Application of a composite preparation of extracts from stems and leaves of tagetes erecta in preventing and treating fruit rust

Abstract

The application discloses application of a composite preparation of marigold stem and leaf extract in prevention and treatment of fruit rust. The composite preparation comprises 15-20 parts of marigold stem and leaf extract, 0.1-0.5 parts of melatonin, 0.2-1.2 parts of zinc glycinate, 0.01-0.05 parts of sodium dodecyl benzene sulfonate, 0.03-0.08 parts of organic fluorine auxiliary agent and 70-85 parts of water. The main component of the composite preparation, the marigold stem and leaf extract, is obtained by combining enzymolysis, water extraction and dimethyl sulfoxide ultrasonic extraction, and some nutrient elements required for fruit growth are added in the composite preparation, and sodium dodecyl benzene sulfonate and the organic fluorine auxiliary agent are used as dispersants and wetting agents to improve the spreading property of the composite preparation. The marigold stem and leaf extract composite preparation is creatively applied in prevention and treatment of fruit rust, the preparation method is simple, environment-friendly, the raw material cost is low, the utilization way of the marigold stem and leaf is fully developed, and a new method for green prevention and treatment of fruit rust is provided.

Description

Technical Field

[0001] This invention relates to the field of fruit rust control, specifically to the application of a compound preparation of marigold stem and leaf extracts in the control of fruit rust. Background Technology

[0002] Fruit russeting is a physiological disorder of fruit caused by adverse external conditions. It occurs when the fruit peel, stimulated by unfavorable external factors, develops tiny cracks or star-shaped breaks in the cuticle, exposing subcutaneous cells and forming a cork cambium. This leads to the formation of corky cells, ultimately resulting in a metallic, rust-like substance on the fruit surface, known as fruit russeting. The first occurrence of fruit russeting primarily happens after the fuzz has fallen from young fruit, before the waxy cuticle has formed, making the fruit highly sensitive to external conditions and prone to developing russeting. The second occurrence occurs during the fruit enlargement period, when the internal cells divide and rapidly expand. When the epidermal cells are subjected to adverse stimuli, cork forms, resulting in fruit russeting.

[0003] Fruit rust is a physiological disorder, mainly including frost rust, water rust, and chemical rust. Frost rust is primarily caused by low-temperature damage during the young fruit stage, where the pubescent hairs on the epidermal cells of the young fruit have just fallen off or not completely fallen off, leading to frost damage to the subcutaneous cells. These cells stop developing or develop at a slightly slower rate than the pulp cells, eventually forming frost rust. Water rust often occurs during the rainy season when rainwater cannot evaporate in time and remains on the stem cavity or fruit surface for a long time, causing damage to the subcutaneous cells and resulting in water rust. Chemical rust is brown and consists of many small dots forming irregular strips or lumps, with a rough texture. Improper operation, such as inappropriate pesticide formulation selection during the sensitive growth period of the fruit, excessively high pesticide concentration, large spray particles, and bagging before the pesticide residue on the fruit surface has dried, can all trigger or aggravate the occurrence of fruit rust. Therefore, it is essential to strictly regulate production operations to reduce the occurrence of fruit rust.

[0004] Currently, chemical control plays an important role in controlling fruit rust and is highly effective in agriculture, making it popular among fruit farmers. Examples of such pesticides include triadimefon, cyproconazole, and benzoylurea, as well as plant growth regulators such as gibberellin and carbendazim. However, these chemical agents all have a certain degree of toxicity, and long-term use can pollute the environment. Furthermore, pesticide residues on the surface of fruits pose a serious threat to food safety.

[0005] Marigolds, also known as stinky hibiscus, ten-thousand-lamp, and honeycomb chrysanthemum, are annual herbaceous plants belonging to the genus Marigold in the family Asteraceae. Native to the Americas, they are easy to cultivate and are now widely cultivated in my country. Marigolds are ornamental plants that bloom in summer and autumn, producing fragrant yellow or orange flowers that can be used as a fragrance. They can also be made into tea, a health beverage. The stems and leaves of marigolds mainly contain flavonoids such as borneol, coumarins, and rutin, as well as alkaloids, polysaccharides, amino acids, and vitamins, exhibiting various biological activities including antibacterial, bacteriostatic, and insecticidal properties.

[0006] The article "Effects of Application Time of Different Marigold Stem Crude Extracts on Tobacco Root-Knot Nematode Disease, Soil Chemical Properties, and Flue-Cured Tobacco Yield and Quality", Yang Lingyu, Sang Yinghua, Xu Junju, et al., Jiangxi Journal of Agricultural Sciences, 2022, 34(11), 23-28. The above article discloses the control effect of preparing a certain concentration of marigold stem ethanol extract on root-knot nematode and its effect on flue-cured tobacco yield. The extraction method involved is ethanol extraction, but it does not involve the control of fruit rust.

[0007] The article "Study on the allelopathic effects of marigold extract on seed germination and seedling growth of two vegetables", Qu Wenping, Wang Pengfei, Liu Changming, Shandong Chemical Industry, 2022, 51(1), 44-47. The above article discloses the effects of extracts obtained by ultrasonic extraction of marigold roots, stems, leaves and flowers with distilled water at room temperature on seed germination and seedling growth. It only involves one extraction solvent method and does not involve the prevention and control of fruit rust.

[0008] The article "Preparation of Extracts from Different Parts of Marigold and Their Effects on Citrus Root-knot Nematodes" by Guo Yanjun, Ji Qianhua, Jiang Hui, et al., Guangxi Plant Protection, 2022, (1), 1-6. The article disclosed the toxic effects of extracts obtained from marigold roots, stems, leaves, and flowers using sterile water and different organic solvents on citrus root-knot nematodes. The sterile water extract had the highest extraction rate but the worst toxic effect. The article did not cover the control of fruit rust.

[0009] The article “Extraction and Chemical Composition Analysis of Marigold Leaf Essential Oil”, Li Jian, Song Shuaidi, Liu Ning, et al., Food Science, 2010, 31(18), 359-362. The above article uses steam distillation to extract marigold leaf essential oil, which only involves one extraction solvent method and does not cover the prevention of fruit rust.

[0010] The article "Analysis of Flavonoid Content and Antioxidant Properties in Marigold Flowers, Leaves and Stems" by Hou Dongyan, Hui Ruihua, Liu Xiaoyuan, et al., Journal of Anshan Normal University, 2008, 10(4), 15-18. The article describes the extraction of flavonoids from marigold flowers, leaves and stems using 95% ethanol, but no further applied research was conducted.

[0011] Currently, the main methods for preparing marigold stem and leaf extracts are single water immersion extraction or organic solvent extraction, which have poor extraction effects. The compound preparation described in this invention, whose main component is marigold stem and leaf extract, employs a combination of enzymatic hydrolysis, water extraction, and dimethyl leaf ultrasonic extraction to improve the extraction rate of active ingredients. The product contains multiple active ingredients and is also fortified with nutrients needed for fruit growth, exhibiting excellent control of fruit rust and providing a new method for green control of fruit rust. Summary of the Invention

[0012] The purpose of this invention is to provide an application of a compound preparation of marigold stem and leaf extracts in the prevention and control of fruit rust. Its characteristics include a simple and environmentally friendly preparation method, low raw material costs, and full utilization of marigold stems and leaves, providing a new method for the green prevention and control of fruit rust.

[0013] To achieve the above-mentioned objectives, this invention uses marigold stems and leaves as raw materials, and obtains the main component of the compound preparation, marigold stem and leaf extract, through a combination of crushing, sieving, enzymatic hydrolysis, water extraction, and dimethyl sulfoxide ultrasonic extraction. At the same time, some nutrients required for fruit growth are added to the composition, and sodium dodecylbenzenesulfonate and organic fluorine adjuvants are used as dispersants and wetting agents to improve the spreadability of the compound preparation.

[0014] The specific technical solution steps are as follows:

[0015] Step 1: Dry the marigold stems and leaves at low temperature, then pulverize them through a 60-mesh sieve to obtain marigold stem and leaf powder;

[0016] Step 2: Mix marigold stem and leaf powder with water at a ratio of 1:40 to 1:50 until homogeneous, add 0.02 to 0.05% (by weight of powder) of cellulase for enzymatic hydrolysis to obtain the hydrolysate;

[0017] Step 3: The marigold stem and leaf enzymatic hydrolysate from Step 2 is heated and extracted at 70-80℃ for 1.5-2.5h, filtered through a filter membrane with a pore size of 0.5-1.0μm, and the filter residue is re-added with dimethyl sulfoxide and ultrasonically extracted for 1-1.5h, then filtered.

[0018] Step 4: Combine the filtrates from the two extractions, concentrate under vacuum to obtain marigold stem and leaf extract;

[0019] Step 5: Take 15-20 parts of marigold stem and leaf extract, 0.1-0.5 parts of melatonin, 0.2-1.2 parts of zinc glycine, 0.01-0.05 parts of sodium dodecylbenzenesulfonate, 0.03-0.08 parts of organofluorine adjuvant, and 70-85 parts of water, mix them evenly, and package them to obtain a compound preparation of marigold stem and leaf extract for preventing fruit rust.

[0020] Furthermore, the marigold stem and leaf extract compound preparation is characterized in that, in step three, the amount of dimethyl arugula added is 10 to 15 times the weight of the filter residue, the ultrasonic extraction power is 350 to 400 W, and the extraction temperature is 65 to 75°C.

[0021] Furthermore, the marigold stem and leaf extract compound preparation is characterized in that, in step four, the vacuum concentration temperature is 50-65°C, the concentration time is 1-2 hours, and the concentration is reduced to 1 / 4-1 / 3 of the original extract volume.

[0022] Furthermore, the marigold stem and leaf extract compound preparation is characterized in that the compound preparation is diluted 100 to 300 times and sprayed on the leaves 2 to 3 times during the flowering period and before bagging after flowering.

[0023] The main component of the compound formulation of this invention, marigold stem and leaf extract, is obtained by first adding cellulase to the marigold stems and leaves for enzymatic hydrolysis, followed by water extraction to extract the effective components. The extracted substances are mainly water-soluble flavonoid glycosides, amino acids, vitamins, and other effective components. The filtered residue is then subjected to ultrasonic extraction with dimethyl sulfoxide to extract fat-soluble flavonoids such as borneol, rutin, and coumarins, as well as alkaloids and other effective components. The final marigold stem and leaf extract exhibits various biological activities, including antibacterial, bacteriostatic, and insecticidal effects.

[0024] Melatonin, an indoleamine compound added to the compound preparation, promotes seed germination, root development, flowering, fruit setting, and fruit ripening. Zinc glycine is a chelate formed from glycine and zinc. It is a nutrient fortifier with a relatively small molecular weight and good absorption by crops. It is a new generation of safe and efficient organic chemical zinc supplement that does not inhibit the growth of other nutrients.

[0025] Sodium dodecylbenzenesulfonate can increase the adhesion of the compound preparation to the plant surface and promote the penetration of the preparation into the plant interior; organofluorine adjuvants can not only improve the spreadability of the compound preparation, form a protective film on the surface of leaves and fruits, and prolong the evaporation time of the preparation, but also have a certain effect in eliminating pests and diseases, which is very good for fruit growth.

[0026] The present invention has the following beneficial effects:

[0027] 1. This invention creatively develops a compound preparation of marigold stem and leaf extracts for the prevention and control of fruit rust. It is applied to the leaves during the flowering period and after flowering but before bagging to prevent and control fruit rust, ultimately improving fruit quality, reducing the use of fruit rust inhibitors, eliminating food safety hazards, and improving food quality and safety.

[0028] 2. The compound preparation of this invention uses marigold stems and leaves as the main raw material. The marigold stem and leaf extract is obtained by a combination of enzymatic hydrolysis, water extraction and dimethyl sulfoxide extraction. Combined with a certain amount of melatonin and zinc glycine, it can prevent fruit rust and provide certain nutrients for fruit growth.

[0029] 3. The raw materials for the compound formulation of this invention are abundant, inexpensive and readily available, with low cost, suitable for large-scale production, and the preparation method is simple and environmentally friendly. It fully utilizes the stems and leaves of marigolds, providing a new method for green prevention and control of fruit rust. Detailed Implementation

[0030] The following examples further describe specific embodiments of the present invention. These examples are only used to explain the present invention and are not intended to limit the scope of the present invention.

[0031] Example 1

[0032] A compound preparation of marigold stem and leaf extracts for preventing fruit rust, the preparation method of which includes the following steps:

[0033] Step 1: Dry the marigold stems and leaves at low temperature, then pulverize them through a 60-mesh sieve to obtain marigold stem and leaf powder.

[0034] Step 2: Mix 8 kg of marigold stem and leaf powder with 400 kg of water until homogeneous, add 1.6 g of cellulase to obtain the enzymatic hydrolysate.

[0035] Step 3: Heat the enzymatic hydrolysate from Step 2 for 2 hours, filter it through a 1.0 μm filter membrane, add 10 times the amount of dimethyl sulfoxide to the filter residue, and extract it ultrasonically for 1 hour at an extraction temperature of 70℃, then filter it.

[0036] Step 4: Combine the two extraction filtrates, concentrate under vacuum for 1.5 hours, and concentrate to 1 / 4 of the original extract volume to obtain marigold stem and leaf extract.

[0037] Step 5: Take 15 parts of marigold stem and leaf extract, 0.2 parts of melatonin, 0.6 parts of zinc glycine, and 84.2 parts of water, mix them evenly, and package them to obtain a marigold stem and leaf extract compound preparation.

[0038] Example 2

[0039] A compound preparation of marigold stem and leaf extracts for preventing fruit rust, the preparation method of which includes the following steps:

[0040] Step 1: Dry the marigold stems and leaves at low temperature, then pulverize them through a 60-mesh sieve to obtain marigold stem and leaf powder.

[0041] Step 2: Mix 10kg of marigold stem and leaf powder with 450kg of water until homogeneous, add 3g of cellulase to obtain enzymatic hydrolysate.

[0042] Step 3: Heat and extract the enzymatic hydrolysate from Step 2 for 2.5 hours. After filtration through a 1.0 μm filter membrane, add 15 times the amount of dimethyl sulfoxide to the filter residue and extract ultrasonically for 1.5 hours at an extraction temperature of 75°C. Then filter.

[0043] Step 4: Combine the two extraction filtrates, concentrate under vacuum for 2 hours, and concentrate to 1 / 4 of the original extract volume to obtain marigold stem and leaf extract.

[0044] Step 5: Take 20 parts of marigold stem and leaf extract, 0.4 parts of melatonin, 1 part of zinc glycine, and 78.6 parts of water, mix them evenly, and package them to obtain a marigold stem and leaf extract compound preparation.

[0045] Comparative Example 1

[0046] The compound preparation was prepared according to Example 1, except that the marigold stem and leaf extract was obtained by water extraction only.

[0047] The remaining technical features are the same as in Example 1.

[0048] Comparative Example 2

[0049] The compound preparation was prepared according to Example 1, except that the marigold stem and leaf extract was obtained by ultrasonic extraction using only dimethyl sulfoxide.

[0050] The remaining technical features are the same as in Example 1.

[0051] Comparative Example 3

[0052] The compound preparation was prepared according to Example 1, except that marigold extract was not added to the compound preparation.

[0053] The remaining technical features are the same as in Example 1.

[0054] Comparative Example 4

[0055] The extract of marigold stems and leaves was obtained by water extraction using distilled water from marigold stems and leaves in the reference article "Study on allelopathic effects of marigold extract on germination and seedling growth of two kinds of vegetables".

[0056] The remaining technical features are the same as in Example 1.

[0057] Comparative Example 5

[0058] The extracts of marigold stems and leaves were extracted using the sterile water-water extraction method described in the article "Preparation of extracts from different parts of marigold and their toxic effects on citrus root nematodes".

[0059] The remaining technical features are the same as in Example 1.

[0060] Comparative Example 6

[0061] Marigold stem and leaf extracts were extracted using steam distillation, referring to the article "Extraction and Chemical Composition Analysis of Marigold Leaf Essential Oil".

[0062] The remaining technical features are the same as in Example 1.

[0063] Comparative Example 7

[0064] The extract of marigold stems and leaves was obtained by 95% ethanol extraction, as described in the article "Analysis of Flavonoid Content and Antioxidant Properties in Marigold Flowers, Leaves and Stems".

[0065] The remaining technical features are the same as in Example 1.

[0066] Experiment 1

[0067] The "Golden Delicious" apple orchard in Muping District, Yantai City, Shandong Province, was selected. Rows were spaced 3.0m x 4.5m apart. Apple trees with uniform growth and free from pests and diseases were chosen as test materials. A randomized block design was used, with three trees in each block. The trees were divided into Example 1 group, Example 2 group, Comparison 1-7 groups, and a control group. The Example 1 and Comparison 2 groups used a 200-fold diluted aqueous solution of the prepared compound formulation, while the control group used only water. Foliar spraying was applied three times, once during flowering and again after flowering but before bagging. After bagging, 400 apples were randomly selected to investigate the rate of russeting, classify russeted apples into grades, and calculate the russeting index. The results are shown in Table 1.

[0068] Table 1. Effects of different compound formulations on fruit rust in Golden Delicious apples.

[0069]

[0070]

[0071] The rust-stained fruit is graded as follows: Grade 1, no rust spots; Grade 2, rust spot area ≤ 1.0 cm². 2 Rust spots ≥ 1.0 cm² 2 The apple russeting rate, russeting index, and control effect of each treatment were calculated using the following formula:

[0072] Rust rate (%) = (Number of rusted fruits / Total number of fruits) × 100

[0073] Fruit rust index (%) = ∑(number of rusted fruits at each grade × number of fruits at that grade) × 100 / (grade number × total number of fruits)

[0074] Control efficacy (%) = (Control rust rate - Treatment rust rate) × 100 / Control rust rate.

[0075] As shown in Table 1, the data from Examples 1-2 and Comparative Example 3 indicate that the marigold stem and leaf extract plays a major role in the compound formulation. The data from Examples 1-2 and Comparative Examples 1-2 and 4-7 show that the compound formulation prepared from marigold stem and leaf extracts extracted solely by water or organic solvents is significantly less effective at controlling russeting in Golden Delicious apples than the compound formulation prepared by combining water extraction with dimethyl sulfoxide. Therefore, the marigold stem and leaf extract compound formulation prepared in this invention has a significant effect on controlling russeting in Golden Delicious apples.

[0076] Experiment 2

[0077] The "Sunshine Rose" grape growing area in Qixia City, Yantai City, Shandong Province was selected, with a row spacing of 1.5m × 2.5m. Grapes with uniform growth and free from pests and diseases were chosen as test materials. A randomized block design was adopted, with 3 vines in each block, divided into Example 1 group, Example 2 group, Comparison 1-7 groups, and a control group. The formulations used in the Example 1 and Comparison groups were aqueous solutions of the corresponding prepared compound formulations diluted 200 times, while the control group used plain water. Foliar spraying was performed three times, once during flowering and again after flowering but before bagging. After removing the bags, 700 grapes were randomly picked to investigate the russeting rate, classify russeted fruit grades, and calculate the russeting index. The results are shown in Table 2.

[0078] Table 2. Effects of different compound formulations on rust on Sunflower rose fruit.

[0079]

[0080] The rust-resistant fruit is graded as follows: Grade 1, no rust spots; Grade 2, rust spot area ≤ 0.5cm². 2 Rust spots ≥ 0.5 cm² 2 The apple russeting rate, russeting index, and control effect of each treatment were calculated using the following formula:

[0081] Rust rate (%) = (Number of rusted fruits / Total number of fruits) × 100

[0082] Fruit rust index (%) = ∑(number of rusted fruits at each grade × number of fruits at that grade) × 100 / (grade number × total number of fruits)

[0083] Control efficacy (%) = (Control rust rate - Treatment rust rate) × 100 / Control rust rate.

[0084] As shown in Table 2, the data from Examples 1-2 and Comparative Example 3 indicate that the marigold stem and leaf extract plays a major role in the compound formulation. The data from Examples 1-2 and Comparative Examples 1-2 and 4-7 show that the compound formulation prepared from marigold stem and leaf extracts extracted solely by water or organic solvents is significantly less effective against russeting in Shine Muscat grapes than the compound formulation prepared by combining water extraction with dimethyl sulfoxide. Therefore, the marigold stem and leaf extract compound formulation prepared in this invention has a significant effect on controlling russeting in Shine Muscat grapes.

[0085] Experiment 3

[0086] The "Akizuki Pear" grape-growing area in Laiyang City, Yantai City, Shandong Province was selected, with a row spacing of 4.5m × 5.0m. Pear trees of uniform growth and free from pests and diseases were chosen as test materials. A randomized block design was adopted, with 3 trees in each block, divided into Example 1 group, Example 2 group, Comparison 1-7 groups, and a control group. The formulations used in the Example 1 and Comparison groups were aqueous solutions diluted 200 times with the corresponding prepared compound formulations, while the control group used plain water. Foliar spraying was applied three times, once during flowering and again after flowering before bagging. After removing the bags, 250 pears were randomly selected to investigate the russeting rate, classify russeted fruit grades, and calculate the russeting index. The results are shown in Table 3.

[0087] Table 3. Effects of different compound preparations on fruit rust in Akizuki pear.

[0088]

[0089] The rust-stained fruit is graded as follows: Grade 1, no rust spots; Grade 2, rust spot area ≤ 2.0 cm². 2 Rust spots ≥ 2.0 cm² 2 The apple russeting rate, russeting index, and control effect of each treatment were calculated using the following formula:

[0090] Rust rate (%) = (Number of rusted fruits / Total number of fruits) × 100

[0091] Fruit rust index (%) = ∑(number of rusted fruits at each grade × number of fruits at that grade) × 100 / (grade number × total number of fruits)

[0092] Control efficacy (%) = (Control rust rate - Treatment rust rate) × 100 / Control rust rate.

[0093] As shown in Table 3, the data from Examples 1-2 and Comparative Example 3 indicate that the marigold stem and leaf extract plays a major role in the compound formulation. The data from Examples 1-2 and Comparative Examples 1-2 and 4-7 show that the compound formulation prepared from marigold stem and leaf extracts extracted solely by water or organic solvents has a significantly weaker effect on preventing russeting in Akizuki pears and apples than the compound formulation prepared by combining water extraction with dimethyl sulfoxide. Therefore, the marigold stem and leaf extract compound formulation prepared in this invention has a significant effect on preventing russeting in Akizuki pears.

[0094] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. The application of a compound preparation of marigold stem and leaf extracts in the prevention and control of fruit rust, characterized in that, The compound preparation of marigold stem and leaf extracts is used for the prevention and control of rust on apple, grape and pear fruits. The preparation method of the marigold stem and leaf extract compound formulation includes the following process steps: Step 1: Dry the marigold stems and leaves at low temperature, then pulverize them through a 60-mesh sieve to obtain marigold stem and leaf powder; Step 2: Mix marigold stem and leaf powder with water at a ratio of 1:40 to 1:50 until homogeneous, add 0.02 to 0.05% (by weight of powder) of cellulase for enzymatic hydrolysis to obtain the hydrolysate; Step 3: The marigold stem and leaf enzymatic hydrolysate from Step 2 is heated and extracted at 70-80℃ for 1.5-2.5h, filtered through a filter membrane with a pore size of 0.5-1.0μm, and the filter residue is re-added with dimethyl sulfoxide and ultrasonically extracted for 1-1.5h, then filtered. Step 4: Combine the filtrates from the two extractions, concentrate under vacuum to obtain marigold stem and leaf extract; Step 5: Take 15-20 parts of marigold stem and leaf extract, 0.1-0.5 parts of melatonin, 0.2-1.2 parts of zinc glycine, and 70-85 parts of water, mix them evenly, and package them to obtain a compound preparation of marigold stem and leaf extract for preventing fruit rust.

2. The application of the marigold stem and leaf extract compound preparation according to claim 1 in the prevention and control of fruit rust, characterized in that, In step three, the amount of dimethyl pine needles added is 10 to 15 times the weight of the filter residue, the ultrasonic extraction power is 350 to 400 W, and the extraction temperature is 65 to 75℃.

3. The application of the marigold stem and leaf extract compound preparation according to claim 1 in the prevention and control of fruit rust, characterized in that, In step four, the vacuum concentration temperature is 50~65℃, the concentration time is 1~2h, and the concentration is reduced to 1 / 4~1 / 3 of the original solution volume.

4. The application of the marigold stem and leaf extract compound preparation according to claim 1 in the prevention and control of fruit rust, characterized in that, Dilute the compound preparation 100-300 times and spray the leaves 2-3 times during the flowering period and before bagging after flowering.

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