CN103555614A

CN103555614A - Bacillus thuringiensis for inhibiting stem bleeding disease of coconut and killing lepidoptera pests and application thereof

Info

Publication number: CN103555614A
Application number: CN201310488408.3A
Authority: CN
Inventors: 孙晓东; 余凤玉; 覃伟权; 吕朝军; 刘丽
Original assignee: Coconut Research Institute of Chinese Academy of Tropical Agricultural Sciences
Current assignee: Coconut Research Institute of Chinese Academy of Tropical Agricultural Sciences
Priority date: 2013-10-18
Filing date: 2013-10-18
Publication date: 2014-02-05
Anticipated expiration: 2033-10-18
Also published as: CN103555614B

Abstract

The invention discloses bacillus thuringiensis for inhibiting stem bleeding disease of coconut and killing lepidoptera pests and application thereof. The bacillus thuringiensis has a collection number of CGMCC No.7967. A bacillus thuringiensis strain WENCHANG-BT20 is directly separated and screened from a soil sample on the coast of Hainan and carries four insecticidal crystal protein encoding genes of crylAa, crylBe, cryllia and cry2Aa. The bacillus thuringiensis has high insecticidal capacity and good insecticidal effect and has a certain effect of inhibiting fungal diseases.

Description

One strain suppresses bacillus thuringiensis and the application thereof that coconut stem rushes down blood disease, kills lepidoptera pest

Technical field

The present invention relates to a kind of novel microorganism bacterial strain, be specifically related to a kind of bacillus thuringiensis bacterial strain screening method and active bacterial strain preparation method that coconut stem rushes down blood disease, kills lepidoptera pest small cabbage moth that suppress, belong to field of biological pesticide.

Background technology

Small cabbage moth belongs to lepidopteran diamond-back moth section, is one of insect of the most difficult control in a kind of important pests ，Ye Shi world today of brassicaceous vegetable, it is estimated, the whole world every year for the expense of preventing and treating small cabbage moth over 1,000,000,000 dollars.Due to the harm of life-time service chemical pesticide control small cabbage moth, cause it to dissimilar sterilant, all easily to develop immunity to drugs.At present, control comparatively difficulty of its harm, this worm is serious in south China harm throughout the year.

At present, stem rushes down blood disease has become one of Major Diseases of coconut (Cocos nucifera L.), has a very wide distribution, and nearly all coconut growing area has report.Ohler (1906) has reported that betiding Sri Lankan coconut stem rushes down blood disease the earliest.The coconut growing area such as India (1922), Philippines (1922), Malaysia (1923), Trinidad (1940) and Florida State (1959) has been reported the generation of this disease in succession subsequently.Kartika etc. (1984) and Nambiar etc. (1988) all report that coconut stem rushes down blood disease and can badly influence coconut fruit output; R.N.Dulce etc. (2009) report that the coconut palm outburst coconut stem in Brazilian Sai Erxipei state rushes down blood disease, approximately have the coconut palm performance stem of 50 coconut plantations to rush down blood disease symptoms, and stem rushes down blood disease becomes one of Major Diseases of Brazilian coconut palm.This disease is generally invaded from wound, if do not take prophylactico-therapeutic measures, disease plant is dead in 3～4 months after symptom occurs.Overcast and rainy wet weather is applicable to this disease and occurs popular.

Bacillus thuringiensis (Bacillus thuringiensis is called for short Bt) is a kind for Gram-positive bacillus.In the process of its formation gemma, can form the crystal proteins (ICPs) with insecticidal activity.This insecticidal crystal protein is by cry genes encoding, have been found that now that the most of bacterial strains of bacillus thuringiensis can produce polytype crystallin, to various insects (Faust G M etal, 1983 such as lepidopteran (Lepidoptera), Diptera (Diptera), Coleoptera (Coleoptera), Hymenoptera (Hymenoptera), Homoptera (Homoptera), Orthoptera (Orthoptera), Mallophagas (Mallophaga); Tailor R et al, 1992; Hoffmann A et al, 1998; Pinto L M et al, 2003; Rowell B et al, 2005), and nematode, mite class and protozoon etc. have special insecticidal activity, and the desinsection scope of Bt has been expanded to cover a large amount of agricultural and sanitary insect pest 9 object biologies in interior invertebral 4 doors and Arthropoda.Bt insecticidal activity is harmless to non-target organism, is therefore made into the control (Shu Changlong etc., 2007) that biotic pesticide are widely used in agricultural, forestry and sanitary insect pest.At present, have many countries all Bt to be produced as the U.S., Japan, France, Belgium, wherein amount produced in USA is maximum.China Bt produces and to start from the sixties in 20th century, reaches so far Jin60Jia manufacturer, and annual production reaches more than 60,000 tons, more than 20 provinces be used for grain, cotton, really, 20 various pests (Yang Huaiwen, 2005) of vegetables, woods.The toxic protein of bacillus thuringiensis has also been obtained very large achievement in genetically engineered field, within 1987, Vaeck reported first imports tobacco by crylAb gene, render transgenic tobacco has avoided the infringement of maduca sexta, demonstrates the great potential of Bt gene in transgenic plant application.Through crylAb and the crylAc modifying, in succession plants such as cotton, tobacco, tomato, corn, potato, soybean, white spruces, obtain high level expression subsequently.By 2004, the transgenic Bt crops of 800,000 hectares was planted in the whole world.(Vaeck，et?al，1987；Takeshi?Ito?et?al，2005)。

Summary of the invention

The object of the present invention is to provide and a kind of small cabbage moth is had good disinsection effect, suppresses the bacillus thuringiensis bacterial strain that coconut fungal disease coconut rushes down blood disease simultaneously.

Bacterial strain of the present invention is the bacillus thuringiensis that the strain production traits filtering out by the high grand gulf of collection Wenchang, hainan coastal soil sample separation is good, lepidoptera pest small cabbage moth is had to high virulence, bacterial strain WENCHANG-BT20, in 2013 Nian07 Yue26 China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) preservations, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica, deposit number: CGMCC NO.7967, Classification And Nomenclature bacillus thuringiensis Bacillus thuringiensis; Strain cell and crystal habit are shown in Fig. 1.

Bt inoculation of the present invention, on 1/2LB flat board, is cultivated for 30 ℃ and is observed colony morphology characteristic.

Bacillus thuringiensis bacterial strain WENCHANG-BT20 of the present invention has following beneficial effect:

The bacillus thuringiensis bacterial strain WENCHANG-BT20 of the present invention's direct separation screening from the coastal soil sample of Hainan, carry crylAa, crylBe, crylia, tetra-kinds of insecticidal proteins encoding genes of cry2Aa, insecticidal toxicity is strong, good disinsection effect, has the effect of certain Antifungi disease simultaneously.

Accompanying drawing explanation

Fig. 1: WENCHANG-BT20 strain cell and crystal habit;

Fig. 2: WENCHANG-BT20 rushes down the biocidal property experimental result of blood disease to coconut stem;

Fig. 3: WENCHANG-BT20 is to small cabbage moth indoor insecticidal determination of activity result;

Embodiment

Below in conjunction with specific embodiment, the present invention is described in detail.

If do not specialize, the conventional means that in embodiment, technique means used is well known to those skilled in the art.

The separation of embodiment 1 bacterial strain

Getting appropriate soil sample adds in isolation medium, 30 ℃, 200rpm shaking table is cultivated after 10h, 80 ℃ of water-bath 15min, by four concentration (stostes, 10 times of stoste dilutions, 100 times, 1000 times) respectively inhale 100 μ L painting solid 1/2LB dull and stereotyped (each concentration is coated with two flat boards), pour little granulated glass sphere into and roll, bacterium liquid is evenly coated with, room temperature is placed in 30 ℃ of incubators by flat board after placing 10min, after 48h, observe colonial morphology, select the bacterium colony smear of similar Bt, with detecting and whether have gemma and parasporal crystal under oily mirror after azaleine dyeing.The bacillus thuringiensis 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., every kind of form get a strain carry out single bacterium colony purifying after preservation standby.

Bacterial strain WENCHANG-BT20, in 2013 Nian07 Yue26 China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) preservations, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica, deposit number: CGMCC NO.7967, Classification And Nomenclature Bacillus thuringiensis; Strain cell and crystal habit are shown in Fig. 1.

The evaluation of embodiment 2 bacterial strains

Bt bacterial strain WENCHANG-BT20 is cultivated after 48 hours and forms single bacterium colony on 1/2LB substratum, bacterium colony oyster white, circle or subcircular, neat in edge, thicker in the middle of bacterium colony, to surrounding attenuation gradually, within general about 30 hours, can be observed crystal.Under scanning electron microscope, observing Bt bacterial strain thalline is elongated rod shape, and gemma is oval bar-shaped, and crystal is rhombus (Fig. 1).

16S rDNA sequence amplification, sequencing and the molecular classification of WENCHANG-BT20 bacterial strain:

Extract strain gene group DNA, with bacterium universal primer F (5 '-AGAGTTTGATCCTGGCTCAG-3 ') and R (5'-AAGGAGGTGATCCAGCC-3 '), carry out polymerase chain reaction (pcr amplification), reaction conditions is 94 ℃ of sex change 1 minute, anneal 1 minute for 52 ℃, 72 ℃ are extended 1 minute, 30 circulations, last 72 ℃ are extended 10 minutes, obtain the conserved sequence (seeing sequence table) of the 16S rDNA of this bacterial strain, by NCBI Blast sequence alignment with DNAMAN5.2 software Multiple Sequence Alignment, analyze and constructing system evolutionary tree, be accredited as a bacillus thuringiensis strain bacterial strain.

WENCHANG-BT20 rushes down the mensuration of blood germ bacteriostatic activity to coconut:

Adopt dull and stereotyped face-off culture method, in dull and stereotyped central authorities, connect bacillus thuringiensis bacterium cake (diameter is 6mm), around 35mm place is equidistant connects 4 strains equally for examination pathogen bacterium cake (diameter is 6mm).With central authorities, only connect pathogenic bacteria, do not connect Bt bacterium and contrast, every processing repeats 3 times, and 25 ℃ of cultivations, cover with after flat board until each control strain.Observe bacillus thuringiensis and coconut stem is rushed down to the fungistatic effect of blood germ, and measure statistics.Through measuring the antibacterial circle diameter of this bacterial strain, be 22mm.

The extraction of WENCHANG-BT20 parasporal crystal protein is with quantitative:

First inoculate beef-protein medium to the cracking of thalline fat (30 ℃ of constant temperature, 39h is cultivated in 230rpm/min concussion); The centrifugal 8min of cryogenic freezing whizzer 10000rpm collects thalline; With the centrifugal 8min of 10000rpm after 1M NaCl suspension thalline, remove supernatant; Aseptic distillation washing precipitation; 50mM Na ₂cO ₃, the 50mM EDTA volume ratio mercaptoethanol pH9.5-10.5 that is 5% the precipitation ice bath 100rpm that suspends, more than 4 hours; The centrifugal 10min of 10000rpm, gets supernatant; Supernatant adds the 3MNaAc of 1/10 volume to adjust PH to 4.5-5.0, mixes, and ice bath precipitates more than 2 hours; The centrifugal 10min of 10000rpm, gets precipitation; With sterile purified water washing protein to without till mercaptoethanol; Appropriate 50mM Na for precipitation ₂cO ₃dissolve pH10.2, dissolve on ice 1 hour.After dissolving, SDS-PAGE and BSA compare quantitatively, and measuring protein dissolution liquid concentration is 1.6 μ g/mL.

The indoor insecticidal determination of activity of embodiment 3WENCHANG-BT20 to small cabbage moth

Adopt leaf dipping method, the protein solution of WENCHANG-BT20 is arranged to 8 experimental concentration gradients, be respectively 1.6 μ g/mL, 0.8 μ g/mL, 0.4 μ g/mL, 0.2 μ g/mL, 0.1 μ g/mL, 0.05 μ g/mL, 0.025 μ g/mL, 0.0125 μ g/mL.Cabbage leaves is cleaned and dried with clear water, choose fresh and tender consistent cabbage leaves and be cut into the bulk that size is close, in the protein solution of each concentration, soak 10min, dry, put into raw bottle, 20 of the every bottle graft 2-3 instar larvaes surveyed, each is processed in triplicate, in 25 ℃ of biochemical cultivation cases, be incubated, cultivate dead, the alive borer population of 72h " Invest, Then Investigate ", and observe larval feeding situation.

Bioassay results shows that WENCHANG-BT20 albumen has high insecticidal activity to small cabbage moth, and the obvious blackening of poisoning small cabbage moth is dead, and whole polypide is black, rot, and contrast is larger for examination worm performance appetite, and worm is active, grows normal (Fig. 3).Its protein liquid LC ₅₀be 0.088 μ g/mL, degree of confidence is 95%, and fiducial interval is 0.046-0.127 μ g/mL (table 1)

Table 1.WENCHANG-BT20 is to small cabbage moth insecticidal activity (72hr)

Embodiment 4WENCHANG-BT20 bacterial strain produces spore characteristic

By concentration, be 1 * 10 ⁸the WENCHANG-BT20 thallus suspension liquid of cfu/mL is (100 μ L/ ware) on 1/2LB substratum, sporulation quantity cultivate 15 days under 20 ℃ of differing tempss, the culture condition of 22 ℃, 24 ℃, 25 ℃, 26 ℃, 27 ℃, 28 ℃, 29 ℃, 30 ℃, 35 ℃ after on analytical unit area, determines that WENCHANG-BT20 bacterial strain produces spore amount for best at 30 ℃.

Should be understood that, for those of ordinary skills, can be improved according to the above description or convert, and all these improvement and conversion all should belong to the protection domain of claims of the present invention.

Claims

1. a strain inhibition coconut stem rushes down blood disease, kills the bacillus thuringiensis of lepidoptera pest, and deposit number is CGMCC NO.7967.

2. bacillus thuringiensis according to claim 1 rushes down blood disease, kills the application in lepidoptera pest at inhibition coconut stem.

3. bacillus thuringiensis according to claim 1 is rushing down blood disease, is killing the application in lepidoptera pest agricultural chemicals for the preparation of suppressing coconut stem.