Uses of extracts from Lycoris and Zephyranthes species in the control of rice blast

Abstract

The use of extracts from plants of the genera *Lycoris* and *Zephyranthes* in the control of rice blast belongs to the field of plant-derived fungicide technology. This paper discloses the uses of extracts from seven *Lycoris* species and one *Zephyranthes* species in the control of rice blast. These extracts can effectively inhibit the mycelial growth and spore germination of *Bacillus oryzae*, and have a good control effect on rice blast. The use of these extracts can delay the development of fungicide resistance in *Bacillus oryzae*.

Description

Technical Field

[0001] This invention belongs to the field of plant-derived fungicide technology, specifically relating to the use of extracts from plants of the genus Lycoris and the genus Zephyranthes in the prevention and control of rice blast. Background Technology

[0002] Rice blast is one of the most serious diseases affecting rice, causing huge economic losses to agricultural production once it occurs. Currently, chemical fungicides and the planting of disease-resistant rice varieties are mainly used for prevention and control. However, long-term use of chemical fungicides has led to certain resistance problems, and the resistance of disease-resistant rice varieties often becomes ineffective due to mutations in the rice blast fungus. Therefore, it is necessary to develop new control agents for alternating use.

[0003] Amaryllidaceae plants are rich in lycolic alkaloids, which possess abundant biological activity. Some lycolic alkaloids exhibit good antibacterial activity against certain plant pathogens, making Amaryllidaceae plants ideal for developing novel control agents. Both Lycoris and Zephyranthes species belong to the Amaryllidaceae family. Currently available literature and patents disclose the use of Lycoris for insect control in patents such as "An Insecticide from Lycoris Plants and Its Preparation Method" (patent application number: CN201710402002.7) and "A Pure Plant-Based Insecticide and Its Preparation Method" (patent application number: CN01129510.4); and "An Insecticide from Lycoris Plants Resistant to Rice Blast Fungus (LrPR4) and Its Encoding Gene and Application" (patent application number: CN200810124655.4). The use of garlic plant proteins in controlling plant fungal diseases; the use of *Zephyranthes candida* extract with antitumor activity (patent application number: CN201710285181.0) and the use of *Zephyranthes candida* for antitumor purposes (patent application number: CN201310559346.0) (patent application number: CN201310559346.0); and the use of *Zephyranthes candida* for treating corns (patent application number: CN201710074944.7) ... Summary of the Invention

[0004] To address the above shortcomings, this invention provides the use of extracts from Lycoris genus and Zephyranthes genus in the prevention and control of rice blast.

[0005] This invention is achieved through the following technical solution.

[0006] The extract was prepared by extracting the bulbs of seven Lycoris species and one Zephyranthes species with methanol and dichloromethane and ethyl acetate.

[0007] The aforementioned Lycoris species include Lycoris chinensis (Chinese Lycoris).Lycoris chinensis Traub), Lycoris radiata ( Lycoris longituba Y. Xu et GJ Fan), Lycoris rosea ( Lycoris rasea Traub & Moldenke), Huanjinhua ( Lycoris sprengeri Comes ex Baker), Garlic ( Lycoris incarnata Comes ex C. Sprenger), suddenly laughing ( Lycoris aurea (L'Her.) Herb.) and Lycoris radiata ( Lycoris radiata (L'Her.) Herb.), the plant in the Zephyranthes genus mentioned is Zephyranthes ( ). Zephyranthes candida (Lindl.)Herb.).

[0008] The stated use refers to its application in the prevention and control of rice blast.

[0009] This invention demonstrates through research that extracts from plants of the genera *Lycoris* and *Zephyranthes* have significant inhibitory activity against mycelial growth and spore germination of *Oryza sativa*, and exhibit good control effects against rice blast. They have the potential to be developed into pesticides for rice blast control and can alleviate the problem of pathogen resistance. Detailed Implementation

[0010] The specific embodiments of the present invention will be described in further detail below, but the content of the present invention is not limited to the contents included in the following embodiments.

[0011] Example 1:

[0012] Bulbs from seven species of the genus *Lycoris* and one species of the genus *Zephyranthes* were collected. The plant species included: *Lycoris chinensis* (…). Lycoris chinensis Traub), Lycoris radiata ( Lycoris longituba Y. Xu et GJ Fan), Lycoris rosea ( Lycoris rasea Traub & Moldenke), Huanjinhua ( Lycoris sprengeri Comes ex Baker), Garlic ( Lycoris incarnata Comes ex C. Sprenger), suddenly laughing ( Lycoris aurea (L'Her.)Herb.), Lycoris radiata ( Lycoris radiata (L'Her.) Herb.) and onion lotus ( Zephyranthes candida(Lindl.) Herb.). The plant material was freeze-dried in a vacuum freeze dryer and then pulverized. 100 g of powder was weighed and ultrasonically extracted three times in 500 mL of methanol. The three methanol extracts were combined and concentrated under reduced pressure to 150 mL of fluid extract, which was then suspended in 150 mL of ultrapure water. Then, equal volumes of dichloromethane and ethyl acetate were extracted sequentially, three times each. The extracts were combined and concentrated under reduced pressure to approximately 10 mL. The solution was then purged with nitrogen at 50 °C to constant weight, yielding dichloromethane and ethyl acetate extracts, respectively.

[0013] Example 2:

[0014] The extract has an effect on rice blast fungus ( Magnaporthe oryzae Determination of mycelial growth inhibitory activity:

[0015] Sixteen extracts were dissolved in DMSO to prepare a stock solution with a concentration of 100 mg / mL. Extract solutions (final concentration 400 μg / mL) or an equal volume of DMSO were added to PSA medium to prepare drug-containing plates. Using a sterile punch, 5 mm diameter bacterial discs were inoculated into the center of each drug-containing plate and incubated at 25°C for 5-8 days. Colony diameters (mm) were measured, and mycelial growth inhibition rates were calculated. Each treatment was repeated three times, and the experiment was repeated three times.

[0016] Experimental results: The inhibitory activities of 16 extracts on the mycelial growth of *Oryza sativa* are shown in Table 1. The results in the table show that the 16 extracts exhibited good inhibitory activity against *Oryza sativa* mycelial growth at a concentration of 400 μg / mL.

[0017] Table 1. Inhibitory activity of 16 plant extracts against mycelial growth of rice blast fungus.

[0018] Extract Name Inhibition rate (%) Lycoris rosea dichloromethane layer 58.11±2.26 Ethyl acetate layer of Lycoris radiata 74.26±6.01 Chinese Lycoris radiata dichloromethane layer 44.57±2.24 Ethyl acetate layer of Lycoris radiata 56.41±5.35 Long-tube Lycoris dichloromethane layer 54.43±2.19 Ethyl acetate layer of Lycoris radiata 53.78±3.00 Lycoris dichloromethane layer 64.40±2.31 Ethyl acetate layer of Lycoris radiata 71.12±4.86 Suddenly laughing dichloromethane layer 58.35±3.56 Suddenly laughing ethyl acetate layer 74.73±6.48 Replace the dichloromethane layer of the brocade flower 53.44±0.99 Replace the ethyl acetate layer of brocade 51.93±1.17 Lycoris dichloromethane layer 41.98±3.38 Ethyl acetate layer of Lycoris radiata 77.70±4.67 Onion and lotus dichloromethane layer 62.22±7.07 Ethyl acetate layer of celery lotus 66.11±3.54

[0019] Example 3:

[0020] Determination of the inhibitory activity of the extract against spore germination of rice blast fungus:

[0021] Sixteen extracts were dissolved in DMSO to prepare a stock solution with a concentration of 100 mg / mL. The extract solution (final concentration 400 μg / mL) or an equal volume of DMSO was added to water agar medium to prepare drug-containing plates. A certain concentration of spore suspension was evenly spread on the plates and incubated in a constant temperature incubator at 25℃. Each treatment was repeated 4 times.

[0022] Experimental results: The inhibitory activities of 16 extracts on the germination of rice blast fungus spores are shown in Table 2. The results in the table show that the 16 extracts exhibited good inhibitory activity against rice blast fungus spore germination at a concentration of 400 μg / mL.

[0023] Table 2. Inhibitory activity of Lycoris genus extracts against spore germination of rice blast fungus.

[0024] Extract Name Inhibition rate (%) Lycoris rosea dichloromethane layer 84.34±1.63 Ethyl acetate layer of Lycoris radiata 95.06±0.23 Chinese Lycoris radiata dichloromethane layer 87.85±2.45 Ethyl acetate layer of Lycoris radiata 99.40±0.24 Long-tube Lycoris dichloromethane layer 87.25±1.23 Ethyl acetate layer of Lycoris radiata 98.15±0.84 Lycoris dichloromethane layer 99.06±0.32 Ethyl acetate layer of Lycoris radiata 99.01±0.14 Suddenly laughing dichloromethane layer 94.08±2.92 Suddenly laughing ethyl acetate layer 92.63±2.84 Replace the dichloromethane layer of the brocade flower 74.04±5.87 Replace the ethyl acetate layer of brocade 98.99±0.44 Lycoris dichloromethane layer 85.23±2.45 Ethyl acetate layer of Lycoris radiata 99.35±0.20 Onion and lotus dichloromethane layer 91.42±0.48 Ethyl acetate layer of celery lotus 95.27±1.39

[0025] Example 4:

[0026] The effect of the extract on the control of rice blast:

[0027] The ethyl acetate extract of Lycoris radiata was dissolved in DMSO to prepare a stock solution with a concentration of 100 mg / mL, which was then diluted to 400 μg / mL with ultrapure water, and 0.1% Tween-20 was added for later use. Rice at the three-leaf stage was cultured in a 25℃ light incubator, and a suspension of rice blast fungus spores (2×10⁻⁶) was used. 5 Spraying the rice leaves with spores (per mL) and keeping them moist, followed by 24 h of dark treatment, was then carried out with alternating 12 h light / 12 h dark conditions. Before or after spraying the spore suspension, the leaves were sprayed with a 400 μg / mL extract solution or a 200 μg / mL methyl thiophanate solution. The control was sprayed with an aqueous solution containing an equal amount of solvent and Tween-20. After the control group developed sufficient disease, the disease incidence was statistically analyzed according to the rice blast grading standards, and the control effect was calculated.

[0028] Experimental results: As shown in Table 3, the ethyl acetate extract of Lycoris radiata had no adverse effects on rice growth and had protective and therapeutic effects on rice blast.

[0029] Table 3. Control efficacy of Lycoris radiata ethyl acetate extract against rice blast.

[0030]

Claims

1. Application of extracts from plants of the genera *Lycoris* and *Zephyranthes* in the control of rice blast, wherein the *Lycoris* plants include *Lycoris chinensis* Traub, *Lycoris longituba* Y. Xu et GJFan, *Lycoris rasea* Traub & Moldenke, *Lycoris sprengeri* Comesex Baker, *Lycoris incarnata* Comes ex C. Sprenger, and *Lycoris aurea* (L'Her.) Herb.; and the *Zephyranthes* plant is *Zephyranthes candida* (Lindl.) Herb. The extract was prepared by the following method: plant material was freeze-dried in a vacuum freeze dryer and then pulverized; 100g of powder was weighed and ultrasonically extracted three times in 500mL of methanol, the three methanol extracts were combined and concentrated under reduced pressure to 150mL of fluid extract, and then suspended in 150mL of ultrapure water; then it was extracted three times with equal volumes of dichloromethane and ethyl acetate, the extracts were combined and concentrated under reduced pressure to 10mL, and then purged with nitrogen at 50℃ to constant mass to obtain dichloromethane layer and ethyl acetate layer extracts, respectively.

2. The application according to claim 1, characterized in that, The extracts from the Lycoris and Zephyranthes species mentioned above are used to inhibit the mycelial growth and spore germination of rice blast fungus.

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