CN113841698B

CN113841698B - Application of citral or plant extract containing citral in preparing plant resistance inducer

Info

Publication number: CN113841698B
Application number: CN202111056526.8A
Authority: CN
Inventors: 闫合; 姜悦; 何姗; 姬晓兰; 罗伟; 刘斌; 马志卿; 冯俊涛
Original assignee: Northwest A&F University
Current assignee: Northwest A&F University
Priority date: 2021-09-09
Filing date: 2021-09-09
Publication date: 2023-04-18
Anticipated expiration: 2041-09-09
Also published as: CN113841698A

Abstract

The invention discloses application of citral and/or a plant extract containing citral in preparation of a plant resistance inducer, wherein the application range of the plant resistance inducer comprises the induction of plant disease resistance, the induction of plant stress resistance or the promotion of plant growth. The invention utilizes the methods of biological activity determination, physiological and biochemical tests and the like to determine the plant disease resistance induction activity, the plant stress resistance induction activity and the plant growth promotion activity of the citral and the plant essential oil or the extract containing the citral, and prepares a pesticide preparation, an auxiliary agent and a biostimulant which take the citral and the plant essential oil or the extract containing the citral as active ingredients and applies the pesticide preparation, the auxiliary agent and the biostimulant in fields.

Description

Application of citral or plant extract containing citral in preparing plant resistance inducer

Technical Field

The invention belongs to the field of plants and application thereof, and particularly relates to application of citral or a plant extract containing citral in preparation of a plant resistance inducer.

Background

Plant essential oils are widely found in the natural world, and most plant families contain plant essential oils, such as Magnoliaceae (Magnoliaceae), gramineae (Grannieae), piperaceae (Piperaceae), and the like, which are rich in essential oils. Plant essential oils are complex mixtures, usually composed of a wide variety of organic compounds, ranging from large polar, small polar and non-polar substances. Terpene substances consisting of monoterpenes and sesquiterpenes are common in essential oils, but the specific ingredients are different from one essential oil to another. It is the main component of various plant essential oils; the content of aromatic compounds (aldehyde, alcohol, methoxyl derivatives, etc.) is second to that of terpene compounds, such as cinnamon essential oil with cinnamaldehyde content of about 80%, litsea cubeba essential oil with citral content of about 70%, etc.

Citral is one of the most important representatives of open-chain monoterpenes, of formula C ₁₀ H ₁₆ O with molecular weight of 152.23, boiling point of 229 ℃ and density of 0.889g/cm ³ The citral is colorless or yellowish liquid, has strong lemon flavor, no optical activity, and has two cis-trans isomers. Citral is soluble in oil, propylene glycol and ethanol, insoluble in glycerol and water, and naturally occurring in lemongrass oil (70-80%), litsea cubeba oil (about 70%), lemon oil, lime oil, citrus leaf oil, etc. Citral has a wide range of uses, and is used in various aspects requiring lemon aroma. The essence is important spices of lemon type essence, deodorant wood type essence, artificially prepared lemon oil, bergamot oil and orange leaf oil, is a raw material for synthesizing ionones and methyl ionones, can be used for covering bad smell in industrial production, can be used as edible essences of ginger, lemon, white lemon, sweet orange, grapefruit, apple, cherry, grape, strawberry, spice and the like, and can also be used as essence for wine. Pharmacological research in recent years shows that citral has various pharmacological effects such as antitumor effect, pain relieving effect, and spasmolysis. In the agricultural aspect, a certain bacteriostatic action of citral on plant pathogenic fungi and the like is reported in documents, and related research reports on the effects of inducing plant disease resistance activity, inducing plant stress resistance activity and promoting plant growth activity are not available, so that the application of citral in the fields of agriculture, horticulture and forestry is to be further developed.

Disclosure of Invention

The invention utilizes the methods of biological activity determination, physiological and biochemical tests and the like to determine the plant disease resistance induction activity, the plant stress resistance induction activity and the plant growth promotion activity of the citral and the plant extract taking the citral as the main component, and prepares the pesticide preparation, the auxiliary agent and the biostimulant taking the citral and/or the plant extract taking the citral as the main component as the active components, and the pesticide preparation, the auxiliary agent and the biostimulant are applied to the field.

The invention discloses a plant resistance inducer prepared by using citral and/or plant extract with citral as main component and its application in agricultural production. The application range of the plant growth promoter comprises the activities of inducing plant disease resistance, inducing plant stress resistance and promoting plant growth. The plant resistance inducer provided by the invention can induce and improve the resistance of plants to fungal diseases, bacterial diseases, nematode diseases, pests, virus diseases and high-temperature, low-temperature and drought stress, and can promote the growth of the plants.

The invention discloses a pesticide adjuvant prepared by using citral and/or a plant extract taking citral as a main component and application of the pesticide adjuvant in agricultural production.

The invention discloses a method for preparing biostimulant by using citral and/or plant extract with citral as main ingredient, and application of biostimulant in agricultural production.

Optionally, the citral is one or a combination of cis-citral neral and trans-citral geranial, wherein the citral neral and trans-citral geranial have the following structures:

optionally, the plant extract containing citral is selected from Litsea cubeba essential oil, verbena officinalis essential oil, cinnamomum camphora essential oil, cymbopogon citratus essential oil, and Ocimum gratissimum essential oil; one or a combination of fructus Litseae extract, litsea cubeba extract, herba Verbenae extract, cinnamomum camphora extract, cymbopogon citratus extract and Ocimum gratissimum extract.

A plant resistance inducer comprises citral and/or plant extract containing citral, and is processed by adding adjuvants, and the preparation form is soluble powder, water dispersible granule, soluble liquid, water emulsion, microemulsion and all agriculturally acceptable preparation types;

the content of the citral is 1-100% by mass.

according to the mass percentage, the content of the citral is 0.1-1%.

the content of citral is 30% by mass.

A composition having an activity of inducing resistance in plants, comprising citral and/or a citral-containing plant extract and a mixture of one or more other commercial pesticides, wherein the citral content is 0.1-99% by mass; the commercial pesticides include plant decoys, fungicides, bactericides, antivirals, insecticides, nematicides, acaricides and combinations thereof.

A pesticide adjuvant comprises citral and/or a plant extract containing citral, wherein the content of the citral is 0.1-100 wt%.

A biostimulant comprising citral and/or a citral-containing plant extract. According to the mass percentage, the content of the citral is 0.1-100%.

A method for enhancing the immunocompetence of a plant, said method comprising the steps of: the product according to the invention is applied to at least one of the plant, the area adjacent to the plant, the soil suitable for supporting the growth of the plant, the roots and the leaves of the plant.

The induced plant disease-resistant activity is essentially to improve the disease-resistant capability of the plant, is broad-spectrum disease-resistant activity, and can be used for enhancing the disease-resistant capability of the plant to bacterial, fungal, viral, oomycete and nematode infection. The nature of the stress resistance activity of the induced plant is to induce the stress resistance potential of the plant, and can be used for enhancing the drought resistance, cold resistance, salt resistance, disease resistance and other capabilities of the plant under the stress of biological factors such as plant diseases and insect pests, weeds and the like and physicochemical factors such as temperature, moisture, salt and alkali, chemical factors, weather and the like. The plant resistance inducer is one of pesticides, can also be called as a plant immune activator and the like, has no direct bactericidal activity, can control and prevent the invasion of harmful organisms such as fungi, bacteria, viruses, nematodes, insects and the like on plants by inducing the plants to generate disease-resistant activity or stress-resistant activity, has the advantages of difficult generation of resistance by pathogenic bacteria, relatively wide control spectrum, capability of being mixed with chemical agents and the like, and is a pesticide meeting the requirements of green prevention and control.

Drawings

The accompanying drawings, which are included to provide a further understanding of the disclosure and are incorporated in and constitute a part of this specification, illustrate embodiments of the disclosure and together with the description serve to explain the disclosure, but do not constitute a limitation of the disclosure. In the drawings:

FIG. 1 shows the change of POD activity in vivo after citral spraying on tobacco leaves;

FIG. 2 is the activity change of PAL in vivo after the tobacco leaves are sprayed with citral;

FIG. 3 shows the in vivo H of tobacco leaves sprayed with citral ₂ O ₂ A change in content;

FIG. 4 shows the change of in vivo disease-resistant gene expression after spraying citral on tobacco leaves;

FIG. 5 shows the activity of tobacco mosaic virus resistance of tobacco leaves after spraying citral, note: a-CK; b-citral 200. Mu.g/ml; c-400 mu g/ml; d-800. Mu.g/ml;

FIG. 6 shows that after the citral is sprayed on strawberry leaves, the strawberry botrytis cinerea resistance activity is as follows: a-CK; b-citral 200. Mu.g/ml; c-400 mug/ml; d-800. Mu.g/ml; e-carbendazim 400 mu g/ml;

fig. 7 shows cucumber powdery mildew resistance activity of cucumber leaves sprayed with citral, note: a-CK; b-citral 200. Mu.g/ml; c-400 mug/ml; d-800. Mu.g/ml; e-carbendazim 400 mu g/ml;

FIG. 8 shows the change of root growth after spraying citral to wheat, note: a-CK; b-citral 200. Mu.g/ml; c-400 mu g/ml; d-800. Mu.g/ml;

fig. 9 shows that the apple is sprayed with citral biostimulant to promote precocity, and the apple is injected with the following components: a-CK; b-citral 200. Mu.g/ml; c-400 mu g/ml; d-800. Mu.g/ml.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The compound citral of the present disclosure can be obtained by a variety of techniques, such as chemical synthesis methods; as another example, from plants containing citral, the extraction method can be by any of a variety of known techniques, such as: supercritical fluid extraction, solvent (ethanol, methanol, acetone, etc.) thermal reflux extraction, solvent (ethanol, methanol, acetone, etc.) percolation, etc. Citral used in the present disclosure was purchased from a chemical platform.

In another embodiment of the invention, the litsea cubeba extract, the verbena extract, the cinnamomum camphora extract, the lemongrass extract and the Ocimum gratissimum extract are respectively prepared by an ethanol percolation method; respectively preparing Litsea cubeba essential oil, verbena essential oil, cinnamomum camphora essential oil, cymbopogon citratus essential oil and Ocimum gratissimum essential oil by supercritical fluid extraction, and measuring citral content. Plant essential oils also belong to one of the plant extracts.

The biological activity test result and the physiological and biochemical test result in the embodiment of the invention prove that the citral, the plant essential oil containing the citral and the extract of the citral have the activities of inducing plant disease resistance, inducing plant stress resistance and promoting plant growth. One skilled in the art will appreciate that the results of the biological activity assay and physiological and biochemical experiments establish the general utility of citral and citral-containing plant extracts as elicitors.

The citral and citral-containing plant extracts of the present disclosure can be applied by any of a variety of known techniques as a formulation comprising the citral and/or citral-containing plant extracts. For example, the compounds may be applied to the roots or foliage of plants to induce plant resistance or to promote plant growth without compromising the commercial value of the plant. Any of the citral and/or citral-containing plant extracts can be applied in the form of any of the commonly used formulation types, for example as a solution, powder, suspension, wettable powder, soluble liquor, flowable concentrate, or emulsifiable concentrate, including but not limited to: the seed treatment emulsion, the aqueous emulsion, the macrogranule, the microemulsion, the water-soluble emulsion, the water-dispersible granule, the poison valley, the aerosol, the block poison bait, the slow-release block, the concentrated poison bait, the capsule granule, the microcapsule suspension, the dry-mixed seed powder, the missible oil, the electrostatic spray, the water-in-oil emulsion, the oil-in-water emulsion, the smoke tank, the fine granule, the smoke candle, the smoke cylinder, the smoke rod, the seed treatment suspension, the smoke tablet, the smoke pill, the granular poison bait, the hot fogging concentrate, the medical paint, the fine granule, the oil suspension, the oil-dispersible powder, the flaky poison bait, the concentrated colloid, the pouring agent, the seed coating agent, the smearing agent, the suspension emulsion, the film-forming oil agent, the soluble powder, the seed treatment water-soluble powder, the ultra-low-capacity suspension, the tracing powder, the ultra-low-capacity liquid and the wet-mixed seed water-dispersible powder.

Preferably, the citral and citral-containing plant extracts of the present disclosure are applied in the form of a formulation comprising citral and/or citral-containing plant extracts with a phytologically acceptable carrier. The concentrated formulation may be dispersed in water or other liquid for application, or the formulation may be dusty or granular. The formulations may be prepared according to procedures conventional in the agrochemical art. The present disclosure contemplates all vehicles by which plant extracts containing citral and/or citral as a major component can be formulated for delivery and use as decoy agents, including all phytologically acceptable inert carriers, surfactants, emulsifiers, organic solvents or water, and the like.

The formulations may optionally include combinations containing other pesticidal or other compounds having decoy activity. Such additional pesticidal compounds or other compounds having decoy activity may be fungicides, insecticides, herbicides, nematocides, miticides, arthropodicides, bactericides, plant decoys or combinations thereof that are compatible and not antagonistic with citral and/or the citral-containing plant extract in the medium selected for application. Thus, in such embodiments, another pesticidal compound or inducer active compound is used as a supplemental agent. The citral and/or the citral-containing plant extract and the further compound in the combination may generally be present in a ratio of 1:100 to 100:1 is present in a weight ratio.

Another embodiment of the invention is to use citral and/or plant extracts containing citral as main ingredient to prepare pesticide adjuvant and its application in agricultural production. The essence of preparing the pesticide adjuvant by using the citral and/or the plant extract taking the citral as the main component lies in that the resistance inducing activity of the citral and/or the plant extract taking the citral as the main component is utilized. When the commercial pesticide including plant inducer, fungicide, bactericide, antiviral agent, insecticide, nematicide and acaricide is applied in the field, citral and/or plant extract with citral as main component are added, so that the plant resistance can be improved while the plant diseases and insect pests are directly killed, and the control effect of other commercial pesticides is increased. The mode of addition may be tank mixing.

Another embodiment of the invention is the use of citral and/or plant extracts containing citral as the main ingredient for the preparation of biostimulant and its application in agricultural production. It is also essential to utilize the resistance-inducing activity of citral and/or a plant extract containing citral as a main ingredient.

Another embodiment of the invention is a method of applying citral and/or a plant extract with citral as a major component for protecting plants from infestation by pests, comprising applying citral and/or a plant extract with citral as a major component to at least one of the plants, areas adjacent to the plants, soil suitable for supporting the growth of the plants, roots, and leaves of the plants.

For better understanding of the essence of the invention, the technical contents of the invention will be described in detail with examples, but the invention is not limited to these examples.

Example 1: measurement of anti-TMV Activity of citral and citral-containing plant extract

(1) Protective Activity

Preparing 0.02mg/mL, 0.1mg/mL and 0.5mg/mL citral, and essential oil and extract solution containing citral, selecting healthy 5-6 leaf-stage heart-leaf tobacco, spraying the medicinal preparation for 48h, inoculating TMV solution diluted 2000 times, treating blank with clear water, and treating positive control with 0.1mg/mL chitosan oligosaccharide. 3 leaves were inoculated per treatment, repeated 3 times, and after 3 days, the number of scorched spots was counted and the inhibition rate was calculated.

(2) In vitro inactivation Activity

Preparing 0.02mg/mL, 0.1mg/mL and 0.5mg/mL citral, and citral-containing essential oil and extract solution, and mixing with 1000 times diluted TMV solution in equal volume. Standing at room temperature for 1h, and inoculating to healthy 5-6 leaf stage heart leaf tobacco. The blank control is clear water treatment, and the positive control is 0.1mg/mL chitosan oligosaccharide. 3 leaves were inoculated per treatment, repeated 3 times, and after 3 days, the number of scorched spots was counted and the inhibition rate was calculated.

(3) Therapeutic Activity

Preparing 0.02mg/mL, 0.1mg/mL and 0.5mg/mL citral, and essential oil and extract solution containing citral, selecting healthy 5-6 leaf-stage heart-leaf tobacco, inoculating 2000 times of diluted TMV solution for 48h, and spraying medicinal preparation. The blank control is clear water treatment, and the positive control is 0.1mg/mL chitosan oligosaccharide. 3 leaves were inoculated per treatment, repeated 3 times, and after 3 days, the number of scorched spots was counted to calculate the inhibition rate.

Inhibition (%) = (number of control scorched spots-number of treated scorched spots)/number of control scorched spots × 100

The results are shown in Table 1.

Table 1: protective, deactivating and therapeutic effects of citral and essential oils and extracts containing citral on TMV

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Note: data are mean ± sem, significant differences between 3 activities were determined by Duncan Multiple Range Test (DMRT) in SPSS software, with different letters indicating significant differences at the 0.05 level between data.

As can be seen from Table 1, citral has good protective effect on TMV, and the disease prevention effect is as high as 76.27%, which indicates that advance application of the composition can make tobacco have certain disease prevention effect. The common passivation effect of citral on TMV indicates that citral does not cause direct damage or has weak damage effect on TMV mitochondria, and is far less suitable for disease resistance caused by early application. The plant extracts containing citral also have certain antiviral activity, wherein the litsea cubeba essential oil and the extracts are highlighted in the embodiment 2: measurement of anti-TMV activity of tobacco induced by citral, essential oil containing citral and extract

The heart-leaf tobacco is selected to be subjected to induced disease-resistant activity verification, the heart-leaf tobacco can form virus withered spots, the symptoms of common tobacco are flower leaves, and different symptoms are counted by different methods. Spraying 0.02mg/mL, 0.1mg/mL and 0.5mg/mL citral and essential oil containing citral and the extract to the lower three leaves of tobacco with consistent growth and 6-7 leaf stage. TMV was inoculated to the non-sprayed upper leaves after 48 h. The blank control is treated with clear water, and the positive control is chitosan oligosaccharide solution. Each plant was inoculated with 2-3 leaves, each treatment included 10 tobacco plants, and the entire experiment was repeated 3 times. And after 3d, counting the disease index of the number of the heart-leaf tobacco withered spots, wherein the formula for calculating the inhibition rate is as above, and the formula for calculating the prevention and treatment effect is as below.

Disease index = (number of disease stages × number of diseased plants)/(highest disease stage × total number of treated plants) × 100;

control effect = (control disease means-treatment disease means)/control disease means × 100%;

the results are shown in Table 2.

Table 2: induced disease resistance effect of citral and essential oil and extract containing citral on TMV

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Note: data are mean ± sem, significant differences between 3 activities were tested by Duncan Multiple Range Test (DMRT) in SPSS software, and different letters indicated significant differences at the 0.05 level between data.

As can be seen from Table 2, on the tobacco leaf, citral and the citral-containing plant extract showed significant effects in inducing plants to resist TMV, indicating that topical application of citral can induce disease resistance in non-applied parts of tobacco.

Example 3: citral causes activity change of tobacco defense enzyme POD

After spraying 0.02mg/mL, 0.1mg/mL and 0.5mg/mL citral to the leaves, the leaves were harvested at 1,3,5,7,9 and 11 days, respectively, and the activity changes of the defense enzymes PAL and POD were examined. Weighing 5g of plant leaves, shearing the plant leaves into pieces, placing the pieces into a frozen mortar, adding a small amount of quartz sand, and adding 5mL of 0.1mol/L acetic acid-sodium acetate buffer solution with the pH value of 5.5 for 2-3 times. Grinding into homogenate, and centrifuging at 12,000r/min for 15min at 4 deg.C to obtain supernatant as crude enzyme extract. Adding 3mL 25mmol/L Guoguephenol and 5mL enzyme extract into test tube, adding 200 μ L,5mol/L H ₂ O ₂ The solution was mixed rapidly to start the reaction. Starting to record the value of the reaction system at 470nm wavelength at the time of reaction for 15s, using distilled water as referenceThe measurement was continuously performed every 1min, and data was obtained at 6 points at least. The experiment was repeated three times. The enzyme activity was calculated as follows: OD ₄₇₀ ＝OD _470F —OD _470I Tp to ti, where OD _470F -reaction liquid end value; OD _470I -a reaction liquid initial value; tp-reaction termination time, min; ti-reaction start time, min. 1 peroxidase Activity Unit at 1 increase in the change in absorbance per minute per gram of sample, Δ OD ₄₇₀ Min g. The calculation formula is as follows: u (Delta A) ₄₇₀ ·g ^-1 min ^-1 )＝[ΔA ₄₇₀ X total amount of enzyme extract]/[ fresh weight of sample X amount of enzyme solution at the time of measurement]

The results are shown in FIG. 1.

Example 4: citral causes changes in the activity of the tobacco defense enzyme PAL

Taking 1.25g tobacco leaf, adding 5mL enzyme extract (0.1 mol/L pH8.8 Tris-H) ₂ SO ₄ Buffer) and 5g of polyvinylpyrrolidine (PVP), homogenized with a mortar or tissue triturator. After filtration, the filtrate was centrifuged at 10,000 g at 4 ℃ for 30min, and the supernatant was taken to measure the volume. Taking 1mL of 0.1mol/L phenylalanine solution and 2mL of 0.1mol/L Tris-H ₂ SO ₄ Buffer (pH 8.8) (3 mL of buffer was directly taken in a control tube without substrate phenylalanine) was placed in the test tube and incubated for 3min in a 30 ℃ water bath. 5mL of enzyme solution to be detected is added into each test tube, no enzyme solution is added into a blank tube, the initial value is measured at the wavelength of 209nm immediately after shaking up, the blank tube is used for zero setting, and the time is accurately counted. Placing each test tube in 30 deg.C water bath, keeping temperature, reacting for 30min, and measuring A again ₂₀₉ . The amount of enzyme required to increase the optical density at 290nm per hour by 0.01 is 1 unit U (g. Fw. H). U = (Δ a × total volume of extract)/(0.01 × T × W × amount of enzyme solution used measured), where Δ a is the difference in absorbance between 2 measurements before and after; w is the sample fresh weight (g); t is the reaction time (30 min).

Citral causes significant changes in the activity of defensive enzymes in tobacco leaves (see figures 1, 2). The POD activity remained stable or increased slowly over 1 to 3 days, and peaked at 5 to 7 days. POD was reported to eliminate peroxides in plants, indicating that POD begins to exert its activity in large amounts after 5 days,counteract excess H ₂ O ₂ And other active oxygen, so as to avoid active oxygen damage to the plants. PAL is a key enzyme in defense, and has been reported to have a key effect on the development of disease resistance. PAL shows a rapid and obvious increasing trend within 0-3 days, reaches the highest value at day 3, and causes the generation and transmission of disease resistance reaction and other disease resistance signals in plants.

Example 5: citral causes tobacco H ₂ O ₂ Content change of

5g of the leaf was taken, 3mL of precooled acetone and a little quartz sand were added, ground into a slurry on an ice bath, and centrifuged at 4 ℃ for 20min under the condition of 12,000g. Taking 1mL of supernatant, adding the reagents according to the following table 4 in sequence, repeatedly washing and centrifuging the obtained precipitate with precooled acetone for 2-3 times (3000 g, 10min each time, discarding the supernatant and retaining the precipitate) until the precipitate has no color of the photosynthetic pigment. Then, 3mL of sulfuric acid was added to the precipitate to dissolve the precipitate for colorimetric determination. Establishing standard curve to calculate H in blade ₂ O ₂ The calculation formula is as follows:

H ₂ O ₂ (nmol/g·Fw)＝(n×V)/(v×m)

wherein n is H calculated from the standard curve ₂ O ₂ The amount (nmol) of (C); v is sample supernatant volume (mL); v is the volume of sample supernatant (mL) used for color development, 1mL in this experiment; and m is the fresh weight (g) of the sample.

Making a standard curve: taking 6 test tubes, numbering, adding each reagent according to the table 3 in a fume hood, mixing uniformly, reacting for 5min, centrifuging at 12,000g under centrifugal force for 15min at 4 ℃, and leaving precipitate. Hydrochloric acid (3 mL) was added and shaken to dissolve the precipitate. The absorbance of the solution was measured colorimetrically at a wavelength of 412nm, using a 0-th tube as a control and zeroing. With H ₂ O ₂ The amount (nmol) of (A) is plotted on the abscissa and the OD value is plotted on the ordinate to prepare a calibration curve.

Table 3: preparation of H ₂ O ₂ Standard curve of assay

The results are shown in FIG. 3, where citral can cause H in tobacco plants ₂ O ₂ The content was significantly changed. 3d after application, H ₂ O ₂ The content increases rapidly and reaches the peak value, and after 5d, the content gradually decreases.

Example 6: changes of tobacco disease-resistant gene expression quantity caused by citral

Spraying a citral solution on the tobacco in the 4-6 leaf stage, and collecting samples every other day within 1-10 days after treatment. Extracting total RNA of tobacco by a liquid nitrogen method, and determining the expression change of disease-resistant related genes NPR1, PR1 and PR2 by real-time fluorescent quantitative PCR.

The result is shown in figure 4, the citral can cause the transcription level of PR protein of the disease-resistant related gene to be obviously changed (wherein, the expression levels of NPR1, PR1 and PR2 are respectively increased by 5.25,6.57 and 5.81 times compared with the control group, which indicates that the citral induces the tobacco to generate disease-resistant defense behavior in vivo.

Example 7: citral and essential oil and extract preparations containing citral for field plot control of tobacco mosaic virus

The cell test is randomly arranged and repeated for 3 times, the area of the cell is equal to 60 square meters, the selection of test areas requires uniform fertility and consistent crop planting and management level, and protection rows are arranged among treatment rooms and around the test areas. Spraying onto leaf surface, spraying onto leaf surface with 500 times of Altailing solution as control agent, and setting clear water as control. All test agents must be diluted twice. Spraying the medicine from the 4-5 leaf stage of the heart leaf tobacco, and then spraying the medicine for 3 times at 4d intervals. And after spraying for 2d for the last time, taking the whole top leaf, performing friction inoculation on TMV, inoculating 3 leaves on each plant, repeating the steps for three times when 10 plants are treated, examining the disease index of each treatment after 10d of virus inoculation, and calculating the control effect.

Disease grading standard a tobacco mosaic virus severity grading survey method (YC/T39-1996) according to the national tobacco industry standard:

stage 0: the whole plant is disease-free;

level 1: the heart and leaf vein is clear or the flower and leaf are slight, or the upper 1/3 of the leaf leaves are not deformed, and the plant is not obviously dwarfed;

stage 2: 1/3 to 1/2 of the leaf or a few leaves are deformed; or the main pulse is changed into black, the plant is dwarfed by more than 2/3 of the normal plant height;

and 3, level: when flowers bloom 1/2 to 2/3, or the main side pulse is changed to black, the plant is dwarfed to 1/2 to 2/3 of the normal plant height;

4, level: the whole leaf has severe deformity or necrosis, and the diseased plant is dwarfed to l/3 to 1/2 of the normal plant height. For refining the investigation result, on the basis of the above severity grading, the grading standard is refined, i + grade is added between 1 and 2 grades, 2+ grade is added between 2 and 3 grades, 3+ grade is added between 3 and 4 grades, and the grades are marked as 1.5, 2.5 and 3.5:

stage 1 +: the vein of heart leaves is clear or slight flower leaves, or the upper 1/3 leaf leaves are slightly shriveled, and plants are not obviously dwarf;

2+ level: l/3 to 1/2 of leaf leaves, leaf deformation or main pulse blackening, and dwarfing the plants to more than 2/3 of normal plants;

3+ level: 1/2 to 2/3 of leaf mosaic, or deformation or necrosis of main side vein, or plant dwarfing to 1/2 of normal plant height.

And calculating the disease index according to the severity, and taking the prevention and treatment effect as the measure of the effect of different treatments.

Disease index = ∑[ (number of infected plants × graded representative value of severity)/(total number of examined plants × highest representative value of severity) ] × 100%

Control effect% = ((control average disease index-treatment average disease index)/control average disease index) × 100%

The results are shown in Table 4 and FIG. 5.

Table 4: citral preparation and field plot pesticide effect test of citral-containing essential oil and extract preparation for preventing and treating tobacco virus diseases

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From the above table, it can be seen that the citral preparation and the preparations containing the essential oil and extract of citral have good control effect on tobacco mosaic virus.

Example 8: test of field plot pesticide effect of citral preparation, and citral-containing essential oil and extract on strawberry gray mold

Greenhouse strawberries are selected for plot experiments. The greenhouse test field has uniform fertility, consistent planting level, uniform disease occurrence and harm degree, and convenient control and management. Protection rows are required to be arranged between each treatment room and around the test area, the cell surface is 60 square meters, and the test is repeated for 3 times. The liquid consumption is 10kg/60m ² . And respectively carrying out leaf surface spraying by taking clear water and 500 times of the Altailing solution as negative and positive controls. All test agents must be diluted twice. Spraying the pesticide from the strawberry growing to 2 months of age on the 1 st day after the shed is sealed, and spraying the pesticide for 3 times at intervals of 5 days. And (5) investigating disease incidence and counting disease indexes of diseased leaves 15d after the last pesticide spraying, and calculating the control effect. The disease grading criteria are as follows:

stage 0: no lesion spots;

stage 1: the area of the lesion is less than 5%;

and 3, stage: the area of the lesion is 6 to 10 percent;

and 5, stage: the area of the lesion is 11 to 20 percent;

and 7, stage: the area of the lesion is 21-50 percent;

and 9, stage: the area of the lesion spots is more than 50 percent.

The disease index and the prevention and treatment effect are calculated by the following formulas.

Disease index = ∑[ (number of diseased leaves per grade x relative grade)/(total number of investigated strains x 9) ] × 100%

Control effect% = [ (control average disease index-treatment average disease index)/control average disease index ] × 100%

The results are shown in Table 5 and FIG. 6.

Table 5: citral preparation and field efficacy test of preparation containing citral essential oil and citral extract for preventing and treating strawberry gray mold

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From the above table, it can be seen that the citral preparation and the preparations containing the essential oil and extract of citral have good preventive and protective effects on gray mold of strawberry.

Example 9: plot pesticide effect test of citral preparation on cucumber powdery mildew

The cell test is randomly arranged and repeated for 3 times, the area of the cell is equal to 60 square meters, the selection of test places requires uniform fertility and consistent crop planting and management level, and protection rows are arranged between each treatment room and around the test area. Spraying on leaf surface with clear water and 500 times of liquid as negative and positive control respectively. All test agents must be diluted twice. Spraying the pesticide when 5-6 real leaves of the cucumber are planted, wherein the spraying liquid amount is to uniformly spray the leaves until the pesticide begins to drip. Spraying every 7d for 1 time and 3 times. And (5) investigating the disease index after spraying 15d for the last time, and calculating the control effect. Each cell was randomly investigated at 5 spots, 5 plants were investigated at each spot, and 5 leaves were investigated from the upper, middle and lower parts of each plant. The disease grading criteria are as follows:

level 0: no disease spots;

stage 1: the lesion area accounts for less than 5% of the whole leaf area;

and 3, level: the lesion area accounts for 6 to 10 percent of the whole leaf area;

and 5, stage: the lesion spot area accounts for 11-25% of the whole leaf area;

and 7, stage: the lesion spot area accounts for 26-50% of the whole leaf area;

and 9, stage: the lesion area accounts for more than 50% of the whole leaf area.

Control% = ((control average disease index-treatment average disease index)/control average disease index) × 100%

The results are shown in Table 6 and FIG. 7.

Table 6: field control effect of citral preparation on cucumber powdery mildew

From the above table, it can be seen that the citral preparation has a good preventive effect on cucumber powdery mildew.

Example 10: test of promoting action of citral on growth of wheat roots

And (3) paving filter paper in the culture dish, adding 4mL of citral aqueous solution with a certain concentration, and using clear water as a blank control. And (4) selecting full wheat seeds, uniformly placing 10 grains in each dish, and repeating for 3 times. The culture dish is placed into a constant temperature incubator for culture, the temperature is room temperature, the humidity is 80 percent, and the filter paper is kept moist in the test process. After 5 days, the length of the longest root of wheat was measured, and the promoting activity on the root of wheat was calculated by the following formula:

promotion rate = (length of drug-treated root-length of rinsing control root)/length of rinsing control root × 100%

The results are shown in Table 7 and FIG. 8.

Table 7: promoting activity of citral treatment on growth of wheat root and bud

From the above table, the concentration ranges are: 0.19-12.5 mu g/mL of citral has a good effect of promoting the growth of wheat roots, has an inhibiting effect on the growth of wheat roots at a high concentration, and shows a remarkable plant growth regulating effect.

Example 11: test of effect of citral treatment on yield increase of capsicum

The area of the plastic greenhouse selected in the test is 1200m ² (100m 12m), east and west trend; the method comprises the steps of setting 4 treatments of citral dilution 200 times, 400 times, 800 times and CK in a shed, repeating each treatment 4 times, totaling 16 cells, adopting a random block design, and setting an experimental area of 960m ² Each cell area is 60m ² . Ridging is carried out according to transverse ridges during planting, 83 ridges (166 rows) are planted in the greenhouse, a corridor of 1.5 m is reserved in the middle of the greenhouse, and 8 cells are reserved in the south and the north respectively; and 2 rows of protection rows are arranged between adjacent cells, and 4 rows of protection rows are respectively arranged at the east end and the west end. After the tested peppers are treated by the citral soluble agent with different concentrations, the yield of the peppers in each cell is recorded and counted, the yield of each cell is directly weighed by an electronic scale, and the acre yield is calculated by multiplying the area of the cell to the area of the cell by the area of each mu of land.

The results are shown in Table 8.

Table 8: effect of citral treatment on Pepper yield

As can be seen from the above table, the dilution of the citral soluble solution (90% content) by 200-fold, 400-fold, 800-fold increased the pepper yield by 47%, 33%, and 13%, respectively, as compared to the control. In conclusion, the citral treatment can significantly improve the pepper yield.

Example 12: effect of citral treatment on chlorophyll content of Capsicum annuum

And randomly selecting 10 peppers with consistent growth vigor in each cell, picking up the upper functional leaves for measurement, and repeating the treatment for three times. Fresh, wiped leaves of the pepper were taken, the midrib was removed, chopped and mixed for assay. And calculating the concentrations of chlorophyll a and b of the pepper leaves according to a formula, and converting into the total chlorophyll mass in the leaves.

C _a+b ＝C _a +C _b ＝8.05OD ₆₆₃ +20.29OD ₆₄₅

The results are shown in Table 9.

Table 9: effect of citral treatment on chlorophyll content

From the above table, it can be known that the citral treatment with different concentrations has a certain effect on the chlorophyll content, wherein the chlorophyll content of the citral soluble solution with the content of 90% is respectively improved by 27.58%, 12.31% and 6.54% under the treatment of the dilution times of 200 times, 400 times and 800 times compared with that of the blank control, and thus, the citral treatment can significantly improve the chlorophyll content in the pepper leaves.

Example 13: effect of citral treatment on apple yield

The area of the plastic greenhouse selected in the test is 1200m ² (100m 12m), east and west trend; the method comprises the steps of setting 4 treatments of citral dilution 200 times, 400 times, 800 times and CK in a shed, repeating each treatment 4 times, totaling 16 cells, adopting a random block design, and setting an experimental area of 960m ² Each cell area is 60m ² . Ridging is carried out according to transverse ridges during planting, 83 ridges (166 rows) are planted in the greenhouse, a corridor of 1.5 meters is reserved in the middle of the greenhouse, and each of the south and north areas is 8; and 2 rows of protection rows are arranged between adjacent cells, and 4 rows of protection rows are respectively arranged at the east end and the west end. After the apples to be tested are treated by the citral soluble agent with different concentrations, the yield of the apples in each cell is recorded and counted, the yield of the cells is directly weighed by an electronic scale, and the acre yield is calculated by multiplying the area of the cell yield by the area of the cells by the area of each mu of land.

The results are shown in Table 10 and FIG. 9.

Table 10: effect of citral treatment on apple yield

As can be seen from the above table, the dilution of the citral soluble solution (90% content) by 200-fold, 400-fold, 800-fold increased apple yield by 14.17%, 8.36%, and 4.31%, respectively, compared to the control. In conclusion, the citral treatment can significantly improve apple yield.

Example 14: synergistic effect of citral on field control effect of commercial bactericide

The cell test is randomly arranged and repeated for 3 times, the area of the cell is equal to 60 square meters, the selection of test areas requires uniform fertility and consistent crop planting and management level, protective rows are arranged between treatments and around the test area, the area of the cell is equal to 60 square meters, and the test is repeated for 3 times. The liquid consumption is 10kg/60m ² . Diluting 50% carbendazim wettable powder, 80% mancozeb wettable powder and 50% thiram wettable powder by 1000 times, adding citral with different concentrations for foliar spraying, and diluting the ethacryl by 500 times, respectively adding citral with different concentrations for foliar spraying. All test agents must be diluted twice. The disease statistics were investigated as in examples 7, 8 and 9. The disease grading standard is as follows:

level 0: no disease spots;

level 1: the area of the lesion spots is less than 5%;

and 3, stage: the area of the lesion is 6 to 10 percent;

stage 5: the area of the lesion spot is 11 to 20 percent;

and 7, stage: the area of the lesion is 21-50 percent;

and 9, stage: the area of the lesion spots is more than 50 percent.

% control effect = [ (control average disease index-treatment average disease index)/control average disease index ] × 100% results are shown in table 11.

Table 11: citral for improving field control effect of carbendazim and other bactericides

As can be seen from the table above, the addition of citral can significantly improve the control effect of the bactericide such as carbendazim on gray mold of strawberries and powdery mildew of cucumbers; the control effect of the altaicine on tobacco mosaic virus, strawberry gray mold and cucumber powdery mildew is improved.

Example 15: citral for improving adverse resistance activity of capsicum

The stress resistance activity of the plants after the medicament treatment is indirectly reflected by measuring the permeability of leaf cell membranes. And cleaning the blade, and punching by using a puncher. 0.3g of perforated leaf was weighed into a clean 100mL beaker, rinsed 3 times with 80mL of deionized water, 50mL of deionized water was added, left to stand for 3h, and the conductivity was measured with a conductivity meter. After the measurement, the mixture was boiled in a water bath for 15min, and immediately after cooling, the electric conductivity was measured. The calculation is as follows: conductivity (%) = (treatment conductivity/boiling conductivity) × 100

The results are shown in Table 12.

Table 12: effect of citral treatment on conductivity of Capsicum annuum leaves

From the above, the conductivity of the leaves after the citral treatment decreases with the increase of the concentration. The citral has an induction effect on the anti-stress related indexes in the plants, and is specifically shown in that the activity of the protective enzyme is enhanced, and the permeability between membranes is reduced, so that the citral treatment activates an antioxidant system of the plants, the antioxidant enzyme activity of plant leaves is improved, and the adaptability of the plants to the environment is improved.

Example 16:6 measurement of citral content in plant extract and plant essential oil thereof

The litsea cubeba extract, the verbena extract, the cinnamomum camphora extract, the lemongrass extract and the Ocimum gratissimum extract are prepared by an ethanol thermal reflux method. The method comprises the following specific steps: after drying and crushing the plant sample in the shade, precisely weighing 1000g of the plant sample, placing the plant sample in a round-bottom flask, adding 6L of ethanol, refluxing at 70 ℃ for 3h, and filtering. Extracting for 3 times, mixing filtrates, and concentrating under reduced pressure to obtain ethanol extract of each plant sample.

Preparing Litsea cubeba essential oil, verbena essential oil, cinnamomum camphora essential oil, lemon grass essential oil and Ocimum gratissimum essential oil by supercritical fluid extraction. The method comprises the following specific steps: drying the plant sample at 100 deg.C for 3 hr, pulverizing, and sieving with 40 mesh sieve. Accurately weighing 50g, placing into an extraction tank, starting a supercritical fluid extraction device, and introducing CO when the temperature reaches a set value (35-60 deg.C) ₂ Start-up of high pressure CO ₂ And a hydraulic pump, when the extraction pressure reaches a set value (12-24 MPa), pumping a certain amount of 75% (volume fraction) ethanol (entrainer). CO regulation ₂ And (4) flow rate. When the whole supercritical fluid extraction system reaches steady state operation, the ultrasonic wave is started to work in the selected power density, frequency and time. The extraction time is 30min each time, and the extraction is carried out for 2 times.

Table 13:6 measurement of citral content in plant extract and plant essential oil thereof

Note: a is the sum of the content of cis-citral and trans-citral in the extract or the essential oil.

The preferred embodiments of the present disclosure are described in detail with reference to the accompanying drawings, however, the present disclosure is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solution of the present disclosure within the technical idea of the present disclosure, and these simple modifications all belong to the protection scope of the present disclosure.

It should be noted that, in the foregoing embodiments, various features described in the above embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, various combinations that are possible in the present disclosure are not described again.

In addition, any combination of various embodiments of the present disclosure may be made, and the same should be considered as the disclosure of the present disclosure, as long as it does not depart from the spirit of the present disclosure.

Claims

1. The application of the citral as an effective component in preparing a plant resistance inducer, wherein the application range of the plant resistance inducer is to induce plant disease resistance;

the content of the citral is 0.02-0.5 mg/mL;

the plant is tobacco, and the disease resistance is tobacco mosaic virus resistance.