A method for evaluating the effect of a pesticide on the inhibition of a plant infection by a soil-borne pathogen

Abstract

The application belongs to the technical field of plant disease prevention and treatment, and relates to a determination method for visually and rapidly evaluating the effect of pesticides on inhibition of soil-borne pathogen infection on crop root systems. Agar powder is added to deionized water and boiled until dissolved, sterilized by high-pressure steam and cooled to 60-65 DEG C to obtain an agar culture medium, and the agar culture medium is poured into a culture dish and continuously cooled to solidification. A double-layer filter paper is laid on the solidified culture medium, pre-germinated seeds are placed above the filter paper and cultivated until the root system expands. After the root system of the seedling expands, the pathogen is inoculated and pesticide liquid is applied; and the activity of the pesticide on the pathogen is evaluated. The advantages of the application are that rapid inoculation can be realized, symptoms can be observed in real time, nematodes can be rapidly inoculated and dyed, pesticides can be applied at different stages before, during and after pathogen infection, and the protective and therapeutic activities of the pesticide can be rapidly evaluated. The application greatly facilitates the related research on pathogen or nematode infection, and provides a rapid and efficient method for evaluating the activity of the pesticide on the soil-borne pathogen.

Description

Technical Field

[0001] This invention belongs to the field of plant disease control technology, and relates to a method for visually and rapidly evaluating the effectiveness of pesticides in inhibiting soil-borne pathogens from infecting crop roots. Background Technology

[0002] Root-knot nematodes are a major disease affecting crops in the Solanaceae and Cucurbitaceae families, occurring in both northern and southern vegetable-growing regions of my country. Particularly in greenhouse agriculture, the long-term cultivation of nematode-prone crops leads to widespread root-knot nematode disease and severe economic losses. Reports indicate that economic losses caused by plant parasites have surpassed those from viral or bacterial diseases, becoming the second largest plant disease after fungal diseases. Nematode infestation also provides entry points for secondary pathogens, frequently resulting in compound diseases with fungi such as Fusarium, Phytophthora, Verticillium, and Rhizoctonia.

[0003] Tomato neck rot and root rot are mainly caused by the soil-borne fungus Fusarium oxysporum (Fusarium oxysporum). Fusarium oxysporum Caused by [unspecified pathogen], this is one of the most destructive soil-borne diseases of tomatoes and can also infect other crops such as cucumbers. The pathogen spreads through various routes, including soil, contact, and seeds, and can enter through wounds or natural openings. As the disease progresses, it leads to necrosis of the vascular bundles in the roots, cessation of new root development, stem wilting, and cessation of plant growth, seriously threatening greenhouse tomato production in my country.

[0004] Currently, the main methods for evaluating pesticide toxicity against these pathogens are the in vitro mycelial growth rate method and the insect inoculation method. The evaluation of pesticide activity against pathogen infection in plants can be divided into two stages: pathogen inoculation and efficacy evaluation. Pathogen inoculation is a crucial step in evaluating the pesticide's activity against pathogen infection in plants. Currently, commonly used methods for inoculating tomato neck rot and root rot include root dipping, corn kernel inoculation, stem base injection, cotton ball inoculation, and root drenching. However, these methods are conducted in pot experiments using soil as a substrate. Due to unstable environmental conditions, pathogen infection is difficult, the cycle is long, and the symptoms are not readily apparent, making it unsuitable for rapid and accurate evaluation of pesticide activity. Nematode inoculation methods also present similar difficulties. Besides pot inoculation, there are also visual inoculation methods such as seedling bag inoculation and glass tube visualization inoculation. However, the seedling bag method is time-consuming, prone to contamination, and prone to uneven inoculation, while the glass tube inoculation method suffers from difficulties in removing seedling roots and uneven inoculation. Currently, there is a lack of visual and rapid bioassay methods for evaluating pesticide activity against soil-borne disease pathogens. It is still necessary to develop methods that are easy to inoculate, easy to observe, short in time, uniform inoculation, and easy to separate roots for pathogen inoculation and efficacy evaluation experiments. Summary of the Invention

[0005] This invention addresses the problems existing in traditional methods for evaluating the effectiveness of pesticides in inhibiting soil-borne disease pathogens from infecting crop roots by proposing a novel method for evaluating the effectiveness of pesticides in inhibiting soil-borne disease pathogens from infecting plants.

[0006] To achieve the above objectives, the present invention is implemented using the following technical solution: A method for evaluating the effectiveness of pesticides in inhibiting the infection of soil-borne disease pathogens on plants, comprising the following steps: (1) Add agar powder to deionized water and boil until dissolved. After high pressure steam sterilization, cool to 60-65℃ to obtain agar medium. Pour the agar medium into a petri dish and continue to cool naturally until solidified.

[0007] (2) Lay the double-layered filter paper flat on the solidified culture medium, and place the pre-germinated seeds on top of the filter paper to cultivate until the root system expands.

[0008] (3) After the seedling roots have expanded, inoculate with pathogens and apply pesticide solution; evaluate the activity of pesticides against pathogens.

[0009] The advantages of this invention lie in its ability to enable rapid inoculation and real-time symptom observation, facilitating rapid inoculation and staining of nematodes, allowing for application of pesticides at different stages before, during, and after pathogen infection, and enabling rapid evaluation of the protective and curative activities of pesticides. This greatly facilitates research related to pathogen or nematode infection and provides a rapid and efficient method for evaluating pesticide activity against soil-borne disease pathogens.

[0010] The lower layer of the double-layer filter paper is wood fiber filter paper, and the upper layer filter paper can be any one of wood fiber, polypropylene, nylon, or glass fiber filter paper. Preferably, a combination of one layer of glass fiber filter paper and one layer of wood fiber filter paper is used, with the wood fiber filter paper placed below the glass fiber filter paper.

[0011] Preferably, the wood fiber filter paper has a pore size of 15-20 μm. The culture dish is preferably a glass culture dish with an outer diameter of 7-20 cm, allowing the plant roots to expand and cover the entire dish, facilitating uniform inoculation. In the device described in this invention, the culture medium is preferably poured in a volume of 5-25 mL. The device is preferably a 9 cm culture dish, and 15 mL of culture medium is preferably added. After pouring in the culture medium, allow it to cool and solidify completely before use. The culture medium helps retain moisture, providing hydration to the plant roots and ensuring normal root growth. The device containing the culture medium is generally easy to inoculate, facilitates real-time observation, provides clear symptoms, has a short experimental cycle, high repeatability, and high accuracy. The visualization of Fusarium oxysporum and nematode infection is significantly improved.

[0012] As a preferred method, the pre-germination treatment of seeds is as follows: after disinfecting the seeds in 75% alcohol for 30 seconds, rinse the seeds with water until there is no alcohol odor; after disinfecting the seeds in 1% NaClO solution for 15 minutes, rinse the seeds with water until there is no odor; after heating in a 55℃ water bath for 15 minutes, transfer the seeds between two layers of filter paper, add 4 ml of deionized water to the petri dish, seal the dish, and culture the seeds in the dark at 25℃ for 2 days until the sprouts emerge and the sprouts are 0.1-1 cm long; the conditions for further cultivation of the pre-germinated seeds are as follows: place the pre-germinated seeds on top of glass fiber filter paper and cultivate them alternately for 7-10 days with 16 hours of light and 8 hours of darkness at a cultivation temperature of 25℃.

[0013] Preferably, the agar content in the agar medium is 0.8-2.5% by mass. The agar medium is prepared by dissolving agar powder in deionized water according to a certain ratio and boiling until completely dissolved. The medium is then autoclaved, cooled to about 60°C, and poured into a device to solidify before use. In this invention, the preferred autoclaving conditions are 121°C for 30 minutes.

[0014] Preferably, the inoculated pathogens include pathogenic spores or fungal cakes, nematode suspensions, and mixtures of pathogenic spores and nematodes, wherein the root-knot nematodes are second-instar larvae. After root extension, nematodes are inoculated alone, and visual observation is performed 1-7 days after inoculation, preferably 3 days later; pathogenic fungal cakes or spore suspensions are inoculated alone, and symptom observation is performed 1-14 days after inoculation, preferably 1, 3, 5, 7, or 14 days later; for combined infection (inoculation with a mixture of nematode suspension and pathogenic spore suspension), visual observation is performed 1-7 days after inoculation, preferably 7 days later. In this invention, the obvious root extension is preferred, with secondary lateral roots growing and the root system completely covering the culture dish, which facilitates uniform infection of nematodes and pathogens and the observation of results.

[0015] In this invention, the drug solution can be applied before, during, or after inoculation of the pathogen, with an application amount of 0.5-5 mL, preferably 2 mL.

[0016] This invention provides a visual observation method including observing Fusarium oxysporum infection under a stereomicroscope and observing nematode infection using acid fuchsin staining. In this invention, the observation of Fusarium oxysporum infection under a stereomicroscope preferably includes disease severity grading, with the following grading standards: Grade 0: Healthy plant, no disease occurrence; Grade 1: Root base turns brown, not soft rot, not constricted, leaves are healthy, roots have no obvious lesions; Grade 2: Root base turns brown with obvious constriction, leaf tips or leaves turn yellow, roots turn brown; Grade 3: Root base turns brown and rots, leaves turn yellow, roots turn brown or even black; Grade 4: Roots and root base rot, the entire seedling dies. Disease index (%) = ∑(number of infected plants at each level × representative value of disease grade) / (highest disease grade × total number of surveys) × 100. In this invention, the nematode infection preferably includes the amount of nematodes infected. The present invention preferably involves staining the infected roots with acid fuchsin to statistically count the infection amount of second-instar larvae. Specifically, the present invention preferably involves removing the roots above the filter paper 3 days after inoculation, rinsing them with distilled water, and then staining them with acid fuchsin. The preferred specific staining steps are as follows: Rinse cucumber roots thoroughly with clean water and place them on absorbent paper for later use; rinse the cleaned cucumber roots in a 5.25% NaClO aqueous solution for 30 minutes; rinse the rinsed roots repeatedly with clean water until there is almost no NaClO smell; dilute the acidic fuchsin solution at a ratio of 2-3 mL / 100 mL water (adjust as needed depending on the staining situation) and boil; place the rinsed cucumber roots in the boiled acidic fuchsin solution and boil for 1-2 minutes; place the stained cucumber roots on absorbent paper to dry, and rinse them in slightly heated glycerol to decolorize them. The glycerol should be heated until it is just slightly steaming to avoid scorching the roots; place the rinsed cucumber roots in a petri dish, add glycerol, seal and preserve, or place them under a dissecting microscope to examine the number of nematodes in the roots (nematodes are short red threads in the roots). In this invention, the preferred specific preparation of the acid fuchsin is: 3.5 g fuchsin + 250 mL acetic acid + 750 mL distilled water, mixed thoroughly. For the roots stained with acid fuchsin, this invention preferably involves observing the infection amount of second-instar larvae under a 40x microscope after pressing them onto a glass slide and counting the infection.

[0017] In this invention, the method for evaluating the activity of pesticides against pathogens is to calculate the inhibition rate of pesticides against pathogens according to the formula. The formula for evaluating the activity of pesticides against nematodes is: Inhibition rate = (Number of nematodes infected in the control group - Number of nematodes infected in the pesticide treatment group) / Number of nematodes infected in the control group × 100; the formula for evaluating the activity of pesticides against pathogens is: Control effect (%) = (Disease index of control group - Disease index of treatment group) / Disease index of control group × 100.

[0018] Verification showed that agar medium with a 2% agar content and 9cm glass petri dishes yielded good detection results. This promotes full root expansion in seedlings, resulting in vigorous root growth, facilitating nematode infection, and highlighting symptoms. The 2% agar medium provides moisture for cucumber growth, maintaining humidity and ensuring Fusarium oxysporum infection, enabling rapid inoculation and real-time, clear observation of symptoms. Filter paper separates the agar medium from the cucumber roots, facilitating uniform nematode infection and symptom observation. This overcomes the poor mobility of nematodes in water agar, allowing for complete separation of roots from the apparatus and staining to statistically determine nematode infection levels. This greatly facilitates research on co-infection of Fusarium oxysporum and nematodes, providing a rapid and efficient method for screening agents that inhibit both Fusarium oxysporum and nematode infection. Furthermore, the lower filter paper in a double-layered system can be replaced to control humidity without affecting root distribution. Applying pesticides before, during, and after pathogen infection allows for rapid investigation of the protective and curative effects of pesticides on soil-borne diseases.

[0019] Compared with the prior art, the advantages and positive effects of the present invention are as follows: The device described in this invention can also be used to investigate the infection efficiency and disease symptoms of Fusarium oxysporum and nematodes under different temperatures and inoculation amounts, thereby exploring the optimal inoculation conditions and shortening the inoculation time. Using this device to conduct inoculation experiments on different seeds allows for more efficient screening of plant materials with certain resistance. Furthermore, the device allows for the application of pesticides before, during, and after pathogen infection, enabling the investigation of the protective or curative activities of different pesticides against certain soil-borne disease pathogens or combined infections. Attached Figure Description

[0020] Figure 1 This is a schematic diagram of the device of the present invention. Detailed Implementation

[0021] To better understand the above-mentioned objectives, features, and advantages of the present invention, the present invention will be further described below with reference to specific embodiments. It should be noted that, unless otherwise specified, the embodiments and features described in these embodiments can be combined with each other.

[0022] Numerous specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways than those described herein, and therefore the invention is not limited to the specific embodiments disclosed in the following specification.

[0023] Example 1 1. Sterilization and germination of plant seeds The seeds used in this embodiment were "Zhai Bubai" cucumber seeds and "Jinhe 50" tomato seeds. Both types of seeds were disinfected by soaking in 75% alcohol for 30 seconds, then rinsed with water until no alcohol odor remained. Next, they were disinfected by soaking in 1% NaClO solution for 15 minutes, then rinsed with water until no odor remained. After heating in a 55°C water bath for 15 minutes, the seeds were transferred to a double layer of moistened filter paper sprayed with sterile water, sealed, and cultured in the dark for 2 days until germination.

[0024] 2. Preparation of agar medium Add 20g of agar powder to 1000ml of water and heat until dissolved. Then autoclave at 121℃ for 30min. After the culture medium cools to about 60℃, pour it into several pre-prepared 9cm glass petri dishes, 15mL per dish. Allow it to cool and solidify completely before use. Cover the surface of the cooled culture medium with sterile water-soaked filter paper.

[0025] In this embodiment, the agar concentration of the agar medium is 2%, which provides good coagulation and moisture retention.

[0026] 3. Experiments on plant growth, quantitative analysis of second-instar larvae J2, and visualization of Fusarium oxysporum infection levels. Germinated cucumber seeds were placed on agar medium lined with double-layered filter paper using sterile forceps. The lower layer was 15-20 μm wood fiber qualitative filter paper, and the upper layer was glass filter paper, 3 seeds / plate. The seeds were then placed in an artificial climate incubator for 7 days. A suspension of Southern Root-Knot Nematode J2s was mixed with an abamectin solution. Immediately afterward, 2 mL of the mixture was added to the roots using a disposable plastic pipette. The abamectin concentrations were 0.005, 0.01, and 0.02 mg / L, respectively. The concentration of the Southern Root-Knot Nematode J2s suspension could be adjusted according to experimental conditions. Approximately 3 days after inoculation with the nematodes and the reagent, the roots were removed, rinsed thoroughly with distilled water, and stained with acid fuchsin. The acid fuchsin-stained roots were then pressed onto glass slides and observed under a 40x microscope. The number of J2 nematodes was counted using a counter. Each treatment was repeated three times.

[0027] The artificial climate incubator was set up under the following conditions: alternating periods of 16 hours of light (25°C) and 8 hours of darkness (25°C).

[0028] The specific process for acid fuchsin staining is as follows: First, prepare an acid fuchsin solution by mixing 3.5g fuchsin, 250mL acetic acid, and 750mL distilled water thoroughly. Rinse cucumber roots thoroughly with clean water and place them on absorbent paper. Soak the cleaned cucumber roots in a 5.25% NaClO aqueous solution for 30 minutes. Rinse the rinsed roots repeatedly with clean water until almost no NaClO taste remains. Dilute acidic fuchsin solution at a ratio of 2-3 mL / 100 mL water (adjust as needed based on staining conditions) and boil. Place the rinsed cucumber roots in the boiled acidic fuchsin solution and boil for 1-2 minutes. Pat the stained cucumber roots dry on absorbent paper and rinse them in slightly warm glycerin to decolorize. The glycerin should be heated until it just begins to steam to avoid scorching the roots. Place the rinsed cucumber roots in a petri dish, add glycerin, and seal for preservation, or examine under a dissecting microscope to check for the number of nematodes (short red threads within the roots). For roots stained with acid fuchsin, this invention preferably involves observing the infection levels of second-instar larvae under a 40x microscope after pressing them onto a glass slide and counting the infection amounts under different treatments. As shown in Table 1, avermectin (AV) at 0.02 mg / L can almost completely inhibit nematode infection.

[0029] Inhibition rate = (Number of nematodes infected in the control group - Number of nematodes infected in the pesticide treatment group) / Number of nematodes infected in the control group × 100.

[0030] Table 1. Inhibitory effect of avermectin on nematode infection. Note: Different letters indicate significance analysis, meaning the difference is significant at the p-value < 0.05 level according to Fisher's LSD test. Since the final result is the average of multiple trials, the final number may not be an integer.

[0031] Example 2 Set up three different petri dish sizes: 6, 9, and 15 cm.

[0032] Unless otherwise specified, the methods are the same as in Example 1.

[0033] After treatment using the same method as in Example 1, the time required for the seedling roots to completely cover the culture dish was statistically analyzed. The results showed that when the culture dish size was 6 cm, the roots covered the entire dish relatively quickly, but the space was small, insufficient to cover the roots of 3 cucumber seedlings; when the culture dish size was 15 cm, the space was large, and the roots could not be completely covered, resulting in nematode inoculation flowing to areas without roots, leading to uneven nematode inoculation between different plates and poor experimental repeatability; when using a 9 cm culture dish, the roots covered the dish in 7 days, and the space was sufficient, resulting in more uniform nematode inoculation. Therefore, the optimal culture dish size was 9 cm.

[0034] Example 3 Three different agar concentrations were set: 0.8%, 1%, and 2%.

[0035] Unless otherwise specified, the methods are the same as in Example 1.

[0036] After treatment using the same method as in Example 1, the time required for the seedling roots to completely cover the petri dish was statistically analyzed. The results showed that agar concentrations of 0.8% and 1% resulted in poor agar coagulation and moisture retention. A 2% agar medium exhibited the best moisture retention and coagulation properties, which was beneficial for the growth of cucumber and tomato seedlings. The roots covered the entire petri dish in just 7 days; therefore, a 2% agar concentration was the optimal choice.

[0037] Example 4 Unless otherwise specified, the methods for setting 0, 1, 2, and 3 layers of filter paper are the same as in Example 1.

[0038] After treatment using the same method as in Example 1, the time required for the seedling roots to cover the entire culture dish was statistically analyzed, and the results are shown in Table 2. Without filter paper, the seedling roots penetrated deep into the agar medium, making inoculation difficult and uneven. Nematodes exhibited poor mobility within the agar medium, and it was difficult to isolate the roots from the agar medium for staining to check the number of infected nematodes. With one layer of filter paper, the filter paper easily broke when wet, allowing the roots to penetrate the filter paper and penetrate the agar medium. With two layers of filter paper, the roots could not break through the filter paper and could fully spread on the surface of the upper layer. If the humidity was too high, the humidity could be adjusted by replacing the lower layer of filter paper without affecting the spread of the roots on the filter paper surface. Roots reached 8 cm in length after 3 days and covered the entire culture dish in 7 days. With three layers of filter paper, the filter paper was too thick, hindering water absorption and retention; roots reached 5 cm in length after 3 days and took 10 days to cover the entire culture dish. Therefore, two layers of filter paper were the optimal choice.

[0039] Table 2 Seedling cultivation effect of filter paper with different layers Example 5 Three different types of filter paper were selected: wood fiber filter paper, nylon filter paper, and glass fiber filter paper.

[0040] Use "Jinhe 50" tomato seeds, 1 seed / plate. Add a suspension of Fusarium oxysporum spores (10 g / L) to the root system using a disposable plastic pipette. 7 / mL), for a total of 1mL. Each treatment was repeated four times.

[0041] Incidence rate (%) = Number of infected plants / Total number of plants × 100 Unless otherwise specified, the methods are the same as in Example 1.

[0042] After treatment using the same method as in Example 1, the time required for the seedling roots to completely cover the culture dish was statistically analyzed, and the incidence rate was statistically analyzed 7 days after Fusarium oxysporum infection. The results showed that the wood fiber filter paper has certain water absorption properties, which can help maintain humidity; and it has a certain tolerance under humid conditions, which can absorb water and maintain structural stability; at the same time, it also has good air permeability, which is beneficial to seed germination and seedling growth.

[0043] In comparison, glass fiber filter paper and nylon filter paper have lower air and water permeability than wood fiber filter paper, and are also more expensive, making them less suitable for seedling cultivation. Overall, wood fiber filter paper is more suitable for seedling cultivation, but it's important to choose products of appropriate thickness to ensure adequate air permeability and water retention. Simultaneously, it's crucial to control factors such as irrigation volume and temperature to ensure healthy seedling growth.

[0044] At 7 days, the incidence rate of Fusarium oxysporum reached over 75.5±1.25%, which was higher than that of nylon filter paper and glass fiber filter paper, at 45.25±2.18% and 58.17±3.45%, respectively. Therefore, wood fiber filter paper is the most preferred among the filter papers, with a pore size of 10-25μm, and wood pulp is selected as the material for the wood fiber filter paper.

[0045] Table 3. Basic indicators of several types of filter paper and the incidence of Fusarium oxysporum. Note: Different letters indicate significant differences at the P < 0.05 level according to Fisher's LSD test.

[0046] Example 6 Filter paper with different pore sizes (10-15μm, 15-20μm and 20-25μm) was prepared.

[0047] Unless otherwise specified, cucumber seedlings were used for nematode inoculation, as per Example 1; tomato seedlings were used for Fusarium oxysporum inoculation, as per Example 5.

[0048] After treatment using the same methods as in Examples 1 and 5, the time required for the seedling roots to completely cover the culture dish was statistically analyzed, as were the infection rates after 3 days of nematode infection and 7 days of Fusarium oxysporum infection. The results are shown in Table 4. Filter paper with a pore size of 15-20 μm showed faster water absorption and no paper damage, resulting in the highest inoculation incidence rate (nearly 100% nematode infection rate) and a Fusarium oxysporum infection rate of 78.17 ± 3.45%. Therefore, filter paper with a pore size of 15-20 μm is the optimal choice.

[0049] Table 4. Inoculum rates of filter paper with different pore sizes Note: Different letters indicate significant differences at the P < 0.05 level according to Fisher's LSD test.

[0050] Example 7 Three different filter paper combinations were set up: wood fiber filter paper, nylon filter paper, and glass fiber filter paper.

[0051] Unless otherwise specified, the methods are the same as in Example 5.

[0052] After treatment using the same method as in Example 5, the time required for the seedling roots to completely cover the culture dish was statistically analyzed, and the infection rate was statistically analyzed 7 days after Fusarium oxysporum infection. The results are shown in Table 5. Compared with the single-type filter paper treatment (Table 3), the combination of wood fiber filter paper and glass filter paper resulted in a shorter seedling time and a higher Fusarium oxysporum inoculation rate. The optimal choice was an upper layer of glass filter paper and a lower layer of wood fiber filter paper. Seedlings only needed 5 days, and the Fusarium oxysporum inoculation rate reached 90.83±1.44%. This may be because the wood fiber filter paper absorbs water faster but has a smaller water absorption capacity, while the glass filter paper absorbs water slower but has a larger water absorption capacity. The two complement each other. After absorbing water, the wood fiber filter paper is stored in the glass filter paper, providing a moist environment for root growth and Fusarium oxysporum infection.

[0053] Table 5. Inoculation rates of treatments using two layers of filter paper of different materials Note: Different letters indicate significant differences at the p-value < 0.05 level according to Fisher's LSD test. Example 8 One tomato seed per dish was used. After the tomato root system had expanded, the main root was punctured with a syringe needle, and a 7mm Fusarium oxysporum mycelium cake was inoculated at the wound. Tetracycline solution was then applied at a concentration of 1, 2, or 4 mg / L, at a dosage of 1 mL. Seven days after inoculation and treatment, the disease index was assessed under a stereomicroscope, and the control effect was calculated.

[0054] The disease classification standards are as follows: Grade 0: Healthy plants, no diseases occurring; Grade 1: The base of the roots is brown, but not soft or rotten, and the leaves are healthy with no obvious lesions on the roots; Grade 2: The base of the root turns brown and has obvious constriction; the leaf tips or leaves turn yellow; and the roots turn brown. Grade 3: The base of the roots turns brown and rots, the leaves turn yellow, and the roots turn brown or even black; Grade 4: The roots and root base are rotten, and the entire seedling is necrotic.

[0055] Disease index (%) = (∑ number of diseased plants at each level × representative value of disease grade) / (highest disease grade × total number of surveys) × 100.

[0056] Prevention and control effect (%) = (control disease index - treatment disease index) / control disease index × 100.

[0057] Unless otherwise specified, the methods are the same as in Example 5.

[0058] The results are shown in Table 6. 4 mg / L tetracycline showed a 67.26 ± 3.29% inhibitory effect on Fusarium oxysporum infection. This method can be used for rapid indoor screening of agents for controlling soil-borne diseases.

[0059] Table 6. Inhibitory effect of tetracycline on Fusarium oxysporum infection. Note: Different letters indicate significant differences at the P < 0.05 level according to Fisher's LSD test.

[0060] Example 9 First, the pesticide solution was dripped onto the roots. Three days later, the roots were inoculated with nematode solution, and the results were checked three days later. The pesticides and concentrations used were tetracycline at 1, 2, and 4 mg / L. Unless otherwise specified, the methods were the same as in Example 1.

[0061] The results are shown in Table 7. Tetramycin was able to inhibit nematode infection, with an inhibition rate of 98.43% at 4 mg / L.

[0062] Table 7. Inhibitory effect of tetracycline on nematode infection. Note: Different letters indicate significant differences at the P < 0.05 level according to Fisher's LSD test.

[0063] Comparing Examples 8 and 9, it can be seen that the method established by the present invention can be used to evaluate pesticide activity at different stages of pathogen infection.

[0064] Example 10 Cucumber seeds were used to evaluate the activity of pesticides against nematode and pathogen co-infection, with one seed per dish. A 1 mL aliquot of *Fusarium oxysporum* spore suspension, nematode suspension, and pesticide was mixed thoroughly and then added to all roots. The pesticide concentrations were: avermectin (0.02 mg / L) + tetracycline (1 mg / L), avermectin (0.02 mg / L) + tetracycline (2 mg / L), and avermectin (0.02 mg / L) + tetracycline (4 mg / L). The severity of neck rot and root rot was assessed under a stereomicroscope, followed by staining with acid fuchsin and observation of the number of nematodes infecting the roots under a stereomicroscope.

[0065] Unless otherwise specified, the methods are the same as in Example 1.

[0066] The results are shown in Table 8. The combination of avermectin and tetracycline can inhibit the co-infection of the two pathogens.

[0067] Table 8. Combination of abamectin and tetracycline for Fusarium oxysporum and nematodes Inhibition of multiple infections Note: Different letters indicate significant differences at the P < 0.05 level according to Fisher's LSD test.

[0068] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any other way. Any person skilled in the art may make changes or modifications to the above-disclosed technical content to create equivalent embodiments for application in other fields. However, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the protection scope of the present invention.

Claims

1. A method for evaluating the efficacy of pesticides in inhibiting the infection of soil-borne disease pathogens on plants, characterized in that, The steps are as follows: (1) Add agar powder to deionized water and boil until dissolved. After autoclaving, cool to 60-65℃ to obtain agar medium. Pour the agar medium into a petri dish and continue to cool naturally until solidified. (2) Lay the double-layered filter paper flat on the solidified culture medium, place the pre-germinated seeds on top of the filter paper and cultivate until the root system expands. The root system only grows on the surface of the filter paper and does not penetrate into the agar culture medium. (3) Simultaneously inoculate with pathogenic fungal spores and second-instar larvae of root-knot nematodes to construct a fungal-nematode complex infection system. (4) Apply pesticide solution, and determine the inhibitory effect of pesticide on fungal infection and nematode infection by root staining and disease grading. The upper filter paper is glass fiber filter paper; The lower layer of the double-layer filter paper is wood fiber filter paper, and the pore size of the wood fiber filter paper is 15-20μm.

2. The method for evaluating the effect of pesticides on inhibiting the infection of soil-borne disease pathogens on plants according to claim 1, characterized in that, The agar content in the agar medium is 0.8-2.5% by mass, and the autoclaving conditions are 121℃ for 30 min.

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