Application of luteolin in preventing and controlling plant root rot

Abstract

The present invention discloses the application of luteolin in preventing and treating plant root rot, and belongs to the technical field of plant disease prevention and treatment. The present invention has found that luteolin can inhibit the radial growth of the hyphae of soybean root rot pathogens. At the same time, luteolin reduces the length of lesions on the hypocotyls of soybean etiolated seedlings infected by soybean root rot pathogens, increases the germination rate of soybean seeds in soil infected by soybean root rot pathogens, and reduces the incidence of root rot of soybean seedlings, and can be used for the prevention and treatment of soybean root rot. In summary, luteolin can be used as a plant antibacterial agent for disease prevention and control of soybean root rot.

Description

Technical Field

[0001] The invention relates to the technical field of plant disease prevention and control, and particularly to application of luteolin in preventing and controlling plant root rot. Background Art

[0002] Luteolin is a flavonoid compound, its chemical name is 3,4,5,7-tetrahydroxyflavone, and its molecular formula is C 15 H 10 O6, the molecular structure is as follows:

[0003]

[0004] Luteolin exists in the form of glycosides in a variety of medicinal materials and vegetable fruits, such as honeysuckle, chrysanthemum and peanut, and is one of the most biologically active flavonoids. Studies have shown that luteolin has multiple pharmacological activities such as anti-inflammatory, antibacterial, anti-tumor, antioxidant and antiviral; and luteolin is safe to eat and has beneficial properties for human health. However, current research on luteolin rarely focuses on its role in plant diseases.

[0005] Soybean root rot is a typical soil-borne disease that occurs throughout the entire growth period of legumes, causing extremely serious losses and is a devastating disease. The causative bacteria of soybean root rot are soybean root rot bacteria, which belong to the class Oomycetes. The strains have evolved in a variety of ways and are included in the national list of agricultural plant quarantine pests. At present, the prevention and control of soybean root rot bacteria still mainly relies on the large-scale use of chemical pesticides, but the effect is generally poor and the resistance of strains to chemical agents has been strengthened with the popularization of their use. Therefore, there is an urgent need to seek and develop safe alternatives to efficient broad-spectrum chemical fungicides that can prevent and control soybean blight, so as to achieve green, environmentally friendly and sustainable development of agricultural production. Summary of the invention

[0006] In view of the above-mentioned prior art, the object of the present invention is to provide the application of luteolin in preventing and treating plant root rot.

[0007] To achieve the above object, the present invention adopts the following technical solution:

[0008] The first aspect of the present invention provides the use of luteolin in the following (1) or (2):

[0009] (1) Application in the prevention and treatment of plant oomycetes;

[0010] (2) Preparation of drugs for preventing and treating plant oomycete diseases.

[0011] In the above application, the plant oomycete is a pathogen of soybean root rot.

[0012] In the above application, the working concentration of luteolin is 20-100 μg / mL.

[0013] The second aspect of the present invention provides the use of luteolin in the preparation of a fungicide for soybean root rot fungi.

[0014] In the above application, the luteolin inhibits the infection of soybean root rot pathogens through at least one of the following pathways (1)-(4):

[0015] (1) Inhibit the radial growth of mycelium of soybean root rot pathogen;

[0016] (2) Inhibit the formation and release of zoosporangium of soybean root rot pathogen;

[0017] (3) Reduce the activity of zoospores of soybean root rot pathogens;

[0018] (4) Shorten the time of oospore formation of soybean root rot fungus and inactivate the oospores.

[0019] The third aspect of the present invention provides the use of luteolin in improving the resistance of plants to soybean root rot pathogens.

[0020] A fourth aspect of the present invention provides a method for preventing and controlling plant phytophthora, comprising the following steps:

[0021] During the entire growth period of the plant, luteolin is applied to the leaves or rhizomes of the plant.

[0022] Preferably, luteolin is applied to plant leaves by spraying; or applied to plant roots by mixing with soil.

[0023] In the above method, the plant phytophthora disease is caused by infection by soybean root rot fungi.

[0024] Beneficial effects of the present invention:

[0025] (1) The use of luteolin to control soybean root rot pathogens is harmless to the health of plants and humans, and has little burden on the environment.

[0026] (2) The use of luteolin to control soybean root rot can effectively reduce the amount of pesticides used and make it less likely for the strains to develop drug resistance. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The effect of luteolin on the radial growth of mycelium of soybean root rot pathogen.

[0028] Figure 2 The effect of luteolin on the zoosporangium formation of soybean root rot pathogen.

[0029] Figure 3The effect of luteolin on the zoospore activity of soybean root rot pathogen.

[0030] Figure 4 The effect of luteolin on the oospore formation of soybean root rot pathogen; in the figure, A: the effect of different treatments on the oospore formation time of soybean root rot pathogen; B: the effect of different treatments on the oospore activity of soybean root rot pathogen.

[0031] Figure 5 The effect of spraying luteolin on improving the resistance of plants to soybean root rot pathogens.

[0032] Figure 6 The effect of luteolin on soybean root rot pathogen in a pot experiment. DETAILED DESCRIPTION

[0033] It should be noted that the following detailed descriptions are illustrative and are intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present application belongs.

[0034] In order to enable those skilled in the art to more clearly understand the technical solution of the present application, the technical solution of the present application will be described in detail below in conjunction with specific embodiments.

[0035] The test materials used in the examples of the present invention are all conventional test materials in the field and can be purchased through commercial channels. The experimental methods without detailed conditions are carried out in accordance with conventional test methods or the operating instructions recommended by the supplier. Among them:

[0036] Luteolin was purchased from LOCAL Company with product number GC74397, CAS no 491-70-3.

[0037] The soybean root rot fungus used in the embodiments of the present invention is recorded in the document “TAN Xin-wei, JIN Yu-ting, LIU Mei-tong, WANG Qun-qing. Molecular Recognition and Counter-recognition Mechanism in Phytophthora sojae-host Interactions [J]. Biotechnology Bulletin, 2018, 34 (2): 2-9.”.

[0038] Test plants: susceptible soybean variety Williams.

[0039] Test culture medium: 10% V8 medium (20 mL V8 vegetable juice + 3 g agar powder + 180 mL H2O).

[0040] Example 1: Plate antibacterial test on soybean root rot pathogen

[0041] 1. Test method:

[0042] On the clean bench, first, the soybean root rot bacteria block was inoculated into 10% V8 medium. After culturing at 25°C in the dark for 7 days, the mycelium grew nearly full of the plate. The test soybean root rot bacteria block was taken along the edge of the colony with a hole puncher (d = 5 mm), and placed in 10% V8 medium containing drugs (containing 0, 20, 40, 80, 100 μg / mL (whether the space before the unit should be unified) luteolin), and cultured at 25°C in the dark for 7 days. The 10% V8 culture dish without drugs was used as a control.

[0043] 2. Test results:

[0044] The results of luteolin's inhibition on soybean root rot pathogens are shown in Tables 1 and Figure 1 The diameter of mycelium of soybean root rot pathogen on the medium containing luteolin was statistically analyzed, and it was found that 20, 40, 80, and 100 μg / mL luteolin had different degrees of inhibitory effect on the growth of soybean root rot pathogen.

[0045] Table 1: Inhibition of soybean root rot pathogen growth by different concentrations of luteolin

[0046] Treatment concentration CK 20 μg / mL 40 μg / mL 80 μg / mL 100 μg / mL Colony diameter (cm) 7.93±0.04 7.07±0.09 6.90±0.07 5.63±0.09 5.60±0.07

[0047] Example 2: Experiment on the formation of zoosporangium of soybean root rot pathogen

[0048] 1. Test method:

[0049] The tested soybean root rot pathogen was taken along the edge of the colony with a hole puncher (d = 5mm) and placed in a 10% V8 medium containing drugs (containing 0, 20, 40, 80, 100μg / mL of luteolin, respectively), with 5 bacterial blocks transferred to each dish. The 10% V8 culture dish without drugs was used as a control. After culturing in the dark at 25℃ for 72h, the liquid in the dish was carefully aspirated (be careful not to touch the colony), and 20mL ddH2O was added. Repeat three times. After culturing in the dark at 25℃ for 3h, the production of zoosporangium was examined under a microscope. All bacterial blocks were homogenized (3 000r / m 1min), and then the volume was adjusted to 50mL with ddH2O (whether the space before the unit should be unified). 40μL of the homogenate was drawn on a clean slide to draw a water strip, and the zoosporangium was observed under a 4× microscope. Each concentration was repeated 3 times.

[0050] 2. Test results:

[0051] The results of luteolin inhibiting zoosporangium formation of soybean root rot pathogen are shown in Table 2 and Figure 2The number of zoosporangia was counted and it was found that 20, 40, 80, and 100 μg / mL of luteolin had different degrees of inhibitory effect on the formation of zoosporangia of soybean root rot pathogen.

[0052] Table 2: Effects of different concentrations of luteolin on the number of zoosporangia produced by soybean root rot pathogen

[0053]

[0054] Example 3: Test on the activity of zoospores of soybean root rot pathogen

[0055] 1. Test method:

[0056] The tested soybean root rot pathogen was taken along the edge of the colony with a hole puncher (d = 5mm) and placed in a 10% V8 medium containing drugs (containing 0, 20, 40, 80, 100μg / mL of luteolin, respectively), and 5 bacterial blocks were transferred to each dish. The 10% V8 culture dish without drugs was used as a control. After culturing in the dark at 25℃ for 72h, the liquid in the dish was carefully aspirated (be careful not to touch the colony), and 20mL ddH2O was added, and repeated three times. After culturing in the dark at 25℃ for 6h, 1mL of the zoospore suspension was aspirated and mixed in a 2ml EP tube, and then 2μL of the zoospore suspension was aspirated to draw a water strip on a clean slide, and the number of zoospores and dormant spores was counted under a 4× microscope. Each concentration was repeated 3 times, and the zoospore dormant rate was calculated. The formula: dormant rate = number of dormant spores / (number of dormant spores + number of zoospores).

[0057] 2. Test results:

[0058] The results of luteolin improving the zoospore resting rate of soybean root rot pathogen are shown in Table 3 and Figure 3 Statistical calculation results showed that 20, 40, 80, and 100 μg / mL of luteolin had different degrees of inhibitory effects on the activity of zoospores of soybean root rot pathogens, and the zoospore resting rate increased with the increase of luteolin concentration.

[0059] Table 3: Effects of different concentrations of luteolin on the zoospore resting rate of soybean root rot pathogen

[0060] Treatment concentration CK 20 μg / mL 40 μg / mL 80 μg / mL 100 μg / mL Rest rate (%) 5.73±0.42 7.78±1.18 10.76±1.43 45.66±3.63 76.32±4.63

[0061] Example 4: Experiment on the time of production of soybean root rot pathogenic bacteria oospores

[0062] 1. Test method:

[0063] The tested soybean root rot pathogen was punched with a hole puncher (d = 5 mm) along the edge of the colony and placed on a 10% V8 medium containing 80 μg / mL luteolin. A 10% V8 culture dish without drug was used as a control. After a period of incubation at 25°C in the dark, the entire colony was homogenized (3 000r / m 1min), and then the volume was fixed to 50mL with ddH2O. 40μL of the homogenate was drawn on a clean slide to draw a water strip, and the oospores were observed under a 4× microscope, and repeated 3 times.

[0064] 2. Test results:

[0065] The results of luteolin promoting the oospore formation of soybean root rot pathogen are shown in Table 4 and Figure 4 . The number of oospores was counted and it was found that 80μg / mL luteolin could shorten the generation time of soybean root rot fungus oospores. MTT assay showed that the generated oospores were inactive oospores.

[0066] Table 4: Effects of different concentrations of luteolin on the oospore production time of soybean root rot pathogen

[0067]

[0068] Example 5: Test on inducing plant resistance to soybean root rot pathogen

[0069] 1. Test method:

[0070] Several yellowing seedlings of susceptible soybean (Willianms) were planted and cultured in the dark at 30℃ for 5 days. Luteolin was prepared into two different concentration solutions of 40μg / mL and 80μg / mL, and H2O solution was used as the control (CK). Then three groups of yellowing soybean seedlings were sprayed with 2ml of the solution per yellowing soybean seedling.

[0071] Prepare fresh soybean root rot pathogen zoospore suspension, dilute the concentration to 100 / μL, and take 2μl of the diluted soybean root rot pathogen zoospore suspension and drop it on the hypocotyl of each yellow soybean seedling 2 hours after spraying. After 48 hours, the incidence of each group of yellow soybean seedlings was counted and the length of the lesions was recorded.

[0072] 2. Test results:

[0073] The results of luteolin improving the resistance of soybean root rot pathogens are shown in Table 5 and Figure 5 It was found that the spraying of 40 and 80 μg / mL of luteolin had a significant inhibitory effect on the incidence of soybean root rot pathogens on yellow soybean seedlings, indicating that the spraying of luteolin improved the resistance of plants to soybean root rot pathogens.

[0074] Table 5: Study on the effect of luteolin on improving plant resistance to soybean root rot pathogen

[0075] Treatment concentration CK 40μg / ml 80μg / ml Lesion length (cm) 3.50±0.20 2.80±0.13 1.67±0.11

[0076] Example 6: Potted plant test on soybean root rot pathogen

[0077] 1. Test method:

[0078] The soybean root rot bacteria were punched out along the edge of the colony using a borer (d = 5 mm), mixed into the nutrient soil (at a ratio of 10 nutrient soil blocks per 50 g nutrient soil), and cultured in the dark at 25°C for 7 days.

[0079] Luteolin was prepared into a 80 μg / mL solution, and 0.5 L of luteolin solution was mixed into the nutrient soil containing soybean root rot pathogens; 0.5 L of H2O solution was mixed into the nutrient soil containing soybean root rot pathogens as a control.

[0080] The susceptible soybean varieties (Willianms) were sown in equal amounts in the two groups of treated soils. The germination rate of soybean plants in each group and the soybean root rot index were statistically analyzed after 7 days, where:

[0081] Occurrence index = ∑ (number of diseased plants at each level × representative value at each level) / (total number of plants surveyed × highest representative value) × 100%

[0082] No disease: level 0; 0-25%: level 1; 26-50%: level 2; 51-75%: level 3; 76-100%: level 4.

[0083] 2. Test results:

[0084] The results of the application of luteolin in the prevention and treatment of soybean root rot are shown in Tables 6 and Figure 6 It was found that the germination rate of soybean plants in the bacterial nutrient soil containing 80 μg / mL luteolin was significantly higher than that of the control, while the occurrence index of soybean root rot was significantly lower than that of the control, indicating that luteolin can prevent and treat soybean root rot.

[0085] Table 6: The results of luteolin on the control of soybean root rot

[0086] Treatment concentration CK 80μg / ml Germination rate (%) 41.00±3.33 79.67±2.22 Occurrence index (%) 92.33±2.44 53.00±2.00

[0087] The above description is only the preferred embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. The application of luteolin in preventing and controlling soybean root rot pathogens, characterized in that: The soybean root rot pathogen is Phytophthora sojae.

2. The use according to claim 1, characterized in that: The working concentration of the luteolin is 20-100 mg / mL.

3. The use according to claim 1, characterized in that: The applications include: (1) Application in inhibiting the radial growth of mycelium of soybean root rot pathogen; (2) Application in inhibiting the formation and release of zoosporangium of soybean root rot pathogen; (3) Application in reducing the activity of zoospores of soybean root rot pathogens; (4) Application in shortening the time of soybean root rot oospore formation and inactivating the oospores.

4. Application of luteolin in improving plant resistance to soybean root rot pathogen, wherein the soybean root rot pathogen is Phytophthora sojae.

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