CN104046576A - Culture method of bacillus amyloliquefaciens and application - Google Patents
Culture method of bacillus amyloliquefaciens and application Download PDFInfo
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- CN104046576A CN104046576A CN201310083560.3A CN201310083560A CN104046576A CN 104046576 A CN104046576 A CN 104046576A CN 201310083560 A CN201310083560 A CN 201310083560A CN 104046576 A CN104046576 A CN 104046576A
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Abstract
The invention provides a culture method of bacillus amyloliquefaciens, comprising the following steps: (1) solid plate culture; (2) liquid shake-flask culture; (3) preparation of a seed solution; (4) preparation of a fermentation broth; (5) fermentation broth condensation; (6) spray drying of a concentrate; and (7) preparation of 20 billion living spore/gram of a bacillus amyloliquefaciens wettable powder. The bacillus amyloliquefaciens can be used for preventing and curing bacterial wilt of crops such as tobacco, tomato, capsicum and the like, is applied in agriculture, has high safety for people and livestock, has good environmental compatibility, is not easy to generate resistance to pests, has cause little pollution.
Description
Technical field
The invention belongs to microorganism field, be specifically related to a kind of cultural method and purposes of bacillus amyloliquefaciens.
Background technology
Bacterial wilt is by Ralstonia solanacearum, claims again the microbial worldwide bacillary soil-borne disease of eggplant material Lei Er.Pathogenic bacteria is very easily invaded people plant from root system, stem's wound, and then endangers whole vascular system and cause that plant wither is withered.This pathogenic bacteria has host range and regional distribution widely, can infect more than 50 300 various plants of planting, comprise many cultivated plants that Important Economic is worth that have, wherein tobacco, potato, peanut, tomato, corn, sweet potato, eggplant, capsicum, banana, Cao Mu, mulberry, Horsetail Beefwood, Fructus oleae europaeae, comfrey only and the hazard of plant such as some valuable medicinals and flowers the most serious, to crop production, bring tremendous economic loss.Tobacco particularly, China is tobacco planting and consumption big country, is also one of most important economic plants.
For a long time, the bacterial disease control of the crops such as tobacco bacterial wilt all be take the antibiotics agricultural chemicals such as agricultural streptomycin as main, this class agricultural chemicals preventive effect is unstable, take effect slow and be difficult to the preventive effect that reaches desirable, the more important thing is, along with the continuous lifting of human security understanding, China began to forbid that various antibioticses and severe toxicity are for humans and animals from 2008, hypertoxic type pesticides application is in agriculture production, thereby caused the famine of the crop bacterial disease agricultural chemicals such as control tobacco.In microbial control, become gradually today of control of plant disease study hotspot, research and develop and the bacterial disease that utilizes microbial pesticide to prevent and treat the crops such as tobacco is undoubtedly best outlet.
Summary of the invention
The technical problem to be solved in the present invention be to provide a kind of can be for preventing and treating the cultural method of the bacillus amyloliquefaciens of tobacco bacterial wilt.
In order to solve the problems of the technologies described above, the invention provides a kind of bacillus amyloliquefaciens, its preservation name is called: Baeillus amgloliquefaciens.
The present invention's separation from flue-cured tobacco blade face, the Qinling Mountains obtains a strain advantage genus bacillus as test strain, it has been carried out to 16SrRNA, atPA gene sequencing, qualification result shows, this bacterium belongs to bacillus amyloliquefaciens, and its Classification And Nomenclature is Bacillus amgloliquefaciens.
Further, morphology and the physiology nature and characteristic of bacillus amyloliquefaciens (Bacillus amgloliquefaciens) are: thalline is shaft-like, Gram-positive, the ellipse circular of gemma, middle life or end are raw, do not expand, size is 0.7-0.8 μ m, long 2-3 μ m..Conventional Physiology and biochemistry experiment shows, is aerobic bacteria, catalase, and oxydase, VP test is positive, in 7% NaCl, grows, PH5.7 growth, hydrolyzed starch, reduction nitrate becomes nitrite.
The cultural method that the invention provides above-mentioned bacillus amyloliquefaciens, comprises the steps:
(1) solid plate is cultivated: with transfering loop, dip bacillus amyloliquefaciens streak inoculation on NA solid medium, in 30 ℃ of cultivations 72 hours, obtain bacterial classification I, described bacterial classification I can be 4 ℃ of following preservations three months.
(2) liquid shaking bottle is cultivated: get a ring bacterial classification I access and be equipped with in the triangular flask of liquid NA substratum, in 30 ℃, 200r/min, cultivate 24 hours, obtain bacterial classification II.
(3) preparation of seed liquor: according to grain weight requirement, select suitable triangular flask, according to the ratio of 0.05%-0.5%, to being equipped with in the triangular flask of liquid NA substratum, access bacterial classification II, in 30 ℃, 200r/min cultivates 24 hours or the longer time, the spore count of seed liquor reaches 0.6 * 10
9left and right, is seed liquor.
(4) preparation of fermented liquid: select 50L fermentor tank, after disappearing, sky adds self-control substratum, coefficient is 0.6, and charging 30L, with steam real disappearing after 0.5 hour under 121 ℃ of conditions, 30 ± 1 ℃ of controlled fermentation temperature, ventilation 20-40L/min, rotating speed 150-200r/min, inoculum size 0.05%-1%, fermentation 40-48 hour, fermented liquid spore amount is 2 * 10
9during individual spore/ml alive, put tank, obtain bacillus amyloliquefaciens fermented liquid.
The component of wherein said self-control substratum and weight percent thereof are: yeast powder 1%-3%, and Semen Maydis powder 1%-5%, glucose 0.8%-3%, calcium chloride 0.1%-1%, bean cake powder 1%-4%, defoamer 0.1-0.3%, all the other are water.
(5) fermented liquid is concentrated: Ceramics membrane filtration is concentrated, membrane pore size 0.05-0.2 μ m, and membrane area 0.2-0.5 ㎡, carries out membrane filtration by above-mentioned fermented liquid 27kg, obtains concentrated solution 7.0kg.
(6) concentrated solution spraying is dry: selecting dehydrating amount per hour is the centrifugal spray drying tower of 3kg, concentrated solution 7.0kg is sprayed dry, inlet temperature 120-220 ℃, air outlet temperature 60-90 ℃, after approximately three hours, can obtain 370 grams, dry powder, be and separate starch bacillus subtilis starter medicine, its spore amount is 2 * 10
11individual spore alive/gram.
(7) 200 hundred million preparations of living spore/gram bacillus amyloliquefaciens wettable powder: the female medicine of above-mentioned bacillus amyloliquefaciens and various auxiliary agent are mixed, obtain 20,000,000,000 spores/gram the separate starch bud bacillus wettable powder products of living after comminution by gas stream.
The invention has the beneficial effects as follows: bacillus amyloliquefaciens, can produce gemma, there is extremely strong resistance, be applied to agricultural high to person poultry safety's property, Environmental compatibility good, harmful organism is difficult for developing immunity to drugs, less pollution.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.
Embodiment 1: the cultural method of bacillus amyloliquefaciens
The acquisition of 1.1 bacillus amyloliquefaciens seed liquor:
1.1.1 bacillus amyloliquefaciens actication of culture: use transfering loop picking
Three ring bacillus amyloliquefaciens freeze-drying lactobacillus, in 250ml triangular flask, adopt NA substratum, cultivate 18h for 30 ℃.
NA culture medium prescription: 10 grams of peptones, 3 grams of extractum carniss, Nacl5 gram, agar
15 grams, distilled water 1000ml, pH value 7.3.
1.1.2 method of scoring solid plate is cultivated: 30 ℃ of cultivations on NA culture medium flat plate, training
Support 72-120 hour, grow single bacterium colony.
1.1.3 test tube slant is cultivated: NA test tube slant, the above-mentioned single bacterium colony of picking is connected to test tube
Inclined-plane, cultivates 72-96 hour for 30 ℃, until spore is scattered.
From above-mentioned test tube slant, select good bacterial classification, with sterilized water, test tube slant seed is rinsed, be connected in the 750mL triangular flask that NA substratum is housed, in 30 ℃, shaking speed 200r/min, cultivate 24-26 hour, thalline content reaches 0.6 * 10
9individual/during mL left and right, to can be used as seed liquor.
The preparation of 1.2 bacillus amyloliquefaciens fermented liquids:
Bacillus amyloliquefaciens fermention medium: yeast powder 2.8%, W-Gum 1.4%, glucose 1.0%, calcium chloride 0.3%, bean cake powder 3.0%, defoamer 0.14%, all the other are water, regulate PH 8.1～8.3.
50 liters of full automatic control stainless steel fermentor tanks carry out disinfection: with 121 ℃ of sterilizations of steam
1 hour.
To fermentor tank, feed: coefficient is 0.6, add 840 grams of yeast powders, corn forms sediment
420 grams, powder, 300 grams of glucose, 90 grams, calcium chloride, 900 grams of bean cake powders, 42 grams of defoamers, add 22 liters, tap water, pass into steam, and in 121 ℃ of sterilizations 1.0 hours, volume reached 30 liters, looks into without miscellaneous bacteria.
Fermentation optimum parameter: 30 ± 1 ℃ of leavening temperatures, initial p H value is 8.1～8.3, logical
Tolerance is 1 ︰ 1, mixing speed 200r/min, and inoculum size is 1%, is warming up to 30 ℃, fermentation 46-48 hour, spore amount is 0.6 * 10
9during/mL, arrive terminal, obtain the fermented liquid of 26.5 kilograms.
Concentrating of 1.3 fermented liquids:
The material fluid bath that the fermented liquid of 26.5kg is packed into membrane separation unit, adopting aperture is the ceramic membrane of 0.1 μ m, and membrane area is 0.25 ㎡, and ON cycle pump carries out separation, circulates and obtains 7.0kg concentrated solution after 3 hours.
1.4 concentrated solution sprayings are dry:
Adopt the Centrafugal spray drying tower that dehydrating amount is 3kg/h, inlet temperature 140
℃, 70 ℃ of air outlet temperatures, spray 7.0kg bacillus amyloliquefaciens concentrated solution dry, obtain the female powder of 375g bacillus amyloliquefaciens through three hours, and spore amount is 3.4 * 10 after testing
11live spore/gram the female medicine of bacillus amyloliquefaciens.
Embodiment 2: bacillus amyloliquefaciens indoor antibacterial test
The pathogenic bacteria of selecting has: tobacco ralstonia solanacearum, capsicum scab bacterium, citrus scab bacterium, soft rot of cabbage bacterium, rice leaf spot bacteria, Fusarium Solani, Potato Ring Rot, Prospect on Kiwifruit Bacterial Canker bacterium, common bacterial blight of bean bacterium, cucumber bacterial angular leaf spot bacterium.
Selecting mass volume ratio is 100mg/L, 10mg/L, and the bacillus amyloliquefaciens of 1mg/L carries out bacteriostatic test to above-mentioned pathogenic bacteria respectively, and test-results is as following table 1:
The antibacterial stave 1 of bacillus amyloliquefaciens
Note: +++: inhibiting rate (preventive effect)=75%-100% ++: inhibiting rate (preventive effect)=45%-75%
+: inhibiting rate (preventive effect)=30%-45%-: inhibiting rate (preventive effect) < 30%
Embodiment 3: bacillus amyloliquefaciens carries out Toxicity Determination to tobacco ralstonia solanacearum (pseudomonas solanacearum)
Select Vetstrep (treptomycin sulfate) for contrast medicine, adopt Oxford agar diffusion method to measure.
72% Vetstrep (treptomycin sulfate), North China Pharmaceutical Group Preparation Co., Ltd. provides.This medicament is dissolved and makes 1.00 * 10 with sterilized water
4the storing solution of mg/L effective concentration, is placed in 4 ℃ of Refrigerator stores standby.
Tobacco ralstonia solanacearum (Pseudomonas solanacearum), disease system is planted by China Agricultural University's agronomy and Biotechnology Institute to be provided.
LB substratum: Tryptones 10g, yeast extract 5g, NaCL 5g, agar powder 12g, distilled water 1000mL.
YM liquid nutrient medium: N.F,USP MANNITOL 14g, yeast extract 5g, MgSO
40.1g, K
2hPO
40.4g, NaCL 0.4g, CaCL
20.01g.
By bacillus amyloliquefaciens on LB flat board 28 ℃ activation 48h after, with 0.85% physiological saline, making concentration is 10
9the bacteria suspension of cfu/ml, then the ratio that the volume ratio of take is 1% is inoculated in YM liquid nutrient medium, nutrient solution is placed on to 30 ℃, on the constant-temperature table of 150rpm, shake training 96h, finally will shake training liquid with 10000rpm high speed centrifugation 10min, remove thalline, what the supernatant liquor obtaining was bacillus amyloliquefaciens shakes training liquid.
Tobacco ralstonia solanacearum is seeded in after the dull and stereotyped upper 28 ℃ of activation 48h of LB, and with 0.85% physiological saline, making concentration is 10
9the bacteria suspension of cfu/ml, standby.
Two Oxford cups that diameter is 1cm of symmetrical insertion on LB culture medium flat plate then add respectively the bacillus amyloliquefaciens of 150 μ l to shake training liquid and Vetstrep liquid, standing 24h in the cup of Oxford.After 0.05% water agar heating and melting, when its temperature is down to 45 ℃～50 ℃, the ratio that according to volume ratio is 1 ︰ 50 is sneaked into tobacco ralstonia solanacearum bacteria suspension in water agar, after concussion evenly, tiling is to the LB culture medium flat plate of the 5ml/ ware through standing processing, form upper strata and connect bacterium layer, finally flat board is placed in to dark culturing at 28 ℃.Every processing repeats 4 times and measures.
Cultivate after 24h, investigation Oxford cup is the size of inhibition zone around, calculates the mean value repeating for 4 times.
Result is as shown in table 2, and the 96h of bacillus amyloliquefaciens shakes training liquid has obvious restraining effect to tobacco ralstonia solanacearum, and average diameter of inhibition zone is 25.4mm, and the contrast medicament Vetstrep that concentration is 50mg/L is 26.9mm to the inhibition zone of tobacco ralstonia solanacearum.Show that bacillus amyloliquefaciens has the making of significantly facing upward to use to tobacco ralstonia solanacearum.
The restraining effect table 2 of bacillus amyloliquefaciens to tobacco ralstonia solanacearum
Embodiment 4: different chemical control tobacco bacterial wilt field control effectiveness tests
Select three kinds of conventional medicaments: blue or green withered spirit, Vetstrep, dazomet and bacillus amyloliquefaciens carry out field control effectiveness test to tobacco bacterial wilt, result is as shown in table 3, show that bacillus amyloliquefaciens (300 grams/acre) preventive effect is up to 84.78%, dazomet and blue or green withered spirit are respectively 76.05% and 75.53%, and Vetstrep is minimum is 68.26%.
4 kinds of different chemical control tobacco bacterial wilt field control effectiveness test result tables 3
Embodiment 5: the prevention effect of bacillus amyloliquefaciens different times medication to tobacco bacterial wilt
Test
Bacillus amyloliquefaciens is watered to 4000 times of dilutions, standby.
Choose 9 groups of identical tobaccos, do respectively following processing: first group at every strain in seedling stage diluent liquid irrigating root of 400ml; Second group at the latter 10 days diluent liquid irrigating roots with 400ml of transplanting; The 3rd group at the latter 30 days diluent liquid irrigating roots with 400ml of transplanting; The 4th group at the latter 50 days diluent liquid irrigating roots with 400ml of transplanting; BSA is not at seedling stage and latter 10 days each diluent liquid irrigating roots with 400ml of transplanting; The 6th group respectively at seedling stage and latter 30 days each diluent liquid irrigating roots with 400ml of transplanting; The 7th group respectively at latter 10 days of transplanting and latter 30 days each diluent liquid irrigating roots with 400ml of transplanting; The 8th group respectively in seedling stage, each is with the diluent liquid irrigating root of 400ml to transplant latter 10 days and transplant latter 30 days; The 9th group is control group, not dispenser liquid; Every group of experiment repeats 3 times.Then after binding, pinching 25 days, observe respectively its sick pearl situation while adopting roasting end, and record.
Experimental result is as table 4, and test shows, control is more early better period.
Each processes preventive effect statistics table 4
Note: in table, each data are the mean value repeating for 3 times; Small letter English alphabet represents the significance of difference of 5% level.
Claims (5)
1. a bacillus amyloliquefaciens, preservation name is called: Bacillus amyloliquefaciens, it is characterized in that, morphology and the physiology nature and characteristic of bacillus amyloliquefaciens (Bacillus amgloliquefaciens) are: thalline is shaft-like, Gram-positive, the ellipse circular of gemma, middle life or end are raw, do not expand, size is 0.7-0.8 μ m, long 2-3 μ m.Conventional Physiology and biochemistry experiment shows, is aerobic bacteria, catalase, and oxydase, VP test is positive, in 7% NaCl, grows, PH5.7 growth, hydrolyzed starch, reduction nitrate becomes nitrite;
Further, the cultural method of bacillus amyloliquefaciens comprises the following steps:
(1) solid plate is cultivated: with transfering loop, dip bacillus amyloliquefaciens streak inoculation on NA solid medium, in 30 ℃ of cultivations 72 hours, obtain bacterial classification I.
(2) liquid shaking bottle is cultivated: get a ring bacterial classification I access and be equipped with in the triangular flask of liquid NA substratum, in 30 ℃, 200r/min, cultivate 24 hours, obtain bacterial classification II.
(3) preparation of seed liquor: according to grain weight requirement, select suitable triangular flask, according to the ratio of 0.05%-0.5%, to being equipped with in the triangular flask of liquid NA substratum, access bacterial classification II, in 30 ℃, 200r/min cultivates 24 hours or the longer time, the spore count of seed liquor reaches 0.6 * 10
9left and right, is seed liquor.
(4) preparation of fermented liquid: select 50L fermentor tank, after disappearing, sky adds self-control substratum, coefficient is 0.6, and charging 30L, with steam real disappearing after 0.5-1 hour under 121 ℃ of conditions, 30 ± 1 ℃ of controlled fermentation temperature, ventilation 20-40L/min, rotating speed 150-200r/min, inoculum size 0.05%-1%, fermentation 40-48 hour, fermented liquid spore amount is 2 * 10
9during individual spore/ml alive, put tank, obtain bacillus amyloliquefaciens fermented liquid.
(5) fermented liquid is concentrated: Ceramics membrane filtration is concentrated, membrane pore size 0.05-0.2 μ m, and membrane area 0.2-0.5 ㎡, carries out membrane filtration by above-mentioned fermented liquid 27kg, obtains concentrated solution 7.0kg.
(6) concentrated solution spraying is dry: selecting dehydrating amount per hour is the centrifugal spray drying tower of 3kg, concentrated solution 7.0kg is sprayed dry, inlet temperature 120-220 ℃, air outlet temperature 60-90 ℃, after approximately three hours, can obtain 370 grams, dry powder, be and separate starch bacillus subtilis starter medicine, its spore amount is about 2 * 10
11individual spore alive/gram.
(7) 200 hundred million preparations of living spore/gram bacillus amyloliquefaciens wettable powder: the female medicine of above-mentioned bacillus amyloliquefaciens and various auxiliary agent are mixed, obtain 20,000,000,000 spores/gram the separate starch bud bacillus wettable powder products of living after comminution by gas stream.
2. bacillus amyloliquefaciens according to claim 1, it is characterized in that: component and the weight percent thereof of described self-control substratum are: yeast powder 1%-3%, Semen Maydis powder 1%-5%, glucose 0.8%-3%, calcium chloride 0.1%-1%, bean cake powder 1%-4%, defoamer 0.1%-0.3%, all the other are water, regulate PH 8.1～8.3.
3. bacillus amyloliquefaciens according to claim 1, is characterized in that: described NA culture medium prescription: 10 grams of peptones, 3 grams of extractum carniss, Nacl5 gram, 15 grams, agar, distilled water 1000ml, pH value 7.3.
4. bacillus amyloliquefaciens according to claim 1, is characterized in that: described bacterial classification I can be 4 ℃ of following preservations three months.
5. bacillus amyloliquefaciens according to claim 1, is characterized in that: described bacillus amyloliquefaciens can be for the bacterial wilt of the crops such as control tobacco, tomato and capsicum.
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| CN105039220A (en) * | 2015-07-30 | 2015-11-11 | 武汉华恩绿农生物科技有限公司 | Bacillus methylotrophicus AR3, bacillus subtilis AR4 and bacillus amyloliquefaciens AR10 and application thereof |
| CN105296386A (en) * | 2015-10-22 | 2016-02-03 | 武汉骏安生物科技有限公司 | Endophytic bacillus amyloliquefaciens capable of generating a large number of antagonistic tobacco bacterial wilt active materials |
| CN109797117A (en) * | 2019-01-30 | 2019-05-24 | 贵州大学 | It is a kind of produce multienzyme double bacteria preparations preparation and its application method |
| CN112175885A (en) * | 2020-10-30 | 2021-01-05 | 江西顺泉生物科技有限公司 | Preparation and application of bacillus amyloliquefaciens capable of preventing and treating tobacco bacterial wilt |
| CN116240149A (en) * | 2023-04-07 | 2023-06-09 | 黑龙江八一农垦大学 | Bacillus amyloliquefaciens and application thereof in preventing and treating common bacterial blight of kidney beans |
| WO2023115809A1 (en) * | 2021-12-22 | 2023-06-29 | 武汉科诺生物科技股份有限公司 | High-content resuspendable microbial bulk drug, and preparation thereof, preparation method therefor, and use thereof |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105039220A (en) * | 2015-07-30 | 2015-11-11 | 武汉华恩绿农生物科技有限公司 | Bacillus methylotrophicus AR3, bacillus subtilis AR4 and bacillus amyloliquefaciens AR10 and application thereof |
| CN105039220B (en) * | 2015-07-30 | 2019-01-11 | 武汉华恩绿农生物科技有限公司 | Methylotrophic bacillus AR3, bacillus subtilis AR4, bacillus amyloliquefaciens AR10 and application |
| CN105296386A (en) * | 2015-10-22 | 2016-02-03 | 武汉骏安生物科技有限公司 | Endophytic bacillus amyloliquefaciens capable of generating a large number of antagonistic tobacco bacterial wilt active materials |
| CN109797117A (en) * | 2019-01-30 | 2019-05-24 | 贵州大学 | It is a kind of produce multienzyme double bacteria preparations preparation and its application method |
| CN112175885A (en) * | 2020-10-30 | 2021-01-05 | 江西顺泉生物科技有限公司 | Preparation and application of bacillus amyloliquefaciens capable of preventing and treating tobacco bacterial wilt |
| WO2023115809A1 (en) * | 2021-12-22 | 2023-06-29 | 武汉科诺生物科技股份有限公司 | High-content resuspendable microbial bulk drug, and preparation thereof, preparation method therefor, and use thereof |
| CN116240149A (en) * | 2023-04-07 | 2023-06-09 | 黑龙江八一农垦大学 | Bacillus amyloliquefaciens and application thereof in preventing and treating common bacterial blight of kidney beans |
| CN116240149B (en) * | 2023-04-07 | 2023-10-17 | 黑龙江八一农垦大学 | Bacillus amyloliquefaciens and application thereof in preventing and treating common bacterial blight of kidney beans |
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Application publication date: 20140917 |