CN1128212C - Parasite-killing thuricinbaillus strain YBT-1532 and parasite killing crystallin - Google Patents
Parasite-killing thuricinbaillus strain YBT-1532 and parasite killing crystallin Download PDFInfo
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- CN1128212C CN1128212C CN 00116062 CN00116062A CN1128212C CN 1128212 C CN1128212 C CN 1128212C CN 00116062 CN00116062 CN 00116062 CN 00116062 A CN00116062 A CN 00116062A CN 1128212 C CN1128212 C CN 1128212C
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Abstract
The present invention relates to a breeding method of a high-toxicity strain of bacillus thuringiensis for killing plant eelworms and parazoons, which belongs to the technical field of microbe disinsection pesticides. The present invention is characterized in that a bacillus thuringiensis strain YBT-1532 having toxicity on plant eelworms and parazoons is separated, and the type of flagellar antigen blood serum is H10; rhombic parasporal crystals are generated; the molecular weight of insecticidal crystal protein forming the parasporal crystals is 130kDa, and the protein has toxic killing action on the plant eelworms and the parazoons. Compared with the prior art, the strain of the present invention and the insecticidal crystal protein thereof have the obvious advantages of high toxicity, strong specificity, etc.
Description
Technical field
The invention belongs to the microbial insecticide technical field of pesticide, be specifically related to the seed selection of Bacillus thuringiensis bacterial strain of a kind of plant nematocide and zooparasite and the exploitation of insecticidal crystal protein thereof.Its international Patent classificating number is A01N 63/00.
Background technology
Plant nematode is one of important pests in the agriculture production.Cash crop such as their dangers to vegetables, corn, cotton, tobacco and paddy rice.Can cause the financial loss that equates with insect.Though the control of chemistry nematocides has certain effect, chemical pesticide is huge to ecotope and to the pollution of soil with to the influence of insect natural enemy, and therefore developing biological pesticide control nematode is problem anxious to be solved in the current Agricultural pests comprehensive regulation.
Haemonchus contortus, filaria, plasmodium and schistosomicide are the parasites of poultry trouble, the mankind or infecting both domestic animals and human, and the control of chemicals often has stronger toxic side effect, and often use also makes pathogenic agent develop immunity to drugs easily.
That biotic pesticide have is free from environmental pollution, person poultry safety, insect are difficult for developing immunity to drugs and produce advantages such as simple, and insecticide Bacillus thuringiensis is present most important biotic pesticide.
Since 1988, U.S. Mycogen company is separated to some to the deleterious Tribactur of animal and plant parasitic nematode, effective object comprises Pratylenchidae, caenorhabditis elegant (Caenorhabditiselegans), protozoon, filaria, be serotype H4, H7, H3ab etc., do not find to root knot nematode (the most serious plant nematode of damage to crops in the world) and haemonchus contortus and bilharzial virulence (
Http:// www.patents.ibm.comFeitelson etc., Bio/Technology 10:271-275,1992).
Summary of the invention
The objective of the invention is to overcome the prior art deficiency, seed selection has the Bacillus thuringiensis bacterial strain and the insecticidal crystal protein thereof of specificity virulence to plant and animal.
The Tribactur of a kind of plant nematocide and zooparasite (Bacillus thuringiensis) bacterial strain YBT-1532 (CCTCC, NO.M94068, preservation date on November 3rd, 1994), it comprises: a. flagellar antigen serotype is H10; B. this bacterial strain produces the rhombus parasporal crystal; C. the insecticidal crystal protein molecular weight of forming parasporal crystal is 130kDa; D. the-terminal amino acid sequence of this insecticidal crystal protein is: Met-Glu-Ile-Val-Asn-Asn-Gln-Asn-Gln-Cys.
2, according to Bacillus thuringiensis bacterial strain YBT-1532 (a kind of Tribactur of described plant nematocide of claim l and zooparasite, CCTCC NO.M94068, preservation date on November 3rd, 1994), it is characterized in that described insecticidal crystal protein has:
A. plant nematode there is toxic action;
B. zooparasite there is toxic action.
Bacterial strain YBT-1532 of the present invention is to the high virulence of plant root-knot nematode tool, belongs to H10, i.e. serotype tool specificity, and the insecticidal crystal protein of its generation is different with external report, and effective object is difference also.
Separation, the screening of embodiment (one) Bacillus thuringiensis bacterial strain YBT-1532
Adopt acetate to select substratum (being called for short the BPA substratum), separate from soil, and be that the examination worm carries out toxicity test with the diamondback moth larvae, screening obtains bacterial strain of the present invention.
1, BPA culture medium prescription: extractum carnis 5 grams, peptone 10 grams, sodium-acetate 34 grams, 1000 milliliters of distilled water, pH value 7.2-7.4.
2, separate
Take by weighing 1 gram pedotheque in the BPA substratum, fully after the vibration, put 30 ℃ of shaking tables and cultivated 4 hours, take out in 75-80 ℃ of water-bath thermal treatment 10-15 minute, after leaving standstill slightly, inhale 0.5 milliliter and place on the BPA plate culture medium, coating evenly is inverted in 30 ℃ of incubators and cultivated 24 hours, the colony inoculation of selecting 3-5 similar Tribactur is to the BPA inclined-plane, cultivate more than 72 hours for 30 ℃, the carbolfuchsin dyeing microscopic examination will have the chorista of parasporal crystal to be defined as Tribactur.
3, spawn culture, go down to posterity and preservation
Beef-protein medium (extractum carnis 0.5%, peptone 1.0%, NaCl0.5%, agar 2.0%, pH7.2) inclined-plane (18 * 180mm) go up in 30 ℃ of cultivations, can be in 4 ℃ of short term storages, with freeze pipe, or the phase preservation of sand pipe range, as original strain.Producing bacterial classification needs to transfer from original strain, is inoculated on the beef-protein medium inclined-plane.
4, the biology of bacterial strain YBT-1532 and genetics characteristic
1. biological characteristics: thalline direct rod shape, nourishing body is chain or Dan Sheng, long 3-5 micron, wide 1-1.2 micron, Gram-positive, the brood cell is cylindric or near ellipse, and end is given birth to partially, and brood cell's capsule does not expand, bacterium colony circle on beef-protein medium, edge-smoothing, the plentiful wax shape that is of lawn, growth temperature 10-45 ℃, optimal pH 6.8-7.4, facultative suspicion oxygen is grown in the beef-protein medium that contains 7% NaCl, at ribose, glucose, fructose, produce acid in maltose and the Zulkovsky starch substratum, the clark and Lubsreaction positive, the brood cell produces parasporal crystal at later stage of growing, and parasporal crystal assumes diamond in shape, and is not of uniform size.
2. genetics characteristic: Bacillus thuringiensis bacterial strain YBT-1532 is the isolating specific strains of the applicant, flagellar antigen serotype is H10, the parasporal crystal rhombus, parasporal crystal albumen is 130kDa, and its-terminal amino acid sequence is Met-Glu-Ile-Val-Asn-Asn-Gln-Asn-Gln-Cys.
(2) toxicity test (effect of the present invention)
1, specimen preparation
The Tribactur chorista is inoculated on the beef-protein medium inclined-plane, cultivates more than 72 hours for 30 ℃, add the 10ml sterilized water and wash lawn, be broken into even bacteria suspension as stoste.The purification parasporal crystal obtains insecticidal crystal protein with the alkali dissolution parasporal crystal.
2, to the virulence of parasitic nematode
With toxin protein feeding nematode, measure virulence LC to northern root knot nematode (Meloidogyne hapla) (2 instar larvae), Meloidogyne incognita (Meloidogyne incognita) (2 instar larvae), sweet potato stem nematode (becoming larva to mix), Pratylenchidae (Pratylenchus spp.) and ramie Pratylenchidae plant nematodes such as (becoming larva to mix)
50Be respectively 66.5 μ g/ml, 70.6 μ g/ml, 183.2 μ g/ml and 134.7 μ g/ml: zooparasite is had toxic action, to the LC of haemonchus contortus (Haemonchuscontortus) (2 phase larva)
50Be 31.5 μ g/ml; 0.11mg/mlq insecticidal crystal protein is to Wuchereria malayi tool 95% killing rate; Can prolong 6 day life-span to infecting the plasmodial mouse of Bai Shi; Lethality rate to the virgin worm of Schistosoma japonicum (Schistosoma japonicum) is respectively 67.8% and 96.8% at 0.49 μ g/ml and 0.98 μ g/ml.
Application prospect of the present invention:
Compared with the prior art, the present invention has opened up the new way of Plant nematode and parazoon control, and it is not Only enlarged the scope of nematode and parazoon high-efficiency low-toxicity biological pesticide, and reduced because in the past a large amount of Use the chemical pesticide to the pollution of environment and nematode and the parasite resistance to chemical agent, be conducive to environment Protection and reduce the residual hazard of agricultural chemicals on the agricultural product is a kind of scheme of preventing and treating of brand-new animals and plants disease and pest.
Generally speaking, the nematode that control colonizes in plant is difficulty relatively, and is particularly such for root-knot nematode The sucking nematode is especially true. Implement the present invention except directly killing plant, the parazoon, also can kill The worm transgenosis is expressed insecticidal crystal protein in plant, thereby develops a kind of important answering that have Method with the control nematode of prospect.
In addition, in view of the insecticidal crystal protein of thuringiensis has higher desinsection specificity, to people and animals Safety non-toxic, the present invention will provide the extremely albumen of parazoon, be used for controlling animal parasites.
Claims (2)
1, the Tribactur of a kind of plant nematocide and zooparasite (Bacillus thuringiensis) bacterial strain YBT-1532 (CCTCC, NO.M94068, preservation date on November 3rd, 1994), it comprises:
A. flagellar antigen serotype is H10;
B. this bacterial strain produces the rhombus parasporal crystal;
C. the insecticidal crystal protein molecular weight of forming parasporal crystal is 130kDa;
D. the-terminal amino acid sequence of this insecticidal crystal protein is: Met-Glu-Ile-Val-Asn-Asn-Gln-Asn-Gln-Cys.
2, Bacillus thuringiensis bacterial strain YBT-1532 (a kind of Tribactur of plant nematocide according to claim 1 and zooparasite, CCTCC NO.M94068, preservation date on November 3rd, 1994), it is characterized in that described insecticidal crystal protein has:
A. plant nematode there is toxic action;
B. zooparasite there is toxic action.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 00116062 CN1128212C (en) | 2000-09-30 | 2000-09-30 | Parasite-killing thuricinbaillus strain YBT-1532 and parasite killing crystallin |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 00116062 CN1128212C (en) | 2000-09-30 | 2000-09-30 | Parasite-killing thuricinbaillus strain YBT-1532 and parasite killing crystallin |
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| CN1345928A CN1345928A (en) | 2002-04-24 |
| CN1128212C true CN1128212C (en) | 2003-11-19 |
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Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| ES2378040B1 (en) * | 2010-03-31 | 2013-02-18 | Probelte, S.A | A BIONEMATICIDE BIOLOGICAL PREPARATION AND STIMULATOR OF THE VEGETABLE GROWTH AND PURE CROPS OF THE NAMES N11, SR11 AND ALO1, CONTAINED IN THE SAME. |
| CN102703338B (en) * | 2011-03-28 | 2014-05-14 | 华中农业大学 | Bacillus thuringiensis YBT-008 for killing ditylenchus destructor and application thereof |
| CN102311936B (en) * | 2011-09-06 | 2012-12-12 | 湖南省植物保护研究所 | Bacillus thuringiensis strain and parasporal crystal thereof, extraction method of parasporal crystal, and application of the two in controlling plant nematode disease |
| CN114606155B (en) * | 2022-01-25 | 2023-08-04 | 云南大学 | Application of bacillus thuringiensis Bt79 and/or Bt80 strain in preventing and controlling plant southern root-knot nematode |
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