CN1122445C - Plant resistance inductor - Google Patents
Plant resistance inductor Download PDFInfo
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- CN1122445C CN1122445C CN 99122582 CN99122582A CN1122445C CN 1122445 C CN1122445 C CN 1122445C CN 99122582 CN99122582 CN 99122582 CN 99122582 A CN99122582 A CN 99122582A CN 1122445 C CN1122445 C CN 1122445C
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- plant
- enzyme
- resistance agent
- induced resistance
- plant resistance
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Abstract
The present invention relates to a plant resistance inductor which is characterized in that the plant resistance inductor mainly comprises oligosaccharide obtained by the following process: F. graminearum schw used as bacteria microorganism is fermented for obtaining a large number of mycelia; the fermented mycelia are degraded by enzymes in a salt solution, the enzyme dosage is from 0.5 to 3.0 wt%, the salt solution concentration is from 0.01 to 0.5%, the pH value is from 3.0 to 7.0, the reaction temperature is from 15 to 60 DEG C, and the reaction time is from 1 to 10 hours; the solution is heated and hot water is extracted. The inductor has high induced plant resistance and activity for promoting growth and development of plants, has the advantages of low production cost and less pollution, and is suitable for large scale production.
Description
The present invention relates to microbial technique, a kind of plant induced resistance agent that obtains to have bioactive ingredients such as oligosaccharides by microbial fermentation in conjunction with the enzymic degradation under the certain condition is provided especially, and has been applied to control of crop disease technology such as soybean, cotton.
Active oligomeric sugar is that a class is made up of 2-15 monose, tool one fixed structure and bioactive polymer, it is generally acknowledged that active oligomeric sugar has regulating plant growth, grow, breeding, the function of diseases prevention and aspect such as disease-resistant, immune system response that can stimulating plant, every kind of active oligomeric sugar can send the information of regulating specific function, activate defense reaction and regulating plant growth, generation has the active substance of disease resistance, suppress the formation of disease, particularly the oligosaccharide of separate sources can be at different pathogen (comprising ecological microspecies), thereby can develop serial oligosaccharide agricultural chemicals, solve the problem that the gene engineering genetic breeding also is difficult to the pathogen ecocline microspecies of solution at all kinds of diseases.Studying more in the world is the oligosaccharides segment of plant and the degraded of microorganism wall polysaccharide.Increasing experimental result shows that plant cell wall not only plays the structural barriers effect of defence, and can play the active defense reaction when being subjected to courses of infection.In plant and cause of disease interaction, one side excreting beta-1,3 glucolase, chitosan enzyme and chitinase directly suppress fungi growth; Secrete the cell wall of endopolygalacturonase and inscribe pectase degrading plant on the other hand again.Can obtain these oligosaccharinses at the interface of plant and cause of disease with lyases and confirm that these fragments play effect really in live body.The cell wall degradation of plant and fungi can be become active oligosaccharins at external these glycosidic bond lyases, thereby the regulation mechanism of plant has related to certainly and has suppressed that these enzymes parts are active to make it the disease resistance response that in longer time generation oligosaccharins is beneficial to plant.So the inhibitor of glycosidic bond lyases oligosaccharide induce may play a part in the regulation mechanism certain.Enzymatic fragment-pathogen that is discharged in this process and the cell wall oligosaccharide of plant are exactly the generting machanism of oligosaccharins in live body probably.When inducer by after the host plant identification, plant must produce the second messenger and come signal on the transport membranes acceptor to cell nucleus, to cause gene transcription.Difference between the disease-resistant or susceptible plant variety is in the difference of their safeguard systems, and is initial identification and the matter that produces of signaling molecule or the difference on the amount thereafter, and oligosaccharins can dwindle this difference.
The existing preparation that contains active oligomeric sugar plant induced resistance agent, the technology that adopts plant extract or Plant Tissue Breeding to extract usually since be subjected to land area, throughout the year, the restriction of factor such as poor growth, large-scale batch production almost is impossible.
The purpose of this invention is to provide a kind of plant induced resistance agent, the activity that it has higher inducing plant resistance and promotes growth and development of plants, and low production cost are polluted for a short time, are suitable for large-scale production.
The invention provides a kind of plant induced resistance agent, it is characterized in that this plant induced resistance agent mainly contains oligosaccharide, obtains by following method:
(1) be that the bacterial classification microbial fermentation obtains in a large number with Fusarium graminearum (F.graminearum Schw)
Mycelium;
(2) with fermentation mycelium at MgSO
4Use chitinase, cellulase, bromelain in the salting liquid
One or more of enzyme, lysozyme, lipase, chitosan enzyme carry out enzyme degraded, enzyme dosage
0.5-3.0% weight, the concentration of salting liquid are 0.01-0.5%, pH3.0-7.0, reaction temperature
15-60 ℃, reaction time 1-10 hour;
(3) be warming up to 80-110 ℃, hot-water extraction 5-20 hour.
The Fusarium graminearum of bacterial classification described in the present invention (F.graminearum Schw) is a kind of common wheat fungal bacterium, belong to sac fungi, this bacterium macroconidium is born on the subsphaeroidal adnation phialide of Dan Sheng or is born on the phialide of complicated branch stigma 10-14um * 3.5-4.5um..The macroconidium sickleshaped, the abdomen back of the body obviously.Conidium is colourless, pink during gathering.Media surface produces light red sometimes to orange adhesive material (pionnotes).The used bacterial classification of the present invention is common known bacterial classification, derives from the preservation strain F23 of Institute of Plant Protection, academy of agricultural sciences, Jiangsu Province Fusarium graminearum respectively as the bacterial classification that adopts among the embodiment; The China's ACCC30213 of DSMZ F.graminearum schw bacterial strain (F.graminearum Schw); The GF Fusarium graminearum of Institute of Plant Protection, academy of agricultural sciences, Shaanxi Province.Bacterial strain uses therefor of the present invention can be grown on general fungi culture medium, as: PDA medium (peeling section potato 200g, little fire boiled 30 minutes, filtered, and collected filtrate, added 2.0% glucose and water 1000ml) Czapek's medium (sucrose 2%, KH
2PO
40.1%, KCl0.05%, MgSO
40.05%, FeSO
40.001%, NaNO
30.3%, PH6.7), be that its biomass is variant under the different culture medium condition.
In order to improve biomass, the preferred fermentation medium of the present invention is as follows:
Peptone 0.3-2.0%, analysis for soybean powder 0.5-3.5%,
Dusty yeast 0.2-1.5%, corn steep liquor 0.2-2.0%,
Sucrose 0.5-2.0%, KH
2PO
40.6-1.5%,
MgSO
4 0.1-1.0％， NaCl 0.01-0.5％。
When plant induced resistance agent of the present invention is used to prevent and treat soybean, tobacco, cotton, eggplant plant disease, can be with this product seed soaking, foliage-spray or root irrigation, working concentration is 10-2000ppm.
The present invention has following advantage:
1. adopt microorganism, can stably obtain to prepare active oligosaccharide raw material by fermentation process with certain cell wall structure.
2. utilize the biological enzymolysis reaction can obtain higher bioactive oligosaccharide fragment.
3. the disease control that is applied to crops has efficient, nontoxic, free from environmental pollution and characteristics such as do not develop immunity to drugs.
4. can solve the corps diseases that virus disease, verticillium wilt etc. still do not have effective pesticide control to a certain extent.
5. raw material sources are abundant, and production cost helps realizing suitability for industrialized production the end of than.
Below by example technology of the present invention is further specified:
Embodiment 1 fermentation of ACCC30213 Fusarium graminearum on different medium
PDA: peeled potatoes 20%, (w/v) slivering, little fire boiled 30 minutes, and eight layers of filtered through gauze are collected filtrate, add 2.0% glucose and get final product.F
1: peptone 0.3%, analysis for soybean powder 2.0%, dusty yeast 0.2%, corn steep liquor 0.2%, sucrose 2.0%, corn starch 1.5%, KH
2PO
40.6%, MgSO
40.1%, NaCl 0.01%.F
2: peptone 0.3%, analysis for soybean powder 3.5%, dusty yeast 0.2%, corn steep liquor 0.2%, sucrose 2.0%, KH
2PO
40.6%, MgS4 0.1%, and NaCl 0.01%.Cultivated 10 days, 25 ℃, its cultivation results of 80rpm is: at medium PDA, F
1, F
2The middle biomass of cultivating (mycelia dry weight/zymotic fluid) is respectively 0.57 1.70 2.13.
Embodiment 2 fermentations of GF Fusarium graminearum on different medium
F
2Medium: peptone 0.3%, analysis for soybean powder 3.5%, dusty yeast 0.2%, corn steep liquor 0.2%, sucrose 2.0%, KH
2PO
40.6%, MgSO
40.1%, NaCl 0.01%.F
3Culture medium protein peptone 0.3%, analysis for soybean powder 0.5%, dusty yeast 0.2%, corn steep liquor 0.2%, sucrose 2.0%, corn starch 3.0%, KH
2PO
40.6%, MgSO
40.1%, NaCl 0.01%.F
4Medium: peptone 0.3%, analysis for soybean powder 0%, dusty yeast 0.2%, corn steep liquor 0.2%, sucrose 3.5%, corn starch 1.0%, KH
2PO
40.6%, MgSO
40.1%, NaCl 0.01%.Cultivated 10 days, 25 ℃, 80rpm.The fermentation result shows F
2The medium biomass is higher than medium F
3And F
4
Embodiment 3 F
23The test of Fusarium graminearum fermentation amplification culture
In the airlift fermentor of 14L, with this inoculation of 800mL in the F of 12L
225 ℃ of constant temperature were cultivated 3 days in the medium, and when cultivating 40 hours, biomass can reach 1.4%, and the breeding with initial 24 hours bacterium is the fastest especially, after 40 hours propagation slowly, cultivate 3 days very littler than biomass increase in 40 hours.This shows, obtains the required time of same biomass, and scale-up is cultivated than shaking table and shortened dramatically.But the maximum biomass that scale-up can reach is cultivated less than shaking table.This may be because due to the difference of oxygen supply situation.The scale-up continuous culture three batches, the result repeats substantially, finds no microbiological contamination, shows F
23Fusarium graminearum has very strong competitive growth ability, and growth is suitable for suitability for industrialized production rapidly.
Embodiment 4 GF Fusarium graminearums and the preparation of ACCC30213 Fusarium graminearum enzymolysis product
In the 250mL triangular flask, add the 100mL culture fluid, respectively with the liquid-spawn inoculation of GF Fusarium graminearum and ACCC30213 Fusarium graminearum to F
2In the medium, inoculum concentration 10mL, under 25 ℃, the 80rpm shaking table was cultivated 10 days, centrifugal respectively, collection mycelium, water is rinsed well, and vacuum filtration is dried the back crushing screening then, obtains the bacterium powder.Gained bacterium powder is added respectively in the magnesium sulfate of 0.6M, add chitinase, chitosan enzyme, cellulase complex enzyme enzyme by 1.0% (to substrate), 40 ℃ of reactions down, after reaction finishes, heating deactivation in 10 minutes in 100 ℃ water-bath, 70 ℃ of lixiviates, centrifugal the going of 4000rpm precipitated, and obtains the enzymolysis product of GF Fusarium graminearum and ACCC30213 Fusarium graminearum respectively.
Embodiment 5 F
23Fusarium graminearum zymophyte powder enzyme degraded scale-up
In the stirring-type reactor of 25L, throw zymophyte powder 260 grams, complex enzyme 26 gram and an amount of 0.6M magnesium sulfate of chitinase, chitosan enzyme, cellulase, bromelain, lipase, mixing speed 100rpm, 37 ℃ of temperature, enzymolysis 3 hours.Find that this enlarge-effect that shows F23 Fusarium graminearum powder enzyme digestion reaction is less in scale-up gained enzymolysis liquid inducing wheat PAL (phenylalnine ammonialyase) active (characterizing the disease resistance of plant index) and the 100mL reaction bulb consistent (table 1).
Table 1 enzymolysis scale-up and the PAL activity of shaking the bottle reaction
Active increase/the contrast (%) of enzyme digestion reaction PAL
Scale-up 39.7
100mL shakes bottle 41.4
Embodiment 6 F
23Fusarium graminearum enzyme degradation product is to the influence of plant PAL resistance enzyme bioactivity
After wheat, soya seeds cleaned up, add 8%NaC10 sterilization 10min, stand-by behind the aseptic water washing 3 times.Cotton seeds is with after the concentrated sulfuric acid lint, and water cleans up 8%NaC10 sterilization 30min, behind the aseptic water washing 3 times, soaks 30min in 55 ℃ of warm water, dries standby.
Get enzyme extract 0.4ml in clean test tube, add the cold 0.1M borate buffer of 2ml (PH8.8 includes mercaptoethanol 5mmol/L).Extract adds the L-phenyl alanine of 1ml20mM, and contrast adds 1ml water, after fully mixing, reacts deactivation mensuration after 45 minutes in 35 ℃ water-bath, with product-trans-cinnamic acid (t-cinnamic acid) OD hourly
290Be 0.01 to be an active unit (U).
After variable concentrations was handled wheat, soybean, cotton seeds vernalization growth, phenylalnine ammonialyase (PAL) activity had had raising in various degree compared with the control in its seedling body.F
23Six concentration of Fusarium graminearum enzyme degradation product are handled wheat PAL activity and all compared according to high, and are wherein best with 20ug/ml and 40ug/ml concentration effect.F
23Three kinds of concentration of Fusarium graminearum enzyme degradation product are handled soybean PAL, and with the 50ug/ml best results, so amplification is little compared with the control for the PAL activity, but the active concentration scope should be between 10-100ug/ml.
The application experiment of embodiment 7 GF Fusarium graminearum enzymolysis products in the cotton verticillium wilt control
Every processing kind amount 800g adds the 1600ml soup and soaked cotton seed 24 hours, and contrast is stirred 2 times during the seed soaking with clear water seed soaking 24 hours, so that handle evenly.Pull seed drop water to the greatest extent after the processing out, be seeded in the nutritive cube, 3 seeds of every alms bowl, 1000 nutritive cubes of every processing are provided with the sub-district by 5 processing, each situation of handling of emerging of investigation.Nutrition pot seedling is moved to cotton verticillium wilt retransmit in the field, every processing sub-district area is 50 square metres, 4 repetitions, randomized arrangement.Mid-April investigation is emerged and to the situation of preventing and treating of seedling diseases, early September investigation verticillium wilt incidence.
Result of the test shows that GF Fusarium graminearum powder enzyme extract has certain preventive effect to cotton verticillium wilt, insecticide-applying way and concentration difference, control efficiency difference.The mode of spray medicine handles with 50ug/ml that to investigate preventive effect 63.99% seedling stage the highest, and the 800 times of soup preventive effects in methyl mop Tianjin are 43.98%.Cut open the bar investigation and be respectively 65.82% and 50%.The seed soaking mode, the investigation in seedling stage, it is the highest to handle preventive effect 100% with 50ug/ml, and the 800 times of soup preventive effects in methyl mop Tianjin are 55.5%.Cut open the bar investigation and be respectively 81.8% and 63.65%.Seed soaking mode effect is better than spraying the preventive effect of prescription formula.
Embodiment 8 Fusarium graminearums (ACCC30213) enzymolysis liquid preparation is to the preventive and therapeutic effect of soybean mosaic
It is fermentation strain that present embodiment adopts Fusarium graminearum (ACCC30213), presses F
23The fermentation condition of Fusarium graminearum, the mycelial enzymolysis liquid preparation that is obtained has carried out the standardization field experiment of soybean mosaic at Heilongjiang Province's plant protection unit, and its preventive effect reaches 70%, volume increase 13%.
Embodiment 9 F
23Fusarium graminearum enzyme degradation product is to the influence of endophyte of plant
To be sowed in the nutritive cube of 15 centimetres of diameters through the cotton seed of persulfuric acid lint, treat to use F after cotton emerges
23Fusarium graminearum enzyme degradation product (diluting 200 times) spraying, every spray in 3 days once.Spray is 5 times continuously.After last the spray, divide different time to get cotton stem and separate.Treat that a large amount of dead seedling " Invest, Then Investigate "s appear in contrast.With the spray clear water is contrast.Select the beef peptone medium for use, adopt the gradient dilution flat band method to separate.Experimental result is as follows:
F
23Fusarium graminearum enzyme degradation product is to the influence of bacterial community in the cotton caulome
| Handle | Extension rate | Sprayed back 10 days (cfu/g) | Sprayed back 30 days (cfu/g) | Sprayed back 45 days (cfu/g) |
| Degradation product | 200 | 8.0×10 7 | 5.0×10 7 | 1.8×10 10 |
| CK | 4.2×10 9 | 3.0×10 8 | 2.0×10 10 |
As seen from table, the interior bacterial community quantity of cotton caulome significantly descends behind the spray oligosaccharide, waits until that the later stage has rise again.
Claims (3)
1, a kind of plant induced resistance agent is characterized in that this plant induced resistance agent mainly contains active oligomeric sugar, obtains by following method: (1) is that the bacterial classification microbial fermentation obtains a large amount of mycelium with F.graminearum schw F.graminearum Schw; (2) with fermentation mycelium at MgSO
4Degrade with one or more compound enzymes that carry out of chitinase, cellulase, bromelain, lysozyme, lipase, chitosan enzyme in the salting liquid, every kind of enzyme proportion is 1%--51%, enzyme dosage 0.5-3.0% weight, the concentration of salting liquid is 0.01-0.5%, pH3.0-7.0, reaction temperature 15-60 ℃, reaction time 1-10 hour; (3) be warming up to 80-110 ℃, hot-water extraction 5-20 hour.
2, according to right 1 described plant induced resistance agent, it is characterized in that: described inoculation is in the fermentation medium of following prescription:
Peptone 0.3-2.0%, analysis for soybean powder 0.5-3.5%,
Dusty yeast 0.2-1.5%, corn steep liquor 0.2-2.0%,
Sucrose 0.5-2.0%, KH
2PO
40.6-1.5%,
MgSO
4 0.1-1.0％， NaCl 0.01-0.5％。
3, the described plant induced resistance agent of claim 1 is used to prevent and treat soybean, tobacco, cotton, eggplant plant disease, it is characterized in that with this product seed soaking, foliage-spray or root irrigation, working concentration is 10-2000ppm.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 99122582 CN1122445C (en) | 1999-12-01 | 1999-12-01 | Plant resistance inductor |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 99122582 CN1122445C (en) | 1999-12-01 | 1999-12-01 | Plant resistance inductor |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1303602A CN1303602A (en) | 2001-07-18 |
| CN1122445C true CN1122445C (en) | 2003-10-01 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 99122582 Expired - Fee Related CN1122445C (en) | 1999-12-01 | 1999-12-01 | Plant resistance inductor |
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|---|---|
| CN (1) | CN1122445C (en) |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| ATE495671T1 (en) * | 2007-07-19 | 2011-02-15 | Univ Montana State | FUNGAL ISOLATES AND THEIR USE FOR PROMOTING SALT AND DRY TOLERANCE IN PLANTS |
| US20120084886A1 (en) * | 2010-06-16 | 2012-04-05 | Agrinos AS | Microbial process and composition for agricultural use |
| CN106399132B (en) * | 2016-12-16 | 2019-09-20 | 山东蓬勃生物科技有限公司 | One plant of Irpex lacteus and its application |
| CN107974423B (en) * | 2017-12-25 | 2021-04-27 | 广东杰士农业科技有限公司 | Soil biological activator and preparation method thereof |
| CN108378061B (en) * | 2018-05-08 | 2020-06-09 | 菏泽学院 | Oil-used peony leaf spot inducer |
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1999
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