CN100595272C - Bacillus thuringiensis bacterial strain for strain insect disinfestations, restraining epiphyte and uses thereof - Google Patents
Bacillus thuringiensis bacterial strain for strain insect disinfestations, restraining epiphyte and uses thereof Download PDFInfo
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- CN100595272C CN100595272C CN200810052439A CN200810052439A CN100595272C CN 100595272 C CN100595272 C CN 100595272C CN 200810052439 A CN200810052439 A CN 200810052439A CN 200810052439 A CN200810052439 A CN 200810052439A CN 100595272 C CN100595272 C CN 100595272C
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Abstract
The invention relates to an insecticidal and antifungal bacillus thuringiensis bacterial strain and the application. The bacterial strain is bacillus thuringiensis 519-1, which is called as Bt 519-1 for short, the collection number is CGMCC No. 2327, the bacterial strain carries the insecticidal protein coding genes of cry1Aa, cry1Ab, cry1Ac, cry1I, cry2 and the vip3A and 70 kDa chitinase coding gene chiB. The semi-lethal concentration (LC50) to spodoptera exigua of the spore crystal complex freeze-dried powder of the bacterial strain is only 8.8Mug/mL, and the bacterial strain can inhibit thegrowth of 8 common plant pathogen fungal myceliums and inhibit the germination of the fungal spores completely. The usage of the bacterial strain can produce the insecticidal and anti-disease multifunctional biological pesticides, which is used for the prevention and the treatment of lepidoptera major pest spodoptera exigua and bollworm; at the same time, the bacterial strain also has the inhibition function of the plant pathogen fungi.
Description
[technical field]:
The invention belongs to the biological control technical field of plant pest, particularly a kind ofly not only killed lepidoptera pest but also suppressed the screening method of bacillus thuringiensis of plant pathogenic fungi and the preparation method of active bacterial strain.
[background technology]:
Beet armyworm belongs to the lepidopteran Noctuidae, is the very strong polyphagous pest-insect of a kind of reproductivity, and desirable food host plant reaches the hundreds of kind, is the important pests of harm wheat, cotton and various vegetables.Bring tremendous loss for every year China's agriculture production.Beet armyworm is insensitive to biological pesticide, and domestic and international many scholars utilize biological pesticide and chemical pesticide preparation method, though play certain preventive and therapeutic effect, harm that chemical pesticide brings to the mankind and insect to the resistance of chemical pesticide in continuous increase.Therefore it is very necessary to screen the bacterial strain with specific activity.
The yield and quality that the Plant diseases that is caused by pathogenic fungi has reduced plant prod brings tremendous loss to agriculture production.In recent years along with the popularizing of chamber planting method, hot and humid environment is more conducive to the breeding of fungi, and fungal disease is on the rise.
Sporeine preparation as biotic pesticide, be widely used in since the 1950's farming, woods, really, the control of vegetables various pests and sanitary insect pest, be the main biotic pesticide of control lepidoptera pest, it is free from environmental pollution, to the person poultry safety, production technique is simple, low production cost.
The insecticidal activity of bacillus thuringiensis (Bacillus thuringiensis is called for short Bt) is from himself synthetic insecticidal crystal protein, and these albumen have specificity to insect.In commercial Bt bacterial strain, be to bollworm bacterial strain efficiently mostly, active high less to beet armyworm.So in recent years, the scholar is screening highly active bacterial strain always.Bt WY-190 bacterial strain was 3～4 times of domestic and international supper toxic strain to the beet armyworm virulence at that time, but patent is not announced its LC
50(Yan builds equality ZL02115581.X, 2002).The efficient Bt 15A3 bacterial strain of tool excellent genes combination is to the toxic limit medium dose (LC of beet armyworm
50) be 15.63ppm (ZL01104421.7 such as Chen Yuehua, 2001).But these bacterial strains all do not suppress the activity of fungi.
Yang Qian (CN 1778886A) utilizes the Trichoderma of the pest-resistant diseases prevention of plasmid construction that contains the Bt gene.Marrone etc. (US patent6077506) thus find that a strain novel B t strains A Q52 can produce microbiotic and suppress various plants pathogenic fungi and bacterium.Moar etc. (US patent 6280722) are separated to a strain Bt mutant strain, and the several plant pathogenic fungi is had restraining effect, and find to have new chitinase gene in the bacterial strain.Above-mentioned bacterial strains does not possess the characteristic of efficient killing beet noctuids.
Chitinase has great and the potential using value in fields such as environment protection, biological control and agriculturals.Except that oomycetes, all contain chitin in all fungal cell walls.Thereby the chitin that chitinase can the hydrolysis fungal cell wall suppresses fungal growth.Chitin still is the peritrophic primary structure of insect midgut, after being destroyed by chitinase, quickens the process of falling ill of insect, improves mortality ratio, therefore sterilant is had the notable synergistic effect.Genus bacillus is an important source of chitinase.Therefore, screening has the chitinase of producing and the high bacterial strain of enzymic activity, is suppressing have important use to be worth aspect the disease and pest.
[summary of the invention]:
The purpose of this invention is to provide a strain wide spectrum suppresses fungi, lepidopteran Noctuidae important pests beet armyworm is had the bacillus thuringiensis bacterial strain of high cytotoxicity and an application of this bacterial strain simultaneously.
This bacterial strain can be used for the preparation of antimycotic, insecticidal multifunction biological pesticide.
Provided by the invention have desinsection, a mycostatic bacillus thuringiensis bacterial strain, be preserved in " China Committee for Culture Collection of Microorganisms's common micro-organisms " center " (Datun Road, Chaoyang District, Beijing City 100101; Institute of Microorganism, Academia Sinica); its preserving number is: CGMCC No.2327; classification called after bacillus thuringiensis Bacillus thuringiensis 519-1, abbreviation Bt 519-1 on January 8th, 2008.(Bt519-1 strain cell and crystal habit are seen Fig. 1 photo.)
Bt519-1 of the present invention, carry cry1Aa,, cry1Ab, cry1Ac, cry1I, cry2 and six kinds of insecticidal proteins encoding genes of vip3A, and coding 70kDa chitinase gene chiB, be a kind of bacillus thuringiensis that wide spectrum suppresses the plant pathogenic fungi activity and lepidoptera pest had high virulence that has.
Described plant pathogenic fungi is: fusarium graminearum, cotton rhizoctonia solani, ring rot of apple bacterium, aspergillus niger, Penicillium chrysogenum, botrytis cinerea pers, Fusarium oxysporum and bread mould; Described lepidoptera pest is: beet armyworm, bollworm.
Described Bt 519-1 has the obvious suppression effect to 8 kind of plant pathogenic fungies, 100% suppresses fungus spore germination, to beet armyworm toxic limit medium dose (LC
50) only be the bacillus thuringiensis bacterial strain of 8.8ug/mL, called after bacillus thuringiensis Bacillusthuringiensis519-1 is called for short Bt519-1 (CGMCCNo.2327).
Above-mentioned bacterial classification can be applied to suppress plant pathogenic fungi and control lepidoptera pest.
Advantage of the present invention and positively effect:
Compared with prior art, the present invention is directed to the weak tendency that Bacillus thuringiensis is narrow to the general action spectrum of insect, not high to the beet armyworm activity and the unrestraint fungi acts on, filter out not only desinsection but also suppress the multi-functional bacterial strain of fungi, the brilliant mixture lyophilized powder of its spore is to the toxic limit medium dose (LC of beet armyworm
50) only be 8.8ug/mL.8 kinds of frequently seen plants pathogeny fungies there is the obvious suppression effect, can 100% inhibition fungus spore germination.Utilize this bacterial strain can produce the multifunctional bio agricultural chemicals of killing pests and preventing diseases, be used to prevent and treat the great insect beet armyworm of lepidopteran section, simultaneously plant pathogenic fungi is also had restraining effect.
[description of drawings]:
Fig. 1 is Bt519-1 strain cell and gemma crystal habit photo.
Fig. 2 is that the Bt519-1 bacterial strain suppresses 3 kind of plant pathogeny fungus spore germinations diagrams, A, is the bread mould diagram, and B, for the ring rot of apple bacterium illustrates, is that Fusarium oxysporum illustrates at C.Show among the figure that the detection cup that adds Bt519-1 bacterium liquid and fungal spore does not have the fungal growth phenomenon, prove that spore germination is suppressed, and fungal growth is normal around adding the contrast cup of sterilized water and fungal spore.
Fig. 3 is the electrophoresis detection result of Bt519-1 bacterial strain vip3A full-length gene PCR product, the 1.PCR product; 2. standard molecular weight DNAMarker.
Fig. 4 is the electrophoresis detection result of Bt519-1 bacterial strain chiB full-length gene PCR product, the 1.PCR product; 2. standard molecular weight DNAMarker.
[embodiment]:
The screening of embodiment 1, multi-functional bacterial strain Bt519-1 and antifungal determination of activity
1.1. the collection pedotheque utilizes the heat-stable principle of genus bacillus, and sample is carried out 80 ℃ of processing in 20min minute, uses microscopic examination after cultivating, and selects to have gemma and crystalline bacterial strain, called after Bt519-1.
1.2. design a pair of special primer according to the disclosed chiB gene order of GenBank, the PCR that carries out the 70kDa chitinase gene detects, the Bt519-1 bacterial strain is a chitinase chiB gene masculine.
1.3. preparation is the solid medium of only carbon source with tobacco brown spot pathogen, its prescription is (g/L): tobacco brown spot pathogen 20, KH
2PO
41, Na
3PO
42, MgSO
47H
2O 0.5, and KCl 0.5, agar powder 15, and pH 7.2.Be used to detect the chitinase activity.
1.4. with aseptic toothpick Bt519-1 bacterial strain point is connected on the above-mentioned substratum, cultivated 5～7 days for 30 ℃.Big hydrolysis circle can be grown and produce to the Bt519-1 bacterial strain on substratum.
1.5. suppressing 8 kind of plant pathogeny fungi activities detects.Method is: be the 8 kind of plant pathogeny fungi bacterium piece of 8mm at the dull and stereotyped central authorities of the PDA for preparing inoculation diameter, each flat board is followed a kind of fungi, cultivated 2～4 days, at distance hypha,hyphae 2cm place inoculation Bt519-1 bacterial strain, cultivated 5 days for 30 ℃, observe the restraining effect of Bt519-1, and measure antibacterial circle diameter and bacterial colony diameter ratio (R value) every kind of fungi.Table 1 is Bt519-1 and reference bacterial strain Bt15A3 (ZL01104421.7) the inhibition effect to 8 kinds of fungies.
Table 1, Bt519-1 bacterial strain and reference bacterial strain are to the inhibition effect of 8 kinds of fungies
a
A: "+" "-" represents mycostatic power.1 "+" representative has slight restraining effect; 2 "+" represents R=1.1; 3 "+" represents R 〉=1.2.R is an antibacterial circle diameter and the ratio of colony diameter.
Use Oxford cup-flat band method (Oxford cup-Plate) to detect the restraining effect of bacterial strain Bt519-1 to fungus spore germination.Concrete grammar is:
2.1. get the PDA flat board for preparing, place 2 aseptic Oxford cups on the every flat board.One of them is for detecting cup, and one is the contrast cup.Oxford cup material is a stainless steel, high 10mm, external diameter 8mm, internal diameter 6mm.
(contain 10 approximately 2.2. get the spore suspension 20ul of the bread mould, ring rot of apple bacterium and the Fusarium oxysporum that prepare
3Spore), join in the cup of whole Oxfords.
2.3. in detecting cup, add 180uL Bt519-1 thalline suspension (OD again
600=1.530), the contrast cup adds the 180uL sterilized water.
2.4. flat board is positioned over to cultivate in 28 ℃ of incubators to observe after 2～3 days suppresses the effect that gemma is sprouted.The detection cup that adds Bt519-1 bacterium liquid and fungal spore does not have the fungal growth phenomenon, proves that spore germination is suppressed fully, and adds fungal growth normal (seeing accompanying drawing 2) around the contrast cup of sterilized water and fungal spore.
Embodiment 3, Bt519-1 bacterial strain are to bollworm and beet armyworm toxic limit medium dose (LC
50) measure
3.1. for aimed strain Bt519-1 that accurate calculation screened toxic limit medium dose to bollworm and beet armyworm, with this strain culturing on common NB solid medium, cultivate after 72 hours for 30 ℃, collect the lawn that includes gemma and crystallin, make dry powder through freeze-drying.
3.2. with this chamber preservation, to bollworm and beet armyworm efficiently patented strain Bt15A3 (ZL01104421.7) handle equally as the reference bacterial strain.
3.3. take by weighing the above-mentioned lyophilized powder of identical weight, serial dilution becomes 5 concentration respectively, the infection feed of preparation respective concentration.
3.4. insert bollworm and beet armyworm newly hatched larvae respectively, each concentration 48 larvas of feeding, and establish the natural death contrast.30 ℃ of incubators were cultivated 72 hours, checked larva in the per cent death loss that each concentration infects feed, be converted into dead machine value after statistical technique calculate the LC of each bacterial strain to each worm
50
3.5. each bacterial strain of above-mentioned biological assay is to each worm replication 3 times.3 results' mean value is listed in the table 3.Bt519-1 is about 50% of 15A3 insecticidal toxicity to the virulence of bollworm.And be Bt519-1 to the minimum bacterial strain of the toxic limit medium dose of beet exigua larvae, only be 8.8ug/mL, higher 2 times than 15A3 bacterial strain virulence.
Table 2, Bt519-1 bacterial strain are to the LC of bollworm and beet armyworm
50Measurement result
The biological property of embodiment 4, Bt519-1 bacterial strain
The Bt519-1 strains separation is in the soil of Kunming, Yunnan, and it is characterized in that: somatic cells is shaft-like, and size is 1～1.2umX3～5um, the blunt circle in two ends.Can form endogenous spore and parasporal crystal, sporangium is not expanded, and gemma is oval.Crystal shape and size are varied, square, oval, spherical, rhombus is arranged, inlay shape and irregular shape (see figure 1).Crystal records molecular weight by the SDS-PAGE method and is approximately 130kDa.
Detect through specific gene PCR, the insecticidal proteins encoding gene that the Bt519-1 bacterial strain carries has: cry1Aa, and cry1Ab, cry1Ac, cry1I, cry2 and vip3A also have the about 70kDa chitinase gene chiB of coding.
Fig. 3, Fig. 4 are respectively agarose gel electrophoresis detected Bt519-1 bacterial strain vip3A and the correct PCR product of chiB gene size.
The fermentation culture of embodiment 5, Bt519-1 bacterial strain
5.1 slant strains NB medium slant (g/L): extractum carnis 5, peptone 10, NaCl 5, agar powder 18, pH7.2～7.4.Medium preparation is back packing test tube well, 121 ℃ of autoclavings 30 minutes, put into the inclined-plane after the taking-up, put 30 ℃ of incubators and cultivated 24～48 hours, no varied bacteria growing just can be inoculated: go bail for and hide the fresh inclined-plane of bacterial classification Bt519-1-articulating kind, cultivated 10～12 hours for 30 ℃, microscopic examination behind the smear staining, thalline be for slightly shaft-like, the blunt circle in two ends, protoplasma is even, does not have assorted bacterium.
5.2 getting an articulating with above-mentioned activatory bacterial classification, the ferment-seeded cultivation goes in the liquid NB substratum, 30 ℃, and 220rpm/min shaking culture 12 hours.
5.3 fermentation culture fermentative medium formula (g/L): beans and powder 40, W-Gum 15, yeast powder 10, and K
2HPO
4MgSO
4CaCl
2MnSO
4Four kinds of inorganic salts are total to 5g.All raw material need reach the fineness of 180 μ m sieve, pH8.5～9.0.Put into fermention medium by fermentation culture bottle volumetrical 1/10th volumes, about sterilization postcooling to 35 ℃, press 3% of culture volume and insert the seed liquor that goes up the step, revolution is 230～250 commentaries on classics, 30 ℃ ± 1 ℃ of temperature, fermentation period 35～45 hours is looked the gemma crystal detachment about 20～30%, and fermented liquid pH reaches and stops fermentation more than 8.0.
5.4 utilize aforesaid method to cultivate, fermented liquid bacterium number can reach 7 * 10
9CFU/mL, the synchronous release rate of gemma crystal reaches 95%.
5.5 after the fermentation ends, add hydrochloric acid and reach about 5.0 to fermented liquid pH, add a small amount of sanitas and other auxiliary material again.
5.6 above-mentioned fermentative medium formula and cultural method are scalable is tonnage level fermentor cultivation.
Claims (3)
1, a strain insect disinfestations, mycostatic bacillus thuringiensis (Bacillus thuringiensis) bacterial strain, be preserved in " China Committee for Culture Collection of Microorganisms's common micro-organisms " center "; its preserving number is: CGMCC No.2327; classification called after bacillus thuringiensis Bacillus thuringiensis 519-1, abbreviation Bt 519-1.
2, the described bacillus thuringiensis bacterial strain of claim 1, be a kind of bacillus thuringiensis that wide spectrum suppresses the plant pathogenic fungi activity and lepidoptera pest is had high virulence that has, described plant pathogenic fungi is: fusarium graminearum, cotton rhizoctonia solani, ring rot of apple bacterium, aspergillus niger, Penicillium chrysogenum, botrytis cinerea pers, Fusarium oxysporum and bread mould; Described lepidoptera pest is: beet armyworm and bollworm.
3, the described bacterial classification of claim 1 is suppressing the described 8 kind of plant pathogenic fungies of claim 2 and is preventing and treating application in the described lepidoptera pest of claim 2.
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Cited By (1)
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|---|---|---|---|---|
| CN102643773A (en) * | 2011-02-18 | 2012-08-22 | 华中农业大学 | Screening and identification of genetically engineered Bacillus thuringiensis YBT-881-L1 of overexpressed CodY protein |
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| CN105039207B (en) * | 2015-06-26 | 2017-10-31 | 滨州学院 | A kind of bacillus (Bacillus sp.) ZY and its application |
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| CN111793580B (en) * | 2020-07-17 | 2021-04-16 | 江西省农业科学院农业应用微生物研究所（江西省农村能源研究中心） | Bacillus thuringiensis JXBT-0301 with insecticidal activity on ganoderma lucidum armyworm and application thereof |
| CN113373091A (en) * | 2021-06-28 | 2021-09-10 | 福建农林大学 | Biocontrol strain bacillus thuringiensis FJ2B-25 for preventing and treating rice sheath blight |
| CN113980866B (en) * | 2021-11-30 | 2023-05-30 | 桂林理工大学 | Endophytic bacterium of lime and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102643773A (en) * | 2011-02-18 | 2012-08-22 | 华中农业大学 | Screening and identification of genetically engineered Bacillus thuringiensis YBT-881-L1 of overexpressed CodY protein |
| CN102643773B (en) * | 2011-02-18 | 2013-10-16 | 华中农业大学 | Screening and identification of genetically engineered Bacillus thuringiensis YBT-881-L1 of overexpressed CodY protein |
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