3-methyl-2-[[2-[3-oxo-2-[pent-2-enyl]cyclopentyl]acetyl]amino]hexanoic acid, method of preparation and use

Abstract

The present application relates to the technical field of pesticides, and in particular to 3-methyl-2-[[2-[3-oxo-2-[pent-2-enyl]cyclopentyl]acetyl]amino]hexanoic acid, a preparation method and applications thereof.The compound is a novel compound synthesized for the first time, and the structure thereof is identified, and biological activity, application range and crop safety thereof are researched.The results show that the compound can be used as or prepared into a novel plant immune elicitor and growth regulator, and has good application value.

Description

Technical Field

[0001] This invention relates to the field of pesticide technology, and in particular to a 3-methyl-2-[[2-[3-oxo-2-[pent-2-enyl]cyclopentyl]acetyl]amino]hexanoic acid, its preparation method, and its application. Background Technology

[0002] In agricultural production, plants often suffer from both biotic and abiotic stresses simultaneously at various stages of growth and development. Abiotic stresses, including salt stress, low-temperature stress (cold stress), and high-temperature stress (heat stress), severely disrupt plant physiological homeostasis, activate multi-level stress response mechanisms, and thus limit growth and development, sometimes even leading to reduced yields or crop failure. Cold stress refers to temperatures below the plant's optimal growth temperature, typically divided into cold stress at 0–15℃ and frost stress at <0℃. Heat stress refers to temperatures exceeding the plant's optimal growth temperature by 5–6℃, with severe heat stress occurring when temperatures exceed 10–15℃. Currently, soil salinization is also a growing problem. my country has one of the largest areas of saline-alkali land in the world, accounting for approximately 10% of the global total, severely restricting the realization of agricultural production potential. Furthermore, pests and diseases remain a significant factor affecting agricultural production safety; large-scale outbreaks often result in significant yield reductions. Currently, disease control in agricultural production still primarily relies on chemical pesticides. However, long-term and excessive use of fungicides can easily induce pathogens to develop drug resistance, and can also lead to a series of problems such as excessive pesticide residues and environmental pollution. This poses a risk of failure to the traditional prevention and control strategy centered on "directly killing pathogens", and is a serious challenge to sustainable agricultural development and food security.

[0003] Plant immune inducers, as a novel class of pesticides, enhance plants' resistance to diseases and abiotic stresses by activating their own immune system and regulating related metabolic processes. Unlike traditional chemical pesticides, plant immune inducers typically do not directly exhibit bactericidal activity but rather exert their control effect by inducing the plant's natural defense mechanisms. Therefore, pathogens are less likely to develop resistance, aligning with the direction of green pest control and sustainable agricultural development. Furthermore, under natural conditions, plants often face multiple stresses simultaneously, such as high temperatures and drought, which can cause more severe damage. Since the plant's own immune system has limitations in its resilience, applying plant immune inducers helps improve the plant's overall stress resistance. However, research and development of plant immune inducers in my country is still in its early stages, with a limited number of officially registered products. Therefore, developing green, efficient, and multifunctional plant immune inducers is of great significance for meeting the needs of green agricultural production in my country and enhancing crop resistance to stress and disease.

[0004] Plant growth regulators are generally prepared through chemical synthesis or bio-fermentation. They can be added exogenously to alter the plant's growth and development process, thereby increasing yield and improving quality. However, existing growth regulators suffer from drawbacks such as relatively singular effects and difficulty in simultaneously addressing the dual needs of promoting growth and enhancing stress resistance, thus limiting their widespread application. Therefore, providing novel and effective growth regulators is of great significance to agricultural production.

[0005] Jasmonic acid (JA) is an important lipoxygenase signaling molecule widely found in plants, belonging to the plant hormone family. It plays a crucial role in regulating plant growth and development, responding to abiotic stress, and defense responses. Jasmonoyl-isoleucine (JA-Ile) is one of the important active forms of jasmonic acid in plants. It can activate the jasmonic acid signaling pathway, is specifically recognized by the COI1-JAZ receptor complex, and mediates various physiological processes such as plant defense responses, damage responses, and abiotic responses. Methyl jasmonate (MeJA) is a volatile derivative of JA. Due to its high volatility, it affects the persistence of its effects and the stability of its application, which is not conducive to its widespread application in agricultural production. Therefore, developing a novel jasmonic acid derivative to further improve its application performance while retaining or enhancing its biological functions is of significant research importance and application value. Summary of the Invention

[0006] In view of this, the technical problem to be solved by the present invention is to provide 3-methyl-2-[[2-[3-oxo-2-[pent-2-enyl]cyclopentyl]acetyl]amino]hexanoic acid, its preparation method and application.

[0007] The technical solution of the present invention to solve the above-mentioned technical problems is as follows:

[0008] This invention provides a novel compound, the Chinese name of which is 3-methyl-2-[[2-[3-oxo-2-[pent-2-enyl]cyclopentyl]acetyl]amino]hexanoic acid, the English name of which is 3-methyl-2-[[2-[3-oxo-2-[pent-2-enyl]cyclopentyl]acetyl]amino]hexanoic acid, and the compound has the structure shown in formula (1) or a salt thereof:

[0009]

[0010] Equation (1).

[0011] This compound is a novel compound synthesized for the first time in this invention. The invention studies its biological activity, scope of application and crop safety. The results show that this compound can be used as or to prepare novel plant immune inducers and growth regulators. It can be used for plant disease control, biotic stress and abiotic stress relief, and has great application value.

[0012] On the other hand, the present invention provides a method for preparing the above-mentioned 3-methyl-2-[[2-[3-oxo-2-[pent-2-enyl]cyclopentyl]acetyl]amino]hexanoic acid, comprising the following steps:

[0013] Using 2-amino-3-methylhexanoic acid and methyl jasmonate as raw materials, an ammonolysis reaction was carried out under the action of a strong organic base to obtain 3-methyl-2-[[2-[3-oxo-2-[pent-2-enyl]cyclopentyl]acetyl]amino]hexanoic acid.

[0014] The molar ratio of 2-amino-3-methylhexanoic acid to methyl jasmonate is preferably 1:(1~2), more preferably 1:(1.2~1.8), and for example, it can be 1:1.2, 1:1.5, or 1:1.8.

[0015] The molar ratio of the 2-amino-3-methylhexanoic acid to the strong organic base is preferably 1:(1~2), more preferably 1:(1.2~1.8), and for example, it can be 1:1.2, 1:1.5, or 1:1.8.

[0016] The organic strong base is preferably one or more of sodium alkoxide, potassium alkoxide, sodium amino group, and alkyl lithium.

[0017] Preferably, the strong organic base is selected from sodium tert-butoxide.

[0018] The preferred temperature for the ammonolysis reaction is room temperature, specifically 15~35℃.

[0019] The ammonolysis reaction time is preferably 12-48 hours, more preferably 12-24 hours.

[0020] In some specific embodiments, the preparation method includes the following steps:

[0021] The reaction was carried out using 2-amino-3-methylhexanoic acid, methyl jasmonate, and sodium tert-butoxide as raw materials; the reaction was quenched; the system was concentrated, water was added to make the mixture turbid, and the pH was adjusted to acidic, preferably to pH 2; the mixture was extracted with ethyl acetate, the organic phases were combined, dried and the solvent was removed to obtain a yellow clear oily liquid, namely compound 3-methyl-2-[[2-[3-oxo-2-[pent-2-enyl]cyclopentyl]acetyl]amino]hexanoic acid.

[0022] On the other hand, the present invention provides an agricultural composition comprising the above-mentioned 3-methyl-2-[[2-[3-oxo-2-[pent-2-enyl]cyclopentyl]acetyl]amino]hexanoic acid or a salt thereof and an adjuvant.

[0023] The formulations of the agricultural compositions include, but are not limited to:

[0024] Liquid dosage forms, solid dosage forms, wettable powders, microemulsions or water-in-oil emulsions, granules, and ultra-low volume liquids.

[0025] The liquid formulations include, but are not limited to, soluble agents and suspending agents.

[0026] The solid formulations include, but are not limited to, water-dispersible granules, soluble powders / granules, and seed treatment suspensions.

[0027] The present invention does not impose any particular limitation on the excipients, and those skilled in the art can select them according to the dosage form of the composition. In some specific embodiments, the excipients may include surfactants, etc. As a further preferred embodiment of the present invention, the surfactant may be Tween 20. The concentration of Tween 20 in the composition is preferably 0.02% (v / v).

[0028] On the other hand, the present invention provides the application of the above-mentioned 3-methyl-2-[[2-[3-oxo-2-[pent-2-enyl]cyclopentyl]acetyl]amino]hexanoic acid or its salt (hereinafter referred to as JG-1) or the above-mentioned agricultural composition in improving plant stress resistance, improving disease and pest resistance and / or improving growth performance.

[0029] Specifically, the present invention provides the use of the above-mentioned JG-1 or the above-mentioned agricultural composition as and / or in the preparation of plant immune inducers and / or plant growth regulators.

[0030] The improvement of plant stress resistance includes at least one of the following:

[0031] I) Improve resistance to high-temperature stress;

[0032] II) Improve resistance to low-temperature stress;

[0033] III) Improve resistance to salt stress;

[0034] ), and improve the ability to resist pesticide damage to seeds and / or seedlings.

[0035] This invention provides the application of JG-1 in enhancing plant resistance to abiotic and / or biotic stresses. As a novel derivative, this compound exhibits significant structural innovation and functional advantages. It demonstrates excellent activity in immune-induced resistance experiments, significantly enhancing plant resistance to both biotic and abiotic stresses.

[0036] Specifically, this invention provides the application of JG-1 in improving the resistance of plants to one or more of high temperature stress, low temperature stress, salt stress and pesticide damage stress.

[0037] The improvement of resistance to high temperature stress includes, but is not limited to: increasing the dry and fresh weight of the aboveground parts of the plant, reducing the heat damage index, alleviating the inhibitory effect of high temperature stress on plant growth and the degree of plant damage, and reducing tissue damage caused by high temperature.

[0038] When used to improve resistance to high-temperature stress, the working concentration of JG-1 is 0.1~1000 nM, preferably 0.1~100 nM, and more preferably 1 nM. Experimental results show that when inducing wheat resistance to high-temperature stress, the heat injury index decreased by 10%, 39%, and 34% when the JG-1 concentration was 0.1 nM, 1 nM, and 10 nM, respectively. When inducing tomato resistance to high-temperature stress, the heat injury index decreased by 3%, 25%, 21%, and 15% when the JG-1 concentration was 0.1 nM, 1 nM, 10 nM, and 100 nM, respectively.

[0039] The improvement of resistance to low temperature stress includes, but is not limited to: increasing the root length and / or stem length of seedlings to alleviate the adverse effects of low temperature stress on the growth and development of plant seedlings.

[0040] When used to improve resistance to low-temperature stress, the working concentration of JG-1 is 0.1~1000 nM, preferably 0.1~100 nM, and more preferably 10 nM. Experimental results show that when inducing resistance to low-temperature stress in rice, a JG-1 concentration of 10 nM significantly promotes root length, with an increase of 31%; a JG-1 concentration of 1 nM significantly promotes stem length, with an increase of 30%.

[0041] The improvement of salt stress resistance includes, but is not limited to: increasing seed germination rate, increasing plant shoot length and / or root length.

[0042] When used to improve salt stress resistance, the working concentration of JG-1 is 0.1~1000 nM, preferably 0.1~100 nM, and more preferably 10~100 nM. When used to induce salt stress resistance in rice, JG-1 treatment at concentrations of 10 nM and 100 nM showed the best results, with germination rate, shoot length, and root length reaching 87%, 81%, and 70% (10 nM) and 87%, 77%, and 73% (100 nM) of the control group, respectively.

[0043] Enhancing resistance to pesticide damage to seeds and / or seedlings includes, but is not limited to: increasing plant germination rate, shoot length, and root length.

[0044] Preferably, the pesticide includes one or more of herbicides, insecticides, and fungicides.

[0045] Preferably, the pesticide includes one or more of the following: fluroxypyr·pentyl·methoxypyr, methoxypyr·ethoxypyr, and quinclorac.

[0046] When used to improve resistance to pesticide damage to seeds and / or seedlings, the working concentration of JG-1 is 0.1~1000 nM, preferably 0.1~100 nM, and more preferably 10~100 nM. When used to alleviate damage to rice seed germination and seedlings caused by fluazinam·pentyl·methrin, the 10 nM JG-1 treatment showed the most significant effect, increasing the germination rate by 24%, and shoot and root length by 18% and 29%, respectively. When used to alleviate damage to rice seed germination and seedlings caused by methamidophos·ethoxysulfuron, the 100 nM JG-1 treatment showed the most significant mitigation effect, increasing the germination rate by 22%, root length by 91%, and shoot length by 5%. When used to alleviate the damage of dichloroquinoline to tobacco seedlings, the 100 nM JG-1 treatment showed the best mitigation effect, with the length and width of the third to last leaf increasing by 27% and 64%, respectively, and the length and width of the fourth to last leaf increasing by 24% and 33%, respectively; plant height increased by 19%, above-ground fresh weight increased by 32%, and fresh weight of the phytotoxic part increased by 79%.

[0047] The diseases and pests mentioned include, but are not limited to, fungal diseases, preferably those caused by the genera *Erythromyces*, *Erythromyces bryonis*, *Monophyllum*, or *Uncaria*; more preferably, powdery mildew of gramineous crops, for example, wheat powdery mildew.

[0048] The improvement of disease and pest resistance includes increasing the expression level of plant disease resistance genes, activating disease resistance-related signal transduction pathways, reducing leaf lesion area, mitigating damage to the leaf photosynthetic system, activating the plant's innate immune response, and limiting the colonization and spread of pathogens.

[0049] The disease-resistant genes include, but are not limited to, PR1 and MPK3.

[0050] When used to improve resistance to pests and diseases, the working concentration of JG-1 is preferably 0.01~1mM, more preferably 0.1~1mM.

[0051] The improvement in growth performance includes promoting seed germination and / or promoting seedling growth.

[0052] The promotion of seedling growth includes, but is not limited to: improving the cold resistance of seedlings and alleviating the effects of low temperature stress on seedlings; inducing the expression of disease resistance genes in seedlings; and promoting the development and growth of plant seedling roots.

[0053] The disease-resistant genes include, but are not limited to: RbohD and PR1.

[0054] The promotion of seed germination includes, but is not limited to: increasing the germination rate of seeds and increasing the root and stem length of seedlings formed from germinating plant seeds.

[0055] When used to improve growth performance, the working concentration of JG-1 is preferably 0.1~1000 nM, more preferably 1~100 nM, and most preferably 1 nM or 10 nM. Experimental results show that 10 nM JG-1 has the most significant promoting effect on root length, while 100 nM JG-1 has the best promoting effect on stem length. When used for tomato seed soaking, treatments with 0.1 nM, 0.5 nM, 1 nM, and 5 nM JG-1 increased the stem length of tomato seedlings by 9%, 8%, 18%, and 26%, respectively, and the root length by 9%, 11%, 13%, and 29%, respectively, with the 5 nM treatment showing the most significant promoting effect.

[0056] The present invention does not impose any special limitation on the variety of the plant. Optionally, the plant is a grass, a cucurbit, or a solanaceous plant. Preferably, the grass includes rice, wheat, and / or corn; the cucurbit includes cucumber; and the solanaceous includes tobacco and / or tomato.

[0057] On the other hand, the present invention provides a method for plant breeding and / or cultivation, comprising at least one of the following:

[0058] i) Soaking, dressing, coating and / or mulching seeds with the above-mentioned 3-methyl-2-[[2-[3-oxo-2-[pent-2-enyl]cyclopentyl]acetyl]amino]hexanoic acid or its salt or the above-mentioned agricultural composition;

[0059] ii) Spray the above-mentioned 3-methyl-2-[[2-[3-oxo-2-[pent-2-enyl]cyclopentyl]acetyl]amino]hexanoic acid or its salt or the above-mentioned agricultural composition on the leaves or plants;

[0060] iii) Apply the above-mentioned 3-methyl-2-[[2-[3-oxo-2-[pent-2-enyl]cyclopentyl]acetyl]amino]hexanoic acid or its salt or the above-mentioned agricultural composition to the roots or root zone soil of plants.

[0061] Based on this, the present invention provides a method for preventing and controlling plant diseases, comprising the following steps: using the above-mentioned 3-methyl-2-[[2-[3-oxo-2-[pent-2-enyl]cyclopentyl]acetyl]amino]hexanoic acid or its salt or the above-mentioned agricultural composition to prevent and control plant diseases.

[0062] The present invention provides a method for improving plant stress resistance, comprising the following steps: using the above-mentioned 3-methyl-2-[[2-[3-oxo-2-[pent-2-enyl]cyclopentyl]acetyl]amino]hexanoic acid or its salt or the above-mentioned agricultural composition to improve plant stress resistance.

[0063] This invention provides a method for improving plant resistance to biotic stress, comprising the following steps: applying 3-methyl-2-[[2-[3-oxo-2-[pent-2-enyl]cyclopentyl]acetyl]amino]hexanoic acid or a salt thereof or the above-mentioned agricultural composition to the plant. The biotic stress may be fungal stress.

[0064] This invention provides a method for improving plant resistance to abiotic stresses, comprising the following steps: applying 3-methyl-2-[[2-[3-oxo-2-[pent-2-enyl]cyclopentyl]acetyl]amino]hexanoic acid or a salt thereof or the above-mentioned agricultural composition to the plant. The abiotic stresses may be selected from any one or more of high-temperature stress, low-temperature stress, salt stress, and / or pesticide stress.

[0065] The advantages and positive effects of this invention include:

[0066] This invention provides a novel compound, 3-methyl-2-[[2-[3-oxo-2-[pent-2-enyl]cyclopentyl]acetyl]amino]hexanoic acid. This invention confirms that this compound possesses significant immunoinducing activity, effectively enhancing plant immune responses to common agricultural diseases and improving plant resistance to various abiotic stresses (such as high temperature, low temperature, and salinity). Therefore, this compound has great potential as a natural plant immune inducer, contributing to improved crop yield and quality and promoting sustainable agricultural development.

[0067] This invention discovers that 3-methyl-2-[[2-[3-oxo-2-[pent-2-enyl]cyclopentyl]acetyl]amino]hexanoic acid possesses highly efficient broad-spectrum immunoinducing activity. At a concentration of 1 mM, it can significantly induce an immune response in wheat against powdery mildew, achieving a relative immunogenicity of 70.5%. Regarding responses to abiotic stresses, experimental results show that: at a concentration of 10 nM, it can significantly improve the resistance of rice to low temperatures; at a concentration of 1 nM, it can enhance the resistance of wheat and tomatoes to high temperatures; at a concentration of 100 nM, it can significantly improve the tolerance of rice to salt; at a concentration of 10 nM, seed soaking treatment can effectively alleviate the damage to rice seeds caused by fluroxypyr•pentyl•carbendazim; at a concentration of 100 nM, it can alleviate the damage to rice seeds caused by carbendazim•ethoxysulfuron; and at a concentration of 100 nM, it can effectively reduce the damage to tobacco seedlings caused by quinclorac. 3-Methyl-2-[[2-[3-oxo-2-[pent-2-enyl]cyclopentyl]acetyl]amino]hexanoic acid has the characteristics of low dosage and no pollution. As a highly efficient and environmentally friendly new plant immune inducer, it has broad application prospects and important agricultural utilization value.

[0068] The compound described in this invention can effectively control major fungal diseases in agriculture, such as wheat powdery mildew, indicating that it can induce an immune response in plants to combat the disease. Simultaneously, 3-methyl-2-[[2-[3-oxo-2-[pent-2-enyl]cyclopentyl]acetyl]amino]hexanoic acid can also enhance plant resistance to abiotic stresses such as high temperature stress, low temperature stress, salt stress, and pesticide damage, providing technical support for mitigating the damage caused by these stresses to plants.

[0069] This invention discovers that 3-methyl-2-[[2-[3-oxo-2-[pent-2-enyl]cyclopentyl]acetyl]amino]hexanoic acid, through seed soaking, foliar application, and root irrigation, can prevent the occurrence and spread of various major agricultural diseases and alleviate the inhibition caused by various abiotic stresses on crops during growth and development. 3-methyl-2-[[2-[3-oxo-2-[pent-2-enyl]cyclopentyl]acetyl]amino]hexanoic acid is easy to use, can provide early prevention, reduces the level of plant damage caused by various biotic and abiotic stresses, reduces pesticide use, and saves production costs.

[0070] 3-Methyl-2-[[2-[3-oxo-2-[pent-2-enyl]cyclopentyl]acetyl]amino]hexanoic acid is a novel structure that is easy to produce industrially. This invention confirms that 3-methyl-2-[[2-[3-oxo-2-[pent-2-enyl]cyclopentyl]acetyl]amino]hexanoic acid can promote plant growth and has the potential to be developed into a novel plant immune inducer and growth regulator.

[0071] This invention discovers that 3-methyl-2-[[2-[3-oxo-2-[pent-2-enyl]cyclopentyl]acetyl]amino]hexanoic acid can effectively promote plant growth through seed soaking and root irrigation. At a concentration of 10 nM, seed soaking promotes rice seed germination and significantly improves the root and stem growth of rice seedlings; at a concentration of 100 nM, seed soaking promotes corn seed germination and significantly improves the root and stem growth of corn seedlings; at a concentration of 5 nM, seed soaking promotes tomato seed germination and significantly improves the growth of tomato seedlings; at a concentration of 1 nM, seed soaking promotes cucumber seed germination and significantly improves the root length of cucumber seedlings; and at a concentration of 1 nM, root irrigation of soil-grown cucumber seedlings promotes cucumber seedling growth, significantly increasing leaf area, plant height, root length, and fresh weight.

[0072] This invention discovers that 3-methyl-2-[[2-[3-oxo-2-[pent-2-enyl]cyclopentyl]acetyl]amino]hexanoic acid can promote plant growth and development through seed soaking and root irrigation. 3-methyl-2-[[2-[3-oxo-2-[pent-2-enyl]cyclopentyl]acetyl]amino]hexanoic acid is easy to use, solves production problems that traditional agronomic methods cannot address, and saves production costs.

[0073] In this invention, for ease of description, 3-methyl-2-[[2-[3-oxo-2-[pent-2-enyl]cyclopentyl]acetyl]amino]hexanoic acid can be abbreviated as JG-1. Attached Figure Description

[0074] Figure 1 The 1H NMR spectrum of JG-1 ( 1 H NMR);

[0075] Figure 2 The carbon NMR spectrum of JG-1 ( 13 (C NMR).

[0076] Figure 3 This is the first-order mass spectrum of JG-1;

[0077] Figure 4 The disease development process of wheat powdery mildew in living leaves induced by JG-1;

[0078] Figure 5 The effects of JG-1 on the accumulation of O2.- and H2O2 in wheat leaves during the process of inducing wheat resistance to powdery mildew;

[0079] Figure 6 The effect of JG-1 on the expression levels of wheat PR1 and MPK3 disease resistance genes;

[0080] Figure 7The effects of different concentrations of JG-1 on wheat phenotypes under high temperature stress;

[0081] Figure 8 The effects of different concentrations of JG-1 on tomato phenotypes under high temperature stress;

[0082] Figure 9 The effects of different concentrations of JG-1 and methyl jasmonate on rice seed germination under low temperature stress;

[0083] Figure 10 The effects of different concentrations of JG-1, methyl jasmonate, and mixed soaking of fluazinam·pentyl·carbendazim on rice seed germination;

[0084] Figure 11 The effects of different concentrations of JG-1, methyl jasmonate, and a mixture of fenitrothion and ethoxysulfuron on rice seed germination;

[0085] Figure 12 The study aimed to mitigate the phytotoxicity of tobacco seedlings caused by dichloroquinoline acid by different concentrations of JG-1.

[0086] Figure 13 Effects of JG-1 and methyl jasmonate on rice seed germination under salt stress;

[0087] Figure 14 The effects of JG-1 and jasmonic acid derivatives on the phenotype of cucumber seedlings under low temperature stress;

[0088] Figure 15 The effects of JG-1 and jasmonic acid derivatives on the expression levels of cucumber PR1 and RbohD defense genes, respectively;

[0089] Figure 16 The effect of different concentrations of JG-1 soaking on rice seed germination;

[0090] Figure 17 The effect of different concentrations of JG-1 soaking on maize seed germination;

[0091] Figure 18 The effect of different concentrations of JG-1 soaking on tomato seed germination;

[0092] Figure 19 The effect of different concentrations of JG-1 soaking on cucumber seed germination;

[0093] Figure 20 The effects of different concentrations of JG-1 root irrigation on the growth of cucumber seedlings. Detailed Implementation

[0094] The principles and features of the present invention are described below with reference to the accompanying drawings. The examples given are only for explaining the present invention and are not intended to limit the scope of the present invention.

[0095] This invention synthesizes a novel compound, 3-methyl-2-[[2-[3-oxo-2-[pent-2-enyl]cyclopentyl]acetyl]amino]hexanoic acid (which can be abbreviated as JG-1). This compound, its preparation method, and its applications have not been reported in any domestic or international literature. Systematic activity screening using this invention revealed that this compound possesses excellent plant immune-inducing and growth-regulating activities. It not only effectively inhibits the occurrence and spread of fungi on plant leaves but also significantly alleviates damage to plants caused by high temperature, low temperature, salinity, and pesticide poisoning, and promotes plant growth.

[0096] This invention provides the application of JG-1 as or in the preparation of novel plant immune inducers. Through a series of experiments, JG-1 demonstrated significant activity in inducing wheat resistance to powdery mildew, rice resistance to low-temperature stress, wheat and tomato resistance to high-temperature stress, and rice resistance to salt stress. Simultaneously, JG-1 can alleviate phytotoxic damage caused by various pesticides, including damage to rice seed germination and seedlings caused by fluazinam·pentyl·methoxyfenozide, damage to rice caused by methoxyfenozide·ethoxyquin, and damage to tobacco seedlings caused by quinclorac. Experimental results indicate that JG-1 possesses multiple functions, including inducing disease resistance, stress resistance, and alleviating phytotoxicity, and has broad application prospects.

[0097] This invention provides a method for controlling plant diseases and improving plant stress resistance using the synthesized JG-1. The specific implementation scheme is as follows: When controlling plant viral diseases, the application concentration of JG-1 is 0.01-1 mM (prepared with 0.02% by volume of the surfactant Tween 20). Under these conditions, JG-1 can significantly inhibit the infection and spread of viruses on plant leaves, thereby effectively suppressing the occurrence and spread of diseases. When improving plant resistance to abiotic stresses, the application concentration of JG-1 is 0.1-1000 nM. Within this concentration range, JG-1 can significantly improve plant resistance to abiotic stresses such as high temperature, low temperature, and salt stress, thereby enhancing the overall stress resistance of crops.

[0098] This invention provides a method for improving plant resistance to biotic stress, comprising applying the plant immune inducer described in this invention to the plant in advance; wherein the biotic stress is fungal stress.

[0099] This invention provides a method for controlling wheat powdery mildew using JG-1, which is applied at a concentration range of 0.01-1 mM (prepared with 0.02% by volume of the surfactant Tween 20) and investigated 5 days after wheat inoculation with powdery mildew fungus. The results show that as the treatment concentration increases, the disease index of wheat infected with powdery mildew significantly decreases, and the relative immune effect improves. At a high concentration of 1 mM, the disease index was 23.3, and the relative immune effect was 70.5%.

[0100] This invention provides a method for improving plant resistance to abiotic stresses, comprising applying the plant immune inducer described in this invention to the plant; wherein the abiotic stresses are selected from any one or more of high temperature, low temperature, salt and / or pesticide stress.

[0101] This invention provides a method for using JG-1 to improve plant resistance to low temperatures. Rice seeds are soaked in JG-1 solutions with concentrations ranging from 0.1 to 100 nM. After 3 days of low-temperature stress at 4°C, the results showed that the root and stem lengths of rice seedlings treated with 0.1 nM, 1 nM, 10 nM, and 100 nM were significantly higher than those of the control group, indicating that this compound can effectively alleviate the damage of low temperatures to rice seedlings and improve the resistance of rice to low-temperature stress.

[0102] This invention provides a method for improving plant resistance to high temperatures using JG-1. Wheat and tomato plants were treated with a JG-1 solution (concentrated in the range of 0.1-100 nM, prepared with 0.02% by volume of the surfactant Tween 20). After treatment, the plants were subjected to high temperatures of 45℃ for 15 hours and 42℃ for 36 hours, respectively, followed by a 5-day recovery period at room temperature. The results showed that the heat damage index of the treated groups was significantly lower than that of the control group, and the biomass was higher. This indicates that exogenous application of JG-1 solution can effectively alleviate the damage caused by high temperatures to seedlings.

[0103] This invention provides a method for using JG-1 to improve the resistance of plants to salt stress, wherein rice seeds are soaked in a JG-1 solution with a concentration range of 0.1-100 nM. Under 50 mM NaCl stress, the rice seeds in the treated group showed significantly higher germination rate, shoot length, and root length than the control group, indicating that this compound can significantly improve the tolerance of rice to salt stress.

[0104] This invention provides a method for using JG-1 to alleviate phytotoxicity to rice caused by fluazinam•pentyl•methoxyfenozide and methoxyfenozide•ethoxyfenozide. The method involves treating rice seedlings with a JG-1 solution at concentrations ranging from 0.1 to 100 nM, adding pesticides, and conducting experiments using a seed soaking treatment. Results showed that compared to the control group without JG-1, the germination rate, shoot length, and root length of the experimental group were significantly increased. This indicates that JG-1 can effectively alleviate the damage to rice seed germination and seedling growth caused by fluazinam•pentyl•methoxyfenozide and methoxyfenozide•ethoxyfenozide.

[0105] This invention provides a method for alleviating the phytotoxicity of quinclorac acid (QNC) in tobacco using JG-1, employing a JG-1 solution with a concentration range of 1-1000 nM for root drenching treatment. Results showed that, compared with the untreated control group, the experimental group of tobacco seedlings exhibited significantly increased leaf length, leaf width, plant height, and fresh weight, and a marked reduction in leaf curling. This indicates that JG-1 can effectively alleviate the phytotoxicity of quinclorac acid in tobacco seedlings, significantly improving leaf growth, plant height, fresh weight, and leaf morphology.

[0106] This invention studies the bioactivity, applicability, and crop safety of JG-1, and finds that JG-1 also has a very unique role in regulating crop growth, can promote plant growth and increase yield, and has the advantages of being environmentally friendly, widely applicable, and safe to use.

[0107] This invention provides a method for promoting seed germination and plant growth by applying a 1-1000 nM JG-1 solution to a target plant; the target plant is selected from grain crops and vegetables; the grain crops are preferably rice and corn, and the vegetables are preferably tomatoes and cucumbers.

[0108] This invention provides a method for promoting rice seed germination and seedling growth. Rice seeds are treated by soaking them with JG-1 at a concentration of 1-1000 nM, which can significantly promote rice seed germination. In particular, when the concentration of JG-1 is 10 nM, the root length of rice can be increased by 31%.

[0109] This invention provides a method for promoting corn seed germination and seedling growth. Treating corn seeds with JG-1 at a concentration of 0.1-1000 nM by soaking can significantly promote corn seed germination. In particular, when the concentration of JG-1 is 100 nM, the germination rate of corn can be increased by 56%, the stem length can be increased by 19%, and the root length can be increased by 22%.

[0110] This invention provides a method for promoting tomato seed germination and seedling growth. Using a JG-1 solution with a concentration of 0.1-100 nM can significantly promote the growth of tomato seedlings. Preferably, the effect is better when the JG-1 concentration is between 0.1-5 nM. Most preferably, when the JG-1 concentration is 5 nM, the effect on promoting the growth of rice seedlings is the most significant, and the stem length and root length can be increased by 26% and 29%, respectively.

[0111] This invention provides a method for promoting rooting of cucumber seedlings. Treating cucumber seeds with JG-1 at a concentration of 0.1-100 nM by soaking can significantly promote cucumber seed germination; especially when the JG-1 concentration is 1 nM, the root length of cucumber seedlings can increase by 19%.

[0112] This invention provides a method for promoting cucumber seedling growth. Treating cucumber seedlings grown in soil with JG-1 at a concentration of 0.1-100 nM via root irrigation significantly promotes seedling growth. Specifically, at a JG-1 concentration of 1 nM, the area of ​​the first true leaf can increase by 58%, 11%, and 2% at 6, 12, and 18 days, respectively; the area of ​​the second true leaf can increase by 139% and 33% at 12 and 18 days, respectively; and at 18 days, the area of ​​the third true leaf can increase by 56%, root length by 29%, above-ground fresh weight by 43%, and root fresh weight by 66%.

[0113] To further illustrate the present invention, a detailed description is provided below with reference to embodiments. However, it should be understood that these descriptions are merely for further illustrating the features and advantages of the present invention, and not for limiting the scope of the claims.

[0114] There are no particular restrictions on the source of any raw materials used in this invention; they can be purchased from the market or prepared using conventional methods known to those skilled in the art.

[0115] JA-Ile, JA-Leu, JA-Glu, and JA-Val were synthesized in our laboratory using a NaOtBu-promoted direct amidation method for unactivated esters with amines, as reported in the literature. The esters were then purified to a purity ≥95%. Reference: Zhang R, Yao WZ, Qian L, et al. A practical and sustainable protocol for direct amidation of unactivated esters under transition-metal-free and solvent-free conditions[J]. Green Chemistry, 2021, 23: 3972–3982. DOI: 10.1039 / D1GC00720C.

[0116] Unless otherwise specified, all techniques or conditions used in the examples are conventional methods or performed according to techniques or conditions described in the literature in this field, or according to product instructions. Reagents used, unless otherwise specified, are all conventional products that can be purchased from legitimate channels or prepared according to conventional methods in this field. Instruments used, unless otherwise specified, are all conventional products that can be purchased from legitimate channels.

[0117] Unless otherwise specified, all solutions in this invention are prepared using water as the solvent. The room temperature in this invention is 26°C. All primers in this invention were synthesized by Shanghai Genscript Biotech Co., Ltd.

[0118] The 0.02% Tween-20 solution in this invention is prepared with distilled water, and the volume percentage of Tween-20 to distilled water is 0.02%.

[0119] The following is a description through specific embodiments.

[0120] Example 1

[0121] The chemical synthesis of JG-1 includes the following steps: 0.726 g (5 mmol) of 2-amino-3-methylhexanoic acid, 1.68 g (7.5 mmol) of methyl jasmonate, and 0.72 g (7.5 mmol) of sodium tert-butoxide were sequentially added to a dry 250 mL three-necked flask. The reaction mixture was stirred at room temperature for 24 h. After the reaction was complete, 20 mL of cold water was slowly added dropwise at 4 °C to quench the reaction, and stirring was continued for 1 h. Then, the reaction system was subjected to rotary evaporation under reduced pressure at 65 °C until nearly dry (approximately 10–20 min). After rotary evaporation, 100 mL of water was added to make the mixture turbid, followed by the addition of 6 M hydrochloric acid to adjust the pH to 2. Subsequently, the mixture was extracted three times with ethyl acetate, with 100 mL of ethyl acetate added for each extraction. The organic phases were combined. The organic phase was transferred to a beaker and dried with anhydrous magnesium sulfate until the anhydrous magnesium sulfate was in a loose powder. Finally, magnesium sulfate was removed by vacuum filtration, and the solvent was removed again by rotary evaporation under reduced pressure at 65°C (about 15-20 min) to obtain a yellow, clear, oily liquid, namely compound JG-1.

[0122] Example 2

[0123] The structure of the obtained compound JG-1 was identified by nuclear magnetic resonance spectroscopy. 15 mg of purified JG-1 (prepared according to the method in Example 1) was dissolved in 0.5 mL of deuterated chloroform (CDCl3) and transferred to an NMR tube. The structure was recorded on a Bruker NMR spectrometer. 1 H NMR and 13 C NMR spectrum, in which 1 The H NMR test frequency was 500 MHz. 13 The C NMR test frequency was 126 MHz. Chemical shift (δ) is expressed in ppm, and coupling constant (J) is expressed in Hz. The obtained NMR data were processed and analyzed using MestReNova 11.0 software (Mestrelab Research SL, Santiago de Compostela, Spain).

[0124] The 1H NMR spectrum of JG-1 ( 1 H NMR) such as Figure 1 As shown, the carbon NMR spectrum of JG-1 ( 13 (C NMR) such as Figure 2 As shown, the first-order mass spectrum of JG-1 is as follows: Figure 3 As shown. The test results are as follows:

[0125] 1 H NMR (500 MHz, Chloroform-d) δ 5.46-5.21 (m, 2H, CH=CH) , 4.15-4.11(m, H, CHNH), 2.77-2.68 (m, H, CH3CH), 2.39-2.37 (m, 2H, COCH2), 2.33-2.29 (m,2H, CH2CH), 2.25-2.12 (m, 2H, CH2CH), 2.09-2.06 (m, 1H, CH2CH), 2.06-2.05 (m,2H, CCH2), 2.05-2.03 (m, 1H, CH2CH), 1.98-1.82 (m, 2H, CH2CH3), 1.54-1.44 (m,2H, CH2CH2CH3), 1.25 (t, J=10 Hz, 3H, CH2CH3) 1.16-0.92 (m, 2H, CH2CH2CH3), 0.89 (d, J=10 Hz, 3H, CHCH3) , 0.86 (t, J=10 Hz, 3H, CH2CH3).

[0126] 13 C NMR (126 MHz, Chloroform-d) δ 220.28 (COCH2), 173.57 (CONH), 171.82 (COOH), 134.21 (CH=CH), 124.46 (CH=CH), 60.51 (CHNH), 54.09 (CHCH2), 39.18 (CHCH2), 38.71 (CHCH2), 38.08 (COCH2), 37.96 (CHCH3), 37.85 (CH2CH2CH3), 27.32 (CH2CH3), 27.27 (CH2CH2), 25.51 (CHCH2), 20.69 (CH2CH2CH3), 14.34(CH2CH2CH3), 14.16 (CH2CH3), 14.06 (CH2CH3).

[0127] Mass spectrometry showed that the molecular ion peak of this compound was at 338.2368 [M+H]. + Its molecular formula was determined to be C 19 H 31NO4. Based on the results of the 1H NMR and 1C NMR spectra, the structural formula of this compound is shown in formula (1).

[0128]

[0129] Equation (1)

[0130] Example 3: JG-1 induces resistance to powdery mildew in wheat seedlings

[0131] The tested wheat variety was "Nannong 0686," which is highly susceptible to powdery mildew. Healthy, plump, and uniformly sized seeds were selected, surface-sterilized with 75% ethanol for 1 min, rinsed three times with sterile water, and then soaked in tap water. The seeds were then treated in the dark at 25℃ for 8 h. After imbibition, the seeds were removed, wrapped in moist gauze, and germinated in a dark environment at 25℃ for 24 h. Once the seeds showed signs of sprouting, they were sown in 10 cm diameter pots. The pots were pre-filled with sterilized mixed potting soil (nutrient soil and vermiculite mixed in a 1:1 volume ratio), and 36 seeds were evenly sown in each pot. The pots were then placed in a plant light cultivation chamber under the following conditions: temperature 22-24℃, relative humidity 60%-70%, and light intensity 200 μmol / m². -2 s -1 The photoperiod was 16 h light / 8 h dark. Accurately weigh JG-1, dissolve it in anhydrous ethanol to prepare a JG-1 stock solution with a concentration of 100 mM. Before use, dilute the JG-1 stock solution sequentially with 0.02% Tween-20 solution to 0.01 mM, 0.1 mM, and 1 mM, respectively. Specifically, for disease index and chlorophyll fluorescence assays, the JG-1 concentrations were 0.1 mM and 1 mM, respectively; for histochemical staining and gene expression analysis, the concentrations were 0.01 mM, 0.1 mM, and 1 mM, respectively. When wheat seedlings reached the one-leaf-one-heart stage, spray the different concentrations of diluted JG-1 solution using a foliar spray method. Spray until the leaf surface is evenly moistened and no dripping occurs. Spray once every 24 hours, for a total of two applications. Twenty-four hours after the second spraying, the plants were transferred to a dedicated wheat powdery mildew culture room for inoculation. Powdery mildew spores (Blumeria graminis f. sp. tritici) were propagated on seedlings 7-10 days prior to inoculation to ensure spore freshness and viability. For inoculation, fresh spore-bearing leaves were collected and immersed in an aqueous solution containing 0.01% (w / w) SDS, stirring thoroughly to release the spores, preparing a solution with a concentration of 1×10⁻⁶. 5A spore suspension of 100 spores / mL was prepared. The suspension was sprayed evenly onto the surface of the tested wheat leaves, ensuring full contact. After inoculation, the plants were placed in isolation areas according to treatment groups and cultured in a powdery mildew culture room under the following conditions: temperature 20-22℃, relative humidity 60%-70%, and light intensity 200 μmol / m². -2 s -1 The photoperiod is 12 hours of light followed by 12 hours of darkness. Each group is repeated 3 times.

[0132] Control group (i.e. blank control): sprayed with 0.02% Tween-20 solution instead of JG-1 solution, and the rest were the same as the experimental group.

[0133] Methyl jasmonate (MeJA) treatment group: spray an equal amount of 1 mM MeJA solution instead of JG-1 solution, and the rest are the same as the experimental group.

[0134] The method for preparing a 1 mM MeJA solution is as follows: dissolve MeJA in anhydrous ethanol to prepare a 100 mM MeJA stock solution, and then use 0.02% Tween-20 solution to prepare a 1 mM MeJA solution.

[0135] (1) After 5 days of 12-hour light / 12-hour dark incubation, the disease severity of wheat in each treatment was investigated. The severity of disease was recorded according to the wheat powdery mildew grading standard (Table 1) in the "Guidelines for Field Efficacy Trials of Pesticides (I)". The disease index and relative immune effect were calculated using the following formula:

[0136]

[0137]

[0138] Table 1 Grading standards for wheat powdery mildew (by leaf)

[0139]

[0140] Five days after inoculation with powdery mildew spores, when the powdery mildew lesions on the leaves of the control group (sprayed with 0.02% Tween-20 solution) were fully visible and the disease was stable, the severity of the disease in each treatment group was observed and photographed. After a 30-minute dark adaptation treatment (temperature 22-25℃), the photosynthetic physiological response during disease development was observed using a modulated chlorophyll fluorescence imaging system (Imaging-PAM). During measurement, three measurement points were randomly selected on each leaf, chlorophyll fluorescence parameters were recorded, and the maximum photochemical quantum yield (F) of photosystem II (PSII) was calculated. V / F M The imaging system parameters are set as follows: measured light intensity 0.25 μmol / m².-2 s -1 Photochemical light intensity 110 μmol m -2 s -1 Saturated pulsed light intensity 6000 μmol m -2 s -1 .

[0141] Experimental results are as follows Figure 4 As shown in Table 2, 5 days after inoculation with powdery mildew, the wheat leaves in the control group turned yellow overall and showed obvious powdery mildew spots. In both the 0.1 mM and 1 mM JG-1 treatment groups, the disease condition of wheat leaves was significantly alleviated, with the 1 mM JG-1 treatment showing better results than the methyl jasmonate treatment. Five days after inoculation, the lesion area in the control group significantly increased, and the lesions merged into patches, resulting in severe yellowing of the leaves. In contrast, the wheat leaves in the 1 mM JG-1 treatment group had smaller lesions, which did not merge into patches, and the leaves remained green, indicating that JG-1 had a significant inhibitory effect on the development of powdery mildew. Further analysis showed that the relative immunity effect of the 1 mM JG-1 treatment was 70.5%, higher than the 64.1% of the MeJA treatment. With the infection of powdery mildew, the degree of leaf damage in the treatment groups was significantly reduced, and with the increase of JG-1 concentration, the damage to the photosynthetic system of the leaves gradually slowed down. This indicates that JG-1 can effectively induce resistance to powdery mildew in wheat, and the higher the concentration, the better the effect. V / F M The F-value is a key indicator for measuring the photosynthetic potential of plant leaves, typically between 0.7 and 0.8. When plants are under stress, the F-value... V / F M The value will decrease, therefore F V / F M The F value can serve as a sensitive indicator of plant stress. Five days after inoculation, the F value of leaves in each treatment group was measured and analyzed. V / F M Value. The results showed that the F value of the 1 mM JG-1 treatment group was... V / F M The F value was significantly higher than that of the control group and the MeJA group; compared with the control group, the F value of the 1 mMJG-1 treatment was significantly higher. V / F M The value increased by 19.0%, and the MeJA treatment increased by 15.9%. This further demonstrates that JG-1 can induce wheat to improve its resistance to powdery mildew and effectively reduce the damage of powdery mildew infection to the photosynthetic system.

[0142] Table 2. Effects of different concentrations of JG-1 on wheat powdery mildew virus infection.

[0143]

[0144] (2) Wheat leaves (leaf segments about 4 cm long) were collected from each treatment group on days 1, 3 and 5 after inoculation with powdery mildew spores. Three leaves were randomly selected from each treatment and nitrile blue tetrazolium (NBT) and diaminobenzidine (DAB) were used for histochemical staining to qualitatively detect the accumulation of superoxide anions (O2.-) and hydrogen peroxide (H2O2) during the JG-1-induced wheat resistance to powdery mildew. NBT staining procedure: Prepare 2 mg / mL NBT staining solution (prepared with 50 mM sodium phosphate buffer, pH 7.8) and dispense into 50 mL centrifuge tubes; immerse the leaves in the staining solution and stain in the dark for more than 8 h; discard the staining solution, add decolorizing solution (ethanol, glacial acetic acid and glycerol in a volume ratio of 3:1:1) to immerse the sample, and decolorize in a 100℃ water bath until the green color of the leaves completely fades; preserve the decolorized leaves in 50% glycerol and observe and photograph them under a stereomicroscope. DAB staining procedure: Prepare a 1 mg / mL DAB staining solution (pH 3.8) and dissolve it in a 42℃ water bath; immerse the leaves in the staining solution and stain in the dark for more than 12 hours; discard the staining solution, add destaining solution, and destain in a 100℃ water bath; after destaining, store in 50% glycerol and observe and photograph under a stereomicroscope.

[0145] To further analyze the possible mechanism by which JG-1 induces resistance to powdery mildew in wheat, the accumulation of reactive oxygen species in leaves after inoculation was detected. The experimental results are as follows: Figure 5As shown, on day 1 (1 dpi) after powdery mildew inoculation, compared with the control group, wheat leaves pretreated with different concentrations of JG-1 showed significantly increased NBT and DAB staining depths, manifested as the accumulation of blue to dark blue precipitates (O2.-) and brownish-red precipitates (H2O2). The 1 mM JG-1 treatment group showed the deepest staining, indicating the strongest induced reactive oxygen species (ROS) burst. This result indicates that JG-1 treatment can activate the innate immune response in wheat, inducing a moderate oxidative burst in the early stages of pathogen infection, laying the foundation for the subsequent defense response. By day 3 (3 dpi) after inoculation, the disease rapidly developed in the control group, with ROS accumulation continuously increasing; while in the JG-1 treatment group, especially the 1 mM concentration treatment, although the ROS level was still higher than the control group, the increase slowed down, indicating that the JG-1-induced defense response had begun to effectively limit the spread of the pathogen. By day 5 post-inoculation (5 dpi), the control group exhibited severe powdery mildew infection, leading to increased tissue necrosis and a strong outbreak of pathological ROS, with extremely deep NBT and DAB staining. In contrast, the ROS accumulation level in the JG-1 treatment group was significantly lower than that in the control group, with the 1 mM JG-1 treatment group showing the best effect. This result further confirms that the JG-1-induced systemic acquired resistance successfully limited the colonization and spread of the pathogen, thus avoiding excessive ROS accumulation caused by cell necrosis in the later stages of infection. In summary, JG-1's regulation of reactive oxygen species exhibits distinct phased characteristics: in the early stage of infection (1 dpi), it promotes rapid ROS accumulation to activate defenses and inhibit pathogen invasion; in the middle stage of infection (3 dpi), it maintains a moderate ROS level to continuously control disease development; and in the late stage of infection (5 dpi), it effectively avoids pathological ROS outbreaks and protects leaf photosynthetic tissues. This dynamic ROS regulation mechanism of early activation, middle maintenance, and late-stage stability endows wheat with durable resistance to powdery mildew.

[0146] (3) 24 h after the second spraying, the middle part of the first leaf of wheat in each treatment group was cut, flash-frozen in liquid nitrogen, and stored at -80℃ for later use. Each treatment was set up with three biological replicates. Total RNA was extracted from the leaves using the Trizol method. The specific operation was as follows: after grinding the sample in liquid nitrogen, Trizol reagent was added to homogenize the sample, followed by chloroform extraction, isopropanol precipitation, washing with 75% ethanol, drying the precipitate, and dissolving it in RNase-free ddH2O. The RNA concentration and purity were determined using an ultra-micro UV spectrophotometer. 1 µg of total RNA was taken and the system was prepared according to the reverse transcription kit instructions. Reverse transcription was performed on a PCR instrument (37℃ for 30 min, 85℃ for 5 sec) to synthesize cDNA. The product was stored at -20℃ for later use.

[0147] Using the wheat GAPDH gene as an internal control, the expression levels of PR1 (pathogenesis-associated protein) and MPK3 (mitogen-activated protein kinase 3) genes were detected. Primers were synthesized by Shanghai Genscript Biotech Co., Ltd., and their sequences are shown in Table 3.

[0148] Table 3. Primer sequences for qRT-PCR amplification

[0149]

[0150] qRT-PCR reactions were performed using the SYBR Green chimeric fluorescence assay on a real-time PCR instrument. The reaction mixture (20 µL) contained: 10 µL SYBR qPCR Master Mix, 0.4 µL each of the upstream and downstream primers, 2 µL cDNA template, and ddH2O to a final volume of 20 µL. Three technical replicates were performed for each sample. -ΔΔCt The relative expression level of the target gene is calculated using the following formula:

[0151] ΔΔC t =(C t目的基因 -C t内参基因 ) Processing Group - (C t目的基因 -C t内参基因 control group

[0152] Using the control group sprayed with 0.02% Tween-20 solution as a baseline, the relative fold increase of PR1 and MPK3 genes in the JG-1 treatment group was calculated.

[0153] To further elucidate the molecular mechanism by which JG-1 induces wheat resistance to powdery mildew, the expression levels of defense-related genes PR1 and MPK3 were examined. PR1 is a key marker gene in the salicylic acid (SA) signaling pathway and plays an important role in the systematic acquisition of resistance in plants. Figure 6As shown, different concentrations of JG-1 treatment significantly affected PR1 expression in wheat leaves. Compared with the control group, the relative expression level of PR1 decreased after treatment with 0.01 mM JG-1, only about 0.43 times that of the control group. When the treatment concentration was increased to 0.1 mM and 1 mM, JG-1 could significantly induce PR1 expression, with relative expression levels approximately 2 times and 1.8 times that of the control group, respectively. Notably, 0.1 mM JG-1 could increase PR1 expression to approximately 2.0 times that of the control group, indicating that JG-1 can strongly induce the expression of the SA-related marker gene PR1 at an appropriate concentration. This result suggests that the SA-related defense response may be involved in the JG-1-induced wheat resistance to powdery mildew. MPK3 is a key component of the mitogen-activated protein kinase (MAPK) cascade, involved in transducing extracellular stress signals into the cell and regulating the expression of downstream defense genes. Compared with the control group, both 0.1 mM and 1 mM JG-1 treatments significantly induced the upregulation of MPK3 gene expression, with relative expression levels reaching approximately 1.6-fold and 1.8-fold, respectively, compared to the control group, and were comparable to the 1 mM MeJA treatment group (approximately 1.6-fold). This result indicates that JG-1 can activate the MAPK signaling pathway, rapidly transducing immune signals downstream through the upregulation of MPK3 expression, thus coordinating the plant's defense response. In conclusion, JG-1 can activate disease-resistance-related signal transduction pathways by inducing the expression of defense-related genes PR1 and MPK3, thereby providing a molecular basis for improving wheat resistance to powdery mildew.

[0154] Example 4: JG-1 induces wheat resistance to high temperature stress

[0155] The test material was wheat (variety "Yangmai 39"). JG-1 was dissolved in anhydrous ethanol to prepare a 10 mM JG-1 stock solution. Immediately before use, the JG-1 stock solution was serially diluted with 0.02% Tween-20 solution to three working solutions of 10 nM, 1 nM, and 0.1 nM concentrations. MeJA was dissolved in anhydrous ethanol to prepare a 10 mM stock solution, which was then diluted with 0.02% Tween-20 solution to a 10 µM MeJA solution as a control inducer. All percentages are volume percentages.

[0156] The experimental setup included: a control group containing only 0.02% Tween 20 solution, a 0.1 nM JG-1 treatment group, a 1 nM JG-1 treatment group, a 10 nM JG-1 treatment group, and a 10 µM MeJA control group. Each treatment was performed in triplicate.

[0157] The wheat seed pretreatment steps are as follows: First, rinse the seed surface with distilled water to remove impurities; then disinfect with 75% anhydrous ethanol for 3 minutes; rinse three times with distilled water; then disinfect with 5% (mass percentage) NaClO solution for about 10 minutes; after disinfection, rinse thoroughly with distilled water. Soak the treated seeds in distilled water for 9 hours, then spread them evenly on gauze and cover with four layers of moist gauze for light-protected germination. Place the seeds in a 25℃ incubation room in the dark for 24 hours. After germination, select seeds with consistent germination for sowing. Sow 36 seeds per pot in a sterilized culture medium of nutrient soil and vermiculite mixed in a 1:1 volume ratio. The plants are then cultured in an artificial climate incubator under the following conditions: temperature 26-28℃, relative humidity 60%-70%, and light intensity 200 μmol / m². -2 s -1 The photoperiod was 16 h light / 8 h dark. Foliar spraying was applied to wheat seedlings 10 days after the initial growth stage, with the amount sprayed enough to fully wet the leaves without dripping. Spraying was done once every 24 hours, for a total of two sprays. 24 hours after the second spray, the plants were transferred to a 45℃ incubator for 15 hours of high-temperature stress treatment. After the high-temperature treatment, the plants were moved to a 25℃ greenhouse for 5 days to recover and grow, for subsequent physiological index determination and heat resistance evaluation. The damage to the plants was observed and recorded, and the plants were classified according to the degree of damage (refer to: Han Ruihong, Zhao Dahua, Chen Jingjing, et al. Comprehensive evaluation of heat resistance in seedlings of different alfalfa germplasm resources [J]. Chinese Journal of Grassland Science, 2015, 37(03):48-54). The heat damage classification standards are shown in Table 4, and the heat damage index calculation formula is as follows.

[0158]

[0159] Table 4 Classification Standards for Heat Damage

[0160]

[0161] After the statistics were completed, the fresh weight of the above-ground parts of the plant was measured. The plant was then completely immersed in water for at least 8 hours, and its weight was measured every hour afterward, until the weight no longer changed, at which point the saturated fresh weight was determined. Finally, the plant was placed in a 55℃ oven for 48 hours to dry, and its dry weight was measured. The formula RWC = (W f - W d ) / (W t - W d ) × 100 to calculate relative water content (W) f Fresh weight of the plant, in grams; W d Plant dry weight, in g; W t (Weight of the plant after it is fully saturated with water, in grams).

[0162] Experimental results are as follows Figure 7 As shown in Table 5, a high temperature of 45℃ significantly inhibited wheat growth; after 5 days of recovery at 27℃, all leaves in the control group (Control) were wilted and unable to grow normally; treatment with different concentrations of JG-1 improved the growth status and heat resistance of wheat to varying degrees. Compared with the control group, treatment with different concentrations of JG-1 increased both the dry and fresh weight of the aboveground parts of wheat, while the heat damage index showed a downward trend. When the JG-1 concentrations were 0.1 nM, 1 nM, and 10 nM, the fresh weight of wheat aboveground parts significantly increased by 23%, 44%, and 12%, respectively, and the dry weight significantly increased by 27%, 37%, and 12%, respectively. In contrast, treatment with 10 µM MeJA solution significantly increased fresh weight by 29%, while dry weight showed no significant change. Treatment with 1 nM JG-1 solution increased relative moisture content by 23%, while treatment with 10 µM MeJA solution increased relative moisture content by 11%. When the JG-1 concentrations were 0.1 nM, 1 nM, and 10 nM, the heat damage index decreased by 10%, 39%, and 34%, respectively, while treatment with 10 µM MeJA solution decreased the heat damage index by 24%. These results indicate that JG-1 treatment can effectively alleviate the inhibitory effect of high-temperature stress on wheat growth and the degree of plant damage, and the effect of JG-1 concentration at 1 nM on inducing wheat resistance to high-temperature stress is most significant.

[0163] Table 5. Effects of different concentrations of JG-1 on wheat biomass and heat stress index under high temperature stress.

[0164]

[0165] Example 5: JG-1 induces resistance to high temperature stress in tomatoes

[0166] The test material was tomato (variety "Ailsa Craig"). The experimental setup included a control group (sprayed with 0.02% Tween 20 solution, i.e., Control), 10 µM MeJA treatment group, 0.1 nM JG-1 treatment group, 1 nM JG-1 treatment group, 10 nM JG-1 treatment group, and 100 nM JG-1 treatment group, with three replicates for each treatment. The JG-1 stock solution was prepared with anhydrous ethanol, with a concentration of 10 mM; the MeJA stock solution was also prepared with anhydrous ethanol, with a MeJA concentration of 10 mM. Before use, both the JG-1 and MeJA stock solutions were diluted with 0.02% Tween 20 solution. Tomato seeds were placed in petri dishes lined with moistened filter paper and germinated at 25°C for 2 days. After germination, the seeds were sown in seedling trays. The culture medium consisted of a mixture of potting soil, vermiculite, and perlite in a volume ratio of 7:3:1, and was sterilized. After 14 days of seedling growth, seedlings with uniform growth were transplanted into larger pots for further cultivation. When the seedlings reached 40 days of growth, foliar spraying was applied. The amount of spray was sufficient to fully moisten the leaves without dripping, applied every 24 hours for a total of two applications. Twenty-four hours after the second spray, the plants were transferred to a 42℃ incubator for 36 hours of high-temperature stress treatment. After the stress treatment, the plants were moved to a 25℃ greenhouse for 5 days to recover and grow, which was then used for subsequent physiological index measurements and heat resistance evaluation.

[0167] Experimental results are as follows Figure 8 As shown in Table 6, under high-temperature stress, the JG-1 treatment significantly improved the growth recovery of tomato plants, with all indicators significantly better than the Control. Compared with the Control, the fresh weight of tomatoes recovered increased by 32%, 160%, 114%, and 58% after treatments with 0.1 nM, 1 nM, 10 nM, and 100 nM JG-1, respectively. Among them, the 1 nM JG-1 treatment showed the largest increase, with a 160% increase in recovered fresh weight, significantly higher than the 10 µM MeJA treatment (90% increase). Regarding the heat damage index, all JG-1 concentrations showed varying degrees of reduction. The heat damage index decreased by 3%, 25%, 21%, and 15% after treatments with 0.1 nM, 1 nM, 10 nM, and 100 nM JG-1, respectively, while the 10 µM MeJA treatment reduced it by 20%. The 1 nM JG-1 treatment showed the largest reduction, significantly better than the MeJA treatment, indicating that it was more effective in mitigating tissue damage caused by high temperatures. In summary, JG-1 can effectively alleviate the damage to tomato plants caused by high temperature stress. The optimal concentration of JG-1 is 1 nM, which is most effective in improving biomass recovery and reducing the heat damage index.

[0168] Table 6. Effects of different concentrations of JG-1 on the recovery of fresh weight and heat damage index of tomatoes under high temperature stress.

[0169]

[0170] Example 6: JG-1 induces rice resistance to low temperature stress

[0171] Rice seeds (variety "Nangeng 46") were rinsed with distilled water, disinfected with 75% ethanol for 3 min, and rinsed three times with distilled water. They were then disinfected with 5% (w / w) NaClO solution for approximately 10 min, rinsed thoroughly with distilled water, and blotted dry with clean filter paper until completely dry. JG-1 was dissolved in anhydrous ethanol to prepare a 10 mM JG-1 stock solution. Before use, the JG-1 stock solution was serially diluted with water to 0.1 nM, 1 nM, 10 nM, and 100 nM working solutions. MeJA was also dissolved in anhydrous ethanol to prepare a 10 mM stock solution, which was then diluted with water to a 10 μM MeJA working solution. The control group (CK) was treated with an equal volume of water; all other procedures were the same as for the other treatment groups. Four g of healthy, plump, and uniformly sized seeds were selected and placed in 50 mL centrifuge tubes. Eight mL each of the following solutions were added to each tube: water (CK), 0.1 nM JG-1 solution, 1 nM JG-1 solution, 10 nM JG-1 solution, 100 nM JG-1 solution, and 10 μM MeJA solution. The seeds were stirred to ensure complete immersion in the solutions and soaked at room temperature for 48 h. After soaking, the seeds were not rinsed and were directly subjected to germination treatment. Each experiment was conducted in triplicate. For each replicate, 30 rice seeds were randomly selected using tweezers and placed in a petri dish lined with two layers of moistened filter paper. The control group was placed at 28℃, and the treatment group at 4℃, incubated in the dark to allow germination. After 3 days of culture, the seed sprout length and root length were measured and recorded.

[0172] Experimental results are as follows Figure 9As shown in Table 7, under low-temperature stress, rice seedlings treated with different concentrations of JG-1 solution (0.1 nM, 1 nM, 10 nM, and 100 nM) significantly improved growth indicators compared to the control group (CK), demonstrating a clear effect of low-temperature resistance and growth promotion. Compared with the control group (CK), the root length of seedlings in each treatment group increased by 15%, 24%, 31%, and 19%, respectively, and the stem length increased by 8%, 30%, 28%, and 17%, respectively. Overall, different concentrations of JG-1 showed a certain promoting effect on seedling growth, with the 10 nM treatment showing the most significant promotion on root length, reaching an increase of 31%; while the 1 nM JG-1 treatment showed the best effect on stem length, with an increase of 30%. The comparative experiment results further showed that although the 10 μM MeJA treatment could increase root length and stem length by 21% and 20%, respectively, its overall promoting effect was lower than that of the 10 nM JG-1 treatment. The above results fully demonstrate that under nanomolar-level low-dose conditions, JG-1 can significantly alleviate the adverse effects of low-temperature stress on the growth and development of rice seedlings. Its growth-promoting effect is superior to that of the control inducer, and it has higher activity efficiency and application potential.

[0173] Table 7. Effects of different concentrations of JG-1 soaking on root and stem length of rice seedlings under low temperature.

[0174]

[0175] Example 7: JG-1 alleviates damage to rice seed germination and seedlings caused by fluroxypyr·pentyl·methrin.

[0176] The test material was the rice variety "Nangeng 46". After thoroughly rinsing the rice seeds with distilled water, they were first disinfected with 75% ethanol for 3 minutes, followed by rinsing three times with distilled water. Then, they were disinfected with 5% (w / w) sodium hypochlorite (NaClO) solution for approximately 10 minutes. After disinfection, they were repeatedly rinsed with distilled water until no disinfectant residue remained. The seeds were then blotted dry with sterile filter paper until completely dry. 4 g of healthy, plump, and uniformly sized seeds were weighed and placed in separate 50 mL centrifuge tubes. The JG-1 stock solution was prepared with anhydrous ethanol, with a JG-1 concentration of 10 mM; the MeJA stock solution was also prepared with anhydrous ethanol, with a MeJA concentration of 10 mM. Before use, both the JG-1 and MeJA stock solutions were diluted with water. Add 8 mL each of the following solutions to the treatment tubes: water, 0.1 nM JG-1 solution, 1 nM JG-1 solution, 10 nM JG-1 solution, 100 nM JG-1 solution, and 10 nM MeJA solution. Then add 0.02 g of fluazinam·pentyl·methoxyfenozide (purchased from Hebei Bojia Agricultural Co., Ltd., diluted 400 times) to each treatment solution. 400Mix the seeds thoroughly to completely immerse them in the solution and soak at room temperature for 48 hours. After soaking, do not rinse; proceed directly to germination treatment. Each treatment has three replicates. For each replicate, randomly select 30 seeds and evenly place them in a petri dish pre-lined with moistened filter paper using tweezers. Germinate the seeds in a 28°C incubator. After 3 days of cultivation, measure and record seed sprout length and root length, among other parameters.

[0177] Meanwhile, a control group (CK) was set up: 8 mL of water was added to the treatment tube, and an equal amount of water was used to replace fluazinam·pentine·methrin, and the rest of the operation was the same as above.

[0178] Experimental results are as follows Figure 10 As shown in Table 8, the 400-fold dilution of fluazinam·pentyl·methamidophos (F... 400 Soaking seeds significantly inhibited rice seed germination and seedling growth; compared with the control group (CK), F 400 After treatment, the germination rate decreased by 5%, and the shoot length and root length decreased by 35% and 43%, respectively, with significant differences, indicating that the agent has a significant inhibitory effect on seed vigor and early seedling growth, especially root elongation. In F 400 Adding different concentrations of JG-1 to the treatment base alleviated the phytotoxic effects to varying degrees, exhibiting a certain dose-dependent effect. (Compared to F) 400 Compared with the previous treatment, the 0.1 nM JG-1 treatment increased germination rate, shoot length, and root length by 5%, 5%, and 7%, respectively; the 1 nM JG-1 treatment increased germination rate by 10%, shoot length by 5%, and root length by 26%, showing a more significant promoting effect on root growth; the 10 nM JG-1 treatment had the most significant effect, increasing germination rate by 24%, and shoot and root length by 18% and 29%, respectively, all significantly better than the previous treatment. 400 Treatment group. In contrast, the 100 nM JG-1 treatment did not show a significant alleviating effect, and all indicators were consistent with F. 400 The differences in treatment were not significant; the 10 nM MeJA treatment increased germination rate by 3%, decreased shoot length by 2%, and increased root length by 11%, showing limited overall improvement, significantly lower than the 10 nM JG-1 treatment. In conclusion, JG-1, under suitable low-concentration conditions, can significantly alleviate the inhibitory effects of fluazinam·pentyl·methrin on rice seed germination and seedling growth, with the 10 nM treatment showing the best effect, demonstrating the optimal ability to alleviate phytotoxicity in improving germination rate and promoting seedling growth.

[0179] Table 8. Effects of different concentrations of JG-1 and fluazinam·pentazocine·carbendazim seed soaking on rice seed germination rate, shoot length and root length.

[0180]

[0181] Example 8: JG-1 alleviates the damage of methamidophos and ethoxysulfuron to rice seed germination and seedlings.

[0182] Rinse rice seeds (variety "Nangeng 46") with distilled water, disinfect with 75% alcohol for 3 minutes, rinse three times with distilled water, and then disinfect with NaClO (5% by mass) solution for about 10 minutes. Afterward, rinse thoroughly with distilled water and use clean filter paper to absorb any remaining moisture on the seed surface until dry. Select 4 g of healthy, plump, and uniform seeds and place them in a 50 mL centrifuge tube. Prepare the JG-1 stock solution with anhydrous ethanol, with a JG-1 concentration of 10 mM; prepare the MeJA stock solution with anhydrous ethanol, with a MeJA concentration of 10 mM. Before use, dilute the JG-1 and MeJA stock solutions with water. Add 8 mL each of the following solutions to each tube: 0.1 nM JG-1 solution, 1 nM JG-1 solution, 10 nM JG-1 solution, 100 nM JG-1 solution, and 10 nM MeJA solution. Then add 0.053 g of methamidophos-ethyl (purchased from Jiangsu Green Shield Plant Protection Agriculture Co., Ltd., diluted 150 times) to each tube. 150 Stir to completely submerge the seeds in the solution. Soak at room temperature for 48 hours, then proceed directly to germination without rinsing. Each experiment has three replicates. For each replicate, 30 rice seeds are randomly selected using tweezers and placed in a petri dish pre-lined with moistened filter paper. The dishes are then placed in a 28°C incubator for germination. After 2-3 days, record the germination rate, shoot length, and root length.

[0183] Meanwhile, a control group (CK) was set up: 8 mL of water was added to the treatment tube, and an equal amount of water was used to replace the methamidophos-ethyl. The rest of the operation was the same as described above.

[0184] Experimental results are as follows Figure 11 As shown in Table 9, the 150-fold dilution of methamidophos·ethoxysulfuron (S) 150 Soaking seeds significantly inhibited rice seed germination and seedling growth; compared with the control group (CK), S 150 After treatment, the germination rate decreased by 13% and the root length decreased by 59%, with significant differences, indicating that the agent has a significant inhibitory effect on seed vigor and seedling root elongation. In S 150 After applying different concentrations of JG-1 to the treatment, the phytotoxic effects were alleviated to varying degrees, showing a certain dose-response relationship. (Compared to S...) 150Compared to individual treatments, 0.1 nM JG-1 increased germination rate by 9% and root length by 38%; 1 nM JG-1 increased germination rate by 11% and root length by 48%; and 10 nM JG-1 increased germination rate by 12%, shoot length by 10%, and root length by 52%. The 100 nM JG-1 treatment showed the most significant mitigation effect, increasing germination rate by 22%, root length by 91%, and shoot length by 5%, demonstrating a clear recovery trend. In contrast, the 10 nM MeJA treatment did not significantly improve germination rate; although shoot length increased by 12%, root length decreased by 5%. In conclusion, JG-1, within an appropriate concentration range, can effectively alleviate the inhibitory effect of methamidophos and ethoxysulfuron on rice seed germination and early seedling growth, and the mitigating effect strengthens with increasing concentration. The 100 nM JG-1 treatment exhibited the best regulatory effect, showing significant advantages in restoring germination ability and promoting root growth. Under this treatment, the germination rate, shoot length, and root length reached 105%, 107%, and 78% of the control group, respectively, indicating that the JG-1 seed soaking treatment can effectively alleviate the phytotoxicity problem of the rice seed soaking agent thiophanate-methyl.

[0185] Table 9. Effects of different concentrations of JG-1 and chlorpyrifos-ethyl on seed germination rate, shoot length, and root length of rice.

[0186]

[0187] Example 9: JG-1 alleviates the damage of dichloroquinoline acid to tobacco seedlings.

[0188] The test plant was tobacco (Nicotiana benthamiana). Seeds underwent surface disinfection before sowing: first, they were soaked in 75% ethanol for 30 seconds, then treated with a 5% (w / w) sodium hypochlorite solution for 10 minutes with gentle agitation during treatment. Afterward, they were rinsed 5–6 times with sterile water to thoroughly remove residual disinfectant. The disinfected seeds were evenly sown on the surface of a moist, sterilized seedling substrate, lightly sprayed with sterile water, covered with plastic wrap to maintain moisture, and then cultured in an artificial climate chamber. The culture conditions were 22℃, relative humidity 70%–80%, and light intensity 200 μmol / m². -2 s -1The photoperiod is 16 h light / 8 h darkness. Seeds germinate approximately 10 days after sowing. Once the cotyledons unfold, remove the plastic wrap and continue cultivation. When seedlings reach 4 true leaves, select healthy seedlings with uniform growth and transplant one seedling per pot. The cultivation substrate is a 1:1 (v / v) mixture of organic nutrient soil and vermiculite, with approximately 0.1 kg of soil per pot. After transplanting, continue cultivation in an artificial climate chamber until the plants reach 5–6 true leaves, at which point they will be used for subsequent treatments. JG-1 stock solution is prepared with anhydrous ethanol, with a JG-1 concentration of 10 mM. Before use, dilute the JG-1 stock solution with water. The plants were treated with water, 1 nM JG-1 solution, 10 nM JG-1 solution, 100 nM JG-1 solution, and 1000 nM JG-1 solution, respectively. 10 mL of the solution was applied to each pot via root drenching, slowly watering along the base of the stem to ensure thorough infiltration of the rhizosphere soil. After 48 h of JG-1 pretreatment, quinclorac acid (QNC) stress was applied. The QNC stock solution concentration was prepared at 0.5 mg / L, calculated based on a soil mass of 0.1 kg per pot, a target final concentration of 0.05 mg / kg soil, and a root drenching volume of 10 mL per pot. In the JG-1 treatment group, 10 mL of QNC stock solution was applied to each pot to ensure even distribution of the solution in the soil around the roots. The control group was drenched with an equal volume of water without QNC. Each treatment had three independent biological replicates, each containing five tobacco seedlings. After the phytotoxicity treatment, the plants continued to be cultured normally in an artificial climate chamber, and their growth status was observed daily. When more than 60% of the plants show obvious symptoms of pesticide damage, immediately investigate and record the phenotypic changes and related morphological indicators of the plants. The main symptoms of pesticide damage are wrinkled central leaves, curled leaves, and deformed new leaves, specifically cup-shaped or ribbon-shaped leaves.

[0189] Experimental results are as follows Figure 12As shown in Table 10, quinclorac acid (QNC) treatment significantly inhibited the vegetative growth of Nicotiana benthamiana. Compared with the control group, QNC treatment reduced the length and width of the third-to-last leaf by 51% and 77%, respectively, and the length and width of the fourth-to-last leaf by 41% and 52%, respectively. Simultaneously, plant height decreased by 20%, aboveground fresh weight by 20%, and the fresh weight of the phytotoxic portion decreased by 44%, all with significant differences. Under QNC stress, the application of different concentrations of JG-1 resulted in varying degrees of recovery in all growth indicators, showing a concentration-dependent trend. Compared with QNC alone, 1 nMJG-1 treatment increased the length of the third-to-last leaf by 2%, while decreasing its width by 7%; the length and width of the fourth-to-last leaf increased by 8% and 5%, respectively; plant height increased by 2%; aboveground fresh weight increased by 10%; and the fresh weight of the phytotoxic portion increased by 6%. Statistical analysis showed that the differences compared with QNC alone were not statistically significant. The 10 nM JG-1 treatment significantly reduced pesticide damage: the length and width of the third-to-last leaf increased by 13% and 53% respectively compared to the QNC treatment alone, while the length and width of the fourth-to-last leaf increased by 25% and 20% respectively; plant height increased by 15%, aboveground fresh weight increased by 27%, and the fresh weight of the pesticide-damaged portion increased by 18%. The 100 nM JG-1 treatment showed the best mitigation effect. Compared with the QNC treatment alone, the length and width of the third-to-last leaf increased by 27% and 64% respectively, while the length and width of the fourth-to-last leaf increased by 24% and 33% respectively; plant height increased by 19%, aboveground fresh weight increased by 32%, and the fresh weight of the pesticide-damaged portion increased by 79%. Several growth parameters recovered to near the control group levels, and statistical analysis showed no significant difference compared to the control group. The 1000 nM JG-1 treatment also had a certain mitigating effect on QNC pesticide damage. Compared with QNC alone, the length and width of the third-to-last leaf increased by 21% and 44%, respectively, while the length and width of the fourth-to-last leaf increased by 20% and 26%, respectively. Plant height increased by 21%, aboveground fresh weight increased by 25%, and the fresh weight of the phytotoxic portion increased by 64%. In summary, QNC significantly reduced leaf morphological indicators, plant height, and biomass accumulation in Nicotiana benthamiana, while JG-1, within an appropriate concentration range, significantly increased leaf length and width, plant height, and fresh weight, effectively alleviating the growth inhibition caused by QNC. The 100 nM treatment showed the best overall effect, particularly in promoting leaf expansion and restoring root growth, indicating that JG-1 has a mitigating effect on dichloroquinoline acid phytotoxicity in tobacco.

[0190] Table 10 Effects of different concentrations of JG-1 on vegetative growth indicators of Nicotiana benthamiana under quinclorac acid (QNC) stress

[0191]

[0192]

[0193] Example 10 JG-1 induces salt stress resistance in rice

[0194] After rinsing the seeds of the rice variety "Nangeng 46" with distilled water, disinfect them in 75% alcohol for 3 minutes, then rinse them three times with distilled water. Next, disinfect them by soaking them in a 5% (w / w) sodium hypochlorite (NaClO) solution for about 10 minutes. After removing them, rinse them thoroughly with distilled water and blot dry with clean filter paper until the seed surface is completely dry. Select healthy, plump, and uniformly sized seeds, weigh 4 g, and place them in a 50 mL centrifuge tube. The JG-1 stock solution was prepared with anhydrous ethanol, with a JG-1 concentration of 10 mM; the MeJA stock solution was also prepared with anhydrous ethanol, with a MeJA concentration of 10 mM. Before use, dilute both the JG-1 and MeJA stock solutions with water. Add 8 mL of water, 0.1 nM JG-1 solution, 1 nM JG-1 solution, 10 nM JG-1 solution, 100 nM JG-1 solution, and 10 nM MeJA solution to each tube, respectively. Gently stir to completely submerge the seeds. After soaking at room temperature for 48 h, do not rinse and proceed directly to the germination step. Each treatment group has three replicates. In each replicate, 30 soaked seeds are randomly selected with tweezers and evenly placed in a petri dish lined with moistened filter paper. Add 10 mL of 50 mM NaCl solution to the petri dish. Place all petri dishes in a 28℃ incubation room for germination culture. After 3 days of culture, measure and record the seed germination rate, shoot length, and root length.

[0195] Control group (CK): Add water to centrifuge tubes, replacing the NaCl solution with an equal amount of water, and follow the same procedure as above.

[0196] Experimental results are as follows Figure 13As shown in Table 11, salt stress severely inhibited rice seed germination and seedling growth. Compared with the control group (CK), the germination rate decreased by 37%, the shoot length decreased by 40%, and the root length decreased by 49%. After treatment with different concentrations of JG-1 solution and NaCl, the damage of NaCl to rice seed germination rate, shoot length, and root length was significantly reduced. Compared with the water + NaCl treatment, the germination rate of rice seeds increased by 15%, 35%, 38%, and 38% respectively in the presence of 0.1 nM, 1 nM, 10 nM, and 100 nM JG-1, with the 10 nM and 100 nM treatments showing the most significant effects. The shoot length increased by 24%, 36%, and 29% in the presence of 0.1 nM, 10 nM, and 100 nM JG-1, respectively, while the increase in shoot length was smaller in the 1 nM JG-1 treatment, only 7%. The root length increased by 32%, 35%, and 42% in the presence of 1 nM, 10 nM, and 100 nM JG-1, respectively, showing a trend of increasing promotion effect with increasing concentration, but there was no significant difference in root length between the 0.1 nM JG-1 treatment and the NaCl treatment. The positive control, 10 nM MeJA treatment, increased the germination rate by 44%, shoot length by 36%, and root length by 34%, comparable to the 10 nM JG-1 treatment. Clearly, all JG-1 treatments significantly alleviated the phytotoxicity of NaCl to rice, with the 10 nM and 100 nM concentrations showing the most stable effects. Overall, JG-1 treatments at 10 nM and 100 nM concentrations yielded the best results, with germination rate, shoot length, and root length reaching 87%, 81%, and 70% (10 nM) and 87%, 77%, and 73% (100 nM) of the control group, respectively.

[0197] Table 11 Effects of different concentrations of JG-1 soaking on rice seed germination rate, shoot length, and root length under salt stress.

[0198]

[0199] Example 11 Evaluation of the cold tolerance induced by JG-1 and different JA-AAs in cucumber seedlings

[0200] Select healthy, plump cucumber seeds of uniform size, rinse them thoroughly with distilled water, and then evenly spread them in a petri dish lined with four layers of moist gauze. Place the dish in a 27℃ incubation room in the dark for 24 hours to promote germination. After germination, select seeds with uniform white sprouts and sow them into the culture soil at a depth of 2-3 cm. The culture medium is prepared by mixing Suqian "Lvzhuang" substrate and vermiculite in a 1:1 (v / v) ratio, thoroughly mixing, and then filling the pots for later use. After sowing, place the pots in an artificial incubation room with the following conditions: temperature 26℃, relative humidity 60%-70%, and light intensity 200 μmol / m². -2 s -1The photoperiod was 16 h light / 8 h dark. When cucumber seedlings reached 18 days of age, vigorous seedlings with uniform growth were selected for subsequent treatments. JG-1, JA-Ile, JA-Leu, JA-Glu, and JA-Val were dissolved in anhydrous ethanol to prepare 10 mM stock solutions; before use, they were diluted to 1 nM working solutions with 0.02% Tween-20 solution. A 0.02% Tween-20 solution was used as a control. Cucumber seedlings were treated by foliar spraying, once every 24 h, for a total of two sprays. The amount of spray was sufficient to fully wet the leaf surface without dripping. 24 h after the second spray, the cucumber seedlings in each treatment group were transferred to 4℃ for low-temperature stress treatment for 36 h. After the low-temperature treatment, the chilling injury phenotypes of plants in each treatment group were observed, and the damage to the plants was investigated to evaluate the induction effect of JG-1 and different JA-AAs on the cold tolerance of cucumber seedlings.

[0201] Experimental results are as follows Figure 14As shown in Table 12, the alleviating effects of different JA-AAs on cucumber seedlings under low-temperature stress varied significantly, with JG-1 showing the best effect. Compared with the control group, JG-1 treatment increased the aboveground fresh weight of cucumber seedlings by 113%, the aboveground dry weight by 29%, and reduced the damaged area by 61%, indicating that JG-1 can significantly alleviate the inhibition of cucumber seedling growth by low temperature and reduce chilling injury. Among the other JA-AAs treatments, JA-Ile was the second most effective. Compared with the control group, JA-Ile treatment increased the aboveground fresh weight by 77%, the aboveground dry weight by 24%, and reduced the damaged area by 58%, indicating that JA-Ile also has a good effect in inducing cold tolerance, but its overall effect is still lower than that of JG-1. JA-Leu also showed a certain alleviating effect on low-temperature damage to cucumber seedlings. JA-Leu treatment increased the aboveground fresh weight by 14%, but decreased the aboveground dry weight by 5% and reduced the damaged area by 48%, showing a certain ability to alleviate low-temperature damage, but its overall effect of promoting growth and reducing damage was weaker than that of JG-1 and JA-Ile. In comparison, the JA-Glu treatment was less effective, increasing aboveground fresh weight by only 4%, decreasing aboveground dry weight by 5%, and reducing damaged area by 23%. However, the JA-Val treatment actually decreased aboveground fresh weight by 13%, aboveground dry weight by 10%, and increased damaged area by 11%, indicating that JA-Val not only failed to improve the cold tolerance of cucumber seedlings but may have exacerbated low-temperature damage. Considering the three indicators of fresh weight, dry weight, and damaged area, the order of effectiveness in inducing cold tolerance in cucumber seedlings was: JG-1 > JA-Ile > JA-Leu > JA-Glu > Control > JA-Val. Among these, JG-1 showed the best activity in promoting cucumber seedling growth under low-temperature stress and mitigating cold damage, indicating that JG-1 has a significant effect in inducing cold tolerance in cucumber seedlings.

[0202] Table 12 Effects of different JA-AAs on cucumber growth and damaged area under low temperature stress

[0203]

[0204] Example 12: Induction of cucumber disease resistance gene expression by JG-1 and different JA-AAs treatments

[0205] Vigorous cucumber seedlings with uniform growth were selected as experimental materials. Treatment solutions of 1 mM JG-1, 1 mM JA-Ile, 1 mM JA-Leu, 1 mM JA-Glu, and 1 mM JA-Val were prepared using 0.02% Tween-20 solution. A control group (control) was used, sprayed with 0.02% Tween-20 solution. The cucumber seedlings were treated by foliar spraying, ensuring the leaf surface was fully moistened without dripping. Leaf samples were collected 24 h after spraying, flash-frozen in liquid nitrogen, and stored at -80℃ for later use. RNA was extracted from the leaves using the Trizol method, and its concentration and purity were determined. 1 µg of total RNA was reverse transcribed according to the reverse transcription kit instructions to obtain cDNA. Using cDNA as a template, real-time quantitative PCR was used to detect the expression levels of cucumber defense-related genes PR1 and RbohD. qRT-PCR was performed using the SYBR Green fluorescence assay. The reaction volume was 20 µL: 10 µL SYBR qPCR Master Mix, 0.4 µL each of upstream and downstream primers, 2 µL cDNA template, and ddH2O to a final volume of 20 µL. Primer sequences are shown in Table 13. Three biological replicates were performed for each treatment, and three technical replicates were performed for each sample. Normalization was performed using an internal control gene as the standard. -ΔΔCt The method calculates the relative expression level of the target gene in each treatment group.

[0206] Table 13 qRT-PCR amplification primer sequences

[0207]

[0208] Experimental results are as follows Figure 15As shown, 24 h after spraying, different JA-AAs could induce the expression of cucumber defense-related genes RbohD and PR1 to varying degrees, but the induction intensity differed significantly. Regarding RbohD expression, the relative expression levels of the JA-Leu, JA-Val, JA-Ile, JA-Glu, and JG-1 treatment groups were 1.28 times, 1.11 times, 1.94 times, 1.87 times, and 2.27 times that of the control group, respectively. Among them, JG-1 showed the strongest induction effect on RbohD, followed by JA-Ile and JA-Glu, while JA-Leu and JA-Val showed relatively weak induction effects. Regarding PR1 expression, the relative expression levels of the JA-Leu, JA-Val, JA-Ile, JA-Glu, and JG-1 treatment groups were 26.23 times, 15.15 times, 22.19 times, 19.40 times, and 34.30 times that of the control group, respectively. Among the treatments, JG-1 showed the strongest induction effect on PR1, significantly higher than other treatments. JA-Leu and JA-Ile also exhibited strong induction effects, while JA-Val and JA-Glu, although significantly increasing PR1 expression levels, had lower induction abilities than JG-1. In summary, JG-1 showed the strongest induction effect on cucumber defense-related genes RbohD and PR1, indicating that JG-1 can more effectively activate the expression of defense-related genes in cucumber seedlings, thereby inducing plant defense responses and improving plant stress resistance.

[0209] Example 13 Effects of JG-1 soaking on rice seed germination and seedling growth

[0210] Rice seeds (variety "Dengliangyou 2108") were rinsed with distilled water, disinfected with 75% alcohol for 3 minutes, rinsed three times with distilled water, and then disinfected with 5% (w / w) NaClO for about 10 minutes. After removal, they were rinsed with distilled water, and then the residual moisture on the seed surface was absorbed with clean filter paper until the surface was dry. 4 g of healthy, plump, and uniformly sized seeds were selected and placed in 50 mL centrifuge tubes as soaking material. JG-1 was dissolved in anhydrous ethanol to prepare a 10 mM JG-1 stock solution; before use, it was diluted with water to 1 nM, 10 nM, 100 nM, and 1000 nM JG-1 working solutions. MeJA was dissolved in anhydrous ethanol to prepare a 10 mM MeJA stock solution; before use, it was diluted with water to 10 nM MeJA working solution. Water was used as the control group. Add 8 mL of treatment solution to each centrifuge tube, including water (control group), 1 nM JG-1 solution, 10 nM JG-1 solution, 100 nM JG-1 solution, 1000 nM JG-1 solution, and 10 nM methyl jasmonate (MeJA) solution. Gently shake the centrifuge tube to completely submerge the seeds in the treatment solution, cover the tube but do not tighten the cap, and soak the seeds at 25℃ in the dark for 48 h. After soaking, do not rinse and proceed directly to the germination step. Each treatment has 3 replicates. In each replicate, randomly select 30 soaked rice seeds, evenly place them in a petri dish pre-lined with moistened filter paper, and add 8 mL of water. Then place the petri dish in a 28℃ incubator in the dark for germination culture. After 3 days of culture, measure and record the sprout length and root length of the rice seeds.

[0211] Experimental results are as follows Figure 16As shown in Table 14, rice seedlings treated with different concentrations of JG-1 exhibited certain differences in phenotype and growth indicators compared to the control group. At concentrations of 1 nM, 10 nM, and 100 nM, JG-1 showed varying degrees of promoting effect on rice seedling growth. Compared to the control group, treatment with 1 nM, 10 nM, and 100 nM JG-1 increased seedling stem length by 4%, 6%, and 16%, respectively, and root length by 7%, 31%, and 12%, respectively. The 10 nM JG-1 concentration showed the most significant promoting effect on root length, while the 100 nM JG-1 concentration showed the best promoting effect on stem length, indicating that JG-1 can effectively promote early rice seedling growth within a suitable concentration range. When the JG-1 concentration increased to 1000 nM, its growth-promoting effect weakened and exhibited a certain inhibitory effect. Compared with the control group, the stem and root lengths of rice seedlings decreased by 14% and 7% respectively after treatment with 1000 nM JG-1, indicating that excessively high concentrations of JG-1 are detrimental to the normal growth of rice seedlings. This suggests that the growth-promoting effect of JG-1 is concentration-dependent; beyond the optimal concentration range, its promoting effect no longer increases and may even have a negative impact on seedling growth. Furthermore, the stem and root lengths of rice seedlings increased by 5% and 6% respectively after treatment with 10 nM MeJA compared to the control group, indicating that MeJA also has a certain promoting effect on rice seedling growth, but the overall promoting effect is weaker than that of JG-1 treatment within the optimal concentration range. A comprehensive comparison of the results shows that JG-1 seed soaking can effectively promote rice seedling growth within a certain concentration range, especially in promoting root development. Among them, the 10 nM treatment showed a relatively better overall growth-promoting effect, demonstrating the good application potential of JG-1 in rice seedling cultivation and early growth regulation.

[0212] Table 14 Effects of different concentrations of JG-1 on rice seedlings

[0213]

[0214] Example 14 Effects of JG-1 soaking on maize seed germination and seedlings

[0215] Corn seeds (variety "Mingtian 695D") were rinsed with distilled water, then sterilized in a 5% (w / w) sodium hypochlorite (NaClO) solution for about 10 minutes. They were then rinsed thoroughly with distilled water and blotted dry with sterile filter paper until the seeds were dry. Healthy, plump, and uniformly sized corn seeds were selected and placed in round petri dishes for soaking. JG-1 was dissolved in anhydrous ethanol to prepare a 10 mM JG-1 stock solution; before use, it was diluted with water to prepare working solutions of different concentrations. 20 mL of treatment solution was added to each petri dish, representing water (control group), 0.1 nM JG-1 solution, 1 nM JG-1 solution, 10 nM JG-1 solution, 100 nM JG-1 solution, and 1000 nM JG-1 solution, respectively. 50 corn seeds per dish were completely immersed in the treatment solution and soaked at 25°C in the dark for 12 hours. After soaking, two layers of filter paper were placed in a square petri dish and moistened with 5 mL of deionized water. Fifteen soaked seeds were randomly selected and evenly arranged on the filter paper in a 3×5 pattern, with the embryo facing the same side and appropriate spacing between seeds. A layer of filter paper was then placed on top of the seeds, and another 5 mL of deionized water was added to keep the filter paper moist. The petri dish was then covered and incubated. Each treatment was repeated in triplicate, and the moisture level of the filter paper in the petri dish was checked daily, with appropriate amounts of deionized water added as needed. The petri dishes were then placed in a 28℃ incubator under dark conditions for germination. On day 3 of incubation, the seed germination rate was recorded, and the shoot and root lengths of maize seedlings were measured to evaluate the promoting effect of JG-1 on maize seed germination and early seedling growth.

[0216] Experimental results are as follows Figure 17As shown in Table 15, significant changes occurred in maize seed germination and seedling growth after treatment with different concentrations of JG-1. All JG-1 concentrations promoted maize seed germination to varying degrees. Compared with the control group, treatments with 0.1 nM, 1 nM, 10 nM, 100 nM, and 1000 nM JG-1 increased the germination rate by 48%, 40%, 31%, 27%, and 56%, respectively, indicating that JG-1 has a good promoting effect on maize seed germination. Regarding seedling growth, treatments with 0.1 nM, 1 nM, 10 nM, and 100 nM JG-1 all showed certain growth-promoting effects. Compared with the control group, the stem length of seedlings in each treatment group increased by 13%, 13%, 14%, and 19%, respectively, and the root length increased by 7%, 3%, 4%, and 22%, respectively. The 100 nM JG-1 treatment significantly promoted both stem and root length in maize seedlings, with the best effect on root growth, indicating that this concentration is more beneficial for early seedling growth. When the JG-1 concentration increased to 1000 nM, its growth-promoting effect weakened, and it showed a certain inhibitory effect on seedling growth. Compared with the control group, the 1000 nM JG-1 treatment resulted in a 9% decrease in stem length and an 11% decrease in root length, indicating that excessively high concentrations of JG-1 are detrimental to normal seedling growth. In summary, JG-1 has a significant concentration effect on maize seed germination and early seedling growth, and can effectively promote seedling growth within a suitable concentration range. The 100 nM treatment showed the best overall effect, demonstrating the good application potential of JG-1 in promoting maize seed germination and seedling growth.

[0217] Table 15 Effects of different concentrations of JG-1 on maize seed germination rate, stem length, and root length

[0218]

[0219] Example 15 Effects of JG-1 soaking on tomato seed germination and seedlings

[0220] Tomato seeds (variety "Ailsa Craig") were first thoroughly rinsed with distilled water, then sterilized in 75% (v / v) ethanol for 3 min, followed by rinsing three times with distilled water and blotting dry with sterile filter paper until the seed surface was dry. JG-1 stock solution was prepared with anhydrous ethanol, with a JG-1 concentration of 10 mM; MeJA stock solution was also prepared with anhydrous ethanol, with a MeJA concentration of 10 mM. Before use, both JG-1 and MeJA stock solutions were diluted with water. Round petri dishes were used as soaking containers. 100 nM JG-1 solution, 10 nM JG-1 solution, 1 nM JG-1 solution, 0.1 nM JG-1 solution, and 1 nM methyl jasmonate (MeJA) solution were added to each dish, with a total volume of 25 mL for each solution. Sixty healthy, plump, and uniformly sized tomato seeds were selected and placed in a petri dish. The dish was gently shaken to ensure complete immersion in the treatment solution, and the seeds were soaked at 25°C in the dark for 8 hours. After soaking, no rinsing was performed; the seeds proceeded directly to the germination culture stage. Each treatment was performed in triplicate. For each replicate, 20 soaked seeds were randomly selected and evenly arranged in a 4×5 pattern in a round petri dish lined with two layers of filter paper. 8 mL of water was added to keep the filter paper moist. The petri dishes were then placed in a 28°C incubator in the dark for germination culture. After 5 days of culture, seed germination was photographed and recorded, and the germination rate was calculated. Hypocotyl length and root length were also measured. Germination was considered complete when the radicle length reached 2 mm or more. Hypocotyl length and root length were measured and analyzed using ImageJ software to preliminarily evaluate the promoting effect of JG-1 on tomato seed germination.

[0221] Control group: Use an equal volume of water instead of JG-1 solution, and follow the same procedure as above.

[0222] Experimental results are as follows Figure 18As shown in Table A and Table 16, different concentrations of JG-1 treatments had varying degrees of effect on tomato seed germination and seedling growth. Overall, JG-1 had a relatively small effect on tomato seed germination rate. Compared with the control group, the germination rates of tomato seeds increased by 1%, 6%, 4%, and 8%, respectively, after treatment with 0.1 nM, 1 nM, 10 nM, and 100 nM JG-1. Regarding seedling growth, different concentrations of JG-1 treatments showed significant differences. Compared with the control group, the stem length of tomato seedlings increased by 19%, 36%, and 24%, respectively, after treatment with 0.1 nM, 1 nM, and 10 nM JG-1, with the 1 nM treatment showing the most significant promoting effect; however, when the JG-1 concentration was increased to 100 nM, the stem length decreased by 31% compared to the control group, showing a significant inhibitory effect. The root length change trend was basically consistent with that of the stem length. Compared with the control group, root length increased by 10% and 23% after treatment with 0.1 nM and 1 nM, respectively, with the 1 nM treatment showing the best effect. The 10 nM treatment showed no significant change, while the 100 nM treatment resulted in a 55% decrease in root length, indicating that high concentrations of JG-1 significantly inhibited root growth in tomato seedlings. Furthermore, after treatment with 1 nM MeJA, the tomato seed germination rate decreased by 8% compared to the control group, stem length increased by 8%, but root length decreased by 7%, indicating that the overall promoting effect was not significant. In summary, all indicators show that JG-1 can promote early growth of tomato seedlings to some extent under low concentration conditions, with the 1 nM treatment showing the best overall effect; while high concentration treatments significantly inhibited seedling growth, indicating that the effect of JG-1 in tomato seed soaking is significantly concentration-dependent.

[0223] Table 16 Effects of different concentrations of JG-1 on tomato seed germination rate, stem length, and root length

[0224]

[0225] Based on the initial concentration screening experiment, the effective concentration range of JG-1 was further optimized. The JG-1 treatment concentrations were set at 0.1 nM, 0.5 nM, 1 nM, and 5 nM, with 5 nM MeJA as a positive control. Seed soaking and germination culture were performed according to the above method. After 5 days of culture, the seed germination rate, hypocotyl length, and root length were measured to determine the optimal concentration of JG-1 for promoting tomato germination.

[0226] Control group: Use an equal amount of water instead of JG-1 treatment solution, and the rest is the same as the above method.

[0227] Based on the results of the first round of experiments, JG-1 showed a certain promoting effect on the early growth of tomato seedlings. However, due to the large concentration gradient in the initial treatments, the optimal concentration was difficult to determine accurately. Therefore, based on the results of the previous round of experiments, this experiment further narrowed the treatment concentration range of JG-1, setting treatments of 0.1 nM, 0.5 nM, 1 nM, and 5 nM, and continued to conduct tomato seed germination experiments to screen for a more suitable concentration. The experimental results are as follows: Figure 18 As shown in Table B and Table 17, the differences in germination rate among the treatments were generally small. Regarding seedling growth, all treatments with different concentrations of JG-1 showed a certain promoting effect. Compared with the control group, the stem length of tomato seedlings increased by 9%, 8%, 18%, and 26% respectively after treatments with 0.1 nM, 0.5 nM, 1 nM, and 5 nM JG-1, with the 5 nM treatment showing the most significant promoting effect. The stem length increased by 10% after treatment with 5 nM MeJA, but its promoting effect was lower than that of the 5 nM JG-1 treatment. The trend in root length was basically consistent with that of stem length. Compared with the control group, the root length increased by 9%, 11%, 13%, and 29% respectively after treatments with 0.1 nM, 0.5 nM, 1 nM, and 5 nM JG-1, with the 5 nM treatment showing the most significant promoting effect. The root length increased by 16% after treatment with 5 nM MeJA, but was still lower than that of the 5 nM JG-1 treatment. In summary, this experiment further clarified the promoting effect of JG-1 on the early growth of tomato seedlings in the low concentration range, and showed that 5 nM JG-1 performed best in promoting stem and root length of tomato seedlings, indicating that this concentration is more conducive to the early growth of tomato seedlings and can be used as a suitable treatment concentration for subsequent related experiments.

[0228] Table 17 Effects of JG-1 on tomato seed germination rate, stem length, and root length within the optimized concentration range.

[0229]

[0230] Example 16 Effect of JG-1 soaking on cucumber seed germination

[0231] A study was conducted on the plant growth-promoting activity of JG-1 in cucumber (variety 'Lufeng'). Healthy, plump, and uniformly sized cucumber seeds were selected. JG-1 stock solution was prepared with anhydrous ethanol, with a JG-1 concentration of 10 mM; MeJA stock solution was also prepared with anhydrous ethanol, with a MeJA concentration of 10 mM. Both JG-1 and MeJA stock solutions were diluted with water before use. Circular petri dishes were used as soaking containers. Each dish contained 25 mL of treatment solutions: water, 0.1 nM JG-1 solution, 1 nM JG-1 solution, 10 nM JG-1 solution, 100 nM JG-1 solution, and 1 nM methyl jasmonate (MeJA) solution. Cucumber seeds were placed in these petri dishes containing different treatment solutions and soaked at 4℃ in the dark for 72 h. After soaking, 18 cucumber seeds were randomly selected using tweezers and evenly arranged in a 3×6 pattern in a square petri dish lined with moistened filter paper. The dishes were then placed in a 28℃ incubator for germination. Each treatment had three replicates. After 2 days of incubation, seed germination was photographed and recorded, and the root length and hypocotyl length of cucumber seedlings were measured to evaluate the promoting effect of JG-1 on cucumber seed germination and early seedling growth.

[0232] Control group: Use an equal volume of water instead of JG-1 solution, and follow the same procedure as above.

[0233] Experimental results are as follows Figure 19 As shown in Table 18, cucumber seedlings treated with JG-1 exhibited different growth characteristics compared to the control group (CK). Under four different concentrations of JG-1 (0.1 nM, 1 nM, 10 nM, and 100 nM), the hypocotyl length and root length of cucumber seedlings changed to varying degrees. Compared to the control group (CK), the hypocotyl length increased by 5%, 8%, and 2% after treatment with 0.1 nM, 1 nM, and 100 nM JG-1, respectively, while it decreased by 5% after treatment with 10 nM. Root length increased by 14%, 19%, 8%, and 11%, respectively, with the 1 nM JG-1 treatment showing the most significant promoting effect. In contrast, the 1 nM MeJA treatment decreased the hypocotyl length and root length of cucumber seedlings by 19% and 8%, respectively, showing a certain inhibitory effect. In summary, JG-1 can promote early growth of cucumber seedlings to a certain extent within a low concentration range, with a more pronounced promoting effect on root growth. Among them, the 1 nM JG-1 treatment showed better overall growth-promoting effect, indicating that this concentration is more conducive to the early growth of cucumber seedlings.

[0234] Table 18 Effects of different concentrations of JG-1 on cucumber seed germination

[0235]

[0236] Example 17 Effect of JG-1 seed soaking on cucumber seedling growth

[0237] To further investigate the effects of JG-1 on cucumber seedling growth, healthy, plump, and uniformly sized cucumber seeds (variety 'Lufeng') were selected. After being rinsed with distilled water, they were evenly sown in petri dishes lined with four layers of moist gauze, and then covered with four more layers of moist gauze. The seeds were then germinated in a 27℃ incubator in the dark for 24 hours. After germination, cucumber seeds with uniform white sprouts were selected and planted in flowerpots containing a culture medium at a depth of 2-3 cm. The culture medium consisted of a 1:1 (v / v) mixture of Suqian 'Lvzhuang' substrate and vermiculite. The flowerpots were then placed in an incubator with the following conditions: temperature 26℃, relative humidity 60%–70%, and light intensity 200 µmol m⁻². 2 s⁻ 1 The photoperiod was 16 h / 8 h. After 5 days of cultivation, robust cucumber seedlings with two fully expanded cotyledons and uniform growth were selected for treatment. JG-1 was dissolved in anhydrous ethanol to prepare a 10 mM stock solution; before use, it was diluted with water to prepare working solutions of different concentrations (JG-1 concentrations of 0.1 nM, 1 nM, 10 nM, and 100 nM). Using a 50 mL syringe without a needle, 25 mL of water or different concentrations of JG-1 treatment solution were slowly injected along the base of the seedling stem, with 4 replicates for each treatment. On days 6, 12, and 18 after treatment, the growth of cucumber seedlings was photographed and the plant height and true leaf growth were measured. After 18 days of treatment, the entire cucumber seedling was removed, the roots were washed, and the plant height, root length, whole plant fresh weight, above-ground fresh weight, and root fresh weight were measured. At the same time, the leaf length and leaf width were measured, and the leaf area was calculated according to the formula: Leaf area (cm²) 2 = Leaf length (cm) × Leaf width (cm) × K, where K is the cucumber leaf area correction coefficient, taken as 0.743 (Reference: Pei Xiaobo, Li Shicheng, Zhang Fuman, et al. Calculation of greenhouse cucumber leaf area and its correlation with plant height [J]. Chinese Agricultural Science Bulletin, 2005, (08): 80-82.).

[0238] Control group: Use an equal amount of water instead of JG-1 treatment solution, and the rest is the same as the above method.

[0239] Experimental results are as follows Figure 20As shown in Table 19, cucumber seedlings treated with JG-1 root irrigation exhibited significant differences in phenotypic and biomass accumulation compared to the control group. Compared to the control group, different concentrations of JG-1 treatment resulted in varying degrees of changes in leaf area, root growth, and fresh weight of cucumber seedlings, with the 1 nM JG-1 treatment showing the most significant promoting effect. Specifically, compared to the control group, after 1 nM JG-1 treatment, the area of ​​the first true leaf increased by 58%, 11%, and 2% on days 6, 12, and 18, respectively; the area of ​​the second true leaf increased by 139% and 33% on days 12 and 18, respectively; and the area of ​​the third true leaf increased by 56% on day 18. Meanwhile, after 18 days of treatment, the height of cucumber seedlings did not increase significantly, but root length increased by 29%, aboveground fresh weight by 43%, and root fresh weight by 66%, all at relatively high levels among the treatments. This indicates that 1 nM JG-1 can effectively promote leaf expansion, root development, and biomass accumulation in cucumber seedlings. Furthermore, the 10 nM JG-1 treatment also showed a certain promoting effect. In summary, JG-1 can promote cucumber seedling growth within a suitable concentration range, with the 1 nM treatment showing the most significant promoting effect on leaf growth, root development, and biomass accumulation, indicating that this concentration is more beneficial for cucumber seedling growth.

[0240] Table 19 Effects of different concentrations of JG-1 on cucumber seedling growth

[0241]

[0242] The above description of the embodiments is only for the purpose of helping to understand the method and core ideas of the present invention. It should be noted that those skilled in the art can make several improvements and modifications to the present invention without departing from the principles of the present invention, and these improvements and modifications also fall within the protection scope of the claims of the present invention.

Claims

1. 3-Methyl-2-[[2-[3-oxo-2-[pent-2-enyl]cyclopentyl]acetyl]amino]hexanoic acid, characterized in that, Having the structure shown in formula (1) or a salt thereof: Equation (1).

2. The method for preparing 3-methyl-2-[[2-[3-oxo-2-[pent-2-enyl]cyclopentyl]acetyl]amino]hexanoic acid according to claim 1, characterized in that, Includes the following steps: Using 2-amino-3-methylhexanoic acid and methyl jasmonate as raw materials, an ammonolysis reaction was carried out under the action of a strong organic base to obtain 3-methyl-2-[[2-[3-oxo-2-[pent-2-enyl]cyclopentyl]acetyl]amino]hexanoic acid.

3. The preparation method according to claim 2, characterized in that, The organic strong base is selected from one or more of sodium alkoxide, potassium alkoxide, sodium amino group, and alkyl lithium.

4. The preparation method according to claim 3, characterized in that, The strong organic base is selected from sodium tert-butoxide.

5. An agricultural composition comprising 3-methyl-2-[[2-[3-oxo-2-[pent-2-enyl]cyclopentyl]acetyl]amino]hexanoic acid as described in claim 1, or a salt thereof, and an adjuvant.

6. The use of the 3-methyl-2-[[2-[3-oxo-2-[pent-2-enyl]cyclopentyl]acetyl]amino]hexanoic acid or its salt as described in claim 1, or the agricultural composition as described in claim 5, in improving plant stress resistance, disease and pest resistance, and / or growth performance.

7. The application according to claim 6, characterized in that, The improvement of plant stress resistance includes at least one of the following: I) Improve resistance to high-temperature stress; II) Improve resistance to low-temperature stress; III) Improve resistance to salt stress; IV) Enhance the ability to resist pesticide damage to seeds and / or seedlings; And / or, the pests and diseases are fungal diseases; And / or, the improvement in growth performance includes promoting seed germination and / or promoting seedling growth.

8. The application according to claim 7, characterized in that, The pesticides include one or more of herbicides, insecticides, and fungicides; The fungal diseases mentioned include those caused by the genera *Erythrina*, *Erythrinae bryonis*, *Monophyllum*, or *Uncaria*.

9. The application according to claim 8, characterized in that, The pesticides include one or more of the following: fluroxypyr·pentyl·carbendazim, carbendazim·ethoxysulfuron, and quinclorac acid; The fungal diseases mentioned include powdery mildew in grass crops.

10. The application according to claim 6 or 7, characterized in that, The plants mentioned are grasses, cucurbits, and / or solanaceae.

11. The application according to claim 10, characterized in that, The grasses include rice, wheat, and / or maize; The Cucurbitaceae plants include cucumber; The Solanaceae plants include tobacco and / or tomato.

12. Plant breeding and / or cultivation methods, comprising at least one of the following: i) Soaking, dressing, coating and / or sedating seeds with 3-methyl-2-[[2-[3-oxo-2-[pent-2-enyl]cyclopentyl]acetyl]amino]hexanoic acid or a salt thereof as described in claim 1 or the agricultural composition as described in claim 5; ii) Spraying the leaves or the plant with the 3-methyl-2-[[2-[3-oxo-2-[pent-2-enyl]cyclopentyl]acetyl]amino]hexanoic acid or a salt thereof as described in claim 1, or the agricultural composition as described in claim 5; iii) Applying the 3-methyl-2-[[2-[3-oxo-2-[pent-2-enyl]cyclopentyl]acetyl]amino]hexanoic acid or a salt thereof, or the agricultural composition of claim 5, to the roots or root zone soil of plants.

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