CN1316882C - Chitosan oligosaccharide containging pesticide composition and its application - Google Patents
Chitosan oligosaccharide containging pesticide composition and its application Download PDFInfo
- Publication number
- CN1316882C CN1316882C CNB031152767A CN03115276A CN1316882C CN 1316882 C CN1316882 C CN 1316882C CN B031152767 A CNB031152767 A CN B031152767A CN 03115276 A CN03115276 A CN 03115276A CN 1316882 C CN1316882 C CN 1316882C
- Authority
- CN
- China
- Prior art keywords
- chitosan oligosaccharide
- plant
- composition
- purposes
- rice
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Images
Landscapes
- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
The present invention discloses a pesticide composition which contains oligosaccharide with effective prevention and cure amount and a carrier. The present invention also discloses a preparation method for the pesticide composition, and a purpose of the pesticide composition in the aspect of providing plant disease resistance. The pesticide composition of the present invention can strongly activate the plant defense response of paddy rice, etc., and thereby, disease resistance is enhanced.
Description
Technical field
The present invention relates to agriculture field, relate more specifically to composition pesticide that contains chitosan oligosaccharide and preparation method thereof.
Background technology
Between plant and the pathogen according to the pattern of " gene pairs gene ", discern mutually and conduct (Flor by signal, H.H. (1971) Current status of the gene-for-gene concept.Annu.Rev.Phytopathol.9,275-296), and transfer answering system separately, its result who vies each other causes plant to susceptible, the anti-disease of cause of disease or disease-resistant.
From signal transduction path, the corresponding R and the mutual work of Avr albumen meet mechanism (Baker B, Zambryski P, the Staskawicz B of receptor-ligand, Dinesh-Kumar SP (1997) Signalingin plant-microbe interactions.Science, 276:726-733; Lamb CJ (1996) Ligand-receptor mechanism in plant-pathogen recognition.Science, 274:2038-2039; Martin GB (1999) Functional analysis of plant diseaseresistance genes and their downstream effectors.Curr Opin Plant Biol, 2:273-279; Staskawicz BJ (2001) Genetics of Plant-Pathogen Interactionsspecifying plant disease resistance.Plant Physiol, 125:73-76).
The mutual biological process that shows as high complexity between plant and the pathogen, cell death can further cause to organize at a distance and make systems response, i.e. systemic acquired resistance as the characteristic feature of the hypersensitivity that takes place in the plant infection site.This cell death is considered to the suicide sexual behavior of a kind of active of plant antagonism pathogen.
Think at present, the related cell death of hypersensitivity has similarity (Gilchrist with said apoptosis in the animal in the plant, D.G. (1998) Programmed cell death in plantdisease:the purpose and promise of cellular suicide.Annu.Rev.Phytopathol.36,393-414).Defense response was divided into for three steps to be finished: conduction of the mutual identification between plant and the pathogen, signal and answering.Therefore, probe into the detailed mechanism in this three steps, become and illustrated plant one of key of the resistance mechanism of pathogenic microorganism.
It is generally acknowledged that plant produces defense response by the conduction of elicitor signal.Elicitor (elicitor) is that inducing plant produces protective plant protecting agent (phytoalexins) and allergy (hypersensitive reaction, factor HR) in the plant disease-resistant physiology course.Include various large biological molecules (coming from oligosaccharide, glycoprotein, albumen and the polypeptide etc. of bacterium, fungi and plant) and small-molecule substance (salicylic acid, jasmonic etc.).Elicitor can be induced the expression of specific gene highly single-minded, fast in the interaction of plant and microorganism.
Chitin is the main component that constitutes the disease fungus cell wall, there are some researches show that its degraded produces oligomerization osamine (chitosan oligosaccharide) and can excite plant to produce defense response.Yet, up to now, still chitosan oligosaccharide is not used for agricultural report.
On the other hand, various chemical pesticides all cause various adverse effects more or less to the environment and the mankind, and therefore, this area chases after to be essential will develop the new biopesticide that can improve disease resistance of plant.
Summary of the invention
An object of the present invention is to provide a kind of biopesticide of novel raising disease resistance of plant.
Another object of the present invention provides described biopesticide preparation method and application thereof.
In a first aspect of the present invention, a kind of composition pesticide is provided, it contains chitosan oligosaccharide and the carrier of preventing and treating effective dose.
In a preference, the content of chitosan oligosaccharide is the 0.0005-99% of composition total weight, preferably is 0.005-95%, more preferably is 0.01-90%.
In another preference, described composition is the formulation that is selected from down group: wettable powder, emulsifiable concentrate, the aqueous solution, emulsion, but spray solution, oil-or-water-based dispersion, suspending agent, pulvis, granule, microcapsules.
In another preference, described composition is a pulvis, and the content of described chitosan oligosaccharide is the 80-90% of composition total weight.
In another preference, described composition is the aqueous solution, and wherein the content of chitosan oligosaccharide is the 0.0005-0.05% of composition total weight.
In another preference, described composition also contains the material that is selected from down group: unimolecule osamine, sodium chloride, Tween-20 and water.
In a second aspect of the present invention, a kind of method that improves disease resistance of plant is provided, comprise step: use the chitosan oligosaccharide of effective dose for described plant.
In a preference, described plant is selected from down group: paddy rice, wheat, vegetables, flowers, and described disease resistance is a plant fungal disease resistance.
In another preference, the effective dose of using of chitosan oligosaccharide is that 5ppm (0.005g/L) is to 1000ppm (1g/L).
In a third aspect of the present invention, a kind of method for preparing chitosan oligosaccharide is provided, may further comprise the steps:
(a) provide solid chitin or clam shell, the powder of shrimp shell after cleaning-drying is pulverized;
(b) in sealing and have in the agitator of cooling installation in 1: 5-1: 10 ratio adds concentrated hydrochloric acid;
(c) 30-45 ℃ is stirred and all to dissolve to chitin in 3-12 hour;
(d) in low-temperature (low temperature) vessel, cool off supernatant suspension to 10-20 ℃;
(e) add NaOH to pH3.0 ± 0.5, produce the NaCl precipitation, and the soluble oligomeric osamine is in supernatant;
(f) filtration is desalted, and reclaims the oligosaccharide amine aqueous solution;
(g) dehydrate.
Description of drawings
The dose effect of Fig. 1 chitosan oligosaccharide inducing paddy rice cell death.
The time effect of Fig. 2 chitosan oligosaccharide inducing paddy rice cell death.
The generation of Fig. 3 different disposal inducing paddy rice cytoactive oxygen.
Fig. 4 variable concentrations chitosan oligosaccharide induced defense expression of gene.
The time of Fig. 5 chitosan oligosaccharide induced defense gene expression is dynamic.
Rice leaf Trypan blue coloration result after Fig. 6 variable concentrations chitosan oligosaccharide is handled.
Rice leaf DAB-uptaking result after Fig. 7 variable concentrations chitosan oligosaccharide is handled.
The anti-pest result of the not same date after Fig. 8 chitosan oligosaccharide is handled.
Fig. 9 chitosan oligosaccharide is handled back rice plant resistance and is strengthened.
Embodiment
The present invention has carried out extensive and deep research to the mechanism of action of chitosan oligosaccharide, finds that first chitosan oligosaccharide can cause the death of paddy rice hypersensitivity like cell, activates the defense response of plants such as paddy rice strongly, thereby has strengthened the disease resistance of plant.Therefore, chitosan oligosaccharide can be used as the disease-resistant derivant of inducing plant and is used for agricultural.Finished the present invention on this basis.
The active component of agriculturally useful compositions of the present invention is chitosan oligosaccharide (Oligo-GlcNAc).With the paddy rice is example, and chitosan oligosaccharide can be induced the rice cell and the leaf cells generation allergic reaction death of suspension culture, and activates expression such as defense response gene RCH10, PAL strongly, produces oxygen and bursts out.The rice plant of handling has obviously strengthened the resistance to rice blast etc.
The preparation method of chitosan oligosaccharide is known, a kind of preferred scheme is as follows: solid chitin or clam shell, the powder of shrimp shell after cleaning-drying is pulverized → in sealing and have in the agitator of cooling installation in 1: 10 ratio add concentrated hydrochloric acid → 30-45 ℃ stir 3-12 hour to chitin all dissolve → in low-temperature (low temperature) vessel cooling supernatant suspension to 10-20 ℃ → progressively add 5N NaOH to pH3.0 to produce the NaCl precipitation, and the soluble oligomeric osamine in supernatant → filter (salt is recyclable as the industrial salt) → oligosaccharide amine aqueous solution that desalts to dehydrate and be finished product → powder for drying and preserve.
The present composition contains the active component (being chitosan oligosaccharide) of total amount 0.0005-99wt% usually.The content of active component is higher in the agricultural chemicals of concentrated type, and as 80-90%, and active component content is lower in dilution type agricultural chemicals and the actual composition that uses, and is generally 0.0005-0.05%.In addition, can also comprise other suitable chemical bactericides,, usual component such as synergist, trace element, stabilizing agent, adhesive, wetting agent, dispersant, emulsifier, bleeding agent, solvent, filler.
For example can also containing in the present composition, selective herbicide has the material of insecticidal activity, chemical fertilizer and/or growth regulator with other.
The present composition is in the disease resistance that is used to improve plant.The form that composition is suitable for comprises aerosol and water-based or the non-aqueous solution or the disperse system of suitable sprinkling, spraying and low capacity or ultra-low volume spraying.Optimal way is aqueous mother liquor and particle form soluble in water.
Suitable solid diluent comprises diatomite, shuck, tricalcium phosphate, cork powder, clays such as kaolin, bentonite or Attagel, and water-soluble polymer when the preparation composition.
In addition, solid composite can also contain one or more compatibility wetting agents, dispersant, and emulsifier or pigment, these compositions also can play thinner when solid-state.
Such solid composite can be a pulvis, the form of granule or wettable powder.Usually obtain pulvis by grinding, obtain granule, tablet or the agent of brick type by granulation or compressing tablet.
The form of fluid composition can be a solution, suspension and emulsion, also can wrap in it natural or synthetic polymer in, and can comprise wetting agent, dispersant or emulsifier.Such emulsion, suspension or solution can prepare water-soluble polymer (and mixture of above-mentioned thinner) with water-based, organic or water-organic diluent.In addition, can contain for example the above ionic or wetting agent, dispersant or the emulsifier of nonionic or their mixture in the described thinner.
Available several different methods is made into preparation with component.Appropriate formulation for example can be selected from: the aqueous solution (SL), wettable powder (WP), emulsifiable concentrate (EC), but spray solution or emulsion, oiliness or aqueous dispersion, suspending agent, pulvis (DP), granule.
The principle of various preparations all is known, and is for example describing to some extent in the following document: Winnacker-Kuchler, " Chemische Technologie "
Chemical technology, Vol.7, C.Hauser Verlag Munich, the 4th edition, 1986; Van Valkenburg, " formulations of pesticide ", Marcel Dekker N.Y., the 2nd edition, 1972-73; K.Martens, " atomized drying handbook ", the 3rd edition, G.Goodwin Ltd.London.
The required formulation aid that is used for the present composition, (inert substance for example, surfactant, solvent and other additives), also be known, it is for example described: Watkins " insecticidal dust thinner and carrier handbook " the 2nd edition, Darland Books, Caldwell N.J.; H.v.Olphen, " guiding of clay colloidal chemistry " the 2nd edition, J.Wiley; Sons, N.Y., Marsden, " solvent guide " the 2nd edition, Interscience, N.Y.1950; McCutcheon ' s, " scale remover and emulsifier annual ", MC Publ.Corp., RidgewoodN.J.; Sisley and Wood, " surfactant encyclopedia ", Chem.Publ.Co.Inc., N.Y.1964; Schonfelt, " Grenzflachenaktive Athylenoxidaddukte "
The addition of surface-active oxirane is produced Thing, Wiss.Verlagsgesell., Stuttgart 1976; Winnacker-Kuchler, " ChemischeTechnologie "
Chemical technology, Vol.7, C.Hauser Verlag Munich, the 4th edition, 1986.
Wettable powder can be dispersed in water.Except that active substance, wettable powder also can comprise wetting agent, dispersant, the material of no environment public hazards such as thinner.The preparation of pulvis can be: solid matters such as the talcum after active substance and the meticulous pulverizing, kaolin, bentonite and so on natural clay or diatomite are together ground.The preparation of granule can be to be adsorbed in inert substance particle with the active substance spraying, or active substance solution put on carrier (for example sand, kaolin or inert substance particle) surface by adhesive (for example polyvinyl alcohol, Sodium Polyacrylate, or mineral oil).Use if desire to mix, then suitable actives matter can be resembled to prepare and be prepared into particle the chemical fertilizer granule with chemical fertilizer.
In an example of the present invention, provide a kind of mother liquor.It prepares with the following method: the chitosan oligosaccharide powder → by 10 or 20mg/ml (1% or 2%) make the aqueous solution → filtration sterilization → room temperature preservation term of validity and can reach more than half a year, low temperature is preserved the term of validity for 4 ℃ and is reached more than 2 years.
In principle, Agrotechnical formulation of the present invention comprises the 0.0005-2wt% chitosan oligosaccharide, preferably 0.005-1wt%, more preferably 0.05-0.5wt%.For example, in the wettable powder, the concentration of active substance is about 0.005-2wt%; All the other are the conventional formulation component.If emulsifiable concentrate, the concentration of active substance can be about 0.5-2wt%, is good with 0.5-1wt%.In the powder-type preparation, active substance accounts for about 0.005-2wt%, is good with 0.005-0.5wt%; But in the spray solution, active substance accounts for about 0.0005-2wt%, is good with 0.0052-1wt%.If granule, in principle, the water dispersible granules contains about 0.005-1wt% active substance, sows to contain 0.005-0.5wt% in the particle.Application concentration at 5ppm (0.005g/L) between the 1000ppm (1g/L), preferably 50-200ppm, more preferably 100-500ppm.
The floristics of available present composition protection
But the plant of the present composition and method protection is not particularly limited, and can be cereal (as corn, wheat, rice, Chinese sorghum), especially rice classes; The field, woodland, plantation institute, greenhouse, ornamental crops in orchard and the vineyard, plantation institute crop and forest, for example: cotton, tobacco, vegetables (beans for example, rape, cucurbit, lettuce, onion, tomato and pepper), field crops (as potato, beet, peanut, soybean, rape), sugarcane, meadow and forest (as corn, Chinese sorghum, alfalfa), plantation institute crop (tealeaves, coffee, cocoa, banana, oil palm, coconut, rubber, spices), orchard and hurst are (as drupe, oranges and tangerines, kiwi fruit, avocado, mango, olive and English walnut), the vineyard, greenhouse, ornamental crops in garden or the park, flowers and shrub, the woods, forest in plantation institute and the flower nursery (comprising fallen leaves forest and evergreen forest).
The disease species that can prevent and treat
The disease of available the inventive method control mainly is all kinds of fungus-caused diseases, for example rice blast.
Prevent and treat method
The appropriate method that composition is applied to the crop in the growth comprises: the foliage spray agent, and pulvis, granule is handled soil and root with liquid irrigation, pulvis, granule and foaming agent.In addition, the present composition can be used for preventing and treating and is subjected to plant disease to invade the plant position of food, for example The compounds of this invention is applied to rice leaf and can prevents and treats rice blast.Preferred method of application is that foliar spray is used.
Those skilled in the art as can be seen, using than need of composition changes with for example external conditions such as temperature, humidity, pending zone and pending plant.Therefore, use than should in the scope of a broad, changing.The frequency of utilization of the present composition can be selected according to Expected Results by peasant, disease control professional person or other those skilled in the art.
A kind of preferred scheme is as follows: for the control of fungal diseases of plants, the mother liquor with 1% or 2% was made into use liquid by 1: 100 to 1: 1000, and seedling stage is to becoming the strain phase all can spray.General crop is sprayed 3 times with this soup altogether at seedling stage to duration of flowering, and sprayed once in 15 days at every interval, and the diseases prevention rate reaches more than 50%.It is better to be used a small amount of chemical pesticide effect between period of disease.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example is usually according to the normal condition or the condition of advising according to manufacturer.
The preparation of rice suspension cell
Rice suspension cell is cultivated in 26 ℃ of shaking tables (80rpm) are dark in the N6 liquid nutrient medium, changes one time fresh medium in per 7～10 days.
N6 liquid culture based formulas is as follows:
KNO
3 2.83g/L (NH
4)
2SO
4 0.463g/L
KH
2PO
4 0.4g/L MgSO
4·7H
2O 0.185g/L
CaCl
2·2H
2O 0.166g/L MnSO
4·H
2O 3.3mg/L
ZnSO
4·7H
2O 1.5mg/L H
3BO
3 1.6mg/L
KI 0.8mg/L
FeSO4·7H
2O 28mg/L Na
2EDTA 37mg/L
Nicotinic acid (B5) 0.5mg/L thiamine HCl (B1) 0.1mg/L
Pyridoxic acid HCl (B6) 0.5mg/L mesoinositol 100mg/L
Sucrose 30g/L 2,4-D 2mg/L
Proline 2.3g/L tryptophan 50mg/L
pH5.8
The preparation of chitosan oligosaccharide and mother liquor
The chitosan oligosaccharide preparation process is as follows:
Powder after cleaning-drying is pulverized of solid chitin or clam shell, shrimp shell → in sealing and have in the agitator of cooling installation in 1: 10 ratio add concentrated hydrochloric acid → 45 ℃ stir 3-12 hour to chitin all dissolve → in low-temperature (low temperature) vessel cooling supernatant suspension to 10-20 ℃ → progressively add 5N NaOH to pH3.0 to produce the NaCl precipitation, and the soluble oligomeric osamine in supernatant → filter (salt is recyclable as the industrial salt) → oligosaccharide amine aqueous solution that desalts to dehydrate and be finished product → powder for drying and preserve.
For example, chitin (Sigma) 0.2g spends the night through the persalt degraded, with the NaOH adjusting pH3-4 of 5N, after the process filtration is desalted and dehydrated, makes the water-soluble mother liquor of oligosaccharide of 10mg/ml, and the filtering with microporous membrane of 0.45um is standby in-20 ℃ of preservations.
The death of chitosan oligosaccharide inducing paddy rice suspension cell
Respectively with 0,100,200,500, the chitosan oligosaccharide of 1000ng/ml concentration handles and changes the rice suspension cell of medium after 4 days, secretly cultivated 24 hours in 26 ℃ of shaking tables (80rpm), Evans blue dyeing 15min through 0.05% washes 6 times with unconjugated dyestuff removal.Therefrom take by weighing the 100mg cell, the methyl alcohol with 50% (containing 1%SDS) 1.5ml is in 50 ℃ of water-bath 30min, during to constantly shake so that fully decolouring, measure the absorbance value of eluent 600nm.Simultaneously, with the contrast of 80 ℃ of water bath processing 10min as the complete death of cell, the dose effect of analysis of cells death.Chitosan oligosaccharide with 1 μ g/ml concentration is done same processing to rice suspension cell, after 12 hours and 24 hours, handles through dyeing and decolouring respectively, measures the absorbance value of eluent 600nm, the dead process that occurs of analysis of cells.
The result as depicted in figs. 1 and 2.The hypersensitivity cell death is the characteristic feature of plant and the non-affine mutual work of pathogen.Handle rice cell by the chitosan oligosaccharide of various dose and find after 24 hours, the death of cell is handled rice cell along with the increase of chitosan oligosaccharide concentration increases with the chitosan oligosaccharide of 1 μ g/ml, just it can be killed (see figure 1) fully.Respectively rice cell is carried out 12 and 24 hours processing with 1 μ g/ml chitosan oligosaccharide, the result shows: handling just obviously generation of cell death after 12 hours, was complete dead (see figure 2) to 24 hours.Therefore draw, chitosan oligosaccharide is a kind of strong exciton of rice cell death.
The generation of disease-resistant signal and the activation of defending gene
(a) H
2O
2Generation
Fresh rice suspension cell (transferring the 4th day) is used for the mensuration of active oxygen.The rice suspension cell of handling with the chitosan oligosaccharide of 1 μ g/ml concentration, respectively at 26 ℃ of shaking tables (80rpm) secretly cultivate 0,30min, 1hr, 3hr, 6hr, 12hr, 24hr, syringe-type funnel with 0.2 μ m aperture filters culture fluid, get its 200 μ l and add 5 μ l 1mmol/LHPTS substrates, after mixing in 5 minutes, go up the concentration (parameter is: excitation wavelength 405nm, emission wavelength 512nm) of measuring active oxygen at luminoscope (Perkin ElmerLS50B).
The result as shown in Figure 3.Oxygen production is one of main defense response of plant.Experimental result shows that the rice suspension cell so that the chitosan oligosaccharide of 1 μ g/ml concentration is handled begins to produce a H about 0.5 hour
2O
2Small peak reached another peak (see figure 3) in 6 hours.After this, the chitosan oligosaccharide active oxygen of inducing produces and weakens gradually.Twice oxygen that the very similar pathogen of this result or exciton the produce phenomenon that bursts out.
(b) defence expression of gene
Chitosan oligosaccharide with variable concentrations is handled rice suspension cell, extracts total RNA (Trizol method) of cell after 26 ℃ of shaking tables (80rpm) are secretly cultivated 12 hours, and Northern blot detects defence disease-resistant gene RCH10, the expression of PAL.Chitosan oligosaccharide with 1 μ g/ml concentration is handled rice cell, after secretly cultivating 0min, 30min, 1hr, 3hr, 6hr, 12hr, 24hr respectively at 26 ℃ of shaking tables (80rpm), extract total RNA (Trizol method) of cell, Northern blot detects defence gene RCH10, PAL, the expression of CatB is dynamic.
The result is shown in Figure 4 and 5.Chitosan oligosaccharide with variable concentrations is handled rice suspension cell, and the chitosan oligosaccharide of 0.01ug/ml just can activate the expression of defence gene RCH10 and PAL strongly after 12 hours, and the treating capacity of 0.5ug/ml just can reach the maximum (see figure 4) of defence expression of gene amount.Chitosan oligosaccharide with 1 μ g/ml concentration is handled rice cell, defends the time dynamic studies of gene activation.Can activate the expression of defence gene RCH10 and PAL after 2 hours, just can reach maximum at 8-12 hour expression; Defence gene C atB can be suppressed the expression (see figure 5) after chitosan oligosaccharide is handled 2 hours, reconfirm that CatB may be the negative regulatory factor (He of a disease resistance response, Z.Et al. (2000) Perception of brassinosteroidsby the extracellular domain of the receptor kinase BRI1.Science.288,2360-2363).
Chitosan oligosaccharide inducing paddy rice blade produces allergic reaction
(a) chitosan oligosaccharide inducing paddy rice blade cell death
Respectively with 0,1,5,10, the chitosan oligosaccharide of 100ug/ml concentration is sprayed onto on the plant of susceptible paddy rice H7S in tri-leaf period, after 24 hours, the clip blade, put into the Trypan blue dyeing liquor (lactic acid: glycerine: phenol: water=1: 1: 1: 1 of the 0.1mg/ml under the boiling water bath state, add isopyknic 96% ethanol again) middle dyeing 5min, place 1hr under the room temperature, blade is decoloured in the chloraldurate of 2.5mg/ml spend the night.Be stored in 50% the glycerine dead production of observation of cell and Taking Pictures recording.
The rice seedling in tri-leaf period that the various dose chitosan oligosaccharide is handled, its vanes Trypan blue dyeing, discovery has tangible cell death spot, and along with the generation of the different cell deaths of treatment dosage has certain difference, the rice leaf that the 5ug/ml chitosan oligosaccharide is handled promptly produces tangible cell death (see figure 6).The result shows that the cell death of chitosan oligosaccharide inducer blade produces required dosage and will be higher than suspension cell.
(b) chitosan oligosaccharide inducing paddy rice blade cell produces H
2O
2Deposition
In the presence of peroxidase, DAB (diaminobenzidine) is in case and H
2O
2Contact forms the bronzing polymer deposition rapidly.Respectively with 0,1,5,10, the chitosan oligosaccharide of 100ug/ml concentration is sprayed onto on the plant of susceptible paddy rice H7S in tri-leaf period, after 24 hours, cut blade and be immersed in 3 of 1mg/ml, mid-25 ℃ of 3 '-diaminobenzidine-HCl (pH3.8), illumination 8 hours, taking-up is placed in 96% ethanol boiling water bath and boils the 10min decolouring, and reddish-brown precipitation and the Taking Pictures recording in the blade observed in 4 hours (25 ℃) backs of bubble in fresh ethanol.
Paddy rice after the variable concentrations chitosan oligosaccharide is handled, its vanes DAB dyeing finds to have tangible H
2O
2The spot (see figure 7) of deposition produces allergic reaction (Hypersensitive Response, oxygen HR) burst out (Oxidative burst) thereby the proof chitosan oligosaccharide can cause rice cell.Thereby can conclude tentatively that the generation of above-mentioned cell death is based on a kind of mechanism of allergic reaction.
Chitosan oligosaccharide inducing paddy rice disease resistance detects
Chitosan oligosaccharide (0.01%Tween20) with 5 μ g/ml is handled commercially available rice plant H7S, and water treatment is as negative control.Inoculate rice blast (is about 50 for good with the spore count of each visual field BLAST) after 8 hours, 28 ℃ of dark cultivations after 24 hours, continued to cultivate 6 days illumination 14 hours in dark 10 hours, observed disease resistance and also added up incidence.Simultaneously, with disease-resistant strain H7R as disease-resistant contrast.
The result is shown in Fig. 8 and 9.Repeated experiments confirms that the rice plant that the chitosan oligosaccharide of process 5ug/ml is handled obviously strengthens the resistance of rice blast, and its scab progression is the 2-3 level, and disease-resistant plant is the 1-2 level, and the very big part scab of undressed plant is susceptible scab, and morbidity progression is in the 4-5 level.The result shows that the processing protection effect of 5ug/ml chitosan oligosaccharide can reach more than 50% and (sees Fig. 8,9).
Discuss
The reaction of plant inducing anti-disease is a quite complicated process.Studies show that as one of main component of fungal cell wall, chitosan oligosaccharide can induced strong rice cell generation defense response, and then the enhancing of initiating system disease resistance.Confirmed these reactions that chitosan oligosaccharide brings out on cell of the present invention, biochemistry and the molecular biology, be very similar to the microbial allergic reaction of cause of disease: comprised that cell death, oxygen burst out and defends the activation of gene.
Research conclusion of the present invention thinks that chitosan oligosaccharide can be used as a kind of strong cell death and the exciton of system induction disease resistance.This excitation of chitosan oligosaccharide may show different on different plants, is necessary different plants are carried out corresponding mechanism research.
After the chitosan oligosaccharide processing, the rice plant disease resistance obviously strengthens.Show as rice leaf and the cell death spot occurs, the HR reaction has also promptly taken place; And the rice leaf through DAB dyeing can be owing to have produced H in defense response
2O
2And the bronzing spot appears; After the rice blast inoculation, obviously strengthen through the rice plant resistance after the chitosan oligosaccharide processing, its scab rank descends, and the speed of infecting slows down.Compare with susceptible contrast, the disease index has descended 50%.The present invention shows that chitosan oligosaccharide has broad application prospects as biopesticide as a kind of organic formulations of wide material sources, but estimates that inducing plant produces at the different fungi bacteriums and the resistance of virus disease.
All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned instruction content of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Claims (12)
1. the purposes of a shitosan, it is characterized in that, be used to prepare the composition pesticide that the inducing plant blade cell produces allergic reaction, wherein said composition pesticide contains chitosan oligosaccharide and carrier, and the content of chitosan oligosaccharide is the 0.0005-0.05% of composition total weight.
2. purposes as claimed in claim 1 is characterized in that, described allergic reaction is that blade cell death or blade cell produce H
2O
2Deposition.
3. purposes as claimed in claim 1 is characterized in that, the content of chitosan oligosaccharide is more than 0.005% of composition total weight.
4. purposes according to claim 1 is characterized in that, described composition pesticide is the formulation that is selected from down group: wettable powder, emulsifiable concentrate, the aqueous solution, emulsion, but spray solution, oil-or-water-based dispersion, suspending agent, pulvis, granule, or microcapsules.
5. purposes according to claim 1 is characterized in that, described composition is the aqueous solution.
6. purposes according to claim 1 is characterized in that, described composition also contains the material that is selected from down group: unimolecule osamine, sodium chloride, Tween-20 and water.
7. purposes as claimed in claim 1 is characterized in that, described plant is selected from down group: paddy rice, wheat, vegetables or flowers.
8. purposes as claimed in claim 1 is characterized in that, described chitosan oligosaccharide is to prepare with the method that may further comprise the steps:
(a) provide solid chitin or clam shell, the powder of shrimp shell after cleaning-drying is pulverized;
(b) in sealing and have in the agitator of cooling installation in 1: 5-1: 10 ratio adds concentrated hydrochloric acid;
(c) 30-45 ℃ is stirred and all to dissolve to chitin in 3-12 hour;
(d) in low-temperature (low temperature) vessel, cool off supernatant suspension to 10-20 ℃;
(e) add NaOH to pH3.0 ± 0.5, produce the NaCl precipitation, and the soluble oligomeric osamine is in supernatant;
(f) filtration is desalted, and reclaims the oligosaccharide amine aqueous solution;
(g) dehydrate.
9. a composition pesticide is characterized in that, it contains chitosan oligosaccharide and carrier, and wherein the content of chitosan oligosaccharide is the 0.0005-0.05% of composition total weight.
10. the method that inducing plant is disease-resistant is characterized in that, comprises step: the shitosan that to give described plant application concentration be 5ppm-500ppm.
11. method as claimed in claim 10 is characterized in that, the application concentration of shitosan is 50-200ppm.
12., it is characterized in that described plant is selected from down group: paddy rice, wheat, vegetables or flowers as claim 10 or 11 described methods.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB031152767A CN1316882C (en) | 2003-01-30 | 2003-01-30 | Chitosan oligosaccharide containging pesticide composition and its application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB031152767A CN1316882C (en) | 2003-01-30 | 2003-01-30 | Chitosan oligosaccharide containging pesticide composition and its application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1520723A CN1520723A (en) | 2004-08-18 |
| CN1316882C true CN1316882C (en) | 2007-05-23 |
Family
ID=34284213
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB031152767A Expired - Fee Related CN1316882C (en) | 2003-01-30 | 2003-01-30 | Chitosan oligosaccharide containging pesticide composition and its application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1316882C (en) |
Families Citing this family (30)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100334952C (en) * | 2005-08-31 | 2007-09-05 | 山东靠山生物科技有限公司 | Biologica land biochemical preparation and its preparing method and use |
| CN101433236B (en) * | 2008-12-12 | 2013-08-28 | 青岛安芙兰芳香制品有限公司 | Amino-oligosacchride composite antistaling agent and preparation method thereof |
| CN102365942B (en) * | 2011-10-28 | 2013-10-23 | 海南正业中农高科股份有限公司 | Chitosan oligosaccharide composition for preventing Chinese cabbage soft rot disease, purpose thereof and method thereof |
| CN102388878A (en) * | 2011-10-28 | 2012-03-28 | 海南正业中农高科股份有限公司 | Chitosan oligosaccharide composition used for resisting tobacco common mosaic as well as use and method thereof |
| CN102365948B (en) * | 2011-10-28 | 2013-03-20 | 海南正业中农高科股份有限公司 | Chitosan oligosaccharide composition for resisting rice blast, application and method thereof |
| CN102365946B (en) * | 2011-10-28 | 2013-08-21 | 海南正业中农高科股份有限公司 | Chitosan oligosaccharide composition for killing fruit flies, and application and method of chitosan oligosaccharide composition |
| CN102415383B (en) * | 2011-10-28 | 2013-11-20 | 海南正业中农高科股份有限公司 | Chitosan oligosaccharide composition for resisting rice black streaked dwarf as well as purpose and method thereof |
| CN102365949B (en) * | 2011-10-28 | 2013-03-27 | 海南正业中农高科股份有限公司 | Chitosan oligosaccharide composition for resisting hot pepper virus disease as well application and method thereof |
| CN102365938B (en) * | 2011-10-28 | 2013-03-27 | 海南正业中农高科股份有限公司 | Chitosan oligosaccharide composition for reducing content of residual pesticides in crops |
| CN102365947B (en) * | 2011-10-28 | 2013-04-03 | 海南正业中农高科股份有限公司 | Chitosan oligosaccharide composition for killing aphids, purpose thereof and method thereof |
| CN102365943B (en) * | 2011-10-28 | 2013-10-23 | 海南正业中农高科股份有限公司 | Chitosan oligosaccharide composition for killing cnaphalocrocis medinalis, and application and method of chitosan oligosaccharide composition |
| CN102885044B (en) * | 2011-10-28 | 2014-03-19 | 海南正业中农高科股份有限公司 | Chitosan oligosaccharide composition for preventing and controlling rice plant hoppers and use and using method thereof |
| CN102365944B (en) * | 2011-10-28 | 2013-08-21 | 海南正业中农高科股份有限公司 | Chitosan oligosaccharide composition for killing diamond back moths, its application and method |
| CN102405905B (en) * | 2011-10-28 | 2014-04-16 | 海南正业中农高科股份有限公司 | Chitosan oligosaccharide for resisting watermelon virus disease, use and method thereof |
| CN102369930B (en) * | 2011-10-28 | 2013-01-30 | 海南正业中农高科股份有限公司 | Chitosan oligosaccharide combination for keeping flower and fruit and use and use method thereof |
| CN102405906B (en) * | 2011-10-28 | 2013-10-23 | 海南正业中农高科股份有限公司 | Chitooligo saccharide composition used for resisting pepper virus disease and application and method thereof |
| CN102388882B (en) * | 2011-10-28 | 2013-11-20 | 海南正业中农高科股份有限公司 | Chitosan oligosaccharide composition used for resisting corn stunt as well as use and method thereof |
| CN102440249B (en) * | 2011-10-28 | 2013-10-23 | 海南正业中农高科股份有限公司 | Purpose of chitosan oligosaccharide and composition thereof in low temperature growth promotion |
| CN102365952B (en) * | 2011-10-28 | 2013-06-26 | 海南正业中农高科股份有限公司 | Chitosan oligosaccharide composition for resisting Venturia pirina, application and method thereof |
| CN102369929B (en) * | 2011-10-28 | 2015-03-18 | 海南正业中农高科股份有限公司 | Chitosan oligosaccharide combination for resisting marssonina mali and use and use method thereof |
| CN102405907A (en) * | 2011-10-28 | 2012-04-11 | 海南正业中农高科股份有限公司 | Anti-wheat rosette stunt disease chitosan oligosaccharide composition, use and method thereof |
| CN102613197B (en) * | 2012-03-07 | 2014-04-30 | 海南正业中农高科股份有限公司 | Application of chitosan oligosaccharide for resisting premature senility |
| CN102613192B (en) * | 2012-03-07 | 2014-04-30 | 海南正业中农高科股份有限公司 | Application of chitosan oligosaccharides in promotion of shortness and stoutness of new branches |
| CN102757287A (en) * | 2012-07-27 | 2012-10-31 | 陕西西大华特科技实业有限公司 | Chitosan oligosaccharide-containing gibberellin compound plant growth regulator |
| CN103109811A (en) * | 2013-02-27 | 2013-05-22 | 苏州市农业科学院 | Vegetable disease prevention and control preparation containing plant inducer |
| CN103340201B (en) * | 2013-07-26 | 2014-12-17 | 海南正业中农高科股份有限公司 | Application of chitosan oligosaccharide aqueous solution in promotion of plant tillering |
| CN103461353B (en) * | 2013-09-05 | 2016-08-17 | 惠州长龙生物技术有限公司 | A kind of composition pesticide containing chitosan oligosaccharide, insecticide and application thereof |
| CN105481572A (en) * | 2015-12-28 | 2016-04-13 | 芜湖润蓝生物科技有限公司 | Efficient fertilizer for enhancing low-temperature stress resistance of cabbage seedlings by virtue of modified oligosaccharin and preparation method thereof |
| CN109691449A (en) * | 2017-10-23 | 2019-04-30 | 青岛胜恩全成生物科技有限公司 | A kind of fungicide and preparation method thereof for preventing and treating large cherry tree bleeding disease |
| CN108378061B (en) * | 2018-05-08 | 2020-06-09 | 菏泽学院 | Oil-used peony leaf spot inducer |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS63216804A (en) * | 1987-03-05 | 1988-09-09 | Meiji Seika Kaisha Ltd | Growth control of plant |
| JPH09143013A (en) * | 1995-11-20 | 1997-06-03 | Yaizu Suisan Kagaku Kogyo Kk | Plant-vitalizing agent |
| KR19990009589A (en) * | 1997-07-10 | 1999-02-05 | 박승림 | Plant growth promotion and pest control agent containing water-soluble low molecular chitin and chitosan oligosaccharide |
| CN1363215A (en) * | 2002-01-25 | 2002-08-14 | 华东理工大学 | Coating agent containing chitosan for vegetable seeds |
-
2003
- 2003-01-30 CN CNB031152767A patent/CN1316882C/en not_active Expired - Fee Related
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS63216804A (en) * | 1987-03-05 | 1988-09-09 | Meiji Seika Kaisha Ltd | Growth control of plant |
| JPH09143013A (en) * | 1995-11-20 | 1997-06-03 | Yaizu Suisan Kagaku Kogyo Kk | Plant-vitalizing agent |
| KR19990009589A (en) * | 1997-07-10 | 1999-02-05 | 박승림 | Plant growth promotion and pest control agent containing water-soluble low molecular chitin and chitosan oligosaccharide |
| CN1363215A (en) * | 2002-01-25 | 2002-08-14 | 华东理工大学 | Coating agent containing chitosan for vegetable seeds |
Non-Patent Citations (5)
| Title |
|---|
| 壳寡糖对水稻纹枯病不同抗性品种几丁质酶诱导的研究 胡键，陈云，陈宗祥，江苏农业研究，第21卷第4期 2000 * |
| 壳寡糖的制备及其对黄瓜的促进生长作用 张文清，隋雪燕，夏玮，张元兴，林彩玲，功能高分子学报，第15卷第2期 2002 * |
| 壳甲素/壳聚糖的研究进展及其在食品工业中的应用 郭开宇，赵谋明，食品与发酵工业，第26卷第1期 2000 * |
| 壳聚糖制备研究进展 严钦，沈月新，王V，上海水产大学学报，第11卷第4期 2002 * |
| 壳聚糖抑制植物病原菌生长的研究 胡键，姜涌明，殷士学，扬州大学学报，第3卷第2期 2000 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN1520723A (en) | 2004-08-18 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN1316882C (en) | Chitosan oligosaccharide containging pesticide composition and its application | |
| TWI617244B (en) | Fungicidal composition and the use thereof | |
| WO2014026504A1 (en) | Use of metal salt of 2-mercaptobenzothiazole | |
| CN104738094A (en) | Plant immune induced resistance protein composition as well as preparation method and application thereof | |
| CN106508991A (en) | Biological pesticide and preparation method thereof | |
| CN104430357A (en) | Bactericide composition containing coumoxystrobin with synergistic effect | |
| WO2020262612A1 (en) | Plant disease control agent and plant disease control method | |
| US20070093387A1 (en) | Anti-microbial/anti-viral composition | |
| CN106922703A (en) | A kind of bactericidal composition | |
| EP2389806A2 (en) | A natural composition for the treatment of plants, preparation and uses thereof | |
| JP5372066B2 (en) | Nematode control agent | |
| WO2015135422A1 (en) | Method for protecting plant against plant disease | |
| CN1957713A (en) | New composition of pesticide, preparation method, and purpose | |
| KR101143033B1 (en) | Compositions for protecting Plant diseases | |
| CN103444721B (en) | A kind of bactericidal pesticide composition, pesticidal preparations and application thereof | |
| CN107114369B (en) | Application of phytosulfokine-alpha in improving gray mold resistance of plants | |
| WO2008007778A1 (en) | Parasitic plant control agent and use thereof | |
| CN107318865A (en) | The composition pesticide of captan and furametpyr compounding | |
| CN103719125A (en) | Bactericide composition and application thereof | |
| CN109497066B (en) | Insect antifeedant 15 beta-hydroxykalainanenone for in-vivo delivery of plant and application thereof | |
| CN1565182A (en) | Bactericidal compositions of flumorph and enostroburin and synergist | |
| KR101915411B1 (en) | Herbicide compositions for controlling pests of horticultural products comprising rare-earth elements as active ingredients | |
| CN107242238B (en) | Application of isocyanuric acid in preventing and treating corn top rot | |
| CN1275527C (en) | Multiple-effect plant source agricultural chemicals and its production method | |
| KR100753371B1 (en) | Germicide Compositon for Agiculture and Horiculture Containing Salt Radiating Far-Infrared ray and Germicidal Method Using the Same |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| C17 | Cessation of patent right | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20070523 Termination date: 20120130 |