CN113875752A - Application of limonene in preparation of plant resistance inducer - Google Patents
Application of limonene in preparation of plant resistance inducer Download PDFInfo
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- CN113875752A CN113875752A CN202111222000.2A CN202111222000A CN113875752A CN 113875752 A CN113875752 A CN 113875752A CN 202111222000 A CN202111222000 A CN 202111222000A CN 113875752 A CN113875752 A CN 113875752A
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- limonene
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Images
Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N27/00—Biocides, pest repellants or attractants, or plant growth regulators containing hydrocarbons
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Agronomy & Crop Science (AREA)
- Pest Control & Pesticides (AREA)
- Plant Pathology (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Dentistry (AREA)
- General Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Environmental Sciences (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
The invention discloses application of limonene for preparing a plant resistance inducer, wherein the application range of the plant resistance inducer comprises plant disease resistance induction and plant stress resistance induction. The invention utilizes the methods of biological activity determination, physiological and biochemical tests and the like to determine the plant disease resistance induction activity and the plant stress resistance induction activity of the limonene and the plant essential oil or the extract containing the limonene, prepares the plant resistance inducer and the biostimulant which take the limonene as the active component and applies the plant resistance inducer and the biostimulant in the field.
Description
Technical Field
The invention belongs to the field of plants and application thereof, and particularly relates to application of limonene in preparation of a plant resistance inducer.
Background
Limonene (limonene) is a natural monoterpene widely present in rutaceae plants and is often a key substance in the action of rutaceae plant volatile oil. Due to wide sources of limonene, simple extraction, wide application range and high practical value, the limonene is gradually researched more and deeper in recent years, the application scene is gradually expanded, and the limonene is applicable to the aspects of food, medicine, agriculture and the like.
Limonene (also called limonene, dipentene, etc.) is 1-methyl 4-isopropyl cyclohexene, is a natural functional monoterpene, is colorless to light yellow oily liquid at normal temperature, has pleasant lemon fragrance, and has molecular formula C10H16The molecular weight is 136.23, is easily soluble in ethanol and most of nonvolatile oil, is stored under the conditions of drying at 2-8 ℃ and avoiding light, is easily oxidized under the condition of illumination, and is not suitable for being stored for too long time so as not to polymerize. The limonene exists in three existing forms, namely D-limonene, L-limonene and DL-limonene, widely exists in plant essential oil, wherein the D-limonene mainly exists in citrus plants, the content of the D-limonene in the essential oil is as high as 90%, and the content of the D-limonene in the essential oil is even as high as 95%, so that the D-limonene is mainly used for extraction and application of the limonene. In the agricultural field, documents report that limonene has a certain bacteriostatic action on plant pathogenic fungi and the like and has a certain insecticidal activity on various pests, but related research reports on plant disease resistance induction activity and plant stress resistance induction activity are not available, and the application of limonene in the fields of agriculture, horticulture and forestry needs 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 inducing activity and the plant stress resistance inducing activity of the limonene, prepares the plant resistance inducer and the biological stimulin which take the limonene as the active component and applies the plant resistance inducer and the biological stimulin in the field.
The invention discloses a plant resistance inducer prepared from limonene and application of the plant resistance inducer in agricultural production. The application range of the plant growth regulator comprises the induction of plant disease resistance activity and the induction of plant stress resistance activity. 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.
The invention discloses a method for preparing biostimulant by utilizing limonene and application of the biostimulant in agricultural production.
Specifically, the method comprises the following steps:
the application of the limonene for preparing the plant resistance inducer comprises the application range of inducing plant disease resistance or inducing plant stress resistance.
Optionally, the limonene is one or a combination of D-limonene and L-limonene, wherein the structures of the D-limonene and the L-limonene are shown as follows:
a plant resistance inducer comprises 1-100% of limonene by mass percentage.
Optionally, the content of limonene is 0.1-1% by mass percent.
Optionally, the content of limonene is 30% by mass percentage.
Optionally, the limonene is one or a combination of D-limonene and L-limonene, wherein the structures of the D-limonene and the L-limonene are shown as follows:
a composition with activity of inducing plant resistance comprises a mixture of limonene and another or more than two commercial pesticides, wherein the content of the limonene is 0.1-99% in percentage by mass; the commercial pesticides include plant elicitors, fungicides, bactericides, antivirals, insecticides, nematicides, miticides, and combinations thereof.
Optionally, the content of limonene is 0.02-0.08% by mass percent.
Optionally, the limonene is one or a combination of D-limonene and L-limonene, wherein the structures of the D-limonene and the L-limonene are shown as follows:
a method for enhancing the immunocompetence of a plant, the method comprising the steps of: the plant resistance inducer according to the present invention or the composition having the activity of inducing plant resistance according to the present invention is applied to at least one of the area adjacent to the plant, soil suitable for supporting plant growth, roots and 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. The bio-stimulants mentioned in the present invention are neither pesticides nor traditional fertilizers. The target of the bio-stimulin is the crop itself, which can improve the physiological and biochemical state of the plant, improve the pesticide effect and the utilization rate of the fertilizer, improve the level of the crop for resisting the adverse environment, and also improve the final yield of the crop and the quality of agricultural products.
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 without limiting the disclosure. In the drawings:
FIG. 1 shows the change of POD activity in vivo after the limonene is sprayed on tobacco leaves;
FIG. 2 is the activity change of PAL in tobacco leaves after limonene spraying;
FIG. 3 shows that the tobacco leaves are sprayed with limonene to form H in vivo2O2A change in content;
FIG. 4 shows the change of the expression level of the disease-resistant gene in the tobacco leaves sprayed with limonene.
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 limonene of the present disclosure can be obtained by a variety of techniques, such as chemical synthesis methods; also, for example, the extract can be obtained by extraction and preparation from plants containing limonene by any of a variety of known techniques, such as: squeezing, supercritical fluid extraction, hot reflux extraction with solvent (ethanol, methanol, acetone, etc.), percolation with solvent (ethanol, methanol, acetone, etc.), etc. Plant materials that can be used to extract limonene include, but are not limited to, orange peel and leaves, citrus peel and leaf peel, citrus peel and leaves, grapefruit peel and leaves, and the like. Limonene is used in the present disclosure as purchased from a chemical reagent platform.
The biological activity determination result and the physiological and biochemical experiment result in the embodiment of the invention prove that the limonene has the functions of inducing plant disease resistance and inducing plant stress resistance. Example 1 demonstrates that limonene has significant antiviral effects on tobacco mosaic virus, and D-limonene and L-limonene are equivalent in activity, with the main mode of action being protection; example 2 proves that limonene has significant induced disease resistance activity on tobacco mosaic virus, and local application of limonene can induce non-application parts of tobacco to generate disease resistance; examples 3, 4, 5 and 6 prove that the activity of related defense enzymes, the content of hydrogen peroxide and the expression level of disease-resistant genes in the tobacco body are improved after the limonene treatment, which indicates that the limonene treatment induces the disease-resistant defense behaviors in the tobacco body; examples 7, 8 and 9 prove that limonene has good prevention and protection effects on tobacco mosaic virus, strawberry gray mold and cucumber powdery mildew, and the limonene has broad spectrum on virus diseases and fungal diseases; examples 10 and 11 prove that the chlorophyll content of the pepper leaves is remarkably improved after limonene treatment, the conductivity of the leaves is reduced, and the limonene has the activity of inducing plant stress resistance; example 12 demonstrates that limonene formulated with a variety of fungicides exhibits synergistic effects. One skilled in the art will appreciate that the biological activity assay results and physiological and biochemical experimental results establish the general utility of limonene as a decoy.
The limonene of the present disclosure can be applied as a formulation comprising said limonene by any of a variety of known techniques. 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. The limonene can be applied in any of the commonly used formulation types, for example, as a solution, powder, suspension, wettable powder, soluble liquor, flowable concentrate or emulsifiable concentrate, including in particular 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 limonene of the present disclosure is applied in a formulation comprising limonene 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 limonene can be formulated for delivery and used 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 with limonene and not antagonistic in the medium selected for application. Thus, in such embodiments, another pesticidal compound or inducer active compound is used as a supplemental agent. The limonene and the other 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 the use of limonene for the preparation of biostimulant and its application in agricultural production. It is also essential to utilize the resistance inducing activity of limonene.
Another embodiment of the invention is a method of applying limonene for enhancing plant immunity and protecting plants from infestation by pests, comprising applying limonene to at least one of a plant, an area adjacent to a plant, soil suitable for supporting the growth of a plant, a root, and a leaf of a plant.
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: determination of limonene anti-TMV Activity
(1) Protective Activity
Preparing 0.02mg/mL, 0.1mg/mL and 0.5mg/mL limonene solutions, selecting healthy 5-6-leaf stage heart-leaf tobacco with consistent growth vigor, spraying a medicament for 48 hours, then inoculating a TMV solution diluted 2000 times, treating a blank control by clear water, and treating a positive control by 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.
(2) In vitro inactivation Activity
Limonene solutions at concentrations of 0.02mg/mL, 0.1mg/mL, and 0.5mg/mL were prepared and mixed with 1000-fold 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 to calculate the inhibition rate.
(3) Therapeutic Activity
Preparing 0.02mg/mL, 0.1mg/mL and 0.5mg/mL limonene solutions, selecting healthy 5-6-leaf-stage heart-leaf tobacco with consistent growth vigor, inoculating 2000-fold diluted TMV solution for 48 hours, and spraying the medicinal agent for treatment. 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 (%) - (control number of scorched spots-number of treated scorched spots)/control number of scorched spots × 100
The results are shown in Table 1.
Table 1: protective, inactivating, and therapeutic effects of limonene on TMV
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 1, D-limonene and L-limonene have good protective effects on TMV infected plants, and the fact that the tobacco can have a certain disease-resistant effect by applying the pesticide in advance is shown. The passivation effect of limonene on TMV is general, which shows that limonene does not cause direct damage or has weak damage effect on TMV mitochondria and is not as good as the disease resistance effect caused by early application.
Example 2: determination of anti-TMV activity of limonene-induced tobacco
The heart-leaf tobacco is selected to be subjected to induced disease resistance activity test, 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. And spraying 0.02mg/mL, 0.1mg/mL and 0.5mg/mL of limonene to the lower three leaves of the tobacco with the same growth vigor and at the 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 ═ Σ (number of disease stages × number of diseased plants)/(highest number of disease stages × total number of treated plants) × 100;
the control effect is (control disease-treatment disease)/control disease x 100%;
the results are shown in Table 2.
Table 2: induced disease resistance effect of limonene on TMV
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, D-limonene and L-limonene showed significant effects of inducing plants to resist TMV, indicating that the local application of limonene can induce the disease resistance of the non-application part of the tobacco.
Example 3: limonene causes activity change of tobacco defense enzyme POD
0.02mg/mL, 0.1mg/mL and 0.5mg/mLDAfter spraying limonene and L-limonene on the leaves, taking the leaves on days 1, 3, 5, 7, 9 and 11 respectively, and detecting the activity changes of the defense enzymes PAL and POD. Weighing 5g of plant leaves, shearing the plant leaves, placing the plant leaves into a frozen mortar, adding a small amount of quartz sand, and adding 5mL of 0.1mol/L (molar ratio) acetic acid-sodium acetate buffer solution with pH of 5.5 in 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 of 25mmol/L of guaiacol and 5mL of enzyme extract into a test tube, and adding 200 μ L of 5mol/L H2O2The solution was mixed rapidly to start the reaction. The value of the reaction system at the wavelength of 470nm is recorded at the beginning of the reaction for 15s by using distilled water as a reference, and the value is recorded every 1min and continuously measured, and at least data of 6 points are obtained. The experiment was repeated three times. The enzyme activity was calculated as follows: OD470＝OD470F—OD470ITp to ti, where OD470F-reaction liquid end value; OD470I-initial values of the reaction solution; tp-reaction termination time, min; ti-reaction start time, min. 1 peroxidase Activity Unit at 1 increase in Absorbance Change per gram of sample per minute (. DELTA.OD)470G/min. The calculation formula is as follows: u (Delta A)470·g-1min-1)＝[ΔA470X 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: limonene caused activity changes of tobacco defense enzyme PAL
Taking 1.25g tobacco leaf, adding 5mL enzyme extract (0.1mol/L Tris-H with pH8.8)2SO4Buffer) and 5g of polyvinylpyrrolidine (PVP), homogenized with a mortar or tissue triturator. After filtration, the filtrate was centrifuged at 10,000g 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-H2SO4Buffer (pH 8.8) (3 mL of buffer was directly taken without substrate phenylalanine in the control 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. Place each test tube at 3Keeping the temperature of the reaction solution in water bath at 0 ℃ for 30min, and measuring A again209. 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 ═ 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 the previous and subsequent 2 measurements; w is the sample fresh weight (g); t is the reaction time (30 min).
D-limonene and L-limonene caused significant changes in the activity of defensive enzymes in tobacco leaves (see FIG. 2). POD activity remained stable or increased slowly at 1-3 days, and peaked at day 5. POD is reported to eliminate peroxides in plants, indicating that POD starts to exert a large amount of activity after 5 days, and eliminates excessive H2O2And 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 play a key role in the development of disease resistance. PAL shows a rapid and obvious increasing trend within 0-5 days, and reaches the highest value on the 5 th day, causing the generation and transmission of disease-resistant reaction and other disease-resistant signals in the plant body.
Example 5: limonene induces tobacco H2O2Content 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 12,000g at 4 ℃ for 20 min. Taking 1mL of supernatant, sequentially adding reagents according to the following table 4, repeatedly washing and centrifuging the obtained precipitate with precooled acetone for 2-3 times (3000g, 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 blade2O2The calculation formula is as follows:
H2O2(nmol/g·Fw)＝(n×V)/(v×m)
wherein n is H calculated from the standard curve2O2The amount (nmol); 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: 6 test tubes were numbered and each reagent was added to the fume hood according to Table 3Mixing, reacting for 5min, centrifuging at 12,000g centrifugal force at 4 deg.C for 15min, and collecting 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-tube as a control and zero adjustment. With H2O2The 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 H2O2Standard curve of content determination
The results are shown in FIG. 3, where limonene can cause H in tobacco plants2O2The content was significantly changed. 3 days after application, H2O2The content increases rapidly and reaches a peak value, and after 5d, the content gradually decreases.
Example 6: variation of tobacco disease-resistant gene expression quantity caused by limonene
And spraying a limonene solution on the tobacco in the leaf stage of 4-6, 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 quantity change of disease resistance related genes NPR1, PR1 and PR2 by real-time fluorescent quantitative PCR.
The result is shown in figure 4, D-limonene and L-limonene can cause the transcription level of PR protein of disease resistance related genes to be obviously changed. (wherein, the expression levels of NPR1, PR1 and PR2 are respectively increased by 8.3 times, 3.2 times and 3.5 times compared with the control group, which shows that D-limonene and L-limonene induce the disease-resistant defense behavior in tobacco bodies.
Example 7: field plot control effect of limonene preparation on 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 in constant amount, spraying onto leaf surface in 500 times of liquid as control agent as positive control, and setting clear water control. All test agents must be diluted twice. Spraying the pesticide in 4-5 leaf periods of the heart leaf tobacco, and then spraying the pesticide for 1 time every 4 days for 3 times. 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, investigating 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 investigation method (YC/T39-1996) according to the tobacco industry standard of the people's republic of China:
level 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 leaf and leaf of the upper 1/3 leaves are not deformed, and the plant is not obviously dwarfed;
and 2, stage: 1/3 to 1/2 leaf lobes, or a few leaf lobes, are deformed; or the main pulse is blackened, the plant is dwarfed to be higher than 2/3 with normal plant height;
and 3, level: 1/2-2/3, or the main side pulse is changed to black, the plant is dwarfed to the normal plant height of 1/2-2/3;
4, level: the whole leaf has severe deformity or necrosis, and the diseased plant is dwarfed to the normal plant height of l/3 to 1/2. 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:
1+ level: the heart leaves have bright veins or slight flower leaves, or the upper 1/3 leaf leaves are slightly shrunken, and plants are not obviously dwarfed;
2+ level: l/3 to 1/2 leaves and leaves are deformed or the main pulse is blackened, and the plant is dwarfed to be more than 2/3 of a normal plant;
3+ level: 1/2-2/3 leaf mosaic, or deformed or primary side vein necrotic, or plant dwarfing to 1/2 of normal plant height.
And calculating the disease index according to the severity, and measuring the effect of different treatments by using the prevention and treatment effect.
Disease index ═ Σ [ (number of infected plants × graded representative value of severity)/(number of total investigated plants × highest representative value of severity) ] × 100%
Control effect%
The results are shown in Table 4.
Table 4: test of pesticide effect of limonene preparation in preventing and treating tobacco virus diseases in field plot
From the above table, it can be seen that the limonene preparation has a good effect of preventing and controlling tobacco mosaic virus.
Example 8: test of field plot pesticide effect of limonene preparation on strawberry gray mold
Greenhouse strawberries are selected for a plot experiment. The greenhouse test field has uniform fertility, consistent planting level, uniform disease occurrence and harm degree and convenient control and management. Protective rows are 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/60m2. 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 1 time every 5 days, wherein the spraying time is 3 times. 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:
level 0: no disease spots;
level 1: the area of the lesion is less than 5%;
and 3, level: 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 is more than 50%.
The disease index and the prevention and treatment effect are calculated by the following formula.
Disease index ═ Σ [ (number of diseased leaves per grade x relative grade)/(number of total investigated strains x 9) ] × 100%
Percent control efficacy [ (control average disease index-treatment average disease index)/control average disease index ] × 100%
The results are shown in Table 5.
Table 5: limonene preparation, limonene-containing essential oil and limonene-containing preparation for preventing and treating strawberry gray mold in field efficacy test
From the above table, it can be seen that the limonene preparation has a good effect of preventing and protecting gray mold of strawberry.
Example 9: plot pesticide effect test of limonene 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 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 on leaf surface with constant amount, and spraying on leaf surface with clear water and 500 times of Tailing solution as negative and positive control respectively. All test agents must be diluted twice. Spraying the cucumber with 5-6 main leaves after field planting, wherein the spraying amount is equal to that of spraying the cucumber onto the leaves until the liquid medicine begins to drip. Spraying every 7d for 1 time and 3 times. And (5) investigating disease indexes after spraying the pesticide for 15d for the last time, and calculating the control effect. Each cell was randomly investigated at 5 points, 5 plants were investigated at each point, 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;
level 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 area accounts for 11 to 25 percent of the whole leaf area;
and 7, stage: the lesion 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.
Disease index ═ Σ [ (number of diseased leaves per grade x relative grade)/(number of total investigated strains x 9) ] × 100%
Control effect%
The results are shown in Table 6.
Table 6: limonene preparation for preventing and controlling cucumber powdery mildew in field
From the above table, it can be seen that the limonene preparation has a good effect of preventing cucumber powdery mildew.
Example 10: effect of limonene treatment on chlorophyll content of Capsicum annuum
Randomly selecting 10 peppers with consistent growth vigor in each cell, picking up the upper functional leaves for determination, and repeating the treatment three times. Fresh, wiped leaves of the pepper were removed, cut to pieces 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.
Ca+b＝Ca+Cb＝8.05OD663+20.29OD645
The results are shown in Table 7.
Table 7: effect of limonene treatment on chlorophyll content
From the above table, it can be seen that the treatment of D-limonene and L-limonene with different concentrations has a certain effect on the chlorophyll content, wherein the chlorophyll content of the D-limonene and L-limonene soluble solution with the content of 100% is respectively increased by 25.52% and 25.59%, 9.77% and 10.6%, 4.17% and 5.10% compared with the blank control under the treatment with the dilution times of 200 times, 400 times and 800 times. Therefore, the content of chlorophyll in the pepper leaves can be obviously improved by limonene treatment.
Example 11: limonene for improving stress resistance activity of hot pepper
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 8.
Table 8: effect of limonene treatment on Pepper leaf conductivity
From the above, the conductivity of the leaves treated by the D-limonene and the L-limonene is reduced along with the increase of the concentration. The limonene has an induction effect on the anti-stress related indexes in the plants, and the concrete expression is that the activity of the protective enzyme is enhanced, and the permeability between membranes is reduced, so that the limonene treatment activates the 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 12: synergistic effect of limonene on field control effect of commercial fungicide
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/60m2. Diluting with 50% carbendazim wettable powder, 80% mancozeb wettable powder and 50% thiram wettable powder 1000 times, and addingAnd d, carrying out foliar spraying on limonene with the same concentration, and respectively adding limonene with different concentrations to carry out foliar spraying after the abiline is diluted by 500 times. All test agents must be diluted twice. The disease statistics were investigated as in examples 7, 8 and 9. The disease grading criteria are as follows:
level 0: no disease spots;
level 1: the area of the lesion is less than 5%;
and 3, level: 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 is more than 50%.
The disease index and the prevention and treatment effect are calculated by the following formula.
Disease index ═ Σ [ (number of diseased leaves per grade x relative grade)/(number of total investigated strains x 9) ] × 100%
Percent control efficacy [ (control average disease index-treatment average disease index)/control average disease index ] × 100%
The results are shown in Table 9.
Table 9: limonene for improving field pesticide effect of bactericide
From the table above, the addition of D-limonene can significantly improve the control effect of fungicides 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.
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 (10)
1. The application of the limonene for preparing the plant resistance inducer comprises the application range of inducing plant disease resistance or inducing plant stress resistance.
2. The use of claim 1, wherein the limonene is one or a combination of D-limonene and L-limonene, wherein the structures of the D-limonene and the L-limonene are as follows:
3. the plant resistance inducer is characterized by comprising 1-100% of limonene by mass percent.
4. The plant resistance inducer according to claim 3, wherein the content of limonene is 0.1-1% by mass.
5. The plant resistance inducer according to claim 3, wherein the content of limonene is 30% by mass.
6. A plant resistance inducer according to claim 3, 4 or 5, wherein the limonene is one or a combination of D-limonene and L-limonene, wherein the structures of the D-limonene and the L-limonene are as follows:
7. a composition with activity of inducing plant resistance is characterized in that the composition comprises a mixture of limonene and one or more than two commercial pesticides, wherein the content of the limonene is 0.1-99% in percentage by mass; the commercial pesticides include plant elicitors, fungicides, bactericides, antivirals, insecticides, nematicides, miticides, and combinations thereof.
8. The composition having activity of inducing plant resistance according to claim 7, wherein the content of limonene is 0.02-0.08% by weight.
9. The composition with plant resistance inducing activity according to claim 7 or 8, wherein the limonene is one or a combination of D-limonene and L-limonene, the structures of the D-limonene and the L-limonene are as follows:
10. a method for enhancing the immunocompetence of a plant, comprising the steps of:
applying the plant resistance inducer of any one of claims 3-6 or the composition having plant resistance-inducing activity of any one of claims 7-9 to at least one of an area adjacent to a plant, soil suitable for supporting plant growth, roots and leaves of a plant.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111222000.2A CN113875752A (en) | 2021-10-20 | 2021-10-20 | Application of limonene in preparation of plant resistance inducer |
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| CN116019099B (en) * | 2023-01-06 | 2023-11-14 | 河南农业大学 | Plant source synergistic composition for preventing and treating peanut southern blight |
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