CN104164378A - Bacillus atrophaeus having poisoning and controlling effects on potato beetles - Google Patents

Abstract

Bacillus atrophaeus CPB072 having poisoning and controlling effects on potato beetles is separated from natural environment, can grow and massively produce spores under conventional culture conditions, an optimum culture medium is a Luria-Bertani culturing medium, an optimum temperature is 30DEG C, and an optimal pH value is 7.0; and an optimum condition of fungal infection pathogenicity is that the inoculation concentration is more than 10<8>/mL. The above bacterium has strong pathogenic poisoning effects on potato beetle larvae, has strong insecticidal activity on lepidopteran insects, can be processed through a bacterium fermentation technology to produce a safe and environmentally-friendly bacterial pesticide without pollution or residual, can also clone other insecticidal functional genes for research and development of transgenic beetle-resistant potato, and has latent commercial development and application values.

Description

One strain has the atrophy genus bacillus of poisoning and control action kou to colorado potato bug

Technical field

The invention belongs to the biological control technical field of Agricultural pests, particularly relate to a strain colorado potato bug is had to poisoning and the atrophy genus bacillus of control action kou.

Background technology

Colorado potato bug ( l. decemlineata, english abbreviation CPB) and be one of ten War Torn insects of generally acknowledging in the world, one of the great Quarantine Objects of Ye Shi China and important Invasive Alien Species.This worm is with the harm of adult and larval feeding, the host of hobby be cultivation potato ( solanum tuberosum) and eggplant ( s. melongena), often, by whole strain potato leaf food light, cause serious harm and production loss.With the harm investigation of China colorado potato bug generating area, be generally 20 ~ 50% because colorado potato bug endangers the production loss causing according to the literature, severe patient can reach more than 80%, even causes total crop failure.Simultaneously CPB also can propagate sprain disease ( alternaria alternata) and ring rot ( clavibacter michiganense subsp. sepedonicum) etc. multiple diseases.Ecological plasticity and the adaptability of colorado potato bug are extremely strong, more than 40 countries that progressively propagated in the past North America, Europe, Asia and Africa in a century and a half by U.S.'s Rocky Mountains east to northern Mexico region, produce and have formed serious threat world potato.According to incompletely statistics, the whole world exceedes US $ 15,000M because colorado potato bug endangers the financial loss causing every year.

China in 1993 finds colorado potato bug first in Xinjiang, over more than 20 year, its distance of propagating from west to east expansion has exceeded 850km, and the northern most regions of Xinjiang of China are distributed with colorado potato bug up to now, epidemic-stricken area enlarged areas hundreds of times.According to the propagation Diffusion Law of colorado potato bug, harm feature and suitable environment condition analysis, it further can be crossed In The Eastern Xinjiang propagation eastwards and diffuses to China Inner Mongol and Gansu Province and continue to go down south, and southwestern potato producing region will face grave danger.And at China's Northeast Regional, the risk that colorado potato bug is invaded by Russian coastal region is very high.Therefore there is from China's periphery epidemic situation the possibility increase that state imports in colorado potato bug.And the frequency that China's sanitary authority intercepts colorado potato bug by port quarantine in recent years is also increasing year by year.As can be seen here, the situation of the generation of colorado potato bug harm and further invasion diffusion is very severe, and China potato is produced and formed great potential threat, makes China carry out the work of colorado potato bug monitoring and prevention and control extremely urgent.

Potato is important in the world grain, Vegetable and forage dual-purpose crop and industrial raw material, especially potato provides abundant nutrition for human body, irreplaceable effect is being brought into play in raising grain security and energy security, the aspect of promoting economic development, be subject to very big concern and the attention of countries in the world.According to Food and Argriculture OrganizationFAO (FAO) statistics in 2011, there were more than 150 countries and regions plantation potato, potato planting area approximately 250,000 km in the whole world ², in global staple food crop, only after paddy rice, wheat, corn, occupy the 4th.China's potato planting industry development is in recent years very swift and violent, and according to statistics, nineteen eighty-three, national potato planting area was 25,600 km ², within 2010, reach record-breaking 53,000 km ², output exceedes 82MT, leaps to the first in the world, and wherein more than 70% potato planting area distributions is in NORTHWEST CHINA and southwestern poverty-stricken area; To " 12 " latter stage, cultivated area will exceed 70,000km ².China's potato outlet increases also very rapid in recent years, and outlet 0.7MT in 2003, reaches 1.0 MT for 2006, within 2011, exceedes 1.2 MT.China has not only become potato production, consumption and big export country, and progressively develops into potato and produce power.Be not difficult to find out, the development of potato planting industry not only has vital role for shaking off poverty and setting out on the road to prosperity of the vast poverty-stricken area of China, is also undertaking more and more important role with the related industries of potato planting industry in China's regional economy and industrial economy development.

Mainly adopt cultural control and use the chemical insecticide controls such as pyrethroid and neonicotine Xinjiang of China epidemic-stricken area prevention and control colorado potato bug at present, cultural control is time-consuming and be difficult to the effect that reaches desirable, and uses too much chemical insecticide may threaten the mankind's health and destroy ecotope etc.; The biological control of Agricultural pests has been subject to the attention of height in recent years.Natural enemy and pathogenic bacteria to colorado potato bug carried out more research both at home and abroad, successively reported more than the 20 kind of predator such as ladybug, tiger beetle, the sugared many spores actinomycetes of utilization thorn ( saccharopolyspora spinsada) pleocidin (spinsad) that is developed into etc., utilize the phototaxis of colorado potato bug also to have certain control effect to colorado potato bug, but these technology still have a serious limitation, fail widespread use aborning; Bacillus thuringiensis is because its toxalbumin has good prevention effect to farmland pest, now existing Bt commercial formulation obtains large-scale popularization application aborning, but it is reported that colorado potato bug also easily develops immunity to drugs to Bt preparation, under laboratory condition, just can produce very strong resistance (Guo Wenchao to Bt preparation through 6 ~ 12 generations, Tan Wanzhong etc., 2013; Wraight & Ramos, 2005); Although Bt preparation still can be controlled the population density of colorado potato bug now effectively, can find a kind of Bt substitute to alleviate resistance pressure and remain numerous scientific workers' problem demanding prompt solution.

In recent years, control along with the development of biochemical and molecular engineering technology and with bacterium the development that worm is studied, Agricultural pests scientist and chemistry of pesticide men pay close attention to the insecticidal activity of microbial metabolites more, for finding new natural compounds sterilant or providing important evidence for bionical synthesizing new sterilant.At present, various countries scholar has culture condition, sphere of action, separation and purification, insecticidal activity, active ingredient and the molecular structure etc. of the pathogenic bacteria of better control action kou to be studied some to insect.Utilize pathogenic micro-organism, particularly some host selection germ carrys out the growth and breeding of Control pests, has demonstrated good development prospect, is also the important channel of finding novel pesticide.

Colorado potato bug in the generation area of China still in continuous expansion, endanger and the financial loss that causes also more and more serious, and its invasion is very high to other vast potato producing region risks of China, and national potato crop has been formed safely to very large threat.Therefore, seek the pernicious insect of this quarantine to have the pathogenic micro-organism of strong cytotoxicity and biological and ecological methods to prevent plant disease, pests, and erosion control action kou, further develop thus noresidue, biotic pesticide free from environmental pollution, important business development using value will not only be there is, and can control safely and effectively causing harm of colorado potato bug, with the tremendous economic loss of avoiding this worm to cause.

Except Bacillus anthracis ( b. anthracis) and waxy Bacillus ( b. cereus) minority strain be the pathogenic bacterium of humans and animals body and outer, some other bacillus ( bacillus) kind of bacterium mostly is the biocontrol microorganisms of crop pest and pathogenic bacteria, by large quantity research both at home and abroad, wherein by Bacillus thuringiensis ( b. thuringiensis) pest-resistant cultivar such as the sterilant of producing and cotton, corn and the Caulis et Folium Brassicae capitatae of cultivating from a series of insecticidal function genes of this bacterium clone has been widely used in preventing and treating the Severe pests such as pine moth, cabbage caterpillar and bollworm in agriculture, forestry; With subtilis ( b. substilis) biological bactericide of fermentative production has also been used to the crop pests such as large area control rice sheath blight disease and bacterial leaf-blight in China.Atrophy genus bacillus is a kind of Gram-positive and aerobic bacteria, and its form is similar to subtilis, and key distinction feature is on the substratum that contains organic nitrogen source, to form a large amount of melanochrome.Once some strains of this kind of bacterium were accredited as in the past " mutation of subtilis black " ( b. substilisvar. niger), black genus bacillus ( b. niger) or ball spore genus bacillus ( b. globogii), Nakamura (1989) proposes first " subtilis " strain called after atrophy genus bacillus that some are produced to pigment after having compared the product pigment strain of a large amount of subtilises and not produced pigment strain, and Fritze and Pukall (2001) also name other several bacterial strains into atrophy genus bacillus; The strain having in this bacterium is so far produced bacterium at industrial surface sterilization bacterium or the restriction enzyme of being used as, the strain having in addition be also used as research people Bacillus anthracis ( b.anthracis) nontoxic alternative bacterium.But, pathogenic effects and the biological and ecological methods to prevent plant disease, pests, and erosion application of atrophy genus bacillus to Agricultural pests, home and abroad all has no research report so far.

Summary of the invention

The object of this invention is to provide a strain has poisoning and control action kou atrophy Bacillus strain (CPB072) to colorado potato bug.The present invention atrophy genus bacillus that separation and purification obtains from physical environment ( bacillus atrophaeus) bacterial strain, be a new biocontrol strain of finding first from colorado potato bug and identifying, according to morphological feature, physio-biochemical characteristics and this bacterium 16S rDNA conserved sequence analysis be accredited as atrophy genus bacillus ( bacillus atrophaeus), determine its growth product spore and infected pathogenic optimum condition by test determination.

The described thalline Strategy of Alien Invasive Species colorado potato bug important to China ( leptinotarsa decemlineata(Say)) have very strong pathogenic toxic action, its pathogenic control effect to 1 age and 2 instar larvaes is best, the existing bacillus thuringiensis that uses in field and shop experiment are all better than producing ( bacillus thuringensisbt) pesticide product, lepidoptera pest is also had to very strong toxic action, can produce by zymophyte body technique the bacillary sterilant of safe environment protection type of pollution-free noresidue, also may clone its insecticidal function gene for the anti-beetle potato of research and development transgenosis, there is potential business development and using value.

the separation and purification of bacterial strain:gather the colorado potato bug of natural death in the potato plot of not spraying any agricultural chemicals in Xinjiang Yili of China, Urumchi and area, Changji, be put in clean test tube and number for subsequent use, adopt Luria-Bertani substratum (LB) and bacterium separation method, obtain 128 Bacillus strains through indoor tissue culture and purifying, demonstrate,prove disease method with Ke He Shi and determine pathogenic bacterium wherein, relatively screen and obtained the atrophy genus bacillus CPB072 bacterial strain that colorado potato bug is had to strong toxic action finally by mistake pathogenicity.

morphological specificity, physio-biochemical characteristics and the Molecular Identification of bacterial strain: upper at Luria-Bertani substratum (LB), colony growth rate is very fast, and this bacterium colony oyster white is moistening, slightly rounded, flats, and edge is more neat, without mucus, more abundant, has wax layer, easily provokes (Fig. 2).Gramstaining is observed, and cell is shaft-like, and thalline is purple, and reaction is positive; Tool cylindricality gemma, the not painted rarely seen refractive body (Fig. 2) with profile that has of gemma.Thalline size is 1.79 ~ 2.19 × 6.83 ~ 7.29 μ m, according to these morphological specificitys and " uncle Jie Shi systematic bacteriology handbook " (the 8th edition) identify this bacterium be genus bacillus ( bacillussp.).

The physio-biochemical characteristics of bacterial strain: measured the physio-biochemical characteristics (table 1) of CPB072 bacterial strain, result shows the characteristic close of itself and atrophy genus bacillus.

table 1 CPB072 bacterial strain physio-biochemical characteristics

Note: "+" represents positive, "-" represents negative

Extract strain gene group DNA, with bacterium universal primer 27F (5 '-AGAGTTTGATCCTGGCTCAG-3 ') and 1541R (5 '-AAGGAGGTGATCCAGCC3 ') pcr amplification, amplified production is connected to PMD18-T carrier, be transformed into bacillus coli DH 5 alpha, order-checking obtains the 16S rDNA conserved sequence of this bacterium, its length is 1511bp, is KF751643 in the accession number of GenBank.With Blast sequence alignment with DNAStar software analysis and set up systematic evolution tree with MegAlign and be accredited as atrophy genus bacillus.

According to above morphology and physiological and biochemical property, in conjunction with 16S rDNA conservative gene Sequence Identification result and physio-biochemical characteristics, this bacterial isolates to be named as atrophy genus bacillus, Latin name is bacillus atrophaeus.This bacterial classification has been deposited in (address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, China Committee for Culture Collection of Microorganisms's common micro-organisms center on November 12nd, 2013, postcode: 100101, phone: 010-64807355), preserving number is CGMCC No. 8449.

the optimum growh breeding of atrophy genus bacillus CPB072 bacterial strain and inoculation infection condition: this bacterium grows the fastest on LB substratum, 30 DEG C of growth optimum temperutures, and Optimum pH is pH7.0, under these conditions, colony growth is fast.The pathogenic top condition of infection process is that inoculum density is 10 ⁸individual/mL order of magnitude, moisturizing 3 ~ 4h after inoculation, and keep 25 ~ 30 DEG C of temperature in the whole phase of infecting.

the insecticidal effect of atrophy genus bacillus CPB072 bacterial strain: atrophy genus bacillus has good pathogenic effects and growth inhibitory effect to colorado potato bug 1 and 2 instar larvaes.Mainly cause that food ingestion declines, and grows slowly, and causes very soon polypide death.After indoor bacterial suspension Direct spraying inoculation colorado potato bug polypide, after 12h, can fall ill, after 7d, larval mortality reaches 52.60%, median lethal concentration(LC&-{50}) (LC ₅₀) be 3.279 × 10 ⁸individual/mL, median lethal time (LT ₅₀) be 4.8d, dead polypide blackening is also flowed out tawny liquid (Fig. 3); Use after this suspension in field, and 7d " Invest, Then Investigate " colorado potato bug mortality ratio reaches 37.80%, is significantly higher than the insecticidal effect (25.78 %) of conventional sporeine preparation in local production.In addition, CPB072 is stronger to the insecticidal activity of lepidopterous insects, and after indoor inoculation lepidopteran represents insect silkworm larva 7d, mortality ratio reaches 72.56%.

advantage of the present invention is: from physical environment, separate the atrophy Bacillus strain CPB072 obtaining, it is the biocontrol strain that China finds first and identifies on colorado potato bug, its thalline has very strong causing a disease and control action kou to the important Strategy of Alien Invasive Species colorado potato bug of China, can pass through the bacillary sterilant of environment-friendly type of thalline fermentation technique production noresidue, can also clone its insecticidal function gene for the anti-beetle potato of research and development transgenosis, alleviate the resistance that insect produces bacillus thuringiensis insecticidal proteins.There is potential development and application values and business promotion benefit.

Brief description of the drawings

fig. 1. colorado potato bug and harm thereof

Note: a is larval feeding harm; B is imago feeding harm

fig. 2. bacterium colony and the thalli morphology of atrophy genus bacillus CPB072

Note: a is the colonial morphology of (24h) on LB substratum; B is gramstaining thalli morphology

fig. 3. the insecticidal effect of atrophy genus bacillus CPB072: lethal colorado potato bug

Note: a is the inoculation test of the indoor larva of feeding; B is that dead larva is flowed out with brown liquid

fig. 4. the toxic effect of atrophy genus bacillus CPB072 to lepidopteran (silkworm) insect

Note: a is the inoculation test of the indoor silkworm of feeding; B is dead polypide, and polypide blackout has brown liquid to flow out.

Embodiment

embodiment 1: the separation and purification of atrophy genus bacillus and screening scheme

atrophy genus bacillus separation and Culture: with Luria-Bertani substratum (LB), its formula is Tryptones 1%, yeast powder 0.5%, NaCl1%, agar 2%, tap water 1000mL, pH7.0.By the agar weighing up, heat fused; Separately take yeast powder, Tryptones and NaCl, be dissolved in after a small amount of hot water and add and mix, then add water and be settled to 1000 mL.Regulate pH with NaOH.After packing test tube or triangular flask, 121 DEG C of sterilizing 25 min.Heating and melting before using, pours in sterilizing culture dish or test tube and makes flat board or slant medium, cultivates or bacterial classification preservation for strains separation.

separation and Culture and purifying: from natural condition, gather dead colorado potato bug sample, get dead worm with aseptic tweezers and immerse about 2s in 70% alcohol, after polypide surface wettability, moved into surface sterilization 1～2min in the 0.1% mercuric chloride aqueous solution.By sterile water wash 3 times, then polypide is proceeded in the finger line pipe that 0.85% stroke-physiological saline solution is housed, gently crush with aseptic glass stick, brown liquid is flowed out and make bacteria suspension.By 75 DEG C of water-bath 15min of bacteria suspension, under aseptic condition, get 0.1mL bacteria suspension on solid medium flat board, after coated plate, cultivate 48h for 30 DEG C, after bacterium colony grows, PHENOL 99.8 MIN ((CARBOLIC ACID)) azaleine microscopy for the similar atrophy genus bacillus of picking, by the atrophy genus bacillus line detecting, separation and purification, transfer to again on LB test tube slant substratum, and numbering is indicated and is saved backup bacterial strain.

strong pathogenic bacterium screening: by measuring the active preliminary screening biocontrol strain of degrading enzyme (chitinase and proteolytic enzyme) of bacterial strain, by chitin agar (CA) substratum, (formula is: NH ₄h ₂pO ₄1 g, KCl 0.2 g, MgSO ₄7H ₂o 0.2 g, chitin quality 1 % (w/v), agar 20 g, distilled water 1000 mL, pH 7.0, fully dissolve, 121 DEG C of sterilizing 25 min), milk nutrient agar (formula is: 55 DEG C time, 50 mL 4 % water agar and 50 mL skimmed milks fully vibrate evenly) is made flat board after melting under aseptic condition, be seeded in flat board under aseptic condition separating the bacterial strain obtaining, the symmetrical inoculation of every ware 3 points, each bacterial strain repeated inoculation 3 times, then be placed in constant incubator (RH 100 %, 28 DEG C) cultivation 3d, observe periphery of bacterial colonies and whether have Clear & Transparent annulus, having the bacterial strain of Clear & Transparent annulus screened is potential colorado potato bug biocontrol bacteria.

By the colorado potato bug in the different length of times be positioned over diameter be 9cm(beetle length of time large be 15cm) culture dish in, every ware is placed 10 of the close and healthy beetles of size in same length of time.All pathogenic bacterias in above-mentioned test with degrading enzymatic activity are made to 10 ⁸the bacteria suspension body of individual/mL order of magnitude, is evenly sprayed at polypide upper, fully to contact and to be advisable, add fresh potato blade, moisturizing 4 ~ 6h, maintains the temperature at 25 ~ 30 DEG C, records dead polypide every day and the beetle of survival is moved into and in another sterilizing culture dish, adds appropriate fresh potato blade.Under same concentration, the first meeting death time is shorter, and what beetle mortality ratio was the highest is strong pathogenic strains.

the qualification of pathogenic bacteria: 1) morphological observation and qualification. on LB flat board, cultivate pathogenic bacteria, observe colonial morphology, colony growth 36h left and right in good time and carry out gramstaining; 2) physio-biochemical characteristics test. according to the method for the elegant pearl in east etc., measure altogether 12 kinds of physio-biochemical characteristics of atrophy genus bacillus; 3) molecular biology identification. the technology of setting up according to Weisburg, extract the genomic dna of bacterial strain, with bacterium universal primer 27F (5 '-AGAGTTTGATCCTGGCTCAG-3 ') and 1541R (5 '-AAGGAGGTGATCCAGCC3 ') pcr amplification, amplified production is connected to PMD18-T, transform bacillus coli DH 5 alpha, order-checking obtains the 16S rDNA conserved sequence of this bacterium, and sequence is signed in in GenBank; This sequence is done to Blast and analyze, adopt DNAStar software analysis and the genus bacillus kind that similarity is high in Blast contrast to compare simultaneously, determine the classification position of this genus bacillus.

embodiment 2: the cultivation of atrophy genus bacillus and inoculation condition testing program

humid test: fresh bacterial classification (inoculation culture 12 ~ 24h) is seeded on LB solid culture flat board, (more than 37 DEG C) that temperature is higher are inoculated in liquid nutrient medium, establish respectively 4 DEG C, 20 DEG C, 30 DEG C, 37 DEG C, 41 DEG C, 45 DEG C and 65 DEG C of 8 temperature preliminary treatments, each processing repeats 3 times, and 37 DEG C are placed in above water-bath and cultivate.Be placed in pre-set temperature required incubator and constant temperature water bath and cultivate, and observe growing state day by day.Choose after temperature range, carry out further humid test, according to tentative experiment situation, different processing is set.Determine thus the optimum growth temperature of atrophy genus bacillus.

potential of hydrogen experiment:experiment adopts LB liquid nutrient medium, and 11 potential of hydrogen processing are set altogether.With NaOH and the HCl solution of 1.0 mol/L, the pH value of substratum is adjusted to respectively to 2.0,3.0,4.0,5.0,6.0,7.0,8.0,9.0,10.0,11.0 and 12.0 (by electronics pH meter mensuration) respectively, each processing repeats 3 times; Germ inoculation and observation procedure are with 2.1.Connect bacterium processing and be placed on 250rpm shaking table cultivation at 30 DEG C.Determine thus the optimal ph of atrophy genus bacillus growth.

the screening of kinds of culture medium: the impact of six kinds of substratum on atrophy genus bacillus colony growth compared in experiment altogether, except LB substratum, other five kinds of substratum respectively: 1) WA substratum: agar 20g, distilled water 1000mL; 2) PDA substratum: potato 200g, glucose 20g, agar 20g, distilled water 1000mL; 3) PSA substratum: potato 200g, sucrose 20g, agar 20g, distilled water 1000mL; 4) PPDA substratum: potato 200g, glucose 20g, agar 20g, peptone 10g, distilled water 1000mL; 5) PPSA: potato 200g, sucrose 20g, agar 20g, peptone 10g, distilled water 1000mL.Preparation PDA, PSA, first the potato of aequum is shredded and put into water well-done (approximately boiling 30min), then cross and filter out residue and be settled to 1000mL, finally add again the chemical substance composition of aequum, stir, contain respectively in triangular flask for subsequent use after moist heat sterilization.The pH value of various substratum is about 6.8-7.0,30 DEG C of culture temperature.Determine thus the optimal medium of Pathogen culture.

gramstaining reaction: 1) reagent: A: solution I: Viola crystallina 20g, 95% ethanol 20mL; Solution II: ammonium oxalate crystallization 0.8g, distilled water 80mL, solution I and solution II are mixed into ammonium oxalate crystal violet; B:Lugol iodine liquid (being stored in coloured ground glass stoppered bottle): iodine 1g, potassiumiodide 2g, distilled water 300mL; C: counterstain: sarranine (2.5% 95% spirituous solution) 10mL, distilled water 100mL; 2) staining procedure: 1. smear: get a clean slide, with extraordinary pen bacterium number on the note of the left and right sides of slide glass, and at two ends an each droplet distilled water, paint very thin water membrane with a small amount of thalline of aseptic inoculation ring picking, on spirit lamp flame, cross 2～3 times dry fixing; 2. just dye: slide glass is placed on level attitude, on thalline, drips ammonium oxalate crystal violet liquid, dyeing 1min, the dye liquor that inclines, it is colourless that rill water rinses to elutant; 3. mordant dyeing: wash away the water on painting face with the Lugol iodine liquid of newly joining, then dye 1min with the topped painting face of Lugol iodine liquid, wash; 4. decolouring: hand is held slide glass, drips 95% ethanol decolorization 20 ~ 30s, and while disappearance to elutant purple crystal, water rinses, and stops decolouring; 5. redye: get rid of the water unloading on slide, on painting face, drip sarranine dye liquor, dyeing 2～3min, washing, dry; 6. microscopy: detect coloration result under oily mirror.Determine thus the gram character of atrophy genus bacillus and measure thalline size.

Biophysical and biochemical tests: experiment determines 12 kinds of physio-biochemical characteristics of atrophy bacillus altogether, they and assay method thereof respectively: 1) utilization of carbon source: Culture medium: (NH ₄) ₂SO ₄2.0g, NaH ₂PO ₄H ₂O 0.5g, MgSO ₄7H ₂O 0.2g, CaCl ₂2H ₂O 0.1g, carbon source (determine four kinds, D-Glucose, D-sucrose, D-wood sugar, sweet mellow wine etc. altogether) 5g, distilled water 1000mL; Inoculation with observe: with fresh bacterial classification (bacteria suspension) inoculation, cultivate at 30 DEG C, raw elder is positive; 2) nitrogenous source utilization: Culture medium: KH ₂PO ₄1.36g,Na ₂HPO ₄2.13g, MgSO ₄7H ₂O 0.5g, NaCl 2.5g, glucose 10.0g, distilled water 1000mL, nitrogenous source (determine NH altogether ₄H ₂PO ₄And KaNO ₃Two kinds) 2.5g; Inoculation with observe: with fresh bacterial classification (bacteria suspension) inoculation, cultivate at 30 DEG C, raw elder is positive; 3) citrate utilization: Culture medium: NaCl1g, MgSO ₄7H ₂O 0.2g, NH ₄H ₂PO ₄0.5g, natrium citricum 2g, distilled water 1000mL, 0.04% phenol red solution 20mL, Inoculation with observe: with fresh bacterial classification (bacteria suspension) inoculation, cultivate at 30 DEG C,Nutrient solution becomes blueness or pink positive, otherwise negative 4) catalase: Reagent: 3% hydrogen peroxide, Inoculation with observe: the slant strains that 24h is cultivated, get a little ring with platinum filament oese and be applied in and drip on the slide that has 3% hydrogen peroxide, produce then positive if any bubble, bubble-free is negative; 5) glycitols fermentation: Culture medium: (NH ₄) ₂HPO ₄1.0g, KCl 0.2g, MgSO ₄0.2g, yeast extract 0.2g, agar 5.5g, sugar or alcohols (determine four kinds, D-Glucose, D-Fructose, D-wood sugar, sweet mellow wine etc. altogether) 10.0g, distilled water 1000mL, 0.04% bromocresol purple solution 15mL, pH7.0, packing test tube, highly about 5cm; Inoculation with observe: with children age slant culture percutaneous puncture-inoculation in culture medium, thermophilic cultivate, 1,3 and 5d after observe, indicator turn yellow represent produce acid positive; Constant or to become blue (purple) negative;6) methyl red (M-R) test: Culture medium: peptone 5g, glucose 5g, NaCl 5g, distilled water 1000mL, pH7.0, packing test tube, it is high that every pipe is about 5cm; Reagent: methyl red 0.1g, 95% ethanol 300mL, distilled water 20mL; Inoculation and observation: inoculation test bacterium, in culture medium, is put thermophilic cultivation 2,6d, adds a methyl red reagent in nutrient solution, red is methyl red test positive reaction, yellow negative reaction; 7) V-P measures: Culture medium: test identical with methyl red (M-R); Reagent: 0.3% creatine, 40%NaOH; Inoculation and observation: inoculation test bacterium is in culture medium, and put thermophilic cultivation 2,6d, get nutrient solution and 40% NaOH mixed in equal amounts, add a little creatine, 10min occurs that as nutrient solution redness is positive reaction, constant negative; 8) nitrate reduction: Culture medium: MgSO ₄0.5g, NaCl 0.5g, K ₂HPO ₄0.5g, KNO ₃1g, sucrose 20g, distilled water 1000mL, pH7.2; Reagent: Griess reagent (A liquid: sulfanilic acid 0.5g, 10% spirit of vinegar 150mL; B liquid: а-naphthylamines 0.1g, distilled water 20mL, 10% spirit of vinegar 150mL); Diphenylamines reagent: 0.5g diphenylamines is dissolved in the 100mL concentrated sulfuric acid, uses 20mL distilled water diluting; Inoculation and observation: strain inoculation will be measured in culture medium, nutrient solution imports in clean empty test tube after cultivating 1,3 and 5d by thermophilic, respectively adds an A liquid and B liquid,Solution becomes pink, rose, orange, brown etc. represents the nitrate reduction positive, if redfree occurs, then adding one, two diphenylamines reagent, now to represent nitrate reduction in blueness negative, if not in blueness, positive; 9) urease test: Culture medium: KH ₂PO ₄2g, peptone 1g, NaCl 5g, glucose 1g, 0.2% phenol red aqueous solution 6mL, agar 20g, distilled water 1000mL, pH6.8 ~ 6.9 after sterilizing, are adjusted to make culture medium in crocus or micro-band pink, packing test tube, add 20% sterilizing urea liquid, make the urea total concentration be 2%, be then put into larger inclined-plane for subsequent use; Inoculation with observe: fresh slant culture is inoculated on inclined-plane, and respectively at 2,4h observes, culture medium is positive in pink, and culture medium color is constant negative; 10) indole reaction: Culture medium: the 1% tryptone aqueous solution, pH7.3, packing 1/3 test tube; Reagent: Paradimethylaminobenzaldehyde 8g, 95% ethanol 760mL, dense HCl 160mL; Inoculation and observation: strain inoculation will be measured in culture medium, thermophilic is cultivated 1,2,4 and 7d, slowly add the reagent that 3 ~ 5cm is high in nutrient solution surface along tube wall, then positive reaction of redness is there is at liquid layer interface, negative without change color, if color is not obvious, 4 ~ 5 droplet ether then can be added, then color is obvious to shake rear static a moment; 11) gelatin liquefaction: Culture medium: peptone 5g, glucose 20g, gelatin 200g, distilled water 1000mL, pH7.3; Inoculation and observation: fresh slant culture is inoculated in media surface, under thermophilic, cultivates, observe its Degree of Liquefaction at 5,10,20 and 30d respectively, if liquefaction, positive, do not liquefy negative; 12) H ₂S produces: Culture medium: peptone 10g, ironic citrate 0.5g, distilled water 1000mL, pH7.2; Inoculation and observation: strain inoculation, in culture medium, is produced melanin and then indicates H after thermophilic is cultivated a period of time ₂S is produced as the positive, is produced as feminine gender without melanin.Above substratum is moist heat sterilization 25min at 121 DEG C all.

embodiment 3: the indoor pathogenicity of germ: the bacterium colony of activation is washed till in triangular flask with sterilized water, and fully vibration is even, and does suitably dilution and make spore suspension, and its concentration is 10 ⁸cells/mL and the above order of magnitude thereof.The sterilizing culture dish that 1 age of colorado potato bug and 2 instar larvaes is placed in to diameter 15cm (is lined with one deck thieving paper in culture dish, 10, every ware) in, draw with liquid-transfering gun the spore suspension having configured and be evenly sprayed at around polypide body wall, fully to contact and to be advisable.Treat that polypide body wall is more dry, when thieving paper is more dry, add certain clean fresh potato blade, then cover gauze, be placed in room temperature, allow its growth.Separately spray examination worm in contrast with sterilizing clear water.Each bacterial strain is processed and contrast arranges 3 repetitions.

embodiment 4: pathogenecity top condition: the shadow that General Influence insect pathogenic bacteria infects mainly contains concentration, temperature and moisture preserving time.5 concentration (3.76 × 10 are set ⁸individual/mL, 0.75 × 10 ⁸individual/mL, 0.15 × 10 ⁸individual/mL, 0.30 × 10 ⁷individual/mL and 0.60 × 10 ⁶individual/mL), and 5 temperature between 20 ~ 30 DEG C are set, moisture preserving time 0,2,4,6 and 8h, each concentration, temperature and moisture preserving time are processed 30 colorado potato bugs.The suitableeest inoculation onset condition (inoculum density, temperature, moisture preserving time) and the LC of Analysis deterrmination germ thus ₅₀.

embodiment 5: effect test is controlled in field: field test is experimental field carried out Institute of Plant Protection, Xinjiang Academy of Agricultural Science's experiment and demonstration base potato (kind is pale reddish brown white, and conventional production management, does not spray any agricultural chemicals, and potato growing way is basically identical).Field test examination worm be 1 age and 2 age CPB larva.Be different communities by experimental plot random division, potato 5 strains are chosen in each community.Test before by every strain potato plant people for being divided into 3 layers of upper, middle and lower, each plant connects worm consistent larva in age, after 1 d, larva grows surely, determines that each instar larvae insect population radix is 15 every layer, 45 of every strains, each community covers with homemade gauze.The spore suspension preparing is loaded on respectively in hand sprayer, be sprayed on plant and polypide surface, taking sporeine preparation (32000IU/mg WP) as experimental control, each bacterial strain and control treatment San Ge community, quantitative check insect population quantity and potato plant extent of injury.After dispenser, temperature continues at 25 ~ 35 DEG C, and one week interior without rainfall.From spray medicine day, investigate once every day, and 3 strain potatos are arbitrarily chosen in each community, and the insect population quantity of investigation colorado potato bug, records every strain potato plant extent of injury, according to severity Scaling criterion calculation insect population decline rate and prevention effect, and continuous observation 10 d.

embodiment 6: to the toxic effect test of lepidoptera pest: atrophy genus bacillus is made to cell suspending liquid, and its concentration is 3.76 × 10 ⁸cells/mL.The representative test worm (because easily obtaining for examination worm source) taking silkworm as lepidopteran, select structure in the length of time healthy silkworm larva consistent with upgrowth situation, the sterilizing culture dish that is placed in diameter 15cm (is lined with one deck thieving paper in culture dish, 15, every ware) in new fresh mulberry leaf on, drawing with liquid-transfering gun the cell suspending liquid preparing is evenly sprayed at silkworm body body wall around and on mulberry leaf blade, is advisable fully to contact polypide.After dry on silkworm body, blade and thieving paper surface, cover gauze, be placed in room temperature and grow.Separately spray and process in contrast with sterilizing clear water, each bacterial strain is processed and contrast arranges 3 repetitions.

Claims (1)

1. a strain has the atrophy genus bacillus of poisoning and control action kou to colorado potato bug bacillus atrophaeus cPB072,its preserving number is CGMCC No. 8449.