CN103898025B - A kind of thuringiensis killing Meloidogyne incognita and cultural method thereof - Google Patents
A kind of thuringiensis killing Meloidogyne incognita and cultural method thereof Download PDFInfo
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Abstract
The present invention relates to a kind of thuringiensis killing Meloidogyne incognita and cultural method thereof, kill the thuringiensis NBIN 863Bacillus thuringiensis NBIN 863 of Meloidogyne incognita, depositary institution: China typical culture collection center, address: China, Wuhan, Wuhan University, November 29 2013 preservation day, survival November 29 2013 date, preservation center number CCTCC NO:M2013612.Cultural method, takes near Anhui Province's Jiuhuashan, east longitude 117 degree 29 points, the soil that north latitude is 30 degree 39 points, suspends with sterilized water, is coated with flat board by ladder concentration dilution, cultivates with LB solid medium, and dilution lines and cultivates on LB flat board；Single colony inoculation is cultivated in shaking flask, mixes with glycerol, puts Storage in refrigerator, and single bacterium colony is transferred in fluid medium cultivate, and smear, violet staining observation by light microscope determine.
Description
Technical field
The present invention relates to a kind of thuringiensis killing Meloidogyne incognita and cultural method thereof.
Background technology
According to FAO conservative estimation, the loss that grain and fibre crops are caused because of eelworm harm by the whole world every year is about
Be 12%, the loss that vegetable, Semen arachidis hypogaeae, Nicotiana tabacum L. and some fruit tree are caused will more than 20%, even up to 50%.
According to authoritative expert, plant nematode causes the loss about 150,000,000,000 dollars that world agriculture produces every year.I
The loss about 3,500,000,000 dollars that state causes because of nematicide every year.
Nematicide to plant pest there are about kind more than 3000 at present, and has kind more than 40, wherein to work what China found
One of nematicide that thing harm is the most serious is root-knot nematode.Root-knot nematode polypide is small, lives in plant interior or native
In earth, can shift along with rainwater, the operation of labor thing and the transfer of diseased plant, prevent and treat extremely difficult.
The main method of preventing and treating plant nematode disease has chemical prevention and Biological control.Chemical agent major part
For severe toxicity medicine, serious environment pollution.Biological control is that the natural enemy utilizing nematicide is to control population number and restriction
The loss that nematicide causes, and the most comparatively safe to people, animal and environment, have a extensive future.In Biological control
The most most widely used is thuringiensis (Bacillus thuringiensis is called for short Bt), in addition
Also have puncture pasteurella (Psteuriapenetrans), Pseudomonas alba (Pseudomonas spp.) with
And the strong bacillus (Bacillus firmus) etc. in recent years found.
As far back as 1972, first Prasad etc. found that Bt β-extracellular toxin is to root-knot nematode (Root-knot
Nematode, RKN) ovum and larva have the highest activity.Find that Bt β--extracellular toxin is to radopholus line afterwards
Worm (Radopholus similis), soybean cyst nematode Heterodera glycines and Chinese lantern plant nematicide (Panagrellus redivivus)
Deng nematicide have the highest insecticidal activity (Ignoff et al., 1977；Devidas et al.,1988).20
Centuries the mid-80 Bone etc. find Bt Kurstaki (subsp.kurstaki) and Israel subclass
(subsp.israelensis) to animal parasitic nematodes trichostrongylus colubriformis (Trichostronglus
Colubriformis) ovum and larva have the highest insecticidal activity, and within 1988, Mycogen company of the U.S. finds
Bt crystalline protein is to Meloidogyne (Meloidogyne spp.), Tylenchorhynchus (Tylenchs spp.), short
The plants such as body Turbatrix (Pratylenchus spp.) and Aphelenchoides (Aphlenchoides spp.).The U.S.
Mycogen company screens a lot of Bt bacterial strain having insecticidal activity to plant nematode, the most also application
Patent.Within 1993, our Preliminary screening has the Bt bacterial strain of insecticidal activity to 9 strains to plant nematode, these
Bt bacterial strain to sweet potato stem nematode (Ditylenchus destructor), M hapla (M.hapla) and
Boehmeria Pratylenchidae (P.scribneri) has the highest insecticidal activity, has the most applied for that the bacterial strain of national patent is
YBT-1532 (ZL00-1-16062.1) (minor congruence, 2007b).
Summary of the invention
It is an object of the invention to for above-mentioned present situation, it is desirable to provide a kind of Su Yun gold brood cell killing Meloidogyne incognita
Bacillus and cultural method thereof.
The implementation of the object of the invention is, a kind of thuringiensis killing Meloidogyne incognita, Su Yun gold bud
Born of the same parents bacillus NBIN-863Bacillus thuringiensis NBIN-863, depositary institution: Chinese Typical Representative is cultivated
Thing preservation center, address: China, Wuhan, Wuhan University, preservation center number CCTCC NO:M2013612,
November 27 2013 preservation day, survival November 29 2013 date.
The cultural method of a kind of thuringiensis killing Meloidogyne incognita, specifically comprises the following steps that
1) take near Anhui Province's Jiuhuashan, east longitude 117 degree 29 points, the pedotheque of 30 degree of 39 points of positions of north latitude
1g, suspends at use for laboratory 1ml sterilized water, according to 10-3、10-4、10-5、10-6Concentration is diluted being coated with and puts down
Plate, cultivates 24h with being inverted in 28 DEG C of constant incubators of LB solid culture liquid, and from flat board, picking list bacterium colony is again
Secondary dilution lines on LB flat board, cultivates 24h under the conditions of 28 DEG C；
LB solid culture formula of liquid: peptone 10g, yeast powder 5g, sodium chloride 10g, agar powder 20g,
It is dissolved in 1L distilled water, 121 DEG C, 30min, moist heat sterilization；
2) single colony inoculation repeated isolation being purified to is in 5ml LB fluid medium shaking flask, 28 DEG C, and 200
Rpm, cultivates 36h,
Described LB liquid culture based formulas: peptone 10g, yeast powder 5g, sodium chloride 10g, is dissolved in 1L
In distilled water, 121 DEG C, 30min, moist heat sterilization；
3) first glycerol is configured with deionized water, 121 DEG C of moist heat sterilization 30min, water: glycerol volume ratio=1:1；
4) by step 2) cultivate the bodies such as the glycerol with 500 μ l50% of sterilizing of the 500 μ l bacterium solution after 36h
Long-pending mixing, is put in preservation in-80 DEG C of refrigerators after mixing；
5) by LB flat board isolated and purified to single bacterium colony be transferred in LB liquid medium, 28 DEG C, 200rpm,
Cultivate after 48h, after carrying out smear, fixing, violet staining, under optical microscope oil lens head, carry out shape
State is viewed as Bacillus thuringiensis bacterial strain.
Accompanying drawing explanation
Fig. 1 is the thuringiensis NBIN-863Bacillus killing Meloidogyne incognita
Thuringiensis NBIN-863 grows the microscopic examination picture after 48h in LB liquid medium；
Fig. 2 is the thuringiensis strain Bacillus thuringiensis killing Meloidogyne incognita
The phylogenetic analysis figure of NBIN-863；
Fig. 3 is the thuringiensis strain Bacillus thuringiensis killing Meloidogyne incognita
The precipitation PAGE gel electrophoretogram of NBIN-863；
Detailed description of the invention
Kill the thuringiensis NBIN-863Bacillus thuringiensis of Meloidogyne incognita
NBIN-863, depositary institution: China typical culture collection center, address: China, Wuhan, Wuhan University,
Preservation center number CCTCC NO:M2013612, November 27 2013 preservation day, survival date 2013
On November 29, in.
Kill the Bacillus thuringiensis bacterial strain form of Meloidogyne incognita: on flat board, single bacterium colony of growth is milky
Flakes, rough surface, it is viewed as shaft-like under growth 24h Electronic Speculum, is stain-fast bud after growth 36h
Born of the same parents and the rhomboidan catching color；After growth 48h, supernatant produces 6 kinds of albumen, and wherein 3 kinds is insecticidal crystal
Albumen, is Cry9Aa albumen (130kDa), Cry2Aa albumen (69kDa) and Cry1Ac albumen (130kDa) respectively
(Fig. 3).Thuringiensis have kill Meloidogyne incognita activity.
The applicant on May 10th, 2012 near Anhui Province's Jiuhuashan, east longitude 117 degree 29 points, north latitude 30
Pedotheque 1g is taked in the position spending 39 points, suspends at use for laboratory 1ml sterilized water, according to 10-3、10-4、
10-5、10-6Concentration is diluted being coated with flat board, with LB solid culture liquid (peptone 10g, yeast powder 5g, chlorine
Change sodium 10g, agar powder 20g, be dissolved in 1L distilled water, 121 DEG C, 30min, moist heat sterilization), 28 DEG C
Being inverted in constant incubator and cultivate 24h, from flat board, picking list bacterium colony again dilutes and lines on LB flat board, 28 DEG C
Under the conditions of cultivate 24h.Single colony inoculation that repeated isolation is purified to in 5ml LB fluid medium shaking flask,
28 DEG C, 200rpm, cultivates 36h.First with deionized water configuration 50% glycerol (water: glycerol volume ratio=1:1,
121 DEG C of moist heat sterilization 30min), will cultivate the 500 μ l50%'s with sterilizing of the 500 μ l bacterium solution after 36h
Glycerol equal-volume mixes, and is put in preservation in-80 DEG C of refrigerators after mixing.By on LB flat board isolated and purified to list
Bacterium colony is transferred in LB liquid medium, 28 DEG C, 200rpm, cultivates after 48h, carry out smear, fixing,
After violet staining, under optical microscope oil lens head, carry out morphologic observation, microscopic examination picture such as Fig. 1
Shown in, it was demonstrated that for Bacillus thuringiensis bacterial strain.
Utilize and separate the Bacillus thuringiensis bacterial strain acquisition 16S rRNA gene killing Meloidogyne incognita, build system
System grows tree, specific implementation process: Bacillus thuringiensis NBIN-863 bacterial strain activated overnight is cultivated,
1% (V/V) inoculum concentration is seeded to fresh LB (Sambrook et al., 1989), cultivates to OD600=0.6,
Centrifugal collecting cell, according to SBS Genetech resin typeTMGenomic DNA purifies test kit operational approach and extracts genome
DNA.With this genomic DNA as template, employing bacterial 16 S rRNA universal primer (16S-27f:
AGAGTTTGATCCTGGCTCAG；16S-1429R:GGTTACCTTGTTACGACTT) carry out PCR amplification.PCR
Reaction system is (25 μ l): 10 × amplification buffer is (containing Mg2+1.5mM) 2.5 μ l, dNTP1 μ l, draws
Thing each 1 μ l, template DNA 1 μ l, Taq archaeal dna polymerase 0.2 μ l, add deionized water and complement to 25 μ l；PCR
Amplification condition: 94 DEG C of denaturations 2min, 94 DEG C of degeneration 30s, 55 DEG C of annealing 1min, 72 DEG C extend 1.5min,
25 circulations, 72 DEG C extend 5min.The 16S rDNA of PCR amplification detects through agarose gel electrophoresis, uses
DNA purification kit is purified according to operating instruction, and is cloned on pMD19-T carrier, converts escherichia coli
DH5 α competent cell, chooses positive colony, surveys in Shanghai Sangon Biological Engineering Technology And Service Co., Ltd
Sequence.
The 16S rDNA sequence that order-checking obtains is carried out BLAST comparison analysis in GenBank, chooses homology
The bacillus 16S rDNA sequence of similar temperament, utilizes Clustal X v2.0 and MEGA5.1 software to compare
To analyzing and building the systematic evolution tree shown in Fig. 2.
Sequence table is the Bacillus thuringiensis bacterial strain 16S rDNA sequence separating and killing Meloidogyne incognita, is
System cladogram display NBIN-863 Yu Bacillus thuringiensis L15 sibship is relatively near, and sequence is same
Source property is 100%, forms a cluster, shows that the bacterial strain being separated to soil near Anhui Province's Jiuhuashan belongs to
Plant Bacillus thuringiensis, further named NBIN-863Bacillus thuringiensis
NBIN-863, the accession number being submitted in GenBank data base obtain is KF935650.
Meloidogyne incognita second instar larvae is carried out bioassay, result display NBIN-863Bacillus
Thuringiensis NBIN-863 bacterial strain has the highest virulence to Meloidogyne incognita second instar larvae, processes 24h
After, Meloidogyne incognita mortality rate has reached more than 80%.Fermentation liquid is 0.1 to the LC50 value of root-knot nematode
μ g/ml, supernatant LC50 value is 0.6 μ g/ml, and precipitation LC50 value is 1.17 μ g/ml, as shown in Table 1.
The form 1 bacterium NBIN-863Bacillus thuringiensis NBIN-863 LC50 to Meloidogyne incognita second instar larvae
Value
The applicant apply on the same day " kill the thuringiensis of Meloidogyne incognita active substance and
Application ", file is mentioned: the active substance of the thuringiensis of Meloidogyne incognita is for killing Root Knot line
The crystal protein gene cry9Aa-like of worm, is a kind of insecticidal crystal protein, and molecular weight is 98kDa.
Cry9Aa-like protein-coding region, by 2223 base compositions, has the core shown in sequence table SEQ ID NO:1
Nucleotide sequence；It is made up of 741 amino acid residues, there is the aminoacid sequence shown in sequence table SEQ ID NO:1
Row.Kill the bacillus active substance Biological control for Meloidogyne incognita of Meloidogyne incognita.Nematicide
Crystalline protein Cry9Aa-like is 5.92 μ g/mL to Meloidogyne incognita half lethal concentration；After pouring root kind
Radix Solani Melongenae portion root knot quantity greatly reduces, well developed root system, and prevention effect is 50%.
Claims (3)
1. the bacillus thuringiensis killing Meloidogyne incognita, it is characterised in that kill Meloidogyne incognita bacillus thuringiensis bacterial strain (Bacillus thuringiensis) NBIN-863, deposit number is CCTCC NO:M 2013612.
2. cultivate the method for bacillus thuringiensis described in claim 1, it is characterised in that specifically comprise the following steps that
Take bacillus thuringiensis NBIN-863 described in claim 1, be inoculated in LB liquid medium, 28 DEG C, 200rpm, cultivate 48h.
3. the application in killing Meloidogyne incognita of the bacillus thuringiensis NBIN-863 described in claim 1.
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| CN109749970B (en) * | 2016-04-20 | 2020-03-31 | 北京市农林科学院 | Application of endophytic bacillus in resisting plant parasitic nematode |
| CN106417345B (en) * | 2016-09-08 | 2019-06-25 | 湖南省植物保护研究所 | Insecticidal crystal proteins composition and its application for preventing and treating Plant nematode |
| CN109810920B (en) * | 2019-01-30 | 2022-11-11 | 湖南师范大学 | Bacillus thuringiensis and application thereof |
| CN111849839A (en) * | 2020-08-11 | 2020-10-30 | 江西顺泉生物科技有限公司 | Preparation and application of two composite bacterial liquids for preventing and treating root knot nematode disease |
| CN112868672A (en) * | 2021-01-28 | 2021-06-01 | 湖北省生物农药工程研究中心 | Bacillus thuringiensis suspending agent for killing meloidogyne incognita and application thereof |
| CN113980866B (en) * | 2021-11-30 | 2023-05-30 | 桂林理工大学 | Endophytic bacterium of lime and application thereof |
| CN114606155B (en) * | 2022-01-25 | 2023-08-04 | 云南大学 | Application of bacillus thuringiensis Bt79 and/or Bt80 strain in preventing and controlling plant southern root-knot nematode |
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| CN103125516B (en) * | 2011-11-24 | 2014-12-17 | 华中农业大学 | Protein composition Cry6A/Cry55A having insecticidal synergistic effect for meloidogyne incognita, and application |
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