CN1141383C - Waxy bacillus and its prepn - Google Patents
Waxy bacillus and its prepn Download PDFInfo
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- CN1141383C CN1141383C CNB021157065A CN02115706A CN1141383C CN 1141383 C CN1141383 C CN 1141383C CN B021157065 A CNB021157065 A CN B021157065A CN 02115706 A CN02115706 A CN 02115706A CN 1141383 C CN1141383 C CN 1141383C
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- bacillus cereus
- bacillus
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Abstract
The present invention discloses bacillus cereus and a preparation method. The preparation method of the Bacillus cereus Bc, YM-2102, CCTCC No. M202011 comprises the following steps: firstly, putting a soil sample in a nutrition culture solution test tube to pass through a water bath of 75 to 78 DEG C, and carrying out shaking culture for a certain time; diluting a nutrition culture solution to be coated on an LB culture medium, carrying out reverse culture in a thermostat for a certain time, and inspecting a single colony by a microscope; purifying the single colony on the LB culture medium again to be inoculated on a fermentation culture medium, and carrying out shaking fermentation to desquamate spores. The method of the present invention has the advantages of simplicity, convenience, easy operation and low cost, and is suitable for industrial production; the present invention can conspicuously improve the pesticidal activity of Entobakterin pesticide.
Description
Technical field
The present invention relates to Bacillus thuringiensis (Bacillus thuringiensis, the Bt) screening of the synergistic agent of class biological pesticide, particularly relate to a kind of can (Bacillus cereus Bc), also relates to the preparation method of this bacterium to Bt synergic bacillus cereus.
Technical background
(Bacillus thuringiensis, Bt) preparation is that current application is the widest to bacillus thuringiensis, the most effective microbial pesticide.Belong to gram-positive microorganism, the parasporal crystal that produces during its toxic effect is mainly derived from gemma and forms became commodity first in France in 1938.Since commercialization, the Bt product just with its highly effective and safe, to the specificity of target pest and enjoy favor, and become the most deep, the most widely used biotic pesticide of research gradually.Although Bt has plurality of advantages, it also exist when using in the field insecticidal effect undesirable, to problem such as some target pest is insensitive.One of main path that addresses this problem is to greatly develop various synergistic agent and synergistic factor, improves the insecticidal activity of existing high toxic bacterial strain.It is composite to add appropriate amount of addition agent as (1996) such as Zhang Xunhao with Bt and bollworm NPV, has obviously improved the insecticidal effect of Bt; Wen Zhiqiang etc. (1999) add inorganic salt in the Bt preparation, also can improve its insecticidal effect to the pickles goose; (1998) such as woods beginning of springs are screened by inquiry and have been obtained three strains have the notable synergistic effect to the Bt preparation bacterial isolates 34-16,30-3 and C16.All these synergistic factors can both improve the prevention effect to target pest of Bt preparation to a certain extent, but can not significantly improve its insecticidal activity.
Summary of the invention
The object of the present invention is to provide a kind of bacillus cereus, adopted this bacterium that the insecticidal activity of these biotic pesticide is significantly improved.
Another object of the present invention is to provide a kind of method for preparing bacillus cereus.
For reaching above purpose, the present invention adopts following technical measures:
Strain name: bacillus cereus Bacillus cereus, Bc, Ym-2102, depositary institution: Chinese typical culture collection center, deposit number: CCTCC No.M202011.The steps include:
1, a little puts into the nutrient medium test tube that 4.5ml is housed with the soil sample of gathering, and through 75～78 ℃, water-bath is after 14～20 minutes, 28～30 ℃ of shaking culture 48～52 hours;
2, nutrient medium is diluted to 10
-8～10
-9Doubly, be applied on the LB substratum, be inverted in 28～30 ℃ of thermostat containers and cultivated 3 days, microscopy is selected the single bacterium colony that produces gemma but do not have parasporal crystal;
3, with single bacterium colony purifying on the LB substratum, be inoculated in fermention medium and carry out shake flask fermentation,, began in 8～10 hours to take a sample respectively in two hours at interval to the different steps that gemma comes off fully from fermenting to bacillus exfoliation;
4, with the bacillus thuringiensis DL5789 (preservation of the fermented liquid of different steps and shake flask fermentation to bacillus exfoliation 15～20%, depositary institution: Chinese typical culture collection center, deposit number: CCTCC No.M202003.) fermented liquid is pressed the different ratios mixing, carries out biological assay;
5, strain morphology is learned and observed: cell is shaft-like, the garden, two ends.Thalline long-chain shape, splayed is arranged, and produces gemma in the process of growth; Bacterium colony is greyish white, and is opaque, drying, and the garden, the edge is irregular;
6, physicochemical property: Gram-positive; The methylene blue stained positive; Can not hydrolyzed starch; Can utilize glucose fermentation; Can grow containing on the 7%NaCl substratum; Can utilize seminose; The indole test feminine gender; VP tests positive, VP culture PH＜6; The energy anaerobic growth has the nitrate reduction activity;
7, synergistic activity: obtain Bt bacterial strain DL5789 synergic one strain bacillus cereus Ym-2102 by above-mentioned approach.Carrying out biological assay separately with the DL5789 fermented liquid, serves as that LC50 is the 1.047ml/g feed for the examination worm with the beet armyworm newly hatched larvae; After DL5789 and Ym-2102 fermented respectively, by volume after 4: 1 the mixed beet armyworm is carried out biological assay, its LC50 was the 0.58ml/g feed with fermented liquid; Synergistic effect is 80.52%.
The present invention compares with other synergisting methods, has following advantage: 1 method is simple and easy to operate; 2 synergistic effects are obvious, can significantly improve the insecticidal activity of Biotrol BTV; 3 is with low cost, little to the production cost increase of sterilant, is applicable to industrial production.
Embodiment
Embodiment 1: strain separating is cultivated: a little puts into the nutrient medium test tube that 4.5ml is housed with the soil sample of gathering, nutrient medium is peptone 1% extractum carnis 0.3%NaCl 0.5%PH7.5, through 76 ℃, water-bath is after 15 minutes, 30 ℃ of shaking culture 51 hours.To cultivate nutrient solution and be diluted to 10
-8Doubly, be applied on the isolation medium, isolation medium is peptone 1% extractum carnis 0.3%NaCl 0.5% agar 2%PH7.5, is inverted in 30 ℃ of thermostat containers and cultivates 3 days, microscopy is selected the single bacterium colony that produces gemma but do not have parasporal crystal, obtains a strain bacillus cereus strain Ym-2102.With its single bacterium colony purifying on the LB substratum, the LB substratum is NaCl 1%, peptone 1%, yeast powder 0.5%, agar 1.5%, PH7.0 is inoculated in fermention medium then and carries out shake flask fermentation, and fermention medium is a starch 2%, bean powder 4%, yeast powder 0.5%, peptone 0.5%, KH
2PO
40.1%, K
2HPO
40.2%, PH8.5 scrapes from isolation medium and to get a ring bacterium, adds in the test tube that 5ml distilled water is housed to shake up, and 76 ℃ of water-baths 15 minutes are taken out and are cooled to normal temperature; Get above-mentioned synchronized bacteria suspension 1ml and be inoculated in the big triangular flask of 500ml, wherein adorn substratum 40ml.The bacillus thuringiensis strains DL5789 of LB medium slant preservation presses with the quadrat method manipulation, and 30 ℃, the 200rpm fermentation culture.Began in 10 hours from fermenting the different steps that comes off fully to gemma at interval two hours respectively sampling observe, stop to cultivate to 48 hours bacillus exfoliations.
Biological assay: the fermented liquid of different steps is mixed by different ratios with the bacillus thuringiensis DL5789 fermented liquid of shake flask fermentation to bacillus exfoliation 15～20%, incubate just with beet armyworm that to exhale larva serve as to carry out biological assay for examination.The Ym-2102 fermented liquid of bacillus exfoliation 10% mixed the back in 1: 4 by volume with the bacillus thuringiensis DL5789 of bacillus exfoliation 15% beet armyworm is carried out biological assay, and its LC50 is the 0.58ml/g feed; And DL5789 fermented liquid in contrast carries out biological assay separately, and LC50 is the 1.047ml/g feed, and synergistic effect is 80.52%.
Embodiment 2:
Strain separating is cultivated: a little puts into the nutrient medium test tube that 4.5ml is housed with the soil sample of gathering, through 78 ℃, and water-bath 12 minutes, shaking culture 48 hours.To cultivate nutrient solution and be diluted to 10
-9Doubly, be applied on the isolation medium, be inverted in 29 ℃ of thermostat containers and cultivated 3 days, microscopy is selected the single bacterium colony that produces gemma but do not have parasporal crystal, obtains a strain bacillus cereus strain Ym-2102.With its single bacterium colony purifying on the LB substratum, be inoculated in fermention medium and carry out shake flask fermentation: scrape from isolation medium and get a ring bacterium, add in the test tube that 5ml distilled water is housed and shake up, 76 ℃ of water-baths 12 minutes are taken out and are cooled to normal temperature; Get above-mentioned synchronized bacteria suspension 1ml and be inoculated in the big triangular flask of 500ml, wherein adorn substratum 40ml.The bacillus thuringiensis strains DL5789 of LB medium slant preservation presses with the quadrat method manipulation, 30 ℃, the 200rpm shake flask fermentation is cultivated, began in 9 hours from fermenting the different steps that comes off fully to gemma at interval two hours respectively sampling observe, stop to cultivate to 50 hours bacillus exfoliations.
Biological assay: the fermented liquid of different steps is mixed by different ratios with the bacillus thuringiensis DL5789 fermented liquid of shake flask fermentation to bacillus exfoliation 20%, serves as to carry out biological assay for examination with the beet armyworm newly hatched larvae.The Ym-2102 fermented liquid of bacillus exfoliation 15% mixed the back in 1: 4 by volume with the bacillus thuringiensis DL5789 of bacillus exfoliation 20% beet armyworm is carried out biological assay, and its LC50 is the 0.58ml/g feed; And DL5789 fermented liquid in contrast carries out biological assay separately, and LC50 is the 1.047ml/g feed, and synergistic effect is 80.52%.Nutrient medium, isolation medium, LB substratum, fermentative medium formula ratio are identical with embodiment 1.
Claims (3)
1, a kind of bacillus cereus, it is characterized in that bacillus cereus (Bacillus cereus, Bc), Ym-2102, CCTCC No.M202011.
2, a kind of preparation method of bacillus cereus as claimed in claim 1 comprises the following steps:
A, soil sample is put into the nutrient medium test tube, through 75～78 ℃, water-bath 14～20 minutes, 28～30 ℃ of shaking culture 48～52 hours;
B, nutrient medium is diluted to 10
-8～10
-9Doubly, be applied on the LB substratum, be inverted in 28～30 ℃ of thermostat containers and cultivated microscopy list bacterium colony 3 days;
C, with single bacterium colony purifying on the LB substratum, be inoculated in fermention medium and carry out shake flask fermentation, to bacillus exfoliation.
3, the preparation method of a kind of bacillus cereus according to claim 2 is characterized in that:
Nutrient medium: peptone 1% extractum carnis 0.3%NaCl 0.5%PH7.5
Isolation medium: peptone 1% extractum carnis 0.3%NaCl 0.5% agar 2%PH7.5
LB substratum: NaCl 1%, peptone 1%, yeast powder 0.5%, agar 1.5%, PH7.0
Fermention medium: starch 2%, bean powder 4%, yeast powder 0.5%, peptone 0.5%, KH
2PO
40.1%, K
2HPO
40.2%, PH8.5.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB021157065A CN1141383C (en) | 2002-04-11 | 2002-04-11 | Waxy bacillus and its prepn |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB021157065A CN1141383C (en) | 2002-04-11 | 2002-04-11 | Waxy bacillus and its prepn |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1374399A CN1374399A (en) | 2002-10-16 |
| CN1141383C true CN1141383C (en) | 2004-03-10 |
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Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB021157065A Expired - Fee Related CN1141383C (en) | 2002-04-11 | 2002-04-11 | Waxy bacillus and its prepn |
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Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102757921B (en) * | 2012-07-27 | 2013-10-16 | 长安大学 | Bacillus cereus and application thereof |
| CN103555602B (en) * | 2012-09-26 | 2014-09-24 | 中国人民解放军总医院 | Bacillus cereus LCT-BC235 strain under space environment |
| CN103555603B (en) * | 2012-09-26 | 2014-09-17 | 中国人民解放军总医院 | Space bacillus cereus strain LCT-BC25 |
| CN103088093B (en) * | 2013-01-08 | 2014-10-29 | 南京工业大学 | Method for purifying antibacterial protein in bacillus cereus fermentation broth |
| CN104126614A (en) * | 2014-08-18 | 2014-11-05 | 熊吉元 | Preparation method for biological pesticide |
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2002
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| CN1374399A (en) | 2002-10-16 |
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