CN109601559A - It is a kind of prevent and treat phytophthora mother chrysanthemum essential oil and its application - Google Patents
It is a kind of prevent and treat phytophthora mother chrysanthemum essential oil and its application Download PDFInfo
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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
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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 present invention provides a kind of mother chrysanthemum essential oil for preventing and treating phytophthora and its application, is related to phytophthora germ Prevention Technique field, the mother chrysanthemum essential oil, which includes the following steps, to be prepared: wild chrysanthemum is mixed with water, standing, ultrasonic extraction obtains ultrasonic extraction mixture；Ultrasonic extraction mixture is subjected to steam distillation, collects distillate；With n-hexane extraction distillate, organic phase and drying are separated, mother chrysanthemum essential oil is obtained.The mycelia growth and spore germination of Phytophthora nicotianae can be effectively suppressed in mother chrysanthemum essential oil provided by the invention, and the stifling lower bacteriostasis rate of concentration that minimum inhibitory concentration can reach 200 μ L/L, 157.48 μ L/L can reach 92.68%.The antibacterial mechanism of action of the mother chrysanthemum essential oil may be related with cell wall, the integrality of cell membrane is destroyed.Mother chrysanthemum essential oil of the present invention is to prevent and treat the botanical pesticide exploitation of phytophthora or even soil-borne disease to lay a good foundation.
Description
Technical field
The present invention relates to phytophthora germ Prevention Technique field more particularly to a kind of mother chrysanthemum essential oil for preventing and treating phytophthora and its answer
With.
Background technique
Phytophthora nicotianae Phytophthora nicotianae van Breda de Haan (syn.P.parasitica
Dastur) it is typical soil-borne pathogen, is presently considered to be one of most crushing oomycetes disease, can infect more than 255
Kind plant, tobacco (Nicotiana spp.), tomato (Lycopersicum esculentum) and citrus including serious harm
(Citrusspp.) etc..Since host's diversity and ecological zone are widely distributed, caused by massive losses be difficult to estimate, be plant
The continuing challenge of object disease control.The disease relies primarily on the chemical pesticide controls such as metalaxyl, Propamocarb, Mancozeb at present,
Medicament service life is long, is easy to cause pathogen drug resistance, causes pesticide residue and environmental pollution.Therefore, it needs to develop newly
Control measure reduces the use of chemical pesticide.
Essential oil is the complicated secondary metabolite obtained using special extracting method from plant, with high volatile and by force
Penetrability.Before this research shows that essential oil has substitution chemical pesticide to prevent and treat the potentiality of crop disease, have low toxicity, low-residual and
Environmental-friendly advantage.Recently, Phytophthora pathogen inhibitory activity has been reported in related plants essential oil.Such as blue gum
(Eucalyptus globulus), takes lemon (Citrus aurantifolia essential oil) and be able to suppress taro phytophthora
P.colocasiae.The discovery clove oil such as Jing Changliang is able to suppress Phytophthora nicotianae, and identifies that its main active is cloves
Phenol.Although related they act on Phytophthora pathogen it is found that the ingredient and bioactivity of a large amount of plants essential oils
Mechanism is unclear.The drug of effective phytophthora bacteria-treating can not effectively be found.
Compositae plant mother chrysanthemum Dendranthema indicum (L.) Des Moul is in extensive branch, China, including strand salt
Alkali.Its flower is traditional Chinese medicine, has apparent antiviral, blood pressure lowering, improving eyesight and other effects.A small amount of research reports mother chrysanthemum essence
Oil, which includes staphylococcus aureus and corynebacterium diphtheriae to several human pathogen's bacterium bags, has antibacterial action, still, to phytopathogen
Antibacterial activity has not been reported.Phytophthora nicotianae is considered as most destructive plant Oomycete disease pathogen, relies primarily on chemistry
Pesticide control, there is an urgent need to environmentally friendly surrogate-data techniques.
Summary of the invention
The present invention lacks the defect of vegetalitas prevention and treatment phytophthora pathogen in order to overcome in the prior art, provides a kind of prevention and treatment
The mother chrysanthemum essential oil of phytophthora, it is significant to the fungistatic effect of Phytophthora nicotianae, it is good to fumigate antibacterial effect.
In order to achieve the above-mentioned object of the invention, the present invention the following technical schemes are provided:
The present invention provides a kind of mother chrysanthemum essential oils for preventing and treating phytophthora, which is characterized in that the mother chrysanthemum essential oil is by including following
Step is prepared:
(1) wild chrysanthemum is mixed with water, is stood, ultrasonic extraction obtains ultrasonic extraction mixture；The ultrasonic power is
100~150W；
(2) ultrasonic extraction mixture is subjected to steam distillation, collects distillate；
(3) with n-hexane extraction distillate, n-hexane phase is separated, it is dry, obtain mother chrysanthemum essential oil.
Preferably, in step (1), the wild chrysanthemum is the dry product of the wild chrysanthemum grown in salt-soda soil.
Preferably, in step (1), the ratio between dry weight and the volume of water of the wild chrysanthemum are 1g:8~12mL.
Preferably, in step (1), the time of the standing is 2~5h.
Preferably, in step (1), the time of the ultrasonic extraction is 15~50min, and the temperature of the ultrasonic extraction is 45
~60 DEG C.
Preferably, in step (2), the time of the steam distillation is 4~6h.
Preferably, in step (3), the volume ratio of the distillate and n-hexane is 8~15:1.
The present invention also provides application of the mother chrysanthemum essential oil described in above-mentioned technical proposal in prevention and treatment Phytophthora nicotianae.
Preferably, the dosage form of the mother chrysanthemum essential oil is fumigant, microemulsion or slow-releasing microcapsule.
Compared with prior art, beneficial effects of the present invention:
(1) the mycelia growth and spore germination of Phytophthora nicotianae, minimum suppression can be effectively suppressed in mother chrysanthemum essential oil provided by the invention
Concentration processed can reach 200 μ L/L, while have good fumigating effect, and the stifling lower bacteriostasis rate of the concentration of 157.48 μ L/L can reach
92.68%.
(2) mycelia of the Phytophthora nicotianae handled through mother chrysanthemum essential oil of the present invention occurs obvious lopsided, such as ultimate swelling,
Mycelia shrinkage and local mycelia rupture, antibacterial mechanism of action may be related with cell wall, the integrality of cell membrane is destroyed.
(3) mother chrysanthemum essential oil provided by the invention is prepared via ad hoc approach, the mother chrysanthemum essential oil that this method is extracted
In monoterpenes and sesquiterpenoids rich in, these compounds cause hyphal cell membrane permeability to increase, mycelia the third two
Aldehyde increases and Apoptosis occurs.
(4) mother chrysanthemum essential oil of the present invention is pure natural plant ingredients, for the plant source of prevention and treatment phytophthora or even soil-borne disease
Pesticide developing is laid a good foundation.
Detailed description of the invention
Fig. 1 is mother chrysanthemum essential oil to Phytophthora nicotianae mycelia growth measurement result；Wherein, a, agar diffusion method processing；B, agar
Diffusion method control；C, fumigating system processing；D, fumigating system control；
Fig. 2 is influence of the mother chrysanthemum essential oil to Phytophthora nicotianae hypha form；Wherein: A, essential oil handle mycelia, left arrow institute
Refer to mycelia ultimate swelling, right arrow mycelia shrinkage, the rupture of middle arrow meaning mycelia；B compares mycelia；
Fig. 3 is mother chrysanthemum essential oil GC-MS total ion chromatogram；Wherein: a, n-hexane dilute sample introduction；B, solid phase microextraction head space
Sample introduction；
Fig. 4 is influence of the mother chrysanthemum essential oil to Phytophthora nicotianae mycelia conductivity；
Fig. 5 is influence of the mother chrysanthemum essential oil processing to Phytophthora nicotianae mycelia mda content；
Fig. 6 is influence of the mother chrysanthemum essential oil to Phytophthora nicotianae Apoptosis；Wherein, a, the processing of 160 μ L/L mother chrysanthemum essential oils；B is right
According to mycelia.
Specific embodiment
The present invention provides it is a kind of prevent and treat phytophthora mother chrysanthemum essential oil, the mother chrysanthemum essential oil by include the following steps preparation and
At:
(1) wild chrysanthemum is mixed with water, is stood, ultrasonic extraction obtains ultrasonic extraction mixture；The ultrasonic power is
100~150W；
(2) ultrasonic extraction mixture is subjected to steam distillation, collects distillate；
(3) with n-hexane extraction distillate, organic phase and drying is separated, mother chrysanthemum essential oil is obtained.
The raw material of mother chrysanthemum essential oil of the present invention is compositae plant mother chrysanthemum (Dendranthema indicum (L.)
DesMoul)；It is currently preferred using the mother chrysanthemum grown in salt-soda soil as raw material, mother chrysanthemum can generate this more hair under Saline Alkali Stress
Active principle needed for the bright mother chrysanthemum essential oil；The mother chrysanthemum that the present invention is more preferably grown with salt-soda soil under the monsoon climate of warm temperate zone
To select the mother chrysanthemum grown in the salt-soda soil of Shandong District as extraction raw material in raw material, such as specific embodiments of the present invention.
The present invention mixes wild chrysanthemum with water, stands, and ultrasonic extraction obtains ultrasonic extraction mixture；The ultrasonic power
For 100~150W.Wild chrysanthemum of the present invention is preferably the dry product of the wild chrysanthemum grown in salt-soda soil.In the present invention, described
It before wild chrysanthemum is mixed with water, is preferably crushed, the partial size of the crushing is preferably 20~60 mesh, more preferably 40 mesh.
In the present invention, the ratio between the dry weight of the wild chrysanthemum and the volume of water are preferably 1g:8~12mL, more preferably 1g:
10mL.In the present invention, the time of the standing is preferably 2~5h, more preferably 3h.In the present invention, when the standing
Environment temperature is preferably 20~25 DEG C.The purpose that the present invention is stood is in order to keep dry wild chrysanthemum sufficiently wetted, with after an action of the bowels
It is continuous to extract.
In the present invention, the power of the ultrasound is preferably 120~140W.In the present invention, the ultrasonic extraction when
Between preferably 15~50min, more preferably 20~35min.In the present invention, the temperature of the ultrasonic extraction is preferably 45~60
DEG C, more preferably 50 DEG C.The present invention first destroys eucaryotic cell structure by the way of ultrasonic extraction, is easier to the active principle in mother chrysanthemum
Dissolution.The present invention carries out ultrasonic extraction at 45~60 DEG C can further increase ultrasonic extraction efficiency.
After obtaining ultrasonic extraction mixture, ultrasonic extraction mixture is carried out steam distillation by the present invention, collects distillate.
The present invention is when carrying out steam distillation without adding moisture.
In the present invention, the time of the steam distillation is preferably 4~6h, more preferably 5h.The present invention to how into
Row steam distillation is not particularly limited, and collects distillate using steam distillation known in the art.In the present invention,
When collecting distillate, 20~25 DEG C of oil water separator temperature in half an hour of boiling, it is maintained at 30~35 DEG C later.Institute of the present invention
The number for stating steam distillation is preferably 1 time.
After obtaining distillate, the present invention is separated organic phase and drying, is obtained mother chrysanthemum essential oil with n-hexane extraction distillate.
In the present invention, the volume ratio of the distillate and n-hexane is preferably 8~15:1, more preferably 10:1.
Active principle is extracted into n-hexane phase by the present invention by n-hexane extraction, and n-hexane is separated after stratification
Mutually to get organic phase.The present invention is to remove remaining n-hexane to the dry purpose of organic phase.It extracts in order to prevent
Active principle volatilization, those skilled in the art, which preferably use anhydrous sodium sulfate dry and volatilize, removes n-hexane, obtains yellowish
The mother chrysanthemum essential oil of color, i.e. mother chrysanthemum essential oil.
Prepare mother chrysanthemum essential oil according to the method described above, recovery rate 0.15% or so, can obtain containing abundant hemiterpene and times
The mother chrysanthemum essential oil of hemiterpene has significant inhibiting effect for Phytophthora nicotianae.
The present invention also provides application of the mother chrysanthemum essential oil described in above-mentioned technical proposal in prevention and treatment Phytophthora nicotianae.Such as the present invention
Shown in embodiment, the mother chrysanthemum essential oil has significant inhibiting effect to Phytophthora nicotianae.Preferably, the mother chrysanthemum essential oil dosage form is preferred
Include fumigant, microemulsion and slow-releasing microcapsule；When the mother chrysanthemum essential oil is fumigant, pass through stifling progress Phytophthora nicotianae
Prevention and treatment has good result.Before tobacco transplant, modeling is used immediately after spraying mother chrysanthemum (the 500 times of dilutions) application that 4000ml is prepared per acre
Expect that film (0.08cm is thick) is 7 days stifling by plastic casing sealing, takes off film, sunning can transplant tobacco seedlings after 7 days.
Technical solution provided by the invention is described in detail below with reference to embodiment, but they cannot be understood
For limiting the scope of the present invention.
Embodiment 1
1. vegetable material
Fresh mother chrysanthemum acquires in October, 2017 in Shandong Seashore beach, takes back laboratory with nylon mesh sack, yin
It does spare.
2. extracting
Dry mother chrysanthemum bud grinds, it is sieved through with 40 meshes, weighs 100g, put into 2000mL beaker, by solid-liquid ratio
Distilled water is added in 1g:10mL, and room temperature stands 3 hours and is placed in ultrasonic container, ultrasonic power 120W, ultrasonic time
30min, ultrasonic temperature are 50 DEG C, obtain ultrasonic extraction mixture.Steam distillation is carried out to ultrasonic extraction mixture, when distillation
Between be 5h, boil half an hour in 20 DEG C of oil water separator temperature, be maintained at 30 DEG C later, distillate obtained after distillation.It will
Distillate and n-hexane separate organic phase and anhydrous sodium sulfate are dry, after vaporing away n-hexane according to volume 10:1 hybrid extraction
Up to flaxen mother chrysanthemum essential oil, precise weighing, calculating recovery rate is 0.15%.
Embodiment 2
One, process is tested:
1. Phytophthora nicotianae culture
To tobacco High pathogenicity Phytophthora nicotianae JM01 strain culturing and spore preparation method reference literature method (Han, T.；
You,C.；Zhang,L.；Feng,C.；Zhang,C.；Wang,J.；Kong,F.Biocontrol potential of
antagonist Bacillus subtilis Tpb55 against tobacco black shank.BioControl
2016,61,195–205.)。
2. Antibacterial Activity
2.1. Joan-board diffusion method
After Phytophthora nicotianae covers on oat medium, colony edge picking bacteria cake (5mm) is placed in plate side, in correspondence
Another side perforating, the mother chrysanthemum essential oil (similarly hereinafter, repeating no more) (10 μ l/mL) that is prepared of 100 μ L embodiments 1 of addition, DMSO
28 DEG C of culture 5d are then set in triplicate for control, measure antibacterial circle diameter, and become using scanning electron microscopic observation hypha form
Change.
2.2. test is fumigated
Using sealing plate make-up method measurement mother chrysanthemum essential oil to the fumigation activity of Phytophthora nicotianae.The Phytophthora nicotianae of diameter 5mm
On pure culture biscuits involvng inoculation to OA culture medium, by 1.25,2.5,5,10,15,20 and 25 μ L (be respectively equivalent to 10,20,40,60,80,
120, and 160 μ L/L) mother chrysanthemum essential oil be added drop-wise to culture ware lid center, sealed with sealed membrane, 28 DEG C of culture 3d are repeated 3 times.It adopts
Colony diameter is measured with crossing method, is not added with the plate of mother chrysanthemum essential oil as control.
Mycelial growth inhibition rate is calculated using following for examination:
2.3. mycelial growth rate method measures
Mother chrysanthemum essential oil is made into OA (45 DEG C) plate of various concentration with DMSO (0.5%, v/v), and mother chrysanthemum essential oil is final concentration of
10,20,40,80,120,160 and 200 μ L/L.It is inoculated with Phytophthora nicotianae bacteria cake (5mm) and arrives the OA containing various concentration mother chrysanthemum essential oil
On culture medium, 28 DEG C of culture 5d, the DMSO of identical dilution ratio is control, and crossing method measures colony diameter, calculates mycelia
Growth inhibition ratio.
2.4. spore germination inhibits test
Spore germination inhibits test to use liquid serial dilutions.By the mother chrysanthemum essential oil of 500mL various concentration (10,20,
40,80,120,160 and 200 μ L/L) and isometric Phytophthora nicotianae spore suspension (106Cfu/mL it) mixes, drop is in band groove
Glass slide on, 8 DEG C, 80% relative humidity culture 6h set microscopically observation spore germination situation, calculate spore germination and inhibit
Rate.
3. Essential Oil
3.1. Gc-mss (GC-MS)
Essential oil is diluted to 100g/L with n-hexyl alcohol, takes 1 μ L sample introduction to Agilent 7890-5975GC-MS, using 30 ×
0.25mm i.d, 0.25 μm, DB-5MS Fused-silica capillary column (Agilent technology).Intake air temperature is 250 DEG C, is with helium
Carrier gas, flow velocity 1.0mL/min.Initial oven temperature keeps the temperature 2 minutes at 45 DEG C, 200 DEG C is risen at 5 DEG C/min, heat preservation 2
Minute, 320 DEG C are risen at 15 DEG C/min, keep the temperature 5 minutes.Ionization mode is 70eV electron collision.Mould is monitored using full scan
Formula, mass scan range are 35~500m/z, obtain mass spectrogram, open acceleration voltage after solvent delay 5min.
3.2. the headspace solid-phase microextraction (HS-SPME) of volatile compound
Based on previous studies, using sandwich type (DVB/CAR/PDMS) SPME fiber (Supelco Analytical,
Sigma-Aldrich, St.Louis, MO, USA) volatile compound is absorbed from EOD.SPME fiber is inserted at room temperature
Sample bottle headspace containing 0.1g EOD 20 minutes, then takes out SPME fiber from sample bottle, and immediately with 250 DEG C
It is inserted into GC syringe 5 minutes with the split ratio of 10:1.Before lower sub-sampling, SPME fiber is improved 5 minutes at 250 DEG C.
Ionization mode is 70eV electron collision.Using full scan monitoring pattern, mass scan range is 35~500m/z, obtains mass spectrum
Scheme, opens acceleration voltage after solvent delay 5min.
3.3. the identification of compound
Retention index (RI) and their mass spectrum and NIST 14 database of the identification of single compound based on calculating
Available reference compound is mass spectrographic in (https: //www.nist.gov/srd/nist-special-database-14)
Compare；Threshold value matching ratio is arranged to 90.Under the same operating conditions, it calculates and n-alkane (C7-C30) homologue system
Arrange related RI.
4. cell leakage
Permeability of cell membrane is indicated by its conductivity (EC).5 are transferred to from the bacteria cake that tobacco OA plate edge extracts
In liquid OA culture medium, cultivated 3 days under 28 DEG C and 175rpm.The EOD of 1 gram of mycelia and 0,80,120 or 160 μ L/L are (with 2%
DMSO dilution) 20mL mixing, (20 ± 3 DEG C) of room temperature preservations.0.5 after processing, 1.0,1.5,2.0,2.5,3.0,3.5,
4.0,4.5 and 5.0h measures EC value.
Mycelium malonaldehyde 5. (MDA) content
The content of malonaldehyde (MDA) in Phytophthora nicotianae mycelium is determined with thiobarbituricacidα- (TBA) method.By 5 bacterium
Silk disk is inoculated in 90mL liquid OA culture medium, cultivates 48h under 28 DEG C and 175rpm.Adding 10 milliliters of EOD, (concentration is
80,120 or 160 μ L/L), it is further cultured for 24 hours, then harvests (2.6) as described above.Mycelium is placed in pre-cooling mortar, is used
Liquid nitrogen grinding is at powder.Erinaceus mycelium powder (1g) is transferred in the centrifuge tube containing 10mL 10% (w/v) TCA, is vortexed at 4 DEG C
30s is centrifuged 20min at 5000 × g, and supernatant (2_mL) is mixed with the 2mL containing 0.6% (w/v) TBA, boils 20min, cold
But absorbance of the mixture at 450nm, 532nm and 600nm is measured afterwards.nm.Following formula for calculate MDA content (C, with
Nmol/g fresh weight indicates):
C=6.45 × (OD532-OD600)-0.56×OD450
6. cell death experiments
Damage with propidium iodide fluorescence analytic approach evaluation EOD to cell membrane.Phytophthora nicotianae is handled with the EOD of 160 μ L/L
Mycelium is for 24 hours.Mycelium (100 μ L) is transferred in 1-mL test tube, dyes 30 in the dark situation of 1 μ g/mL with PI (400 μ L)
Minute.After dyeing, it is washed with distilled water mycelia three times, then in fluorescence microscope (excitation wavelength 535nm, launch wavelength
It is shot under 615nm).
7. statistical analysis
Data are analyzed using Excel 2003 and SPSS 22.0.Bacteriostatic activity, permeability of cell membrane, mycelium
The result of MDA assay is indicated with the Mean +/- SE of triple experiments.Pass through the logarithm and corresponding suppression to EOD concentration
The regression analysis of the probability value of rate processed calculates half maximum suppression concentration (IC50) value.Variance analysis and multiple is carried out with Duncan method
Compare (P < 0.05).
Two, test result
2.1. inhibit mycelia growth
Agar diffusion method measurement result show mother chrysanthemum essential oil (10 μ L/L) can strong inhibition Phytophthora nicotianae growth, can
Apparent inhibition zone is formed, entire bacterium colony growth is also obviously suppressed (Fig. 1 a), and mycelia growth is sparse, and compares mycelia growth
Uniformly, fine and close (Fig. 1 b).
Make-up it is stifling the experimental results showed that, mother chrysanthemum essential oil volatile component can obviously inhibit Phytophthora nicotianae mycelia to grow (table
1).As shown in figs. lc and ld, it is handled using 10 μ L/L mother chrysanthemum essential oils, Phytophthora nicotianae mycelia slow growth and sparse.It is raw to mycelia
Concentration IC in long inhibition50For 20.0445 μ L/L (y=2.7097+1.7591x, r=0.9977).Mother chrysanthemum essential oil concentration is 160
When μ L/L, mycelia growth is suppressed completely.Our result indicate that mother chrysanthemum essential oil and its volatile matter can strong inhibition tobaccos
The growth of phytophthora mycelia.
1 mother chrysanthemum essential oil of table is grown to Phytophthora nicotianae mycelia and the inhibiting effect of spore germination
* in same column the mean difference of same letter without significant property (P > 0.05), and Duncan ' s multi-way examine difference without
Significant property (P > 0.05).
* compared with the control, inhibiting rate (mycelium) is reacted with dimethyl sulfoxide (dimethyl sulfoxide).
Scanning electron microscope the result shows that, mother chrysanthemum essential oil causes Phytophthora nicotianae mycelia that apparent metamorphosis occurs, and shows as bacterium
Silk shrinkage, deformation even rupture (Fig. 2 a), and compare mycelia then smooth plentiful (Fig. 2 b).
Table 1 lists mother chrysanthemum essential oil in the inhibitory effect of mycelia difference growth time.Phytophthora nicotianae mycelia grows by bright
It is aobvious to inhibit, and increase as concentration for the treatment of increases.Percentage mycelial inhibition is about 6.46% when 10 μ L/L, the then bacterium in 200 μ L/L
Silk growth is suppressed completely.Concentration (IC in inhibition50) and minimum inhibitory concentration (MIC) be respectively 48.4531 μ L/L (y=
1.4264+2.1204x, r=0.9824) and 200 μ L/L.
2.2. spore germination inhibiting effect
As shown in table 1, mother chrysanthemum essential oil obviously inhibits Phytophthora nicotianae spore germination, and under 40~160 μ L/L concentration, inhibiting rate is about
For 8.52~92.48%, IC50For 55.0148 μ L/L (y=1.5628+1.9750x, r=0.9429), the spore in 200 μ L/L
Sprouting is suppressed completely.
2.3. Chemical Composition of The Essential Oil
Extraction of essential oil rate is 0.15%.GC-MS analysis identifies 55 kinds of chemical components altogether, accounts for the 88.2% of all the components.
The chemical component and ion figure of identification are shown in Table 2 and Fig. 3.These components include 27 kinds of monoterpenes compounds, 5 kinds of aromatic compounds
Object, a kind of lipid, 19 kinds of sesquiterpenoids and 3 kinds of ester type compounds.Wherein monoterpenes compound and sesquiterpenoids
For main component, the 25.77% and 54.14% of total peak area is accounted for respectively.α-selinene accounting is maximum (12.08%), is secondly
δ-cadinene (9.26%), α-Alaska alkene (7.09%), β-eudesmene (5.39%) and curcumin (5.06%).Above-mentioned 5
Ingredient accounting is planted up to 38.88%.In addition there are 6 kinds of non-principal components, accountings nearly 11.8%.
HS-SPME GC-MS the result shows that, essential oil main component at normal temperature all have good volatility, 61 kinds of ingredients
In there are 52 kinds to can detecte (table 2).The lower monoterpenes compound of boiling point is that major volatile constituents (account for total peak area
74.39%), a small amount of sesquiterpenoids also is detected.The biggish compound of accounting includes 1,8- Cineole
(17.12%), camphor (+) -2-Bornanone (11.52%), limonene (6.87%), anethene (6.51%) and α-pine tar
Alcohol (6.02%).
2 mother chrysanthemum essential oil main chemical compositions of table
* a, retention time；B, peak area percentage；C, solvent dilute sampling method；D, headspace solid-phase microextraction sampling method；
E, it is undetected；" Unknown " refers to unknown ingredient.
ClassI, II, III, IV and V respectively refer to monoterpene, aromatic compound, lipid, sesquiterpenoids and esters；" NA " refers to
It is unavailable.
2.4. to the influence of Phytophthora nicotianae cell leakage
Fig. 4 shows mother chrysanthemum essential oil to the measurement result of Phytophthora nicotianae cell permeability, with the increasing of processing time and concentration
Add, cell leakage gradually increases.Control conductivity value gently increase, tend to be steady after 3 hours, and mother chrysanthemum essential oil processing then by
It cumulative plus (80 μ L/L and 120 μ L/L) or sharply increases (160 μ L/L).3 hours after processing, 80 μ L/L, 120 μ L/L and 160 μ L/
The processing of L mother chrysanthemum essential oil causes Phytophthora nicotianae mycelia conductivity to increase separately 127.31%, 146.63% and 621.92%.
2.5. to the influence of Phytophthora nicotianae mda content
As shown in figure 5, the processing of mother chrysanthemum essential oil obviously increases Phytophthora nicotianae mycelia mda content, and as concentration for the treatment of increases
Add and gradually rises.It is wherein handled with 160 μ L/L and increases the most obvious, relatively control increase by 117.36%.
2.6. Apoptosis measures
PI dye test the result shows that, the processing of mother chrysanthemum essential oil can destroy the integrality of cell membrane.As shown in fig. 6,160 μ
The processing of L/L mother chrysanthemum essential oil, Phytophthora nicotianae mycelia issues obvious feux rouges, and compares almost without being colored.
Three, it discusses
The present invention is prepared for the mother chrysanthemum essential oil rich in monoterpene and sesquiterpenoid, which can have strong inhibition tobacco
The growth of phytophthora mycelia and spore germination, the mode of action may be related with somatic cells film is destroyed, and the above results show mother chrysanthemum essence
Oil, which can be used as the new bio-control factors of one kind, leads to the prevention and treatment of disease for Phytophthora nicotianae.
Other than the high inhibition effect to mycelia growth and spore germination, mother chrysanthemum essential oil has Phytophthora nicotianae good smoked
Effect is steamed, showing some chemical components at room temperature has good volatility.Since penetration power is strong and is evenly distributed, antibacterial is waved
Hair object inhibits soil-borne disease to have a clear superiority.Our result indicate that mother chrysanthemum essential oil has the good of prevention and treatment plant soil-borne diseases
Good potentiality.
Mother chrysanthemum essential oil mother chrysanthemum essential oil main component prepared by the present invention is then that α-selinene accounting is maximum (12.08%),
Secondary is δ-cadinene (9.26%), α-Alaska alkene (7.09%), β-eudesmene (5.39%) and curcumin (5.06%).I
Research simultaneously show that mother chrysanthemum essential oil majority ingredient has good volatility at normal temperature, achieve good to Phytophthora nicotianae
Fumigating effect.This research is to utilize the volatile component of HS-SPME GC-MS measurement mother chrysanthemum essential oil for the first time.
The fumigating effect of essential oil is related with its volatile component, it is presumed that the antibacterial activity of mother chrysanthemum essential oil may with it is main
Ingredient and submember joint or the result of synergistic effect.
Studies have shown that MDA content, conductivity variations and PI dye test of the invention confirms that mother chrysanthemum essential oil can destroy
Phytophthora nicotianae cell membrane.Mother chrysanthemum essential oil increases Phytophthora nicotianae mycelia cell leakage, aggravates membrane damage, withers so as to cause cell
It dies.In addition, the processing of mother chrysanthemum essential oil also results in Phytophthora nicotianae mycelia deformity, including ultimate swelling, mycelia shrinkage and rupture etc., table
Bright morphological change may be also the mechanism of action of the anti-Phytophthora nicotianae of mother chrysanthemum essential oil.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Claims (9)
1. a kind of mother chrysanthemum essential oil for preventing and treating phytophthora, which is characterized in that the mother chrysanthemum essential oil is prepared by including the following steps:
(1) wild chrysanthemum is mixed with water, is stood, ultrasonic extraction obtains ultrasonic extraction mixture；The ultrasonic power be 100~
150W；
(2) ultrasonic extraction mixture is subjected to steam distillation, collects distillate；
(3) with n-hexane extraction distillate, n-hexane phase is separated, it is dry, obtain mother chrysanthemum essential oil.
2. mother chrysanthemum essential oil according to claim 1, which is characterized in that in step (1), the wild chrysanthemum is raw in salt-soda soil
The dry product of long wild chrysanthemum.
3. mother chrysanthemum essential oil according to claim 1 or 2, which is characterized in that in step (1), the dry weight of the wild chrysanthemum with
The ratio between volume of water is 1g:8~12mL.
4. mother chrysanthemum essential oil according to claim 1 or 2, which is characterized in that in step (1), time of the standing is 2~
5h。
5. mother chrysanthemum essential oil according to claim 1, which is characterized in that in step (1), the time of the ultrasonic extraction is 15
~50min, the temperature of the ultrasonic extraction are 45~60 DEG C.
6. mother chrysanthemum essential oil according to claim 1, which is characterized in that in step (2), the time of the steam distillation is
4~6h.
7. mother chrysanthemum essential oil according to claim 1, which is characterized in that in step (3), the body of the distillate and n-hexane
Product is than being 8~15:1.
8. application of the mother chrysanthemum essential oil described in claim 1~7 any one in prevention and treatment Phytophthora nicotianae.
9. application according to claim 8, which is characterized in that the dosage form of the mother chrysanthemum essential oil is fumigant, microemulsion, delays
Release micro-capsule etc..
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| CN114854489A (en) * | 2022-05-10 | 2022-08-05 | 北京市农林科学院 | Essential oil of Mao Huaju, preparation method and application thereof |
| CN114854489B (en) * | 2022-05-10 | 2024-03-22 | 北京市农林科学院 | Chamomile essential oil and preparation method and application thereof |
| CN114847309A (en) * | 2022-05-20 | 2022-08-05 | 湖北工程学院 | Application of mixed extract of lindera glauca leaves and fruits, wild chrysanthemum extract and mixture thereof in prevention and treatment of strawberry diseases |
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