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

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 their applications, and particularly relates to the application of citral or a plant extract containing citral in preparing a plant resistance inducer.

Background Art

Plant essential oils are widely present in nature. Most plant families contain plant essential oils. For example, Magnoliaceae, Granineae, Piperaceae and other plant families are rich in essential oil substances. Plant essential oils are complex mixtures, usually composed of many kinds of organic compounds, and the range of compounds covers highly polar, less polar and non-polar substances. Terpenes composed of monoterpenes and sesquiterpenes are more common in essential oils, but the specific composition varies with different essential oils. It is the main component of various plant essential oils; aromatic compounds (aldehydes, alcohols, methoxy derivatives, etc.), the content is second only to terpenes, such as cinnamaldehyde content in cinnamon essential oil is about 80%, citral content in Litsea cubeba essential oil is about 70%, etc.

Citral is one of the most important representatives of open-chain monoterpenes, with a chemical formula of C ₁₀ H ₁₆ O, a molecular weight of 152.23, a boiling point of 229°C, and a density of 0.889 g/cm ^3. Citral is a colorless or slightly yellow liquid with a strong lemon aroma, no optical activity, and has two cis and trans isomers. Citral is soluble in oils, propylene glycol, and ethanol, but insoluble in glycerin and water. Natural products exist in lemongrass oil (70% to 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 that require lemon aroma. It is an important spice for lemon-type, deodorizing wood-type flavors, artificial lemon oil, bergamot oil and orange leaf oil. It is the raw material for synthesizing ionones and methyl ionones. It can also be used to cover up bad smells in industrial production. It can also be used in edible flavors such as ginger, lemon, white lemon, sweet orange, grapefruit, apple, cherry, grape, strawberry and spicy flavors, and wine flavors. Pharmacological studies in recent years have shown that citral has anti-tumor, analgesic, antispasmodic and other pharmacological effects. In terms of agricultural use, there are literature reports that citral has a certain antibacterial effect on plant pathogenic fungi, etc. There is no relevant research report on its induction of plant disease resistance activity, induction of plant stress resistance activity and promotion of plant growth activity. Its application in the fields of agriculture, horticulture and forestry needs to be further developed.

Summary of the invention

The present invention clarifies the plant disease resistance-inducing activity, plant stress resistance-inducing activity and plant growth promotion activity of citral and a plant extract with citral as the main component by using methods such as biological activity determination and physiological and biochemical tests, and prepares pesticide preparations, adjuvants and biostimulants with citral and/or a plant extract with citral as the main component as active ingredients, and applies them in the field.

The present invention discloses the use of citral and/or a plant extract with citral as the main component to prepare a plant resistance inducer and apply it in agricultural production. Its application scope includes inducing plant disease resistance activity, inducing plant stress resistance activity and promoting plant growth activity. The plant resistance inducer provided by the present invention can induce and improve the resistance of plants to fungal diseases, bacterial diseases, nematode diseases, pests, viral diseases and high temperature, low temperature and drought adversity, and can also promote plant growth.

The invention discloses the preparation of pesticide adjuvants by utilizing citral and/or a plant extract with citral as a main component and the application of the adjuvants in agricultural production.

The invention discloses the preparation of biostimulant by utilizing citral and/or a plant extract with citral as a main component and the application of the biostimulant in agricultural production.

Optionally, the citral is one of cis-citral neral and trans-citral geranial or a combination thereof, wherein the structures of citral neral and trans-citral geranial are shown in the following formula:

Optionally, the plant extract containing citral is selected from the group consisting of cassia bark essential oil, litsea cubeba essential oil, verbena essential oil, camphor essential oil, lemongrass essential oil and basil essential oil; one of cassia bark extract, litsea cubeba extract, verbena extract, camphor extract, lemongrass extract and basil extract, and combinations thereof.

A plant resistance inducer, which comprises citral and/or a plant extract containing citral and is processed by adding an auxiliary agent, and the preparation form is a soluble powder, a water-dispersible granule, a soluble liquid, an aqueous emulsion, a microemulsion and all preparation types acceptable in agriculture;

Calculated by mass percentage, the content of citral is 1% to 100%.

A plant resistance inducer, which comprises citral and/or a plant extract containing citral and is processed by adding an auxiliary agent, and the preparation form is a soluble powder, a water-dispersible granule, a soluble liquid, an aqueous emulsion, a microemulsion and all preparation types acceptable in agriculture;

Calculated by mass percentage, the content of citral is 0.1% to 1%.

A plant resistance inducer, which comprises citral and/or a plant extract containing citral and is processed by adding an auxiliary agent, and the preparation form is a soluble powder, a water-dispersible granule, a soluble liquid, an aqueous emulsion, a microemulsion and all preparation types acceptable in agriculture;

Calculated by mass percentage, the content of citral is 30%.

A composition with plant resistance inducing activity, the composition comprising citral and/or a plant extract containing citral and another or a mixture of two or more commercial pesticides, wherein the content of citral is 0.1% to 99% by mass; the commercial pesticides include plant resistance inducers, 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 citral is 0.1% to 100% by mass.

A biostimulant comprises citral and/or a plant extract containing citral, wherein the content of citral is 0.1% to 100% by mass.

A method for improving plant immunity, characterized in that the method comprises the following steps: applying the product of the present invention to at least one of plants, areas adjacent to plants, soil suitable for supporting plant growth, roots and leaves of plants.

The induced plant disease resistance activity mentioned in the present invention is essentially to enhance the disease resistance of the plant itself, which is a broad-spectrum disease resistance activity and can be used to enhance the disease resistance of plants to bacterial, fungal, viral, oomycete and nematode infections. The induced plant stress resistance activity mentioned in the present invention is essentially to induce the stress resistance potential of plants, which can be used to enhance the drought resistance, cold resistance, salt resistance, disease resistance and other abilities of plants when they are coerced by biological factors such as pests and diseases, weeds, and physical and chemical factors such as temperature, moisture, salinity, chemical factors and weather. The plant resistance inducer described in the present invention is a kind of pesticide, which can also be called a plant immune activator, etc. It has no direct bactericidal activity, but can control and prevent the invasion of fungi, bacteria, viruses, nematodes, insects and other harmful organisms on plants by inducing plants to produce disease resistance or stress resistance. It has the advantages that pathogens are not easy to produce resistance, the prevention and control spectrum is relatively wide, and it can be mixed with chemical agents. It is a pesticide that meets the requirements of green prevention and control.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings are used to provide a further understanding of the present disclosure and constitute a part of the specification. Together with the following specific embodiments, they are used to explain the present disclosure but do not constitute a limitation of the present disclosure. In the accompanying drawings:

Figure 1 shows the changes in the activity of POD in tobacco leaves after spraying citral;

Figure 2 shows the changes in the activity of PAL in tobacco leaves after spraying citral;

Figure 3 shows the changes in H ₂ O ₂ content in tobacco leaves after spraying citral;

FIG4 is a graph showing changes in the expression of disease resistance genes in tobacco leaves after spraying citral;

Figure 5 shows the anti-tobacco mosaic virus activity of tobacco leaves after spraying citral, Note: a-CK; b-citral 200μg/ml; c-400μg/ml; d-800μg/ml;

Figure 6 shows the activity of strawberry leaves sprayed with citral against gray mold, Note: a-CK; b-citral 200μg/ml; c-400μg/ml; d-800μg/ml; e-carbendazim 400μg/ml;

Figure 7 shows the activity of cucumber leaves sprayed with citral against cucumber powdery mildew, Note: a-CK; b-citral 200μg/ml; c-400μg/ml; d-800μg/ml; e-carbendazim 400μg/ml;

Figure 8 shows the changes in root growth of wheat after spraying citral, Note: a-CK; b-citral 200μg/ml; c-400μg/ml; d-800μg/ml;

Figure 9 shows the early maturity of apples after spraying citral biostimulant, Note: a-CK; b-citral 200μg/ml; c-400μg/ml; d-800μg/ml.

DETAILED DESCRIPTION

The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

The compound citral of the present disclosure can be obtained by various techniques, such as chemical synthesis method; for example, it can be extracted from plants containing citral, and the extraction method can be any of various known techniques, such as: supercritical fluid extraction, solvent (ethanol, methanol, acetone, etc.) hot reflux extraction, solvent (ethanol, methanol, acetone, etc.) percolation method, etc. The citral used in the present disclosure is purchased from a chemical reagent platform.

Another embodiment of the present invention uses ethanol percolation to prepare Litsea cubeba extract, Litsea cubeba extract, Verbena extract, Cinnamomum camphora extract, Lemongrass extract and Ocimum basilicum extract respectively; uses supercritical fluid extraction to prepare Litsea cubeba essential oil, Litsea cubeba essential oil, Verbena essential oil, Cinnamomum camphora essential oil, Lemongrass essential oil and Ocimum basilicum essential oil respectively, and determines the content of citral therein. Plant essential oil is also a kind of plant extract.

The results of biological activity determination and physiological and biochemical experiments in the embodiments of the present invention prove that citral and plant essential oils and extracts containing citral have the activity of inducing plant disease resistance, inducing plant stress resistance and promoting plant growth. Those skilled in the art will understand that the results of biological activity determination and physiological and biochemical experiments establish the general utility of citral and plant extracts containing citral as inducers.

The citral and citral-containing plant extracts of the present disclosure can be applied as a formulation comprising the citral and/or citral-containing plant extracts by any of a variety of known techniques. For example, the compound can be applied to the roots or leaves of the plant to induce plant resistance or promote plant growth without damaging the commercial value of the plant. Any of the citral and/or citral-containing plant extracts can be applied in the form of any commonly used formulation type, such as a solution, powder, suspension, wettable powder, soluble liquid, flowable concentrate or emulsifiable concentrate, specifically including but not limited to: seed treatment emulsions, water emulsions, large granules, microemulsions, water-soluble emulsions, soluble emulsions, water-dispersible granules, poison valleys, aerosols, block baits, slow-release blocks, concentrated baits, capsule granules, microcapsule suspensions, dry seed powders , emulsions, electrostatic sprays, water-in-oil emulsions, oil-in-water emulsions, smoke cans, fine granules, smoke candles, smoke tubes, smoke sticks, seed treatment suspensions, smoke tablets, smoke pills, granular poison baits, thermal fog agents, lacquers, microgranules, oil suspensions, oil-dispersible powders, flaky poison baits, concentrated glues, pouring agents, seed dressing agents, smears, suspension emulsions, film-forming oil agents, soluble powders, water-soluble powders for seed treatment, ultra-low volume suspensions, tracking powders, ultra-low volume liquids, and water-dispersible powders for wet seed dressing.

Preferably, the citral and citral-containing plant extracts of the present disclosure are applied in the form of a preparation comprising citral and/or a citral-containing plant extract and a botany-acceptable carrier. The concentrated preparation can be dispersed in water or other liquids for application, or the preparation can be dust-filled or granular. The preparation can be prepared according to conventional procedures in the field of agricultural chemistry. The present disclosure contemplates all vehicles by which citral and/or a plant extract with citral as the main component can be prepared for delivery and use as an inducer, including all botany-acceptable inert carriers, surfactants, emulsifiers, organic solvents or water, etc.

The formulation may optionally include a combination containing other pesticidal or other compounds with resistant activity. Such additional pesticidal compounds or other compounds with resistant activity may be fungicides, insecticides, herbicides, nematicides, acaricides, arthropodicides, bactericides, plant resistant agents or combinations thereof that are compatible and non-antagonistic with citral and/or a plant extract containing citral in the medium selected for application. Therefore, in such an embodiment, another pesticidal compound or resistant activity compound is used as a supplemental agent. Citral and/or a plant extract containing citral and another compound may be present in a weight ratio of 1:100 to 100:1 in the combination.

Another embodiment of the present invention is to prepare pesticide adjuvants using citral and/or plant extracts with citral as the main component and their application in agricultural production. The essence of preparing pesticide adjuvants using citral and/or plant extracts with citral as the main component is to utilize the inducing activity of citral and/or plant extracts with citral as the main component. When commercial pesticides including plant inducers, fungicides, bactericides, antivirals, insecticides, nematicides, and acaricides are applied in the field, citral and/or plant extracts with citral as the main component are added, which can directly kill pests and diseases while improving plant resistance, thereby increasing the prevention effect of other commercial pesticides. The addition method can be barrel mixing.

Another embodiment of the present invention is to prepare biostimulants using citral and/or plant extracts with citral as the main component and use them in agricultural production. Its essence is also to utilize the inducing activity of citral and/or plant extracts with citral as the main component.

Another embodiment of the present invention is a method for applying citral and/or a plant extract with citral as the main component to protect plants from infection by harmful organisms, comprising applying citral and/or a plant extract with citral as the main component to at least one of the plant, an area adjacent to the plant, soil suitable for supporting the growth of the plant, the root of the plant, and the leaf.

In order to better understand the essence of the invention, the technical content of the invention is described in detail with examples below, but the invention is not limited to these examples.

Example 1: Determination of anti-TMV activity of citral and citral-containing plant extracts

(1) Protective activity

0.02mg/mL, 0.1mg/mL and 0.5mg/mL citral and essential oil and extract solutions containing citral were prepared, and healthy 5-6 leaf heart-leaf tobacco with consistent growth was selected. 48 hours after spraying the agent, a 2000-fold diluted TMV solution was inoculated. The blank control was water treatment, and the positive control was 0.1mg/mL chitosan oligosaccharide. Three leaves were inoculated for each treatment, repeated 3 times, and the number of dead spots was counted after 3 days to calculate the inhibition rate.

(2) In vitro inactivation activity

Prepare 0.02mg/mL, 0.1mg/mL and 0.5mg/mL citral and essential oil and extract solutions containing citral, and mix with 1000 times diluted TMV solution in equal volumes. After leaving at room temperature for 1 hour, inoculate on healthy 5-6 leaf heartleaf tobacco with uniform growth. The blank control is water treatment, and the positive control is 0.1mg/mL chitosan oligosaccharide. Inoculate 3 leaves for each treatment, repeat 3 times, count the number of dead spots after 3 days, and calculate the inhibition rate.

(3) Therapeutic activity

0.02mg/mL, 0.1mg/mL and 0.5mg/mL citral and essential oil and extract solutions containing citral were prepared, and healthy 5-6 leaf heart-leaf tobacco with consistent growth was selected. After inoculation with 2000 times diluted TMV solution, the pesticide was sprayed 48 hours later. The blank control was treated with water, and the positive control was 0.1mg/mL chitosan oligosaccharide. Three leaves were inoculated for each treatment, repeated 3 times, and the number of dead spots was counted after 3 days to calculate the inhibition rate.

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

The results are shown in Table 1.

Table 1: Protective, inactivating and therapeutic effects of citral and essential oils and extracts containing citral on TMV

Note: Data are mean ± standard error. The significant differences among the three activities were analyzed by Duncan multiple range test (DMRT) in SPSS software. Different letters indicate significant differences between the data at the 0.05 level.

As shown in Table 1, citral has a good protective effect on TMV, with a disease prevention effect of up to 76.27%, indicating that applying the drug in advance can make tobacco have a certain disease resistance effect. However, the passivation effect of citral on TMV is general, indicating that citral does not cause direct damage to TMV particles or the damage effect is weak, far less than the disease resistance caused by early application of the drug. Plant extracts containing citral also have certain antiviral activity, among which Litsea cubeba essential oil and extract are the most prominent. Example 2: Determination of tobacco anti-TMV activity induced by citral and essential oils and extracts containing citral

Heartleaf tobacco was selected for verification of induced disease resistance activity. Heartleaf tobacco can form virus dead spots, and the symptoms on ordinary tobacco are mosaic. Different symptoms are statistically tested using different methods for disease resistance. Spray 0.02mg/mL, 0.1mg/mL and 0.5mg/mL citral and essential oils and extracts containing citral to the three lower leaves of tobacco in the 6-7 leaf stage with consistent growth. After 48h, inoculate TMV to the unsprayed upper leaves. The blank control is treated with clean water, and the positive control is a chitosan oligosaccharide solution. Each plant is inoculated with 2 to 3 leaves, each treatment includes 10 tobacco plants, and the entire experiment is repeated 3 times. After 3d, the disease index of the number of dead spots on the heartleaf tobacco is counted, and the inhibition rate is calculated as above, and the control effect is calculated as follows.

Disease index = ∑ (disease level × number of diseased plants) / (highest disease level × total number of plants in each treatment) × 100;

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

The results are shown in Table 2.

Table 2: Induction of anti-disease effects of citral and essential oils and extracts containing citral on TMV

Note: Data are mean ± standard error. The significant differences among the three activities were analyzed by Duncan multiple range test (DMRT) in SPSS software. Different letters indicate significant differences between the data at the 0.05 level.

As shown in Table 2, citral and citral-containing plant extracts showed significant effects in inducing plant resistance to TMV in heartleaf tobacco, indicating that local application of citral can induce disease resistance in untreated parts of tobacco.

Example 3: Citral causes changes in the activity of tobacco defense enzyme POD

After spraying 0.02mg/mL, 0.1mg/mL and 0.5mg/mL citral on the leaves, the leaves were taken on the 1st, 3rd, 5th, 7th, 9th and 11th days to detect the activity changes of defense enzymes PAL and POD. Weigh 5g of plant leaves, cut them into pieces and put them in a frozen mortar, add a small amount of quartz sand, and add a total of 5mL of 0.1mol/L, pH5.5 acetic acid-sodium acetate buffer in 2-3 times. After grinding into a homogenate, centrifuge at 4℃, 12,000r/min for 15min, and the supernatant is the crude enzyme extract. Take a test tube and add 3mL of 25mmol/L guaiacol and 5mL of enzyme extract, then add 200μL, 5mol/LH ₂ O ₂ solution and mix quickly to start the reaction. Using distilled water as a reference, start recording the value of the reaction system at a wavelength of 470nm at 15s of the reaction, record it every 1min, and measure continuously to obtain data at least 6 points. Repeat the experiment three times. The enzyme activity calculation formula is as follows: OD ₄₇₀ = OD _470F - OD _470I / tp ~ ti, where OD _470F - reaction solution end value; OD _470I - reaction solution initial value; tp-reaction end time, min; ti-reaction start time, min. The absorbance change value per gram of sample per minute increases by 1 time as 1 unit of peroxidase activity, the unit is ΔOD ₄₇₀ / min·g. The calculation formula is as follows: U (ΔA ₄₇₀ ·g ^-1 min ^-1 ) = [ΔA ₄₇₀ × total amount of enzyme extract] / [sample fresh weight × enzyme solution volume at the time of measurement]

The results are shown in Figure 1.

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

Take 1.25g of tobacco leaves, add 5mL of enzyme extract (0.1mol/L pH8.8 Tris-H ₂ SO ₄ buffer) and 5g of polyvinylpyrrolidine (PVP), and homogenize with a mortar or a tissue crusher. After filtering, centrifuge the filtrate at 10,000 g for 30min at 4℃, and take the supernatant to measure the volume. Take 1mL of 0.1mol/L phenylalanine solution and 2mL of 0.1mol/L Tris-H ₂ SO ₄ buffer (pH 8.8) (the control tube does not add substrate phenylalanine, and directly takes 3mL of buffer) into a test tube and keep it warm in a 30℃ water bath for 3min. Add 5mL of the enzyme solution to be tested to each test tube, and do not add enzyme solution to the blank tube. After shaking well, immediately measure the starting value at a wavelength of 209nm, and use the blank tube to zero. And accurately time it. Put each test tube in a 30℃ water bath for 30min, and measure A ₂₀₉ again. The amount of enzyme required to increase the optical density at 290 nm by 0.01 per hour is 1 unit U (g·Fw·h). U = (ΔA×total volume of extract)/(0.01×T×W×amount of enzyme solution used for determination), where ΔA is the difference in absorbance between the two measurements; W is the fresh weight of the sample (g); and T is the reaction time (30 min).

Citral caused significant changes in the activity of defense enzymes in tobacco leaves (see Figures 1 and 2). The activity of POD remained stable or increased slowly from 1 to 3 days, and reached a peak value from 5 to 7 days. It is reported that POD can eliminate peroxides in plants, indicating that POD began to exert a large amount of activity after 5 days, eliminating excessive _H2O2 and other active _oxygen , and avoiding active oxygen damage to plants. PAL is a key enzyme in defense, and it is reported to play a key role in the production of disease resistance. PAL showed a rapid and obvious increase trend within 0 to 3 days, reaching the highest value on the 3rd day, causing disease resistance reactions in plants and the production and transmission of other disease resistance signals.

Example 5: Changes in H ₂ O ₂ Content in Tobacco Caused by Citral

Take 5g of leaves, add 3mL of pre-cooled acetone and a little quartz sand, grind into slurry on an ice bath, and centrifuge at 12,000g for 20min at 4℃. Take 1mL of supernatant, add reagents in sequence according to Table 4 below, and wash the resulting precipitate repeatedly with pre-cooled acetone and centrifuge for 2 to 3 times (3000g, 10min each time, discard the supernatant and keep the precipitate) until the precipitate has no photosynthetic pigment color. Add 3mL of sulfuric acid to the precipitate to dissolve the _precipitate for colorimetric determination. Establish a standard curve to calculate the amount of _H2O2 in the leaves, the calculation formula is as follows:

H ₂ O ₂ (nmol/g·Fw)=(n×V)/(v×m)

Wherein, n is the amount of H ₂ O ₂ calculated according to the standard curve (nmol); V is the volume of the sample supernatant (mL); v is the volume of the sample supernatant used for color development (mL), which is 1 mL in this experiment; and m is the fresh weight of the sample (g).

Prepare standard curve: Take 6 test tubes, number them, add each reagent according to Table 3 in a fume hood, mix well, react for 5 minutes, centrifuge at 12,000g for 15 minutes at 4℃, and keep the precipitate. Add 3mL of hydrochloric acid and shake to dissolve the precipitate. Use tube No. 0 as a reference to adjust to zero, and colorimetrically measure the absorbance of the solution at a wavelength of 412nm. Prepare a standard curve with the amount of H ₂ O ₂ (nmol) as the horizontal axis and the OD value as the vertical axis.

Table 3: Preparation of standard curve for determination of H ₂ O ₂ content

The results are shown in Figure 3. Citral can cause significant changes in the H ₂ O ₂ content in tobacco plants. The H ₂ O ₂ content increased rapidly and reached a peak 3 days after application, and gradually decreased after 5 days.

Example 6: Citral causes changes in the expression of tobacco disease resistance genes

Tobacco leaves at the 4-6 leaf stage were sprayed with citral solution, and samples were collected every other day within 1-10 days after treatment. Total RNA of tobacco leaves was extracted by liquid nitrogen method, and the expression changes of disease resistance-related genes NPR1, PR1 and PR2 were determined by real-time fluorescence quantitative PCR.

The results are shown in Figure 4. Citral can cause significant changes in the transcription level of disease resistance-related gene PR protein. Among them, the expression levels of NPR1, PR1, and PR2 increased by 5.25, 6.57, and 5.81 times compared with the control group, respectively. This shows that citral induces disease resistance in tobacco.

Example 7: Field plot control effect of citral and essential oils and extracts containing citral on tobacco mosaic virus

The plot test was randomly arranged and repeated 3 times. The plot area was equal to 60 m2. The selection of the test site required uniform fertility, consistent crop planting and management levels, and protection rows should be set up between each treatment and around the test area. Foliar spraying was performed at a constant rate, and 500 times of Atai Ling was used as a control agent for foliar spraying as a positive agent control, and a water control was set up. All test agents must be diluted twice. Self-core leaf tobacco was sprayed at the 4-5 leaf stage, and then sprayed once every 4 days, for a total of 3 times. 2 days after the last spraying, the top whole leaf was taken and rubbed with TMV, 3 leaves were inoculated for each plant, 10 plants were treated for each treatment, and repeated three times. After 10 days of virus inoculation, the disease index of each treatment was investigated and the prevention effect was calculated.

The disease classification standard is in accordance with the tobacco industry standard of the People's Republic of China-tobacco mosaic virus severity classification survey method (YC/T 39-1996):

Level 0: The whole plant is disease-free;

Level 1: The heart leaf veins are clear or slightly mosaic, or the upper 1/3 of the leaves are mosaic but not deformed, and the plant is not obviously dwarfed;

Level 2: 1/3 to 1/2 of the leaves are mosaic, or a few leaves are deformed; or the main veins turn black, and the plant is dwarfed by more than 2/3 of the normal height;

Level 3: 1/2 to 2/3 of the flowers are in bloom, or the plant is deformed or the main and side veins turn black, and the plant is dwarfed to 1/2 to 2/3 of the normal height;

Level 4: All leaves of the plant are mosaic, severely deformed or necrotic, and the diseased plant is dwarfed to 1/3 to 1/2 of the normal plant height. In order to refine the survey results, based on the above severity classification, the classification standards are refined, adding level 1+ between levels 1 and 2, level 2+ between levels 2 and 3, and level 3+ between levels 3 and 4, and the levels are recorded as 1.5, 2.5, and 3.5:

Grade 1+: The heart leaves have clear veins or slight mosaic, or the upper 1/3 leaves are mosaic or slightly wrinkled, and the plant has no obvious dwarfing;

Grade 2+: 1/3 to 1/2 of the leaves are mosaic, deformed, or the main veins are black, and the plant is dwarfed to more than 2/3 of the normal plant;

Grade 3+: 1/2 to 2/3 of the leaves are mosaic, or deformed, or the main and lateral veins are necrotic, or the plant is dwarfed to 1/2 of the normal height.

The disease index is calculated based on the severity, and the effectiveness of different treatments is measured by the effectiveness of prevention and treatment.

Disease index = ∑[(number of susceptible plants × representative value of severity level)/(total number of investigated plants × highest representative value of severity level)] × 100%

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

The results are shown in Table 4 and Figure 5.

Table 4: Field plot efficacy test of citral preparations and preparations containing citral essential oils and extracts for preventing and controlling tobacco virus diseases

As can be seen from the above table, citral preparations and preparations of essential oils and extracts containing citral have good prevention and control effects on tobacco mosaic virus.

Example 8: Field plot efficacy test of citral preparations and essential oils and extracts containing citral against strawberry gray mold

Greenhouse strawberries were selected for plot tests. Greenhouse test plots generally have uniform fertility, consistent planting levels, and relatively uniform disease occurrence and degree of damage, making them easy to control and manage. Protective rows should be set up around each treatment room and test area. The plot area is 60 ㎡, and the test is repeated 3 times. The liquid volume is 10kg/ ^60m2 . Use clean water and 500 times diluted Atailing as negative and positive controls for foliar spraying. All test agents must be diluted twice. When the strawberries grow to 2 months old, spraying begins on the first day after the greenhouse is closed, and then sprays once every 5 days, for a total of 3 times. 15 days after the last spraying, investigate the incidence of diseased leaves and calculate the disease index to calculate the prevention effect. The disease grading standards are as follows:

Level 0: no lesions;

Level 1: The lesion area is less than 5%;

Level 3: lesion area 6% to 10%;

Level 5: lesion area 11% to 20%;

Level 7: lesion area 21% to 50%;

Level 9: The lesion area is more than 50%.

The disease index and control effect were calculated using the following formula.

Disease index = ∑[(number of diseased leaves at each level × relative level)/(total number of investigated plants × 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 Figure 6.

Table 5: Field efficacy test of citral preparations and preparations containing citral essential oils and extracts for controlling strawberry gray mold

As can be seen from the above table, citral preparations and preparations of essential oils and extracts containing citral have good preventive and protective effects on strawberry gray mold.

Example 9: Field plot test of the efficacy of citral preparation against cucumber powdery mildew

The plot test was randomly arranged and repeated 3 times. The plot area was equal to 60 m2. The selection of the test site required uniform fertility, consistent crop planting and management levels, and protective rows should be set up around each treatment room and the test area. Foliar spray was performed at a constant rate, with clean water and 500 times diluted Atailing as negative and positive controls for foliar spraying respectively. All test agents must be diluted twice. Spray the cucumber when there are 5 to 6 true leaves after planting. The spray amount should be enough to evenly wet the leaf surface until the liquid begins to drip. Spray once every 7 days, for a total of 3 times. Investigate the disease index 15 days after the last spraying and calculate the prevention effect. Randomly select 5 points for investigation in each plot, investigate 5 plants at each point, and investigate 5 leaves of each plant in the upper, middle and lower parts respectively. The disease grading standards are as follows:

Level 0: no lesions;

Level 1: The lesion area accounts for less than 5% of the entire leaf area;

Level 3: The lesion area accounts for 6% to 10% of the entire leaf area;

Level 5: The lesion area accounts for 11% to 25% of the entire leaf area;

Level 7: The lesion area accounts for 26% to 50% of the entire leaf area;

Level 9: The lesion area accounts for more than 50% of the total leaf area.

Disease index = ∑[(number of diseased leaves at each level × relative level)/(total number of investigated plants × 9)] × 100%

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

The results are shown in Table 6 and Figure 7.

Table 6: Field efficacy of citral preparations in controlling cucumber powdery mildew

It can be seen from the above table that citral preparations have a good preventive effect on cucumber powdery mildew.

Example 10: Experiment on the promoting effect of citral on wheat root growth

Place filter paper in the culture dish, add 4 mL of a certain concentration of citral aqueous solution, and use clean water as a blank control. Select full wheat seeds, 10 seeds per dish, and place them evenly in the culture dish, and repeat 3 times. Place the culture dish in a constant temperature incubator for culture, the temperature is room temperature, the humidity is 80%, and the filter paper is kept moist during the test. Measure the longest root length of wheat after 5 days, and calculate the promoting activity on wheat roots with the following formula:

Promotion rate = (root length of drug treatment - root length of water control) / root length of water control × 100%

The results are shown in Table 7 and Figure 8.

Table 7: Citral treatment promoting the growth of wheat roots and shoots

From the above table, we can see that citral has a good promoting effect on wheat root growth in the concentration range of 0.19-12.5 μg/mL, and has an inhibitory effect on wheat root growth at high concentrations, showing a significant plant growth regulating effect.

Example 11: Experiment on the effect of citral treatment on the yield of pepper

The plastic greenhouse used in the experiment has an area of ^1200m2 (100m*12m), which is east-west. There are 4 treatments in the greenhouse, including 200-fold, 400-fold, 800-fold and CK dilution of citral. Each treatment is repeated 4 times, with a total of 16 plots. A random block design is adopted, the experimental area is ^960m2 , and the area of each plot is ^60m2 . When planting, ridges are formed according to horizontal ridges. A total of 83 ridges (166 rows) are planted in the greenhouse. A 1.5-meter corridor is left in the middle of the greenhouse, with 8 plots in the south and north. Two rows of protection rows are set between adjacent plots, and 4 rows of protection rows are set at the east and west ends. After the peppers for the test are treated with different concentrations of citral soluble solution, the yield of peppers in each plot is recorded and counted. The plot yield is directly weighed with an electronic scale, and the per mu yield is calculated by multiplying the plot yield by the plot area and then multiplying the area per mu.

The results are shown in Table 8.

Table 8: Effect of citral treatment on pepper yield

As shown in the table above, compared with the control, the dilution of citral soluble solution (90% content) by 200 times, 400 times, and 800 times can increase the pepper yield by 47%, 33%, and 13%, respectively. In summary, citral treatment can significantly increase pepper yield.

Example 12: Effect of citral treatment on chlorophyll content of pepper

Ten pepper plants with the same growth potential were randomly selected from each plot, and the functional leaves on the top were picked for determination. Each treatment was repeated three times. Freshly cleaned pepper leaves were taken, the midribs were removed, cut into pieces, and mixed for determination. The concentrations of chlorophyll a and b in pepper leaves were calculated according to the formula and converted 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, we can see that different concentrations of citral treatment have a certain effect on the chlorophyll content. Among them, the chlorophyll content of 90% citral soluble solution increased by 27.58%, 12.31% and 6.54% respectively compared with the blank control when the dilution ratio was 200 times, 400 times and 800 times. It can be seen that citral treatment can significantly increase the chlorophyll content in pepper leaves.

Example 13: Effect of citral treatment on apple yield

The plastic greenhouse used in the experiment has an area of ^1200m2 (100m*12m), which is east-west. There are 4 treatments in the greenhouse, including 200-fold, 400-fold, 800-fold and CK dilutions of citral. Each treatment is repeated 4 times, with a total of 16 plots. A random block design is adopted, the experimental area is ^960m2 , and the area of each plot is ^60m2 . When planting, ridges are formed according to horizontal ridges. A total of 83 ridges (166 rows) are planted in the greenhouse. A 1.5-meter corridor is left in the middle of the greenhouse, with 8 plots in the south and north. Two rows of protection rows are set between adjacent plots, and 4 rows of protection rows are set at the east and west ends. After the test apples are treated with different concentrations of citral soluble solution, the yield of apples in each plot is recorded and counted. The plot yield is directly weighed with an electronic scale, and the per mu yield is calculated by multiplying the plot yield by the plot area and then multiplying the area per mu.

The results are shown in Table 10 and Figure 9.

Table 10: Effects of citral treatment on apple yield

As can be seen from the table above, compared with the control, the dilution of citral soluble solution (90% content) by 200 times, 400 times, and 800 times can increase the apple yield by 14.17%, 8.36%, and 4.31%, respectively. In summary, citral treatment can significantly increase apple yield.

Example 14: The synergistic effect of citral on the field efficacy of commercial fungicides

The plot test is randomly arranged and repeated 3 times. The plot area is equal to 60㎡. The selection of the test site requires uniform fertility, consistent crop planting and management levels. Protective rows should be set up around each treatment room and the test area. The plot area is 60㎡ and the test is repeated 3 times. The liquid volume is 10kg/ ^60m2 . 50% carbendazim wettable powder, 80% mancozeb wettable powder, and 50% thiram wettable powder are diluted 1000 times and then added with different concentrations of citral for foliar spraying. After Atailing is diluted 500 times, different concentrations of citral are added for foliar spraying. All test agents must be diluted twice. The disease statistical survey is the same as Examples 7, 8, and 9. The disease classification standards are as follows:

Level 0: no lesions;

Level 1: The lesion area is less than 5%;

Level 3: lesion area 6% to 10%;

Level 5: lesion area 11% to 20%;

Level 7: lesion area 21% to 50%;

Level 9: The lesion area is more than 50%.

The disease index and control effect were calculated using the following formula.

Disease index = ∑[(number of diseased leaves at each level × relative level)/(total number of investigated plants × 9)] × 100%

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

Table 11: Citral improves the field efficacy of carbendazim and other fungicides

As can be seen from the above table, the addition of citral can significantly improve the control effect of fungicides such as carbendazim on strawberry gray mold and cucumber powdery mildew; and improve the control effect of Ata Ling on tobacco mosaic virus disease, strawberry gray mold and cucumber powdery mildew.

Example 15: Citral improves the stress resistance of pepper

By measuring the permeability of leaf cell membranes, the stress resistance of plants after treatment with chemicals can be indirectly reflected. Wash the leaves and punch holes with a hole puncher. Weigh 0.3g of the punched leaves and put them into a clean 100mL beaker. Rinse with 80mL of deionized water 3 times, add 50mL of deionized water, let it stand for 3h, and measure the conductivity with a conductivity meter. After the measurement, boil it in a water bath for 15min, and measure its conductivity immediately after cooling. Calculation is as follows: Conductivity (%) = (treatment conductivity/boiling conductivity) × 100

The results are shown in Table 12.

Table 12: Effects of citral treatment on the electrical conductivity of pepper leaves

From the above, it can be seen that the conductivity of leaves after citral treatment decreased with the increase of concentration. Citral has an inductive effect on stress resistance related indicators in plants, which is specifically manifested as enhanced protective enzyme activity and decreased membrane permeability. Therefore, citral treatment activates the antioxidant system of plants, increases the activity of antioxidant enzymes in plant leaves, and improves the adaptability of plants to the environment.

Example 16: Determination of citral content in 6 plant extracts and plant essential oils

The extracts of Litsea cubeba, Litsea cubeba, Verbena officinalis, Cinnamomum camphora, Lemongrass and Ocimum basilicum were prepared by ethanol hot reflux method. The specific steps are as follows: After the plant samples are dried in the shade and crushed, 1000 g is accurately weighed and placed in a round-bottom flask, 6 L of ethanol is added, refluxed at 70 degrees Celsius for 3 hours, and filtered. Extract three times, combine the filtrate and concentrate under reduced pressure to obtain the ethanol extract of each plant sample.

The essential oils of Litsea cubeba, Litsea cubeba, Verbena, Cinnamomum camphora, Lemongrass and Ocimum basilicum were prepared by supercritical fluid extraction. The specific steps are as follows: Dry the plant sample at 100°C for 3h, crush it and pass it through a 40-mesh sieve. Accurately weigh 50g and put it into the extraction tank. Turn on the supercritical fluid extraction device. When the temperature reaches the set value (35-60°C), pass _CO2 , start the high-pressure _CO2 hydraulic pump, and when the extraction pressure reaches the set value (12-24MPa), pump in a certain amount of 75% (volume fraction) ethanol (entrainer). Adjust the _CO2 flow rate. When the entire supercritical fluid extraction system reaches steady-state operation, turn on the ultrasonic wave and make it work at the selected power density, frequency and time. The extraction time is 30min each time, and the extraction is repeated twice.

Table 13: Determination of citral content in 6 plant extracts and essential oils

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

The preferred embodiments of the present disclosure are described in detail above in conjunction with the accompanying drawings; however, the present disclosure is not limited to the specific details in the above embodiments. Within the technical concept of the present disclosure, a variety of simple modifications can be made to the technical solution of the present disclosure, and these simple modifications all fall within the protection scope of the present disclosure.

It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present disclosure will not further describe various possible combinations.

In addition, various embodiments of the present disclosure may be arbitrarily combined, and as long as they do not violate the concept of the present disclosure, they should also be regarded as the contents disclosed by the present disclosure.

Claims (1)

1. The application of citral as an effective ingredient in the preparation of a plant resistance inducer, wherein the application scope of the plant resistance inducer is to induce plant disease resistance; The content of citral is 0.02-0.5 mg/mL; The plant is tobacco, and the disease resistance is resistance to tobacco mosaic virus disease.

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