CN100372462C - Microbiological agent resisting rice bakanae disease, preparation method and application thereof - Google Patents
Microbiological agent resisting rice bakanae disease, preparation method and application thereof Download PDFInfo
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- CN100372462C CN100372462C CNB2006100486602A CN200610048660A CN100372462C CN 100372462 C CN100372462 C CN 100372462C CN B2006100486602 A CNB2006100486602 A CN B2006100486602A CN 200610048660 A CN200610048660 A CN 200610048660A CN 100372462 C CN100372462 C CN 100372462C
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- bakanae disease
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Abstract
The invention relates to a microbiological agent against rice banakae disease, its preparing process and use, wherein the preparing process comprises steps of microbiological strains test tube seedling culture, bacterial strain fluid seed culture, and bacterial strain fluid fermentation culture. The produced bacterial strain being Streptomyces avermitilis ECO 00005 which was preserved in China General Microbiological Culture Collection Center on April 17, 2006 with a docket number of CGMCC No.1681. The produced preparation can be used for controlling rice banakae disease.
Description
Technical field the present invention relates to a kind of microbiological agent resisting rice bakanae disease and its production and application, the microorganism belonging to genus technical field of pesticide.
Background technology bakanae disease of rice pathogen (Fusarium monilifome) is a seed-borne disease important in the Rice Production, and the pathogen conidium of carrying on the rice paddy seed is main primary source of infection.The popular of this disease causes the paddy rice underproduction 10%～20%, but the serious underproduction because the long-term chemical pesticide that uses identical type causes pathogen to develop immunity to drugs, makes the trend that growth is arranged of this disease more than 50% in recent years.Simultaneously owing to use chemical agent in a large number; make persticide residue increase in the crop, natural enemy is killed and wounded in a large number, and the ecological balance in farmland is destroyed and environment is contaminated; along with China joined WTO and the people attention day by day to environmental and ecological protection, China's agricultural chemicals already is being faced with severe situation.
Biological control is pollution-free with it, noresidue, and no eco-toxicity and safety are good etc., and advantage plays leading role in the control of plant pest.Greatly develop biopesticide China is had very important realistic meaning.Microbial pesticide is the important source of novel pesticide initiative, have weak point research cycle, drop into low, as to be easy to industrialization and commercialized development advantage, and to the person poultry safety, be easy to decompose, with environmentally compatible, from eighties of last century since the nineties, microbial pesticide every year has become one of research and development focus in the agricultural biotechnologies industry with 20% speed increase.China's microbial resources are abundant, and the development microbial pesticide has sufficient advantage.People are by a large amount of screenings with utilize beneficial microbe and the metabolite thereof of resisting rice bakanae disease pathogen in recent years, make microbial control become the important and valid approach of in the bakanae disease of rice control one just day by day.
Summary of the invention the objective of the invention is to study by a strain that screens is had the streptomycete that prevents and treats the bakanae disease of rice activity, the exploitation microbiological agent resisting rice bakanae disease.
The present invention screens a strain bakanae disease of rice is had deinsectization streptomycete (Streptomyces avermitilis) ECO 00005 of fine preventive and therapeutic effect, and ECO 00005 bacterial strain has been deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center; Address: China. Beijing; Preservation date: on April 17th, 2006; Preservation is registered on the books and is numbered CGMCC No.1681.
Streptomyces avermitilis ECO 00005 strain morphology of the present invention is characterized as: bacteria colony white, circle, surperficial lint shape, there is more mycelial growth at the edge, bacterium colony central protrusion, rare gas silk, form the no gas silk circle of central authorities, the inhomogeneous projection of bacterium colony presents the petal-shaped of 3-4 lobe; Substrate mycelium and aerial hyphae are thread, and irregular branch does not generally rupture; Form long spore chain on the aerial hyphae, straight shape or waveform or spirality, spore circle, oval to shaft-like.
The present invention is achieved in that
The preparation microbiological agent resisting rice bakanae disease:
1, the test tube kind is cultivated
Bacterial strain ECO 00005 is inoculated on the test tube culture medium slant, and culture medium prescription is: yeast extract 4g; Glucose 4g; Malt extract 5g; B B-complex 1ml; Agar 20g; Water 1000ml; PH7.2 cultivated 6 days down in 28 ℃, obtained the test tube kind.(B B-complex consists of: Cobastab
11.0mg/L, Cobastab
61.0mg/L, vitamin b3 1.0mg/L, nicotinic acid 1.0mg/L, phenyl alanine 1.0mg/L, vitamin h 1.0mg/L, alanine 0.3mg/L, down together)
2, liquid seeds is cultivated
Adopt 500ml triangular flask shake-flask culture, method is first obtaining liq seed culture medium, and the liquid seed culture medium prescription is: yeast extract 4g; Glucose 4g; Malt extract 5g; B B-complex 1ml; Water 1000ml; PH7.2, the 100ml liquid nutrient medium of packing in each triangular flask was sterilized 30 minutes for 120 ℃, and 1cm is inserted in the cooling back
2Big test tube kind was cultivated 2 days on rotating speed is the shaking table of 220rpm under 28 ℃.
3, preparation microbiological agent resisting rice bakanae disease
Adopt 500ml triangular flask shake-flask culture, method is first obtaining liq fermentation medium, and the liquid fermentation medium prescription is: soybean meal 20g; Mannitol 20g; Water 1000ml; PH7.8, the 100ml liquid fermentation medium of in each triangular flask, packing into, 120 ℃ of sterilizations 30 minutes, the seed liquor that obtains in the 10ml said method 2 is inserted in cooling back, on rotating speed is the shaking table of 220rpm, cultivated 6 days under 28 ℃ zymotic fluid.In zymotic fluid, add equal-volume ethanol, after shaking table vibration 8 hours, be centrifugal 10 minutes of the centrifuge of 3000rpm with rotating speed, get supernatant and on Rotary Evaporators 50 ℃ boil off ethanol, be diluted with water to the original fermentation liquor volume, promptly become operational microbiological agent resisting rice bakanae disease.
The microbiological agent resisting rice bakanae disease that method for preparing is obtained, the method that adopts the Toxicity Determination (suppressing spore germination method, dull and stereotyped bacteriostatic test method) and the live body test of pesticide effectiveness (greenhouse pot culture live test) to combine, the test said preparation is to the preventive and therapeutic effect of bakanae disease of rice.
Toxicity Determination result shows: bacterial strain ECO 00005 agent resisting rice bakanae disease for preparing with said method is 100% to the inhibiting rate of bakanae disease of rice pathogen spore germination in suppressing the test of spore germination method; In dull and stereotyped bacteriostatic test method, to the antibacterial circle diameter average out to 18.7mm (3 repetitions) of bakanae disease of rice pathogen, two tests show that said preparation has stronger inhibition activity to the bakanae disease of rice pathogen.
The greenhouse pot culture live test is the result show: bacterial strain ECO 00005 agent resisting rice bakanae disease has shown the better prevention effect to the pot rice seedling, and its control efficiency to bakanae disease of rice is 90.56% as calculated.
The preparation that utilizes the present invention to produce has stripped preferably and live body control efficiency to bakanae disease of rice, can be used for the control of bakanae disease of rice.The present invention has that cost of material is low, and production technology is simple, and the advantage that control efficiency is good has application promise in clinical practice.
Embodiment:
Embodiment one: suppress the test of spore germination method
1, preparation test agent resisting rice bakanae disease
Microbiological agent resisting rice bakanae disease of the present invention by producing bacterial strain (Streptomyces avermitilis) ECO 00005 bacterial strain, is pressed the liquid fermentation production method preparation.It is identical that concrete preparation process and condition and summary of the invention are described part.
2, preparation bakanae disease of rice pathogen
Known bakanae disease of rice pathogen (Fusarium monilifome) is inoculated into 28 ℃ of cultivations down on the test tube culture medium slant, culture medium prescription is PDA medium commonly used, this medium method for making is to be cut into small pieces after getting potato 200g peeling, adding water 1000ml boiled 20 minutes, obtain 1000ml filtrate after the filtration, add glucose 20g then, agar 20g, the pH nature, treat that mycelia covers with medium, produced spores in 6-7 days after, wash spore with distilled water, and being diluted to concentration under 10 * 10 low-powered microscopes, the bakanae disease cause of disease bacterium spore suspension of each visual field 40-50 spore is standby.
3, suppress the spore germination test method:
Agent resisting rice bakanae disease is mixed with the bakanae disease of rice pathogen spore suspension equal-volume for preparing for test agent, be tiled on the water agar, culture medium prescription is glucose 10g, agar 10g, water 1000ml, pH nature, 3 repetitions of every processing, establishing clear water simultaneously is blank.In the sprouting situation that the cultivation of preserving moisture under 28 ℃ was watched the blank spore after 24 hours, surpassing conidium end diameter one half with germ tube length is sprouting.After the germination rate of blank spore reaches 95%, watch agent resisting rice bakanae disease for the inhibition situation of test agent to the spore germination of bakanae disease of rice pathogen.
Calculate the spore germination inhibiting rate as follows:
Spore germination inhibiting rate (%)=(contrast spore germination rate-processing spore germination rate)/(contrast spore germination rate) * 100
Experimental result shows: it is 100% to the sprouting inhibiting rate of bakanae disease of rice pathogen spore that bacterial strain ECO 00005 agent resisting rice bakanae disease supplies test agent.
Embodiment two: dull and stereotyped bacteriostatic test
Microbiological agent resisting rice bakanae disease of the present invention by producing bacterial strain (Streptomyces avermitilis) ECO 00005 bacterial strain, is pressed the liquid fermentation production method preparation.It is identical that concrete preparation process and condition and summary of the invention are described part, and the preparation of bakanae disease of rice pathogen is with the method in the inhibition spore germination method.
Dull and stereotyped bacteriostatic test: cultured bakanae disease of rice pathogen is got the big test tube kind inclined-plane of 0.5cm2 mix with 10ml PDA medium (method for making is with the method in the inhibition spore germination method), pour internal diameter into and be in the culture dish of sterilization of 9cm and make the flat board that carries disease germs, bacteriostatic test plate adopts conventional filter paper method, promptly on carrying disease germs flat board, each places 3 sterilization filter papers that diameter is 5mm, the positive contrast of Shi Baoke (prochloraz) medicament of drop 45ppm on 1 filter paper wherein, 1 aseptic clear water of filter paper drop is as blank in addition, remain the microbiological agent resisting rice bakanae disease that dropping prepares on 2 filter papers and supply test agent 15 μ L, 3 repetitions of every processing, be positioned over and cultivate in 28 ℃ of incubators after 48 hours, measure antibacterial circle diameter with the right-angled intersection method.
Table 1. microbiological agent resisting rice bakanae disease is to the antibacterial result of bakanae disease of rice pathogen
| Pathogen | Agent resisting rice bakanae disease is for test agent antibacterial circle diameter (mm) | Shi Baoke antibacterial circle diameter (mm) | Clear water contrast antibacterial circle diameter (mm) |
| Rice bakanae disease cause of disease bacterium | 18.4 | 20.0 | 0 |
| Rice bakanae disease cause of disease bacterium | 19.0 | 20.4 | 0 |
| Rice bakanae disease cause of disease bacterium | 18.6 | 19.7 | 0 |
| On average | 18.7 | 20.0 | 0 |
Experimental result shows: bacterial strain ECO 00005 agent resisting rice bakanae disease is for the antibacterial circle diameter average out to 18.7mm of test agent to the bakanae disease of rice pathogen.
Embodiment three: the greenhouse pot culture live test
Microbiological agent resisting rice bakanae disease of the present invention by producing bacterial strain (Streptomyces avermitilis) ECO 00005 bacterial strain, is pressed the liquid fermentation production method preparation.It is identical that concrete preparation process and condition and summary of the invention are described part, and the preparation of bakanae disease of rice pathogen is with the method in the inhibition spore germination method.Concrete test procedure is:
The rice varieties that this experiment is adopted be pick up from Songming County, Kunming, Yunnan Province infection the Mongolian rice that carries disease germs of Fusarium moniliforme Sheld.
Soaked seed 48 hours for test agent with agent resisting rice bakanae disease, take out seed rice, place clear water, 30 ℃ of following vernalization 24 hours, when treating seed germination, evenly being sowed at the length and width that rice seedling bed soil is housed is 5 * 15cm, in the plastic seeding culturing box of dark 10cm, and the surface coverage thin soil, every box 50 seedlings, in temperature is 28-30 ℃ greenhouse, cultivate, impose urea, execute 2-3 time altogether by 0.2 gram/box.Carry out the diseased plant rate investigation in 3 leaf phases and transplanting to the land for growing field crops respectively, record diseased plant number calculates control efficiency.
Control efficiency (%)=(control group diseased plant rate-processed group diseased plant rate)/control group diseased plant rate * 100.
The greenhouse pot culture live test is the result show: bacterial strain ECO 00005 agent resisting rice bakanae disease has shown the better prevention effect for test agent to bakanae disease of rice, and its control efficiency to bakanae disease of rice is 90.56% as calculated.
Claims (3)
1. microbiological agent resisting rice bakanae disease, the production bacterial strain that it is characterized in that said preparation is deinsectization streptomycete (Streptomyces avermitilis) ECO 00005, ECO 00005 bacterial strain is deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center on April 17th, 2006, and deposit number is CGMCC No.1681.
2. the preparation method of the described microbiological agent resisting rice bakanae disease of claim 1 is characterized in that:
(1) the test tube kind is cultivated and is: bacterial strain ECO 00005 is inoculated on the test tube culture medium slant, and culture medium prescription is: yeast extract 4g; Glucose 4g; Malt extract 5g; B B-complex 1ml; Agar 20g; Water 1000ml; PH7.2 cultivated 6 days down in 28 ℃, obtained the test tube kind;
(2) liquid seeds is cultivated to adopting 500ml triangular flask shake-flask culture, and method is first obtaining liq seed culture medium, and the liquid seed culture medium prescription is: yeast extract 4g; Glucose 4g; Malt extract 5g; B B-complex 1ml; Water 1000ml; PH7.2, the 100ml liquid nutrient medium of packing in each triangular flask was sterilized 30 minutes for 120 ℃, and 1cm is inserted in the cooling back
2Big test tube kind was cultivated 2 days on rotating speed is the shaking table of 220rpm under 28 ℃;
(3) the preparation microbiological agent resisting rice bakanae disease adopts 500ml triangular flask shake-flask culture, and method is first obtaining liq fermentation medium, and the liquid fermentation medium prescription is: soybean meal 20g; Mannitol 20g; Water 1000ml; PH7.8, the 100ml liquid fermentation medium of in each triangular flask, packing into, sterilized 30 minutes for 120 ℃, the seed liquor that obtains in the 10ml said method (2) is inserted in the cooling back, under 28 ℃ in rotating speed be cultivated 6 days on the shaking table of 220rpm zymotic fluid, in zymotic fluid, add equal-volume ethanol, after shaking table vibrated 8 hours, with rotating speed is centrifugal 10 minutes of the centrifuge of 3000rpm, get supernatant and on Rotary Evaporators 50 ℃ boil off ethanol, be diluted with water to the original fermentation liquor volume, promptly become operational microbiological agent resisting rice bakanae disease.
3. the described microbiological agent resisting rice bakanae disease of claim 1 is applied to prevent and treat the purposes of bakanae disease of rice.
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| CNB2006100486602A CN100372462C (en) | 2006-09-06 | 2006-09-06 | Microbiological agent resisting rice bakanae disease, preparation method and application thereof |
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| CNB2006100486602A CN100372462C (en) | 2006-09-06 | 2006-09-06 | Microbiological agent resisting rice bakanae disease, preparation method and application thereof |
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| CN100372462C true CN100372462C (en) | 2008-03-05 |
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| CN108617666A (en) * | 2018-07-23 | 2018-10-09 | 南京轩凯生物科技有限公司 | A kind of fungicide and its preparation method and application containing epsilon-polylysine and epoxiconazole |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1021708C (en) * | 1989-08-08 | 1993-07-28 | 林中 | Diamond composition material and drill bit made of it |
| CN1757744A (en) * | 2004-10-09 | 2006-04-12 | 武汉绿世纪生物工程有限责任公司 | Production bacterial strain of abamectin with high yield and high secreation rate and new method of extracting AVM |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1021708C (en) * | 1989-08-08 | 1993-07-28 | 林中 | Diamond composition material and drill bit made of it |
| CN1757744A (en) * | 2004-10-09 | 2006-04-12 | 武汉绿世纪生物工程有限责任公司 | Production bacterial strain of abamectin with high yield and high secreation rate and new method of extracting AVM |
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