CN108902149A - Scopoletin excites the purposes and application method of the broad spectrum resistance of plant immune - Google Patents
Scopoletin excites the purposes and application method of the broad spectrum resistance of plant immune Download PDFInfo
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- A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
- A01N43/02—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms
- A01N43/04—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom
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Abstract
The present invention relates to the purposes and application method of the broad spectrum resistance of Scopoletin excitation plant immune, the method that Scopoletin passes through seed treatment, Plant nematode resistance can be improved, can be used for the biological control of plant nematode, and plant growth and yield is promoted to increase；It can induce plant by the method for seed treatment and generate resistance of wide spectrum, prevent and treat grey speck of soybean, fusarium root rot of maize, rice blast；The beneficial effects of the invention are as follows；Scopoletin has efficient induced activity to plant nematode, and can with broad spectrum activity induce Different Crop Disease Resistance, provides new resource for research and development Novel seed inorganic agent and inducer.
Description
Technical field
The invention belongs to field of biological pesticide, and in particular to Scopoletin excites the use of the broad spectrum resistance of plant immune
Way and application method.
Background technique
Anisodus luridus chlorins compound is a kind of with phenylpropyl alcohol α-pyranone core Cis-hydroxyl groups cortex cinnamomi acid lactone.Recently
A large amount of scientific research shows that Scopoletin and its derivative are played an important role in agricultural and pharmaceutical sector.In fungi or
Scopoletin can be accumulated largely in the plant of microbial infection, this shows that Scopoletin may be anti-with the defense of plant stress-resistance
It should there is connections；Many Chinese traditional herbs, such as the root of purple-flowered peucedanum, frutus cnidii, girald daphne bark, Chinese prickly ash, the bark of ash, the dried immature fruit of citron orange, active constituent
It is exactly Scopoletin and its derivative, they are in anti-inflammatory, antibacterial, inhibition thrombosis, anticoagulant, antitumor, inhibition maincenter
Nervous system and the clinical efficacy that reduces blood pressure etc. are significant.
Many countries and regions occur and cause harm plant nematode in the world, and type is varied.In the world
Interior, the generation of plant nematode is got worse with the development of agriculture and forestry and the variation of tillage and cultivation system with endangering, according to
Esser statistics, the whole world has reported that discovery plant nematode 207 belongs to 4832 kinds of (Liu Weizhi, section imperial jade seals until nineteen ninety
.2000. Beijing plant pathogeny line insect：Chinese agriculture publishing house, P.1-2).It is estimated that plant nematode causes the world
The year loss late of staple crops be 12.3%, more than 100,000,000,000 dollars, and it is actual lose considerably beyond estimation, reason is
(the Wang Shou China Pomology that do not arouse attention because not having field Visual symptoms or specialization feature is endangered caused by many nematodes
Learn the Beijing:Chinese agriculture Science Press, 1994.P.1-5), in addition, also existing in world wide to the new discovery that nematode is caused harm
It is continuously increased, and the agricultural cultivation system of some updates keeps nematode problem more prominent, furthermore, nematode and other biofacies
Interaction and on plant generate it is direct or indirect influence also sharply increasing.In this sense, it is endangered caused by nematode
More other biologies are more hidden！At present to the nematode of plant pest there are about more than 3000 kinds, mainly there is soybean cyst line in China
Worm, root-knot nematode, Bursaphelenchus xylophilus, sweet potato stem nematode etc..Soybean cyst nematode Heterodera glycines (Soybean cyst nematode) are agriculturally
Cause harm maximum a kind of nematode, after disease occurs, the general underproduction 10% or so, serious up to 75% or more, or even total crop failure
(A.G.Whitehead,1998.Plant Nematode Control. ISBN0851991882CAB Intertiol).Mesh
Before, still based on chemical prevention, but in recent years, the application waste of nematocide is big for the prevention and treatment of plant nematode, pollutes ring
Border, poor selectivity, natural disposition of going out is strong, destroys the drawbacks such as Soil bio-environment and increasingly payes attention to (Zhang Keqin, He Shichuan, week by people
The effect and its progress induction of resistance microorganism .3 of the .1991. such as common vetch fungi and bacterium in nematode biological and ecological methods to prevent plant disease, pests, and erosion:55-66),
21 century is known as environmentally friendly century, and the application of some effective nematocides is gradually restricted, therefore to plant nematode
Biological control research become hot issue naturally.
Summary of the invention
To make up for the shortcomings of the above existing technologies, the purpose of the present invention is to propose to Scopoletin excitation plant immunes
The purposes and application method of broad spectrum resistance measure Scopoletin to its preventive and therapeutic effect, together using plant nematode as target
When searched out from biological metabolic product have efficiently induction Genes For Plant Tolerance nematode active material Scopoletin, and broad spectrum activity lure
Different Crop disease resistance is led, research and development Novel seed inorganic agent is intended to be and inducer provides new resource.
The purpose of the present invention is what is be achieved through the following technical solutions, Scopoletin excites the broad-spectrum disease resistance of plant immune
The purposes and application method of property, are characterized in that：
The Scopoletin can be improved Plant nematode resistance, can be used for phytotrophy by the method for seed treatment
The biological control of nematode, and plant growth and yield is promoted to increase.
The Scopoletin can induce plant by the method for seed treatment and generate resistance of wide spectrum, prevent and treat soybean greyness
Disease, fusarium root rot of maize, rice blast.
Further, the Scopoletin is for the method for the biological control of plant nematode：By Seed sterilization,
The Scopoletin solution and seed that prepare are used 1:70 ratios (i.e. 1g Scopoletin solution handles 70g seed) are mixed
Even, the concentration of Scopoletin solution is 20.25mg/L.
Further, Scopoletin prevention and treatment grey speck of soybean, fusarium root rot of maize, rice blast method be：Anisodus luridus
Plain solution concentration is 20.25mg/L, and liquid kind 1 is respectively adopted:70 ratios (i.e. 1g Scopoletin solution handle 70g seed) into
Row soya seeds processing, using liquid kind 1:50 ratios (i.e. 1g Scopoletin solution handles 50g seed) carry out at corn seed
Reason, using liquid kind 1:10 ratios (i.e. 1g Scopoletin solution handles 10g seed) carry out rice paddy seed processing.
Further, the Scopoletin can be prepared by biological metabolic product, and the specific method is as follows：
(1) prepared by biological metabolic product crude extract
A. prepared by microbial metabolic products crude extract
Penicillium chrysogenum (Penicillium Chrysogenum) Snef1216, depositary institution：Chinese microorganism strain is protected
Hide administration committee's common micro-organisms center；Preservation address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, the Chinese Academy of Sciences
Institute of microbiology；Preservation date：On December 14th, 2010；Deposit number:CGMCC No.4469.
Microassembly robot (Penicillium janthinellum) Snef1650, depositary institution：Chinese microorganism strain is protected
Hide administration committee's common micro-organisms center；Preservation address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, the Chinese Academy of Sciences
Institute of microbiology；Preservation date：On May 11st, 2015；Deposit number:CGMCC No.10487.
Penicillium chrysogenum, microassembly robot pure culture, using PDA culture medium：Potato 200g, glucose 20g, agar 17g,
Water l000mL.
Penicillium chrysogenum, microassembly robot liquid fermentation medium are improvement Cha Shi fluid nutrient medium：(NH4)2S043.67g
KCl 0.96g, FeSO40.02g, K2HPO40.64g, MgSO4·5H20 1.04g, sucrose 28.3g, distilled water 1000mL.
The preparation of penicillium chrysogenum, microassembly robot fermentation liquid：
Bacterial strain penicillium chrysogenum and microassembly robot are cultivated into 7d respectively at PDA at 25 DEG C, is inoculated in and ferments equipped with sterilized liquid
In culture medium, 25 DEG C of shaking table shake culture (120r/min) 8d, cultured fermentation liquid is filtered with filter paper, to remove thick mycelia
Body, supercentrifuge 12000r/min are centrifuged l0min, are placed in 4 DEG C of refrigerators and save backup, and select full-automatic mechanical stirring not
The steel fermentor that becomes rusty carries out a large amount of preparations of fermentation liquid, by penicillium chrysogenum and microassembly robot fermentation liquid respectively with anhydrous methanol 1:7 ratios
Example, which is placed on ice cube, to be sufficiently pre-chilled and mixes, and is taken out mixed liquor and is removed precipitating and evaporated methanol with Rotary Evaporators, by gained
Pretreatment fluid with freeze drier be freeze-dried at the pigment in powder macroporous absorbent resin adsorption sample, deionized water
Elution is collected liquid and is freeze-dried up to crude extract.
B. plant extract
Plant is：Soybean；Rice；Corn；Arabidopsis；
Extracting method：By soybean；Rice；Corn；Arabidopsis seed uses liquor natrii hypochloritis's surface sterilization respectively, then uses
Aseptic water washing aseptically dries, and the seed dried is seeded into respectively in seedling alms bowl, takes soybean, corn, rice and
The blade and root system of arabidopsis are placed in mortar on ice plus anhydrous methanol grinding, sample and anhydrous methanol 1:7 ratios are sufficiently mixed
It is even, methanol is evaporated with filter paper impurity screening, then with Rotary Evaporators, resulting pretreatment fluid is freezed with freeze drier
It is dried to powder, with the pigment in macroporous absorbent resin adsorption sample, deionized water elution, collects liquid and freeze-drying is
Obtain crude extract.
(2) in biological metabolic product crude extract Scopoletin detection
The extracted biological metabolic product crude extract of step (1) is crossed chromatographic column HPLC to prepare, standard items is collected and goes out
The sample of peak time, then the sample of preparation is carried out HPLC detection, chromatography Detection wavelength are 254nm, 25 DEG C of column temperature, sample volume 10
μ L, mobile phase 37%-48%CH3OH·H2Sterling in extracted biological metabolic product crude extract is crossed high-resolution liquid phase matter by O
Spectrometer carries out relative molecular mass analysis.
Beneficial effects of the present invention：The present invention provides the purposes of the broad spectrum resistance of Scopoletin excitation plant immune
And application method, there is efficient induced activity to plant nematode, and can with broad spectrum activity induce Different Crop disease anti-
Property, new resource is provided for research and development Novel seed inorganic agent and inducer.
Detailed description of the invention
Fig. 1 is high performance liquid chromatography detection Scopoletin standard items；
Fig. 2 is the purifying substance in high performance liquid chromatography detection biological metabolite；
Fig. 3 is the purifying substance that high performance liquid chromatography and mass spectrometry detect biological metabolite.
Specific embodiment
Embodiment 1
(1) prepared by biological metabolic product crude extract
A. microculture and fermentation
Penicillium chrysogenum (Penicillium Chrysogenum) Snef1216, depositary institution：Chinese microorganism strain is protected
Hide administration committee's common micro-organisms center；Preservation address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, the Chinese Academy of Sciences
Institute of microbiology；Preservation date：On December 14th, 2010；Deposit number:CGMCC No.4469.
Microassembly robot (Penicillium janthinellum) Snef1650, depositary institution：Chinese microorganism strain is protected
Hide administration committee's common micro-organisms center；Preservation address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, the Chinese Academy of Sciences
Institute of microbiology；Deposit number:CGMCC No.10487.
Penicillium chrysogenum, microassembly robot pure culture, using PDA culture medium：Potato 200g, glucose 20g, agar 17g,
Water l000mL.
Penicillium chrysogenum, microassembly robot liquid fermentation medium are improvement Cha Shi fluid nutrient medium：(NH4)2S043.67g
KCl 0.96g, FeSO40.02g, K2HPO40.64g, MgSO4·5H20 1.04g, sucrose 28.3g, distilled water 1000mL.
The preparation of penicillium chrysogenum, microassembly robot fermentation liquid：
Bacterial strain penicillium chrysogenum Snef1216 and microassembly robot Snef1650 is cultivated into 7d respectively at PDA at 25 DEG C, uses 5mm
The punch of diameter beats bacteria cake 3, is inoculated in the triangular flask equipped with 50mL sterilized liquid fermentation medium, 25 DEG C of shaking table shakes
Culture (120r/min) 8d is swung, cultured fermentation liquid is filtered with filter paper, to remove thick mycelium, supercentrifuge 12000r/
Min is centrifuged l0min, is placed in 4 DEG C of refrigerators and saves backup, and full-automatic mechanical stirring stainless steel fermentation tank is selected to carry out 25L fermentation
A large amount of preparations of liquid, by Snef1216 and Snef1650 fermentation liquid respectively with anhydrous methanol 1:7 ratios are placed on ice cube sufficiently pre-
Cold and mixing takes out mixed liquor removing precipitating and is simultaneously evaporated methanol with Rotary Evaporators, and resulting pretreatment fluid is dry with freezing
Dry instrument freeze-drying is collected liquid and is freezed at pigment of the powder in macroporous absorbent resin adsorption sample, deionized water elution
It is drying to obtain crude extract.
B. plant extract
Extracting method：By soybean；Rice；Corn；Arabidopsis seed uses liquor natrii hypochloritis's surface sterilization respectively, then uses
Aseptic water washing aseptically dries, and the seed dried is seeded into respectively in seedling alms bowl, takes soybean, corn, rice and
The blade and root system of arabidopsis are placed in mortar on ice plus anhydrous methanol grinding, sample and anhydrous methanol 1:7 ratios are sufficiently mixed
It is even, methanol is evaporated with filter paper impurity screening, then with Rotary Evaporators, resulting pretreatment fluid is freezed with freeze drier
It is dried to powder, with the pigment in macroporous absorbent resin adsorption sample, deionized water elution, collects liquid and freeze-drying is
Obtain crude extract.
(2) in biological metabolic product crude extract Scopoletin detection
Biological metabolic product is filtered with filter paper, then is filtered with miillpore filter, takes filtrate that equal volume is added
Acquired solution is evaporated by methanol with Rotary Evaporators after evenly mixing, and the remaining 1.5mL substance obtained is biological metabolic product
Crude extract.
The HPLC analysis of Scopoletin in biological metabolic product：Extracted biological metabolic product crude extract is crossed into chromatography
It is prepared by column HPLC.Chromatography Detection wavelength be 254nm, 25 DEG C of column temperature, sample volume 10 μ L, mobile phase 37%-48%
CH3OH.H2O。
The spectrometer analysis of Scopoletin in biological metabolite：Extracted biological metabolic product crude extract is crossed into high score
Distinguish that liquid phase mass spectrograph carries out relative molecular mass analysis.
By finding in standard items appearance time, only one unimodal, and the peak to biological metabolite quality detection
Appearance time is identical as standard items appearance time, illustrates that the biological metabolism substance collected is a kind of pure material (Fig. 1 and Fig. 2), can
It can be same substance with standard items；And high-resolution liquid phase mass spectrograph detection and analysis average molecular matter is carried out to the pure material
Amount, the relative molecular mass for obtaining the pure material is 192.168, molecular formula C10H8O4, detect that the substance is Scopoletin
(scopoletin) (Fig. 3).
2 field Scopoletin of embodiment prevents and treats nematode test
(1) test medicine
A：Sterling Scopoletin (SIGMA company), being configured to concentration with 60% methanol is 20.25mg/L Scopoletin
Solution；
B：The Scopoletin solution that penicillium chrysogenum ferments, preparation method are shown in embodiment 1, are configured with 60% methanol
It is 20.25mg/L Scopoletin solution at concentration；
C：The Scopoletin solution that microassembly robot ferments, preparation method are shown in embodiment 1, are configured with 60% methanol
It is 20.25mg/L Scopoletin solution at concentration；
D：Soybean extracts gained Scopoletin solution, and preparation method is shown in embodiment 1, is configured to concentration with 60% methanol
For 20.25mg/L Scopoletin solution；
E：Corn extracts gained Scopoletin solution, and preparation method is shown in embodiment 1, is configured to concentration with 60% methanol
For 20.25mg/L Scopoletin solution；
F：Arabidopsis extracts gained Scopoletin solution, and preparation method is shown in embodiment 1, is configured to 60% methanol dense
Degree is 20.25mg/L Scopoletin solution；
(2) seed and method for treating seeds
By soya seeds (kind be Ji beans 17, seed sale market purchase) surface sterilization, then use aseptic water washing, general
Above-mentioned Scopoletin solution uses liquid kind 1:70 ratios (i.e. 1g Scopoletin solution handles 70g seed) carry out seed treatment,
It is dry under aseptic condition, it is spare.
(3) test method
Soybean in field is tested respectively in Shenyang City, Liaoning Province Agricultural University Of Shenyang experimental plot and Shenyang City, Liaoning Province Kangping town
Experimental plot carries out field efficacy experiment, and experimental plot soybean cyst nematode Heterodera glycines are very serious at two, main cultivation crop soybean and corn etc..
It is control with the soya seeds of untreated soya seeds (CK) and 60% methyl alcohol process, 45d is investigated after cultivation,
Soybean plant strain is connected root to dig out, keeps root system complete, investigates soybean root system cyst number, calculates preventive effect, 120d is adjusted after cultivation
It looks into, investigates the 100-grain weight of each processing, calculate rate of growth.
(4) test result
The soybean handled using Scopoletin, soybean root system cyst number, which is substantially less than, compares (CK), in Agricultural University Of Shenyang
Field efficacy with Kangping is respectively 59.72% and 72.37% or more, and treated, and soybean shows with the presence of untreated soybean
It writes difference (table 1)；The soybean of Scopoletin processing, soybean 100-grain weight is significantly higher than control (CK), in Agricultural University Of Shenyang and health
Flat rate of growth is respectively 16.16% and 16.52% or more, and there are significant differences with untreated soybean for treated soybean
(table 2).
The field efficacy of 1 Scopoletin processed soybeans seed of table
Note：Data are average ± standard error/difference with letters different after column data in result table after investigation after 45d
It indicates in P<0.01 level difference is significant (Duncan " sShi duncan's new multiple range method),
The field effect of increasing production of 2 Scopoletin processed soybeans seed of table
Note：The result after investigation after 120d
Scopoletin prevention and treatment nematode test in embodiment Room 3
(1) test medicine
With test medicine in embodiment 2, i.e. medicament A-F.
(2) seed and method for treating seeds
By soya seeds (kind is Ji beans 17, the purchase of seed sale market) surface sterilization, then use aseptic water washing.It will
The fermentation liquid prepared uses liquid kind 1:70 mass percents carry out seed treatment, aseptically dry, spare.
(3) for trying nematode
The rhizosphere cyst soil for picking up from Agricultural University Of Shenyang experimental plot, is identified as soybean cyst nematode Heterodera glycines (Soybean cyst
Nematode), by eluriating the cyst of the full maturation of crossing sieve method picking, it is put into the sterilizes culture dish for filling sterile water, then
It is sterilized with NACLO, and aseptic water washing, therefrom the complete and full cyst of picking, merging fills the sterilizing training of appropriate amounts of sterilized water
It supports in ware, puts it into insulating box and cultivate, the second instar larvae J2 hatched is inoculated into soybean easily susceptible in greenhouse after 3d
Root expand numerous.The cyst for expanding numerous acquisition is hatched into J2 using tray method, is prepared into 200/mL of nematode suspension.
(4) test method
Scopoletin solution (processing A-F) is used into liquid kind 1:70 ratios (i.e. 1g Scopoletin solution handles 70g seed)
Seed treatment is carried out, the soya seeds dried after processing are seeded into pot for growing seedlings, every basin is inoculated with 2000 soybean cyst nematode Heterodera glycines
Second instar larvae J2.Be control with untreated soya seeds and the soya seeds of 60% methyl alcohol process, after transplanting 9d into
Row investigation, measurement soybean plant height, root long, root fresh weight and root interior lines borer population, and calculate preventive effect.
5. test result
The soybean handled using Scopoletin solution, root interior lines borer population is significantly lower than control, soybean plant height, root long, stalk
There are notable difference, potting preventive effect is 40.91% or more for weight, root fresh weight and untreated soybean.
Potting control efficiency of 3 Scopoletin of the table processing to soybean nematode
4 Scopoletin of embodiment induces plant generation system resistant proof
(1) test medicine
With test medicine in embodiment 2, i.e. medicament A-F.
(2) seed and method for treating seeds
Soya seeds (kind is Ji beans 17, the purchase of seed sale market) surface sterilization, then use aseptic water washing.It will be eastern
Henbane element solution (processing A-F) uses liquid kind 1:70 mass percents carry out seed treatment, aseptically dry, spare.
(3) for examination nematode and its processing
The rhizosphere cyst soil for picking up from Agricultural University Of Shenyang experimental plot, is identified as soybean cyst nematode Heterodera glycines (Soybean cyst
Nematode), by eluriating the cyst of the full maturation of crossing sieve method picking, it is put into the sterilizes culture dish for filling sterile water, then
It is sterilized with NACLO, and aseptic water washing, therefrom the complete and full cyst of picking, merging fills the sterilizing training of appropriate amounts of sterilized water
It supports in ware, puts it into insulating box and cultivate, the second instar larvae J2 hatched is inoculated into soybean easily susceptible in greenhouse after 3d
Root expand numerous.The cyst for expanding numerous acquisition is hatched into J2 using tray method, is prepared into 200/mL of nematode suspension.
(4) test method
Soil matrix (matrix, sand, native ratio 1:1:1) it is fitted into alms bowl after 165 DEG C of hot air sterilization 3h, is implanted into four leaf stage
Soybean seedling, soybean root system is divided into impartial two parts, is placed at triangle disposition, diameter 3cm is opened in the bottom of alms bowl at top
Circular aperture, soybean seedling is placed in the alms bowl at top, and separated root system is made to arrive separately at two of lower part by circular aperture
In alms bowl, the root system grown in the alms bowl of bottom two is respectively to challenge root system and response root system.System is placed in greenhouse, in challenge root
Scopoletin solution is poured in the alms bowl of system below 2cm soil, response root system inoculation second instar larvae suspension 5mL is total about after 2d
It 1000, is repeated 6 times.The sterile water of equivalent, response root system inoculation two are added in the challenge root system of same method contradistinction system
Instar larvae suspension 5mL does blank control.
Detection nematode invades quantity after inoculation nematode 1 week, and root system is cleaned up with tap water, claims root fresh weight, then
The dyeing that nematode in root is carried out with sodium hypochlorite-acid fuchsin staining observes and records root interior lines borer population under stereomicroscope
Amount.
(5) test result
It is tested by crack root method, Scopoletin shows the induction of resistance to soybean root system.It is being inoculated with J2 after a week, east
J2 quantity is significantly lower than control in the root system of henbane element inoculation.By dyeing it can be observed that the response root of Scopoletin processing
Inhibiting rate is up to 41.14% or more in system.
Induction of resistance of 4 Scopoletin of the table processing to soybean root system
| Processing | Root interior lines borer population (item) | Inhibiting rate |
| A | 91.67±2.37B | 41.98% |
| B | 90.33±2.08B | 42.83% |
| C | 92.37±1.98B | 41.54% |
| D | 91.67±2.37B | 41.98% |
| E | 93.00±2.21B | 41.14% |
| F | 91.67±2.37B | 41.98% |
| CK | 158.00±2.37A | —— |
5 Scopoletin seed treatment of embodiment generates resistance of wide spectrum test
(1) prepared by Scopoletin solution
Scopoletin solution preparation method is the same as 1 test medicine of embodiment.
(2) seed and processing method
Soybean, distant beans 15；Rice, Liaoxing No.1；Corn, Shen He 128 (the above seed is purchased from seed sale market)
The surface of the seed disinfection, is aseptically dried, spare.
(3) for examination pathogen and its culture
Germ To Soybean Frogeye Leaf Spot (Cercosporidium sofinum)；Fusarium root rot of maize bacterium (Fusarium
graminearum)；Pyricularia oryzae (Magnaporthe grisea)
Using potato culture (PDA) activation culture pathogen, the Germ To Soybean Frogeye Leaf Spot strain deposited of going bail for, by corn
Pine root fungus and Germ To Soybean Frogeye Leaf Spot part mycelia are inoculated in respectively in PDA culture medium cultivate 5d and 15d, 5d and 15d after with spitting
The conidium that temperature 20 washes lower surface is adjusted with optics microexamination spore concentration to spore concentration 1 × 106A/mL is standby
With.Pyricularia oryzae (M.grisea) uses soybean oat medium activation culture, and part mycelium inoculation is taken to cultivate in soybean oat
5d is cultivated on base, washes the conidium of lower surface after 5d with polysorbas20, with optics microexamination spore concentration, is adjusted to spore
Sub- concentration 1 × 106A/mL is spare.
(4) test method
The anti-grey speck of soybean indoor pot experiment of Scopoletin inducing soybean.It is carried out in 28 DEG C of constant temperature of greenhouse, for examination
Scopoletin solution is used liquid kind 1 in seedling alms bowl by soil 2h sterilization treatment at 165 DEG C:70 ratios (i.e. 1g Anisodus luridus
Plain solution handles 70g seed) seed treatment is carried out, the soya seeds dried are seeded into seedling alms bowl, when soybean tri-leaf period, are adopted
With the mode of spray inoculation, the spore suspension of Germ To Soybean Frogeye Leaf Spot is uniformly sprayed on blade.With untreated soybean
Seed (CK) and the soya seeds of 60% methyl alcohol process are control, are investigated after being inoculated with 10d, investigation grey speck of soybean hair
State of an illness condition, the sick grade of statistics grey speck of soybean morbidity, sick grade grade scale reference《Plant sick research method》, and calculate the state of an illness and refer to
Several and preventive effect size.
The anti-fusarium root rot of maize indoor pot experiment of Scopoletin inducing maize.Soil disinfection is same as above, in seedling alms bowl, then
According to 3, every alms bowl, using liquid kind 1:50 ratios (i.e. 1g Scopoletin solution handles 50g seed) carry out seed treatment processing, with
The corn seed of untreated corn seed (CK) and 60% methyl alcohol process is control, if 3 repetitions.To corn seedling
When length to the 1 leaf phase, by the way of pouring root inoculation, the spore suspension of fusarium root rot of maize bacterium is poured and is filled in corn root system week
It encloses, each processing is inoculated with same amount of suspension.Humidity is kept, it is preceding to use black-out cloth dark processing, rear normal illumination culture for 24 hours.
After being inoculated with 5d, fusarium root rot of maize incidence, the sick grade of statistics fusarium root rot of maize morbidity, sick grade grade scale reference are investigated《It plants
Sick research method》, and calculate disease index and preventive effect size.
Scopoletin inducing paddy rice blast resisting indoor pot experiment.Soil disinfection is same as above, in seed plate, according still further to
3, every cave, using liquid kind 1:10 ratios (i.e. 1g Scopoletin solution handles 10g seed) carry out seed treatment processing, with not
The rice of treated rice paddy seed (CK) and 60% methyl alcohol process is control, 50 caves of each processing, if 3 repetitions.To water
When rice seedling is grown to three leaves wholeheartedly, by the way of spray inoculation, the spore suspension of Pyricularia oryzae is uniformly sprayed on rice children
On seedling leaf, make to be covered with droplet on blade, each processing is inoculated with same amount of suspension.Humidity is kept, it is preceding to use black-out cloth for 24 hours
Dark processing, rear normal illumination culture.After being inoculated with 7d, rice blast incidence, the disease of statistics rice blast morbidity are investigated
Grade, sick grade grade scale reference《Plant sick research method》, and calculate disease index and preventive effect size.
(6) test result
Indoor anti-grey speck of soybean efficiency test is carried out with Scopoletin solution and 60% methyl alcohol process soya seeds,
Scopoletin solution can the significantly anti-grey speck of soybean of inducing soybean, and Scopoletin solution processed soybeans compared with control (CK)
Seed is 57.29% to the preventive effect of grey speck of soybean；60% methyl alcohol process is only to the control efficiency of grey speck of soybean
14.07%.In summary conclusion, Scopoletin are shown significantly to the control effect of grey speck of soybean (being shown in Table 5).
Indoor anti-fusarium root rot of maize efficiency test is carried out with Scopoletin solution and 60% methyl alcohol process corn seed,
Scopoletin solution can significantly inducing paddy rice blast resisting, and Scopoletin solution processing corn seed compared with control (CK)
Preventive effect to fusarium root rot of maize is 87.17% or more；60% methyl alcohol process is only to the control efficiency of fusarium root rot of maize
12.4%, it may be said that can not have preventive effect to fusarium root rot of maize, the generation of fusarium root rot of maize can not be inhibited.In summary it ties
By Scopoletin solution is shown significantly to the control effect of fusarium root rot of maize (being shown in Table 6).
Indoor blast resisting efficiency test is carried out with Scopoletin solution and 60% methyl alcohol process rice paddy seed, and it is right
According to (CK) compared to the significant inducing paddy rice blast resisting of Scopoletin solution energy, and Scopoletin solution Rice seeds treated is to rice
The preventive effect of seasonal febrile diseases is 51.75% or more；60% methyl alcohol process is 0.29% or more to the control efficiency of rice blast.It is comprehensive with
Upper conclusion, Scopoletin are shown significantly to the control effect of rice blast (being shown in Table 7).
5 Scopoletin of table measures the control efficiency of grey speck of soybean
| Processing | Disease index | Control efficiency |
| A | 28.33±0.36c | 57.29% |
| B | 27.33±0.46c | 58.80% |
| C | 28.33±0.36c | 57.29% |
| D | 27.33±0.46c | 58.80% |
| E | 27.33±0.46c | 58.80% |
| F | 28.33±0.36c | 57.29% |
| 60% methanol | 57±0.53b | 14.07% |
| Water | 66.33±0.89a | — |
Note：Data are that average ± standard error/difference is indicated with letters different after column data in P in table<0.01 level difference
Significantly (Duncan " sShi duncan's new multiple range method), similarly hereinafter.
6 Scopoletin of table measures the control efficiency of fusarium root rot of maize
| Processing | Disease index | Control efficiency |
| A | 10.33±1.78c | 87.60% |
| B | 11.33±1.33c | 87.17% |
| C | 10.33±1.78c | 87.60% |
| D | 11.33±1.33c | 87.17% |
| E | 11.33±1.33c | 87.17% |
| F | 10.33±1.78c | 87.60% |
| 60% methanol | 73±0.69b | 12.40% |
| Water | 88.33±1.36a | — |
7 Scopoletin of table measures the control efficiency of rice blast
| Processing | Disease index | Control efficiency |
| A | 32.8±0.79b | 52.00% |
| B | 32.6±0.67b | 52.34% |
| C | 32.8±0.79b | 52.00% |
| D | 32.9±0.79b | 51.90% |
| E | 33.0±0.33b | 51.75% |
| F | 32.7±0.67b | 52.19% |
| 60% methanol | 68.2±0.66a | 0.29% |
| Water | 68.4±1.67a | — |
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, without departing from the principle of the present invention, it can also make several improvements and retouch, these improvements and modifications
It should be regarded as protection scope of the present invention.
Claims (5)
1. Scopoletin excites the purposes and method of the broad spectrum resistance of plant immune, it is characterized in that：The Scopoletin is logical
Plant nematode resistance can be improved in the method for crossing seed treatment, can be used for the biological control of plant nematode, and promotes plant
Growth and yield increase.
2. Scopoletin excites the purposes and method of the broad spectrum resistance of plant immune, it is characterized in that：The Scopoletin is logical
The method for crossing seed treatment can induce plant and generate resistance of wide spectrum, prevent and treat grey speck of soybean, fusarium root rot of maize, rice blast.
3. the purposes and method of the broad spectrum resistance of plant immune are excited according to Scopoletin described in right 1, it is characterized in that：Institute
The method of biological control that Scopoletin is stated for plant nematode is：It is by Seed sterilization, the Scopoletin prepared is molten
Liquid and seed use 1:70 ratios（I.e. 1g Scopoletin solution handles 70g seed）It is mixed, the concentration of Scopoletin solution
For 20.25mg/L.
4. the purposes and method of the broad spectrum resistance of plant immune are excited according to Scopoletin described in right 2, it is characterized in that：Institute
State Scopoletin prevention and treatment grey speck of soybean, fusarium root rot of maize, rice blast method be：Scopoletin solution concentration is
Liquid kind 1 is respectively adopted in 20.25mg/L:70 ratios（I.e. 1g Scopoletin solution handles 70g seed）Soya seeds processing is carried out,
Using liquid kind 1:50 ratios（I.e. 1g Scopoletin solution handles 50g seed）Corn seed processing is carried out, using liquid kind 1:10 ratios
Example（I.e. 1g Scopoletin solution handles 10g seed）Carry out rice paddy seed processing.
5. the Scopoletin according to right 1 or 2 excites the purposes and method of the broad spectrum resistance of plant immune, feature
It is：The Scopoletin can be prepared by biological metabolic product, and the specific method is as follows：
（1）The preparation of biological metabolic product crude extract
A. prepared by microbial metabolic products crude extract
Penicillium chrysogenum（Penicillium Chrysogenum）Snef1216, depositary institution：Chinese microorganism strain preservation management
Committee's common micro-organisms center；Preservation address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Chinese Academy of Sciences microorganism are ground
Study carefully institute；Preservation date：On December 14th, 2010；Deposit number: CGMCC No. 4469；
Microassembly robot（Penicillium janthinellum）Snef1650, depositary institution：Chinese microorganism strain preservation pipe
Reason committee common micro-organisms center；Preservation address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Chinese Academy of Sciences microorganism
Research institute；Deposit number: CGMCC No.10487；
Penicillium chrysogenum, microassembly robot pure culture, using PDA culture medium：Potato 200g, glucose 20g, agar 17g, water
l000mL；
Penicillium chrysogenum, microassembly robot liquid fermentation medium are improvement Cha Shi fluid nutrient medium：(NH4)2S043.67g KCl
0.96g, FeSO40.02g, K2HPO40.64g, MgSO4·5H20 1.04g, sucrose 28.3g, distilled water 1000mL；
The preparation of penicillium chrysogenum, microassembly robot fermentation liquid：
Bacterial strain penicillium chrysogenum and microassembly robot are cultivated into 7d respectively at PDA at 25 DEG C, are inoculated in equipped with sterilized liquid fermented and cultured
In base, 25 °C of shaking table shake cultures（120r/min）8d, cultured fermentation liquid are filtered with filter paper, high to remove thick mycelium
Fast centrifuge 12000r/min is centrifuged l0min, is placed in 4 DEG C of refrigerators and saves backup, and selects full-automatic mechanical stirring stainless steel hair
Fermentation tank carries out a large amount of preparations of fermentation liquid, by penicillium chrysogenum and microassembly robot fermentation liquid respectively with anhydrous methanol 1:7 ratios are set
It is sufficiently pre-chilled and mixes on ice cube, take out mixed liquor and remove precipitating and evaporated methanol with Rotary Evaporators, it will be resulting pre-
Treatment fluid freeze drier is freeze-dried into the pigment in powder macroporous absorbent resin adsorption sample, deionized water elution,
It collects liquid and is freeze-dried up to crude extract；
B. plant extract
Plant is：Soybean；Rice；Corn；Arabidopsis；
Extracting method：By soybean；Rice；Corn；Arabidopsis seed uses liquor natrii hypochloritis's surface sterilization respectively, then uses sterile water
It rinses, aseptically dries, the seed dried is seeded into respectively in seedling alms bowl, take soybean, corn, rice and arabidopsis
Blade and root system are placed in mortar on ice plus anhydrous methanol grinding, sample and anhydrous methanol 1:7 ratios mix well, with filter
Paper impurity screening, then evaporated methanol with Rotary Evaporators, resulting pretreatment fluid freeze drier is freeze-dried into powder
End, with the pigment in macroporous absorbent resin adsorption sample, deionized water elution is collected liquid and is freeze-dried up to crude extract；
（2）The detection of Scopoletin in biological metabolic product crude extract
By step（1）Extracted biological metabolic product crude extract is crossed chromatographic column HPLC and is prepared, when collecting standard items appearance
Between sample, then the sample of preparation is subjected to HPLC detection, chromatography Detection wavelength is 254nm, 25 DEG C of column temperature, 10 μ L of sample volume,
Mobile phase 37%-48%CH3OH·H2Sterling in extracted biological metabolic product crude extract is crossed high-resolution liquid phase mass spectrograph by O
Carry out relative molecular mass analysis.
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