CN103160450A - Broad-spectrum and highly effective new Bacillus thuringiensis strain, its bacterial agent and application - Google Patents
Broad-spectrum and highly effective new Bacillus thuringiensis strain, its bacterial agent and application Download PDFInfo
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Abstract
Belonging to the field of biological control of pests, the invention discloses a Bacillus thuringiensis strain that has a broad spectrum and is highly effective on lepidoptera pests, preparation of its wettable powder and application. The Bacillus thuringiensis YX-1 strain involved in the invention is preserved in China General Microbiological Culture Collection Center, and the preservation number is CGMCC No. 5256. Containing a plurality of insecticidal genes, the strain has a highly effective lethal effect on Cydia molesta, cotton bollworm, fall webworms, Adoxophyes orana Fisher von Roslerstamm, Phalera flavescens Bremer et Grey, carpocapsa pomonella, prodenia litura, plutella xylostella, beet armyworm and other lepidoptera pests. The invention also discloses a method for preparation of wettable powder through the YX-1 strain. The Bacillus thuringiensis YX-1 strain and its wettable powder provided in the invention are mainly used for prevention and treatment of lepidoptera pests on various fruit trees and vegetables, also can be used for prevention and treatment of lepidoptera pests on field crops and garden plants, and have the characteristics of broad spectrum, high efficiency, safety to people, livestock as well as animals and plants, and no environmental pollution, etc.
Description
Technical field
The present invention relates to a bacillus thuringiensis strain, also relate to the microbial pesticide, the preparation method and application that utilize the preparation of this bacillus thuringiensis, belong to the biological control of insect pests field.
Background technology
Lepidopteran belongs to Pterygota, Holometabola, known approximately 200,000 kinds of the whole world, and China is known approximately more than 8000 plants.This order is the 2nd the large order that is only second to Coleoptera in Insecta, and distribution range is extremely wide, and is the abundantest with tropical kind.This order comprises moth, butterfly two class insects, the various plants such as harm grain, cotton, oil plant, vegetables, tobacco, flowers, herbage, lawn, fruit tree, and almost each farm crop all is subjected to the wherein harm of one or more lepidoptera pests.In order to control the harm of these insects, the a large amount of high poison of annual immoderate use, high residue chemical insecticide, the environmental pollution that causes thus, residually increase, the series of problems such as resistance enhancing, have a strong impact on agricultural products in China and eating safely production, be badly in need of development in production to the effective non-environmental pollution control technology of lepidoptera pest.
Bacillus thuringiensis (
Bacillus thuringiensis, be called for short Bt) and owing to having good disinsection effect, characteristics such as high specificity is produced and easy to use, and is free from environmental pollution and brought into play vital role in biological control of insect pests.Different Bt bacterial strain produces different insecticidal crystal protein matter (Insecticidal Cyrstal Proteins is called for short ICPs), make insecticidal spectrum between bacterial strain and insecticidal activity exist notable difference (
Bacillus thuringiensisAnd its pesticidal crystal proteins.Microbiology and Molecular Biology Reviews.1998,62 (3): 775-806), this just provides the broad space for screening for the new bacterial strain that the different sorts lepidoptera pest has the specificity insecticidal activity.isolation identification bacillus thuringiensis, the screening aspect, document " the separation of bacillus thuringiensis and the evaluation " isolation identification of orchard, Hebei province bacillus thuringiensis bacterial strain (North China agronomy newspaper, 2010, 25 (4): 201-205) ", " separation of China's Bacillus thuringiensis from Soil and genotypic evaluation (are used and the environmental organism journal, 2005, 11 (6): 756-759) " reach " the multifarious research of Bacillus thuringiensis in Some Areas of Hebei province (Agriculture of Anhui science, 2007, 35 (18): 5540-5541) ", " screening of bacillus thuringiensis and preliminary evaluation (biotechnology, 2003, 13 (6): 34-36) " etc. China bacillus thuringiensis is separated, identify, characteristic distributions, crystal habit, insecticidal properties etc. are studied.efficiently, the screening aspect of specific strains, document " the separation of the efficient Bt bacterial strain of small cabbage moth, biochemical characteristic and genotype identification (applied entomology newspaper, 2011, 48 (2): 285-290) ", " strain is identified (Chinese agronomy circular to the killing gene of the high virulence bacillus thuringiensis of fall webworms, 2010, 26 (3): 242-244) ", " biological characteristic research (the JOURNAL OF MICROBIOLOGY of anti-Noctuidae bacillus thuringiensis WY-197 bacterial strain, 2005, 25 (1): 25-28) ", " separation and evaluation (the Sichuan Agricultural University journal of a strain to the Bacillus thuringiensis subspecies of the high virulence of lepidopteran, 2006, 24 (2): 152-155) " cry genetic analysis and insecticidal properties (the microorganism journal of the bacillus thuringiensis Gadamer subspecies 15A3 of section strain, 2002, 42 (2): 169-174) " philosophy has screened the efficient specific strains of killing lepidoptera pest from numerous bacillus thuringiensiss.aspect the fermentation and dosage form research of bacterial strain, document " optimization (the life science of Bacillus thuringiensis Bt0601 strain fermentation substratum, 2007, 11 (4): 323-327) ", the optimization of Bt Medium Proportion and parasporal crystal purification condition (East China University of Science's journal (natural science edition), 2006, 32 (3): 285-289) ", " research (the JOURNAL OF MICROBIOLOGY of Bacillus thuringiensis "79007" strain fermentation process, 2006, 26 (3): 5-8) ", " optimization research (chemistry and the biotechnology of High Concentration Liquid Culture for Bacillus Thuringiensis, 2006, 23 (9): 38-39) ", " screening (Hua Zhong Agriculture University's journal of bacillus thuringiensis finish synergistic factor, 2000, 19 (2): 134-137) ", " development (the hubei agricultural science of 17600 IU/mg bacillus thuringiensis suspension concentrates, 2010, 49 (12): 3048-3051) " etc. the fermentation of bacillus thuringiensis and formulation aspect are studied.
Thuringiensis cladosporioides bacillus insecticide is a kind of free of contamination biological pesticide, for insecticidal spectrum and the raising insecticidal activity that enlarges bacillus thuringiensis, all is devoted to the isolation identification of new bacterial strain and the exploitation of new killing gene both at home and abroad.But, the separation of the bacillus thuringiensis of broad-spectrum high efficacy and use still seldom report, particularly for the Bt bacterial strain of lepidoptera pest on fruit tree and vegetables still less.Bt YX-1 is that a natural broad-spectrum high efficacy of strain that is separated at present kills the bacillus thuringiensis of lepidoptera pest, contain a plurality of insect-killing protein encoding genes, the insects such as multiple lepidoptera pest such as fall webworms, bollworm, prodenia litura, beet armyworm, Pyrausta nubilalis (Hubern)., small cabbage moth, oriental fruit months, apple olethreutid, Phalera flavescens and carpocapsa pononella on fruit tree and vegetables all there is the efficiently poisoning effect, leavening property is good, is the natural bacillus thuringiensis that a strain has development prospect.
Summary of the invention
One of purpose of the present invention is to provide the bacillus thuringiensis YX-1(that a strain has higher virulence to multiple lepidoptera pests such as fall webworms, bollworm, prodenia litura, beet armyworm, Pyrausta nubilalis (Hubern)., small cabbage moth, oriental fruit months, apple olethreutid, Phalera flavescens and carpocapsa pononellas
Bacillus thuringiensisYX-1).Another object of the present invention is that short, unstable properties of the lasting period that exists for thuricade-1 in the past, onset wait problem slowly, a kind of preparation method of Bt YX-1 wettable powder is provided, can be used for the control of fruit tree and vegetables and other plant lepidoptera pest with the sterilant of the method preparation, be characterized in that insecticidal spectrum is wide, lasting period is long, and efficacy stability does not does not kill and wound Natural Enemies of Insects, nontoxic to people, animal, environmentally safe.
The present invention is achieved through the following technical solutions:
Bacterial strain of the present invention is to separate the new bacterial strain of the bacillus thuringiensis (Bacillus thuringiensis) that obtains in peanut ground, Yi County, Baoding, Hebei province soil, (be called for short CGMCC on September 17th, 2011 at China Committee for Culture Collection of Microorganisms's common micro-organisms center, the address is: No. 1, North Star West Road, Chaoyang District, BeiJing, China city institute, Institute of Microorganism, Academia Sinica, postcode 100101) preservation, numbering: CGMCC № .5256, name is called bacillus thuringiensis YX-1(Bacillus thuringiensis YX-1).
Bacillus thuringiensis YX-1 specifically obtains by the following method: adopt sodium-acetate-Antibiotics separation method, take the 10g soil sample and put into the shaking flask that 50mL sodium-acetate substratum is housed, add penicillin sodium salt 400 μ g/mL, 200rpm/min, 30 ℃ of shaking tables are cultivated 4h.The earth suspension 10mL that fetches earth after cultivate finishing adds the centrifugal 15min of 308g in aseptic centrifuge tube, gets upper strata turbid solution 1.5mL in 65 ℃ of water-bath water-bath 15min, and the turbid solution 0.2mL after heat-obtaining is processed is coated with flat board, flat board is put in 30 ℃ of incubators cultivated.After 3-4d from the flat board the bacterial strain smear of the similar Bt of picking, find that a strain contains the Bt bacterial strain of rhomboidan form, form code YX according to the initial of the title (Yi County) in its pedotheque source place, again because of this bacterial strain be from screen in the many bacillus thuringiensis strains that separate a collection of pedotheque and be numbered a strain of 1, therefore with this bacillus thuringiensis called after YX-1.
Through identifying, comprise about the information of this bacterial strain: can form gemma, can form rhombus parasporal crystal (see figure 1) simultaneously; Its crystallin begins to express at 14h, and growth curve shows that its 14h has entered (see figure 2) stationary phase; Analyze through PCR-RFLP, the insecticidal crystal protein plasmagene of Bt YX-1 is
Cry1Ac,
Cry2Ac,
Cry1I,
Vip3Aa,
Cry34-35(seeing Fig. 3 and Fig. 4); The SDS-PAGE electrophoresis shows, Bt YX-1 mainly produces approximately 130kD а and two kinds of insecticidal crystal protein matter (see figure 5)s of 65kD а; Bioassay results shows (seeing Table l), Bt YX-1 has higher insecticidal activity to multiple lepidoptera pests such as fall webworms, bollworm, prodenia litura, beet armyworm, Pyrausta nubilalis (Hubern)., small cabbage moth, oriental fruit months, apple olethreutid, Phalera flavescenses, wherein responsive to fall webworms and small cabbage moth, LC
50Value is respectively 14.48mg/L, 15.03mg/L, and is the poorest to the susceptibility of prodenia litura, LC
50Value is 6.24 * 10
4Mg/L, the LC of this bacterium to these insects
50Value is all lower than the LC of reference culture Bt HD-1 to these insects
50Value.Bt YX-1 also has insecticidal activity to the carpocapsa pononella larva, and the 72h corrected mortality reaches 86.5 ± 6.8%.
Median lethal dosage (the LC of table 1 Bt YX-1 to 9 kinds of insects of lepidopteran
50)
Shake flask fermentation: the purpose of shake flask fermentation is to select best fermentating formula, and optimization of fermentation conditions takes different solid contents, different C/N ratio and different inorganic salt to carry out orthogonal test.
1. the selection of fermention medium:
(1) carbon source, nitrogenous source screening
Select 7 factors such as soybean cake powder, groundnut meal, fish meal, peptone, Semen Maydis powder, yeast powder, sucrose, each factor selects high, medium and low 3 concentration to carry out orthogonal experimental design, KH in different culture media
2PO
4Be fixed as 1, pass through L
18(3
7) orthogonal test is optimized each component.Take soybean cake powder, groundnut meal as major nitrogen source, take Semen Maydis powder as main carbon source, investigate the different culture media component to the impact of fermented liquid virulence and living spores number, orthogonal design sees Table 2.
Table 2 L
18(3
7) orthogonal test level of factor table (g/L)
| Level | Soybean cake powder | Groundnut meal | Fish meal | Peptone | Semen Maydis powder | | Sucrose | |
| 1 | 30 | 30 | 10 | 3 | 16 | 6 | 1 | |
| 2 | 32 | 32 | 12 | 6 | 14 | 3 | 2 | |
| 3 | 34 | 34 | 16 | 9 | 18 | 9 | 3 |
(2) inorganic salt screening
With L
18(3
7) the better substratum determined of orthogonal test is basic medium, add respectively sal epsom, potassium primary phosphate, calcium carbonate, ferric sulfate of different concns etc., investigate inorganic salts to the impact of fermented liquid virulence, every kind of inorganic salt select high, medium and low 3 concentration, adopt L
9(3
4) orthogonal table carries out test design, the experimental design scheme sees Table 3.
Table 3 L
9(3
4) orthogonal test level of factor table (g/L)
| Level | MgSO 4·7H 2O | KH 2PO 4 | CaCO 3 | FeSO 4·7H 2O |
| 1 | 0.2 | 1 | 0.1 | 0.02 |
| 2 | 0.4 | 2 | 0.5 | 0.06 |
| 3 | 0.6 | 4 | 1 | 0.04 |
According to orthogonal test, the optimal medium formula that obtains Bt YX-1 is: soybean cake powder 32g/L, groundnut meal 30g/L, fish meal 16g/L, peptone 6g/L, Semen Maydis powder 20g/L, yeast powder 6g/L, sucrose 1g/L, MgSO
47H
2O 0.2g/L, KH
2PO
41g/L, CaCO
30.5g/L, FeSO
47H
2O 0.06g/L, remaining person's water are supplied.
2. fermentation culture step:
(1) actication of culture: the bacterial strain YX-1 that will be stored in-20 ℃ activates (30 ℃) on the LB plate culture medium, and picking list bacterium colony expands numerous (30 ℃) on the LB slant medium standby, and gained is as the inoculation bacterium;
(2) preparation of seed liquor: make according to a conventional method the LB liquid nutrient medium, the 100mL LB nutrient solution of packing in the 250mL triangular flask, the high pressure moist heat sterilization, after temperature drops to room temperature, every bottle graft enters step (1) the one good YX-1 bacterial strain of the above-mentioned activation of transfering loop, carry out shaking culture on shaking table, 30 ℃, 180rpm are cultivated 24h, and gained is as seed liquor;
(3) preparation fermention medium: according to the formulated substratum of above-mentioned fermention medium, be sub-packed in the 500mL triangular flask, every bottle of 200mL, 121 ℃ of sterilization 30min are down to after room temperature standby;
(4) fermentation culture: carry out fermentation culture under inoculation step (2) gained seed liquor 2mL in every bottle of fermention medium of step (3) gained, 30 ℃, 180rpm condition;
(5) after 24h, carry out microscopy every 1h from the triangular flask of step (4), the gemma in the visual field and total thalline number are counted, and calculated the gemma rate; The gemma rate reaches 90% can stop fermentation culture; Both got bacillus thuringiensis YX-1 liquid preparation.
3. formulation preparation:
A kind of Bacillus thuringiensis wettable powder mainly is comprised of the brilliant former powder of mixture of quantitative bacillus thuringiensis spore and quantitative inactive substance, wherein:
(1) formula of this wettable powder is: the former powder 40% ~ 50% of Bacillus thuringiensis, dispersion agent 5%～10%, wetting agent 5%～10%, stablizer 1%, ultraviolet radiation protectant 1%, carrier 28%～48%.
(2) described dispersion agent can be sodium lignosulfonate, CNF, HK-2302, NNO or dispersing agent MF; Wetting agent can be Morwet EFW, washing powder, excrementum bombycis meal, dodecyl phenenyl sulfate, Sodium dodecylbenzene sulfonate or pull open powder; Stablizer can be xanthan gum or Xylo-Mucine; Ultraviolet radiation protectant can be skim-milk, humic acid, vitamins C or carboxymethyl cellulose; Carrier can be white carbon black, kaolin, diatomite, light calcium carbonate or talcum powder; The former powder of described Bacillus thuringiensis is that Bacillus thuringiensis is through freeze-drying after centrifugal concentrating or the spray-dried former powder of the brilliant mixing of spore;
(3) preparation method: the former powder of Bt and above-mentioned other raw material is good according to the said ratio weighing, after pulverizing with the pulverize at low temperature airflow machine, mix, sieve and make the following fine particle of 44 μ m, both got Bt YX-1 wettable powder;
(4) Bt YX-1 wettable powder toxicity evaluation measuring method: with Bt standard substance (the ProductName green 16000IU/mg Bacillus thuringiensis wettable powder of making a living, available from Hubei health novel pesticide technology company limited) and the Bt testing sample dilute successively, select 5 suitable concentration gradients, carry out biological assay take small cabbage moth as the examination worm by immersion method.Be about to the fresh cabbage leaves of in the same size and flood 10s in individual concentration for the treatment of solution, put into after naturally drying to give birth to and survey box, the consistent second instar larvae of every box access 15 head growths, each is processed and repeats 3 times, does blank with sterilized water.The examination worm is placed under (26 ± 1) ℃, 65% relative humidity, 14h illumination condition to be cultivated, and records 72h and respectively processes the dead borer population of larva.Then utilize DPS 7.05 softwares to calculate respectively Bt standard substance LC
50The LC of value and Bt testing sample
50Be worth, be calculated as follows the toxicity evaluation of testing sample:
In formula: X---testing sample toxicity evaluation
S---standard substance LC
50Value
The P---standard substance are tired
Y---testing sample LC
50Value
The present invention compared with prior art, have the following advantages: this invention is for lepidoptera pest important on fruit tree, vegetables, the insects such as fall webworms, bollworm, prodenia litura, beet armyworm, Pyrausta nubilalis (Hubern)., small cabbage moth, oriental fruit months, apple olethreutid, Phalera flavescens and carpocapsa pononella all there is good preventive effect, insecticidal spectrum is wide, and its virulence is all higher than reference culture Bt HD-1.Utilize this advantage of bacterial strain, the Bt YX-1 wettable powder of developing both can be used for preventing and treating the lepidoptera pest on multiple fruit tree, vegetables, also can be used for preventing and treating the lepidoptera pest on field crop and ornamental plant.
Description of drawings:
Fig. 1: the opticmicroscope picture of the gemma of Bt YX-1 and crystal
Fig. 2: the growth curve of Bt YX-1 and pH value change curve
Fig. 3: the pcr amplification collection of illustrative plates of Bt YX-1, wherein:
M:DNA molecular weight standard (8kb, 5kb, 3kb, 2kb, 1kb, 0.75kb, 0.5kb, 0.25kb, 0.1kb)
1-6:Bt YX-1
Cry1,
Cry2,
Cry1I,
Vip3A,
Cry34-35The PCR product
Fig. 4: the restriction analysis collection of illustrative plates of Bt YX-1, wherein:
M:DNA molecular weight standard (8kb, 5kb, 3kb, 2kb, 1kb, 0.75kb, 0.5kb, 0.25kb, 0.1kb)
1：
cry1-KUN2/PstI＆EcoRI
2：
cry1-KUN3/PstI＆XbaI
3：cry2/MspI＆HincⅡ
4：Vip3A/HindⅢ＆EcoRI
Fig. 5: Bt YX-1 crystallin SDS-PAGE collection of illustrative plates.
Embodiment
Following example is further to explanation of the present invention, should not become limitation of the present invention.
In the following example, if no special instructions, be ordinary method.
In the following example, described percentage composition is the quality percentage composition if no special instructions.
The separation of embodiment 1, bacillus thuringiensis YX-1 and evaluation
The inventor has separated Bt bacterial strain YX-1 voluntarily from soil, soil derives from Yi County, Baoding, Hebei province.
Taking the 10g soil sample puts into 50mL sodium-acetate liquid nutrient medium (peptone 0.5g is housed, yeast powder 0.25g, NaCl 0.5g, sodium-acetate 1.8g, water 50mL, adjust pH 7.2-7.4,121 ℃, autoclaving 30min) in shaking flask, add penicillin sodium salt 400 μ g/mL, shaking table is cultivated (200rpm/min, 30 ℃) 4h.Earth suspension 10mL fetches earth after cultivation finishes, add the centrifugal 15min of 308g in aseptic centrifuge tube, get upper strata turbid solution 1.5mL in 65 ℃ of water-bath water-bath 15min, the turbid solution 0.2mL after heat-obtaining is processed is coated with LB solid medium (peptone 2g, yeast powder 1g, NaCl 2g, agar powder 3.2g, water 200mL, adjust pH 7.2-7.4,121 ℃, autoclaving 30min) flat board, flat board is put in 30 ℃ of incubators cultivated.The bacterial strain smear of 3-4d similar Bt of picking from the flat board with basic fuchsin and Victoria Green WPB dyeing, at the separation rate of oily Microscopic observation Jing bodily form Zhuan ﹑ size and gemma and crystal, finds that a strain contains the bacterial strain of rhomboidan, called after YX-1.
Bt YX-1 in the LB solid medium on 30 ℃ cultivate 24h, form the light yellow bacterium colony of needle point size, edge-smoothing is cultivated 72h, forms discoid bacterium colony, oyster white, the edge is irregular, there is wrinkle on the surface like obscure glass.The brilliant mixture of spore is coated on slide glass, with spirit lamp heat fixation, Victoria Green WPB and basic fuchsin dyeing, the shape (see figure 1) of optical microphotograph Microscopic observation crystal, as seen the nourishing body cell is shaft-like, single or one-tenth short chain shape, and sporocyst has no and expands, gemma is oval, and parasporal crystal assumes diamond in shape.
Above-mentioned bacillus thuringiensis (Bacillus thuringiensis) YX-1, be preserved in Chinese microorganism strain preservation board of trustee reason person on September 17th, 2011 and understand common micro-organisms center (abbreviation CGMCC, address: No. 1, North Star West Road, Chaoyang District, BeiJing, China city institute, Institute of Microorganism, Academia Sinica, postcode 100101), preserving number is CGMCC № .5256, title bacillus thuringiensis YX-1(Bacillus thuringiensis YX-1).
The growth characteristics of embodiment 2, bacillus thuringiensis YX-1 are observed
Bt YX-1 is inoculated in to activate in the LB liquid nutrient medium spends the night, the inoculum size by 1% connects bacterium in the LB liquid nutrient medium, and shaking culture (28 ℃, 230rpm/min) is every 2h sampling and measuring OD
600, survey altogether 24h.Measure simultaneously the pH value of nutrient solution, and observe the growth and development state of bacterial strain in its fermenting process.
Bacillus thuringiensis YX-1 is well-grown in the LB liquid nutrient medium, in growth cycle 24h, can normally produce brood cell and parasporal crystal.OD every 2h sampling and measuring nutrient solution
600Value and pH value are drawn growth curve and pH change curve (seeing Fig. 2), and observe the development condition of bacterial strain in its fermenting process.Wherein 0～4h is latent period, and the pH value changes little; 4～14h is logarithmic phase, and the 14h nourishing body begins to occur endogenous spore, and gemma is positioned at the central authorities of bacillus, and the pH value is first decline rapidly, the phenomenon of rear slow rise, and pH 5.89 touches the bottom when 10h; 14～24h is stationary phase, has about 80% nourishing body gemma to occur, and gemma is positioned at the central authorities of bacillus, and crystallin is positioned at the two ends of bacillus, but occurs that spore is brilliant to be separated, and this stage pH value is rising, and during to 24h, pH reaches 7.53.
The genotype identification of embodiment 3, bacillus thuringiensis YX-1
The extraction of the total DNA of Bt YX-1 is with reference to Narva (Novel
Bacillus thuringiensisMicrobes active against nematodes, and genes encoding novel nematodes-active toocin from
Bacillus thuringiensisIsolates. European Patent Office.1991, EP0462721) method.
Utilize Bt cry gene identification system (Identification of novel cry-type genes from
Bacillus thuringiensisStrains on the basis of restriction fragment length polymorphism of the PCR-amplified DNA. Appli-ed and Environmental Microbiology.1996,62:1369-1377; The foundation of bacillus thuringiensis cry gene PCR-RFLP identification system. Scientia Agricultura Sinica, 1998,31 (3): 13-18), take total DNA of Bt YX-1 as template, carry out pcr amplification with 31 pairs of universal primers, result shows that Bt YX-1 contains
Cry1,
Cry2,
Cry1I,
Vip3A,
Cry34-35The gene (see figure 3).Increase with K5un2/K3un2, K5un3/K3un3, S5un2/S3un2, four pairs of primers of vip3A-1/vip3A-2, amplified production carries out the restriction analysis (see figure 4), judges according to this result, and Bt YX-1 contains
Cry1Ac,
Cry2Ac,
Cry1I,
Vip3Aa,
Cry34-35Gene.
The SDS-PAGE of embodiment 4, bacillus thuringiensis YX-1 crystallin analyzes
bacterium colony in the brilliant separation of spore of oily Microscopic observation, scraping the lawn that takes a morsel fully is dissolved in 100 μ L aqua sterilisas, vibrate resuspended, centrifugal (4 ℃, 15294g, 5min), abandon supernatant, after resuspended with 200mL 1M NaCl, centrifugal (4 ℃, 15294g, 5min), abandon supernatant, after using again aqua sterilisa resuspended, centrifugal (4 ℃, 15294g, 5min), abandon supernatant, precipitation is resuspended in 20 μ L aqua sterilisas, 2 * the sample-loading buffer that adds equal volume, after mixing, 100 ℃ are boiled 5min, centrifugal (4 ℃, 15294g, 1min), get supernatant and be used for the SDS-PAGE detection, adopt 5% concentrated glue, 8% separation gel, adopt the constant current electrophoresis, electrophoresis is used coomassie brilliant blue staining after finishing.Simultaneously make positive control with reference culture HD-1, make test sample with same method, the results are shown in Figure 5.
As seen from Figure 5, Bt YX-1 contains two albumen master bands that molecular weight is approximately 130kDa and 65kDa.
Spore is brilliant mixes the mother liquor preparation: respectively Bt YX-1 and Bt HD-1 are inoculated on LB solid plate substratum 28 ℃ and cultivate microscopy after 72h, when producing a large amount of crystal, add appropriate aqua sterilisa, thalline is scraped from flat board, put into centrifuge tube, 15294g is centrifugal, and 5min abandons supernatant, gets to be precipitated as the brilliant mixture of spore, and adding the spore that is prepared into uniform 500mg/mL after the abundant mixing of a certain amount of sterilized water after weighing, brilliant to mix mother liquor standby.
Respectively the brilliant mother liquor that mixes of the spore of Bt YX-1 and Bt HD-1 is diluted to the series concentration gradient, feed for the examination insect by following two kinds of methods, and each is processed under the condition that examination worm is placed in (26 ± 1) ℃, 60%～70% relative humidity, 14h illumination cultivate, observe the upgrowth situation of examination worm after 72h, record dead borer population, adopt the DPS data handling system to calculate LC
50Value.
To the biological activity determination of 3 instar larvaes of 2 instar larvaes of bollworm, beet armyworm, Pyrausta nubilalis (Hubern). and oriental fruit months with artificial diet mixed feeding method.Add the brilliant plastc ring of 1mL spore by every 10g feed, fully mix, average mark is loaded in 24 orifice plates, 1 examination worm of every hole access.Each is processed and repeats 3 times, repeats 24 for the examination insect at every turn, processes feed in contrast with sterilized water.
Biological activity determination leaf dipping method to 3 instar larvaes of 2 instar larvaes of fall webworms, prodenia litura, small cabbage moth and Phalera flavescens and apple olethreutid.Get fresh Eucommia ulmoides Oliv. leaves, castor-oil plant blade, cabbage leaves and Apple Leaves, dipping 10s in the brilliant plastc ring of the Bt spore for preparing (including 0.3% POLYSORBATE 80), put into 4 * 9cm and give birth to the survey box after taking-up is so dried respectively, 20 of every box access larvas.Each is processed and repeats 3 times, repeats 20 examination worms at every turn, does blank with sterilized water (including 0.3% POLYSORBATE 80) dipping blade.
Adopt feed mixed feeding method to measure Bt YX-1 to the insecticidal activity of carpocapsa pononella 3 instar larvaes.Utilize the artificial diet of oriental fruit months, add the brilliant mixture (20mg/mL) of 1mL spore by the 10g feed, be distributed into 24 holes after mixing and give birth in drafting board, 1 examination worm of every hole access.Each is processed and repeats 3 times, repeats 24 for the examination insect at every turn, processes feed in contrast with sterilized water.Observe the upgrowth situation of examination worm after 72h, record dead borer population.
As can be seen from Table 1, this bacterial strain all has insecticidal activity to the larva of fall webworms, bollworm, prodenia litura, beet armyworm, Pyrausta nubilalis (Hubern)., small cabbage moth, oriental fruit months, apple olethreutid and Phalera flavescens, and to the median lethal dosage (LC of these insects
50Value) all lower than the LC of reference culture Bt HD-1 to these insects
50Value.
Bt YX-1 bacterial strain also has insecticidal activity to the carpocapsa pononella larva, and the 72h corrected mortality reaches 86.5 ± 6.8%.
The shake flask fermentation of embodiment 6, bacillus thuringiensis YX-1
1. the selection of fermention medium:
The purpose of shake flask fermentation is to select best fermentating formula, and optimization of fermentation conditions takes different solid contents, different C/N ratio and different inorganic salt to carry out orthogonal test.
(1) carbon source, nitrogenous source screening
Select 7 factors such as soybean cake powder, groundnut meal, fish meal, peptone, Semen Maydis powder, yeast powder, sucrose, each factor selects high, medium and low 3 concentration to carry out orthogonal experimental design, KH in different culture media
2PO
4Be fixed as 1, pass through L
18(3
7) orthogonal test is optimized each component.Take soybean cake powder, groundnut meal as major nitrogen source, take Semen Maydis powder as main carbon source, investigate the different culture media component to the impact of fermented liquid virulence and living spores number, orthogonal design sees Table 2.
(2) inorganic salt screening
With L
18(3
7) the better substratum determined of orthogonal test is basic medium, add respectively sal epsom, potassium primary phosphate, calcium carbonate, ferric sulfate of different concns etc., investigate inorganic salts to the impact of fermented liquid virulence, every kind of inorganic salt select high, medium and low 3 concentration, adopt L
9(3
4) orthogonal table carries out test design, the experimental design scheme sees Table 3.
According to orthogonal test, the optimal medium formula that obtains Bt YX-1 is: soybean cake powder 32g/L, groundnut meal 30g/L, fish meal 16g/L, peptone 6g/L, Semen Maydis powder 20g/L, yeast powder 6g/L, sucrose 1g/L, MgSO
47H
2O 0.2g/L, KH
2PO
41g/L, CaCO
30.5g/L, FeSO
47H
2O 0.06g/L, remaining person's water are supplied.
2. fermentation culture step:
(1) actication of culture: the bacterial strain YX-1 that will be stored in-20 ℃ activates (30 ℃) on the LB plate culture medium, and picking list bacterium colony expands numerous (30 ℃) on the LB slant medium standby, and gained is as inoculation liquid;
(2) preparation of seed liquor: make according to a conventional method the LB liquid nutrient medium, the 100mL LB nutrient solution of packing in the 250mL triangular flask, the high pressure moist heat sterilization, after temperature drops to room temperature, every bottle graft enters step (1) the one good YX-1 bacterial strain of the above-mentioned activation of transfering loop, carry out shaking culture on shaking table, 30 ℃, 180rpm are cultivated 24h, and gained is as seed liquor;
(3) preparation fermention medium: according to the formulated substratum of above-mentioned fermention medium, be sub-packed in 500 mL triangular flasks, every bottle of 200mL, 121 ℃ of sterilization 30min are down to after room temperature standby;
(4) fermentation culture: carry out fermentation culture under inoculation step (2) gained seed liquor 2mL in every bottle of fermention medium of step (3) gained, 30 ℃, 180rpm condition and cultivate;
(5) after 24h, carry out microscopy every 1h from the triangular flask of step (4), the gemma in the visual field and total thalline number are counted, and calculated the gemma rate; The gemma rate reaches 90% can stop fermentation culture; Both got bacillus thuringiensis YX-1 liquid preparation.
The former powder preparation of embodiment 7, bacillus thuringiensis YX-1
The preparation method of Bt YX-1 lyophilized powder completes realization: a according to the following steps, the Bacillus thuringiensis YX-1 that hides that goes bail for is inoculated on LB solid slant culture base, cultivate 24h under 30 ℃ of conditions, and then transferring in 5ml LB liquid nutrient medium with transfering loop picking lawn on LB solid slant culture base, under 30 ℃ of conditions, rotating speed with 200rpm/min shakes bacterium 12h, as Bacillus thuringiensis kind daughter bacteria liquid; B, will plant daughter bacteria liquid by volume mark 4%-5% be inoculated in the 50ml fermention medium, under 30 ℃ of conditions, shake bacterium 72-96h with the rotating speed of 200rpm/min, until the bacillus exfoliation amount is 85%-95%, obtain Broth of Bacillus Thuringiensis; C, with nutrient solution at the centrifugal 10min of 6082g, abandon supernatant liquor, collecting precipitation namely gets the brilliant mixture of Bt spore; D, the brilliant mixture of Bt spore is sub-packed in the 50ml triangular flask, freeze-drying thickness is 1cm-1.5cm, and rearmounted Ultralow Temperature Freezer carries out pre-freeze 5h, takes out to put into the vacuum-freeze-dry machine and carry out frozen dried.In vacuum-freeze-dry machine control storehouse, Temperature Setting is-60 ℃ of left and right, and vacuum keep is between 60 ~ 100Pa, and after 48h, dry the end, namely obtain the former powder of Bt in the present invention.
The preparation of embodiment 8, the 18420 IU/mg Bt YX-1 wettable powders of tiring
Get the former powder 40g of Bt YX-1, sodium lignosulfonate 5g, sodium laurylsulfonate 10g, xanthan gum 1g, skim-milk 1g, white carbon black 43g, each component mixes, and then grinding and sieving is made fine particle below 44 μ m in micronizer mill, is Bt YX-1 wettable powder.Take standard substance as reference, detecting its biological value is 18420 IU/mg.
The preparation of embodiment 9, the 24800 IU/mg Bt YX-1 wettable powders of tiring
Get the former powder 50g of Bt YX-1, sodium lignosulfonate 5g, sodium laurylsulfonate 10g, xanthan gum 1g, skim-milk 1g, white carbon black 33g, each component mixes, and then grinding and sieving is made fine particle below 44 μ m in micronizer mill, is Bt YX-1 wettable powder.Take standard substance as reference, detecting its biological value is 24800 IU/mg.
Because the combination of each composition and adopting parameters is too numerous to mention; therefore the various embodiments described above are only for reference; the various embodiments described above can some variations in addition under not departing from the scope of the present invention, therefore should being considered as of comprising of above explanation is exemplary, but not in order to limit the protection domain of the present patent application patent.
Claims (13)
1. a bacillus thuringiensis strain YX-1 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and preserving number is CGMCC № .5256.
2. bacillus thuringiensis bacterial strain YX-1 according to claim 1, is characterized in that, this bacterial strain has following feature:
(1) have at least
Cry1Ac,
Cry2Ac,
Cry1I,
Cry34-35,
Vip3AaThe killing gene combination; (2) lepidopterous oriental fruit months, bollworm, fall webworms, apple olethreutid, boat type caterpillar, carpocapsa pononella, prodenia litura, bollworm, small cabbage moth, beet armyworm all there is higher lethal effect.
3. the microbial pesticide produced of bacillus thuringiensis YX-1 according to claim 1, is characterized in that described sterilant is wettable powder.
4. wettable powder according to claim 3, is characterized in that, its component and content thereof are: the former powder 40% ~ 50% of Bacillus thuringiensis, dispersion agent 5%～10%, wetting agent 5%～10%, stablizer 1%, ultraviolet radiation protectant 1%, carrier 28～48%.
5. wettable powder according to claim 4, is characterized in that, YX-1 bacterium powder is gemma and mixed crystal, is that bacillus thuringiensis YX-1 is cultured to the brilliant separation period of spore by fermention medium, through the concentrate drying gained.
6. wettable powder according to claim 4, is characterized in that, described dispersion agent is any in sodium lignosulfonate, CNF, HK-2302, NNO, dispersing agent MF.
7. wettable powder according to claim 4, is characterized in that, described wetting agent is Morwet EFW, washing powder, excrementum bombycis meal, dodecyl phenenyl sulfate, Sodium dodecylbenzene sulfonate, pulls open one or more mixtures in powder.
8. wettable powder according to claim 4, is characterized in that, described stablizer is one or more mixtures in xanthan gum, Xylo-Mucine.
9. wettable powder according to claim 4, is characterized in that, described ultraviolet radiation protectant can be one or more mixtures of skim-milk, humic acid, vitamins C, carboxymethyl cellulose.
10. wettable powder according to claim 4, is characterized in that, described carrier is one or more mixtures in white carbon black, kaolin, diatomite, light calcium carbonate, talcum powder.
11. fermention medium according to claim 4 is characterized in that, each component of fermention medium is: soybean cake powder 32g/L, groundnut meal 30g/L, fish meal 16g/L, peptone 6g/L, Semen Maydis powder 20g/L, yeast powder 6g/L, sucrose 1g/L, MgSO
47H
2O 0.2g/L, KH
2PO
41g/L, CaCO
30.5g/L, FeSO
47H
2O 0.06g/L, remaining person's water are supplied.
12. claim 1 described bacillus thuringiensis YX-1 bacterial strain or the described bacillus thuringiensis YX-1 of the claim 3 wettable powder application in plant insect biological control field.
13. application as claimed in claim 12 is characterized in that, described plant insect is fruit tree lepidoptera pest, vegetables lepidoptera pest, field crop lepidoptera pest and ornamental plant lepidoptera pest.
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