A kind of smilax china leaf spot disease prevention and control agent and prevention and control method

Abstract

The present application relates to the technical field of plant disease control, and particularly discloses a kind of smilax bracteata leaf spot disease control agent and control method, the present application provides a kind of smilax bracteata leaf spot disease control agent, the control agent contains the ethanol extract of big fruit of soil chestnut fruit, the present application is indicated by indoor virulence test, the ethanol extract of big fruit of soil chestnut fruit has significant inhibitory effect on smilax bracteata leaf spot disease various pathogenic bacteria;Field test shows that after spraying the ethanol extract of big fruit of soil chestnut fruit with mass concentration of 800mg / L, smilax bracteata leaf spot disease is effectively controlled, and the highest control effect reaches 80.04%, and the present application provides a theoretical basis and technical support for the sustainable prevention and control of smilax bracteata disease.

Description

Technical Field

[0001] This invention relates to the field of medicinal plant disease control technology, specifically providing a control agent and method for controlling leaf spot disease in Polygonatum yunnanense. Background Technology

[0002] *Polygonatum kingianum*, a perennial herb belonging to the genus *Polygonatum* in the family Liliaceae, is an important traditional Chinese medicine in Yunnan Province with a long history of medicinal use. To meet market demand, large-scale cultivation of *Polygonatum kingianum* has been initially established in many parts of Yunnan. However, it is worth noting that the frequent occurrence of leaf spot disease accompanying this large-scale cultivation has become a major bottleneck restricting the industry's development. Leaf spot disease is most severe in spring and autumn, mainly manifesting as localized necrotic spots on the leaf surface. The disease incidence rate can reach over 50%, causing yield reductions of 10% to 30%. This disease leads to a significant decrease in plant yield and deterioration in quality, resulting in substantial economic losses and seriously threatening the sustainable development of the *Polygonatum kingianum* industry. There are multiple pathogens causing leaf spot disease in Polygonatum kingianum. In the main planting areas of Polygonatum kingianum in Yunnan, the pathogens that have been isolated and identified for leaf spot disease in Polygonatum kingianum include Stemphylium lycopersici (Jianxin C, Yuqian W, Zejia L, et al. First report of Stemphylium lycopersici causing leaf spot on Polygonatum kingianum in China. [J]. Plant disease, 2021) and Neopestaltiopsisacrostichi (Xu Rong, Wang Siting, Wang Zhandi, et al. Isolation and identification of pathogens causing leaf spot disease in Polygonatum kingianum [J]. Acta Phytopathologica Sinica, 2024, 54 (06): 1252-1256.).

[0003] Currently, the control of this disease mainly relies on chemical agents such as carbendazim and chlorothalonil. Although these can inhibit the spread of pathogens in the short term, long-term use can easily lead to drug resistance in pathogens, reducing the control effect and even potentially disrupting the balance of soil microbial communities, affecting the ecological adaptability of *Polygonatum yunnanense*, which is inconsistent with the development trend of modern green agriculture and organic farming. Plant-derived extracts have advantages such as low toxicity, environmental friendliness, easy degradation, and low resistance to drug development. Plant-derived antibacterial substances, such as flavonoids, alkaloids, phenolic acids, and terpenoids, can exert antibacterial effects through multiple mechanisms, such as interfering with the integrity of pathogen cell membranes, inhibiting mycelial growth, or spore germination. In addition, some plant extracts can induce systemic resistance in plants, enhancing their own immunity, thereby achieving a more lasting disease control effect. Therefore, screening for highly efficient, low-residue, and environmentally compatible plant-derived control agents, and combining them with green control technologies such as biological control and agricultural ecological regulation, to construct a comprehensive control system for *Polygonatum yunnanense* leaf spot disease is a key breakthrough to ensure the sustainable development of the *Polygonatum yunnanense* industry. Summary of the Invention

[0004] The purpose of this invention is to provide a plant extract-based biocontrol agent that can effectively control leaf spot disease in Polygonatum yunnanense, reduce environmental pollution, and improve the yield and quality of Polygonatum yunnanense.

[0005] To achieve the above objectives, the present invention provides the following technical solution:

[0006] This invention provides a control agent for leaf spot disease of Polygonatum yunnanense, wherein the control agent contains an ethanol extract of Phoebe bournei fruit.

[0007] This invention also provides a method for controlling leaf spot disease in Polygonatum yunnanense, which involves spraying a fungicide containing an ethanol extract of the fruit of *Phoebe zhennan* onto the Polygonatum yunnanense. In a preferred embodiment, the pathogens causing leaf spot disease in Polygonatum yunnanense include *Phyllostacta capitalensis*, *Neopestalotiopsis acrostichi*, *Stemphylium lycopersici*, or *Boeremiaexigua*.

[0008] Furthermore, the concentration of the ethanol extract of the fruit of *Phoebe zhennan* is not less than 116.48 mg / L.

[0009] Furthermore, the present invention also provides the application of an ethanol extract of the fruit of *Phyllostacta capitalensis* in the prevention and control of plant pathogens, wherein the pathogens include *Phyllostacta capitalensis*, *Neopestalotiopsis acrostichi*, *Stemphylium lycopersici*, or *Boeremia exigua*.

[0010] In one embodiment, the present invention also provides the application of a fungicide in controlling plant pathogens, said pathogens including *Phyllostacta capitalensis*, *Neopestalotiopsis acrostichi*, *Stemphylium lycopersici*, or *Boeremia exigua*, wherein the fungicide contains an ethanol extract of *Phyllostacta capitalensis* fruit. In some embodiments, the fungicide further contains adjuvants, carriers, preservatives, pH adjusters, or buffers, etc. In some embodiments, the adjuvants include surfactants, emulsifiers, dispersants, or stabilizers, etc.

[0011] In one embodiment, the present invention provides an application of a bactericidal composition for the control of plant pathogens, including *Phyllostacta capitalensis*, *Neopestalotiopsis acrostichi*, *Stemphylium lycopersici*, or *Boeremia exigua*, wherein the bactericidal composition contains an ethanol extract of *Phyllostacta capitalensis* fruit. In some embodiments, the bactericidal composition includes any other bactericidal active ingredient that can synergistically interact with the ethanol extract of *Phyllostacta capitalensis* fruit.

[0012] The technical effects achieved by this invention are as follows:

[0013] This invention demonstrates through indoor toxicity tests that the ethanol extract of *Phoebe zhennan* fruit has a significant inhibitory effect on multiple pathogens causing leaf spot disease in *Polygonatum yunnanense*, particularly showing an inhibition rate of up to 96.56% against *Neopestalotiopsis acrostichi*. Field trials show that spraying with an ethanol extract of *Phoebe zhennan* fruit at a concentration of 800 mg / L effectively controls leaf spot disease in *Polygonatum yunnanense*, with a control effect reaching up to 80.04%. The plant-derived fungicide provided by this invention can rapidly degrade into harmless small molecules in the environment, avoiding the persistent pollution of chemical pesticides and laying the foundation for the development of organic agriculture. Detailed Implementation

[0014] The present invention is further illustrated below through embodiments. It should be emphasized that these embodiments are merely examples to illustrate the principles and effects of the present invention and do not constitute a limitation on the scope of protection of the present invention. In the following embodiments, the experimental methods mentioned are, unless otherwise specified, conventional methods commonly used in the art. Similarly, the materials and reagents used in the embodiments, unless otherwise specified, can be obtained through conventional commercial channels.

[0015] Example 1: Extraction of active ingredients from the tested plant source

[0016] 1. Plant materials

[0017] The list of plants and the parts from which they were collected is shown in Table 1.

[0018] Table 1 Test Plants

[0019]

[0020] The term "fruit" refers to the reproductive organ that develops from the ovary (and other floral structures that may be involved) of angiosperms after fertilization, including the pericarp and the pit.

[0021] 2. Extraction Method

[0022] Ultrasonic extraction was employed. First, dried plant samples were pulverized and filtered through a 100-mesh sieve, collecting the homogeneous powder for later use. Then, the dried powder sample was weighed into an Erlenmeyer flask at a material-to-liquid ratio of 1:10 (w / v), and 95% ethanol solution was added. The flask was then fixed in an ultrasonic cleaner, and extraction was performed at 40 °C for 45 min with an ultrasonic power of 300 W and a frequency of 40 kHz. After extraction, the sample was immediately vacuum-filtered through double-layer qualitative filter paper (pore size 10-12 μm), and the filtrate was collected. The residue was extracted twice more, and the three filtrates were combined and centrifuged at 8000 r / min for 10 min at 4 °C. The filtrate was then concentrated by rotary evaporation to obtain the ethanol extract of the plant fruit. Anhydrous ethanol was added to the extract for redissolving, followed by distilled water to prepare a 50 mg / mL stock solution, which was stored at 4 °C for later use.

[0023] Example 2 Indoor toxicity experiment

[0024] 1. Test strains and culture media

[0025] The pathogens of leaf spot disease in Solomon's seal of Yunnan Province are Stemphylium lycopersici, Neopestalotiopsisacrostichi, Phyllostacta capitalensis, and Boeremia exigua. These pathogens were provided by the Yunnan Provincial Institute of Microbiology's Culture Collection Center. The culture medium was PDA medium (200 g potato, 20 g glucose, 15 g agar, diluted to 1 L with distilled water, pH 6.8, sterilized at 121℃ for 20 min).

[0026] 2. Experimental Methods

[0027] Strain activation: The pathogenic bacterial strain preserved at −80℃ was taken out and inoculated into PDA agar plates under aseptic conditions, and incubated in a dark incubator at 28℃ for 3 days. After incubation, mycelial blocks were aseptically collected from the edge of the colony for subsequent inoculation.

[0028] Preparation of drug-containing plates: Dilute the stock solution with sterile distilled water to the set concentration. Take 1 mL of the drug solution and add it to 49 mL of sterile PDA medium cooled to 40–45 °C. Mix thoroughly under aseptic conditions and pour into 90 mm sterile petri dishes. Set up 3 replicates for each concentration. Use an equal volume of sterile distilled water instead of PDA medium as a blank control.

[0029] Virulence assay method: After the culture medium solidifies, use a sterile punch (5 mm diameter) to collect mycelial cakes from the edge of colonies of the pathogen of *Polygonatum yunnanense* leaf spot disease activated for 5 days. Inoculate the mycelial cakes, hyphae facing down, onto the center of drug-containing PDA plates and control plates, and place them in a 28℃ incubator. After 5 days of continuous culture, measure the colony diameter (mm) using the cross-cross method. Calculate the mycelial growth inhibition rate of each treatment on the pathogen, converting the inhibition rate into an inhibition probability value. Convert the concentration of each tested agent into the logarithm of the agent concentration. Plot the logarithm of each tested agent concentration on the x-axis and the probability value on the y-axis. Use Excel software to calculate the virulence regression equation, correlation coefficient, and EC50 for each agent. 50 Value, and with the lowest toxicity EC 50 The value is used as a baseline to calculate the relative toxicity index of other drugs.

[0030]

[0031] 3. Experimental Results

[0032] (1) Inhibitory activity of ethanol extracts from different plant fruits against the pathogen of leaf spot disease in Polygonatum yunnanense.

[0033] As shown in Table 2, the ethanol extracts of different plant fruits exhibited different inhibitory activities at a mass concentration of 1 mg / mL. Among them, the ethanol extract of *Machilus maculatus* fruit showed significant inhibitory effects on various pathogens, with inhibition rates exceeding 60% for each pathogen. *Machilus maculatus* was selected for the next stage of the experiment.

[0034] Table 2. Inhibitory activity of ethanol extracts from different plant fruits against the pathogen causing leaf spot disease of Polygonatum odoratum.

[0035]

[0036] Note: The mass concentration of the tested extract was 1 mg / ml; "-" indicates an inhibition rate of less than 10%.

[0037] (2) Virulence determination of ethanol extract of Phoebe zhennan fruit against the pathogen of leaf spot disease of Polygonatum yunnanense

[0038] The antibacterial effects and virulence of the ethanol extract of *Neopestalotiopsis acrostichi* fruit against various pathogens are shown in Table 3. The results indicate that there are significant differences in the sensitivity of different pathogens to the agents. *Neopestalotiopsis acrostichi* was the most sensitive, with its EC50 value being [missing data]. 50 The value was 116.48 mg / L; followed by the EC of Phyllostachys capitalensis. 50 EC with 156.92 mg / L and Stemphylium lycopersici 50 The value was 239.47 mg / L; finally, the EC of Boeremia exigua was... 50 The value was 371.82 mg / L.

[0039] Table 3. Antibacterial effects and virulence of ethanol extracts from *Phoebe zhennan* fruit against different pathogens.

[0040]

[0041] Example 3: Field efficacy test of leaf spot disease control in Polygonatum yunnanense.

[0042] 1. Experimental Design

[0043] Three-year-old *Polygonatum yunnanense* plants with uniform growth were selected from Jianxing Township, Xinping County, Yuxi City (referred to as "XP") and Dalishu Township, Maguan County, Wenshan Prefecture (referred to as "MG"), respectively, for the experiment. The experiment included two treatments, with three replicates for each treatment. Each plot was 20 m². 2 A randomized block design was used, with guard rows between subgroups. The ethanol extract of *Machilus thunbergii* fruit was diluted with distilled water containing 0.1% Tween-80 before use. A carbendazim treatment group and a control group (CK) were also established. The CK group was sprayed with distilled water containing 0.1% Tween-80 ("0.1%" is by volume) using a foliar spray method, ensuring sufficient plant coverage without dripping, with a water volume of 450 L·hm². -2 Spray once every 5 days. Conduct disease surveys before the first application and 5 days after the second application to calculate the disease index and control effect.

[0044] Grading standards for leaf spot disease of Polygonatum yunnanense:

[0045] Grade 0: The entire plant is disease-free;

[0046] Grade 1: The lesion area accounts for less than 5% of the total leaf area;

[0047] Grade 3: Lesions cover 6%-10% of the total leaf area;

[0048] Level 5: Lesions cover 11%-25% of the total leaf area;

[0049] Grade 7: Lesions cover 26%-50% of the total leaf area;

[0050] Grade 9: The lesion area accounts for more than 51% of the total leaf area.

[0051] Methods for calculating drug efficacy:

[0052] Disease index = ∑(relative grade value × number of diseased leaves at each grade) / (9 × total number of leaves surveyed) × 100

[0053] Prevention efficacy (%) = (Disease index in control area - Disease index in treatment area) / Disease index in control area × 100

[0054] 2. Test Results

[0055] Table 4 shows that each pesticide had a significant effect on the control of leaf spot disease of Polygonatum yunnanense. In the XP experimental field, the ethanol extract of Phoebe zhennan fruit showed a relative control efficacy of 80.04±2.1% on the 10th day after application, significantly higher than the 65.03±1.8% of the carbendazim treatment group. In the MG experimental field, the control efficacy on the 10th day after application was 64.93±1.9%, slightly lower than the 69.50±1.6% of carbendazim.

[0056] Table 4. Control efficacy of the tested agents against leaf spot disease of Polygonatum yunnanense.

[0057]

[0058] In summary, this invention, through systematic screening and evaluation, clarified the inhibitory effects of the ethanol extracts of the fruits of five plants—*Machilus yunnanensis*, *Machilus dauricus*, *Cinnamomum yunnanensis*, *Cinnamomum camphora*, and *Cinnamomum cassia*—on multiple pathogens causing leaf spot disease of *Polygonatum yunnanensis*. Indoor antibacterial tests showed that the ethanol extracts of the tested plants' fruits exhibited varying degrees of antibacterial activity against different pathogens. At a concentration of 1 mg / mL, the ethanol extract of *Machilus dauricus* fruit showed significant antibacterial effects against all pathogens, with inhibition rates exceeding 60% for all pathogens. Indoor toxicity tests indicated that the ethanol extract of *Machilus dauricus* fruit was most sensitive to *Neopestalotiopsis acrostichi*, with an EC50 value of [missing value]. 50 The value was 116.48 mg / L. Field trials showed that when the mass concentration was 800 mg / L, the ethanol extract of *Phoebe zhennan* fruit had varying effects on the control of leaf spot disease of *Polygonatum yunnanense* in different experimental fields, but all achieved effective control after spraying. In particular, in the XP experimental field, the effect was significantly better than that of the conventional fungicide carbendazim, with a control efficacy of 80.04%.

[0059] Polygonatum yunnanense is widely cultivated in different regions of Yunnan. As a native medicinal herb of Yunnan, the long-term use of chemical pesticides during cultivation not only exacerbates the resistance of pathogens to pesticides but also leads to chemical residues. Cultivating high-quality medicinal materials free of pesticide residues is currently a key focus in agricultural production. Extracts from the fruit of Endiandra macrocarpa (Lauraceae family) can be considered a preferred plant-derived fungicide for the green control of leaf spot disease in Polygonatum yunnanense. This invention provides a theoretical basis and technical support for the sustainable control of diseases in Polygonatum yunnanense, and lays an important foundation for the development and application of plant-derived fungicides.

[0060] The above description is merely a preferred embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent transformations made using the content of the present invention under the concept of the present invention, or direct / indirect applications in other related technical fields, are included within the patent protection scope of the present invention.

Claims

1. A leaf spot control agent for Polygonatum yunnanense, characterized in that, The control agent contains an ethanol extract of the fruit of *Phoebe zhennan*.

2. A method for controlling leaf spot disease in Polygonatum yunnanense, characterized in that, Spray the fungicide containing ethanol extract of Phoebe bournei fruit onto Polygonatum yunnanensis.

3. The prevention and control method according to claim 2, characterized in that, The pathogens that cause leaf spot disease in Polygonatum yunnanense include Phyllostacta capitalensis , Neopestalotiopsis acrostichi , Stemphylium lycopersici or Boeremia exigua .

4. The method according to claim 2 or 3, characterized in that, The concentration of the ethanol extract of the fruit of *Phoebe zhennan* is not less than 116.48 mg / L.

5. The application of ethanol extract of *Phoebe zhennan* fruit in the prevention and control of plant pathogens, characterized in that... The pathogens include Phyllostacta capitalensis , Neopestalotiopsis acrostichi , Stemphylium lycopersici or Boeremia exigua.

6. The application of a fungicide in the control of plant pathogens, characterized in that, The pathogens include Phyllostacta capitalensis , Neopestalotiopsis acrostichi , Stemphylium lycopersici or Boeremia exigua. The bactericide contains an ethanol extract of the fruit of *Phoebe zhennan*. 。 7. The application of a bactericidal composition in the control of plant pathogens, characterized in that, The pathogens include Phyllostacta capitalensis , Neopestalotiopsis acrostichi , Stemphylium lycopersici or Boeremia exigua. The bactericidal composition contains an ethanol extract of the fruit of *Phoebe zhennan*. 。

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