An oligosaccharide biostimulant and its application method

Abstract

This invention belongs to the field of biostimulant technology, specifically relating to an oligosaccharide biostimulant, the active ingredients of which include component A, component B and component C; component A is chitin oligosaccharide, component B is fucoidan oligosaccharide, and component C is one or more of pectin oligosaccharide, cellulose oligosaccharide, and xylooligosaccharide in any proportion. Using this biostimulant can improve the vitality of crops, increase the yield and quality of agricultural products, and at the same time solve the problems of growth inhibition, excessive growth or weak immunity caused by using a single oligosaccharide.

Description

Technical Field

[0001] This invention belongs to the field of biostimulant technology, specifically relating to an oligosaccharide biostimulant and its application method. Background Technology

[0002] For many years, abiotic stresses such as drought, flooding, low temperatures, high temperatures, and insufficient sunlight, as well as biotic stresses such as diseases, pests, and weeds, have had a significant negative impact on the yield and quality of agricultural products. While the use of fertilizers and chemical pesticides can mitigate the losses caused by these stresses to some extent, the long-term irrational use of fertilizers and pesticides poses risks to the environment and human safety. Therefore, the application of safer and more effective bioactive substances—biostimulants—has attracted attention.

[0003] Biostimulants, also known as plant growth promoters, are substances containing certain components and microorganisms. When applied to plants or their rhizosphere, these components and microorganisms stimulate the plant's natural processes, including enhancing / benefiting nutrient absorption, nutrient efficiency, resistance to abiotic stresses, and crop quality, regardless of the nutrient content. Although biostimulants are not plant hormones or nutrients, even trace amounts can stimulate plant vitality and promote growth and development.

[0004] Chitosan oligosaccharides are typically obtained from shrimp shells, crab shells, or fungal mycelia through a series of physical, chemical, and / or biological treatments. These two types of oligosaccharides are exogenous biostimulants for plants. Pectin oligosaccharides, cellulosic oligosaccharides, and xylooligosaccharides are all derived from the plant itself and are endogenous biostimulants. Both of these types of biostimulants can activate the immune response of plant cells, but their receptors on the cell membrane are different, and their effects on plant cell physiological and biochemical processes also differ significantly. Alginate-derived oligosaccharides (ADO) are small molecular weight fragments obtained from alginate in brown algae through oxidative degradation, acid hydrolysis, or degradation by lyases. ADO can promote the production of endogenous hormones in plants, increase the net photosynthetic rate, and thus promote crop growth.

[0005] Currently, scientists have discovered receptors for chitin oligosaccharides on the cell membranes of various crops such as Arabidopsis thaliana and rice, and have elucidated their mechanisms of action in detail. Oligogalacturonic acid can interact with cell wall-linked receptor-like kinases. These kinases contain extracellular, transmembrane, and intracellular domains. The extracellular domain is directly linked to cell wall components, while the intracellular domain exhibits serine / threonine protein kinase activity, thus enabling transmembrane signal transduction. Glucosamine oligosaccharides (including cellulosic oligosaccharides) were the first oligosaccharide inducers discovered, and therefore the first oligosaccharide inducers to undergo receptor research. As early as 1983, scientists used isotope labeling to label branched β-1,3-glucheptaose extracted from Phytophthora indicum and discovered its specific binding to soybean cotyledons. Subsequent studies found that glucheptaose specifically binds to soybean suspension cells, protoplasts, and root-derived cells, with this binding primarily occurring on the plasma membrane.

[0006] Plant immunity involves multiple signaling pathways, and different crops exhibit varying abilities to recognize different oligosaccharide biostimulants. Furthermore, different immune signaling pathways may exhibit effects of efficacy cancellation or superposition amplification. Therefore, studying the effects of different oligosaccharide compositions on crop immunity, growth, and other physiological and phenotypic aspects can deepen our understanding of these products from an application perspective and offers a significant opportunity to develop new products with broad immune activity and more pronounced efficacy in crops. Summary of the Invention

[0007] To address the aforementioned problems, this invention provides an oligosaccharide biostimulant and its application method. Using this biostimulant can improve crop vigor, increase agricultural product yield and quality, while simultaneously solving the problems of growth inhibition, excessively rapid growth, or weakened immunity caused by using single oligosaccharides.

[0008] In order to solve the existing problems, the inventors conducted in-depth research and repeated experiments. The results showed that when the mixture of chitosan oligosaccharide and fucoidan oligosaccharide is combined with any one or more of the three oligosaccharides, namely pectin oligosaccharide, cellulose oligosaccharide and xylooligosaccharide, it can significantly enhance the activity of the single oligosaccharide, promote crop growth, and improve the yield and quality of agricultural products.

[0009] The technical solution of the present invention is as follows:

[0010] An oligosaccharide biostimulant, the active ingredients of which include component A, component B and component C; wherein component A is chitin oligosaccharide, component B is fucoidan oligosaccharide, and component C is one or more of pectin oligosaccharide, cellulose oligosaccharide and xylooligosaccharide in any proportion.

[0011] Preferably, the stimulant comprises, by weight percentage, 1-5 wt% of component A, 1-5 wt% of component B, 1-5 wt% of component C, 1.1-6 wt% of excipients, and the balance of solvent, with a pH value of 4.0-8.0.

[0012] Preferably, the degree of polymerization of the active ingredients is 2-10. More preferably, it is 4-10.

[0013] The above-mentioned oligosaccharide biostimulants are applied by spraying on the leaves or applying to the roots of crops. Alternatively, the biostimulants and fertilizers can be applied simultaneously by spraying on the leaves or applying to the roots of crops.

[0014] Preferably, the concentration of the active ingredient in the biostimulant used on crops is 0.5-200 ppm. More preferably, the concentration of the active ingredient in the biostimulant used on crops is 20-200 ppm for foliar spraying and 10-120 ppm for root application.

[0015] Preferably, the crop is one of the following: Asteraceae, Solanaceae, Brassicaceae, Poaceae, Fabaceae, Rosaceae, Cucurbitaceae, Convolvulaceae, Chenopodiaceae, Liliaceae, Apiaceae, Malvaceae, Zingiberaceae, or Nelumbo nucifera.

[0016] Preferably, the excipients include a surfactant and a preservative, and the solvent is water. More preferably, by weight percentage, the surfactant is 1-5 wt% and the preservative is 0.1-1.0 wt%, wherein the surfactant is betaine and the preservative is sodium diacetate.

[0017] Specifically, crops can include cruciferous plants such as cabbage, kale, broccoli, cauliflower, radish, and turnip; solanaceous plants such as potato, tomato, eggplant, bell pepper, chili pepper, and tobacco; Asteraceae plants such as garland chrysanthemum, lettuce, and burdock; Cucurbitaceae plants such as watermelon, cantaloupe, pumpkin, cucumber, bitter melon, loofah, and gourd; Chenopodiaceae plants such as spinach and beet; Apiaceae plants such as carrot, celery, parsley, and parsley; Fabaceae plants such as soybean, red bean, kidney bean, broad bean, pea, winged bean, and peanut; Convolvulaceae plants such as sweet potato; Liliaceae plants such as leek, onion, garlic, and garlic; Rosaceae plants such as strawberry, apple, pear, and loquat; Malvaceae plants such as okra and cotton; Zingiberaceae plants such as ginger; Nelumbo nucifera plants such as lotus; and Gramineae plants such as corn, rice, barley, wheat, and sugarcane.

[0018] In this invention, chitosan oligosaccharides are obtained by degrading chitin, which is a polysaccharide composed of β-N-acetyl-D-glucosamine linked by β-1,4 glycosidic bonds. It is the second most abundant natural polysaccharide in nature after cellulose and is widely found in shrimp and crab shells, insect exoskeletons and fungal cell walls. Compared with chitosan oligosaccharides with a degree of polymerization of 2-3, chitosan oligosaccharides with a degree of polymerization of 4-8 have higher immune-inducing activity.

[0019] The chitosan oligosaccharides used in this invention are particularly preferred to be substances having the following chemical structure.

[0020]

[0021] In addition, it also includes substances in which the acetyl group (COCH3) in the formula is partially removed and transformed into NH2, and the proportion of such deacetylation is preferably less than 30% of the total chitosan oligosaccharide.

[0022] Applying the stimulant of this invention can produce plants or parts thereof (e.g., roots, stems, leaves, flowers, fruits, seeds, tissues, cells, etc.) with "plant vaccine" activity, thereby improving crop vigor, enhancing its resistance to biotic and abiotic stresses, and significantly improving yield, quality, and post-harvest shelf life. The biostimulant of this invention can strongly stimulate crop resistance to biotic stresses, enabling crops to maintain a good condition under multiple adversities, thus laying a solid foundation for stabilizing / improving yield and quality.

[0023] The activity of biostimulants is primarily determined by measuring the production of chitinase in plants. Other monitored indicators include root and aboveground biomass, root activity, chlorophyll content, and endogenous hormone levels. Chitinase production is used as one of the characterizations of biostimulant effectiveness, reflecting the crop's defense capabilities under external stress. Analyzing the chitinase activity of a portion of the leaf sample from cultivated plants allows for the evaluation of different treatments. In this invention, by measuring chitinase activity, root and aboveground biomass, root activity, chlorophyll content, and endogenous hormone levels, it can be demonstrated that the biostimulant provided in this invention, compared to other biostimulants, has significant growth-promoting and immune-activating capabilities, effectively balances the relationship between crop growth and immunity, promotes crop growth without sacrificing disease resistance, and improves product quality and yield.

[0024] This invention provides an oligosaccharide biostimulant and its application method. The active ingredient of the biostimulant is composed of different oligosaccharides, which can improve crop stress resistance, promote crop growth, and improve quality and yield. Using this biostimulant can improve crop vitality, increase agricultural product yield and quality, and avoid the problems of growth inhibition, excessive growth or weak immunity caused by using single oligosaccharides. Attached Figure Description

[0025] Figure 1 This is a standard curve for chitinase content. Detailed Implementation

[0026] The technical solution of the present invention will be illustrated below through specific embodiments. All raw materials and reagents used in the present invention are commercially available, and the percentages below are by weight.

[0027] The specific technical specifications are as follows:

[0028]

[0029] When the above-mentioned single oligosaccharides or combinations are applied to crops, they can affect the intracellular physiological metabolic activities of crops, such as inducing chitinase production and promoting the synthesis of chlorophyll and endogenous hormones. The chitinase content in leaves was determined using a kit (Solarbio, Beijing, China). Chitinase activity is defined as the amount of enzyme that, at 37°C, produces 1 μg of N-acetylglucosamine per gram of tissue per hour by breaking down colloidal chitin; this is defined as one unit of enzyme activity. The main steps are as follows:

[0030] (I) Preparation of colloidal chitin: Weigh 20g of powdered chitin and slowly pour it into 200mL of concentrated hydrochloric acid. Then add 200mL of distilled water and stir continuously with a magnetic stirrer for 12 hours. Add 2L of 95% ethanol at 4℃, mix well, and let stand at room temperature for 12 hours. Centrifuge at 5000r / min for 20 minutes at 4℃. Wash the precipitate repeatedly with distilled water until neutral, and finally dilute to 1L with distilled water. Store at 4℃ for later use.

[0031] (II) Preparation of chitinase content standard curve: Based on the concentration (x, μg / mL) of N-acetylglucosamine standard tube and absorbance ΔA standard (y, ΔA standard), a standard curve is established.

[0032] (III) Preparation of leaf extract: Add 1.0 mL of protein extraction buffer to a 2.0 mL centrifuge tube, and add 0.10 g (denoted as W) of leaves cut into approximately 3 × 3 mm pieces with scissors. Repeat this process three times for each sample. Use a glass rod to further crush the leaves until no solids are visible to the naked eye. Centrifuge at 10000 r / min and 4 °C for 20 minutes. Collect the water layer into a new 2.0 mL centrifuge tube to prepare the extract. Place the extract in ice water for testing.

[0033] (IV) Based on the standard curve, substitute ΔA into the equation to obtain the chitinase content x (μg / mL) in the sample; then calculate the enzyme activity (U / mg) based on the sample mass = X ÷ W ÷ 1000.

[0034] Example 1

[0035] An oligosaccharide biostimulant: 1.7% chitosan oligosaccharide, 1.7% fucoidan oligosaccharide, 1.7% cellulose oligosaccharide, 1.0% betaine, 0.3% sodium diacetate, and water as the balance;

[0036] The specific preparation steps of the solution are as follows: Chitosan oligosaccharide, fucoidan oligosaccharide, cellulose oligosaccharide, sodium diacetate and water are mixed according to the above formula, and the mixture is mixed evenly by high-speed shearing. Betaine is added, and the mixture is further sheared and mixed evenly until the pH is 7.0, thus completing the preparation of the solution.

[0037] Example 2

[0038] An oligosaccharide biostimulant: 1.7% chitosan oligosaccharide, 1.7% fucoidan oligosaccharide, 1.7% pectin oligosaccharide, 1.0% betaine, 0.3% sodium diacetate, and water as the remainder;

[0039] The specific preparation steps of the solution are as follows: Chitosan oligosaccharide, fucoidan oligosaccharide, pectin oligosaccharide, sodium diacetate and water are mixed according to the above formula, and the mixture is mixed evenly by high-speed shearing. Betaine is added, and the mixture is further sheared and mixed evenly until the pH is 7.0, thus completing the preparation of the solution.

[0040] Example 3

[0041] An oligosaccharide biostimulant: 1.7% chitosan oligosaccharide, 1.7% fucoidan oligosaccharide, 1.7% xylooligosaccharide, 1.0% betaine, 0.3% sodium diacetate, and water as the balance;

[0042] The specific preparation steps of the solution are as follows: Chitosan oligosaccharide, fucoidan oligosaccharide, xylooligosaccharide, sodium diacetate and water are mixed according to the above formula, and the mixture is mixed evenly by high-speed shearing. Betaine is added, and the mixture is further sheared and mixed evenly until the pH is 7.0, thus completing the preparation of the solution.

[0043] Example 4

[0044] An oligosaccharide biostimulant: 1% chitosan oligosaccharide, 1% fucoidan oligosaccharide, 2% cellulose oligosaccharide, 1.0% betaine, 0.3% sodium diacetate, and water as the remainder;

[0045] The specific preparation steps of the solution are as follows: Chitosan oligosaccharide, fucoidan oligosaccharide, cellulose oligosaccharide, sodium diacetate and water are mixed according to the above formula, and the mixture is mixed evenly by high-speed shearing. Betaine is added, and the mixture is further sheared and mixed evenly until the pH is 7.0, thus completing the preparation of the solution.

[0046] Example 5

[0047] An oligosaccharide biostimulant: 2% chitosan oligosaccharide, 4% fucoidan oligosaccharide, 1% pectin oligosaccharide, 1.0% betaine, 0.3% sodium diacetate, and water as the remainder;

[0048] The specific preparation steps of the solution are as follows: Chitosan oligosaccharide, fucoidan oligosaccharide, pectin oligosaccharide, sodium diacetate and water are mixed according to the above formula, and the mixture is mixed evenly by high-speed shearing. Betaine is added, and the mixture is further sheared and mixed evenly until the pH is 7.0, thus completing the preparation of the solution.

[0049] Example 6

[0050] An oligosaccharide biostimulant: 5% chitosan oligosaccharide, 1% fucoidan oligosaccharide, 3% xylooligosaccharide, 1.0% betaine, 0.3% sodium diacetate, and water as the remainder;

[0051] The specific preparation steps of the solution are as follows: Chitosan oligosaccharide, fucoidan oligosaccharide, xylooligosaccharide, sodium diacetate and water are mixed according to the above formula, and the mixture is mixed evenly by high-speed shearing. Betaine is added, and the mixture is further sheared and mixed evenly until the pH is 7.0, thus completing the preparation of the solution.

[0052] Comparative Example 1

[0053] An oligosaccharide biostimulant: 5.0% chitosan oligosaccharide, 1.0% betaine, 0.3% sodium diacetate, and water as the remainder.

[0054] The specific preparation steps of the solution are as follows: Chitosan oligosaccharide, sodium diacetate and water are mixed according to the above formula, and then mixed evenly by high-speed shearing. Betaine is added, and shearing and mixing are continued until the pH is 7.0, thus completing the preparation of the solution.

[0055] Comparative Example 2

[0056] An oligosaccharide biostimulant: 5.0% brown algae oligosaccharide, 1.0% betaine, 0.3% sodium diacetate, and water as the remainder.

[0057] The specific preparation steps of the solution are as follows: Mix the alginate oligosaccharide, sodium diacetate and water according to the above formula, mix them evenly by high-speed shearing, add betaine, continue to shear and mix evenly, and the pH is 7.2 to complete the preparation of the solution.

[0058] Comparative Example 3

[0059] An oligosaccharide biostimulant, comprising 5.0% fiber oligosaccharide, 1.0% betaine, 0.3% sodium diacetate, and the remainder water.

[0060] The specific preparation steps of the solution are as follows: mix the cellulose oligosaccharide, sodium diacetate and water according to the above formula, mix them evenly by high-speed shearing, add betaine, continue to shear and mix evenly, the pH is 7.1, and the solution preparation is completed.

[0061] Comparative Example 4

[0062] An oligosaccharide biostimulant: 5.0% pectin oligosaccharide, 1.0% betaine, 0.3% sodium diacetate, and the remainder water.

[0063] The specific preparation steps of the solution are as follows: Pectin oligosaccharide, sodium diacetate and water are mixed according to the above formula, and then mixed evenly by high-speed shearing. Betaine is added, and shearing and mixing are continued until the pH is 6.8, thus completing the preparation of the solution.

[0064] Comparative Example 5

[0065] An oligosaccharide biostimulant: xylooligosaccharide 5.0%, betaine 1.0%, sodium diacetate 0.3%, water balance.

[0066] The specific preparation steps of the solution are as follows: Xylooligosaccharide, sodium diacetate and water are mixed according to the above formula, and then mixed evenly by high-speed shearing. Betaine is added, and shearing and mixing are continued until the pH is 6.6, thus completing the preparation of the solution.

[0067] Comparative Example 6

[0068] An oligosaccharide biostimulant: 2.5% chitosan oligosaccharide, 2.5% fucoidan oligosaccharide, 1.0% betaine, 0.3% sodium diacetate, and water as the remainder.

[0069] The specific preparation steps of the solution are as follows: Chitosan oligosaccharide, fucoidan oligosaccharide, sodium diacetate and water are mixed according to the above formula, and the mixture is mixed evenly by high-speed shearing. Betaine is added, and the mixture is further sheared and mixed evenly until the pH is 7.0, thus completing the preparation of the solution.

[0070] Comparative Example 7

[0071] An oligosaccharide biostimulant: 2.5% chitosan oligosaccharide, 2.5% cellulose oligosaccharide, 1.0% betaine, 0.3% sodium diacetate, and water as the remainder.

[0072] The specific preparation steps of the solution are as follows: Chitosan oligosaccharide, cellulose oligosaccharide, sodium diacetate and water are mixed according to the above formula, and the mixture is mixed evenly by high-speed shearing. Betaine is added, and the mixture is further sheared and mixed evenly until the pH is 7.0, thus completing the preparation of the solution.

[0073] Comparative Example 8

[0074] An oligosaccharide biostimulant: 2.5% chitosan oligosaccharide, 2.5% pectin oligosaccharide, 1.0% betaine, 0.3% sodium diacetate, and water as the remainder.

[0075] The specific preparation steps of the solution are as follows: Chitosan oligosaccharide, pectin oligosaccharide, sodium diacetate and water are mixed according to the above formula, and the mixture is mixed evenly by high-speed shearing. Betaine is added, and the mixture is further sheared and mixed evenly until the pH is 7.0, thus completing the preparation of the solution.

[0076] Comparative Example 9

[0077] An oligosaccharide biostimulant: 2.5% chitosan oligosaccharide, 2.5% xylooligosaccharide, 1.0% betaine, 0.3% sodium diacetate, and water as the remainder.

[0078] The specific preparation steps of the solution are as follows: Chitosan oligosaccharide, xylooligosaccharide, sodium diacetate and water are mixed according to the above formula, and the mixture is mixed evenly by high-speed shearing. Betaine is added, and the mixture is further sheared and mixed evenly until the pH is 7.0, thus completing the preparation of the solution.

[0079] Experimental Example 1: Promoting Effect on the Growth of Tomato Roots and Aboveground Parts

[0080] The experiment was conducted in 2023 in a glass greenhouse at Shandong Jingbo Agricultural Chemical Technology Co., Ltd., using cherry tomatoes as the experimental material. Peat moss (purchased from a flower market) and sand were mixed in a 1:1 ratio and placed into 54cm×27cm×6cm seedling trays. Seeds were evenly sown in the trays and covered with approximately 0.5cm of soil. Watering was done from the bottom up, and seedlings were germinated under ample sunlight at 24–28℃. Once the tomato plants had grown to 6-8 leaves, seedlings of uniform growth were selected and transplanted into pots (20cm in diameter, 15cm in height, each containing 4.0kg of field soil). One seedling was transplanted into each pot, ensuring consistent growing conditions. After the seedlings had recovered, a water-soluble fertilizer (N-P2O5-K2O: 17-17-17) was applied at a rate of 30kg / hm². 2 Application: The biostimulant was applied together with the water-soluble fertilizer containing macroelements. Specifically, the products prepared in the examples and comparative examples were first dispersed in the water-soluble fertilizer solution containing macroelements. The active ingredients of the products were diluted as shown in the table below. The solution was then applied to the roots at a rate of 100 mL per plant. The water control group received only the same amount and concentration of the water-soluble fertilizer solution. The experiment consisted of 13 treatments, with 18 seedlings per treatment, and 6 seedlings per group, for a total of three replicates. The experimental design is shown in Table 1.

[0081] Table 1 Experimental Design

[0082]

[0083] Twenty days after applying the above samples, six plants were randomly selected from each treatment group. The soil was carefully washed away from the roots, and the root dry weight (after draining and treatment at 80℃ for 6 hours) and aboveground fresh weight were measured. Three additional plants from each treatment were selected, and their roots were washed in an ice-water bath. 0.5g of fresh root tip samples were collected, and root vigor in different treatment groups was determined using the TTC staining method. Three additional plants from each treatment were selected, and the chitinase activity, chlorophyll content (chlorophyll meter), and indoleacetic acid (refer to DB 22 / T 25989-2016) levels in leaf cells were measured. Specific results are shown in Table 2.

[0084] Table 2 Comparison of tomato growth and physiological indicators among different treatment groups

[0085]

[0086]

[0087] According to the data in Table 2, chitosan oligosaccharides can efficiently activate the expression of chitinase in crop cells, thereby improving the crop's resistance to external stress. However, this ability can affect biomass accumulation to some extent. Fucoidan oligosaccharides have excellent root-promoting and growth-promoting abilities, but their immune-inducing activity is low. The chitosan oligosaccharide + fucoidan oligosaccharide treatment group not only promoted tomato growth but also demonstrated a high ability to cope with external stress. When applied alone, cellulose oligosaccharides, pectin oligosaccharides, and xylooligosaccharides did not perform as well as chitosan oligosaccharides. However, when combined with chitosan oligosaccharides and fucoidan oligosaccharides, their growth-promoting and immune-inducing activities showed a synergistic effect. In particular, the chitosan oligosaccharide + fucoidan oligosaccharide + cellulose oligosaccharide combination treatment group showed the most significant growth-promoting and immune-activating abilities.

[0088] Experimental Example 2: Different treatments enhance the resistance of plants to cucumber gray mold.

[0089] The treatment groups with higher growth-promoting and chitinase activities (Table 2) were selected to further investigate their ability to prevent gray mold disease in cucumbers.

[0090] Table 3 Experimental Design

[0091]

[0092] The specific steps include: (1) Preventive application of pesticides. The samples prepared according to the examples and comparative examples were diluted with water at a certain multiple to obtain the pesticide concentration shown in Table 3. The crop in each block was sprayed with a backpack sprayer. The control group used an equal amount of water. The pesticide was applied twice consecutively, with an interval of 7 days. The disease incidence was investigated 5 days after the first application and 10 days after the second application. The disease severity of cucumber leaves was recorded according to GB / T 17980.28-2000. (2) Gray mold infection of cucumber leaves. The isolated and preserved *Botrytis cinerea* fungus was transferred to PDA medium for activation culture (static culture at 28℃ for 3 days). Then, agar blocks containing mycelia were taken using a 10mm punch and inoculated onto PDB medium. The medium was cultured at 28℃ and 180 rpm with shaking for 3 days. The culture medium was filtered through sterile cotton to remove the mycelia, yielding a *Botrytis cinerea* spore suspension. The spore concentration was determined by hemocytocyte counting and adjusted to a spore count of 10-1 with sterile water. 8 Two days after preventative application, the spore suspension was diluted 500 times and sprayed evenly onto cucumber leaves to induce disease.

[0093] Table 4 Comparison of disease index among different treatment groups

[0094]

[0095]

[0096] Table 4 shows that applying chitosan oligosaccharide or a binary combination of chitosan oligosaccharide and four other oligosaccharides effectively inhibited disease development. The combination of chitosan oligosaccharide and fucoidan oligosaccharide with fiber oligosaccharide, pectin oligosaccharide, or xylooligosaccharide showed the best inhibitory effect on disease development. The chitosan oligosaccharide + fucoidan + fiber oligosaccharide treatment group showed the best effect in preventing cucumber gray mold. Combined with the data in Table 3, this treatment group also exhibited outstanding root-promoting and growth-promoting effects. This treatment group effectively balanced the relationship between crop growth and immunity, promoting crop growth without sacrificing its disease resistance.

[0097] Experimental Example 3: Oligosaccharide Biostimulants Improve Tomato Quality

[0098] (1) Experimental samples and usage dosage

[0099] The concentrations of active ingredients in each sample and the samples used are shown in Table 5. The experimental group received diluted solutions of the products from the examples and comparative examples, diluted to the concentrations shown in the table below, in addition to the treatment of the control group. Fertilizer and pesticide application were the same as conventional management. The diluted solutions were applied to the roots 10 days after transplanting and allowing the tomato seedlings to establish themselves, and were applied three times consecutively, with an interval of 20 days between each application.

[0100] Table 5 Samples and Usage / Dosage

[0101] Processing group Dosage and administration control group Routine Management Chitosan oligosaccharides (Comparative Example 1) 100ppm, root filling, 100mL / plant Chitosan oligosaccharide + fucoidan oligosaccharide (Comparative Example 6) 50+50ppm, root filling, 100mL / plant Chitosan oligosaccharides + fiber oligosaccharides (Comparative Example 7) 50+50ppm, root filling, 100mL / plant Chitosan oligosaccharides + pectin oligosaccharides (comparative ratio 8) 50+50ppm, root filling, 100mL / plant Chitosan oligosaccharides + xylooligosaccharides (Comparative Example 9) 50+50ppm, root filling, 100mL / plant Chitosan oligosaccharide + fucoidan oligosaccharide + cellulose oligosaccharide (Example 1) 33+33+33ppm, root filling, 100mL / plant Chitosan oligosaccharide + fucoidan oligosaccharide + pectin oligosaccharide (Example 2) 33+33+33ppm, root filling, 100mL / plant Chitosan oligosaccharide + fucoidan oligosaccharide + xylooligosaccharide (Example 3) 33+33+33ppm, root filling, 100mL / plant

[0102] (2) Survey Methods

[0103] 1) Field management data

[0104] The growing environment and soil of greenhouse crops are kept consistent.

[0105] 2) Visual inspection and measurement methods are used to observe the effects of pesticides on crop growth and vigor. By measuring plant height, quality, and yield, the effects of each treatment on crop growth, yield, fruit quality, and disease control are clarified.

[0106] ① Throughout the crop growth period, observe and record the crop growth and disease status before and after each application of pesticides, measure the plant height and stem diameter, and determine the relative growth.

[0107] ② During the harvest stage, record the fruit yield and quality, and evaluate the taste.

[0108] ③ Tomato quality determination: The total sugar, total acid and vitamin C content of mature fruits were measured to determine the effect of each treatment on tomato quality.

[0109] ④ Yield determination: Three rows (6 rows) of tomatoes were selected from both the experimental group and the control group. Mature tomatoes were harvested and their weights were measured and tallied to determine the effect of each treatment on tomato yield.

[0110] Table 6. Effects of each treatment on tomato quality and yield.

[0111]

[0112] As shown in Table 6, compared with the control group, the experimental group of tomatoes showed an increase in total sugar content of 4.9%-17.1%, a decrease in total acid content of 9.2%-18.3%, a maximum increase in vitamin C of 75%, and an increase in yield of 2.2%-9.3%. The treatment groups with chitosan oligosaccharide + fucoidan oligosaccharide combined with fiber oligosaccharide, pectin oligosaccharide, or xylooligosaccharide exhibited better quality and yield, with the chitosan oligosaccharide + fucoidan oligosaccharide + fiber oligosaccharide treatment group showing the best performance in both quality and yield.

[0113] This invention provides an oligosaccharide biostimulant and its application method. The active ingredient of the biostimulant is composed of different oligosaccharides, which can improve crop stress resistance, promote crop growth, and improve quality and yield. Using this biostimulant can improve crop vitality, increase agricultural product yield and quality, and avoid the problems of growth inhibition, excessive growth or weak immunity caused by using single oligosaccharides.

[0114] 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. An oligosaccharide biosfimulant, characterized in that, The active ingredients include component A, component B and component C; the component A is chitinous oligosaccharide, the component B is brown algal oligosaccharide, and the component C is one of pectic oligosaccharide, fibrous oligosaccharide and xylo-oligosaccharide or any proportion of several thereof; The oligosaccharide biological stimulant includes component A 1-5wt%, component B 1-5wt%, component C 1-5wt%, auxiliary 1.1-6wt% and solvent the rest, in percentage by weight, pH value is 4.0-8.0; The polymerization degree of the active ingredients is 2-10.

2. An oligosaccharide biospasmogen according to claim 1, characterized in that, The polymerization degree of the active ingredients is 4-10.

3. The use of an oligosaccharide biological stimulator according to claim 1 or 2, characterized in that, The oligosaccharide biological stimulant is sprayed on the leaves or applied to the roots of the crops.

4. The method of using an oligosaccharide biological stimulator according to claim 3, wherein The active ingredient concentration of the biological stimulant for crops is 0.5-200ppm.

5. The method of using an oligosaccharide biological stimulator according to claim 4, wherein The active ingredient concentration of the biological stimulant for crops, sprayed on the leaves, is 20-200ppm.

6. The method of using an oligosaccharide biological stimulator of claim 4, wherein, The active ingredient concentration of the biological stimulant for crops, applied to the roots, is 10-120ppm.

7. The method of using an oligosaccharide biological stimulator of claim 3, wherein, The crops are one of chrysanthemum family, solanaceae, cruciferous family, gramineae, leguminous family, rosaceae, cucurbitaceae, convolvulaceae, chenopodiaceae, liliaceae, umbelliferae, malvaceae, zingiberaceae, nelumbonaceae.

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