CN102618467A - Bacillus thuringiensis, as well as preparation method and application thereof - Google Patents
Bacillus thuringiensis, as well as preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a bacillus thuringiensis, as well as a preparation method and an application thereof. The collection number of the bacillus thuringiensis is CCTCC M2012040. The method comprises the following steps: A, according to the characteristic that a spore of the bacillus thuringiensis can resist 78 to 82 DEG C high temperature and can form a parasporal crystal, the bacillus thuringiensis is separated according to an earth bomb method; and B, a just hatched beet armyworm or a cotton bollworm is adopted as a test worm for virulence bioassay, a middle quantity of LC50 is killed through virulence comparison, and a high-virulence strain BtCS36 is obtained by screening. The formula of a fermentation culture medium and a fermentation process are defined by utilizing an orthogonal experiment and small scale fermentation. The fermentation period is about 30 h by utilizing the bacillus thuringiensis and the defined fermentation process; and the virulence of the fermentation liquid works on cotton bollworms with 3000 to 5000IU/Mul. The production of the novel Bt pesticide not only can efficiently control various lepidoptera pests and ensure high and stable yield of crops, but also has important practical significance and urgency on the safety of the ecological environment.
Description
Technical field
The present invention relates to the microbial pesticide technical field, be specifically related to a kind of bacillus thuringiensis, more relate to the preparation method of a kind of bacillus thuringiensis, also relate to the application of a kind of bacillus thuringiensis in the preparation of sterilant fermented liquid.
Background technology
For the insecticidal spectrum that enlarges bacillus thuringiensis with improve insecticidal activity, all be devoted to the isolation identification of new bacterial strain and the exploitation of new killing gene at present both at home and abroad.Aspect the isolation identification of bacillus thuringiensis bacterial strain, screening; Document " screening of three subregion bacillus thuringiensiss and biological activity determination (Anhui agricultural sciences; 2009; 37 (28): 13660-13661) ", " area, Da Mao mountain bacillus thuringiensis distribute with Study on Diversity (Chinese agronomy circular, 2011,27 (01): 166-169) ", " isolation identification of Guilin ' landforms range of distribution Su Yun gold bud pole bacteria strain (Chinese biological control; 2009; 25 (2): 138-142) ", " isolation identification of orchard, Hebei province bacillus thuringiensis bacterial strain (North China agronomy newspaper, 2010,25 (4): 201-205) ", and " collection of cold water wilderness area, Heilungkiang bacillus thuringiensis and evaluation (genomics and applied biology; 2009,284 (4): 685-690) " etc. the separation of China bacillus thuringiensis, evaluation, characteristic distributions, crystal proteins form, insecticidal properties etc. are studied.Aspect the screening of efficient, specific strains; Document " 1 strain to the highly active Bacillus thuringiensis BtCYZ-4 of various pests (Northeast Forestry University's journal; 2009; 37 (9): 104-105) ", " strain to the separation of the bacillus thuringiensis of the high virulence of lasioderma serricorne and evaluation (chemistry and biotechnology, 2009,26 (5): 42-45) ", " to the screening of the high virulence bacillus thuringiensis strains of fall webworms (Agricultural University Of Hebei's journal; 2009; 32 (2): 89-92) ", " to the separation of the high virulence Bacillus thuringiensis of beet armyworm and biological assay (Yanbian University's agronomy journal, 2010,34 (2): 238-242) ", " the high virulence bacillus thuringiensis research of grub (modern commerce and trade industry; 2010; 9:353-355) ", and " bacillus thuringiensis to pine wood nematode kill line active (Chinese biological control, 2010,26 (1): 85-89) " wait in the bacillus thuringiensis bacterial strain how of comforming respectively and screened efficient specific strains insects such as lasioderma serricorne, fall webworms, beet armyworm, grub, nematodes; Aspect killing gene clone and engineering bacteria structure; The gene of having cloned 71 types of nearly more than 600 bacillus thuringiensiss in the whole world at present; Document " progress of bacillus thuringiensis and insecticidal crystalline gene resource thereof (Shanghai Agricultural journal, 2010,26 (4): 135-139) ", " clone, expression and the activation analysis of bacillus thuringiensis cry9Aa gene (plant protection journal; 2010; 37 (6): 541-546) ", " clone of bacillus thuringiensis cry1Ac28 gene and prokaryotic expression (Northeast Agricultural University's journal, 2010,41 (10): 61-67) ", " strain to the killing gene of the high virulence bacillus thuringiensis of fall webworms identify (Chinese agronomy circular; 2010; 26 (3): 242-244) ", " research of Tribactur cry1Ia17 gene clone, expression (Northeast Agricultural University's journal, 2011,42 (4): 93-97) ", " clonal expression of Bacillus thuringiensis cry7Ab8 gene and insecticidal activity thereof (North China agronomy newspaper; 2011; 26 (2): 35-38) ", and " strain contain the bacillus thuringiensis strains of the new gene of cry research (North China agronomy newspaper, 2011,26 (1): 54-57) " etc. separate, identify, expressed a series of new active gene respectively.Aspect preparation manufacture; Document " preparation technology of uv degradation Tribactur microcapsule (Hua Zhong Agriculture University's journal; 2009; 28 (3): 281-285) ", " development of 17600IU/mg bacillus thuringiensis suspension concentrates (hubei agricultural science; 2010,49 (12): 3048-3051) ", " 0.18% Avrmectin, 10,000,000,000 living spores/gram thuringiensis bacillus powder Control Dendrolimus punctatus test (modern agriculture science and technology, 2011 the 13rd phases: 143-144) ", " Bacillus thuringiensis and the combined action of mythimna separata granulosis virus(GV) to the influence of beet armyworm (Yangzhou University's journal; 2011; 32 (2): 64-68) ", " potentiating compounds of Tribactur engineering bacteria WG-001 wettable powder screen (plant protection journal, 2011,38 (3): 271-275) " etc. are studied to the preparationization of thuringiensis cladosporioides bacillus insecticide and with the combined utilization technology of other pesticide preparations respectively.Integral body from ecotope and social development; The Application and Development of the biological pesticide of highly effective and safe all has great significance to the Sustainable development of the mankind, environment and agricultural; Thuringiensis cladosporioides bacillus insecticide is most widely used a kind of harmless boilogical sterilant in the biological pesticide, and vast potential for future development is arranged.But the thuringiensis cladosporioides bacillus insecticide of production application at present still exists desinsection narrow range, effective constituent to be prone to degrade, to be subject to shortcomings such as such environmental effects.And in production practice; Because artificial continuous passage; Bacillus thuringiensis gemma and crystal diminish, the speed of growth is slack-off, the low inferior shortcoming of insecticidal effect, so still need constantly to be used for developing sterilant from occurring in nature direct separation bacillus thuringiensis efficient more, wide spectrum.
Summary of the invention
The objective of the invention is to be to provide a kind of bacillus thuringiensis (Bacillus thuringiensis), called after BtCS36 (preserving number: CCTCCM2012040) of the present invention.This bacterium is from the nature direct separation; Contain a plurality of insect-killing protein encoding soxs; Various pests such as lepidopterous bollworm, tea geometrid, small cabbage moth, beet armyworm, cabbage caterpillar are had the efficiently poisoning effect, and to be a strain efficient to lepidoptera pest and the natural bacillus thuringiensis of wide spectrum.This bacterium leavening property is good, is the natural bacterial strain of bacillus thuringiensis that a strain has exploitation, application prospect.
Another object of the present invention is the preparation method who has been to provide a kind of bacillus thuringiensis; The characteristic of utilizing the bacillus thuringiensis gemma can anti-78-82 ℃ of pyritous characteristic and can form parasporal crystal; Adopt and play local method separation bacillus thuringiensis; Just to incubate beet armyworm and bollworm is the examination worm, carries out virulence bioassay, through comparing virulence median lethal dosage LC
50, screen supper toxic strain BtCS36.
Purpose in addition of the present invention is to be to provide the application method of a kind of bacillus thuringiensis in the preparation of sterilant fermented liquid, and this method fermentation condition is reasonable, simple, fermentation period short, the fermented liquid toxicity evaluation is high.Utilize its production novel B t sterilant not only can the multiple lepidoptera pest of high-efficiency prevention and control, guarantee the farm crop high and stable yields, and to ecological environment security, therefore have important practical significance and urgent.
In order to realize above-mentioned purpose, the present invention adopts following technical measures:
The preparation method of a kind of bacillus thuringiensis the steps include:
A, the characteristic of utilizing the bacillus thuringiensis gemma can anti-78-82 ℃ of pyritous characteristic and can form parasporal crystal; Adopt and play local method (Acta Microbiologica Sinica 30; 380-388), bacillus thuringiensis is separated in (Changsha) from Chinese soil.
B, serve as the examination worm, carry out virulence bioassay, through virulence median lethal dosage LC relatively just to incubate beet armyworm and bollworm
50, screen supper toxic strain BtCS36.
C, employing morphologic observation and classical Physiology and biochemistry authentication method have been established the basic physio-biochemical characteristics of BtCS36.The basic physio-biochemical characteristics of BtCS36 are seen table 1.
Its concrete physio-biochemical characteristics are following:
1.H serotype: carry out cross agglutination and cross absorption agglutination reaction test with the flagellar antigen of BtCS36 and the standard antiserum(antisera) of Bt, the H serotype of BtCS36 is H7.
2. biochemical characteristic: the classical way by Barjac carries out physiological and biochemical test.
The biochemical character of table 1 bacillus thuringiensis BtCS36 bacterial strain
The application of a kind of bacillus thuringiensis in the preparation of sterilant fermented liquid the steps include:
A, shake flask fermentation:
The purpose of shake flask fermentation is to select best fermentating formula, and optimization of fermentation conditions adopts different solid contents to carry out orthogonal test with different C/N ratios.Cultivate flat board more than 3 days and scrape culture and be dissolved in and process spore suspension in the sterilized water, handle for 75 ℃ and be used for inoculation in 20 minutes.20/250ml loading amount, 30 ℃, 250rpm shake-flask culture.The Optimal compositions of fermentation medium prescription that obtains bacterial strain BtCS36 is seen table 2.
Table 2 fermention medium is formed
B, adopt the Optimal compositions of fermentation medium prescription screen, in automatic fermenter, carry out fermentation test more than 10 times, established its best zymotechnique: the substratum preceding pH8.1 that disappears, the back pH that disappears is about 7.0.80 ℃ of slant strains of handling 20 minutes of warp of every jar of inoculation, 30 ℃ of inoculation temps.Adopt alternating temperature, speed change, transformation (0-11 hour, 0.6 cubic metre/hour of 32 ℃, 500 rev/min, air flow; 12-21 hour, 0.8 cubic metre/hour of 29 ℃, 600 rev/min, air flow; 22 hours-fermentation ends, 0.8 cubic metre/hour of 32 ℃, 600 rev/min, air flow) the fermentation means, confirmed the best zymotechnique of a cover.Fermentation period is about 30 hours, and fermentation ends secondary fermentation liquid is acidified to pH5.0, and fermented liquid reaches 3000-5000IU/ μ l to the toxicity evaluation of bollworm.
Compared with prior art, the present invention has the following advantages and effect:
1. bacillus thuringiensis BtCS36 has the advantages that to kill multiple lepidoptera pest; Domestic still do not have the natural lepidopteran bacillus thuringiensis commodity preparation that kills of wide spectrum at present; Thereby BtCS36 capable of using produces novel thuringiensis cladosporioides bacillus insecticide, makes the product diversification and the seriation of thuringiensis cladosporioides bacillus insecticide, enlarges the use range of thuringiensis cladosporioides bacillus insecticide; Reduce the pesticide control expense, reduce the pollution of chemical pesticide, increase crop yield environment.
2. as wild bacillus thuringiensis, it is harmless to people, animal and plant-animal, and is free from environmental pollution, and good economy and ecological benefits are arranged.Thereby,, suitability for industrialized production technology maturation low, aspects such as people and plant-animal safety, the free from environmental pollution and market requirement are considered that bacillus thuringiensis BtCS36 has good popularizing application prospect from superior and unique insecticidal properties, production and use cost.And because this strain hereditary characteristic is highly stable, so this bacterium is suitable as the recipient bacterium of industrial production bacterial classification or structure broad-spectrum high efficacy engineering bacteria.
3. the natural bacillus thuringiensis of preparation has very high virulence and tangible control effect to farming, woods insects such as the bollworms of harm cotton, tea tree, vegetables, tea geometrid, small cabbage moth, beet armyworm, cabbage caterpillars.
4. utilize fermentation condition provided by the invention and method to produce thuringiensis cladosporioides bacillus insecticide and have with short production cycle, fermented liquid virulence advantages of higher.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Used experiment material among the following embodiment like no specified otherwise, is to buy from routine biochemistry reagent shop and obtains, and unreceipted concrete experiment condition and method in the following example are cultivated with separation method according to routine usually and obtained.
Embodiment 1:
The preparation method of a kind of bacillus thuringiensis the steps include:
A, utilize the bacillus thuringiensis gemma can anti-78-82 ℃ of pyritous characteristic; Adopt and play local method: the dry back of 5g soil (Changsha) grinds and is powder; Be sprinkling upon on the sterilization filter paper of 8 * 8cm size; Make to be stained with one deck sample powder on the filter paper face uniformly, make to be stained with one of sample and to flick under the 1-2, pour 80 ℃ of nutrient agar mediums in the petridish into facing to petridish.(Acta Microbiologica Sinica 30,380-388), single bacterium colony of formation separates bacillus thuringiensis through microscopic examination to cultivate 5-7 days in 30 ℃.
B, serve as the examination worm, carry out virulence bioassay,, screen supper toxic strain BtCS36 through virulence median lethal dosage LC50 relatively with reference to GB/T19567.2-2004 just to incubate beet armyworm and bollworm.
C, employing morphologic observation and classical Physiology and biochemistry authentication method have been established the basic physio-biochemical characteristics of BtCS36.
The preservation of D, bacterial strain: deposit number: CCTCCM2012040; Depositary institution: Chinese typical culture collection center; Address: China. Wuhan. Wuhan University, preservation date: on February 27th, 2012, classification name: Bacillus thuringiensis BtCS36;
Its concrete physio-biochemical characteristics are following:
1.H serotype: carry out cross agglutination and cross absorption agglutination reaction test with the flagellar antigen of BTCS36 and the standard antiserum(antisera) of Bt, the H serotype of BTCS36 is H7.
2. form and physio-biochemical characteristics: adopt microscopic examination and carry out physiological and biochemical test by the classical way of Barjac.The biochemical character of bacillus thuringiensis BtCS36 bacterial strain is seen table 1
E. (method is with reference to Biological Control, and 2008,44 (3): 380-388), the insecticidal crystal protein plasmagene of bacterial strain BtCS36 is Cry1Aa, Cry1Ab, Cry1Ac, Cry1C, Cry2 in process PCR detection.
F. (method is with reference to Biological Control, and 2008,44 (3): 380-388), the insecticidal crystal protein matter of bacterial strain BtCS36 is 138kD through the SDS-PAGE detection; 127kD; 116kD; 86kD; 76kD.
Embodiment 2:
The application of a kind of bacillus thuringiensis in the preparation of sterilant fermented liquid the steps include:
A, shake flask fermentation
The purpose of shake flask fermentation is to select best fermentating formula, and optimization of fermentation conditions adopts different solid contents to carry out orthogonal test with different C/N ratios.Cultivate flat board more than 3 days and scrape culture and be dissolved in and process spore suspension in the sterilized water, handle for 75 ℃ and be used for inoculation in 20 minutes.20/250ml loading amount, 30 ℃, 250rpm shake-flask culture.The optimal medium prescription of the BtCS36 that obtains is seen table 2.
B, the lab scale in automatic fermenter
The best fermentating formula that employing screens carries out fermentation test more than 10 times in automatic fermenter, confirm best zymotechnique: the substratum preceding pH8.1 that disappears, the back pH that disappears is about 7.0.80 ℃ of slant strains of handling 20 minutes of warp of every jar of inoculation, 30 ℃ of inoculation temps.Adopt alternating temperature, speed change, transformation (0-11 hour, 0.6 cubic metre/hour of 32 ℃, 500 rev/min, air flow; 12-21 hour, 0.8 cubic metre/hour of 29 ℃, 600 rev/min, air flow; 22 hours-fermentation ends, 0.8 cubic metre/hour of 32 ℃, 600 rev/min, air flow) the fermentation means, confirmed the best zymotechnique of a cover.Fermentation period is about 30 hours, and fermentation ends secondary fermentation liquid is acidified to pH5.0.
Carry out virulence bioassay with reference to GB/T19567.2-2004, fermented liquid reaches 3000-5000IU/ μ l to the toxicity evaluation of bollworm.
Claims (3)
1. bacillus thuringiensis is characterized in that:
Bacillus thuringiensisBtCS36 CCTCCM2012040.
2. the preparation method of the described a kind of bacillus thuringiensis of claim 1 the steps include:
A, the characteristic of utilizing the anti-78-82 of bacillus thuringiensis gemma ℃ high temperature in the soil and forming parasporal crystal adopt to play local method and separate bacillus thuringiensis;
B, serve as the examination worm, carry out virulence bioassay, through virulence median lethal dosage LC relatively just to incubate beet armyworm and bollworm
50, screen supper toxic strain BtCS36.
3. the application of the described a kind of bacillus thuringiensis of claim 1 in the preparation of sterilant fermented liquid:
A, the different solid content of employing carry out orthogonal test with different C/N ratios, obtain fermentative medium formula, and fermention medium consists of:
Component content unit
W-Gum 3.5 g
Cottonseed cake powder 2.0 g
Soybean cake powder 2.0 g
Steeping water 2.0 g
Yeast powder 0.4 g
Peptone 0.6 g
MgSO4 0.04 g
CaCO3 0.25 g
AMS 100 U
Zero(ppm) water 100 mL;
The fermentative medium formula that screens in B, the employing steps A; In automatic fermenter, carry out fermentation test more than 10 times, established its zymotechnique: the substratum preceding pH8.1 that disappears, the back pH that disappears is about 7.0; 80 ℃ of slant strains of handling 20 minutes of warp of every jar of inoculation; 30 ℃ of inoculation temps adopt alternating temperature, speed change, transformation: 0-11 hour, 0.6 cubic metre/hour of 32 ℃, 500 rev/min, air flow; 12-21 hour, 0.8 cubic metre/hour of 29 ℃, 600 rev/min, air flow; 22 hours-fermentation ends; The fermentation means that 32 ℃, 600 rev/mins, air flow are 0.8 cubic metre/hour have been confirmed zymotechnique, and fermentation period is 30 hours; Fermentation ends secondary fermentation liquid is acidified to pH5.0, and fermented liquid reaches 3000-5000IU/ μ l to the toxicity evaluation of bollworm.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104611277A (en) * | 2015-02-15 | 2015-05-13 | 中国热带农业科学院椰子研究所 | Bacillus thuringiensis for controlling opisina arenosella walkers, serving as palm pests, and application of Bacillus thuringiensis |
| CN105340975A (en) * | 2015-11-26 | 2016-02-24 | 常州乐农生物科技有限公司 | Preparation process for bacillus thuringiensis biopesticide wettable powder |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1329830A (en) * | 2001-06-15 | 2002-01-09 | 中国科学院武汉病毒研究所 | Process for preparing insecticide by recombining broad-spectrum Thuricide |
| CN1392248A (en) * | 2002-06-26 | 2003-01-22 | 中国科学院武汉病毒研究所 | Broad spectrum bacillus thuringiensis and the method for preparing pesticide therewith |
-
2012
- 2012-03-26 CN CN 201210082251 patent/CN102618467B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1329830A (en) * | 2001-06-15 | 2002-01-09 | 中国科学院武汉病毒研究所 | Process for preparing insecticide by recombining broad-spectrum Thuricide |
| CN1392248A (en) * | 2002-06-26 | 2003-01-22 | 中国科学院武汉病毒研究所 | Broad spectrum bacillus thuringiensis and the method for preparing pesticide therewith |
Non-Patent Citations (2)
| Title |
|---|
| 彭克凡等: "苏云金芽孢杆菌的分离与鉴定及其高毒力菌株的筛选", 《武汉大学学报（理学版）》 * |
| 蔡亚君等: "1株广谱苏云金芽孢杆菌及其发酵条件的研究", 《华中农业大学学报》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104611277A (en) * | 2015-02-15 | 2015-05-13 | 中国热带农业科学院椰子研究所 | Bacillus thuringiensis for controlling opisina arenosella walkers, serving as palm pests, and application of Bacillus thuringiensis |
| CN104611277B (en) * | 2015-02-15 | 2017-07-04 | 中国热带农业科学院椰子研究所 | One plant of preventing and treating palm insect coconut knits bacillus thuringiensis and the application of moth |
| CN105340975A (en) * | 2015-11-26 | 2016-02-24 | 常州乐农生物科技有限公司 | Preparation process for bacillus thuringiensis biopesticide wettable powder |
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| Publication number | Publication date |
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| CN102618467B (en) | 2013-05-01 |
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