Compositions based on methylcarboline and their application in plant stress resistance and growth promotion

Abstract

This invention belongs to the field of biotechnology, specifically disclosing the application of several methylcarboline acid-based compositions in plant stress resistance and growth promotion. This invention solves the problems of commercially available plant growth regulators, such as limited functionality, poor efficacy, large dosage, and high cost. The combined use of methylcarboline acid, paclobutrazol, fucoidan oligosaccharides, and aminoethyl esters at specific concentrations can significantly promote crop growth and improve crop yield and quality. The combined use of methylcarboline acid and γ-aminobutyric acid at specific concentrations can significantly alleviate the effects of salt-alkali stress on the growth of crops such as wheat and cucumber; the combined use of methylcarboline acid and 5-aminolevulinic acid at specific concentrations can promote the growth of crops such as tomatoes and peanuts under low temperatures. The stress resistance and growth promotion compositions provided by this invention can significantly improve plant resistance at appropriate concentrations, effectively promote plant growth and development, and improve crop yield and quality.

Description

Technical Field

[0001] This invention belongs to the field of plant growth regulator technology, and in particular relates to compositions based on methylcarboline acid and their application in plant stress resistance and growth promotion. Background Technology

[0002] Plants are susceptible to abiotic stresses such as low temperature and salinity during their growth, which affect plant growth, reduce crop yields, and have a significant impact on the distribution and productivity of agricultural crops. This impact further poses a challenge to food safety and sustainable agricultural development. Moreover, with the development of modern agriculture, people's increasing demand for agricultural product yield and quality, as well as the harm caused to the ecological environment and food security by the long-term unreasonable use of traditional pesticides and fertilizers, safe, green, and efficient plant cultivation products have become an inevitable trend in agricultural development.

[0003] Plant growth regulators include plant hormones, natural compounds, and synthetic compounds that have physiological effects on plant growth and development. There are numerous types of plant growth regulators; currently, hundreds of varieties are registered in my country. However, the quality of plant growth regulator products on the market varies greatly, with problems such as limited functionality, poor efficacy, high dosage, and high cost. Methylcarboline acid, as a newly discovered and prepared plant growth regulator, has received relatively little research attention, and studies on its combination with other plant growth regulators for plant stress resistance and growth promotion are even scarcer.

[0004] Chinese patent document CN117204433A (application number: CN202310972464.8) discloses the application and preparation method of compound 1-methyl-1,234tetrahydro-β-carboline 3-carboxylic acid. This patent document discloses the preparation and application of methylcarboline acid, a compound isolated and purified from Agaricus polylipidus YXI. The patent document discloses that the compound has the effects of drought resistance, antiviral activity, and promoting plant growth and increasing yield when combined with fertilizers. However, it does not conduct in-depth research on the stress resistance and growth-promoting effects of methylcarboline acid combined with other plant growth regulators. Summary of the Invention

[0005] To overcome the shortcomings of existing technologies, this invention employs a compound application of the plant growth regulator methylcarboline acid with conventional stress-resistant and growth-promoting plant growth regulators. This combination enhances plant resistance, effectively promotes plant growth, and exhibits a significant synergistic effect. The technical solution provided by this invention is as follows:

[0006] As a first aspect of the invention, it is to provide a composition based on methylcarboline acid, comprising methylcarboline acid and other plant growth regulators, said other plant growth regulators being selected from one or more of fucoidan, paclobutrazol, aminoethyl esters, γ-aminobutyric acid (GABA), and 5-aminolevulinic acid. The effective concentration of methylcarboline acid is 1–200 ng / mL.

[0007] In another aspect, the present invention also provides the application of methylcarboline acid-based compositions in plant stress resistance and growth promotion.

[0008] Preferably, the methylcarboline acid is produced by *Amanita muscaria* strain (…). Pholiota adiposa YX1 was isolated and purified, and it is a polylipidated agaric strain ( Pholiota adiposa YX1 has accession number CGMCC No. 21077. The prior patent "Application and preparation method of compound 1-methyl-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid" (application number: CN202310972464.8) has the effects of promoting crop growth, increasing crop yield, and improving crop resistance to drought stress.

[0009] This invention verifies the application of methylcarboline acid (1-methyl-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid) in combination with different plant growth regulators in terms of plant stress resistance, growth promotion and efficiency enhancement.

[0010] In one embodiment of the present invention, a composition comprising methylcarboline acid, fucoidan oligosaccharide, paclobutrazol, and aminoethyl ester is provided, wherein the effective concentration of methylcarboline acid is 1-200 ng / mL, preferably 10 ng / mL, the effective concentration of paclobutrazol (15%) is 0.5-10 mg / mL, preferably 2.50 mg / mL, the effective concentration of fucoidan oligosaccharide is 0.25-5 mg / mL, preferably 0.5 mg / mL, and the effective concentration of aminoethyl ester DA-6 is 5-30 mg / L, preferably 10 mg / L.

[0011] Furthermore, the present invention provides the effect of a composition comprising methylcarboline acid, fucoidan oligosaccharide, paclobutrazol, and aminoethyl ester in promoting plant growth, wherein the plants include bok choy, lettuce, etc.

[0012] Fucoidan, a highly active seaweed extract obtained by degrading macromolecular alginate, can act as an elicitor to induce excessive reactive oxygen species (ROS) in tobacco suspension cells within a short period of time. This initiates the intracellular ROS signal transduction program, stimulates the increase of antioxidant enzyme activity in plants, maintains intracellular free radicals at a low level, thereby reducing plant cell membrane damage, decreasing electrolyte leakage, and improving plant stress resistance. Adding 0.2% to 1.0% fucoidan during corn planting can significantly improve fertilizer utilization and corn yield. Exogenous fucoidan can promote crop growth and development, thereby increasing yield and quality.

[0013] Paclobutrazol is a plant growth regulator that can delay basal leaf growth, promote tillering, promote carbohydrate output, and increase the content of chlorophyll, protein, and nucleic acid in plants by regulating the levels of endogenous hormones and carbon and nitrogen nutrition in plants. It has the effects of delaying plant growth, inhibiting stem elongation, shortening internodes, promoting plant tillering, increasing plant stress resistance, and increasing yield.

[0014] DA-6 is a broad-spectrum plant growth regulator. After being absorbed by plants, it can increase the content of chlorophyll, protein, and nucleic acid in plants; improve photosynthetic efficiency; enhance carbon and nitrogen metabolism in plants; strengthen the absorption of water and fertilizer by plants; and regulate the water balance in plants. It has significant effects on increasing yield, improving resistance, and improving quality in many crops.

[0015] In one embodiment of the present invention, a composition comprising methylcarboline acid and γ-aminobutyric acid is provided, wherein the effective concentration of methylcarboline acid is 1 to 200 ng / mL, preferably 10 ng / mL, and the effective concentration of γ-aminobutyric acid is 0.1 mg / mL to 5.0 mg / mL, preferably 0.5 mg / mL.

[0016] Furthermore, the present invention provides the application of compositions containing methylcarboline acid and γ-aminobutyric acid in the resistance of plants to salt and alkali stress, including cucumber and wheat.

[0017] Gamma-aminobutyric acid (GABA) is a four-carbon non-protein amino acid widely found in animals, plants, and microorganisms. Numerous studies have shown that GABA can act as a signaling or energy source, participating in various physiological and biochemical reactions in plants, regulating plant growth and development and responses to abiotic stresses. Exogenous GABA can also be absorbed by plants as a substitute for organic nitrogen fertilizer, promoting photosynthesis and influencing energy metabolism, promoting vegetative and reproductive growth, enhancing plant resistance to various abiotic stresses, and promoting the absorption of micronutrients by plants.

[0018] In one embodiment of the present invention, a composition comprising methylcarboline acid and 5-aminolevulinic acid is provided, wherein the effective concentration of methylcarboline acid is 1 to 200 ng / mL, preferably 10 ng / mL, and the effective concentration of 5-aminolevulinic acid is 0.05 to 10 mg / mL, preferably 0.20 mg / mL.

[0019] Furthermore, the present invention provides the application of methylcarboline acid-based compositions containing 5-aminolevulinic acid in plant resistance to low-temperature stress, said plants including tomatoes and peanuts.

[0020] 5-Aminolevulinic acid (ALA), first discovered in the culture medium of common Chlorella, is a naturally occurring amino acid widely found in living cells of bacteria, fungi, animals, and plants. ALA can enhance plant tolerance to low temperatures through multiple mechanisms. Exogenous ALA can increase the activity of antioxidant enzyme systems and the efficiency of photosynthesis, increase the content of osmotic substances and polyamines, activate NO and γ-aminobutyric acid signal transduction, protect organelles, enhance chlorophyll synthesis and xanthophyll cycling, and regulate plant secondary metabolism and mineral element absorption, thereby improving plant cold tolerance. Exogenous ALA can also enhance the cold tolerance of plant seedlings by regulating glutathione metabolism, β-alanine metabolism, phenylalanine metabolism, and starch content.

[0021] After adopting this technical solution, 1-methyl-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid (methylcarboline acid) has a good effect on plant stress resistance and growth promotion.

[0022] The low-temperature resistance mechanism of methylcarboline acid (MCA) is as follows: It increases the activity of oxidases such as superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) in plants under low-temperature conditions, reduces the content of hydrogen peroxide and malondialdehyde (MDA), alleviates cell membrane damage, stabilizes cell membrane structure, enhances the antioxidant capacity of plant membranes, reduces cell osmotic regulation, and mitigates the degree of cell membrane damage. The salt-alkali tolerance mechanism of MCA is as follows: It increases the content of free proline and soluble sugars in plants, enhances their osmotic regulation capacity, reduces MDA content, increases the activity of antioxidant enzymes, and regulates the content of abscisic acid in plants, thereby improving the salt-alkali tolerance of plants.

[0023] Methylcarboline acid, fucoidan, γ-aminobutyric acid, 5-aminolevulinic acid, paclobutrazol, and aminoethyl esters are all plant growth regulators with effects such as promoting growth, resisting stress, and controlling excessive growth. Their compound formulation can not only make up for shortcomings, but also has its own unique functions. The compound formulation enhances the effect, expands the function, and reduces the dosage of single agents to save costs. They all show synergistic effects under plant growth and stress.

[0024] The beneficial effects of this invention are as follows:

[0025] 1. This invention uses multiple experiments to determine the optimal ratio of methylcarboline acid to three plant growth regulators: paclobutrazol (15%), fucoidan oligosaccharide, and amino acid ester DA-6, and verifies their significant role in promoting plant growth, increasing yield, and improving quality.

[0026] 2. The combined application of two plant growth regulators, methylcarboline acid and γ-aminobutyric acid and 5-aminolevulinic acid, was discovered to improve the plant's resistance to low temperature and salinity.

[0027] 3. Low absolute dosage: Experiments show that 0.1 g of methylcarboline acid, combined with paclobutrazol (15%), alginate oligosaccharide, aminoethyl ester DA-6, γ-aminobutyric acid and 5-aminolevulinic acid, can be sprayed on 1 acre of crops and has a significant effect on promoting growth and enhancing stress resistance. Attached Figure Description

[0028] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0029] Figure 1 The figure shows the growth-promoting phenotypes of lettuce after the combination of methylcarboline acid and three other compositions in Example 3 (the first row, left 1 is the water control, left 2 is the methylcarboline acid single agent treatment, and left 3 is the paclobutrazol single agent treatment; the second row, left 1 is the fucoidan single agent treatment, left 2 is the aminoethyl ester single agent treatment, and left 3 is the combined treatment of methylcarboline acid with fucoidan, paclobutrazol, and aminoethyl ester).

[0030] Figure 2 The figure shows the effect of the combined use of methylcarboline acid and γ-aminobutyric acid on the germination characteristics of wheat seeds under salt stress in Example 4 (from left to right in the figure: water control under normal conditions, water control under salt stress, γ-aminobutyric acid single agent treatment under salt conditions, methylcarboline acid single agent treatment under salt conditions, and γ-aminobutyric acid combined with methylcarboline acid treatment under salt conditions).

[0031] Figure 3 The figure shows the phenotypes of cucumber seedlings under salt-alkali stress after the combined use of methylcarboline acid and γ-aminobutyric acid in Example 4 (left 1 is the water control under normal conditions, left 2 is the water control under salt-alkali conditions, left 3 is the γ-aminobutyric acid single agent treatment under salt-alkali conditions, left 4 is the methylcarboline acid single agent treatment, and left 5 is the treatment of γ-aminobutyric acid combined with methylcarboline acid).

[0032] Figure 4 The figure shows the effect of the combination of methylcarboline acid and 5-aminolevulinic acid on the phenotypic effect of tomatoes under low temperature stress in Example 5 (left 1 is the water control at room temperature, left 2 is the water control at low temperature, left 3 is the 5-aminolevulinic acid single agent treatment at low temperature, left 4 is the methylcarboline acid single agent treatment at low temperature, and left 5 is the treatment of the combination of 5-aminolevulinic acid and methylcarboline acid at low temperature).

[0033] Figure 5The image shows the phenotype of peanut plants under low-temperature stress after the combined use of methylcarboline and 5-aminolevulinic acid in Example 5 (left 1 is the water control, left 2 is the 5-aminolevulinic acid single treatment, left 3 is the methylcarboline acid single treatment, and left 4 is the 5-aminolevulinic acid and methylcarboline acid combined treatment).

[0034] Figure 6 The figure shows the contents of MDA, proline, soluble sugar, and SOD in cucumber leaves under salt-alkali stress after the combination of methylcarboline acid and γ-aminobutyric acid in Example 4.

[0035] Figure 7 The figure shows the content of MDA, proline, soluble sugar, and SOD in peanut seedlings under low-temperature stress after the combination of methylcarboline acid and 5-aminolevulinic acid in Example 5. Detailed Implementation

[0036] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0037] The earliest disclosure of *Agaricus polylipidus* YX1 was in patent CN113637594B, entitled "A *Agaricus polylipidus* YX1, its culture method and its application." It was collected from wild forests in Mount Tai, and *Agaricus polylipidus* was obtained through isolation, purification, and culture. The *Agaricus polylipidus* (…) Pholiotaadiposa YX1 was deposited on December 1, 2020, at the China General Microbiological Culture Collection Center (CGMCC, address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences), with accession number CGMCC NO. 21077.

[0038] Example 1: Preparation of methylcarboline acid

[0039] Fermentation broth was obtained by fermenting *Agaricus polylipidus* YX1 at 24℃ and 180 r / min. The fermentation broth was centrifuged to obtain fermentation mycelia. After washing, the mycelia were dried, weighed, and pulverized. The pulverized mycelia were mixed with ethanol and ultrasonically treated to obtain an extract. The extract was then freeze-dried, dissolved in water to prepare a solution, and adsorbed using a macroporous resin. The resin adsorbate was eluted with ethanol, and then the adsorbate was separated using 80% acetonitrile. The supernatant was collected and separated by HPLC to obtain the compound 1-methyl-1,2,3,4-tetrahydro-β-carboxylic acid.

[0040] Example 2: Experiment on the effective concentration of methylcarboline acid

[0041] (1) Experimental design:

[0042] This experiment was conducted in indoor pots. Arabidopsis thaliana, wheat, corn, rice, tomato, pepper, soybean, and peanut were selected as experimental materials. Ten treatments were set up: a water control (CK), and methylcarboline acid at concentrations of 1 ng / mL, 5 ng / mL, 10 ng / mL, 20 ng / mL, 50 ng / mL, 100 ng / mL, 150 ng / mL, 200 ng / mL, and 250 ng / mL. A suitable amount of sterilized potting soil was placed in flowerpots (15 cm in diameter and 20 cm in height). Five seeds or tubers of the aforementioned plants were sown in each pot, and a layer of potting soil was evenly covered. All treatments were managed uniformly. One week after emergence, each pot was watered with 50 mL of different concentrations of methylcarboline acid, while the control treatment was watered with the same amount of water. Watering was carried out every two weeks. The fresh weight of the plants was measured one month after emergence.

[0043] (2) Experimental results:

[0044] The results are shown in Table 1. Methylcarboline acid (MCCA) concentrations within the range of 1-200 ng / mL showed varying degrees of promoting effect on the growth of different crops. At a concentration of 250 ng / mL, MCCA inhibited the growth of a few plants but had no significant growth-promoting effect on most. Within the 1-200 ng / mL range, most plants achieved optimal fresh weight at an MCCA concentration of 10 ng / mL. At this concentration, soybeans, peanuts, and...

[0045] The fresh weights of wheat, corn, and chili peppers increased by 13.81%, 24.54%, 30.99%, 23.75%, and 34.72% respectively compared to the control (CK).

[0046] Table 1. Effects of different concentrations of methylcarboline acid on the fresh weight (g) of different plants.

[0047] plant Arabidopsis soybeans peanut wheat corn rice tomato chili CK processing 3.02 7.89 9.25 5.26 11.24 6.03 7.89 5.04 Carboxylic Acid-1 3.88 8.12 10.91 6.49 13.01 6.61 8.68 6.24 Carboxylic Acid-5 3.72 8.23 10.25 6.51 12.89 6.79 8.29 6.36 Carboxylic Acid-10 4.27 8.98 11.52 6.89 13.91 7.04 9.25 6.79 Carboxylic Acid-20 4.35 8.45 11.38 6.74 13.73 7.12 9.32 6.53 Carboxylic Acid-50 4.01 8.22 11.26 6.75 13.51 6.98 9.41 6.15 Carboxylic Acid-100 3.58 8.01 11.21 6.21 12.67 6.63 8.55 6.03 Carboxylic Acid-150 3.60 7.92 11.04 5.87 12.12 6.32 8.34 5.88 Carboxylic Acid-200 3.24 7.88 10.85 5.46 11.87 6.25 8.01 5.74 Carboxylic Acid-250 2.69 7.64 10.56 5.28 11.42 6.04 7.57 5.23

[0048] Example 3: Growth-promoting effect of a combination of methylcarboline acid and various plant growth regulators on plants.

[0049] 1. Concentration screening of compound formulations of methylcarboline acid with fucoidan, paclobutrazol, and aminoethyl ester

[0050] (1) Experimental design:

[0051] After two weeks of direct seeding and seedling cultivation, the plastic film was removed, and seedlings of uniform size were transplanted into small pink pots, with six pots placed in a tray. Five days after transplanting, a treatment solution was sprayed onto the lettuce leaves, ensuring even coverage. Three different concentrations of methylcarbolic acid, fucoidan, paclobutrazol, and aminoethyl ester were used. After harvest, the yield of the lettuce was measured to determine the optimal combination of these four plant growth regulators for promoting growth and increasing yield in lettuce.

[0052] (2) Test results

[0053] Table 2. Effects of different concentrations of compound pesticides on the growth of Chinese cabbage.

[0054] Methylcarboline (ng / mL) Paclobutrazol (15%) (mg / mL) Fucoidan (mg / mL) DA-6 (mg / L) Fresh weight of bok choy (g) 0 0 0 0 7.62 10 0 0 0 10.01 0 2.5 0 0 8.69 0 0 0.5 0 8.97 0 0 0 20 9.51 5 5 0.5 10 9.4 20 2.5 0.5 5 10.51 10 2.5 1 5 9.88 20 1 0.5 10 9.84 10 5 0.5 20 9.98 20 5 0.5 10 10.78 20 2.5 0.5 20 10.25 10 1 0.5 20 9.68 5 2.5 1 10 9.38 5 2.5 0.5 20 9.39 10 5 0.5 5 11.00 20 2.5 1 10 10.68 10 2.5 0.25 20 9.75 10 5 0.25 10 10.64 10 2.5 1 20 10.75 10 2.5 0.5 10 11.31 10 1 1 10 9.35 10 5 1 10 10.34 20 2.5 0.25 10 10.19

[0055] As shown in Table 2, compared with the control (CK), the single application of the four agents and the combination of different concentrations all promoted the growth of pak choi to varying degrees. Among them, the combination of 10 ng / mL methylcarbolic acid, 2.50 mg / mL 15% paclobutrazol, 0.5 mg / mL fucoidan, and 10 mg / L aminoethyl ester had the most significant promoting effect on pak choi, with the fresh weight of pak choi reaching 11.31 g, which was 48.23%, 12.99%, 30.15%, 26.09%, and 18.18% higher than that of the CK treatment and the single application of methylcarbolic acid, 15% paclobutrazol, fucoidan, and aminoethyl ester, respectively.

[0056] 2. Compound agents promote the growth and development of lettuce.

[0057] (1) Experimental design

[0058] The optimal compound concentrations of four plant growth regulators from the previous experiments were selected for subsequent verification experiments. Six treatments were set up: water control (CK), 10 ng / mL methylcarboline acid, 2.5 mg / mL 15% paclobutrazol, 0.5 mg / mL fucoidan, 10 mg / L aminoethyl ester, and a combination of methylcarboline acid + fucoidan + paclobutrazol + aminoethyl ester. Lettuce was selected as the test crop. After harvest, growth and quality indicators such as leaf number, maximum leaf area, leaf SPAD value, and fresh weight were measured and recorded.

[0059] (2) Test results

[0060] As shown in Table 3 and Figure 1 As shown, compared with the control (CK), the application of the four agents promoted lettuce growth and improved lettuce quality to varying degrees. Methylcarbophosphate and fucoidan showed the best growth-promoting effect, while paclobutrazol and methylcarbohydrate had the best yield-promoting effect, and aminoethyl ester had the best quality-promoting effect. The combination of methylcarbohydrate, fucoidan, paclobutrazol, and aminoethyl ester combined the advantages of each plant growth regulator, significantly promoting lettuce growth, yield, and quality. The number of lettuce leaves, leaf area, leaf fresh weight, root fresh weight, chlorophyll content, and vitamin C content increased by 23.57%, 21.62%, 39.88%, 34.07%, 30.42%, and 67.53%, respectively, compared to the control (CK).

[0061] Table 3 Effects of different treatments on lettuce growth

[0062] deal with Number of leaves / blades <![CDATA[Leaf area / cm 2 > Leaf fresh weight / g Fresh weight of roots / g Chlorophyll content / SPAD value <![CDATA[VC content / mg·100g -1 > CK 11.33b 112.82c 18.28c 3.64b 22.62c 6.16c Methylcarboline 13.83a 135.99ab 24.38ab 4.36ab 24.27bc 8.06abc Paclobutrazol 13.0a 128.55b 24.55ab 4.2ab 24.83bc 7.64bc Brown algae oligosaccharides 13.67a 134.96ab 23.28ab 4.28ab 23.57c 7.37bc amino acid ester 13.67a 133.83ab 23.23b 4.11ab 27.27ab 9.74ab Composition 14.00a 137.21a 25.57a 4.88a 29.50a 10.32a

[0063] Note: Different lowercase letters indicate significant differences at the 0.05 level.

[0064] Example 4: Methylcarboline acid combined with γ-aminobutyric acid improves crop resistance to salt and alkali stress

[0065] 1. Promotes wheat seed germination characteristics under salt stress

[0066] (1) Screening of NaCl salt stress concentration

[0067] Six NaCl concentration gradients were set up: 0, 50, 100, 150, 200, and 250 g / L. Uniformly sized and plump wheat seeds (Jimai 22) were selected, sterilized, and dried before soaking for 20 h. Two layers of sterile filter paper were placed in each petri dish, and 10 mL of salt solution of different concentrations was added to each dish. The filter paper was replaced daily, and 10 mL of solution was added again. Each treatment was repeated three times, with 30 soaked seeds per replicate. The culture conditions were 23℃ (16 / 8 h) in the dark. From the day of seed germination, the total number of germinated seeds and the number of newly germinated seeds were recorded daily. On day 7, various indicators were measured, and the experimental concentration of NaCl solution was finally determined to be 200 mmol / L.

[0068] (2) Experimental design

[0069] Five treatment groups were established: normal treatment (CK0), salt treatment group (CK1), 0.5 mg / mL γ-aminobutyric acid (GABA), 10 ng / mL methylcarboline acid (MCCA), and 0.5 mg / mL γ-aminobutyric acid + 10 ng / mL MCCA (GABA + methylcarboline acid). CK0 and CK1 were treated with water soaking, while the other three groups were treated with the chemical treatments. After 20 h, the seeds in the CK0 group were treated with 10 ml of water, while the seeds in the CK1 group and the other three chemical treatment groups were treated with an equal volume of 200 mmol / L NaCl solution. Each dish contained 30 seeds, and the treatment was repeated three times. The total number of germinated seeds and the number of newly germinated seeds were recorded daily. After 7 days, 10 seedlings from each dish were taken to measure root length and shoot length, and the results were calculated using the following formula:

[0070] Germination rate = Number of seeds that germinate within 7 days / Total number of seeds

[0071] Germination potential = Number of seeds germinating within 3 days / Total number of seeds

[0072] Germination index = ∑ Daily germination count / Corresponding germination days

[0073] Seedling activity index = germination index x 7 days of shoot length

[0074] Salt tolerance germination index = germination index of treatment / germination index of control

[0075] (3) Test results

[0076] The germination of wheat seeds in different treatment groups is shown in Table 4. (From Table 4 and...) Figure 2 It can be seen that the germination and growth of wheat seeds were significantly inhibited under salt stress. All indicators of CK1 were significantly reduced. Compared with CK1, all three treatments alleviated the effects of salt stress on wheat to some extent. Among them, the wheat treated with methylcarboline acid combined with γ-aminobutyric acid showed significant improvement in all indicators. The germination potential, germination rate, radicle length, plumule length, germination index, vigor index, drought resistance germination index, and drought resistance activity index of wheat were 31.10%, 8.74%, 35.42%, 37.76%, 7.05%, 47.46%, 6.59%, and 47.92% higher than those of CK1, respectively. From the perspective of the germination indicators of wheat seeds under salt stress, the germination indicators of methylcarboline acid combined with GABA were significantly improved compared with GABA and methylcarboline acid alone. This indicates that the effect of methylcarboline acid combined with γ-aminobutyric acid on improving the salt tolerance of plants is significantly higher than that of GABA and methylcarboline acid alone.

[0077] Table 4 Germination indices of wheat seeds under different treatments under salt stress

[0078] deal with Germination potential / % Germination rate / % Radicle length / cm Embryo length / cm Germination Index Activity index Salt tolerance germination index CK0 90.86 a 99.86 a 12.1 a 7.29 a 17.49 a 127.50 a 1.00 a CK1 65.33 c 90.02 b 6.07 d 3.84 c 15.89 c 61.02 d 0.91 b GABA 72.45 b 95.58 ab 7.24 c 4.89 bc 16.24 b 79.41 c 0.93 b Methylcarboline 70.89 bc 94.33 ab 7.85 bc 5.05 b 16.58 b 83.73 b 0.95 a GABA+methyl 85.65 ab 97.89 a 8.22 b 5.29 b 17.01 a 89.98 b 0.97

[0079] 2. Promote the growth and development of cucumber seedlings under salt and alkali stress

[0080] (1) Experimental design

[0081] Cucumbers were sown and raised normally. Once they reached the four-leaf stage, uniformly growing cucumber seedlings were selected for transplanting. The experiment included five treatments: normal treatment (CK0), saline-alkali treatment (CK1), saline-alkali + 10 ng / mL methylcarboline acid (MCCA), saline-alkali + 0.5 mg / mL γ-aminobutyric acid (GABA), and saline-alkali + 10 ng / mL MCCA + 0.5 mg / mL γ-aminobutyric acid (GABA + methylcarboline). Each treatment was replicated five times. The normal treatment group was treated with water, while the saline-alkali treatment group was treated with a 150 mM saline-alkali solution (NaCl:Na2SO4:NaHCO3:NaCO3 = 9:1:9:1). Simultaneously, the saline-alkali experimental group was irrigated with the corresponding treatment solution. The yellowing growth was observed and recorded regularly, and growth indicators were measured. Malondialdehyde (MDA) content, proline content, catalase activity, and abscisic acid content were measured on cucumber leaves.

[0082] (2) Test results

[0083] The experimental results showed that cucumber seedlings in the normal treatment group (CK0) grew normally, while cucumbers in all salt stress treatment groups grew slowly, and their leaves showed wilting and yellowing. After 28 days of cultivation, the cucumber seedlings were harvested and their indicators were measured. (See Table 5 and...) Figure 3 It was found that, compared with CK1, both methylcarboline and γ-aminobutyric acid (GABA) alone promoted the growth of cucumber seedlings under salt-alkali stress, and all growth indicators of cucumber were generally improved, with the largest leaf area and above-ground fresh weight showing the most significant increases. The combination of methylcarboline and GABA had a better effect on the growth of cucumber seedlings under salt stress than either alone. Specifically, the largest leaf area, above-ground fresh weight, and number of flower buds were significantly increased, by 48.97%, 58.20%, and 38.89% compared to CK, and by 28.61%, 42.88%, and 25.00% compared to GABA, and by 14.74%, 9.50%, and 51.52% compared to methylcarboline. This indicates that the combination of methylcarboline and GABA has a significant synergistic effect on the growth of cucumber seedlings under salt stress.

[0084] Table 5 Growth indicators of cucumber seedlings under different treatments under salt-alkali stress

[0085] deal with Plant height / cm Root length / cm <![CDATA[Maximum leaf area / cm 2 > Leaf fresh weight / g Fresh weight of roots / g Number of flower buds / Chlorophyll / SPAD CK0 13.52 a 49.98 a 91.19 a 14.48 a 4.12 a 13.0 a 45.48 a CK1 6.66 b 17.36 b 39.62 c 6.34 d 0.72 b 7.0 c 43.00 a GABA 7.10 b 19.74 b 45.89 bc 7.02 CD 0.88 b 8.0 bc 43.06 a Methylcarboline 7.82 b 19.80 b 51.44 bc 9.16 bc 1.01 b 6.6 c 42.52 a GABA+methyl 8.00 b 20.60 b 59.02 b 10.03 b 1.00 b 10.0 ab 47.58 a

[0086] like Figure 6 As shown, compared with CK0, the MDA content in cucumber leaves increased under salt-alkali stress conditions. The highest MDA content was found in cucumber seedlings treated with CK1, reaching 0.1056 μmol·L⁻¹. -1 The MDA content in cucumber seedlings treated with methylcarboline acid combined with γ-aminobutyric acid was significantly lower than that in CK1, decreasing by 59.94%. The MDA content was 10.98% and 16.08% lower than that of methylcarboline acid and γ-aminobutyric acid alone, respectively. The proline, CAT activity, and abscisic acid content in cucumber seedlings treated with methylcarboline acid combined with γ-aminobutyric acid were all significantly increased compared with CK1, increasing by 64.56%, 40.46%, and 624.32%, respectively. This indicates that the combination of methylcarboline acid and γ-aminobutyric acid can improve the osmotic regulation capacity of cucumber, reduce cellular oxidative damage, regulate abscisic acid content, and thus improve the salt and alkali resistance of cucumber.

[0087] Example 5: Effects of the combination of methylcarboline acid and 5-aminolevulinic acid on crop growth under low temperature stress

[0088] 1. Promotes tomato growth under low temperature stress

[0089] (1) Experimental Design

[0090] After sterilizing the substrate, mix it thoroughly and fill it into seedling trays. Spread the surface evenly and then sow tomato seeds at a depth of approximately 0.5 cm. Cultivate the seedlings in a culture room at 23 ℃, 50%-60% humidity, and 6000-7000 lx light. Transplant healthy, pest-free seedlings with uniform growth when they reach the four-leaf stage. After a 3-day recovery period, conduct experimental spraying treatments. Five treatments were established: room temperature treatment (CK0), low temperature treatment (CK1), low temperature + 10 ng / mL methylcarboline acid (methylcarboline acid), low temperature + 5-aminolevulinic acid - 0.2 mg / mL (ALA), and low temperature + 10 ng / mL methylcarboline acid + 0.2 mg / mL 5-aminolevulinic acid (ALA + methyl). Spraying was performed every two days, with the low temperature treatment occurring 12 hours after the second spray. The seedlings were placed in two separate intelligent incubators at the following temperatures: room temperature: 23℃ (16 / 8); low temperature treatment: 4℃ (16 / 8). Each treatment had four replicates, and various growth indicators of tomatoes were measured after harvest.

[0091] (2) Test Results

[0092] From Table 6 and Figure 4 It can be seen that under low temperature stress, tomato growth is inhibited, plants are stunted, and leaves show wilting and yellowing. Compared with the application of methylcarboline or 5-aminolevulinic acid alone, the combination of methylcarboline and 5-aminolevulinic acid improved various indicators of tomatoes to varying degrees. The combination of methylcarboline and 5-aminolevulinic acid had a significant synergistic effect, with tomato plant height, root length, and SPAD value increasing by 28.54%, 38.55%, and 75.93%, respectively, compared with CK1.

[0093] Table 6. Tomato growth indicators under different treatments under low temperature stress

[0094] deal with Leaf length / cm Leaf width / cm Plant height / cm Root length / cm Leaf fresh weight / g Chlorophyll / SPAD value CK0 11.68 a 7.94 a 10.82 a 17.65 a 7.54 a 62.85 a CK1 5.37 c 3.89 c 5.08 c 9.08 c 0.60 d 21.02 c ALA 6.54 b 4.86 b 6.03 b 12.30 ab 1.63 c 35.34 b Methylcarboline 6.79 b 4.45 bc 5.88 b 10.63 b 1.61 c 33.75 b ALA+methyl 6.91 b 5.36 b 6.53 b 12.58 ab 2.20 b 36.98 b

[0095] 2. Promote peanut growth under low temperature stress

[0096] (1) Experimental design

[0097] Nutrient substrate was placed in the planting baskets of the vigorous model, and four peanut seeds from each treatment (after soaking) were sown in each basket, with each treatment replicated four times. A certain amount of water was added to the culture bottle, maintaining the water level approximately 2 cm below the bottom of the planting basket. The baskets were placed in a 10 / 4℃ incubator for 7 days for low-temperature treatment, and then transferred to room temperature for recovery culture. Peanut germination was recorded, and germination rate, plant height, root length, fresh weight, and other growth indicators were measured. Peanut leaf samples were collected to determine the content of malondialdehyde, proline, soluble sugar, superoxide dismutase activity, and catalase activity.

[0098] (2) Data statistics and analysis

[0099] Table 7. Growth of peanut seedlings under different treatments under low temperature stress.

[0100] deal with Plant height / cm Root length / cm Stem diameter / mm Fresh weight of above-ground parts (g) Fresh weight of underground part (g) CK 4.76 c 4.96 d 5.57b 5.89 c 2.22 b ALA 5.91 b 5.95 c 5.84ab 7.19 b 2.47 b Methylcarboline 6.59 ab 7.37 b 5.96ab 8.02 a 2.98 b ALA+methyl 7.34 a 9.42 a 6.11a 8.74 a 3.06 a

[0101] From Table 7 and Figure 5 As shown, both single and combined applications of 5-aminolevulinic acid (5-ALA) and methylcarboline acid (MCCA) can alleviate the effects of low temperature on peanut seedling growth. The combined application of the two was significantly more effective than single application. After 5-ALA was combined with MCCA, the plant height, root length, stem diameter, aboveground fresh weight, and underground fresh weight increased by 54.20%, 89.92%, 9.69%, 48.39%, and 37.84%, respectively, compared to the control (CK). The peanut plant height treated with 5-ALA combined with MCCA increased by 24.20% and 11.38% compared to single application, the root length increased by 58.32% and 27.82%, respectively, and the stem diameter increased by 4.62% and 2.52%, respectively. Therefore, compared to single application, 5-ALA combined with MCCA significantly improved the plant height, root length, and stem diameter of peanut seedlings under low temperature conditions, promoting their growth.

[0102] like Figure 7 As shown, under low-temperature stress, the MDA content in peanut seedlings treated with 5-aminolevulinic acid (5-aminolevulinic acid) combined with methylcarboline acid was significantly reduced, decreasing by 44.70% compared to the control (CK). The proline and soluble sugar contents in peanut seedlings treated with 5-aminolevulinic acid combined with methylcarboline acid were significantly increased, increasing by 96.90% and 84.85% respectively compared to the CK. The SOD and CAT activities of the combined product increased by 68.21% and 62.56% respectively compared to the CK, while the SOD and CAT activities of 5-aminolevulinic acid alone increased by 51.40% and 27.68% respectively compared to the CK, and the SOD and CAT activities of methylcarboline acid alone increased by 59.26% and 34.24% respectively compared to the CK. This indicates that compared to applying either product alone, the combination of 5-aminolevulinic acid and methylcarboline acid can improve the cold resistance of peanuts and promote the growth and development of peanut seedlings under low-temperature stress by increasing the activity of peanut antioxidant enzymes, reducing cellular oxidative damage, and maintaining cell osmotic pressure balance.

[0103] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A composition based on methylcarboline, characterized in that, It is composed of methylcarboline acid, fucoidan oligosaccharide, paclobutrazol and aminoethyl ester, wherein the effective concentration of methylcarboline acid is 1-200 ng / mL, the effective concentration of paclobutrazol 15% is 0.5-10 mg / mL, the effective concentration of fucoidan oligosaccharide is 0.25-5 mg / mL, and the effective concentration of aminoethyl ester DA-6 is 5-30 mg / L.

2. The composition based on methylcarboline acid according to claim 1, characterized in that, The methylcarboline acid is produced by *Amanita muscaria* strain (…). Pholiota adiposa YX1 was isolated and purified, and it is a polylipidated agaric strain ( Pholiota adiposa The accession number for YX1 is CGMCC No. 21077.

3. The effect of the methylcarboline acid-based composition of claim 1 in promoting plant growth, wherein the plants include bok choy and lettuce.

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