CN109601559B - Wild chrysanthemum essential oil for preventing and treating phytophthora and application thereof - Google Patents
Wild chrysanthemum essential oil for preventing and treating phytophthora and application thereof Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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- A01N65/00—Biocides, pest repellants or attractants, or plant growth regulators containing material from algae, lichens, bryophyta, multi-cellular fungi or plants, or extracts thereof
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N65/00—Biocides, pest repellants or attractants, or plant growth regulators containing material from algae, lichens, bryophyta, multi-cellular fungi or plants, or extracts thereof
- A01N65/08—Magnoliopsida [dicotyledons]
- A01N65/12—Asteraceae or Compositae [Aster or Sunflower family], e.g. daisy, pyrethrum, artichoke, lettuce, sunflower, wormwood or tarragon
Abstract
The invention provides wild chrysanthemum essential oil for preventing and treating phytophthora and application thereof, and relates to the technical field of phytophthora prevention and treatment, wherein the wild chrysanthemum essential oil is prepared by the following steps: mixing flos Chrysanthemi Indici with water, standing, and ultrasonic extracting to obtain ultrasonic extraction mixture; performing steam distillation on the ultrasonic extraction mixture, and collecting distillate; extracting the distillate with n-hexane, separating the organic phase, and drying to obtain flos Chrysanthemi Indici essential oil. The wild chrysanthemum essential oil provided by the invention can effectively inhibit the hypha growth and spore germination of phytophthora nicotianae, the minimum inhibition concentration can reach 200 mu L/L, and the bacteriostasis rate under the fumigation of 157.48 mu L/L can reach 92.68%. The bacteriostatic action mechanism of the chrysanthemum indicum essential oil can be related to the integrity of cell walls and cell membranes. The wild chrysanthemum essential oil lays a foundation for the development of botanical pesticides for preventing and treating phytophthora and even soil-borne diseases.
Description
Technical Field
The invention relates to the technical field of phytophthora prevention and control, and in particular relates to a wild chrysanthemum essential oil for preventing and controlling phytophthora and application thereof.
Background
Phytophthora Nicotiana van Breda de Haan (syn. p. parasitica Dastur) is a typical soil-borne pathogen, now considered one of the most devastating oomycete diseases, capable of infecting over 255 plants, including severely compromised tobacco (Nicotiana spp.), tomatoes (Lycopersicum esculentum), citrus (citrus spp.), and the like. Due to the diversity of hosts and the wide distribution of ecological regions, the huge loss caused by the hosts is difficult to estimate and is a continuous challenge for controlling plant diseases. The disease is mainly controlled by chemical pesticides such as metalaxyl, propamocarb, mancozeb and the like at present, the medicament has long service life, and is easy to cause the drug resistance of pathogenic bacteria, pesticide residue and environmental pollution. Therefore, there is a great need to develop new control measures to reduce the use of chemical pesticides.
Essential oils are complex secondary metabolites obtained from plants using special extraction methods, and have high volatility and strong penetration. Previous researches show that the essential oil has the potential of replacing chemical pesticides to prevent and control crop diseases and has the advantages of low toxicity, low residue and environmental friendliness. Recently, the inhibitory activity of plant essential oils against phytophthora pathogens has been reported. Such as Eucalyptus globulus (Eucalyptus globulus) and Citrus aurantifolia (Citrus aurantifolia essential oil) can inhibit Phytophthora tara P. The syzygium aromaticum essential oil can inhibit phytophthora nicotianae and is identified to be eugenol as a main active ingredient. Although a number of plant essential oils have been discovered for their composition and biological activity, the mechanism of action on phytophthora pathogens is unclear. Effective phytophthora bacteria prevention and treatment medicines cannot be effectively found.
Dendranthema indicum (L.) Des Moul, a family of Compositae, is widely distributed in China, including coastal saline-alkali land. Its flower is a traditional Chinese medicine, and has the obvious functions of resisting virus, reducing blood pressure and improving eyesight. A small number of studies report that wild chrysanthemum essential oil has an antibacterial effect on several human pathogenic bacteria including Staphylococcus aureus and Bacillus diphtheriae, but no antibacterial activity on plant pathogenic bacteria is reported. Phytophthora nicotianae is considered as the most destructive pathogenic bacteria of plant oomycete diseases, mainly depends on chemical pesticides for control, and urgently needs environment-friendly alternative medicaments.
Disclosure of Invention
The invention provides the wild chrysanthemum essential oil for preventing and treating phytophthora in order to overcome the defect that phytophthora pathogenic bacteria are absent in the prior art, and the wild chrysanthemum essential oil has an obvious bacteriostatic effect on phytophthora nicotianae and a good fumigating bacteriostatic effect.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides wild chrysanthemum essential oil for preventing and treating phytophthora, which is characterized by being prepared by the following steps:
(1) mixing flos Chrysanthemi Indici with water, standing, and ultrasonic extracting to obtain ultrasonic extraction mixture; the ultrasonic power is 100-150W;
(2) performing steam distillation on the ultrasonic extraction mixture, and collecting distillate;
(3) extracting the distillate with n-hexane, separating n-hexane phase, and drying to obtain flos Chrysanthemi Indici essential oil.
Preferably, in the step (1), the wild chrysanthemum is a dried wild chrysanthemum growing in the saline-alkali soil.
Preferably, in the step (1), the volume ratio of the dry weight of the wild chrysanthemum flower to the water is 1g: 8-12 mL.
Preferably, in the step (1), the standing time is 2-5 h.
Preferably, in the step (1), the ultrasonic extraction time is 15-50 min, and the ultrasonic extraction temperature is 45-60 ℃.
Preferably, in the step (2), the steam distillation time is 4-6 h.
Preferably, in the step (3), the volume ratio of the distillate to the n-hexane is 8-15: 1.
The invention also provides application of the chrysanthemum indicum essential oil in the technical scheme in prevention and treatment of phytophthora nicotianae.
Preferably, the dosage form of the chrysanthemum indicum essential oil is fumigant, microemulsion or sustained-release microcapsule.
Compared with the prior art, the invention has the beneficial effects that:
(1) the wild chrysanthemum essential oil provided by the invention can effectively inhibit the hypha growth and spore germination of phytophthora nicotianae, the minimum inhibition concentration can reach 200 mu L/L, and simultaneously, the wild chrysanthemum essential oil has a good fumigating effect, and the bacteriostasis rate can reach 92.68% under the fumigation of 157.48 mu L/L.
(2) The phytophthora nicotianae treated by the chrysanthemum indicum essential oil has obvious deformity of hypha, such as tip enlargement, hypha shrinkage and local hypha rupture, and the action mechanism of bacteriostasis of the phytophthora nicotianae essential oil is probably related to the integrity of cell walls and cell membranes.
(3) The wild chrysanthemum essential oil provided by the invention is prepared by a specific method, and the wild chrysanthemum essential oil extracted by the method contains abundant monoterpene and sesquiterpene compounds, which cause the increase of hyphal cell membrane permeability, the increase of hyphal malondialdehyde content and apoptosis.
(4) The wild chrysanthemum essential oil provided by the invention is a pure natural plant component, and lays a foundation for development of plant-derived pesticides for preventing and treating phytophthora and even soil-borne diseases.
Drawings
FIG. 1 shows the result of measurement of the growth of phytophthora nicotianae hyphae by wild chrysanthemum essential oil; wherein, a, agar diffusion treatment; b, agar diffusion method control; c, carrying out fumigation treatment; d, fumigation control;
FIG. 2 is a graph showing the effect of wild chrysanthemum essential oil on the morphology of phytophthora nicotianae hyphae; wherein: treating hyphae with essential oil, wherein the top end of the hyphae indicated by an arrow on the left expands, the hyphae indicated by an arrow on the right shrinks, and the hyphae indicated by an arrow in the middle breaks; b, control hyphae;
FIG. 3 is a GC-MS total ion chromatogram of wild chrysanthemum flower essential oil; wherein: a, diluting and injecting normal hexane; b, solid phase microextraction headspace sample introduction;
FIG. 4 is a graph showing the effect of wild chrysanthemum essential oil on the conductivity of phytophthora nicotianae hyphae;
FIG. 5 is a graph showing the effect of wild chrysanthemum essential oil treatment on the malondialdehyde content of phytophthora nicotianae hyphae;
FIG. 6 is a graph showing the effect of wild chrysanthemum essential oil on apoptosis of phytophthora nicotianae cells; wherein, a, 160 mu L/L of wild chrysanthemum essential oil is treated; b, control hyphae.
Detailed Description
The invention provides wild chrysanthemum essential oil for preventing and treating phytophthora, which is prepared by the following steps:
(1) mixing flos Chrysanthemi Indici with water, standing, and ultrasonic extracting to obtain ultrasonic extraction mixture; the ultrasonic power is 100-150W;
(2) performing steam distillation on the ultrasonic extraction mixture, and collecting distillate;
(3) extracting the distillate with n-hexane, separating the organic phase, and drying to obtain flos Chrysanthemi Indici essential oil.
The raw material of the chrysanthemum indicum essential oil is chrysanthemum indicum (Dendranthemama indicum (L.) DesMoul) of the Compositae; the preferred wild chrysanthemum in the invention takes the wild chrysanthemum growing in saline-alkali soil as the raw material, and the wild chrysanthemum can generate more effective substances required by the wild chrysanthemum essential oil under the saline-alkali stress; the wild chrysanthemum growing in the saline-alkali soil in the warm-temperate zone monsoon climate is more preferably used as the raw material, for example, the wild chrysanthemum growing in the saline-alkali soil in the Shandong area is selected as the extraction raw material in the specific embodiment of the invention.
Mixing wild chrysanthemum flower with water, standing, and performing ultrasonic extraction to obtain an ultrasonic extraction mixture; the ultrasonic power is 100-150W. The wild chrysanthemum is preferably a dried wild chrysanthemum growing in saline-alkali soil. In the invention, before the wild chrysanthemum flowers are mixed with water, the wild chrysanthemum flowers are preferably crushed, and the crushed particle size is preferably 20-60 meshes, and more preferably 40 meshes.
In the invention, the volume ratio of the dry weight of the wild chrysanthemum flower to the volume of water is preferably 1g: 8-12 mL, and more preferably 1g:10 mL. In the invention, the standing time is preferably 2-5 h, and more preferably 3 h. In the invention, the environment temperature during standing is preferably 20-25 ℃. The purpose of standing is to fully wet the dried wild chrysanthemum for subsequent extraction.
In the invention, the power of the ultrasonic wave is preferably 120-140W. In the invention, the time for ultrasonic extraction is preferably 15-50 min, and more preferably 20-35 min. In the invention, the temperature of ultrasonic extraction is preferably 45-60 ℃, and more preferably 50 ℃. The invention firstly adopts an ultrasonic extraction mode to destroy the cell structure, so that the effective substances in the wild chrysanthemum can be dissolved out more easily. The ultrasonic extraction is carried out at the temperature of 45-60 ℃, so that the ultrasonic extraction efficiency can be further improved.
After the ultrasonic extraction mixture is obtained, the ultrasonic extraction mixture is subjected to steam distillation, and distillate is collected. The invention does not need to add water when steam distillation is carried out.
In the invention, the steam distillation time is preferably 4-6 h, and more preferably 5 h. The present invention is not particularly limited as to how the steam distillation is carried out, and the distillate may be collected by a steam distillation method known in the art. In the invention, when the distillate is collected, the temperature of the oil-water separator is 20-25 ℃ within half an hour of boiling, and then is kept at 30-35 ℃. The number of steam distillations described in the present invention is preferably 1.
After the distillate is obtained, the invention uses normal hexane to extract the distillate, separates the organic phase and dries to obtain the chrysanthemum indicum essential oil. In the invention, the volume ratio of the distillate to the n-hexane is preferably 8-15: 1, and more preferably 10: 1.
According to the invention, effective substances are extracted into the n-hexane phase through n-hexane extraction, and the n-hexane phase is separated after standing and layering, so that an organic phase is obtained. The purpose of the present invention for drying the organic phase is to remove residual n-hexane. In order to prevent the extracted effective substances from volatilizing, those skilled in the art preferably dry and volatilize n-hexane with anhydrous sodium sulfate to obtain pale yellow chrysanthemum indicum essential oil, i.e. chrysanthemum indicum essential oil.
The wild chrysanthemum essential oil prepared by the method has the extraction rate of about 0.15 percent, can obtain the wild chrysanthemum essential oil rich in hemiterpene and sesquiterpene, and has a remarkable inhibiting effect on phytophthora nicotianae.
The invention also provides application of the chrysanthemum indicum essential oil in the technical scheme in prevention and treatment of phytophthora nicotianae. As shown in the embodiment of the invention, the wild chrysanthemum essential oil has a remarkable inhibiting effect on phytophthora nicotianae. Preferably, the wild chrysanthemum essential oil dosage form preferably comprises a fumigant, a microemulsion and a slow-release microcapsule; when the wild chrysanthemum essential oil is a fumigant, the control of phytophthora nicotianae by fumigation has a good effect. Before tobacco transplantation, 4000ml of prepared wild chrysanthemum (500 times of diluent) is sprayed per mu, a plastic film (0.08cm thick) is used for sealing and fumigating a plastic box for 7 days immediately after pesticide application, the plastic film is uncovered, and tobacco seedlings can be transplanted after drying for 7 days.
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1
1. Plant material
Collecting fresh flos Chrysanthemi Indici in 2017 in 10 months on Shandong coast beach, taking back to laboratory with nylon mesh bag, and drying in shade.
2. Extraction of
Grinding dried chrysanthemum buds of the wild chrysanthemum into powder, sieving the powder by a 40-mesh sieve, weighing 100g of the powder, putting the powder into a 2000mL beaker, adding distilled water according to the material-liquid ratio of 1g to 10mL, standing the mixture for 3 hours at normal temperature, and putting the mixture into an ultrasonic container, wherein the ultrasonic power is 120W, the ultrasonic time is 30min, and the ultrasonic temperature is 50 ℃ to obtain an ultrasonic extraction mixture. And (3) performing steam distillation on the ultrasonic extraction mixture for 5h, wherein the temperature of the oil-water separator is 20 ℃ within half an hour of boiling, then the temperature is kept at 30 ℃, and a distillate is obtained after the distillation is finished. Mixing the distillate with n-hexane according to a volume of 10:1, extracting, separating organic phase, drying with anhydrous sodium sulfate, volatilizing n-hexane to obtain yellowish wild chrysanthemum essential oil, precisely weighing, and calculating the extraction rate to be 0.15%.
Example 2
Firstly, a test process:
1. phytophthora nicotianae culture
Reference is made to the literature methods for culturing and preparing the spores of the virulent tobacco phytophthora nicotianae JM01 strain (Han, t.; You, c.; Zhang, l.; Feng, c.; Zhang, c.; Wang, j.; Kong, f. biocontrol potential of anticagonist Bacillus subtilis Tpb55 againt tobaco black sank. biocontrol 2016,61, 195-.
2. Determination of bacteriostatic Activity
2.1. Plate agar diffusion method
After phytophthora nicotianae grew on the oat culture medium, bacterial cake (5mm) was picked at the edge of the bacterial colony and placed on one side of the plate, holes were made on the other side, 100 μ L of wild chrysanthemum essential oil (same as below, not described in detail) prepared in example 1 was added, DMSO was used as a control, the procedure was repeated three times, and then cultured at 28 ℃ for 5 days, the diameter of the zone of inhibition was measured, and the change in the hypha morphology was observed using a scanning electron microscope.
2.2. Fumigation test
And (3) determining the fumigating activity of the wild chrysanthemum essential oil on the phytophthora nicotianae by adopting a sealed flat plate buckling method. A phytophthora nicotianae cake having a diameter of 5mm was inoculated on OA medium, 1.25, 2.5, 5, 10, 15, 20, and 25. mu.L (corresponding to 10, 20, 40, 60, 80, 120, and 160. mu.L/L, respectively) of wild chrysanthemum essential oil was dropped into the center of the lid of the dish, sealed with a sealing film, cultured at 28 ℃ for 3 days, and repeated 3 times. The colony diameter was measured by the cross method, and the plate without added wild chrysanthemum essential oil was used as a control.
The hyphal growth inhibition rate was calculated using the following test:
2.3. determination of hyphal growth Rate by the method
The wild chrysanthemum essential oil was plated in DMSO (0.5%, v/v) at varying concentrations on OA (45 ℃) plates to final concentrations of 10, 20, 40, 80, 120, 160 and 200. mu.L/L. Inoculating phytophthora nicotianae cake (5mm) to OA culture medium containing different concentrations of chrysanthemum indicum essential oil, culturing at 28 ℃ for 5d, taking DMSO with the same dilution ratio as a control, measuring the colony diameter by a cross method, and calculating the hypha growth inhibition rate.
2.4. Spore germination inhibition assay
The spore germination inhibition test adopts a liquid serial dilution method. 500mL of wild chrysanthemum essential oil (10, 20, 40, 80, 120, 160 and 200. mu.L/L) at different concentrations were mixed with an equal volume of phytophthora nicotianae spore suspension (10. mu.L/L)6cfu/mL), dropping on a glass slide with a groove, culturing for 6h at 8 ℃ and 80% relative humidity, observing the spore germination condition under a microscope, and calculating the spore germination inhibition rate.
3. Analysis of chemical composition of essential oil
3.1. Gas chromatography-Mass spectrometer analysis (GC-MS)
The essential oil is diluted to 100g/L by n-hexanol, 1 mu L of the essential oil is injected into Agilent 7890-. The inlet temperature was 250 ℃ and helium gas was used as carrier gas at a flow rate of 1.0 mL/min. The initial oven temperature was held at 45 ℃ for 2 minutes, at 5 ℃/min up to 200 ℃, for 2 minutes, at 15 ℃/min up to 320 ℃, for 5 minutes. The ionization mode is 70eV electron impact. And (3) adopting a full-scanning monitoring mode, wherein the mass scanning range is 35-500 m/z, obtaining a mass spectrum, and starting an accelerating voltage after the solvent is delayed for 5 min.
3.2. Headspace solid phase microextraction of volatile Compounds (HS-SPME)
Based on previous studies, sandwich-type (DVB/CAR/PDMS) SPME fibers (Supelco Analytical, Sigma-Aldrich, St. Louis, Mo., USA) were used to absorb volatile compounds from EOD. SPME fiber was inserted into the headspace of a sample vial containing 0.1g of EOD for 20 minutes at room temperature, then the SPME fiber was removed from the sample vial and immediately inserted into a GC syringe at 250 ℃ for 5 minutes at a split ratio of 10: 1. SPME fibers were conditioned at 250 ℃ for 5 minutes before the next sampling. The ionization mode is 70eV electron impact. And (3) adopting a full-scanning monitoring mode, wherein the mass scanning range is 35-500 m/z, obtaining a mass spectrum, and starting an accelerating voltage after the solvent is delayed for 5 min.
3.3. Identification of Compounds
The identification of individual compounds was based on calculated Retention Indices (RI) and their mass spectra compared to mass spectra of reference compounds available in the NIST 14 database (https:// www.nist.gov/srd/NIST-specific-database-14); the threshold matching ratio is set to 90. Under the same operating conditions, RI was calculated for the family of normal alkane (C7-C30) homologs.
4. Cell membrane permeability
Cell membrane permeability is indicated by its Electrical Conductivity (EC). 5 of the cakes extracted from the edge of the tobacco OA plate were transferred to liquid OA medium and incubated at 28 ℃ and 175rpm for 3 days. 1g of hyphae was mixed with 20mL of EOD (diluted with 2% DMSO) at 0, 80, 120 or 160. mu.L/L and stored at room temperature (20. + -. 3 ℃). EC values were determined at 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5 and 5.0h after treatment, respectively.
5. Malondialdehyde (MDA) content of mycelium
The content of Malondialdehyde (MDA) in the phytophthora nicotianae mycelia is determined by a thiobarbituric acid (TBA) method. 5 discs of hyphae were inoculated into 90mL of liquid OA medium and cultured at 28 ℃ and 175rpm for 48 h. 10ml of EOD ( concentration 80, 120 or 160. mu.L/L) was added, incubated for an additional 24 hours and then harvested as described above (2.6). The mycelium was placed in a pre-cooled mortar and ground into powder with liquid nitrogen. The mycelium powder (1g) was transferred to a centrifuge tube containing 10mL of 10% (w/v) TCA, vortexed at 4 ℃ for 30s, centrifuged at 5000 Xg for 20min, the supernatant (2_ mL) was mixed with 2mL of 0.6% (w/v) TBA, boiled for 20min, and after cooling, the absorbance of the mixture at 450nm, 532nm and 600nm was measured. And (5) nm. The following formula was used to calculate the MDA content (C, expressed in nmol/g fresh weight):
C＝6.45×(OD532-OD600)-0.56×OD450
6. cell death assay
Damage to cell membranes by EOD was assessed by propidium iodide fluorescence analysis. The P.nicotianae mycelia were treated with 160. mu.L/L EOD for 24 h. Mycelium (100. mu.L) was transferred to a 1-mL tube and stained with PI (400. mu.L) for 30 minutes in a dark environment of 1. mu.g/mL. After staining, the mycelia were washed three times with distilled water and then photographed under a fluorescence microscope (excitation wavelength of 535nm, emission wavelength of 615 nm).
7. Statistical analysis
Data were analyzed using Excel 2003 and SPSS 22.0. The results of the determination of the bacteriostatic activity, the cell membrane permeability and the mycelium MDA content are expressed by the average +/-standard error of the triple experiments. Calculating the half maximal Inhibitory Concentration (IC) by regression analysis of the logarithmic value of the EOD concentration and the probability value of the corresponding inhibition50) The value is obtained. Analysis of variance and multiple comparisons (P) with the Duncan method<0.05)。
Second, test results
2.1. Inhibiting hypha growth
The agar diffusion method determination result shows that the wild chrysanthemum essential oil (10 muL/L) can strongly inhibit the growth of phytophthora nicotianae, can form an obvious inhibition zone, the growth of the whole colony is also obviously inhibited (figure 1a), the hypha grows sparsely, and the control hypha grows uniformly and compactly (figure 1 b).
The results of the buckling fumigation experiments show that the volatile components of the wild chrysanthemum essential oil can obviously inhibit the growth of phytophthora nicotianae hyphae (table 1). As shown in FIGS. 1c and 1d, phytophthora nicotianae hyphae grew slowly and sparsely with treatment with 10. mu.L/L of wild chrysanthemum essential oil. Inhibitory Medium concentration IC on hypha growth5020.0445 μ L/L (y 2.7097+1.7591x, r 0.9977). When the concentration of the wild chrysanthemum essential oil is 160 mu L/L, the hypha growth is completely inhibited. Our results show thatThe chrysanthemum essential oil and the volatile matter thereof can strongly inhibit the growth of phytophthora nicotianae hyphae.
TABLE 1 inhibition of Phytophthora nicotianae hypha growth and spore germination by Chrysanthemum indicum essential oil
Mean differences of the same letters in the same column were not significant (P >0.05), whereas Duncan's multipass test differences were not significant (P > 0.05).
Inhibition (mycelium) reacted with dimethyl sulfoxide (dimethyl sulfoxide) compared to control.
Scanning electron microscope results show that the wild chrysanthemum essential oil causes obvious morphological changes of phytophthora nicotianae hyphae, which are represented by hypha shrinkage, deformation and even rupture (fig. 2a), while the control hyphae are smooth and full (fig. 2 b).
Table 1 shows the inhibitory effect of wild chrysanthemum essential oil on different growth times of hyphae. The growth of phytophthora nicotianae hyphae is significantly inhibited and increases with increasing treatment concentration. The hyphal inhibition rate was about 6.46% at 10. mu.L/L, and hyphal growth was completely inhibited at 200. mu.L/L. Inhibition of Intermediate Concentration (IC)50) And Minimum Inhibitory Concentrations (MICs) of 48.4531 μ L/L (y 1.4264+2.1204x, r 0.9824) and 200 μ L/L, respectively.
2.2. Spore germination inhibiting action
As shown in Table 1, the wild chrysanthemum essential oil obviously inhibits phytophthora nicotianae spore germination, the inhibition rate is about 8.52-92.48% at the concentration of 40-160 mu L/L, and IC is50At 55.0148 μ L/L (y 1.5628+1.9750x, r 0.9429), spore germination was completely inhibited at 200 μ L/L.
2.3. Chemical composition of essential oil
The extraction rate of essential oil is 0.15%. A total of 55 chemical components were identified by GC-MS analysis, representing 88.2% of all components. The chemical compositions and ion patterns identified are shown in table 2 and fig. 3. These components include 27 monoterpenes, 5 aromatics, 1 lipid, 19 sesquiterpenes and 3 esters. Wherein the monoterpene compound and the sesquiterpene compound are main components, and respectively account for 25.77% and 54.14% of the total peak area. Alpha-apiecene was the largest in percentage (12.08%), followed by delta-cadinene (9.26%), alpha-alaskene (7.09%), beta-eucalyptene (5.39%) and curcumin (5.06%). The proportion of the 5 components is 38.88%. In addition, 6 unknown components account for nearly 11.8 percent.
The results of HS-SPME GC-MS show that the essential oil has good volatility at normal temperature, and 52 of 61 components can be detected (Table 2). The monoterpenes with lower boiling points were the major volatile component (74.39% of the total peak area) and a small amount of sesquiterpenes were detected. The larger compounds include 1, 8-cineole (17.12%), camphor (+) -2-Bornanone (11.52%), limonene (6.87%), anethole (6.51%), and alpha-terpineol (6.02%).
TABLE 2 Chrysanthemum indicum essential oil major chemical composition
A, retention time; b, peak area percentage; c, solvent dilution sampling method; d, headspace solid phase microextraction sampling; e, not detected; "Unknown" means an unidentified element.
ClassI, II, III, IV and V refer to monoterpenes, aromatics, lipids, sesquiterpenes and esters, respectively; "NA" means unavailable.
2.4. Influence on the cell membrane permeability of Phytophthora nicotianae
FIG. 4 shows the results of the measurement of the permeability of the wild chrysanthemum essential oil to the phytophthora nicotianae cells, and the cell membrane permeability gradually increases with the treatment time and concentration. The control conductivity values increased gently and leveled off after 3 hours, while the wild chrysanthemum essential oil treatment increased gradually (80. mu.L/L and 120. mu.L/L) or sharply (160. mu.L/L). 3 hours after treatment, 80. mu.L/L, 120. mu.L/L and 160. mu.L/L wild chrysanthemum essential oil treatment resulted in 127.31%, 146.63% and 621.92% increase in conductivity of phytophthora nicotianae hyphae, respectively.
2.5. Influence on the malondialdehyde content of Phytophthora nicotianae
As shown in FIG. 5, treatment with wild chrysanthemum essential oil significantly increased the malondialdehyde content of phytophthora nicotianae hyphae, and gradually increased as the treatment concentration increased. The increase was most pronounced with 160. mu.L/L treatment, 117.36% greater than the control.
2.6. Apoptosis assay
PI staining test results show that the chrysanthemum indicum essential oil treatment can destroy the integrity of cell membranes. As shown in FIG. 6, 160. mu.L/L of wild chrysanthemum essential oil treatment gave a distinct red light on Phytophthora nicotianae hyphae, whereas the control was barely stained.
Third, discuss
The wild chrysanthemum essential oil rich in monoterpene and sesquiterpene compounds is prepared, the essential oil can strongly inhibit the growth of phytophthora nicotianae hyphae and the spore germination, the action mode of the essential oil is probably related to the damage of a bacterial cell membrane, and the result shows that the wild chrysanthemum essential oil can be used as a new biocontrol factor for preventing and treating diseases caused by the phytophthora nicotianae.
Besides strong inhibition effect on hypha growth and spore germination, the wild chrysanthemum essential oil has good fumigation effect on phytophthora nicotianae, and shows that some chemical components have good volatility at room temperature. Because of strong penetrating power and even distribution, the antibacterial volatile substance has obvious advantage in inhibiting soil-borne diseases. Our results show that the wild chrysanthemum essential oil has good potential for preventing and controlling plant soil-borne diseases.
The main components of the chrysanthemum indicum essential oil prepared by the invention are alpha-apiene with the largest proportion (12.08%), and delta-cadinene (9.26%), alpha-alaskene (7.09%), beta-eucalyptene (5.39%) and curcumin (5.06%). The research shows that most of components of the wild chrysanthemum essential oil have good volatility at normal temperature, and the good fumigating effect on the tobacco phytophthora is achieved. The research firstly utilizes HS-SPME GC-MS to determine the volatile components of the wild chrysanthemum essential oil.
The fumigating effect of the essential oil is related to its volatile components, and we speculate that the antibacterial activity of the wild chrysanthemum essential oil may be combined with the main and secondary components or as a result of synergistic effect.
The research of the invention shows that the content of MDA, the change of conductivity and the PI staining test prove that the wild chrysanthemum essential oil can destroy the cell membrane of the phytophthora nicotianae. The wild chrysanthemum essential oil increases the permeability of the phytophthora nicotianae filamentous cell membrane and aggravates membrane damage, thereby causing cell apoptosis. In addition, the treatment of the wild chrysanthemum essential oil also causes the phytophthora nicotianae hypha malformation, including tip enlargement, hypha shrinkage, rupture and the like, and indicates that the morphological change is possible to be the action mechanism of the wild chrysanthemum essential oil for resisting the phytophthora nicotianae.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (8)
1. The application of the chrysanthemum indicum essential oil in preventing and treating phytophthora nicotianae is prepared by the following steps:
(1) mixing flos Chrysanthemi Indici with water, standing, and ultrasonic extracting to obtain ultrasonic extraction mixture; the ultrasonic power is 100-150W;
(2) performing steam distillation on the ultrasonic extraction mixture, and collecting distillate;
(3) extracting the distillate with n-hexane, separating n-hexane phase, and drying to obtain flos Chrysanthemi Indici essential oil.
2. The use of claim 1, wherein in the step (1), the wild chrysanthemum flower is a dried wild chrysanthemum flower growing in saline-alkali soil.
3. The use according to claim 1 or 2, wherein in the step (1), the ratio of the dry weight of the wild chrysanthemum flower to the volume of water is 1g: 8-12 mL.
4. The use according to claim 1 or 2, wherein in the step (1), the standing time is 2-5 h.
5. The use according to claim 1, wherein in the step (1), the ultrasonic extraction time is 15-50 min, and the ultrasonic extraction temperature is 45-60 ℃.
6. The use according to claim 1, wherein in the step (2), the steam distillation time is 4-6 h.
7. The use according to claim 1, wherein in the step (3), the volume ratio of the distillate to the n-hexane is 8-15: 1.
8. The use according to claim 1, wherein the wild chrysanthemum essential oil is in the form of a fumigant, a microemulsion or a slow release microcapsule.
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