WO2023103931A1 - 2-氨基-3-吲哚基丁酸或3-甲基吡咯烷-2-羧酸作为植物免疫诱抗剂的应用 - Google Patents
2-氨基-3-吲哚基丁酸或3-甲基吡咯烷-2-羧酸作为植物免疫诱抗剂的应用 Download PDFInfo
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- WO2023103931A1 WO2023103931A1 PCT/CN2022/136464 CN2022136464W WO2023103931A1 WO 2023103931 A1 WO2023103931 A1 WO 2023103931A1 CN 2022136464 W CN2022136464 W CN 2022136464W WO 2023103931 A1 WO2023103931 A1 WO 2023103931A1
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- amino
- methylpyrrolidine
- carboxylic acid
- acid
- stress
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- UVZZDDLIOJPDKX-UHFFFAOYSA-N paraherquamide A Natural products O1C(C)(C)C=COC2=C1C=CC1=C2NC(=O)C11C(C)(C)C2CC3(N(C4)CCC3(C)O)C(=O)N(C)C42C1 UVZZDDLIOJPDKX-UHFFFAOYSA-N 0.000 description 1
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Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
- A01N43/34—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one nitrogen atom as the only ring hetero atom
- A01N43/36—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one nitrogen atom as the only ring hetero atom five-membered rings
- A01N43/38—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one nitrogen atom as the only ring hetero atom five-membered rings condensed with carbocyclic rings
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G7/00—Botany in general
- A01G7/06—Treatment of growing trees or plants, e.g. for preventing decay of wood, for tingeing flowers or wood, for prolonging the life of plants
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
- A01P21/00—Plant growth regulators
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
Definitions
- the invention belongs to the field of agricultural biological pesticides and relates to the application of 2-amino-3-indolyl butyric acid or 3-methylpyrrolidine-2-carboxylic acid as a plant immune inducer.
- soil salinization is the main abiotic limiting factor that hinders global crop growth and productivity, and has a great harmful impact on the biosphere and ecological structure.
- the area of saline-alkali land in China ranks third in the world, accounting for about 10% of the world's saline-alkali land area. Therefore, in view of the main abiotic stress conditions faced by different crops in actual production, it is particularly urgent to develop products and technologies aimed at reducing the level of plant hazards to ensure safe agricultural production.
- Plant immune inducers are a new class of pesticides, which can enhance plant disease resistance and stress resistance by activating the plant's immune system and regulating plant metabolism.
- the plant immune inducer itself has no direct bactericidal activity, mainly by promoting the plant to use its own natural immune system to prevent and control the disease, without relying on exogenous pesticides to directly kill the pathogen, so the bacteria are not easy to develop resistance to it, which is in line with the effective protection of agricultural The idea of realizing green prevention and control under the condition of biodiversity.
- the growth of plants is usually not only subject to a single stress, but multiple stresses coexist, such as drought and high temperature stress often occur at the same time, causing more serious damage to plants.
- plant immune inducers can increase the stress resistance level of plants. Therefore, plant immune inducers, as a category of emerging pesticides, provide new development ideas for the sustainable development of agriculture and the effective green prevention and control of diseases, and are the main direction for the future development of green plant protection.
- 2-Amino-3-indolylbutyric acid the molecular formula is C 12 H 14 N 2 O 2 , the molecular weight is 218 g/mol, and it is light brown crystal.
- 2-amino-3-indolylbutyric acid is an intermediate product in the biosynthetic pathway of some natural products such as Maremycin and Streptonigrin with anticancer activity (Zou et al.,2013; Kong et al ., 2016).
- the first step in Streptomyces flocculus' synthesis of streptavidin may be the synthesis of 2-amino-3-indolebutyric acid (Gould & Chaug, 1977).
- Hartley et al. used S. flocculus enzyme to conduct in vitro enzyme catalysis experiments and found that the methyl group of S-adenosylmethionine (S-adenosylmethionine) can be transferred to tryptophan to synthesize 2-amino-3-indolebutyric acid (Hartley & Speedie, 1984).
- 3-Methylpyrrolidine-2-carboxylic acid the molecular formula is C 6 H 11 NO 2 , the molecular weight is 129 g/mol, and it is a colorless crystal.
- the earliest report on this compound was in 1964, and 3-methylpyrrolidine-2-carboxylic acid was obtained for the first time by chemical synthesis. Subsequent activity studies found that the compound could inhibit the synthesis of actinomycin in Streptomyces antibioticus (Yoshida et al., 1964; Mauger et al., 1966; Katz et al., 1968; Yoshida et al., 1968).
- Tan et al. isolated two new cyclic heptapeptides, Scytalidamides A and B, from the fermentation broth of the marine fungus Scytalidium.sp., and found that 3-methylpyrrole could be obtained by hydrolyzing Scytalidamides B Alkane-2-carboxylic acids (Tan et al., 2003). Fredenhagen et al.
- the object of the present invention is to address the above-mentioned deficiencies of the prior art, and to provide the application of 2-amino-3-indolyl butyric acid or 3-methylpyrrolidine-2-carboxylic acid as a plant immune inducer.
- Another object of the present invention is to provide an immune inducer.
- Another object of the present invention is to provide a method for increasing the resistance of plants to biotic and/or abiotic stress.
- 2-Amino-3-indolylbutyric acid and 3-methylpyrrolidine-2-carboxylic acid are natural products isolated from Alternaria sp.
- the fungal disease is preferably wheat powdery mildew; the bacterial disease is preferably Pseudomonas syringae disease; and the viral disease is preferably tomato spotted wilt.
- the plants are selected from food crops, economic crops and vegetables.
- the food crops are preferably wheat, the economic crops are preferably tea and cotton, and the vegetables are preferably tomatoes.
- a plant immunity inducer comprising any one or both of 2-amino-3-indolyl butyric acid or 3-methylpyrrolidine-2-carboxylic acid.
- the plant immune inducer includes any one of component A: 2-amino-3-indolyl butyric acid or 3-methylpyrrolidine-2-carboxylic acid or two, component B: surfactant.
- the surfactant is Tween 20, and the concentration of Tween 20 in the plant immune inducer is preferably 0.02% (v/v).
- the concentration of 2-amino-3-indolylbutyric acid or 3-methylpyrrolidine-2-carboxylic acid in the plant immune inducer is 0.1-10000nM.
- the natural metabolite 2-amino-3-indolylbutyric acid or 3-methylpyrrolidine-2-carboxylic acid isolated from the saprotrophic fungus Alternaria is used for the prevention and treatment of diseases, its details and embodiments As follows: in the range of 0.1-10000nM concentration (addition of 0.02% surfactant Tween 20 by volume percentage), it can effectively inhibit the infection and spread of viruses, bacteria and fungi on plants, inhibit the occurrence and spread of diseases, and improve plant health. Resistance to high temperature, low temperature, drought and salt stress.
- a method for improving plant resistance to biological stress comprising applying the plant immune inducer of the present invention to plants in advance; the biological stress is selected from any one or more of fungal, bacterial, and viral stresses.
- 2-amino-3-indolyl butyric acid or 3-methylpyrrolidine-2-carboxylic acid is used for the method for preventing and treating tomato spotted wilt, under the concentration of 0.1-10nM (adding volume percent is 0.02% surfactant Tween 20), can significantly inhibit the spread of the virus 3 days after tobacco inoculation with tomato spotted wilt virus (TSWV). After 15 days, tobacco disease was investigated, and it was found that the disease index of tobacco plants treated with 2-amino-3-indolylbutyric acid and 3-methylpyrrolidine-2-carboxylic acid was significantly reduced.
- 2-amino-3-indolebutyric acid can effectively inhibit the expression of TSWV in tobacco leaves, and its disease index, relative immune effect and virus content are 30.42, 67.36% and 0.17, respectively.
- 3-methylpyrrolidine-2-carboxylic acid can also effectively inhibit the expression of TSWV on tobacco leaves, and its disease index, relative immune effect and virus content are 21.44, 75.73% and 0.16, respectively.
- 2-amino-3-indole butyric acid or 3-methylpyrrolidine-2-carboxylic acid is used for the method of preventing and treating bacterial diseases, and it is 100-10000nM concentration range (adding volume percent is 0.02% surface active Tween 20), with the increase of treatment concentration, the accumulation of bacteria PstDC3000 in Arabidopsis leaves gradually decreased, and when the treatment concentration of 2-amino-3-indolebutyric acid was 10000nM, the number of bacteria per mg of leaves Compared with the blank control , the number of bacteria decreased by 85.039%, and the disease index was 29.58.
- 2-amino-3-indolyl butyric acid or 3-methylpyrrolidine-2-carboxylic acid is used for the method for preventing and treating wheat powdery mildew, and it is in the range of concentration 100-10000nM (adding volume percent is 0.02% surfactant Tween 20), investigated after wheat was inoculated with powdery mildew bacterium 10 days, found that with the increase of treatment concentration, the disease index of wheat infection with powdery mildew decreased, the relative immune effect improved, and 2-amino-3-indole butyric acid was in When treated at a high concentration of 10000nM, the disease index was 29.70, and the relative immune effect was 68.42%. When 3-methylpyrrolidine-2-carboxylic acid was treated at a high concentration of 10000nM, the disease index was 30.26, and the relative immune effect was 68.57%.
- 2-Amino-3-indolebutyric acid is used in the field to prevent wheat powdery mildew.
- the disease index, relative immune effect and thousand-grain weight of wheat under 1000nM treatment concentration are 45.44, 40.55% and 27.39g respectively, which are all obviously good In the treatment of Atailing and the auxiliary control group.
- 2-amino-3-indolebutyric acid has a significant inhibitory effect on the occurrence and spread of wheat powdery mildew.
- a method for improving plant resistance to abiotic stress comprising applying the plant immune inducer of the present invention to plants; the abiotic stress is selected from any one of high temperature, low temperature, drought and/or salt stress one or more species.
- 2-amino-3-indolyl butyric acid or 3-methylpyrrolidine-2-carboxylic acid is used to improve the method of high temperature resistance of plants, and its concentration is between 1-1000nM 2-amino-3-ind Indyl butyric acid solution (adding 0.02% surfactant Tween 20 by volume) was used to induce ryegrass seedlings and Arabidopsis thaliana, and it was found that the plants in the treatment group were treated at 45°C for 12 hours and then recovered at room temperature for 7 days.
- the photosynthetic performance index PI ABS was higher than that of the control group, and the heat damage index was lower than that of the control group. This result indicated that exogenous spraying of 2-amino-3-indolylbutyric acid or 3-methylpyrrolidine-2-carboxylic acid solution effectively alleviated the level of damage caused by high temperature to seedlings.
- 2-amino-3-indolyl butyric acid or 3-methylpyrrolidine-2-carboxylic acid is used to improve the method of plant resistance to low temperature, with 2-amino-3-indolyl of 1-1000nM concentration Butyric acid or 3-methylpyrrolidine-2-carboxylic acid solution (surfactant Tween 20 with a volume percentage of 0.02%) was sprayed on the leaves of tea seedlings, and it was found that after 24 hours of low temperature stress at -4°C, The photosynthetic performance index PI ABS of tea seedlings treated with 1nM, 10nM, 100nM and 1000nM were significantly higher than that of the control group, and the chilling injury index was significantly lower than that of the control group, indicating that 2-amino-3-indolebutyric acid and 3-methyl Pyrrolidine-2-carboxylic acid can effectively alleviate the damage caused by low temperature to tea seedlings, and improve the resistance of tea to low temperature stress.
- 2-Amino-3-indolylbutyric acid or 3-methylpyrrolidine-2-carboxylic acid is used to improve the method of plant resistance to drought stress, with 100 and 1000nM of 2-amino-3-indolyl Butyric acid or 3-methylpyrrolidine-2-carboxylic acid solution (adding volume percentage is 0.02% surfactant Tween 20) was carried out foliar spraying treatment to the hydroponic wheat of two leaves one heart, found that in 25% poly Under ethylene glycol-6000 (PEG-6000) stress, the biomass of wheat treated with 100nM and 1000nM was significantly higher than that of the control group. This result shows that 2-amino-3-indolylbutyric acid or 3-methylpyrrole Alkane-2-carboxylic acids increase resistance to drought stress in wheat.
- PEG-6000 poly Under ethylene glycol-6000
- 2-amino-3-indole butyric acid or 3-methylpyrrolidine-2-carboxylic acid is used for improving the method for the resistance ability of plant to salt stress, uses the 2-amino-3-indole of 1-1000nM concentration Butyric acid or 3-methylpyrrolidine-2-carboxylic acid solution (adding 0.02% surfactant Tween 20 by volume percentage) was used to spray the hydroponic cotton in the two true leaf stages, and it was found that Under 100mM NaCl stress, the cotton mortality rate and salt damage index of the treatment groups sprayed with 2-amino-3-indolylbutyric acid or 3-methylpyrrolidine-2-carboxylic acid were lower than those of the control group, which indicated that The results indicated that 2-amino-3-indolylbutyric acid and 3-methylpyrrolidine-2-carboxylic acid increased the tolerance level of cotton to salt.
- Both 2-amino-3-indolylbutyric acid and 3-methylpyrrolidine-2-carboxylic acid are natural products with simple structures and convenient biological extraction methods. Since the present invention confirms that 2-amino-3-indolylbutyric acid and 3-methylpyrrolidine-2-carboxylic acid can induce plants to produce immune activity to some serious diseases existing in agricultural production, and can induce plant It has the potential to be developed as a natural plant immune inducer for the relatively prominent abiotic stress faced in the current agricultural production.
- the present invention has found that 2-amino-3-indolyl butyric acid and 3-methylpyrrolidine-2-carboxylic acid have higher broad-spectrum immune-inducing activity, and can induce tobacco to produce immunity at a low concentration of 0.1nM
- the reaction prevents the occurrence and spread of tomato spotted wilt; when the concentration is 100nM, it can inhibit the accumulation of Pseudomonas syringae PstDC3000 in Arabidopsis leaves and reduce the disease index of Arabidopsis; when the concentration is 1000nM, it can Induced wheat has a relative immune effect of 53.58% against powdery mildew.
- the invention can be used to control the main fungal diseases in farmland, such as wheat powdery mildew; viral diseases, such as tomato spotted wilt; bacterial diseases, such as diseases caused by Pseudomonas syringae and the like.
- farmland such as wheat powdery mildew
- viral diseases such as tomato spotted wilt
- bacterial diseases such as diseases caused by Pseudomonas syringae and the like.
- farmland such as wheat powdery mildew
- viral diseases such as tomato spotted wilt
- bacterial diseases such as diseases caused by Pseudomonas syringae and the like.
- the present invention has discovered that 2-amino-3-indolebutyric acid or 3-methylpyrrolidine-2-carboxylic acid can prevent the occurrence and spread of major diseases in various agricultural productions, and can reduce the growth rate of crops. Inhibition by various abiotic stresses during development. 2-Amino-3-indolebutyric acid and 3-methylpyrrolidine-2-carboxylic acid are easy to use, can play a preventive role in advance, and reduce the damage level of plants caused by various biotic and abiotic stresses, Reduce the use of pesticides and save production costs.
- 2-amino-3-indolylbutyric acid and 3-methylpyrrolidine-2-carboxylic acid are naturally occurring metabolites with simple structure and belong to ⁇ -amino acids, they have high environmental and biological It is safe and belongs to the category of green and efficient biological pesticides.
- the inventors isolated and purified 2-amino-3-indolylbutyric acid and 3-methylpyrrolidine-2-carboxylic acid from the saprophytic plant pathogenic fungus Alternaria, and identified their structures. Subsequently, the biological activity, scope of application and crop safety research were carried out on it, and it was found that the substance is a natural plant immune inducer and has the potential to be developed as a biological pesticide. At the same time, its research ideas provide a new direction for the development of biopesticides, disease control and abiotic stress mitigation.
- the substantive features of the present invention can be realized from the following embodiments and examples, but these should not be regarded as any limitation to the invention.
- Embodiment 1 biological synthesis, extraction method and structural identification of the compound of the present invention
- Glucose sodium nitrate medium glucose, 40.0g; NaNO 3 , 1.0g; NH 4 Cl, 0.25g; KH 2 PO 4 , 1.0g; KCl , 0.25g; NaCl, 0.25g; g; FeSO 4 ⁇ 7H 2 O, 0.01g; ZnSO 4 ⁇ 7H 2 O, 0.01g; yeast extract, 1g, add water to 1L, adjust pH to 5.5.
- the Alternaria culture method is as follows: PDA medium activates the preserved strains, and after 7 days, select colonies with consistent growth, take a 5mm diameter bacterial cake, and inoculate it into 500mL medium, and the inoculation amount is one bacterial cake per 100mL. Place the medium inoculated with the bacterial blocks into a constant temperature shaker, and the culture conditions are: 140 rpm, 25°C, and dark culture for 7 days.
- the mycelium was separated from the fermentation broth after 7 days of cultivation.
- a centrifuge is used for separation, and the centrifugation condition is 10000 rpm for 5 min.
- the supernatant was removed, and the mycelium was taken out from the bottom of the bottle and put into a mortar, and quickly ground into a uniform powder with liquid nitrogen. Put the powder into a centrifuge tube, add 5mL of water, shake well, and let it stand for extraction for 1h.
- the precipitate was removed by centrifugation, and the centrifugation condition was 10000 rpm for 5 min.
- the obtained supernatant is the crude extract of amino acids.
- the crude extract of the compound was separated and purified by high performance liquid chromatography, and the dual mobile phase method was used for elution.
- the elution conditions are A: 60% water (containing 0.1% formic acid), B: 40% acetonitrile, the ultraviolet detection wavelength is 256nm, and the flow rate is 2mL min -1
- impurities in the crude extract can be removed to obtain a single component 2-Amino-3-indolebutyric acid, peak time is 9.6min, this method can effectively separate the compound in Alternaria.
- the structure of the isolated light brown crystal was identified by NMR and mass spectrometry.
- the mass spectrum showed that the molecular ion peak of the compound was 219.1028[M+H] + , and its molecular formula was determined to be: C 12 H 14 N 2 O 2 .
- the compound was determined to be 2-amino-3-indolebutyric acid by combining the results of H NMR and C NMR.
- the amino acid crude extract was separated and purified by high performance liquid chromatography, and eluted by a dual mobile phase method.
- the elution conditions are A: 60% water (containing 0.1% formic acid), B: 40% acetonitrile, the ultraviolet detection wavelength is 256nm, and the flow rate is 2mL min -1
- impurities in the crude extract can be removed to obtain a single component 3-Methylpyrrolidine-2-carboxylic acid, the elution time is 5.9min, this method can effectively separate the compound in Alternaria.
- Tomato spotted wilt virus was obtained from Yunnan province, China.
- the initial virus source was stored in a -80°C refrigerator.
- the virus was inoculated on the leaves of Nicotiana benthamiana by friction inoculation to activate the virus, and the virus plasmid was extracted using Escherichia coli competent cells. Transformation, smeared on resistant plates and cultured, picked a single colony for PCR screening, selected positive colonies for sequencing and subsequent plasmid extraction, added the plasmids with normal sequencing to Agrobacterium competent cells, and carried out agronomy by electroshock method. For Bacillus transformation, the transformed Agrobacterium solution was spread on the corresponding resistance screening plate, and cultured at 28°C ( ⁇ 1) for 48h.
- Agrobacterium treatment buffer (10mM MgCl 2 , 10mM MES, 10 ⁇ M Acetosyringone) to make the OD 600 value of the suspension 0.5, and treated in the dark at 28°C for 3 hours before use.
- 2-Amino-3-indolebutyric acid was dissolved in distilled water and then gradiently diluted with distilled water to form 0nM, 0.1nM, 1nM and 10nM solutions. Sow the seeds of Nicotiana benthamiana in small pots, and cultivate them for 5 weeks at 22( ⁇ 1)°C and 12h/12h light. Select healthy tobacco plants (8-10 leaves are advisable) spray their stems and leaves with the above-mentioned concentration of 2-amino-3-indolebutyric acid solution, and repeat the treatment once every 24 hours, twice in total deal with.
- Grade 0 The whole plant is disease-free
- Grade 3 One-third of the leaves are mosaic but the leaves are not deformed, or the plants are dwarfed to more than three-quarters of the normal plant height;
- Grade 5 One-third to one-half of the leaves are mosaic, or a few leaves are deformed, or the main veins are black, or the plants are dwarfed to two-thirds to three-quarters of the normal plant height;
- Grade 7 One-half to two-thirds of the leaves are mosaic, or deformed or a few main lateral veins are necrotic, or the plants are dwarfed to one-half to two-thirds of the normal plant height;
- Grade 9 The leaves of the whole plant are mosaic, severely deformed or necrotic, or the diseased plant is dwarfed to more than half of the normal plant height.
- Table 1 result shows, when 2-amino-3-indole butyric acid concentration scope is 0.1-10nM, each treatment all can significantly reduce the infection of tomato spotted wilt virus to tobacco, the disease index of tobacco infection tomato spotted wilt virus If it is lower than 50, the relative immune effect is more than 45%, and within this concentration range, as the concentration increases, the disease index of tobacco infected with tomato spotted wilt virus is significantly reduced, and the relative immune effect is significantly improved compared with the control. The protein content of the virus in the virus decreased significantly. When the treatment concentration was 10nM, the immune effect of tobacco to tomato spotted wilt virus was the best, and the disease index, relative immune effect and virus content were 30.42, 67.36% and 0.17, respectively. The above results indicated that 2-amino-3-indolebutyric acid can improve the immunity of tobacco to tomato spotted wilt virus and effectively inhibit the spread of tomato spotted wilt virus in tobacco.
- Table 2 result shows, when 3-methylpyrrolidine-2-carboxylic acid concentration scope is 0.1-10nM, each treatment all can significantly reduce the infection of tomato spotted wilt virus to tobacco, the disease index of tobacco infection tomato spotted wilt virus If it is lower than 50, the relative immune effect is more than 50%, and within this concentration range, with the increase of the concentration, the disease index of tobacco infected with tomato spotted wilt virus is significantly reduced, and the relative immune effect is significantly improved compared with the control. The protein content of the virus in the virus decreased significantly. When the treatment concentration was 10nM, the immune effect of tobacco to tomato spotted wilt virus was the best, and the disease index, relative immune effect and virus content were 21.44, 75.73% and 0.16, respectively. The above results show that 3-methylpyrrolidine-2-carboxylic acid can improve the immunity of tobacco to tomato spotted wilt virus and effectively inhibit the spread of tomato spotted wilt virus in tobacco.
- Example 3 (2-amino-3-indole butyric acid and 3-methylpyrrolidine-2-carboxylic acid induce Arabidopsis resistance to Pseudomonas syringae infection)
- Grade 1 The lesion area accounts for 0%-10% of the entire leaf area
- Level 2 The lesion area accounts for 10%-25% of the entire leaf area
- Grade 3 The lesion area accounts for 25%-50% of the entire leaf area
- Grade 4 Lesions account for 50%-75% of the entire leaf area
- Grade 5 The lesion area accounts for 75%-100% of the entire leaf area.
- 2-Amino-3-indolebutyric acid was dissolved in distilled water and then diluted with distilled water to form 100nM, 1000nM and 10000nM solutions, and a blank control was set up.
- Wheat (NAU0686) seeds were germinated, planted in sterilized soil cultivation pots, and placed in a greenhouse at 23( ⁇ 1)°C for 12h under light for cultivation. When the seedlings grow to the stage of 1 leaf and 1 heart, spray the stems and leaves of the wheat seedlings with the 2-amino-3-indolebutyric acid solution of the above-mentioned concentration, and repeat the treatment once at intervals of 24 hours, and carry out two treatments altogether.
- Grade 1 Lesion area accounts for less than 5% of the entire leaf area
- Grade 3 The lesion area accounts for 6%-15% of the entire leaf area
- Grade 5 Lesions account for 16%-25% of the entire leaf area
- Grade 7 Lesions account for 26%-50% of the entire leaf area
- Grade 9 The lesion area accounts for more than 50% of the entire leaf area.
- the results in Table 6 show that with the increase of the concentration of 3-methylpyrrolidine-2-carboxylic acid, the disease index of susceptible varieties of wheat decreases, and the relative immune effect increases. There were significant differences in the condition index of each treatment. When the concentrations were 10nM, 100nM, 1000nM and 10000nM, the disease index were 72.16, 54.17, 40.41, 30.26, and the relative immune effects were 25.04%, 43.73%, 58.02 and 68.57%. When the concentration of 3-methylpyrrolidine-2-carboxylic acid is greater than 1000nM, the disease index of susceptible varieties of wheat infected with powdery mildew is lower than 50, while the relative immune effect exceeds 50%, and the effect is the best at the concentration of 10000nM.
- Embodiment 5 field test of 2-amino-3-indolyl butyric acid and 3-methylpyrrolidine-2-carboxylic acid inducing wheat resistance to powdery mildew infection
- Grade 1 Lesion area accounts for less than 5% of the entire leaf area
- Grade 3 The lesion area accounts for 6%-15% of the entire leaf area
- Grade 5 Lesions account for 16%-25% of the entire leaf area
- Grade 7 Lesions account for 26%-50% of the entire leaf area
- Grade 9 The lesion area accounts for more than 50% of the entire leaf area.
- 2-Amino-3-indolebutyric acid was dissolved in distilled water and then gradually diluted with distilled water into 1nM, 10nM, 100nM and 1000nM solutions.
- a blank control was also set up, and 0.02% Tween 20 was added as a surfactant.
- Four groups of repetitions were set for each concentration, and a normal temperature blank control was set at the same time.
- the ryegrass seeds were weighed according to 0.8g per pot, and sowed in pots with a diameter of 8.5 cm, at a temperature of 25°C, a humidity of 60%-70%, and a light intensity of 200 ⁇ mol m -2 s -1 (12h light/12h dark) grown in a greenhouse. The ryegrass was treated after 7 days of growth.
- the treatment method was to spray 2-amino-3-indolebutyric acid solution on the leaves, and spray twice in 24 hours. 24 hours after the second spraying, it was transferred to a light incubator with a temperature of 45°C for 12 hours of high temperature stress treatment, and the plants were taken out and transferred to a greenhouse at 25°C for 7 days of recovery.
- the heat damage classification standard is shown in Table 8, and the heat damage index calculation formula is as follows. The heat damage results are shown in Table 6.
- Seeds of Arabidopsis thaliana were sown in pots with a diameter of 8.5 cm according to about 50 seeds per pot . planted in.
- the treatment was started when Arabidopsis grew normally for 21 days.
- the experiment was set at 0, 1, 10, 100 and 1000 nM, and 0.02% Tween 20 was added as a surfactant at the same time, and four sets of repetitions were set.
- spray treatment method is identical with the treatment method of ryegrass among the embodiment 5.
- After 24 hours of the second treatment they were transferred to a light incubator at 45°C for 12 hours of high temperature stress treatment, and the chlorophyll fluorescence of the Arabidopsis leaf discs was measured with Plant Efficiency Handy-PEA.
- the plants were taken out and transferred to a greenhouse at 25°C for 7 days, and the damage of the plants was observed and counted, and the heat damage classification was calculated. The results are shown in Table 11.
- the tea tree tested was Baiye No. 1 cutting seedling. Select tea seedlings with consistent growth and move them into plastic pots with a diameter of 18 cm, and place them in a greenhouse with a temperature of 25° C. and a humidity of 60%-70% to make them suitable for growth for about a week for experimentation. Experimental settings were 0, 1, 10, 100 and 1000 nM, while 0.02% Tween 20 was added as a surfactant. Wherein the spraying treatment method is the same as that of the ryegrass in Example 1, the time of low temperature stress is 24 hours, and the temperature is set at -4°C.
- the performance characteristics of leaves after drought damage and salt damage are similar, and the drought damage rate and drought damage index are introduced by using the evaluation index of salt damage.
- the formula of drought damage index is as follows, and the drought damage classification standard is shown in Table 16.
- Table 17 show that with the increase of treatment concentration, the resistance ability of wheat to drought stress is gradually enhanced.
- the fresh weight, dry weight and root length of wheat under the two treatment concentrations were higher than those of the control group, which made the drought damage index of wheat significantly lower.
- the 2-amino-3-indole butyric acid treatment of 1000nM concentration significantly increased the root length of wheat seedlings by 10.86%, and the fresh weight of aboveground and underground parts increased by 44.93% and 54.98%, respectively.
- the index is down 44%. This shows that 2-amino-3-indolebutyric acid can improve the ability of wheat to resist drought stress.
- the experimental material is "Sikang No. 1" cotton, which is hydroponically grown in a 500mL plastic cup, and the 1/2 Hoagland nutrient solution is replaced every two days.
- the 2-amino-3-indole butyric acid solution is sprayed on the leaves.
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Abstract
本发明公开了2-氨基-3-吲哚基丁酸或3-甲基吡咯烷-2-羧酸作为植物免疫诱抗剂的应用。将2-氨基-3-吲哚基丁酸或3-甲基吡咯烷-2-羧酸作为天然活性物质开发为植物免疫诱抗剂可用于提高植物对生物胁迫和非生物胁迫的抵抗能力，有效阻止真菌、细菌和病毒对植物的侵染，进而降低致病水平；同时，能显著提高植物对高温、低温、干旱和盐胁迫的耐受能力。2-氨基-3-吲哚基丁酸或3-甲基吡咯烷-2-羧酸具有安全、环保、高效的特点。
Description
本发明属于农业生物农药领域，涉及2-氨基-3-吲哚基丁酸或3-甲基吡咯烷-2-羧酸作为植物免疫诱抗剂的应用。
在农业生产中，由于高温、低温、干旱和盐等非生物胁迫所造成的损失非常巨大。近年来，全球极端天气频繁出现，农业植物所面临的胁迫也日益严重。高温和低温严重影响植物的生长发育，进而影响植物的产量和品质。干旱是影响植物生存、生长和分布最重要的逆境胁迫因素之一，当前全球干旱、半干旱地区面积约占总耕地面积的40％以上，近年来由于全球性气候恶化使得干旱发生周期越来越短，干旱程度越来越重，对粮食生产构成的威胁也越来越大。其次，土壤盐碱化是阻碍全球作物生长和生产力的主要非生物限制因素，对生物圈及生态结构造成的有害影响很大，中国盐碱地面积位居世界第三，占世界盐碱地面积的10％左右。因此，针对当前实际生产中不同作物面临的主要非生物胁迫状况，开发旨在减轻植物危害水平的产品和技术对于保障农业安全生产显得尤为迫切。
除了非生物胁迫外，植物在生长发育过程当中还不断受到各种病虫害的威胁。农业生产中众多病害一旦发生，往往会造成作物大面积严重减产甚至绝收。因此，重要农业病虫害的预防就显得尤为重要。目前，植物病害的防治主要采取施用农药直接杀死病原菌的策略，但长期、大量使用杀菌剂，加之施药方法不够科学，不仅带来了农产品残留超标、作物药害、病原菌抗药性、环境污染、生物多样性降低等一系列问题，也使传统植保的“杀灭”策略面临失效的风险，严重威胁农业的可持续发展和粮食生产安全。所以，开发环保、高效、经济的植物免疫剂，在作物发病前或早期阶段通过增强植物自身的抵抗能力来降低或者抑制作物发病水平，从而实现少用或不用化学杀菌剂的目标，对于实现农业绿色生产具有十分重要的意义。
植物免疫诱抗剂是一类新概念农药，其通过激活植物的免疫系统并调节植物的新陈代谢，从而增强植物抗病和抗逆能力。植物免疫诱抗剂本身没有直接的杀菌活性，主要通过促使植物利用自身的天然免疫系统来防治病害，而不依赖外源农药直接杀死病原体，因此病菌不易对其产生抗药性，符合有效保护农业生物多样性的条件下实现绿色防控的思路。此外，在自然界，植物的生长通常并不只是受到单一胁迫，而是多种胁迫并存，如干旱和高温胁迫常常同时发生， 对植物造成更严重的危害。植物自身虽然存在免疫系统，但其抵抗逆境胁迫的能力是有限的，通过植物免疫诱抗剂的使用能够增加植物的抗逆水平。因此，植物免疫诱抗剂作为新兴农药的一类，为农业可持续发展和病害的有效绿色防治和提供了新的发展思路，是绿色植保未来发展的主要方向。
2-氨基-3-吲哚基丁酸，分子式为C
12H
14N
2O
2，分子量218克/摩尔，为浅棕色晶体。该化合物的化学合成方法很多，但是过程都比较繁琐(Han et al.,2001；Liu et al.,2012)。有研究表明2-氨基-3-吲哚基丁酸是一些天然产物如Maremycin和具有抗癌活性的链霉黑素(Streptonigrin)生物合成途径的中间产物(Zou et al.,2013；Kong et al.,2016)。小叶链霉菌(Streptomyces flocculus)合成链霉黑素的第一步可能就是合成2-氨基-3-吲哚基丁酸(Gould&Chaug,1977)。Hartley等人利用S.flocculus酶进行体外酶催化试验发现S-腺苷甲硫氨酸(S-adenosylmethionine)的甲基可以转移到色氨酸上而合成2-氨基-3-吲哚基丁酸(Hartley&Speedie,1984)。此外，科学家对一种超嗜热古菌—强烈火球菌(Pyrococcus furiosus)的色氨酸合酶亚基进行改造后发现，该酶可以利用苏氨酸与吲哚结合直接反应合成2-氨基-3-吲哚基丁酸(Herger et al.,2016；Boville et al.,2018)。截至目前，由于2-氨基-3-吲哚基丁酸是合成一些抗病毒和抗肿瘤天然产物的中间物，因此对于它的研究主要集中在化学合成方法，体外酶催化和生物合成途径的方面。而对于生物合成的研究又仅限于原核生物小叶链霉菌。在广泛的真核生物中是否存在该化合物、以及其生物活性等方面到目前为止还是空白，未有过报道。
3-甲基吡咯烷-2-羧酸，分子式为C
6H
11NO
2，分子量为129克/摩尔，为无色晶体。最早关于该化合物的报道在1964年，化学合成的方式首次得到了3-甲基吡咯烷-2-羧酸。随后的活性研究发现，该化合物可以抑制抗生素链霉菌(Streptomyces antibioticus)中放线菌素(actinomycin)的合成(Yoshida et al.,1964；Mauger et al.,1966；Katz et al.,1968；Yoshida et al.,1968)。对来自青霉菌(Penicillium sp.)环状七肽Paraherquamide A的研究发现，其结构中含有β-甲基-β-羟基脯氨酸组分(Stocking et al.,2001)。2003年，Tan等人从海洋真菌柱顶孢霉(Scytalidium.sp.)的发酵液中分离得到两种新环状七肽Scytalidamides A和B，发现对Scytalidamides B进行水解可得到3-甲基吡咯烷-2-羧酸(Tan et al.,2003)。Fredenhagen等人对白地霉(Geotrichum candidum)合成的多种多肽neoefrapeptins A-N进行了水解，发现其中4种多肽含有3-甲基吡咯烷-2-羧酸的结构(Fredenhagen et al.,2006)。截至目前，对于3-甲基吡咯烷-2-羧酸的研究多集中在多肽水解和生物合成途径方面，还没有关于其游离存在的报道的，并且其生物活性等方面的研究到目前为止也还是空白。
发明内容
本发明的目的是针对现有技术的上述不足，提供2-氨基-3-吲哚基丁酸或3-甲基吡咯烷-2-羧酸作为植物免疫诱抗剂的应用。
本发明的另一目的是提供一种免疫诱抗剂。
本发明的又一目的是提供一种提高植物对生物和/或非生物胁迫抗性的方法。
本发明的目的可通过以下技术方案实现：
2-氨基-3-吲哚基丁酸和3-甲基吡咯烷-2-羧酸是从链格孢菌中分离得到的天然产物。
2-氨基-3-吲哚基丁酸结构式如下：
3-甲基吡咯烷-2-羧酸结构式如下：
2-氨基-3-吲哚基丁酸和/或3-甲基吡咯烷-2-羧酸在制备植物免疫诱抗剂中的应用。
2-氨基-3-吲哚基丁酸和/或3-甲基吡咯烷-2-羧酸在提高植物对非生物胁迫和/或生物胁迫中的应用。
2-氨基-3-吲哚基丁酸和/或3-甲基吡咯烷-2-羧酸在提高植物对高温、低温、干旱和/或盐胁迫中的应用。
2-氨基-3-吲哚基丁酸和/或3-甲基吡咯烷-2-羧酸在提高植物对真菌、细菌、病毒胁迫中的应用。
2-氨基-3-吲哚基丁酸和/或3-甲基吡咯烷-2-羧酸在防治植物真菌性病害、细菌性病害和/或病毒性病害中的应用。
所述的真菌性病害优选小麦白粉病；所述的细菌性病害优选丁香假单胞菌病害；所述的病毒性病害优选番茄斑萎病。
所述的植物，选自粮食作物、经济作物、蔬菜。所述的粮食作物优选小麦，所述的经济作物优选茶叶、棉花，所述的蔬菜优选番茄。
一种植物免疫诱抗剂，包含2-氨基-3-吲哚基丁酸或3-甲基吡咯烷-2-羧酸中的任意一种或 两种。
作为本发明的一种优选，所述的植物免疫诱抗剂，包含组分A：2-氨基-3-吲哚基丁酸或3-甲基吡咯烷-2-羧酸中的任意一种或两种，组分B：表面活性剂。作为本发明的进一步优选，所述的表面活性剂为吐温20，吐温20在植物免疫诱抗剂中的浓度优选0.02％(v/v)。
作为本发明的进一步优选，所述的植物免疫诱抗剂中2-氨基-3-吲哚基丁酸或3-甲基吡咯烷-2-羧酸的浓度为0.1-10000nM浓度。
2-氨基-3-吲哚基丁酸和3-甲基吡咯烷-2-羧酸的已有相关研究并未涉及天然微生物代谢产物和生物农药领域的报道。植物免疫诱抗剂属于新型农药，是未来植保领域绿色防控的主要发展方向。我国免疫诱抗剂的发展处于刚起步阶段，获得正式登记的产品屈指可数。因此，发展天然植物免疫诱抗剂，并推动其产业化，对于保障农业生产安全、提高农产品竞争力具有重要的意义。2-氨基-3-吲哚基丁酸和3-甲基吡咯烷-2-羧酸在相关诱导免疫抗逆性实验中均表现良好，能够提高植物对生物胁迫和非生物胁迫的抵抗能力。
由腐生型真菌链格孢菌中分离的天然代谢产物2-氨基-3-吲哚基丁酸或3-甲基吡咯烷-2-羧酸用于防治病害的方法，其详细内容和实施方案如下：在0.1-10000nM浓度(加入体积百分比为0.02％的表面活性剂吐温20)范围，可有效抑制病毒、细菌和真菌在植物上的侵染和扩散，抑制病害的发生与蔓延，提高植物对高温、低温、干旱和盐胁迫的抵抗能力。
一种提高植物对生物胁迫抗性的方法，包括提前向植物施加本发明所述的植物免疫诱抗剂；所述的生物胁迫选自真菌、细菌、病毒胁迫中的任意一种或多种。
2-氨基-3-吲哚基丁酸或3-甲基吡咯烷-2-羧酸用于防治番茄斑萎病的方法，在0.1-10nM浓度下(加入体积百分比为0.02％的表面活性剂吐温20)，可在烟草接种番茄斑萎病毒(TSWV)3天后显著抑制病毒的扩散。15天后调查烟草病害情况，发现2-氨基-3-吲哚基丁酸和3-甲基吡咯烷-2-羧酸处理的烟草植株的病情指数均显著降低。在低浓度10nM时，2-氨基-3-吲哚基丁酸可有效抑制TSWV在烟草叶片上的表达，其病情指数、相对免疫效果和病毒含量分别为30.42、67.36％和0.17。在低浓度10nM时，3-甲基吡咯烷-2-羧酸也可有效抑制TSWV在烟草叶片上的表达，其病情指数、相对免疫效果和病毒含量分别为21.44、75.73％和0.16。
2-氨基-3-吲哚基丁酸或3-甲基吡咯烷-2-羧酸用于防治细菌病害的方法，其在浓度为100-10000nM浓度范围(加入体积百分比为0.02％的表面活性剂吐温20)，随着处理浓度的提高，拟南芥叶片中细菌PstDC3000积累量逐渐下降，当2-氨基-3-吲哚基丁酸处理浓度为10000nM时，每毫克叶片中细菌个数为4.79×10
5，与空白对照相比细菌个数减少85.039％，病情指 数为29.58。当3-甲基吡咯烷-2-羧酸处理浓度为10000nM时，每毫克叶片中细菌个数为1.64×10
5，与空白对照相比细菌个数减少94.92％，病情指数为23.26。这一结果说明，2-氨基-3-吲哚基丁酸和3-甲基吡咯烷-2-羧酸均能够激发拟南芥自身免疫，抑制细菌在植物体内的繁殖，降低细菌积累量，延缓并抑制病害的发展。
2-氨基-3-吲哚基丁酸或3-甲基吡咯烷-2-羧酸用于防治小麦白粉病的方法，其在浓度100-10000nM范围(加入体积百分比为0.02％的表面活性剂吐温20)，在小麦接种白粉病菌10天后进行调查，发现随着处理浓度的升高，小麦感染白粉病的病情指数下降，相对免疫效果提高，2-氨基-3-吲哚基丁酸在高浓度10000nM处理时，病情指数为29.70，相对免疫效果为68.42％。3-甲基吡咯烷-2-羧酸在高浓度10000nM处理时，病情指数为30.26，相对免疫效果为68.57％。
2-氨基-3-吲哚基丁酸用于田间预防小麦白粉病的方法，其在1000nM处理浓度下小麦的病情指数、相对免疫效果和千粒重分别为45.44、40.55％和27.39g，均明显好于阿泰灵处理和助剂对照组。综上2-氨基-3-吲哚基丁酸对小麦白粉病的发生与扩散起到显著的抑制作用。
一种提高植物对非生物胁迫抗性的方法,包括向植物施加本发明所述的植物免疫诱抗剂；所述的非生物胁迫选自高温、低温、干旱和/或盐胁迫中的任意一种或多种。
2-氨基-3-吲哚基丁酸或3-甲基吡咯烷-2-羧酸用于提高植物对高温抵抗能力的方法，其浓度在1-1000nM之间的2-氨基-3-吲哚基丁酸溶液(加入体积百分比为0.02％的表面活性剂吐温20)对黑麦草幼苗和拟南芥进行处理诱导，发现处理组的植株经45℃高温处理12h后在室温恢复7d后，光合性能指数PI
ABS均高于对照组，热害指数均低于对照组。这一结果说明通过外源喷施2-氨基-3-吲哚基丁酸或3-甲基吡咯烷-2-羧酸溶液有效缓解了高温对幼苗造成的伤害水平。
2-氨基-3-吲哚基丁酸或3-甲基吡咯烷-2-羧酸用于提高植物对低温的抵抗能力的方法，用1-1000nM浓度的2-氨基-3-吲哚基丁酸或3-甲基吡咯烷-2-羧酸溶液(加入体积百分比为0.02％的表面活性剂吐温20)对茶叶幼苗进行叶面喷施处理，发现在-4℃低温胁迫24h后，经过1nM、10nM、100nM和1000nM处理的茶叶幼苗的光合性能指数PI
ABS均显著高于对照组，冷害指数明显低于对照组，说明2-氨基-3-吲哚基丁酸和3-甲基吡咯烷-2-羧酸均能够有效缓解了低温对茶叶幼苗造成的伤害，提高了茶叶对低温胁迫的抵抗能力。
2-氨基-3-吲哚基丁酸或3-甲基吡咯烷-2-羧酸用于提高植物对干旱胁迫的抵抗能力的方法，用100和1000nM的2-氨基-3-吲哚基丁酸或3-甲基吡咯烷-2-羧酸溶液(加入体积百分比 为0.02％的表面活性剂吐温20)对两叶一心的水培小麦进行叶面喷施处理，发现在25％聚乙二醇-6000(PEG-6000)胁迫下，经过100nM和1000nM处理的小麦各生物量显著高于对照组，这一结果说明2-氨基-3-吲哚基丁酸或3-甲基吡咯烷-2-羧酸提高了小麦对干旱胁迫的抗性。
2-氨基-3-吲哚基丁酸或3-甲基吡咯烷-2-羧酸用于提高植物对盐胁迫的抵抗能力的方法，用1-1000nM浓度的2-氨基-3-吲哚基丁酸或3-甲基吡咯烷-2-羧酸溶液(加入体积百分比为0.02％的表面活性剂吐温20)对两片真叶期的水培棉花进行叶面喷施处理，发现在100mM NaCl胁迫下，各喷施了2-氨基-3-吲哚基丁酸或3-甲基吡咯烷-2-羧酸的处理组，棉花死亡率及盐害指数均低于对照组，这一结果说明2-氨基-3-吲哚基丁酸和3-甲基吡咯烷-2-羧酸提高了棉花对盐的耐性水平。
本发明的主要优点和积极效果如下：
2-氨基-3-吲哚基丁酸和3-甲基吡咯烷-2-羧酸均为天然产物，结构简单，生物提取方式简便。由于本发明确认了2-氨基-3-吲哚基丁酸和3-甲基吡咯烷-2-羧酸能够诱导植物对农业生产中存在的部分危害严重的病害产生免疫活性，以及能够诱导植物对于目前农业生产中所面临的较为突出的非生物胁迫产生抗逆性，具有开发成天然植物免疫诱抗剂的潜力。
本发明发现了2-氨基-3-吲哚基丁酸和3-甲基吡咯烷-2-羧酸具有较高的广谱免疫诱导活性，在0.1nM的低浓度下就能诱导烟草产生免疫反应阻止番茄斑萎病的发生和蔓延；在浓度为100nM时，能够抑制丁香假单胞菌PstDC3000在拟南芥叶片中的积累，降低拟南芥的病情指数；在浓度为1000nM时，就能够诱导小麦对白粉病产生53.58％的相对免疫效果。在应对非生物胁迫方面，在浓度为1-1000nM时，能够诱导黑麦草和拟南芥提高对高温的抵抗能力，以及小麦对干旱和茶叶对低温的抵抗能力；在浓度为100nM时，能够显著提高棉花对盐渍的抵抗能力。2-氨基-3-吲哚基丁酸和3-甲基吡咯烷-2-羧酸用量低，对环境无污染，是高效、环保的生物农药，这指明了该物质在农业生产上的巨大利用价值和广阔应用前景。
本发明可以用于控制发生于农田主要的真菌性病害，如小麦白粉病；病毒性病害，如番茄斑萎病；细菌性病害，如丁香假单胞菌引起的病害等。这表明该化合物能够诱导植物对多类型病害产生免疫反应。同时，其能够诱导植物抵抗自然界多种非生物胁迫，如高温、低温、干旱和盐胁迫，为缓解各类胁迫对植株产生的伤害提供技术参考。
本发明发现了2-氨基-3-吲哚基丁酸或3-甲基吡咯烷-2-羧酸做茎叶处理可以阻止多种农业生产中主要病害的发生和蔓延，能够减轻作物在生长发育过程中所受的多种非生物胁迫的抑 制。2-氨基-3-吲哚基丁酸和3-甲基吡咯烷-2-羧酸使用方便，可以起到提前预防的作用，降低多种生物和非生物胁迫所引起的植物的伤害水平，减少农药的使用量，节约生产成本。此外，由于2-氨基-3-吲哚基丁酸和3-甲基吡咯烷-2-羧酸是天然存在的一种结构简单的代谢产物，属于α-氨基酸，具有很高的环境和生物安全性，属于绿色、高效的生物农药范畴。
发明人从腐生型植物病源真菌—链格孢菌中分离提纯得到了2-氨基-3-吲哚基丁酸和3-甲基吡咯烷-2-羧酸，对其结构进行了鉴定。随后对其进行了生物活性、适用范围及作物安全性研究，发现该物质为天然植物免疫诱抗剂，具备开发为生物农药的潜力。同时，其研究思路为生物农药的发展以及病害的防治和非生物胁迫的缓解提供了新的发展方向。本发明的实质性特点可以从下述的实施方案和实施例中得以体现，但这些不应视为是对发明的任何限制。
实施例1(本发明化合物的生物合成、提取方法及结构鉴定)
(1)链格孢菌的培养
葡萄糖硝酸钠培养基：葡萄糖，40.0g；NaNO
3，1.0g；NH
4Cl，0.25g；KH
2PO
4，1.0g；KCl，0.25g；NaCl，0.25g；MgSO
4·7H
2O，0.5g；FeSO
4·7H
2O，0.01g；ZnSO
4·7H
2O，0.01g；酵母提取物，1g，加水定容至1L，调pH到5.5。
链格孢菌培养方法为：PDA培养基活化保存的菌株，7d后，选取生长一致的菌落，打取直径为5mm的菌饼，接种到500mL培养基内，接种量为每100mL一个菌饼。将接种菌块的培养基放置到恒温摇床内，培养条件为：140rpm，25℃，黑暗培养7d。
(2)化合物的提取
从培养7d后的发酵液中将菌丝分离出来。采用离心机进行分离，离心条件为10000rpm，5min。除去上清液，将菌丝从瓶底取出放入研钵中，用液氮快速将其研磨成均匀粉末。粉末装入离心管中，加5mL水后摇匀，静置萃取1h。采用离心的方式去除沉淀，离心条件为10000rpm，5min。所得到的上清液即为氨基酸的粗提物。
(3)HPLC法分离提纯2-氨基-3-吲哚基丁酸：
利用高效液相色谱对化合物粗提物进行分离提纯，采用双流动相法进行洗脱。洗脱条件为A:60％水(含0.1％甲酸)，B：40％乙腈，紫外检测波长为256nm，流速2mL min
-1经过分离，可以除去粗提物中的杂质，得到单一组分的2-氨基-3-吲哚基丁酸，出峰时间为9.6min，此方法可以有效的分离链格孢菌中的该化合物。
分离得到的浅棕色晶体利用核磁共振和质谱的方式对其结构进行鉴定。
核磁结果如下：
1H NMR(500MHz,Deuterium Oxide)δ7.67-7.56(m,1H,Ph),7.23-7.16(d,J＝10Hz,1H,NHCH),7.19-7.12(m,1H,Ph),7.11-7.00(m,1H,Ph),4.18-4.01(dd,J
1＝5Hz,J
2＝5Hz,1H,CH-NH
2),3.89-3.69(m,1H,CHCH
3),1.42-1.28(d,J
1＝10Hz,J
2＝5Hz,3H,CHCH
3).
13C NMR(125MHz,Deuterium Oxide)δ173.24(CHCOOH),136.61(Ph),129.53(Ph),123.94(NHCH),122.38(Ph),119.54(Ph),118.47(Ph),113.16(Ph),112.08(CHNH),62.55(CHNH
2),31.31(CHCH
3),13.11(CHCH
3)。
质谱显示该化合物的分子离子峰为：219.1028[M+H]
+，确定其分子式为：C
12H
14N
2O
2。结合核磁氢谱和碳谱的结果确定该化合物为2-氨基-3-吲哚基丁酸。
(4)HPLC法分离提纯：
利用高效液相色谱对氨基酸粗提物进行分离提纯，采用双流动相法进行洗脱。洗脱条件为A:60％水(含0.1％甲酸)，B：40％乙腈，紫外检测波长为256nm，流速2mL min
-1经过分离，可以除去粗提物中的杂质，得到单一组分的3-甲基吡咯烷-2-羧酸，出峰时间为5.9min，此方法可以有效的分离链格孢菌中的该化合物。
分离得到的3-甲基吡咯烷-2-羧酸利用核磁和质谱的方式对其结构进行鉴定,
核磁结果如下：
1H NMR(500MHz,Deuterium Oxide)δ12.39(br,1H,OH),3.52(d,J＝10Hz,1H,CH-NH),2.75-2.49(m,2H,CH
2NH),2.05-1.98(m,1H,CHCH
3),1.66-1.41(m,2H,CH
2CH),1.11(d,J＝10Hz,3H,CHCH
3)。
13C NMR(125MHz,Deuterium Oxide)δ174.56(CHCOOH),73.73(CHCOOH),43.51(CH2NH),38.26(CHCH
3),35.93(CH2CH),14.87(CHCH
3)。
质谱显示该化合物的分子离子峰为：130.0802[M+H]
+，确定其分子式为：C
6H
11NO
2。结合核磁氢谱和碳谱的结果确定该化合物为3-甲基吡咯烷-2-羧酸。
实施例2(2-氨基-3-吲哚基丁酸和3-甲基吡咯烷-2-羧酸诱导烟草抗番茄斑萎病毒侵染)
番茄斑萎病毒取自中国云南省，初始毒源放于-80℃冰箱进行保存，采取摩擦接种的方法将其接种在本氏烟叶片上对病毒进行活化，提取病毒质粒利用大肠杆菌感受态细胞进行转化，涂布到抗性平板上培养，挑取单菌落进行PCR筛选，选取阳性菌落用于测序和后续的质粒提取，将测序正常的质粒加入到农杆菌感受态细胞中，采用电击法进行农杆菌转化，取转化后的农杆菌菌液涂布于相应抗性的筛选平板上，28℃(±1)培养48h。挑取转化平板上的农杆菌 单菌落，置于5mL含有相应抗性的LB培养基中，28℃，180rpm过夜培养。6000rpm离心2min收集菌体，用农杆菌处理缓冲液(10mM MgCl
2、10mM MES、10μM Acetosyringone)悬浮菌体，使悬浮液的OD
600值为0.5，28℃避光处理3h后待用。
取2-氨基-3-吲哚基丁酸用蒸馏水溶解后再用蒸馏水梯度稀释成0nM、0.1nM、1nM和10nM的溶液。将本氏烟草种子播于小盆中，22(±1)℃，12h/12h光照，培养5周。挑选健康的烟草植株(8-10片叶为宜)用上述浓度的2-氨基-3-吲哚基丁酸溶液对其行茎叶喷雾处理，间隔24小时重复进行处理一次，共进行两次处理。24小时后，用1mL注射器抽取浓度均一的农杆菌菌液，将注射器的注射口直接压在烟草叶片背面的小孔上，缓慢推进菌液，使整个叶片浸润。将浸润后的烟草移到22(±1)℃，12h/12h光照的条件下培养。3d后进行显微镜观察记录；同时进行取样，采用Western-blot结合Image J软件对蛋白条带的灰度进行分析，测定叶片内病毒相对蛋白含量。15d后观察烟草叶片发病情况，参照GB/T23222—2008《烟草病虫害分级及调查方法》，记录病情指数，公式如下：
番茄斑萎病毒病分级标准(以株为单位分级调查)：
0级：全株无病；
1级：心叶脉明或轻微花叶，病株无明显矮化；
3级：三分之一叶片花叶但叶片不变形，或植株矮化成正常株高的四分之三以上；
5级：三分之一至二分之一叶片花叶，或少数叶片变形，或主脉变黑，或植株矮化成正常株高的三分之二至四分之三；
7级：二分之一至三分之二叶片花叶，或变形或少数主侧脉坏死，或植株矮化成正常株高的二分之一至三分之二；
9级：全株叶片花叶，严重变形或坏死，或病株矮化成正常株高的二分之一以上。
表1不同浓度2-氨基-3-吲哚基丁酸对番茄斑萎病毒侵染烟草的影响
表1结果表明，在2-氨基-3-吲哚基丁酸浓度范围为0.1-10nM时，各处理均能显著降低番茄斑萎病毒对烟草的侵染，烟草感染番茄斑萎病毒的病情指数低于50，相对免疫效果为45％以上，且在本浓度范围内随着浓度的升高，烟草感染番茄斑萎病毒的病情指数显著降低，与对照相比相对免疫效果均显著提高，烟草叶片内病毒蛋白含量均显著下降。在处理浓度为10nM时，烟草对番茄斑萎病毒的免疫效果最佳，病情指数、相对免疫效果和病毒含量分别为30.42、67.36％和0.17。以上结果说明，2-氨基-3-吲哚基丁酸能够提高烟草对番茄斑萎病毒的免疫能力，有效抑制番茄斑萎病毒在烟草中的扩散。
按照相同方法考察3-甲基吡咯烷-2-羧酸诱导烟草抗番茄斑萎病毒侵染效果，结果见表2：
表:2不同浓度3-甲基吡咯烷-2-羧酸对番茄斑萎病毒侵染烟草的影响
表2结果表明，在3-甲基吡咯烷-2-羧酸浓度范围为0.1-10nM时，各处理均能显著降低番茄斑萎病毒对烟草的侵染，烟草感染番茄斑萎病毒的病情指数低于50，相对免疫效果为50％以上，且在本浓度范围内随着浓度的升高，烟草感染番茄斑萎病毒的病情指数显著降低，与对照相比相对免疫效果均显著提高，烟草叶片内病毒蛋白含量均显著下降。在处理浓度为10nM时，烟草对番茄斑萎病毒的免疫效果最佳，病情指数、相对免疫效果和病毒含量分别为21.44、75.73％和0.16。以上结果说明，3-甲基吡咯烷-2-羧酸能够提高烟草对番茄斑萎病毒的免疫能力，有效抑制番茄斑萎病毒在烟草中的扩散。
实施例3(2-氨基-3-吲哚基丁酸和3-甲基吡咯烷-2-羧酸诱导拟南芥抗丁香假单胞菌侵染)
取2-氨基-3-吲哚基丁酸用无菌水溶解后再用无菌水梯度稀释成100nM、1000nM和10000nM的溶液，另设空白对照，同时加入0.02％吐温20作为表面活性剂。将丁香假单胞菌PstDC3000涂布于LB平板上，28℃培养48h；挑取单克隆菌落接种到含有2mL培养基的50mL离心管中，28℃，250rpm在摇床上培养，每1-2h监测菌液OD
600值变化，在OD
600值达 到0.8之前停止培养细菌；转移1mL菌液至无菌的1.5mL离心管中，8000rpm离心2min，收集沉淀；去掉上清，用10mM的氯化镁洗涤沉淀3次并离心，最后将PstDC3000重悬在10mM氯化镁，使其OD
600值达到0.001备用。将拟南芥种子用75％酒精浸泡3min，然后用无菌水洗涤4次，种在装有1/2MS培养基的培养皿中，每个培养皿播12颗种子，将带种子的1/2MS培养皿在4℃下春化3d以打破休眠，然后置于22℃、光照强度为100μE m
-2s-
1(16h光/8h黑暗)的培养室中，幼苗生长2周时将上述不同浓度的2-氨基-3-吲哚基丁酸缓慢倒入培养皿中，直至淹没整个拟南芥幼苗，保持2-3分钟，然后将处理液从培养皿中倾倒干净，每隔24h处理一次，共处理2次，第2次处理24h后用同样的淹没方法将PstDC3000悬浮液(OD
600＝0.01)接种到拟南芥叶片上，接种后用医用透气胶贴将培养皿封闭好，放置于培养室中继续培养。3d后测定不同处理细菌个数，并观察拟南芥发病情况，计算病情指数，计算方式与实施例2病情指数计算公式相同。
PstDC3000引起的病害分级标准(以叶片为单位)：
0级：叶面上无病斑；
1级：病斑面积占整片叶面积的0％-10％；
2级：病斑面积占整片叶面积的10％-25％；
3级：病斑面积占整片叶面积的25％-50％；
4级：病斑面积占整片叶面积的50％-75％；
5级：病斑面积占整片叶面积的75％-100％。
表3不同浓度2-氨基-3-吲哚基丁酸对叶片中细菌个数和病情指数的影响
表3结果表明，随着2-氨基-3-吲哚基丁酸浓度的上升，每毫克叶片中的细菌个数逐渐下降。处理浓度为100nM、1000nM和10000nM时，每毫克叶片中细菌个数减少76.75％、80.97％和85.03％，病情指数分别降低51.25％、53.92％和64.42％。说明2-氨基-3-吲哚基丁酸能够激发植物产生对丁香假单胞菌的免疫能力，抑制细菌在植物叶片中的积累，降低植株发病水平。
按照相同方法考察3-甲基吡咯烷-2-羧酸诱导拟南芥抗丁香假单胞菌侵染效果，结果见表 4：
表4不同浓度3-甲基吡咯烷-2-羧酸对叶片中细菌个数和病情指数的影响
表4结果表明，随着3-甲基吡咯烷-2-羧酸浓度的上升，每毫克叶片中的细菌个数逐渐下降。处理浓度为100nM、1000nM和10000nM时，每毫克叶片中细菌个数减少70.78％、80.90％和94.90％，病情指数分别降低41.13％、52.47％和72.69％。说明3-甲基吡咯烷-2-羧酸能够激发植物产生对丁香假单胞菌的免疫能力，抑制细菌在植物叶片中的积累，降低植株发病水平。
实施例4(2-氨基-3-吲哚基丁酸和3-甲基吡咯烷-2-羧酸诱导小麦抗白粉病菌侵染)
取2-氨基-3-吲哚基丁酸用蒸馏水溶解后再用蒸馏水梯度稀释成100nM、1000nM和10000nM的溶液，另设空白对照。小麦(NAU0686)种子催芽后，种于灭菌土培养钵中，放入23(±1)℃光照12h的温室中培养。当幼苗长至1叶1心期时，用上述浓度的2-氨基-3-吲哚基丁酸溶液对小麦幼苗进行茎叶喷雾处理，间隔24小时重复进行处理一次，共进行两次处理，24h后将新鲜的小麦白粉菌孢子均匀的撒在小麦叶片上，每处理3盆，每盆20株。10d后调查各处理的小麦病情等级，按《农药田间药效试验准则》(一)中的小麦白粉病分级标准记载发病程度，并计算病情指数和相对免疫效果，计算方式与番茄斑萎病病情指数和相对免疫效果计算公式相同，结果如表5所示。
小麦白粉病分级标准(以叶片为单位)：
1级：病斑面积占整片叶面积的5％以下；
3级：病斑面积占整片叶面积的6％-15％；
5级：病斑面积占整片叶面积的16％-25％；
7级：病斑面积占整片叶面积的26％-50％；
9级：病斑面积占整片叶面积的50％以上。
表5不同浓度2-氨基-3-吲哚基丁酸对小麦病情指数和相对免疫效果的影响
表5的结果表明，随着2-氨基-3-吲哚基丁酸浓度的上升，易感病品种小麦的病情指数下降，相对免疫效果提高。各处理的病情指数均存在显著性差异。当浓度分别为10nM、100nM、1000nM和10000nM时，病情指数分别为73.34、63.41、43.67、29.70，相对免疫效果为22.04％、32.59％、53.58和68.43％。当2-氨基-3-吲哚基丁酸浓度大于1000nM时，易感病品种小麦感染白粉病的病情指数低于50，而相对免疫效果超过50％，在浓度10000nM时效果最佳。以上结果说明2-氨基-3-吲哚基丁酸能够提高小麦对真菌病害白粉病的免疫能力，从而抑制白粉病菌在小麦叶片中的侵染和扩散，阻止小麦白粉病的发展与蔓延。
按照相同方法考察3-甲基吡咯烷-2-羧酸诱导小麦抗白粉病菌侵染效果，结果见表6：
表6不同浓度3-甲基吡咯烷-2-羧酸对小麦病情指数和相对免疫效果的影响
表6的结果表明，随着3-甲基吡咯烷-2-羧酸浓度的上升，易感病品种小麦的病情指数下降，相对免疫效果提高。各处理的病情指数均存在显著性差异。当浓度分别为10nM、100nM、1000nM和10000nM时，病情指数分别为72.16、54.17、40.41、30.26，相对免疫效果为25.04％、43.73％、58.02和68.57％。当3-甲基吡咯烷-2-羧酸浓度大于1000nM时，易感病品种小麦感染白粉病的病情指数低于50，而相对免疫效果超过50％，在浓度10000nM时效果最佳。以上结果说明3-甲基吡咯烷-2-羧酸能够提高小麦对真菌病害白粉病的免疫能力，从而抑制白粉病菌在小麦叶片中的侵染和扩散，阻止小麦白粉病的发展与蔓延。
实施例5(2-氨基-3-吲哚基丁酸和3-甲基吡咯烷-2-羧酸诱导小麦抗白粉病菌侵染的田间试验)
用1000nM浓度的2-氨基-3-吲哚基丁酸溶液(加入体积百分比为0.02％的表面活性剂吐温20)在田间进行茎叶喷雾处理，以喷施体积百分比为0.02％的表面活性剂吐温20为助剂对照，以喷施30g/亩的阿泰灵为阳性对照，每个处理三次重复。调查各处理的小麦病情等级，按《农药田间药效试验准则》(一)中的小麦白粉病分级标准记载发病程度，并计算病情指数和相对免疫效果，计算方式与番茄斑萎病病情指数和相对免疫效果计算公式相同。待收获的小麦种子晾干后，对不同处理小麦种子的千粒重进行测定。
小麦白粉病分级标准(以叶片为单位)：
1级：病斑面积占整片叶面积的5％以下；
3级：病斑面积占整片叶面积的6％-15％；
5级：病斑面积占整片叶面积的16％-25％；
7级：病斑面积占整片叶面积的26％-50％；
9级：病斑面积占整片叶面积的50％以上。
表7 1000nM浓度的2-氨基-3-吲哚基丁酸对小麦病情指数和相对免疫效果及千粒重的影响
由表7结果可以看出，1000nM浓度的2-氨基-3-吲哚基丁酸溶液处理可有效提高小麦对真菌病害白粉病的免疫能力，经过该浓度处理的小麦的病情指数降低了40.55％，显著低于助剂对照组；与助剂对照比，相对免疫效果提高了40％、千粒重增加了10.80％。与阿泰灵对照组相比，喷施1000nM 2-氨基-3-吲哚基丁酸后，小麦的相对免疫效果提高12％，说明喷施2-氨基-3-吲哚基丁酸能够有效提高小麦对真菌病害白粉病的免疫能力。
实施例6(2-氨基-3-吲哚基丁酸和3-甲基吡咯烷-2-羧酸诱导黑麦草抵抗高温胁迫)
取2-氨基-3-吲哚基丁酸用蒸馏水溶解后再用蒸馏水梯度稀释成1nM、10nM、100nM和1000nM的溶液，另设空白对照，同时加入0.02％吐温20作为表面活性剂。每个浓度设置4组重复，同时设置常温空白对照。黑麦草种子按照每盆0.8g称重，并播种于直径8.5cm的盆钵中，在温度25℃、湿度60％-70％、光强200μmol m
-2s
-1(12h光照/12h黑暗)的温室中种 植。黑麦草生长7d后开始进行处理，处理的方法为叶面喷施2-氨基-3-吲哚基丁酸溶液，24h喷施两次。在第二次喷施24h后，将其转移至温度45℃的光照培养箱进行高温胁迫处理12h，将植株取出转移至25℃的温室恢复7d，观察统计植株的受害情况并计算热害分级。其中热害分级标准见表8，热害指数计算公式如下。热害结果见表6。
表8热害分级标准
表9 2-氨基-3-吲哚基丁酸对高温胁迫下黑麦草的影响
表9的结果表明，经过2-氨基-3-吲哚基丁酸处理的黑麦草在高温胁迫后的热害指数明显低于对照组，且随着处理浓度的增加，热害指数逐渐降低。处理浓度上升到1000nM时，黑麦草的热害指数降低了65％。可见，2-氨基-3-吲哚基丁酸能够缓解高温胁迫对黑麦草植株的损伤，提高黑麦草对高温胁迫的抵抗能力。
按照相同方法考察3-甲基吡咯烷-2-羧酸诱导黑麦草抵抗高温胁迫想过，结果见表10：
表10 3-甲基吡咯烷-2-羧酸对高温胁迫下黑麦草的影响
表10的结果表明，经过3-甲基吡咯烷-2-羧酸处理的黑麦草在高温胁迫后的热害指数明显低于对照组，且随着处理浓度的增加，热害指数逐渐降低。处理浓度上升到1000nM时，黑麦草的热害指数降低了68％。可见，3-甲基吡咯烷-2-羧酸能够缓解高温胁迫对黑麦草植株的损伤，提高黑麦草对高温胁迫的抵抗能力。
实施例7(2-氨基-3-吲哚基丁酸和3-甲基吡咯烷-2-羧酸诱导拟南芥抵抗高温胁迫)
拟南芥种子按照每盆50粒左右播种于直径8.5cm的盆钵中，在温度25℃、湿度60％-70％、光强100μmol m
-2s
-1(16h光照/8h黑暗)的温室中种植。在拟南芥正常生长21d时开始进行处理。实验设置0、1、10、100和1000nM，同时加入0.02％吐温20作为表面活性剂，设置四组重复。其中喷雾处理方法与实施例5中黑麦草的处理方法相同。在第二次处理24h后，将其转移至温度45℃的光照培养箱进行高温胁迫处理12h，用植物效率Handy-PEA测定拟南芥叶圆片的叶绿素荧光。将植株取出转移至25℃的温室恢复7d，观察统计植株的受害情况并计算热害分级，结果如表11所示。
表11 2-氨基-3-吲哚基丁酸处理对高温胁迫下拟南芥的影响
表11的结果表明，在高温胁迫条件下，经过2-氨基-3-吲哚基丁酸处理的拟南芥光合性能指数PI
ABS显著上升，热害指数明显下降。随着2-氨基-3-吲哚基丁酸浓度的升高，拟南芥的热害指数逐渐降低，同时光合性能指数PI
ABS相较于对照组明显上升，尤其是1000nM浓度下拟南芥的光合性能指数PI
ABS大幅提高，相较于对照提高了20倍，而热害指数降低了65％。由此可见，2-氨基-3-吲哚基丁酸能够缓解高温胁迫对拟南芥光合作用活性造成的伤害，提高拟南芥对高温胁迫的抵抗能力。
按照相同方法考察3-甲基吡咯烷-2-羧酸诱导拟南芥抵抗高温胁迫的效果，结果见表12：
表12 3-甲基吡咯烷-2-羧酸处理对高温胁迫下拟南芥的影响
表12的结果表明，在高温胁迫条件下，经过3-甲基吡咯烷-2-羧酸处理的拟南芥光合性能指数PI
ABS显著上升，热害指数明显下降。随着3-甲基吡咯烷-2-羧酸浓度的升高，拟南芥的热害指数逐渐降低，同时光合性能指数PI
ABS相较于对照组明显上升，尤其是1000nM浓度下拟南芥的光合性能指数PI
ABS大幅提高，相较于对照提高了23倍，而热害指数降低了68％。由此可见，3-甲基吡咯烷-2-羧酸能够缓解高温胁迫对拟南芥光合作用活性造成的伤害，提高拟南芥对高温胁迫的抵抗能力。
实施例8(2-氨基-3-吲哚基丁酸和3-甲基吡咯烷-2-羧酸诱导茶树抗低温胁迫)
供试茶树为扦插苗白叶1号。选择长势较一致的茶苗移入直径18cm的塑料盆钵中，置于温度25℃，湿度60％-70％的温室使之适应生长一周左右进行实验。实验设置0、1、10、100和1000nM，同时加入0.02％吐温20作为表面活性剂。其中喷雾处理方法与实施例1中黑麦草的处理方法相同，低温胁迫的时间为24h，温度设置为-4℃。胁迫完成后将茶苗取出，常温暗处理30min后用植物效率Handy-PEA测定茶苗顶部叶片的叶绿素荧光，随后置于25℃ 温室恢复3d，观察统计其冷害状况并进行分级。其中冷害指数统计分级标准见表13，计算公式如下，结果如表14所示。
表13冷害分级标准
表14 2-氨基-3-吲哚基丁酸处理对低温胁迫下茶叶的影响
表14的结果表明，经过2-氨基-3-吲哚基丁酸处理，茶叶在低温胁迫条件的光合性能指数PI
ABS显著上升，冷害指数明显下降。其中以1000nM的效果最佳，该浓度处理的茶叶PI
ABS提高了52％，冷害指数降低了40％。由此可见，2-氨基-3-吲哚基丁酸能够缓解低温胁迫对茶叶幼苗光合系统的伤害，提高茶叶对低温胁迫的抵抗能力。
按照相同方法考察3-甲基吡咯烷-2-羧酸诱导茶树抗低温胁迫的效果，结果见表15
表15 3-甲基吡咯烷-2-羧酸处理对低温胁迫下茶叶的影响
表15的结果表明，经过3-甲基吡咯烷-2-羧酸处理，茶叶在低温胁迫条件的光合性能指数PI
ABS显著上升，冷害指数明显下降。其中以1000nM的效果最佳，该浓度处理的茶叶PI
ABS提高了138％，冷害指数降低了48％。由此可见，3-甲基吡咯烷-2-羧酸能够缓解低温胁迫对茶叶幼苗光合系统的伤害，提高茶叶对低温胁迫的抵抗能力。
实施例9(2-氨基-3-吲哚基丁酸和3-甲基吡咯烷-2-羧酸诱导小麦抗干旱胁迫)
用6目的网筛作为容器水培小麦，每筛50粒，每隔两天更换一次1/2Hoagland营养液，待小麦生长到两叶一心时期开始叶面喷施2-氨基-3-吲哚基丁酸溶液，2-氨基-3-吲哚基丁酸浓度为0、100和1000nM，同时加入0.02％吐温20作为表面活性剂；连续喷施两天后，在第三天将水培营养液替换成含有25％PEG-6000的1/2Hoagland营养液进行胁迫处理，干旱胁迫6d后复水处理，在正常的营养液中恢复生长7d后观察测定旱害指数，并测定其根长和生物量。结果如表17所示。
叶片旱害与盐害后的表现特征类似，借用盐害的评估指标引入旱害率及旱害指数，旱害指数公式如下，旱害分级标准见表16。
表16旱害分级标准
表17 2-氨基-3-吲哚基丁酸处理对干旱胁迫下小麦生物量与旱害指数的影响
表17的结果表明，随着处理浓度的增加小麦对干旱胁迫的抵御能力在逐步增强。两个处理浓度下小麦的鲜重、干重、根长均高于对照组，这使得小麦旱害指数明显降低。例如和对照组相比，1000nM浓度的2-氨基-3-吲哚基丁酸处理使小麦幼苗根长显著增加了10.86％，地上和地下部分鲜重分别提高了44.93％和54.98％，旱害指数降低了44％。这说明2-氨基-3-吲哚基丁酸能提高小麦抗干旱胁迫的能力。
按照相同方法考察3-甲基吡咯烷-2-羧酸诱导小麦抗干旱胁迫的效果，结果见表18：
表18 3-甲基吡咯烷-2-羧酸处理对干旱胁迫下小麦生物量与旱害指数的影响
表18的结果表明，随着处理浓度的增加小麦对干旱胁迫的抵御能力在逐步增强。两个处理浓度下小麦的鲜重、干重、根长均高于对照组，这使得小麦旱害指数明显降低。例如和对照组相比，1000nM浓度的3-甲基吡咯烷-2-羧酸处理使小麦幼苗根长显著增加了12.58％，地上和地下部分鲜重分别提高了44.08％和36.55％，旱害指数降低了61％。这说明3-甲基吡咯烷-2-羧酸能提高小麦抗干旱胁迫的能力。
实施例10(2-氨基-3-吲哚基丁酸和3-甲基吡咯烷-2-羧酸诱导棉花抗盐胁迫)
实验材料为“泗抗一号”棉花，用500mL塑料杯进行水培，每隔两天更换一次1/2Hoagland营养液。当棉花幼苗生长至第二片真叶完全展开时用2-氨基-3-吲哚基丁酸溶液进行叶面喷施， 实验设置0、1、10、100和1000nM浓度，同时加入0.02％吐温20作为表面活性剂。每24h喷施一次，共2次，处理后第二天向1/2Hoagland营养液加入NaCl使其终浓度为100mM，进行盐胁迫处理。每个处理三次重复。盐胁迫三天后复水处理，观察棉花的盐害症状，并计算盐害指数，计算公式如下：
表19盐害分级标准
表20 2-氨基-3-吲哚基丁酸处理对盐胁迫下棉花的影响
表20的结果表明，棉花的盐害指数随着2-氨基-3-吲哚基丁酸浓度的升高而下降，各处理的植株死亡率均低于对照。当浓度为1000nM时，盐害指数和死亡率均最低，分别为44％和27％。以上结果说明2-氨基-3-吲哚基丁酸能够诱导棉花对盐胁迫产生较好的抗性。
按照相同方法考察3-甲基吡咯烷-2-羧酸诱导棉花抗盐胁迫的效果，结果见表21：
表:21 3-甲基吡咯烷-2-羧酸处理对盐胁迫下棉花的影响
表21的结果表明，棉花的盐害指数随着3-甲基吡咯烷-2-羧酸浓度的升高而下降，各处理的植株死亡率均低于对照。当浓度为1000nM时，盐害指数和死亡率均最低，分别为39％和24％。以上结果说明3-甲基吡咯烷-2-羧酸能够诱导棉花对盐胁迫产生较好的抗性。
化学合成的2-氨基-3-吲哚基丁酸和3-甲基吡咯烷-2-羧酸也具有与生物提取的2-氨基-3-吲哚基丁酸和3-甲基吡咯烷-2-羧酸相同的效果。2-氨基-3-羟基-3-甲基丁酸和3-甲基吡咯烷-2-羧酸的制备方法及来源不影响其作为免疫诱抗剂的应用和效果。
Claims (10)
- 2-氨基-3-吲哚基丁酸和/或3-甲基吡咯烷-2-羧酸在制备植物免疫诱抗剂中的应用。
- 2-氨基-3-吲哚基丁酸和/或3-甲基吡咯烷-2-羧酸在提高植物对非生物胁迫和/或生物胁迫中的应用,所述的非生物胁迫选自高温、低温、干旱和/或盐胁迫中的任意一种或几种,所述的生物胁迫选自真菌、细菌、病毒胁迫中的任意一种或几种，所述的真菌性病害为小麦白粉病；所述的细菌性病害为丁香假单胞菌病害；所述的病毒性病害为番茄斑萎病。
- 根据权利要求1或2所述的应用，其特征在于，所述的植物，选自粮食作物、经济作物、蔬菜。
- 根据权利要求3所述的应用，其特征在于，所述的粮食作物为小麦，所述的经济作物为黑麦草、茶叶、棉花，所述的蔬菜为番茄。
- 一种植物免疫诱抗剂，其特征在于，包含组分A：2-氨基-3-吲哚基丁酸或3-甲基吡咯烷-2-羧酸中的任意一种或两种，组分B：表面活性剂。
- 根据权利要求5所述的植物免疫诱抗剂，其特征在于，所述的表面活性剂为吐温20。
- 根据权利要求6所述的植物免疫诱抗剂，其特征在于，吐温20在植物免疫诱抗剂中的浓度为0.01～0.05％(v/v)，优选0.02％(v/v)。
- 根据权利要求5所述的植物免疫诱抗剂，其特征在于，所述的植物免疫诱抗剂中2-氨基-3-吲哚基丁酸或3-甲基吡咯烷-2-羧酸的浓度为0.1-10000nM浓度。
- 一种提高植物对生物和/或非生物胁迫抗性的方法，其特征在于向目标植物施加0.1-10000nM的2-氨基-3-吲哚基丁酸或3-甲基吡咯烷-2-羧酸。
- 根据权利要求9所述的方法，其特征在于，所述的非生物胁迫选自高温、低温、干旱和/或盐胁迫中的任意一种或几种,所述的生物胁迫选自真菌、细菌、病毒胁迫中的任意一种或几种，所述的真菌性病害为小麦白粉病；所述的细菌性病害为丁香假单胞菌病害；所述的病毒性病害为番茄斑萎病。
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| CN114946863A (zh) | 2022-08-30 |
| CN114128716B (zh) | 2022-09-06 |
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