Oligosaccharide-based multifunctional plant immunomodulator

Abstract

Provided in the present invention is an oligosaccharide-based multifunctional plant immunomodulator. Specifically, the plant immunomodulator has a structure represented by formula I below. The plant immunomodulator of the present invention can not only regulate the growth and development of crops, but also induce crops to resist against biological stress and abiotic stress, thereby improving the yield and quality of crops. By using the plant immunomodulator of the present invention, not only can multiple plant growth and development processes be positively regulated, including promoting seed germination, seedling morphogenesis and plant vigor at the mature stage, thereby increasing the crop yield and fruit quality, but also improving the resistance of plants against biological stress (diseases, insects and weeds) and abiotic stress (a frozen or near-frozen soil environment, drought, waterlogging, salinity and alkalinity, extremely low / high temperature, etc.). In addition, the plant immunomodulator of the present invention has the advantages of being non-toxic, free of pesticide residues, environmentally friendly, incapable of inducing pesticide resistance in pathogens, etc.

Description

A multifunctional plant immunomodulator based on oligosaccharides Technical Field

[0001] This invention belongs to the field of oligosaccharide plant growth regulators, specifically relating to the field of plant growth and development regulation and plant immune regulators, and more specifically to an oligosaccharide multifunctional plant immune regulator. Background Technology

[0002] In 1985, Albertsheim, director of the Carbohydrate Research Center in the United States, first proposed the concept of oligosaccharins, suggesting that certain bioactive oligosaccharides can stimulate the immune system response of plants and have functions in regulating plant growth, development, reproduction, disease prevention, and disease resistance. Currently, researchers have identified several structurally well-defined sugars with clear targets and possessing plant immunomodulatory activity, such as trehalose and chitosan.

[0003] In regulating plant growth and development, oligosaccharide regulators not only provide energy and nutrients but also function as signaling molecules. As signaling molecules, oligosaccharides activate multiple signaling pathways within cells, including plant hormones and calmodulin, regulating the expression of a series of downstream target genes and causing corresponding physiological and biochemical changes. This regulates plant growth and development, including seed germination, axillary bud growth, and leaf senescence. Simultaneously, it enhances the plant's resistance to abiotic stresses (salt-alkali soil, drought, frozen or near-frozen soil environments, extreme temperatures, etc.), ultimately improving crop yield and quality. On the other hand, oligosaccharide plant immunomodulators stimulate plant immune defense by mimicking signaling molecules triggered when pathogens attack plants. During the interaction between plants and pathogens, a series of signaling cascades that specifically or broadly recognize and stimulate plant immune responses are achieved through the release of oligomers derived from the cell walls of plants and pathogens (including pectin and chitin derivatives, respectively), resulting in "glycoimmunity" and "glycoenhanced defense."

[0004] Oligosaccharide plant immunomodulators, as novel plant growth regulators, have a wide range of effects. They not only promote plant growth and development and improve plant resistance to abiotic stress, but also possess broad-spectrum antibacterial properties. Oligosaccharide plant immunomodulators offer numerous advantages, such as low application concentrations, harmlessness to humans, no residue, environmental friendliness, significant disease resistance, long-lasting effects, and no development of drug resistance in pathogens, while having no impact on non-pathogenic microorganisms. Furthermore, oligosaccharide plant immunomodulators can be used in conjunction with conventional chemical fungicides to reduce the amount of chemical pesticides used, thus contributing to ecological balance and environmental protection.

[0005] Currently, commercially available biopesticides (such as sugar-based plant growth regulators) are mainly based on chitosan oligosaccharides, primarily targeting pathogens. Their functions are generally limited and their usage costs are relatively high compared to traditional pesticides. While their structures contain multiple amino sugars, their structural diversity is limited due to their mostly linear structures, resulting in slow progress in modifying their structures and optimizing their biological activities. Summary of the Invention

[0006] The purpose of this invention is to provide an oligosaccharide-based multifunctional plant immunomodulator.

[0007] In a first aspect, the present invention provides a plant immunomodulator having the structure shown in Formula I:

[0008] In formula I,

[0009] R1 is selected from H and R3-C(O)-;

[0010] R2 is selected from H, C1-C6 alkyl groups;

[0011] R3 is selected from H, C1-C6 alkyl, and C6-C14 aryl.

[0012] In a second aspect, the present invention provides a pesticide composition comprising the plant immunomodulator described in the first aspect of the present invention and a pesticide-acceptable carrier.

[0013] A third aspect of the present invention provides the application of the plant immune modulator described in the first aspect of the present invention and the pesticide composition described in the second aspect of the present invention in regulating plant growth, development, reproduction, disease prevention and / or disease resistance.

[0014] A fourth aspect of the present invention provides a method for regulating plant growth, development, reproduction, disease prevention and / or disease resistance, the method comprising applying to the plant the plant immunomodulator described in the first aspect of the present invention or the pesticide composition described in the second aspect of the present invention.

[0015] In one or more embodiments, the application includes soaking and / or spraying. Attached Figure Description

[0016] Figure 1(a) shows the phenotypic changes of dicotyledonous plants (soybean, peanut) and monocotyledonous plants (corn, wheat) after soaking in XT2401 (pentasaccharide) for 1 h; Figure 1(b) shows the phenotypic changes of dicotyledonous plants (soybean, peanut) and monocotyledonous plants (corn, wheat) after soaking in XT2401 (pentasaccharide) for 6 h.

[0017] Figure 2 shows the phenotypic time-series changes induced by soaking rapeseed seeds in XT2401 (pentasugar).

[0018] Figure 3 shows the dynamic changes in seed germination of 'Zhongmai 578' after soaking in different concentrations of XT2401 (pentasugar).

[0019] Figure 4 shows the germination dynamics of wheat variety 'Zhongmai 578' seeds treated with XT2401 (pentasugar) soaking.

[0020] Figure 5 shows the repair of diseased areas on cucumbers using XT2401.

[0021] Figure 6 shows how XT2401 treatment improves cucumber's tolerance to salt and alkali stress.

[0022] Figure 7 shows a comparative experiment on the promotion of rapeseed seed germination by XT2401 (pentasaccharide) and GAP9805 (disaccharide).

[0023] Figure 8 shows the 1H NMR spectrum of the prepared XT2401 (pentasaccharide).

[0024] Figure 9 shows the control group after rice seeds were soaked in warm water for 192 hours in Example 9.

[0025] Figure 10 shows the situation of the XT2401 intervention group in Example 9, where rice seeds were soaked for 24 hours and then left to stand for 12 hours.

[0026] Figure 11 shows the emergence of seedlings in the control group 7 days after sowing in Example 9.

[0027] Figure 12 shows the emergence of seedlings in the experimental group (XT2401 treatment group) 7 days after sowing in Example 9. Detailed Implementation

[0028] To enable those skilled in the art to understand the features and effects of the present invention, the terms and expressions used in the specification and claims are explained and defined in general below. Unless otherwise specified, all technical and scientific terms used herein have the ordinary meaning understood by those skilled in the art regarding the present invention, and in case of conflict, the definitions in this specification shall prevail.

[0029] The theories or mechanisms described and disclosed herein, whether right or wrong, should not in any way limit the scope of the invention, that is, the contents of the invention can be implemented without being limited by any particular theory or mechanism.

[0030] The applicant prepared a series of Lewis compounds through chemical synthesis. X Oligosaccharides were studied, and their biological activities were investigated in detail. Lewis series antigens containing fucose modification were also investigated. X Oligosaccharides are common structural units of glycan chains on cell surfaces, playing a crucial role in many physiological and pathological processes. Studies have shown that Lewis... XOligosaccharides significantly promote seed germination rate, plant growth, and crop root growth, increase yield, and significantly improve crop quality, making them a natural plant growth regulator. Therefore, for Lewis... X The development and widespread application of oligosaccharide biopesticides will undoubtedly play a unique role in the sustainable development of agriculture and human health in my country. They will not only bring certain economic benefits to agriculture, but also generate far-reaching environmental and social benefits, and have broad development prospects.

[0031] In this document, the terms “contains,” “includes,” “containing,” and similar terms encompass the meanings of “basically composed of” and “composed of.” For example, when this document discloses “A contains B and C,” “A is basically composed of B and C” and “A is composed of B and C” should be considered as having been disclosed in this document.

[0032] In this document, all features defined by numerical ranges or percentage ranges, such as numerical values, quantities, contents, and concentrations, are for the sake of brevity and convenience only. Accordingly, descriptions of numerical ranges or percentage ranges should be considered as covering and specifically disclosing all possible sub-ranges and individual numerical values ​​(including integers and fractions) within those ranges.

[0033] Unless otherwise specified, percentages refer to mass percentages and proportions refer to mass ratios in this article.

[0034] In this document, when describing embodiments or examples, it should be understood that it is not intended to limit the invention to those embodiments or examples. Rather, all alternatives, modifications, and equivalents of the methods and materials described herein are covered within the scope defined by the claims.

[0035] For the sake of brevity, not all possible combinations of the technical features in each implementation scheme or embodiment are described herein. Therefore, as long as there is no contradiction in the combination of these technical features, the technical features in each implementation scheme or embodiment can be combined arbitrarily, and all possible combinations should be considered within the scope of this specification.

[0036] In this document, as used herein, "alkyl" refers to a straight-chain or branched monovalent saturated hydrocarbon group having a specified number of carbon atoms. Specifically, alkyl groups are those having 1 to 6 carbon atoms ("C1-C6 alkyl"), typically containing 1 to 5 carbon atoms (C1-C5 alkyl), preferably containing 1 to 4 carbon atoms (C1-C4 alkyl), and more preferably containing 1 to 3 carbon atoms (C1-C3 alkyl). Examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, n-hexyl, etc.

[0037] In this document, "aryl" or "Ar" refers to an unsaturated aromatic carbocyclic group having a single ring (e.g., phenyl) or multiple fused rings (e.g., naphthyl or anthracene), wherein the fused rings may or may not be aromatic. In one variation, the aryl group comprises 6 to 14 ring carbon atoms, preferably a C6-C10 aryl group. Examples of aryl groups include phenyl (Ph), naphthyl, phenanthryl, anthracene, indene, azulel, biphenyl, biphenylene, and genus.

[0038] In this article, "Ac" represents acetyl group.

[0039] This invention is a plant immune modulator.

[0040] This invention provides a plant immune modulator having the structure shown in Formula I:

[0041] In formula I,

[0042] R1 is selected from H and R3-C(O)-;

[0043] R2 is selected from H, C1-C6 alkyl groups;

[0044] R3 is selected from H, C1-C6 alkyl, and C6-C14 aryl.

[0045] In some embodiments, R1 is selected from H, HC(O)-, C1-C4 alkyl-C(O)-, and C6-C10 aryl-C(O)-. Preferably, R1 is selected from HC(O)-, C1-C2 alkyl-C(O)-, and C6-C10 aryl-C(O)-. More preferably, R1 is selected from CH3-C(O)- and Ph-C(O)-.

[0046] In some embodiments, R2 is a C1-C4 alkyl group, such as a C1-C2 alkyl group, preferably methyl.

[0047] In some embodiments, the plant immune modulator of the present invention is XT2401:

[0048] pesticide compositions

[0049] This invention provides a plant immunomodulator that can positively regulate multiple plant growth and development processes, including promoting seed germination, seedling morphogenesis, and mature plant growth, thereby increasing crop yield and fruit quality. It also enhances plant resistance to biotic stresses (diseases, insects, weeds) and abiotic stresses (frozen or near-frozen soil environments, drought, waterlogging, salinity, extreme low / high temperatures, etc.). Therefore, this invention provides a pesticide composition containing the plant immunomodulator of this invention. The pesticide composition of this invention comprises the plant immunomodulator of this invention and a pesticide-acceptable carrier. In some embodiments, the pesticide-acceptable carrier is water, Hoagland nutrient solution, or Murashige and Skoog medium-MS medium.

[0050] The pesticide composition of the present invention can be processed into soluble powder or granules using methods commonly used in the art.

[0051] In some embodiments, the pesticide composition of the present invention further includes other pesticides and / or fertilizers. In some embodiments, the other pesticides may be plant growth regulators. Plant growth regulators may be synthesized artificially, extracted artificially, or bio-fermented. Plant growth regulators are active substances similar to or opposite to endogenous plant hormones, which can affect the synthesis, transport, metabolism, and physiological functions of endogenous plant hormones at low concentrations, and can be used to regulate plant growth and development. Plant growth regulators may be selected from one or more of 2,4-D (2,4-dichlorophenoxyacetic acid), naphthaleneacetic acid, polyamines (e.g., spermine, spermidine, butanediamine), cytokinins (e.g., 6-BA (benzylaminopurine), ZT (zeatin), KT30, CPPU (chlorpyrifos), or TDZ (thiafenuron)), mepiquat chloride, quinacrine, paclobutrazol, chlormequat chloride, and abscisic acid. Excessive or improper use of existing plant growth regulators may damage the safety and effectiveness of crops and have adverse effects on soil and water bodies in the production area. Using the plant immune modulator of this invention in combination with plant growth regulators can greatly reduce the amount of plant growth regulators in pesticide compositions, thereby reducing the adverse effects of plant growth regulators.

[0052] application

[0053] The present invention also provides the use of the plant immune modulators and / or pesticide compositions of the present invention in regulating plant growth, development, reproduction, disease prevention and / or disease resistance.

[0054] In some implementations, the plant may be selected from one or more of the following: soybean, peanut, rapeseed, corn, wheat, cucumber, and rice.

[0055] In some implementation schemes, regulating plant growth, development, reproduction, disease prevention and / or disease resistance is selected from: promoting seed germination, promoting seedling morphogenesis, promoting plant growth in the mature stage, promoting plant damage repair, increasing crop yield, improving fruit quality, improving plant resistance to biotic stress, and improving plant resistance to abiotic stress.

[0056] In some implementations, the biological stress includes one or more of diseases, pests, and weeds.

[0057] In some embodiments, the abiotic stress includes one or more of drought, waterlogging, salinity, extreme low temperature, and extreme high temperature. In some embodiments, the abiotic stress includes one or more of permafrost or near-permafrost environments, drought, waterlogging, salinity, extreme low temperature, and extreme high temperature. In some embodiments, the abiotic stress includes permafrost or near-permafrost environments.

[0058] In this article, permafrost refers to soil or rock layers with a temperature of 0°C or below that contain ice and moisture (including ice and unfrozen water).

[0059] In this article, "near-permafrost" refers to soil or rock layers where the temperature fluctuates frequently around 0°C (e.g., -20~20°C, -15~15°C, -10~10°C, -5~5°C, -20~15°C, or -10~5°C), causing repeated freeze-thaw phase changes in the water (ice) within the soil or rock mass. Controlling the temperature to fluctuate frequently around 0°C (e.g., -20~20°C, -15~15°C, -10~10°C, -5~5°C, -20~15°C, or -10~5°C) (for up to 1 week, 2 weeks, 3 weeks, or 5-10 days) is a recognized method in this field for simulating near-permafrost environments. For example, this can be achieved for 7 consecutive days in a freeze-thaw cycle environment with daytime temperatures of 5°C and nighttime temperatures of -10°C.

[0060] In this article, "extreme low temperature" and "extreme high temperature" refer to temperatures outside the optimal temperature range for plant growth and development, severely affecting and / or inhibiting plant growth, development, or reproduction. Extreme low temperature is below the optimal temperature range for plant growth and development, while extreme high temperature is above the optimal temperature range. "Extreme low temperature," "extreme high temperature," and the optimal temperature range for plant growth and development are related, and therefore also to the plant variety. For example, the optimal temperature range for rapeseed is 20-25℃; temperatures below 10℃ can severely inhibit growth, and temperatures above 35℃ also severely affect its growth. Therefore, for rapeseed, extreme low temperature refers to ≤10℃, and extreme high temperature refers to ≥35℃. The optimal temperature range for rice is 25-30℃. When the ambient temperature is below 15℃, rice growth and development are affected; therefore, for rice, extreme low temperature refers to ≤15℃. In this field, the optimal temperature range for the growth of most plants is 15-30℃; temperatures exceeding this range are considered extreme low or extreme high temperatures. In this article, extreme low temperature is ≤15℃. In this article, extreme high temperature is ≥30℃.

[0061] In some embodiments, the present invention provides the use of the plant immunomodulators and pesticide compositions of the present invention in promoting seed germination. Seed germination hereinspection refers to seed sprouting. The plants described in any embodiment of the present invention are preferably selected from one or more of rice, cucumber, soybean, peanut, rapeseed, corn, and wheat.

[0062] This invention provides the application of the plant immune modulator and pesticide composition of this invention in promoting plant seed germination. The plant, as described in any embodiment of this invention, is preferably selected from one or more of corn, wheat, and peanut.

[0063] This invention provides the application of the plant immunomodulator and pesticide composition of this invention in increasing plant yield. The plant, as described in any embodiment of this invention, is preferably selected from one or more of corn, wheat, rice, rapeseed, cucumber, and peanut.

[0064] This invention provides the application of the plant immune modulator and pesticide composition of this invention in promoting plant damage repair. The plant is as described in any embodiment of this invention, preferably cucumber.

[0065] This invention provides the application of the plant immunomodulator and pesticide composition of this invention in enhancing plant responses to abiotic stress. The plant is as described in any embodiment of this invention, preferably one or more of rapeseed, cucumber, and rice. The abiotic stress includes growth in saline-alkali soil and / or exposure to extreme low temperatures. Extreme low temperatures are defined as temperatures ≤15°C, for example, temperatures of 0-15°C, 5-15°C, or 7-15°C.

[0066] This invention also provides the application of the plant immunomodulator and pesticide composition of this invention in improving seed germination rate, seedling chlorophyll content, rhizosphere microbial abundance, cold resistance, seedling height, root activity, and freeze-thaw survival rate in permafrost or near-permafrost environments; and / or, in reducing the seed germination initiation time. In this document, the criteria for determining plant root activity are as follows. The definitions of the plant immunomodulator, pesticide composition, permafrost, and near-permafrost are as described in any embodiment herein. In some embodiments, the plant includes seeds and seedlings selected from plants of the Poaceae, Leguminosae, Brassicaceae, and Asteraceae families, such as rice (including rice seeds and rice seedlings).

[0067] method

[0068] The plant immunomodulators and / or pesticide compositions of the present invention can regulate plant growth, development, reproduction, disease prevention, and / or disease resistance. Therefore, the present invention also provides a method for regulating plant growth, development, reproduction, disease prevention, and / or disease resistance. This method includes applying the plant immunomodulators or pesticide compositions of the present invention to the plant. In some embodiments, the application includes seed soaking and / or spraying.

[0069] In some embodiments, the plant, and the regulators of plant growth, development, reproduction, disease prevention, and / or disease resistance, are as described in any of the embodiments herein.

[0070] In some implementation schemes, the spraying method is as follows: after the plant cotyledons have expanded, spray the plant immunomodulator solvent or pesticide composition evenly on both sides of the cotyledons until the solution drips down. Spray once a day for 4-8 days.

[0071] In some implementation methods, the seed soaking method is as follows: the plant seeds are soaked in a plant immunomodulator solvent or pesticide composition, dried, and then sown directly. The soaking time can be adjusted according to the plant variety.

[0072] This invention provides a method for promoting plant seed germination. The method includes soaking plant seeds in a plant immunomodulator solvent or pesticide composition. The concentration of the plant immunomodulator in the solvent or pesticide composition may be 0.08-0.12 mg / L, for example 0.09-0.11 mg / L or 0.1 mg / L. The plant, as described in any embodiment of this invention, is preferably selected from one or more of rice, cucumber, soybean, peanut, rapeseed, corn, and wheat. In some embodiments, rice seeds are soaked in the plant immunomodulator solvent or pesticide composition for 20-28 hours, for example 24-25 hours, at a concentration of 0.09-0.11 mg / L. In some embodiments, cucumber seeds are soaked in the plant immunomodulator solvent or pesticide composition for 20-28 hours, for example 24-25 hours, at a concentration of 0.09-0.11 mg / L. In some embodiments, peanut seeds are soaked in a plant immunomodulator solvent or pesticide composition for 3-3.5 hours, with the concentration of the plant immunomodulator in the solvent or pesticide composition being 0.09-0.11 mg / L. In some embodiments, corn seeds are soaked in a plant immunomodulator solvent or pesticide composition for 5-7 hours, preferably 6 hours, with the concentration of the plant immunomodulator in the solvent or pesticide composition being 0.09-0.11 mg / L. In some embodiments, wheat seeds are soaked in a plant immunomodulator solvent or pesticide composition for 5-7 hours, preferably 6 hours, with the concentration of the plant immunomodulator in the solvent or pesticide composition being 0.09-0.11 mg / L. In some embodiments, soybean seeds are soaked in a plant immunomodulator solvent or pesticide composition for 20-30 minutes, with the concentration of the plant immunomodulator in the solvent or pesticide composition being 0.09-0.11 mg / L. In some embodiments, rapeseed seeds are soaked in a plant immunomodulator solvent or pesticide composition for 15-20 minutes, wherein the concentration of the plant immunomodulator in the solvent or pesticide composition is 0.09-0.11 mg / L. In some embodiments, wheat seeds are soaked in a plant immunomodulator solvent or pesticide composition for 6.5-7.5 hours, wherein the concentration of the plant immunomodulator in the solvent or pesticide composition is 0.10-0.13 mg / L. In some embodiments, wheat seeds are soaked in a plant immunomodulator solvent or pesticide composition for 6.5-7.5 hours, wherein the concentration of the plant immunomodulator in the solvent or pesticide composition is 0.05-0.08 mg / L.

[0073] This invention provides a method for promoting the germination of plant seeds. The method includes soaking plant seeds in a plant immunomodulator solvent or pesticide composition. The plant, as described in any embodiment of this invention, is preferably selected from one or more of corn, wheat, and peanut. The concentration of the plant immunomodulator in the solvent or pesticide composition may be 0.10-0.13 mg / L, for example, 0.11-0.12 mg / L. In some embodiments, wheat seeds are soaked in the solvent or pesticide composition for 5.5-6.5 hours at a concentration of 0.10-0.13 mg / L. In some embodiments, peanut seeds are soaked in the solvent or pesticide composition for 5.5-6.5 hours at a concentration of 0.10-0.13 mg / L. In some embodiments, corn seeds are soaked in a plant immunomodulator solvent or pesticide composition for 7.5-8.5 hours, wherein the concentration of the plant immunomodulator in the solvent or pesticide composition is 0.10-0.13 mg / L.

[0074] This invention provides a method for increasing plant yield. The method includes soaking plant seeds in a plant immunomodulator solvent or pesticide composition and / or spraying the plant with the plant immunomodulator solvent or pesticide composition. The plant is the above-ground part of the plant, such as a seedling. The plant, as described in any embodiment of this invention, is preferably selected from one or more of corn, wheat, rice, rapeseed, cucumber, and peanut. The concentration of the plant immunomodulator in the plant immunomodulator solvent or pesticide composition used for soaking the plant seeds may be 0.08-0.13 mg / L, for example, 0.10-0.12 mg / L or 0.11 mg / L. The concentration of the plant immunomodulator in the plant immunomodulator solvent or pesticide composition used for spraying the plant may be 0.0005-0.0006 mg / L. In some embodiments, the plant immunomodulator solvent or pesticide composition used for spraying the plant is a 1800-2000 times dilution of a stock solution with a plant immunomodulator concentration of 1 mg / L. In some embodiments, the plant immunomodulator solvent or pesticide composition used for soaking plant seeds is a 9-10 times dilution of a stock solution of a plant immunomodulator at a concentration of 1 mg / L.

[0075] This invention provides a method for promoting plant damage repair. The method includes spraying the damaged area of ​​the plant with a plant immunomodulator solvent or pesticide composition according to this invention. The plant immunomodulator solvent or pesticide composition may be an 800-2000 times dilution of a stock solution of a plant immunomodulator at a concentration of 1 mg / L. The plant is as described in any embodiment of this invention, preferably cucumber. The damaged area is the part of the plant after the diseased tissue has been removed. In some embodiments, the diseased tissue is a leaf.

[0076] This invention provides a method for improving plant response to abiotic stress. The method includes soaking plant seeds in a plant immunomodulator solvent or pesticide composition and / or spraying the plant with the plant immunomodulator solvent or pesticide composition. The plant, abiotic stress, and plant are as described in any embodiment of this invention. In some embodiments, the concentration of the plant immunomodulator in the plant immunomodulator solvent or pesticide composition used for soaking the plant seeds is 0.1-1.2 mg / L, preferably 0.1-1.0 mg / L. In some embodiments, the concentration of the plant immunomodulator in the plant immunomodulator solvent or pesticide composition used for spraying the plant is 0.005-0.03 mg / L, preferably 0.01-0.02 mg / L.

[0077] The inventors have discovered that soaking plant seeds in the plant immunomodulator solvent or pesticide composition of this invention can enhance the thickness of the sugar chain layer on the cell membrane surface, thereby improving the stability and frost resistance of the cell membrane. Therefore, this invention provides a method for improving seed germination and seedling growth in permafrost or near-permafrost environments. The method includes soaking plant seeds in a plant immunomodulator solvent or pesticide composition. The plant immunomodulator, pesticide composition, permafrost, and near-permafrost are defined as described in any embodiment of this invention. In some embodiments, the plant includes seeds and seedlings selected from plants of the Poaceae, Leguminosae, Brassicaceae, and Asteraceae families, such as rice (including rice seeds and rice seedlings). Preferably, the concentration of the plant immunomodulator in the plant immunomodulator solvent or pesticide composition is 0.1-100 mg / L, more preferably 0.1-1 mg / L, 0.1-0.5 mg / L, or 1-10 mg / L. In some embodiments, the soaking time is 6-48 hours, for example 12-24 hours, 20-30 hours, or 24-30 hours. In some implementations, improving plant seed germination and seedling growth includes: increasing plant seed germination rate, seedling chlorophyll content, rhizosphere microbial abundance, cold resistance, seedling height, root activity, and freeze-thaw survival rate; and / or, reducing plant seed germination initiation time.

[0078] The present invention has the following beneficial effects:

[0079] This invention provides a novel, non-toxic, and harmless plant immune regulator that can regulate crop growth and development and induce crops to develop resistance to biotic and abiotic stresses, thereby improving crop yield and quality.

[0080] Using the plant immunomodulator of this invention, multiple plant growth and development processes can be positively regulated, including promoting seed germination, seedling morphology, and mature plant growth, thereby increasing crop yield and fruit quality. It can also enhance plant resistance to biotic stresses (diseases, insects, weeds) and abiotic stresses (frozen or near-frozen soil environments, drought, waterlogging, salinity, extreme low / high temperatures, etc.). Furthermore, the plant immunomodulator of this invention is non-toxic, pesticide-free, environmentally friendly, and does not induce drug resistance in pathogens. The plant immunomodulator of this invention is convenient to use; it can be applied directly to seeds or sprayed onto the plants.

[0081] This invention provides a novel broad-spectrum Lewis X Oligosaccharide plant growth regulators can effectively stimulate the plant's immune defense system to improve the plant's basic immune response to diseases and extreme environmental stresses, and can simultaneously participate in multiple processes affecting plant immune defense and growth and development. Lewis X Oligosaccharides contain multiple monosaccharide fragments, making structural modification and activity optimization easier. As a novel biopesticide, Lewis... X Oligosaccharides exhibit unprecedented structural and functional advantages.

[0082] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer. Percentages and parts are by weight unless otherwise stated.

[0083] In this paper, the solvent for XT2401 (pentasaccharide) mother liquor, XT2401 (pentasaccharide) solution and GAP9805 (disaccharide) solution is water.

[0084] In this study, the soybean variety used in the experiment was 'Zhonghuang 29' (Glycine max L.).

[0085] In this paper, the peanut variety used in the experiment was 'Bianhua No. 7' (Arachis hypogaea L.).

[0086] In this paper, the rapeseed variety used in the experiment was 'Qinsheng 800' (Brassica napus L.).

[0087] In this paper, the maize variety used in the experiment was 'Jinhai 188' (Zea mays L.).

[0088] In this paper, the wheat variety used in the experiment was 'Zhongmai 578' (Triticum aestivum L.).

[0089] The cucumber variety used in this experiment was 'Early Spring No. 1' (Cucumis sativus L.).

[0090] The rice variety used in this experiment was 'Nanjing 46' (Oryza sativa L.).

[0091] Unless otherwise specified, the soaking and spraying experiments in this article were conducted at room temperature (25°C).

[0092] Preparation Example

[0093] Preparation of XT-23:

[0094] Compound XT-22 (2.3 g, 1.05 mmol), hydrazine hydrate (4 mL), and water (4 mL) were added to ethanol (90 mL), and the mixture was stirred and refluxed at 80 °C for 14 hours. After the reaction was complete, the mixture was concentrated and dried. Pyridine (20 mL) and acetic anhydride (10 mL) were added, and the mixture was stirred overnight at room temperature. After the reaction was complete, the mixture was concentrated and dried. Methanol (25 mL) and sodium (60 mg) were added, and the mixture was reacted at room temperature for 12 hours. After the reaction was complete, the mixture was analyzed using Amberlite IR 120 (H) reagent. + The resin was neutralized to pH 7, filtered, and the solvent was evaporated. The crude product was purified by silica gel column chromatography (eluent was toluene / acetone, volume ratio 1 / 1.2) to give a white powdery compound XT-23 (81%, three steps).

[0095] 1H MMR (400MHz, CDCl3): δ7.5-7.2(m,45H),5.78(d,J=7.1Hz,1H),5.18(d,J=7.4Hz,1H),5.08(d,J=3.5Hz,1H) ,5.02,4.77(2d,J=10.7Hz,2H),4.95,4.59(2d,J=11.5Hz,2H),4.91,4.68(2d,J=11.8Hz,2H),4.89,4.74(2 d,J=10.9Hz,2H),4.52-4.41(m,5H),4.29(d,J=7.7Hz,1H),4.15(m,1H),4.08(d,J=10.2Hz,1H),3.92(d,J= 2.6Hz,1H),3.70(m,1H),3.58(s,3H),3.47(dd,J=3.6Hz,J=10.1Hz,1H),1.40(s,3H),1.14(d,J=8.0Hz,3H).

[0096] Mass spectrometry (CI, NH3): m / z 1695.8 (M+NH4) + .

[0097] Preparation of XT2401:

[0098] Compound XT-23 (1.08 g) was dissolved in methanol (45 mL), and 10 wt% palladium on carbon (Pd / C, 0.2 g) was added. The mixture was stirred at room temperature under hydrogen (150 kPa) for 12 hours. After the reaction was completed, the mixture was filtered, concentrated, and dried. The crude product was purified by dextran gel column chromatography (eluent: water) to give a freeze-dried white amorphous solid XT2401 (98%).

[0099] 1 H MMR (400MHz, D2O): δ5.02(d,J=4.0Hz,1H), 4.61(d,J=8.2Hz,1H), 4.40–4.27(m,3H), 4.05(d,J=3.3Hz, 1H),3.94–3.43(m,28H),3.38(d,J=9.7Hz,1H),3.25–3.17(m,1H),1.92(s,3H),1.07(d,J=6.6Hz,3H).

[0100] Mass spectrometry (ESI): m / z 890.3 (M+Na) + .

[0101] Example 1: Seed soaking using 0.1 mg / L XT2401 (pentasaccharide) solution

[0102] Seeds of several widely planted crops (dicotyledonous varieties: soybean 'Zhonghuang 29', peanut 'Bianhua 7', rapeseed '560'; monocotyledonous varieties: corn 'Jinhai 188', wheat 'Zhongmai 578') were selected as experimental materials. Twenty uniform soybean, peanut, and corn seeds, and 50 wheat seeds were selected and soaked in water or 0.1 mg / L XT2401 (pentasaccharide) for seed priming. The seeds were completely submerged in water or XT2401 (pentasaccharide) solution for soaking priming.

[0103] The phenotypic changes of water absorption and swelling in seeds of various crops were observed through experiments (Table 1, Figure 1) to determine the optimal soaking time for promoting germination in different crops: peanuts for 3-3.5 hours; corn for 6 hours; and wheat for 6 hours. During the experiment, it was found that soybeans were not suitable for prolonged soaking in a 0.1 mg / L XT2401 (pentasaccharide) solution. To further explore the suitable soaking time for initiating germination in soybeans, the soaking time was shortened to within 1 hour. It was observed that soaking in a 0.1 mg / L XT2401 (pentasaccharide) solution for 20-30 minutes was beneficial for soybean seed germination. For rapeseed seed soaking, it was found that most rapeseed seeds began to absorb water and germinate after 8 minutes of soaking, with a few radicles emerging; after 15 minutes of soaking, most seeds absorbed water and germinated, and the number of germinating seeds increased; after 20 minutes of soaking and drying, the seeds continued to germinate (see Figure 2). Compared to the 8-hour soaking time required for regular rapeseed germination, XT2401 (pentasaccharide) solution allows rapeseed seeds to germinate in just 15-20 minutes, significantly reducing the soaking time. Table 2 shows the optimal soaking time for germination of soybeans, peanuts, corn, wheat, and rapeseed using 0.1 mg / L XT2401 (pentasaccharide) solution.

[0104] Table 1: Phenotypic temporal changes induced by soaking seeds of XT2401 (pentasaccharide) in dicotyledonous plants (soybean, peanut) and monocotyledonous plants (maize, wheat).

[0105] Table 2: Optimal soaking time for inducing germination of soybeans, peanuts, corn, wheat, and rapeseed using 0.1 mg / L XT2401 (pentasaccharide) solution.

[0106] In conclusion, soaking seeds in 0.1 mg / L XT2401 (pentasaccharide) can shorten the germination time.

[0107] Example 2: Seed soaking using different concentrations of XT2401 (pentasaccharide)

[0108] To further clarify the optimal application concentration of XT2401 (pentasaccharide) for a specific species, wheat variety 'Zhongmai 578' was used as the test material. Three seed soaking concentration gradients were set up: control group (water), low concentration group (1 mg / L stock solution diluted 15 times), and high concentration group (1 mg / L stock solution diluted 9 times). 5g of uniformly sized and plump seeds were placed in beakers and soaked in XT2401 (pentasaccharide) solutions of different concentrations, ensuring that the seeds were completely submerged in the solution.

[0109] Phenotypic differences began to appear after 5 hours of soaking. Clear endosperm was visible in all five sugar treatment groups (regardless of low or high concentration). After 7 hours of soaking, seeds in the high-concentration group began to show white sprouts, revealing the embryo; seeds in the low-concentration group remained intact, with the embryo faintly visible; and no embryo was visible in the control group. Soaking was stopped after 7 hours, and the seeds were placed in a drying beaker for re-drying. After 21 hours of re-drying, almost all seeds in the high-concentration group showed fully visible embryos, some seeds in the low-concentration group showed visible embryos, and the control group seeds only swelled without showing embryos. After 39 hours of re-drying, all seeds in the five sugar treatment groups survived. The difference in embryo development between the low-concentration and high-concentration groups gradually narrowed, while no significant changes were observed in the control group seeds. After 63 hours of re-drying, all seeds in the five sugar treatment groups survived. The embryo development of the low-concentration group gradually surpassed that of the high-concentration group, while the embryo development of the high-concentration group slowed down. No significant changes were observed in the control group seeds (see Figure 3).

[0110] Therefore, in the wheat variety 'Zhongmai 578', soaking seeds in a higher concentration of XT2401 (pentasaccharide) solution (1 mg / L mother liquor diluted 9 times) can rapidly promote seed germination; soaking seeds in a lower concentration of XT2401 (pentasaccharide) solution (1 mg / L mother liquor diluted 15 times) is more conducive to the continuous germination and subsequent growth and development of wheat seeds.

[0111] Example 3: Using XT2401 (pentasugar) to promote seedling emergence

[0112] Working solutions were prepared by diluting 1 mg / L XT2401 (pentasaccharide) stock solution 9 times. Corn, wheat, and peanut seeds were soaked for 8 h, 6 h, and 6 h, respectively, and the emergence rate of each species in the field was observed. The results showed that XT2401 (pentasaccharide) soaking treatment improved the emergence rate of corn, wheat, and peanut (Table 3). The emergence rate of corn increased by 24.33%; the emergence rate of wheat increased by 7.35% in 2021 and 10.78% in 2022; and the emergence rate of peanut increased by 5.96%. Furthermore, dynamic monitoring of the wheat seed emergence process in the field revealed that the XT2401 (pentasaccharide) treatment group was superior to the control group (soaked in water) in terms of emergence rate, emergence rate, and seedling growth (Figure 4).

[0113] Table 3: XT2401 (five sugars) seed soaking improves emergence rate of corn, wheat and peanut.

[0114] Example 4: Using XT2401 (pentasugar) to increase yield

[0115] Experiments were conducted in different regions (Shanghai, Henan) and different years (2020-2023) to investigate the effects of XT2401 (pentasugar) on the yield of different crops (corn, wheat, rice, rapeseed, peanut and cucumber). The intervention of XT2401 (pentasugar) was divided into two treatment methods: (1) Seed soaking + spraying: 0.001 cubic meters of 1 mg / L mother liquor was diluted 9 times to prepare a seed soaking working solution for seed soaking treatment (the corresponding soaking time is shown in Table 4 below). After treatment, the recovered liquid was further diluted 200 times to prepare a spraying working solution for spraying on seedlings and above-ground parts of plants in the later stage of growth. Spraying was carried out evenly on both sides of the leaves until water droplets fell; (2) Spraying: 0.001 cubic meters of 1 mg / L mother liquor was diluted 1800 times and sprayed on the above-ground parts of plants directly, without seed soaking treatment. The control group was treated with water soaking for 6 hours. The results showed that the XT2401 (pentasugar) treatment group could increase the yield of corn, wheat, rice, rapeseed, peanut and cucumber (Table 4). Among them, the yield of corn, wheat, rice, peanut and rapeseed refers to the seed yield, and the yield of cucumber refers to the fruit yield. The yield increase percentages ranged from 10% to 52.34%.

[0116] Taking corn as an example, compared to the control group, the corn plants in the experimental group treated with XT2401 (pentasugar) solution were less sensitive to changes in soil pH, temperature, and humidity, exhibited stronger tolerance to extreme environmental stress, and had a lower incidence of pests and diseases. In terms of growth rate, the experimental group flowered and set fruit earlier than the control group, shortening the growth period by one-third to one-half. Regarding yield, the control group mostly produced single ears per plant, with a few producing two ears, while the experimental group mostly produced double or triple ears per plant, resulting in a 2-fold increase in the total ear-setting rate. In terms of product quality and taste, the experimental group was sweeter and more palatable, making it more popular with consumers.

[0117] Table 4: Yield-increasing effects of XT2401 on maize, wheat, rice, rapeseed, peanut, and cucumber.

[0118] Example 5: Using XT2401 (pentasugar) to promote damage repair

[0119] Infected cucumber leaves were pruned, and the pruned areas were sprayed with a 1000-fold dilution of XT2401 (diluted 1000 times with a 1 mg / L stock solution). 0.01 mL of XT2401 was sprayed on each pruned area. The control group was sprayed with the same volume of water. Compared to the control, the infected areas treated with the five-sugar solution sprouted new leaves within 24 hours (with supplemental lighting), subsequently producing mainly female flowers and fruits (see Figure 5). The control group failed to sprout new leaves and did not flower. Therefore, spraying with the five-sugar solution can rapidly repair damaged areas.

[0120] Example 6: Enhancing Abiotic Stress Response Using XT2401 (Pentasaccharide)

[0121] XT2401 was used to treat cucumbers grown in saline-alkali soil. The treatment methods for the XT2401 treatment group were (1) soaking (concentration 1 mg / L, time 6 hours) + spraying (0.01 mg / L, volume 0.001 cubic meters); (2) soaking (concentration 1 mg / L, time 6 hours). The control group was soaked in the same volume of water and sprayed with the same volume of water. It was found that the XT2401 treatment group was significantly better than the control group in terms of emergence rate and growth potential under saline-alkali stress (Figure 6); and the XT2401 soaking and spraying group could rapidly promote emergence and subsequent growth and development, indicating that XT2401 can improve the resistance of cucumbers to saline-alkali stress.

[0122] Example 7: Comparison of the effects of XT2401 (pentasaccharide) and GAP9805 (disaccharide) products

[0123] Using cucumber variety 'Early Spring No. 1' as the test material, seeds were treated with water (control group), XT2401 (pentasaccharide), and GAP9805 (disaccharide) solutions, with 30 seeds in each group. The XT2401 (pentasaccharide) and GAP9805 (disaccharide) soaking solutions were working solutions prepared by diluting the 1 mg / L stock solution 10 times. After soaking for 7 hours and then drying for 30 minutes, the XT2401 (pentasaccharide) and GAP9805 (disaccharide) treatment groups showed obvious signs of germination compared to the control group. After soaking for 24 hours and then drying for 30 minutes, the germination rate of the control group was 13.33%, the germination rate of the GAP9805 (disaccharide) treatment group reached 56.67%, while all seeds in the XT2401 (pentasaccharide) group germinated. The results showed (Table 5, Figure 7) that both XT2401 (pentasaccharide) and GAP9805 (disaccharide) could promote cucumber seed germination, with XT2401 (pentasaccharide) showing better results.

[0124] Table 5: Differences in the effects of XT2401 (pentasaccharide) and GAP9805 (disaccharide) products on cucumber seed soaking

[0125] In addition, using rice 'Nanjing 46' as the test material, seeds were soaked in XT2401 (pentasaccharide) and GAP9805 (disaccharide) solutions (working solution prepared by diluting 1 mg / L mother liquor 10 times) for 24 h and then dried for 30 min. It was found that the treatment of XT2401 (pentasaccharide) and GAP9805 (disaccharide) caused most seeds to germinate. After being exposed to low temperature for 5 days (March 17-21, 2023) (the experiment was carried out at a low temperature of ≤15℃, and the temperature is shown in Table 6 below), the XT2401 (pentasaccharide) treatment group had better results than the GAP9805 (disaccharide) treatment group.

[0126] Table 6: Highest and lowest temperatures during the test, March 17-21, 2023

[0127] Similarly, in rapeseed, XT2401 (pentasaccharide) and GAP9805 (disaccharide) were diluted 10 times with 1 mg / L stock solution to induce germination. After soaking for 20 minutes and drying for 2 hours, the seeds in the XT2401 (pentasaccharide) and GAP9805 (disaccharide) treatment groups showed obvious signs of germination, and XT2401 (pentasaccharide) had a better germination-promoting effect than GAP9805 (disaccharide).

[0128] Example 8

[0129] Control group: Corn seeds were not treated with XT2401 (pentasugar). Seeds were sown and raised into seedlings. They were transplanted at the same time as the experimental group into soil plots with a pH of 5.6. No fertilizers or pesticides were used during the period. New leaves emerged 12 days after transplanting, ears appeared 65 days after transplanting, and ears matured and were harvested 130 days after transplanting. 95% of the plants had single ears, 5% of the plants had double ears, and 28% of the plants showed varying degrees of wilting and disease.

[0130] Experimental group: Corn seeds were soaked in XT2401 (pentasugar) at a concentration of 0.1 mg / L for 8 hours and then evenly sown in the same plot as the control group. No other fertilizers or pesticides were used during this period. Seedlings emerged 4 days after sowing, new leaves appeared 8 days later, ears emerged 45 days later, and ears matured and were harvested 79 days later. 90% of the plants in this group produced double ears, and 10% of the plants produced triple ears. There were no cases of wilting or disease.

[0131] Example 9

[0132] Control group: Rice seeds were soaked in warm water for 192 hours, drained and sown, and then placed in a freeze-thaw cycle environment of 5℃ during the day and -10℃ at night for 7 consecutive days.

[0133] Experimental group: Rice seeds were soaked in 0.1 mg / L XT2401 solution for 24 hours, then left to stand for 12 hours, drained and sown, and then placed in a freeze-thaw cycle environment of 5℃ during the day and -10℃ at night for 7 consecutive days.

[0134] The results showed that the germination rate of the experimental group was 99%, while that of the control group was 60%. The chlorophyll content of the seedlings in the experimental group increased by about 30%. Under simulated near-permafrost conditions, treatment with XT2401 solution promoted an increase of about 1.8 times in the number of rhizosphere microorganisms (Bacillus subtilis, Pseudomonas fluorescens), indicating that it has the effect of improving rhizosphere microecology and synergistic cold resistance.

[0135] Compared to the experimental group, the experimental group treated with XT2401 solution showed the following significant effects in near-permafrost environments, as shown in Table 7 below.

[0136] Table 7

Claims

1. A plant immunomodulator having the structure shown in Formula I: In formula I, R1 is selected from H and R3-C(O)-; R2 is selected from H, C1-C6 alkyl groups; R3 is selected from H, C1-C6 alkyl, and C6-C14 aryl.

2. The plant immune modulator as described in claim 1, characterized in that: R1 is selected from H, HC(O)-, C1-C4 alkyl-C(O)-, and C6-C10 aryl-C(O)-; preferably, R1 is selected from HC(O)-, C1-C2 alkyl-C(O)-, and C6-C10 aryl-C(O)-; and / or, R2 is a C1-C4 alkyl group.

3. The plant immune modulator as described in claim 1, characterized in that, The plant immunomodulator has the structure shown in XT2401:

4. A pesticide composition, characterized in that, The pesticide composition comprises the plant immunomodulator of any one of claims 1-3 and a pesticide-acceptable carrier.

5. The pesticide composition according to claim 4, characterized in that, The pesticide composition further includes other pesticides and / or fertilizers; preferably, the other pesticides are plant growth regulators.

6. The use of the plant immunomodulator of any one of claims 1-3, or the pesticide composition of claim 4 or 5, in regulating plant growth, development, reproduction, disease prevention and / or disease resistance.

7. The application as described in claim 6, characterized in that, The plant is selected from one or more of soybean, peanut, rapeseed, corn, wheat, cucumber and rice; and / or, the regulation of plant growth, development, reproduction, disease prevention and / or disease resistance is selected from: promoting seed germination, promoting seedling morphogenesis, promoting plant growth in the mature stage, promoting plant damage repair, increasing crop yield, improving fruit quality, improving plant resistance to biotic stress, and improving plant resistance to abiotic stress.

8. The application as described in claim 7, characterized in that, The biological stresses include one or more of diseases, pests, and weeds; and / or the abiotic stresses include one or more of drought, waterlogging, salinity, frozen or near-frozen soil environments, extreme low temperatures, and extreme high temperatures, preferably, extreme low temperatures are ≤15℃ and extreme high temperatures are ≥30℃.

9. The application as described in any one of claims 6-8, characterized in that, The applications are selected from the following group: (1) The use of the plant immunomodulator of any one of claims 1-3, or the pesticide composition of claim 4 or 5, in promoting the germination of plant seeds; (2) The use of the plant immune modulator according to any one of claims 1-3, or the pesticide composition according to claim 4 or 5, in promoting the emergence of plant seeds; (3) The application of the plant immunomodulator of any one of claims 1-3 and the pesticide composition of claim 4 or 5 in improving plant yield; (4) The use of the plant immune modulator of any one of claims 1-3, or the pesticide composition of claim 4 or 5, in promoting plant damage repair; (5) The use of the plant immunomodulator of any one of claims 1-3, or the pesticide composition of claim 4 or 5, in improving the plant response to abiotic stress.

10. A method for regulating plant growth, development, reproduction, disease prevention, and / or disease resistance, characterized in that, The method includes applying the plant immunomodulator of any one of claims 1-3 or the pesticide composition of claim 4 or 5 to the plant; preferably, the application includes seed soaking and / or spraying.

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