Botanical biological control agent for controlling cucumber mosaic virus and application of botanical biological control agent

Abstract

The invention relates to a plant source biological control agent for controlling cucumber mosaic virus and application thereof. The plant source biological control agent is one or a combination of any two or three of garlic, wedelia chinensis and mikania micrantha. Researches show that the combination of one, any two or three of garlic, wedelia chinensis and mikania micrantha has a remarkable inhibition effect on cucumber mosaic virus, and the combined use effect of garlic, wedelia chinensis and mikania micrantha is remarkably improved. According to the invention, more safe and efficient plant source control agents are provided for controlling plant diseases caused by cucumber mosaic virus, and a foundation is laid for subsequent research and development of novel cucumber mosaic virus resistant plant source pesticides. Moreover, the garlic, the wedelia chinensis and the mikania micrantha are wide in source and low in cost, and have very good application value.

Description

Technical Field

[0001] This invention relates to the field of biological control technology for plant diseases, specifically to a plant-derived biological control agent for controlling cucumber mosaic virus and its application. Background Technology

[0002] Cucumber mosaic virus (CMV) is listed as one of the world's top ten plant viruses, and is one of the most common and economically destructive plant viruses. CMV has a wide host range, infecting more than 1200 plant species, primarily affecting legumes, cruciferous plants, cucurbits, and solanaceous crops (Palukaitis and García-Arenal, 2003). CMV's diverse transmission methods give it a strong ability to spread in the field, posing a significant challenge to disease control. Statistics show that in China, CMV infection can lead to a 25%-50% reduction in tomato yield and a 20% reduction in pepper yield. Furthermore, diseases caused by CMV also cause huge economic losses to other cash crops and agricultural crops in China, as well as to crops worldwide. With global warming, diseases caused by CMV are becoming increasingly serious and difficult to control.

[0003] Currently, compared with chemically synthesized pesticides, the control of CMV faces severe challenges. Traditional control methods are clearly insufficient. For example, the application of chemical agents is limited due to environmental safety concerns; the efficacy of biological agents fluctuates greatly, making them difficult to promote; conventional breeding cycles are long, and some methods only yield resistant varieties; existing resistance genes offer limited protection, making them vulnerable to being overcome by new strains. The current lack of specific CMV control agents and effective control methods is seriously impacting agricultural production.

[0004] Developing plant-derived pesticides using plant active ingredients is a new approach to agricultural pest control (Guan Yun et al., 2024). Plant-derived pesticides have advantages such as environmental friendliness, strong targeting, easy degradation, and low residue (Liu Shuangqing et al., 2016). These agents not only meet the requirements for safe agricultural production but are also the preferred plant protection products for organic farming and ecological agriculture, and their economic value and sustainable development potential are becoming increasingly prominent (Wang Junping, 2018). Currently, the development of plant-derived pesticides focuses on the application in controlling tobacco mosaic virus (TMV) (Lin Zhongzheng, 2012), with limited research on the control of CMV. Chinese patent CN116725041A discloses a plant-derived biocontrol agent for controlling cucumber mosaic virus, which contains one or more of the following as active ingredients: Alternanthera philoxeroides, Bidens pilosa, Rhizoma Scutellariae, Houttuynia cordata, or Mentha haplocalyx. Therefore, screening plants with inhibitory activity against CMV from herbaceous plants and garlic is of high value and can lay the foundation for the subsequent development of novel CMV-resistant plant-derived pesticides. Summary of the Invention

[0005] This invention aims to explore new plant-derived control agents against cucumber mosaic virus.

[0006] The primary objective of this invention is to provide the application of plant-derived biocontrol agents in the treatment of cucumber mosaic virus or in the preparation of products resistant to cucumber mosaic virus.

[0007] The second objective of this invention is the application of plant-derived biological control agents in the prevention and control of plant diseases caused by cucumber mosaic virus or in the preparation of products for the prevention and control of plant diseases caused by cucumber mosaic virus.

[0008] The third objective of this invention is to provide a plant-derived biological control agent for the prevention and control of cucumber mosaic virus.

[0009] A fourth objective of this invention is to provide the application of the above-mentioned biocontrol agent in the treatment of cucumber mosaic virus or in the prevention and control of cucumber mosaic virus disease.

[0010] The fifth objective of this invention is to provide a method for combating cucumber mosaic virus.

[0011] The sixth objective of this invention is to provide a method for preventing and controlling plant diseases caused by cucumber mosaic virus.

[0012] To achieve the above objectives, the present invention is implemented through the following technical solution: This invention demonstrates that one or more of garlic, wedelia triloba, and Mikania micrantha have significant inhibitory effects on cucumber mosaic virus, and the combined use significantly enhances the effect. Therefore, this invention provides the following application scheme: The present invention primarily provides the application of plant-derived biocontrol agents in the treatment of cucumber mosaic virus or in the preparation of products resistant to cucumber mosaic virus, wherein the plant-derived biocontrol agents are one, any combination of two or three of garlic, wedelia triloba, and Mikania micrantha.

[0013] The present invention also provides the application of plant-derived biocontrol agents in the control of plant diseases caused by cucumber mosaic virus or in the preparation of products for the control of plant diseases caused by cucumber mosaic virus, wherein the plant-derived biocontrol agents are one, any combination of two or three of garlic, wedelia triloba, and Mikania micrantha.

[0014] Based on this, the present invention also provides a plant-derived biological control agent for controlling cucumber mosaic virus containing any two or three of garlic, wedelia triloba, and Mikania micrantha.

[0015] Preferably, when it contains Wedelia triloba and Mikania micrantha, the mass ratio of the two is (4~6):(3~5).

[0016] More preferably, when it contains Wedelia triloba and Mikania micrantha, the mass ratio of the two is 5:4.

[0017] Preferably, when the product contains garlic, wedelia trifoliata, and Mikania micrantha, the mass ratio of the three is (4~6):(4~6):(3~5).

[0018] More preferably, when the product contains garlic, wedelia trifoliata, and Mikania micrantha, the mass ratio of the three is 5:5:4.

[0019] The application of the aforementioned biological control agents in the control of cucumber mosaic virus or in the prevention and treatment of cucumber mosaic virus disease should also be within the scope of protection of this invention.

[0020] Based on the above-mentioned control agents, the present invention also provides a method for resisting cucumber mosaic virus, which is also a method for preventing and controlling plant diseases caused by cucumber mosaic virus. Specifically, the method involves using one or more of the above-mentioned biological control agents containing garlic, wedelia triloba, and Mikania micrantha to inhibit cucumber mosaic virus and thus prevent and control plant diseases caused by cucumber mosaic virus.

[0021] In the solution provided by the present invention, the garlic is garlic cloves, and the leaves of Wedelia can be ground into coarse juice.

[0022] garlic( Allium sativum Garlic (Allium tuberosum) is the bulb of the plant *Allium chinense*, belonging to the Amaryllidaceae family. It is a perennial herb with a strong, pungent odor. The underground bulb is spherical or flattened-spherical, composed of 6-10 fleshy, lobed bulblets, covered with a dry, grayish-white or pale purplish-red membranous scale. It has several basal leaves, flat and solid, linear-lanceolate, grayish-green, about 2.5 cm wide and up to 50 cm long. It blooms with pale pink flowers in summer; the flower stalk is about 60 cm tall, erect, cylindrical, with a terminal umbel-like inflorescence, bearing 1-3 long membranous bracts; the flowers are often replaced by bulbils, usually sterile, and have long pedicels; the perianth has 6 segments, and there are 6 stamens; the ovary is superior and 3-locular. The fruit is a capsule. It is cultivated. It is distributed throughout China. Allicin in garlic is the main substance that produces its pungent flavor, which can remove fishy smells and greasiness, and awaken the taste buds. During cooking, garlic also produces various flavor substances, including sulfur compounds, adding rich layers of flavor to dishes. It is also a widely used medicinal herb, with functions of relieving indigestion, killing bacteria and parasites. It is commonly used for carbuncles, boils, scabies, pulmonary tuberculosis, whooping cough, diarrhea, and dysentery.

[0023] Insect chrysanthemum ( Sphagneticola calendulacea*Linnaeus* Pruski is a perennial herbaceous plant belonging to the genus *Linnaeus* in the family Asteraceae. Its stem is cylindrical and curved, grayish-green or pale purple with wrinkles, and sometimes fine roots at the nodes. Leaves are opposite, greenish-brown on the upper surface and grayish-green on the lower surface, both surfaces covered with short white hairs. The flower heads are solitary, with grayish-green bracts. Flowering occurs from March to September, and fruiting from July to October. *Linnaeus* is widely adaptable and highly invasive, competing with many weeds. It is not sensitive to light. It can grow in various types of poor soil, but thrives best in fertile and moist soil. The entire plant is medicinal; it is slightly bitter, sweet, and cool in nature. It is used to clear heat and detoxify, cool the blood, and stop bleeding. It is used to treat colds and fever, diphtheria, pharyngitis, tonsillitis, bronchitis, pneumonia, lung abscess, pertussis, epistaxis, hemoptysis, hematuria, dysentery, measles, hepatitis, rheumatoid arthritis, hemorrhoids, gingivitis, boils and carbuncles, and snake bites.

[0024] Mikania ( Mikania micrantha Mikania micrantha (also known as 'Kunth') is a perennial herbaceous or shrub-like climbing vine belonging to the Asteraceae family and the Mikania genus. It is one of the first invasive alien species in my country. The stems of Mikania micrantha are cylindrical, sometimes tubular, and angular; the leaves are thin, pale green, ovate-cordate or hastate, and acuminate; the panicles are terminal or lateral, with compound inflorescences branching in umbels; the flower heads are small, with white corollas, a bell-shaped throat, and long, curved teeth; the achenes are black, with scattered granular protuberances on the surface; the pappus is white when fresh. Mikania micrantha has now evolved into an ineradicable and difficult-to-eradicate harmful plant. Its seeds are small, light, and numerous, with dandelion-like pappus, allowing for three-dimensional dispersal by wind, water, and animal and human activities, achieving spread across land, sea, and air.

[0025] Therefore, garlic, wedelia triloba, and Mikania micrantha are widely available, inexpensive, environmentally friendly, and have excellent application value.

[0026] Compared with the prior art, the present invention has the following beneficial effects: This invention demonstrates that garlic, wedelia triloba, mitochondritis, and their combined use significantly inhibit CMV, with the combined effect being significantly enhanced. This invention provides more safe and efficient plant-derived biological control agents for the prevention and control of plant diseases caused by cucumber mosaic virus, laying the foundation for the subsequent development of novel CMV-resistant plant-derived pesticides. Furthermore, garlic, wedelia triloba, and mitochondritis are widely available and inexpensive, possessing significant application value. Attached Figure Description

[0027] Figure 1 Symptoms of CMV inoculation with zucchini in the aloe vera treatment group: a: negative control; b: CMV control; c: aloe vera treatment.

[0028] Figure 2Symptoms of CMV inoculation in zucchini in the cottonseed seed treatment group: a: negative control; b: CMV control; c: cottonseed seed treatment.

[0029] Figure 3 Symptoms of CMV inoculation of zucchini in the purslane-treated group: a: negative control; b: CMV control; c: purslane treatment.

[0030] Figure 4 Symptoms of CMV inoculation in zucchini in the oleander treatment group: a: negative control; b: CMV control; c: yellow oleander treatment.

[0031] Figure 5 Symptoms of CMV inoculation of zucchini in the taro treatment group: a: negative control; b: CMV control; c: taro treatment.

[0032] Figure 6 Symptoms of CMV inoculation of zucchini in the Wedelia candel treatment group: a: negative control; b: CMV control; c: Wedelia candel treatment.

[0033] Figure 7 Symptoms of CMV inoculation in zucchini treated with Ageratum conyzoides: a: negative control; b: CMV control; c: Ageratum conyzoides treatment.

[0034] Figure 8 Symptoms of CMV inoculation with zucchini in the Mikania treatment group: a: negative control; b: CMV control; c: Mikania treatment.

[0035] Figure 9 Symptoms of CMV inoculation in zucchini in the garlic treatment group: a: negative control; b: CMV control; c: garlic treatment.

[0036] Figure 10 Symptoms of CMV inoculation in zucchini in the mixed treatment group of Wedelia trifoliata and Mikania micrantha, and in the CMV inoculation group of zucchini in the triple treatment group of garlic, Wedelia trifoliata and Mikania micrantha: a: negative control; b: CMV control; c: mixed treatment of Wedelia trifoliata and Mikania micrantha; d: mixed treatment of garlic, Wedelia trifoliata and Mikania micrantha.

[0037] Figure 11 CMV concentrations of zucchini in different treatment groups on day 14 after inoculation; Note: Error bars represent the standard error of biological replication; *: P <0.05; **: P <0.01. Detailed Implementation

[0038] To make the objectives, technical solutions, and advantages of this application clearer, the following detailed description is provided in conjunction with the accompanying drawings and embodiments.

[0039] It should be understood that the specific embodiments described herein are merely illustrative of this application and are not intended to limit this application.

[0040] Unless otherwise specified, the reagents, methods and equipment used in this invention are conventional reagents, methods and equipment in this technical field.

[0041] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0042] Example 1: Inhibitory effects of different plants on CMV I. Materials and Methods 1. Experimental materials Zucchini seeds were purchased from Beijing Zhinong Tiandi Network Technology Co., Ltd.

[0043] The seeds of *Ceiba speciosa* were collected from the campus of South China Agricultural University.

[0044] Aloe vera, purslane, yellow oleander, taro, wedelia triloba, ageratum, and mikanella were all grown using leaves, and were all collected from within the campus of South China Agricultural University.

[0045] The garlic was purchased from the South China Agricultural University market.

[0046] CMV-infected leaves were obtained by inoculating zucchini with CMV virus preserved by the Plant Virus Research Laboratory of South China Agricultural University.

[0047] 2. Experimental Methods When planting zucchini seedlings, inoculate uniformly growing zucchini seedlings when the seedlings have developed two cotyledons and are fully expanded. Inoculate using the standard sap friction method, grouping them as follows: (1) Prepare the inoculation solution: Nine treatment groups: Aloe vera, purslane, yellow oleander, taro, wedelia triloba, Ageratum conyzoides, Mikania micrantha, garlic cloves, and Bombax ceiba seeds were ground into crude juices; then each crude juice was ground together with CMV diseased leaves at a ratio of (8~10) mL: 1 g to obtain the inoculation solution for each group.

[0048] CMV control group: CMV diseased leaves were ground to obtain crude sap, which was used as inoculation solution.

[0049] Negative control group: Phosphate buffer was used as the inoculum.

[0050] A small amount of phosphate buffer was added during the grinding process described above.

[0051] (2) Inoculation: Each group of inoculum was inoculated into healthy zucchini leaves that were growing uniformly.

[0052] Each group consisted of 10 zucchini plants, and each treatment was repeated 3 times.

[0053] (3) Observational and statistical results: Place the inoculated zucchini in a sunny location and water it regularly.

[0054] Six days after inoculation, leaves from the same position were selected for observation, and the disease incidence in zucchini was recorded.

[0055] II. Experimental Results 1. The inhibitory effect of aloe vera on CMV The aloe vera treatment group showed curled and slightly wrinkled leaf edges, exhibiting obvious mosaic symptoms; the negative control group showed normal leaf growth; the CMV control group showed chlorosis and more severe mosaic symptoms. Figure 1 This indicates that aloe vera has no significant inhibitory effect on CMV.

[0056] 2. The effect of Ceiba speciosa seeds on CMV The leaves of the *Ceiba speciosa* seed treatment group showed slight yellowing at the leaf margins and mosaic patterns; the leaves of the negative control group grew normally; the leaves of the CMV control group showed chlorosis and more severe mosaic patterns. Figure 2 This indicates that *Ceiba speciosa* seeds have no significant inhibitory effect on CMV.

[0057] 3. The effect of purslane on CMV Purslane treatment group plants were stunted, with small and wrinkled leaves; severely affected leaves even died. The negative control group showed normal leaf growth. The CMV control group showed chlorosis and severe mosaic patterns. Figure 3 This indicates that a certain substance in purslane can inhibit the growth of zucchini and even cause its death. Its effect on CMV needs further observation.

[0058] 4. The effect of yellow oleander on CMV The yellow oleander treatment group showed stunted growth, small and wrinkled leaves, and some leaves even died; the negative control group showed normal leaf growth; the CMV control group showed chlorosis and severe mosaic patterns. Figure 4 This indicates that a certain substance in yellow oleander can inhibit the growth of zucchini and even cause its death. Its effect on CMV needs further observation.

[0059] 5. The impact of taro on CMV The leaves of the Alocasia treatment group showed severe mosaic patterns and significant leaf wrinkling; the leaves of the negative control group grew normally; the leaves of the CMV control group showed chlorosis and severe mosaic patterns, with wrinkled leaf edges. Figure 5 This indicates that taro does not have a significant inhibitory effect on CMV.

[0060] 6. The inhibitory effect of Wedelia triloba on CMV The leaves of the *Wedelia candel* treatment group showed slight mosaic symptoms; the leaves of the negative control group grew normally; while the mosaic symptoms were more severe in the CMV control group. Figure 6This indicates that *Wedelia triloba* has a significant inhibitory effect on CMV.

[0061] 7. The effect of Ageratum conyzoides on CMV The leaves of the Ageratum conyzoides treatment group showed symptoms such as mosaic patterns, chlorosis, and leaf curling. Figure 7 The negative control group showed normal leaf growth; the CMV control group showed chlorosis, curled leaf edges, smaller leaves, and more severe symptoms. This indicates that *Ageratum conyzoides* has no significant inhibitory effect on CMV.

[0062] 8. The inhibitory effect of Mikania micrantha on CMV The zucchini treatment group showed milder mosaic symptoms; the negative control plants grew normally; the CMV control group showed obvious mosaic symptoms and stunted growth. Figure 8 This indicates that Mikania micrantha has a significant inhibitory effect on CMV.

[0063] 9. The inhibitory effect of garlic on CMV The garlic treatment group showed milder mosaic symptoms in zucchini; the negative control group showed normal leaf growth; the CMV control group showed obvious mosaic symptoms and stunted growth. Figure 9 This indicates that garlic has a significant inhibitory effect on CMV.

[0064] The results in summary indicate that garlic, wedelia triloba, and Mikania micrantha significantly inhibited CMV without adversely affecting zucchini growth; while aloe vera, taro, Bombax ceiba seeds, and Ageratum conyzoides did not significantly inhibit CMV, and purslane and yellow oleander inhibited zucchini growth.

[0065] Example 2: Inhibitory effect of different compositions on CMV I. Materials and Methods 1. Experimental Materials and Methods The crude juice in Example 1 was replaced with crude juice obtained by mixing Wedelia triloba and Mikania micrantha in a mass ratio of 5:4, and this was designated as the Wedelia triloba and Mikania micrantha mixed treatment group; The crude juice in Example 1 was replaced with crude juice obtained by mixing garlic, wedelia trifoliata and Mikania micrantha in a mass ratio of 5:5:4, and this was designated as the garlic, wedelia trifoliata and Mikania micrantha triple treatment group.

[0066] All other operations are the same as in Example 1.

[0067] 2. ELISA testing The CMV concentration in zucchini was measured on day 14 post-inoculation using an indirect ELISA method (referencing the method of Rao Xueqin and Lan Cuiyu (2003)). Each sample was tested twice. When the sample OD... 490nm Value ≥ negative control OD 490nm If the value is twice that of the positive sample, the sample is considered positive.

[0068] 3. Calculation formulas and statistical analysis The symptom grading of zucchini was based on Liu Junfeng's (2012) disease grading standards, and the disease index was calculated. The following formulas were used to calculate the zucchini disease incidence rate and disease index: Incidence rate (%) = Number of infected plants / Total number of plants × 100%; Disease index = ∑ (number of diseased leaves at each level × representative value at each level) / (total number of leaves surveyed × highest representative value at the highest level) × 100.

[0069] Statistical data are expressed as mean ± standard error (SE) of three replicates. Analysis of variance was performed using Duncan's multiple range test.

[0070] II. Experimental Results (1) Day 6 after vaccination: The negative control group showed normal plant growth and flat leaves. The CMV control group plants showed obvious mosaic patterns and stunted growth, with a disease incidence rate of 100% and a disease index of 67.2. The zucchini in the mixed treatment group of Wedelia candel and Mikania micrantha only showed mild mosaic symptoms. Figure 10 The incidence rate was 75%, and the disease index was 25.9. The disease index was significantly different from that of the CMV control group, indicating that the disease was significantly controlled. The zucchini in the triple treatment group of garlic, wedelia trifoliata, and Mikania micrantha showed only milder mosaic symptoms. Figure 10 The incidence rate was further reduced to 53.3%, and the disease index was only 18.9, which was not only significantly lower than the CMV control group, but also significantly lower than the mixed treatment group of Wedelia candel and Mikania micrantha.

[0071] The results indicate that the mixed treatment of Wedelia candel and Mikania micrantha, as well as the triple treatment of garlic, Wedelia candel and Mikania micrantha, all have significant inhibitory effects on CMV, and the triple treatment of garlic, Wedelia candel and Mikania micrantha is more effective than the mixed treatment of Wedelia candel and Mikania micrantha.

[0072] (2) To quantitatively analyze CMV concentration, the CMV concentration in zucchini treated with different CMV inhibitory effects was determined by indirect ELISA on day 14 after inoculation. The results showed that, compared with the CMV control group, the garlic treatment group, the Mikania micrantha treatment group, and the mixed treatment group of Wedelia trifoliata and Mikania micrantha showed significant differences at the 0.05 level. The Wedelia trifoliata treatment group and the triple mixed treatment group of garlic, Wedelia trifoliata, and Mikania micrantha showed significant differences at the 0.01 level. The OD values ​​of different treatment groups were... 490nm The values ​​were all twice that of the negative control. Figure 11 The results indicate that different treatments are beneficial for controlling CMV, with the triple treatment of garlic, wedelia trifoliata, and Mikania micrantha showing the best effect in controlling CMV.

[0073] The foregoing examples are merely illustrative, used to explain some features of the method described in this invention. The appended claims are intended to claim the broadest possible scope, and the embodiments presented herein are demonstrated by the applicant's actual experimental results. Therefore, the applicant intends that the appended claims are not limited by the selection of examples illustrating the features of the invention. Some numerical ranges used in the claims also include sub-ranges within them, and variations within these ranges should also be interpreted as being covered by the appended claims where possible.

Claims

1. The application of plant-derived biocontrol agents in the treatment of cucumber mosaic virus or in the preparation of products resistant to cucumber mosaic virus, wherein the plant-derived biocontrol agent is one, any combination of two or three of garlic, wedelia triloba, and Mikania micrantha.

2. The application of plant-derived biological control agents in the control of plant diseases caused by cucumber mosaic virus or in the preparation of products for the control of plant diseases caused by cucumber mosaic virus, wherein the plant-derived biological control agent is one, any combination of two or three of garlic, wedelia triloba, and Mikania micrantha.

3. A plant-derived biological control agent for controlling cucumber mosaic virus, characterized in that, It contains any two or three of the following: garlic, wedelia trifoliata, and Mikania micrantha.

4. The biological control agent according to claim 3, characterized in that, When it contains Wedelia trifoliata and Mikania micrantha, the mass ratio of the two is (4~6):(3~5).

5. The biological control agent according to claim 4, characterized in that, When it contains Wedelia trifoliata and Mikania micrantha, the mass ratio of the two is 5:

4.

6. The biological control agent according to claim 3, characterized in that, When the product contains garlic, wedelia trifoliata, and Mikania micrantha, the mass ratio of the three is (4~6):(4~6):(3~5).

7. The biological control agent according to claim 6, characterized in that, The mass ratio of garlic, wedelia trifoliata, and Mikania micrantha is 5:5:

4.

8. The use of the biocontrol agent according to any one of claims 3 to 7 in the control of cucumber mosaic virus or in the prevention and control of cucumber mosaic virus disease.

9. A method for combating cucumber mosaic virus, characterized in that, The biological control agent described in any one of claims 3 to 7 is used to inhibit cucumber mosaic virus.

10. A method for preventing and controlling plant diseases caused by cucumber mosaic virus, characterized in that, Control measures may be taken using any of the biological control agents described in claims 3 to 7.

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