Application of calendula officinalis extract in prevention and treatment of sclerotiniose of brassicaceous vegetables

Abstract

The invention discloses application of a calendula officinalis extract in prevention and treatment of sclerotiniose of brassicaceous vegetables. A preparation method of the calendula officinalis extract comprises the steps of calendula officinalis raw material treatment, organic solvent extraction and concentration. The calendula officinalis extract can obviously inhibit the activity of brassicaceous vegetable sclerotinia sclerotiorum, is a plant extract with good biocontrol effect and no pollution, and has wide development and application prospects.

Description

Technical Field

[0001] The invention relates to the technical field of agricultural planting disease prevention and control, and in particular to application of a calendula extract in preventing and controlling sclerotinia rot of cruciferous vegetables. Background Art

[0002] Cruciferae ( Brassicaceae ) vegetables include Chinese cabbage, Chinese cabbage, Chinese mustard greens and many other vegetables. They are widely planted in my country, with short cultivation cycles and high yields. They are important vegetable crops in my country. Sclerotinia is a fungal disease that harms cruciferous vegetables worldwide. Its pathogen is Sclerotinia sclerotiorum. Sclerotiniasclerotiorum ), which is mainly transmitted through soil, develops rapidly, causes serious harm, and has a devastating impact on the vegetable industry.

[0003] Calendula officinalis L., also known as marigold, golden marigold, etc., is an annual herbaceous plant of the genus Calendula in the Asteraceae family. Calendula is widely used and can be used as a Chinese herbal medicine. It has anti-inflammatory, antioxidant, and antibacterial effects. It is often used as a raw material for Chinese medicine and cosmetics for antibacterial and anti-inflammatory effects. At present, it has been reported that marigold extract has antibacterial effects on animal pathogens including Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, and Salmonella, but there are no reports on the antibacterial effect on plant pathogens, and there are no reports on the antibacterial effect on cruciferous vegetables.

[0004] In the existing technology, the inhibition of plant pathogens is mainly based on spraying pesticides. Pesticides are highly targeted and will not cause harm to non-target organisms, but they have obvious problems, such as long-term harm to the soil, ecological environment, and human and animal health.

[0005] In addition, the use of biological agents to prevent plants from being infected with Sclerotinia sclerotiorum is also a research and development hotspot. For example, patent CN201710420782.8 discloses a biological agent for preventing and controlling cabbage sclerotinia. The pH value of the biological agent is in the range of 5.5-6.5, and includes the following components by volume: 33.3-75 parts of straw pyrolysis liquid and 66.7-25 parts of bacillus fermentation liquid, wherein the bacillus fermentation liquid is a sterile filtrate obtained by centrifugation of the bacillus culture liquid, and the bacillus is a bacillus separated from soybean paste; the bacillus is methyl bacillus, bacillus subtilis or bacillus; the straw pyrolysis liquid is prepared by the following steps: crushing the straw and pyrolyzing it at 450-550°C to obtain bio-oil; stirring and mixing the bio-oil with water and letting it stand, distilling the obtained aqueous phase at normal pressure, collecting the distillate at 95-120°C, which is the straw pyrolysis liquid; the straw is rice straw, corn straw or soybean straw. The biological agent has a significant antibacterial effect on cabbage sclerotinia fungus, can effectively prevent and control cabbage sclerotinia fungus and the diseases caused by it, and is a non-toxic and residue-free biological agent. However, this biological agent needs to be in a slightly acidic pH environment (5.5~6.5) to work. If the pH environment of the soil is not within this range, the final antibacterial effect will be greatly reduced, and the preparation process of this biological agent is complicated.

[0006] Therefore, it is of great significance to study new components that inhibit Sclerotinia sclerotiorum, as the use of pesticides for the current cruciferous vegetable Sclerotinia sclerotiorum can easily affect the crop soil and ecological environment, and the existing bacterial agents are sensitive to soil pH, their use areas are limited, and they are difficult to promote and apply on a large scale. Summary of the invention

[0007] The purpose of the present invention is to provide an application of a calendula extract in preventing and treating cruciferous vegetable sclerotinia disease, so as to solve the problems that existing pesticides are prone to affect crop soil and ecological environment, the existing biological microbial agent preparation process is complicated, and is relatively sensitive to the pH of the use environment, and the soil in different regions is prone to different prevention and control effects when used.

[0008] The purpose of the present invention is achieved through the following technical solutions: The invention discloses a use of a calendula extract in preparing a preparation for preventing and treating sclerotinia disease of cruciferous vegetables, wherein the sclerotinia disease of cruciferous vegetables includes sclerotinia disease of Chinese mustard and sclerotinia disease of Chinese cabbage.

[0009] Furthermore, the preparation method of the calendula extract comprises: calendula raw material processing, organic solvent extraction, and concentration.

[0010] Furthermore, the preparation method of the calendula officinalis extract comprises: (1) Processing of Calendula officinalis raw materials: The petals of Calendula officinalis are washed with water, dried and crushed, and sieved through a 40-mesh sieve to obtain Calendula officinalis powder; (2) Organic solvent extraction: add organic solvent to the calendula powder for impregnation, and filter after impregnation; (3) Concentration: The filtrate after filtering in step (2) is evaporated and concentrated to prepare a calendula officinalis extract.

[0011] Furthermore, the organic solvent used in step (2) is ethanol.

[0012] Further, ethanol is 95% ethanol. Here, 95% ethanol refers to 95% ethanol aqueous solution.

[0013] Furthermore, the soaking method in step (2) is: adding 10 times the volume of ethanol to the calendula powder to fully soak it, shaking it at a constant temperature of 30° C. for 8-24 hours, and filtering to obtain a filtrate and a filter residue.

[0014] Furthermore, the number of impregnations is 3 times, and the specific impregnation method is as follows: first impregnation: add 10 times the volume of ethanol to the calendula powder to fully soak it, oscillate at a constant temperature of 30°C for 24 hours, and filter to obtain the first filtrate and the first filter residue; second impregnation: add 5-10 times the volume of ethanol to the first filter residue to fully soak it, oscillate at a constant temperature of 30°C for 12 hours, and filter to obtain the second filtrate and the second filter residue; third impregnation: add 5-10 times the volume of ethanol to the second filter residue to fully soak it, oscillate at a constant temperature of 30°C for 8 hours, and filter to obtain the third filtrate and the third filter residue.

[0015] Furthermore, the temperature of evaporation and concentration in step (3) is 45-55°C.

[0016] A method for preventing and controlling sclerotinia of cruciferous vegetables comprises contacting leaves of cruciferous vegetables with a calendula extract for prevention and control.

[0017] The beneficial effects of the present invention are: 1. The present invention provides an application of a calendula extract in preventing and treating sclerotinia rot of cruciferous vegetables. The calendula extract can inhibit the activity of Sclerotinia sclerotiorum of cruciferous vegetables, effectively prevent and treat sclerotinia rot of cruciferous vegetables caused by Sclerotinia sclerotiorum, and provide a new idea for the subsequent development of preparations of calendula extract for preventing and treating sclerotinia rot of cruciferous vegetables.

[0018] 2. The present invention provides an application of a calendula extract in preventing and treating sclerotinia of cruciferous vegetables. The calendula extract is applied to inhibit the activity of sclerotinia of cruciferous vegetables. The calendula extract is a plant secondary metabolite and can participate in the natural material and energy metabolism. When applied to plants or the environment, it will not cause biological enrichment, environmental pollution, or residue, and is not affected by the pH of the soil environment.

[0019] 3. The present invention provides an application of a calendula extract in preventing and treating cruciferous vegetable sclerotinia disease. The extract is prepared using calendula as a raw material. The raw material source is wide, the price is low, the production cost is low, the production process is simple, and it is suitable for production of various scales. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a diagram showing the antibacterial effect of the Calendula officinalis extract on Sclerotinia sclerotiorum according to Example 2 of the present invention; Figure 2 This is a graph showing the effect of the Calendula officinalis extract of Example 3 of the present invention on the prevention and treatment of Brassica chinensis sclerotinia; Figure 3 This is a diagram showing the effect of the Calendula officinalis extract of Example 4 of the present invention on the prevention and treatment of Sclerotinia sclerotiorum of Brassica juncea. DETAILED DESCRIPTION

[0021] Definitions of terms used in the present invention: Unless otherwise stated, the initial definitions provided for the terms in this document apply to the terms throughout the specification; for terms that are not specifically defined in this document, the meaning that a person skilled in the art would give them should be given based on the disclosure and context.

[0022] The reagents and equipment used in the present invention are all known products or obtained by purchasing commercially available products. The pathogens used in the following embodiments are described as follows: Sclerotiniasclerotiorum ), provided by the Vegetable Institute of Ningbo Academy of Agricultural Sciences. The public can contact the unit to obtain the corresponding pathogens. The biological material is only used to repeat the relevant experiments of the present invention and cannot be used for other purposes.

[0023] In order to more clearly illustrate the technical solution of the present invention, the specific implementation methods are described as follows: Embodiment 1 Preparation of Calendula officinalis Extract (1) Wash the petals of calendula officinalis with water, dry them, and then crush them. Then, sieve them through a 40-mesh sieve to obtain calendula officinalis powder.

[0024] (2) The extraction was performed by three immersion methods. Specifically, 50 g of calendula powder was weighed and placed in a sealed conical flask, and then fully immersed in 10 times the volume of 95% ethanol, and then oscillated at a constant temperature of 30°C and 120 r / min for 24 h. After the oscillation, the first filtrate and the first filter residue were obtained by vacuum filtration. Five times the volume of 95% ethanol was added to the first filter residue and fully immersed, and then oscillated at a constant temperature of 30°C and 120 r / min for 12 h. After the oscillation, the second filtrate and the second filter residue were obtained by vacuum filtration. Five times the volume of 95% ethanol was added to the second filter residue and fully immersed, and then oscillated at a constant temperature of 30°C and 120 r / min for 8 h. After the oscillation, the third filtrate and the third filter residue were obtained by vacuum filtration.

[0025] (3) The three filtrates obtained by the three immersion methods in step (2), namely the first filtrate, the second filtrate and the third filtrate, are combined by rotary evaporation and concentrated by evaporation at 50° C. to obtain a calendula extract with a concentration of 1 g / mL. The 1 g / mL calendula extract herein means that 1 ml of calendula extract contains 1 g of calendula sample material. The calendula sample material herein refers to the calendula petal sample before washing in the above step (1).

[0026] Embodiment 2 Determination of antibacterial activity of Calendula officinalis extract against Sclerotinia sclerotiorum of cruciferous vegetables The antibacterial activity of Sclerotinia sclerotiorum on cruciferous vegetables was determined by growth rate method.

[0027] Preparation of Calendula officinalis extract verification solution: Using distilled water as solvent, dissolve the 1 g / mL Calendula officinalis extract prepared in Example 1 to a final concentration of 10% Calendula officinalis extract (0.1 g / mL) for testing, where 0.1 g / mL means that 1 ml of Calendula officinalis extract contains 1 g of Calendula officinalis sample substance.

[0028] The prepared Calendula officinalis extract was sterilized and filtered through a 0.22 μm microporous filter membrane. The PDA medium was heated and melted and then cooled to about 50°C. The culture plates were poured into 15 ml plates each. Different volumes (750 μl, 500 μl, 250 μl, 100 μl, 50 μl) of Calendula officinalis extract verification solution were added and mixed evenly, so that the final concentrations of Calendula officinalis extract in each culture plate were: 4.76 mg / ml, 3.23 mg / ml, 1.64 mg / ml, 0.66 mg / ml, 0.33 mg / ml, respectively. After solidification, the Cruciferae vegetable Sclerotinia sclerotiorum expanded on PDA was cut with a 6 mm diameter puncher and transferred to the center of the culture medium with Calendula officinalis alcohol extract. Each concentration treatment was repeated for 3 plates, and the Cruciferae vegetable Sclerotinia sclerotiorum cultured on the culture medium with sterile distilled water was used as the control.

[0029] The culture was placed at a constant temperature of 20°C. On the third day, the colony diameter was measured by the ruler cross method to obtain the inhibition rate of different concentrations of Calendula officinalis extract on the growth of Cruciferae Sclerotinia sclerotiorum. The inhibition rate calculation formula is: Inhibition rate (%) = (control group colony diameter - treatment group colony diameter) / treatment group colony diameter × 100%. The specific antibacterial effect of Calendula officinalis extract on Cruciferae Sclerotinia sclerotiorum is as follows: Figure 1 The specific results of the inhibition rate of cruciferous vegetables against the growth of Sclerotinia sclerotiorum are shown in Table 1. Figure 1 CK in Table 1 means the control experimental group.

[0030] Table 1 Inhibition of different concentrations of Calendula officinalis extract on the growth of Sclerotinia sclerotiorum on cruciferous vegetables Calendula officinalis extract mass concentration (mg / ml) Colony diameter (cm) Antibacterial rate (%) 4.76 1.02±0.23 88.37±2.62 3.23 1.84±0.33 79.07±3.83 1.64 2.77±0.55 67.44±6.40 0.66 5.73±0.42 33.72±4.84 0.33 7.53±0.34 12.79±3.92 0 (CK) 8.60 / According to the results in Table 1, the antibacterial effect of 4.76 mg / ml of Calendula officinalis extract can reach 88.37%.

[0031] Embodiment 3 To test the efficacy of Calendula officinalis extract on the control of Sclerotinia sclerotiorum of Chinese cabbage.

[0032] The in vitro leaf method was used to detect the control effect of calendula extract on sclerotinia rot of Chinese cabbage. The Chinese cabbage variety was Yongqing No. 8, a variety bred by Ningbo Academy of Agricultural Sciences. Place absorbent paper in a rectangular plastic dish and spray water to soak it. Select the leaves of Chinese cabbage in the mature stage and place them on the absorbent paper. Inoculate a 6mm mycelium block of Sclerotinia sclerotiorum in the center of the leaf. Use a pipette to draw 300μl of the calendula extract verification solution prepared in Example 2 (that is, 10% calendula extract) and drop it on the mycelium block. Add clean water as a control. Each treatment was repeated 3 times. Cover the treated plastic dish with plastic wrap to keep it moist, and place it in an incubator at 20°C. After 48 hours, the diameter of the sclerotinia lesions was counted. The specific control effect of calendula extract on sclerotinia rot of Chinese cabbage is shown in the figure below. Figure 2 The specific results of the sclerotinia lesion diameter are shown in Table 2 below. Figure 2 CK in Table 2 means control group.

[0033] Table 2 The effect of Calendula officinalis extract on the control of Brassica chinensis sclerotinia Processing Name Diameter of lesion (cm) 10% Calendula officinalis extract 0 CK 4.56±0.34 According to the results in Table 2, after treatment with 10% calendula extract, the diameter of the sclerotinia lesions of Pakchoi was 0 cm, showing a good control effect.

[0034] Embodiment 4 To test the efficacy of Calendula officinalis extract on Sclerotinia sclerotiorum of Brassica juncea.

[0035] The in vitro leaf method was used to detect the control effect of calendula extract on sclerotinia sclerotiorum of Chinese potherb mustard. The Chinese potherb mustard variety was Yongxue No. 5, a variety bred by Ningbo Academy of Agricultural Sciences. Place absorbent paper in a rectangular plastic dish and spray water to soak it. Select leaves of Chinese potherb mustard in the mature stage and place them on the absorbent paper. Inoculate a 6mm mycelium block of Sclerotinia sclerotiorum in the center of the leaf. Use a pipette to draw 300μl of the calendula extract verification solution prepared in Example 2 (that is, 10% calendula extract) and drop it on the mycelium block. Add clean water as a control. Each treatment was repeated 3 times. Cover the treated plastic dish with plastic wrap to keep it moist, and place it in an incubator at 20°C. After 48 hours, the diameter of the sclerotinia lesions was counted. The specific control effect of calendula extract on sclerotinia sclerotiorum is shown in the figure below. Figure 3The specific results of the sclerotinia lesion diameter are shown in Table 3 below. Figure 3 CK in Table 3 means control group.

[0036] Table 3 The effect of Calendula officinalis extract on the control of Sclerotinia sclerotiorum Processing Name Diameter of lesion (cm) 10% Calendula officinalis extract 0 CK 2.43±0.25 According to the results in Table 2, after treatment with 10% of Calendula officinalis extract, the diameter of the lesions of Sclerotinia sclerotiorum was 0 cm, showing a good control effect.

Claims

1. Use of a calendula extract in preparing a preparation for preventing and treating cruciferous vegetable sclerotinia disease, wherein the cruciferous vegetable sclerotinia disease includes sclerotinia disease of Chinese mustard and sclerotinia disease of Chinese cabbage.

2. Use of the Calendula officinalis extract according to claim 1 in preparing a preparation for preventing and treating sclerotinia sclerotiorum of cruciferous vegetables, characterized in that: The preparation method of the calendula officinalis extract comprises: calendula officinalis raw material processing, organic solvent extraction and concentration.

3. Use of the Calendula officinalis extract according to claim 2 in preparing a preparation for preventing and treating sclerotinia sclerotiorum of cruciferous vegetables, characterized in that: The preparation method of the calendula officinalis extract comprises: (1) Processing of Calendula officinalis raw materials: The petals of Calendula officinalis are washed with water, dried and crushed, and sieved through a 40-mesh sieve to obtain Calendula officinalis powder; (2) Organic solvent extraction: add organic solvent to the calendula powder for impregnation, and filter after impregnation; (3) Concentration: The filtrate after filtering in step (2) is evaporated and concentrated to prepare a calendula officinalis extract.

4. Use of the Calendula officinalis extract according to claim 3 in preparing a preparation for preventing and treating sclerotinia sclerotiorum of cruciferous vegetables, characterized in that: The organic solvent used in step (2) is ethanol.

5. Use of the Calendula officinalis extract according to claim 4 in preparing a preparation for preventing and treating sclerotinia sclerotiorum of cruciferous vegetables, characterized in that: The ethanol is 95% ethanol.

6. Use of the Calendula officinalis extract according to claim 3 in preparing a preparation for preventing and treating sclerotinia sclerotiorum of cruciferous vegetables, characterized in that: The soaking method in step (2) is as follows: add 10 times the volume of ethanol to the calendula powder and fully soak it, shake it at a constant temperature of 30° C. for 8-24 hours, and filter to obtain a filtrate and a filter residue.

7. Use of the Calendula officinalis extract according to claim 3 in preparing a preparation for preventing and treating sclerotinia sclerotiorum of cruciferous vegetables, characterized in that: The number of immersions is 3 times; The specific impregnation method is as follows: first impregnation: add 10 times the volume of ethanol to the calendula powder to fully soak it, oscillate at a constant temperature of 30°C for 24 hours, and filter to obtain the first filtrate and the first filter residue; second impregnation: add 5-10 times the volume of ethanol to the first filter residue to fully soak it, oscillate at a constant temperature of 30°C for 12 hours, and filter to obtain the second filtrate and the second filter residue; third impregnation: add 5-10 times the volume of ethanol to the second filter residue to fully soak it, oscillate at a constant temperature of 30°C for 8 hours, and filter to obtain the third filtrate and the third filter residue.

8. Use of the Calendula officinalis extract according to claim 3 in preparing a preparation for preventing and treating sclerotinia sclerotiorum of cruciferous vegetables, characterized in that: The temperature of evaporation and concentration in step (3) is 45-55°C.

9. A method for preventing and treating sclerotinia sclerotiorum of cruciferous vegetables, characterized in that: It includes using calendula extract to contact the leaves of Chinese mustard and Chinese cabbage for prevention and control.

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