CN101481668A - Bacillus thuringiensis suspending agent for killing cotton spider mites, preparation and use - Google Patents
Bacillus thuringiensis suspending agent for killing cotton spider mites, preparation and use Download PDFInfo
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- CN101481668A CN101481668A CNA2009100138504A CN200910013850A CN101481668A CN 101481668 A CN101481668 A CN 101481668A CN A2009100138504 A CNA2009100138504 A CN A2009100138504A CN 200910013850 A CN200910013850 A CN 200910013850A CN 101481668 A CN101481668 A CN 101481668A
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Abstract
The invention relates to a suspending agent sterilizing two-spotted spider mite Bacillus thuringiensis, belonging to the field of biological pesticides. The suspending agent is prepared by mixing fermentation liquid of the Bacillus thuringiensis BtR05 with a surfactant, an antiseptic agent, a suspending auxiliary agent and a thickening agent, wherein, the suspending auxiliary agent is ammonium sulphate. The invention also provides preparation methods and uses of the suspending agent, the suspending agent prepared by the BtR05 bacterium and the fermentation liquid of the bacterium is quite poisonous against two-spotted spider mite, American white moth, diamond-back moth, chive maggot, grub, maggot galerucella and Meloidogyne incognita, which is an efficient and broad-spectrum biological pesticide.
Description
Technical field
The present invention relates to a strain has bacillus thuringiensis (Bacillus thuringiensis) BtR05 bacterial strain and the suspension agent preparation method and the application of efficient acaricidal activity to cotton spider mites, belongs to biological pesticide technical field.
Background technology
Cotton spider mites claims cotton red spider again, belongs to Arachnida, acarina, section pest mite, and all there is generation in each cotton region, except that the harm cotton, also endangers corn, Chinese sorghum, wheat, soybean etc., and the host is extensive.Cotton spider mites is mainly inhaled juice in back, cotton blade face thorn, makes the blade face hazard symptoms such as macula lutea, red autumnal leaves and fallen leaves occur, and likeness in form is burnt, and is commonly called as flue.Break out the time, cause to drop in production over a large area even have no harvest.Generation and Prevention Technique (Jing County agricultural information network) referring to cotton spider mites.Fall webworms belong to the lepidopteran Arctiidae, it is a kind of leaf-feeding insect, it is worldwide Quarantine Objects, be the significant risk insect of agricultural, forestry, gardens, gardening plant, have the advantages that feeding habits are assorted, breeding is fast, the route of transmission is wide, the suitable ability of giving birth to by force, is easily broken out and caused disaster, can eat up all green plantss in the extremely short time.Last century end, fall webworms are at first imported northeast into through Dandong, very fast in the Huanghe valley, Bohai Rim takes place, and is listed in the important harmful organism that disturbs " green Games ", should give special concern, monitor closely, whole people's joint defence in Beijing.From in June, 2008, the fall webworms insect pest breaks out in the Shandong Province comprehensively, and enters the hazard boom phase, and its tempo exceeds anticipation.To in July, 2008, in 17 cities of Shandong province, except that Zaozhuang, all become epidemic-stricken area (2008-10-0609:17:00 author: dzwww Web-Shandong evening paper).Small cabbage moth belongs to lepidopteran diamond-back moth section, be the important pests of worldwide brassicaceous vegetable, endanger throughout the year, the about 15%-30% of its from generation to generation main natural hazard loss in the subtropical zone, serious reach 70% or more even ruin product, vegetables production has been constituted very big threat.Referring to Wu Yonghan, main vegetable-growing area, Wenzhou City small cabbage moth resistance preliminary study (Wenzhou agricultural science and technology, 2004 (3): 79-81).The fragrant-flowered garlic maggot belongs to Diptera, and Lycoriidae is the primary pest of harm leek, it is caused harm not only influences the leek quality, and causes output to reduce significantly, the killed strain rate 20%～30% in general plot, serious then up to 100%, cause production loss 30%～80%.Along with the leek big area is planted year after year, the fragrant-flowered garlic maggot is caused harm and increases the weight of gradually.Referring to Zhang Yi, and the measure of fragrant-flowered garlic maggot non-environmental pollution control (northwest gardening, 2008,09:36-37).Grub belongs to Coleoptera, is the general designation of chafer, is one of important subterranean pest-insect of farm crop.Subterraneous root, stem that it causes harm multiple kinds of crops such as wheat, corn, peanut, Ipomoea batatas cause seedling withered.Its adult farm crop blade of also can causing harm influences photosynthesis, makes crop cause the underproduction significantly.Particularly serious to causing harm of peanut.It stings the food peanut, makes peanut form ghost, and the lighter loses one two one-tenth, heavy person seven, most probably, even total crop failure.Referring to Chai Guanqing, grub is caused harm and Preventing Countermeasures (plant protection, 2002,21) to peanut.Plant nematode has become the important pathogen of a class of plant, and nematodiasiss has become the major issue in farming, the woods production.According to the FAO conservative estimation, whole world every year accounts for 12% of total losses greatly because of the loss that nematodiasiss causes grain and fibre crops, and the loss that vegetables, peanut, tobacco and some fruit tree are caused then surpasses 20%.Meloidogyne incognita belongs to nematode door, Tylenchida, golden nematode section, Meloidogyne, is the common and very serious a kind of insect of harm in vegetable crops such as Curcurbitaceae, Solanaceae, Cruciferae.Referring to Zhang Nan, press down screening (Gansu Agriculture University,'s journal, 2008,43 (4): 87-90) of the vegetable active extracting solution that kills Meloidogyne incognita.The above insect all has generation every year to the harm of farm crop, vegetables and afforestation, and chemical pesticide is all had in various degree resistance, for delaying their resistance, presses for effective biological pesticide and prevents and treats.
Bacillus thuringiensis (Bacillus thuringiensis is called for short Bt) sterilant is to study maximum, most widely used microbial pesticide at present both at home and abroad.Up to the present the Bt sterilant is mainly used to prevent and treat the insect of some kind of lepidopteran, Diptera and Coleoptera.Referring to Liu Shiquan, tire progress (the microbiology circular of sterilant of bacillus thuringiensis height, 2008,35 (7): 1091-1095), and cotton spider mites is had efficient acaricidal activity, the bacterial strain that part insect and the Plant nematode of lepidopteran, Diptera and Coleoptera all had insecticidal activity does not appear in the newspapers as yet simultaneously.Bacterial strain BtR05 of the present invention has further enlarged the insecticidal spectrum of Bt, for important basis has been established in the widespread use of Bt sterilant.
Summary of the invention
Deficiency at existing bacillus thuringiensis (Bt) sterilant insecticidal spectrum, the invention provides a kind of bacillus thuringiensis suspending agent and preparation method thereof and application of the wide spectrum that cotton spider mites is had the bacterial strain of high-efficiency broad spectrum acaricidal activity, the present invention also to provide to utilize this bacterial strain preparation.
One, bacterial strain
The invention provides a kind of bacterial strain that cotton spider mites is had the high-efficiency broad spectrum acaricidal activity, classification called after bacillus thuringiensis (Bacillus thuringiensis) BtR05, its preserving number is CGMCCN0.2823, the preservation mouth phase is on December 23rd, 2008, depositary institution: China Committee for Culture Collection of Microorganisms common micro-organisms center (being called for short CGMCC).
1 strains separation and evaluation
(1) the bacterial strain method that adopts substratum to add sodium-acetate is screened gained in soil.
(2) identification of strains employing physiological and biochemical property mensuration and 16SrDNA sequence alignment analysis bonded method are bacillus thuringiensis (Bacillus thuringiensis) with this identification of strains, called after BtR05.
2 pairs of cotton spider mites pathogenic
(1) acquisition of susceptible polypide: to the LB shake flask fermentation liquid (viable count 2.10 * 10 of potted plant band mite cotton spray BtR05
9CfumL
-1), take blade after four days, collect susceptible polypide.
(2) separation and Culture of pathogenic bacterium BtR05: the separation and Culture of pathogenic bacterium is carried out on the aseptic technique platform.Get the flat board of a sterilization, put a filter paper on the flat board.45 susceptible tetranychids are placed on the filter paper, alcohol flushing 2min with liquid-transfering gun absorption 75%, change clean filter paper then, sick mite brushed down gently with writing brush change new filter paper over to, wash 3～5min with 0.1% mercuric chloride then, wash 3～5min with 10% hypo solution again after changing filter paper according to the method described above, place for 4 times with aseptic water washing then and blot surface water on the sterilizing paper, be inoculated on the substratum with the line partition method again, put into 30 ℃ of constant incubators and cultivate, and observe its morphological specificity., on slide glass, fixedly be placed under the oily mirror its form of observation and take pictures at single bacterium colony of picking line separation and purification gained on the Bechtop through gramstaining.
(3) evaluation of pathogenic bacterium: single bacterium colony of separation and purification is carried out the LB liquid fermenting, carry out the evaluation of seven key characters such as V.P test, mycoderm formation test, Nagler's reaction, starch hydrolysis experiment, casein hydrolysis test, polychrom, cellobiose simultaneously, measure according to " the common bacteria system identification handbook " of all writing with reference to eastern elegant pearl, Cai Miaoying etc.
(4) virulence of BtR05 test (seeing Table 1): the fermented liquid stoste that adopts LB to cultivate 2 days is done acaricidal activity and is measured (leaf dipping method), is contrast with the clear water.Check each handling dead, borer population alive respectively at different time.The result shows that twice isolating pathogenic bacterium 72h corrected mortality after connecing bacterium all reaches more than 50%, proves that this bacterium has toxic action to cotton spider mites.
Table 1 BtR05 becomes the pathogenic measurement result of mite to cotton spider mites
Annotate: there have alphabetical identical person to represent through Duncan duncan's new multiple range method check difference on 0.05 conspicuous level with the column data heel in the table to be not remarkable.
(5) separation and Culture once more and the evaluation of pathogenic bacterium: cotton spider mites dead in (4) is handled with the method for (2), identified by the step of (3) then; Qualification result shows that twice isolating pathogenic bacterium is BtR05.
Two, the preparation of fermentation liquid of BtR05 bacterium
The fermentation condition of BtR05 bacterium is: inclined-plane and shake-flask culture base are LB.Fermention medium is by mass percentage: 2% Semen Maydis powder, 6% beans paper tinsel powder, 1% sucrose, 0.5% (NH
4)
2SO
4, 0.2% K
2HPO
4, 0.2% KH
2PO
4, 0.03% MgSO
47H
2O, pH7.0-7.5.Ferment in the 10L fermentor tank, per minute air flow and fermentor tank volumetric ratio are 1.2～1.5:1,30～32 ℃ of culture temperature, inoculum size 5%wt, rotating speed 150～400r/min, fermentation time are 36～48h, spore production rate is 70～72%, live bacterial count 8.33 * 10
9CfumL
-1
Illustrate: substratum LB is this area regular bevel and shakes bottle and use substratum that its composition is: 1% peptone, 0.5% yeast extract, 1% sodium-chlor, 1.2% agar (inclined-plane), pH7.0-7.5.
Three, utilize the bacillus thuringiensis suspending agent (being called for short the BTD suspension agent) of bacterial strain BtR05 preparation
Fermented liquid and tensio-active agent, sanitas, suspension aids and the thickening material etc. of the BtR05 bacterium of above-mentioned two, middle preparation are mixed with the BTD suspension agent, and wherein suspension aids is an ammonium sulfate.
The suspension aids of BTD suspension agent of the present invention selects ammonium sulfate to substitute sodium-chlor commonly used now, can reduce the content of sodium-chlor in the edatope in actual applications, prevents salting of soil, and while ammonium sulfate can be used as nitrogenous fertilizer and can be absorbed by crop.
Preferably, suspension aids of the present invention is an ammonium sulfate, and sanitas is a Sodium Benzoate, and tensio-active agent is a fatty alcohol-polyoxyethylene ether, and thickening material is an xanthan gum.
Preferably, bacillus thuringiensis suspending agent of the present invention, it is characterized in that, in the fermented liquid of BtR05 bacterium, add ammonium sulfate 5%～8% by mass percentage, Sodium Benzoate 0.1%～0.2%, fatty alcohol-polyoxyethylene ether 0.1%～0.3%, xanthan gum 0.2%～0.3% makes, BtR05 live bacterial count 8.33 * 10 in the gained suspension agent
9CfumL
-1The consumption of above each component is a radix with the quality of the fermented liquid of BtR05 bacterium all.
2, bacillus thuringiensis suspending agent preparation method:
Earlier ammonium sulfate is stirred in the fermented liquid that adds the BtR05 bacterium down, treat that ammonium sulfate all after the dissolving, under agitation adds preservative sodium benzoate, tensio-active agent fatty alcohol-polyoxyethylene ether, thickening material xanthan gum successively, stirring gets final product.
3, the insecticidal spectrum of BTD suspension agent is measured
For whether checking BTD suspension agent has the insecticidal activity of wide spectrum, we have carried out insecticidal test to following insect (seeing Table 2).Found that the BTD suspension agent all has certain insecticidal effect to cotton spider mites, the chrysomelid larva of fragrant-flowered garlic firefly, grub, fall webworms, diamondback moth larvae, fragrant-flowered garlic maggot, the hatching to Meloidogyne incognita egg capsule and cotton spider mites ovum simultaneously has restraining effect.
The insecticidal spectrum of table 2 BTD suspension agent
Four, use
BtR05 bacterial strain of the present invention is used to prepare sterilant.Each growth period to cotton spider mites has toxic action, also insect and the Plant nematode (Meloidogyne incognita) of lepidopteran (fall webworms and small cabbage moth), Diptera (fragrant-flowered garlic maggot), Coleoptera (grub and fragrant-flowered garlic firefly are chrysomelid) is had toxic action simultaneously.
The application of the BTD suspension agent of BtR05 bacterial strain of the present invention preparation, as the sterilant of following plant insect:
Each budding cotton spider mites, fall webworms, small cabbage moth, the fragrant-flowered garlic maggot, grub, the fragrant-flowered garlic firefly is chrysomelid, Meloidogyne incognita.
Advantage compared with prior art of the present invention is:
BtR05 bacterial strain among the present invention is the bacillus thuringiensis with efficient acaricidal activity, utilize this bacterial strain to develop a kind of BTD suspension agent of high-efficiency broad spectrum, the active bacteria formulation biological pesticide of this suspension agent mechanism of action uniqueness, have the stomach toxicity action of contace poison of holding concurrently, can avoid producing cross resistance with existing business-like sterilant.Test-results shows, this suspension agent not only has toxic action to each growth period of cotton spider mites, also insect and the Plant nematode (Meloidogyne incognita) of lepidopteran (fall webworms and small cabbage moth), Diptera (fragrant-flowered garlic maggot), Coleoptera (grub and fragrant-flowered garlic firefly are chrysomelid) is had toxic action simultaneously; The discovery of bacterial strain BtR05 has further enlarged the insecticidal spectrum of bacillus thuringiensis, for important basis has been established in the widespread use of thuringiensis cladosporioides bacillus insecticide.BTD suspension agent of the present invention is a kind of biological pesticide of high-efficiency broad spectrum, can be widely used in the green agriculture production.
Description of drawings
Fig. 1-3 is respectively nourishing body, crystal and the gemma of BtR05 in embodiment 1 step 2 (2).
Embodiment
Following examples are to further specify of the present invention, but the present invention is not limited thereto.
Embodiment 1: the separation of bacterial strain BtR05 and evaluation
1, the separating step of bacterial strain BtR05 is as follows:
(1) sample collecting: collected specimens is a vegetables field soil, and the place is Jinan, gets the dark topsoil of 0～10cm with spades, installs with polyethylene bag, takes back the laboratory.
(2) separation method: the earth sample 1g that fetches earth is in BPA substratum (BPA substratum: extractum carnis 5g, peptone 10g, sodium-acetate 34g, water 1000mL, pH7.2～7.5) fully after the vibration, place 30 ℃ of shaking tables to cultivate 6 hours, taking-up places 75～80 ℃ of thermal treatments 10 minutes, static slightly after, get 0.5mL in BP substratum (extractum carnis 3g, peptone 5g, sodium-chlor 5g, agar 15g, distilled water 1000mL, pH7.0～7.2) on the flat board, coating evenly; Put 30 ℃ and cultivated 24 hours, the colony inoculation of selecting the seemingly plain cloud gold of feature class genus bacillus is on the BP inclined-plane; Place 30 ℃ to cultivate more than 72 hours, the film-making microscopy, reservation can be produced the bacterial strain of parasporal crystal.
2, identification of strains
Adopting physiological and biochemical property mensuration and 16SrDNA sequence alignment to analyze the bonded method is bacillus thuringiensis (Bacillus thuringiensis) with this identification of strains, called after BtR05.Concrete steps are as follows:
(1) 16SrDNA sequential analysis
The extraction of a strain gene group DNA: extract the genomic dna of bacterium in a small amount by common bacteria genome extracting method
The pcr amplification of b 16SrDNA: according to conservative region design universal primer A and the B of bacterial 16 S rDNA, with total DNA is template, with primer A (5 '-AGAGTTTGATCCTGGCTCAG-3 ') and primer B (5 '-TACGGCTACCTTGTTACGACT-3 ') amplification 16SrDNA.PCR reaction cumulative volume is 25 μ L: add 10 * amplification buffer respectively and (contain Mg
2+) 2.5 μ L; 4 kinds of dNTP mixtures (2.5mM), 2 μ L; Each 1 μ L of primer (10 μ M) A and B; Template DNA 1 μ L; Taq archaeal dna polymerase 1 μ L; Add distilled water then to 25uL.The PCR reaction conditions is: 94 ℃ of pre-sex change 2min at first, and 94 ℃ of sex change 45sec then, 53 ℃ of annealing 45sec, 72 ℃ are extended 45sec, and after 30 circulations, 72 ℃ are extended 15min.The agarose of employing 1% is to the pcr amplification product electrophoresis detection, and the result shows that the amplified production size is about 1500bp.Adopt the silica gel PCR of TIANGEN Biotech (Beijing) Co., Ltd. product purification test kit, carry out the recovery of pcr amplification product.Shanghai is given birth to the worker pMD18T of Bioisystech Co., Ltd clone test kit and is carried out the connection of pcr amplification product.To transform good thalline liquid culture, serve the Hai Shenggong order-checking.
C order-checking and Phylogenetic Analysis: the 16SrDNA nucleotide sequence total length that obtains bacterial strain BtR05 through order-checking is 1525bp, and login GenBank sequence number is EU702408.Existing bacterial 16 S rDNA sequence is carried out the similarity comparative analysis in application BLAST and the database, the result shows that the 16SrDNA sequence homology of the 16SrDNA sequence of BtR05 and bacillus thuringiensis (Bacillus thuringiensis) is very high, reaches 99%.
(2) morphological specificity
The BtR05 bacterial strain adopts the LB solid plate to cultivate.The inoculation back is cultivated 48h and is grown up to the thicker lawn of one deck under 28 ± 2 ℃ constant temperature, after the abundant maturation of thalline, and most of gemma and crystal of forming.The bacterial strain Gram-reaction is positive, and moves with peritrichous.The nourishing body that can be observed BtR05 under oily mirror is shaft-like (shown in Figure 1), the blunt circle in two ends, and size is about 3-6 μ m, generally is cascaded more than 2.Gemma is at a sporangial end, and oblong (shown in Figure 2) forms parasporal crystal at the other end, and crystal is rhombus or irregular shape (shown in Figure 3).Grow on the LB substratum, top temperature is 40 ℃, and minimum temperature is 10 ℃, and the most suitable temperature is 30 ℃.The appropriate pH value of bacterial strain is 7.0-8.0, and the lawn smooth surface is glossy, and bacterium colony oyster white and edge are root shape branch.
(3) physiological and biochemical property
The VP reaction+; Nagler's reaction-; The saligenin test-; Gelatin hydrolysis+; Chromogenesis-; The sucrose utilization-; Urease test-; The polychrom hydrolysis+; The seminose test-; The starch hydrolysis+; The cellobiose test+; Mycoderm formation+; Arginine decarboxylase+; Caseinhydrolysate+;
Morphological specificity of above-mentioned steps (2) and (3) bacterial strain and physiological and biochemical property are measured according to " the common bacteria system identification handbook " of all writing with reference to eastern elegant pearl, Cai Miaoying etc.
Embodiment 2: the liquid fermenting of bacterial strain BtR05, described per-cent is mass percent.
Bacillus thuringiensis BtR05, preserving number is CGMCCNO.2823, separation from soil, screening obtain, and find that through the Study on Pathogenicity to cotton spider mites this bacterial strain acaricidal ability is strong; Easily cultivate, pollution-free.
The liquid fermenting of bacterial strain BtR05, carry out according to the following steps:
(1) slant strains: adopt solid LB substratum, bacillus thuringiensis BtR05 is seeded on the test tube substratum, cultivated 24 hours for 30 ℃.
(2) shake a bottle bacterial classification: adopt liquid LB substratum, test tube strains is seeded in the liquid LB substratum, put on the shaking table 30 ℃ of shaking culture 18 hours.
(3) liquid fermenting: adopt substratum, 2% Semen Maydis powder, 6% beans paper tinsel powder, 1% sucrose, 0.5% (NH
4)
2SO
4, 0.2% K
2HPO
4, 0.2% KH
2PO
4, 0.03% MgSO
47H
2O, pH7.0～7.5, pH7.5,121 ℃ of sterilizations 20 minutes, seed that will (2) is inoculated in 10 liters fermentor tank interior (inoculum size is 5%wt), 30 ℃ of cultivations, per minute air flow and fermentor tank volumetric ratio are 1.2～1.5:1, and stirring velocity is 400r/min, and incubation time is 36～48 hours; When bacillus exfoliation 70%, stop fermentation, the pH of fermented liquid is transferred to 4.5～5.0 with HCL.
The preparation of embodiment 3:BTD suspension agent, described per-cent is mass percent, is radix with the amount of fermented liquid.
Add 5% ammonium sulfate, 0.15% Sodium Benzoate, 0.1% fatty alcohol-polyoxyethylene ether, 0.20% xanthan gum in the fermented liquid in embodiment 2.Attention will slowly and not stop ammonium sulfate to stir ground and add in the fermented liquid in suspension agent configuration, treat that ammonium sulfate all after the dissolving, slowly adds Sodium Benzoate, fatty alcohol-polyoxyethylene ether, xanthan gum more successively.
The insecticidal spectrum of embodiment 4:BTD suspension agent is measured
The BTD suspension agent finds that through the Toxicity Determination test this suspending agent disinfestation spectrum is wide, and prevention effect is good.
(1) the BTD suspension agent is to the different budding Toxicity Determination (seeing Table 3) of cotton spider mites
The leaf dipping method that adopts international resistance Action Committee (IRAC) to propose is handled cotton spider mites.It is stand-by that the BTD suspension agent is diluted with water to different concns.Fresh clean cotton leaf is flooded 30sec in the medicament diluent of different concns, taking-up is dried, insert cotton spider mites then and become 30 of mites (or if mite), each is handled three times and repeats, be put in the constant incubator under (25 ± 1) ℃ condition and cultivate, respectively at checking each handling dead, borer population alive behind the different time, be blank with the clear water.Polypide death standard: touch examination worm polypide with the writing brush point, do not have obvious autonomic response person and be judged as death.Calculate the corrected mortality that each is handled with following formula.
Table 3 is as nextpage.
(2) the BTD suspension agent is to the Toxicity Determination (seeing Table 4) of chrysomelid 3 instar larvaes of fragrant-flowered garlic firefly
Adopt leaf dipping method to handle chrysomelid 3 instar larvaes of fragrant-flowered garlic firefly.It is stand-by that the BTD suspension agent is diluted with water to different concns.Fresh clean leek is flooded 30sec in the medicament diluent of different concns, taking-up is dried, insert chrysomelid 3 instar larvaes of fragrant-flowered garlic firefly then, be divided into 4 processing, 3 repetitions are established in every processing, each 45 larva, place in the insectary, keep natural temperature (April early and middle ten days, 10～20 ℃), handle back observation every day once, write down dead borer population.
Table 4 BTD suspension agent is to the Toxicity Determination of chrysomelid 3 instar larvaes of fragrant-flowered garlic firefly
Annotate: there have alphabetical identical person to represent through Ducan duncan's new multiple range method check difference on 0.05 conspicuous level with the column data heel in the table to be not remarkable.
(3) the BTD suspension agent is to the Toxicity Determination (seeing Table 5) of fall webworms 2 instar larvaes
Adopt spray method to handle fall webworms 2 instar larvaes.Win band worm blade (30～50/leaf) from oriental plant, respectively the BTD suspension agent of different concns is sprayed at on examination blade and the polypide with manual sprayer then, be divided into 6 processing, 3 repetitions are established in every processing, are contrast with the clear water, place in the insectary, keep natural temperature (mid or late September, 20～30 ℃), handle back observation every day once, write down dead borer population.
Table 5 BTD suspension agent is to the Toxicity Determination of fall webworms 2 instar larvaes
Annotate: there have alphabetical identical person to represent through Ducan duncan's new multiple range method check difference on 0.05 conspicuous level with the column data heel in the table to be not remarkable.
(4) the BTD suspension agent is to the Toxicity Determination (seeing Table 6) of grub 3 instar larvaes
Adopt spray method to handle grub 3 instar larvaes.Respectively with the BTD suspension agent spray time grub polypide of different concns, allowing it freely creep moments later with manual sprayer, move into and adorn native the raising in the Cans, is contrast with the clear water.Raise a grub for every bottle, place in the insectary, keep natural temperature (October, 20～30 ℃), suitably preserve moisture.30 grubs of every processing, 3 repetitions.Handle back observation every day once, write down dead borer population.
Table 6 BTD suspension agent is to the Toxicity Determination of grub 3 instar larvaes
Annotate: there have alphabetical identical person to represent through Ducan duncan's new multiple range method check difference on 0.05 conspicuous level with the column data heel in the table to be not remarkable.
(5) the BTD suspension agent is to the Toxicity Determination (seeing Table 7) of fragrant-flowered garlic maggot
Adopt leaf dipping method to handle the fragrant-flowered garlic maggot.It is stand-by that the BTD suspension agent is diluted with water to different concns.Fresh clean leek is flooded 30sec in the medicament diluent of different concns, taking-up is dried, and inserts the fragrant-flowered garlic maggot then, is divided into 5 processing, and 3 repetitions are established in every processing, and each 45 cephalont places in the insectary, keeps 25 ± 1 ℃ of temperature, relative humidity 80%.Handle back 72h and write down dead borer population.
Table 7 BTD suspension agent is to the Toxicity Determination of fragrant-flowered garlic maggot
Annotate: there have alphabetical identical person to represent through Ducan duncan's new multiple range method check difference on 0.05 conspicuous level with the column data heel in the table to be not remarkable.
(6) the BTD suspension agent is to the Toxicity Determination (seeing Table 8) of small cabbage moth
Adopt leaf dipping method: it is stand-by that the BTD suspension agent is diluted with water to different concns.Fresh clean cabbage leaves is flooded 30sec in the medicament diluent of different concns, taking-up is dried, insert 45 of small cabbage moth 3 instar larvaes then, each is handled secondary and repeats, be put in the constant incubator under (25 ± 1) ℃ condition and cultivate, respectively at checking each handling dead, borer population alive behind the different time, be blank with the clear water.Polypide death standard: touch examination worm polypide with the writing brush point, do not have obvious autonomic response person and be judged as death.
Table 8 BTD suspension agent is to the toxic action of small cabbage moth
Annotate: there have alphabetical identical person to represent through Ducan duncan's new multiple range method check difference on 0.05 conspicuous level with the column data heel in the table to be not remarkable.
(7) the BTD suspension agent is to the Toxicity Determination (seeing Table 9) of Meloidogyne incognita egg capsule hatching
The BTD suspension agent is become 25 times with distilled water diluting, and 50 times, 100 times, 200 times, 400 times, other establishes the clear water contrast, and totally 6 processing are standby.To gather the segment that the tomato root of morbidity is cut into 1-2cm from the field and place anatomical lens to observe, 108 egg capsules of picking are in culture dish.Pour 10mL respectively into and respectively handle bacterium liquid in culture dish, put 6 egg capsules in every culture dish, every processing repeats 3 times, culture dish usefulness rubber belt sealing microscopically observations after 4 days.
Table 9 BTD suspension agent is to the restraining effect of Meloidogyne incognita egg capsule hatching
Annotate: there have alphabetical identical person to represent through Ducan duncan's new multiple range method check difference on 0.05 conspicuous level with the column data heel in the table to be not remarkable.
Above-mentioned testing data shows that the BTD suspension agent has the insecticidal activity of wide spectrum.Cotton spider mites desinsection result is shown the concentration of BTD suspension agent dilution below 500 times is to just incubating young mite, better with the toxic effect that becomes mite as if mite, the 96h corrected mortality reaches more than 80%, 75% and 60% respectively behind the medicine; Test-results to the chrysomelid larva of fragrant-flowered garlic firefly shows that the dilution of BTD suspension agent is better to the insecticidal effect of chrysomelid 3 instar larvaes of fragrant-flowered garlic firefly more than 300 times, and the 72h corrected mortality all reaches more than 75%; Test-results to the fragrant-flowered garlic maggot shows, the dilution of BTD suspension agent is better to the insecticidal effect of fragrant-flowered garlic maggot more than 100 times, the test-results that the 72h corrected mortality all reaches more than 60% grub shows that the dilution of BTD suspension agent is better to the insecticidal effect of grub 3 instar larvaes more than 200 times, and the 72h corrected mortality all reaches more than 60%; Test-results to fall webworms shows that the dilution of BTD suspension agent is better to the insecticidal effect of fall webworms 2 instar larvaes more than 200 times, and the 72h corrected mortality all reaches more than 69%; Test-results to small cabbage moth shows that the dilution of BTD suspension agent is better to the insecticidal effect of small cabbage moth 3 instar larvaes more than 100 times, and the 96h corrected mortality all reaches more than 65%; Restraining effect result to the hatching of Meloidogyne incognita egg capsule shows that the dilution of BTD suspension agent reaches more than 77% the restraining effect of egg capsule hatching more than 200 times.
(8) BTD suspension agent control cotton spider mites field control effectiveness test (seeing Table 10)
Test method and condition are all in strict accordance with national field experiment criterion.
Table 10 BTD suspension agent control cotton spider mites field test results
Annotate: the preventive effect (%) that goes up in the table repeats mean value for each, adopts Deng Kenshi new multipole poor (DMRT) method (a=0.05) that testing data is carried out statistical study.
BTD suspension agent consumption is under 450mL/ mu and the 150mL/ mu condition cotton spider mites to be had the better prevention effect, respectively handles preventive effect in seven days behind the medicine and all reaches maximum, is respectively 93.50% and 84.00%.
The preventive effect that three days 450mL/ mu and 150mL/ mu two are handled behind the BTD suspension agent control cotton spider mites medicine is respectively 70.29%, 63.07%, is lower than 91.24% of Avrmectin, illustrates that the quick-acting of this medicine is lower than Avrmectin; The 7th day BTD suspension agent respectively handled preventive effect behind the medicine all had obvious rising than the 3rd day, and wherein 450mL/ mu dosage preventive effect rises to 93.50%, and the preventive effect of Avrmectin drops to 84.52%, illustrates that the long-lasting of this medicine is higher than Avrmectin; Respectively handling preventive effect on the tenth day behind the medicine all has decline, shows that this medicine is seven to ten days in the following lasting period of test conditions.
The BTD suspension agent can be promoted the use of, and it is 150～450mL/ mu that the control cotton spider mites is recommended the preparation consumption.Should select to try the dispenser of worm early period of origination, should evenly, thoughtfully look insect pest situation about seven days and carry out the dispenser second time during dispenser.
Claims (8)
1. bacillus thuringiensis (Bacillus thuringiensis) BtR05 bacterial strain, culture presevation CGMCCNO.2823.
2. the application of the described bacillus thuringiensis BtR05 of claim 1 bacterial strain is used to prepare sterilant.
3. the suspension agent of a bacillus thuringiensis, the fermented liquid of the described BtR05 bacterium of claim 1 and tensio-active agent, sanitas, suspension aids and thickening material is mixed, and wherein suspension aids is an ammonium sulfate.
4. the suspension agent of bacillus thuringiensis as claimed in claim 3 is characterized in that sanitas is a Sodium Benzoate, and tensio-active agent is a fatty alcohol-polyoxyethylene ether, and thickening material is an xanthan gum.
5. the suspension agent of bacillus thuringiensis as claimed in claim 4, it is characterized in that the fermented liquid quality with the BtR05 bacterium is a radix: ammonium sulfate 5%～8%, Sodium Benzoate 0.1%～0.2%, fatty alcohol-polyoxyethylene ether 0.1%～0.3%, xanthan gum 0.2%～0.3% is mass percent.
6. the preparation method of the suspension agent of the described bacillus thuringiensis of claim 3, it is characterized in that, earlier ammonium sulfate is stirred in the fermented liquid that adds the BtR05 bacterium down, treat that ammonium sulfate is all after the dissolving, under agitation add sanitas, tensio-active agent, thickening material successively, stirring gets final product.
7. the preparation method of the suspension agent of bacillus thuringiensis as claimed in claim 6 is characterized in that, the preparation of fermentation liquid condition of described BtR05 bacterium is as follows:
Inclined-plane of BtR05 bacterium and shake-flask culture base are LB;
Fermention medium is by mass percentage: 2% Semen Maydis powder, 6% beans paper tinsel powder, 1% sucrose, 0.5% (NH
4)
2SO
4, 0.2%K
2HPO
4, 0.2% KH
2PO
4, 0.03% MgSO
47H
2O, pH7.0-7.5;
Ferment in the 10L fermentor tank, per minute air flow and fermentor tank volumetric ratio are 1.2～1.5:1,30～32 ℃ of culture temperature, inoculum size 5%wt, rotating speed 150～400r/min, fermentation time are 36～48h, spore production rate is 70～72%, live bacterial count 8.33 * 10
9CfumL
-1
8. the application of the suspension agent of the described bacillus thuringiensis of claim 3, as the sterilant of following plant insect:
Cotton spider mites, fall webworms, small cabbage moth, the fragrant-flowered garlic maggot, grub, the fragrant-flowered garlic firefly is chrysomelid, Meloidogyne incognita.
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