CN1802927A - Method for preparing bactericidal agent using antagonizing bacteria M18 strain - Google Patents
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Abstract
The invention relates to a method for preparing bactericide by using derivative bacterial of growth promoting antagonist bacterial M18, preparing fermentation liquor with high concentration of under phenazine-1-carboxyl acid and pyoluteorin under optimized culture medium and condition by using derivative bacterial M18G and M18R of growth promoting antagonist bacterial M18, preparing the fermentation liquor M18G and M18R into dry powder, double crossing physically the phenazine-1-carboxyl acid in M18G powder and pyoluteorin in M18R according to the weight ratio of 90%-10% and 10%-90%, and finally getting bactericide of high efficiency for preventing plant disease. Compared with current technology, the bactericide provided in this invention is wet powder taking metabolite of microorgsanism as active element but not live bacterial agent, as a result of which the product is characterized by high stability, not easy to be influenced by environment and better prevention and curing effect for multiple plant disease under low consumption.
Description
Technical field
The present invention relates to a kind of preparation method of microbial source bactericide, relate in particular to the method that a kind of metabolite that utilizes antagonizing bacteria M 18 strain prepares the bactericide of controlling plant diseases.Belong to the microbial pesticide technical field.
Background technology
The crop production reduction amplitude that plant disease causes accounts for 25～75% of gross yield, causes the tremendous economic loss.Staple food crop paddy rice with China is an example, about 400,000,000 mu of annual cultivated area, and by 400 kilograms of per mu yields, rice disease causes the underproduction 10%, causes every year economic loss to reach 30,000,000,000 yuans.At present, the controlling plant disease is except selecting breeding for use and improving the cultivation step in the production, and main dependence sprayed chemical bactericide.But mostly chemical bactericide has in various degree toxic action to human body and animal, remains in the potential threat that the oxious component of plant edible part causes health, has caused the concern of government and all orders of society; Moreover, chemical pesticide generally is difficult for decomposing, and can be accumulated in chronically in the ecosystem, causes the pollution to environment, is unfavorable for society and economic sustainable development; And existing chemical pesticide is not in full force and effect to the certain plants disease.Therefore, need development high-efficiency low-toxicity of new generation, the chemical pesticide good, also need research and development biogenic pesticide energetically simultaneously Environmental compatibility.
1992, world environments once proposed with the development conference, and before the new century, the usage amount of biogenic pesticide will reach the target that agricultural chemicals uses total amount 60%, yet, even to this day, also only account for about 15% as U.S. of developed country, and China accounts for 5%.At present, compare with chemical pesticide, the biogenic pesticide of on producing, having promoted the use of, kind and quantity are all less, and some kind has caused that phytopathogen produces pesticide resistance in various degree owing to use year for a long time, and control efficiency is not ideal enough.With the rice sheath blight disease is example, this disease and rice blast, bacterial leaf-blight are listed as the big main disease of three in the Rice Production, the medicament of preventing and treating this disease mainly depends on the biogenic pesticide jinggangmycin, but, through single chronically use over more than 30 years, the fusion group of part Rhizoctonia solani Kuhn develops immunity to drugs; And jinggangmycin is only effective to Rhizoctonia solani Kuhn, and other pathogens are not had tangible control efficiency, and the scope of application has very big limitation.
Growth-promoting antagonistic bacterium M 18 for biologic agricultural chemical, to the bactericidal action of plant disease have efficiently, safety, wide spectrum, with features such as Environmental compatibility is good, obtained national inventing patent, the patent No. is 00119857.2.This growth-promoting antagonistic bacterium M18 is in the depositary institution of on June 27th, 2000 in Patent Office of the People's Republic of China's appointment: Beijing, and China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation, preserving number is CGMCC NO.0462.But growth-promoting antagonistic bacterium M 18 for biologic agricultural chemical is a kind of viable bacteria microbial inoculum, and the active component content of its synthetic anti-plant disease is subjected to the influence of environmental condition easily, thereby can influence the ability of anti-plant disease and the stability of control efficiency.
Find out that the dominant mechanism of growth-promoting antagonistic bacterium M18 controlling plant diseases is two kinds of antibacterial materials of secretion, is respectively phenazine-1-carboxylic acid and pyoluteorin.In recent years, the present patent application people uses molecular biological technology, to the regulatory mechanism of synthetic these the two kinds of antibacterial materials of growth-promoting antagonistic bacterium M18, has carried out research in depth.The utilization genetic engineering means, double base controlling gene gacA in the growth-promoting antagonistic bacterium M18 genome has been carried out orthomutation, obtained M18 derivative strain M18G, can improve the output of phenazine-1-carboxylic acid, the technical method of this achievement in research has been disclosed in 2004, and " microbiology journal " 44 rolled up the 761st～765 page, and thesis topic is " pseudomonad gacA inserts sudden change to phenazine-1-carboxylic acid and the anabolic otherness regulation and control of pyoluteorin ".Simultaneously, another controlling gene rsmA in the growth-promoting antagonistic bacterium M18 genome is carried out orthomutation, obtained M18 derivative strain M18R, can improve the output of pyoluteorin, the technical method of this achievement in research also has been disclosed in 2004 " microbiology journal " 44 volume 189-193 pages or leaves, thesis topic be " structure of pseudomonas fluorescens rsmA mutant strain and the distinctiveness regulating and controlling effect that pyoluteorin and phenazine-1-carboxylic acid are synthesized thereof ".Microbial bactericide is a new research topic how further to utilize the characteristic of the above two kinds of M18 derivative strain to prepare efficiently, has important practical significance.
Summary of the invention
The objective of the invention is at the defective that exists in the prior art, a kind of method of utilizing antagonizing bacteria M 18 strain to prepare bactericide is provided, utilize the metabolite of microorganism and the non-microorganism live body prepares bactericide, can stablize more, effective controlling plant disease.
The present invention can be achieved through the following technical solutions: under medium of optimizing and condition of culture, utilize derivative strain M18G and the M18R of growth-promoting antagonistic bacterium M18, produce the zymotic fluid of high-load phenazine-1-carboxylic acid and pyoluteorin respectively, respectively M18G zymotic fluid and M18R zymotic fluid are prepared into dry powder again, and be that 90%～10% and 10%～90% to carry out physics composite with the percentage by weight of the pyoluteorin content in phenazine-1-carboxylic acid in the M18G dry powder and the M18R dry powder, be prepared into the microbial source bactericide of high-efficiency prevention and control plant disease.
Method of the present invention specifically may further comprise the steps:
1, with the M18G inoculation on the flat board of glycerin medium, activate growth 20～24 hours down at 26～30 ℃, then the M18G bacterial strain that activates is transferred in the triangular flask that contains the glycerine culture fluid and amplifies, shaken cultivation is 10～14 hours in 26～30 ℃ shaking table, and shaking speed is 210～230 rev/mins; Continue switching 2～3 times then, in the glycerine culture fluid, carry out multistage amplification fermented and cultured; Be transferred at last and amplify fermented and cultured in the glucose culture solution, temperature and rotating speed are constant, and fermentation time is 70～74 hours, and obtaining phenazine-1-carboxylic acid content is the M18G bacterial strain fermentation liquor of 1500～1700 mg/litre.Wherein, the weight percentages of components that contains in described glycerin medium and the glycerine culture fluid is: peptone 1.8～2.2%, glycerine 1.3～1.7%, magnesium sulfate 0.05～0.1%, potassium dihydrogen phosphate 0.01～0.05%, and pH 6.8～7.2; The weight percentages of components of described glucose culture solution is: analysis for soybean powder 4.5～5.5%, glucose 3.8～4.2%, soya-bean oil 0.3～0.5%, urea 0.1～0.3%, all the other are water, and pH 6.8～7.2.
2, with the M18R inoculation on the flat board of glycerin medium, 26～30 ℃ of down activation growths 18 to 24 hours; Then the M18R bacterial strain that activates is transferred in the triangular flask that contains the glycerine culture fluid and amplifies, shaken cultivation is 10～14 hours in 26～30 ℃ shaking table, and shaking speed is 230～250 rev/mins; Continue switching 3～4 times then, carry out multistage amplification fermented and cultured in the glycerine culture fluid, the fermentation time of afterbody is 68～72 hours, and temperature and rotating speed are constant, and obtaining pyoluteorin content is the M18R bacterial strain fermentation liquor of 750～850 mg/litre.Wherein, the weight percentages of components that contains in described glycerin medium and the glycerine culture fluid is with identical described in the step 1.
3, respectively with M18G bacterial strain fermentation liquor and M18R bacterial strain fermentation liquor, technology is dried to powder routinely, is prepared into M18G dry powder and M18R dry powder.
4, with M18G dry powder and M18R dry powder blend, make wetting powder, be bactericide of the present invention, wherein, the pyoluteorin percentage by weight in phenazine-1-carboxylic acid in the M18G dry powder and the M18R dry powder is respectively 90%～10% and 10%～90%.
The microbial source bactericide that utilizes the present invention to prepare is sprayed to plant or is irritated root and use, working concentration is 15 mg/litre, mu consumption 0.3 gram can effectively be prevented and treated the mould and epidemic disease of rice sheath blight disease, cucumber fusarium axysporum, watermelon blight, gummy stem blight of melon, cotton wilt, anthracnose, damping off and the various corruption plurality of plant diseases that causes such as mould.
The control demonstration extend trial of carrying out on suburb of Shanghai, Jiangxi, Zhejiang, Jiangsu and other places to rice sheath blight disease has been obtained the control efficiency more than 80%, applies area and reaches 70,000 mu.
The bactericide of the present invention preparation kept the original safety of growth-promoting antagonistic bacterium M18, wide spectrum and with features such as Environmental compatibility is good outside, also have the following advantages:
1, the content of active component phenazine-1-carboxylic acid in the bactericide and pyoluteorin is comparatively stable.M18 compares with growth-promoting antagonistic bacterium, above-mentioned two kinds of active components of bactericide produce under controlled condition, and the active component of growth-promoting antagonistic bacterium M18 is to produce under the environmental condition when being used in the field by this viable bacteria, and its active component content is subjected to the influence of environmental condition easily.
2, the active component phenazine-1-carboxylic acid in the bactericide and the content of pyoluteorin increase substantially.Tiring of synthetic these the two kinds of antibacterial materials of growth-promoting antagonistic bacterium M18 bacterial strain is all lower, and under usual conditions, in every liter of zymotic fluid, tiring of phenazine-1-carboxylic acid is 100 to 150 milligrams, and tiring of pyoluteorin only is about 20 to 40 milligrams.Utilize the inventive method, under medium of optimizing and fermentation condition, by the synthetic phenazine-1-carboxylic acid of derivative strain M18G, the content in every liter of zymotic fluid can reach more than 1500 milligrams, compares with former technology, and fermentation titer has improved about 10 times; Simultaneously, under the fermentation condition of suitable culture base and optimization, by the synthetic pyoluteorin of derivative strain M18R, content can reach more than 750 milligrams in every liter of zymotic fluid, compares with former technology, and fermentation titer has improved about 20 times.
3, the antibacterial effect of bactericide improves exponentially.M18G dry powder and M18R dry powder are carried out composite, can produce addition, thereby the inhibitory action to the plant disease that comprises rice sheath blight disease improves 5 to 10 times than the drug effect with the agent of dosage list.The rice sheath blight is an example in case harness the river, single with the inhibitory action of phenazine-1-carboxylic acid to Rhizoctonia solani Kuhn, concentration when reaching 50% effect, be EC50, be 8.4 mg/litre, single is 53.4 mg/litre with pyoluteorin to the EC50 of Rhizoctonia solani Kuhn, carry out M18G dry powder and M18R dry powder composite, the percentage by weight that makes phenazine-1-carboxylic acid and pyoluteorin is 90%: 10% o'clock, EC50 to Rhizoctonia solani Kuhn drops to 0.74 mg/litre, has improved more than 10 times and 70 times with phenazine-1-carboxylic acid and pyoluteorin inhibition effect than single respectively.
4, the formulation of bactericide is superior, can prolong storage life.The bactericide that the present invention makes is wetting powder.It is characterized in that its active component is the microbial metabolic products that produces, rather than viable bacteria under controlled condition, be prepared into pulvis after, content of effective is more stable, can long preservation.By contrast, growth-promoting antagonistic bacterium M18 active bacteria formulation, the viable count in its zymotic fluid can reduce along with the prolongation of storage life, and storage life is shorter.
5, bactericide is easy to use.Because the bactericide that the inventive method obtains is stable wetting powder, and the on-liquid active bacteria formulation, thereby simple and easy to do aspect packing, storage, transportation and use.
6, the production cost of bactericide and use cost are low.Compare with relevant active bacteria formulation, reduce more than 50% with the production cost of dosage and use cost.
Embodiment
Below by specific embodiment technical scheme of the present invention and technique effect are further described.Following examples do not constitute limitation of the invention.
Embodiment 1:
The derivative strain M18G of growth-promoting antagonistic bacterium M18 is seeded on the flat board of glycerin medium, activate growth 24 hours down at 26 ℃, M18G bacterial strain with activation is transferred in the triangular flask that contains the glycerine culture fluid then, and shaken cultivation is 14 hours in 26 ℃ shaking table, and shaking speed is 210 rev/mins.Continue switching then, in the glycerine culture fluid, carry out 3 grades and amplify fermented and cultured.At last, be transferred to and amplify fermented and cultured in the glucose culture solution, temperature and rotating speed are constant, and fermentation time is 74 hours, obtains the M18G bacterial strain fermentation liquor.Wherein, the weight percentages of components that contains in described glycerin medium and the glycerine culture fluid is: peptone 1.8%, glycerine 1.3%, magnesium sulfate 0.07%, potassium dihydrogen phosphate 0.03%, pH 7.0; The weight percentages of components of described glucose culture solution is: analysis for soybean powder 5.0%, glucose 3.8%, soya-bean oil 0.4%, urea 0.2%, surplus are water, and pH 7.0.Measuring the 74 hours phenazine-1-carboxylic acid content in the M18G bacterial strain fermentation liquor is 1500 mg/litre.Dry technology is prepared into M18G dry powder with zymotic fluid routinely.
The derivative strain M18R of growth-promoting antagonistic bacterium M18 is seeded on the flat board of glycerin medium, activates growth 24 hours down at 26 ℃; Then the M18R bacterial strain that activates is transferred in the triangular flask that contains the glycerine culture fluid and amplifies, shaken cultivation is 14 hours in 26 ℃ shaking table, and shaking speed is 230 rev/mins.Continue switching then, carry out 3 grades and amplify fermented and cultured in the glycerine culture fluid, the fermented incubation time of the third level is extended for 72 hours, and temperature and rotating speed are constant, obtain the M18R bacterial strain fermentation liquor; Wherein, the weight percentages of components that contains in described glycerin medium and the glycerine culture fluid is: peptone 1.8%, glycerine 1.3%, magnesium sulfate 0.07%, potassium dihydrogen phosphate 0.03%, pH7.0.Measuring the 72 hours pyoluteorin content in the M18R bacterial strain fermentation liquor is 800 mg/litre.Dry technology is prepared into M18R dry powder with zymotic fluid routinely.
With M18G dry powder and M18R dry powder blend, make the bactericide of wetting powder, wherein, the pyoluteorin percentage by weight in phenazine-1-carboxylic acid in the M18G dry powder and the M18R dry powder is respectively 90% and 10%.
The bactericide for preparing under this proportioning to rice sheath blight disease, has maximum synergistic effect, improves more than 10 times than the drug effect with the agent of dosage list.
Embodiment 2:
With the derivative strain M18G inoculation of growth-promoting antagonistic bacterium M18 on the flat board of glycerin medium, activate growth 22 hours down at 28 ℃, then the M18G bacterial strain that activates is transferred in the triangular flask that contains the glycerine culture fluid and amplifies, shaken cultivation is 12 hours in 28 ℃ shaking table, and shaking speed is 220 rev/mins.Continue switching then, carry out 3 grades and amplify fermented and cultured in the glycerine culture fluid, be transferred at last and amplify fermented and cultured in the glucose culture solution, fermentation temperature and rotating speed are constant, and fermentation time is extended for 72 hours, obtains the M18G bacterial strain fermentation liquor.Wherein, the weight percentages of components that contains in described glycerin medium and the glycerine culture fluid is: peptone 2.0%, glycerine 1.7%, magnesium sulfate 0.1%, potassium dihydrogen phosphate 0.05%, pH7.2; The weight percentages of components of described glucose culture solution is: analysis for soybean powder 5.5%, glucose 4.0%, soya-bean oil 0.5%, urea 0.3%, surplus are water, and pH 7.2.Measuring the 72 hours phenazine-1-carboxylic acid content in the M18G bacterial strain fermentation liquor is 1700 mg/litre.Dry technology is prepared into M18G dry powder with zymotic fluid routinely.
The derivative strain M18R of growth-promoting antagonistic bacterium M18 is seeded on the flat board that contains glycerin medium, activate growth 22 hours down at 28 ℃, then the M18R bacterial strain that activates is transferred in the triangular flask that contains the glycerine culture fluid and amplifies, shaken cultivation is 12 hours in 28 ℃ shaking table, and shaking speed is 240 rev/mins; Continue switching then, carry out 3 grades and amplify fermented and cultured in the glycerine culture fluid, the fermentation time of the third level is 70 hours, and temperature and rotating speed are constant, obtain the M18R bacterial strain fermentation liquor.Wherein, the weight percentages of components that contains in described glycerin medium and the glycerine culture fluid is: peptone 2.0%, glycerine 1.7%, magnesium sulfate 0.1%, potassium dihydrogen phosphate 0.05%, pH 6.8.
Measuring the 70 hours pyoluteorin content in the M18R bacterial strain fermentation liquor is 850 mg/litre.The dry zymotic fluid of technology is prepared into M18R dry powder routinely.
With M18G dry powder and M18R dry powder blend, make the bactericide of wetting powder, wherein, in the M18G dry powder in contained phenazine-1-carboxylic acid and the M18R dry powder percentage by weight of contained pyoluteorin be respectively 16% and 84%.
The bactericide for preparing under this proportioning to the mould synergistic effect with maximum of melon and fruit epidemic disease, improves more than 5 times than the drug effect with the agent of dosage list.
Embodiment 3:
With the derivative strain M18G inoculation of growth-promoting antagonistic bacterium M18 on the flat board of glycerin medium, activate growth 20 hours down at 30 ℃, then the M18G bacterial strain that activates is transferred in the triangular flask that contains the glycerine culture fluid and amplifies, shaken cultivation is 12 hours in 28 ℃ shaking table, and shaking speed is 220 rev/mins; Continue switching then, in the glycerine culture fluid, carry out 3 grades and amplify fermented and cultured.At last, be transferred to and amplify fermented and cultured in the glucose culture solution, temperature and rotating speed are constant, and fermentation time is 70 hours, obtains the M18G bacterial strain fermentation liquor.Wherein, the weight percentages of components that contains in described glycerin medium and the glycerine culture fluid is: peptone 2.2%, glycerine 1.7%, magnesium sulfate 0.05%, potassium dihydrogen phosphate 0.01%, and pH 6.8; The weight percentages of components of described glucose culture solution is: analysis for soybean powder 4.5%, glucose 4.2%, soya-bean oil 0.3%, urea 0.1%, pH 6.8.Measure the 70 hours phenazine-1-carboxylic acid content in the M18G bacterial strain fermentation liquor and reach 1600 mg/litre.The technology drying is prepared into M18G dry powder with zymotic fluid routinely.
With the derivative strain M18R inoculation of growth-promoting antagonistic bacterium M18 on the flat board of glycerin medium, activate growth 18 hours down at 32 ℃, then the M18R bacterial strain that activates is transferred in the triangular flask that contains the glycerine culture fluid and amplifies, shaken cultivation is 12 hours in 32 ℃ shaking table, and shaking speed is 2250 rev/mins.Continue switching then, carry out 3 grades and amplify fermented and cultured in the glycerine culture fluid, the fermentation time of the third level is 68 hours, and temperature and rotating speed are constant, obtain the M18R bacterial strain fermentation liquor.Wherein, the weight percentages of components that contains in described glycerin medium and the glycerine culture fluid is: peptone 2.2%, glycerine 17%, magnesium sulfate 0.05%, potassium dihydrogen phosphate 0.01%, pH6.8.
Measuring the 68 hours pyoluteorin content in the M18R bacterial strain fermentation liquor is 750 mg/litre.The technology drying is prepared into M18R dry powder with zymotic fluid routinely.
With M18G dry powder and M18R dry powder blend, make Wettable powdered disinfectant, wherein, the percentage by weight of the pyoluteorin in phenazine-1-carboxylic acid in the M18G dry powder and the M18R dry powder is 50%.
The bactericide for preparing under this proportioning to various fusarium wilts, has maximum synergistic effect, improves more than 8 times than the drug effect with the agent of dosage list.
Claims (2)
1, a kind of method of utilizing antagonizing bacteria M 18 strain to prepare bactericide is characterized in that comprising the steps:
1) with the M18G inoculation on the flat board of glycerin medium, activate growth 20～24 hours down at 26～30 ℃, then the M18G bacterial strain that activates is transferred in the triangular flask that contains the glycerine culture fluid and amplifies, shaken cultivation is 10～14 hours in 26～30 ℃ shaking table, and shaking speed is 210～230 rev/mins; Continue switching 2～3 times then, in the glycerine culture fluid, carry out multistage amplification fermented and cultured; Be transferred at last and amplify fermented and cultured in the glucose culture solution, temperature and rotating speed are constant, and fermentation time is 70～74 hours, and obtaining phenazine-1-carboxylic acid content is the M18G bacterial strain fermentation liquor of 1500～1700 mg/litre; Wherein, the weight percentages of components that contains in described glycerin medium and the glycerine culture fluid is: peptone 1.8～2.2%, glycerine 1.3～1.7%, magnesium sulfate 0.05～0.1%, potassium dihydrogen phosphate 0.01～0.05%, pH 6.8～7.2; The weight percentages of components of described glucose culture solution is: analysis for soybean powder 4.5～5.5%, glucose 3.8～4.2%, soya-bean oil 0.3～0.5%, urea 0.1～0.3%, surplus are water, and pH 6.8～7.2;
2) with the M18R inoculation on the flat board of glycerin medium, 26～30 ℃ of down activation growths 18 to 24 hours; Then the M18R bacterial strain that activates is transferred in the triangular flask that contains the glycerine culture fluid and amplifies, shaken cultivation is 10～14 hours in 26～30 ℃ shaking table, and shaking speed is 230～250 rev/mins; Continue switching 3～4 times then, carry out multistage amplification fermented and cultured in the glycerine culture fluid, the fermentation time of afterbody is 68～72 hours, and temperature and rotating speed are constant, and obtaining pyoluteorin content is the M18R bacterial strain fermentation liquor of 750～850 mg/litre; Wherein, the weight percentages of components that contains in described glycerin medium and the glycerine culture fluid is with identical described in the step 1;
3) respectively with M18G bacterial strain fermentation liquor and M18R bacterial strain fermentation liquor, be dried to powder, be prepared into M18G dry powder and M18R dry powder;
4) with M18G dry powder and M18R dry powder blend, make wetting powder, be bactericide, wherein, the percentage by weight of the pyoluteorin in phenazine-1-carboxylic acid in the M18G dry powder and the M18R dry powder is respectively 90%～10% and 10%～90%.
2, a kind of application of bactericide of the method preparation of adopting claim 1, it is characterized in that being used for plant is sprayed or irritates root and use, prevent and treat the mould and mould plant disease that causes of epidemic disease of rice sheath blight disease, cucumber fusarium axysporum, watermelon blight, gummy stem blight of melon, cotton wilt, anthracnose, damping off and various corruption.
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| CN1141379C (en) * | 2000-08-31 | 2004-03-10 | 上海交通大学 | Growth-promoting antagonistic bacterium M18 for biologic agricultural chemical and its preparing process |
| CN1539959A (en) * | 2003-10-30 | 2004-10-27 | 上海交通大学 | Method for inserting gene of pseudomonas fluorescens M18 into mutation strain H18G |
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| CN100411521C (en) | 2008-08-20 |
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