A high-efficiency and broad-spectrum anti-plant virus disease agent composition and its application

Abstract

The present invention relates to a highly effective, broad-spectrum anti-plant viral disease pharmaceutical composition and its application. The active ingredients of the anti-plant viral disease pharmaceutical composition include chlorin iron and lentinan; wherein the weight ratio of chlorin iron to lentinan is 1:6000 to 4:1. When a certain concentration of the chlorin iron and lentinan pharmaceutical composition is applied to plants, the two active ingredients exert a synergistic effect, alleviating symptoms of diseased plants, reducing disease indexes, and reducing viral accumulation in the plants. The plant anti-viral disease pharmaceutical composition is a biological agent that is environmentally friendly, safe, and environmentally friendly. It has important application prospects in combating plant viral diseases, preventing and controlling plant diseases caused by plant viruses, and improving plant quality.

Description

Technical Field

[0001] The present invention relates to the technical field of agricultural pest control, and specifically to a highly effective and broad-spectrum anti-plant viral disease agent composition, and the application of the above-mentioned composition in preventing and treating plant viral diseases. Specifically, the active ingredients of the above-mentioned anti-plant viral disease agent composition include dihydrochlorin iron and lentinan. Background Art

[0002] Plant viral diseases are the second most common plant disease after fungal diseases, characterized by a wide variety of species, a wide host range, severe damage, and difficulty in prevention and control. Viral infections often cause severe declines in crop yield and quality, posing a significant threat to crop production safety. Adjustments in cropping structures and climate change have led to an increasing prevalence of plant viral diseases in my country, with sudden, explosive, and recurring outbreaks, severely impacting grain production and the security of the supply chain of important agricultural products. Currently, the prevention and control of plant viral diseases in production mostly relies on agricultural measures and disease-resistant varieties. However, the prevention and control of many plant viral diseases still faces prominent problems such as a lack of resistant varieties and the degeneration of resistance. Antiviral agents remain the ideal, most direct and effective means of preventing and controlling plant viral diseases. At present, there is an extreme shortage of pesticides for the prevention and control of plant viral diseases. Although there are some antiviral products on the market, such as guanidine hydrochloride, amino oligosaccharides, chlorfenapyr, and cinclorac, plant viruses are complex and infect a variety of crops. Registered antiviral products account for less than 0.5% of all registered pesticides. Therefore, the development of more efficient, broad-spectrum, and green anti-plant viral disease agents is still of great significance in agricultural development.

[0003] Iron chlorine, ISO common name: iron chlorine e6, is a green, loose powdered solid extracted from silkworm excrement. It is a new natural plant growth regulator that regulates crop growth. It is environmentally friendly and safe to use. Iron chlorine inhibits chlorophyllase, delaying chlorophyll degradation and enhancing photosynthesis. It can also promote root growth and enhance plant stress resistance. Currently, large-scale trials of iron chlorine have been conducted on crops such as rice, wheat, and rapeseed, demonstrating promising growth-promoting and yield-increasing effects. Recent research and experiments have shown that iron chlorine has excellent immune-inducing properties. By activating the plant immune system and regulating defense-related signaling and metabolic pathways, it can induce plant resistance to biotic and abiotic stresses, thereby improving plant disease and stress resistance. Previous studies have shown that iron chlorine can enhance crop resistance to various viruses. However, to date, only single-agent iron chlorine has been used to combat plant viral diseases, and its effectiveness has been suboptimal.

[0004] Lentinan is a fungal polysaccharide, an active ingredient extracted from high-quality shiitake mushroom fruiting bodies. It is the primary active ingredient and a host immune enhancer. Medical research has shown that lentinan has antiviral, anti-tumor, immune-modulating, and interferon-stimulating properties. In the pesticide field, lentinan is a safe and green fungicide that also has some inhibitory effects on plant viral diseases. Lentinan promotes protein synthesis in plants, making crops stronger and significantly enhancing their disease resistance. It has excellent wetting and permeability on plant surfaces, allowing it to be rapidly absorbed and degraded by plants. Lentinan is commonly used in the market as a plant growth regulator. There are also some documented uses of plant polysaccharides to combat plant viral diseases, but the effectiveness is limited.

[0005] While some plant virus disease control agents are currently available on the market, plant viruses are complex and infect a wide variety of crops, making chemical control the most effective and ideal method. However, the frequent occurrence of various viral diseases in production and the lack of relevant control agents hinder control of plant virus diseases. The development of new, highly effective, broad-spectrum antiviral agents and their combination compositions is of great significance for addressing the harm of plant virus diseases in the industry, improving the level of plant virus disease control in my country, and promoting the green and low-carbon development of the agricultural industry.

[0006] There is currently no effective solution to the demand for efficient, green, and broad-spectrum anti-plant viral disease agents in agricultural development, as well as the problem that the use of related technical agents is not ideal in treating plant viral diseases. Summary of the Invention

[0007] In order to overcome the shortcomings and deficiencies in the prior art, the present invention provides a highly efficient and broad-spectrum anti-plant viral disease agent composition, which uses dihydrochlorin iron and lentinan as active ingredients, and provides the application of the above-mentioned agent composition in resisting plant viral diseases (for example, tobacco mosaic virus, wheat yellow mosaic virus, etc.), so as to at least solve the problem in the prior art that the anti-plant viral disease effect of a single dose is not obvious, and provide a more efficient, broad-spectrum, green anti-plant viral disease agent for the field of pesticide technology.

[0008] To achieve the above object, the present invention adopts the following technical solutions:

[0009] The first aspect of the present invention is to provide an anti-plant viral disease pharmaceutical composition, wherein the active ingredients of the anti-plant viral disease pharmaceutical composition include dihydrochlorin iron and lentinan. Preferably, the active ingredients of the anti-plant viral disease pharmaceutical composition are composed of dihydrochlorin iron and lentinan.

[0010] Furthermore, the active component comprises 0.02 to 2 parts of dihydrochlorin iron and 0.5 to 60 parts of lentinan, that is, the weight ratio of dihydrochlorin iron to lentinan is 1:6000 to 4:1, preferably 1:3000 to 4:1, 1:1250 to 1:125, 1:1500 to 1:150 or 1:2500 to 1:250, etc., and more preferably 1:2000 to 1:100.

[0011] Furthermore, the active component accounts for 0.51% to 62% of the total weight of the composition, preferably 2.52% to 60.48%, and more preferably 0.67% to 24.01%.

[0012] Furthermore, the anti-plant virus disease composition further includes auxiliary ingredients, including at least one of a solvent, an emulsifier, a wetting agent, a dispersant, a stabilizer, an antifreeze agent, a thickener, a binder, a disintegrant, a defoaming agent, a penetrant, a polymeric capsule wall material, a synergist, a preservative, and a filler. It is understood that the aforementioned auxiliary ingredients are all commonly used or permitted ingredients in pesticide formulations and are not particularly limited. The specific ingredients and amounts can be adjusted and selected according to the formulation requirements.

[0013] Furthermore, the composition can be prepared in formulations including suspension concentrates, aqueous emulsions, microcapsule suspensions, wettable powders, water-dispersible granules, and microemulsions. It is understood that the anti-plant virus disease composition can be prepared in any suitable formulation suitable for agricultural use, and the formulation to be selected for specific use can be determined based on the intended use, usage environment, and other factors.

[0014] In a specific embodiment, the auxiliary components include β-cyclodextrin, distilled water (deionized water), etc. For example, when preparing a powder, β-cyclodextrin is used; when preparing a suspension, β-cyclodextrin and distilled water or only distilled water is used.

[0015] In one embodiment, the composition is prepared in the form of a powder, and the preparation steps include: uniformly mixing a predetermined amount of dihydrochlorin iron (mother drug) and auxiliary ingredients, and then adding a predetermined amount of Lentinan powder and mixing them uniformly.

[0016] In a specific embodiment, the preparation form of the composition is a suspension, and its preparation steps include: mixing a predetermined amount of dihydrochlorin iron (mother drug) with auxiliary ingredients to form component A; mixing a predetermined amount of lentinan (mother drug) with distilled water, stirring and dissolving to form component B, and mixing components A and B according to a predetermined weight ratio and stirring evenly.

[0017] The second aspect of the present invention is to provide an application of any pharmaceutical composition described in the first aspect of the present invention, wherein the application is selected from at least one of the following applications: application in inhibiting plant virus replication, application in resisting plant viral diseases, application in preventing and controlling plant diseases caused by the plant viruses, and application in improving plant quality.

[0018] Furthermore, in the above application, the plant viruses include tobacco mosaic virus and wheat yellow mosaic virus.

[0019] Furthermore, in the above application, the tobacco mosaic virus includes tomato mottle mosaic virus.

[0020] Furthermore, in the above application, the plant diseases include tobacco mosaic disease, wheat yellow mosaic disease, and tomato virus disease.

[0021] Furthermore, in the above application, improving plant quality includes improving tomato quality.

[0022] Furthermore, in the above application, the pharmaceutical composition inhibits the replication of plant viruses, alleviates viral symptoms of plants, and alleviates brown spot symptoms on fruits.

[0023] Furthermore, in the above application, the anti-plant virus disease pharmaceutical composition is used after dilution, and the specific dilution multiple can be: 100~4000 times. The above dilution multiple can be adaptively adjusted according to the content of each active ingredient in the prepared pharmaceutical composition.

[0024] Furthermore, in the above application, the anti-plant virus disease agent composition is applied by spraying it onto the leaves or fruit surfaces of the plants, with the number of spraying being no less than 2 times, and the interval between each spraying being 3 to 10 days. The application concentration is preferably 0.05 to 0.2 mg / L of dihydrochlorin iron and 16 to 100 mg / L of lentinan. The above-mentioned number of spraying and application concentration can be adaptively adjusted according to the different plant viruses used for prevention and control.

[0025] The present invention adopts the above technical solution, which has the following technical effects compared with the prior art:

[0026] The present invention provides an effective active ingredient for the anti-plant virus disease composition of chlorin iron and lentinan, chlorin iron and lentinan are both natural compounds, belong to biopesticides, are environmentally friendly, safe and environmentally friendly, meet the requirements of green and low-carbon development of the agricultural industry, and have good prospects. Compared with a single dose, the composition to which the present invention relates is composed of two effective ingredients chlorin iron and lentinan with different mechanisms of action, which is conducive to giving full play to their respective antiviral functions, and chlorin iron and lentinan have obvious synergistic effects, improve the viral disease prevention and control effect, have broad-spectrum resistance to plant viral diseases, and are suitable for the prevention and control of various types of crop viral diseases. The present invention provides an efficient and broad-spectrum pharmaceutical composition, which embodies significant disease resistance in anti-plant viral diseases, and its prevention and control method is simple, economical, green, efficient, and can be directly applied to agricultural production and related research fields, more efficiently and environmentally friendly to ensure the healthy growth of crops, and provide a good solution for the anti-plant viral disease problem in agricultural development. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 These are the symptoms of plants treated with a chlorin iron•lentinan composite composition (partial concentrations) after inoculation with Tomato Mottle Mosaic Virus (ToMMV) in one embodiment of the present invention; wherein, 1 is a healthy plant; 2 is a plant treated with clear water as a control; 3 is a plant treated with a 1000-fold dilution of 0.02% chlorin iron soluble powder; 4 is a plant treated with a 200-fold dilution of 1% lentinan aqueous solution; 5 is a plant treated with a 167-fold dilution of 0.837% chlorin iron•lentinan suspension; and 6 is a plant treated with a 2609-fold dilution of 13.06% chlorin iron•lentinan powder.

[0028] Figure 2 The present invention shows the symptoms of the fruits of tomato virus disease plants in the field after applying the combination of dihydrochlorin iron and lentinan in one embodiment of the present invention; among them, 1-2 are fruits of the pure water control; 3 is the fruit of the fruit using 3333 times of 0.02% dihydrochlorin iron soluble powder; 4 is the fruit using 200 times of 1% lentinan aqueous solution; 5-7 are the fruits using 2609 times of 13.06% dihydrochlorin iron and lentinan powder. DETAILED DESCRIPTION

[0029] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. It is obvious that the described embodiments are only some of the embodiments of the present invention, and are not all of the embodiments. All other embodiments obtained by persons of ordinary skill in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention. The experimental methods in the following examples, for which specific conditions are not specified, are generally measured in accordance with national standards. The experimental materials in the following examples, for which the sources are not specified, are all commercially available raw materials. The equipment used in each step of the following examples is conventional equipment. If there are no corresponding national standards, the steps are carried out in accordance with general international standards, conventional conditions, or conditions recommended by the manufacturer. Unless otherwise specified, all parts are by weight, and all percentages are by mass percentages. Unless otherwise defined or specified, all professional and scientific terms used in the present invention have the same meanings as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein can be applied to the methods of the present invention.

[0030] It should be noted that, in the absence of conflict, the embodiments and features of the embodiments of the present invention can be combined with each other. The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but they are not intended to limit the present invention.

[0031] The tomato mottle mosaic virus ( 番茄斑驳花叶病毒 The ToMMV (ToMMV) isolate was collected from diseased tomato samples in Shenyang, Liaoning Province, by the inventor's laboratory in 2015. The virus isolate was purified by infectious cloning and named ToMMV-LN (Genbank accession number: MN853592); dihydrochlorin iron (mother drug, 2%) was purchased from Anqing Baxter Bioengineering Co., Ltd., lentinan powder (60%) was purchased from Xi'an Ruihe Bioengineering Technology Co., Ltd., and lentinan (mother drug, 10%) was purchased from Shandong Shengpeng Technology Co., Ltd.

[0032] In certain specific embodiments of the present invention, the active ingredients of the anti-plant viral disease agent composition include dihydrochlorin iron and lentinan (specifically, composed only of dihydrochlorin iron and lentinan), and the composition of the above active ingredients is 0.02-2 parts of dihydrochlorin iron and 0.5-60 parts of lentinan, preferably the weight ratio of dihydrochlorin iron to lentinan is 1:2000-1:100; the above active ingredients account for 0.51%-62% of the total weight of the composition, preferably 0.67%-24.01%; when prepared into different dosage forms, the above anti-plant viral disease agent composition also includes auxiliary ingredients commonly used in the art, including but not limited to: solvents, emulsifiers, wetting agents, dispersants, stabilizers, antifreeze agents, thickeners, binders, disintegrants, defoaming agents, penetrants, polymer capsule wall materials, synergists, preservatives and fillers, etc.

[0033] The following is an illustrative description of the anti-plant virus disease pharmaceutical composition and its application provided by the present invention.

[0034] Example 1

[0035] This example describes the preparation of a preferred anti-plant viral disease agent composition, comprising a 13.056% chlorin iron and lentinan powder. The raw materials, by weight, consist of: 0.016% chlorin iron, 13.04% lentinan, and the remainder, the auxiliary ingredient, β-cyclodextrin. The process steps are as follows: 0.78 parts chlorin iron (2% parent drug) and 77.48 parts β-cyclodextrin are uniformly mixed in a powder mixer. Then, 21.74 parts lentinan powder (60%) are added and mixed uniformly in a powder mixer to produce the finished product.

[0036] Example 2

[0037] This example describes the preparation of a preferred anti-plant viral disease agent composition. The composition comprises an 8.588% chlorin iron and lentinan powder. The raw materials are proportioned by weight as follows: chlorin iron: 0.017%; lentinan: 8.571%; and the remainder is the auxiliary ingredient, β-cyclodextrin. The process steps are the same as in Example 1.

[0038] Example 3

[0039] This example describes the preparation of a preferred anti-plant viral disease agent composition, comprising a 0.945% chlorin iron and lentinan suspension concentrate. The raw materials, by weight, are as follows: chlorin iron: 0.015%; lentinan: 0.93%; β-cyclodextrin (excipient): 7.39%; and distilled water to 100%. The process steps are as follows: 0.074 parts chlorin iron (2% parent drug) and 7.33 parts β-cyclodextrin are mixed in a powder mixer to produce a 0.02% chlorin iron powder (A). Simultaneously, 9.26 parts lentinan (10% parent drug) and 83.33 parts distilled water are mixed and stirred to dissolve the mixture into a 1% solution (B). A and B are then mixed and stirred until uniformly distributed (the weight ratio of A to B is 1:12.5). This results in the finished product.

[0040] Example 4

[0041] This example describes the preparation of a preferred anti-plant viral disease agent composition. The composition is used to prepare a 0.8333% chlorin iron and lentinan suspension concentrate. The raw materials, by weight, are as follows: chlorin iron: 0.0033%; lentinan: 0.83%; β-cyclodextrin (excipient): 16.67%; and distilled water: made up to 100%. The process steps are as follows: 0.17 parts chlorin iron (the parent drug) and 16.5 parts β-cyclodextrin are uniformly mixed in a powder mixer to produce a 0.02% chlorin iron powder (A). Simultaneously, 8.33 parts lentinan (the parent drug, 10%) are mixed with 75 parts distilled water and stirred to dissolve, producing a 1% solution (B). A and B are then mixed and stirred until uniformly distributed (A:B weight ratio: 1:5) to produce the finished product.

[0042] Example 5

[0043] This embodiment is a composition of some preferred anti-plant virus disease agents. The process steps thereof refer to Examples 1 to 4, and the component contents are shown in the following table:

[0044]

[0045] Example 6 - Indoor Experiment on Control of Tomato Mottle Mosaic Virus with the Compound Composition

[0046] In this example, the tomato mottle mosaic virus (TMV) is a member of the genus Tobacco Mosaic Virus. 番茄斑驳 花叶病毒 , ToMMV) infected tobacco system indoors to evaluate the efficacy of the compound composition in controlling plant viral diseases.

[0047] 1. Preparation of test agents

[0048] The reagents prepared in Example 1 and Example 4 were diluted according to the following ratios and methods to prepare test reagents of different concentrations:

[0049] (1) 13.056% chlorin iron·Lentinan powder 1304 times diluted (diluted and dissolved in water in proportion, the same below)

[0050] (2) 13.056% chlorin iron·Lentinan powder 1957 times diluted;

[0051] (3) 13.056% chlorin iron·Lentinan powder 2609 times diluted;

[0052] (4) 13.056% chlorin iron·Lentinan powder 3130 times diluted;

[0053] (5) 167 times dilution of 0.8333% chlorin iron·Lentinan suspension concentrate;

[0054] (6) 333 times dilution of 0.8333% chlorin iron·Lentinan suspension;

[0055] (7) 500 times dilution of 0.8333% dihydrochlorin iron·Lentinan suspension;

[0056] (8) 1000-fold dilution of 0.02% chlorin iron soluble powder (0.2 mg / L);

[0057] (9) 3333 times dilution of 0.02% chlorin iron soluble powder (0.06 mg / L);

[0058] (10) 200 times dilution of 1% Lentinan aqueous solution (50 mg / L);

[0059] (11) Clean water.

[0060] 2. Acquisition of plant materials

[0061] The tobacco used in the experiment was Nicotiana benthamiana ( 本氏烟 ), the plant acquisition method and steps are as follows:

[0062] Germination: Sprinkle the seeds evenly on the surface of moist soil (vermiculite and nutrient soil mixed in a ratio of 3:1) and place them in a 28℃ plant culture room for seed germination.

[0063] Transplanting: After 7 days, take a plastic cup and fill it with soil. Move the tobacco seedlings to the center of the cup and flatten the surrounding soil. Transplant one seedling per cup. Place the plastic cup in a water tray and allow the soil to absorb water through the holes at the bottom of the cup.

[0064] Tobacco seedlings were grown in a 28°C plant culture room with a photoperiod of 16 h light and 8 h dark. They were watered once 7 days before the 4-leaf stage and once 4 days thereafter. Nutrient solution was replenished every other week.

[0065] 3. Virus preparation and inoculation methods

[0066] The ToMMV isolate used in the experiment was collected by the inventor's laboratory from diseased tomato samples in Shenyang, Liaoning Province in 2015. The virus isolate was purified by infectious cloning preparation and named ToMMV-LN (Genbank accession number: MN853592).

[0067] Tobacco plants were inoculated with ToMMV using the Agrobacterium infiltration method. Approximately one month after sowing, tobacco plants with three to four large leaves and no flowers were selected for infiltration. Laboratory-prepared Agrobacterium containing a ToMMV infectious clone was cultured with shaking until the OD value reached 0.8-1.2. The cells were harvested by centrifugation at 6000 g for 6 minutes, and the infiltration solution was added to adjust the OD value to 0.6. After standing at room temperature for 2 hours, the underside of a thick, flat leaf was infiltrated using a needleless syringe, with 0.5 mL of infiltration solution applied to each tobacco plant.

[0068] 4. Pesticide treatment

[0069] Two days after the tobacco plant was inoculated with the virus, different concentrations of the test agent were evenly sprayed onto the tobacco leaves. Five days later, the second application was carried out, for a total of two applications. A control was sprayed with water using the same method and timing. Each treatment group consisted of 10 tobacco plants, and the treatment was repeated three times.

[0070] 5. Plant symptom record and prevention effect calculation

[0071] Three days after the second application of the pesticide (i.e., 10 days after inoculation), tobacco plant symptoms were observed in each group. The severity of disease on the top five or six leaves of each plant was recorded, and the disease index was calculated to determine the control efficacy. The Duncan's new multiple range method using the DPS data processing system was used to compare the efficacy of different treatments against viral diseases. The data recorded are shown in Table 1.

[0072] The grading standards are as follows:

[0073] Level 0: no mosaic lesions;

[0074] Level 1: The lesion area accounts for less than 5% of the entire leaf area;

[0075] Level 3: The lesion area accounts for 5.1-15% of the entire leaf area;

[0076] Level 5: The lesion area accounts for 15.1-25% of the entire leaf area;

[0077] Level 7: The lesion area accounts for 25.1-50% of the entire leaf area;

[0078] Level 9: The lesion area accounts for more than 50.1% of the entire leaf area.

[0079] Calculation formula for disease index and disease index prevention effect:

[0080]

[0081] 6. Detection of virus accumulation in plants

[0082] Three days after the second drug application to each group of tobacco plants (i.e., 10 days after inoculation), the tobacco leaves of each treatment group that had been inoculated as described above were taken, with 4 leaves from each treatment group taken as 4 replicates.

[0083] Total RNA was extracted from leaves using the Trizol method and reverse-transcribed to synthesize first-strand cDNA. Quantitative PCR was then performed to detect the accumulation of ToMMV in the plants. The ToMMV CP gene was used as the target, and the Nicotiana benthamiana UBC gene was used as the internal control. The primers used were as follows: qToM-cp-F: 5'-CATTGCTGGGAACTTTCGAT-3', qToM-cp-R: 5'-CAGGCCAACCCAGACATACT-3', qUBC-F: 5'-TTTCGGTCCTGATGATACT CCC-3', qUBC-R: 5'-CACAGAGCAAAGACTGGA TTGA-3'. The reaction system (20 μL) consisted of 10 μL 2×SYBR Green PCR Master Mix, 1 μL cDNA, 1 μL upstream primer, 1 μL downstream primer, and 7 μL ddH2O. The reaction conditions were pre-denaturation at 95°C for 30 seconds, followed by 40 cycles of denaturation at 95°C for 5 seconds and annealing at 60°C for 15 seconds. Melting curve analysis was performed from 60°C to 95°C in increments of 0.1°C / second. After obtaining the Ct values ​​for each sample, the relative levels of the CP gene in each treatment group were calculated using the 2-ΔΔCt method. The accumulation of ToMMV in tobacco leaves from each treatment group was determined using the above assay method. The results are shown in Table 1.

[0084] 7. Prevention results

[0085] Table 1 Indoor combined efficacy of chlorin iron and lentinan against ToMMV virus

[0086]

[0087] According to the disease index, disease prevention effect, and ToMMV accumulation in different groups of tobacco plants recorded and counted in Table 1, data comparison found that: compared with the water control group, it can be seen that if no drug was given, the disease index of tobacco leaves was 87.72%, and the disease index decreased after drug administration. The drug treatment played a role in inhibiting the development of tomato mottle mosaic virus.

[0088] Results of infiltration and inoculation: The leaves of the control group showed obvious chlorosis and yellowing symptoms. Compared with the control group, the disease control efficiency of the groups treated with 1000 times and 3333 times of 0.02% dihydrochlorin iron soluble powder was 41.12% and 12.18%, respectively. The disease control efficiency of the group treated with 200 times of 1% lentinan aqueous solution was 32.02%. In the groups treated with different concentrations of dihydrochlorin iron and lentinan compound composition, the virus disease symptoms of the plants were significantly alleviated (such as Figure 1The results showed that the control efficacy against the diseased fingers of ToMMV was significantly improved, with each treatment group achieving over 60%, demonstrating a synergistic effect (as shown in Table 1). The treatments with a 167-fold dilution of 0.8333% chlorin iron and lentinan suspension (chlorin iron concentration: 0.2 mg / L, lentinan concentration: 50 mg / L) and a 2609-fold dilution of 13.056% chlorin iron and lentinan powder (chlorin iron concentration: 0.06 mg / L, lentinan concentration: 50 mg / L) achieved the highest control efficacy, at 80.09% and 78.89%, respectively, demonstrating a more pronounced synergistic effect.

[0089] Virus Accumulation: Compared to the single-dose treatments in terms of virus replication inhibition rate and disease index control efficacy, the iron-chlorin and lentinan combination treatments significantly reduced ToMMV accumulation in plants, with virus replication inhibition rates reaching 64-86%, demonstrating a synergistic effect. The largest reductions in virus accumulation were observed in the treatments with a 167-fold dilution of the 0.8333% iron-chlorin · lentinan suspension (0.2 mg / L iron, 50 mg / L chlorin) and a 2609-fold dilution of the 13.056% iron-chlorin · lentinan powder (0.06 mg / L iron, 50 mg / L chlorin), at 86% and 82%, respectively, demonstrating a more pronounced synergistic effect.

[0090] From the above results, it can be concluded that the composite composition of dihydrochlorin iron and lentinan can synergistically enhance resistance to plant viral diseases and can be used to prevent and control tomato mottle mosaic virus disease. The preferred weight ratio of the active ingredients of the two is 1:2000 to 1:100 (specifically about 1:800 to 1:250).

[0091] Example 7 - Field Control of Wheat Yellow Mosaic Virus

[0092] This example demonstrates the effectiveness of the compound composition in controlling wheat yellow mosaic virus in the field.

[0093] Wheat yellow mosaic virus ( 小麦黄花叶病毒 WYMV) is a soil-borne virus. In the early spring when wheat yellow mosaic virus symptoms appeared, a compound composition of dihydrochlorin iron and lentinan was applied in Baimahu Farm, Jiangsu Province to prevent and control wheat yellow mosaic disease, and to evaluate the effect of the compound composition in preventing and controlling plant viral diseases.

[0094] 1. Virus detection

[0095] Before the experiment, diseased wheat plants were collected from the diseased fields, RNA was extracted, and RT-PCR detection was performed. The primers used were as follows: WYMV-CP1: 5'-AAGCCAGGGACCTCACAGCAACC-3', WYMV-CP2: 5'- CGTCTCAAGCCACCATTCAACCT-3'. The amplified bands were sequenced to confirm that the wheat plants in each group were infected with WYMV.

[0096] 2. Preparation of test agents

[0097] The reagents prepared in Example 1 and Example 3 were diluted according to the following ratios and methods to prepare test reagents of different concentrations:

[0098] (1) 2609 times dilution of 13.056% chlorin iron·Lentinan powder (dilute and dissolve with water in proportion, the same below);

[0099] (2) 185 times dilution of 0.945% chlorin iron·Lentinan suspension concentrate;

[0100] (3) 3333 times dilution of 0.02% dihydrochlorin iron soluble powder;

[0101] (4) 200 times dilution of 1% Lentinan aqueous solution;

[0102] (5) Clean water.

[0103] 3. Pesticide treatment

[0104] The compounded combination of different concentrations was evenly sprayed on wheat leaves. After an interval of 8 days, the second application was carried out, with the drug used twice in total. A blank control was sprayed with water. Four plots (four replicates) were set up for each treatment group, each plot being 20 square meters.

[0105] 4. Plant symptom record and prevention effect calculation

[0106] Seven days after the second application of the pesticide to each group of wheat leaves, wheat plant symptoms were observed in the field. 100 plants were randomly selected from each plot, and the severity of disease on the upper six leaves of each plant was recorded. The disease index was calculated and the control efficacy was calculated. The differences in the control efficacy of different treatments against viral diseases were compared using Duncan's new multiple range method using the DPS data processing system. The corresponding calculated data are recorded in Table 2. The grading criteria are as follows:

[0107] Level 0: no flower leaves, purple leaves;

[0108] Level 1: 1 mosaic leaf, purple leaf;

[0109] Level 3: 2 mosaic leaves, purple leaves;

[0110] Level 5: 3 mosaic leaves, purple leaves;

[0111] Level 7: 4 or 5 mosaic leaves, purple leaves;

[0112] Level 9: 6 variegated leaves, purple leaves.

[0113] Calculation formula for disease index and disease index prevention effect:

[0114]

[0115] 5. Prevention and control effects

[0116] Table 2 Field efficacy of chlorin iron and lentinan against WYMV

[0117]

[0118] From the data in Table 2, it can be seen that the average disease index of the wheat yellow mosaic virus disease in the drug treatment group was 65.25% when no drug was given. After drug administration, the disease index decreased, and the drug played a role in inhibiting the development of the disease. Among them, the average disease index of the drug treatment group with a single dose of 0.02% dihydrochlorin iron soluble powder 3333 times (0.06 mg / L) was 56.03%, and the disease index control effect was only 14.13%, which had a low effect in inhibiting the development of the disease. The single dose of 1% lentinan polysaccharide aqueous solution 200 times (50 mg / L) of the pesticide treatment group had an average disease index of 55.31%, and the disease index prevention efficiency was only 15.24%, which had a low effect in inhibiting the development of the disease; after treatment with the compound composition of dihydrochlorin iron and lentinan at the same concentration as the single agent, the symptoms of yellow mosaic virus disease in wheat plants were significantly alleviated, which showed that the compound composition of dihydrochlorin iron and lentinan had a synergistic effect; among them, the disease index prevention efficiency of the group treated with 2609 times of 13.056% dihydrochlorin iron•lentinan powder (dihydrochlorin iron 0.06 mg / L, lentinan 50 mg / L) was 63.80%, and the disease index prevention efficiency of the group treated with 185 times of 0.945% dihydrochlorin iron•lentinan suspension (dihydrochlorin iron 0.08 mg / L, lentinan 50 mg / L) was 67.22%.

[0119] It can be seen that the dihydrochlorin iron and lentinan (3:250~2:125) in the compound composition of dihydrochlorin iron and lentinan can significantly inhibit wheat yellow mosaic virus disease. The dihydrochlorin iron and lentinan agent composition in this concentration range has important application value in preventing and controlling wheat yellow mosaic virus.

[0120] Example 8 - Field Trial on Controlling Tomato Viruses

[0121] This example verifies the effectiveness of the compound composition in preventing and controlling tomato viruses in the field.

[0122] In this embodiment, tobacco mosaic virus genus ( 烟草花叶病毒属) virus is an important virus that harms tomatoes in production. When tomato virus disease occurs in greenhouses in Shouguang, Shandong, and symptoms of harming fruits appear, a compound composition of dihydrochlorin iron and lentinan polysaccharide is used to prevent and control the virus disease, and the effect of the compound composition in preventing and controlling tomato virus disease is evaluated.

[0123] 1. Preparation of test agents

[0124] The test agent is the same as that in Example 7.

[0125] 2. Pesticide treatment

[0126] Different concentrations of the compound combination were evenly sprayed on the surface of tomato leaves and fruits. After an interval of 8 days, the second application was carried out, and the drug was used twice in total. The blank control was sprayed with water. Each treatment group was set up with 4 replicates, and each replicate had 20 tomato plants.

[0127] 3. Plant symptom record and prevention effect calculation

[0128] Tomato leaves and fruits in each group were observed in the field 20 days after the second application of the drug. Fruits from the fourth and fifth ears of each replicate of 20 seedlings were counted, and the number of diseased fruits (i.e., fruits with brown spots) on each plant was recorded. The disease index was calculated and the control efficacy was calculated. Differences in control efficacy against viral diseases between treatments were statistically compared using Duncan's new multiple range method using the DPS data processing system. The data recorded are shown in Table 3. The grading criteria are as follows:

[0129] Level 0: No lesions on the fruit;

[0130] Level 1: 1 lesion;

[0131] Level 3: 2 lesions;

[0132] Level 5: 3 lesions;

[0133] Level 7: 4 lesions;

[0134] Level 9: 5 or more lesions.

[0135] Calculation formula for disease index and disease index prevention effect:

[0136]

[0137] 4. Prevention and control effects

[0138] Table 3 Effects of chlorin iron and lentinan on tomato fruit 烟草花叶病毒属 Field test of virus control efficacy

[0139]

[0140] From the data in Table 3, it can be seen that the average disease index of the tomato fruit in the drug treatment group was 143.58% when compared with the water control group without drug administration. After drug administration, the disease index decreased, and the drug played a role in inhibiting the development of the disease; among them, the average disease index of the group treated with a single dose of 0.02% dihydrochlorin iron soluble powder 3333 times (0.06 mg / L) was 88.50%, and the disease index prevention effect was only 38.36%, which had a low effect of inhibiting the development of the disease; the average disease index of the group treated with a single dose of 1% edodes polysaccharide aqueous solution 200 times (50 mg / L) was 103.58%, and the disease index prevention effect was only 27.86%, which had a low effect of inhibiting the development of the disease; after treatment with a compound composition of dihydrochlorin iron and edodes polysaccharide at the same concentration as a single dose of the drug, the tomato fruit showed infection 烟草花叶病毒属 The virus symptoms were significantly alleviated. 烟草花叶病毒属 The two drugs have a synergistic effect on the diseases caused by them; among them, the disease prevention efficiency of the group treated with 2609 times of 13.056% dihydrochlorin iron•Linenin polysaccharide powder (dihydrochlorin iron 0.06 mg / L, lentinan polysaccharide 50 mg / L) was 90.85%, and the disease prevention efficiency of the group treated with 185 times of 0.945% dihydrochlorin iron•Linenin polysaccharide suspension (dihydrochlorin iron 0.08 mg / L, lentinan polysaccharide 50 mg / L) was 90.87%.

[0141] The results of the disease index survey showed that the drug had no adverse effects on tomato growth. The brown patch symptoms of the 4th and 5th tomato ears in the blank control group were not alleviated. Compared with the control group, the control groups treated with 3333 times of 0.02% dihydrochlorin iron soluble powder and 200 times of 1% lentinan aqueous solution had a protective effect of 38.36% and 27.86% respectively. After treatment with the compound composition of dihydrochlorin iron and lentinan, the brown patch symptoms of the 4th and 5th tomato ears were significantly alleviated (such as Figure 2 (As shown in the figure, Grade 7 and 9 diseased fruits were almost nonexistent, demonstrating a clear synergistic effect.) The control efficacy of the treatment groups with a 2609-fold dilution of 13.056% chlorin iron and lentinan powder and a 185-fold dilution of 0.945% chlorin iron and lentinan suspension exceeded 90%.

[0142] It can be seen that the dihydrochlorin iron and lentinan (3:2500~2:1250) in the composite composition of dihydrochlorin iron and lentinan has important application value in preventing and controlling tomato virus diseases and improving tomato fruits.

[0143] The above describes the specific embodiments of the present invention in detail, but they are only examples. The present invention is not limited to the specific embodiments described above, and the implementation methods and protection scope of the present invention are not limited thereto. For those skilled in the art, equivalent changes and modifications made without departing from the spirit and scope of the present invention should be included in the protection scope of the present invention.

Claims

1. A plant virus disease resistance composition, characterized in that: The active components of the anti-plant virus disease pharmaceutical composition are dihydrochlorin iron and lentinan; wherein the weight ratio of dihydrochlorin iron to lentinan is 1:1250 to 1:

125.

2. The anti-plant virus disease pharmaceutical composition according to claim 1, characterized in that: The active component accounts for 0.51% to 62% of the total weight of the composition.

3. The anti-plant virus disease pharmaceutical composition according to claim 2, characterized in that: The active component accounts for 0.67% to 24.01% of the total weight of the composition.

4. The anti-plant virus disease pharmaceutical composition according to claim 1, characterized in that: The anti-plant virus disease drug composition also includes auxiliary ingredients, which are at least one of solvents, emulsifiers, wetting agents, dispersants, stabilizers, antifreeze agents, thickeners, binders, disintegrants, defoaming agents, penetrants, polymer capsule wall materials, synergists, preservatives and fillers.

5. The anti-plant virus disease pharmaceutical composition according to claim 1, characterized in that: The preparation form of the composition is suspension, aqueous emulsion, microcapsule suspension, wettable powder, water dispersible granule or microemulsion.

6. Use of the anti-plant virus disease pharmaceutical composition according to any one of claims 1 to 5, characterized in that: The application is an application in preventing and treating plant diseases caused by the plant virus, wherein the plant virus is tomato mottle mosaic virus ( Tomato mottled mosaic virus ), wheat yellow mosaic virus ( Wheat yellow mosaic virus ); in the application, the anti-plant virus disease pharmaceutical composition is applied by spraying it onto the leaves or fruit surfaces of the plants, the number of spraying is not less than 2 times, the interval between each spraying is 3 to 10 days, and the application concentration is 0.05 to 0.2 mg / L of dihydrochlorin iron and 16 to 100 mg / L of lentinan.

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