention provides a biocontrol fungus, Trichoderma asperellum ZYC2023, with accession number CGMCC No.40528.

[0007] The present invention also provides a biocontrol agent, comprising Trichoderma echinosporum ZYC2023 as described in the above technical solution.

[0008] Preferably, the biocontrol agent is in the form of a liquid or solid formulation;

[0009] The spore concentration of Trichoderma echinocandes ZYC2023 in the liquid formulation is 10. 6 ~10 8 CFU / mL;

[0010] The spore concentration of Trichoderma echinocandes ZYC2023 in the solid dosage form is 1×10⁻⁶. 9 ~50×10 9 CFU / g.

[0011] The present invention also provides a method for preparing a biocontrol agent, comprising the following steps: inoculating Trichoderma echinosporum ZYC2023 as described in the above technical solution into a solid fermentation medium for fermentation culture, drying the obtained solid fermentation product, and obtaining the biocontrol agent;

[0012] The solid fermentation culture medium includes fermentation substrate and water; the fermentation substrate includes wheat bran, rice husks and corn flour; the mass ratio of wheat bran, rice husks and corn flour is 2-5:1-4:1.5-2.5.

[0013] Preferably, the fermentation culture temperature is 26-28℃, the time is 5-9 days, and the pH is 5.0-6.5; the drying temperature is 37-40℃.

[0014] The present invention also provides the application of the biocontrol agent prepared by the above-described Trichoderma echinocandi ZYC2023 or by the above-described preparation method in inhibiting the mycelial growth and / or sclerotium formation of the pathogen of white rot.

[0015] The present invention also provides the application of the *Trichoderma hygroscopicum* ZYC2023 described in the above technical solution or the biocontrol agent prepared by the preparation method described in the above technical solution in the control of plant white rot disease.

[0016] Preferably, the plant includes Aconitum carmichaelii.

[0017] The present invention also provides a method for preventing and treating Aconitum carmichaelii white rot, comprising one or more steps of the following I to III:

[0018] I: On the second day after Aconitum carmichaelii is infected with the pathogen of white rot, drench the roots with the biocontrol agent described in the above technical solution or the biocontrol agent prepared by the preparation method described in the above technical solution;

[0019] II: During the root pruning period of Aconitum carmichaelii, apply the biocontrol agent described in the above technical solution or the biocontrol agent prepared by the preparation method described in the above technical solution;

[0020] III: After applying base fertilizer, sow Aconitum carmichaelii; the base fertilizer includes the biocontrol agent described in the above technical solution or the biocontrol agent prepared by the preparation method described in the above technical solution.

[0021] Preferably, in step I, the biocontrol agent is a liquid preparation with a spore concentration of 10. 6 ~10 8 CFU / mL, root irrigation volume is 50-100 mL / plant;

[0022] In section II, the biocontrol agent is a solid preparation with a spore concentration of 1×10⁻⁶. 9 ~50×10 9 CFU / g, diluted and applied as a root drench, with the amount applied based on the mass of the solid formulation being 2.5 kg / mu;

[0023] In section III, the biocontrol agent is a solid preparation with a spore concentration of 1×10⁻⁶. 9 ~50×10 9 CFU / g, application rate is 2.5 kg / mu.

[0024] Beneficial effects:

[0025] This invention provides a biocontrol fungus, *Trichoderma echinocandii* ZYC2023, with preservation number CGMCCNo.40528. *Trichoderma echinocandii* ZYC2023 provided by this invention can inhibit the mycelial growth and sclerotium formation of the pathogen causing *Aconitum carmichaelii* scabies, and shows excellent antifungal effects in both pot and field trials. *Trichoderma echinocandii* ZYC2023 provided by this invention has a significant inhibitory effect on *Aconitum carmichaelii* scabies, and its inhibitory effect is superior to commercially available Bacillus agents and *Trichoderma harzianum*, making it applicable for the control of *Aconitum carmichaelii* scabies. *Trichoderma echinocandii* ZYC2023 provided by this invention originates from the rhizosphere of *Aconitum carmichaelii*, has a high affinity for *Aconitum carmichaelii*, and avoids the problems of environmental pollution, pathogen resistance, and disease resurgence caused by pesticide overuse, showing broad application prospects in the control of diseases in traditional Chinese medicinal materials.

[0026] Biological Preservation Information

[0027] Trichoderma asperellum ZYC2023 was deposited on March 23, 2023, at the China General Microbiological Culture Collection Center (CGMCC), No. 1, Beichen West Road, Chaoyang District, Beijing, 100101, China, with accession number CGMCC No. 40528. Attached Figure Description

[0028] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0029] Figure 1 The growth of fungi in soil suspension on Czapek's medium;

[0030] Figure 2 The colony morphology of strain T15;

[0031] Figure 3 The results of the plate confrontation test between strain T15 and the pathogen of white sclerotium wilt;

[0032] Figure 4 Phylogenetic tree of strain T15;

[0033] Figure 5 The inhibitory effect of Trichoderma echinococcus ZYC2023 on the sclerotium growth of the pathogen of white rot;

[0034] Figure 6 The effect of the pathogen of white rot on the growth of Aconitum carmichaelii in different potted treatment groups;

[0035] Figure 7 The disease index of white mold disease in different potted plant treatment groups; where different lowercase letters represent significant differences;

[0036] Figure 8 The incidence of white rot in different treatment groups during the field experiment in Dakang Town in 2023; different lowercase letters indicate significant differences.

[0037] Figure 9 The incidence of white rot in different treatment groups during the field experiment in Taiping Town in 2023; different lowercase letters indicate significant differences.

[0038] Figure 10 The growth of Aconitum carmichaelii in different treatment groups during the field experiment in Taiping Town in 2024;

[0039] Figure 11 The incidence of white rot in the T15 treatment group at different time points during the field experiment in Taiping Town in 2024;

[0040] Figure 12 The incidence of white mold disease in the conventional organic fertilizer treatment groups at different times during the field experiment in Taiping Town in 2024. Detailed Implementation

[0041] This invention provides a biocontrol fungus, Trichoderma asperellum ZYC2023, with accession number CGMCC No.40528.

[0042] This invention involves collecting soil samples from the roots of healthy Aconitum carmichaelii plants in fields infected with white sclerotium rot. A fungal strain was isolated from these samples. After 72 hours of culture on PDA plates, the fungus exhibited a circular distribution with regular edges. The central colony was light gray (a dense area of ​​newly formed hyphae), the transition zone was grayish-green (an active sporulation zone), and the peripheral zone was dark grayish-green (a layer rich in mature spores). The surface displayed typical "cotton-like" ridges (aerial hyphae approximately 1-2 mm in height). It was identified as *Trichoderma echinosporum*. The *Trichoderma echinosporum* ZYC2023 provided by this invention significantly inhibits the mycelial growth and sclerotium formation of the pathogen causing white sclerotium rot in Aconitum carmichaelii, effectively reducing the incidence of the disease.

[0043] The present invention also provides a biocontrol agent, comprising Trichoderma echinosporum ZYC2023 as described in the above technical solution.

[0044] In one embodiment, the biocontrol agent of the present invention is in the form of a liquid or solid formulation. In one embodiment, the spore concentration of *Trichoderma echinocandes* ZYC2023 in the liquid formulation of the present invention is 10... 6 ~10 8 CFU / mL; As another embodiment, the spore concentration of Trichoderma echinocandes ZYC2023 in the liquid formulation of the present invention is 5 × 10⁻⁶ CFU / mL; 6 ~5×10 7 CFU / mL; As another embodiment, the spore concentration of Trichoderma echinocandes ZYC2023 in the liquid formulation of the present invention is 1×10⁻⁶ CFU / mL. 7 CFU / mL. As one embodiment, the spore concentration of *Trichoderma echinocandes* ZYC2023 in the solid formulation of the present invention is 1×10⁻⁶ CFU / mL. 9 ~50×10 9 CFU / g; As another embodiment, the spore concentration of Trichoderma echinocandes ZYC2023 in the solid formulation of the present invention is 2×10⁻⁶ CFU / g; 9 ~10×10 9 CFU / g; As another embodiment, the spore concentration of Trichoderma echinocandes ZYC2023 in the solid formulation of the present invention is 5 × 10⁻⁶ CFU / g; 9 ~8×10 9 CFU / g.

[0045] The present invention also provides a method for preparing a biocontrol agent, comprising the following steps: inoculating Trichoderma echinocandes ZYC2023 as described in the above technical solution into a solid fermentation medium for fermentation culture, drying the obtained solid fermentation product, and obtaining the biocontrol agent; the solid fermentation medium includes fermentation substrate and water; the fermentation substrate includes wheat bran, rice husk and corn flour; the mass ratio of wheat bran, rice husk and corn flour is 2-5:1-4:1.5-2.5.

[0046] In one embodiment, the mass ratio of wheat bran, rice husk, and corn flour in the fermentation substrate of the present invention is 4:3:2. In another embodiment, the mass ratio of *Trichoderma echinococcus* ZYC2023 to the fermentation substrate of the present invention is 1:9, and the mass of *Trichoderma echinococcus* ZYC2023 is based on the mass of the *Trichoderma echinococcus* ZYC2023 spore suspension; the concentration of the *Trichoderma echinococcus* ZYC2023 spore suspension is 10. 6 ~10 8 CFU / mL; As another embodiment, the concentration of the spore suspension of Trichoderma echinocandes ZYC2023 described in this invention is 5 × 10⁻⁶ CFU / mL. 6 ~5×10 7 CFU / mL; As another embodiment, the concentration of the spore suspension of Trichoderma echinocandes ZYC2023 described in this invention is 1×10⁻⁶ CFU / mL. 7 CFU / mL. In one embodiment, the mass ratio of fermentation substrate to water is 0.9:0.8–1.4; in another embodiment, the mass ratio is 0.9:1.3. In one embodiment, the pH of the solid fermentation medium is 5.0–6.5; in another embodiment, the pH is 5.5. The fermentation temperature is 26–28°C, and the time is 5–9 days; in another embodiment, the fermentation temperature is 26–28°C, and the time is 7 days. In one embodiment, the drying temperature is 37–40°C.

[0047] The present invention also provides the application of the biocontrol agent prepared by the above-described Trichoderma echinocandi ZYC2023 or by the above-described preparation method in inhibiting the mycelial growth and / or sclerotium formation of the pathogen of white rot.

[0048] The present invention also provides the application of the *Trichoderma hygroscopicum* ZYC2023 described in the above technical solution or the biocontrol agent prepared by the preparation method described in the above technical solution in the control of plant white rot disease.

[0049] In one embodiment, the plant described in this invention includes Aconitum carmichaelii.

[0050] The present invention also provides a method for preventing and treating Aconitum carmichaelii white rot, comprising one or more steps of the following I to III:

[0051] I: On the second day after Aconitum carmichaelii is infected with the pathogen of white rot, drench the roots with the biocontrol agent described in the above technical solution or the biocontrol agent prepared by the preparation method described in the above technical solution;

[0052] II: During the root pruning period of Aconitum carmichaelii, apply the biocontrol agent described in the above technical solution or the biocontrol agent prepared by the preparation method described in the above technical solution;

[0053] III: After applying base fertilizer, sow Aconitum carmichaelii; the base fertilizer includes the biocontrol agent described in the above technical solution or the biocontrol agent prepared by the preparation method described in the above technical solution.

[0054] In one embodiment, in step I of the present invention, the biocontrol agent is a liquid preparation with a spore concentration of 10. 6 ~10 8 CFU / mL; as another embodiment, the spore concentration of the liquid formulation of the present invention is 5 × 10⁻⁶ CFU / mL. 6 ~5×10 7 CFU / mL; as another embodiment, the spore concentration of the liquid formulation of the present invention is 1×10⁻⁶. 7 CFU / mL. In one embodiment, in step I of the present invention, the amount of biocontrol agent applied to the roots is 50-100 mL / plant; in another embodiment, in step I of the present invention, the amount of biocontrol agent applied to the roots is 100 mL / plant.

[0055] In one embodiment, in section II of the present invention, the biocontrol agent is a solid preparation with a spore concentration of 1×10⁻⁶. 9 ~50×10 9 CFU / g; As another embodiment, the spore concentration of the solid formulation of the present invention is 2 × 10⁻⁶. 9 ~10×10 9 CFU / g; As another embodiment, the spore concentration of Trichoderma echinocandes ZYC2023 in the solid formulation of the present invention is 5 × 10⁻⁶ CFU / g; 9 ~8×10 9 CFU / g. In one embodiment, in section II of this invention, the biocontrol agent is diluted and applied as a root drench, with the drench volume based on the mass of the solid formulation being 2.5 kg / mu. In another embodiment, the biocontrol agent of this invention is diluted 20 to 80 times.

[0056] In one embodiment, in section III of the present invention, the biocontrol agent is a solid preparation with a spore concentration of 1×10⁻⁶. 9 ~50×10 9 CFU / g; As another embodiment, the spore concentration of the solid formulation of the present invention is 2 × 10⁻⁶. 9 ~10×10 9 CFU / g; As another embodiment, the spore concentration of Trichoderma echinocandes ZYC2023 in the solid formulation of the present invention is 5 × 10⁻⁶ CFU / g; 9 ~8×10 9 CFU / g. In one embodiment, in section III of this invention, the application rate of basal fertilizer is 2.5 kg / mu.

[0057] The biocontrol agent described in this invention can be used in various ways, such as root irrigation or as a base fertilizer mixed with soil. It inhibits the mycelial growth and sclerotium formation of the pathogen causing Aconitum carmichaelii, significantly reducing the incidence of Aconitum carmichaelii and lowering the disease index.

[0058] To further illustrate the present invention, the following detailed description, in conjunction with the accompanying drawings and embodiments, describes a biocontrol fungus, Trichoderma echinocandii ZYC2023, and its application in the prevention and control of Aconitum carmichaelii white rot. However, these descriptions should not be construed as limiting the scope of protection of the present invention.

[0059] Example 1

[0060] Isolation and screening of rhizosphere microorganisms from Aconitum carmichaelii

[0061] (1) Soil collection from the roots of healthy Aconitum carmichaelii plants in fields affected by white mold disease: Aconitum carmichaelii plants with fresh soil attached were collected from Jiangyou, Sichuan. Excess soil was shaken off from the roots of the Aconitum carmichaelii plants, leaving the soil tightly attached to the root surface. The surface was gently brushed with a toothbrush until the soil tightly attached to the root surface of the fresh Aconitum carmichaelii plants fell onto a prepared sterile preservation bag. After collecting enough soil, excess impurities were removed by sieving through a 100-mesh sieve. The rhizosphere soil with appropriate particle size was collected by sterile filter paper and stored in 10ml centrifuge tubes in time to prevent excessive evaporation of water from the soil from affecting the activity of the strains in the rhizosphere soil. The tubes were divided into 10 portions for later use, and the region and date were marked respectively. They were temporarily stored in a -4℃ refrigerator for the isolation of soil microorganisms.

[0062] (2) Isolation of fungi from soil using the plate dilution spread method: Weigh 0.1g of soil sample and add it to a 50mL Erlenmeyer flask containing 0.9mL of sterile water. Shake and incubate at 300r / min for 10min. In a clean bench, pipette 0.1mL of the soil suspension into a sterile test tube containing 0.9mL of sterile water. Shake thoroughly to obtain a soil suspension with a concentration of 0.01g / mL. Spread the suspension evenly on Czapek's agar using a sterile triangular glass rod. Then, incubate in the dark and upside down in a constant temperature incubator at 28℃. After 2 days, observe the growth of fungi, take photos and record the results. Figure 1 Select a single colony from the plate and immediately purify and preserve the strain. This step screened one fungus; after 72 hours of culture on a PDA plate, the colonies were circular with regular edges. The central area of ​​the colony was light gray (dense area of ​​newly formed hyphae), the transition zone was grayish-green (active sporulation zone), and the edge area was dark grayish-green (rich layer of mature spores). The surface exhibited typical "cotton-like" ridges (aerial hyphae approximately 1–2 mm in height). Figure 2 It was named T15.

[0063] Example 2

[0064] Plate confrontation test with pathogens

[0065] A piece of strain T15, isolated and purified in Example 1, was cultured on PDA medium. A 2mm diameter mycelial cake of the pathogen of *Sclerotium affine* was inoculated into the center of the PDA solid medium. A 5mm piece of strain T15 was inoculated 2.0cm away from the mycelial cake. After 4 days of incubation at 28℃, the diameter of the inhibition zone was measured, and the inhibition rate (%) was calculated. Inhibition rate = (Coronavirus colony diameter in control group - Coronavirus colony diameter in experimental group) / Coronavirus colony diameter in control group × 100%. Results are as follows: Figure 3 As shown, the left side represents the control treatment with a single inoculation of the pathogen; the right side represents the experimental group inoculated with strain T15 and the pathogen. According to... Figure 3 It can be seen that strain T15 has a significant inhibitory effect on the growth of the pathogen of white rot on the plate. The diameter of the pathogen colony in the control group was 90 mm, and the diameter of the pathogen colony in the experimental group was 14.4 mm. The inhibition rate of strain T15 against the pathogen was 84%.

[0066] Example 3

[0067] Identification of strain T15

[0068] (1) Genomic DNA was extracted from strain T15, which was isolated and purified in Example 1, using the Omega Fungi DNA kit provided by Kunming Shuoqingke Biotechnology Co., Ltd. Then, PCR amplification was performed using universal primers for fungal identification: ITS 1: 5'-TCCGTAGGTGACCTGCGG-3' (SEQ ID NO:1) and ITS 4: 5'-TCCTCCGC TTATTGATATGC-3' (SEQ ID NO:2);

[0069] The PCR amplification system consisted of: 10 μL Gold Mix (containing dye), 1 μL upstream primer (ITS1), 1 μL downstream primer (ITS4), 1 μL template DNA, and 7 μL ddH2O.

[0070] PCR amplification program: 94℃ pre-denaturation for 1.5 min; 94℃ denaturation for 30 s; 57℃ annealing for 30 s; 72℃ extension for 30 s; 30 cycles; 72℃ extension for 15 min.

[0071] (2) The PCR amplification results obtained in step (1) were sent to Kunming Shuoqing Biotechnology Co., Ltd. for sequencing. The ITS gene sequence of strain T15 was: 5'-TCGTAACAAGGTCTCCGTTGGTGAAC-3' (SEQ ID NO:3). The obtained sequence was compared with the NCBI database. Reference strains were selected based on the comparison results, and a phylogenetic tree was constructed using MEGA 7.0 software. Figure 4 ), clarifying its classification status. According to Figure 4 It can be seen that strain T15 clusters with Trichoderma asperellum in one branch, with a similarity of 98%. At the same time, according to the identification criteria of China General Microbiological Culture Collection Center (CGMCC), strain T15 is identified as Trichoderma asperellum, renamed Trichoderma asperellum ZYC2023, and biologically preserved.

[0072] Example 4

[0073] Inhibitory effect of Trichoderma echinococcus ZYC2023 on sclerotium growth of the pathogen causing white rot of Aconitum carmichaelii.

[0074] (1) Inoculation material for pathogens: The pathogenic plants of Aconitum carmichaelii were activated and inoculated onto PDA solid medium and cultured at 25°C for 4 days before use. When using, cut the fungal cakes into 5mm long circular pieces with a blade.

[0075] (2) In the treatment group, a pathogenic fungal cake with a diameter of 5 mm was inoculated on one side of a PDA plate with the mycelial side facing down, and a Trichoderma echinocandi ZYC2023 fungal block with a diameter of 5 mm was inoculated at equal distances on the other side of the PDA plate. A control group was set up (the Trichoderma echinocandi ZYC2023 fungal block was replaced with a PDA fungal cake). After incubation at 28℃ for 4 days, the diameter of the inhibition zone was measured. Three replicates were set up.

[0076] (3) When the control colonies fully covered the culture dish, the diameter of the *Trichoderma echinococcus* ZYC2023 mycelial block was measured using the plate confrontation method. When the control colonies had 30–40 sclerotia, the number of sclerotia in the treatment group was recorded, and the sclerotia formation inhibition rate of the treatment group was calculated. The sclerotia formation inhibition rate = (average number of sclerotia in the control group - average number of sclerotia in the treatment group) / average number of sclerotia in the control group × 100%. Results are as follows: Figure 5 As shown, the left side represents the sclerotium formation in the control group; the right side represents the sclerotium formation in the treatment group. According to... Figure 5 It can be seen that Trichoderma echinococcus ZYC2023 can significantly inhibit the formation of sclerotia in plants infected with white sclerotium wilt. The average number of sclerotia in the control group was 62, while the average number of sclerotia in the treatment group was 3. The inhibition rate of Trichoderma echinococcus ZYC2023 on the formation of sclerotia in plants infected with white sclerotium wilt was 95.16%.

[0077] Example 5

[0078] Inhibitory effect of Trichoderma echinococcus ZYC2023 on white rot disease

[0079] 1. Referring to existing technology (Xu Rong, Yao Jiahuan, Qiu Xinyue, et al. Field control effect of Trichoderma on maize stalk rot and its influence on maize yield [J]. Shanxi Agricultural Sciences, 2024, 52(04):133-141.), Trichoderma echinosporum ZYC2023 in Example 4 was cultured to obtain a concentration of 5×10 6 A spore suspension of CFU / mL.

[0080] 2. Prepare 120 flower pots with a diameter of 20cm, fill them with sterilized aconite planting soil at high temperature to about 80% of their capacity, and plant one healthy aconite seedling with a height of 25cm in each flower pot; set up CK, white mold fungus block and white mold fungus block + T153 treatments, and carry out the following treatments in accordance with existing technology (Su Chang, Lian Hua, Ma Guangshu, et al. Effects of different application methods of Trichoderma on peroxidation of cucumber membrane lipids, activity of protective enzymes and control efficacy of wilt disease [J]. Plant Protection, 2021, 47(02):142-149+155.DOI:10.16688 / j.zwbh.2019671.):

[0081] Group CK: No processing performed;

[0082] White rot fungal block group: First, use a fine needle sterilized at high temperature to puncture the junction of Aconitum carmichaelii root and stem, and then inoculate the white rot pathogen C201909 fungal block into the puncture site;

[0083] White rot pathogen blocks + T15 group: First, use a fine needle sterilized at high temperature to puncture the junction of Aconitum carmichaelii root and stem, and then inoculate the white rot pathogen blocks at the puncture site; the day after inoculation, drench each plant with 100mL of the spore suspension obtained in step 1.

[0084] 3. After 14 days of treatment in step 2, observe the growth of Aconitum carmichaelii in each treatment group and calculate the disease index. The results are as follows: Figure 6 and Figure 7 As shown.

[0085] according to Figure 6 and Figure 7 It can be seen that no plants died in the group inoculated with *Trichoderma harzianum* var. *spinosa* + T15, while all plants in the group inoculated with *Trichoderma harzianum* var. *spinosa ...

[0086] Example 6

[0087] Inhibitory effect of Trichoderma echinococcus ZYC2023 on white rot disease

[0088] 1. Preparation of solid-state fermentation products

[0089] (1) Referring to existing technology (Xu Rong, Yao Jiahuan, Qiu Xinyue, et al. Field control effect of Trichoderma on maize stalk rot and its influence on maize yield [J]. Shanxi Agricultural Sciences, 2024, 52(04):133-141.), Trichoderma echinosporum ZYC2023 in Example 4 was cultured to obtain a concentration of 1×10 6 CFU / mL spore suspension;

[0090] Wheat bran, whole rice husks, corn flour, and spore suspension were mixed in a mass ratio of 4:3:2:1 to obtain the fermentation substrate. The fermentation substrate and water were mixed in a mass ratio of 1:1.3, the pH was adjusted to 5.5, and the mixture was incubated at 26–28℃ for 7 days. It was then dried at 37℃, not exceeding 40℃, to obtain a solid fermented product with a concentration of 20 × 10⁻⁶. 9 CFU / g.

[0091] 2. In 2023, a comparative experiment was conducted in Dakang Town and Taiping Town, Jiangyou City, Sichuan Province, to control white mold disease in Aconitum carmichaelii. Aconitum carmichaelii was planted in fields with severe white mold disease in the previous year, and treatment began after root pruning.

[0092] Six treatments were set up in Dakang Town: Control group (CK): no treatment; T1 treatment group (T15): using solid fermentation material obtained in step 1 (spore count of 2 billion / g); T2 treatment group: using copper oxychloride·copper oxychloride; T3 treatment group: using oligosaccharide·allicin; T4 treatment group: using fludioxonil·thifluzamide; T5 treatment group: using Bacillus subtilis·Trichoderma harzianum. Among them, copper oxychloride·copper oxychloride, oligosaccharide·allicin, fludioxonil·thifluzamide, and Bacillus subtilis·Trichoderma harzianum are all finished products and are used according to the recommended dosage of commercial products. The dosage of solid fermentation material is 2.5 kg / mu, diluted with 2.5 kg to 200 L of water and then applied to the roots.

[0093] Six treatments were set up in Taiping Town: Control group (CK): no treatment; T1 treatment group (T15): using solid fermentation material obtained in step 1; T2 treatment group: using fludioxonil; T3 treatment group: using pyraclostrobin; T4 treatment group: using sclerotinib; T5 treatment group: using hymexazol. Fludioxonil, pyraclostrobin, sclerotinib, and hymexazol are all commercially available pesticides and should be used according to the recommended dosage. The dosage of solid fermentation material is 2.5 kg / mu, diluted in 200 L of water and then applied to the roots.

[0094] 3. When Aconitum carmichaelii develops white mold disease, the root and stem gradually rot. Initially, the leaves appear normal, but as the rot worsens, the leaves wilt and droop around midday on sunny days. In severe cases, the above-ground parts collapse, the leaves wither, but the stem does not break, and the mother root remains connected to the stem. White, silky mycelium and blackish-brown, rapeseed-sized sclerotia appear on the surface of the rotten roots, the base of the stem, and the surrounding soil. In severe cases, the entire plant dies. At harvest, the number of Aconitum carmichaelii plants infected with white mold disease was counted in each treatment group. The incidence rate and biological control rate of Aconitum carmichaelii were calculated: Incidence rate = Number of infected plants / Total number of plants × 100%; Biological control rate = (Incidence rate in control group - Incidence rate in experimental group) / Incidence rate in control group × 100%. Results are as follows: Figure 8 and Figure 9 As shown.

[0095] according to Figure 8 and Figure 9It can be seen that the application of *Trichoderma echinocandus* ZYC2023 during the root pruning period of *Aconitum carmichaelii* can significantly reduce the incidence of white mold disease. In Dakang Town, the incidence rate can be controlled below 20%, which is better than other treatments. The CK incidence rate was over 27% in the same year, and the biocontrol rate of white mold disease using *Trichoderma echinocandus* ZYC2023 was 55%. In Taiping Town, the incidence rate of white mold disease in *Aconitum carmichaelii* can be controlled below 10%, while other treatments are all between 10% and 15%. Among them, the CK incidence rate was over 14%, and the biocontrol rate of white mold disease using *Trichoderma echinocandus* ZYC2023 was 53%. The results prove that the application of *Trichoderma echinocandus* ZYC2023 during the root pruning period of *Aconitum carmichaelii* can control the occurrence of white mold disease.

[0096] Example 7

[0097] A field trial was conducted in 2024 at Puzhao Temple, Taiping Town, Jiangyou, Sichuan Province. The plots with the most severe disease outbreaks in the previous year were selected and randomly divided into two treatments: the T15 treatment group and the production standard treatment group. Each treatment had 12 replicates. The specific treatment methods are as follows:

[0098] T15 treatment group: Before planting Aconitum carmichaelii, the solid fermentation product obtained in Example 6 (spore quantity 20 × 10⁻⁶) was applied as a base fertilizer. 9 Apply CFU / g evenly at a dosage of 2.5 kg / mu.

[0099] Production routine treatment group: Before planting Aconitum carmichaelii, apply commercial organic fertilizer (purchased from the fertilizer distribution point of Sichuan Zhongnong Runze Biotechnology Co., Ltd.) evenly according to the recommended dosage of the commercial agent, following the method of applying base fertilizer;

[0100] In the later stages, the T15 treatment group and the conventional organic fertilizer treatment group were treated according to the local conventional aconite production method.

[0101] At harvest, the number of plants infected with Aconitum carmichaelii in the T15 treatment group and the conventional organic fertilizer treatment group was counted, and the incidence rate and biological control rate of Aconitum carmichaelii were calculated: Incidence rate = Number of infected plants / Total number of plants × 100%, Biological control rate = (Incidence rate in control group - Incidence rate in experimental group) / Incidence rate in control group × 100%. Results are as follows: Figures 10-12 As shown.

[0102] according to Figures 10-12It can be seen that there are significant differences between the T15 treatment group and the conventional organic fertilizer treatment group. In the T15 treatment group, the incidence of white mold disease in all 12 replicates (T1-T12) treated with *Trichoderma echinococcus* ZYC2023 was significantly lower than that in the conventional organic fertilizer treatment group. The incidence rate of white mold disease was also significantly lower in the T15 treatment group than in the conventional organic fertilizer treatment group. The average incidence rate in the T15 treatment group was below 10%, while the average incidence rate in the conventional organic fertilizer treatment group was above 20%. The biocontrol rate of white mold disease using *Trichoderma echinococcus* ZYC2023 was 57%. These results demonstrate that applying *Trichoderma echinococcus* ZYC2023 as a basal fertilizer can significantly reduce the incidence of white mold disease in *Trichoderma echinococcus*.

[0103] As can be seen from the above, the technical solution provided by the present invention can inhibit the mycelial growth and sclerotium formation of the pathogen of Aconitum carmichaelii, significantly reduce the incidence of Aconitum carmichaelii, and reduce the disease index.

[0104] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A biocontrol fungus Trichoderma asperellum ZYC2023, with accession number CGMCCNo.40528.

2. A biocontrol agent, characterized in that, Includes Trichoderma hygroscopicum ZYC2023 as described in claim 1.

3. The biocontrol agent according to claim 2, characterized in that, The biocontrol agent is in the form of a liquid or solid preparation; The spore concentration of Trichoderma echinocandes ZYC2023 in the liquid formulation is 10. 6 ~10 8 CFU / mL; The spore concentration of Trichoderma echinocandes ZYC2023 in the solid dosage form is 1×10⁻⁶. 9 ~50×10 9 CFU / g.

4. A method for preparing a biocontrol agent, characterized in that, The process includes the following steps: inoculating Trichoderma hygroscopicum ZYC2023 according to claim 1 into a solid fermentation medium for fermentation culture, drying the solid fermentation product, and obtaining the biocontrol agent; The solid fermentation culture medium includes fermentation substrate and water; the fermentation substrate includes wheat bran, rice husks and corn flour; the mass ratio of wheat bran, rice husks and corn flour is 2-5:1-4:1.5-2.

5.

5. The biocontrol agent according to claim 4, characterized in that, The fermentation culture was carried out at a temperature of 26–28°C for 5–9 days, with a pH of 5.0–6.5; the drying temperature was 37–40°C.

6. The application of the *Trichoderma hygroscopicum* ZYC2023 according to claim 1, or the biocontrol agent according to claim 2 or 3, or the biocontrol agent prepared by the preparation method according to claim 4, in inhibiting the mycelial growth and / or sclerotium formation of the pathogen of *Trichoderma hygroscopicum*.

7. The application of the Trichoderma hygroscopicum ZYC2023 according to claim 1, or the biocontrol agent according to claim 2 or 3, or the biocontrol agent prepared by the preparation method according to claim 4, in the control of plant white mold disease.

8. The application according to claim 7, characterized in that, The plant mentioned includes Aconitum carmichaelii.

9. A method for preventing and treating Aconitum carmichaelii white rot, characterized in that, Includes one or more of the following steps I to III: I: On the second day after Aconitum carmichaelii is infected with the pathogen of white rot, drench the roots with the biocontrol agent as described in claim 2 or 3 or the biocontrol agent prepared by the preparation method described in claim 4; II: During the root pruning period of Aconitum carmichaelii, apply the biocontrol agent as described in claim 2 or 3 or the biocontrol agent prepared by the preparation method described in claim 4; III: After applying basal fertilizer, sow Aconitum carmichaelii; the basal fertilizer includes the biocontrol agent as described in claim 2 or 3 or the biocontrol agent prepared by the preparation method described in claim 4.

10. The method according to claim 9, characterized in that, In the case of I, the biocontrol agent is a liquid preparation with a spore concentration of 10. 6 ~10 8 CFU / mL, root irrigation volume is 50-100 mL / plant; In section II, the biocontrol agent is a solid preparation with a spore concentration of 1×10⁻⁶. 9 ~50×10 9 CFU / g, diluted and applied as a root drench, with the amount applied based on the mass of the solid formulation being 2.5 kg / mu; In section III, the biocontrol agent is a solid preparation with a spore concentration of 1×10⁻⁶. 9 ~50×10 9 CFU / g, application rate is 2.5 kg / mu.

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