Application of quinazoline carboxylic acid compound in plant virus resistance

Abstract

The invention relates to the technical field of plant disease control, in particular to application of a quinazoline carboxylic acid compound in plant virus resistance. The invention discovers that 1, 5-dioxo-2, 3, 4, 5-tetrahydropyrrolo [1, 2-a] quinazoline-3a (1H)-carboxylic acid (DHCA) has the effect of resisting plant viruses for the first time. In the specific embodiment of the invention, the 1, 5-dioxo-2, 3, 4, 5-tetrahydropyrrolo [1, 2-a] quinazoline-3a (1H)-carboxylic acid is used for preventing and treating the cucumber virus disease and the pepper virus disease, and the result shows that the 1, 5-dioxo-2, 3, 4, 5-tetrahydropyrrolo [1, 2-a] quinazoline-3a (1H)-carboxylic acid and the 1, 5-dioxo-2, 3, 4, 5-tetrahydropyrrolo [1, 2-a] quinazoline-3a (1H)-carboxylic acid are used for preventing and treating the cucumber virus disease and the pepper virus disease. And the composition has a good control effect by combining the 2, 2-a] quinazoline-3a (1H)-carboxylic acid with wuyiencin.

Description

Technical Field

[0001] This invention relates to the field of plant disease control technology, and in particular to the application of a quinazoline carboxylic acid compound in the fight against plant viruses. Background Technology

[0002] Plant viral diseases are a significant biological stressor hindering the sustainable development of the global vegetable industry. Current control methods primarily rely on chemical pesticides to manage virus vectors, but these methods suffer from limitations such as pesticide residues, resistance, and poor direct inactivation of viruses. Microbial antiviral agents have become a research hotspot due to their environmental friendliness and diverse mechanisms of action, with Streptomyces species receiving particular attention due to their abundant secondary metabolites.

[0003] In recent years, with in-depth research into plant immune mechanisms and biocontrol technologies, an increasing number of microorganisms have been found to enhance plant disease resistance through various pathways, including inducing systemic resistance, inhibiting viral replication, and promoting healthy plant growth by secreting plant growth regulators (such as indole-3-acetic acid, IAA). *Streptomyces roseum* NKZ-259 (CGMCC No. 13416) is a microorganism with potential application value, exhibiting good characteristics in plant growth promotion and disease control. *Streptomyces roseum* NKZ-259 can produce IAA and other heterocyclic amide compounds that promote the growth of plant roots and aboveground parts. It has also been shown to have certain fungicidal activity, especially showing good control efficacy against gray mold in crops such as tomatoes. Its application in plant growth promotion and disease control has been partially protected by patents. However, as a biocontrol microorganism, there are currently few reports on the application of its metabolites' antiviral properties against plant viruses. Summary of the Invention

[0004] The purpose of this invention is to provide an application of quinazoline carboxylic acid compounds in the treatment of plant viruses, thereby solving the problems existing in the prior art.

[0005] To achieve the above objectives, the present invention provides the following solution:

[0006] This invention provides the application of 1,5-dioxo-2,3,4,5-tetrahydropyrrolo[1,2-a]quinazoline-3a(1H)-carboxylic acid (DHCA or heterocyclic amide compound) in the fight against plant viruses.

[0007] Preferably, the plant virus includes one or more of the following: cucumber mosaic virus (CMV), pepper mild mottle virus (PepMoV), cucumber green mottle mosaic virus (CGMMV), tomato yellow leaf curl virus (ToMV), tobacco mosaic virus (TMV), and turnip mosaic virus (TuMV).

[0008] Pepper viral diseases are mainly caused by cucumber mosaic virus, pepper mild mottle virus, etc., while cucumber viral diseases are mainly caused by cucumber mosaic virus, cucumber green mottle mosaic virus, etc.

[0009] This invention provides the application of 1,5-dioxo-2,3,4,5-tetrahydropyrrolo[1,2-a]quinazolin-3a(1H)-carboxylic acid in the preparation of antiviral formulations for plants.

[0010] Preferably, the plant virus includes one or more of the following: cucumber mosaic virus, pepper mild mottle virus, cucumber green mottle mosaic virus, tomato yellow leaf curl virus, tobacco mosaic virus, and turnip mosaic virus.

[0011] This invention provides an antiviral formulation for plants, wherein the active ingredient of the formulation comprises 1,5-dioxo-2,3,4,5-tetrahydropyrrolo[1,2-a]quinazoline-3a(1H)-carboxylic acid.

[0012] Preferably, the active ingredient also includes Wuyi mycin.

[0013] Preferably, the plant virus includes one or more of the following: cucumber mosaic virus, pepper mild mottle virus, cucumber green mottle mosaic virus, tomato yellow leaf curl virus, tobacco mosaic virus, and turnip mosaic virus.

[0014] Preferably, the formulation further includes excipients.

[0015] This invention provides the application of the above-mentioned formulation in the prevention and control of plant viral diseases or in the preparation of formulations for the prevention and control of plant viral diseases.

[0016] More preferably, the plant viral diseases include one or more of cucumber virus disease, pepper virus disease, tomato virus disease, tobacco virus disease, and turnip virus disease.

[0017] This invention provides a method for controlling plant viral diseases, comprising the step of treating plants with 1,5-dioxo-2,3,4,5-tetrahydropyrrolo[1,2-a]quinazoline-3a(1H)-carboxylic acid or the above-mentioned preparations.

[0018] More preferably, the plant viral diseases include one or more of cucumber virus disease, pepper virus disease, tomato virus disease, tobacco virus disease, and turnip virus disease.

[0019] As an optional addition, the present invention provides an agent for controlling plant viral diseases, the active ingredient of which comprises 1,5-dioxo-2,3,4,5-tetrahydropyrrolo[1,2-a]quinazoline-3a(1H)-carboxylic acid.

[0020] More preferably, the active ingredient also includes Wuyi mycin.

[0021] More preferably, the plant viral diseases include one or more of cucumber virus disease, pepper virus disease, tomato virus disease, tobacco virus disease, and turnip virus disease.

[0022] The present invention discloses the following technical effects:

[0023] This invention discloses for the first time that a quinazoline carboxylic acid compound (a metabolite of *Streptomyces roseum* NKZ-259)—1,5-dioxo-2,3,4,5-tetrahydropyrrolo[1,2-a]quinazoline-3a(1H)-carboxylic acid (DHCA)—has antiviral activity against plant viruses. In specific embodiments of this invention, 1,5-dioxo-2,3,4,5-tetrahydropyrrolo[1,2-a]quinazoline-3a(1H)-carboxylic acid was used to control cucumber virus diseases and pepper virus diseases. Results showed that 1,5-dioxo-2,3,4,5-tetrahydropyrrolo[1,2-a]quinazoline-3a(1H)-carboxylic acid, and 1,5-dioxo-2,3,4,5-tetrahydropyrrolo[1,2-a]quinazoline-3a(1H)-carboxylic acid combined with Wuyi mycin exhibited good control efficacy. Meanwhile, 1,5-dioxo-2,3,4,5-tetrahydropyrrolo[1,2-a]quinazoline-3a(1H)-carboxylic acid exhibits significant inhibitory activity against typical vegetable plant viruses under in vitro conditions. Developing the application of metabolites of Streptomyces roseum NKZ-259 in the prevention and control of plant viral diseases not only fills the gap in existing technologies but also provides an environmentally friendly solution for green agriculture to address the challenges of crop viral disease control, which has significant theoretical and practical value. Attached Figure Description

[0024] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0025] Figure 1 This is a field survey map of Example 3;

[0026] Figure 2 This is a field survey map of Example 5;

[0027] Figure 3 The figure shows the experimental results of Example 6. Detailed Implementation

[0028] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0029] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0030] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0031] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0032] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0033] Example 1. Preparation of 1,5-dioxo-2,3,4,5-tetrahydropyrrolo[1,2-a]quinazoline-3a(1H)-carboxylic acid

[0034] The preparation method of 1,5-dioxo-2,3,4,5-tetrahydropyrrolo[1,2-a]quinazoline-3a(1H)-carboxylic acid is described in Chinese Patent Application No. 202410925761.1, entitled "Heterocyclic amide compound, preparation method and application thereof", and the steps are as follows:

[0035] (1) Preparation of fermentation broth for Streptomyces roseoflavus NKZ-259: Streptomyces roseoflavus NKZ-259 was inoculated onto MS medium and cultured at 28°C in the dark for 7 days. A 1cm sample was scraped off with a sterile toothpick. 2Fermentation broth of *Streptomyces roseoflavus* NKZ-259 was obtained by inoculating small-sized mycelial blocks into fermentation medium and incubating at 28℃ and 220 rpm for 96 h on a shaker. The broth was then filtered through filter paper. The accession number for *Streptomyces roseoflavus* NKZ-259 is CGMCC NO.13416. This strain, classified as *Streptomyces roseoflavus*, was deposited on December 2, 2016, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, China. This strain is described in invention patent ZL201710044242.4, entitled "A *Streptomyces roseoflavus* strain and its application."

[0036] MS medium formulation includes: mannitol 20 g / L, soybean flour 20 g / L, agar powder 20 g / L, prepared with water.

[0037] The fermentation medium consists of: 10 g / L glucose, 4 g / L peptone, 2 g / L yeast extract, 0.5 g / L MgSO4, and 2 g / L KH2PO4, prepared with water.

[0038] (2) Extraction of compounds: 80L of Streptomyces roseum NKZ-259 fermentation broth was centrifuged in batches at 25℃ and 8000rpm for 15min. After centrifugation, the cells and supernatant were collected separately. An equal volume of ethyl acetate was added to the supernatant and extracted at 150rpm for 1h. After standing until the layers separated, the organic phase and aqueous phase were collected separately. The organic phase was rotary evaporated at 40℃ and 25rpm until dry and the fraction (i.e., ethyl acetate extract) was collected and stored in a refrigerator at 4℃.

[0039] (3) Isolation and purification of compounds: The ethyl acetate extract was dissolved in a small amount of methanol, and then silica gel (100-200 mesh) was added and stirred until homogeneous. The mass ratio of ethyl acetate extract to silica gel was 1:1. After the methanol evaporated, 10 times the mass of the extract was packed into a 200-300 mesh silica gel column (60×400mm glass column). Dry loading was used, and gradient elution was performed sequentially with petroleum ether-ethyl acetate as the eluent. The volume ratios of petroleum ether to ethyl acetate were 1:1, 1:2, 1:4, and 0:1, respectively. That is, first, elution was performed with a volume ratio of petroleum ether to ethyl acetate of 1:1 to obtain component 1; then, elution was performed with a volume ratio of petroleum ether to ethyl acetate of 1:2 to obtain component 2; then, elution was performed with a volume ratio of petroleum ether to ethyl acetate of 1:4 to obtain component 3; and finally, elution was performed with a volume ratio of petroleum ether to ethyl acetate of 0:1 to obtain component 4.

[0040] The fractions (fractions 1 to 4) obtained by gradient elution were analyzed by UPLC-MS. The analytical conditions were: Waters Xevo TQD ultra-high performance liquid chromatography-mass spectrometry, ACQUITY BEH C18 column (2.1 × 100 mm, 1.7 μm), mobile phase 5%–100% acetonitrile / water (volume percentage), and elution time 7 min. The results showed that only fraction 3 (i.e., the fraction eluted with petroleum ether / ethyl acetate at a 1:4 ratio) contained the compound 1,5-dioxo-2,3,4,5-tetrahydropyrrolo[1,2-a]quinazoline-3a(1H)-carboxylic acid.

[0041] The above components were prepared by preparative liquid chromatography. The preparation conditions were a Waters 2767 high-performance liquid chromatograph and a Sunfire C10 ... 18 OBD preparative column (5 μm, 19 × 250 mm), eluted with a gradient of 5%–60% (v / v) acetonitrile / water for 30 min. 1,5-dioxo-2,3,4,5-tetrahydropyrrolo[1,2-a]quinazoline-3a(1H)-carboxylic acid was obtained at a retention time of 11.57 min.

[0042] The English name of 1,5-dioxo-1,2,3,3a,4,5-hexahydropyrrolo[1,2-a]quinazoline-3a(1H)-carboxylic acid is 1,5-Dioxo-1,2,3,3a,4,5-hexahydropyrrolo[1,2-a]quinazoline-3a-carboxylic acid, and its molecular formula is C1. 12 H 10 N₂O₄, with a molecular weight of 246 g / mol, is a white crystalline solid. For ease of description, this compound can be referred to as DHCA. Its chemical formula is shown in Formula I, where R in Formula I is selected from OH.

[0043]

[0044] Example 2: Treatment trial of DHCA on cucumber viral diseases (Cucumber viral diseases are mainly caused by cucumber mosaic virus, cucumber green mottle mosaic virus, etc.)

[0045] 1. Experimental site, application time, cucumber variety, and management

[0046] 1) Site selection: A cold-frame cucumber experimental field with flat terrain, convenient transportation, and fertile soil was selected. Prior to the experiment, sporadic viral diseases were observed in the experimental field. 2) Site environment: The daytime temperature inside the greenhouse was approximately 45℃, and the nighttime temperature was approximately 20℃. Drip irrigation under mulch was used for cultivation (soil conditions, fertilizer application, and watering were uniform). The cultivation management of the experimental field conformed to local production realities. 3) The tested cucumber variety was a non-viral-resistant variety.

[0047] The specific experimental location for this embodiment was as follows: the experimental field was set up in a cold-frame cucumber field in Guojia Village, Jiyang District, Jinan City, Shandong Province. The cucumber variety used for the experiment was Jinyou 35. Before the experiment, sporadic viral diseases occurred in the experimental field. The daytime temperature inside the greenhouse was around 45℃, and the nighttime temperature was around 20℃. The terrain inside the greenhouse was flat, and the cultivation method of drip irrigation under film was used, resulting in uniform soil conditions, fertilizer application, and watering. The cultivation management of the experimental field was in line with local production realities.

[0048] 2. Meteorological and Soil Data

[0049] The weather was sunny that day, with an indoor temperature of 35℃ and a relative humidity of 43%. The soil type in the experimental field was sandy loam. The organic matter content in the topsoil layer (0-0.25m) was approximately 26.0 g / kg, available phosphorus content was 170.8 mg / kg, available potassium content was 399 mg / kg, and available nitrogen content was 268 mg / kg. The soil was slightly alkaline, with a pH of 7.87. The high multiple cropping index in the greenhouse led to a decreasing trend in soil organic matter content and a reduced soil pH buffering capacity.

[0050] 3. Test reagents, dosage, and application methods

[0051] Treatment agents: DHCA, developed by the Institute of Plant Protection, Chinese Academy of Agricultural Sciences, dosage 900 g / ha; Wuyi mycin aqueous solution, developed by the Institute of Plant Protection, Chinese Academy of Agricultural Sciences, dosage 120 g / ha.

[0052] Control reagents: 0.5% chitosan AS, Taikang Chitosan, manufactured by Chengdu Tepu Biotechnology Co., Ltd.; 200 million / mL methyltrophic Bacillus, Taikang Woyou, manufactured by Chengdu Tepu Biotechnology Co., Ltd.; Tepu Micro-Aid, manufactured by Chengdu Tepu Biotechnology Co., Ltd.

[0053] The dosage of the test reagents and the content of the active ingredients are shown in Table 1.

[0054] Table 1 Experimental Design of Test Reagents

[0055]

[0056]

[0057] First, spray the pesticide with clean water using a sprayer, ensuring that the leaves are evenly covered with the pesticide without dripping. Calculate the water consumption and spraying speed for a single plot area. When spraying, prepare the stock solution of the test agent and the control agent and measure the solution using a 50mL graduated cylinder. Before application, stir and shake the solution thoroughly. Dilute the solution twice. Clean the sprayer twice before each pesticide preparation.

[0058] Water consumption per hectare: 450L.

[0059] Application equipment: The sprayer model is 3WBD-20, with a lithium-ion battery, working pressure of 0.2-0.4MPa, capacity of 20L, and a conical atomizing nozzle with dimensions of 395×205×495mm.

[0060] The pesticide was applied on May 9, 2024 (10 days after cucumber transplanting), during the cucumber seedling stage, and once.

[0061] 4. Community Setup

[0062] Four treatments were set up (DHCA, DHCA + Wuyi mycin, 0.5% chitosan AS + 200 million / mL methyltrophic Bacillus + TEP Micro-Aid, and blank treatment). The plot area of ​​each treatment was 30 square meters (8 meters long × 3.75 meters wide), and each treatment was replicated 4 times.

[0063] 5. Experimental investigation and analysis

[0064] The field survey method for cucumber virus diseases involves selecting half of the plants in each plot for investigation, recording the total number of plants and the number of plants at each disease level. Diseased plants are recorded according to the following 9-level grading method. The grading method for cucumber virus diseases (by plant) is as follows:

[0065] Level 0: Asymptomatic;

[0066] Grade 1: Heart-shaped leaves with clear veins, lightly patterned leaves;

[0067] Grade 3: Mottled leaves in the heart and middle leaves;

[0068] Grade 5: Mottled leaves in the heart and middle leaves, a few leaves are deformed, wrinkled or the plant is slightly stunted;

[0069] Level 7: Heavy foliage, most leaves are deformed, wrinkled or the plant is stunted;

[0070] Level 9: Heavy foliage, with obviously deformed and linear leaves, severely stunted plants, and even death.

[0071] All plants in each plot were surveyed, starting from the cucumber seedling stage, for pre-application surveys; secondary and tertiary surveys were conducted at different times after application to calculate the disease index and the control effect on the plants.

[0072] The formula for calculating the disease index (DIA) is as follows:

[0073]

[0074] Without prior disease indicators, the formula for calculating the prevention and treatment effect is as follows:

[0075]

[0076] Results analysis: Based on the calculation results of disease index and plant control effect, the field efficacy of the tested pesticides for controlling cucumber virus diseases was evaluated.

[0077] 6. Survey time and number of times

[0078] On May 9, 2024, during the cucumber seedling stage, a pre-treatment survey was conducted and pesticide was sprayed; an investigation was conducted 8 days after the pesticide application (May 16, 2024).

[0079] 7. Test Results

[0080] The disease index and control efficacy of the plants in the field are shown in Table 2 below.

[0081] Table 2. Results of field efficacy trials of different treatments for controlling cucumber virus disease.

[0082]

[0083] The results of the post-application survey showed that: after applying DHCA, the average disease index of plants in the greenhouse was 7.95; after applying DHCA + Wuyi mycin, the average disease index of plants in the greenhouse was 8.35; after applying 0.5% chitosan AS + 200 million / mL methyltrophic Bacillus + TEP Micro-Adjuvant, the average disease index of plants in the greenhouse was 8.09; and the average disease index of the control treatment was 13.56. The control efficacy of DHCA-treated plants in the field was 41.40%, and that of DHCA + Wuyi mycin-treated plants in the field was 38.40%, which was not significantly different from the control efficacy of 0.5% chitosan AS + 200 million / mL methyltrophic Bacillus + TEP Micro-Adjuvant (40.37%). Therefore, DHCA treatment and DHCA + Wuyi mycin can achieve the control efficacy of the control agents (Table 2).

[0084] Example 3: DHCA Prevention Experiment on Pepper Virus Diseases (Pepper virus diseases are mainly caused by cucumber mosaic virus, pepper mild mottle virus, etc.)

[0085] 1. Test site, application time, chili variety, and management

[0086] The experimental field was set up in an open-field chili pepper field in Gongyuan Village, Luorong Town, Liuzhou City, Guangxi Province. The tested chili pepper variety was Tongle 23188. Before the experiment, sporadic viral diseases occurred in the experimental field. During the experiment, the daytime temperature was around 28℃ and the nighttime temperature was around 22℃. The experimental field was flat and uniformly cultivated using mulch film (soil conditions, fertilizer application, watering, etc.). The cultivation and management of the experimental field were in line with local production realities.

[0087] 2. Meteorological and Soil Data

[0088] At 4 PM on April 23, 2024, the weather was sunny, the field temperature was 28℃, and the relative humidity was 81%.

[0089] The soil type of the experimental field was red clay, ranging from red to reddish-brown in color, with a high clay content and a heavy clay texture; it exhibited an acidic reaction, with a pH of around 5.0. The acidity gradually increased from top to bottom; in the topsoil layer (0-0.5m), the organic matter content was 2.51%, total nitrogen 0.119%, total phosphorus 0.061%, available phosphorus 2.2ppm, and available potassium 37ppm. The experimental field was used for crop rotation with chili peppers, resulting in very few root diseases and vigorous chili pepper growth.

[0090] 3. Test reagents, dosage, and application methods

[0091] Treatment agents: DHCA, developed by the Institute of Plant Protection, Chinese Academy of Agricultural Sciences, dosage 900 g / ha; Wuyi mycin, developed by the Institute of Plant Protection, Chinese Academy of Agricultural Sciences, dosage 120 g / ha.

[0092] Control reagent: 15% cyclophosphamide·morpholine guanidine hydrochloride wettable powder, produced by Guangxi Tianyuan Biochemical Co., Ltd.

[0093] The dosage of the test reagents and the content of the active ingredients are shown in Table 4.

[0094] Table 4 Experimental Design of Test Reagents

[0095] medicine Dosage Active ingredient content (g / hectare) DHCA Dilute 500 times 900 DHCA+Wuyi mycin Dilute 500 times + Dilute 75 times 900+120 15% Flufenoxam·Morphine Hydrochloride Wettable Powder Diluted 750 times 600 Blank processing - -

[0096] First, spray the pesticide with clean water using a sprayer, ensuring that the leaves are evenly covered with the pesticide without dripping. Calculate the water consumption and spraying speed for a single plot area. When spraying, prepare the stock solution of the test and control pesticides and measure the solution using a 100mL graduated cylinder. Before application, stir and shake the solution thoroughly. Dilute the solution twice. Clean the sprayer twice before each pesticide preparation.

[0097] Application equipment: Sprayer model 3WBD-20, lithium-ion battery, working pressure 0.2-0.4MPa, capacity 20L, nozzle is a conical atomizing nozzle, size: 395×205×495mm. Water consumption per hectare: 450L.

[0098] The first application of the pesticide was on April 23, 2024, when the chili peppers were in the early fruit-setting stage (57 days after transplanting). The pesticide was then applied again on May 5, 2024, May 16, 2024, and May 26, 2024, for a total of 4 applications.

[0099] 4. Community Setup

[0100] Four treatments were set up (DHCA, DHCA + Wuyi mycin, 15% flufenoxuron-methyl hydrochloride wettable powder and blank treatment), with a plot area of ​​24 square meters (16 meters long × 1.5 meters wide) for each treatment, and each treatment was replicated 4 times.

[0101] 5. Experimental investigation and analysis

[0102] The field survey method for pepper virus diseases involves selecting half of the plants in each plot for investigation, recording the total number of plants and the number of plants at each disease level. Diseased plants are recorded according to the following 9-level grading method. Pepper virus disease grading method (per plant):

[0103] Level 0: Asymptomatic;

[0104] Grade 1: Heart-shaped leaves with clear veins, lightly patterned leaves;

[0105] Grade 3: Mottled leaves in the heart and middle leaves;

[0106] Grade 5: Mottled leaves in the heart and middle leaves, a few leaves are deformed, wrinkled or the plant is slightly stunted;

[0107] Level 7: Heavy foliage, most leaves are deformed, wrinkled or the plant is stunted;

[0108] Level 9: Heavy foliage, with obviously deformed and linear leaves, severely stunted plants, and even death.

[0109] All plants in each plot were surveyed, starting from the seedling stage of chili peppers. A pre-treatment survey was conducted, followed by a second and third survey after the application of pesticides. The disease index and the control effect on the plants were calculated.

[0110] The formula for calculating the disease index (DIA) is as follows:

[0111]

[0112] Before administering medication, a disease index is established. The formula for calculating the prevention and treatment effect is as follows:

[0113]

[0114] Results analysis: Based on the disease index and the calculated plant control effect, the field efficacy of the tested pesticides for controlling pepper viral diseases was evaluated.

[0115] 6. Survey time and number of times

[0116] On April 23, 2024, during the early fruiting stage of the chili peppers, a pre-treatment survey was conducted and pesticides were sprayed. A second survey was conducted 12 days after the first treatment, on May 5, 2024. A third survey was conducted 11 days after the second treatment, on May 16, 2024. A fourth survey was conducted 10 days after the third treatment, on May 26, 2024. A fifth survey was conducted 16 days after the fourth treatment, on June 11, 2024.

[0117] 7. Test Results

[0118] The disease index and control efficacy of the plants in the field are shown in Table 5 below. Figure 1 As shown.

[0119] Table 5. Results of field efficacy trials of different treatments for controlling pepper virus disease.

[0120]

[0121] In this embodiment, viral disease prevention treatment was initiated at the early fruit-setting stage of peppers, and four consecutive applications were made at approximately 10-day intervals. A district-wide survey was conducted before each application. The control efficacy of each treatment against pepper viral diseases was assessed in the final survey of this experiment, and the results are as follows:

[0122] The treatment with DHCA + Wuyi mycin showed the best control efficacy, at 89.81%. There was no significant difference in the control efficacy against pepper virus disease among the different treatments tested. This example is for preventative application. Before the fourth application, the disease indices for all treatments were 0.17, 0.56, and 0.09, respectively, while the control group had a disease index of 0.9. After the fourth application, the disease indices for each treatment were 11.00, 9.19, 12.03, and 14.72, respectively. In fields with a high incidence of pepper virus disease, timely and repeated spraying with DHCA or DHCA + Wuyi mycin after pepper transplanting can effectively control the occurrence of viral diseases (Table 5).

[0123] Example 4: Treatment trial of DHCA against pepper virus disease

[0124] 1. Test site, application time, chili variety, and management

[0125] The experimental field was set up in an open-field chili pepper field next to Juyou Farm in Luorong Town, Liuzhou City. The chili pepper variety used for the experiment was Tongle 23188. Before the experiment, the incidence of viral diseases in the experimental field was about 10%. During the experiment, the daytime temperature was around 30℃ and the nighttime temperature was around 21℃. The experimental field was flat and used mulching cultivation (soil conditions, fertilizer application, watering, etc.) were uniform. The cultivation and management of the experimental field were in line with local production realities.

[0126] 2. Meteorological and Soil Data

[0127] At 4 PM on April 23, 2024, the weather was sunny, the field temperature was 29℃, and the relative humidity was 90%.

[0128] The soil type of the experimental field was red clay, ranging from red to reddish-brown in color, with a high clay content and a heavy clay texture; it exhibited an acidic reaction, with a pH of around 5.0. The acidity gradually increased from top to bottom; in the topsoil layer (0-0.5m), the organic matter content was 2.5%, total nitrogen 0.12%, total phosphorus 0.06%, available phosphorus 2.2ppm, and available potassium 37ppm. The experimental field was used for crop rotation with chili peppers, resulting in very few root diseases and vigorous chili pepper growth.

[0129] 3. Test reagents, dosage, and application methods

[0130] The test reagents and dosages were the same as in Example 4.

[0131] The first application of the pesticide was on April 24, 2024, when the chili peppers were in the early stage of fruit setting (54 days after transplanting). The pesticide was applied again on May 5, 2024, for a total of two applications.

[0132] 4. Community Setup

[0133] Four treatments were set up (DHCA, DHCA + Wuyi mycin, 15% flufenoxuron-methyl hydrochloride wettable powder and blank treatment), with a plot area of ​​22.5 square meters (15 meters long × 1.5 meters wide) for each treatment, and each treatment was replicated 4 times.

[0134] 5. Experimental investigation and analysis

[0135] Same as Example 4.

[0136] 6. Survey time and number of times

[0137] On April 24, 2024, at the initial stage of fruit setting in chili peppers, a pre-treatment survey was conducted and pesticides were sprayed. Eleven days after the first treatment, on May 5, 2024, a second survey was conducted. Eleven days after the second treatment, on May 16, 2024, a third survey was conducted.

[0138] 7. Test Results

[0139] The disease index and control efficacy of the plants in the field are shown in Table 6 below.

[0140] Table 6. Results of field efficacy trials of different treatments for controlling pepper virus disease.

[0141]

[0142]

[0143] This example describes the therapeutic application of the drug. Before treatment, the disease indexes for all treatments were 3.21, 3.19, and 2.6, respectively, while the blank treatment had a disease index of 3.77, indicating an overall disease incidence rate of approximately 10% in the experimental field. After two applications, the disease incidence rate in the experimental field was approximately 63%, indicating a severe disease condition. The disease indices for DHCA, DHCA + Wuyi mycin, 15% flufenoxuron-methyl hydrochloride wettable powder, and the blank treatment were 4.24, 3.25, 3.59, and 23.45, respectively. There was no significant difference in the control efficacy against pepper virus disease among the different treatments. Compared with the control agent, DHCA and DHCA + Wuyi mycin treatments showed better antiviral effects against pepper virus disease. Eleven days after the second application, DHCA achieved a control efficacy of approximately 78.76% against pepper virus disease, while DHCA + Wuyi mycin achieved a control efficacy of approximately 83%. Both the DHCA and DHCA + Wuyi mycin treatments were superior to the control, indicating that DHCA and its compound products can effectively prevent and control the occurrence of pepper virus disease (Table 6).

[0144] Example 5: Treatment trial of DHCA against pepper virus disease

[0145] 1. Test site, application time, chili variety, and management

[0146] The experimental field was set up in an open-field chili pepper field in Dongxiaohe Village, Luorong Town, Liuzhou City, Guangxi Province. The tested chili pepper variety was Tongle 23188. Before the experiment, a small number of viral diseases occurred in the experimental field. During the experiment, the daytime temperature was around 30℃ and the nighttime temperature was around 21℃. The experimental field was flat and uniformly cultivated using mulch film (soil conditions, fertilizer application, watering, etc.). The cultivation and management of the experimental field were in line with local production realities.

[0147] 2. Meteorological and Soil Data

[0148] At 4 PM on April 24, 2024, the weather was cloudy, the field temperature was 29℃, and the relative humidity was 90%.

[0149] The soil type used in the experimental field was red clay, ranging from red to reddish-brown in color, with a high clay content and a heavy clay texture; it exhibited an acidic reaction, with a pH of around 5.0. The acidity gradually increased from top to bottom; in the topsoil layer (0-0.5m), the organic matter content was 2.6%, total nitrogen 0.118%, total phosphorus 0.063%, available phosphorus 2.2ppm, and available potassium 36ppm. The experimental field was used for crop rotation of chili peppers, resulting in very few root diseases and healthy chili pepper growth.

[0150] 3. Test reagents, dosage, and application methods

[0151] Treatment agents: DHCA, developed by the Institute of Plant Protection, Chinese Academy of Agricultural Sciences, dosage 900 g / ha; Wuyi mycin, developed by the Institute of Plant Protection, Chinese Academy of Agricultural Sciences, dosage 120 g / ha.

[0152] Control agent: 40% methyl hydroxymorpholine guanidine, produced by Hebei Zhongbaolv Agricultural Products Co., Ltd.

[0153] The dosage of the test reagents and the content of the active ingredients are shown in Table 7.

[0154] Table 7 Experimental Design of Test Reagents

[0155]

[0156]

[0157] The first application of the pesticide was on April 24, 2024, when the chili peppers were in the early stage of fruit setting (52 days after transplanting). The pesticide was then applied again on May 5, 2024, and May 16, 2024, for a total of 3 applications.

[0158] 4. Community Setup

[0159] Four treatments were set up (DHCA, DHCA + Wuyi mycin, 40% methyl hydroxymorpholine guanidine and blank treatment), with a plot area of ​​18 square meters (12 meters long × 1.5 meters wide) for each treatment, and each treatment was replicated 4 times.

[0160] 5. Experimental investigation and analysis

[0161] Same as Example 4.

[0162] 6. Survey time and number of times

[0163] On April 24, 2024, during the early fruit setting stage of chili peppers, a pre-treatment survey was conducted and pesticides were sprayed. A second survey was conducted 11 days after the first treatment, on May 5, 2024. A third survey was conducted 11 days after the second treatment, on May 16, 2024.

[0164] 7. Test Results

[0165] The disease index and control efficacy of the plants in the field are shown in Table 8 below. Figure 2 As shown.

[0166] Table 8. Results of field efficacy trials of different treatments for controlling pepper virus disease.

[0167]

[0168] In this embodiment, viral disease control treatment was initiated 52 days after chili pepper transplanting, at the early fruit-setting stage, with two consecutive applications approximately 10 days apart. A district-wide survey was conducted before each application. The control efficacy of each treatment against chili pepper viral diseases in the final survey of this embodiment is as follows: 11 days after the second application, the control efficacy of DHCA and DHCA + Wuyi mycin against chili pepper viral diseases reached approximately 90%, significantly higher than the control agent, indicating that DHCA and DHCA + Wuyi mycin can effectively control the occurrence of chili pepper viral diseases (Table 8).

[0169] Example 6: Inhibitory effect of DHCA on other crop viral diseases

[0170] Plant material: 4-6 week old Nicotiana benthamiana (growing conditions: 25±2℃, 16h light / 8h dark).

[0171] Virus species: Tobacco Mosaic Virus (TMV): Its pathogenicity was first reported by Mayer (1886); Beijerinck (1898) proposed the concept of 'virus'; Stanley (1935, Science 81:644-645) first obtained TMV crystals and published its biochemical characteristics in the literature "Isolation of a Crystalline Protein Possessing the Properties of Tobacco-Mosaic Virus", and promised to release them to the public for 20 years. It belongs to the genus Tobamovirus of the family Virgaviridae. The virus particle is rod-shaped, about 18 nanometers in diameter and about 300 nanometers in length. It is a single-stranded RNA virus, and its genome encodes at least four proteins, including replicase, mobile protein, and capsid protein. The genome RNA is about 6395 nucleotides long and is wrapped by 2130 protein subunits. These protein subunits are arranged in a helical manner around the RNA to form a helical symmetrical capsid. It has a wide host range, infecting various Solanaceae plants such as tobacco and tomato, as well as 198 species of plants from 22 families, including monocotyledons.

[0172] Tomato Yellow Leaf Curl Virus (ToMV): First described by Ainsworth (1935, Ann. Appl. Biol. 22:55-67) in the paper "Mosaic disease of the tomato," with a commitment to release it to the public for 20 years; its complete genome sequence has been entered into GenBank NC_002692.1. Representative isolates in China, CH1 and CH2, have been published in the *Acta Phytopathologica Sinica* (1990), with a note indicating that they are available to the public upon request. Belonging to the genus *Golden Mosaic Virus* of the family Geminiviridae, the virus particles are twinned granules, composed of two nearly twinned spheres, approximately 22 nanometers in diameter, and lack an envelope. The genome is a single-stranded circular DNA (ssDNA), typically containing multiple open reading frames, encoding proteins involved in viral replication, transcription, motility, and pathogenicity. It has a wide host range, affecting various Solanaceae economic crops such as tomatoes and peppers.

[0173] Turnip mosaic virus (TuMV): First publicly described by Yoshii (1963, Ann. Phytopathol. Soc. Jpn. 28:226-233), published in the paper "Studies on turnip mosaic virus," with a commitment to release it publicly for 20 years; its complete genome sequence has been entered into GenBank NC_002509.1. The representative isolate in China, C1, is a filamentous, non-enveloped virus, typically 700-800 nanometers in length. The genome is a single-stranded, positive-sense RNA (ssRNA) containing a large open reading frame encoding a polyprotein, which, after cleavage, produces multiple functional proteins, such as capsid proteins and replicase. It primarily affects cruciferous plants, but can also infect some other families. Symptoms include mosaic patterns on leaves, i.e., yellow-green patches; in severe cases, leaves become wrinkled and deformed, plant growth is stunted, and yield and quality decrease. The virus can be transmitted non-persistently by aphids and through sap contact.

[0174] Prepare 1L of Agrobacterium inoculation buffer. Take some Agrobacterium bacteria stored at -80℃ and streak them on an antibiotic-treated plate, then incubate at 28℃. Pick out the newly grown strain and place it in 4mL of LB broth with antibiotics, then incubate on a shaker at 28℃ and 220rpm for 16h. Centrifuge the bacterial suspension at 4000rpm for 10min, discard the supernatant, resuspend the bacteria in 1mL of Agrobacterium inoculation buffer, then mix with the Agrobacterium inoculation buffer and adjust the OD values: adjust the OD value to 0.4 for Tomato Yellow Leaf Curl Virus (ToMV), 0.8 for Tobacco Mosaic Virus (TMV), and 0.4 for Turnip Mosaic Virus (TuMV). Inject the inoculation solution into the tobacco leaves using a 1mL syringe (avoiding the midrib) to complete the inoculation on the underside of the leaves. Observe the virus infection under UV light 48 hours after inoculation. Take photos based on the fluorescence display to preliminarily evaluate the virus control effect. The results are as follows: Figure 3 As shown.

[0175] The results showed that spraying DHCA could significantly inhibit the occurrence of viruses, and it was effective against tomato yellow leaf curl virus, tobacco mosaic virus, and turnip mosaic virus, indicating that DHCA has a good inhibitory effect on the control of RNA viruses.

[0176] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. Application of 1,5-dioxo-2,3,4,5-tetrahydropyrrolo[1,2-a]quinazoline-3a(1H)-carboxylic acid in antiviral activity against plant viruses.

2. The application according to claim 1, characterized in that, The plant viruses include one or more of the following: cucumber mosaic virus, pepper mild mottle virus, cucumber green mottle mosaic virus, tomato yellow leaf curl virus, tobacco mosaic virus, and turnip mosaic virus. 3.1,5-Dioxo-2,3,4,5-Tetrahydropyrrolo[1,2-a]quinazolin-3a(1H)-carboxylic acid in the preparation of antiviral agents for plants.

4. The application according to claim 3, characterized in that, The plant viruses include one or more of the following: cucumber mosaic virus, pepper mild mottle virus, cucumber green mottle mosaic virus, tomato yellow leaf curl virus, tobacco mosaic virus, and turnip mosaic virus.

5. An antiviral agent for plants, characterized in that, The active ingredient of the formulation includes 1,5-dioxo-2,3,4,5-tetrahydropyrrolo[1,2-a]quinazoline-3a(1H)-carboxylic acid.

6. The formulation according to claim 5, characterized in that, The active ingredient also includes Wuyi mycin.

7. The formulation according to claim 5, characterized in that, The plant viruses include one or more of the following: cucumber mosaic virus, pepper mild mottle virus, cucumber green mottle mosaic virus, tomato yellow leaf curl virus, tobacco mosaic virus, and turnip mosaic virus.

8. The formulation according to claim 5, characterized in that, The formulation also includes excipients.

9. The use of the formulation according to any one of claims 5-8 in the prevention and control of plant viral diseases or in the preparation of formulations for the prevention and control of plant viral diseases.

10. A method for controlling plant viral diseases, characterized in that, The steps include treating plants with 1,5-dioxo-2,3,4,5-tetrahydropyrrolo[1,2-a]quinazoline-3a(1H)-carboxylic acid or the formulation according to any one of claims 5-8.

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