CN102388921A - Bacillus subtilis Tpb55 wettable powder and application - Google Patents
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Abstract
The invention discloses wettable powder in which bacillus subtilis Tpb55 is used as active ingredients, and which consists of bacillus subtilis Tpb55 raw powder, white carbon black, KH2PO4, K2HPO4, an emulsifying agent lauryl sodium sulfate, a dispersing agent melamine-formaldehyde (MF) and a dispersing agent sodium salt of polynaphthalene sulphonic acid (NNO). The bacillus subtilis wettable powder overcomes the defects that the bacillus subtilis Tpb55 fermentation liquor and raw powder cannot be placed for a long time and cannot be used conveniently. Through experiments, the preparation has stable performance, can be stored for a long time, and has an obvious effect on brown spot and a good effect of controlling black shank. The wettable powder is expected to have wide application prospect in the field of biological control.
Description
Technical field
The present invention relates to a kind of is the wetting powder of active component with bacillus subtilis (Bacillus subtilis) Tpb55, and uses, and belongs to the biological control field.
Background technology
Fungal disease such as Alternaria alternate, balck shank distributes extensively, harm is heavy.Plantation disease-resistant variety, reinforcement cultivation management and chemical control are the major measure that China prevents and treats Alternaria alternate at present.But good resistant variety is difficult to obtain, and forfeiture easily, and the equal resistance of main breed on producing is relatively poor.Utilize plastic mulching, efficent rotation is early transplanted and suitable measures such as planting density in good time, can alleviate the occurrence degree of disease to a certain extent, but often effect is also not obvious when the disease selection pressure is big, can only bring into play booster action.Therefore, the use of chemical agent is still main means, and wherein, dimethachlon is to use medicament the most widely.The control of balck shank is main with chemical control equally, and medicament commonly used mainly contains metalaxyl series, aliette and aliette MnZn, fenaminosulf, Wakil MnZn, zineb, tmtd, mancozeb etc.Mostly these chemical agents are that phenylamide and aliette are equal to several kinds of systemic fungicides a kind of or that mechanism of action is identical, are easy to produce drug resistance, thereby cause that drug effect descends.
Visible by foregoing description, the control of black shank and rust seriously relies on chemical control, has many drawbacks.The one,, cause environmental pollution and residue of pesticide.The 2nd, develop immunity to drugs, control efficiency descends, and the control cost increases.The 3rd, the use of medicament is affected by environment big, and control efficiency is undesirable, is beneficial to the outburst of Alternaria alternate like high temperature and rainy weather, and chemical agent is difficult to use.Biological control is because its all unique advantages, becomes the field of tool development prospect, also is the focus of domestic and international researcher research.
Therefore, screening some new bactericidal agent for preventing and treating is a very urgent job.But it is high that new chemical pesticide is researched and developed not only cost, and difficulty is big, and result of use is undesirable sometimes, and biological control environmental friendliness not only, and effect is comparatively desirable sometimes, becomes the Recent study focus.The application's applicant is at patent " bacillus subtilis and the application in the fungal disease control thereof " (number of patent application: 201110093415.4; The applying date: disclose the new bacillus subtilis strain Tpb55 of a strain on April 14th, 2011); It separates acquisition from Qingzhou, Shandong vega tobacco leaf is looked unfamiliar the border; In on December 29th, 2008 by (address: Datun Road, Chaoyang District, Beijing City, Chinese microbial preservation administration committee common micro-organisms center; Institute of Microorganism, Academia Sinica) carry out preservation, deposit number is CGMCC No.2843.This bacterial strain has the disease prevention growth-promoting effect to tobacco, can be used as bio-fertilizer and biopesticide, and remarkable through verification experimental verification control Alternaria alternate effect, and control efficiency reaches 74.22%, and no poisoning produces, and the upper tobacco leaf quality is had certain improvement effect.But the former powder stability of simple bacillus subtilis Tpb55 zymotic fluid and bacillus subtilis Tpb55 is bad, can not place for a long time, also is not suitable for the production large-area applications.
Formulation processing is one of biopesticide key application technology, just with use and improve drug effect, also be convenient to standardized production and management.But biopesticide especially or body preparation must obtain stable physical behavior, the survival and control efficiency certain hour will be arranged.Since the particularity of microorganism formulation, present selectable 10 kinds of formulations such as wetting powder, wettable tablet, water dispersible granules, dried seed dressing, granule that have.
Summary of the invention
To above-mentioned prior art, bacillus subtilis Tpb55 zymotic fluid and former powder can not be placed for a long time and the shortcoming of inconvenience and use in order to overcome, and the invention provides a kind of bacillus subtilis Tpb55 wetting powder of good stability.Through experiment showed, that this preparation nature is stable, be convenient to long-term storage, and remarkable to the Alternaria alternate control efficiency, black shank there is the better prevention effect.Through retrieval, in the patent documentation that all have been applied for, all do not relate to the bibliographical information of theme of the present invention.
The present invention realizes through following technical scheme:
A kind of bacillus subtilis Tpb55 wetting powder; Form by the component of following weight portion: 40～80 parts in the former powder of bacillus subtilis Tpb55,10～50 parts of white carbons, 1～3 part of emulsifier sodium lauryl sulfate; 0.5～2 part of potassium dihydrogen phosphate; 0.5～2 part of dipotassium hydrogen phosphate, 1～3 part of dispersant NNO, 1～3 part of Dispersant MF.
The chemical composition of said dispersant NNO is: di-2-ethylhexylphosphine oxide bitter edible plant sodium sulfonate, and molecular formula: CH2 (C10H6SO3Na) 2 or C21H14O6S2Na2, CAS number: 36290-04-7, can buy from market.Proterties: the brown powdery of dispersant NNO rice, be soluble in the water of any hardness, anionic, acidproof, alkaline-resisting, heat-resisting, anti-hard water, anti-mineral salt are soluble in the water of any hardness; Dispersant NNO has superior proliferating property and protecting colloid performance, but does not have surface-active such as infiltration foaming etc., and protein and Fypro are had affinity, and fibers such as cotton, fiber crops are not had affinity.
The chemical composition of said Dispersant MF is: dispersing agent MF (methylene dimethyl sodium dinaphthalenesulfonate), and molecular formula: C23H18O6S2Na2 or (C10H8O3SCH2O) xxNa, CAS number: 9084-06-4, can buy from market.Proterties: Dispersant MF is brown ceramic powder, and is soluble in water, is prone to the moisture absorption, do not fire, and has good diffusibility and heat endurance, do not have miserable passing through property and foaming characteristic, and acid and alkali-resistance, hard water and mineral salt do not have affinity to fibers such as linen-cottons; Protein and Fypro there is affinity; Can use simultaneously with anionic and nonionic surface active agent, but can not mix use with cationic dyes or surfactant; Is good to disperseing the reducing dye particle agglomeration preventing ability of being heated than dispersant NNO.Dispersant MF belongs to anion surfactant.
Preferably, form: 60 parts in the former powder of bacillus subtilis Tpb55,32.5 parts of white carbons, 2 parts of emulsifier sodium lauryl sulfates, 0.5 part of potassium dihydrogen phosphate, 1 part of dipotassium hydrogen phosphate, 2 parts of dispersant NNO, 2 parts of Dispersant MFs by the component of following weight portion.
The bacillus subtilis Tpb55 wetting powder for preparing, bacteria containing amount (cfu/g)>=10
10, viable bacteria rate (%)>=85%, loss on drying (%)≤3.5, suspensibility＞80%, wetting time (s)＜30s, fineness (through 50 μ m sieve)＞98%, pH value 6-8, bin stability: store 12 months viable bacteria rate＞85% for 20 ℃.
The examinaing method of sample and data are announced as follows:
Capillary mensuration:
1. take by weighing the wetting powder powder that 0.05g makes, join in the 50g water, be stirred to evenly.
2. surface tension instrument is connected, regulate liquid level, make the capillary exit and the water surface tangent, open piston and bleed.It is 6s～10s that adjusting makes the formation time of single isolated bubbles.The maximum pressure differential of record absolute value three times, is averaged totally.
3. by r=kp, calculate surface tension coefficient.
The surface tension test result is as shown in table 1, and the maximum pressure difference is 0.679kPa, surface tension coefficient γ=k* Δ P=0.07469N/m, and instrumental constant is k=0.11.
Table 1 surface tension test result
| Number of times | 1 | 2 | 3 | Mean value |
| Maximum pressure poor (kPa) | 0.677 | 0.679 | 0.681 | 0.679 |
The mensuration of blade face hold-up:
1. the blade of intercepting 1cm * 1cm on fresh pears leaf is weighed and is designated as m1 with ten thousand/balance.
2. in the one thousandth dilution of wetting powder, soak 15s～30s, take out blade, and keep the water on the blade face not drip automatically.Weigh once more and be designated as m2.
3. triplicate, and average.
It is as shown in table 2 that the blade hold-up is measured the result, and average hold-up is 0.0020g.
Table 2 blade hold-up
Dispersed mensuration:
1. take by weighing the 0.5g wetting powder, join in the 200g distilled water, stir 1min with glass bar.
2. observe the record phenomenon after leaving standstill about 5min.
Measure the result: better dispersed, beaker bottom has a small amount of deposition.
Wetting experiment:
1. take by weighing the 0.5g wetting powder, and use the 250ml beaker, measure the 200ml deionized water.
2. pour into rapidly in the deionized water wetting powder is disposable, use stopwatch to note the time that wetting powder soaked fully simultaneously and be t.
Wetting time: t=221 second.
Particle size determination: D
10=3.46um, D
50=9.20um, D
90=25.40um, D
Av=9.20um.
Bacillus subtilis Tpb55 content is 100～1,000 hundred million living spores/grams in the former powder of said bacillus subtilis Tpb55.
The former powder of said bacillus subtilis Tpb55 prepares through following method:
(1) bacillus subtilis Bacillus subtilis Tpb55 test tube kind is inoculated in broth bouillon (be conventional medium, its prescription repeats no more at this for known) and shakes in the bottle shaken cultivation to logarithmic phase;
(2) with the volume ratio 10% inoculum concentration access 30L seeding tank of above-mentioned cultured bacterial classification by the seed tank culture base, be cultured to exponential phase, the used culture medium prescription of seeding tank is: glucose 10g/L, peptone 3g/L, MgSO
40.5g/L, yeast extract 1.5g/L, beef extract 1.5g/L; Condition of culture: dissolved oxygen amount is 15-18%, mixing speed 200rpm, 31 ℃ of fermentation temperatures;
10% the inoculum concentration of (3) seed liquor being produced jar medium inserts to produce and jar cultivates, and produces a jar culture medium prescription and is: corn flour 20g/L, soybean cake powder 50g/L, MgSO
47H
2O 0.5g/L, MnSO
4H
2O 10g/L; Condition of culture: dissolved oxygen amount is 15-18%, mixing speed 200rpm, 31 ℃ of fermentation temperatures; Being cultured to final biomass is 109cfu/g, and the gemma formation rate reaches more than 97%, and fermentation is accomplished;
(4) after fermentation was accomplished, culture fluid went out jar, filtered, and atomized drying promptly gets former powder.
The preparation method of said bacillus subtilis Tpb55 wetting powder is: each component is mixed, and the control processing temperature is pulverized in airslide disintegrating mill at 0～50 ℃, crosses 150 mesh sieves, promptly gets.
Said bacillus subtilis Tpb55 wetting powder can be used to prevent and treat Alternaria alternate, balck shank.During application, method for using is: during the control Alternaria alternate, can use 800～1000 times of liquid foliar sprays of bacillus subtilis Tpb55 wetting powder.During the control black shank, mix soil (1 weight portion preparation and 99 weight portions soil) with 1% of bacillus subtilis Tpb55 wetting powder amount of formulation during transplanting and spread manuer in holes, the 3-5 gram is executed in every strain; After the transplanting, 800-1000 times of liquid irrigating root of available bacillus subtilis Tpb55 wetting powder, every strain 30-50 milliliter.
In the foregoing description, said Alternaria alternate is caused by alternaric bacteria tobacco specialized form (Altternaria altternata f.sp.nicotianae).Said black shank is caused by phytophthora parasitica oronoco (Phytophthora parasitica var.nicotianae).
Bacillus subtilis Tpb55 involved in the present invention, its isolation identification, cultural method, biological nature, fermentation condition etc., open; Be documented in Chinese document " tobacco leaf encloses the evaluation and the bacteriostatic activity thereof of bacterium Tpb55 bacterial strain, Chinese biological control, 2008; 24 (1): 63-68 " and " screening of Alternaria alternate antagonistic bacterium Tpb55 conditions of flask fermentation; Chinese tobacco science 2009,30 (1): 54-57 ", repeat no more at this.
The present invention has overcome bacillus subtilis Tpb55 zymotic fluid and former powder can not be placed for a long time and the shortcoming of inconvenience and use; Through experiment showed, that preparation nature of the present invention is stable, is convenient to long-term storage; And remarkable to the Alternaria alternate control efficiency, black shank there is the better prevention effect.Can predict, the present invention will have wide application prospect in the biological control field.
Embodiment
Below in conjunction with embodiment the present invention is further described.
Embodiment 1 preparation bacillus subtilis Tpb55 wetting powder
Prescription is: 40 kilograms in the former powder of bacillus subtilis Tpb55, and 50 kilograms of white carbons, 3 kilograms of lauryl sodium sulfate, 1 kilogram of potassium dihydrogen phosphate, 2 kilograms of dipotassium hydrogen phosphates, NNO are 2 kilograms, MF is 2 kilograms.
The preparation method: above-mentioned raw materials is mixed, and the control processing temperature is pulverized in airslide disintegrating mill about 25 ℃, crosses 150 mesh sieves, promptly gets.
The former powder of said bacillus subtilis Tpb55 prepares (down together) through following method:
(1) bacillus subtilis Bacillus subtilis Tpb55 test tube kind is inoculated in broth bouillon and shakes in the bottle, shaken cultivation is to logarithmic phase;
(2) with the volume ratio 10% inoculum concentration access 30L seeding tank of above-mentioned cultured bacterial classification by the seed tank culture base, be cultured to exponential phase, the used culture medium prescription of seeding tank is: glucose 10g/L, peptone 3g/L, MgSO
40.5g/L, yeast extract 1.5g/L, beef extract 1.5g/L; Condition of culture: dissolved oxygen amount is 15-18%, mixing speed 200rpm, 31 ℃ of fermentation temperatures;
10% the inoculum concentration of (3) seed liquor being produced jar medium inserts to produce and jar cultivates, and produces a jar culture medium prescription and is: corn flour 20g/L, soybean cake powder 50g/L, MgSO
47H
2O 0.5g/L, MnSO
4H
2O 10g/L; Condition of culture: dissolved oxygen amount is 15-18%, mixing speed 200rpm, 31 ℃ of fermentation temperatures; Being cultured to final biomass is 10
9Cfu/g, the gemma formation rate reaches more than 97%, and fermentation is accomplished;
(4) after fermentation was accomplished, culture fluid went out jar, filtered, and atomized drying promptly gets former powder.
Embodiment 2 preparation bacillus subtilis Tpb55 wetting powders
Prescription is: 50 kilograms in the former powder of bacillus subtilis Tpb55, and 40 kilograms of white carbons, 3 kilograms of lauryl sodium sulfate, 0.5 kilogram of potassium dihydrogen phosphate, 0.5 kilogram of dipotassium hydrogen phosphate, NNO are 3 kilograms, MF is 3 kilograms.
The preparation method is with embodiment 1.
Embodiment 3 preparation bacillus subtilis Tpb55 wetting powders
Prescription is: 60 kilograms in the former powder of bacillus subtilis Tpb55, and 32.5 kilograms of white carbons, 2 kilograms of lauryl sodium sulfate, 0.5 kilogram of potassium dihydrogen phosphate, 1 kilogram of dipotassium hydrogen phosphate, NNO are 2 kilograms, MF is 2 kilograms.
The preparation method is with embodiment 1.
Embodiment 4 preparation bacillus subtilis Tpb55 wetting powders
Prescription is: 80 kilograms in the former powder of bacillus subtilis Tpb55, and 11.5 kilograms of white carbons, 3 kilograms of lauryl sodium sulfate, 0.5 kilogram of potassium dihydrogen phosphate, 1 kilogram of dipotassium hydrogen phosphate, NNO are 2 kilograms, MF is 2 kilograms.
The preparation method is with embodiment 1.
The test of embodiment 5 bacillus subtilis Tpb55 wetting powders control Alternaria alternate
Bacillus subtilis Tpb55 wetting powder (embodiment 1 preparation) control Alternaria alternate test arrangement is little one hundred houses vega in the Luliang, Yunnan Province; The test tobacco bred is K326; The field trial time is 3 days-July 28 July in 2008; The test spraying medicine concentration is respectively 150,180 and 210 gram/hectares, prevent and treat medicament dimethachlon (Lucheng City, Zhejiang plant protection laboratory) with conventional rust and contrast as medicament, with clear water as experimental control.Experimental result shows; Tpb55 wetting powder 180 gram/hectare preventive effects are preferably 94.45%; Through the new multipole difference analysis of Dancan; Preventive effect under 5% significance level, handles with other concentration and the contrast dimethachlon is handled significant difference, is illustrated under the 180 gram/hectare dispenser dosage, and preventive effect significantly is superior to other concentration and handles (table 3).
The different spraying medicine concentration efficiency test of table 3 bacillus subtilis Tpb55 result
| Handle | Disease index before the dispenser | 10d disease index after the secondary dispenser | Preventive effect/% |
| 150 gram/hectare Tpb55 wetting powders | 2.01 | ?3.9 | 82.78Ab |
| 180 gram/hectare Tpb55 wetting powders | 6.05 | ?7.32 | 94.45Aa |
| 210 gram/hectare Tpb55 wetting powders | 8.21 | ?10.46 | 83.65Ab |
| 125 gram/hectare dimethachlon wetting powders | 6.76 | ?9.40 | 79.85Ab |
| The clear water contrast | 9.53 | ?24.72 |
Through the yield and quality data analysis; During 3 variable concentrations of bacillus subtilis Tpb55 wetting powder are handled; 210 gram/hectares are handled with clear water contrast (CK) and are compared, increase of production 14.37%, and last medium grade cigarette ratio improves 4.37%; First-class cigarette ratio improves 18.36%, and the output value increases by 15.69% (table 4).
Table 4 bacillus subtilis Tpb55 is different, and spraying medicine concentration influences the result to the tobacco production and value amount
Bacillus subtilis Tpb55 prevents and treats Alternaria alternate, and under 180 gram/hectare spraying medicine concentrations, control efficiency is best, but under 210 gram/hectare spraying medicine concentrations, to yield of tobacco and quality, tangible promotion and raising is arranged all, and does not find have poisoning to produce.The result shows that under the suitable spraying medicine concentration, bacillus subtilis has the better prevention effect to Alternaria alternate, and can improve by a relatively large margin yield of tobacco and quality.Bacillus subtilis Tpb55 wetting powder is a microorganism formulation, and is environmentally safe pollution-free to crop safety, considers from preventive effect with to the influence of yield and quality, has broad application prospects.
The test of embodiment 6 bacillus subtilis Tpb55 wetting powders control black shank
Control black shank test arrangement is in Tobacco Institute, Chinese Academy of Agricultural Science Jimo proving ground for bacillus subtilis Tpb55 wetting powder (embodiment 1 preparation), and be 20 days-August 15 July in 2009 date of test.Experimental concentration is 150,180 and 210 gram/hectares, and 58% first frost mancozeb wettable powder 100 gram/mus (Jiangsu Baoling Chemical Co., Ltd) are the medicament contrast, and blank does not use any medicament.Repeat every sub-district area 3,0m2 four times.Insecticide-applying way is for irritating root.Investigation before the medication on July 20, July 28 was investigated once, and last investigation was carried out August 15.Under this experimental condition and experimental concentration, not finding has poisoning to tobacco.Last investigation result shows; During bacillus subtilis Tpb55 wetting powder variable concentrations is handled; Remarkable with 210 gram/mus to the control efficiency of black shank; Preventive effect to black shank is 62.40%, with contrast medicament 58% metalaxyl-mn-zn (59.80%) difference not significantly (as shown in table 5).
Table 5 bacillus subtilis Tpb55 wetting powder is to the control efficiency of black shank
Claims (6)
1. a bacillus subtilis Tpb55 wetting powder is characterized in that, is made up of the component of following weight portion: 40～80 parts in the former powder of bacillus subtilis Tpb55; 10～50 parts of white carbons; 1～3 part of lauryl sodium sulfate, 0.5～2 part of potassium dihydrogen phosphate, 0.5～2 part of dipotassium hydrogen phosphate; 1～3 part of dispersant NNO, 1～3 part of Dispersant MF.
2. a kind of bacillus subtilis Tpb55 wetting powder according to claim 1; It is characterized in that: form: 60 parts in the former powder of bacillus subtilis Tpb55,32.5 parts of white carbons, 2 parts of emulsifier sodium lauryl sulfates by the component of following weight portion; 0.5 part of potassium dihydrogen phosphate; 1 part of dipotassium hydrogen phosphate, 2 parts of dispersant NNO, 2 parts of Dispersant MFs.
3. a kind of bacillus subtilis Tpb55 wetting powder according to claim 1 is characterized in that: bacillus subtilis Tpb55 content is 100～1,000 hundred million living spores/grams in the former powder of said bacillus subtilis Tpb55.
4. a kind of bacillus subtilis Tpb55 wetting powder according to claim 1 is characterized in that: the former powder of said bacillus subtilis Tpb55 prepares through following method:
(1) bacillus subtilis Bacillus subtilis Tpb55 test tube kind is inoculated in broth bouillon and shakes in the bottle, shaken cultivation is to logarithmic phase;
(2) with the volume ratio 10% inoculum concentration access 30L seeding tank of above-mentioned cultured bacterial classification by the seed tank culture base, be cultured to exponential phase, the used culture medium prescription of seeding tank is: glucose 10g/L, peptone 3g/L, MgSO
40.5g/L, yeast extract 1.5g/L, beef extract 1.5g/L; Condition of culture: dissolved oxygen amount is 15-18%, mixing speed 200rpm, 31 ℃ of fermentation temperatures;
10% the inoculum concentration of (3) seed liquor being produced jar medium inserts to produce and jar cultivates, and produces a jar culture medium prescription and is: corn flour 20g/L, soybean cake powder 50g/L, MgSO
47H
2O 0.5g/L, MnSO
4H
2O 10g/L; Condition of culture: dissolved oxygen amount is 15-18%, mixing speed 200rpm, 31 ℃ of fermentation temperatures; Being cultured to final biomass is 10
9Cfu/g, the gemma formation rate reaches more than 97%, and fermentation is accomplished;
(4) after fermentation was accomplished, culture fluid went out jar, filtered, and atomized drying promptly gets former powder.
5. the preparation method of each described a kind of bacillus subtilis Tpb55 wetting powder in the claim 1～4 is characterized in that: each component is mixed, control processing temperature at 0～50 ℃, in airslide disintegrating mill, pulverize, cross 150 mesh sieves, promptly get.
6. the application of each described a kind of bacillus subtilis Tpb55 wetting powder in Alternaria alternate, balck shank control in the claim 1～4.
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| CN109997450A (en) * | 2019-04-04 | 2019-07-12 | 青岛农业大学 | A kind of production method promoting cigarette quality using addition probiotics |
| CN110106115A (en) * | 2019-05-13 | 2019-08-09 | 中国农业科学院烟草研究所 | A kind of bacillus subtilis synergist and its preparing the application in bacillus subtilis microbial agent |
| CN110106115B (en) * | 2019-05-13 | 2020-11-06 | 中国农业科学院烟草研究所 | Bacillus subtilis synergist and application thereof in preparation of bacillus subtilis microbial inoculum |
| CN110564655A (en) * | 2019-10-17 | 2019-12-13 | 中国烟草总公司陕西省公司 | Preparation and application of bacillus subtilis SL-19 microbial inoculum for biological control of alternaria alternate |
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| CN113604394B (en) * | 2021-08-19 | 2024-04-09 | 山东京青农业科技有限公司 | Bacillus subtilis water dispersible granule and preparation method and application thereof |
| CN117511749A (en) * | 2023-10-31 | 2024-02-06 | 中国农业科学院烟草研究所(中国烟草总公司青州烟草研究所) | Solid state fermentation method for trichoderma asperellum HG1 and bacillus subtilis Tpb55 and application thereof |
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