Use of chlorin iron combined with volatile oil of traditional Chinese medicine in preparing pesticide for preventing and / or treating rice blast

Abstract

The present invention provides the use of chlorin iron combined with Chinese medicine volatile oil in the preparation of pesticides for preventing and / or treating rice blast, belonging to the field of plant pesticides. The present invention uses chlorin iron in combination with Chinese medicine volatile oil, patchouli volatile oil or litsea cubeba volatile oil, to form a novel rice blast prevention and treatment agent with all Chinese medicine ingredients, breaking the current research and development ideas for rice blast prevention and treatment pesticides, innovatively broadening the application scope of Chinese medicine theory, and providing Chinese medicine wisdom and practice for practical agricultural problems.

Description

Technical Field

[0001] The invention belongs to the field of botanical medicines, and particularly relates to the use of chlorin iron combined with traditional Chinese medicine volatile oil in the preparation of pesticides for preventing and / or treating rice blast. Background Art

[0002] Rice blast is a disease of rice caused by Magnaporthe oryzae. It is the main disease in rice production and has caused great harm to my country's food security. At present, the main measures for the prevention and control of rice blast are to cultivate disease-resistant varieties and use pesticides for prevention and control. However, the long-term and large-scale use of chemical pesticides not only causes environmental and food pollution, but also causes pathogens to develop drug resistance in long-term adaptive evolution, resulting in inefficient prevention and control. The cultivation and promotion of disease-resistant varieties is currently the most economical and effective method for the prevention and control of rice blast. However, due to the complexity and high variation of the physiological subspecies of rice blast fungi, a disease-resistant variety often loses its resistance after several years of promotion due to the emergence of new dominant subspecies. Since the operating mechanism of resistance genes is not very clear at present, although it is possible to cultivate disease-resistant varieties with broad spectrum and durability, it takes a long time and a lot of work. Therefore, seeking new methods or ways to prevent and control rice blast has very important practical significance and broad application prospects.

[0003] In recent years, botanical pesticides have become a hot topic for new pesticides because they are more efficient, residue-free, low-toxic, broad-spectrum, and non-resistant than traditional chemical pesticides. my country has a long history of research on botanical pesticides. In ancient times, there were records of using mugwort to kill ants. A large number of studies have shown that many plant extracts can effectively prevent and treat diseases. For example, allicin extracted from garlic can effectively prevent and treat tomato leaf mold and inhibit cucumber black spot. [7] and wilt [8] , cabbage black spot, tomato gray mold and early blight, pepper anthracnose and other plant diseases, and the effect is far better than a variety of chemical pesticides. Using plant extracts to prevent and control diseases or establish disease-free seed fields and cultivate disease-free seedlings can effectively reduce soil and crop pollution.

[0004] With the rise of plant-derived pesticides in the agricultural field, Chinese medicine-derived pesticides have gradually received more and more attention. Finding effective ingredients with agricultural activity from Chinese medicinal materials with medical effects is an innovative direction for the research of plant-derived pesticides. my country is rich in Chinese medicine resources, which can provide a variety of raw materials for scientific research. Studies have shown that Chinese herbal medicine extracts can effectively inhibit plant diseases. Existing studies have found that ethanol extracts of six Chinese medicines, including cloves, Schisandra chinensis, Root of Beans, Asarum, Pulsatilla striata, and Lantern Festival, also have a good inhibitory effect on the pathogen of ginseng gray mold; matrine has a certain inhibitory effect on rice blast. A team used 38 Chinese medicine acetone extracts as materials, and four plant pathogens, including cotton wilt, wheat fusarium, poplar canker, and rapeseed sclerotinia, as the test strains. The biological activity of Chinese medicine extracts against the four pathogens was determined, and it was found that some Chinese medicine extracts had high antibacterial activity.

[0005] Chlorin iron is a new type of plant immune inducer extracted from the traditional Chinese medicine sandworm. It is a plant growth regulator with independent intellectual property rights in my country. It obtained national pesticide registration in February 2019. After application on rice, wheat, rapeseed, grapes, soybeans and other crops, it has obvious stress resistance, yield increase, and quality improvement effects. Chlorin iron inhibits the activity of chlorophyllase, delays the degradation of chlorophyll, and increases the chlorophyll content, thereby increasing the maximum photochemical efficiency of the plant's light system. At the same time, it can also promote the production of nitric oxide in root cells, inhibit the activity of indoleacetic acid oxidase, and promote plant root growth. Chlorin iron has a good effect in enhancing rice's resistance to stress, such as lodging resistance. After repeated purification and refining, the pure natural plant-active plant growth regulator obtained by dihydrochlorin iron is very similar to chlorophyll and heme in structure. It is safe for the environment and aquatic animals, slightly toxic, and has ultra-high physiological activity at a low dosage. Dihydrochlorin iron has a very good effect on enhancing plant resistance. It has been proven that it can significantly improve the ability of rice to resist rice blast. Combining it with Chinese herbal volatile oils with good antibacterial effects can enhance the resistance of rice to inhibit bacteria. And combined with green and organic Chinese herbal volatile oils, it can achieve the effects of high efficiency, low toxicity, low residue, and resistance to drugs.

[0006] There is no report on the combined use of dihydrochlorin iron and volatile oil of traditional Chinese medicine to improve the control effect of rice blast. Summary of the invention

[0007] To solve the above problems, the present invention provides the use of dihydrochlorin iron combined with traditional Chinese medicine volatile oil in the preparation of pesticides for preventing and / or treating rice blast, wherein the traditional Chinese medicine volatile oil is patchouli volatile oil, coptis volatile oil, litsea cubeba volatile oil and / or Xanthium sibiricum volatile oil.

[0008] Furthermore, the mass volume ratio of the dihydrochlorin iron to the traditional Chinese medicine volatile oil is 0.03g:0.02~0.2ml.

[0009] Furthermore, the mass volume ratio of the dihydrochlorin iron to the patchouli volatile oil is 0.03g: 0.02~0.2ml;

[0010] The mass volume ratio of the chlorin iron to the volatile oil of Litsea cubeba is 0.03g: 0.05~0.15ml;

[0011] The mass volume ratio of the dihydrochlorin iron and the volatile oil of Coptis chinensis is 0.03g: 0.1~0.2ml;

[0012] The mass volume ratio of the dihydrochlorin iron to the Xanthium sibiricum volatile oil is 0.03g:0.05-0.15ml.

[0013] The present invention also provides a combined pesticide for preventing and / or treating rice blast, which contains chlorin iron and Chinese herbal medicine volatile oil for simultaneous or separate administration;

[0014] The mass volume ratio of the dihydrochlorin iron to the volatile oil of traditional Chinese medicine is 0.03g: 0.02~0.2ml;

[0015] The traditional Chinese medicine volatile oil is patchouli volatile oil, coptis chinensis volatile oil, litsea cubeba volatile oil and / or xanthium sibiricum volatile oil.

[0016] Further, the mass volume ratio of the dihydrochlorin iron to patchouli volatile oil is 0.03g: 0.02~0.2ml;

[0017] The mass volume ratio of the chlorin iron to the volatile oil of Litsea cubeba is 0.03g: 0.05~0.15ml;

[0018] The mass volume ratio of the dihydrochlorin iron and the volatile oil of Coptis chinensis is 0.03g: 0.1~0.2ml;

[0019] The mass volume ratio of the dihydrochlorin iron to the Xanthium sibiricum volatile oil is 0.03g:0.05-0.15ml.

[0020] The present invention also provides a composition for preventing and / or treating rice blast, which is a preparation prepared by taking chlorin iron and Chinese herbal medicine volatile oil as active ingredients and adding acceptable auxiliary materials;

[0021] The mass volume ratio of the dihydrochlorin iron to the volatile oil of traditional Chinese medicine is 0.03g: 0.02~0.2ml;

[0022] The Chinese medicine volatile oil is patchouli volatile oil, coptis chinensis volatile oil, litsea cubeba volatile oil or xanthium sibiricum volatile oil.

[0023] Further, the mass volume ratio of the dihydrochlorin iron to patchouli volatile oil is 0.03g: 0.02~0.2ml;

[0024] The mass volume ratio of the chlorin iron to the volatile oil of Litsea cubeba is 0.03g: 0.05~0.15ml;

[0025] The mass volume ratio of the dihydrochlorin iron and the volatile oil of Coptis chinensis is 0.03g: 0.1~0.2ml;

[0026] The mass volume ratio of the dihydrochlorin iron to the Xanthium sibiricum volatile oil is 0.03g:0.05-0.15ml.

[0027] The present invention also provides a method for preparing the above-mentioned composition, which comprises the following steps:

[0028] 1) Take dihydrochlorin iron and dissolve it in water to prepare a 0.03% dihydrochlorin iron solution;

[0029] 2) Take Tween 80 and add water to make 5% Tween 80; take Chinese herbal volatile oil and add 5% Tween 80 to make 0.02%~0.2% Chinese herbal volatile oil solution;

[0030] 3) Mix the dihydrochlorin iron solution obtained in step 1) and the Chinese medicine volatile oil solution obtained in step 2) according to a ratio, then add acceptable excipients or auxiliary ingredients, mix well, and obtain the product.

[0031] The chlorin iron of the present invention is combined with the Chinese medicine volatile oil of patchouli volatile oil, litsea cubeba volatile oil, coptis volatile oil or Xanthium sibiricum volatile oil, from the perspective of supporting the positive energy, the resistance of rice is enhanced by chlorin iron, from the perspective of eliminating external evil, the Chinese medicine volatile oil has the effect of blocking the spread of pathogens and efficiently killing pathogens through the high-efficiency antibacterial effect, leaf dehumidification, and volatile odor insect repellent of the Chinese medicine volatile oil, and the good prevention and control effect of rice blast fungi is achieved together, and the effect is better than that of either of the two alone at the same dosage, and the effect of coordination and synergy is achieved. The two are used in combination to form a new type of rice blast prevention and control agent with all Chinese medicine ingredients, breaking the current research and development ideas for rice blast prevention and control pesticides, innovatively broadening the application scope of traditional Chinese medicine theory, and providing traditional Chinese medicine wisdom and traditional Chinese medicine practice for practical agricultural problems.

[0032] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0033] The above contents of the present invention are further described in detail below through specific implementation methods in the form of examples. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 Inhibitory effects of different concentrations of Chinese herbal volatile oil on the mycelial growth of rice blast fungus (a, Pogostemon cablin volatile oil treatment, the concentrations from left to right are 0.02%, 0.05%, 0.08%; b, Litsea cubeba volatile oil treatment, the concentrations from left to right are 0.10%, 0.15%, 0.20%; c, Xanthium sibiricum volatile oil treatment, the concentrations from left to right are 0.05%, 0.10%, 0.20%; d, Coptis chinensis volatile oil treatment, the concentrations from left to right are 0.20%, 0.25%, 0.30%; e, control group, from left to right are negative Tween control and positive rice blast control; scale bar: 1 cm);

[0035] Figure 2 Microscopic observation of the effects of Chinese herbal medicine extracts on rice blast fungus (a, low-power microscopic observation, from left to right, negative Tween, positive rice blast, patchouli volatile oil treatment, scale bar: 100 μm; b, high-power microscopic observation, from left to right, mycelial morphology of the negative group, spore suspension of the negative group; mycelial morphology of the patchouli treatment group, spore suspension of the patchouli treatment group, scale bar: 20 μm);

[0036] Figure 3 The inhibitory effect of the compound agent and rice blast-resistant on rice leaf lesions (a, lesions in the negative Tween group; b, lesions in the group treated with 0.15% Litsea cubeba volatile oil combined with dihydrochlorin iron; scale bar: 1 cm)

[0037] Figure 4 High performance liquid chromatograms of volatile oils from traditional Chinese medicines (A, chromatogram of volatile oil from Patchouli, B, chromatogram of volatile oil from Litsea cubeba) DETAILED DESCRIPTION

[0038] The raw materials and equipment used in the present invention are all known products, which are obtained by purchasing commercially available products.

[0039] Embodiment 1, Chinese medicine compound medicine of the present invention

[0040] 1. Solution preparation

[0041] 0.03% dihydrochlorin iron: Take 0.3g of dihydrochlorin iron and add 1000ml of water to dissolve it.

[0042] 5% Tween 80: Take 5 ml of Tween 80 and add water to 100 ml of water to make it miscible.

[0043] 0.02% Patchouli volatile oil: Take 0.02 ml of Patchouli volatile oil and add 100 ml of 5% Tween 80 to mix.

[0044] 2. Preparation of Chinese medicine compound medicine

[0045] Take 0.03% dihydrochlorin iron and 0.02% patchouli volatile oil, mix them in a volume ratio of 1:1, and add pharmaceutically acceptable excipients to obtain the product.

[0046] Embodiment 2, Chinese medicine compound medicine of the present invention

[0047] 1. Solution preparation

[0048] 0.03% dihydrochlorin iron: Take 0.3g of dihydrochlorin iron and add 1000ml of water to dissolve it.

[0049] 5% Tween 80: Take 5 ml of Tween 80 and add water to 100 ml of water to make it miscible.

[0050] 0.05% Patchouli volatile oil: Take 0.05 ml of Patchouli volatile oil and add 100 ml of 5% Tween 80 to mix.

[0051] 2. Preparation of Chinese medicine compound medicine

[0052] Take 0.03% dihydrochlorin iron and 0.05% patchouli volatile oil, mix them in a volume ratio of 1:1, and add pharmaceutically acceptable excipients to obtain the product.

[0053] Embodiment 3, Chinese medicine compound medicine of the present invention

[0054] 1. Solution preparation

[0055] 0.03% dihydrochlorin iron: Take 0.3g of dihydrochlorin iron and add 1000ml of water to dissolve it.

[0056] 5% Tween 80: Take 5 ml of Tween 80 and add water to 100 ml of water to make it miscible.

[0057] 0.15% Patchouli volatile oil: Take 0.15 ml of Patchouli volatile oil and add 100 ml of 5% Tween 80 to mix.

[0058] 2. Preparation of Chinese medicine compound medicine

[0059] Take 0.03% dihydrochlorin iron and 0.15% patchouli volatile oil, mix them in a volume ratio of 1:1, and add pharmaceutically acceptable excipients to obtain the product.

[0060] Embodiment 4, Chinese medicine compound medicine of the present invention

[0061] 1. Solution preparation

[0062] 0.03% dihydrochlorin iron: Take 0.3g of dihydrochlorin iron and add 1000ml of water to dissolve it.

[0063] 5% Tween 80: Take 5 ml of Tween 80 and add water to 100 ml of water to make it miscible.

[0064] 0.20% Patchouli volatile oil: Take 0.20ml of Patchouli volatile oil and add 100ml of 5% Tween 80 to mix.

[0065] 2. Preparation of Chinese medicine compound medicine

[0066] Take 0.03% dihydrochlorin iron and 0.20% patchouli volatile oil, mix them in a volume ratio of 1:1, and add pharmaceutically acceptable excipients to obtain the product.

[0067] Embodiment 5, Chinese medicine compound medicine of the present invention

[0068] 1. Solution preparation

[0069] 0.03% dihydrochlorin iron: Take 0.3g of dihydrochlorin iron and add 1000ml of water to dissolve it.

[0070] 5% Tween 80: Take 5 ml of Tween 80 and add water to 100 ml of water to make it miscible.

[0071] 0.15% Litsea cubeba essential oil: Take 0.15ml of Litsea cubeba essential oil and add 100ml of 5% Tween 80 to mix.

[0072] 2. Preparation of Chinese medicine compound medicine

[0073] Take 0.03% dihydrochlorin iron and 0.15% Litsea cubeba essential oil, mix them in a volume ratio of 1:1, and add pharmaceutically acceptable excipients to obtain the product.

[0074] Embodiment 6, Chinese medicine compound medicament of the present invention

[0075] 1. Solution preparation

[0076] 0.03% dihydrochlorin iron: Take 0.3g of dihydrochlorin iron and add 1000ml of water to dissolve it.

[0077] 5% Tween 80: Take 5 ml of Tween 80 and add water to 100 ml of water to make it miscible.

[0078] 0.05% Litsea cubeba volatile oil: Take 0.05 ml of Litsea cubeba volatile oil and add 100 ml of 5% Tween 80 to mix.

[0079] 2. Preparation of Chinese medicine compound medicine

[0080] Take 0.03% dihydrochlorin iron and 0.05% Litsea cubeba essential oil, mix them in a volume ratio of 1:1, and add pharmaceutically acceptable excipients to obtain the product.

[0081] Embodiment 7, Chinese medicine compound medicine of the present invention

[0082] 1. Solution preparation

[0083] 0.03% dihydrochlorin iron: Take 0.3g of dihydrochlorin iron and add 1000ml of water to dissolve it.

[0084] 5% Tween 80: Take 5 ml of Tween 80 and add water to 100 ml of water to make it miscible.

[0085] 0.10% Litsea cubeba essential oil: Take 0.10ml of Litsea cubeba essential oil and add 100ml of 5% Tween 80 to mix.

[0086] 2. Preparation of Chinese medicine compound medicine

[0087] Take 0.03% dihydrochlorin iron and 0.10% Litsea cubeba essential oil, mix them in a volume ratio of 1:1, and add pharmaceutically acceptable excipients to obtain the product.

[0088] Embodiment 8, Chinese medicine compound medicament of the present invention

[0089] 1. Solution preparation

[0090] 0.03% dihydrochlorin iron: Take 0.3g of dihydrochlorin iron and add 1000ml of water to dissolve it.

[0091] 5% Tween 80: Take 5 ml of Tween 80 and add water to 100 ml of water to make it miscible.

[0092] 0.1% Coptis chinensis volatile oil: Take 0.1ml of Coptis chinensis volatile oil and add 100ml of 5% Tween 80 to mix.

[0093] 2. Preparation of Chinese medicine compound medicine

[0094] Take 0.03% dihydrochlorin iron and 0.1% coptis chinensis volatile oil, mix them in a volume ratio of 1:1, and add pharmaceutically acceptable excipients to obtain the product.

[0095] Embodiment 9, Chinese medicine compound medicine of the present invention

[0096] 1. Solution preparation

[0097] 0.03% dihydrochlorin iron: Take 0.3g of dihydrochlorin iron and add 1000ml of water to dissolve it.

[0098] 5% Tween 80: Take 5 ml of Tween 80 and add water to 100 ml of water to make it miscible.

[0099] 0.15% Coptis chinensis volatile oil: Take 0.15ml of Coptis chinensis volatile oil and add 100ml of 5% Tween 80 to mix.

[0100] 2. Preparation of Chinese medicine compound medicine

[0101] Take 0.03% dihydrochlorin iron and 0.15% coptis chinensis essential oil, mix them in a volume ratio of 1:1, and add pharmaceutically acceptable excipients to obtain the product.

[0102] Embodiment 10, Chinese medicine compound medicament of the present invention

[0103] 1. Solution preparation

[0104] 0.03% dihydrochlorin iron: Take 0.3g of dihydrochlorin iron and add 1000ml of water to dissolve it.

[0105] 5% Tween 80: Take 5 ml of Tween 80 and add water to 100 ml of water to make it miscible.

[0106] 0.2% Coptis chinensis volatile oil: Take 0.2ml of Coptis chinensis volatile oil and add 100ml of 5% Tween 80 to mix.

[0107] 2. Preparation of Chinese medicine compound medicine

[0108] Take 0.03% dihydrochlorin iron and 0.2% coptis chinensis essential oil, mix them in a volume ratio of 1:1, and add pharmaceutically acceptable excipients to obtain the product.

[0109] Embodiment 11, Chinese medicine compound medicine of the present invention

[0110] 1. Solution preparation

[0111] 0.03% dihydrochlorin iron: Take 0.3g of dihydrochlorin iron and add 1000ml of water to dissolve it.

[0112] 5% Tween 80: Take 5 ml of Tween 80 and add water to 100 ml of water to make it miscible.

[0113] 0.1% Xanthium sibiricum volatile oil: Take 0.1ml of Xanthium sibiricum volatile oil and add 100ml of 5% Tween 80 to mix.

[0114] 2. Preparation of Chinese medicine compound medicine

[0115] Take 0.03% dihydrochlorin iron and 0.1% Xanthium sibiricum volatile oil, mix them in a volume ratio of 1:1, and add pharmaceutically acceptable excipients to obtain the product.

[0116] Embodiment 12, Chinese medicine compound medicine of the present invention

[0117] 1. Solution preparation

[0118] 0.03% dihydrochlorin iron: Take 0.3g of dihydrochlorin iron and add 1000ml of water to dissolve it.

[0119] 5% Tween 80: Take 5 ml of Tween 80 and add water to 100 ml of water to make it miscible.

[0120] 0.2% Xanthium sibiricum volatile oil: Take 0.2ml of Xanthium sibiricum volatile oil and add 100ml of 5% Tween 80 to mix.

[0121] 2. Preparation of Chinese medicine compound medicine

[0122] Take 0.03% dihydrochlorin iron and 0.2% Xanthium sibiricum volatile oil, mix them in a volume ratio of 1:1, and add pharmaceutically acceptable excipients to obtain the product.

[0123] Embodiment 13, Chinese medicine compound medicine of the present invention

[0124] 1. Solution preparation

[0125] 0.03% dihydrochlorin iron: Take 0.3g of dihydrochlorin iron and add 1000ml of water to dissolve it.

[0126] 5% Tween 80: Take 5 ml of Tween 80 and add water to 100 ml of water to make it miscible.

[0127] 0.05% Xanthium sibiricum volatile oil: Take 0.05ml of Xanthium sibiricum volatile oil and add 100ml of 5% Tween 80 to mix.

[0128] 2. Preparation of Chinese medicine compound medicine

[0129] Take 0.03% dihydrochlorin iron and 0.05% Xanthium sibiricum volatile oil, mix them in a volume ratio of 1:1, and add pharmaceutically acceptable excipients to obtain the product.

[0130] Example 14: Combined pesticide for controlling rice blast of the present invention

[0131] 1. Solution preparation

[0132] 0.03% dihydrochlorin iron: Take 0.3g of dihydrochlorin iron and add 1000ml of water to dissolve it.

[0133] 5% Tween 80: Take 5 ml of Tween 80 and add water to 100 ml of water to make it miscible.

[0134] 0.02% Patchouli volatile oil: Take 0.02 ml of Patchouli volatile oil and add 100 ml of 5% Tween 80 to mix.

[0135] 2. Preparation of combined pesticides

[0136] Take equal volumes of 0.03% dihydrochlorin iron and 0.02% patchouli volatile oil and package them separately.

[0137] The beneficial effects of the present invention are demonstrated by experimental examples below.

[0138] Experimental Example 1 Research on a new type of Chinese medicine for the prevention and treatment of rice blast based on traditional Chinese medicine theory

[0139] 1 Materials and methods

[0140] 1.1 Materials

[0141] Volatile oil of Chinese herbal medicine: Patchouli ( Pogostemon cablin (Blanco) Benth . )、Coptis chinensis( Coptis chinensis Franch . ), Litsea cubeba ( Litsea cubeba (Lour.) Pers . ) and Xanthium sibiricum ( Xanthium sibiricum Patr . ) Volatile oil was extracted by the State Key Laboratory of Southwestern Characteristic Traditional Chinese Medicine Resources of Chengdu University of Traditional Chinese Medicine. The extraction method was as follows: take each Chinese medicine separately and extract it according to the method A of the volatile oil determination method in Part IV 2204 of the Pharmacopoeia 2020;

[0142] Specifically: Take the Chinese medicine, add water and glass beads, shake and mix, and then connect the volatile oil tester to the reflux condenser. Add water from the upper end of the condenser until it fills the scale part of the volatile oil tester and overflows into the flask. Place it in an electric heating mantle and slowly heat it to boiling, and keep it at a slight boil for 5 hours until the amount of oil in the tester no longer increases. Stop heating, leave it for a while, open the piston at the lower end of the tester, and slowly release the water until the upper end of the oil layer reaches 5mm above the scale 0 line. Leave it for more than 1 hour, then open the piston and collect the volatile oil.

[0143] Chlorin iron: provided by Anqing Better Biotechnology Co., Ltd.; 0.03% chlorin iron is prepared by diluting the purchased chlorin iron powder with water to the use concentration.

[0144] Test pathogens: Guy11 The rice blast strain was provided by Professor Yin Junjie from the State Key Laboratory of Discovery and Utilization of Southwestern Crop Gene Resources, Sichuan Agricultural University.

[0145] The control agent used in the test was 40% Isoprothiolane EC, which was purchased from Chongqing Shurong Crop Science Co., Ltd.

[0146] Test rice: provided by Rice Research Institute of Sichuan Agricultural University.

[0147] CM medium: 20 × Nitrate Salts 50 mL, Trace Elements 1 mL, D-Glucose 10 g, Peptone 2 g, Yeast Extract 1 g, Casamino Acids 1 g, Vitamin Solution 1 mL, PH 6.5

[0148] Instruments: DNP-9082A electric constant temperature incubator, Shanghai Jiangren Experimental Equipment Co., Ltd.; Eppendorf pipette; Leica EZ4 optical microscope

[0149] 1.2 Test methods

[0150] In order to explore the antibacterial activity and control effect of Chinese herbal volatile oil on rice blast fungus, the study was divided into two parts: indoor toxic medium method test and field test. In the indoor toxic medium test, the inhibitory effect of different concentrations and types of Chinese herbal volatile oil and the combination of dihydrochlorin iron on the mycelial growth of rice blast fungus was obtained, and the combination of agents with better effects was screened out, and then field tests were carried out in rice fields to verify the actual control effect of the drugs.

[0151] 1.2.1 Preparation of culture medium for rice blast fungus

[0152] Take 22.4 g / L of CM medium powder and dissolve it, and adjust the pH to 6.5. For solid medium, add 15 g / L of agar powder and heat until the solution is clear. Then sterilize it with high pressure steam at 121℃ for 15 min. After completion, cool it to 50-60℃, add 50 mg / L of carbenicillin and 50 mg / L of streptomycin sulfate to prepare the rice blast fungus culture medium.

[0153] 1.2.2 Preparation of four Chinese herbal medicine volatile oil solutions with different concentrations

[0154] Using 5% Tween 80 as solvent, the mother solutions of four kinds of Chinese herbal volatile oils were used to prepare the drug solution. The specific preparation concentrations are shown in Table 1.

[0155] Table 1 Concentration ratio of each drug in the toxic medium test

[0156]

[0157] 1.2.3 Antibacterial test of Chinese herbal medicine extracts in toxic media

[0158] The antibacterial activity of Chinese herbal medicine extracts was determined by the indoor toxic medium method. 15 ml of each concentration of volatile oil, negative control Tween 80, positive control rice blast and dihydrochlorin iron were added to 20 mL of 50 ℃ rice blast culture medium and mixed thoroughly to prepare drug-containing plates. After the plates solidified, 0.2 cm 2 The fungus cakes were cultured in a constant temperature incubator at 25 ℃. The growth of mycelium of rice blast fungus was observed uniformly after three weeks, and the colony diameter was calculated using Image J software.

[0159] The antibacterial activity of Chinese herbal medicine extracts combined with dihydrochlorin iron against rice blast fungus was determined by the indoor toxic medium method. Take 7.5 ml of 0.03% dihydrochlorin iron and 7.5 ml of Chinese herbal medicine volatile oil (0.02% Patchouli volatile oil, 0.20% Coptis chinensis volatile oil, 0.15% Litsea cubeba volatile oil or 0.10% Xanthium sibiricum volatile oil) to make 15 ml of mixed solution, add it to 20 mL of rice blast fungus culture medium at about 50 ℃, and mix it thoroughly to make a drug-containing plate. After the plate solidifies, inoculate 0.2 cm long stalks of uniform growth. 2 The fungus cakes were cultured in a constant temperature incubator at 25 ℃. The growth of mycelium of rice blast fungus was observed uniformly after three weeks, and the colony diameter was calculated using Image J software.

[0160] Inhibition rate (%) = [ (colony growth diameter of blank control group - colony growth diameter of treatment group) / colony growth diameter of control group] × 100, colony growth diameter = colony diameter - bacterial cake diameter (0.5 cm).

[0161] 1.2.4 Field control trials of Chinese herbal medicine extracts

[0162] Field tests were conducted to verify the actual control effect of the composite agent (i.e., a mixture of different concentrations of Chinese herbal volatile oil and 0.03% dihydrochlorin iron in a volume ratio of 1:1) on rice blast. The test site is located in the experimental field of Sichuan Agricultural University in Chengdu, Sichuan Province, with geographical coordinates of 103°8'N, 30°72'E. The altitude is between 510m and 650m, the terrain is flat, and it belongs to the subtropical humid monsoon climate zone. Rice seeds were sown and raised on the seedbed. The seedbed was not fertilized and transplanted to the field on April 16, 2024. There were a total of 120 plots, each with an area of ​​0.08 m 2, 50-60 plants in each area. In the later stage, all the test rice were uniformly inoculated to ensure that all rice was at the same disease level. The random block arrangement method was adopted. The prevention group and the treatment group were sprayed twice at a frequency of 12h and 24h, respectively. The prevention group started the first spraying 24h before inoculation and then the second spraying 12h before inoculation. The treatment group started the first spraying 12h after inoculation and then the second spraying 24h. The volume of liquid sprayed in each medication group was the same, and sampling was carried out on May 21, 2024. The third leaf of rice in the prevention group and the experimental group was randomly adhered to the printing paper, and the black spots on the leaves were counted by taking pictures. The inhibitory effect of different drugs on rice blast fungus was analyzed by the formula:

[0163] Disease index = ∑ (number of diseased leaves at each level × representative value at each level) / (total number of leaves surveyed × highest representative value) × 100

[0164] Control effect (%) = [(disease index of control area - disease index of drug-treated area) / disease index of control area] × 100

[0165] Lesion inhibition effect (%) = [(number of lesions per leaf in negative group - number of lesions per leaf in treatment group) / number of lesions per leaf in negative group] × 100

[0166] Leaf blight (based on leaves): Level 0: disease-free; Level 1: less than 5 leaf spots, less than 1 cm in length; Level 3: 6 to 10 leaf spots, some of which are greater than 1 cm in length; Level 5: 11 to 25 leaf spots, some of which are connected, accounting for 10% to 25% of the leaf area; Level 7: more than 26 leaf spots, which are connected, accounting for 26% to 50% of the leaf surface; Level 9: The spots are connected, accounting for more than 50% of the leaf area or the entire leaf is dead.

[0167] 1.3 Determination of active ingredients in volatile oils of traditional Chinese medicine

[0168] After completing the field test, the active ingredients and content analysis of the volatile oils of traditional Chinese medicines with the best control effect were carried out using high-performance liquid chromatography.

[0169] HPLC chromatographic conditions: Shimadzu high performance liquid chromatograph, SPD-20A UV detector, ODS-C18 reverse phase column (250×4.6 mm, 5 μm); mobile phase: 90% methanol in water, flow rate 1.0 mL / min, isocratic elution mode 15 min; column temperature 37 °C. Detection wavelength: 210 nm; sample solution filtered through 0.20 μm microporous membrane, injection volume 10 L.

[0170] 1.4 Data Analysis Methods

[0171] The experimental results were analyzed using Excel software, Image J software, and DPS (Data Processing Synstem) statistical software, and the statistical method used was Duncan's Multiple Range Test.

[0172] 2 Results and Analysis

[0173] 2.1 Inhibitory effect of Chinese herbal medicine extracts on mycelial growth of rice blast

[0174] The study conducted a toxic medium experiment on the volatile oils of four traditional Chinese medicines, namely, Patchouli, Litsea cubeba, Coptis chinensis, and Xanthium sibiricum, with the solvent Tween as the negative control and the chemical drug Rice Blast as the positive control. As the concentration of the four traditional Chinese medicine volatile oils increased, the inhibition rate increased and the diameter of the rice blast fungus colony decreased. The results showed that the four traditional Chinese medicine volatile oils had an inhibitory effect on the growth of the mycelium of the rice blast fungus ( Figure 1 ). When the concentration (volume fraction) of patchouli extract was 0.02%, 0.05%, and 0.08%, the inhibition rates were 17.38%, 37.03%, and 100%, respectively (Picture 1a); when the concentration (volume fraction) of Litsea cubeba extract was 0.10%, 0.15%, and 0.20%, the inhibition rates were 25.33%, 44.38%, and 100%, respectively (Picture 1b); when the concentration (volume fraction) of Xanthium sibiricum extract was 0.05%, 0.10%, and 0.20%, the inhibition rates were 4.94%, 34.45%, and 100%, respectively (Picture 1c); when the concentration (volume fraction) of Coptis chinensis extract was 0.20%, 0.25%, and 0.30%, the inhibition rates were 67.39%, 100%, and 100%, respectively (Picture 1d); among them, the best effect was patchouli volatile oil, which could achieve 100% inhibition of colony growth at extremely low concentrations. Based on the data of the toxic medium method, the toxicity regression curve and EC of the Chinese herbal medicine extract were calculated. 50 (Table 2), EC of each extract 50 They are 0.269 mg / mL for Patchouli, 0.695 mg / mL for Litsea cubeba, 0.989 mg / mL for Coptis chinensis, and 1.266 mg / mL for Xanthium sibiricum. Among them, the slope of the toxicity curve of Coptis chinensis is the largest, indicating that within the set concentration gradient range, the change in concentration has the greatest effect on mycelial growth. The slope of the toxicity curve of Patchouli is the smallest, and the change in concentration has the smallest effect on mycelial growth.

[0175] Table 2 Inhibitory effect of Chinese herbal extracts on mycelial growth of rice blast fungus

[0176]

[0177] 2.2 Inhibitory effect of Chinese herbal extracts combined with dihydrochlorin iron on mycelial growth of rice blast fungus

[0178] As can be seen from Table 3, dihydrochlorin iron itself has a certain antibacterial effect. At the concentration used, the inhibition rate of dihydrochlorin iron on mycelial growth can reach 35.74%. After dihydrochlorin iron and four kinds of Chinese herbal volatile oils were used in combination, the antibacterial effect was greater than that of dihydrochlorin iron and Chinese herbal volatile oils when they were administered separately at the same dosage, producing a synergistic effect. Among them, after Litsea cubeba and Coptis chinensis were used in combination with dihydrochlorin iron, the antibacterial effect was equivalent to that of the chemical pesticide rice blast, and the average concentration of Chinese herbal volatile oils was lower than that of rice blast.

[0179] Table 3 Inhibitory effect of Chinese herbal extracts combined with chlorin iron on mycelial growth of rice blast fungus

[0180]

[0181] 2.3 Effects of Chinese herbal medicine extracts on mycelial morphology of rice blast

[0182] The mycelial morphology of each treatment group was observed under a microscopic microscope. Figure 2 a) Without drug intervention, the mycelium of rice blast fungus is thin and white in color. The mycelium of the positive group of rice blast fungus was found to be obviously black. The patchouli treatment group was analyzed in detail. The mycelium in the patchouli treatment group was observed to be obviously swollen, and the mycelium was obviously thicker and changed in color than that of other groups. The mycelium of the Xanthium sibiricum group, the Litsea cubeba group and the Coptis chinensis group did not have obvious swelling, but some mycelium turned obviously black ( Figure 1 ). Observe under a high-power microscope ( Figure 2 b) The mycelia in the negative group were smooth and had a small bending range. The mycelia in the patchouli group were wrinkled, tortuous, and had more mycelial branches. In the same field of view, the spore suspension was observed to have a higher density in the negative Tween group, while the number of spores in the patchouli treatment group was significantly reduced and the density was significantly decreased, indicating that patchouli volatile oil can affect the morphology of mycelia and spore growth and reproduction of rice blast fungus.

[0183] 2.4 Field control effect of compound pesticides in rice fields

[0184] The study further conducted field trials in rice fields to verify its actual control effect. All test rice were uniformly inoculated with bacteria to ensure that all rice had the same disease level. Field observations found that after the drug was administered, the residual moisture on the rice leaves decreased, and the number of rice insects between the leaves decreased significantly, indicating that the volatile oil of traditional Chinese medicine had a certain effect of drying moisture and repelling insects with its odor, reducing the spread of the disease to a certain extent. The rice leaves were photographed and observed. In the negative group, lesions were seen all over the leaves, and some rice leaves turned yellow and necrotic ( Figure 3 a), the rice leaf spots in the compound pesticide treatment group were significantly reduced ( Figure 3 b). According to the drug lesion inhibition effect, regression equation analysis was performed (Table 4). The equations for the patchouli prevention group and the treatment group were:y =1.2598 x + 8.9772, y =1.1327 x +8.5437, EC 50 is 0.0697%, 0.0744%, the equations of the prevention group and treatment group of Litsea cubeba are y =3.4601 x +15.446, y =3.4731 x +15.474,EC 50 0.0959%, ​​0.0964% (% are volume fractions). 50 The analysis shows that the inhibitory effect of the four Chinese herbal volatile oils on rice leaf lesions is generally higher in the prevention group than in the treatment group. Among them, the volatile oil of patchouli has the best inhibitory effect on lesions, followed by the volatile oil of Litsea cubeba. The control effect was calculated according to the disease index (Table 5). The results showed that the volatile oil of Litsea cubeba with a concentration of 0.15% volume fraction had the best control effect, reaching 72.94%, followed by the volatile oil of patchouli with a volume fraction of 0.2%, with a control effect of 71.82%. The chemical drug rice blast reached 73.95%. After being combined with dihydrochlorin iron, the two produced obvious synergistic synergy. The composite agent composed of 0.20% patchouli volatile oil and dihydrochlorin iron had a control effect of 92.32%, and the control effect of 0.15% Litsea cubeba volatile oil reached 95.43% after the combination. Through one-way analysis of variance (ANOVA): the F value was 1.569 and the p value was 0.272, indicating that there was no significant difference in the control effect between the different Chinese medicine volatile oil treatment groups and the rice blast treatment group. For the different concentration treatment groups of the four Chinese medicine volatile oils, the control effect showed a significant increase with the increase of concentration, and there were significant differences between the control effects of different concentration components.

[0185] Comparing the control effects of four kinds of Chinese herbal volatile oils after compatibility, the study found that the field test and the plate antibacterial test had the same effect, that is, at the same dosage, the antibacterial effect of the compound agent was greater than that of dihydrochlorin iron and Chinese herbal volatile oils administered separately, producing a synergistic effect. Among them, the synergistic effect of the volatile oil of Litsea cubeba and the volatile oil of Patchouli with dihydrochlorin iron was the best, with an average increase of about 30% after compatibility. In the experiment, only one spray was carried out 24 hours before inoculation. If the spraying time is advanced and the frequency is increased, the stress resistance effect of dihydrochlorin iron will be more obvious, and the control effect of the compound agent on rice blast fungi will be more prominent. After calculating the cost, it was found that the average cost per mu of the compound agent was nearly 80% lower than that of the chemical pesticide rice blast, which has a very high cost advantage and has the potential to be developed into a new type of Chinese herbal pesticide.

[0186] Table 4 Inhibitory effect of Chinese herbal extracts on rice blast lesions

[0187]

[0188] Table 5 Field control effect of Chinese herbal medicine extracts combined with dihydrochlorin iron on rice blast

[0189]

[0190] 2.5 Analysis of components of volatile oils from traditional Chinese medicines and determination of active ingredients

[0191] The study found that the volatile oils of Patchouli and Litsea cubeba had the best treatment effect. The components of the two were analyzed by high performance liquid chromatography ( Figure 4 ). First, a chromatographic analysis of patchouli was performed ( Figure 4 A), compared with the standard, the main component of patchouli volatile oil was determined to be patchouli alcohol. Further content determination revealed that patchouli alcohol (C 15 H 26 O) was 26%. Secondly, the chromatogram analysis of Litsea cubeba was performed ( Figure 4 B), the main components of the essential oil of Litsea cubeba were determined to be α-citral, β-citral, and D-limonene, and the proportion of these three components reached 68%.

[0192] 4 Results and discussion

[0193] Based on the above results, it is determined that the best inhibitory effects on rice blast fungi are patchouli and litsea cubeba volatile oils among the four types of Chinese medicine volatile oils for drying and removing dampness. Both showed good control effects in field trials. Therefore, patchouli volatile oil and litsea cubeba volatile oil are respectively matched with dihydrochlorin iron, and the three are matched together to form three composite agents for preventing and treating rice blast. Under the guidance of the theory of strengthening the body and expelling evil spirits in traditional Chinese medicine, the composite agent prepared by compounding the Chinese medicine volatile oil and dihydrochlorin iron of the present invention is innovatively improved by improving the resistance of rice to strengthen the body, high-efficiency antibacterial, smell insect repellent, and leaf drying and expelling evil spirits, and the Chinese medicine volatile oil and dihydrochlorin iron produce obvious synergistic effects, and the average control effect is enhanced by about 30%. At the same time, the composite agent has the advantages of green safety, low residue, and low drug resistance, and the composite agent is cost-effective, and it is further developed into a Chinese medicine pesticide product with very good operability and practical significance.

Claims

1. Use of chlorin iron combined with volatile oil of traditional Chinese medicine in the preparation of pesticides for preventing and / or treating rice blast, characterized in that: The Chinese medicine volatile oil is Patchouli volatile oil, Coptis chinensis volatile oil, Litsea cubeba volatile oil or Xanthium sibiricum volatile oil; The mass volume ratio of the dihydrochlorin iron to patchouli volatile oil is 0.03g: 0.02~0.2ml; The mass volume ratio of the chlorin iron to the volatile oil of Litsea cubeba is 0.03g: 0.05~0.15ml; The mass volume ratio of the dihydrochlorin iron and the volatile oil of Coptis chinensis is 0.03g: 0.1~0.2ml; The mass volume ratio of the dihydrochlorin iron to the Xanthium sibiricum volatile oil is 0.03g:0.05-0.15ml.

2. A combined pesticide for preventing and / or treating rice blast, characterized in that: It contains chlorin iron and Chinese herbal volatile oil for simultaneous or separate administration; The Chinese medicine volatile oil is Patchouli volatile oil, Coptis chinensis volatile oil, Litsea cubeba volatile oil or Xanthium sibiricum volatile oil; The mass volume ratio of the dihydrochlorin iron to patchouli volatile oil is 0.03g: 0.02~0.2ml; The mass volume ratio of the chlorin iron to the volatile oil of Litsea cubeba is 0.03g: 0.05~0.15ml; The mass volume ratio of the dihydrochlorin iron and the volatile oil of Coptis chinensis is 0.03g: 0.1~0.2ml; The mass volume ratio of the dihydrochlorin iron to the Xanthium sibiricum volatile oil is 0.03g:0.05-0.15ml.

3. A composition for preventing and / or treating rice blast, characterized in that: The preparation is prepared by using dihydrochlorin iron and volatile oil of traditional Chinese medicine as active ingredients and adding auxiliary materials acceptable to pesticides; The Chinese medicine volatile oil is Patchouli volatile oil, Coptis chinensis volatile oil, Litsea cubeba volatile oil or Xanthium sibiricum volatile oil; The mass volume ratio of the dihydrochlorin iron to the patchouli volatile oil is 0.03g:0.02-0.2ml; The mass volume ratio of the chlorin iron to the volatile oil of Litsea cubeba is 0.03g: 0.05~0.15ml; The mass volume ratio of the dihydrochlorin iron and the volatile oil of Coptis chinensis is 0.03g: 0.1~0.2ml; The mass volume ratio of the dihydrochlorin iron to the Xanthium sibiricum volatile oil is 0.03g:0.05-0.15ml.

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