CN101148683A - Method for screening Bacillus thuringiensis nematocidal strain - Google Patents
Method for screening Bacillus thuringiensis nematocidal strain Download PDFInfo
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- CN101148683A CN101148683A CNA2006100690375A CN200610069037A CN101148683A CN 101148683 A CN101148683 A CN 101148683A CN A2006100690375 A CNA2006100690375 A CN A2006100690375A CN 200610069037 A CN200610069037 A CN 200610069037A CN 101148683 A CN101148683 A CN 101148683A
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Abstract
The process of screening nematocidal Bacillus thuringiensis strain includes the steps of soaking bioassay, culture medium bioassay, protein crystal bioassay, etc. The process adopts easy-to-obtain defect type colibacillus JM109, and has short screening period, low cost, high screening efficiency. The screened nematocidal Bacillus thuringiensis strain IPS has a 24 hr LC50 of 1.008 mcg/ml.
Description
Affiliated technical field the present invention relates to screen the method that the nematocidal effect bacterial strain is arranged in microorganism, particularly the method for screening nematicide supper toxic strain in bacillus thuringiensis.
Background technology
Bacillus thuringiensis (Bacillus thuringiensis, be called for short Bt) be a kind of very important entomiasis indigenous bacteria, extensively the distribution hand all over the world, successively existing thousands of bacterial strains are separated, purifying and its insecticidal activity of test, at present found that Bt comprises lepidopteran to many important Agricultural pests, Coleoptera, Diptera, Hymenoptera, mite class and nematode etc. all have high special insecticidal activity, and to people and animals and non-target insect, as natural enemy insect, as safe as a house, therefore being widely used in preventing and treating many important Agricultural pests, is the most successful up to now biotic pesticide.But the report that rarely has the relevant nematicide Bt bacterial strain of report for a long time.
Because plant nematode can't isolated culture, screening method in the past all need carry out in the land for growing field crops.It is long that general land for growing field crops screening method has the cycle, shortcoming such as is difficult to repeat.Also be subjected to the limitation in time place simultaneously.For example, owing to can not get a large amount of nematode populations of same kind, just can't and cause the production loss measuring and calculating that experimentizes to pathogenic, the virulence of this kind nematode.
Biological assay is the significant process of bacterial strain screening work.Cultivate because plant nematode can have external source,, will seriously hinder the screening of nematicide bacterial strain if can not get a large amount of nematode populations of same kind.Therefore, explore nematode do not have approach that external source cultivates cause close important.Nematode hand-feeding propagation and evaluation of pesticide effectiveness program and technology are numerous and diverse, quick, easy, accurate and economic bioassay method the plate inhibition zone method that shortage is used always in the antimicrobial substance screening makes the discovery of nematicide bacterial strain and research speed be subjected to certain influence.
Summary of the invention
Purpose of the present invention: selecting non-parasitic nematode-Samsonite rhabditis axei (Caenorhabditis elegance) is target pest, utilizes the nematode bioassay to screen the strong bacterial strain of eelworm-killing activity in Bt.
The Samsonite rhabditis axei is a kind of model animals, living in the soil with the microorganism is the non-parasitic nematode of food, can be at indoor cultivation, the about 1mm of this worm is long, generation cycle is short, health is transparent, can examine under a microscope these processes of cell fission, differentiation and allelotaxis and genetic analysis and connect.Nematode only needs 3.5d from the egg development adult, because become body length to have only 1mm, handling a large amount of polypides only needs limited space.Polypide is transparent, and the investigator can grow the intravital effect of polypide by the direct viewing compound.In the general animal toxicity screening experiment,, also need usually to study the influence of compound to the animal organ by operation or postmortem even animal is not sick or dead in the poisonous substance environment.This research has obtained stable health with the defective escherichia coli feed, the beautiful rhabditis axei that can constantly breed and preserve.Overcome nematode and given birth to the problem that is difficult to cultivate that runs in the survey.
The method of screening Bacillus thuringiensis nematocidal strain of the present invention is a strains tested with Bt, is target pest with beautiful rhabditis axei, adopts nematode bioassay screening Bacillus thuringiensis nematocidal strain, it is characterized in that may further comprise the steps:
1, soak to give birth to survey: with ovum, larval stage nematode be target pest, cultivate the Bt strain cultured solution behind the 8-10h, 1000-1500r/min is centrifugal, supernatant carries out indoor infection; The aqueous suspensions that will contain the healthy nematode of 100-150 bar, respectively with isopyknic bacterium liquid to be measured thorough mixing on depression slide, with the deionized water is contrast, in biochemical incubator, preserve moisture, 25 ℃, judge the life or death of nematode behind the 24-36h with needle punching, every processing repeats 3-5 time, selects the strong Bt primary dcreening operation bacterial strain of eelworm-killing activity standby;
2, substratum is given birth to and is surveyed: put e. coli jm109 and shake 8-10h in LB in shaking table, with Bt primary dcreening operation bacterial strain activation 24-36h, collect activatory e. coli jm109 and Bt primary dcreening operation bacterial strain again, centrifugal, bacterium liquid is concentrated into 500-1000ul, is uniformly coated on the NGM flat board, and every processing repeats 3-5 time; Healthy nematode is chosen on the NGM flat board of each coating Bt primary dcreening operation bacterial strain; NGM flat board with the coating e. coli jm109 is contrast, behind the 24-36h, selects the strong Bt bacterial strain of eelworm-killing activity standby; Described e. coli jm109 is used to satisfy the normal growth of nematode available from the Stratagene biotech firm of the U.S..
3, albumin crystal is given birth to and surveyed: the Bt inoculation that eelworm-killing activity is strong is in 1/2 liquid LB, and 28-35 ℃, 250-300r/min cultivates 72-84h, and the centrifugal collection thalline of 10000-12000r/min again with aseptic washing 3-5 time, gets Bt bacterial strain structure cell mixture;
4, the high virulence nematicide bacterial strain of screening: adopt 96 hole microtest plates, in every hole, add the healthy nematode of 100-150 tail; Bt bacterial strain structure cell mixture is joined respectively in the culture hole according to the multiple proportions concentration dilution method, and diluent is DDW (ultrapure water), makes every hole cumulative volume reach 500-1000 μ L.Each bacterial strain is provided with 3-5 repetition, median lethal concentration LC
50Minimum bacterial strain is the Bt bacterial strain that eelworm-killing activity is the strongest in the strains tested.
Described Bt strains tested is the Bt reference culture, available from U.S. Stratagene biotech firm, cultivates according to a conventional method; Described Samsonite rhabditis axei adopts conventional NGM culture method to cultivate; Described LC
50Method of calculation be conventional method of calculation.
The nematode that supplies examination is available from the U.S. Samsonite rhabditis axei biological heredity center (CaenorhabditisGenetics Center).
The invention has the advantages that:
1. select the screening object of Samsonite rhabditis axei for use, overcome nematode and given birth to the problem that is difficult to cultivate that runs in the survey as target pest.Because plant nematode is the biology of parasitics, be difficult to cultivate and breeding, so the biological assay of nematode is a very big difficult point always.The present invention explores and has obtained nematode and do not have the approach that external source is cultivated, and the nematode population that has solved same kind is difficult to a large amount of the cultivation and the problems of breeding, helps the screening of Bt nematicide bacterial strain.The rhabditis axei that the present invention uses has the generation cycle weak point, and advantages such as easy cultivation are utilized this model animals, have finished the foundation of Bt nematicide bacterial strain screening system.
2. adopted the defective escherichia coli JM109 of easier acquisition in beautiful rhabditic cultivation, the whole process of the high virulence nematicide of screening Bt bacterial strain only needs several days time, has not only saved cost, and has improved screening efficiency.
The high virulence nematicide bacterial strain that the method for screening Bacillus thuringiensis nematocidal strain of the present invention screens not only has toxic action to rhabditis axei, and its preparation also has good prevention effect to the tomato nematode.
Specific embodiment is illustrated below in conjunction with embodiment in order fully to disclose the method for screening Bacillus thuringiensis nematocidal supper toxic strain of the present invention.
Embodiment: the screening method of Bacillus thuringiensis nematocidal supper toxic strain is a strains tested with Bt, is target pest with beautiful rhabditis axei, and employing is soaked and given birth to survey, the living survey of substratum and albumin crystal bioassay screening Bacillus thuringiensis nematocidal strain:
1, soak to give birth to survey: with ovum, larval stage nematode be target pest, 1000r/min is centrifugal for Bt bacterial strain incubated overnight liquid, supernatant carries out indoor infection experiment; The aqueous suspensions that will contain 100 healthy nematodes with isopyknic bacterium liquid to be measured thorough mixing on depression slide, is contrast with the deionized water respectively, preserve moisture, 25 ℃ of following 24h judge the life or death of nematode with needle punching, and every processing repeats 4 times, selects the strong Bt primary dcreening operation bacterial strain of eelworm-killing activity standby;
2, substratum is given birth to and is surveyed: put e. coli jm109 and shake 8h in LB in shaking table, with Bt primary dcreening operation bacterial strain activation 24h, collect activatory e. coli jm109 and Bt primary dcreening operation bacterial strain again, centrifugal, bacterium liquid is concentrated into 500ul, is uniformly coated on the NGM flat board, and every processing repeats 3 times; Healthy nematode is chosen on the NGM flat board of each coating Bt primary dcreening operation bacterial strain; NGM flat board with the coating e. coli jm109 is contrast, behind the 24h, selects the strong Bt bacterial strain of eelworm-killing activity standby;
3, albumin crystal is given birth to and surveyed: the Bt inoculation that eelworm-killing activity is strong is in 1/2 liquid LB, and 30 ℃, 250r/min cultivates 72h, and the centrifugal collection thalline of 10000r/min is used aseptic washing 3 times again, gets Bt bacterial strain structure cell mixture;
4, the high virulence nematicide bacterial strain of screening: adopt 96 hole microtest plates, in every hole, add the healthy nematode of 100 tails; Bt bacterial strain structure cell mixture is joined respectively in the culture hole according to the multiple proportions concentration dilution method, and diluent is DDW (ultrapure water), makes every hole cumulative volume reach 500 μ L.Each bacterial strain is provided with 6 concentration gradients (seeing Table 2), 3 repetitions.
Described 96 hole microtest plates are placed 25 ℃ of cultivations of biochemical incubator, and the nematode death toll is as shown in table 1.
Median lethal concentration LC
50Minimum bacterial strain is the Bt bacterial strain that eelworm-killing activity is the strongest in the strains tested.(seeing Table 3)
Table 1 NGM substratum is given birth to and is surveyed the result
| Bacterial strain | 1d | 2d | 3d | 4d | 5d | 6d |
| HD-198 IPS 8010 HD-2 HD-293 HD-298 8311 HD-98 7612 E3 HD-1 HD-98 JM109 | Have no on the plate to have no on the worm plate and have no worm on the worm plate and outside flat board, climb plate and have no worm and outside flat board, climb from connecing worm point and outside flat board, climb and began breeding on the 5th day and outside flat board, climb and began to breed plate on the 4th day and have no the worm plate and have no worm and began breeding on the 3rd day and outside flat board, climb from connecing worm point and outside flat board, climb the sign that breed is obviously arranged from connecing worm point from connecing worm point from connecing worm point from connecing worm point | None None None None None None 5 None 5 None None None breeding | None None None 3 10 15＞10 7 None＞10＞10 breedings | None None 3 None None 57＞10＞10＞10＞10＞10 breedings | 35555 10 5＞10＞10＞10 5＞10 breedings | 5 None, 55＞10＞10＞10＞10＞10＞10 None＞10 breedings |
The numeral borer population can be bred substantially when borer population＞10 just illustrate nematode.
Table 2 is for prelibation power protein concentration gradient
| Bacterial strain | Concentration (μ g/ml) | |||||
| HD-2 IPS HD-98 contrasts (NaOH) | 87.907 84.544 86.053 400.000 | 43.953 42.272 43.026 200.000 | 21.977 21.136 21.513 100.000 | 10.988 10.568 10.257 50.000 | 5.494 5.284 5.128 25.000 | 2.747 2.642 2.068 12.500 |
Table 3 Bt reference culture parasporal crystal is to Samsonite rhabditis axei LC
50Comparison (μ g/ml)
| Bacterial strain | 12 hours | 24 hours | 36 hours | 48 hours |
| HD-2 IPS HD-98 | 2.015 2.050 3.121 | 1.094 1.008 3.009 | 1.131 1.107 2.498 | 2.125 1.036 4.020 |
Give birth to the survey result and show, strain HD-2, IPS has obvious toxic action to the nematode growth, and HD-293 and HD-98 have certain restraining effect to the nematode growth.
The Bt toxin does not influence the Samsonite rhabditis axei in 4h, its mortality ratio straight line rises behind the 8h: from first day to the 6th day, its mortality ratio rose to 28% from 5%.Bt bacterial strain and parasporal crystal toxin concentration thereof are the main factors that influences nematode death, and according to this experimental result, the parasporal crystal of different B t bacterial strain is different to the speed of nematode effect, and efficient bacterial strain speed of action is also fast.Though 2 bacterial strains that use in this experiment all have certain killing effect to nematode, its virulence size and inequality, LC
50Prolongation with action time reduces.
Obtain the nematicide vigor bacterial strain of Bt thus.IPS virulence in reference culture is the strongest, its LC
50When effect 12h, 24h, 36h is respectively 2.050,1.008,1.107 μ g/ml.
The fermentation and the application of high virulence nematicide bacterial strain:
High virulence nematicide bacterial strain IPS with screening ferments according to ordinary method, and its fermented liquid carries out infection experiment in pathogenic tomato is potted plant, and potted plant middle knot goitre rate obviously reduces, and the IPS bacterial strain has good prevention effect to root knot nematode.
Claims (2)
1. a method of screening Bacillus thuringiensis nematocidal strain is a strains tested with Bt, is target pest with beautiful rhabditis axei, it is characterized in that adopting the screening of nematode bioassay, may further comprise the steps:
(1) soak to give birth to survey: with ovum, larval stage nematode be target pest, cultivate the Bt strain cultured solution behind the 8-10h, 1000-1500r/min is centrifugal, supernatant carries out indoor infection; The aqueous suspensions that will contain the healthy nematode of 100-150 bar, respectively with isopyknic bacterium liquid to be measured thorough mixing on depression slide, with the deionized water is contrast, in biochemical incubator, preserve moisture, 25 ℃, judge the life or death of nematode behind the 24-36h with needle punching, every processing repeats 3-5 time, selects the strong Bt primary dcreening operation bacterial strain of eelworm-killing activity standby;
(2) substratum is given birth to and is surveyed: put e. coli jm109 and shake 8-10h in LB in shaking table, again with described Bt primary dcreening operation bacterial strain activation 24-36h, collect activatory e. coli jm109 and Bt primary dcreening operation bacterial strain, centrifugal, bacterium liquid is concentrated into 500-1000ul, be uniformly coated on the NGM flat board, every processing repeats 3-5 time; Healthy nematode is chosen on the NGM flat board of each coating Bt primary dcreening operation bacterial strain; NGM flat board with the coating e. coli jm109 is contrast, behind the 24-36h, selects the strong Bt bacterial strain of eelworm-killing activity standby;
(3) albumin crystal is given birth to and surveyed: the Bt inoculation that described eelworm-killing activity is strong is in 1/2 liquid LB, and 28-35 ℃, 250-300r/min cultivates 72-84h, and the centrifugal collection thalline of 10000-12000r/min again with aseptic washing 3-5 time, gets Bt bacterial strain structure cell mixture;
(4) the high virulence nematicide bacterial strain of screening: adopt 96 hole microtest plates, in every hole, add the healthy nematode of 100-150 tail; Described Bt bacterial strain structure cell mixture is joined respectively in the culture hole according to the multiple proportions concentration dilution method, and diluent is DDW, makes every hole cumulative volume reach 500-1000 μ L.Each bacterial strain is provided with 3-5 repetition, median lethal concentration LC
50Minimum bacterial strain is the Bt bacterial strain that eelworm-killing activity is the strongest in the strains tested.
2. a kind of method of screening Bacillus thuringiensis nematocidal strain according to claim 1 is characterized in that
(1) soak to give birth to survey: with ovum, larval stage nematode be target pest, 1000r/min is centrifugal for Bt bacterial strain incubated overnight liquid, supernatant carries out indoor infection experiment; The aqueous suspensions that will contain 100 healthy nematodes with isopyknic bacterium liquid to be measured thorough mixing on depression slide, is contrast with the deionized water respectively, preserve moisture, 25 ℃ of following 24h judge the life or death of nematode with needle punching, and every processing repeats 4 times, selects the strong Bt primary dcreening operation bacterial strain of eelworm-killing activity standby;
(2) substratum is given birth to and is surveyed: put e. coli jm109 and shake 8h in LB in shaking table, with Bt primary dcreening operation bacterial strain activation 24h, collect activatory e. coli jm109 and Bt primary dcreening operation bacterial strain again, centrifugal, bacterium liquid is concentrated into 500ul, is uniformly coated on the NGM flat board, and every processing repeats 3 times; Healthy nematode is chosen on the NGM flat board of each coating Bt primary dcreening operation bacterial strain; NGM flat board with the coating e. coli jm109 is contrast, behind the 24h, selects the strong Bt bacterial strain of eelworm-killing activity standby;
(3) albumin crystal is given birth to and surveyed: the Bt inoculation that eelworm-killing activity is strong is in 1/2 liquid LB, and 30 ℃, 250r/min cultivates 72h, and the centrifugal collection thalline of 10000r/min is used aseptic washing 3 times again, gets Bt bacterial strain structure cell mixture;
(4) the high virulence nematicide bacterial strain of screening: adopt 96 hole microtest plates, in every hole, add the healthy nematode of 100 tails; Bt bacterial strain structure cell mixture is joined respectively in the culture hole according to the multiple proportions concentration dilution method, and diluent is DDW, makes every hole cumulative volume reach 500 μ L; Each bacterial strain is provided with 6 concentration gradients, 3 repetitions, and LC is chosen in 25 ℃ of cultivations in biochemical incubator
50Minimum bacterial strain.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102311936A (en) * | 2011-09-06 | 2012-01-11 | 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 |
| CN111378715A (en) * | 2020-02-29 | 2020-07-07 | 广西壮族自治区兽医研究所 | Caenorhabditis elegans model for identifying bovine escherichia coli virulence |
-
2006
- 2006-09-20 CN CNA2006100690375A patent/CN101148683A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102311936A (en) * | 2011-09-06 | 2012-01-11 | 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 |
| 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 |
| CN111378715A (en) * | 2020-02-29 | 2020-07-07 | 广西壮族自治区兽医研究所 | Caenorhabditis elegans model for identifying bovine escherichia coli virulence |
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