CN104611277A - Bacillus thuringiensis for controlling opisina arenosella walkers, serving as palm pests, and application of Bacillus thuringiensis - Google Patents

Bacillus thuringiensis for controlling opisina arenosella walkers, serving as palm pests, and application of Bacillus thuringiensis Download PDF

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CN104611277A
CN104611277A CN201510079220.2A CN201510079220A CN104611277A CN 104611277 A CN104611277 A CN 104611277A CN 201510079220 A CN201510079220 A CN 201510079220A CN 104611277 A CN104611277 A CN 104611277A
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bacillus thuringiensis
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CN104611277B (en
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孙晓东
覃伟权
阎伟
李朝绪
吕朝军
牛小庆
余凤玉
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Coconut Research Institute of Chinese Academy of Tropical Agricultural Sciences
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    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates

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Abstract

The invention belongs to the field of microorganisms and relates to Bacillus thuringiensis for controlling opisina arenosella walkers, serving as palm pests, and an application of Bacillus thuringiensis. The strain is named as Bacillus thuringiensis (Bacillus thuringiensis) BAT-10 and is preserved in China Center for Type Culture Collection on December 31, 2014 with the preservation number to be CCTCCNO:M2014679. The strain of the Bacillus thuringiensis (Bacillus thuringiensis) BAT-10 has the purpose of application of the Bacillus thuringiensis for controlling the opisina arenosella walkers. Shown by insecticidal tests, the strain has very good insecticidal activity to the opisina arenosella walkers, is capable of effectively controlling the population number of the opisina arenosella walker so as to reduce loss, is a biocontrol strain with the potential significance of controlling the opisina arenosella walkers, has the characteristics of strong insecticidal toxicity, good insecticidal effect and the like and provides help to development of palmae industry in a country.

Description

One strain control palm insect coconut knits bacillus thuringiensis and the application of moth
Technical field
The invention belongs to microorganism field, relate to a kind of bacillus thuringiensis bacterial strain and application thereof, specifically, relating to moth is knitted in a strain bacillus thuringiensis and application for preventing and treating palm insect coconut.
Background technology
Coconut is knitted moth (Opisina arenosella Walker) and is belonged to lepidopteran (Lepidoptera) Zhi E section (Qecophoridae), English coconut blackheaded caterpillar by name, also known as coconut wood moth, blackhead crawler belt worm, coconut palm post moth, food leaf crawler belt worm etc., be mainly distributed in the ground such as Sri Lanka, Bangladesh of India, Burma, Indonesia, Pakistan, Thailand, Malaysia.The blade that moth larvae takes food babassu knitted by coconut, and time serious, whole tree crown is invaded gnaws thus causes plant development slow, and fruit yield significantly reduces.Moth is knitted to coconut and carries out the display of pest risk analysis result, it belongs to excessive risk harmful organism, easily surely grow in a lot of area of China, threaten very large to the palm crops such as coconut, betel nut, Chinese fan palm, Middle East nipa palm, large yagua of China, in China, there is the serious ecological hazard and economic damage.
Bacillus thuringiensis (Bacillus thuringiensis, be called for short Bt) be a kind of microbial pesticide be most widely used in the world at present, because it is efficient, low toxicity, safety, economical, therefore be the most potential microbial pesticide, have been found that now that the most of bacterial strain of bacillus thuringiensis can produce polytype crystallin, to lepidopteran (Lepidoptera), Diptera (Diptera), Coleoptera (Coleoptera), Hymenoptera (Hymenoptera), Homoptera (Homoptera), Orthoptera (Orthoptera), the various insects such as Mallophaga (Mallophaga), and nematode, mite class and protozoon etc. have specific insecticidal activity, the insecticidal range of Bt has expanded to cover a large amount of agricultural and sanitary insect pest 9 object biologies in interior invertebral 4 doors and Arthropoda.Bacillus thuringiensis as one most important in biocontrol microorganisms, it have in use target strong, can not murder by poisoning be caused to the mankind and livestock, and environmentally friendly feature.Through retrieval, have not yet to see the correlative study and the report that utilize bacillus thuringiensis prevention and control coconut to knit moth.
Background technology
The object of this invention is to provide bacillus thuringiensis and the application that moth knitted by a strain control palm insect coconut.
One bacillus thuringiensis strain strain, called after bacillus thuringiensis (Bacillus thuringiensis) BAT-10, is preserved in China typical culture collection center, address on December 31st, 2014: China. Wuhan. and Wuhan University; Deposit number: CCTCCNO:M 2014679.
Bacillus thuringiensis provided by the present invention (Bacillus thuringiensis) BAT-10 bacterial strain, direct separation screening from Shi Houli village, Li-Miao Autonomous County of Baoting, Hainan Province pedotheque, through morphology, cultural colony, customary physiological biochemistry and 16s-rDNA Sequence Identification, this bacterial strain is bacillus thuringiensis, carries cry1Aa, cry1Gb, cry1Ma, cry1Ia, cry2Af five kinds of insecticidal proteins encoding genes.
The Basic Biological Character of bacillus thuringiensis provided by the present invention (Bacillus thuringiensis) BAT-10 is:
Form single bacterium colony, bacterium colony oyster white after Bt bacterial strain BAT-10 is cultivated 48 hours on 1/2LB substratum, circular or subcircular, neat in edge, thicker in the middle of bacterium colony, thinning gradually to surrounding, general 30 hours can be observed crystal.To observe Bt bacterial strain BAT-10 thalline under scanning electron microscope be elongated rod shape size is (1.1-1.9) μm × (3.1-5.2) μm, and crystal is rhombus, and gemma be oval, and life near, sporangium is micro-expands.
The purposes of bacillus thuringiensis provided by the present invention (Bacillus thuringiensis) BAT-10 is that it knits the application in moth at control coconut.
Also protection scope of the present invention is belonged to the biotechnological formulation that bacillus thuringiensis provided by the present invention (Bacillus thuringiensis) BAT-10 is activeconstituents.When needing, said preparation can comprise common carrier and auxiliary material prepared by biotechnological formulation.
The present invention knits the insecticidal activity of moth by technique study bacillus thuringiensis (Bacillus thuringiensis) BAT-10 directly smearing administration to coconut, experimental result shows that the insecticidal toxicity of bacillus thuringiensis (Bacillus thuringiensis) BAT-10 is strong, good disinsection effect, and knitting moth to coconut has good insecticidal action.Show that this bacterial strain is that a strain knits biocontrol strain moth with potential significance at control coconut.
The present invention is by the screening and separating to Soil Microorganism, obtain bacillus thuringiensis (Bacillus thuringiensis) BAT-10, show that this bacterial strain is knitted moth to coconut and had good insecticidal action through insecticidal test, effectively can control coconut and knit moth population quantity, reduce the loss, be that a strain knits biocontrol strain moth with potential significance at control coconut, there is the features such as insecticidal toxicity is strong, good disinsection effect, for the development of China's Palmae industry is offered help.
Embodiment
Below in conjunction with embodiment, the specific embodiment of the present invention is described in further detail.Following examples for illustration of the present invention, but are not used for limiting the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually conveniently condition, or according to the condition that manufacturer advises.
The screening of embodiment 1 bacterial strain and qualification thereof
1, the screening of bacterial strain
From the pedotheque in Shi Houli village, Bao Tingxian new policies town, Hainan Province, getting appropriate soil sample from collection adds in isolation medium, 30 DEG C, after 10h cultivated by 230rpm shaking table, 80 DEG C of water-bath 15min, by four concentration, (stoste dilutes 10 times, 100 times, 1000 times, 10000 times) each suction 100 μ L painting solid 1/2LB flat board (each concentration is coated with two flat boards), pour little granulated glass sphere into roll, make bacterium liquid even spread, flat board is placed in 30 DEG C of incubators after placing 10min by room temperature, colonial morphology is observed after 48h, select the bacterium colony smear of similar Bt, under oily mirror, detect whether there is gemma and parasporal crystal with after azaleine dyeing.The Bt bacterial strain that same isolate detects is accepted or rejected according to its colonial morphology and color, nourishing body and spore crystalline form state etc., often kind of form get a strain carry out single bacterium colony purifying after preservation for subsequent use.After purifying obtain in the production traits good, the bacterial strain that moth has high virulence is knitted to lepidoptera pest coconut, called after bacillus thuringiensis (Bacillus thuringiensis) BAT-10, be preserved in China typical culture collection center, address on December 31st, 2014: China. Wuhan. Wuhan University; Deposit number: CCTCCNO:M 2014679.
2, the identification of morphology of bacterial strain
Bacterial strain BAT-10 separation and purification obtained cultivates after 48 hours and forms single bacterium colony, bacterium colony oyster white on 1/2LB substratum, circular or subcircular, and neat in edge is thicker in the middle of bacterium colony, thinning gradually to surrounding.Observing Bt bacterial strain thalline under scanning electron microscope is elongated rod shape, and gemma is oval bar-shaped, and crystal is rhombus.
3, the 16S rDNA sequence amplification of bacterial strain, sequencing and molecular classification
Adopt the DNA of alkaline lysis method of extracting bacterial strain BAT-10, by inoculation in 5mL LB liquid medium test tube 30 DEG C, 230rpm activates.Connect bacterium amount by 1% and connect bacterium in triangular flask 37 DEG C, about 4h (OD is cultivated in 230rpm concussion 600=2.0) 5,000rpm, 5min collected by centrifugation thalline.Every 100mL bacterium liquid adds 2mL SI [0.3M Sucrose, 25 μMs of Tris HCl (pH8.0), 25 mM EDTA (sterilizing), Lysozyme20mg/mL (used time now joins)], mixing, places 2-4h for 37 DEG C.Add the SII [2mM EDTA, 0.16 M NaOH, 2%SDS, 20mM Tris HCl (pH8.0) (now with the current)] of 2mL; Add 2mL SIII [3M NaAc, is adjusted to pH5.2 with glacial acetic acid], mix gently, place more than 4h for 0 DEG C.The centrifugal 5min of 12,000rpm, gets supernatant, adds equal-volume Virahol, places more than 30min for-20 DEG C.The centrifugal 5min of 12,000rpm, abandons supernatant, and precipitation uses 70% ethanol rinse.Dry rear 1mL ultrapure water in vacuum drier will be deposited in dissolve.Add 10 μ l RNase, 37 DEG C of effect 1h, removing RNA.After phenol, chloroform, moisturizing, to 10mL, adds 3M NaAC and 2 times of volume dehydrated alcohol of 1/10 volume ,-20 DEG C of precipitations; The centrifugal 5min of 12,000rpm, abandons supernatant, and precipitation uses 70% ethanol rinse, will precipitate (DNA) dry rear 1mL ultrapure water in vacuum drier and dissolve.After electrophoresis detection ,-20 DEG C of preservations are stand-by.
Adopt the 16S rDNA sequence of bacterial universal primers F (5 '-AGAGTTTGATCCTGGCTCAG-3 ') and R (5 '-AAGGAGGTGATCCAGCC-3 ') pcr amplification BAT-10 bacterial strain, primer is synthesized by order-checking portion, Sheng Gong Guangzhou, Shanghai, PCR instrument: Takara company.PCR reaction system (50 μ l): 50ngDNA template 1 μ l, 10 × PCR buffer 5 μ l, 10pmol/L primer each 1 μ l, 10mmol/L dNTPs1 μ l, the Taq DNA polymerase 1 μ l of 4U/ μ l, adds ddH2O and complements to 50 μ l; PCR reaction conditions is that 94 DEG C of denaturations are after 5 minutes, 94 DEG C of sex change 1 minute, anneal 1 minute for 52 DEG C, 72 DEG C extend 1 minute, 30 circulations, last 72 DEG C extend 10 minutes, obtain the conserved sequence of the 16S rDNA of this bacterial strain, analyze and constructing system evolutionary tree by NCBI Blast sequence alignment with DNAMAN5.2 software Multiple Sequence Alignment, be accredited as a bacillus thuringiensis strain bacterial strain.
4, the amplification of cry gene, sequencing and molecular classification
The extracting method of the DNA of BAT-10 is with 3, cry gene identification primer is in table 1, wherein cry1 class primers designed is K5un2/K3un2 and K5un3/K3un3, cry1I class primers designed is S5uni/S3uni, cry2 class primers designed is S5un2/S3un2, and the primers designed of other gene type by that analogy.Carry out the PCR qualification of all kinds of cry gene identification primer respectively, PCR reactant is with 3, pcr amplification product reclaims test kit through gel and (connects carrier T (Takara company) after (Takara company) purifying to clone, the raw work in picking positive monoclonal sample presentation Shanghai checks order, sequence after NCBI BLAST comparison with GenBank known array Sequence ID:gb|AF510713.1, gb|U70725.1, emb|Y09326.1, gb|KF980886.1, the maximum comparability of gb|EF439818.1 is respectively 98%, 99%, 99%, 92%, 99%, determine that this bacterial strain contains cry1Aa, cry1Gb, cry1Ma, cry1Ia, cry2Af five kinds of new type disinsection encoding genes.
Table 1 primers designed sequence
Primer Sequence Primer Sequence
K5un2 AGGACCAGGATTTACAGGAGG S5un5 ATTGGAGGTGGTATTGCTGATAC
K3un2 GCTGTGACACGAAGGATATAGCCAC S3un5 ATAAGATGAAGACAGTGCTGGTGGTGG
K3un3 CCTCCTGTAAATCCTGGTCCT S5un6 CAACAAATCCTAGCAATGGTC
K5un3 CAATGCGTACCTTACAATTGTTTAAGTAAT S3un6 TAGAGAGTGGAACGACTTTACC
S5uni GCTGTCTACCATGATTCGCTTG S5un7 GGATATGAAGATAGTAATAGAAC
S3uni CAGTGCAGTAACCTTCTCTTGC S3un7 GCTGTAGCATGACATAATCGATG
S5un2 GGAAGAACTACTATTTGTGATGC Sn5un8 CGGCAAACTTAGTAGAATGC
S3un2 AATAGTTTGAATTACCGCGAGC Sn3un8 CTGACTGATTTCCACCATCACG
S5un3 CGAACAATCGAAGTGAACATGATAC S5un9 AGGACCAGGATTTACAGGAGG
S3un3 CATCTGTTGTTTCTGGAGGCAAT S3un9 CCCAATGCGAAAGAACTAAG
S5un4-10 GTGTCAAGAGAACCAACAGTATG S5un11 GGTAATCAGCCTGCTACTATGG
S3un4-10 ACTAAGTCTCCTCCTGTATGACCAG S3un11 GTTGCTTGATCTGGTGTATCTTC
Note: primer sequence direction is 5 '-3 '
The preparation of embodiment 2 bacterial strain BAT-10 parasporal crystal protein is with quantitative
First 1/2LB solid medium is inoculated to cellular lysate (30 DEG C of constant temperature, 48hr); Aseptic technique scraping thalline, then uses sterilizing deionized water to be prepared into bacterium suspension, and use Byeotime BAC protein concentration quantification kit (enhancement type) to carry out the protein concentration of bacterium suspension quantitatively, quantitative concentrations is 45.52ug/ml.
Embodiment 3 BAT-10 knits the indoor insecticidal determination of activity of moth to coconut
Adopt leaf dipping method, the protein solution of BAT-10 is arranged 5 experimental concentration gradients, is respectively 45.52 μ g/mL, 22.76 μ g/mL, 11.38 μ g/mL, 5.69 μ g/mL, 2.845 μ g/mL.Coconut blade clear water is cleaned and dries, choose the section shape that fresh and tender consistent coconut blade is cut into 15cm, 5min is soaked in the protein solution (containing 0.1% tween-80) mixed of each concentration, dry, put into raw survey bottle, every bottle graft 3-4 instar larvae 30, each process in triplicate, be incubated in 28 DEG C of biochemical cultivation cases, cultivate dead, the alive borer population of 72hr " Invest, Then Investigate ", and observe larval feeding situation.Bioassay results display BAT-10 albumen is knitted moth larvae to coconut and is had high insecticidal activity, and the obvious blackening death of moth knitted by poisoning coconut, and whole polypide is black, rotten, and contrast shows appetite comparatively greatly for examination worm, and worm enlivens, and grows normal.Its protein liquid LC 50be 9.08 μ g/mL, degree of confidence is 95%, and fiducial interval is 6.72-12.26 μ g/mL (table 1).
Table 2. bacterial strain BAT-10 knits the insecticidal activity (72hr) of moth to coconut
Embodiment 4 BAT-10 bacterial strain sporulating character
Be 1 × 10 by concentration 8the BAT-10 thallus suspension liquid (100 μ L/ ware) on 1/2LB substratum of cfu/mL, differing temps 20 DEG C, 22 DEG C, 24 DEG C, 26 DEG C, cultivate 48h under the culture condition of 28 DEG C, 30 DEG C, 32 DEG C, 34 DEG C, 36 DEG C, 38 DEG C after sporulation quantity on analytical unit area, determine that BAT-10 bacterial strain produces spore amount for best at 30 DEG C.
The outdoor prevention effect of embodiment 5 bacterial strain
The microbial inoculum of bacillus thuringiensis BAT-10 is brought to field (civilian coconut palm No. 3 Tom Thumb coconut bases), be diluted to containing spore 1x10 with the 1 ‰ tween-80 aqueous solution 8cfu/ml, 2x10 8cfu/ml, 4x10 8cfu/ml, the high-pressure sprayer that stirs is sprayed.Start spraying control on January 12nd, 2015, it was cloudy at that time, temperature 25 DEG C, gentle breeze, side angle 50 ° during spraying, and droplet distributes more on tree crown and evenly, coverage density is 27.9/cm 2divided by pilot region every district, 4 regions to choose three similar strain coconut palms of harm shape and use Bt microbial inoculum and the 1 ‰ tween-80 aqueous solution (CK) of above-mentioned three concentration respectively, borer population of living before statistics dispenser is insect population radix, ld, 3d, 5d and 7d borer population alive after statistics dispenser, the full phase investigates 5 times altogether.Each repetition calculates prevention effect respectively, and prevention effect is carried out variance analysis, and compares with LSD method, the results are shown in Table 3.Find out that, in process after 3 days, the survival rate of larvae on tree crown blade starts remarkable decline by result, within 5 days, prevention effect is best, and the higher control effect of spraying medicine concentration is better.
The field test results analysis of moth knitted by table 3 different concns BAT-10 microbial inoculum to coconut
The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the prerequisite not departing from the technology of the present invention principle; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.

Claims (3)

1. a bacillus thuringiensis strain bacterial strain, called after bacillus thuringiensis (Bacillus thuringiensis) BAT-10, be preserved in China typical culture collection center, deposit number on December 31st, 2014: CCTCCNO:M 2014679.
2. bacillus thuringiensis according to claim 1 (Bacillus thuringiensis) BAT-10 knits the application in moth at control coconut.
3. prevent and treat the biotechnological formulation that moth knitted by coconut, bacillus thuringiensis (Bacillus thuringiensis) BAT-10 of its activeconstituents to be deposit number according to claim 1 be CCTCCNO:M 2014679.
CN201510079220.2A 2015-02-15 2015-02-15 One plant of preventing and treating palm insect coconut knits bacillus thuringiensis and the application of moth Expired - Fee Related CN104611277B (en)

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