Ustilaginoidea virens botanical fungicide based on isoliquiritigenin

Abstract

The invention relates to a Ustilaginoidea virens botanical fungicide based on isoliquiritigenin. The invention develops a novel efficient, environment-friendly, safe and cost-controllable biopesticide mainly aiming at false smut by deeply researching the biological activity and action mechanism of plant source secondary metabolites, remarkably improves the field application effect and stability of the biopesticide by scientific proportioning and formula optimization, reduces the production cost and environmental pollution risk, and has a wide application prospect. Powerful support is provided for agricultural sustainable development and grain safety guarantee.

Description

Technical Field

[0001] The invention belongs to the technical field of plant disease prevention and control, and particularly relates to a botanical fungicide for rice false smut based on isoliquiritigenin. Background Art

[0002] Rice false smut is one of the main fungal diseases that occurs on rice, an important food crop. It is caused by the infection of rice panicles by the chlororoblast fungus oryzae to form rice smut balls. In recent years, the incidence of rice false smut has increased year by year; in 2024, the area of ​​rice false smut in my country is expected to be 40 million mu. In addition to yield losses, fusarium smut can cause lesions and deformities in animal cells, seriously threatening food security. At present, chemical pesticides have always been the main means of preventing and controlling rice false smut. Their high efficiency and rapid antibacterial properties provide an important guarantee for increasing crop yields and incomes. However, with the increasing attention paid to food safety, environmental protection and ecological balance, the problems caused by the widespread use of chemical pesticides have become increasingly prominent, prompting the agricultural field to urgently explore more green and sustainable strategies for preventing and controlling rice false smut.

[0003] There are many types of fungicides and biopesticides on the market. Among them, plant-derived secondary metabolites have gradually become a research hotspot due to their natural, degradable and relatively low-toxic properties. These substances are derived from plants and produced through complex biosynthetic pathways. They have a wide range of biological activities, such as antibacterial and antiviral activities, and provide rich resources for the development of biopesticides. For example, plant-derived fungicides such as saxifrage extracts have shown certain application potential.

[0004] Problems and shortcomings in the existing technology:

[0005] 1. Limited bactericidal effect: Although some existing plant-based fungicides such as saxifrage extract have certain antibacterial effects, their antibacterial effects are lower than those of chemical pesticides, reaching only 10%-50% of chemical pesticides and cannot completely replace chemical pesticides.

[0006] 2. Environmental issues: Some existing fungicidal compositions, such as those containing chemical pesticides such as tinostrobin and fluazifop-butyl, can achieve good antibacterial effects, but they are compositions of chemicals, are not environmentally friendly, do not meet the requirements of ecological agriculture, and may also affect food safety.

[0007] 3. Chemical residues and drug resistance: Long-term use of chemical fungicides will lead to increased drug resistance of pathogens. The continuous use of chemical reagents will also cause environmental pollution. The residues of chemical agents on crops may affect human health through the food chain.

[0008] 4. Research on plant-derived secondary metabolites: There are very few studies on plant-derived secondary metabolites against rice smut fungus. Although some scholars have studied their inhibitory activity against rice bacterial diseases, no scholars have been found to have studied the three major fungal diseases of rice, especially rice smut fungus, and few have delved into its mechanism. Summary of the invention

[0009] In view of the many problems and deficiencies in the prior art, the present invention aims to develop a new type of biological pesticide that is efficient, environmentally friendly, safe and cost-controlled by deeply studying the biological activity and mechanism of action of plant-derived secondary metabolites. The pesticide is mainly targeted at rice smut pathogens. Through scientific proportioning and formula optimization, its field application effect and stability are significantly improved, while production costs and environmental pollution risks are reduced, providing strong support for the sustainable development of agriculture. The creation of the present invention not only makes up for the defects and deficiencies of the prior art, but also injects strength into the development of the field of biological pesticides.

[0010] The purpose of the present invention is to provide a botanical fungicide for rice fungal diseases, the effective sources of which are all derived from plants, without causing any harm to plants and not prone to residue, and is an efficient and safe botanical fungicide.

[0011] The invention provides a plant-derived fungicide for preventing rice false smut, which takes isoliquiritigenin as a main active ingredient, and the concentration of the isoliquiritigenin is not less than 12.5 mg / L.

[0012] Furthermore, in practical application, the concentration of isoliquiritigenin is not less than 50 mg / L, preferably 50-100 mg / L.

[0013] Furthermore, the method may include adding an auxiliary agent, nano-silicon hydrogel, during field application.

[0014] The present invention also provides a method for preparing any of the above-mentioned plant-derived fungicides for rice smut fungus, comprising dissolving isoliquiritigenin with DMSO, mixing it with water, and adding nano-silicon hydrogel to finally prepare the plant-derived fungicide for rice smut fungus.

[0015] Furthermore, 300-600 mg of isoliquiritigenin is dissolved in 300-600 mL of DMSO, mixed with 6-12 L of water, and 4.5-9 mL of nano-silicon hydrogel is added to finally prepare a botanical fungicide for rice false smut with an isoliquiritigenin concentration of 50-100 mg / L.

[0016] The present invention also provides the use of isoliquiritigenin in any of the following:

[0017] A1) Control of rice false smut,

[0018] A2) preparing a product for preventing and controlling rice false smut,

[0019] A3) inhibit the growth of rice smut pathogen,

[0020] A4) preparing a product for inhibiting the growth of U.S. smut pathogen.

[0021] The present invention also provides a method for preventing and controlling rice false smut, characterized in that the rice false smut is prevented and controlled using the botanical fungicide described in any one of claims 1 to 3 or the botanical fungicide prepared by the method described in any one of claims 4 to 5.

[0022] Furthermore, the control is to spray a botanical fungicide during the rice booting stage.

[0023] Application of any of the above-mentioned botanical fungicides or the botanical fungicides prepared by any of the above-mentioned methods or any of the above-mentioned methods in preventing and controlling rice false smut.

[0024] Beneficial effects:

[0025] 1. This product is a plant-derived fungicide. The main ingredient, isoliquiritigenin, has a general inhibitory effect on rice blast and rice sheath blight on the plate, but has good inhibitory activity on the mycelial growth of rice smut fungus.

[0026] 2. It also has a good effect on the prevention and control of rice false smut in indoor pot tests and field tests; it is green and environmentally friendly, has a wide source and is not easy to leave residues, has little impact on non-target organisms, has high safety, and reduces chemical pesticide pollution, promoting the recovery of my country's arable land fertility.

[0027] 3. The main ingredient of this product, isoliquiritigenin, can destroy the subcellular structure of the rice false smut fungus, causing confusion and death. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0029] Figure 1 :Inhibitory effects of 11 plant-derived secondary metabolites on the mycelial growth of Aspergillus oryzae.

[0030] Figure 2 : The antibacterial effect of isoliquiritigenin on the three major pathogens of rice diseases: A1: DMSO control + U.S. oryzae, A2: isoliquiritigenin + U.S. oryzae; B1: DMSO control + Sheath Blight, B2: isoliquiritigenin + Sheath Blight; C1: DMSO control + Magnaporthe oryzae, C2: isoliquiritigenin + Magnaporthe oryzae.

[0031] Figure 3:Subcellular observation of the effect of isoliquiritigenin on the mycelial morphology of Aspergillus oryzae: the first row is the control group, and the second row is the isoliquiritigenin group. CW: cell wall; CM: cell membrane; N: cell nucleus; MT: mitochondria; IO: incomplete organelle; NM: nuclear membrane.

[0032] Figure 4 : Statistics of potted plant test results: isoliquiritigenin (YGCS) group, control (CK) group, and DMSO group.

[0033] Figure 5 : Photos of potted plant test results: from left to right: HWD-2 (pathogenic strain of U.S. oryzae) i.e. CK group, DMSO solvent group, and isoliquiritigenin treatment group. DETAILED DESCRIPTION

[0034] The following examples are only used to more clearly illustrate the technical scheme of the present invention, and are therefore only used as examples, and cannot limit the scope of protection of the present invention. It should be noted that, unless otherwise stated, the technical terms or scientific terms used in this application should be the usual meanings understood by those skilled in the art to which the present invention belongs. Unless otherwise stated, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art. Unless otherwise stated, the reagents and materials used in the following examples are commercially available.

[0035] The object of the present invention is to provide a botanical fungicide for rice fungal diseases, the active ingredients of which are derived from plants, will not cause any harm to plants, and are not likely to remain.

[0036] The rice variety selected for the experiment of the present invention is: Wanxian 98, which is a susceptible variety.

[0037] Example 1 Screening of plant-derived secondary metabolites that have an inhibitory effect on the growth of Aspergillus oryzae hyphae

[0038] (1) The present invention screened the following 20 plant-derived secondary metabolites: baicalein, ginsenoside Rd, paeoniflorin, scutellariae, biochanin A, pterostilbene, isoliquiritigenin, crocin I, galangin, magnoligenin, apigenin, chrysin, hesperidin, mangiferin, demethoxycurcumin, psoralen, hypoaconitine, podophyllotoxin, atractylodesin, and ginsenoside Rh1 (S type), wherein the CAS Number of isoliquiritigenin is 961-29-5.

[0039] The screening detection method is the mycelium growth rate method (the following tests are all carried out on a clean bench): First, the secondary metabolites are prepared into a 100 mg / L drug-containing medium PSA with DMSO, and the medium with only an equal amount of solvent is used as the control group. After ensuring full mixing, pour it into 3 sterilized culture dishes, use tweezers to take a new and alive rice koji fungus cake with a diameter of 0.5 cm and insert it into the culture dish, and culture it in a biochemical incubator at a constant temperature of 28±1 degrees. After the rice koji fungus grew for 14 days, the cross-cross method was used to measure the diameter of the rice koji fungus colony in the drug group and the DMSO control group, in mm, and three replicates were used for each treatment. The inhibition rate of isoliquiritigenin on mycelial growth was calculated by the following formula.

[0040]

[0041] The preliminary screening results showed that there were 11 substances that had an inhibitory effect on the mycelial growth of Aspergillus oryzae, accounting for 55% of the total number of samples (such as Figure 1 There are only three metabolites with an inhibition rate of more than 70%, namely pterostilbene, isoliquiritigenin, and biochanin A, accounting for 15% of the total number of samples tested. Among them, isoliquiritigenin has an inhibition rate of 100% on the growth of Aspergillus oryzae hyphae, that is, it has a very strong inhibitory effect on the growth of Aspergillus oryzae hyphae, which is significantly different from other secondary metabolites. The results are shown in Figure 1 .

[0042] (2) To further explore the inhibitory effect of isoliquiritigenin on common rice pathogens, suitable culture media for three common fungal diseases were prepared: PSA medium (rice smut fungus): 200 g potato, 20 g sucrose, 15 g agar, and water to 1 L; PDA medium (rice sheath blight fungus): 200 g potato, 20 g glucose, 15 g agar, and water to 1 L; TOA medium (rice blast fungus): 150 mL tomato juice, 30 g oatmeal, 15 g agar, and water to 1 L.

[0043] The isoliquiritigenin solution was mixed with three culture media to a final concentration of 50 mg / L. Finally, the bacterial cakes of the same size were inoculated on the plate, and the same amount of DMSO solvent was used as the control. The rice blast pathogen, rice blast pathogen, and sheath blight pathogen were cultured at 28℃ for 14 days, 7 days, and 3 days, respectively, and the inhibition rate was recorded and calculated.

[0044] The results are shown in Table 1 and Figure 2 As shown, the inhibition rate of isoliquiritigenin on the pathogen of rice false smut is 100%, which is much better than the inhibition effect on the pathogens of rice sheath blight and rice blast. That is, in the application of fungal disease prevention and control, isoliquiritigenin is a specific fungicide for the pathogen of rice false smut, rather than a broad-spectrum antibacterial agent for rice.

[0045] Table 1: Antibacterial activity of isoliquiritigenin against three major fungal diseases of rice

[0046]

[0047] Example 2: Concentration screening test

[0048] Taking isoliquiritigenin as the object, the relationship between concentration and inhibitory effect was further explored: PSA culture medium with different concentrations of isoliquiritigenin was prepared with a concentration of 12.5-100 mg / L, and DMSO was used as the control group. The results are shown in Table 2. There is a significant positive correlation between the inhibitory effect of isoliquiritigenin and the concentration. Among them, when the concentration of isoliquiritigenin is greater than or equal to 50 mg / L, its inhibitory effect on rice urticaria hyphae is as high as 100%. (The correlation coefficient and EC50 value are calculated from the test concentration of 12.5-22.5 mg / L of the same batch)

[0049]

[0050] Example 3: Subcellular observation of the effect of isoliquiritigenin on the mycelial morphology of Aspergillus oryzae

[0051] The present invention finds that the mycelium and the compound can be better and fully contacted during the shaking culture process of the liquid culture medium PSB, so the inhibition effect is better. In order to obtain sufficient mycelium, isoliquiritigenin is selected to be prepared into a concentration of 5 mg / L, and no isoliquiritigenin is used as a control. The rice koji cake is inoculated into the PDB culture medium containing 5 mg / L isoliquiritigenin and the PDB culture medium without isoliquiritigenin, and is placed in a shaking table at a temperature of 28°C and a rotation speed of 180r / min for 7 days.

[0052] Pick out 1-3 mycelial masses from the control and PDB culture medium with 5mg / L isoliquiritigenin and put them into a 2mL centrifuge tube containing 1mL 2.5% pentanediol fixative. The sample only needs to cover the bottom of the centrifuge tube. Rinse with phosphate buffer for 15min, repeat three times; after rinsing, fix with 1% osmic acid (2h), rinse with double distilled water for many times, dehydrate with 50-100% gradient ethanol solution, each time for 20min; after each sample is dehydrated, treat with 100% acetone for 20min, soak with Spurr resin, embed at 70 degrees for 16h, and make ultra-thin sample slices (60-70nm); finally, staining is required, and the sample is stained with lead citrate and 50% ethanol saturated solution of uranyl acetate in turn, and the time is 15min; after sufficient staining, the subcellular structure of the mycelial cells of Aspergillus oryzae can be observed and photographed using transmission electron microscopy.

[0053] The results are as follows Figure 3As shown, transmission electron microscopy observations found that in the control group, the hyphae had regular morphology, intact internal cell walls, nuclei, and mitochondria, clear nuclear membranes and nucleoli, and regular cells. In the isoliquiritigenin-treated group, the hyphae had different morphologies and thicknesses, incomplete internal organelles, no complete mitochondria and complete nuclei could be seen, and it was suspected that they had dissolved and the contents were chaotic. This shows that isoliquiritigenin can affect the external morphology of rice koji hyphae and destroy internal subcellular structures such as organelles and nuclei, thereby inhibiting the growth of rice koji hyphae.

[0054] Example 4: Potted plant test

[0055] The seeds of Late Indica 98 rice were sterilized with 1 / 1000 potassium permanganate solution, germinated at 37°C for three days, and then transferred to indoor potting soil for further cultivation.

[0056] The rice spores were cultured with 50 mL PSB at 27 °C for 7-8 days until the spore concentration reached 2 × 10 6 / mL, and use a juicer to break up the mycelium to prevent clogging of the syringe.

[0057] At the 7th to 8th stage of rice panicle differentiation, 3 ml of a mixture of isoliquiritigenin and mycelial spores of rice smut pathogen (final concentration 50 mg / L) was injected. After inoculation, the rice was transferred to a greenhouse at 28±2℃ and 95±5% RH for 16 days, and then moved to a greenhouse at 28±2℃ and RH90±5% for further cultivation. The number of rice spores per panicle was observed and counted on the 21st day after inoculation. Each group treated 9 panicles of rice, the experiment was repeated twice, and an equal amount of DMSO solvent was used as a control. The results are shown in Figure 4 and Figure 5 As shown, compared with the control, the mycelium-spore mixture treated with YGCS completely lost its infection ability and did not form rice balls.

[0058] Example 5: Field trial

[0059] 300 mg of isoliquiritigenin was dissolved in DMSO and mixed with 6 L of water to a final concentration of 50 mg / L. 2 mL of nano-silica hydrogel (provided by Wuhan Zhiyun Biotechnology Co., Ltd.) was added per 1.5 L.

[0060] In early August, during the rice heading stage, three replicates were set up with an area of ​​18 square meters per plot, and spraying water was used as a control. The spraying volume in each plot was 1.5 liters.

[0061] The survey was conducted and the results were counted in late September using a five-point sampling method.

[0062] Result calculation: The number of rice balls is graded as follows: 1 is grade 1, 2 are grade 3, 3-5 are grade 5, 6-9 are grade 7, and 10 and above are grade 9. The disease index is calculated according to the following formula, and the average of the three repeated disease indexes is taken as the final result to calculate the disease index control effect.

[0063]

[0064] Table 3: Field test results

[0065]

[0066] The results are shown in Table 3, and the actual control effect of isoliquiritigenin in the field is as high as 80.53%. In summary, the present invention has discovered the inhibitory effect of isoliquiritigenin on rice false smut fungi, and verified it through potted and field experiments. Therefore, isoliquiritigenin can be used as a botanical fungicide for preventing and controlling rice false smut, which not only has excellent control effect, but also can reduce production costs and environmental pollution risks, and has extremely high industrial value.

[0067] The above specific embodiments describe the implementation of the present invention in detail, but the present invention is not limited to the specific details in the above embodiments. Within the scope of the claims and technical concept of the present invention, the technical solution of the present invention can be modified and changed in many simple ways, and these simple modifications all belong to the protection scope of the present invention.

Claims

1. A plant-derived fungicide for controlling rice false smut, characterized in that: The main active ingredient is isoliquiritigenin, and the concentration of the isoliquiritigenin is not less than 12.5 mg / L.

2. The plant-derived fungicide for rice false smut according to claim 1, characterized in that: In practical application, the concentration of isoliquiritigenin is not less than 50 mg / L, preferably 50-100 mg / L.

3. The botanical fungicide according to claim 2, characterized in that It also includes nano silicon hydrogel supplemented with an auxiliary agent.

4. The method for preparing the botanical fungicide for controlling rice false smut according to any one of claims 2 to 3, characterized in that: Isoliquiritigenin was dissolved in DMSO, mixed with water, and nano-silicon hydrogel was added to finally prepare a plant-based fungicide for rice smut.

5. The method according to claim 4, characterized in that 300-600 mg of isoliquiritigenin is dissolved in 3-6 mL of DMSO, mixed with 6-12 L of water, and 4.5-9 mL of nano-silicon hydrogel is added to finally prepare a rice false smut botanical fungicide with an isoliquiritigenin concentration of 50-100 mg / L.

6. Use of isoliquiritigenin in any of the following: A1) Control of rice false smut, A2) preparing a product for preventing and controlling rice false smut, A3) inhibit the growth of rice smut pathogen, A4) preparing a product for inhibiting the growth of U.S. smut pathogen.

7. A method for preventing and controlling rice false smut, characterized in that: The plant-derived fungicide according to any one of claims 2 to 3 or the plant-derived fungicide prepared by the method according to any one of claims 4 to 5 is used to prevent and control rice false smut.

8. The method according to claim 7, characterized in that The control method is spraying botanical fungicide during the rice booting stage.

9. Use of the botanical fungicide according to any one of claims 1 to 3, or the botanical fungicide obtained by the method according to any one of claims 4 to 5, or the method according to any one of claims 7 to 8 in preventing and controlling rice false smut.

Images