WO2023103941A1 - 2-氨基-3-苯基丁酸或2,6-二氨基-3-甲基己酸作为植物免疫诱抗剂的应用 - Google Patents

2-氨基-3-苯基丁酸或2,6-二氨基-3-甲基己酸作为植物免疫诱抗剂的应用 Download PDF

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WO2023103941A1
WO2023103941A1 PCT/CN2022/136490 CN2022136490W WO2023103941A1 WO 2023103941 A1 WO2023103941 A1 WO 2023103941A1 CN 2022136490 W CN2022136490 W CN 2022136490W WO 2023103941 A1 WO2023103941 A1 WO 2023103941A1
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amino
diamino
acid
stress
phenylbutyric acid
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PCT/CN2022/136490
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English (en)
French (fr)
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陈世国
王赫
郭妍婧
米丽汝
房婉萍
郭爱平
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南京天秾生物技术有限公司
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    • 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
    • A01N37/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids
    • A01N37/44—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids containing at least one carboxylic group or a thio analogue, or a derivative thereof, and a nitrogen atom attached to the same carbon skeleton by a single or double bond, this nitrogen atom not being a member of a derivative or of a thio analogue of a carboxylic group, e.g. amino-carboxylic acids
    • 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
    • A01N25/00—Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests
    • A01N25/30—Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests characterised by the surfactants
    • 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
    • A01P1/00—Disinfectants; Antimicrobial compounds or mixtures thereof
    • 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
    • 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
    • A01P3/00—Fungicides
    • 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-phenylbutyric acid and/or 2,6-diamino-3-methylhexanoic acid as a plant immune inducer.
  • soil salinization is the main abiotic limiting factor that hinders global crop growth and productivity.
  • 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 the actual agricultural production, it is particularly urgent to develop products and technologies aimed at reducing the level of plant damage to ensure safe agricultural production.
  • crops are constantly threatened by various pests and diseases during their growth and development.
  • the occurrence and prevalence of some diseases usually cause severe crop yield reduction or even failure on a large scale. Therefore, it is particularly important to establish an integrated management system for important agricultural pests and diseases.
  • the main measure for the prevention and control of agricultural plant diseases and insect pests is to use pesticides to directly kill them.
  • a series of problems also make the traditional "killing" strategy of plant protection face the risk of failure, which seriously threatens the safety of food production and the sustainable development strategy of agriculture.
  • 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. Plant immune inducers themselves have no insecticidal or fungal activity, and are mainly used to stimulate the plant's own natural immune system to prevent and control pests and diseases. Because it does not rely on exogenous pesticides to directly kill pathogens, it is not easy for pests to develop resistance to them, which is in line with the idea of realizing green prevention and control under the condition of effectively protecting agricultural biodiversity. In addition, in nature, 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-phenylbutyric acid with a molecular formula of C 10 H 13 NO 2 , and a molecular weight of 179 g/mol, is a new type of amino acid compound and is a colorless transparent crystal.
  • 2-amino-3-phenylbutyric acid was chemically synthesized for the first time, and activity tests showed that it had an inhibitory effect on the growth of Leuconostoc dextranicum (Edelson & Keeley, 1963).
  • 2-amino-3-phenylbutyric acid can be used as a pharmaceutical adjuvant (carrier or absorption enhancer or humectant), for example, for the pharmaceutical composition of surgical local anesthetic-lidocaine (Liu Li , 2017), injections for the prevention or treatment of multiple trace element deficiencies in humans and mammals (Liu Li, 2018), and compositions of puerarin eye drops for external use (Liu Li, 2021).
  • carrier or absorption enhancer or humectant for example, for the pharmaceutical composition of surgical local anesthetic-lidocaine
  • compositions of puerarin eye drops for external use Liu Li, 2021).
  • 2-amino-3-phenylbutyric acid could alleviate arthritis in rats (Ren et al., 2019). Feng et al.
  • 2-amino-3-phenylbutyric acid may have a therapeutic effect on Parkinson's disease (Feng et al., 2020).
  • 2-amino-3-phenylbutyric acid is obtained by chemical synthesis or hydrolysis. So far, there is no report about the natural free existence of this compound. Therefore, 2-amino-3-phenylbutyric acid is considered an unnatural amino acid.
  • 2,6-Diamino-3-methylhexanoic acid the molecular formula is C 7 H 16 N 2 O 2 , the molecular weight is 160 g/mol, and it is a colorless crystal.
  • This compound The earliest report was 2,6-diamino-3-methylhexanoic acid obtained by chemical synthesis in 1969 (Takehara & Yoshida, 1969). Subsequent studies on the specificity of lysine monooxygenase for this compound showed that 2,6-diamino-3-methylhexanoic acid has no substrate activity for this enzyme (Ohnishi et al., 1976). So far, there are few studies on 2,6-diamino-3-methylhexanoic acid, and there are no related studies, reports and patents involving natural products and plant immune induction activities.
  • the purpose of the present invention is to provide 2-amino-3-phenylbutyric acid and 2,6-diamino-3-methylhexanoic acid as the application of plant immune inducer to the above-mentioned deficiency of prior art.
  • 2-amino-3-phenylbutyric acid and 2,6-diamino-3-methylhexanoic acid can effectively alleviate high temperature, low temperature, drought and Salt damage to plants.
  • 2-amino-3-phenylbutyric acid and 2,6-diamino-3-methylhexanoic acid are natural products isolated from Alternaria sp., and the structural formula of 2-amino-3-phenylbutyric acid is as follows :
  • 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 preferred wheat of the food crops the preferred ryegrass, tea, cotton of the economic crops, the preferred tomato of the vegetables.
  • a plant immune inducer comprising 2-amino-3-phenylbutyric acid and/or 2,6-diamino-3-methylhexanoic acid.
  • the plant immune inducer includes component A: any one of 2-amino-3-phenylbutyric acid or 2,6-diamino-3-methylhexanoic acid One or more; 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-phenylbutyric acid or 2,6-diamino-3-methylhexanoic acid in the plant immune inducer is 0.1-10000 nM.
  • Plant immune inducer is a new type of pesticide, which is the main development direction of green prevention and control in the field of plant protection in the future.
  • the development of immune inducers in my country is in its infancy, and there are only a handful of products that have been officially registered. Therefore, the development of natural plant immune inducers and the promotion of their industrialization are of great significance for ensuring the safety of food production and improving the competitiveness of agricultural products.
  • the research of the present invention shows that: 2-amino-3-phenylbutyric acid and 2,6-diamino-3-methylhexanoic acid perform well in related experiments of inducing immunity and stress resistance, and can improve the resistance of plants to biological stress and non-toxicity. Resistance to biological stress.
  • it 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, fungi and bacteria 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 fungi, bacteria, and virus stress.
  • 2,6-diamino-3-methylhexanoic acid is used for preventing and treating tomato spotted wilt, under the concentration of 0.1-10nM (adding the surfactant Tween 20 that is 0.02% by volume), can inoculate tomato in tobacco Spotted wilt virus (TSWV) significantly inhibited the spread of the virus after 3 days. After 15 days, tobacco disease was investigated, and it was found that the disease index of tobacco plants treated with 2,6-diamino-3-methylhexanoic acid was significantly reduced. At a low concentration of 10nM, it can effectively inhibit the expression of TSWV on tobacco leaves, and its disease index, relative immune effect and virus content are 26.41, 70.94% and 0.12, respectively.
  • TSWV tobacco Spotted wilt virus
  • 2-Amino-3-phenylbutyric acid is used for the method for preventing and treating wheat powdery mildew, and it is in the range of concentration 10-10000nM (adding the surfactant Tween 20 that volume percentage is 0.02%), carries out after wheat is inoculated with powdery mildew bacterium 10 days.
  • concentration 10-10000nM adding the surfactant Tween 20 that volume percentage is 0.02%
  • 2,6-diamino-3-methylhexanoic acid is used for the method for preventing and treating wheat powdery mildew, and it is in the range of concentration 100-10000nM (adding the surfactant Tween 20 that volume percentage is 0.02%), inoculates powdery mildew bacteria in wheat After 10 days of investigation, it was found that with the increase of the treatment concentration, the disease index of wheat infected with powdery mildew decreased, and the relative immune effect increased. When the high concentration was 10000nM, the disease index was 25.64, and the relative immune effect was 73.45%.
  • 2-Amino-3-phenylbutyric acid is used to prevent wheat powdery mildew in the field.
  • the disease index of wheat is 25.30 under the treatment concentration of 1000nM, which is significantly lower than that of Altaline treatment and adjuvant control group.
  • the relative immune effect and thousand-grain weight were 51.72% and 38.87g, respectively, both of which were significantly higher than those treated with Altaline and the auxiliary control group.
  • 2-amino-3-phenylbutyric acid has a significant inhibitory effect on the occurrence and spread of wheat powdery mildew.
  • 2-amino-3-phenylbutyric acid is used for the method for preventing and treating bacterial diseases, and its concentration is 100-10000nM concentration range (adding the surfactant Tween 20 that volume percentage is 0.02%), along with the raising of treatment concentration, The accumulation of bacteria PstDC3000 in Arabidopsis leaves gradually decreased.
  • the treatment concentration was 10000nM
  • the number of bacteria per mg of leaves was 1.34 ⁇ 10 5 , which was 95.56% lower than that of the blank control, and the disease index was 14.58.
  • This result shows that 2-amino-3-phenylbutyric acid can stimulate the autoimmunity of Arabidopsis, inhibit the reproduction of bacteria in plants, reduce the accumulation of bacteria, and delay and inhibit the development of diseases.
  • 2,6-diamino-3-methylhexanoic acid is used for the method for preventing and treating bacterial diseases, and its concentration is 100-10000nM concentration range (adding the surfactant Tween 20 that volume percentage is 0.02%), with treatment concentration
  • the accumulation of bacteria PstDC3000 in leaves of Arabidopsis thaliana gradually decreased.
  • the treatment concentration was 10000nM
  • the number of bacteria per milligram of leaves was 1.58 ⁇ 10 5 , which was 95.11% lower than that of the blank control.
  • the disease index was 19.86. This result shows that 2,6-diamino-3-methylhexanoic acid can stimulate the autoimmunity of Arabidopsis, inhibit the reproduction of bacteria in plants, reduce the accumulation of bacteria, and delay and inhibit the development of diseases.
  • 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-phenylbutyric acid is used to improve the method for high temperature resistance of plants, and its concentration is 2-amino-3-phenylbutyric acid solution between 100-10000nM (adding volume percentage is 0.02% surface
  • the active agent Tween 20 was used to induce Arabidopsis thaliana at the seedling stage, and it was found that the photosynthetic performance index PI ABS of the plants in the treatment group was higher than that of the control group, and the heat damage index was lower in the control group. This result indicated that exogenous spraying of 2-amino-3-phenylbutyric acid solution could effectively alleviate the level of injury caused by high temperature to seedlings.
  • 2,6-diamino-3-methylhexanoic acid is used to improve the method for high temperature resistance of plants, its concentration is between 1-1000nM 2,6-diamino-3-methylhexanoic acid solution (added volume Percentage is 0.02% surfactant Tween 20) to induce ryegrass seedlings and Arabidopsis thaliana, and found that the photosynthetic performance index PI ABS of the plants in the treatment group was higher than The heat damage index of the control group was lower than that of the control group. This result indicated that exogenous spraying of 2,6-diamino-3-methylhexanoic acid solution effectively alleviated the level of injury caused by high temperature to seedlings.
  • 2-amino-3-phenylbutyric acid is used for improving the method for the resistance ability of plant to low temperature, with the 2-amino-3-phenylbutyric acid solution of 100-10000nM concentration (adding volume percent is 0.02% surfactant Tween 20) sprayed the leaves of the tea seedlings, and found that after 24 hours of low temperature stress at -4°C, the photosynthetic performance index PI ABS of the tea seedlings treated with 100nM, 1000nM and 10000nM were significantly higher than that of the control group, and the chilling injury index was significantly higher than that of the control group. It was lower than that of the control group, indicating that 2-amino-3-phenylbutyric acid effectively alleviated the damage caused by low temperature to tea seedlings and improved the resistance of tea to low temperature stress.
  • 2,6-diamino-3-methylhexanoic acid is used for improving the method for the resistance ability of plant to low temperature
  • 2,6-diamino-3-methylhexanoic acid solution of 1-1000nM concentration (addition volume percentage is 0.02% surfactant Tween 20) was sprayed on the leaves of tea seedlings, and it was found that the photosynthetic performance index PI ABS of the tea seedlings treated with 1nM, 10nM, 100nM and 1000nM were all significant after -4°C low temperature stress for 24h.
  • the chilling injury index was significantly lower than that of the control group, indicating that 2,6-diamino-3-methylhexanoic acid effectively alleviated the damage caused by low temperature to tea seedlings and improved the resistance of tea to low temperature stress.
  • 2-amino-3-phenylbutyric acid is used for improving the method for the resistance ability of plant to drought stress, with 100 and 1000nM 2-amino-3-phenylbutyric acid solution (adding volume percent is 0.02% surfactant Tween 20) carried out foliage spraying treatment to the hydroponic wheat with two leaves and one heart, and found that under 25% polyethylene glycol-6000 (PEG-6000) stress, the biomass of wheat treated with 100nM and 1000nM was significantly higher than In the control group, this result indicated that 2-amino-3-phenylbutyric acid improved the resistance of wheat to drought stress.
  • PEG-6000 polyethylene glycol-6000
  • 2,6-diamino-3-methylhexanoic acid is used for improving the method for the resistance ability of plant to drought stress, with 100 and 1000nM 2,6-diamino-3-methylhexanoic acid solution (addition volume percentage is 0.02% surfactant Tween 20) was sprayed on the leaves of hydroponic wheat with two leaves and one heart, and it was found that under the stress of 25% polyethylene glycol-6000 (PEG-6000), the wheat treated with 100nM and 1000nM Each biomass was significantly higher than that of the control group, which indicated that 2,6-diamino-3-methylhexanoic acid improved the resistance of wheat to drought stress.
  • PEG-6000 polyethylene glycol-6000
  • 2-amino-3-phenylbutyric acid is used for improving the method for the resistance ability of plant to salt stress, with the 2-amino-3-phenylbutyric acid solution of 1-1000nM concentration (adding volume percent is 0.02% surfactant Tween 20) carried out foliar spraying treatment to two hydroponic cottons at the true leaf stage, and found that under the stress of 100mM NaCl, each sprayed treatment group of 2-amino-3-phenylbutyric acid had a lower cotton mortality rate. and salt damage index were lower than those of the control group, which indicated that 2-amino-3-phenylbutyric acid improved the tolerance level of cotton to salt.
  • 2,6-diamino-3-methylhexanoic acid is used for improving the method for the resistance ability of plant to salt stress, with the 2,6-diamino-3-methylhexanoic acid solution of 1-1000nM concentration (add volume percentage 0.02% surfactant Tween 20) carried out foliar spraying treatment to two hydroponic cottons in the true leaf stage, and found that under the stress of 100mM NaCl, each sprayed 2,6-diamino-3-methyl In the hexanoic acid treatment group, the cotton mortality rate and salt damage index were lower than those in the control group, which indicated that 2,6-diamino-3-methylhexanoic acid improved the tolerance level of cotton to salt.
  • Both 2-amino-3-phenylbutyric acid and 2,6-diamino-3-methylhexanoic acid are natural products with simple structures and convenient biological extraction methods. Since the present invention confirms that both 2-amino-3-phenylbutyric acid and 2,6-diamino-3-methylhexanoic acid can induce plants to produce immune activity to some serious diseases existing in agricultural production, and can Inducing plants to produce stress resistance to the main abiotic stresses currently faced in agricultural production has the potential to be developed into natural plant immune inducers.
  • the present invention has found that both 2-amino-3-phenylbutyric acid and 2,6-diamino-3-methylhexanoic acid have higher broad-spectrum immune-inducing activity, and can induce tobacco immunity at a low concentration of 0.1nM. Produce an immune response to prevent the occurrence and spread of tomato spotted wilt; when the concentration is 1000nM, it can induce 55.38% and 65.26% relative immune effects of wheat against powdery mildew; when the concentration is 100nM, it can inhibit Pseudomonas syringae The accumulation of PstDC3000 in Arabidopsis leaves reduces the disease index of Arabidopsis.
  • 2-amino-3-phenylbutyric acid and 2,6-diamino-3-methylhexanoic acid can be used to control the main fungal diseases in farmland, such as wheat powdery mildew; viral diseases, such as tomato spotted wilt Diseases; bacterial diseases, such as those caused by Pseudomonas syringae.
  • farmland such as wheat powdery mildew
  • viral diseases such as tomato spotted wilt Diseases
  • bacterial diseases such as those caused by Pseudomonas syringae.
  • 2-amino-3-phenylbutyric acid and 2,6-diamino-3-methylhexanoic acid can prevent the occurrence and spread of major diseases in various agricultural productions, and can reduce crop damage Inhibition of various abiotic stresses during growth and development.
  • 2-Amino-3-phenylbutyric acid and 2,6-diamino-3-methylhexanoic acid are convenient to use and can play a preventive role in advance, reducing the level of plant damage caused by various biotic and abiotic stresses , reduce the use of pesticides, save production costs, and reduce carbon emissions.
  • 2-amino-3-phenylbutyric acid and 2,6-diamino-3-methylhexanoic acid are naturally occurring metabolites with simple structures 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-phenylbutyric acid and 2,6-diamino-3-methylhexanoic acid from 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, the centrifugation condition was 10000rpm, 5min.
  • the obtained supernatant is the crude extract of amino acids.
  • 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 2-amino-3-phenylbutyric acid, the peak time is 7.9min, this method can effectively separate the compound in Alternaria.
  • the structure of the isolated 2-amino-3-phenylbutyric acid was identified by NMR and mass spectrometry.
  • the mass spectrum shows that the molecular ion peak of the compound is: 180.1020[M+H] + , and its molecular formula is determined to be: C 10 H 13 NO 2 .
  • the compound was determined to be 2-amino-3-phenylbutyric 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 210nm, and the flow rate is 2mL min -1
  • impurities in the crude extract can be removed to obtain a single component 2,6-diamino-3-methylhexanoic acid, the peak time is 4.3min, this method can effectively separate the compound in Alternaria.
  • Example 2 (2-amino-3-phenylbutyric acid and 2,6-diamino-3-methylhexanoic acid induce tobacco resistance to tomato spotted wilt virus infection)
  • 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 rubbing inoculation method to activate the virus, and the virus plasmid was extracted and transformed into Escherichia coli competent cells. , smeared on a resistant plate 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 used the electric shock method for Agrobacterium For transformation, the transformed Agrobacterium liquid was spread on the corresponding resistance screening plate, and cultured at 28°C ( ⁇ 1) for 48h.
  • 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 show that when the concentration range of 2-amino-3-phenylbutyric acid is 0.1-10nM, each treatment can significantly reduce the infection of tomato spotted wilt virus to tobacco, and the disease index of tobacco infected with tomato spotted wilt virus is low At 50, the relative immune effect is above 35%. Compared with the control group not sprayed with 2-amino-3-phenylbutyric acid, within this concentration range, with the increase of concentration, the disease index of tobacco infected with tomato spotted wilt virus was significantly reduced, and the relative immune effect was significantly improved , the viral protein content in tobacco leaves decreased significantly.
  • Table 2 show that when 2,6-diamino-3-methylhexanoic acid concentration range is 0.1-10nM, each treatment can significantly reduce the infection of tomato spotted wilt virus to tobacco, and the infection of tobacco with tomato spotted wilt virus
  • the disease index is lower than 50, and the relative immune effect is more than 50%, and within this concentration range, along with the increase of the concentration, the disease index of tobacco infection tomato spotted wilt virus is significantly reduced, and compared with the control, the relative immune effect is significantly improved.
  • the content of virus protein in tobacco leaves decreased significantly.
  • 2-Amino-3-phenylbutyric acid was dissolved in distilled water and then gradually diluted with distilled water to form 10nM, 100nM, 1000nM and 10000nM solutions, and a blank control was also 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 seedling grows to 1 leaf and 1 heart stage: the wheat seedlings are sprayed with stems and leaves with the 2-amino-3-phenylbutyric acid solution of the above-mentioned concentration, and the interval is repeated in 24 hours to process once, and two processes are carried out 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.
  • Example 4 field test of 2-amino-3-phenylbutyric 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-phenylbutyric acid was dissolved in sterile water and then diluted into 100nM, 1000nM and 10000nM solutions with sterile water. A blank control was also added, and 0.02% Tween 20 was added as a surfactant.
  • 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-phenylbutyric acid was dissolved in distilled water and then gradually diluted with distilled water into 100nM, 1000nM and 10000nM 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. 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 at the 21d of the Arabidopsis seedling stage, and the treatment method was to spray the 2-amino-3-phenylbutyric acid solution on the leaves, and spray twice in 24 hours.
  • the experiment set the concentration of 2,6-diamino-3-methylhexanoic acid at 0, 1, 10, 100 and 1000 nM, and added 0.02% Tween 20 as a surfactant at the same time, and set four sets of repetitions.
  • the other methods are the same as 2-amino-3-phenylbutyric acid, and the effect of 2,6-diamino-3-methylhexanoic acid on inducing Arabidopsis to resist high temperature stress is investigated.
  • the results are shown in Table 10:
  • 2,6-diamino-3-methylhexanoic acid can alleviate the damage caused by high temperature stress to the photosynthetic activity of Arabidopsis thaliana, and improve the resistance of Arabidopsis to high temperature stress.
  • Example 7 (2,6-diamino-3-methylhexanoic acid induces ryegrass resistance to high temperature stress)
  • the ryegrass was treated after 7 days of growth, and the treatment method was spraying 2,6-diamino-3-methylhexanoic acid solution on the leaves, and spraying 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 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, 100, 1000 and 10000 nM, while 0.02% Tween 20 was added as a surfactant.
  • the spraying treatment method is the same as that of Arabidopsis thaliana in Example 1, the time of low temperature stress is 24 hours, and the temperature is set at -4°C.
  • Example 8 2-amino-3-phenylbutyric acid and 2,6-diamino-3-methylhexanoic acid induce drought stress resistance in wheat
  • a 6-mesh mesh sieve as a container for hydroponic wheat, sieve 50 grains per sieve, replace 1/2 Hoagland nutrient solution every two days, and spray 2-amino-3-phenylbutyl on the leaves when the wheat grows to two leaves and one heart Acid solution, the concentration of 2-amino-3-phenylbutyric acid is 0, 100 and 1000nM, and 0.02% Tween 20 is added as a surfactant at the same time; after two days of continuous spraying, the hydroponic nutrient solution is replaced with The 1/2 Hoagland nutrient solution containing 25% PEG-6000 was used for stress treatment. After 6 days of drought stress, rewatering treatment was carried out. After 7 days of recovery in normal nutrient solution, the drought index was observed and measured, and the root length and biomass were measured. The results are shown in Table 16.
  • 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 15.
  • Table 1 Effects of 7 2,6-diamino-3-methylhexanoic acid treatments on wheat biomass and drought damage index under drought stress
  • 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.
  • 1000nM concentration of 2,6-diamino-3-methylhexanoic acid treatment significantly increased the root length of wheat seedlings by 9.77%, and the fresh weight of the aboveground and underground parts increased by 31.03% and 34.42%, respectively, Drought index decreased by 59%. This shows that 2,6-diamino-3-methylhexanoic 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.
  • spray the leaves with 2-amino-3-phenylbutyric acid solution and set the concentration of 0, 1, 10, 100 and 1000nM in the experiment, and add 0.02% Tween at the same time 20 as a surfactant.

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Abstract

本发明公开了2-氨基-3-苯基丁酸或2,6-二氨基-3-甲基己酸作为植物免疫诱抗剂的应用。将2-氨基-3-苯基丁酸和/或2,6-二氨基-3-甲基己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、病毒和细菌对植物的侵染和致病水平的降低；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-苯基丁酸和2,6-二氨基-3-甲基己酸具有安全、环保、高效的特点。

Description

2-氨基-3-苯基丁酸或2,6-二氨基-3-甲基己酸作为植物免疫诱抗剂的应用 技术领域
本发明属于农业生物农药领域，涉及2-氨基-3-苯基丁酸和/或2,6-二氨基-3-甲基己酸作为植物免疫诱抗剂的应用。
背景技术
近年来，全球极端天气频繁出现，农业植物所面临的非生物胁迫也日益严重。每年由于高温、低温、干旱和盐等主要非生物胁迫给农业生产所造成的损失非常巨大。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，目前，全球干旱、半干旱地区面积约占总耕地面积的40％以上。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。近年来，由于全球性气候恶化使得干旱、高低温农业灾害发生的频率越来越高，对粮食生产安全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，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，针对当前农业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。
另一方面，农作物在生长发育过程中会不断受到各种病虫害的威胁，一些病害发生和流行，通常会造成作物大面积严重减产甚至绝收。因此，建立重要农业病虫害的综合治理体系显得尤为重要。当前，农业植物病虫害防治的主要措施是利用农药进行直接杀死，但长期、大量使用杀菌、杀虫剂，不仅带来了残留污染、抗药性发生、生物多样性降低、和食品安全性等一系列问题，也使传统植保的“杀灭”策略面临失效的风险，严重威胁粮食生产安全和农业可持续发展战略。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杀虫、菌的活性，主要通过外源施用激发植物自身天然免疫系统来防治病虫害。因为不依赖外源农药直接杀死病原体，因此病虫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-苯基丁酸，分子式为C 10H 13NO 2，分子量179克/摩尔，属于新型氨基酸类化合物，为无色透明晶体。1963年，2-氨基-3-苯基丁酸首次被化学合成，活性试验表明它对葡聚糖明串珠菌(Leuconostoc dextranicum)的生长有抑制作用(Edelson & Keeley,1963)。2002年，He等人从吸水链霉菌(Streptomyces hygroscopicus)次生代谢物—甘露霉素(mannopeptimycin)的水解产物中检测到了2-氨基-3-苯基丁酸，证明该氨基酸是甘露霉素的组成结构之一(He et al.,2002)。一些研究表明2-氨基-3-苯基丁酸可用作药学上的辅料(载体或吸收促进剂或保湿剂)，例如用于外科手术局部麻醉药—利多卡因的药物组合物(刘力，2017)、预防或治疗人类及哺乳动物多种微量元素缺乏时的注射剂(刘力，2018)、外用葛根素眼药的组合物(刘力，2021)。2019年Ren等人发现在100mg/kg和200mg/kg处理浓度下，2-氨基-3-苯基丁酸能缓解大鼠的关节炎(Ren et al.,2019)。Feng等人发现2-氨基-3-苯基丁酸可能对帕金森病具有治疗效果(Feng et al.,2020)。而以上的这些报道中2-氨基-3-苯基丁酸都是通过化学合成，或者水解方式得到。到目前为止，还没有关于该化合物为天然游离存在的报道。因此，2-氨基-3-苯基丁酸被认为是一种非天然的氨基酸。截至目前，关于2-氨基-3-苯基丁酸的研究很少，仅有的研究都聚焦在化学合成及异构体手性拆分(Grobuschek et al.,2002；Vékes et al.,2002)、医药学用途方面，没有涉及作为天然产物和植物活性方面的相关研究、报道和专利。关于2,6-二氨基-3-甲基己酸的研究也很少，没有涉及天然产物和植物免疫诱抗活性方面的相关研究、报道和专利。
2,6-二氨基-3-甲基己酸，分子式为C 7H 16N 2O 2，分子量为160克/摩尔，为无色晶体。目前对于该化合物的研究非常少，最早的报道在1969年，通过化学合成的方式得到的2,6-二氨基-3-甲基己酸(Takehara & Yoshida,1969)。随后研究了赖氨酸单加氧酶对该化合物的特异性，结果表明2,6-二氨基-3-甲基己酸对该酶无底物活性(Ohnishi et al.,1976)。截至目前，关于2,6-二氨基-3-甲基己酸的研究很少，没有涉及天然产物和植物免疫诱抗活性方面的相关研究、报道和专利。
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-苯基丁酸和2,6-二氨基-3-甲基 己酸作为植物免疫诱抗剂的应用。
我们从植物致病真菌—链格孢菌(Alternata sp.)中成功分离、纯化得到2-氨基-3-苯基丁酸和2,6-二氨基-3-甲基己酸。这是首次从天然微生物体中分离得到游离的2-氨基-3-苯基丁酸和2,6-二氨基-3-甲基己酸，且含量较高，证明其是两种新型的天然氨基酸。针对植物的诱抗活性研究发现，在抵抗生物胁迫方面，2-氨基-3-苯基丁酸和2,6-二氨基-3-甲基己酸均能有效抑制病毒、真菌和细菌在植物叶片上的发生与扩散；在诱导植物抗非生物胁迫方面，2-氨基-3-苯基丁酸和2,6-二氨基-3-甲基己酸均能有效缓解高温、低温、干旱和盐渍对植物的伤害。
本发明的目的可通过以下技术方案实现：
2-氨基-3-苯基丁酸和2,6-二氨基-3-甲基己酸均是从链格孢菌中分离得到的天然产物，2-氨基-3-苯基丁酸结构式如下：
Figure PCTCN2022136490-appb-000001
2,6-二氨基-3-甲基己酸结构式如下：
Figure PCTCN2022136490-appb-000002
2-氨基-3-苯基丁酸和/或2,6-二氨基-3-甲基己酸在制备植物免疫诱抗剂中的应用。
2-氨基-3-苯基丁酸和/或2,6-二氨基-3-甲基己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-苯基丁酸和/或2,6-二氨基-3-甲基己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-苯基丁酸和/或2,6-二氨基-3-甲基己酸在提高植物对真菌、细菌、病毒胁迫中的应用。
2-氨基-3-苯基丁酸和/或2,6-二氨基-3-甲基己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 物优选黑麦草、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-苯基丁酸和/或2,6-二氨基-3-甲基己酸。
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-苯基丁酸或2,6-二氨基-3-甲基己酸中的任意一种或多种；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-苯基丁酸或2,6-二氨基-3-甲基己酸的浓度为0.1-10000nM浓度。
2-氨基-3-苯基丁酸和2,6-二氨基-3-甲基己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，对于保障粮食生产安全、提高农产品竞争力具有重要的意义。本发明研究显示：2-氨基-3-苯基丁酸和2,6-二氨基-3-甲基己酸在相关诱导免疫、抗逆性实验中表现良好，能够提高植物对生物胁迫和非生物胁迫的抵抗能力。
由腐生真菌链格孢菌中分离的天然代谢产物2-氨基-3-苯基丁酸和2,6-二氨基-3-甲基己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、真菌和细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-苯基丁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-苯基丁酸处理的烟草植株的病情指数显著降低。在10nM的浓度下，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20.95、69.23％和0.10。
2,6-二氨基-3-甲基己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2,6-二氨基-3-甲基己酸处理的烟草植株的病情指数显著降低。在低浓度10nM时，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26.41、70.94％和0.12。
2-氨基-3-苯基丁酸用于防治小麦白粉病的方法，其在浓度10-10000nM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，在高浓度10000nM处理时，病情指数为31.85，相对免疫效果为66.92％。
2,6-二氨基-3-甲基己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，在高浓度10000nM处理时，病情指数为25.64，相对免疫效果为73.45％。
2-氨基-3-苯基丁酸用于田间预防小麦白粉病的方法，在1000nM处理浓度下小麦的病情指数为25.30，明显低于阿泰灵处理和助剂对照组，相对免疫效果和千粒重分别为51.72％和38.87g，均明显高于阿泰灵处理和助剂对照组。综上，2-氨基-3-苯基丁酸对小麦白粉病的发生与扩散有显著的抑制作用。
2-氨基-3-苯基丁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，当处理浓度为10000nM时，每毫克叶片中细菌个数为1.34×10 5，与空白对照相比细菌个数减少95.56％，病情指数为14.58。这一结果说明，2-氨基-3-苯基丁酸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。
2,6-二氨基-3-甲基己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，当处理浓度为10000nM时，每毫克叶片中细菌个数为1.58×10 5，与空白对照相比细菌个数减少95.11％，病情指数为19.86。这一结果说明，2,6-二氨基-3-甲基己酸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。
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-苯基丁酸用于提高植物对高温抵抗能力的方法，其浓度在100-10000nM之间的2-氨基-3-苯基丁酸溶液(加入体积百分比为0.02％的表面活性剂吐温20)对苗期拟南芥进行处 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-苯基丁酸溶液能有效缓解了高温对幼苗造成的伤害水平。
2,6-二氨基-3-甲基己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2,6-二氨基-3-甲基己酸溶液(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2,6-二氨基-3-甲基己酸溶液有效缓解了高温对幼苗造成的伤害水平。
2-氨基-3-苯基丁酸用于提高植物对低温的抵抗能力的方法，用100-10000nM浓度的2-氨基-3-苯基丁酸溶液(加入体积百分比为0.02％的表面活性剂吐温20)对茶叶幼苗进行叶面喷施处理，发现在-4℃低温胁迫24h后，经过100nM、1000nM和10000nM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-苯基丁酸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。
2,6-二氨基-3-甲基己酸用于提高植物对低温的抵抗能力的方法，用1-1000nM浓度的2,6-二氨基-3-甲基己酸溶液(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2,6-二氨基-3-甲基己酸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-苯基丁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-苯基丁酸溶液(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-苯基丁酸提高了小麦对干旱胁迫的抗性。
2,6-二氨基-3-甲基己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2,6-二氨基-3-甲基己酸溶液(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2,6-二氨基-3-甲基己酸提高了小麦对干旱胁迫的抗性。
2-氨基-3-苯基丁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-苯基丁酸溶液(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-苯基丁酸的处理组，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-苯基丁酸提高了棉花对盐的耐性水平。
2,6-二氨基-3-甲基己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2,6-二氨基-3-甲基己酸溶液(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2,6-二氨基-3-甲基己酸的处理组，棉花死亡率及盐害指数均低于对照组，这一结果说明2,6-二氨基-3-甲基己酸提高了棉花对盐的耐性水平。
技术先进性和有益效果
本发明的主要优点和积极效果如下：
2-氨基-3-苯基丁酸和2,6-二氨基-3-甲基己酸均为天然产物，结构简单，生物提取方式简便。由于本发明确认了2-氨基-3-苯基丁酸和2,6-二氨基-3-甲基己酸均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主要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-苯基丁酸和2,6-二氨基-3-甲基己酸均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1000nM时，就能够分别诱导小麦对白粉病产生55.38％和65.26％的相对免疫效果；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。在应对非生物胁迫方面，在浓度为100-10000nM时，能够诱导拟南芥对高温的抵抗能力，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-苯基丁酸和2,6-二氨基-3-甲基己酸用量低，安全环保，因而是高效生物农药，这表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。
2-氨基-3-苯基丁酸和2,6-二氨基-3-甲基己酸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-苯基丁酸和2,6-二氨基-3-甲基己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-苯基丁酸和2,6-二氨基-3-甲基己酸使用方便，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，减少碳排放。此外，由于2-氨基-3-苯基丁酸和2,6-二氨基-3-甲基己酸均为天然存在的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。
具体实施方式
发明人从链格孢菌中分离提纯得到了2-氨基-3-苯基丁酸和2,6-二氨基-3-甲基己酸，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；酵母膏，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-苯基丁酸：
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2-氨基-3-苯基丁酸,出峰时 间为7.9min，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2-氨基-3-苯基丁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)δ7.33-7.21(m,5H,Ph),3.81-3.66(dd,J 1＝5Hz,J 2＝10Hz,1H,CH-NH 2),3.45-3.09(m,1H,CHCH 3),1.29-1.25(dd,J 1＝10Hz,J 2＝10Hz,3H,CHCH 3)。
13C NMR(125MHz,Deuterium Oxide)δ173.78(CHCOOH),140.23(Ph),129.27(Ph),129.11(Ph),127.98(Ph),127.88(Ph),127.77(Ph),60.98(CHNH 2),40.80(CHCH 3),17.67(CHCH 3)。
质谱显示该化合物的分子离子峰为：180.1020[M+H] +，确定其分子式为：C 10H 13NO 2。结合核磁氢谱和碳谱的结果确定该化合物为2-氨基-3-苯基丁酸。
(4)HPLC法分离提纯2,6-二氨基-3-甲基己酸：
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210nm，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2,6-二氨基-3-甲基己酸，出峰时间为4.3min，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2,6-二氨基-3-甲基己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)δ12.13(br,1H,OH),8.34(br,2H,CHNH 2),3.83(d,J＝5Hz,1H,CHNH 2),2.63(t,J＝5Hz,2H,CH 2NH 2),1.53-1.19(m,4H,CH 2CH 2CH 2NH 2),1.11(d,J＝5Hz,3H,CHCH 3)。
13C NMR(125MHz,Deuterium Oxide)δ175.16(CHCOOH),59.51(CHCOOH),42.62(CH 2NH 2),36.27(CHCH 3),29.93(CH 2CH 2CH 2NH 2),28.82(CH 2CH 2CH 2NH 2),13.41(CHCH 3)。
质谱显示该化合物的分子离子峰为：161.1203[M+H] +，确定其分子式为：C 7H 16N 2O 2。结合核磁氢谱和碳谱的结果确定该化合物为2,6-二氨基-3-甲基己酸。
实施例2(2-氨基-3-苯基丁酸和2,6-二氨基-3-甲基己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，用处理液(10mM MgCl 2、10mM MES、10μM Acetosyringone)悬浮菌体，使悬浮液OD 600值为0.5，28℃避光处理3h后待用。取2-氨基-3-苯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-苯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24(±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：
Figure PCTCN2022136490-appb-000003
Figure PCTCN2022136490-appb-000004
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-苯基丁酸对番茄斑萎病毒侵染烟草的影响
Figure PCTCN2022136490-appb-000005
Figure PCTCN2022136490-appb-000006
表1结果表明，在2-氨基-3-苯基丁酸浓度范围为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35％以上。与未喷施2-氨基-3-苯基丁酸的对照组相比，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，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。例如在处理浓度为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20.95、69.23％和0.10。以上结果说明，2-氨基-3-苯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。
按照相同的方法考察2-氨基-3-苯基丁酸诱导烟草抗番茄斑萎病毒侵染效果，结果如表2：
表2 不同浓度2,6-二氨基-3-甲基己酸对番茄斑萎病毒侵染烟草的影响
Figure PCTCN2022136490-appb-000007
表2结果表明，在2,6-二氨基-3-甲基己酸浓度范围为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26.41、70.94％和0.12。以上结果说明，2,6-二氨基-3-甲基己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-苯基丁酸和2,6-二氨基-3-甲基己酸诱导小麦抗白粉病菌侵染)
取2-氨基-3-苯基丁酸用蒸馏水溶解后再用蒸馏水梯度稀释成10nM、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- 苯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3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3 2-氨基-3-苯基丁酸对小麦病情指数和相对免疫效果的影响
Figure PCTCN2022136490-appb-000008
表3的结果表明，随着2-氨基-3-苯基丁酸浓度的上升，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77.15、66.67、42.96和31.85，相对免疫效果为19.88％、30.77％、55.38％和66.92％。当2-氨基-3-苯基丁酸浓度大于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-苯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。
取2,6-二氨基-3-甲基己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。按照上述方法考察2-氨基-3-苯基丁酸诱导小麦抗白粉病菌侵染效果，结果如表4：
表4 不同浓度2,6-二氨基-3-甲基己酸对小麦病情指数和相对免疫效果的影响
Figure PCTCN2022136490-appb-000009
表4的结果表明，随着2,6-二氨基-3-甲基己酸浓度的上升，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69.41、49.27、33.55、25.64，相对免疫效果为28.13％、48.99％、65.26％和73.45％。当2,6-二氨基-3-甲基己酸浓度大于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2,6-二氨基-3-甲基己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4(2-氨基-3-苯基丁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-苯基丁酸溶液(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。施药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。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。
发现1000nM浓度的2-氨基-3-苯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显著低于助剂对照。且小麦的相对免疫效果及千粒 重显著高于助剂对照组(表5)。1000nM浓度2-氨基-3-苯基丁酸溶液处理后的小麦病情指数、相对免疫效果及千粒重分别为25.30、51.72％和38.87g，均明显好于阿泰灵处理。说明喷施2-氨基-3-苯基丁酸能够有效提高小麦对真菌病害白粉病的免疫能力。
表5 3种处理对小麦病情指数和相对免疫效果及千粒重的影响
Figure PCTCN2022136490-appb-000010
实施例5(2-氨基-3-苯基丁酸和2,6-二氨基-3-甲基己酸诱导拟南芥抗丁香假单胞菌侵染)
取2-氨基-3-苯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光照/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-苯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6 不同浓度2-氨基-3-苯基丁酸对叶片中细菌个数和病情指数的影响
Figure PCTCN2022136490-appb-000011
表6结果表明，随着2-氨基-3-苯基丁酸浓度的上升，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92.05％、92.94％和95.56％，病情指数分别降低了52.57％、58.45％和81.82％。说明2-氨基-3-苯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2-氨基-3-苯基丁酸诱导拟南芥抗丁香假单胞菌侵染效果，结果如表7所示：
表7 不同浓度2,6-二氨基-3-甲基己酸对叶片中细菌个数和病情指数的影响
Figure PCTCN2022136490-appb-000012
表7结果表明，随着2,6-二氨基-3-甲基己酸浓度的上升，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73.93％、84.15％和95.11％，病情指数分别降低50.96％、56.15％和76.82％。说明2,6-二氨基-3-甲基己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6(2-氨基-3-苯基丁酸和2,6-二氨基-3-甲基己酸诱导拟南芥抗高温胁迫)
取2-氨基-3-苯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，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。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22℃、湿度60％-70％、光强100μmol m -2s -1(16h光照/8h黑暗)的温室中种植。在拟南芥苗期21d时开始进行处理，处理的方法为叶面喷施2-氨基-3-苯基丁酸溶液，24h喷施两次。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后常温暗处理30min后用植物效率Handy-PEA测定拟南芥叶片的叶绿素荧光，然后将植株取出转移至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1，热害指数计算公式如下。热害及荧光参数结果见表8。
Figure PCTCN2022136490-appb-000013
表8 热害分级标准
Figure PCTCN2022136490-appb-000014
表9 2-氨基-3-苯基丁酸对高温胁迫下拟南芥的影响
Figure PCTCN2022136490-appb-000015
表9的结果表明，经过2-氨基-3-苯基丁酸处理的拟南芥在高温胁迫后的光合性能指数PI ABS显著高于未经2-氨基-3-苯基丁酸的处理组。热害指数随着处理浓度的增加而降低。其中 10000nM的效果最好，该浓度处理下拟南芥的光合性能指数PI ABS升高134％，热害指数降低了56％。可见，2-氨基-3-苯基丁酸能够缓解高温胁迫对拟南芥植株光合系统的伤害，提高拟南芥对高温胁迫的抵抗能力。
实验设置2,6-二氨基-3-甲基己酸浓度为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。其他方法同2-氨基-3-苯基丁酸，考察2,6-二氨基-3-甲基己酸诱导拟南芥抗高温胁迫的效果，结果见表10：
表10 2,6-二氨基-3-甲基己酸处理对高温胁迫下拟南芥的影响
Figure PCTCN2022136490-appb-000016
表10的结果表明，在高温胁迫条件下，经过2,6-二氨基-3-甲基己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2,6-二氨基-3-甲基己酸浓度的升高，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36倍，而热害指数降低了64％。由此可见，2,6-二氨基-3-甲基己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7(2,6-二氨基-3-甲基己酸诱导黑麦草抵抗高温胁迫)
取2,6-二氨基-3-甲基己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2,6-二氨基-3-甲基己酸溶液，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11。
Figure PCTCN2022136490-appb-000017
表11 2,6-二氨基-3-甲基己酸对高温胁迫下黑麦草的影响
Figure PCTCN2022136490-appb-000018
表11的结果表明，经过2,6-二氨基-3-甲基己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69％。可见，2,6-二氨基-3-甲基己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8(2-氨基-3-苯基丁酸和2,6-二氨基-3-甲基己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、100、1000和10000nM，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中拟南芥的处理方法相同，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12，计算公式如下，结果如表13所示。
Figure PCTCN2022136490-appb-000019
表12 冷害分级标准
Figure PCTCN2022136490-appb-000020
表13 2-氨基-3-苯基丁酸处理对低温胁迫下茶叶的影响
Figure PCTCN2022136490-appb-000021
表13的结果表明，在低温胁迫条件下，经过2-氨基-3-苯基丁酸处理的茶叶光合性能指数PI ABS均显著上升，冷害指数明显下降。其中以10000nM的效果最佳，该浓度处理的茶叶PI ABS提高了147％，冷害指数降低了35％。可见，2-氨基-3-苯基丁酸显著缓解了低温胁迫对茶叶幼苗光合系统结构和功能的伤害，提高了茶叶对低温胁迫的抵抗能力。
实验设置2,6-二氨基-3-甲基己酸浓度分别为0、1、10、100和1000nM，同时加入0.02％吐温20作为表面活性剂，按照上述方法考察2,6-二氨基-3-甲基己酸诱导茶树抗低温胁迫效果，结果如表14：
表14 2,6-二氨基-3-甲基己酸处理对低温胁迫下茶叶的影响
Figure PCTCN2022136490-appb-000022
Figure PCTCN2022136490-appb-000023
表14的结果表明，经过2,6-二氨基-3-甲基己酸处理，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121％，冷害指数降低了45％。由此可见，2,6-二氨基-3-甲基己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8：2-氨基-3-苯基丁酸和2,6-二氨基-3-甲基己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-苯基丁酸溶液，2-氨基-3-苯基丁酸浓度为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16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15。
Figure PCTCN2022136490-appb-000024
表15 旱害分级标准
Figure PCTCN2022136490-appb-000025
表16 2-氨基-3-苯基丁酸处理对干旱胁迫下小麦生物量与旱害指数的影响
Figure PCTCN2022136490-appb-000026
Figure PCTCN2022136490-appb-000027
表16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-苯基丁酸处理使小麦幼苗根长显著增长了11.87％，地上和地下部分鲜重分别增加了46.33％和55.14％，旱害指数降低了46％。这说明2-氨基-3-苯基丁酸能提高小麦抗干旱胁迫的能力。
按照上述相同方法考察2,6-二氨基-3-甲基己酸诱导小麦抗干旱胁迫效果，结果见表17：
表1:7 2,6-二氨基-3-甲基己酸处理对干旱胁迫下小麦生物量与旱害指数的影响
Figure PCTCN2022136490-appb-000028
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2,6-二氨基-3-甲基己酸处理使小麦幼苗根长显著增加了9.77％，地上和地下部分鲜重分别提高了31.03％和34.42％，旱害指数降低了59％。这说明2,6-二氨基-3-甲基己酸能提高小麦抗干旱胁迫的能力。
实施例9：2-氨基-3-苯基丁酸和2,6-二氨基-3-甲基己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-苯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：
Figure PCTCN2022136490-appb-000029
表18 盐害分级标准
Figure PCTCN2022136490-appb-000030
表19 2-氨基-3-苯基丁酸处理对盐胁迫下棉花的影响
Figure PCTCN2022136490-appb-000031
表19的结果表明，棉花的盐害指数随着2-氨基-3-苯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42％和28％。以上结果说明2-氨基-3-苯基丁酸能够诱导棉花对盐胁迫产生较好的抗性。
按照上述方法考察2-氨基-3-苯基丁酸诱导棉花抗盐胁迫的效果，结果见表20：
表20 2,6-二氨基-3-甲基己酸处理对盐胁迫下棉花的影响
Figure PCTCN2022136490-appb-000032
表20的结果表明，棉花的盐害指数随着2,6-二氨基-3-甲基己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36％ 和23％。以上结果说明2,6-二氨基-3-甲基己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-苯基丁酸和2,6-二氨基-3-甲基己酸也具有与生物提取的2-氨基-3-苯基丁酸和2,6-二氨基-3-甲基己酸相同的效果。2-氨基-3-苯基丁酸和2,6-二氨基-3-甲基己酸的制备方法不影响其作为免疫诱抗剂的应用和效果。

Claims (10)

  1. 2-氨基-3-苯基丁酸和/或2,6-二氨基-3-甲基己酸在制备植物免疫诱抗剂中的应用。
  2. 2-氨基-3-苯基丁酸和/或2,6-二氨基-3-甲基己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。
  3. 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。
  4. 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。
  5. 一种植物免疫诱抗剂，其特征在于，包含组分A：2-氨基-3-苯基丁酸和2,6-二氨基-3-甲基己酸中的任意一种或多种；组分B：表面活性剂。
  6. 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。
  7. 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)。
  8. 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-苯基丁酸或2,6-二氨基-3-甲基己酸的浓度为0.1-10000nM浓度。
  9. 一种提高植物对生物和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-苯基丁酸或2,6-二氨基-3-甲基己酸；亦或权利要求5-8中任一项所述的植物免疫诱抗剂。
  10. 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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