A plant-derived fungicide based on isofraxidin for rice sheath blight

Abstract

The present application relates to a kind of based on isoglycyrrhizin's rice sheath blight plant source fungicide.The present application is through in-depth study the biological activity of plant source secondary metabolite, mechanism of action, mainly for rice sheath blight, develops a kind of new biological pesticide of efficient, environmental protection, safe and cost controllable, through scientific proportioning and formula optimization, significantly improves its field application effect and stability, simultaneously reduces production cost and environmental pollution risk, provides strong support for the sustainable development of agriculture and safeguard food security.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of plant disease control, and particularly relates to a plant-derived fungicide for rice smut based on isofraxidin. BACKGROUND

[0002] Rice smut is one of the main fungal diseases on important food crops rice, which is caused by the infection of rice green core bacteria on the panicle of rice to form rice smut balls. In recent years, the incidence of rice smut has been increasing year by year; it is estimated that the occurrence area of rice smut in China will reach 40 million mu in 2024. In addition to yield loss, oryzacins can cause animal cell pathology and deformity, which seriously threatens food security. At present, chemical pesticides have been the main means for controlling rice smut, and their high efficiency and rapid bacteriostatic properties have provided an important guarantee for crop yield increase. However, with the increasing attention 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 explore more green and sustainable strategies for controlling rice smut.

[0003] There are many types of fungicides and biological pesticides on the current market, among which plant-derived secondary metabolites have gradually become a research hotspot due to their natural, degradable and relatively low toxicity characteristics. These substances are derived from plants and produced through complex biosynthetic pathways, and have a wide range of biological activities such as antibacterial and antiviral properties, providing a rich resource for the development of biological pesticides. For example, Saxifraga extract and other plant-derived fungicides have shown certain application potential.

[0004] Problems and deficiencies in the prior art:

[0005] 1. Limited fungicidal effect: Some existing plant-derived fungicides such as Saxifraga extract have certain antibacterial effect, but compared with chemical pesticides, the antibacterial effect is lower, only reaching 10%-50% of that of chemical pesticides, and cannot completely replace chemical pesticides.

[0006] 2. Environmental problems: Some existing fungicidal compositions, such as those containing chemical pesticides such as cinnamycin and fluazinam, can achieve good fungicidal effect, but they are compositions of chemicals, which are not environmentally friendly and 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 can lead to increased drug resistance of pathogens, and continuous use of chemical reagents can also cause environmental pollution, and residues of chemical agents on crops may affect human health through the food chain.

[0008] 4. Plant secondary metabolites related research: there are few studies on the effect of plant secondary metabolites on Ustilaginoidea virens. Although some researchers have studied the inhibitory activity of plant secondary metabolites on bacterial diseases of rice, no one has studied the three major fungal diseases of rice, especially Ustilaginoidea virens, and few have studied the mechanism in depth. SUMMARY

[0009] In view of the many problems and deficiencies in the prior art, the present application aims to develop a new type of biological pesticide with high efficiency, environmental protection, safety and controllable cost by in-depth study of the biological activity and mechanism of plant secondary metabolites. The pesticide is mainly aimed at Ustilaginoidea virens, and through scientific proportioning and formula optimization, the field application effect and stability are significantly improved, the production cost and environmental pollution risk are reduced, and strong support is provided for the sustainable development of agriculture. The creation of the present application 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 present application aims to provide a plant source fungicide for fungal diseases of rice, which is derived entirely from plants and does not cause any harm to plants, and is not easily residual, and is a highly efficient and safe plant source fungicide.

[0011] The present application provides a plant source fungicide for rice smut, which takes glycyrrhizin as the main active ingredient, and the concentration of glycyrrhizin is not less than 12.5 mg / L.

[0012] Further, in actual application, the concentration of glycyrrhizin is not less than 50 mg / L, preferably 50-100 mg / L.

[0013] Further, in field application, it also includes adding an auxiliary agent, nano-silicon hydrogel.

[0014] The present application also provides a preparation method of the above-mentioned plant source fungicide for Ustilaginoidea virens, which dissolves glycyrrhizin with DMSO, then mixes with water, and adds nano-silicon hydrogel, and finally configures into a plant source fungicide for rice smut.

[0015] Further, 300-600 mg of glycyrrhizin is dissolved in 300-600 mL of DMSO, then mixed with 6-12 L of water, and 4.5-9 mL of nano-silicon hydrogel is added, and finally configured into a plant source fungicide for rice smut with a glycyrrhizin concentration of 50-100 mg / L.

[0016] The present application also provides the use of glycyrrhizin in any of the following:

[0017] A1) preventing and treating rice smut,

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

[0019] A3) inhibiting the growth of Ustilaginoidea virens,

[0020] A4) preparing a product for inhibiting the growth of Ustilaginoidea virens.

[0021] The present application also provides a method for preventing and treating Ustilaginoidea virens, characterized in that the Ustilaginoidea virens is prevented and treated by using the plant source fungicide as described above or the plant source fungicide prepared by the method as described above.

[0022] Further, the prevention and treatment is spraying the plant source fungicide at the booting stage of the rice.

[0023] The plant source fungicide as described above or prepared by any of the methods as described above or any of the methods as described above in the application of preventing and treating Ustilaginoidea virens.

[0024] Beneficial effects:

[0025] 1. The product is a plant source fungicide, and the main component, isoglycyrrhizin, has general inhibitory effect on Magnaporthe oryzae and Helminthosporium oryzae, but has good inhibitory activity on the mycelial growth of Ustilaginoidea virens.

[0026] 2. The product also plays a good effect in preventing and treating Ustilaginoidea virens in indoor pot experiments and field experiments, is green and environmentally friendly, has wide sources and is not easy to remain, has little effect on non-target organisms, has high safety, reduces chemical pesticide pollution, and promotes the recovery of the fertility of cultivated land in China.

[0027] 3. The main component of the product, isoglycyrrhizin, can destroy the subcellular structure of Ustilaginoidea virens, cause the content to be chaotic, and kill the Ustilaginoidea virens. BRIEF DESCRIPTION OF DRAWINGS

[0028] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings needed in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present application, and therefore should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can also be obtained without creative labor on the basis of these drawings.

[0029] Figure 1 : Inhibition of mycelial growth of Ustilaginoidea virens by 11 plant secondary metabolites.

[0030] Figure 2 : Antibacterial effect of isoglycyrrhizin on three major disease pathogens of rice: A1: DMSO control + Ustilaginoidea virens, A2: isoglycyrrhizin + Ustilaginoidea virens; B1: DMSO control + Helminthosporium oryzae, B2: isoglycyrrhizin + Helminthosporium oryzae; C1: DMSO control + Magnaporthe oryzae, C2: isoglycyrrhizin + Magnaporthe oryzae.

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

[0032] Figure 4 Statistical results of the pot experiment: isofraxidin (YGCS) group, control (CK) group, and DMSO group.

[0033] Figure 5 Photos of the pot experiment results: from left to right are HWD-2 (rice smut pathogenic strain), i.e. the CK group, the DMSO solvent group, and the isofraxidin treatment group. DETAILED DESCRIPTION

[0034] The following examples are only used to more clearly illustrate the technical solutions of the present application, and thus are only examples and cannot be used to limit the protection scope of the present application. It should be noted that, unless otherwise specified, the technical terms or scientific terms used in the present application should be understood as the common meanings understood by the skilled in the art to which the present application belongs. Unless otherwise specified, the reagents, methods, and devices used in the present application are the common reagents, methods, and devices in the art. Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0035] The purpose of the present application is to provide a plant source fungicide for rice fungal diseases, the effective component of which is derived from plants, does not cause any harm to plants, and is not easy to remain.

[0036] The rice variety selected in the experiment of the present application is late indica 98, which is a susceptible variety.

[0037] Example 1: Screening of plant source secondary metabolites with inhibitory effect on the growth of rice smut hyphae

[0038] (1) The present application screens the following 20 plant source secondary metabolites: wogonin, ginsenoside Rd, paeoniflorin, swertiamarin, biochanin A, pterostilbene, isofraxidin, crocin I, luteolin, magnolol, apigenin, chrysin, hesperidin, mangiferin, demethoxycurcumin, bakuchin, subaconitine, podophyllotoxin, and atractylodin, ginsenoside Rh1 (S type), wherein the CAS Number of isofraxidin is 961-29-5.

[0039] The screening detection method is mycelial growth rate method (all tests are carried out on the clean bench): first, the secondary metabolites are mixed with DMSO to prepare a 100 mg / L drug-containing medium PSA, and a medium containing only an equal amount of solvent is used as a control group, and after ensuring sufficient mixing and uniformity, pour into 3 sterilized culture dishes, use forceps to take a new and active rice fungus cake with a diameter of 0.5 cm and inoculate it into the culture dish, and place it in a 28±1 degree biochemical incubator for constant temperature incubation. After 14 days of growth of the rice fungus, the diameters of the colonies of the drug group and the DMSO control group are measured using the cross method, in units of mm, with three replicates for each treatment. The inhibition rate of isoliquiritigenin on mycelial growth is calculated by the following formula.

[0040]

[0041] The preliminary screening results show that there are 11 substances that inhibit the growth of mycelium of the rice fungus, accounting for 55% of the total number of samples (as shown in Figure 1 ); only three secondary metabolites have an inhibition rate of more than 70%, namely pterostilbene, isoliquiritigenin, and biochanin A, accounting for 15% of the total number of test samples. Among them, isoliquiritigenin has a 100% inhibition rate on the growth of mycelium of the rice fungus, i.e., it has a very strong inhibitory effect on the growth of mycelium of the rice fungus, which is significantly different from other secondary metabolites, and the results are shown in Figure 1 .

[0042] (2) Further explore the inhibitory effect of isoliquiritigenin on common pathogenic fungi of rice, and prepare three suitable media for common fungal diseases respectively: PSA medium (rice fusarium disease): 200 g of potato, 20 g of sucrose, 15 g of agar, and water to 1 L; PDA medium (rice sheath blight fungus): 200 g of potato, 20 g of glucose, 15 g of agar, and water to 1 L; TOA medium (rice blast fungus): 150 mL of tomato juice, 30 g of oatmeal, 15 g of agar, and water to 1 L.

[0043] Mix isoliquiritigenin solution with the three media respectively, with a final concentration of 50 mg / L, and finally inoculate the uniform fungus cake on the plate with an equal amount of DMSO solvent as a control. The rice fusarium, rice blast fungus, and sheath blight fungus are cultured at 28°C for 14d, 7d, and 3d respectively, and the inhibition rate is recorded and calculated.

[0044] The results are shown in Table 1 and Figure 2 , the inhibitory rate of isoliquiritigenin on the rice fusarium pathogen is 100%, which is much better than the inhibitory effect on the rice sheath blight and blast pathogen, i.e., in the application of fungal disease control, isoliquiritigenin is a specific fungicide for the rice fusarium pathogen, but not a broad-spectrum fungicide for rice.

[0045] Table 1: Inhibitory activity of isoliquiritigenin on three major fungal diseases of rice

[0046]

[0047] Example 2: Concentration screening test

[0048] With isoflavone as the object, the relationship between concentration and inhibition effect was further explored: isoflavone PSA medium with different concentrations of 12.5-100 mg / L was prepared, and equal solvent DMSO was used as the control group. The results are shown in Table 2, and there is a clear positive correlation between the inhibition effect of isoflavone and the concentration, and when the concentration of isoflavone is greater than or equal to 50 mg / L, the inhibition effect on the mycelium of Ustilaginoidea virens is as high as 100%. (The correlation coefficient and EC50 value are calculated from the same batch of test concentrations 12.5-22.5 mg / L)

[0049]

[0050] Example 3: Subcellular observation of the effect of isoflavone on the mycelial morphology of Ustilaginoidea virens

[0051] The present application found that the mycelium and the compound can be better contacted in the shake culture process of the liquid medium PSB, so the inhibition effect is better. In order to obtain sufficient amount of mycelium, isoflavone is prepared at a concentration of 5 mg / L, and isoflavone is not added as a control. The Ustilaginoidea virens cake is inoculated into PDB culture solution containing 5 mg / L isoflavone and PDB culture solution without isoflavone, and placed in a 28°C, 180 r / min shaker for 7 days.

[0052] 1-3 mycelial masses are picked from the control and PDB culture solution with a concentration of 5 mg / L of isoflavone and placed in a 2 mL centrifuge tube containing 1 mL of 2.5% glutaraldehyde fixing solution. The sample only needs to cover the bottom of the centrifuge tube. Rinse with phosphate buffer for 15 min, repeat three times; after rinsing, fix with 1% osmium acid (2h), rinse with distilled water several times, dehydrate with 50-100% gradient ethanol solution, 20 min each time; after each sample is dehydrated, 100% acetone is treated for 20 min, Spurr resin is soaked, and embedding is performed at 70 degrees for 16h, ultra-thin sample sections (60-70 nm) are prepared; finally, staining is needed, and the sample is stained with lead citrate, 50% ethanol saturated solution of uranyl acetate, and the time is 15 min; after sufficient staining, the subcellular structure of the Ustilaginoidea virens mycelium cell can be observed and photographed by transmission electron microscopy.

[0053] The results are as follows Figure 3As shown, transmission electron microscopy observation found that in the control group, the hyphal morphology was regular, the internal cell wall, cell nucleus, mitochondria were complete, the nuclear membrane and nucleolus were clear, and the cells were regular. In the isofraxidin treatment group, the hyphal morphology was different, the internal organelle was not complete, the complete mitochondria and complete nucleus could not be seen, suspected to occur dissolution, and the content was chaotic. Therefore, isofraxidin can affect the external morphology of the rice smut fungus hypha and destroy the internal subcellular structures such as organelles and nuclei, thereby playing a role in inhibiting the growth of the rice smut fungus hypha.

[0054] Example 4: Pot experiment

[0055] The late indica 98 rice seeds were disinfected with a thousandth of potassium permanganate solution, and after 37°C germination for three days, they were transferred to indoor potting soil for further culture.

[0056] The rice smut fungus was shaken in 50 mL PSB at 27°C for 7-8 days until the spore concentration reached 2×10 6 The mycelium was broken up with a juicer to prevent the syringe from being blocked.

[0057] At the differentiation stage of the rice young ear, 3 ml of isofraxidin and rice smut fungus mycelium spore mixture (final concentration 50 mg / L) was injected, and after inoculation, the rice was transferred to a 28±2°C, 95±5% RH greenhouse for 16 days, then moved to a 28±2°C, RH 90±5% greenhouse for further culture. On the 21st day after inoculation, the number of rice smut balls per ear was observed and counted. Each group of treatment had 9 ears of rice, the test 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 Compared with the control, the mycelium spore mixture treated with isofraxidin (YGCS) completely lost the ability to infect and did not form rice smut balls.

[0058] Example 5: Field experiment

[0059] 300 mg of isofraxidin was dissolved in DMSO, and then mixed with 6 L of water to give a final concentration of 50 mg / L. 2 mL of nano-silicon hydrogel (provided by Wuhan Zirun Biological Technology Co., Ltd.) was added to each 1.5 L.

[0060] In early August, at the rice booting stage, three replicates were set up according to 18 square meters per plot, and water spraying was used as a control. The spraying amount for each plot was 1.5 liters.

[0061] The results were investigated and counted in late September according to the five-point sampling method.

[0062] Result calculation: according to the number of rice curved ball classification, 1 is 1 level, 2 rice curved ball is 3 level, 3-5 is 5 level, 6-9 is 7 level, 10 and above is 9 level. According to the following formula, the disease index is calculated respectively, and the average value of three repeated disease indexes is taken as the final result to calculate the disease prevention effect.

[0063]

[0064] Table 3: Field test prevention effect results

[0065]

[0066] The results are shown in Table 3, and the actual prevention effect of isofraxidin in the field is as high as 80.53%. In summary, the present application finds that isofraxidin has an inhibitory effect on rice curved bacteria, and is verified by potting and field experiments, so isofraxidin can be used as a plant source fungicide to prevent and control rice curved disease, not only has excellent prevention and control effect, but also can reduce production cost and environmental pollution risk, and has very high industrial value.

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

Claims

1. The use of isoliquiritigenin in any one of: A1) controlling rice smut, A2) preparing a product for controlling rice smut, A3) inhibiting the growth of Ustilaginoidea virens, A4) preparing a product for inhibiting the growth of Ustilaginoidea virens; wherein the concentration of isoliquiritigenin is 50-100 mg / L.

2. A method for controlling rice sheath blight, characterized by, A plant source fungicide is used to control rice smut, the plant source fungicide takes isoliquiritigenin as the main active ingredient, and the concentration of isoliquiritigenin is 50-100 mg / L.

3. The method of claim 2, wherein, The plant source fungicide also includes auxiliary nano-silica hydrogel.

4. The method of claim 2, wherein, The preparation method of the plant source fungicide comprises: dissolving isoliquiritigenin with DMSO, mixing with water, and adding nano-silica hydrogel, and finally configuring into a rice smut plant source fungicide.

5. The method of claim 4, wherein, 300-600 mg of isoliquiritigenin is dissolved in 3-6 mL of DMSO, then mixed with 6-12 L of water, and 4.5-9 mL of nano-silica hydrogel is added, and finally configured into a rice smut plant source fungicide with an isoliquiritigenin concentration of 50-100 mg / L.

6. The method of claim 2, wherein, The control is spraying the plant source fungicide at the rice panicle initiation stage.

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