CN1911043A - Method for preparing biological type seed coating agent for anti-cucumber powdery mildew of system induction type - Google Patents
Method for preparing biological type seed coating agent for anti-cucumber powdery mildew of system induction type Download PDFInfo
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- CN1911043A CN1911043A CN 200610029883 CN200610029883A CN1911043A CN 1911043 A CN1911043 A CN 1911043A CN 200610029883 CN200610029883 CN 200610029883 CN 200610029883 A CN200610029883 A CN 200610029883A CN 1911043 A CN1911043 A CN 1911043A
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Abstract
A biologic seed coating agent for systematically inducing the cucumber to resist against powdery mildew is prepared from the metabolite generated by fermenting the mutant strain of Trichoderma and the endogenic Bacillus subtilis through preparing the suspension of said endogenic Bacillus subtilis and the fermented liquid of mutant Frichoderma strain, preparing the assistant solution from sodium lauryl phenylsulfonate, carboxymethyl cellulose sodium, rhodamine, salicylic acid, inorganic ions and distilled water, and sequentially mixing them together while regulating the concentration.
Description
Technical field
The present invention relates to a kind of preparation method of biological type seed coating agent for anti-cucumber powdery mildew of system induction type, adopt antagonism Trichoderma T23 (Trichoderma atroviride) through the restriction enzyme mediation integrate metabolite that the mutant strain F3 fermentation of back gained produces with a kind of in give birth to type antagonism bacillus subtilis B 2 0-006 (Bacillussubtilis) and be re-dubbed the agent of a kind of new bio type kind clothing, can also improve system's resistance in the growth while of obvious promotion cucumber seedling to powdery mildew of cucumber (Sphaerotheca fuliginea).
Background technology
The biological control agents that Trichoderma is most widely used at present as the world, it prevents and treats disease mechanism except that having to pathogen hyperparasitism, the antibiosis, also have the function of obviously inducing the potential resistance related gene of host plant to express.But Trichoderma or bacillus subtilis inducing cucumber have been proved at present, cotton, tobacco, lettuce and capsicum etc. make deposits yields to multiple diseases such as gray mold (Botrytis cinerea), anthracnose (Colletotrichum lindemuthianum, C.orbicular, C.graminiicolar), phytophthora root rot (Phytophthora capsci), early blight (Alternaria solani), bacteriosis (Pseudomonassyringae pv.lachrymans, Xanthomonas campestris pv.phaseoli, X.oryzae) and the resistance effect of virus disease (Cucumber Green-mottle), do not utilize Trichoderma metabolite and bacillus subtilis compound action to handle cucumber seeds but still report up to now, and then by the resistance of signal transduction inducing cucumber overground part blade generation to powdery mildew.Although having proved, a lot of in the past researchs can obviously induce the potential resistance related gene of host plant to express under Trichoderma or the bacillus subtilis condition of living body, but have some difficulties in actual applications: breed although can constantly enclose with rhizosphere in kind based on the inducer of single live body Trichoderma or bacillus subtilis (1), produce the induction of resistance metabolite, but living microorganism population and activity are vulnerable to various Effect of Environmental in the soil very much in actual applications, cause induction of resistance effect instability.(2) with the microbial metabolic products be biotype kind agent main component, though realize the quantification that inducer uses easily, but can make the effective dose of induction of resistance be difficult to guarantee because of absorption and the decomposition of other microorganism in the soil equally, be difficult to realize the successive induction of host resistance.Therefore how to bring into play living microorganism and its metabolite separately advantage in induction of resistance, it is significant to develop efficient, stable biotype kind clothing agent.
Summary of the invention
The objective of the invention is at the deficiencies in the prior art, a kind of preparation method of biological type seed coating agent for anti-cucumber powdery mildew of system induction type is provided, the biotype kind clothing agent that makes has long, advantages such as preventive effect is high, anti-storage of life-span, and powdery mildew of cucumber is had good control efficiency.
For realizing such purpose, the present invention adopts the mutant strain F3 of antagonism Trichoderma T23 through metabolite and a kind of interior type antagonism bacillus subtilis B 2 0-006 that gives birth to that liquid fermentation produces, and is re-dubbed the agent of a kind of new bio type kind clothing.Prepare endophytic Bacillus subtilis suspension and trichoderma harzianum mutant strain zymotic fluid at first respectively, then auxiliary agents such as neopelex, sodium carboxymethylcellulose, rhodamine, salicylic acid and inorganic ions are joined in the distilled water, stir and be made into compounding agent solution, join bacillus subtilis suspended liquid and trichoderma harzianum mutant strain zymotic fluid in the compounding agent solution successively again and regulate the wherein concentration of active component, obtain required biological type seed coating agent for anti-cucumber powdery mildew of system induction type.This kind clothing agent that contains the trichoderma harzianum mutant strain metabolite and the live body endophytic Bacillus subtilis of induction of resistance function can overcome the short and single unsettled shortcoming of effect of inducing of bacillus subtilis of Trichoderma conidium survival period.
Of the present invention kind of clothing agent key component is:
Biological components: interior living type antagonism bacillus subtilis B 2 0-006; Abiotic component: the zymotic fluid of antagonism trichoderma harzianum mutant strain F3; Auxiliary agent 1: neopelex 0.5%-2.5%; Auxiliary agent 2: sodium carboxymethylcellulose 1%-3%; Auxiliary agent 3: rhodamine 0.5%-1.5%; Salicylic acid 0.005%-0.015%; Inorganic ions comprises K
2HPO
30.5%-1.5%, MgSO
40.05%-0.15%, CaCO
30.05%-0.15%, ZnSO
40.005%-0.015% and dipotassium tetraborate 0.005%-0.015%.
Of the present invention kind of clothing agent preparation method comprises following concrete steps:
1, the preparation of endophytic Bacillus subtilis suspension:
Antagonism endophytic Bacillus subtilis B20-006 is inoculated in the LB liquid nutrient medium, 25 ℃ of 135 rev/mins of shaken cultivation 4 days, 5000 rev/mins centrifugal 5 minutes, abandon supernatant, use the sterile water washing precipitation, 5000 rev/mins were descended centrifugal 5 minutes, and abandoned supernatant, with a small amount of sterile distilled water suspension thalline, it is standby to make endophytic Bacillus subtilis suspension after washing 3 times.
2, trichoderma harzianum mutant strain preparation of fermentation liquid:
Antagonism trichoderma harzianum mutant strain F3 is inoculated on the PDA solid culture medium, cultivated 6 days down for 25 ℃, make the bacterium colony surface produce a large amount of conidiums, spore is washed, and to be made into concentration be 3 * 10 with sterile distilled water
7The spore suspension of individual spore/milliliter, in the PDB liquid nutrient medium, cultivate, connect bacterium amount and be 5 milliliters of spore suspensions of per 100 milliliters of culture medium inoculateds, 141 rev/mins, 28 ℃ condition bottom fermentations 3 days, filter then and remove mycelia, obtain the trichoderma harzianum mutant strain zymotic fluid.
3, the preparation of compounding agent solution:
By weight percentage, with the neopelex of 0.5%-2.5%, the sodium carboxymethylcellulose of 1%-3%, the rhodamine of 0.5%-1.5%, the salicylic acid of 0.005%-0.015%, the K of 0.5%-1.5%
2HPO
3, 0.05%-0.15% MgSO
4, 0.05%-0.15% CaCO
3, 0.005%-0.015% ZnSO
4, 0.005%-0.015% dipotassium tetraborate, join successively in the distilled water, stir into uniform sticky shape liquid, be made into compounding agent solution.
4, the trichoderma harzianum mutant strain zymotic fluid with endophytic Bacillus subtilis suspension and 80-120 milliliter joins in the compounding agent solution successively, and vibration mixes, and regulating antagonism bacillus subtilis final concentration is 10
10Colony forming units cfu/milliliter obtains biological type seed coating agent for anti-cucumber powdery mildew of system induction type.
The kind clothing agent of gained of the present invention is carried out Cotton seeds to cucumber seeds, treat that seedling carries out the challenge inoculation of powdery mildew (Sphaerotheca fuliginea (Schlecht) Poll.) when growing to two true leaves, 6 days " Invest, Then Investigate " disease indexs, found that biotype kind clothing of the present invention agent can also improve the system resistance of cucumber to powdery mildew simultaneously in the growth that obviously promotes cucumber seedling, reaches 70-75% to the relative control efficiency of powdery mildew.
Of the present invention kind of clothing agent contains the trichoderma harzianum mutant strain metabolite and the live body endophytic Bacillus subtilis of induction of resistance function, can overcome the short and single unsettled shortcoming of effect of inducing of bacillus subtilis of Trichoderma conidium survival period in kind of the clothing agent, have long, advantages such as preventive effect is high, anti-storage of life-span, powdery mildew of cucumber is had good control efficiency.The present invention is applicable to most of melon kinds, and the preparation method is simple, is suitable for large-scale industrialized production, and market prospects are wide.
Embodiment
Below by specific embodiment technical scheme of the present invention is further described.
Embodiment 1
1, the preparation of endophytic Bacillus subtilis suspension:
Antagonism bacillus subtilis B 2 0-006 is inoculated in the LB liquid nutrient medium, and nutrient media components is: tryptone 10 grams, sodium chloride 10 grams, yeast extract 5 grams, 1000 milliliters in water, pH7.0.25 ℃ of 135 rev/mins of shaken cultivation 4 days, 5000 rev/mins centrifugal 5 minutes, abandon supernatant, use the sterile water washing precipitation, 5000 rev/mins were descended centrifugal 5 minutes again, abandoned supernatant, with a small amount of sterile distilled water suspension thalline, it is standby to make endophytic Bacillus subtilis suspension after washing 3 times.
2, trichoderma harzianum mutant strain preparation of fermentation liquid:
Antagonism trichoderma harzianum mutant strain F3 is inoculated on the PDA solid culture medium, and nutrient media components is: 1000 milliliters of potato 200 grams, glucose 20 grams, agar 15-20 gram, water.Cultivate down after 6 days for 25 ℃, the bacterium colony surface produces a large amount of conidiums, with sterile distilled water conidium is washed, and to be made into concentration is 3 * 10
7The spore suspension of individual spore/milliliter, liquid fermentation medium adopts the PDB liquid nutrient medium, and nutrient media components is: potato 200 grams, glucose 20 grams, yeast extract 15 grams.Connect the bacterium amount for 5 milliliters of spore suspensions of per 100 milliliters of culture medium inoculateds, under 141 rev/mins, 28 ℃ condition, fermented 3 days, filter then and remove mycelia, obtain the trichoderma harzianum mutant strain zymotic fluid.
3, the preparation of compounding agent solution:
Get neopelex 7.5 grams, sodium carboxymethylcellulose 10 grams, rhodamine 5 grams, salicylic acid 0.05 gram and inorganic ions: K
2HPO
35 grams, MgSO
40.5 gram, CaCO
30.5 gram, ZnSO
40.05 gram, dipotassium tetraborate 0.05 gram; Above-mentioned each component is joined in 300 ml distilled waters successively, stir into uniform sticky shape liquid, be made into compounding agent solution.
4, antagonism bacillus B20-006 suspension and 80 milliliters of antagonism trichoderma harzianum mutant strain zymotic fluids are joined in the ground above-mentioned viscous fluid successively, vibration mixes, regulating antagonism bacillus subtilis final concentration is 1010 colony forming units cfu/milliliters, adding distil water is supplied 500 milliliters, obtains biological type seed coating agent for anti-cucumber powdery mildew of system induction type.
The detection of biotype kind clothing agent induction of resistance effect
Growing seedlings early stage, cucumber seeds to be hoted water treatment of seeds 12-15 minute in 55 ℃ of water-baths, taking-up is dried, then with kind of a clothing agent capsuled seed.The ratio of soaking 50-55 grain seed according to the agent of every milliliter of kind clothing was soaked into about 30 minutes, treat to take out when kind of a clothing agent is attached on the seed fully, fully dry, through kind of clothing agent seeds treated can be in 28 ℃ of incubators vernalization about 24 hours, to guarantee certain humidity during vernalization, also can directly sow without vernalization.
The cucumber seeds sowing that to handle through the agent of biotype kind clothing is 3: 2: 1 a turfy soil filling went out bacterium and volume proportion, vermiculite, in the polypots of ash, every alms bowl 2 strains, place temperature again, illumination, cultivated 12-14 days in the good greenhouse of humidity, treat to carry out after two cotyledons of cucumber fully flatten the challenge inoculation of powdery mildew, 6 days " Invest, Then Investigate " disease indexs, the result shows that the agent of this kind clothing is obviously promoting also to improve the system resistance of cucumber seedling to powdery mildew in the cucumber seedling growth, to the relative control efficiency of powdery mildew more than 70%.
Embodiment 2
1, the preparation of endophytic Bacillus subtilis suspension:
Antagonism bacillus subtilis B 2 0-006 is inoculated in the improved LB liquid nutrient medium, 25 ℃ of 135 rev/mins of shaken cultivation 4 days, 5000 rev/mins centrifugal 5 minutes, abandon supernatant, use the sterile water washing precipitation, 5000 rev/mins were descended centrifugal 5 minutes again, abandoned supernatant, with a small amount of sterile distilled water suspension thalline, it is standby to make endophytic Bacillus subtilis suspension after washing 3 times.
2, trichoderma harzianum mutant strain preparation of fermentation liquid:
Antagonism trichoderma harzianum mutant strain F3 is inoculated on the PDA solid culture medium of improvement, cultivates down after 6 days for 25 ℃, the bacterium colony surface produces a large amount of conidiums, with sterile distilled water conidium is washed, and to be made into concentration is 3 * 10
7The spore suspension of individual spore/milliliter, liquid fermentation medium adopts the PDB liquid nutrient medium, connects the bacterium amount and is 5 milliliters of spore suspensions of per 100 milliliters of culture medium inoculateds, under 141 rev/mins, 28 ℃ condition, fermented 3 days, and filtered then and remove mycelia, obtain the trichoderma harzianum mutant strain zymotic fluid.
3, the preparation of compounding agent solution:
Get neopelex 8.5 grams, sodium carboxymethylcellulose 9 grams, rhodamine 5 grams, salicylic acid 0.06 gram and inorganic ions: K
2HPO
34 grams, MgSO
40.6 gram, CaCO
30.4 gram, ZnSO
40.06 gram, dipotassium tetraborate 0.04 gram; Above-mentioned each component is joined in 300 ml distilled waters successively, stir into uniform sticky shape liquid, be made into compounding agent solution.
4, the antagonism trichoderma harzianum mutant strain zymotic fluid with antagonism bacillus B20-006 suspension and 120 milliliters joins in the ground above-mentioned viscous fluid successively, vibration mixes, regulating antagonism bacillus subtilis final concentration is 1010 colony-forming units/milliliters, adding distil water is supplied 500 milliliters, obtains biological type seed coating agent for anti-cucumber powdery mildew of system induction type.
Susceptible variety cucumber in will handling through the agent of biotype kind clothing, planting seed is 3: 2: 1 a turfy soil filling went out bacterium and volume proportion, vermiculite, in the polypots of ash, every alms bowl 2 strains, place temperature again, illumination, grew seedlings 12-14 days in the greenhouse that humidity is fit to, after treating that two cotyledons of cucumber fully flatten, being transplanted to greenhouse soil, is contrast with what do not carry out kind of clothing agent processing, under the management conditions such as normal field humidity, treat that powdery mildew of cucumber sick leaf rate in check plot reaches 40%, disease index reaches 25%, the preventive effect in the district that investigates, the result show the agent of this kind clothing can inducing cucumber in the field, greenhouse to system's resistance of powdery mildew, to the relative control efficiency of powdery mildew more than 65%.
Claims (1)
1, a kind of preparation method of biological type seed coating agent for anti-cucumber powdery mildew of system induction type is characterized in that comprising following concrete steps:
1) antagonism endophytic Bacillus subtilis B20-006 is inoculated in the LB liquid nutrient medium, 25 ℃ of 135 rev/mins of shaken cultivation 4 days, 5000 rev/mins centrifugal 5 minutes, abandon supernatant, use the sterile water washing precipitation, 5000 rev/mins were descended centrifugal 5 minutes, and abandoned supernatant, washing back is with the sterile distilled water thalline that suspends, and it is standby to make endophytic Bacillus subtilis suspension;
2) antagonism trichoderma harzianum mutant strain F3 is inoculated on the PDA solid culture medium, cultivated 6 days down for 25 ℃, make the bacterium colony surface produce a large amount of conidiums, spore is washed, and to be made into concentration be 3 * 10 with sterile distilled water
7The spore suspension of individual spore/milliliter, in the PDB liquid nutrient medium, cultivate, connect bacterium amount and be 5 milliliters of spore suspensions of per 100 milliliters of culture medium inoculateds, 141 rev/mins, 28 ℃ condition bottom fermentations 3 days, filter then and remove mycelia, obtain the trichoderma harzianum mutant strain zymotic fluid;
3) by weight percentage, with the neopelex of 0.5%-2.5%, the sodium carboxymethylcellulose of 1%-3%, the rhodamine of 0.5%-1.5%, the salicylic acid of 0.005%-0.015%, the K of 0.5%-1.5%
2HPO
3, 0.05%-0.15% MgSO
4, 0.05%-0.15% CaCO
3, 0.005%-0.015% ZnSO
4, 0.005%-0.015% dipotassium tetraborate, join successively in the distilled water, stir into uniform sticky shape liquid, be made into compounding agent solution;
4) the trichoderma harzianum mutant strain zymotic fluid with endophytic Bacillus subtilis suspension and 80-120 milliliter joins in the compounding agent solution successively, and vibration mixes, and regulating antagonism bacillus subtilis final concentration is 10
10Colony forming units cfu/milliliter obtains biological type seed coating agent for anti-cucumber powdery mildew of system induction type.
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| CN 200610029883 CN1911043A (en) | 2006-08-10 | 2006-08-10 | Method for preparing biological type seed coating agent for anti-cucumber powdery mildew of system induction type |
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| CN 200610029883 CN1911043A (en) | 2006-08-10 | 2006-08-10 | Method for preparing biological type seed coating agent for anti-cucumber powdery mildew of system induction type |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| FR2929805A1 (en) * | 2008-02-12 | 2009-10-16 | B2B Sarl | Use of bacteria, proteins or peptides derived from clones of Bacillus subtilis, deposited at american type culture collection, as stimulators of entire natural plant, against pathogen of fungal, bacterial and/or viral type cultures |
| CN100563442C (en) * | 2008-03-28 | 2009-12-02 | 西北农林科技大学 | Seed coating agent for cucumbers |
| CN103039439A (en) * | 2011-10-11 | 2013-04-17 | 上海万力华生物科技有限公司 | Seed coating agent of Trichoderma fungi and preparation method thereof |
| CN103141518A (en) * | 2013-03-04 | 2013-06-12 | 沈阳农业大学 | Preparation method and application of multi-effect compound anti-biological inoculants |
| CN103355357A (en) * | 2012-03-26 | 2013-10-23 | 上海万力华生物科技有限公司 | Potato seed coating |
| CN103461386A (en) * | 2013-08-23 | 2013-12-25 | 河南省农业科学院植物保护研究所 | Composite sesame seed coating agent |
| RU2576534C2 (en) * | 2014-07-10 | 2016-03-10 | Федеральное государственное бюджетное образовательное учреждение высшего профессионального образования "Астраханский государственнный технический университет" | Composition for presowing treatment of cucumber seeds |
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2006
- 2006-08-10 CN CN 200610029883 patent/CN1911043A/en active Pending
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| FR2929805A1 (en) * | 2008-02-12 | 2009-10-16 | B2B Sarl | Use of bacteria, proteins or peptides derived from clones of Bacillus subtilis, deposited at american type culture collection, as stimulators of entire natural plant, against pathogen of fungal, bacterial and/or viral type cultures |
| CN100563442C (en) * | 2008-03-28 | 2009-12-02 | 西北农林科技大学 | Seed coating agent for cucumbers |
| CN103039439A (en) * | 2011-10-11 | 2013-04-17 | 上海万力华生物科技有限公司 | Seed coating agent of Trichoderma fungi and preparation method thereof |
| CN103039439B (en) * | 2011-10-11 | 2015-04-15 | 上海万力华生物科技有限公司 | Seed coating agent of Trichoderma fungi and preparation method thereof |
| CN103355357A (en) * | 2012-03-26 | 2013-10-23 | 上海万力华生物科技有限公司 | Potato seed coating |
| CN103355357B (en) * | 2012-03-26 | 2016-05-11 | 上海万力华生物科技有限公司 | A kind of potato seed-coating agent |
| CN103141518A (en) * | 2013-03-04 | 2013-06-12 | 沈阳农业大学 | Preparation method and application of multi-effect compound anti-biological inoculants |
| CN103461386A (en) * | 2013-08-23 | 2013-12-25 | 河南省农业科学院植物保护研究所 | Composite sesame seed coating agent |
| CN103461386B (en) * | 2013-08-23 | 2015-05-06 | 河南省农业科学院植物保护研究所 | Composite sesame seed coating agent |
| RU2576534C2 (en) * | 2014-07-10 | 2016-03-10 | Федеральное государственное бюджетное образовательное учреждение высшего профессионального образования "Астраханский государственнный технический университет" | Composition for presowing treatment of cucumber seeds |
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