A synergistic agent of plant-derived alkaloids and Trichoderma and its application

Abstract

This invention relates to a synergistic formulation of plant-derived alkaloids and Trichoderma, and its application, belonging to the field of biological control technology. To address the technical problems of existing methods for controlling tobacco mosaic virus, such as long treatment cycles, low control efficiency, and potential harm to the environment and human health, this invention provides a formulation containing Trichoderma tuftedum and celandine. The formulation provided by this invention, at a dilution of 50 times, synergistically enhances the control effect against tobacco mosaic virus through multiple mechanisms. The formulation can be applied to tobacco cultivation and production, effectively improving the control efficiency of tobacco mosaic virus and increasing the economic benefits of tobacco cultivation.

Description

Technical Field

[0001] This invention belongs to the field of biological control technology, and relates to a synergistic agent of plant-derived alkaloids and Trichoderma and its application. Background Technology

[0002] Tobacco mosaic virus (TMV) is a plant disease caused by the tobacco mosaic virus. This disease is extremely detrimental to tobacco production, characterized by its wide host range, strong resistance, and difficulty in control. After infection, plants exhibit browning and necrosis of leaves, shortened internodes, and slowed or even stopped growth, significantly impacting both yield and quality, resulting in substantial economic losses for agricultural production. Currently, the main methods for controlling TMV are breeding resistant varieties, enhancing field management, and applying chemical pesticides. However, breeding resistant varieties is time-consuming, and resistance can become ineffective due to viral mutations. Furthermore, while field management can reduce the incidence of the disease, it cannot completely eliminate it. Although chemical pesticides can quickly control the disease, long-term and excessive use can harm the environment and human health, and can easily lead to increased pesticide resistance in pathogens.

[0003] Trichoderma sp. is a commonly used biocontrol fungus in agriculture. It controls pathogens through parasitism, competition, and inhibition, and can even induce and enhance the plant's own immunity. Among them, Trichoderma tuftedum, as a biocontrol fungus, can produce a variety of bioactive metabolites, such as enzymes, antibiotics, and secondary metabolites, which can directly inhibit viral activity or destroy viral structure.

[0004] Chelidonine, a component of the poppy plant *Chelidonium majus* L., possesses various pharmacological effects, including antitumor, analgesic, and antispasmodic properties. In agricultural applications, literature reports some insecticidal activity against gypsy moth larvae and red pine caterpillars; however, no research reports have been published regarding its other agricultural activities, and its application in agriculture requires further development.

[0005] Because biological control methods can effectively improve disease control, reduce the use of chemical pesticides, and minimize harm to the environment and human health, those skilled in the art are eager to develop a green and efficient biological control method for tobacco mosaic disease. Summary of the Invention

[0006] To address the technical problems of existing methods for controlling tobacco mosaic disease, such as long treatment cycles, low control efficiency, and potential harm to the environment and human health, this invention provides a synergistic agent of plant-derived alkaloids and Trichoderma and its application.

[0007] One of the objectives of this invention is to provide a synergistic formulation of plant-derived alkaloids and Trichoderma, wherein the formulation contains a fermentation extract of Trichoderma tuftedum and a mother liquor of celandine; the fermentation extract of Trichoderma tuftedum is obtained by centrifugation and extraction of the fermentation broth of Trichoderma tuftedum.

[0008] Preferably, the purity of the chelidonine mother liquor used in the formulation is 10%-20%.

[0009] Preferably, the mixing volume ratio of the Trichoderma tuftedilum fermentation extract and the chelidonine mother liquor in the preparation is 1:(5-10).

[0010] The second objective of this invention is to provide the application of the above-mentioned preparation in the prevention and control of tobacco mosaic virus.

[0011] Preferably, the application involves diluting the preparation 50 times and spraying it onto Tobacco Benedict plants for the control of tobacco mosaic virus.

[0012] The beneficial effects of this invention: This invention provides a synergistic formulation of plant-derived alkaloids and Trichoderma, the formulation containing fermentation extract of Trichoderma tuftedum and celandine. This invention uses Trichoderma tuftedum strains as biocontrol fungi to prepare a synergistic formulation, utilizing its secretion of bioactive metabolites, such as enzymes, antibiotics, and polysaccharides, to activate the plant's internal defense system, thereby enhancing the plant's resistance to viruses. This invention utilizes the antibacterial, antiviral, and anti-inflammatory effects of celandine to prepare the formulation. By mixing the fermentation extract of Trichoderma tuftedum with celandine stock solution at a volume ratio of 1:(5-10), and determining the optimal dilution ratio of this synergistic formulation to be 50 times, the control effect against tobacco mosaic virus is improved through multiple synergistic mechanisms.

[0013] The efficacy data show that, under 50-fold dilution, the formulation prepared by this invention at a 1:5 volume ratio exhibits an inhibition rate of up to 82.21% against tobacco mosaic virus, and the formulation prepared at a 1:10 volume ratio exhibits an inhibition rate of up to 80.77% against tobacco mosaic virus. Furthermore, the formulation significantly induces enhanced resistance in tobacco plants. Therefore, the formulation provided by this invention has high prevention and control efficiency against tobacco mosaic virus, effectively improving disease control, reducing the use of chemical pesticides, and minimizing harm to the environment and human health. It is green and safe. The provided formulation can be applied to tobacco planting and production, effectively improving the control efficiency of tobacco mosaic virus and increasing the economic benefits of tobacco cultivation. Attached Figure Description

[0014] Figure 1 The effect of different formulations on the accumulation of malondialdehyde (MDA) in plants is shown in the figure; a is the experimental group without tobacco mosaic virus infection, and b is the experimental group infected with tobacco mosaic virus.

[0015] Figure 2 The graph shows the effect of different formulations on the accumulation of salicylic acid (SA) in plants. Detailed Implementation

[0016] Those skilled in the art can refer to the content of this document and appropriately improve the process parameters to achieve the desired results. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments, and those skilled in the art can obviously make modifications or appropriate alterations and combinations to the methods and applications described herein without departing from the content and scope of this invention to implement and apply the technology of this invention.

[0017] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the materials, reagents, methods, and instruments used are all conventional materials, reagents, methods, and instruments in the art, and can be obtained commercially by those skilled in the art.

[0018] The Trichoderma tuftedum strain involved in the following examples is Trichoderma tuftedum strain YW411, with accession number CGMCCNo.23202, and is disclosed in the invention patent with authorization announcement number CN114134052B.

[0019] Example 1: Preparation of fermentation extract from Trichoderma tuftans strain

[0020] The purified Trichoderma tuftedum strain was inoculated into PDB medium (200 g of potato was boiled in water until soft, filtered, 20 g of glucose was added, water was added to make up to 1 L, and sterilized at 115℃ for 30 min). It was cultured at 28℃ and 150 rpm for 7 days with constant temperature shaking. It was then centrifuged at 4℃ and 8000 r / min for 10 min, and the supernatant was collected. An equal volume of ethyl acetate was added and extracted three times. The extracts were combined. The crude extract was obtained by rotary evaporation at 35℃ (concentrated to about 300 mL). It was then filtered through a 0.22 μm organic phase filter membrane.

[0021] Example 2: Preparation of Chelidonium red alkaloid mother liquor

[0022] This embodiment refers to the preparation of chelidonine mother liquor according to "Wei Qinghui. Study on the inhibitory mechanism of chelidonine and its derivatives against plant pathogens [D]. Northeast Forestry University, 2020." The specific steps include: accurately weighing 15 mg of chelidonine using an analytical balance, accurately measuring 10 mL of dimethyl sulfoxide using a graduated cylinder, and preparing a mother liquor with a final concentration of 0.15 mg / mL using 90 mL of deionized water. The solution is then filtered through a 0.22 μm organic phase filter membrane to obtain the chelidonine mother liquor.

[0023] Example 3: Preparation of a synergistic agent of plant-derived alkaloids and Trichoderma.

[0024] The fermentation extract of Trichoderma tufts obtained in Example 1 and the mother liquor of Celandine alkaloids obtained in Example 2 were mixed according to the volume ratios of formulations 1-7 in Table 1 to obtain preparations with different ratios.

[0025] Table 1

[0026]

[0027] Effect Experiment:

[0028] In this embodiment, the tobacco mosaic virus venom was prepared according to the "Zhou Xiangping, Deng Zhengyu, Xu Qingxiao, et al. Preparation and verification of tobacco mosaic virus (TMV) immune inducer [J]. Hunan Agricultural Sciences, 2024, (04): 72-76." The concentration of the tobacco mosaic virus venom prepared in this embodiment was 10 μg / mL. When inoculating, select tobacco plants with uniform growth, mix the venom with an equal volume of water, and inoculate the tobacco leaves.

[0029] (1) The inhibitory effect of different formulations on tobacco mosaic virus.

[0030] During the experiment, the control group mixed the venom with an equal volume of water and inoculated it onto the left half of the tobacco leaves; the experimental group mixed the venom with an equal volume of the formulations from Example 3 and inoculated it onto the right half of the tobacco leaves. The number of lesions on the left and right leaves of the tobacco leaves was counted, and the inhibition rate was:

[0031] Inhibition rate (%) = [(Number of lesions on the left leaf - Number of lesions on the right leaf) / Number of lesions on the left leaf] × 100%.

[0032] The results are shown in Table 2. The formulation provided by this invention has a certain inhibitory effect on tobacco mosaic virus. Only when the volume ratio of the fermentation broth of *Trichoderma tufteri* to the mother liquor of chelidonine is within the range of 1:(5-10), the inhibition rate of the formulation provided by this invention exceeds 50%, specifically 67.30% and 61.29%. The fermentation broth of *Trichoderma tufteri* and chelidonine in the formulation of this invention must be in a specific mixing ratio to achieve the optimal inhibition rate against tobacco mosaic virus.

[0033] Table 2

[0034]

[0035] (2) Inhibitory effect of different concentrations of formulations on tobacco mosaic virus

[0036] The formulation 6 in Example 3 was diluted at dilution ratios of 5, 10, 50, 100, and 500 times to obtain formulations of different concentrations. In the control group, the toxicant was mixed with an equal volume of water and inoculated onto the left half of tobacco leaves. In the experimental group, the toxicant was mixed with an equal volume of the above-mentioned formulations of different concentrations and inoculated onto the right half of tobacco leaves. The number of lesions on the left and right leaves of the tobacco plants was counted, and the inhibition rate was:

[0037] Inhibition rate (%) = [Number of lesions on the left leaf - Number of lesions on the right leaf] / Number of lesions on the left leaf] × 100%.

[0038] The results are shown in Table 3. When a dilution factor of 50 times is used, the formulation prepared by the present invention at a mixing volume ratio of 1:5 has the best inhibitory effect on tobacco mosaic virus, with an inhibition rate as high as 82.21%.

[0039] Table 3

[0040]

[0041] The formulation 7 in Example 3 was diluted at dilution ratios of 5, 10, 50, 100, and 500 times to obtain formulations of different concentrations. In the control group, the toxicant was mixed with an equal volume of water and inoculated onto the left half of tobacco leaves. In the experimental group, the toxicant was mixed with an equal volume of the above-mentioned formulations of different concentrations and inoculated onto the right half of tobacco leaves. The number of lesions on the left and right leaves of the tobacco plants was counted, and the inhibition rate was:

[0042] Inhibition rate (%) = [Number of lesions on the left leaf - Number of lesions on the right leaf] / Number of lesions on the left leaf] × 100%.

[0043] The results are shown in Table 4. When a dilution factor of 50 times is used, the formulation prepared by the present invention at a mixing volume ratio of 1:10 has the best inhibitory effect on tobacco mosaic virus, with an inhibition rate as high as 80.77%.

[0044] Table 4

[0045]

[0046] Therefore, when the volume ratio of the fermentation broth of Trichoderma tuftans to the mother liquor of Celandine is in the range of 1: (5-10), and the mixture is diluted 50 times, the formulation provided by the present invention can exert the best inhibitory effect on tobacco mosaic virus, thereby achieving a high control effect on tobacco mosaic disease.

[0047] (3) Detection of plant antiviral ability induced by different formulations

[0048] The fermented extract of Trichoderma tufts from Example 1 and the formulations of Formula 6 and Formula 7 from Example 3 were sprayed onto the leaves of Tobacco Benedict (experimental group). The control group mixed tobacco mosaic virus virulence with an equal volume of water and sprayed it onto the leaves of Tobacco Benedict. After 6 days of treatment, the total number of lesions per tobacco plant in each group was counted.

[0049] The results are shown in Table 5. Compared with the control group and the fermented extract of Trichoderma tufts, the formulation provided by the present invention significantly reduced the number of lesions on the plant leaves. It can be seen that the formulation provided by the present invention can induce plant resistance to tobacco mosaic virus and has a good control effect on tobacco mosaic virus.

[0050] Table 5

[0051]

[0052] (4) Effects of different formulations on the accumulation of malondialdehyde (MDA) in plants

[0053] The fermentation extract of Trichoderma tufts from Example 1 and the formulations of Formula 6 and Formula 7 from Example 3 were sprayed on the leaves of Nicotiana benthamiana (divided into infected and uninfected Nicotiana benthamiana) (experimental group). The control group was sprayed with an equal amount of sterile water on the leaves of Nicotiana benthamiana (divided into infected and uninfected Nicotiana benthamiana). After 5 days of treatment, the plant leaves of each group were cut off and the malondialdehyde content was detected.

[0054] The results are as follows Figure 1 As shown, compared with the control group and the Trichoderma tuftedum fermentation extract, the formulation provided by the present invention significantly reduced the malondialdehyde content in plant leaves; it can be seen that the Trichoderma tuftedum fermentation extract provided by the present invention, as well as the formulation containing the Trichoderma tuftedum fermentation extract, have the ability to enhance plant disease resistance and have a good control effect on tobacco mosaic virus.

[0055] (5) Effects of different formulations on the accumulation of salicylic acid (SA) in plants

[0056] The fermentation extract of Trichoderma tufts from Example 1 and the formulations of Formula 6 and Formula 7 from Example 3 were sprayed on the leaves of Nicotiana benthamiana (the experimental group, which was Nicotiana benthamiana without tobacco mosaic virus infection). The control group was sprayed with an equal amount of sterile water on the leaves of Nicotiana benthamiana (the control group, which was Nicotiana benthamiana without tobacco mosaic virus infection). After 2 hours of treatment, the plant leaves of each group were cut off and the salicylic acid content was detected.

[0057] The results are as follows Figure 2As shown, compared with the control group and the Trichoderma tuftedum fermentation extract, the formulation provided by the present invention significantly increased the salicylic acid content in plant leaves; it can be seen that the Trichoderma tuftedum fermentation extract provided by the present invention, as well as the formulation containing the Trichoderma tuftedum fermentation extract, have the ability to enhance plant disease resistance and have a good control effect on tobacco mosaic virus.

[0058] The contents not described in detail in this specification are well-known to those skilled in the art. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A synergistic agent of plant-derived alkaloids and Trichoderma for the prevention and control of tobacco mosaic virus, characterized in that, The preparation contains a fermentation extract of Trichoderma tuftedum and a mother liquor of celandine alkaloids; The Trichoderma tuftedum fermentation extract was obtained by centrifugation and extraction of the Trichoderma tuftedum fermentation broth. The volume ratio of the fermentation extract of Trichoderma tuftana and the mother liquor of Chelidonium majus in the preparation is 1:(5-10); The *Trichoderma tuftedum* strain is *Trichoderma tuftedum* strain YW411, with accession number CGMCC No. 23202; The extractant used in the extraction was ethyl acetate.

2. The formulation according to claim 1, characterized in that, The purity of the chelidonine mother liquor used in the formulation is 10%-20%.

3. The use of the formulation according to any one of claims 1 to 2 in the prevention and control of tobacco mosaic virus, characterized in that, The application involves diluting the preparation 50 times and spraying it onto Tobacco Benedict plants to control tobacco mosaic virus.

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